The studies on hormone replacement therapy (HRT) in females with estrogen deficiency are not conclusive. Thus, non-estrogen therapies, such as atorvastatin (ATO), could be new strategies to substitute or complement HRT. This study evaluated the effects of ATO on mesenteric vascular bed (MVB) function from ovariectomized (OVX) female rats. Female rats were divided into control SHAM, OVX, and OVX treated with 17β-estradiol (EST) or ATO groups. The MVB reactivity was determined in organ chambers, vascular oxidative stress by dihydroethidine staining, and the expression of target proteins by western blot. The reduction in acetylcholine-induced relaxation in OVX rats was restored by ATO or EST treatment. The endothelium-dependent nitric oxide (NO) component was reduced in OVX rats, whereas the endothelium-derived hyperpolarizing factor (EDHF) component or prostanoids were not altered in the MVBs. Endothelial dysfunction in OVX rats was associated with oxidative stress, an up-regulation of iNOS and NADPH oxidase expression and a down-regulation of eNOS expression. Treatment with ATO or EST improved the NO component of the relaxation and normalized oxidative stress and the expression of those signaling pathways enzymes. Thus, the protective effect of ATO on endothelial dysfunction caused by estrogen deficiency highlights a significant therapeutic benefit for statins independent of its effects on cholesterol, thus providing evidence that non-estrogen therapy could be used for cardiovascular benefit in an estrogen-deficient state, such as menopause. The role of estrogens in vascular function has received considerable research interest because epidemiological studies have shown a greater risk of developing cardiovascular disease (CVD) due to reduced 17β-estradiol levels after menopause . One of The main mechanisms involved in the impaired vascular response in estrogen deficiency models are connected decreased nitric oxide (NO) bioavailability and the attenuation of hyperpolarization and relaxation transduced by endothelium-derived hyperpolarizing factor (EDHF) –8. This Recent experimental and clinical evidence has suggested that statins  reductase inhibitors) have cholesterol-independent (“pleiotropic”) effects. Statins are extremely safe but can produce myalgia and rarely rhabdomyolysis . AdditioLike estrogen, statins exert vasoprotective effects that are independent of their lipid-lowering action –20. The Although statins are able to reduce the risk of coronary events and mortality in patients with coronary artery disease ,19, studTherefore, we performed this study to evaluate the effects of atorvastatin on vascular reactivity in mesenteric beds from ovariectomized female rats and the involvement of NO, EDHF and NADPH oxidase in these mesenteric resistance arteries.All of the procedures were conducted in accordance with the biomedical research guidelines for the care and use of laboratory animals, as stated by the Brazilian College of Animal Experimentation (COBEA). The experimental protocol was approved by the Ethics Committee in Animal Experimentation of the Federal University of Espirito Santo under the number 069/2011.ad libitum. Four groups were studied : sham-operated females (SHAM); ovariectomized females (OVX); ovariectomized females treated with 17β-estradiol  or ovariectomized females treated with atorvastatin (ATO: 20 mg/kg/day). The experiments were performed using eight weeks-old female Wistar rats weighting 180 to 200 g. Throughout the experiment, the animals were housed in groups in a temperature- (22 °C) and humidity- (50%) controlled room with a 12-h (light) – 12-h (dark) cycle. Standard rat chow and tap water were available Bilateral ovariectomy was performed in female rats under ketamine (70 mg/kg) and xylazine (10 mg/kg) anesthesia by intraperitoneal injection (i.p). The females were subjected to a muscular incision to open the peritoneal cavity for posterior connection of the uterine tubules and removal of the ovaries. Then, the peritoneal cavity was sutured and cleaned. The female sham group only underwent an incision. Next, the animals were allowed to recover. Twenty-one days after surgery, the ovariectomized female rats were subcutaneously given 17β-estradiol diluted in peanut oil (EST group) or atorvastatin through gavage (ATO group). The sham and ovariectomized (OVX) groups received only the vehicle. Those treatments lasted 14 days. As previously described , the effDaily vaginal smears were taken from each female sham rat as previously described  to config for 10 minutes), and serum was stored at −20 °C. The LDL- and total- serum cholesterol were measured using automated equipment . Blood samples were collected on the day of the vascular reactivity experiments by aortic puncture, after the cannulation of the superior mesenteric artery. All of the samples were centrifuged (1500 in vitro with oxygenated (95% O2- 5% CO2) Krebs-Henseleit solution  at a constant flow rate of 4 ml/min and maintained at 37 °C. The MVB were excised from the intestinal wall, placed in a chamber, and the preparations were allowed to stabilize for 30 min before the beginning of the experiments. Changes in the perfusion pressure, which reflect peripheral resistance, were measured with a pressure transducer, (Spectramed P23XL) connected to an acquisition system , and were calculated as percentage of reduction in the perfusion pressure after noradrenaline (NE)-induced contraction. After a stabilization period, noradrenaline (0.1 to 0.3 mM) was added to the perfusion fluid to increase the tone by approximately 90-120 mmHg. Once a stable tone was established, concentration-response curves to acetylcholine  were determined in the MVB. The ACh curves were performed initially in each MVB without any inhibitors. To evaluate the effect of NO availability on vascular reactivity, the preparations were treated with the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester  and the inducible NO synthase (iNOS) inhibitor aminoguanidine  . For each concentration-response curve, the maximal response (EP<0.05); however, atorvastatin did not alter the BW in the OVX group. Conversely, ovariectomy produced a significant decrease in the uterine weight and the ratio of uterine weight to tibia length compared to the SHAM animals. ATO did not modify this parameter. The uterine weight to tibia length ratio returned to normal values after 17β-estradiol treatment. Ovariectomy neither the treatments used in the present study were able to alter the LDL- and total- serum cholesterol  among the groups , neither in the perfusion pressure after the increase of the vascular tone with noradrenaline . Acetylcholine induces relaxation in the MVB in a dose-dependent manner . In the  studied . Nitric oxide modulation of ACh-induced relaxation was evaluated using AG and L-NAME incubation. The MVB preparations treated with these inhibitors showed similar perfusion pressure values  among the groups. In the OVX rats, ACh relaxation was significantly attenuated by AG, but AG did not modify the response in MVBs from the SHAM group. Treatment with ATO or EST prevented the endothelial alterations observed in the OVX group, as shown by the dAUC values  , Table 22 values after ACh-induced relaxation in the MVBs in any of the groups . When applied concomitantly, these drugs failed to further increase ACh-induced relaxation and Ee groups , Table 2e groups , inset, max or pD2 of the concentration-response curves occurred in the presence of L-NAME plus INDO compared to L-NAME alone (The putative role of prostanoids was assessed by the dAUC (%) values in the presence of L-NAME and after inhibition with both L-NAME and INDO, which showed no differences among the groups . In addiME alone . These rTo evaluate tissue ROS production, DHE staining was performed in the mesenteric arteries. At baseline, DHE red fluorescence analysis revealed an increased production of superoxide anion from the mesenteric vessels in the OVX rats compared to the SHAM rats. Treatment with ATO or EST for 2 weeks corrected the enhanced ROS production in the mesenteric arteries from the OVX rats .Ovariectomy reduced eNOS and increased iNOS protein expression in the mesenteric branches ; in addiThe present study indicates that the treatment with atorvastatin is sufficient to reverse the endothelial dysfunction observed in female rats with estrogen deficiency. Importantly, our experiments also demonstrated that functional changes in the MVBs were associated with molecular adaptations. Namely, our major novel finding is demonstrating that increased ROS production, NADPH oxidase and iNOS overexpression and reduced eNOS expression in OVX mesenteric vessels, which can lead to reduced NO availability, were restored by atorvastatin and estrogen replacement.The decrease in the relaxation response after acetylcholine administration to MVBs from ovariectomized rats is most likely due to increased negative endothelial modulation ,20,43,45There is particular interest in the endothelial NO system and endogenous estrogen deprivation, in light of the relevant functional role of endothelial NO on regulating vascular tone ,52. HoweThe expression of iNOS has been documented in vascular endothelial and smooth muscle cells after either inflammatory or cytokine stimulation –60, in fIn conjunction with our functional data, the DHE analysis revealed that the enhanced production of ROS in the OVX rats was dramatically reduced by atorvastatin. Consistent with these results, we found that statin treatment reduced NADPH oxidase and iNOS expression in the mesenteric vessels. Moreover, ATO-treated OVX rats had normalization of eNOS expression, similar to the estrogen-treated rats. Taken together, these results showed that atorvastatin was able to reverse endothelial dysfunction in resistance vessels from OVX rats by restoring NO availability, preventing oxidative stress and normalizing the expression of important signaling pathways enzymes.The present study agree with and extend previous evidence supporting statins’ effects on modulating oxidative stress ,27,31,62Several reports have also suggested that ROS production during estrogen deprivation may lead to the development of endothelial dysfunction –70, espeDespite these ﬁndings, the involvement of other vasoactive substances that counteract the vasodilatory effect of ACh cannot be ruled out. Our study examines the impact of atorvastatin on endothelial dysfunction in ovariectomized rats and its effect on the NO pathway and intravascular oxidative stress, without modulating EDHF and prostanoid function and COX-2 vascular expression. The inhibitory effect of L-NAME on ACh-induced relaxation was not modified by COX blockade, based on pharmacological analysis using indomethacin. The double blockade (L-NAME plus INDO) revealed that EDHF was not altered by estrogen deficiency or atorvastatin treatment in the MVBs, as also shown by the %dAUC values, suggesting that NO might be reduced in the OVX rats independent of EDHF, which has been reported by others . FurtherIn conclusion, our results indicate that endothelial dysfunction secondary to estrogen deficiency in ovariectomized rats was normalized by atorvastatin through NO-mediated mechanisms and reduced oxidative stress. This experimental evidence partially explains the atheroprotective properties of statins independent of their effects on cholesterol and provides insight into the development of new therapeutic strategies to treat menopausal endothelial dysfunction. Large clinical trials are necessary to confirm if these beneficial effects are maintained in a long period.


