E) of 0.6 V was implemented in pH 6.5 phosphate buffer solution (PBS) that served as the supporting electrolyte. A well-defined oxidation peak has been obtained in studies using PVP as a modifier of CPE based on the oxidation of morin. The present work introduces the LOV technique as a useful tool for amperometric measurement, documents advantages of using programmable flow, and outlines means for miniaturization of assays on the basis of PVP modified CPE. The proposed method was applied successfully to the determination of morin in real samples, and the spiked recoveries were satisfactory.The potential capabilities and analytical performance of lab-on-valve (LOV) manifold as a front end to amperometry have been explored for the on-line determination of morin. Meanwhile, the electrochemical behaviors of morin were investigated based on polyvinylpyrrolidone- (PVP-) doped carbon paste electrode (CPE), which found that PVP can significantly improve its oxidation peak current. The excellent amperometric current response was achieved when the potential difference (Δ As for polyvinylpyrrolidone (PVP) mentioned here, which is a water-soluble synthetic polymer, it is an amphiphilic high molecular-weight compound containing highly polar amide groups in conjunction with apolar methylene and methane (CH) moieties . And itsFlavonoids are the most abundant polyphenolic compounds widely distributed in fruits, vegetables, and plant-derived beverages such as tea and red wine , 7. Due  As far as we know, there is a few information available in the literatures concerning the electrochemical behavior of morin , 12, 19.In recent years, more attention was focused on the development of automated analytical procedures. And the development of sequential injection lab-on-valve (SI-LOV) format provides vast potentials for instrumental miniaturization and automatization and online detection –25. The μL syringe pump , and a six-position selector valve is shown schematically in μL with a three-electrode system, namely, a CPE modified by PVP as working electrode (WE), a platinum wire electrode as counter electrode (CE), and a Ag/AgCl electrode as reference electrode (RE). Moreover, the working electrode is placed in the canal adjacent to the sample inlet in order to have the best contact with the sample from EFC. The amperometric measurements were carried out with a LK2005 electrochemical workstation .The sequential injection lab-on-valve (SI-LOV) system composed of a FIAlab-3000 sequential injection system , a high-precision 2500 −2 mol L−1 morin (Fluka) was prepared and kept at 4°C by dissolving 0.3022 g morin with water in 100 mL volumetric flask where a small quantity of 0.2 mol L−1 NaOH solution was added in order to promote dissolution. The working standard solutions were obtained by stepwise dilution using 0.05 mol L−1 phosphate buffer solution . PBS , and de-ionized water was used that acted as the supporting electrolyte and carrier solution, respectively. The pH value of buffer solution was adjusted by phosphoric acid solution or sodium hydroxide solution. PVP K-30 and graphite powder (CP) were used as electrode materials. All the reagents used were at least of analytical reagent grade and purchased from Sinopharm Chemical Reagent Co., Ltd.  unless otherwise stated. Double deionized water (18.2 MΩ cm) was used throughout the experiments. All the experiments were carried out at room temperature. Stock solution of 1 × 10μL paraffin oil into a mortar to form a homogeneous mixture. And then, the mixture was pressed into a homemade cylindrical glass tube 6.0 mm i.d. with loading height of approximately 3.5 cm, which was connected with a copper wire (diameter: 1.0 mm), and the electrode surface was polished manually. The prepared PVP-CPE was activated at 100 mV/s over the potential range from −1.0 to 0.6 V using HAc-NaAc  until stable cyclic voltammetry curves were obtained. All potentials were against Ag/AgCl in this work. Such pretreatment of the working electrodes allowed us to obtain a maximum sensitivity, and it was repeated after each regeneration of electrode. It is noted that the amount of paraffin oil should be restricted to a moderate amount since insufficient paraffin oil would be unfavorable for the formation of uniform mixture; excessive paraffin oil, however, would result in the decrease of conductivity of the electrode. The PVP-doped CPE was prepared by mixing 2.3 g graphite, 0.2 g PVP, and 500 2. The residue was reconstituted in PBS, which was used for the assay of morin by the proposed methodology.The tea samples were purchased from local market and treated as follows. The tea sample (about 1.0 g) was exactly weighed, and the morin was extracted with 30 mL of ether and acetone , vortexed for 5 min, then centrifuged for 20 min at 2000 rpm. The upper organic layer was transferred into another test tube and evaporated to dryness with NM. alba L.) were purchased from local Chinese pharmacy store and finely pulverized by electric blender before the experiment. Twenty grams of powdered leaves was transferred into a beaker together with de-ionized water and a few drops of 0.2 mol L−1 NaOH solution for 24 hours. After being filtered through a filter paper, the extract was transferred to 100 mL conical beaker, and then the contents of morin were analyzed after appropriate dilution.Mulberry leaves . The electrochemical responses at bare CPE (b) and PVP/CPE (a) surfaces were presented in 6H9NO, which corresponds to the overall composition of the polymer. PVP doped in CPE surface could effectively facilitate the adsorption of morin on the electrode surface and exhibit high accumulation efficiency, which would lead to the enhancement of current responses of morin. This was caused by the fact that PVP had the ability to form hydrogen bonds between imide in the center of polymer and hydroxyl group of morin [The electrochemical characteristics of morin were studied by cyclic voltammetry (CV) using 1.0 × 10of morin . During ΔE of 0.6 V, influences of various buffer systems on resulting currents were tested using 1.0 × 10−5 mol L−1 morin standard solution, namely, 0.05 mol L−1 Na2HPO4-KH2PO4, 0.05 mol L−1 Tris-HCl, and 0.1 mol L−1 HAc-NaAc. The results showed that the current response of morin in phosphate buffer solution was significantly higher than the other two kinds of mediums along with well-shaped peaks and stable current signals. As a result, PBS was chosen as the reaction medium. A more detailed study of the effect of pH on the amperometric response was performed using PBS (0.05 mol L−1) over the pH level of 3.5–8.5. It can be seen that the peak height increased with the pH value of PBS up to about 6.5, above which it decreased, and at which a maximum signal was obtained. Therefore, phosphate buffer solution of pH 6.5 was used throughout the experiments.The supporting electrolyte that had a significant impact on the amperometric analysis was selected based on the effect of its pH on the peak height and peak shape. Under the condition of Ep(V) = 0.6799 − 0.0613 pH with R2 = 0.9949. The slope of 61.3 mV pH−1 was obtained, which suggested that the number of protons and electrons involved in the oxidation of morin was equal. In addition, the current intensity of the oxidation peak p decreased obviously with increase of solution pH. This may be due to the increasing deprotonation of OH groups in morin with increasing pH.Furthermore, the influence of pH of PBS on peak potential was studied by CV over the pH range of 4.5–8.0. It was suggested that the oxidation peak potential shifted negatively with increasing solution pH, obeying the equation −5 mol L−1 morin on scan rate at PVP/CPE was illustrated in ν) over the range of 25 to 300 mV/s. A linear relationship between the peak current and the scan rate was found as Ip(μA) = 0.3482v + 131.76 (R2 = 0.9973), suggesting that the electrochemical reaction process of morin at the PVP/CPE was an adsorption-controlled process.The current responses of morin would be influenced by scan rate, and corresponding electrochemical parameters could be deduced from the relationship between scan rate of potential sweep and current responses of morin. The dependence of oxidation peak current of 1.0 × 10It is noted that the PVP/CPE can remarkably improve the oxidation peak current of morin. However, further studies showed that the amount of PVP contained in the doped CPE also affects the electrochemical response to morin. The influence of PVP amount on the oxidation peak current of morin was examined within the range of 2–14%. As the content of PVP is gradually improving, the oxidation peak current also increased correspondingly and reached its maximum at 8% of the compound electrode material, above which it decreased. As the amount of PVP is gradually rising, more active sites were obtained so that the oxidation peak current greatly increased. With further improvement of the content of PVP, however, the conductivity of PVP/CPE lowered, which clogged the electron transfer of morin and enhanced the background current. Thus, the oxidation peak current of morin contrarily decreased when excessive higher content of PVP was applied. Moreover, high PVP ratio in the composition could lead to its leaching from the electrode because of its solubility in water and other polar solvents. As a result, the best mass content of PVP was found to be 8%.E) appeared to be a predominant parameter in amperometric measurement, which had an influence on sensitivity, selectivity, and peak shape. The influence of the ΔE on amperometric signal magnitude was investigated within the range of 0.1–1.0 V in 1.0 × 10−5 mol L−1 morin solution. With increasing ΔE, the current response was improved along with larger background current. However, when higher ΔE was applied above 0.6 V, the poor-quality peak shape was obtained with unstable baseline. This could be explained that the increasing of the applied ΔE leads to the increase of sensitivity accompanying the decrease of both selectivity and S/N ratio, meaning that possible interference of undesirable species presented in the measuring sample could be suppressed at the lower potential. With ΔE of 0.6 V, the well-shaped peak and more stable baseline were obtained. Therefore, imposed ΔE of 0.6 V was favored as a compromise between the sensitivity and selectivity for the further experiment. The potential difference  within the range of 1.0–10.0 μmol L−1 of morin with a correlation coefficient of 0.9953. And the other one was Ip = 0.1456C + 24.973  within the range of 10.0–400.0 μmol L−1) of morin with a correlation coefficient of 0.9959. The present method provided sufficient sensitivity as reflected by the values of LOD and LOQ. The calculation of LOD/LOQ for morin was yielded by utilizing LOD = 3 σ/s and LOQ = 10 σ/s, where σ is the standard deviation of the blank signals and s is the slope of the linear calibration graph. And the resulting concentrations at LOD and LOQ levels were 0.19 and 0.63 μmol L−1, respectively. The RSDs of the proposed procedure determined by 11 replicates of samples at the levels of 5.0 and 40.0 μmol L−1 were 3.1% and 3.7%, respectively. And the sample throughput of 45 h−1 was achieved.The analytical performance of the LOV procedure was evaluated by means of the application range, reproducibility, limit of detection (LOD), limit of quantitation (LOQ), and sample throughput. Under the selected experimental conditions above described, a calibration graph of the peak height versus concentrations of morin was obtained, which was described in μL of sample volume for each analysis) and high sample throughput (45 samples per hour) with good precision  rank the developed method competitive for routine analysis. The present work introduces the SI-LOV technique in connection with amperometric assay and highlights advantages of miniaturization: reduced sample/reagent consumption and reduction of effluent waste, small size of the apparatus that makes it portable, and advantages of programmable flow.The proposed method shows a lower LOD, a comparable concentration range of calibration curve, and a suitable sampling frequency, which have demonstrated the feasibility of the LOV platform with amperometric detection for the determination of morin. The low reagents/samples consumption . Meanwhile, the spiked recovery experiments were performed with satisfactory results ( The present work introduces the lab-on-valve technique in connection with amperometric measurement, documents advantages of using programmable flow, and outlines means for miniaturization of assays. The feasibility of the established system was demonstrated by morin detection in tea and mulberry leaves ( results , suggestA robust and straightforward automated procedure for the determination of morin in real sample is developed as an alternative analytical technique to other more costly and complicated procedure. With the use of programmable flow, advantages of LOV technique are the small size of the instrument and unprecedented reagent economy, which enriches the apparatus of miniaturization and automatization, broadening its range of application in electrochemical scope. The PVP/CPE integrated to EFC is easy to construct in laboratory and enables the implementation of easy regeneration accomplished by means of a quick mechanical renewal of the electrode surface layer. The PVP/CPE exhibited an excellent electrochemical activity towards the oxidation of morin in PBS, and the electrochemical characteristics of morin oxidation peak were well investigated. PVP could effectively facilitate the adsorption of morin on surface of the modified electrode, which led to the enhancement of the oxidation peak of morin. So above advantages make LOV-amperometric analytical platform a solid candidate for monitoring morin in real sample by the application of PVP/CPE.


