Polymer infiltrated ceramics and nano-ceramic resins are the new restorative materials which have been developed in order to enhance the adverse properties of glass-matrix ceramics and resin composites. The aim of the present in vitro study was to evaluate the characteristics of various CAD/CAM materials through mechanical, microstructural, and SEM analysis.n = 22) were formed by using the indicated CAD/CAM blocks: VITA Enamic (VITA Zahnfabrik), Lava Ultimate (3 M ESPE), IPS e.max CAD (Ivoclar Vivadent), IPS Empress CAD (Ivoclar Vivadent), and VITA Mark II (VITA Zahnfabrik). Two specimens from each test group were used for XRD and EDS analysis. Remaining samples were divided into two subgroups (n = 10). One subgroup specimens were thermocycled  whereas the other were not. All of the specimens were evaluated in terms of flexural strength, Vickers hardness, and fracture toughness. Results were statistically analyzed using two-way ANOVA, one-way ANOVA, Tukey’s HSD, and Student’s t tests (α = .05). Fractured specimens were evaluated using SEM.Five test groups (p < .001), however flexural strength and fracture toughness results were lowest conversely (p < .05). IPS e.max CAD was found to have the highest flexural strength (p < .001). Fracture toughness of IPS e.max CAD was also higher than other tested block materials (p < .001). Lava Ultimate and VITA Enamic’s mechanical properties were affected negatively from thermocycling (p < .05). Microhardness, flexural strength, and fracture toughness values of Lava Ultimate and VITA Enamic were found to be similar to VITA Mark II and IPS Empress CAD groups.The highest Vickers microhardness value was found for VITA Mark II (It should be realised that simulated aging process seem to affect ceramic-polymer composite materials more significantly than glass ceramics. Glass-matrix ceramics and resin composites are frequently used materials for CAD/CAM (Computer Aided Design/Computer Aided Manufacturing) restorations due to enhanced mechanical and optical properties , 2. Alth2O3 and the polymer part contains urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA)  on the ordinate and a corresponding lnσ on the abscissa and calculating the slope of the fitted line, where slope equals m; the Weibull modulus.2, ENA has Al2O3, Na2O and K2O in its inorganic network. Two materials have similar ratios of SiO2. VIT, EMP, and MAX displayed high ratios of SiO2, Al2O3 and other metal oxides.Results of EDS analysis were summarized in Table 2O6) crystals for IPS Empress CAD and lithium disilicate crystals (Li2Si2O5) for IPS e.max CAD.The XRD analysis displayed ULT, ENA and VIT as amorphous materials without different phases Figs. , 2 and 3p < .01) (Table p < .01) (Table p < .01). According to the Student’s t-test, flexural strengths of ceramic-polymer composite materials (ULT and ENA) were significantly decreased after thermocycling (p < .01). No significant decrease in flexural strength of glass ceramics  was detected after thermocycling (p > .05). Weibull distribution of groups are displayed in Fig. Two-way ANOVA showed that the type of tested material and aging, and the interaction between these parameters were significant (1) Table . The res1) Table . MAX grop < .01) (Table p < .01) (Table p < .01). There were no statistically significant difference between microhardness values of VIT, EMP, and MAX groups (p > .05). There were also no statistically difference between the microhardness values of ULT and ENA groups (p > .05). According to the Student’s t test, Vickers hardness of ceramic-polymer composite materials (ULT and ENA) were significantly decreased after thermocycling (p < .01). There was no significant decrease in Vickers hardness of glass ceramics  after thermocycling (p > .05).Two-way ANOVA showed that the type of tested material and aging, and the interaction between these parameters were significant (1) Table . The res1) Table . The micp < .01) (Table p < .01) (Table p < .01). There were no statistically significant difference between fracture toughness of VIT, EMP, and MAX groups (p > .05). There were also no statistically difference between fracture toughness of ULT and ENA groups (p > .05). According to the Student’s t test, fracture toughness of ceramic-polymer composite materials (ULT and ENA) were significantly decreased after thermocycling (p < .01). There was no significant decrease in fracture toughness of glass ceramics  after thermocycling (p > .05).Two-way ANOVA showed that the type of tested material and aging, and the interaction between these parameters were significant (1) Table . The res1) Table . The fraIn the SEM observations, ULT displayed microcracks between the inorganic and organic components in the aged specimen Fig. . ENA imaThe primary hypothesis of the study was rejected; one of the glass-matrix ceramics (MAX) had higher flexural strength compared to polymer infiltrated ceramics and nano-ceramic resins. The secondary hypothesis was accepted; ceramic-polymer composite materials were found to have reduced Vickers hardness and fracture toughness mean values than the glass-matrix ceramics. The tertiary hypothesis of the study was confirmed; ceramic-polymer composite materials were affected by thermocycling.The present study showed that ceramic-resin composite materials are not superior to all types of glass-matrix ceramics in terms of flexural strength. The reason for the MAX group having the highest flexural strength may be the crystal structure of the material. Wang et al. reported that the mechanical properties of dental glass ceramics are closely related to the crystal structure of the material . XRD anap < .001)  Table . AlthougWeibull modulus identifies the strength variability, give information on material’s structural homogeneity and has been reported to range from 5 to 15 for dental ceramics . The WeiSurface hardness is described as relative measure of resistance to permanent surface indentation. Indentation hardness is defined as a factor that affects the capability of getting finished and polished and also resistance of a material to occlusal wear , 29. FraBased on the results of the present study, thermocycling significantly affected the flexural strength, Vickers hardness, and fracture toughness of ULT and ENA but not those of EMP, VIT, and MAX Tables , 5 and 6Present study results are in agreement with results of Thornton who stated that, ULT and ENA were affected by thermocycling but MAX was not affected significantly . In a siOne of the limitations of this study may be that applied aging method might be considered short-coming for imitating real clinical conditions. Thermocycling is only one method for investigating the aging of the materials and the materials’ properties may differ following different in vitro aging protocols . FurtherWithin the limitations of this study, it can be stated that ENA and ULT, also classified as ceramic-polymer composite materials, have significant differences in terms of flexural strength, hardness and fracture toughness when compared to VIT, EMP, and MAX. Clinicians should consider these mechanical properties when deciding on the treatment plan of various clinical situations. It should also be remembered that flexural strength, hardness, and fracture toughness of ENA and ULT are affected negatively by thermocycling and studies investigating their long term success are scarce. Considering that the thermal changes and water absorption are not the only factors that age dental restorative materials, further studies are recommended which would investigate these restorative materials with various aging processes in order to simulate the clinical situation.Within the limitations of the study it can be concluded that; flexural strength, Vickers hardness, and fracture toughness of the evaluated materials have significant differences in addition thermocycling affects the aforementioned properties of ULT and ENA negatively. Clinicians are suggested to take these differences into consideration when planning prosthodontic rehabilitation using these materials.