Redox enzyme maturation proteins (REMPs) bind pre-proteins destined for translocation across the bacterial cytoplasmic membrane via the twin-arginine translocation system and enable the enzymatic incorporation of complex cofactors. Most REMPs recognize one specific pre-protein. The recognition site usually resides in the N-terminal signal sequence. REMP binding protects signal peptides against degradation by proteases. REMPs are also believed to prevent binding of immature pre-proteins to the translocon. The main aim of this work was to better understand the interaction between REMPs and substrate signal sequences. Two REMPs were investigated: DmsD  and TorD . Green fluorescent protein (GFP) was genetically fused behind the signal sequences of TorA and DmsA. This ensures native behavior of the respective signal sequence and excludes any effects mediated by the mature domain of the pre-protein. Surface plasmon resonance analysis revealed that these chimeric pre-proteins specifically bind to the cognate REMP. Furthermore, the region of the signal sequence that is responsible for specific binding to the corresponding REMP was identified by creating region-swapped chimeric signal sequences, containing parts of both the TorA and DmsA signal sequences. Surprisingly, specificity is not encoded in the highly variable positively charged N-terminal region of the signal sequence, but in the more similar hydrophobic C-terminal parts. Interestingly, binding of DmsD to its model substrate reduced membrane binding of the pre-protein. This property could link REMP-signal peptide binding to its reported proofreading function. Escherichia coli Tat system was first identified as a transporter of extracellular redox enzymes that require cofactor insertion and assembly in the cytoplasm prior to transport E. coli Tat substrates. The E. coli Tat translocon consists of the proteins TatA/TatE, TatB, and TatC E. coli DnaK is directly involved in the Tat pathway E. coli chaperones, e.g. GroEL and SlyD, remains obscure owing to their largely overlapping functions.Twin-arginine transport (Tat) systems are present in plant chloroplasts and many prokaryotes. The In addition to general chaperones, a class of substrate-specific redox enzyme maturation proteins (REMPs) has been identified  pathway, Tat substrates bear a transport-essential N-terminal signal sequence that consists of a positively charged N-region, a hydrophobic H-region and a short polar C-region. However Tat signal sequences bear several additional features Tat substrate signal sequences are involved in at least three important steps along the translocation path. First, the signal peptide is involved in the recognition of Tat substrates by REMPs. For example DmsD has been shown to directly bind to the signal peptide of DmsA REMPs are considered bifunctional chaperones, with roles in both the maturation and transport of substrates. The function in maturation is to keep the substrate in a conformation in which it can bind its redox cofactor et al. have analyzed the binding of the TorA signal peptide to TorD by isothermal titration calorimetry (ITC). They found that the twin-arginine motif is probably not required for TorD binding et al. have analyzed the interaction between DmsD and the DmsA signal peptide using docking and molecular dynamics simulations, and concluded that the twin-arginine motif probably interacts with conserved regions on the surface of DmsD An open question is: what is the function of the two arginine residues (RR) in the signal sequence? They are known to be essential for translocation. Even the most conservative mutation to two lysine residues blocks translocation entirely i) how do REMPs discriminate between their cognate substrate(s) and other Tat substrates, which often have many similar features; and (ii) how does REMP binding affect signal-peptide binding to the membrane relating to the next step in the Tat translocation pathway. Chimeric substrates were created based on green fluorescent protein (GFP) fused to different signal sequences. This approach has three important features: (i) GFP reports on the folding state of the pre-protein: it is only fluorescent when fully folded ii) the same mature domain is used in all experiments, which ensures that observed differences in REMP binding are due to the signal sequence, and not to the mature domain; and (iii) such chimeric pre-proteins are functional Tat substrates, since their expression in E. coli results in periplasmic accumulation of GFP The work presented here aims to provide more insight into the nature of the interactions between Tat signal sequences and REMPs, and to better understand the function of REMP binding in the Tat pathway. In particular, we focus on two questions: . Subsequently, signal sequences were amplified from pBSK or pUC57 by PCR, and at the same time given a 3′-extension that corresponds to the first 15 basepairs of the GFP insert. In a second PCR reaction the signal sequences were fused to the GFP insert and cloned into pTYB11  such that a single fusion protein is encoded consisting of an intein domain, a signal sequence and GFP. Mutations in the twin-arginine regions of the signal sequences were subsequently created by site-directed mutagenesis. pGFP6A, encoding Strep-GFP, was made as follows. First, the internal NdeI restriction site in the GFP gene was removed by site-directed mutagenesis. Subsequently, the mutant GFP gene was amplified by pcr and cloned into the NdeI/BamHI restriction sites of pET3a (New England Biolabs), resulting in pGFP4. Finally, pGFP4 was restricted by NdeI and ligated with two oligonucleotides such that a StrepII-tag is encoded upstream of the GFP-gene. An overview of the expression plasmids used is given in Wild-type signal sequences were amplified from the E. coli torD and dmsD genes were amplified from the genome and cloned into vector pET16b (Invitrogen), which encodes an N-terminal His-tag. E. coli strains used in this study were MC4100 (− araD139 Δ(argF-lac)U169 rpsL150 relA1 deoC1 rbsR fthD5301 fruA25 λ−F), DADE * cells and column purified as described in previous work E. coli BL21(DE3)* cells and column purified as recommended in the manual, “The Qiaexpressionist” (Qiagen).For large-scale preparation, the intein-fused signal GFPs were expressed in 2 in running buffer was injected to activate the surface, followed by injection of His-tagged REMP in running buffer, except for the reference surface, to which no protein was bound. For qualitative binding studies, REMPs were immobilized to a readout of 1000 response units (RU) in order to maximize the sensitivity of the experiment. For quantitative experiments in which dissociation constants were determined, a lower level of immobilization (500 RU) was employed, to minimize non-specific binding of the pre-proteins to the reference surface. The concentration of pre-proteins was varied between 10 and 6400 nM. After each interaction measurement, the SPR chip was regenerated with 250 mM EDTA.Surface plasmon resonance measurements were performed on Biacore 2000 or Biacore T100 systems  at 25°C and a flow rate of 50 µl/min, unless indicated otherwise. All solutions were freshly prepared, degassed, and filtered through membranes with 0.22 µm pores. Nitriloacetic acid (NTA) chips providing affinity for Ni-ions were used to study interaction between pre-proteins and REMPs. The running buffer was 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 150 mM NaCl, 50 µM ethylenediaminetetraacetic acid (EDTA), and 0.005% P20 detergent at pH 7.4. In each cycle, 50 µl of 0.5 mM NiClE. coli membranes or from lipids mimicking the E. coli inner membrane composition as described in our previous work N-octyl -D-glucopyranoside.L1 sensor chips (Biacore AB) on which lipophilic groups are covalently attached to the surface via a dextran matrix were used to study protein interactions with membranes. Small unilamellar vesicles (SUVs) were generated from http://www.ch.embnet.org/cgi-bin/clustalw_parser) with default parameters: open gap penalty 10, extending gap penalty 0.05, an end gap penalty 10, and a separation gap penalty 0.05.The signal sequences of TorA (residues 1–42), DmsA (residues 1–45), YnfE (residues 1–43) and YnfF (residues 1–49) were aligned using the online tool ClustalW  and the signal sequence of TorA fused to GFP (ssTorA-GFP) were separately injected over this surface for a time period of 60 s. The observed SPR responses  imply th2+ ions available for unspecific binding to ssTorA-GFP than a surface to which TorD is bound. As a result, subtraction of the control curve results in an over-correction for unspecific binding, which leads to the observed drop in the SPR intensity. Fortunately, the effect is small and mainly seen at the highest pre-protein concentrations. We therefore continued to quantify the corrected SPR curves. The SPR intensity at equilibrium (eqI) was determined as the average of the signal intensity over the shaded region in eqI divided by the concentration of pre-protein is plotted against eqI  was mutated to lysine, resulting in ssTorA(RK)-GFP, ssTorA(KR)-GFP, and ssTorA(KK)-GFP. These mutations are known to affect translocation: ssTorA(RK)-GFP and ssTorA(KR)-GFP show strongly reduced translocation efficiencies, whereas ssTorA(KK)-GFP is not transported at all In the signal sequence of TorA, the residue following the two conserved arginine residues happens to be an arginine as well. In order to rule out that the third arginine takes over any role of the two conserved ones in the binding to TorD, this residue was also mutated to a lysine, resulting in ssTorA(KKK)-GFP. The dissociation constant of 4.9 µM found for ssTorA(KKK)-GFP binding to TorD is similar to that obtained for ssTorA(KR)-GFP and ssTorA(RK)-GFP, and approximately ten-fold higher than for the wild-type signal sequence , Table 2The two arginines in the signal peptide of DmsA were also mutated to lysine. The SPR curve for a 100 nM solution of the pre-protein ssDmsA(KK)-GFP binding to DmsD was qualitatively very similar to that for wild-type ssDmsA-GFP at the same concentration . MoreoveIn conclusion, the conserved arginine pair in the signal peptides of the Tat substrates TorA and DmsA does not absolutely determine binding of the peptides to the cognate REMP. Conservative mutations to lysine residues are tolerated. However, for ssTorA binding to TorD more drastic mutations in which the positively charged residues are removed result in loss of binding.The amino acid sequences of the signal peptides of TorA and DmsA were compared in order to assess which part(s) of the signal sequence are responsible for the specific interaction with the corresponding REMP. The signal sequences of YnfE and YnfF were included in this exercise, since these paralogs of DmsA are also known substrates of DmsD The high similarity of the DmsD substrates in their H- and C-regions and the lack of similarity in the same regions of the TorA signal suggest that these regions might be important for specific recognition by the correct REMP. The validity of this hypothesis was tested by creating region-swapped chimeric signal sequences, in which either the N- or the H- or the C-regions of ssTorA-GFP and ssDmsA-GFP were replaced by the corresponding region from the other signal sequence. The borders between the different regions were delineated according to the scheme shown in In case of ssTorA, the only chimeric signal sequence protein that bound with reasonable affinity to TorD was the DTT-GFP fusion, in which the N-region was replaced for the one of DmsA. When either the H- or the C-region of ssTorA was replaced, binding was hardly detected even at the highest pre-protein concentration, indicating that the binding site for TorD was lost. These TorA-based chimeric pre-proteins were also tested for binding to DmsD. However, none of these TorA-based signal sequences displayed any binding to DmsD .For ssDmsA, similar results were observed. Its N-region could be replaced without affecting DmsD binding, but when either the H- or the C-region was replaced, binding to DmsD could hardly be detected. Apparently, the combined H- and the C-regions but not the N-region of ssTorA and ssDmsA are essential for binding to the corresponding REMP.E. coli inner membrane lipid composition were immobilized on an L1 SPR chip. First we tested whether the REMPs themselves could bind to these membrane surfaces. No interaction of DmsD alone with membranes was found  and other Tat substrates, which often share many similar features. We used an dK = 0.33 µM. The interaction between TorD and the signal sequence of TorA has been investigated previously by various techniques  In conclusion, REMP binding to signal peptides serves at least two purposes. The first purpose, already well established, is that this interaction prevents premature cleavage of signal peptides by peptidases