Although semi-arid and arid regions account for about 40% of terrestrial surface of the Earth and contain approximately 10% of the global soil organic carbon stock, our understanding of soil organic carbon dynamics in these regions is limited.Cleistogenes squarrosa and an adjacent shrub community co-dominated by Reaumuria soongorica and Haloxylon ammodendron, two typical plant life forms in arid ecosystems of saline-alkaline arid regions in northwestern China during the growing season 2010. We found that both fine root biomass and necromass in two life forms varied greatly during the growing season. Annual fine root production in the perennial grasses was 45.6% significantly higher than in the shrubs, and fine root turnover rates were 2.52 and 2.17 yr−1 for the perennial grasses and the shrubs, respectively. Floor mass was significantly higher in the perennial grasses than in the shrubs due to the decomposition rate of leaf litter in the perennial grasses was 61.8% lower than in the shrubs even though no significance was detected in litterfall production. Soil microbial biomass and activity demonstrated a strong seasonal variation with larger values in May and September and minimum values in the dry month of July. Observed higher soil organic carbon stocks in the perennial grasses (1.32 Kg C m−2) than in the shrubs (1.12 Kg C m−2) might be attributed to both greater inputs of poor quality litter that is relatively resistant to decay and the lower ability of microorganism to decompose these organic matter.A field experiment was conducted to compare soil organic carbon dynamics between a perennial grass community dominated by Our results suggest that the perennial grasses might accumulate more soil organic carbon with time than the shrubs because of larger amounts of inputs from litter and slower return of carbon through decomposition. Soil carbon pool, as the major part of the terrestrial carbon reservoir, plays a critical, yet poorly understood role in the global carbon cycle The difference between inputs from primary production and the return of carbon to atmosphere through decomposition of organic matter determines the soil carbon budget in semi-arid and arid ecosystems, and other terrestrial ecosystems Litterfall and its decomposition are critical steps in forming soil organic matter, mineralizing organic nutrients, thus balancing soil carbon in terrestrial ecosystems In arid ecosystems, perennial grass and shrub are two typical plant life forms. Selective grazing leads to the reduction of plant coverage, or even induces the replacement of plant life forms from herbaceous plants, mostly perennial grasses, to long-lived woody shrubs. Different plant life forms forming different micro-environment and providing different quantity and quality of litter may play different in nutrient cycling and ecosystem functioning within the same climatic region Several studies have separately compared the ecological roles of fine root or litterfall between perennial grasses and shrubs P<0.05, P<0.001) and varied greatly among sample dates  differed significantly between life forms in the growing season 2010 P<0.05, . IntegraP<0.001, .−2) was 33.6% significantly higher than the shrubs (38.8 g m−2) in 2010  and the shrubs (20.5 g m−2) in 2010 . Integrated over all depths, fine root production in the perennial grasses (142.2 g m−2 yr−1) was 45.6% higher than the shrubs (77.4 g m−2 yr−1) (−1) and the shrubs (2.17 yr−1) (Fine root production was significantly differed between life forms (−2 yr−1) . Fine ro17 yr−1) .−2 yr−1) and the shrubs (10.9 g m−2 yr−1) in litterfall in the growing season 2010  was 44.2% significantly higher than the shrubs (10.6 g m−2) in the growing season 2010  was significantly lower than the shrubs leaves (54.9% yr−1) (k (mean ± SE), of the perennial grasses was 0.19 and of the shrubs was 0.84  . The maswas 0.84 .P<0.05, P<0.001, qCO2 as a measure of microbial efficiency was significantly lower in the perennial grasses than in the shrubs but showed no variation among the three measured months  did not significantly differ between two life forms, Soil microbial activity (SMA) was significantly lower in the perennial grasses than in the shrubs P<0.05, . More sp P<0.05, , Fig. 5cP<0.001, −2) was 15.2% higher than in the shrubs (1.12 Kg C m−2) in 2010. SOC significantly decreased with soil depth  was 27.8% higher than the shrubs (31.2 g C m−2 yr−1). No significant difference was detected in litterfall carbon input between the perennial grasses and the shrubs.Total SOC was significantly different between two life forms P<0.001, . IntegraP<0.001, . Soil orSeasonal dynamics of fine root biomass and necromass for perennial grasses and shrubs have been reported in different parts of semi-arid and arid regions R. soongorica and H. ammodendron belonged to other root types instead of deep root type R. soongorica and H. ammodendron depended on water in shallow upper soil and when long time lack of effective precipitation generated extreme drought in upper soil, both R. soongorica and H. ammodendron could shrink their roots in upper soil to effectively adjust the ratio of roots absorption area to canopy assimilation area so as to maintain regular carbon uptake This difference might largely be explained by the different responses to water stress between the perennial grasses and the shrubs. It is well established that perennial grasses with shallow-root systems would use more discontinuous and erratic water sources in the upper soil layers while shrubs with deep-root systems would use more stable water in deeper soil layers R. soongorica and H. ammodendron to water stress after a long lack of rainfall in August. Moreover, our estimates of mean fine root biomass revealed that fine root biomass of the perennial grasses was more abundant than the shrubs near the upper soil layers, implying that the former were better competitors for water in spatial dimension, particularly for pulsed rainfall.Therefore, the observed decrease of fine root biomass in the shrubs might be attributed to the strategies of As for fine root necromass, we observed bimodal peaks separately in the beginning and near the end of the growing season in the perennial grasses while in shrubs the necromass slightly increased in the beginning of the growing season and did not obviously change in the remaining . Fine ro−2 yr−1) was greater than in shrubs (77.4 g m−2 yr−1)  . Except −1 for the perennial grasses and the shrubs, respectively) are larger than those reviewed by Gill and Jackson Fine root turnover rate varies within and among species and across ecosystems, also estimates vary depending on the method used. Although our fine root turnover rates (2.54 and 2.17 yrqCO2) for the perennial grasses might be ascribed to lower quality of organic matter inputs derived from perennial grasses Soil microbial biomass (SMB) represents a small but important labile pool of nutrients in soils, and its activity exerts a key controlling influence on the rate which C, N and other nutrients cycle through ecosystems Previous studies have indicated that litterfall could show no distinct seasonal pattern, uni-modal, bimodal, or even multi-modal peaks −2 yr−1) and the shrubs (10.9 m−2 yr−1) in our study, which were much lower than those reported in other semi-arid and arid ecosystems −2 yr−1 reported by Weatherly et al. Annual litterfall production values were similar between the perennial grasses  in our study was comparable to the rate for a perennial grass community in the Patagonian steppe (31% after 20 months) R. soongorica and H. ammodendron are dicotyledon) than in the perennial grasses (C.squarrosa is monocotyledon). Besides, litter chemistry, particularly the concentration of nutrient and carbon compounds, is a major control on litter decomposition Although lack of directly comparable data of leaf litter decomposition rates for −2 for the perennial grasses and the shrubs, respectively) are within the range reported by Feng et al. −2 for 0–20 cm profile). Total soil carbon stocks were higher in the perennial grasses than in the shrubs. This accumulative discrepancy might ascribe to the following two main reasons. First, soil organic carbon inputs through primary production  in the perennial grasses were higher than in the shrubs. Second, our litter bag experiment and measured soil microbial properties suggested that inputs from the perennial grasses are in poor quality and relatively resistant to decay by lower ability of microorganism. Therefore, our results suggested that the perennial grasses might accumulate more soil organic carbon with time than the shrubs because larger amounts of inputs from litter and slower return of carbon through decomposition in the perennial grasses than in the shrubs.Soil organic carbon storage, which represents the accumulated difference between inputs from primary production and outputs through decomposition, is critical to estimate effective carbon sequestration capacity of ecosystems. In our study, the observed total soil organic carbon stocks  of the Chinese Academy of Sciences, which is located at a transitional area of oasis-desert and the southern edge of the Gurbantonggut Desert. This region has a typical continental arid temperate climate, with a long-term mean annual precipitation <150 mm but a potential mean annual evaporation >2000 mm, and has a long cold winter and hot summer, with a long-term mean annual temperature of 4–6°C. Soils at this region are characterized as Yermi-sodi-Haplic Luvisol Cleistogenes squarrosa which is widely distributed in the Eurasian steppe zone C. squarrosa is a perennial bunchgrass with characteristic C4 anatomy and a fibrous root system Reaumuria soongorica and Haloxylon ammodendron. R. soongorica, an extreme xeric semishrub of Tamaricaceae, is the constructive and dominant species of the desert ecosystems in the Central Asia, and forms the vast and distinctive landscape of the salt desert H. ammodendron, a shrubby perennial of Chenopodiaceae with characteristic C4 anatomy, is an important component of old Mediterranean flora H. ammodendron are the most widely distributed vegetation types of Asian deserts −1, 0.14 m and 50%, respectively. In the shrubs sites, mean density, height and ground cover of R. soongorica are about 2500 stems ha−1, 0.34 m and 23%, respectively. For H. ammodendron they are about 500 stems ha−1, 1.07 m and 10%, respectively.We established three plots (each 25 m×25 m) for each plant life form for field measurements during the growing season of 2010. The perennial grasses community is dominated by Soil samples  for soil organic carbon, soil water content, and fine root mass were randomly collected from five cores (8 cm diameter) in each plot per month. Besides, soil samples (0–10 cm) for analyzing microbial properties were done during May, July and September. Fresh samples for determination of soil microbial properties were immediately transported to the laboratory with a portable ice box and stored at 4°C before analysis. In the laboratory, these soil samples for analyzing soil organic carbon, soil physical properties, and soil microbial properties were passed through a 2-mm sieve and manually cleaned off any visible plant tissues.