The MSM prepared at 500°C (MSM–500) had the highest surface area, pore size, and volume, and the results of isotherms and the kinetics of ibuprofen (IBP) removal indicated that MSM–500 had the highest sorption capacity and fastest removal speed vs. SBA–15 and zeolite. Compared with commercial granular activated carbon (GAC), MSM–500 had a ~100 times higher sorption rate at neutral pH. IBP uptake by MSM–500 was thermodynamically favorable at room temperature, which was interpreted as indicating relatively weak bonding because the entropy  was much smaller. Five times recycling tests revealed that MSM–500 had 83–87% recovery efficiencies and slower uptake speeds due to slight deformation of the outer pore structure. In the IBP delivery test, MSM–500 drug loading was 41%, higher than the reported value of SBA–15 (31%). The in vitro release of IBP was faster, almost 100%, reaching equilibrium within a few hours, indicating its effective loading and unloading characteristics. A cost analysis study revealed that the MSM was ~10–70 times cheaper than any other mesoporous silica material for the removal or delivery of IBP.Mesoporous silica materials (MSMs) were synthesized economically using silica (SiO Mesoporous silica materials (MSMs) have large, ordered pores, ranging from 2 to 50 nm, as classified by the International Union of Pure and Applied Chemistry (IUPAC) , 2. MSMs2H5)4) [In addition to environmental applications , mesoporous materials have emerged as suitable candidates for drug delivery systems and other biomedical applications , 7. Refe2H5)4) , 10, whi2H5)4) . Thus, aIbuprofen (IBP) was selected as a model drug for this study because it is one of the most widely consumed drugs worldwide; it is classified as a nonsteroidal anti-inflammatory drug (NSAID) , 13. Ind2 as a framework precursor, replacing TEOS. The main objectives of this investigation were to (i) assess the effects of thermal treatment  through characterization using X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), and N2 gas isotherm; (ii) assess the sorption capacities and rates of IBP in multi-recycle runs; (iii) study IBP loading and unloading by MSMs; and finally (iii) determine the adsorption mechanism.To synthesize MSMs for IBP removal or delivery economically, we developed a modified alkaline fusion method, which broadens the choice of framework precursors. For the first time, we synthesized MSMs using SiO20PO70EO20) and SiO2 were obtained from Sigma-Aldrich  and R&M Chemicals , respectively. Sodium hydroxide (NaOH) and hydrochloric acid (HCl) (37%) were purchased from Merck . Analytical grade IBP was supplied by Alfa Aesar .Pluronic P123 (EO2 in the presence of NaOH and HCl. SiO2 (1 M) was dissolved in a 1 M NaOH solution and stirred using a magnetic stirrer at 45°C for 20 h. The pore-templating agent was prepared separately using 4 g of Pluronic P123, dissolved in 120 mL of a 2 M HCl solution with continuous stirring at 45°C. The two solutions were then mixed and stirred for 3 h at 45°C, and then for an additional 12 h at room temperature. The molar ratio of chemicals used in the synthesis of the MSM was 1 SiO2:1 NaOH: 5.28 HCl: 0.015 Pluronic: 200 H2O. The solution was then aged in a Teflon bottle at 90°C for 20 h. The precipitated solid product was recovered by filtering it using a 0.45 μm pore size cellulose acetate membrane filter. Finally, the material was washed with deionized (DI) water and ethanol (50%) and dried at 60°C for 24 h. A Western furnace was used to calcine the dried samples at temperatures ranging from 500 to 900°C for 4–6 h.MSMs were synthesized by reacting Pluronic P123 with SiOθ from 0.5 to 2.5° using a step size of 0.0070° and a scanning time of 19.9260 s. Nitrogen adsorption and desorption isotherms were measured using a TriStar II 3020 system . The samples were analyzed at 77.35 K. The adsorption and desorption data were calculated using Brunauer–Emmett–Teller (BET) theory to determine the specific surface area of the samples, and the pore-size distributions and pore volumes were determined using the Barrett–Joyner–Halenda (BJH) theory. Infrared (IR) spectra were obtained using a NICOLET IS 10 spectrometer . Microscopic images of nanoscale pore structures were taken using a transmission electron microscope  at 120 kV.XRD analysis was performed using an X-ray diffractometer . The samples were scanned at 23OH; Thermo Fisher Scientific) and dissolved in DI water in a 250 mL volumetric flask. It was then stirred for 12 h, sonicated for 2 h, and filtered using a 0.45 μm pore-size cellulose acetate membrane filter. A sodium chloride (NaCl) solution was added to the filtrate to create ionic strength (0.01 M) in the solution. The pH was adjusted to 7 using a sodium phosphate (Na3PO4) solution.The required amount of IBP powder (200 mg) was dissolved in 10% methanol  were added together. The vials were placed in an electric shaker and agitated at 250 rpm and 25°C. The suspension was filtered using a 0.45 μm pore-size cellulose acetate membrane filter, and the filtrate was analyzed for IBP.In 20 mL vials, 10–500 mg of MSM and 10 mL of IBP solution (150 mg Lqeq is the amount of solute adsorbed per unit weight of adsorbent (mg g–1) at equilibrium, Ceq is the equilibrium concentration of the solute in the bulk solution (mg L–1), Qmax is the maximum adsorption capacity (mg g–1), and KL is the Langmuir constant related to the energy of adsorption.The data were fitted with the Langmuir and Freundlich isotherms. When the adsorption is retained on a uniform monolayer surface, the Langmuir model fits the data of the isotherm. The maximum adsorption capacity is achieved when all sorption sites are saturated. The linear form of the Langmuir model can be written asKF and n are Freundlich isotherm constants related to the adsorption capacity and adsorption intensity, respectively.Assuming that the adsorption is retained on the heterogeneous surface of the adsorbent, the Freundlich model fits the isotherm data better. In the Freundlich model, chemisorption and physisorption are pertinent to monolayer and multilayer adsorption, respectively. The linear form of the Freundlich equation can be expressed as–1 were added to four separate beakers in which 1,000 mg of MSM had been pre-added. These four beakers were stirred constantly at 200 rpm for 200 min at room temperature. At timed intervals, samples from each of the four beakers were collected, filtered, and analyzed for IBP. After obtaining the kinetic data, a pseudo-second-order kinetic model equation was used to obtain the kinetic constants of the reaction,K2 is the rate constant of pseudo-second-order adsorption (g mg–1 min–1), K2qeq2 or v0 (mg g–1 min–1) is the initial adsorption rate, and qt is the amount of adsorbate adsorbed at time t (min).IBP solutions (200 mL) with concentrations of 100 mg LΔadsG0), enthalpy (ΔadsH0), and entropy (ΔadsS0), were determined using the kinetic data. This adsorption process is represented by a reversible heterogeneous equilibrium: IBP in solution ↔ IBP adsorbed.The kinetics of IBP uptake by MSM-500 were examined at 299, 309, and 319 K. Thermodynamic values, such as the Gibbs free energy  at pH 7.4. The solution was stirred at 150 rpm and kept at 36.9°C, similar to body temperature. At each time point, 3 mL of solution was extracted and analyzed, and an equal volume of fresh solution was replaced. The IBP was analyzed to assess the release of IBP. The test was also repeated with the PBS solution at a lower pH, i.e., 4–6.The λmax) was detected at 264 nm. A calibration curve, which was obtained by analyzing various concentrations of the prepared IBP standard solution, showed good linearity, with a determination coefficient (R2) of 0.9997.IBP was analyzed using a UV 2600 spectrophotometer . A standard IBP solution was subjected to a full-spectrum scan, ranging from 900 to 0 nm wavelengths, and maximum absorbance (d(100) reflection [θ angles of 1.08 and 1.58°, indexed at d(110) and d(200), respectively. These types of XRD reflection patterns can be assigned to a well-ordered mesoporous structure with P6mm hexagonal symmetry [d00 reflecflection , 18. Add10) and d00, respesymmetry .θ values with higher calcination temperature shows an increase in the pore sizes of the MSMs, indicating the possibility that MSM–500 had larger pores than MSM–600 and –700. However, MSMs calcined at >700°C showed no XRD diffraction peak, demonstrating the collapse of the pore structure [2 adsorption–desorption analysis.The XRD results indicated that MSM–500 had the highest peak intensity compared with the other MSMs, reflecting the formation of more uniform mesopores , 19. The3–) and acidified Pluronic P123 solutions were mixed together. The precipitate was treated using a hydrothermal process, in which it was heated and condensed to form the MSM. This method allows the excess hydrogen (H+) and chlorine (Cl–) to coordinate electrostatically between the nonionic pore-templating agent (So) and the hydrogen-associated silicic acid 4), which may finally lead to the formation of SoH+Cl–Io. Given these observations, the reaction mechanism of this synthesis can be represented using reactions 1–3.Park et al. reported that synthesis of mesoporous materials using silica glass at pH >10 yielded a white gel that transformed into a mesoporous material due to a hydrothermal process . However2 may lead to the formation of silicate ions (HSiO3–) (R1).First, the mixing of NaOH and SiO4) is formed under acidic conditions (R2).Second, when both the silicate and acidic Pluronic solution are mixed together, silicic acid (Si(OH)Finally, through a hydrothermal process, two silicic acids are condensed and form a silicate dimer, which further polymerizes to form the framework of the MSM (R3).2 adsorption–desorption isotherms of the various