Hematopoieticic stem cell transplantation is the only therapeutic option that can cure thalassemia disease. Reduced intensity hematopoietic stem cell transplantation (RI-HSCT) has demonstrated a high cure rate with minimal complications compared to other options. Because RI-HSCT is very costly, economic justification for its value is needed. This study aimed to estimate the cost-utility of RI-HSCT compared with blood transfusions combined with iron chelating therapy (BT-ICT) for adolescent and young adult with severe thalassemia in Thailand.A Markov model was used to estimate the relevant costs and health outcomes over the patients’ lifetimes using a societal perspective. All future costs and outcomes were discounted at a rate of 3% per annum. The efficacy of RI-HSCT was based a clinical trial including a total of 18 thalassemia patients. Utility values were derived directly from all patients using EQ-5D and SF-6D. Primary outcomes of interest were lifetime costs, quality adjusted life-years (QALYs) gained, and the incremental cost-effectiveness ratio (ICER) in US ($) per QALY gained. One-way and probabilistic sensitivity analyses (PSA) were conducted to investigate the effect of parameter uncertainty.In base case analysis, the RI-HSCT group had a better clinical outcomes and higher lifetime costs. The incremental cost per QALY gained was US $ 3,236 per QALY. The acceptability curve showed that the probability of RI-HSCT being cost-effective was 71% at the willingness to pay of 1 time of Thai Gross domestic product per capita (GDP per capita), approximately US $ 4,210 per QALY gained. The most sensitive parameter was utility of severe thalassemia patients without cardiac complication patients.At a societal willingness to pay of 1 GDP per capita, RI-HSCT was a cost-effective treatment for adolescent and young adult with severe thalassemia in Thailand compared to BT-ICT. Thalassemia is one of the important hematological diseases in Thailand. Thalassaemia patients have produced abnormal haemoglobin, leading to anaemia.The prevalence of thalassemia carriers in Thailand was reported as 30%-40%. About 1% of Thai population, approximately 65,000 people, have thalassemia .At present, the only curative treatment existing for severe thalassemic patients was hematopoietic stem cell transplantation (HSCT) . HoweverReduced intensity hematopoietic stem cell transplantation (RI-HSCT) is a new preparative regimen for high risk thalassemia patients especially adolescent and adult. RI-HSCT has been demonstrated to be an effective strategy to reduce the toxicity of transplantation with low incidence of graft rejection. This novel approach is a promising alternative for reducing the risk of life-threatening complications and increasing the number of patients successfully cured with an allograft . ReducedAlthough RI-HSCT has demonstrated efficacy, its procedure is complex and incurs high expenditure especially during the first three years of treatment. Given the high cost of RI-HSCT, there remains an important question to be answered from a policy maker perspective whether RI-HSCT is worth the money spent and increase quality of life of patients. This study examines the potential cost-utility of RI-HSCT compared to BT-ICT in the treatment of patients with severe adolescent and young adult thalassemia.We used a markov model to simulate severe thalassemia patients receiving RI-HSCT compared to BT-ICT, which is a standard practice of thalassemia treatment. The study was undertaken using a societal perspective as recommended by the Thailand’s health technology assessment guidelines . We perfThe model simulates for the life time horizon. Clinical effectiveness and economic outcomes was directly obtained from an ongoing clinical investigation of RI-HSCT in 18 thalassemia patients in Thailand. The simulated cohort’s characteristics of the model were based on the characteristics of 18 patients. Ethical approval was granted by the Committee on Human Rights Related to Research Involving Human Subjects, Mahidol University.1-Q4], with the length of three months each. The first six months [Q1-Q2] is the period with the highest costs, highest graft rejections and worst quality of life (QoL). We assumed that the rejection rate in this period is consistent. The remaining six months [Q3-Q4] is when the patients needed close follow up in order to monitor side effects, resulting in more frequent visits. No graft rejection occured in this period; (ii) The second and third year of RI-HSCT [Iron chelation] was when patients had higher costs due to either regular phlebotomy or chelation therapy and immunosuppressive therapy; (iii) following years after successful RI-HSCT [Post RI-HSCT] (iv) RI-HSCT failure resulting in a switch to BT-ICT. After graft rejection, patients were to receive blood transfusions and subcutaneous infusion iron chealtion therapy only. Since cardiac disease is the major cause of death, we constructed the BT-ICT health state with two sub-health states: thalassemia patients without complication and thalassemia patients with cardiac complication; and (v) death Blood transfusion dependent patients had two health states (i.e. BT-ICT and death). The arrows represent the possible transitions from one state to the other. Treatment complications were included within all health states as they typically took far less than one year to resolve. The simulation estimated the costs and health outcomes over a lifetime time horizon. All transition probabilities, costs and outcomes variables are shown in Table A Markov model, as shown in Figure 2 for 4 days, fludarabine 35 mg/m2 for 6 days and antithymocyte globulin (Thymoglobulin®) 1.5 mg/kg for 4 days and they also received fludarabine 30 mg/m2 for 5 days and dexamethasone 25 mg/m2 for 5 days 1 month prior to transplant for immunosuppression rationale. The reduced intensity regimen was designed for patients who were classified as having Lucarelli class 3. All these patients were given hydroxyurea 20 mg/kg/d ≥ 3 months before BMT to decrease erythroid expansion and thus prevent graft rejection  and EQ5D (EuroQoL EQ-5D). In Thailand, the validated official Thai version of EQ-5D is available. The answer from Thai version of EQ-5D questionnaire can be converted into 0–1 utility score by Thai preference score for EQ-5D health state . Becausevs BT-ICTPrimary outcomes of interest were lifetime costs, QALYs gained, and ICER in US ($) per QALY gained. For base-case analysis, we calculated the expected lifetime costs and outcomes from each group of treatment. All future costs and outcomes were discounted at a rate of 3% per annum . The resThe interpretation of cost-effectiveness of our findings is based on the statement of the Subcommittee for Development of the National List of Essential Drugs and the Subcommittee for Development of the Health Benefit Package and Service Delivery of the NHSO in 2007. The subcommittee set up the societal willingness to pay (WTP) threshold in Thailand to one to three times per capita Gross Domestic Product (GDP) , approxiOne-way sensitivity analyses were performed to investigate the effects of altering parameters within plausible ranges including survival parameters, time to failure parameters, all costs, utilities, costs to charge ratio and discounting rate. The results of one-way sensitivity were presented using a tornado diagram. For the sensitivity analysis of discounting rate, we used the recommendation from WHO  includinA total of 18 patients receiving RI-HSCT were included in this study. Mean age was 13.7 years; range 9–18 years. Peripheral Blood Stem Cell (PBSC) was used as sources for stem cell transplantation for all patients. Patients were classified as class III according to Lucarelli or Pesaro classification . Of 18 pOur base-case analysis demonstrated the estimated life time cost for BT-ICT and RI-HSCT as US $ 73,928  and US$ 114,000 , respectively, while the estimated QALYs were 14.11, and 26.49 QALYs, respectively.  Figure . Cost efOur findings demonstrate that RI-HSCT is highly effective and very cost-effective for adolescent and adult thalassemia patients given the consideration of local context on the willingness to pay value. The high success rate of this transplantation is highly regarded as a strong evidence for considering RI-HSCT as a therapeutic alternative for this population. On the overall basis, this study provides key relevant information aiding policy makers to make informed decision making regarding resource allocation.Clinical effectiveness of RI-HSCT is a key factor driving this intervention to be cost-effective. The high effectiveness rate, demonstrated as low treatment failure and high survival rate, is attributable to 2 key steps of RI-HSCT. First, we used rigorous criteria to select patients for RI-HSCT. We selected only patients with consistent blood transfusion and iron chelation therapy for 1–2 years before transplantation. Second, we had a good pre-transplantation management which was the use of effective medications to suppress bone marrow expansion or alloimmunization from previous multiple blood transfusions such as hydroxyurea, fludarabine and dexamethasone. Previous studies showed that these medications could increase the success rate of transplantation . At presThe findings of being cost-effective are robust across all sensitivity analyses performed. Our study revealed that the ICERs were sensitive to utility values of BT-ICT patients, discount rate and cost to charge ratio. However, there remains unchanged in terms of direction of overall research findings. The cost-effectiveness acceptability curves suggest that the probability that the RI-HSCT being cost-effectiveness compared to usual care is approximately sixty-four percent using one GDP and ninety-nine percent when using three GDP per capita.To our knowledge, our study is the first study that determine the cost-effectiveness of RI-HSCT in adolescence and young adult with severe thalassemia patients. A number of clinical studies of RI-HSCT have demonstrated its effectiveness in both pediatric and adult thalassemia population -8, but tWe believe that our findings are highly valid and contextually relevant because we used local data as much as possible in