−2) for each soil sample at different depths by measuring bulk density (g m−3) and mass-based soil carbon content (%). Bulk density of soil was determined by weighing air-dried volume samples. Soil carbon content was measured by the dichromate oxidation method 2Cr2O7 and 5 ml of concentrated H2SO4 at 170°C for 10 min, then titrated the digests with 2 M standardized FeSO4.We calculated soil carbon stock (g mGravimetric soil water content (SWC) was measured by oven-drying samples at 105°C for 24 h. Soil pH was measured in the 1∶5 soil/water extract, electrical conductivity in the saturated paste extract (ECsp). The particle size distribution of the <2 mm particle fractions was determined using the laser detection technique.Fine roots (<2 mm) were manually washed free of soil samples and separated into live and dead root fragments by their color, resilience, consistency and the degree of cohesion between the cortex and the periderm. The separated fine roots were oven-dried to constant mass at 65°C for determination of mass and chemical analysis. Fine root production was estimated using the Decision matrix method Litterfall samples in each plot were collected monthly from April to October 2010 using three randomly located litter traps. Each trap (50 cm×50 cm) was made up of 1 mm mesh opening nylon netting suspended from a PVC tube square and held 5 cm above the ground by four PVC tubes. Floor mass was measured on five 50 cm×50 cm subplots randomly located in each plot every month. All plant litter samples were washed and oven-dried to constant mass at 65°C for determination of mass and chemical analysis.t = Y0×ekt−k) and the average rate of leaf litter loss.Decomposition rate of leaf litter was determined using the litterbag method At the onset of the decomposition experiments, we also determined total C and N of leaf litter. Total C was determined by the standard method of wet-combustion, and total N by semi-micro Kjeldahl method 3Cl for 24 hours and non-fumigated soil samples were extracted with 0.5 M K2SO4. The extracts were filtered through 0.45-µm filters and analyzed for extractable organic carbon by dichromate digestion as described by Lovell et al. Soil microbial biomass carbon (SMBC) was estimated using the fumigation-extraction method 2 evolution over a 2-week incubation period. Briefly, 20 g  of soil sample after adjusting to approximate 60% water holding capacity was incubated for 2-week at 25°C. Respired CO2 was captured in 5.0 ml of 0.5 M NaOH contained in a beaker suspended inside a Mason jar 2 evolved, and SMA was expressed as mg CO2 kg−1 d−1.Soil microbial activity (SMA), i.e. soil microbial respiration, was measured by determining COqCO2: soil microbial respiration to microbial biomass carbon, mg CO2-C g−1 Cmic h−1), which representing the microbial respiration per biomass unit, was calculated according to Wardle and Ghani The metabolic quotient .Seasonal mean values used in this study were calculated from the monthly mean values, which were first averaged from all measurements in the same month. Repeated Measures ANOVA (RMANOVA) were used to examine life form and soil layer effects on soil water content, soil organic content, fine root biomass, necromass, and also life form effects on aboveground litterfall, floor mass, soil microbial biomass carbon, soil microbial activity and


Fifteen years after the discovery of hypocretin/orexin a large body of evidence has been collected supporting its critical role in the modulation of several regulatory physiological functions. While reduced levels of hypocretin/orexin were initially associated with narcolepsy, increased levels have been linked in recent years to pathological states of hypervigilance and, in particular, to insomnia. The filing to FDA of the dual-activity orexin receptor antagonist (DORA) suvorexant for the indication of insomnia further corroborates the robustness of such evidences. However, as excessive vigilance is also typical of anxiety and panic episodes, as well as of abstinence and craving in substance misuse disorders. In this review we briefly discuss the evidence supporting the development of hypocretin/orexin receptor 1 (OX1) antagonists for these indications. Experiments using the OX1 antagonist SB-334867 and mutant mice have involved the OX1 receptor in mediating the compulsive reinstatement of drug seeking for ethanol, nicotine, cocaine, cannabinoids and morphine. More recently, data have been generated with the novel selective OX1 antagonists GSK1059865 and ACT-335827 on behavioral and cardiovascular response to stressors and panic-inducing agents in animals. Concluding, while waiting for pharmacologic data to become available in humans, risks and benefits for the development of an OX1 receptor antagonist for Binge Eating and Anxiety Disorders are discussed. Hypocretin/orexin is an hypothalamic neuropeptide consisting of two forms, A and B, containing 33 and 28 amino acids respectively, and binding to two G-protein coupled receptors, OX1 (or hcrt-1) and OX2 (or hcrt-2)  and in the lateral hypothalamic nucleus (LH). Positive nerve fibers and terminals can be found in several areas of the central and peripheral nervous system, including vagus nerve, spinal cord, brainstem, hypothalamus, thalamus, limbic system, and some cortical regions  almorexant  of the hypocretin/orexin peptide, showing reduced signs of withdrawal from morphine -1-(3-fluoro-2-methoxybenzoyl)-5-methylpiperidin-2-yl]methyl-pyridin-2-amine) was characterized within the GSK collection  would increase the activity on hypocretin/orexin neurons, which in turn will release more hypocretin/orexin in their terminal fields located in brain limbic regions that regulate emotion and stress response. Then hypocretin/orexin will be shifting the level of activation of the fear, stress and anxiety circuit toward a higher level of arousal, which includes vegetative, endocrine and behavioral phenomena typical of anxiety and panic states. An abnormal persistence of excessive release of hypocretin/orexin could be seen as a critical factor in the maintenance of high arousal and anxiety, as well as a liability to relapse into panic episodes in predisposed individuals, suggesting a potential critical pathophysiological role for anxiety.Data in humans showed an increased release of extracellular hypocretin/orexin driven by emotional stimuli in the amygdala of subjects suffering from treatment-resistant temporal lobe epilepsy that were implanted with microdialysis probes  and that hypocretin/orexin neurons projects to CRF-rich brain regions suggested the possibility that these two peptidergic systems are functionally entangled in the control of the stress response  (Steiner et al., The rationale for considering OX1 receptors as a possible target in conditions such as Anxiety Disorders, Drug Addiction, and Binge Eating was reviewed. Based on the current knowledge on mechanism of action, OX1 receptor antagonists should have fewer development risks than DORAs. As highlighted by Scammell and Winrow  and BoutInterestingly, treatments with selective OX1 antagonists are not expected to share the same risks of DORAs since only the OX2 receptor is primarily associated with narcolepsy which was demonstrated for dogs carrying a disruptive genetic mutation of this receptor (Wu et al., These considerations are based on a limited dataset of preclinical studies, only recently conducted with the new generation of selective compounds. Chronic dosing studies are missing and therefore information of long term risks and benefits are not available yet. In addition, no selective OX1 receptor antagonists have been tested in humans so far and the translational barrier is still unclear. However, the promising therapeutic potential in the modulation of anxiety and compulsive behaviors is stimulating further basic research and is encouraging active investments of pharmaceutical companies.Emilio Merlo Pich is full time employee of F. Hoffman-La Roche. The other author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The most effective method of controlling nosocomial infection is hand hygiene. Hand hygiene has been monitored by trained nurses’ observation of hygiene practices and assessment of the amount of alcohol-based hand sanitizer used. This study used a web camera to examine hand hygiene adherence.A web camera was installed in the ICU in September 2010, video from the camera was relayed to the Department of Infection Control via the hospital’s intranet, and that video was saved on a server for later observation. Starting in December 2010, personnel on both the day and night shifts were assessed for a total of 100 hours using the Observation Form of the WHO Patient Safety team.Alcohol-based hand sanitizer were used 170L per 1,000 patients. Conventional observation by ward nurses indicated that compliance with hand hygiene before touching a patient was 77%. Direct observation with the web camera indicated that hand hygiene was required 11.6 times/hour for each patient. Adherence to hand hygiene was 22.5%. Compliance with hand hygiene was 25.3? before touching a patient, 25.2? before a clean/aseptic procedure, 14.0? after body fluid exposure risk, 30.6? after touching a patient, and 11.5? after touching patient surroundings. After improved education of and practices by health care practitioners, staff were again observed for 100 hours. Adherence to hand hygiene was found to have improved to 33.8% (P < 0.05).Direct observation with a web camera allowed video to be recorded and saved for long periods and it allowed practices to be assessed. The saved video was circulated among the health care practitioners to allow a more objective intervention.None declared.