Brachionus forficula was selected as the model organism. The differences in developmental durations, life-table demography, starvation resistant time and the competitive ability with Moina macrocopa were compared between B. forficula with long (LPS) and short (SPS) posterior spines. The results showed that LPS showed relatively longer durations of juvenile stage at 1.0 × 106, 2.0 × 106 and 4.0 × 106 cells/ml Scenedesmus obliquus, and longer embryo stage at 2.0 × 106 cells/ml S. obliquus than SPS. The intrinsic rate of population increase and net reproduction rate were lower in LPS than SPS, suggesting the energy input to reproduction decreased. The starvation resistant time was also reduced in LPS, in comparison to SPS, further supporting that LPS consumed more energy, which might be directed to the development of long spines. All these results revealed that LPS spent more energy for individual growth than SPS, which might be used to develop long spines. Moreover, the maximum population density and population growth rate of LPS were always lower than those of SPS, suggesting that LPS might have a weaker competition ability with M. macrocope than SPS.In rotifers, the costs of morphological defenses, especially the development of long spines, have been investigated for several decades. However, the obtained results were inconsistent and the underlying reasons were complicated. Investigations on more species might be helpful to find out the reasons. In the present study,  Daphnia3. The most pronounced phenotypic changes are the morphological defenses against predators, including the formation and enlargement of helmets, development and elongation of spines, enlarged body size, increased thickness and hardness of lorica8. These morphological variations could effectively protect animals from being captured or mechanically injured by increasing the handling time and ingestion time of predators9.Phenotypic plasticity is a common phenomenon in zooplankton, such as rotifers and 11. Lots of publications dealt with the costs of morphological defenses in Daphnia and rotifers. However, the results were contradictory2. Decreased reproduction was observed in Daphnia pulex3, Keratella testudo12, Brachionus havanaensis14, Brachionus calyciflorus16 and Plationus macracanthus16 in response to predators or the kairomone released by predators or among different natural morphs. In comparison, Gilbert revealed that the development of morphological defenses did not affect the reproduction of B. calyciflorus17 and Keratella tropica19. Stemberger20 even observed increased reproduction in long-spined B. calyciflorus compared with non-spined one. Moreover, the results of changes in fitness of individuals were also inconsistent. A high fitness of individuals (such as long average lifespan)15 and a low fitness (such as low survivorship)12 were both observed in strengthened morph (long-spined) compared with basic morph (unspined). The reasons underlying these inconsistent performances might be very complicated. The differences among species and the problems in experimental design are both possible explanations2. Besides, the employed environmental factors might also affect the results. As reported previously, the costs of morphological defenses, reflected as differences in intrinsic rate of population growth between long-spined and unspined morphs, varied in B. calyciflorus among treatments with different temperatures15 and in Kerutella testudo among treatments with different food concentrations12.The inducible morphological defenses, which consume materials and energy, are believed conditional responses to predators and have costs at some aspects; otherwise, the phenotype with constitutive morphological defenses should be retained after natural selectionBrachionus forficula is a common species in freshwater ecosystems. In natural waters, B. forficula with long or short posterior spines  and individually cultured in Gilbert media22 at 25 ± 1 °C under natural light. Rotifers were fed 1.0 × 106 cells/ml Scenedesmus obliquus daily, which was cultured in HB-4 medium as described before15. After 8 weeks, one well-established rotifer population (one strain) was selected. At that time, rotifers had long posterior spines, namely long posterior spine morph (LPS). This strain was continuously cultured for four more weeks and then its posterior spines became shorter . The experiments on LPS and SPS were conducted not simultaneously.During May 2015, 6, 2.0 × 106 and 4.0 × 106 cells/ml S. obliquus. Before the commencement of experiments, rotifers were precultured at the corresponding algal level at 25 ± 1 °C using 10 ml glass test tubes for more than one week. The rotifer population was kept at the exponential growth phase by enlarging the total cultural volume every day. Next, rotifers with mictic eggs were collected, placed in new dishes and continuously cultured at the same condition. They were observed every 2 hours and newly hatched juveniles (<2 hours old) were pipetted into small plastic cavities containing 0.5 ml cultural media with corresponding algal level. In each cavity, only one juvenile was inoculated. Next, the juveniles were observed every 2 hours, the time for the emergence of the first egg and the hatching of the first egg was recorded. Afterwards, rotifers were observed every 12 hours, the number of newly hatched juveniles were recorded and then discarded. The culture media were replaced every 24 hours. The experiments were ended until the last original rotifer died. In each treatment, three replicates were performed with each replicate containing 12 cavities.Three algal densities were set, including 1.0 × 1023. The life-table demongraphic parameters were calculated according to Krebs24 and Pianka25, including age-specific survivorship (lX) and fecundity (mX), net reproductive rate (R0), average lifespan, generation time (T), and intrinsic rate of population growth (rm).The developmental durations were calculated as described previouslym), was first approximated using:Intrinsic rate of population growth  were collected and then placed individually