our analysis, as illustrated by the following examples. First, even though there is a lack of survival data for patients treated with blood transfusions in Thailand, we adjusted the mortality rate of BT-ICT patients  by incorA number of limitations in our study deserved discussion. First, the survival data and transition probabilities for RI-HSCT patients as well as the direct medical costs of RI-HSCT were obtained from the small number of patients. Because RI-HSCT is considered a new and innovative treatment of thalassemia in Thailand, it was offered only to a few numbers of patients receiving care at a couple of hospitals in Bangkok. Second, the sensitivity analysis indicated that the ICER per QALY gained was most sensitive to changes in the utility of BT-ICT patients which this study obtained from foreign data (UK). This is identified as an area where further studies using local data are needed. Third, the direct medical costs of the interventions were obtained from a single hospital. RI-HSCT is the innovation intervention to cure thalassemia patients and need expert to look after these patients. RI-HSCT patients need to be followed up regularly at the same hospital so all costs could be collected in a single hospital database. However, using a single hospital database could however underestimate the true costs of blood transfusions, as patients might receive these in a number of different hospitals.These findings are very favorable and could be interpreted by policy makers as paramount evidence to strongly endorse the decision to support the program; however, most of the decision making generally cannot be made based solely on a cost-effectiveness analysis.Before deciding to support to reimburse RI-HSCT in the any health benefit package, decision makers may be interested in knowing other important issues about the characteristics of the populations that benefit from the transplantation. Then, budget impact analysis should be done along with the implementation suggested, in order to estimate the total budget needed. Moreover, preparing health care system including facilities, resources and treatment knowledge should be transferred to various centers to further expand the chance for the patients to access the treatment. In addition, it is crucial to review the reimbursement system for transplantation in other countries in order to develop appropriate package for the Thai context.RI-HSCT is considered a curative option for adolescence and young adult with severe thalassemia as it is highly effective and cost-effective throughout the patient’s lifetime. Clinicians and policy makers may consider including RI-HSCT in the health-benefit package given the long-term value for money of this intervention.RI-HSCT: Reduced intensity hematopoietic stem cell transplantation;BT-ICT: Blood transfusions combined with iron chelating therapy;SF-36: Short-form 36;QoL: Quality of life;EQ-5D: European quality of life – 5 dimensions;QALYs: Quality adjusted life- years;ICER: Incremental cost-effectiveness ratio;PSA: Probabilistic sensitivity analyses;GDP: Gross domestic product;PBSC: Peripheral blood stem cell;CPI: Consumer price index, Pre-BMT, pre-bone marrow transplantation;WTP: Willingness to pay;CCR: Cost to charge ratio;NMB: Net monetary benefitThe authors declare that they have no competing interests.RS participated in design of the study, developed the cost-effectiveness model, conducted the cost-effectiveness analyses, drafted the manuscript and critically reviewed the manuscript for important intellectual content. NC participated in design of the study, developed the cost-effectiveness models, conducted the cost-effectiveness analysis, and critically reviewed the manuscript for important intellectual content. SP participated in the design of the study, the acquisition of data for use in the cost-effectiveness models and critically reviewed the manuscript for important intellectual content. SS participated in the acquisition of data for use in the cost-effectiveness models and critically reviewed the manuscript for important intellectual content. NS participated in the acquisition of data for use in the cost-effectiveness models and critically reviewed the manuscript for important intellectual content. UB participant in the design of the study and the acquisition of data for use in the cost-effectiveness models and critically reviewed the manuscript for important intellectual content. SH participated in design of the study and critically reviewed the manuscript for important intellectual content. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/13/45/prepub


Females showed a strong preference for wild-type males over miR-124 mutant males when given a choice of mates. These effects were traced to aberrant pheromone production. We identified the sex-specific splicing factor transformer as a functionally significant target of miR-124 in this context, suggesting a role for miR-124 in the control of male sexual differentiation and behavior, by limiting inappropriate expression of the female form of transformer. miR-124 is required to ensure fidelity of gender-appropriate pheromone production in males. Use of a microRNA provides a secondary means of controlling the cascade of sex-specific splicing events that controls sexual differentiation in Drosophila.Many aspects of social behavior are controlled by sex-specific pheromones. Gender-appropriate production of the sexually dimorphic transcription factors doublesex and fruitless controls sexual differentiation and sexual behavior. DOI:http://dx.doi.org/10.7554/eLife.00640.001 Drosophila uses pheromones to influence sexual behaviour, with males and females producing different versions of these chemicals. One of the pheromones produced by male flies, for example, is a chemical called 11-cis-vaccenyl-acetate (cVA), which is an aphrodisiac for female flies and an anti-aphrodisiac for males.Like many animals, the fruit fly The production of the correct pheromones in each sex is genetically controlled using a process called splicing that allows a single gene to be expressed as two or more different proteins. A variety of proteins called splicing factors ensures that splicing results in the production of the correct pheromones for each sex. Sometimes, however, the process by which sex genes are expressed as proteins can be ‘leaky’, which results in the wrong proteins being produced for one or both sexes.miR-124 carries out this function in the developing brain Drosophila. Now, Weng et al. show that miR-124 also helps to regulate sex-specific splicing and thereby to control pheromone production and sexual behaviour.Small RNA molecules called microRNAs act in some genetic pathways to limit the leaky expression of genes, and a microRNA called miR-124 were less successful than wild-type males at mating with female flies, and were almost always rejected if a female fly was given a choice between a mutant male and a wild-type male. Moreover, both wild-type and mutant male flies were more likely to initiate courtship behaviour towards another male if it lacked miR-124 than if it did not.Mutant male flies lacking transformer in the mutant males, but further work is needed to understand this process in detail.The mutant male flies produced less cVA than wild-type males, but more of other pheromones called pentacosenes, which is consistent with the observed behaviour because cVA attracts females and repels males, whereas pentacosenes act as aphrodisiacs for male flies in large amounts. Weng et al. showed that these changes in the production of pheromones were caused by an increased expression of the female version of a splicing factor called DOI:http://dx.doi.org/10.7554/eLife.00640.002 Drosophila melanogaster, the courtship and aggressive behaviors exhibited by male flies are influenced by a cocktail of pheromones produced by males and females , although these compounds are also produced by females . TraF in turn promotes splicing to produce the female form of Doublesex (DsxF). In the absence of TraF, the default male form of Dsx (DsxM) is produced, along with the male form of Fruitless (FruM). Dsx proteins direct male vs female sexual differentiation, including pheromone production, as well as sexual behavior  female virgins in a standard courtship assay . However, miR-124 mutant males achieved copulation significantly less often than CS controls during the 30-min observation period . GC-MS analysis showed that the level of cVA was significantly reduced in miR-124 mutant males  has been reported to reduce cVA levels, whereas DsxF expression in dsx mutant males abolished cVA production completely . 120 µl of hexane  containing 10 μg/ml of hexacosane  standard was added into each vial and incubated at room temperature for 20 min. 100 μl of solvent was transferred into a new vial and evaporated under a gentle stream of nitrogen. Extract was stored at −20°C until analysis. At least three biological replicates were prepared per genotype.Extracts were re-dissolved in 60 µl of hexane and transferred into GC-MS vials (Supelco). Analysis was run in a 5% phenyl-methylpolysiloxane  column and GCMS QP2010 system  with an initial column temperature of 50°C for 2 min and increment to 300°C at a rate of 15 °C/min in splitless mode. The relative signal intensity for each hydrocarbon species was calculated by dividing the area under the chromatography peak by the total area under all of the peaks. The values from 3–6 replicate measurements were averaged.For application of synthetic compounds to target flies, 9 μg of synthetic cVA  was diluted in 200 μl of hexane and introduced into a 1.8-ml glass microvial. The hexane was evaporated under a gentle flow of nitrogen, leaving the compound as a residue coating the bottom of the vial. Flies were briefly anaesthetized on ice, transferred to coated vials in groups of seven, and subjected to three vortex pulses lasting 20 s each, with 10 s pauses between each pulse. The perfumed flies were allowed to recover for about 1 hr in fresh vials with standard food. Six flies from each group were used for behavioral tests and the remaining fly was subjected to hydrocarbon analysis by Direct Analysis in Real Time mass spectrometry to monitor effective transfer of the test compound to the flies.2 gas flow was set to 2.5 l/min. Under these conditions, mostly protonated ([M + H]+) molecules are observed. Using clean forceps, an anaesthetized fly was picked up by both wings, making sure not to damage the fly. The fly was placed in a stream of charged helium gas until peaks of triacylglycerides start to appear. All fly samples were placed approximately in the same location in the DART source for the same amount of time in order to obtain reproducible spectra. Six flies from each genotype were measured. Polyethylene glycol (Sigma-Aldrich) was used as calibrant. Relative quantification of compound abundance was performed by normalizing the areas under the signal corresponding to cVA ([M + H]+ 311.29) to the tricosene signal ([M + H]+ 323.36). DART MS is unable to differentiate isoforms of tricosene therefore the tricosene signal represents the summed signal intensity from 5, 7, and 9-Tricosene. Tricosene was selected as the normalization peak due to the unaltered levels in mutants compared to CS controls in GC-MS.The atmospheric pressure ionization time-of-flight mass spectrometer  was equipped with a DART interface and operated in positive-ion mode at a resolving power of 6000 (FWHM definition). Mass accuracy is within ±15 ppm. The DART interface was operated using the following settings: the gas heater was set to 200°C, the glow discharge needle was set at 3.5 kV. Electrode 1 was set to +150 V and electrode 2 was set to +250 V. HeStatistical analysis for behavior assays and hydrocarbon quantification was done using Prism 4 . For behavior data, a nonparametric Mann–Whitney test was used to compare two samples. Kruskal–Wallis test followed by Dunn’s post-test was used to compare multiple samples. For hydrocarbon analysis, multi-way ANOVA followed by Tukey HSD post-test was performed.S2 cells were transfected in 24-well plates with 250 ng of miRNA expression plasmid or empty vector, 25 ng of firefly luciferase reporter plasmid, and 25 ng of Renilla luciferase DNA as a transfection control. Transfections were performed in triplicate in at least three independent experiments. 