Aspergillus fumigatus. A deletion mutant of nikA showed low conidia production, abnormal hyphae, marked sensitivity to high osmolarity stresses, and resistance to cell wall perturbing reagents such as congo red and calcofluor white, as well as to fungicides such as fludioxonil, iprodione, and pyrrolnitrin. None of these phenotypes were observed in mutants of the SskA response regulator and SakA MAPK, which were thought to be downstream components of NikA. In contrast, in response to fludioxonil treatment, NikA was implicated in the phosphorylation of SakA MAPK and the transcriptional upregulation of catA, dprA, and dprB, which are regulated under the control of SakA. We then tested the idea that not only NikA, but also the other 13 histidine kinases play certain roles in the regulation of the HOG pathway. Interestingly, the expression of fos1, phkA, phkB, fhk5, and fhk6 increased by osmotic shock or fludioxonil treatment in a SakA-dependent manner. However, deletion mutants of the histidine kinases showed no significant defects in growth under the tested conditions. Collectively, although the signal transduction network related to NikA seems complicated, NikA plays a crucial role in several aspects of A. fumigatus physiology and, to a certain extent, modulates the HOG pathway.The fungal high osmolarity glycerol (HOG) pathway is composed of a two-component system (TCS) and Hog1-type mitogen-activated protein kinase (MAPK) cascade. A group III (Nik1-type) histidine kinase plays a major role in the HOG pathway of several filamentous fungi. In this study, we characterized a group III histidine kinase, NikA/TcsC, in the life-threatening pathogenic fungus,  Aspergillus fumigatus is a major causative pathogen of invasive aspergillosis (IA) worldwide. This fungus infects immunocompromised patients, and IA is known for its relatively high mortality The two-component system (TCS) is a signal transduction system that is conserved in a wide range of organisms from bacteria to higher plants, but not in mammals Saccharomyces cerevisiae, in which a single hHK, Sln1p, a single HPt, Ypd1p, and two response regulators, Ssk1p and Skn7p, have been shown to constitute TCS signaling. The TCS is directly linked to the stress-activated mitogen-activated protein kinase  cascade and regulates its activation in response to osmotic conditions in the extracellular environment, resulting in a high osmolarity glycerol (HOG) pathway Neurospora crassa and Aspergillus nidulans, the TCS plays a role in a variety of physiological cellular functions including conidiation, sexual development, oxidative stress response, osmotic adaptation, and sensitivity to fungicides A. fumigatusMolecular analysis of the fungal TCS has been intensively performed in S. cerevisiae (1 hHK), Schizosaccharomyces pombe (3 hHKs), and Candida albicans (3 hHKs) A. fumigatus, 13 HKs have previously been identified, and three  of them have been investigated. A deletion mutant of the fos1 gene showed a moderate resistance to fungicides and attenuated virulence tcsB deletion mutant showed a slight sensitivity to sodium dodecyl sulfate (SDS) and growth inhibition under high temperature conditions A. fumigatus.In this decade, genome data have become available for several fungi, which allowed us to quickly search for components of the TCS and examine the diversity and universality of the fungal TCS. Catlett et al. (2003) revealed that the number of hHKs in filamentous fungi is generally greater than 10, which presented a sharp contrast to the numbers observed in yeast such as os-1/nik1) was initially identified in N. crassa as an osmotic stress-sensitive mutant allele, and later it was identified as a dicarboximide-resistant mutant allele S. cerevisiae has no Nik1-type HK, heterologous expression of Nik1-type HKs from other species made S. cerevisiae responsive to these fungicides Penicillium marneffei, Histoplasma capsulatum, and Blastomyces dermatitidisAmong the different types of HKs, a large amount of attention has been paid to group III (Nik1-type) HKs. This gene  with regard not only to its role in osmotic stress and fungicide responses, development, and morphology, but also with regard to its role in the signaling pathway associated with the SskA response regulator and SakA MAPK, which are downstream components of the HOG pathway. Furthermore, the involvement of the other HKs in the HOG pathway was investigated. Based on our findings, we discussed the molecular mechanism of the TCS circuitry and the stress response mechanism of the HOG pathway in A. fumigatus.Recently, the characterization of TcsC, a Nik1-type HK of nikA gene. Afs35 (akuA::loxP) was used as a host strain (WT), and the nikA gene was replaced with the hygromycin-resistant marker nikA) were obtained through a standard transformation procedure, and the deletion of the nikA gene was confirmed at both the genomic and expression levels (data not shown). First, colony growth and conidia production were examined in the nikA deletion mutant (named ΔnikA hereafter). On a 0.1% yeast extract-containing glucose minimal medium (YGMM) plate, the colony diameter of ΔnikA was 82.5±0.8% of the WT (nikA produced fewer conidia than WT after 48 h and 96 h of incubation (nikA conidia were indistinguishable from those of WT conidia (data not shown). Scanning electron microscopy (SEM) observations showed that although the conidiophore structure was normal in ΔnikA, the number of conidiophores was reduced (nikA-complemented strains (named Co-nikA). The fitness and number of conidia were restored in Co-nikA . Hyphal diameter and cell wall thickness were determined from the images of hyphal cross-sections, which showed that the cell wall of ΔnikA was slightly thinner than that of WT and Co-nikA cells , calcofluor white (CFW), and micafungin (MCFG). ΔnikA showed significant resistance to CR and CFW compared with WT, whereas it only showed slight resistance to MCFG  and alkali-insoluble (AI) fractions. Alpha-1,3-glucan is largely fractionated in AS2, while beta-1,3-glucan and chitin are largely fractionated in AI. Galactomannan is fractionated in both AS2 and AI. The amounts of glucose or glucosamine in the AS2 and AI fractions were largely similar between ΔnikA and WT, indicating that the amounts of alpha-1,3-glucan, beta-1,3-glucan and chitin were indistinguishable  of the WT was determined. Fludioxonil, a phenylpyrrole fungicide, and iprodione, a dicarboximide fungicide, inhibited the radial growth on YGMM plates at concentrations above 0.01 µg/mL and 0.5 µg/mL, respectively, while pyrrolnitrin had a superior inhibiting activity (nikA was examined on plates containing appropriate concentrations of fungicides (roughly estimated concentrations of IC50 and IC90 of the WT strain), revealing that ΔnikA had substantial resistance to the fungicides , and these growth phenotypes were restored in Co-nikA . ΔsakA and ΔsskA showed slightly faster radial growth (ΔsakA: 106.6±0.9% and ΔsskA: 104.0±1.2%) and comparable colony morphology and conidia production on YGMM with those of the WT . We also found that ΔsakA and ΔsskA were not as resistant to CR, CFW, and MCFG as ΔnikA was , antifungal chemicals , and high temperature (42°C) compared with WT (data not shown). Thus, we focused on responses to osmotic stress and fungicides to define the signal transduction from the TCS to the SakA MAPK cascade in A. fumigatus. To monitor the SakA MAPK activation, we examined the phosphorylation level of SakA protein by immunoblot analysis with the commercial antibodies, anti-phospho-p38 MAPK (Thr180/Tyr182) and anti-Hog1 (y-215) . The phosphorylation of SakA was detected 10 min after the addition of sorbitol , and the levels decreased at 20 and 30 min . Hence, we used the corresponding A. fumigatus catA to monitor the activation of the SakA MAPK cascade. In addition, the recently reported SakA-regulated genes dprA and dprB, both encoding a dehydrin-like protein, were also used for the expression analysis catA, dprA, and dprB were upregulated and the peak of expression was at 15–30 min  or fludioxonil (10 µg/mL) treatment  and fludioxonil . When treated with 0.5 M sorbitol, the expression was significantly upregulated in WT as well as in ΔnikA  of fludioxonil through the SskA-SakA pathway.Next, the upregulation of these genes (15 min after adding each stimulus) was investigated in Δreatment . These r on NikA . To furtin ΔnikA . In respnikA cells. To examine the involvement of the other HKs in the HOG pathway, the expression level of all 13 HK genes in response to the osmotic stress and fludioxonil treatment was determined in WT and ΔsakA. fhk3 expression was not detected by our real-time reverse transcriptase-polymerase chain reaction (RT-PCR) system in several trials using different primer sets. In response to osmotic shock, the expression of fos1, phkA, phkB, and fhk6 was markedly upregulated (>4-fold), and this upregulation was dependent on the SakA MAPK . This result led us to assume that none of these HKs were activated, at least at the expression level, in the cells lacking NikA. Therefore, we did not investigate the double deletion mutants in this study. Collectively, these results suggest that whereas NikA plays an important role in modulating the SakA MAPK cascade, none of the other HKs affect the HOG pathway functions.To reveal whether these HKs play a compensative role for NikA in modulating the SakA MAPK cascade, double deletion mutants should be constructed. Prior to this, we investigated the expression profiles of these HKs in ΔA. fumigatus. In A. nidulans, the double gene deletion of SskA and SrrA, which is an ortholog of AfSkn7, resulted in a ΔnikA-level tolerance to the fungicides and a marked sensitivity to osmotic stress Cochliobolus heterostrophus, suggesting that SskA and SrrA/Skn7 are redundantly or cooperatively involved in the adaptations to fungicides and osmotic environment downstream of NikA A. nidulans single deletion mutants of sskA or srrA showed only slight resistance to fungicides and a moderate sensitivity to the osmotic stress afskn7 deletion mutant constructed by our group showed no significant resistance to cell wall-perturbing reagents and fungicides (unpublished data). Thus, to better understand how NikA regulates conidiation, morphology, and responses to chemicals, the generation and investigation of a double deletion mutant of sskA and afskn7 is necessary. This will be the subject of our future study.In this study, we found that the absence of NikA resulted