MSMs. According to the IUPAC classification, the as-synthesized MSM, MSM–500, and MSM–600 had a type-IV sorption pattern [P/Po < 0.2, MSM–500 had more primary micropores (<2 nm) than MSM–600. Reversible sorption of N2 gas for MSM–500 and–600 occurred up to a P/Po of 0.6, whereas that for as-synthesized MSM happened much earlier, at a P/Po of 0.2. These results indicate that MSM–500 and–600 had more uniform pores with a hysteresis loop pattern type H1, similar to SBA–15 [ pattern , 22, wheo SBA–15 , 24. Howo SBA–15 .Using the BJH method, the pore-size distribution was obtained for as-synthesized MSM, MSM–500, and–600 (2 g–1) and pore volume (0.541 cm3 g–1). The BET specific surface area and pore volume decreased substantially when the calcination temperature increased from 500 to 900°C. A significant decrease was noted at 700°C, with the BET specific surface area reduced to <10 m2 g–1. Generally, the MSMs synthesized had smaller BET specific surface areas and pore volumes than reported for SBA–15 [The pore structure characteristics of MSMs and other referenced media are summarized in r SBA–15 .ao) and mean BJH desorption pore diameter were used to determine the wall thickness of the MSMs. The wall thickness was calculated as the difference between ao, where ao = 2 × d(100)/d-spacing and wall thickness decreased from 11.9 to 9.3 nm and 7.6 to 3.3 nm, respectively. This reduction in wall thickness is a result of silicate condensation during calcination [The corresponding unit cell parameter , which could be due to the formation of weaker bonds between the condensed silica walls. In contrast, Tung et al. reported that SBA––15 and a few other mesoporous silica-based materials had high thermal stabilities of up to 850°C [Qeq of IBP by MSM–500 and–600 increased with increasing Ceq, although differences were observed in the adsorption capacity. However, MSM–700 and–900 showed no significant adsorption, consistent with the physical property analyses using XRD and N2 adsorption–desorption analysis. To better understand the adsorption phenomenon of these MSMs, Langmuir and Freundlich isotherms were fitted with the experimental data of MSM–500 and–600.The IBP adsorption isotherms were determined using MSMs calcined at different temperatures (R2) of the linear plots, were calculated. The results are presented in Qmax) for MSM–500 and–600 varied considerably: 64.8 mg g–1 for MSM–500 and 31.9 mg g–1 for MSM–600.The Langmuir and Freundlich parameters, along with determination coefficients , which may increase IBP solubility. As a result, a weaker interaction between the MSM surface and IBP would occur, and the adsorption capacity would decrease significantly. However, the Qmax of MSM–500 was higher than the value reported for SBA–15.At higher pH, two important factors, ionization and surface charge of the adsorbent, can affect the adsorption process. The acid dissociation of IBP molecules occurs at a pH higher than the pKn obtained from the Freundlich model for IBP removal by MSM–500 and–600 were 0.82 and 0.93, respectively. These values were larger than the constant values of other media except commercial granular activated carbon  for SBA–15 [The adsorption densities per unit surface area for MSM–500 and–600 were similar, 0.21 and 0.20 mg mr SBA–15 , 8.t/qtvs. t was plotted to obtain the rate parameters. The results showed that the fit of the model to the data had a high determination coefficient (0.99).The adsorption kinetics were investigated using a pseudo-second-order kinetic model, in which K2 values for MSM–500 and–600 were 0.10 and 0.03 g mg–1 min–1, respectively. The higher K2 value for MSM–500 indicates that the medium was much faster in adsorbing IBP than the other MSMs. Although several types of AC have higher K2 values than MSMs  [∆adsG0 values were –10.4, 1.01, and 11.3 kJ mol–1 at 299, 309, and 319 K, respectively. Thus, the IBP adsorption is a spontaneous physical process at room temperature, but becomes non-spontaneous at higher temperatures. Moreover, ∆adsG0, ∆adsH0, and ∆adsS0 were also calculated for comparison with other references ((MWCNTs) , 38–40. ferences .∆adsH0 was –23.0 kJ mol–1, indicating that the IBP adsorption onto MSM–500 is an exothermic reaction, and ∆adsS0 was found to be –0.07 J mol–1 K–1. Accordingly, as a consequence of IBP adsorption onto the pores of MSM–500, the randomness of IBP decreased because the degree of freedom or mobility of IBP is limited compared with IBP in solution. The ∆adsS0 value was much smaller than other references, indicating that less energy is needed to restore the system back to its original state. The negative ∆adsH0 and ∆adsS0 suggest that the removal process of IBP can be considered an enthalpy-driven process.The calculated qeq, K2, and v0, respectively, at each cycle. The first cycle had the highest qeq (22.8 mg g–1). This result is similar to the kinetic tests conducted previously, suggesting that the experiments were reproducible under the same conditions. All consecutive cycles showed a qeq of 19–19.8 mg g–1, slightly lower than the qeq for the first cycle. Thus, based on the first qeq, MSM–500 had recovery efficiencies of 83–87%.qeq for IBP uptake was achieved at 5 min, but it slowed to ~30 min in the third and fourth cycles. In detail, the values of K2 and v0 were comparable in the first and second cycles, but dropped considerably at the third and decreased further at the fourth and fifth cycles. Accordingly, the reduction trends of IBP uptake capacity and speed were different, and these differences might have been affected by the deformation of the pore structure or the existence of some IBP molecules trapped in the pores after extraction with methanol. AC is composed mostly of micropores, and IBP molecules are adsorbed at the supermicropores, because its critical dimension is 0.72 nm [ex situ using heat or steam, which is a high energy-consuming process. Also, during this process, a large proportion of the AC can be lost [In the first cycle, 90% of the  0.72 nm . Thus, e be lost . In term2 gas isotherm. The TEM image of MSM–500 obtained after the fifth adsorption showed slight deformation at the inlet side of the pore structure, but no major defect. Thus, this result may be linked to the kinetic results for re-adsorption.–1) of all the MSMs prepared at different calcination temperatures, as well as those of IBP-retained MSMs. The as-synthesized MSM had obvious bands for OH , H2O , Si–O–Si (symmetric stretching vibrations (vs.) at 1,057 cm–1 and ~800 cm–1), Si–OH (vs. at ~950 cm–1), and O–Si–O (deformation vibration (δ) at 435cm–1). Si–O–Si , can be attributed to the asymmetric vibration of the carboxylic functional group (COO–). Two more sharp peaks, at 1,435 and 1,500 cm–1, were assigned to the C–H bonding of carbon atoms. The peak at 2,920 cm–1 (box labeled as e′) was assigned to a strong C–H bond, confirming the adsorption of IBP into the channels of MSM–500 [After the adsorption of IBP, the Si–O–Si , the unloading time of MSM–500 was much shorter. MSM–500 has a larger pore size than SBA–15 and MCM–41, which increases the diffusion of IBP molecules through the pores and subsequently increases the loading and unloading rates. The release behavior was also affected by the pH of the solution. At a lower pH, the IBP release was faster and higher, which is advantageous because most tumor cells in the body are slightly acidic and the drug release can be more specific to such target areas. Moreover pure SiO2 was utilized as a precursor, instead of TEOS to synthesis MSMs. This is expected to produce less toxic materials than the conventional mesoporous materials. Nakashima et al. have reported the acute and subchronic inhalation toxicity of TEOS in the synthesis of SBA–15, MCM–41 and MCM–48 [MSM–500 showed a 41% IBP drug loading capacity, comparable to the value reported for SBA–15 (20–30%) . Fig 8 sd MCM–48 .–1 and 1 m3, respectively. As shown in vs. other silica-based mesoporous media.In this study, the initial synthesis costs of materials were calculated to estimate the removal or delivery costs for IBP, for which the IBP concentration and volume were 10 mg L2. The allocated price of TEOS is ~96% of the total costs for synthesis and TEOS is ~200 times more expensive than SiO2. Furthermore, the reusability of MSM will further reduce the cost significantly.For example, the MSM was 67 times cheaper than granular SBA-15. The reduction in cost was mainly due to the replacement of TEOS with the much cheaper silicon source, SiO−of IBP and Si–OH of the pore surface. MSM–500 was also found to be suitable for IBP drug loading and unloading purposes, with a 41% loading capacity and almost 100% unloading efficiency within a few hours. The MSM was synthesized successfully using SiO2, so that its production costs were much lower than that of SBA-15. Thus, the new synthesis route developed in this study could have high potential for cost-effective mass production, and the MSM could be a suitable medium for industrial applications (for water treatment and drug delivery), as shown in the cost analysis study [2 is expected to produce less toxic MSMs, which are more suitable for water treatment and biomedical application.Adsorption isotherms and kinetics revealed that the MSMs synthesized showed relatively higher adsorption capacities and faster adsorption rates than other comparable media. The adsorption of IBP by MSM–500 was thermodynamically favorable at room temperature, but it involved relatively weak bonding because the calculated entropy was much smaller than in other references. In consecutive sorption studies, MSM–500 showed 83–87% recovery efficiencies, although it had a slower pattern of uptake. Based on the FTIR results, the reaction mechanism was identified as a hydrophilic interaction between the COOis study , 46, 47.