into each cavity containing 0.5 ml of media. Next, rotifers were observed every 12 hours and the culture media were replaced daily. The time when rotifers died were recorded. For each algal density, 60 individuals were repeated.M. macropcopa was pre-cultured at the same temperature and algal density as described for rotifers. After three generations, individuals from the third brood were used for the competition experiments. The competition experiments were performed in glass test tubes with the culture system volume of 10 ml. In each test tube, 30 randomly selected rotifers and six M. macrocopa individuals (<24 hours old) were placed. The algal densities were the same as life-table demographic experiments. Next, the density of rotifers was counted, M. macrocopa were replaced by a batch of the same aged individuals, the culture media were changed and fresh alga were added daily. The whole experiments lasted for 7 days. All treatments were repeated for 3 times. The population growth rate (r) were calculated by the quotation: r = (lnN7 − lnN0)/7, where N0 and N7 represents the density of rotifers at the first and seventh day, respectively.For all parameters, the normality and the homogeneity of variances were tested using the one-sample Kolmogórov-Smirnov procedure and the Levene’s test, respectively. Two-factor variance analysis was performed to test the effects of algal density, morph and their interactions on each parameter. One-way ANOVA was conducted to identify the significant effects of algal density on each parameter within the same morph, followed by multiple comparisons (SNK-q test). Student’s T tests were conducted to compare the differences in each parameter between two morphs at the same algal density. All statistical analyses were performed in SPSS 17.0.6 cells/ml, 1.0 × 106 cells/ml and 4.0 × 106 cells/ml S. obliquus, respectively (SNK tests). In comparison, algal density significantly affected the duration of juvenile stage, embryo stage and reproduction stage of SPS. The duration of juvenile and reproduction stage was shorter at 1.0 × 106 cells/ml than that at 2.0 × 106 cells/ml. The duration of embryo stage was longer at 1.0 × 106 cells/ml than that at 4.0 × 106 cells/ml. The duration of reproduction stage was shorter at 2.0 × 106 cells/ml than that at 4.0 × 106 cells/ml  was used, which was 300–500 µm in length. The lorica length of B. forficula was approximately 100–150 µm. Obviously, the threat of M. macropcope on B. forficula in the present study was small due to the limited differences in body size. Secondly, the competition outcome between B. forficula and M. macrocope was integratedly affected by the susceptibility, developmental speed and the population growth rate of rotifers. The high net reproduction rate and the high intrinsic rate of population increase of SPS might offset the loss of individuals caused by M. macrocope injuries.Cladocerans exploitatively and intrusively compete with rotifers. They not only race for food with rotifers, but also mechanically interfere with rotifersB. forficula with long posterior spines revealed longer developmental durations, lower reproduction and less resistance to starvation than that with short posterior spines, which might be due the costs of long spines. Moreover, B. forficula with long posterior spines showed a weaker competition ability with M. macrocope than short-spined morph.In conclusion, Supplementary information


Stress is the most frequently self-reported seizure precipitant in patients with epilepsy. Moreover, a relation between ear stress and epilepsy has been suggested. Although ear stress and stress hormones are known to influence seizure threshold in rodents, effects on the development of epilepsy (epileptogenesis) are still unclear. Therefore, we studied the consequences of ear corticosteroid exposure for epileptogenesis, under highly controlled conditions in an animal model. Experimental febrile seizures (eFS) were elicited in 10-day-old mice by warm-air induced hyperthermia, while a control group was exposed to a normothermic condition. In the following 2 weeks, mice received either seven corticosterone or vehicle injections or were left undisturbed. Specific measures indicative for epileptogenesis were examined at 25 days of age and compared with vehicle injected or untreated mice. We examined structural  and functional (glutamatergic postsynaptic currents and long-term potentiation) plasticity in the dentate gyrus (DG). We found that differences in DG morphology induced by eFS were aggravated by repetitive (mildly stressful) vehicle injections and corticosterone exposure. In the injected groups, eFS were associated with decreases in neurogenesis, and increases in cell proliferation, dendritic length, and spine density. No group differences were found in MFS. Despite these changes in DG morphology, no effects of eFS were found on functional plasticity. We conclude that corticosterone exposure during early epileptogenesis elicited by eFS aggravates morphological, but not functional, changes in the DG, which partly supports the hypothesis that ear stress stimulates epileptogenesis. Epilepsy is a common neurological disorder, especially in childhood where its prevalence is as high as 0.5–1.0% ]. The sedevelopment of epilepsy  are currently unknown.Animal models can provide more insight into the exact mechanisms by which stress influences epilepsy. In various preclinical epilepsy models, stress has been shown to lower the threshold for the induction of epileptic seizures and to increase seizure severity –16. The To improve insight into the impact of stress hormones on epileptogenesis, we studied the effects of corticosterone, an important stress hormone, during early-life epileptogenesis on neuronal morphology and functional plasticity in the rodent brain. Using a controlled design, we elicited experimental febrile seizures (eFS) in young mouse pups by warm-air induced hyperthermia (HT) and subsequently exposed them to repetitive (1) high concentrations of corticosterone, (2) vehicle injections , or (3) no injections. We next examined