in pleiotropic phenotypes such as growth retardation, reduction of conidia, aberrant hyphae, tolerance to cell wall-perturbing reagents and fungicides, and marked sensitivity to high osmolarity stress. However, NikA is likely to function independently of SskA and SakA in these phenotypes except for osmotic adaptation, which raised the question of what alternative component functions downstream of NikA. Because NikA is a component of the His-Asp phosphorelay circuitry in the TCS, NikA is thought to regulate RRs via the YpdA HPt in a phosphorelay-dependent manner. Given that SskA is not involved in the phenotypes, a plausible candidate is AfSkn7, the other type of RR in A. fumigatus SakA MAPK was transiently phosphorylated by osmotic shock and fungicide treatment, which was fully dependent on SskA. This clearly indicates that under the conditions tested here the SakA MAPK cascade is solely regulated by the TCS. Unexpectedly, we found that SakA was phosphorylated in response to osmotic shock in ΔnikA cells, suggesting that another mechanism modulates the SakA phosphorylation instead of NikA, possibly via SskA. On the one hand, NikA seemed to be indispensable for the accumulation of phosphorylated SakA in response to low concentrations of fludioxonil treatment (up to 1 µg/mL), whereas it was dispensable when treated with 10 µg/mL fludioxonil. Judged by the roughly estimated growth ICs , 10 µg/mL of fludioxonil seems high and may cause a leaky effect at a specific interaction between the chemical and the target protein. In C. heterostrophus and N. crassa, the accumulation of phosphorylated Hog1-type MAPK was observed in the cells lacking the Nik1-type HK in the presence of high concentrations of fungicides or high osmolar reagents A. fumigatusAs in other fungi, the nikA was moderately high in the absence of stimuli. This increased phosphorylation level is reminiscent of a role of the S. cerevisiae Sln1p in modulating the Hog1 MAPK phosphorylation, where a defect in the sln1 gene causes abnormal activation (hyper-phosphorylation) of the Hog1 MAPK and leads to growth defects nikA showed a slight defect in radial growth on YGMM plates and increased the phosphorylation of SakA. These findings could support the idea that similarly to Sln1p NikA is a negative regulator of the SakA MAPK, whereas it plays a positive role at least in response to fludioxonil treatment. Here, it should be noted that the expression level of catA, dprA, and dprB in ΔnikA in the absence of stimuli was comparable to that in WT, suggesting that the moderate accumulation of phosphorylated SakA dose not necessarily lead to the high expression of the genes. There may be unknown regulatory mechanisms, for example a negative feedback system, concerning this. In any event, judged by the results of SakA phosphorylation and transcriptional responses to the stimuli, the phosphorylated form of SakA appears to be an active form. Also, we showed a straightforward signaling from SskA to the SakA MAPK in the A. fumigatus HOG pathway both at the transcriptional and post-translational levels.We also found that the phosphorylation level of the SakA MAPK in ΔnikA, ΔsskA, and ΔsakA strains showed growth sensitivity at distinct levels. As has been well studied in S. cerevisiaeA. fumigatus, and the HOG pathway was responsible for this. In addition, DprB is a dehydrin-like protein responsible for growth under hyperosmotic conditions sskA and ΔsakA, in which not only the transcriptional response to osmotic shock but also the basal level of dprB expression were impaired. On the other hand, moderate levels of the transcriptional response and basal expression of dprB were observed in ΔnikA. These findings presented the possibility that the considerable growth impairment of ΔnikA under osmotic stress conditions resulted from not only a shut-off of the SskA-SakA-DprB function but also from a defect in another component. As stated above, the Skn7-type RR and SskA additively function in the osmotic adaptation of several fungi In hyperosmotic stress conditions, the ΔA. fumigatus NikA was indispensable for fungicide activity (growth inhibitory effects). This result is consistent with the results of studies on other fungi. As stated above, relatively high concentrations of fludioxonil may have a leaky effect on HOG pathway activation. With regard to this, we hypothesized that non-specific interactions between fludioxonil and the other HKs occurred during the 10 µg/mL fludioxonil treatment. To examine this, we tried to clarify any implication of other HKs in this effect, but no obvious interplay was observed. These results allowed us to suggest another possibility: fludioxonil interacts directly with YpdA protein or the NikA-YpdA complex to inhibit its function. The reason for this assumption is twofold. First, as in most fungi including S. cerevisiae, N. crassa, Cryptococcus neoformans, and A. nidulansafypdA is likely to be an essential gene (unpublished data). Second, when the expression of afypdA is downregulated, growth is inhibited and the SakA MAPK pathway is activated (unpublished data). These phenomena are reminiscent of how cells react to fludioxonil treatment. The clarification of this hypothesis is under investigation in our group.In general, the fungal TCS is a target of fungicides such as fludioxonil, iprodione, and pyrrolnitrin, and the Nik1-type HK is supposedly the molecular target Candida, Cryptococcus, Aspergillus, and Fusarium species A detailed molecular mechanism of the mode of action of fungicides such as fludioxonil is one of the long-sought topics concerning the biological control of plant pathogenic fungi A. fumigatus, only a few HKs have been characterized so far A. fumigatus TCS, we determined the expression profiles of all HKs. Interestingly, some HKs were upregulated in response to osmotic shock or fludioxonil treatment, and they were dependent on the SakA MAPK. We assumed that these HKs play certain roles in the signaling of the SakA MAPK cascade. From investigations of the deletion mutants, however, we had no clear evidence for this assumption. Notably, Fos1 has been reported to be involved in pathogenicity, sensitivity to fungicides, and cell wall construction fos1 deletion mutant, however, showed no role in sensitivity to fungicide and cell wall stress reagents. We think that this discrepancy of phenotypes is derived from the different background of the strain. In addition, ΔphkA, ΔphkB, Δfhk5, and Δfhk6 showed no visible phenotypes , including susceptibility to hydrogen peroxide, fungicides, and cell wall stressors, when compared with WT (data not shown). To elucidate the physiological role of these HKs, further functional analyses are necessary. Recently, Ji et al. (2012) have reported that TcsB plays an important role in growth in a high-temperature environment and likely modulates the SakA MAPK phosphorylation state in A. fumigatusA. fumigatus.In general, the TCS of filamentous fungi is composed of more than 10 HKs, two to four RRs, and one HPt factor A. fumigatus pathogenicity, molecular insights provided in this study may open possibilities in the development of new antifungal chemicals focusing on the TCS signaling of medically relevant fungal pathogens.In conclusion, we found that NikA plays an important role in conidiation, morphology, and stress responses, and that the SakA MAPK cascade is regulated through SskA (TCS) in response to osmotic shock and fungicide treatment. We also characterized the other HKs including Fos1, PhkA, PhkB, Fhk5, and Fhk6. There were no interactions between these HKs and NikA or SakA, at least under the conditions tested here. Although NikA seems to be dispensable in A. fumigatus strain Afs35 (FGSC A1159) (akuA::loxP) was used to generate the following gene deletion strains: ΔnikA, ΔsakA, ΔsskA, Δfos1, ΔphkB, Δfhk5, and Δfhk6A. fumigatus strain Af293 was used to generate ΔphkA. These genes  were replaced with the hygromycin resistance marker (hph or HygBr), sequences of which were derived from plasmids pSH75  or pCB1004, respectively nikA-complemented strain (Co-nikA) was generated by transformation with a fragment containing nikA on pPTR I  (see below). Fungal strains used in this study are listed in Pseudomonas cepacia), CFW (fluorescent brightener 28), and CR were commercially obtained .A. fumigatus deletion and the reconstituted strains, plasmids for each purpose were generated. DNA manipulation was performed according to standard laboratory procedures for recombinant DNA. To amplify DNA fragments from the genome and prepare gene replacement cassettes using one-step fusion PCR, Platinum Taq DNA Polymerase High Fidelity  or PrimeSTAR HS (Takara BIO) were used. Plasmids for transformation were constructed by the GeneArt system (Invitrogen) or one-step fusion PCR To construct the sakA gene were obtained using the primers sakA-U(Bgl)-FW and sakA-U(Bgl)-RV (for the 5′-region) and sakA-D(Xho)-FW and sakA-D(Xho)-RV (for the 3′-region). The fragments were digested using the restriction enzymes BglII and XhoI and were ligated into pSH75 carrying the hph cassette. The resulting plasmid, pSH75-sakA, was used for transformation to construct the sakA deletion strain.For the generation of pSH75-sakA, 5′- and 3′-flanking regions of the nikA gene were obtained using the primers nikA-U(Pst)-FW and nikA-U-RV-(HygBr) (for the 5′-region) and nikA-D-FW(HygBr) and nikA-D(Pst)-RV (for the 3′-region). The fragment of the HygBr cassette was obtained from the pCB1004 plasmid using the primers HygBr-FW and HygBr-RV. HygBr flanked by the nikA-flanking regions was obtained by one-step fusion PCR. The fragment was digested with the restriction enzyme PstI and was ligated into PstI-linearized pUC119. The resulting plasmid, pUC119-nikA, was used for transformation to construct the nikA deletion strain.For the generation of plasmid pUC119-nikA, 5′- and 3′-flanking regions of the sskA gene were obtained using the primers sskA-U-F(BamHI) and sskA-U-RV(HygBr) (for the 5′-region) and sskA-D-FW(HygBr) and sskA-D-R(EcoRI) (for the 3′-region). HygBr flanked by the sskA-flanking regions was obtained by one-step fusion PCR. The fragment was digested with the restriction enzymes BamHI and EcoRI and was ligated into pUC119 that was linearized with BamHI and EcoRI. The resulting plasmid, pUC119-sskA, was used for transformation to construct the sskA deletion strain.For the generation of plasmid pUC119-sskA, 5′- and 3′- flanking regions of the fos1 gene were obtained using the primers