In humans, optimism is a cognitive construct related to motivation; optimists exert effort, whereas pessimists disengage from effort. In this study, using a recently developed ambiguous-cue interpretation (ACI) paradigm we took the unique opportunity to investigate whether “optimism” as a trait is correlated with motivation in rodents. In a series of ACI tests , we identified rats displaying “pessimistic” and “optimistic” traits. Subsequently, we investigated the trait differences in the motivation of these rats to gain reward and to avoid punishment using a progressive ratio (PR) schedule of reinforcement paradigm. Although “optimistic” and “pessimistic” animals did not differ in their motivation to avoid punishment, the “optimistic” rats were significantly more motivated to gain reward than their “pessimistic” conspecifics. For the first time, we showed an association between cognitive judgment bias and motivation in an animal model. Because both investigated processes are closely related to mental health and wellbeing, our results may be valuable for preclinical modeling of many psychiatric disorders. Optimism is a cognitive construct consisting of expectancy, and it has motivational implications  but also to avoid electric foot shock (motivation to avoid punishment).We hypothesized that in rats, similar to humans, the traits of “optimism” and “pessimism” would be associated and correlated with motivation to gain reward and avoid punishment.All described experimental procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Committee for Ethics in Animal Experiments at the Institute of Pharmacology Polish Academy of Sciences.Sprague Dawley rats  weighing between 175–200 g upon arrival. The animals were housed in groups of 4, in a temperature (21 ± 1°C) and humidity (40–50%) controlled room under a 12/12 h dark/light cycle (lights on at 06:00 h). The animals were habituated to the housing conditions and experimental facility for 2 weeks after arrival and before the start of experiments. In all of the experiments, the animals received 15–20 g of food per rat per day (standard laboratory chow) what corresponds to mild food-deprivation. The food deprivation began 7 days prior to beginning of the training. The water was provided ad libitum. The animals were trained and tested during the light phase of the dark/light cycle. The rats were habituated to the experimental room for 30 min prior to training and testing sessions and were tested once daily.We used 80 male The experiments were conducted in 8 computer-controlled, operant conditioning boxes ; the boxes were equipped with lights, speakers, liquid dispensers (0.1 ml of 5% sucrose solution), electric grid floors, and 2 retractable levers. The levers were located on both sides of the liquid dispenser. The experimental protocols were written in Med State notation code (Med Associates).The experimental training and testing procedures for the ambiguous-cue interpretation (ACI) paradigm used in this study were modified from procedures previously described by Enkel et al.  and haveIn brief, initially the animals were trained to press one lever when a “positive” tone (2000 Hz at 75 dB) signaled reward (5% sucrose solution) availability, and to press second lever when another, “negative” tone (9000 Hz at 75 dB) signaled a forthcoming punishment . By pressing appropriate levers the animals could either receive a reward or avoid punishment. The tone presentations were separated by 10 s intertrial intervals (ITI) and each training session lasted 30 min. The animals had to fulfill the criteria of at least 90% of accurate responses to the tone signaling reward availability maintained over 3 consecutive training sessions and, at least 60% of correct punishment-prevention responses maintained over 3 consecutive training sessions, to proceed to the discrimination training.During the discrimination-training phase, the animals were trained to discriminate between pseudo-randomly presented positive (20) and negative (20) tones, by responding to the appropriate levers (as learned in the previous training stages) to minimize punishment and maximize reward delivery. Each discrimination training session lasted 40 min. The animals had to achieve a minimum of 70% correct responses with each lever, maintained over 3 consecutive discrimination sessions to be qualified for the ACI testing.During the ACI testing sessions the animals were exposed to 20 negative, 20 positive, and 10 ambiguous (5000 Hz at 75 dB) tone presentations. The tones were played in a pseudo-randomized order and were separated by 10 s ITIs. The responses to each tone  during the ACI testing were analyzed as the proportion of the overall number of responses to a given tone. To calculate the cognitive bias index we subtracted the proportion of negative responses to the ambiguous-cues from the proportion of positive responses to the ambiguous-cue, what resulted in values ranging between −1 and 1. The values above 0 indicated an overall positive judgment and “optimistic” interpretation of the ambiguous-cue while the values below 0 indicated overall negative judgment and “pessimism”.The cognitive bias screening (CBS) procedure has been described in detail elsewhere .Because all animals had previously learned that pressing one lever in the operant chamber results in a reward delivery and pressing the other lever prevents punishment, they were switched directly after the CBS procedure to the tests with a progressive-ratio schedule of reinforcement.(0.2 × reward number))−5, rounded to the nearest integer. Thus, the values of the steps were 1, 2, 4, 6, 9, 12, 15, 20, 25, 32, 40, 50, 62, 77, 95, 118, 145, 178, 219, 268, 328, 402, 492, 603, etc. Each reward delivery was accompanied by presentation of the tone ) previously used in the ACI paradigm to signal reward availability. After each reward delivery, both levers retracted for a 10 s ITI. Sessions lasted 30 min. Responding on the “negative” lever, previously associated with avoiding of the punishment, was recorded but had no consequences.At the beginning of the PR session, both levers were extended into the operant chamber, and each rat received one non-contingent, experimenter-delivered reward (0.1 ml of 20% sucrose solution). Later during the session, responding on the previously rewarded “positive” lever was reinforced with sucrose solution delivery, and the number of responses required to produce the next reward increased progressively with each successive reward obtained. The steps of the exponential progression used in our study were the same as those previously developed by Roberts and Bennett  and prevOne week after measurements of motivation to gain reward, the animals were subjected to tests of motivation to avoid punishment in a modified PR paradigm. In this paradigm, extension of the levers was accompanied by presentation of the tone (9000 Hz at 75 dB SPL), which previously, in the ACI paradigm, signaled a forthcoming punishment. The rats had to make a progressively increasing number of responses on the “negative lever”, which was previously (in the ACI paradigm) associated with avoiding punishment, to avoid an electric shock . The steps of the exponential progression were the same as those used previously for measuring motivation to gain reward. Each consecutive lever press during the tone presentation prolonged its duration by 10 s, until the animal reached the number of lever presses required to avoid punishment at that stage. Reaching the required number of lever presses resulted in tone termination, retraction of the levers and a 10 s ITI. Not reaching the required number of lever presses resulted in prolongation of the tone and shock delivery. Pressing the lever after the shock onset terminated the tone and shock and initiated a 10 s ITI. After each shock delivery, both levers retracted for a 10 s ITI. Sessions lasted 30 min. Responding on the other “positive” lever was recorded but had no consequences.The experimental design is schematically presented in Figure 1Bt-tests. Finally, Pearson correlations between cognitive bias index and motivation to gain reward and avoid punishment were determined. For pair-wise comparisons, we adjusted the values using Sidak’s correction factor for multiple comparisons . The distribution of the cognitive bias index data was tested using the Kolmogorov-Smirnov test. The difference between “optimists” and “pessimists” in the frequency of “optimism” was analyzed using the t-test. The differences in the processing of the experimental tones between the “optimists” and “pessimists” were investigated using 4-way analysis of variance (ANOVA) with the between-subject factor of Cognitive judgment bias (2 levels: optimistic and pessimistic) and the within-subjects factors of Test (10 levels: baseline test 1–10), Lever (2 levels: positive and negative) and Tone . The differences between “optimistic” and “pessimistic” animals in motivation to gain reward and to avoid punishment were analyzed separately using  Howell, . All of t(78) = −0.85, NS).All trained animals (80) reached the training criteria, and qualified for CBS. The “optimists” reached the criteria of positive tone, negative tone and discrimination trainings after 5.7 ± 0.33, 6.25 ± 0.71 and 35.88 ± 1.9 days, respectively, whereas the “pessimists” reached the criteria after 6.23 ± 0.25, 7.16 ± 0.60 and 32.50 ± 0.80 days, respectively. We observed no significant differences in the total duration of the training between the “optimists” and “pessimists” .The AVG cognitive bias index of all of the experimental animals established based on the CBS was −0.11 ± 0.028. The distribution of the cognitive bias index data during CBS Figure  was normF = 4.50, p < 0.001), post hoc pairwise comparisons revealed that between-test differences showed no unequivocal pattern.An analysis of the response of animals to the positive and negative levers following reference and ambiguous tones across the screening period indicated no test-retest effects. Although the Test × Lever × Tone interaction was significant  and “pessimists” .Based on the results of the CBS Figure  we dividF = 24.83, p < 0.001)). The “optimists” responded significantly less often to the negative lever in response to the ambiguous tone compared to “pessimists”  compared to after the positive tones  = 12.85, p < 0.001, Figure “Optimists” and “pessimists” did not differ in the numbers of omitted trials (no significant effect of Cognitive bias or Cognitive bias × Tone interaction); however, all of the rats made significantly more omissions following negative and ambiguous tones  revealed that on AVG, rats classified as “optimists” were significantly more frequently “optimistic” than their “pessimistic” conspecifics Figure —inset.F = 4.50, p < 0.001)), the differences between the “optimists” and “pessimists” did not change significantly across the CBS  = 0.73, NS), indicating stability of the traits  = 4.58, p = 0.002). Correlation analysis revealed that the cognitive bias index and motivation to gain reward, measured for the PR schedule of reinforcement, were significantly positively correlated . The “optimistic” and “pessimistic” animals also differed significantly in the numbers of total lever presses  = 4.13, p < 0.001) and percentages of inactive lever presses made during the PR test session  = 3.21, p < 0.01).On a PR schedule, “optimism” has been found to be associated with a significantly higher motivation to gain reward than “pessimism”. t(78) = 0.61, NS). There was also no significant correlation between cognitive bias