alterations in morphological and functional parameters in the dentate gyrus (DG), a hippocampal subarea that is affected by eFS –28 as wen = 99). On postnatal day (P) 1, litters were culled to four to six pups consisting of both males and females. The pups were not weaned during the experiment. Animals were kept in a controlled 12-h light–dark cycle (light on 7 a.m. to 7 p.m.) with a temperature of 22 ± 1°C. Food and water were available ad libitum . All animals were housed in transparent Plexiglas cages (Macrolon type II) with sawdust bedding and paper tissues for nest building. Cages were cleaned at P7 and at P17/18 (in between injection days) by replacing half of the sawdust bedding. All experimental procedures were performed according to the institutional guidelines of the University Medical Center Utrecht and approved by the committee on ethical considerations in animal experiments of Utrecht University . All efforts were made to minimize suffering of the animals. Pups were assigned to multiple treatment groups per litter. A maximum of two pups per litter was used per treatment group per method of analysis to minimize litter effects on outcome measures. All animal experiments were performed within a period of 6 months and animals in all treatment groups were tested across the whole period to control for environmental or seasonal variation. For the purpose of this study, experiments were only performed on male animals.Breeding pairs of C57BL6/J mice were obtained from The Jackson Laboratory  and subsequently bred in-house. Litters used in this experiment were derived from multiple breeding pairs (n = 4–6 per time point per treatment group) were injected intraperitoneally with corticosterone  or vehicle  between 8.30 and 9.00 a.m. Immediately before, or at 15, 30, 60, 120, 180, or 240 min after injection, mice were decapitated and trunk blood was collected. Between injection and decapitation, pups were returned to their home cage and left undisturbed. A separate group of non-injected mice was decapitated at the same time points to control for diurnal corticosterone variability.To evaluate corticosterone levels after injection with corticosterone or vehicle, naïve P12 mice  after the last injection, mice were weighed and decapitated or perfused  according to the manufacturer’s instructions. All samples were processed in the same assay to exclude inter-assay variability.Trunk blood samples were collected immediately after decapitation at P25/26 from all animals subjected to HT/NT except those receiving perfusion fixation. Directly after decapitation, thymus and both adrenals were resected and weighed. Blood samples were centrifuged for 10 min at 4,000 rpm at 4°C. Plasma was stored at −80°C until assayed with an IAll morphological and functional outcome parameters were examined in the DG. This hippocampal area was selected based on its functional and anatomical characteristics. First, the (epileptogenic) changes induced by eFS are located in the hippocampus, including the DG , 36, 37.All following experimental procedures were performed by experimenters unaware of the treatment groups. Per outcome measure, tissue of animals belonging to different treatment groups was ordered in a semi-randomized way to control for environmental or experimenter effects on recording, staining and/or quantification over the total experimental period.n = 6 per group) were decapitated between 9.00 and 10.30 a.m. Brains were dissected, post fixed at 4°C in 4% formaldehyde for 4 h, transferred to 30% sucrose for 24 h, frozen on powdered dry ice and stored at −80°C. Cryostat sections (20 μm) were cut in the coronal plane, collected in series of 15 on Superfrost slides and stored at −80°C until further use.Male mice  or Ki67 ], diluted in supermix (0.25% gelatine and 0.1% Triton in TBS) at room temperature for 1 h and then incubated overnight at 4°C. The next morning, sections were washed and incubated for 2 h with donkey antigoat biotinylated  or goat antirabbit biotinylated  secondary antibody diluted in supermix at room temperature. Sections were washed, incubated in avidin–biotin complex (ABC)  for 2 h (for DCX) or 1.5 h (for Ki67) at room temperature, washed again and incubated with biotinylated tyramide (1:500) in 0.01% H2O2 in TBS for 30 min. Sections were washed and incubated in ABC (1:800 in TBS) for 1.5 h at room temperature and washed in TBS. After washing in 0.05 M Tris HCl pH 7.6 (TB), chromogen development was performed with diaminobenzidine  for 40 min. Sections were washed in TB and stored overnight at 4°C. The next day, sections were washed in distilled water, counterstained with Hematoxylin and shortly rinsed in distilled water. After washing with running tap water, sections were dehydrated using a grading series of ethanol, cleared in xylene and coverslipped using Entallan.As a measure of neurogenesis, tissue was stained for Doublecortin (DCX), a marker for neuronal precursor cells and immature neurons, and Ki67, a marker for proliferating cells using modified protocols. Mounted sections were postfixed in acetone-methanol (1:1) at −20°C for 7.5 min, washed in 0.05 M Tris HCl 0.9% saline (TBS) pH 7.6 and heated in 0.01 M citrate buffer (pH 6.0) in a microwave oven for 10 min at 800 W followed by 5 min at 480 W and 5 min at 260 W. After a cool down period of 20 min, sections were washed in TBS. Endogenous peroxidase activity was blocked with 0.5% (for DCX) or 1.5% (for Ki67) H+ and Ki67+ cells in the granule cell layer and the subgranular zone of the DG were quantified unilaterally in every 15th section in a total of five sections per animal within Bregma range −1.46 to −2.80  without a left/right preference within or between animals. DCX+ cells were quantified stereologically using a StereoInvestigator system  with a ×100 oil-immersion objective of a Zeiss Axiophot microscope and StereoInvestigator software, according to the optical fractionator method. The number of DCX+ cells was estimated using a 25 μm × 25 μm counting frame, with a grid size of 70 μm × 80 μm. Section thickness was 10.5 μm. The estimated total of DCX+ cells