fos1A-U(Pst) and fos1-U-R(HygBr) (for the 5′-region) and fos1-D-F(HygBr) and fos1-D(Pst) (for the 3′-region). HygBr flanked by the fos1-flanking regions was obtained by one-step fusion PCR. The fragment was digested with the restriction enzyme PstI and was ligated into pUC119 that was linearized with PstI. The resulting plasmid, pUC119-fos1, was used for transformation to construct the fos1 deletion strain.For the generation of plasmid pUC119-fos1, 5′- and 3′-flanking regions of the phkA), phkA-D-F(HygBr) and phkA-R(pUC119E) (for the 3′-region of phkA), phkB-F(pUC119B) and phkB-U-R(HygBr) (for the 5′-region of phkB), phkB-D-F(HygBr) and phkB-R(pUC119E) (for the 3′-region of phkB), fhk5-F(pUC119B) and fhk5-U-R(HygBr) (for the 5′-region of fhk5), and fhk5-D-F(HygBr) and fhk5-R(pUC119E) (for the 3′-region of fhk5), fhk6-F2(pUC119B) and fhk6-U-R(HygBr) (for the 5′-region of fhk6), and fhk6-D-F(HygBr) and fhk6-R2(pUC119E) (for the 3′-region of fhk6). These flanking regions and the HygBr fragment were fused into pUC119 using the GeneArt system, resulting in pUC119-phkA, pUC119-phkB, pUC119-fhk5, and pUC119-fhk6, which were used for transformation to construct ΔphkA, ΔphkB, Δfhk5, and Δfhk6, respectively.For the generation of plasmids pUC119-phkA, pUC119-phkB, pUC119-fhk5, and pUC119-fhk6, 5′- and 3′-flanking regions of each gene were obtained using the primers phkA-U2-F(pUC119B) and phkA-U2-R(HygBr) (for the 5′-region of nikA fragments containing 5′- and 3′-flanking regions were obtained using the primers nikA-N-F(pPTR-P) and nikA-N-R (for the fragment containing the 5′-region and N-terminus of nikA), and nikA-C-F and nikA-C-R(pPTR-K) (for the fragment containing the 3′-region and C-terminus of nikA). These two fragments were fused into pPTRI using the GeneArt system, resulting in pPTRI-nikA+, which was used for transformation of ΔnikA to construct the ectopically complemented strain Co-nikA.For the generation of plasmid pPTRI-nikA+, the A. fumigatus transformation was performed according to conventional methods for protoplast-polyethylene glycol (PEG) transformation for Aspergillus. Briefly, a mycelium that was cultured for one night was harvested from YGMM liquid medium and was treated with Lysing Enzymes (Sigma-Aldrich Co.) and Yatalase (Takara BIO) for more than 3 h to obtain a sufficient amount of protoplasts. The DNA was coincubated with the protoplasts, and osmotic treatment with PEG4000 led to the incorporation of the DNA. The protoplast was mixed into agar medium containing 1.2 M sorbitol and appropriate antibiotics for selection.Homologous recombination and gene replacement were confirmed by PCR of the genomic DNA, and the absence of the mRNA of the target gene was verified using real-time RT-PCR analysis. We obtained at least three independent transformants for each gene deletion mutant, and the phenotypes of the multiple strains were clarified.Conidia of each strain were stored in a deep freezer (–80°C) to prevent unexpected mutations. To prepare fresh conidia suspensions, the stored conidia were inoculated on a PDA slant and incubated at 37°C for four to five days. Conidia were harvested with phosphate-buffered saline (PBS) containing 0.1% Tween 20 and kept at 4°C. Throughout this study, conidia were freshly harvested every two weeks. The number of conidia was counted using a hemocytometer .5 conidia/mL) were cultivated in liquid YGMM for 18 h, and mycelia were harvested using Miracloth , washed with distilled water, and transferred onto YGMM plates. These plates were incubated at 37°C (this time point was set as 0 h), and the mycelia were harvested at the appropriate times.For the synchronized induction of asexual development, conidia , and the mean values were calculated. The growth ratio under stressed conditions was calculated by comparing the radial diameter on the YGMM plate with and without antifungal chemicals or osmotic stress reagents. To judge sensitivity or resistance to the stresses, the calculated ratio of the diameter of each strain was compared with that of WT.About 104 conidia/mL). The medium containing conidia was poured into a 6-well plate (3 mL per well), solidified, and then incubated at 37°C. After 48 h or 96 h of incubation, the agar from each well including mycelia and produced conidia was vortexed in 10 mL of PBS containing 0.1% Tween 20 for 1 min. The number of conidia in the suspensions was counted using a hemocytometer.Before the agar solidified, conidia were mixed into YGMM agar medium . The possible contaminating DNA was digested with Deoxyribonuclease for Heat Stop . To obtain cDNA pools from the total RNA, reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit .actin gene was used as the normalization reference  for target gene expression ratios, and WT without stress or at 0 h was set as the calibrator in each experiment. Relative expression ratios were calculated by first calculating the threshold cycle changes in sample and calibrator as Real-time RT-PCR was performed using the 7300 system (Life Technologies Corporation) with SYBR Green detection according to the manufacturer’s instructions. For reaction mixture preparation, the THUNDERBIRD SYBR qPCR Mix was used . The primers listed in 5/mL) were inoculated into liquid YGMM and cultured for 18 h prior to addition of sorbitol or fungicides. Mycelia were collected with Miracloth, frozen in liquid nitrogen, ground to a powder, and immediately suspended in protein extraction buffer containing protease inhibitors . The suspension was immediately boiled for 10 min with an appropriate sample buffer for SDS-polyacrylamide gel electrophoresis (PAGE), and cell debris was then removed by centrifugation for 10 min at 14,000 rpm. The protein concentration of the supernatant was determined using a Pierce BCA Protein Assay Kit–Reducing Agent Compatible . Total protein was loaded onto a NuPAGE Novex Bis-Tris 4–12% gel (Invitrogen) and blotted using the iBlot gel transfer system (Invitrogen). To detect total SakA and phospho-SakA proteins, a rabbit polyclonal IgG antibody against Hog1 y-215  and a rabbit polyclonal IgG antibody against dually phosphorylated p38 MAPK  were used, respectively. To detect these signals on blotted membranes, the ECL Prime Western Blotting Detection System  and LAS1000  were used.Conidia  glutaraldehyde containing 0.1 M phosphate buffer for 24 h, followed by 1% osmium tetraoxide in phosphate buffer for 1 h. Samples were dehydrated through a graded ethanol series to anhydrous 100% ethanol, substituted with isoamyl acetate, and then dried by the critical-point method . After sputter coating with platinum , the samples were visualized under an S-3400 Scanning Electron Microscope (Hitachi).For TEM analysis, hyphae that were cultured for 18 h were prefixed with 2% (w/v) glutaraldehyde containing 0.1 M phosphate buffer at 4°C for 10 h, followed by 1% osmium tetraoxide in phosphate buffer for 1 h. After dehydration, tissues were embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate, and were visualized under a JEM 1400 electron microscope . The hyphal diameter and cell wall thickness were measured using SMileView software (JEOL).nikA strains were cultured in 1 L of YGMM for 24 h, harvested, squeezed, and freeze-dried. One gram of the mycelial powder was used for fractionation of cell walls by alkali treatment. All of the fractions  were freeze-dried, and the weight was measured. Each freeze-dried cell wall fraction was hydrolyzed by H2SO4 at 100°C and neutralized with barium carbonate. Finally, the carbohydrate composition of the samples was determined using a high-performance anion-exchange chromatography (HPAC) system.The cell wall fractionation by alkali treatment and quantitative determination of the carbohydrate composition of the fractions were performed as previously described Figure S1Radial growth rate on plates containing fungicides. Growth inhibitory effects of fludioxonil (Flu), iprodione (Ipr), and pyrrolnitrin (PN) were examined. The WT strain was inoculated on YGMM agar with or without the indicated concentration of chemicals and grown at 37°C for 40 h. The radial growth rates were calculated by comparing the diameter of a colony to that of a colony grown on YGMM without chemicals. Each plot shows the mean based on three replicates.(TIF)Click here for additional data file.Figure S2Growth of the Co-nikA strain under high osmotic or fungicide stress conditions. Conidia of Co-nikA were inoculated onto YGMM containing sorbitol, NaCl, iprodione (Ipr), or fludioxonil (Flu) at the indicated concentrations and were incubated at 37°C for 44 h.(TIF)Click here for additional data file.Figure S3Resistance to cell wall-perturbing reagents. (A) Conidia of ΔsakA, ΔsskA, ΔnikA, and WT were inoculated onto YGMM containing 1 ng/mL micafungin (MCFG), 30 µg/mL congo red (CR), or 15 µg/mL calcofluor white (CFW) and incubated at 37°C for 48 h.(TIF)Click here for additional data file.Figure S4Time-course expression of catA, dprA, and dprB in response to osmotic shock and fungicide. (A and B) The WT strain was grown for 18 h at 37°C; then, sorbitol (A) or fludioxonil (B) was added . The mycelia were harvested at the indicated time points. The expression ratios were investigated by real-time RT-PCR. Relative expression ratios were calculated relative to the 0 min sample. Error bars represent the standard deviations based on three independent replicates.(TIF)Click here for additional data file.Figure S5Comparison of growth of A. fumigatus HK gene deletion mutants under osmotic or fungicide stress conditions. (A) Growth on plates containing hyperosmotic stress. Conidia of WT (Afs35), ΔnikA, Δfos1, ΔphkB, Δfhk5, and Δfhk6 were inoculated onto YGMM containing 1.2 M sorbitol and 1 M NaCl and incubated at 37°C for 72 h. Conidia of WT (Af293) and ΔphkA were inoculated onto YGMM containing 1.2 M sorbitol and were incubated at 37°C for 72 h. (B) Growth on plates containing fungicides. Conidia of WT (Afs35), ΔnikA, Δfos1, ΔphkB, Δfhk5, Δfhk6, WT (Af293), and ΔphkA were inoculated onto YGMM containing 5 µg/mL iprodione (Ipr), 0.1 µg/mL fludioxonil (Flu), or 0.02 µg/mL pyrrolnitrin (PN), and were incubated at 37°C for 48 h.(TIF)Click here for additional data file.Table S1Radial growth of each strain on the plate containing fungicide or osmotic stress.(XLSX)Click here for additional data file.Table S2Cell wall composition of each cell wall fraction.(XLSX)Click here for additional data file.Table S3PCR primers used in this study.(XLSX)Click here for additional data file.