index and motivation to avoid punishment . The “optimists” and “pessimists” did not significantly differ in the number of total lever presses  = 1.33, NS), percentage of inactive lever presses  = 0.61, NS) and omissions made during the PR test session  = 1.20, NS).No significant differences were observed in the motivation to avoid punishment between the “optimistic” and “pessimistic” animals  = 7.77, p < 0.001) more motivated to gain a reward (AVG breakpoint 14.44 ± 0.88) than to avoid punishment (AVG breakpoint 9.10 ± 0.74).On AVG, all animals were found to be significantly (In the present study, we used an animal model to examine whether the traits of “optimism” and “pessimism” are associated with different levels of motivation in rats. Our results indicate that animals displaying the “optimistic” trait were more motivated to obtain a sweet sucrose reward compared to their “pessimistic” conspecifics. There was also a significant positive correlation between the level of “optimism” and motivation to obtain the reward, whereas motivation to avoid punishment did not differ between “optimistic” and “pessimistic” animals.In combination with our previous report (Rygula et al., Motivation is a pervasive and important determinant of behavior. It has been conceptualized as selecting goals based on their predictive value, initiating behavior to achieve goals, and maintaining goal-directed action (Dickinson and Balleine, The concepts of optimism and pessimism concern an individual’s expectations for the future (Carver et al., There are, of course, also limitations to the present study. First, all measurements of cognitive judgment bias and motivation were performed using operant conditioning tasks that required extensive use of punishment to shape the animals’ behavior. Because it is uncertain how the history of such training influenced motivation, it would be desirable to test the association between cognitive judgment bias and motivation using alternative methods. For example, the use of the spatial version of the ACI task with rewards of different magnitude and with no punishment may prove particularly enlightening. Another limitation is that the nature of our studies prevented us from establishing whether it is “optimism” that increases approach motivation or vice versa. The present research clearly demonstrates that cognitive judgment bias is linked to motivational dispositions, and this is consistent with theoretical models that place optimism as a defining feature of motivation (e.g., Carver and Scheier, Taken together, using multiple consecutive ACI tests and a PR schedule of reinforcement based tasks, we demonstrate, for the first time, a link between cognitive judgment bias and motivation in an animal model. Based on the present results, one would predict that cognitive judgment bias would be a powerful shaper of approach motivation in other species, including humans. Evidence of this may be useful for developing therapeutic interventions that target motivational deficits associated with depressive disorders. This would suggest, for example, that reinforcing optimistic judgment bias either pharmacologically or by cognitive bias modification therapy could help to abolish motivational deficits, which are a core symptom of depression in humans.RR, JG, JK and JK conceived and designed the experiments, JG, JK, JK and RR performed the experiments, RR analyzed the data, RR and PP contributed materials/analysis tools, RR wrote the paper, RR, JG, JK, JK and PP revised the paper critically for important intellectual content and gave final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Staphylococcus aureus Sp17, a typical highly virulent (HV) rabbit strain. As current medicine apparently fails to effectively reduce disease and economical losses caused by this organism, it is essential to gain better insight on its genomic arrangement.We report the draft genome sequence of  Staphylococcus aureus strains result in severe clinical conditions; these strains are also frequently resistant to antimicrobials. An outbreak of an HV S. aureus strain hinders profitable production and frequently necessitates culling the entire flock  re flock .S. aureus clone, as it shows the mixed biotype CV-C . This strain belongs to the typical highly virulent rabbit ype CV-C , is sens strains , and hasTotal DNA of the strain was subjected to 2 × 300-bp paired-end Illumina MiSeq sequencing at the Department of Biochemistry, Faculty of Medicine, University of Szeged, Hungary. A total of 3.96 million read pairs were recorded, and the estimated coverage of the whole genome is 700×.de novo using MIRA version 4.0.2 , the genetic code to “11 ,” the annotation scheme to “ClassicRAST,” and “preserve gene calls,” “automatically fix errors,” “fix frameshifts,” and “backfill gaps” to “no.” We obtained 2,395 annotated genes, 50 tRNAs, and 5 rRNAs.Scaffolds were submitted to the RAST annotation server . The taxS. aureus subsp. aureus 21337 (accession no. NZ_JHPZ00000000.1). Pairwise alignment showed 98.56% similarity of these sequences, which indicates a close relationship between these strains.A search for similar sequences deposited in GenBank was conducted using BLAST. The best match was S. aureus infections in rabbits.A detailed analysis of the virulence genes in these sequences will be conducted to identify key elements responsible for the remarkable pathogenicity of this strain, in order to facilitate the development of effective treatment methods and/or preventive measures against HV LBCS00000000. The version described in this paper is version LBCS01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.


Risky decision-making is commonly observed in persons at risk for and infected with HIV and is associated with executive dysfunction. Yet it is currently unknown whether HIV alters brain processing of risk-taking decision-making.This study examined the neural substrate of a risky decision-making task in 21 HIV seropositive (HIV+) and 19 seronegative (HIV-) comparison participants. Functional magnetic resonance imaging was conducted while participants performed the risky-gains task, which involves choosing among safe (20 cents) and risky (40/80 cent win or loss) choices. Linear mixed effects analyses examining group and decision type were conducted. Robust regressions were performed to examine the relationship between nadir CD4 count and Kalichman sexual compulsivity and brain activation in the HIV+ group. The overlap between the task effects and robust regressions was explored.Although there were no serostatus effects in behavioral performance on the risky-gains task, HIV+ individuals exhibited greater activation for risky choices in the basal ganglia, i.e. the caudate nucleus, but also in the anterior cingulate, dorsolateral prefrontal cortex, and insula relative to the HIV- group. The HIV+ group also demonstrated reduced functional responses to safe choices in the anterior cingulate and dorsolateral prefrontal cortex relative to the HIV- group. HIV+ individuals with higher nadir CD4 count and greater sexual compulsivity displayed lower differential responses to safe versus risky choices in many of these regions.This study demonstrated fronto-striatal loop dysfunction associated with HIV infection during risky decision-making. Combined with similar between-group task behavior, this suggests an adaptive functional response in regions critical to reward and behavioral control in the HIV+ group. HIV-infected individuals with higher CD4 nadirs demonstrated activation patterns more similar to seronegative individuals. This suggests that the severity of past immunosuppression (CD4 nadir) may exert a legacy effect on processing of risky choices in the HIV-infected brain. The human immunodeficiency virus (HIV) can cross the blood-brain barrier early in the course of infection and trigger a cascade of functional and structural alterations Risk-taking and reward processing are important processes that influence behavior Risky decision-making is commonly observed in individuals at risk for e.g.,  and infeA number of neuroimaging studies have shown that HIV infection can alter brain function Extensive study of risk-taking in the context of decision-making has revealed a key network of cortical and subcortical brain regions. This network is composed of circuits, two of which, namely the ventral limbic circuit and the dorsal executive circuit, may be important to choice behavior. The ventral circuit includes several striatal structures: the nucleus accumbens, rostromedial caudate, rostroventral putamen, and ventromedial caudate This study aimed to examine whether HIV alters brain processes underlying risk-taking decision-making. We hypothesized that greater activity for risky choices would be observed in grey matter basal ganglia regions. Additionally, we theorized that dorsolateral and anterior cingulate cortex would display greater activity in the HIV+ group relative to HIV- comparisons, and that this may be an adaptive functional response to compensate for aberrant information provided by other cortical and subcortical structures that may have been impacted by HIV infection. Finally, we explored whether nadir cluster of differentiation 4 (CD4) count, a measure of historical immune function that has been shown to predict neurocognitive impairment The University of California, San Diego human research protection program approved this study. Participants gave informed written consent and were compensated for their time and effort.th Edition-Text Revision; DSM-IV-TR) Participants were recruited as part of the Translational Methamphetamine AIDS Research Center and included 21 HIV+ and 19 seronegative comparison adults. HIV status was confirmed by MedMira Multiplo rapid test . All participants were seronegative for Hepatitis C virus (HCV) as determined by the MedMira Multiplo rapid test. Current CD4 T lymphocyte counts (cells/ml) were determined by flow cytometry at a medical center laboratory certified by Clinical Laboratory Improvement Amendments (CLIA), or CLIA equivalent. HIV RNA levels were measured in plasma by reverse transcriptase PCR . CD4 nadir was obtained by self-report, with confirmation by documented prior measurements in a subset of individuals. Participants were excluded if they tested positive for illicit drugs (with the exception of marijuana (MJ)) or alcohol  on the day of scan; had contra-indications for MRI; had a lifetime history of schizophrenia or other primary psychotic disorders; had previous cerebrovascular events, determined by comprehensive neurological exam; head injury with loss of consciousness for greater than 30 minutes or resulting in neurologic complications; seizure disorder; demyelinating diseases from non-HIV neurological disorders; met Diagnostic and Statistical Manual of Mental Disorders  choices over the safe choices (20). While the best response for each trial depended on whether there was a punishment scheduled for that trail or not, a strategy of selecting all 20 s would yield exactly the same winnings as selecting all 40 s or all 80 s.Three different trial types were presented in a predetermined pseudo-randomized order: (1) non-punished , (2) punished 40 (n = 24), (3) punished 80 (n = 18) and six null trials. The relative number of punished and non-punished trials was chosen to guarantee that the strategy of consistently choosing 20-cents or always selecting 40- or 80-cents would yield the exact same winnings. Punishment occurred on a punished trial only if the participant failed to respond to the previous numbers