within the studied range was determined using the optical fractionator method and multiplied by two to correct for unilateral counting. The mean Gundersen coefficient of error of stereological quantification (m = 1) was 0.076 (range 0.06–0.10). Ki67+ cells were counted manually using a light microscope (Olympus BH-2) with ×40 magnification and multiplied by the inverse of the sampling fraction and by two to correct for unilateral counting. The total estimated number of DCX+ or Ki67+ cells per animal was used for analysis.DCXn = 6 per group) were decapitated between 9.00 and 10.30 a.m. Directly after decapitation, brains were dissected. Rapid Golgi staining  was performed according to the manufacturer’s instructions with an impregnation time of 9 days. Vibratome sections (200 μm) were cut in the transversal plane . Images were obtained using Zen 2011 (Carl Zeiss) in combination with an automated stage and focus control connected to the microscope. Golgi-impregnated dentate granule cells, fulfilling the following criteria, were randomly selected: (1) localization in the middle part (relative to the DG curvature and start of the CA3 region) of the suprapyramidal blade of the DG, within Bregma range −2.16 and −3.16 , (2) consistent and dark impregnation along the entire extent for all dendrites, and (3) relative isolation from neighboring impregnated neurons to avoid interference with analysis.Male mice (n = 50 (17%), 5–10 neurons per group, evenly distributed over the groups]. Spines were counted in two segments of ±20 μm per neuron, located on different dendrites. Segments were randomly chosen based on the following criteria: (1) localization at approximately 100 μm (80–120 μm) radial distance from the cell soma; (2) secondary or higher order dendritic branches; and (3) straight and remaining in a single focal-plane. Of each cell, dendritic properties were evaluated by analyzing the total dendritic length and maximum dendritic reach , and the dendritic complexity index . The remaining cells were subdivided into cells located in the inner-most part of the granule cell layer versus cells located in the middle or outer part of the granule cell layer  to obtain a 3D representation of each cell. Numerical analysis and graphical processing were performed with NeuroExplorer (MicroBrightField). Traced dendritic trees were evaluated by two investigators unaware of the treatment, on completeness of staining/tracing and on whether they belonged to a single cell, followed (if required) by a consensus meeting. Dendritic trees that were considered not completely stained/traced or belonging to multiple neurons were excluded from analysis [ndrites) ]. If denll plots  were conayer see . This sun = 6 per group) were killed between 9 and 12 a.m. under deep pentobarbital anesthesia  by transcardial perfusion with 0.1% sodium sulfide for 5 min, followed by 4% formaldehyde for 5 min . Brains were removed from the skull, postfixed at 4°C in 4% formaldehyde/15% sucrose overnight, immersed at 4°C in 30% sucrose in phosphate-buffered saline until they sank and frozen on powered dry ice. Cryostat sections (30 μm) were cut in the coronal plane, mounted on superfrost slides in series of 15 and stored at −80°C until further use. Mossy fibers were stained with Timm histochemistry, according to Danscher  was scored according to the scoring system described by Holmes et al. , rangingn = 6–9 per group) were decapitated between 8.15 and 9.15 a.m., a few minutes after taking the animal out of its home cage. Only the first two mice of each litter were used for electrophysiological analysis to avoid confounding effects of a rise in plasma corticosterone due to acute stress. After rapid dissection, the brain was chilled in ice-cold, carbogenated (95% O2:5% CO2) artificial cerebrospinal fluid (aCSF) containing in mM: NaCl 120, KCl 3.5, MgSO4 5.0, NaH2PO4 1.25, CaCl2 0.2, NaHCO3 25.0, and d-glucose 10.0. After removing frontal lobes and cerebellum, 350 μm transversal hippocampal sections were prepared using a vibratome . Both hemispheres were separated and all sections were incubated at room temperature in continuously carbogenated aCSF for at least 1 h.Male mice A-receptor mediated transmission . Cell patching was performed as described by Pasricha et al.  analog-to-digital converter. Data acquisition was performed with Clampex, version 8.2 (Axon Instruments) at a sampling rate of 50 μs and a 5 kHz Bessel filter. The surface of the section was cleaned to have better vision of the cells in the deeper layers of DG. After establishing a gigaseal, the membrane patch was ruptured and the cell was clamped at a holding potential of −70 mV to allow measurement of currents mediated by the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, as at this potential, the N-methyl-d-aspartic acid or N-methyl-d-aspartate receptor is blocked by Mg2+. sEPSCs were recorded for 5 min, starting 5–10 min after membrane rupture. After administration of tetrodotoxin  for 5 min to block voltage-gated Na channels and consequently also the development of action potentials, miniature EPSCs (mEPSCs) were measured for 5 min. Only one cell was recorded per slice and no more than two recordings were obtained per animal. Only recordings with an uncompensated series resistance of <2.5 times the pipette resistance, <20% variation during the recording period, and with frequencies <2 SD from the mean, were accepted for analysis. For each event, the area under the curve (AUC) and inter-event interval was analyzed using Clampfit version 9.2 (Axon Instruments). The median AUC and inter-event interval per cell were used for statistical analysis.Patching of DG neurons was performed using an upright microscope (Nicon Eclipse E600FN) with differential interference contrast and a water immersion objective (×40) to visually identify the cells. The sections were continuously perfused with carbogenated aCSF containing in mM NaCl 120, KCl 3.5, MgSOa et al. . Briefly4 4.0, NaH2PO4 1.2, CaCl2 4.0, NaHCO3 26.0, d-glucose 10.0, and 20 μM bicuculline . Field excitatory postsynaptic potentials (fEPSPs) were recorded