Only a limited number of polymorphisms were observed. Comparison with homologous mutations in other Plasmodium species showed that these polymorphisms are unlikely to be associated with sulfadoxine resistance.The  Plasmodium falciparum and P. vivax have revealed stepwise increase in antifolate resistance with additional mutations within the dihydrotheorate synthase (dhps) and dihydrofolate reductase (dhfr) genes P. malariae are usually not treated with antifolate drugs, exposure is still likely in endemic areas where sulfadoxine - pyrimethamine is used for treatment of falciparum malaria, because of the high frequency of mixed infections. The previously reported S114N mutation within the dhfr gene of P. malariae supports this P. malariae gene for dhps, the target for sulfadoxine, has not been studied to date.Molecular characterizations of antifolate resistance in dhps gene of 44 P. malariae strains from Asian clinical samples  were isolated and analysed. Genomic DNA of P. brasilianum, a New World monkey malaria parasite considered to be genetically indistinguishable from P. malariae, purified from a cloned line maintained in Saimiri monkeys was obtained from MR4 (MR4-349). Samples were obtained from patients enrolled in a variety of previous treatment studies. All patients provided written informed consent. The protocol for this study was reviewed and approved from the Faculty of Tropical Medicine, Mahidol University, Thailand (reference no. MUTM2011-049-03). To design primers for isolation of the dhps gene from P. malariae and P. brasilianum, the nucleotide sequences of dhps gene from other human Plasmodium spp. were aligned, including P. falciparum (accession number XM_001349382), P. vivax (accession number XM_001617159), and P. knowlesi (accession number XM_002262146). A nested and seminested PCR approach was used to increase sensitivity of the amplification product. The sequences of primers, Mg2+concentrations, annealing temperatures, numbers of cycles, and sizes of products were individually determined for the different primer pairs shown in In this study, the pppk-dhps gene was isolated from both P. malariae  and P. brasilianum . The partial 1,953 bp Pmpppk-dhps included one intron (167 bp) at the carboxyl terminus coding region, which position and size was established by reverse-transcriptase PCR amplification and sequencing from corresponding mRNA. DNA sequences of pppk-dhps are available from 3 other species infecting humans: P. falciparum, P. vivax and P. knowlesi. In all species the pppk-dhps gene has two introns, one of which is located near the N-terminus coding region of the pppk gene and one near the C-terminal. This study revealed an 167 bp intron within dhps gene of P. malariae. AT content of the pppk-dhps gene from P. malariae, P. brasilianum and P. falciparum was high (72.2–76.5%), whereas this is low in P. vivax and P. knowlesi (56.8% and 62.2% respectively). The pppk-dhps gene of P. falciparum appeared to be located on chromosome 8, whereas in P. vivax and P. knowlesi this is on chromosome 14.The partial Pmdhps gene from all 44 P. malariae isolates. In the amplified product 1,140 bp (corresponding to 379 amino acids) of Pmdhps were analyzed, which revealed 2 and 3 positions of synonymous and nonsynonymous mutations which were predicted to change amino acids located outside the binding pocket of the enzyme for sulfadoxine. This was assessed through amino acid alignment with DHPS of P. falciparum and P. vivax . The nonsysnonymous mutation was located at the equivalent position 516 in P. falciparum and 488 in P. vivax, which does not code for the enzyme binding pocket. The dhfr gene sequences were then assessed in the 17 P. malariae isolates from Thailand, none of which showed mutations, and all were classified as haplotype 4 Pmdhfr sequences Plasmodium species suggest that the initial mutation associated with drug pressure may be at position S114N. In order to assess this point mutation more efficiently , a PCR-RFLP method was developed and investigated. Patterns of wild type S114 contain two bands of 401 bp and 172 bp+168 bp product, and mutant type 114N showed two bands of 569 bp and 172 bp product. Forty-four samples of P. malariae were tested and PCR-RFLP results were in accordance with the sequence data. Using PCR-RFLP, we analysed 17 isolates recently collected in Thailand which revealed no mutations, with all strains classified as the common haplotype 4 The current study investigated 27 isolates from which we have previously reported details on dhps genes isolated from P. falciparum, P. vivax, P. knowlesi and P. malariae has some limitations. Amino acid alignment of DHPS in each Plasmodium spp. determined equivalent position of nonsynonnymous mutations in association to sulfadoxine resistance in P. falciparum and P. vivax (P. malariae showed wild type amino acids at these equivalent positions, whereas the three nonsynonymous mutations present in three P. malariae isolates have not been described in these other species. They are equivalent to T537, L516 and E339 in P. falciparum (P. vivax and P. knowlesi at these three residues are the same as in P. falciparum, suggesting that these three residues are conserved among Plasmodium spp. It is therefore interesting to construct an in silico comparative structure model to further explore whether the nonsynonymous mutations found in P. malariae at these positions have an effect on molecular docking of sulfadoxine.Comparing homology of P. vivax . All 44 lciparum . Amino aP. malariae in the region has been lower than on P. falciparum or P. vivax. Mutations in the PmDHPS and PmDHFR genes are rare in Thailand, suggesting absence of selection of SP resistant parasite populations. We plan to extend our observations to P. malariae isolates collected from African countries where SP drug pressure is more prominent and where prevalence of P. malariae is higher compared to Southeast Asia. The nonsynonymous mutations identified in PmDHPS and PmDHFR require further in vivo and in vitro studies to elucidate their significance in SP resistance in P. malariae.Our findings suggest that drug pressure of sulphadoxine-pyrimethamine on


HNF1A-inactivated HCA (35–40%), inflammatory HCA (IHCA), and beta-catenin-mutated inflammatory HCA (b-IHCA) (50–55%), beta-catenin-activated HCA (5–10%), and unclassified HCA (10%) has greatly improved the diagnostic accuracy of benign hepatocellular nodules. If HCA malignant transformation occurs in all HCA subgroups, the risk is by far the highest in the β-catenin-mutated subgroups . In the coming decade the management of HCA will be more dependent on the identification of HCA subtypes, particularly for smaller nodules (<5 cm) in terms of imaging, follow-up, and resection.Focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are benign hepatocellular tumors. The risk of bleeding and malignant transformation of HCA are strong arguments to differentiate HCA from FNH. Despite great progress that has been made in the differential radiological diagnosis of the 2 types of nodules, liver biopsy is sometimes necessary to separate the 2 entities. Identification of HCA subtypes using immunohistochemical techniques, namely,  The knowledge of benign hepatocellular tumors, that is, focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA), has considerably progressed in the last 10 years, thanks to molecular biology, followed by immunohistochemical applications. Following these advances, new classification is now largely used, first in France, and more recently in other European, American, and East countries.The aims of this study are (1) to make a brief general overview of these 2 entities ; (2) to It is the second most frequent benign liver nodule (after hemangioma), occurring in 0.8% of an adult autopsy population and has been reported in 0.6–3% of the general population. In 80–90% of cases, FNH is discovered in women in their third or fourth decade. In countries  where OC use has been less prevalent, FNH tends to be a lesion of adult men or children of either gender.FNH is solitary in 2/3 of cases. Most lesions are asymptomatic and are therefore discovered as incidental findings during surgery, autopsy, or imaging procedures for unrelated symptoms. Large lesions can present with abdominal pain or compression of adjacent organs. Reports of hemorrhage or malignant transformation probably do not exist or are exceptional and require confirmation. FNH lesions may regress with age as shown by disappearance of presumed FNH lesions on serial imaging studies. The background liver is usually normal. FNH is associated with hepatic hemangioma in 20% of cases. Coexistence with HCA is not rare. Associated lesions outside the liver have been reported such as hemihypertrophy, hemangioma of cervix, vascular malformations of brain, and meningioma.(1) Gross Findings. On cut section, classical FNH is a pale, firm mass measuring from a few millimeters to more than 10 centimeters in diameter. The margin is well delimited, and the mass is lobulated and non encapsulated. The lesion is composed of nodules each measuring 2-3 mm, separated by zones of atrophy that give the lesion a multinodular appearance. The lesion characteristically has a central or eccentric stellate fibrous scar with radiating extensions that partially surround some component nodules.(2) Microscopic Findings. FNH lesions are composed of nodules of benign-appearing hepatocytes arranged in plates not more than 2 cells in thickness. Steatosis may occur, usually focal. The central scar is often edematous or congested and contains one or more large dystrophic vessels, accompanied by numerous small arterioles. The large vessels have irregular fibrous thickening of the intima with focal thinning of the media. The internal elastic lamina is poorly formed and reduplicated. The portal vein is absent. The central fibrous region has radiating branches composed of portal tract-like structures that contain an artery unaccompanied by portal veins or ducts. When fibrous septation is prominent, the appearance may be indistinguishable from cirrhosis, especially in biopsy specimens. A lymphocytic or mixed inflammatory infiltrate is frequent in fibrous regions. At the interface between fibrous regions and nodules, there are often features of cholate stasis including feathery degeneration of hepatocytes, Mallory-Denk bodies, and a ductular reaction that may be highlighted with CK7 and CK19 immunostaining. Sinusoids adjacent to arterial sources are lined by CD34-positive endothelium. Glutamine synthetase is a very useful immunostain, showing a characteristic broadband of expression in hepatocytes often near the hepatic veins.Incomplete or early forms may lack a central scar; they have an incompletion (or absence) of multinodular organization and sometimes exhibit more or less prominent regions of congestion.Clonal analysis using the HUMARA test demonstrated the reactive polyclonal nature of liver cells in FNH in 50–100% of the cases , 7. MessThe pathogenesis of FNH is not fully established. The association with conditions having local or systemic vascular anomalies and the presence of unusually large vessels within the lesions has led to the belief that FNH is a nonspecific response to focally increased blood flow. It is a rare benign liver neoplasm composed of hepatocytes. The incidence is around 3-4/100 000 in Europe and North America and is lower in Asia. 85% of cases occur in young women; HCA is rare in children, men, and the elderly.The major risk factor for development of HCA is the exposure to estrogenic or androgenic steroids. In young women, 80% have been users of oral contraceptives (OC). The risk increases with the duration and type of OC usage. The prevalence appears to be declining as low-estrogen preparations have become more widely used. The lesions usually decrease in size after stopping OC or after menopause. The clinical presentation of HCA may include abdominal pain, abdominal mass, intraperitoneal hemorrhage, abnormal liver tests, or space occupying lesion found incidentally on an imaging study. HCA can be single or multiple. When 10 or more adenomas occur, the condition is known as adenomatosis. Clinically significant hemorrhage is observed in 20–25% of cases; the risk is highest when the tumors are larger than 5 cm. Malignant transformation to hepatocellular carcinoma (HCC) is rare, but well documented, occurring in up to 7% of cases as reported from referral centers. The risk of transformation varies with the HCA subtype (see below) and with the clinical association, being higher in patients with glycogenosis or androgenic-anabolic steroid use.