on that trial (i.e. when holding out for 80 they did not respond to either the 20 or 40 stimulus). The relative frequency of safe (20) to risky (40 and 80) was used to quantify baseline risk-taking behavior. To investigate the sensitivity to punishment, defined here as the propensity to alter choice pattern on a trial immediately following a losing trial, the relative frequency of risky responses was examined as a function of the outcome of the previous trial, that is, punished versus non-punished risky trials.This task has previously been used to assess risk-taking behavior in healthy volunteers Functional images were acquired in bottom-up interleaved axial slices using T2* weighted echo planar imaging (EPI). Images were acquired on two scanners: a 3T GE Discovery MR 750   and a 3T GE Signa HDx   axial slices, 3.5×3.5 mm voxels). High-resolution T1-weighted fast spoiled gradient echo anatomical images  were acquired to permit subsequent activation localization and spatial normalization. Gradient echo field-maps were also acquired to permit compensation for geometric distortions caused by magnetic field inhomogeneity  axial slices, 3.5×3.5 mm voxels). Stimuli were projected onto a screen at the participants’ feet and viewed with the aid of a mirror attached to the head coil.0 inhomogeneity Analyses were conducted using AFNI rd order Lagrange polynomial, which accounted for slow signal drift, were included in the baseline. Brain activation was operationally defined as percent signal change relative to baseline.Decision phase regressors were created such that they started at trial onset and ended either when the participant responded or was punished. Five regressors were defined: (1) 20 (safe), (2) win 40, (3) win 80, (4) lose 40, and (5) lose 80. General linear tests (GLT) of the safe (20) versus the risky win  choices were computed for each participant. The baseline comprised all other time-points not accounted for by the regressors of interest. Additionally, six nuisance motion-related regressors  and a 3 = 4.11, p = 0.05, uncorrected) and, to control for multiple comparisons, were required to be part of a cluster of no less than 1685 µL. The volume threshold was determined by a Monte-Carlo simulation that together with the voxel-wise threshold resulted in a 5% probability of a cluster surviving due to chance. The average percent signal change was extracted from the clusters so formed and a series of post hoc t-tests were conducted in R to examine the group and task interaction effects.The within-participant general linear tests of safe versus risky choices were subjected to linear mixed effects (LME) analyses in the R statistical analysis package Since the HIV+ group consisted of more nicotine users than the HIV- group (see below), we conducted an additional analysis to investigate whether there was an effect of nicotine usage  on differential brain activation to risky versus safe choices. This was performed within the HIV+ group and accomplished using linear mixed effects models on the average contrast of risky versus safe choices in each of the clusters identified by the task related group analysis. In this analysis participant was treated as a random effect.Several recent studies have examined the effect of including MR data from multiple sites within the same analysis. Overall, these studies reported that inter-participant variance was anywhere from 7–44 times greater than that generated by site variance Whole-brain voxel-wise Huber robust regressions In all cases, regression coefficients and their corresponding t-values were split according to whether they demonstrated a positive or negative relationship with the GLTs. Thereafter, significant voxels were required to pass a voxel-wise statistical threshold (t(18) = 2.10, p = 0.05, uncorrected) and, to control for multiple comparisons, were required to be part of a cluster of no less than 1685 µL which resulted in a 5% chance of a cluster surviving due to chance. The volume threshold was determined in the same manner as above.To investigate whether between-group differences could potentially be attributed to neuropsychiatric and neuromedical variables, we assessed whether the regions identified from the task related analysis overlapped with those regions identified by the robust regression analysis. To accomplish this we computed the intersection of all those regions from the task related analysis with those from the regression analyses conducted solely within the HIV+ group. Since both of the maps included in this analysis included significantly different clusters, the resultant overlap maps can, therefore, also be regarded as statistically significantly different 2 test of equal proportions. Using Spearman’s rank correlation test, we tested for the presence of a relationship between BDI-II score and the mean percentage signal change from each of the clusters resulting from the task-based whole brain analysis. This correlation was performed both solely within participants with lifetime diagnosis of MDD and within the sample as a whole.All analyses were conducted in R The behavioral data gathered during task performance was subjected to a 3-way ANOVA in R χ2(1) = 0.18, p>0.1), gender (χ2(1) = 1.00, p>0.1), years of education (t(37.97) = −0.85, p>0.1), or ethnicity (χ2(3) = 3.85, p>0.1). The groups differed in terms of sexual orientation, with the HIV+ group composed primarily of men who have sex with men, whereas the HIV- group was predominantly heterosexual (χ2(1) = 13.81, p<0.001). Three participants tested positive for MJ , a proportion that did not differ between groups (χ2(1)≈0, p≈1). None of these participants met DSM-IV criteria for current or lifetime substance abuse or dependence. No participants tested positive for alcohol.There were no significant differences between the serostatus groups in age (t(37.99) = −0.74, p>0.1), handedness (χ2(1) = 0.84, p>0.1). There were no between-group differences observed in any of the speeded information processing, verbal fluency, learning, working memory, executive functions, and motor skills domains . There was a trend for the HIV- group to exhibit a higher WRAT-4 score than the HIV+ group (t(35.81) = 1.85, p = 0.07, g = 0.60). On the subscales of the KS4, the groups did not differ on the sexual sensation seeking subscale (t(34.04) = −1.79, p>0.05, g = −0.60) but the HIV-positives scored higher on sexual compulsivity (t(26.01) = −2.33, p<0.05, g = −0.70) and lower on non-sexual sensation seeking (t(36.32) = 2.02, p<0.05, g = 0.60). The groups showed no differences on the BIS-11 or any of its subscales  (See χ2(1) = 4.37, p<0.05). The HIV+ group was comprised of marginally more nicotine dependent individuals than the HIV- group (χ2(1) = 3.22, p = 0.07). There were no significant between-group differences on nicotine usage measured by the FTND (t(38) = −0.22, p>0.1). There were no significant differences between the two groups on other substance use characteristics  = 14.06, p<0.001), there was no effect of visit  = 1.81, p = 0.18), or an interaction between group and visit  = 1.79, p = 0.18). This suggests that the BDI-II score was stable over time. Finally, no significant between-group differences on the POMS were observed (t(29.02) = −1.75, p>0.05).There was no difference between the groups on GDS . Three HIV- and seven HIV+ were classed as impaired (GDS >0.5); the proportions did not differ between the two groups (.05) See . Three Htics see . Signifi = 337.92, p<0.0001) with safe (M = 0.65) responses more likely than risky (M = 0.16). No significant effects of punishment  = 0.05, p>0.05, punished: M = 0.33, non-punished: M = 0.33) or group  = 0.21, p>0.05; HIV-: M = 0.33, HIV+: M = 0.32) were observed. No significant interactions of punishment × group  = 0.06, p>0.05) or risk × punishment × group  = 0.00, p>0.05) were observed. A marginally significant effect for risk × group  = 3.09, p = 0.08) was observed with the HIV- group selecting risky choices marginally more than the HIV+ group . Consistent with our prior observations on this task  = 11.42, p<0.001) was observed wherein participants were more likely to chose the safe option when the prior trial was punished. See A significant main effect of risk was evident  critical to risk and reward processing despite broadly similar task performance between the two groups. In the overlap between the task-related regions of interest and those resulting from the robust regression analysis, those HIV+ individuals with greater sexual compulsivity measured by the KS4 and higher nadir CD4 count displayed lower differential responses to safe versus risky choices in many of the regions that showed between-group task related differences. Taken together, these results support the hypothesis that HIV alters risk-related processing in the basal ganglia, among other structures.We observed significant between-group differences in left and right hemisphere clusters that included subcortical regions and small insular components. The subcortical constituents of this cluster included the ventral and dorsal striatum. These regions have been heavily implicated in reward related processing There were significant between-group differences in activation level in the right anterior cingulate between the HIV+ and HIV- groups. This dorsal region of the ACC is thought to be important to cognitive processes Finally, consistent with prior finding of this task in DACC and DLPFC are extensively interconnected Since, relative to the seronegative group, the HIV+ group displayed elevated sexual compulsivity – a factor that has been associated with risk of HIV infection Within the HIV+ group, we also investigated whether nadir CD4 counts would show any relationship to differential activation to safe versus risky choices. We observed that subsets of the voxels identified as showing between-group task related differences also showed a relationship to nadir CD4 count. As depicted in This study has several limitations. The data were acquired from the two groups of participants on two different scanners. Though we attempted to minimize the differences between the protocols on both scanners and to account for this source of variation in our models, future studies are required to replicate the results reported here in the absence of this potential confound. This is a cross-sectional study, and thus we cannot address whether the differences reported here arose as a consequence of HIV infection or whether they predated HIV infection. Future longitudinal studies are required to determine whether factors such as duration of infection or the use of anti-retroviral therapy may influence impairment. In light of the recent report that risky decision-making is more prevalent in individuals with HAND In summary, the present study examined the functional neuroanatomy of risky decision-making in HIV+ individuals compared to seronegetative individuals. The HIV+ group displayed altered functional responses to safe and risky choices in several brain regions compared to the seronegetative group. Specifically, these regions included portions of the anterior cingulate, ventral and dorsal striatum, insula, and bilateral DLPFC. These results are consistent with and further support the role of these structures in risky decision-making Table S1Regions showing differences between differential brain responses to risky versus safe choices and neuropsychiatric measures.(XLSX)Click here for additional data file.Table S2Marginal and cell means from the ANOVA for the behavioral analysis.(XLSX)Click here for additional data file.