60 hr after transfection, dual luciferase assays  were performed according to manufacturer’s instructions. review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , a splicing factor that regulates the male-specific splicing of its downstream targets, dsx and fru. Several lines of evidence show tra to be a target of miR-124, and reduction of tra is shown to suppress miR-124 mutant phenotypes. As cVA is a cuticular hydrocarbon of males flies that mediates male–female attraction, as well as male–male repulsion, several of the behavioral defects of miR-124 are proposed to be explained by the dysregulation of one downstream target, tra, and its role in the production of sex-specific pheromones. Together, this supports a model in which endogenous miR-124  act to suppress phenotypes that may result from the leaky regulation of tissue and sex-specific mRNA splicing.This manuscript describes male-specific behavioral defects in eLife. However, several additional experiments and substantive revisions are required to strengthen some key conclusions. The most important of which are essential to convincingly explain the links between altered tra expression, altered pheromone production, and behavior.In terms of novelty and interest, this is appropriate for publication in tra function in sex determination is mediated through its effects on fru and dsx, and a major hypothesis here is that increased levels of tra would result in leaky regulation of sex-specific splicing, it would be valuable to assess how the expression of these transcripts downstream of tra is affected miR-124 mutants. Leaky traF would suggest the male makes less Fru, less DsxM, and some DsxF.1) As miR-124 repression of tra occurs, as relevant to the production of cVA. Is miR-124 expressed in oenocytes? Is reduction of tra in miR-124 oenocytes sufficient to suppress miR-124 mutant phenotypes? Is reduction of tra in the nervous system sufficient , or not, to suppress miR-124 mutant phenotypes?2) It is necessary to establish the cell type in which miR-124 behavioral phenotypes is that increased levels of tra in miR-124 mutants results in altered development/morphology of sex-specific neural circuitry. An analysis of the anatomy of fru or dsx expressing neurons in brains of miR-124 mutant and miR-124 mutant flies with reduced tra levels will allow a deeper understanding of the link between elevated tra expression in miR-124 mutants and associated behavioral phenotypes.3) An alternative hypothesis for miR-124 mutant males. miR-124 mutant male genotype? Complete genotypes are also needed in 4) Throughout the text and figure legends the authors should be explicit about which genotypes have been used, particularly for miR-124 affects male–female courtship.5) miR-124 mutant, rescuing transgene, sponge and miR-124-Gal4 should be provided in the Materials and methods.6) The origins, details, and properties of the  1) As tra function in sex determination is mediated through its effects on fru and dsx, and a major hypothesis here is that increased levels of tra would result in leaky regulation of sex-specific splicing, it would be valuable to assess how the expression of these transcripts downstream of tra is affected miR-124 mutants. Leaky traFwould suggest the male makes less Fru, less DsxM, and some DsxF.dsxM transcript was detected in head samples from miR-124 adult males. However, we did not observe an increase of dsxF transcript. This is somewhat unexpected. The conventional model is that tra should switch splicing between the dsxM and dsxF forms. It is possible that production of the two Dsx splice products does not vary linearly with traF levels. We speculate on a possible molecular explanation in the revised Discussion (with data provided in Less fruM levels by qPCR. None of the 6 pairs of primers tested gave a specific PCR product (multiple products formed).We were unable to measure 2) It is necessary to establish the cell type in which miR-124 repression of tra occurs, as relevant to the production of cVA. Is miR-124 expressed in oenocytes? Is reduction of tra in miR-124 oenocytes sufficient to suppress miR-124 mutant phenotypes? Is reduction of tra in the nervous system sufficient , or not, to suppress miR-124 mutant phenotypes?miR-124 is not expressed in oenocytes.miR-124 mutant.Removing oenocytes does not affect cVA production . It is umiR-124 acts in the nervous system:Several lines of evidence indicate that miR-124 activity in neurons is sufficient to reproduce the male–male courtship phenotype. miR-124 was depleted in neurons by expressing the miR-124 sponge using elav-Gal4. Data are shown in the new (A) Reduction of elav-Gal4 driven expression of TraF increased male–male courtship and reduced completion of male–female courtship (TraF in the CNS is sufficient to reproduce the effects of the miR-124 mutant.(B) ourtship . Elevatetra mRNA in the CNS offset the male–male courtship phenotype in the miR-124 mutant background. The level of male–male courtship was comparable in the elav-Gal4>UAStraRNAi control compared to the elav-Gal4>UAStraRNAi in the miR-124 mutant. Data are shown in the new (C) Selective depletion of 3) An alternative hypothesis for miR-124 behavioral phenotypes is that increased levels of tra in miR-124 mutants results in altered development/morphology of sex-specific neural circuitry. An analysis of the anatomy of fru or dsx expressing neurons in brains of miR-124 mutant and miR-124 mutant flies with reduced tra levels will allow a deeper understanding of the link between elevated tra expression in miR-124 mutants and associated behavioral phenotypes.miR-124 mutant male. The data on courtship behaviour do not support this view.This hypothesis would lead to the expectation of a change in the behaviour of the miR-124 mutant male toward control CS males in the male courtship assay . We saw no obvious differences. For the reasons above, we do not think it would be fruitful to look in depth for subtle changes.We have looked at 4) Throughout the text and figure legends the authors should be explicit about which genotypes have been used, particularly for miR-124 mutant males., what is the deficiency used?, what is the miR-124 mutant male genotype? Complete genotypes are also needed in,, and.We intended to provide full genotype information in the Materials and methods section, but we now see that there were a few details missing. The text has been revised to provide complete information, as requested.5), the CI for the control flies is unusually low: what are the reasons for this? Male–female courtship values seem to show an unacceptably large distribution. It is not clear from these data  whether or not miR-124 affects male–female courtship.The point of this experiment was to ask whether the reason for the low mating success shown in The reviewers are correct to note that the variance is large in this data set. This is true for both the control and the mutant. We think it may be helpful to present the data as a scatter plot to give a better feel for the variance .Author rThe data is borderline significant at p<0.05 for the difference between the median of 56 pairs of flies.  The origins, details, and properties of the miR-124 mutant, rescuing transgene, sponge and miR-124-Gal4 should be provided in the Materials and methods.Done.


During critical illness, oliguria is often used as a biomarker of acute kidney injury (AKI). However, its relationship with the subsequent development of AKI has not been prospectively evaluated.We documented urine output and daily serum creatinine concentration in patients admitted for more than 24 hours in seven intensive care units (ICUs) from six countries over a period of two to four weeks. Oliguria was defined by a urine output < 0.5 ml/kg/hr. Data were collected until the occurrence of creatinine-defined AKI (AKI-Cr), designated by RIFLE-Injury class or greater using creatinine criteria (RIFLE-I[Cr]), or until ICU discharge. Episodes of oliguria were classified by longest duration of consecutive oliguria during each day were correlated with new AKI-Cr the next day, examining cut-offs for oliguria of greater than 1,2,3,4,5,6, or 12 hr duration,P < 0.0001) with negative predictive value of 98% (0.97-0.99). Oliguria preceding AKI-Cr was more likely to be associated with lower blood pressure, higher heart rate and use of vasopressors or inotropes and was more likely to prompt clinical intervention. However, only 30 of 487 individual episodes of oliguria preceded the new occurrence of AKI-Cr the next day.We studied 239 patients during 723 days. Overall, 32 patients had AKI on ICU admission, while in 23, AKI-Cr developed in ICU. Oliguria of greater than one hour was significantly associated with AKI-Cr the next day. On receiver-operator characteristic area under the curve (ROCAUC) analysis, oliguria showed fair predictive ability for AKI-Cr . The presence of 4 hrs or more oliguria provided the best discrimination (sensitivity 52% (0.31-0.73%), specificity 86% (0.84-0.89%), positive likelihood ratio 3.8 (2.2-5.6), Oliguria was significantly associated with the occurrence of new AKI-Cr, however oliguria occurred frequently compared to the small number of patients (~10%) developing AKI-Cr in the ICU, so that most episodes of oliguria were not followed by renal injury. Consequently, the occurrence of short periods (1-6 hr) of oliguria lacked utility in discriminating patients with incipient AKI-Cr . However, oliguria accompanied by hemodynamic compromise or increasing vasopressor dose may represent a clinically useful trigger for other early biomarkers of renal injury. Urine output monitoring is almost universal in critically ill patients worldwide. Historically, maintenance of urine output has been regarded as being synonymous with the preservation of renal function and decreases in urine output regularly prompt a variety of clinical interventions with the aim of preventing or attenuating acute kidney injury (AKI) . AccordiIt is important to note that Risk, Injury, Failure, Loss, End-Stage (RIFLE) and Acute Kidney Injury Network (AKIN) consensus definitions of AKI incorporate lengthy periods of oliguria  by which time significant renal injury may have already occurred. Consequently, much shorter durations of oliguria, such as two hours or less ,6. AccorLocal Ethics Committee approval was obtained for anonymous analysis of routinely collected clinical data with waiver of informed consent. A case report form was generated centrally and distributed to experienced center lead investigators who supervised the local data-collection process. We documented hourly urine output and daily serum creatinine (sCr) in unselected consecutive ICU admissions of at least one calendar day in seven adult ICUs from six