in the DG, using glass microelectrodes filled with aCSF, positioned in the medial perforant pathway over the suprapyramidal blade of the DG, as confirmed by paired pulse stimulation elicited paired pulse inhibition. Minimum and maximum stimulation intensities were identified. After an incubation period of 20 min, an input–output response curve was generated by gradually increasing the stimulus intensity to define the stimulus intensity that generated the half-maximal response in peak-amplitude; this intensity was used for the remainder of the experiment. To measure paired pulse depression, paired pulses were delivered at an inter stimulus interval of 50, 100, and 200 ms. After 15–20 min of stable baseline recordings, sections were tetanized with 4 trains of 50 pulses of supramaximal intensity at 100 Hz  to induce LTP. Field potentials were recorded for 1 h posttetanus at 30 s intervals. At the end of this period, the post-tetanic paired pulse depression and final input–output curve were determined. LTP was quantified by calculating the ratio between fEPSP slopes recorded post tetanization and pre tetanization. Data were acquired and analyzed with Signal 2.0 software .Sections were transferred to a submersion type recording chamber and continuously perfused with carbogenated aCSF containing in mM: NaCl 124, KCl 2.5, MgSOA priori we hypothesized that group differences would be caused by HT treatment or injection type. Therefore, not all possible group comparisons were included in statistical analysis. More specifically, in case of a significant main effect of injection type, post hoc tests were performed comparing injection types with Bonferroni correction for multiple comparison. Additionally, differences between HT and NT animals were analyzed per injection group with an independent samples t-test, Bonferroni corrected for multiple comparison. When data from multiple neurons per animal, or multiple segments per neuron, were analyzed, a linear mixed model was used, including the animal (for dendritic complexity and glutamatergic transmission) or neuron (for spine density) as subject variables. Effects of HT, injection type and their interaction on Sholl distribution were tested with a repeated measure general linear model. Correlation between parameters was assessed with Pearson correlation coefficient. Mean fEPSP slopes pre- and posttetanization were compared with a paired samples t-test.The effect of eFS and injection type on outcome measures was analyzed with a general linear mixed model in a 2 × 3 design, examining the main effects of HT (HT versus NT) and injection type (corticosterone versus vehicle versus none), as well as their interaction. p < 0.05 (two-tailed) after correction for multiple comparison. Differences that were significant before, but not after correction for multiple comparison (0.05 ≤ p < 0.15), were considered trends. Data were analyzed using SPSS 20.0 . Unless stated otherwise, data are presented as mean ± SEM.Normality of residues was evaluated with Q–Q plots, variance of residues was evaluated with error plots. Differences were considered statistically significant at Experimental seizures were elicited in all mice exposed to the HT protocol and the average seizure duration was 26.4 ± 2.0 min. Corticosterone injection resulted in a high peak concentration of corticosterone  with a fast return to baseline in approximately 180 min, while vehicle injection elicited a much smaller (94.6 ± 8.2 ng/ml) and more short-termed increase in corticosterone levels  = 10.73, p = 0.001], but was not influenced by subsequent injections  = 8.7, p < 0.001; corticosterone versus vehicle p = 0.009, corticosterone versus no injection p < 0.001, vehicle versus no injection p = 0.36], indicating that repetitive corticosterone injection indeed affected stress hormone regulation, which confirms successful manipulation.Weight increase between HT/NT treatment and decapitation, a 15 days interval, was significantly lower in HT animals compared to controls   = 4.94, p = 0.034] compared to NT . Analyses per injection type revealed that the effects of HT on both DCX- and Ki67-staining were significant in the vehicle injected animals , while a similar effect was observed at trend level after corticosterone injection . Non-injected HT and NT groups did not differ at all.Neurogenesis significantly differed between HT and NT animals. HT was associated with a decrease in the number of immature DCX-positive neurons , an effect that was only significant in animals receiving no subsequent injections  and was less pronounced after vehicle or corticosterone injection  = 4.47, p = 0.04], an effect that aggravated with injection type, although analyses per injection type only revealed a difference in the corticosterone-injected group at trend level  = 6.18, p = 0.01] and radial distance from the soma  on dendrite length, as well as a HT × injection type × radial distance interaction   Figure A, A1, thF = 7.28, p = 0.008]. This effect was significant after corticosterone injection , at trend level after vehicle injection , while it was not observed in animals receiving no injections   = 7.56, p = 0.06, Figure Hippocampal MFS was assessed in the DG and the CA3 area of the hippocampus. As expected, intense Timm staining was observed in the hilus of the DG and in the stratum lucidum of the CA3 area, the main projection sites of mossy fibers. The amount of infrapyramidal Timm staining, characteristic for MFS, was very low in all treatment groups . HT or injection type did not significantly affect MFS score in both areas, although there was a trend towards an increased MFS score in the CA3 area after HT in animals receiving corticosterone injections   = 3.95, p = 0.03; corticosterone versus vehicle p = 0.65; corticosterone versus no injections p = 0.07, vehicle versus no injections p = 0.01]  = 4.39, p = 0.02; corticosterone versus vehicle p = 0.04, vehicle versus no injections p = 0.004, corticosterone versus no injections not significant] with a smaller AUC after vehicle injection compared to the other groups  = 4.20, p = 0.02; corticosterone