Achieving optimal symptom control with minimal side effects is a major goal in clinical practice. Dual-agent dopamine receptor agonist (DA) therapy in Parkinson’s disease (PD) may represent a promising approach to treatment, as the combination of different pharmacokinetic/pharmacological profiles may result in a lesser need for high dosages and, accordingly, may be well tolerated. The objective of the current study was to investigate safety and efficacy of rotigotine transdermal system as add-on to oral DA in patients with advanced PD inadequately controlled with levodopa and low-dose oral DA.PD0015 was an open-label, multinational study in patients with advanced-PD and sleep disturbance or early-morning motor impairment. Patients were titrated to optimal dose rotigotine (≤8 mg/24 h) over 1–4 weeks and maintained for 4–7 weeks (8-week treatment). Dosage of levodopa and oral DA  was stable. Primary variable was Clinical Global Impressions (CGI) item 4: side effects, assessing safety. Other variables included adverse events (AEs), Patient Global Impressions of Change (PGIC), Unified Parkinson’s Disease Rating Scale (UPDRS) II and III, Parkinson’s Disease Sleep Scale (PDSS-2), Pittsburgh Sleep Quality Index (PSQI), and “off” time.Of 90 patients who received rotigotine, 79 (88%) completed the study; 5 (6%) withdrew due to AEs. Most  had a CGI-4 score <3 indicating that rotigotine add-on therapy did not interfere with functioning; 6 (7%) experienced drug-related AEs that interfered with functioning (score ≥3). AEs occurring in ≥5% were application site pruritus (13%), dizziness (10%), orthostatic hypotension (10%), nausea (8%), dyskinesia (8%), and nasopharyngitis (6%). Numerical improvements in motor function (UPDRS III), activities of daily living (UPDRS II), sleep disturbances , and reduction in “off” time were observed. The majority  improved on PGIC.Addition of rotigotine transdermal system to low-dose oral DA in patients with advanced-PD was feasible and may be associated with clinical benefit.NCT01723904. Trial registration date: November 6, 2012.ClinicalTrials.gov identifier The online version of this article (doi:10.1186/s12883-015-0267-7) contains supplementary material, which is available to authorized users. Treatment with chronic levodopa for the symptoms of advanced Parkinson's disease (PD) is often associated with the development of motor fluctuations and dyskinesia, which gradually worsen as the disease progresses ,2. TheseRotigotine is a non-ergot derived DA with activity across D1 through D5 receptors as well as select adrenergic and serotonergic sites . ContinuThe objective of this study was to investigate the safety and efficacy of rotigotine transdermal system as add-on to therapy with low-dose pramipexole or ropinirole, in patients with advanced PD. Enrolled patients were insufficiently controlled with levodopa and low-dose oral DA, identified as experiencing motor complications and sleep disturbance or early-morning motor impairment.Patients enrolled in the PD0015 study included men and women, aged 30–80 years, with idiopathic PD of longer than 3 years’ duration, and Hoehn and Yahr stage II-IV. PD was defined by the presence of bradykinesia and at least one of the following: resting tremor, rigidity, or impairment of postural reflexes. In addition, all patients included had to be taking levodopa  in combination with benserazide or carbidopa, and a stable low dose of IR or ER pramipexole (≤1.5 mg/day) or ropinirole (≤6.0 mg/day) for at least 28 days before baseline. Patients had to experience motor fluctuations  or dyskinesia, sleep disturbance or early-morning motor impairment, as determined by the investigator, and nocturia for at least 3 nights within 7 days before baseline. At the screening visit, patients were instructed to differentiate between “off” and “on” states and symptoms of “troublesome dyskinesia” for diary recordings. Patients completed 7 days of diary recordings before beginning study treatment; four of the diaries had to be determined “valid” by the investigator for the patient to be eligible. Permitted PD medications included anticholinergics, monoamine oxidase B (MAO-B) inhibitors, N-methyl-D-aspartate (NMDA) antagonists , and entacapone; permitted CNS active drugs included sedatives, antidepressants, anxiolytics, hypnotics. All permitted drugs were required to be at stable doses for at least 28 days prior to baseline, and were to remain stable for the duration of the study.Patients with clinically significant hepatic or renal impairment were excluded. Prohibited medications included DAs other than pramipexole or ropinirole, MAO-A inhibitors, dopamine-releasing substances, dopamine-modulating substances, tolcapone, budipine, dopamine receptor antagonists . The study was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. The study protocol and amendments were approved by the Institutional Review Board of all 21 centers  was a Phase IIIb, open-label, single-arm study in patients with advanced PD (between October 2012 and April 2013). Twenty one centers enrolled patients, who applied at least one rotigotine patch, in South Korea, Malaysia, Taiwan, Australia, and Singapore.2), 4 mg/24 h (20 cm2), or 6 mg/24 h (30 cm2). The study consisted of a 1-4-week titration and 4-7-week maintenance period ; patients were titrated in weekly increments of 2 mg/24 h rotigotine to their optimal  dose (up to 8 mg/24 h)  item 4 score (side effects) at the end of maintenance: 1 = “None”, 2 = “Not significantly interfering with patient’s functioning”, 3 = “Significantly interfering with the patient’s functioning”, and 4 = “Side effects outweigh therapeutic efficacy”. Other safety evaluations included extent of rotigotine exposure, and treatment-emergent adverse events . In addition, AEs of special interest  were pre-identified and assessed. The AEs of special interest were pre-identified as application site reactions, nausea and vomiting, somnolence, psychosis, sleep attacks/sudden onset of sleep, obsessive-compulsive disorder/impulse-control disorder, postural deformities, freezing gait, and perception disturbances that required atypical antipsychotic treatment. In addition, the modified Minnesota Impulsive Disorder Interview was used to prompt the investigators and patients to monitor the emergence of impulse control disorders. Finally, physical and neurological examinations, changes in laboratory tests, vital signs, 12-lead electrocardiography (ECG), and the Columbia-Suicide Severity Rating Scale (C-SSRS) were also assessed.Efficacy variables included outcomes assessing motor symptoms, motor fluctuations, non-motor symptoms (sleep disturbance), and quality of life. Change from baseline to end of maintenance was assessed for Unified Parkinson’s Disease Rating Scale (UPDRS) Parts II , and III ; absolute time spent "off" and absolute time spent "on" without troublesome dyskinesia (assessed from patient diaries); Parkinson's Disease Sleep Scale version 2 (PDSS-2); global score of the Pittsburgh Sleep Quality Index (PSQI); Patient Global Impression of Change , and the 8-item short-form Parkinson’s Disease Questionnaire . Other variables included number of awakenings during the night and number of nocturias, assessed from patient diaries.The primary variable (CGI item 4 score) was analyzed by the safety set , using last observation carried forward (LOCF). With the exception of time spent “off”, efficacy variables were analyzed using the full analysis set , with LOCF. Time spent “off” was analyzed using a subgroup of the FAS comprising patients who recorded time spent “off” at baseline in the patient diary. Efficacy variables were summarized with univariate statistics (mean ± SD), and 95% confidence intervals (CI) were calculated for changes from baseline .Of 112 patients who provided informed consent and were screened, 22 failed screening, and 90 (80%) applied at least one rotigotine patch and were included in the safety set. Of these 90 patients, 79 (88%) completed the study; the remaining 11 patients (12%) withdrew prematurely due to AEs (n = 5) or for other reasons (n = 6). Baseline demographic data are presented in Table The mean (±SD) duration of rotigotine exposure was 58.7 ± 14.9 days. A total of 84 patients (93%) entered maintenance. The maintenance dose was 2 mg/24 h in 14 patients, 4 mg/24 h