countries over periods of two weeks (six centers) or four weeks (one center) during 2008. Patients with end stage renal disease  or without a urinary catheter were excluded. Patient demographics, admission clinical details, Simplified Acute Physiology Score (SAPS II) illness severity scoring  on ICU aFor each individual episode of oliguria basic hemodynamic variables at the beginning of the episode and any clinician response to oliguria  were recorded and comparison was made between episodes of oliguria that were and were not associated with progression to RIFLE I[Cr] the next day. In this analysis individual episodes of oliguria were treated as discrete events. This is because two different episodes of oliguria occurring on the same day might have occurred in different hemodynamic contexts and prompted different interventions. Clinical interventions were deemed to be associated with oliguria if they occurred during or within one hour of the end of a period of oliguria.Data were collected and collated using Microsoft Excel . Categorical variables were compared using Fisher's exact test, continuous data were reported as median with inter-quartile range (IQR) and compared using the Mann-Whitney U test. Receiver-operator characteristic (ROC) curve analysis was used to assess the ability of varying duration of the longest period of oliguria to predict the occurrence of RIFLE I[Cr] or greater the next day. Univariate statistics, ROC curve analysis and area under curve (AUC) calculation was carried out using GraphPad Prism version 5.0 d for Mac OS   additionWe studied 239 patients during 723 ICU days Table . Pre-morP = 0.015) while more patients who developed AKI-Cr in the ICU were surgical than medical (P = 0.05).Overall, 28-day mortality was 10.7% and 54 (22.5%) patients had sCr criteria for RIFLE I[Cr] or greater. Of these, 32 (13.4%) had AKI-Cr on ICU admission and were not considered further, whereas in 23 individuals (9.6%), new AKI-Cr developed in ICU. Overall, of the 293 patients, 21 (8.8%) received RRT in the ICU. Of the 54 patients with AKI-Cr, 21 (39%) received RRT; however, of the 23 patients developing AKI-Cr in the ICU only five received RRT, this is 22% of AKI-Cr in ICU. On the other hand, 50% of patients with AKI-Cr on admission required RRT. The median day of AKI-Cr occurring in the ICU was day three (range second to fourth ICU day). The relation between type of admission , or diagnosis of sepsis and the occurrence of AKI-Cr on admission or in the ICU is shown in Table Episodes of oliguria of one hour or more occurred on 265 of 723 study days and were significantly associated with the occurrence of new AKI-Cr on the next day (Table P < 0.0001). However, even with this degree of oliguria, the positive predictive value for AKI-Cr was only 11% .Looking at oliguria occurring at any stage prior to a diagnosis of AKI-Cr in the ICU, in all 207 patients at risk, 130 (63%) experienced at least one episode of oliguria. Only 20 (15%) of these patients actually developed RIFLE I AKI-Cr. Conversely of the 77 patients with no oliguria, only three (3.9%) developed AKI-Cr  to sCr criteria can improve the ability to predict mortality ,24, but Our study provides the only prospective multicenter assessment of oliguria in ICU to date. It shows that oliguria is, at best, only a fair predictor of subsequent AKI-Cr. Notably all short durations of oliguria (< 12 hours) were associated with positive likelihood ratios for AKI-Cr of only two to four. This finding suggests that the presence of these durations of oliguria during a given day does not usefully increase the post-test probability of AKI-Cr the next day, because, in general, a likelihood ratio of more than 10 is considered necessary for a clinically useful test ,27.Oliguria of 12 hours or more was associated with a high enough likelihood ratio to have clinical utility, in part validating its use in the RIFLE-Injury urine output definition. However, use of this cut-off would result in missing a large majority of cases of AKI-Cr. These observations are important because identifying patients at risk of developing AKI-Cr is the first and necessary step for clinicians to decide which patients should receive specific treatment and which patients should be observed. The crucial importance of these distinctions is supported by evidence that fluid resuscitation might reduce renal dysfunction , but thaOur data indicate that, if, for example, oliguria of two hours or more was used to trigger further fluid resuscitation, some patients might receive unnecessary fluid therapy, whereas others who might benefit from such intervention might not receive it. Thus, while clinically helpful, oliguria should be interpreted with caution in isolation, but may be more usefully interpreted in the clinical context as a screening test to direct additional methods for early detection of renal injury -42.Another important observation from our study is that more patients had biochemically overt AKI on ICU admission than developed it in the ICU. This finding suggests the renal component of critical illness develops outside the ICU in most patients and that oliguria might be a more useful screening test in the pre-ICU environment. However, patients outside the ICU rarely have hourly documentation of urine output. Surgical patients may be more likely to have monitoring of urine output and develop AKI-Cr in the ICU. However, oliguria appeared, if anything, less predictive of AKI-Cr in this group - perhaps because transient postoperative oliguria is a common and relatively benign response to surgery.Strengths of this study include that it is the first study to investigate a common and important aspect of critical illness, its prospective design, and representation of a diverse population of critically ill patients from several countries and a variety of ICU settings. Thus, these results are likely to be widely applicable. On the other hand, a number of patients had their baseline sCr and/or body weight estimated, however, estimates are routinely used in clinical decisionmaking and such use seems appropriate when studying the real-world performance of oliguria and exclusion of all patient with estimated baseline creatinine did not materially affect the predictive ability of oliguria. Some clinicians might consider the choice of RIFLE I or greater as a threshold for diagnosis of AKI-Cr somewhat conservative, as smaller increments in serum creatinine may be associated with adverse outcomes. However, we wished to choose a severity of AKI-Cr that was clearly clinically significant and the potential target of interventional trials. The timing of observation with respect to the diagnosis of AKI-Cr could also be criticised. However, a rising sCr during a 24-hour period implies a fall in GFR. It is during this period that oliguria might be expected to occur most frequently and act as an early biomarker of patients with evolving AKI-Cr. Clinical management of patients was unaffected by any aspect of this study. Thus, episodes of oliguria may have triggered a variety of changes in treatment that might have averted the development of AKI-Cr. However, it would have been unethical to stop interventions in order to study the "natural history" of oliguria.Univariate analysis suggested that patient characteristics such as hemodynamic status and physician response to oliguria were associated with the development of AKI-Cr; however, these factors are highly interdependent and the true significance of individual variables is difficult to assess. We were not able to better define the nature of these associations in multi-variate analysis as the small number of patients developing AKI-Cr and the high degree of co-linearity between hemodynamic variables and interventions preclude development of a statistically meaningful model from our dataset. Oliguria not associated with subsequent AKI based on sCr changes is likely still to be clinically significant and has been associated with adverse outcomes ,24. ThisThe results of this study should be taken not to dismiss, but to strengthen, the existing role of urine output in hemodynamic assessment and management in the ICU. However, they do indicate that, with standard treatment, most cases of oliguria are not associated with subsequent AKI-Cr in the ICU, and consequently, that trials of novel nephro-protective therapies, which aim to intervene early in the process of kidney injury should not use oliguria alone as a trigger for recruitment.Use of oliguria alone may be insufficiently precise to be used as a trigger for intervention in trials of novel protective interventions. However, it may be more significant in the context of concomitant hemodynamic instability. Furthermore, the use of novel blood or urine biomarkers of AKI may increase diagnostic yield. Thus, future studies should focus on determining the combined predictive value of oliguria, clinical variables, standard urinary biochemistry, and novel biomarkers in the early diagnosis of AKI ,43. If tOliguria is significantly associated with the occurrence of new AKI-Cr; however, most episodes of oliguria are not followed by biochemical evidence of renal injury. Oliguria alone is at best only a fair predictor of AKI-Cr. However, in the presence of hemodynamic compromise or increasing vasopressor dose, it may represent a clinically useful screening test to trigger other early biomarkers of renal injury with the goal of achieving a more accurate and timely identification of patients at risk of AKI.• More patients develop AKI outside the ICU and present with it rather than developing AKI while in ICU.• Using oliguria in isolation as a trigger for intervention in ICU might lead to some patients receiving unnecessary intervention and other patients not receiving potentially helpful intervention.• Oliguria is relatively frequent in ICU patients and most episodes are not followed by AKI.• Oliguria has only a fair predictive ability for subsequent AKI and lacks clinical utility as a test at the observed frequencies of AKI in the ICU.• Oliguria accompanied by hemodynamic compromise or increasing vasopressor dose may represent a clinically useful trigger for other early biomarkers of renal injury with the goal of achieving a more accurate and timely identification of patients at risk of AKI.AKI: acute kidney Injury; AKI-Cr: acute kidney injury defined by changes in serum creatinine; AKIN: Acute Kidney Injury Network; AUC: area under the curve; CI: confidence interval; GFR: glomerular filtration rate; IQR: interquartile range; RIFLE: Risk Injury Failure Loss End stage; RIFLE-I: RIFLE-Injury: RIFLE I[Cr]: RIFLE-Injury by serum creatinine criteria; ROC: receiver-operator characteristic; SAPS II: Simplified Acute Physiology Score II; sCr: serum creatinine; SIRS: systemic inflammatory response syndrome.The authors declare that they have no competing interests.JRP participated in study design, collected data, prepared data, performed statistical analysis and wrote the paper. YL and EL participated in study design and collected data. SM collected data and was the center organiser (Edmonton). ME collected data and was the center organiser (Okayama). MH and AH-F collected data and were center organisers (Berlin). JK was the center organiser (Pittsburgh). CR and DC collected data and were center organisers (Vicenza). KT collected data and SU was center organiser (Jikei). RB conceived the study, coordinated study centers, participated in study design, and edited the final manuscript. All authors read and approved the final manuscript.