versus vehicle p = 0.02, corticosterone versus no injections p = 0.44, vehicle versus no injections p = 0.01, other group comparisons not significant]  corticosterone, (2) vehicle injection (a mild stressor), or (3) no injections, and evaluated morphological and functional parameters in the DG. In this latent phase of early epileptogenesis, few effects of eFS were observed in animals that were not subsequently injected. However, in mice that received repetitive corticosterone or vehicle injection (mild stress), eFS induced epileptogenesis was associated with changes in DG morphology, namely a lower number of immature cells, an increased cell proliferation and an increased dendritic length and spine density. These morphological changes associated with epileptogenesis did not translate into differences in glutamatergic functional plasticity.The eFS model is a model of early-life prolonged febrile seizures with close resemblance to the human situation , 31. Thedid downregulate responsiveness of the hypothalamic–pituitary–adrenal axis. The effects of these supraphysiological doses of corticosterone were smaller than expected, which might relate to the young age of the animals, just after the stress hyporesponsive period. Also, the transient increases in corticosterone levels in the morning may not have interfered with the ultradian pulsatility of endogenous hormone levels (We hypothesized the mild epileptogenic changes in the latent phase after eFS to be aggravated by stress (hormone) exposure. As different stressors might differentially affect seizure-susceptibility e levels , benefitnot subsequently injected, HT only affected dendritic complexity, but none of the other outcome measures. The negative results during this early phase of epileptogenesis are in line with previous studies that also reported no differences in MFS  cells, consistent with the gliosis prominent in many epilepsy models  exposure is in line with the vast amount of previous studies reporting stress exposure  in Ref. ]. The ef in Ref. , 26, sugy models , 57.Dendritic length, complexity and spine density are generally reported to decrease after seizures –63, whic+ cells in the vehicle injected compared to non-injected NT group, that is consistent with the literature  injection, the latent phase of early epileptogenesis may be accompanied by an expanded postsynaptic “potential” for synaptic transfer of signals. We tested whether this translated to the functional level; i.e., a higher spine density may result in increased mEPSC frequency and this, in turn, may enhance the ability to induce LTP . HoweverThe increase in morphological differences after HT when followed by corticosteroid and mild stress exposure suggests that stress (hormones) aggravates or accelerates epileptogenesis. Epileptogenesis, i.e., the neurobiological processes leading to epilepsy, is not limited to the time before the onset of spontaneous seizures, but continues during the course of epilepsy, contributing to the progression of the disease , 76. As In conclusion, our results suggest that stress and stress hormones modulate epileptogenesis, indicating that stress reduction strategies and possibly even medication targeting the stress system may have a potential role in reducing epileptogenesis. As studying epileptogenesis apart from seizure frequency is difficult, especially in humans, animal studies could provide valuable information on the effects of stress reduction on epileptogenesis, for example, by studying effects of an enriched environment during the latent phase of epileptogenesis on seizure outcome.All experimental procedures were performed according to the institutional guidelines of the University Medical Center Utrecht and approved by the committee on ethical considerations in animal experiments of Utrecht University .JC, KPJB, PG and MJ contributed conception and design of the study. JC, EH, KB, GR, ST, EU, and GM contributed to data acquisition. JC organized the database, performed the statistical analysis, and wrote the first draft of the manuscript. JC, KB, GR, PL, PG, and MJ contributed to data interpretation. All authors contributed to manuscript revision, read, and approved the submitted version.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.


Indeed, it has been shown that the MSC secretome increases neurogenesis and cell survival, and has numerous neuroprotective actions under different conditions. Additionally, using dynamic culturing conditions (through computer‐controlled bioreactors) can further modulate the MSC secretome, thereby generating a more potent neurotrophic factor cocktail . In this study, we have characterized the MSC secretome by proteomic‐based analysis, investigating its therapeutic effects on the physiological recovery of a 6‐hydroxidopamine (6‐OHDA) PD rat model. For this purpose, we injected MSC secretome into the substantia nigra (SNc) and striatum (STR), characterizing the behavioral performance and determining histological parameters for injected animals versus untreated groups. We observed that the secretome potentiated the increase of dopaminergic neurons  and neuronal terminals in the SNc and STR, respectively, thereby supporting the recovery observed in the Parkinsonian rats’ motor performance outcomes (assessed by rotarod and staircase tests). Finally, proteomic characterization of the MSC secretome  revealed the presence of important neuroregulatory molecules, namely cystatin C, glia‐derived nexin, galectin‐1, pigment epithelium‐derived factor, vascular endothelial growth factor, brain‐derived neurotrophic factor, interleukin‐6, and glial cell line‐derived neurotrophic factor. Overall, we concluded that the use of human MSC secretome alone was able to partially revert the motor phenotype and the neuronal structure of 6‐OHDA PD animals. This indicates that the human MSC secretome could represent a novel therapeutic for the treatment of PD. S It has been suggested that the therapeutic effects of human mesenchymal stem cells (hMSCs) in central nervous system (CNS) regenerative medicine are mediated by the active secretion of