in 19 patients, 6 mg/24 h in 16 patients, and 8 mg/24 h in 35 patients (mean ± SD dose: 5.71 ± 2.28 mg/24 h). Of the 11 patients who discontinued the study, six patients discontinued during the first 2 weeks of titration (at 2 mg/24 h), and the remaining five patients discontinued after between 43 and 56 days  of rotigotine treatment.The dose of concomitant oral DA at baseline (as rotigotine converted dose) was 2 mg/24 h in 30 patients, 4 mg/24 h in 29 patients, and 6 mg/24 h in 31 patients (mean ± SD dose: 4.02 ± 1.66 mg/24 h). There was no obvious relationship between the dose of oral DA and dose of rotigotine  had a score of <3 on CGI item 4  at end of treatment, indicating that rotigotine add-on therapy did not interfere with the patient’s functioning. At end of maintenance, a total of six patients (7%) experienced AEs, considered related to study drug by the investigator, that interfered with the patient’s functioning (score ≥3). Three of these six patients were receiving 2 mg/24 h rotigotine at the time of the AE (Table Fifty eight patients 64%) experienced a total of 147 AEs. AEs occurring with an overall incidence of 5% or higher were application site pruritus reported by 12 patients (13%), dizziness , orthostatic hypotension , nausea , dyskinesia , and nasopharyngitis . The incidence of AEs by 1) rotigotine dose, and 2) oral DA dose is presented in Additional file 4% experiForty four patients (49%) experienced at least one AE during titration, and 25 patients (30%) experienced at least one AE during maintenance. The majority of AEs were mild or moderate in intensity; six patients (7%) experienced at least one severe AE. Six serious AEs were reported in five patients (6%): hallucination, subdural hemorrhage, nasopharyngeal cancer, delirium, confusional state, and urinary retention. Except for the hallucination, all the events were considered unrelated to rotigotine. No deaths were reported.Five patients were prematurely withdrawn from the study due to AEs : dyskinesia (one patient), orthostatic hypotension (one patient), worsening of Parkinson’s disease (one patient), subdural hemorrhage (one patient), dizziness, hyperhidrosis, insomnia, and nausea (one patient). One patient, whose primary reason for withdrawal was due to noncompliance, also discontinued the study due to an AE (nausea). Down-titration of rotigotine was performed in 15 patients due to AEs.Of the AEs of special interest, 22 (24%) patients experienced application site reactions, eight (9%) nausea and vomiting , four (4%) somnolence , four (4%) psychosis , and one (1%) patient experienced obsessive-compulsive disorder/impulse-control disorder.There were no clinically relevant changes in laboratory parameters, vital signs, ECGs, physical and neurological examinations, or the C-SSRS.Motor function (UPDRS III) and ability to carry out ADL (UPDRS II) was improved following the addition of rotigotine to the existing treatment with low oral DA Figure . Mean ±; mean (±Improvements in items relating to disturbed sleep (a non-motor symptom of PD) were observed, including PDSS-2 total score, PSQI global score, number of awakenings during the night, and number of nocturias Figure .Figure 2The majority of patients reported an improvement on the PGIC , with 29/88 (33%) reporting “much improved” or “very much improved”, and only 3% (3/88) reporting a deterioration of “much worse” or “very much worse”. Mean (±SD) PDQ-8 total score at baseline was 29.4 ± 17.0, and change from baseline to the end of titration/maintenance was −6.6 ± 14.2 . The upper limit of 95% CI was below 0, suggesting an improvement in items assessing PD-related quality of life.This open-label study was the first study of rotigotine transdermal system used as an adjunct to treatment with an oral DA. The majority of patients in this study were Hoehn and Yahr stage II-III, and all were inadequately controlled with a treatment regimen of levodopa and low-dose oral pramipexole or ropinirole, presenting with early-morning motor impairment or nocturnal sleep disturbance. The study identified no major safety concerns when rotigotine was added to this treatment regimen. The addition of rotigotine was also associated with numerical improvements in efficacy outcomes, including motor function and sleep disturbances.The addition of rotigotine was generally well tolerated, with the majority of patients (93%) not experiencing drug-related AEs that interfered with functioning, as assessed by the primary outcome (CGI item 4). In the patients experiencing drug-related AEs that interfered with functioning (CGI item 4 ≥ 3), there was no obvious relationship with the total DA dose  or levodopa dose. Of note, the majority of patients experienced an improvement on the PGIC (81%), demonstrating that most patients considered rotigotine add-on therapy as beneficial.The AE profile was similar to previous studies of rotigotine in patients with advanced PD, with typical dopaminergic side-effects and application site reactions observed, and AEs were comparable with those seen with ropinirole or pramipexole -14,18. TNumerical improvements in patients’ motor function (UPDRS III) and ability to carry out ADL (UPDRS II) were observed following rotigotine add-on therapy. A reduction in “off” time was observed, with a corresponding prolongation of time spent “on” without troublesome dyskinesia (patient diary). This suggests that the improvement in “on” time was not at the expense of increased dyskinesia. The current study was not designed to investigate the effect of the oral DA formulation  on the efficacy outcomes; however, the formulation of the oral DA did not appear to have an obvious effect on the improvement in “off” time. Improvements in the non-motor symptom of sleep disturbance , and PD-related quality of life (PDQ-8) were also observed.In some patients with PD, symptoms may not be adequately controlled with existing oral DA treatment, and the dose cannot be sufficiently increased due to adverse drug reactions. In addition, in some cases, night-time symptoms may be observed although the current dose of DA improves day-time symptoms. In the present study, the patients were taking seemingly low doses of pramipexole/ropinirole, yet they were receiving stable doses which were maximal for the individual . The patients were inadequately controlled on these doses, and the addition of rotigotine aimed to further control their symptoms. We observed that concurrent activation of D2 and D3 receptors following dual DA therapy was feasible, at least in the dose ranges used. However, the mechanism/s by which rotigotine added to pramipexole or ropinirole induced potential benefits on efficacy outcomes  can only be speculated. They may result from the increase in DA total dose , the different DA receptor profiles  and/or the different pharmacokinetics.There are some potential limitations to consider. First, as there was no comparator arm or control group, and no statistical significance testing was performed, this limits the conclusions that can be drawn. Second, the ability to generalize the results of this study is restricted by the entry criteria of the study, and so limited to patients with advanced PD inadequately controlled on levodopa and a low-dose oral DA. Third, longer term safety cannot be concluded from this 8-week study. Finally, to prevent potential overstimulation of D2/D3 receptor after concomitant use of rotigotine-pramipexole and rotigotine-ropinirole, less than half of the respective approved maximum doses were used in this study. Therefore, we cannot conclude whether higher doses may provide further benefits or be associated with safety concerns.In summary, this study demonstrates that addition of rotigotine transdermal system to a low-dose oral DA was feasible and may be associated with clinical benefit in patients with advanced PD inadequately controlled on levodopa and a low-dose oral DA. Dual therapy with rotigotine transdermal system and a low-dose oral DA in PD may represent a promising approach to treatment. Equivalent dose should be taken into consideration when the DAs are used concomitantly, and the maximum dose of DAs should not exceed the upper limit of the approved dose of any of the DAs. Double-blind controlled studies are required to determine the significance and clinical relevance of these findings.