This article aims to update the existing systematic review evidence elicited by Mickenautsch et al. up to 18 January 2008  and addressing the review question of whether, in the same dentition and same cavity class, glass-ionomer cement (GIC) restored cavities show less recurrent carious lesions on cavity margins than cavities restored with amalgam.The systematic literature search was extended beyond the original search date and a further hand-search and reference check was done. The quality of accepted trials was assessed, using updated quality criteria, and the risk of bias was investigated in more depth than previously reported. In addition, the focus of quantitative synthesis was shifted to single datasets extracted from the accepted trials.The database search (up to 10 August 2010) identified 1 new trial, in addition to the 9 included in the original systematic review, and 11 further trials were included after a hand-search and reference check. Of these 21 trials, 11 were excluded and 10 were accepted for data extraction and quality assessment. Thirteen dichotomous datasets of primary outcomes and 4 datasets with secondary outcomes were extracted. Meta-analysis and cumulative meta-analysis were used in combining clinically homogenous datasets. The overall results of the computed datasets suggest that GIC has a higher caries-preventive effect than amalgam for restorations in permanent teeth. No difference was found for restorations in the primary dentition.This outcome is in agreement with the conclusions of the original systematic review. Although the findings of the trials identified in this update may be considered to be less affected by attrition- and publication bias, their risk of selection- and detection/performance bias is high. Thus, verification of the currently available results requires further high-quality randomised control trials. Carious lesions associated with the margins of tooth restorations have been defined as recurrent or secondary caries . In receMickenautsch et al. , in a prThe results of this meta-analysis were based on a systematic search of literature until 5 January 2008 . It has Thus, this quantitative systematic review aims to update the existing evidence provided by the original article  regardinIn order to update the existing evidence, the systematic literature search was extended from the original search date and a further hand-search and reference check were done. Updated quality criteria were used in assessing the quality of accepted trials Table  9-12] a-12 a9-12Dental Caries OR Dental Caries Susceptibility OR Root Caries OR Tooth Demineralization AND Glass Ionomer Cements OR Cermet Cements AND Cariostatic Agents OR Dental Caries OR Cariostatic Agents AND Dental Amalgam OR silver mercury amalgam and Ionomer$ and amalg$ and cariosta$. Only the start and cut-off dates were changed. The databases Biomed Central, Cochrane Oral Health Reviews, Cochrane Library, Directory Of Open Access Journals, Expanded Academic ASAP PLUS, Meta Register Of Controlled Trials, PubMed and Science-Direct, were searched for relevant papers published between 8 January 2008  and 10 August 2010. Criteria for trial inclusion were:The search strategy used in the previous review  was repl- 2-arm clinical prospective study design;- Comparison of GIC versus Amalgam;- Year of publication of trials, identified through hand-search/reference check, from 1990.Included trials were excluded after further review if:- No outcome measure related to caries was reported;- No computable data, consisting of number of teeth included at baseline (BSL), number of observed effects (n) and total number of evaluations (N), per treatment group was reported.Included trials that passed the exclusion criteria were accepted for further quality assessment and data extraction. Review, data extraction and quality assessment of the accepted trials was undertaken independently by two reviewers (SM and VY). Differences were resolved through discussion and consensus.In contrast to the original published systematic review , qualityPotential attrition- and publication bias was not investigated in the original systematic review . This upAll data concerning primary and secondary outcomes of accepted trials were extracted as single dichotomous datasets, containing the number of observed effects (n) and the total number of evaluations (N) for both the control and the test groups. The Cochrane RevMan, Version 4.2 software package was used in computing the Relative Risk . Statistical significance was set at α = 0.05.2) was used in assessing statistical heterogeneity [Meta-analysis, using RevMan Version 4.2 statistical software by The Nordic Cochrane Centre, The Cochrane Collaboration , was considered for datasets only if they complied with the previously published criteria for clinical homogeneity . The perogeneity . StatistCumulative meta-analysis, using MIX Version 1.7 meta-analysis software , was perIn addition to the 9 trials included in the original meta-analysis -24, one Thirteen individual computable datasets  of primary outcomes and 4 datasets with secondary outcomes DS 13,15-17) were extracted from the 10 accepted trials. Characteristics of these trials and their datasets are shown in Table ,15-17 weOf the primary datasets, 9 reflected results for restorations in primary teeth, with 3 datasets for single-surface restorations ; 2 datasets for multiple-surface restorations  and 4 datasets  for primary teeth in which single- and multiple-surface restorations were combined. Four datasets showed results in permanent teeth: 3 datasets for single surface restorations  and one dataset for multiple surface restorations (DS 12).The four datasets that showed secondary outcome results were related to caries progression  and regression (DS 17) in approximal tooth surfaces adjacent to restoration surfaces of neighbouring teeth ,29.The computed results of each dataset are shown in Table 2 = 56.7%, p = 0.13). For that reason the decision was made to pool both datasets, using a fixed-effects model. The meta-analysis results shown in Figure Of the four datasets, two  were considered as fulfilling the criteria for clinical homogeneity ,24. AddiOne dataset for multiple-surface restoration (DS 12) was identified. The results of this dataset indicate no difference between the two types of restoration after 2 years  .2 = 0%, p = 0.67). For that reason the decision was made to pool both datasets, using a fixed effect model. The meta-analysis results shown in Figure Of the nine datasets, two DS 02,04) were found to fulfil the criteria for clinical homogeneity ,22. Addi were fou2 = 5.94%; p = 0.36). The cumulative Relative Risk indicates no statistical significant difference between the two materials after 5 years . However, a shift was observed in the cumulative Relative Risk over time, with a continued reduction of the 95% confidence intervals in favour of GIC  after 1 year (DS 07), 2 years (DS 08), 3 years (DS 14) and 5 years (DS 01) were included in a cumulative meta-analysis ,25,26. TThree datasets  reported on caries progression, and one dataset (DS 17) reported on caries regression on approximal surfaces of primary or permanent teeth adjacent to each filling material. The results indicate a statistically significant lower caries progression in surfaces adjacent to GIC and showed no difference between the two materials in caries regression after 3 years Table .The results of the quality assessment regarding selection- and detection/performance bias are shown in Table All datasets were computed under the assumption that either (i) all restored teeth lost to follow-up developed caries on margins or (ii) no restored teeth lost to follow-up developed caries on margins. The results did not change the conclusions for the majority of datasets. However, a possible risk of attrition bias was identified in the results of three datasets  extracted from three trials ,23,26. T(i) For dataset 02 : If it w(ii) For dataset 05 : If it w(iii) For dataset 14 : If it wIn line with the potential influence of attrition bias on datasets 02 and 05, the meta-analysis results Figure  would chIn addition to the risk of bias due to loss-to-follow-up, no trial indicated that a run-in phase was implemented before randomisation Table .Publication bias was investigated, using one funnel plot Figure . The funThe aim of this quantitative systematic review was to update the existing evidence related to the review question about whether, on margins of restored tooth cavities in the same dentition and of same cavity class, GIC-restored cavities show less recurrent carious lesions than cavities restored with amalgam.The new systematic literature search found 12 more trials -36 that In comparison to the original published systematic review , this upAll of the accepted trials appear to be limited by risk of selection- and detection/performance bias. Bias or systematic error may affect studies, causing either an over- or an under-estimation of the treatment effect of an investigated clinical procedure. Overestimation has been observed to be the most common . KjaergaIt has been emphasized that selection bias can be successfully prevented only if the allocation sequence remains truly random and free from potential interference throughout the trial ,10. For From the onset, successful blinding or masking appeared not to have been possible in all trials, owing to the obvious differences between GIC and amalgam in their clinical appearance. For that reason the allocation to either treatment group was visible to patients, operators and evaluators. However, the difficulties of successful blinding still carry the danger of detection-/performance bias, which thus may have affected the results of the trials. Potential knowledge of superiority claims prior to the trial may have led patients to change their oral hygiene habits, operators to place restorations more carefully or evaluators to apply evaluation criteria more subjectively. This in turn may have favoured the outcome of one type of treatment above the other.Sensitivity analysis may be used to establish whether missing data could have affected the trial outcomes, by assuming that the numbers of restoration lost to evaluation were either failures or successes . CompariThe outcome of dataset 14 would be affected by attrition bias, in that no significant differences between GIC and amalgam would be found if all patients lost to follow-up were assumed to show no caries progression in the adjacent teeth . HoweverA run-in phase is considered to be a stage during a trial where all patients receive, for example, the test treatment and only those patients that respond well to the treatment are later used for random allocation in either the control or the test group . Such prPublication bias was investigated by generating a funnel plot Figure . PublicaThe extended scope of this update did not change the overall results of the meta-analysis originally published . HoweverIn eight out of the ten accepted trials that used split-mouth or partial split-mouth design Table  a cross-It also has to be noted that none of the accepted studies reported on fluoride exposure of subjects. It can be assumed that subjects may have been exposed to external fluoride sources and that this may have increased the caries resistance of teeth restored with amalgam, thus confounding the caries-preventive effect of GIC as suggested by Hara et al. . In thisA significantly lower chance of caries development on GIC restoration margins was reported in the permanent dentition only . In the Systematic reviews have been reported as providing the highest form of clinical evidence . HoweverThe results of this update of previous systematic evidence confirm the findings of the original published review regarding whether, on margins of restored tooth cavities in the same dentition and of same cavity class, GIC-restored cavities show less recurrent carious lesions than cavities restored with amalgam. Although the findings in this update may be considered less affected by attrition- and publication bias, their risk of selection- and detection-/performance bias is high. Therefore, further high-quality randomised control trials are needed in order to verify the currently available results.The authors declare that they have no competing interests.Both authors contributed equally to the systematic literature search, review, data extraction and the writing of the manuscript. SM conducted the data analysis.


Nickel-titanium alloy is applied to biomaterials such dental wire, stents and so on, because they have outstanding capacities for corrosion-resistant, adequate mechanical strength and shape-memory ,2. On th2. After that, the viable cell numbers were determined by the trypan blue exclusion assay and counting in a hemacytometer under a phase contrast microscope. Both nickel and titanium concentrations of the extracts were determined by ICP atomic emission spectrometer . Nickel-titanium alloys were shaped into round columns  then sterilized and transplanted to dorsal subcutaneously of mice. After 28 days, a certain amount of nickel solution was injected into the auricularis skin of the mice and the degree of turgid auriculae was measured.Titanium particles were mixed with nickel particles to become 5%, 15%, 25% or 50% titanium by weight and the nickel-titanium alloys were made by arc melting. The components of nickel-titanium alloy were determined by X-ray fluorescence analysis. Nickel plate was used as a control. Nickel plate and nickel-titanium alloys were cut into determined surface area by sharing machine then soaked in 70% ethanol due to sterilize. The sterilized ones were utilized for cellular assay. First, they were soaked in PBS and heated at 36.5 °C for 25 hours in order to make extracts. Next, each extract was added to the culture supernatant of MOLT-3 cells  and the MOLT-3 cells were cultured with 10% FBS containing RPMI for 5 days at 36.5 °C in humidified air containing 5% of COIn cellular assay with MOLT-3, the viable cell number of the cells cultured with the extract of nickel-5% or 50% titanium alloy was significantly more than that of nickel plate Figure . In alleWe studied the relationship between characters of nickel-titanium alloys and biological reactions. Four kinds of nickel-titanium alloys having different titanium contents were tested. Except nickel-50% titanium alloy, the nickel concentration of the extract of nickel-titanium alloy increased with the content rate of titanium. This result suggests that increasing the content rate of titanium will promote leaking nickel from nickel-titanium alloys. Otherwise the viable cell number decreased with the content rate of titanium. The extract of nickel-50% titanium alloy indicated not only the largest viable cell number among all extracts but also the lowest nickel concentration. These results indicate that the amount of nickel would affect cell survival of MOLT-3 cells, and that nickel concentration of the extracts was not correlated with the titanium content rates of nickel-titanium alloys but it was done with the structural states of them. When nickel-titanium alloys were transplanted into mice, only nickel-50% titanium alloy affected the allergic reaction for the better. This result suggests that cellular assay will be more sensitive to detect the material differences of nickel-titanium alloys than animal test.


Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family.Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in  S-acylation refers to the reversible post-translational attachment of an acyl group to a cysteine residue via a thioester linkage S. cerevisiae. In 2002, two independent groups identified Akr1 and Erf2 as PATs for the casein kinase Yck2 and the small GTPase Ras2 Drosophila open reading frame called DNZ1 S-acyl modification of proteins is carried out by S-palmitoyltransferases, referred to as protein acyltransferase (PATs) ArabidopsisArabidopsis have already been confirmed to be PATs So far, many proteins containing the DHHC-CRD domain have been identified in eukaryotes, including 7 in yeast, 24 in mice, 23 in humans and 24 in Arabidopsis mutants resulted in the identification of TIP GROWTH DEFECTIVE1 (TIP1) (At5g20350), which displays a pleiotropic phenotype with defects in cell expansion, root hair and pollen tube growth and an overall dwarf phenotype akr1Δ yeast strain, confirming that TIP1 has a PAT function Arabidopsis thalianaPAT10 function resulted in pleiotropic growth defects, including smaller leaves, dwarfism and sterility Arabidopsis PATs were examined last year. Arabidopsis PATs proteins display a complex targeting pattern, and they have been detected in the endoplasmic reticulum, Golgi, endosomes and at the vacuolar membrane In plants, only two PATs have been characterised in detail, TIP1 and PAT10. A screen of PATs genes of plants using genomes that have only recently been completely sequenced. We first identified 804 putative PAT proteins in 31 species and then analysed the phylogenetic relationships of these proteins. We analysed the structure, chromosome location and expression patterns of PAT genes in Arabidopsis, Oryza sative, Zea mays and Glycine max using bioinformatics data and surveyed the expression patterns of the PATs genes in Zea mays as well as their responses to four phytohormones  and three abiotic stress mimic treatments  using real-time PCR. The results showed that PATs may play important roles in plants growth and development. This study provides a foundation for the cloning and further functional analysis of each member of this protein family.The objective of this study was to identify the complete set of putative S-acyltransferases in plants which the genome sequence is available. With the development of comparative genomics, it is now possible to analyse proteins from the same protein family among different species. Recent draft genome sequences for plants offer the opportunity to investigate the www.phytozome.net/) www.plantgdb.org/) http://blast.ncbi.nlm.nih.gov), which are available from NCBI, were used with an e-value cut-off of 1e-003 http://pfam.sanger.ac.uk/) and SMART (http://smart.embl-heidelberg.de/) with the default cut-off parameters. Second, we analysed the domains of all plant peptide sequences using a Hidden Markov Model (HMM) analysis with PFAM searching http://www.clustal.org/) to verify the sequences that were candidate PAT proteins http://expasy.org/) To identify members of the PAT gene family in plants, we collected the known PAT genes in Arabidopsis and analysed the domains of the PAT peptide sequences using the PFAM search tool http://www.clustal.org/) http://www.megasoftware.net/) using the p-distance and complete deletion option parameters The PAT protein sequences were aligned using the ClustalX program with BLOSUM30 as the protein weight matrix. The MUSCLE program (version 3.52) was also used to perform multiple sequence alignments to confirm the ClustalX results (http://gsds.cbi.pku.edu.cn/) The chromosomal locations and gene structures were retrieved from the genome data downloaded from the Phytozome database and PlantGDB database. The remaining genes were mapped to the chromosomes using MapDraw https://www.genevestigator.com/gv/) Arabidopsis (ATH1: 22k array), Oryza sativa (OS_51k: Rice Genome 51k array), Zea mays (ZM_84k: Nimblegen Maize 385k) and Glycine max (GM_60k: Soybean Genome Array) Gene Chip platforms. Then, the identified PAT-containing gene IDs were used as query sequences to perform searches in the Gene Chip platform of Genevestigator The microarray expression data from various datasets were obtained using Genevestigator (Zea mays) was used for this study. Unless stated otherwise, the seed germination and plant growth conditions were the same as those described by Ganal et al Zea mays) wild type seedlings were transferred to liquid Murashige and Skoog (MS) medium 2 and stored at −80°C.Qi319  following the manufacturer's instructions. The RNA was further purified using a Fermentas RNAeasy mini kit. RNA (1 µg) was used as a template for first strand cDNA synthesis using the SuperScript First-Strand Synthesis system (Transgen). Real-time PCR was performed using gene-specific primers and the UltraSYBR Mixture (With Rox) (CWBio). The 18S ribosomal RNA genes were used as internal normalisation controls Arabidopsis  and analysed the domains of the PAT peptide sequences using PFAM search tool and SMART tool. Then, we used two different approaches to gather extensive information regarding this family. A total of 804 proteins were identified as potential members of the DHHC-CRD-containing PAT family within the 31 plant genomes that have been completely sequenced (Phytozome database: http://www.phytozome.net/). The presence of the DHHC-CRD domain in all of the protein sequences was confirmed using SMART and PFAM searches. We assigned each of these sequences an identity based on the gene identifier  to 52 (Panicum virgatum)  . These rirgatum) .Panicum virgatum, Limum usitatissimum, Populustrichocarpa, Glycine max, Brassjca rapa or Zea mays. In addition, we found that all the species from chloropyta to the green higher plants contained PATs from groups I, V and VII. In contrast, there were no PATs from group II, III or IV in chloropyta. These results suggested that the PATs in group II, III and IV may have originated after the divergence of ferns and chloropyta.To clarify the phylogenetic relationship among the PATs proteins and to infer the evolutionary history of the protein family, the full-length protein sequences of the PATs in plants were used to construct a joint unrooted phylogenetic tree , from whArabidopsis, Oryza sative, Zea mays and Glycine max were examined by aligning their amino acid sequences and implementing the neighbour-joining method in MEGA 5.0. Then, a more extensive bioinformatic analysis of the PAT gene family was subsequently performed. We compared the genomic sequences of all of the genes with the sequences from the Gene Structure Display Server  to generate schematic diagrams of the gene structure. An analysis of the structure of the PATs genes showed that the mosaic structure was different from each member of the DHHC-CRD-containing PATs gene family in Arabidopsis, Oryza sative, Zea mays and Glycine max, respectively. Not only were the positions and lengths of the introns not conserved, but the number of introns was also highly variable . The plant tissues and developmental stages selected for microarray analysis span the entire life cycle. The deep and light colour shading represents the relatively high or low expression levels of the PATs genes in the different tissues, respectively.To investigate the expression profiling of the Arabidopsis, 19 of the 24 AtPATs genes could be identified using microarray. Most AtPATs showed a broad expression pattern at different developmental stages and tissues analysed, except AtPAT2 and AtPAT3, which only exhibited high expression in the stamen. In addition, AtPAT8 and AtPAT21 exhibited very low expression in the developmental stages  were specifically expressed in the flowering stage, while 11 genes  were not expressed in the flowering stage. In addition, GmPAT19 and GmPAT32 were abundantly transcribed in they hypocotyl and cotyledon, respectively  had higher expression signals in the seedlings and flowers than in the other tissues tested, and many genes (17 ZmPATs) showed the lowest expression levels in the endosperm , 6-BA (6-benzylaminopurine), SA  and ABA (abscisic acid). The real-time PCR results showed that 14 ZmPATs were down regulated by all of the phytohormones tested. In additional, 6 ZmPATs  were up-regulated by IAA, 2 ZmPATs were up-regulated by 6-BA (ZmPAT16 and 38) and 2 ZmPATs (ZmPAT3 and 32) were up-regulated by both SA and ABA  were induced or reduced by both NaCl and PEG. Six genes  were up-regulated by NaCl and down-regulated by PEG. On the contrary, 5 genes  were down-regulated by NaCl and up-regulated by PEG  to 52 (Panicum virgatum). Generally, the numbers of PATs is fewer in low plant than which in green higher plant. In average, there are 27.2 PATs in land plants, while only 7.5 PATs in chloropyta. This may be due to substrate specificity and the redundancy of genes that serve important functions in plant evolution processing. The first systematic analysis of how individual DHHC proteins affect cellular palmitoylation was performed in S. cerevisiaeDHHC-CRD-containing proteins mediate the S-acylation of proteins PAT genes were studied. Nine AtPATs  have high expression in flowers, 5 AtPATs  are highly expressed in seedlings and 8 AtPATs  exhibit high expression in siliques. In addition, the cellar localisation of all 24 AtPATs was studied. Most of these proteins are targeted to membrane structures, such as the endoplasmic reticulum, the Golgi, endosomes and the vacuolar membrane To clarify the phylogenetic relationships among the PAT proteins and infer the evolutionary history of the protein family, a phylogenetic tree of PATs in plants was constructed. It could be observed that the proteins fell into 8 groups (group I to VIII). The sequence conservation among the groups of PATs is exceptional low, which is consistent with AtPATsPAT genes may provide clues as to the biological function of PATs. Through the analysis of the expression profiles in Arabidopsis, Oryza sative, Zea mays and Glycine max, we found that PAT gene transcripts accumulated during different developmental stages and in different tissues Click here for additional data file.