(1) Gross Findings. Hepatocellular adenomas are typically large globular tumors with prominent vessels in the overlying hepatic capsule. On cut section, the tumor parenchyma is soft and relatively uniform although areas of congestion, necrosis, hemorrhage, or fibrosis are frequent. The margins of the lesion are ill-defined both grossly and microscopically, with little or no fibrous capsule. Lesions vary in size from microscopic up to 20 cm in diameter. In livers with adenomatosis, there may be hundreds of lesions visible as minute ill-defined nodules visible grossly or only microscopically. HCA may be similar in color and texture to the background liver but are more easily seen when there is lesional steatosis, major congestion and hemorrhage or degenerative changes. The background liver is usually normal, though there may be pallor, fibrosis, or brown pigmentation related respectively to fatty liver disease, glycogen storage disease, iron overload, or other diseases.(2) Microscopic Findings. HCA is typically composed of benign hepatocytes arranged in regular liver cell plates that are usually one, or at most two, cells in width. A pseudoglandular growth pattern may be seen focally. Tumor hepatocytes have cytoplasm that may be normal, clear (glycogen-rich), steatotic, or contain pigment in lysosomes. Nuclear atypia and mitoses are unusual. The tumor parenchyma is supplied by isolated arteries unaccompanied by bile ducts. Variations in this typical pattern are frequently seen in some of the subtypes, as described below. The reader is referred to the paper by Nault et al. in this issue. Briefly,α-Inactivated HCA (H-HCA).(1) HNF1 The HNF1A gene encodes the hepatocyte nuclear factor 1 (HNF1α), a transcription factor that is involved in hepatocyte differentiation. Bi-allelic inactivating mutations of this gene is found in approximately 35–40% of HCA; 90% of HNF1A mutations are somatic; in 10%, they are constitutional (germline). Heterozygous germline mutations in HNF1A are responsible for an autosomal dominant form of diabetes MODY3 (maturity onset diabetes of the young type 3). In patients with MODY 3 and HCA, there is an additional somatic mutation of the second allele in the tumor.(2) Beta-Catenin Activated HCA (b-HCA). An activating β-catenin mutation is found in 10–15% of HCA cases. Glul, a target gene of β-catenin, coding for the protein glutamine synthetase (GS) is also upregulated.(3) Inflammatory HCA. Inflammatory HCA (IHCA), represent more than half of HCA cases. They are characterized by increased expression of inflammation-associated proteins such as serum amyloid A (SAA) and C-reactive protein (CRP), at both the mRNA and protein levels. 60% of these adenomas harbor mutations in gp130. Mutant gp130 activates STAT3 in the absence of its ligand, which is IL-6. Beta-catenin mutations may coexist with gp130 mutations in 10% of IHCA.(4) Unclassified HCA. HCA without distinguishing histological features and without known mutations represent less than 10% of all cases. An accurate diagnosis can be made using imaging techniques in 90% of cases in experienced centers. Contrast enhanced ultrasonography (CEUS) is the first modality of choice for FNH. MRI is the first modality of choice for HCA. In less than 10% of cases, the differential diagnosis of FNH, HCA, and hepatocellular carcinoma (HCC) cannot be solved by imaging alone. A biopsy interpreted by an experienced liver pathologist can resolve most of these problem cases, with standard and/or immunohistochemical stainings. If the biopsy is not definitive, surgery may be advocated.The first task is to be certain that the biopsy includes the lesion. Therefore, it is recommended that a biopsy be accompanied by a sample of non-lesional liver. HCA is the most frequent lesion to be distinguished from FNH. The histologic diagnosis of FNH requires two main criteria. The lesion must be composed of benign-appearing hepatocytes and must be supplied by altered portal tracts. Adenomas are supplied by isolated arteries, not portal tracts. A source of difficulty, in this regard, is the presence of CK7/CK19-positive ductular elements in HCA of the inflammatory type. The key feature is that glutamine synthetase expression shows a distinctive map-like distribution adjacent to hepatic veins in FNH while expression is diffusely positive in beta-catenin-activated HCA and mostly negative in other types of HCA (see the following).Macroregenerative nodule/FNH-like in cirrhotic patients and patients with vascular alterations may be difficult to differentiate from FNH or HCA. These nodules share some similarities with FNH but differ in some ways: the beta-catenin pathway is not activated in cirrhotic FNH-like nodules ; the ANGHCC may mimic FNH including focal scarring, arterialized sinusoids, and residual portal tract remnants. Warning signs of malignancy include nuclear pleomorphism, high N/C ratio, wide plates, and mitotic figures in the lesion and often cirrhosis in the background liver. The differential diagnosis between HCA and well-differentiated HCC remains difficult.At the end of the last century it was thought that HCA will disappear with the use of OC of the third generation. This was not the case. There are several reasons for that: the wider use of modern imaging techniques, the better identification of HCA among hepatocellular tumors, and the emergence of new etiological factors such as obesity , 11. HCAToday the use of the HCA immunohistochemical classification , 14–19 iThe pathological diagnosis of benign liver tumors should take into account the clinical, biological , and radMain informations are summarized in H-HCA represents a homogeneous group of tumors with lobulated contours, showing typically marked and diffuse steatosis, absence of significant inflammation, or nuclear atypia. FABP1, coding for L-FABP (liver fatty acid binding protein), is a gene positively regulated by HNF1A, expressed in normal liver tissue and clearly downregulated in this HCA subtype. By immunohistochemistry, there is a nearly complete absence of LFABP staining, contrasting with the nontumoral surrounding liver which appeared homogeneously stained (even though faintly). Therefore, lack of LFABP expression is a very good diagnostic argument for HNF1-alpha-inactivated HCA, specific of this subtype, since there is a very good concordance between this immunophenotype and HNF1A mutations. Furthermore, the downregulation of LFABP may contribute to the fatty phenotype through impaired fatty acid trafficking. HNF1A-mutated HCA occurs almost exclusively in women. Nodules can be solitary or multiple. Most of adenomatosis is HNF1A mutated. Constitutional mutations can affect both sexes and can be discovered in children, sometimes as a familial form or associated with MODY 3.Briefly B-HCA. This HCA subtype is often associated with specific conditions  and male gender. The lesions are usually solitary (except in glycogenosis) and have an increased risk of malignant transformation, compared to the other subtypes. Steatosis and inflammation are usually absent. Nuclear atypia and a pseudoglandular growth pattern are frequent in this subtype, so that distinction from well-differentiated HCC may be very difficult. By immunohistochemistry, glutamine synthetase is usually strongly expressed in a diffuse pattern, associated with aberrant cytoplasmic and nuclear expression of beta-catenin. When glutamine synthetase staining is heterogeneous and beta-catenin nonconclusive, molecular biology remains the method of choice for identifying beta-catenin mutation.IHCA. Most patients with IHCA are women. Obesity and fatty liver diseases are frequent. In 50% of cases there are elevated serum levels of CRP and increased erythrocyte sedimentation rate, rarely in association with fever and anemia, features which can regress after HCA resection. Nodules can be solitary or multiple. Micronodules can also be detected by SAA and CRP immunostaining in the liver parenchyma, outside the main tumors. Histologically, IHCA typically exhibits focal or diffuse inflammation, sinusoidal dilatation, congestion and peliotic areas, and numerous and thick-walled arteries often associated with ductular reaction, lying in small amount of connective tissues. Steatosis may be seen, mainly focally. Immunohistochemistry demonstrates strong expression of SAA and CRP restricted to tumoral hepatocytes. There is a risk of malignant transformation, particularly for IHCA which is also beta-catenin mutated.Reasons for classifying HCA are summarized in Major advances brought by the classification are (a) individualization of FNH from HCA and FNH from FNH-like, (b) identification of MODY3 and patients at high risk of malignant transformation, (c) demonstration that adenomatosis was not per se a specific HCA subtype, but an entity defined by an arbitrary number of nodules > ten detected by imaging techniques (the high number linked probably to a specific susceptibility), and (d) correction of errors .Practical guidelines for the diagnosis of HCA subtypes are summarized in The HCA classification has changed the way we consider HCA and FNH in our center. The diagnosis accuracy of FNH and HCA has completely changed at the end of the first 2000 decade. There are probably in the literature many unavoidable diagnostic errors concerning benign hepatocellular nodules particularly in the field of hemorrhagic FNH, malignant transformation of FNH, identification of difficult nodules, and so forth. http://dx.doi.org/10.1155/2013/268625) is a good starting point to obtain valuable information.For practical reasons once the diagnosis of liver tumors is made, the patient is often referred to a surgeon. In spite of the vast literature in various domains: imaging, pathology, surgery, complications, and so forth, there is no guidelines for the diagnosis and treatment of HCA. Nevertheless the major contribution of some European centers  and size (2 to 20 cm), mode of discovery, and so forth, therefore it is extremely difficult to define acceptable guidelines based on solid arguments. Most of the series published are very small and it is difficult to make a judgment on case report. β-HCA and β-IHCA).This is the reason why we believe that to make progress we need to collect prospective data from different countries and continents. The identification of subtypes is one of the key factors among others that need to be collected . The ideIn the absence of accepted guidelines, our proposition for the management of HCA subtypes <5 cm is summarized in It is clear that we have entered a new era in the study of benign hepatocellular tumors and perhaps more importantly in the field of HCC developing in nonfibrotic liver. One may still consider that this breach has not yet dramatically changed our strategies—diagnostic and therapeutic . This isBecause HCA is rare, it is necessary to combine data from different centers. The main task is to better define patients at risk of malignant transformation , 59, to Deciphering the molecular pathways leading to nodule formation, one can imagine that the medical treatment of HCA is not out of hand.The diagnosis and prognosis of benign hepatocellular nodules have changed in the recent years. This will have an impact on the management of these nodules including surveillance. Supplementary Table 1: main publications from European centers dealing with hepatocellular nodules.Click here for additional data file.


The degree of this microcirculatory impairment has not been assessed in the early phases of Emergency Department (ED) sepsis management. The purpose of this study was to assess the relationship between tissue oxygen saturation  but there were no significant differences in co-morbidities or conventional vital signs. On arrival in the ED there was no difference in mean StO2 between survivors and nonsurvivors . With treatment, StO2 improved significantly to 78% (P = 0.006) in survivors but remained persistently low in nonsurvivors. The AUROC for StO2 was 0.63 on ED departure and 0.71 after 24 hours of treatment, performing far better than heart rate (0.53), SpO2 (0.50) and systolic blood pressure (0.51). There was no correlation between StO2 and any of the routine vital signs.Forty-nine patients were included in this study, of which 24 (49%) died. Nonsurvivors were significantly older than survivors (79 vs. 64, Our results demonstrate that a consistently low tissue oxygen saturation despite initial sepsis resuscitation is associated with an increased in-hospital mortality. We have further shown that tissue oxygen saturation is a better prognostic indicator than conventional vital signs in severely septic ED patients.