Canine reticulated platelets (r-PLTs) i.e., juvenile PLTs reflecting thrombopoiesis can be measured automatically with the hematology analyzer Sysmex XT-2000iV using manual gating options. However, the impact of interferences on r-PLT measurements performed with the gates published previously  is largely unknown.The aim was to compare different published gates for measurement of r-PLTs with the Sysmex XT-2000iV with an own, optimized gate (“Oellers-gate”) and to establish reference intervals (RIs) in > 120 dogs.Data of 362 measurements of diseased and healthy dogs were analyzed retrospectively. Several gates were applied and RIs for r-PLTs and platelet indices were established for pet dogs and a group of 153 healthy Beagles kept under defined housing conditions. Intra-assay precision (CV) was also assessed.s: 0.88–1.00). RIs for the “Pankraz-gate”, the “Gelain-gate”, and the “Oellers-gate” were 0.0–1.2, 0.2–3.7 and 0.2–3.9 % respectively. CVs were ranging between 22 and 41 %.In 30/362 samples, interferences consistent with small erythrocytes/reticulocytes were seen in the previously published gates but not in the “Oellers-gate”. Good correlation was found between the different gates (rOptimization of previously published gates minimized interferences of small erythrocytes with r-PLT measurements. In contrast, the MPV was significantly higher in dogs with immune mediated thrombocytopenia than in healthy control dogs [Several platelet indices such as mean platelet volume (MPV), platelet distribution width (PDW), platelet large cell ratio (P-LCR) and plateletcrit (PCT) are provided automatically by the XT-2000iV with each measurement. The diagnostic use of platelet indices reflecting platelet size as marker for juvenile platelets has been controversially discussed –17. We prol dogs .The aims of our study were 1) to compare an optimized gate with the two previously published gating methods , 9, 2) tData of 362 canine complete blood cell counts (CBCs) including 14 follow-up samples was analyzed retrospectively. Follow-up samples are defined as blood samples, which have been taken from the same diseased dog but at different dates. The blood samples have been collected between July 2006 and April 2007 in the department of veterinary clinical sciences, Giessen, Germany and between June 2009 and February 2010 in the department of Global Non-Clinical Safety, Merck Serono, Darmstadt, Germany. Overall, 169/362 samples were obtained from diseased dogs and 193/362 samples were taken from healthy dogs respectively.Measurement was performed with ethylenediamine tetra-acetic acid (EDTA) anticoagulated blood samples using the XT-2000iV hematology analyzer and the veterinary software version 00–08 and 00–10. The retrospective analysis and gating was performed using the veterinary software version 00–11. To detect the number of r-PLTs in the blood samples, we created one gate to quantify the whole platelet population Fig.  was compRetrospective analysis of each CBC included the following variables: platelet count by optical (PLT-O) and impedance methods (PLT-I), MPV, platelet distribution width (PDW), platelet large cell ratio (P-LCR), and plateletcrit (PCT).12/L and a reticulocyte count > 150.1 × 109/L was considered regenerative).The analyzer-specific canine reference intervals established previously for the XT-2000iV  were usen = 193, one sample per dog) included mainly samples taken from healthy Beagle dogs (153/193) obtained from one breeder . The Beagle dogs were kept under defined housing conditions: For 20 h a day, they were housed in groups of three to five dogs. Each dog spent the remaining four hours separated from the others in a standard kennel of 6.54 m2 and natural light. The Beagle dogs were fed once a day with 300 g of a commercially available pelleted dry food . Water was offered ad libitum. All Beagle dogs were dewormed and vaccinated against canine distemper, leptospirosis, canine parvovirus, parainfluenza, Bordetella, canine adenovirus 2, and rabies.The CBCs obtained from healthy dogs (n = 17), Border Collies (n = 6), Great Danes (n = 3), West Highland White Terrier (n = 2), and one Beagle dog, Berger Blanc Suisse, Cocker Spaniel, Dalmatian, German Shepherd, Golden Retriever, Groenendael, Jack Russel Terrier, Labrador Retriever, Old German Shepherd Dog, Rough Collie, and Small Munsterlander each.The other 40/193 healthy dogs kept as pet dogs were of various breeds including mixed-breed dogs , and also for the subgroups “Beagles” (n = 153) and “non-Beagles” (n = 40). As recommended by the American Society of Veterinary Clinical Pathology (ASVCP) [n = 193) and the group of Beagle dogs (n = 153) included ≥ 120 individuals, so that the nonparametric method was chosen to calculate reference intervals [Reference intervals for r-PLTs and platelet indices were generated for all healthy dogs independent of the breed ( (ASVCP) , the stantervals . The grontervals . In casentervals , the parThe group of healthy Beagles included dogs with an age ranging from 10 to 14 months and equally distributed sex .In the group of healthy “non-Beagles”, 40 dogs  with a median age of two years (range: 4 months to 15 years) were enrolled.In contrast, the diseased dogs (169/362) had a median age of seven years ranging between 3 months and 16 years. Overall, 60/169 intact males, 25/169 castrated males, 42/169 intact females as well as 42/169 castrated females were included.9/L and 871 × 109/L. The r-PLT count as measured with the “Oellers-gate” ranged between 0.12 and 57.34 % in the whole group of 362 healthy and diseased dogs and between 0.29 and 57.34 % when regarding the group of diseased dogs alone (169/362).In all 362 CBCs obtained from healthy and diseased dogs, the PLT count ranged between 3 × 1012 /L, whereby 19/41 anemic specimen showed regeneration of erythrocytes at the time of sampling with reticulocyte counts (RET) > 150.1 × 109/L.The categories of underlying diseases are shown in Table All three evaluated gates of the XT-2000iV showed good correlation as shown in Table In 30/362 samples, a number of dots exceeding 5 % of the sum of dots in gates 1 and 4 was found in gate 4 Fig.  and e. IFurthermore, in 6/362 samples, visual control of the scattergrams revealed a small number of dots other than r-PLTs displayed in all evaluated r-PLT gates Fig. . These dFor measurement of r-PLTs in all three gates, intra-assay CVs > 22.0 % were obtained. The highest CV of 41.1 % was seen for the “Pankraz-gate”. CVs for PLT-O, PDW and PCT were 5.6, 6.8 and 4.5 % and thus markedly lower than for r-PLT measurement. For the variables MPV, P-LCR and PLT-I, CVs < 3 % were obtained , platelet indices were not reported by the analyzer due to an error in (data) analysis so that these three samples could not be included in the calculation of the respective reference intervals.Furthermore, the analyzer did not report a result for the platelet indices in samples with r-PLT percentages exceeding 11.61 % (19/362) for the “Gelain-gate” and 11.66 % (21/362) for the “Oellers-gate”, respectively. In samples without reported PLT indices, the platelet histogram was characterized by a relatively indistinctive separation  between erythrocytes and platelets so that a reliable differentiation between the cellular populations could not be ensured by the analyzer’s software.Overall, the retrospective analysis of a large number of dog CBCs enabled us to optimize the already existing r-PLT gates by excluding interferences of reticulocytes and small erythrocytes with the r-PLT measurement.The significant difference between the “Pankraz-gate” and the two other gates is presumably a result of its smaller size. Compared to the other gates, r-PLTs need to contain more RNA to be detected in the “Pankraz-gate”. Consequently, the corresponding reference ranges obtained for the “Pankraz-gate” are much narrower than for the other gates. The difference between the “Gelain-gate” and “Oellers-gate” was solely based on samples where erythrocytes and/or reticulocytes interfered with the measurement of platelets and r-PLTs. Using the “Gelain-gate”, these interfering erythrocytes and/or reticulocytes were incorrectly counted as mature platelets, consequently resulting in a false high total platelet count obtained with the manual gate and thus a false low percentage of r-PLTs. This incorrect cell count is only a problem of the manual gating technique; however, the original automated PLT count could not be used for the purposes of r-PLT measurement. In contrast, the effect of interferences, i.e., false low r-PLT counts, is minimized by the novel “Oellers-gate” by excluding the critical group of cellular populations Fig.  and b.Another reason for visual examination of the scattergrams is the occurrence of another type of interfering cells within the r-PLT-gate, which were rarely observed in the present study Fig.  but have9/L) and the “Oellers-gate”  in 193 dogs are comparable with results published previously by Gelain et al. [9/L, n = 86). Furthermore, our reference interval established for the “Pankraz-gate” (0.0–1.2 %) is in accordance to the reference range shown previously by Pankraz et al. for the XT-2000iV [n = 40).The r-PLT reference intervals obtained in this study are consistent with the reference intervals published previously for the XT-2000iV , 9. RefeOur results clearly showed that the reference intervals in Beagle dogs are much smaller than in non-Beagle dogs, which is most likely because of the lower genetic variation, the similar age and the identical housing conditions.9/L, n = 36) with our results. However, when regarding other reference intervals for r-PLTs determined flow-cytometrically with the Thiazol Orange method, there is a large discrepancy between results reported in the literature ranging from 3.4 ± 2.0 % (n = 8 dogs) [n = 20 dogs) [Moreover, the reference intervals published by Smith et al. for flow cytometry with Thiazol Orange and CD61  are also 8 dogs)  to 9.3 ±20 dogs)  and the 20 dogs) .n = 190–193, depending on the variable) showed good accordance with reference ranges published before for the XT-2000iV .However, the study is limited by its retrospective nature so that a comparison with a reference method other than the previously published gates was not performed. For interpretation of the reference intervals, it has to be considered that the group of non-Beagle dogs is also smaller than recommended by the ASVCP  and refeDespite the optimization of the gating method described here, interferences could not be totally avoided. Only flow cytometric assessment of specifically antibody-labeled platelets would allow a definite distinction between platelets and other cellular populations. Another potential limitation of the gating method applied by us and the previous authors evaluating the r-PLT count measured by the Sysmex XT is that the gates are based on visual – and therefore subjective and potentially imprecise – assessment of the dot plots. Additionally, the position and size of the gates is fixed, so that in some samples, the shape of the gates did not entirely match the shape of the total platelet population. Thus, each measurement should be verified by visual examination of the scattergram. Ideally, algorithms would be generated to adapt the gate’s position to the properties of the platelet population.Similar to results of previous studies using the Sysmex XT-2000iV, the platelet indices MPV, PDW, P-LCR and PCT , 19, 25 In conclusion, we managed to optimize the previously published gates by excluding interferences from erythrocytes. However, visual assessment of the scattergrams still remains necessary. Future improvement might be achieved by the generation of algorithms automatically adapting the gate to the position of the platelet population. The use of platelet indices as a marker of PLT regeneration is questionable as their measurement is often not reliably possible especially in samples with evidence of larger numbers of immature PLTs.The reference intervals obtained from a large population of Beagle dogs for r-PLTs and platelet indices might become particularly useful for experimental studies using Beagles.