bioactive molecules, which is known as the secretome. This study demonstrated that the injection of hMSC secretome in a 6‐hydroxidopamine Parkinson's disease (PD) rat model was able to revert the Parkinsonian phenotype, potentiating the recovery of dopaminergic neurons in both the substantia nigra and striatum, thereby supporting the motor recovery observed in the PD rats. This work shows the modulatory role of the hMSC secretome in brain repair, further indicating that such cell‐free therapies could represent the basis of future strategies for the treatment of PD.l‐DOPA) has been considered as the standard treatment for PD and for the reduction of its major symptoms l‐DOPA treatment, the use of dopamine reuptake inhibitors, DA agonists, and muscarinic antagonists also have positive clinical effects Parkinson's disease (PD), the second most prevalent neurodegenerative disorder worldwide, is clinically characterized by a progressive degeneration of dopaminergic (DA) neurons in several dopaminergic networks The use of a human mesenchymal stem cell (hMSC)‐based strategy has emerged as a potential alternative therapy for PD We have observed that the dynamic culture of hMSCs in computer‐controlled suspension bioreactors enriched the neuroregulatory properties of their secretome https://www.eppendorf.com) was used for the expansion of hMSCs. Before inoculating hMSCs into the DASGIP bioreactors, the 500‐ml suspension bioreactors and modified Teflon four‐paddle impellers  were siliconized using Sigmacote  to minimize cell attachment to the sides of the bioreactor vessel and the impeller. After siliconization and autoclaving of the vessels, the DASGIP system was set up and calibrated according to protocols provided by the manufacturer. The bioreactors were maintained at 37°C, using a heating jacket; 21% oxygen in the headspace; pH of 7.4, controlled by a gas mixture connected to oxygen, nitrogen, carbon dioxide, and air tanks; and agitated at 52 rpm using a magnetic stir plate under the bioreactors.A DASGIP Parallel Bioreactor system  were used for this study and were prepared as previously described http://www.thermofisher.com). Following this, 500 ml of Neurobasal‐A medium with 1% of kanamycin (Thermo Fisher Scientific Life Sciences) was added to the bioreactors and the bioreactors were placed back into the DASGIP control system for 24 hours. After 24 hours, the bioreactors were again removed, placed in a biosafety cabinet for 10 minutes to allow the microcarriers to settle, and then the supernatant was harvested and centrifuged at 300g for 10 minutes to remove any cell debris. This supernatant, called the hMSCs CM , was then placed at −80°C until it was required.Cytodex 3 microcarriers  were housed (2 per cage) and maintained in a controlled environment at 22°C–24°C and 55% humidity, on 12‐hour light/dark cycles and fed with regular rodents’ chow and tap water ad libitum. Animals were handled for 1 week before beginning injections to reduce the stress induced by the surgical procedures. All manipulations were done after consent from the Portuguese National Authority for animal experimentation, Direção Geral de Veterinária (ID: DGV28421), and in accordance with the regulations on animal care and experimentation (European Union Directive 2010/63/EU). Thus, for surgical procedures, under combined ketamine (75 mg/kg) and medetomidine anesthesia , animals (n = 36) were placed on a stereotaxic frame  and unilaterally injected, using a Hamilton syringe with a 30‐gauge needle , with either vehicle  or 6‐hydroxidopamine  directly into the medial forebrain bundle (MFB) , or hMSC CM  directly in the SNc  and into four different striatum (STR) coordinates  Five weeks after the injection of 6‐OHDA, we proceeded to inject the hMSC secretome. After anesthesia was administered, as described in the previous section, animals were placed on a stereotaxic frame and unilaterally injected, using an Hamilton syringe with a 30‐gauge needle, with either the vehicle  was evaluated using the TSE RotaRod System  under an accelerating protocol previously described Motor coordination and balance of animals , as previously described n = 11; 6‐OHDA, n = 13; 6‐OHDA_hMSC CM, n = 12) were familiarized with the test and pellets that were available for 5 and 10 minutes on days 1 and 2, respectively. During the test session, animals were kept inside the box and had 15 minutes to reach, retrieve, and eat food pellets present on the steps. All sessions were performed at the same time of day and with food‐restricted animals. After each test interval, animals were removed from the staircase boxes and the uneaten pellets were counted.Skilled paw reaching  was performed with double staircase boxes  for 45 minutes. The number of contralateral rotations of the bowl was digitally recorded, which allowed assessment of the effects of the injection vehicle (0.2 mg/ml of ascorbic acid in 0.9% of NaCl) and 6‐OHDA (with 0.2 mg/ml of ascorbic acid in 0.9% of NaCl). This test was used to validate the model (after 6‐OHDA injections). Apomorphine is a strong dopamine agonist, and its repeated use could lead to an overstimulation of the dopaminergic system, which could impair the adequate interpretation of the impact of hMSC secretome on the functional outcomes of the animals To test apomorphine‐induced turning behavior (rotameter test), animals’ necks were subcutaneously injected with a solution of 0.05 mg/kg apomorphine hydrochloride (Sigma‐Aldrich) dissolved in 1% of ascorbic acid in 0.9% of NaCl, and then placed in metal testing bowls  and transcardially perfused with 4% paraformaldehyde  in 0.1 M phosphate‐buffered saline (PBS). Striatal and mesencephalon coronal sections, 30 μm thick, were obtained with a vibratome . Four series of consecutive sections were obtained and one was processed as a free‐floating tyrosine hydroxylase (TH) immunohistochemistry. Sections were immersed for 20 minutes in 1 M PBS with 3% H2O2, followed by blocking for 2 hours with 5% fetal calf serum  in 1 M PBS. Sections then were incubated overnight (at 4°C) with the rabbit anti‐mouse TH primary antibody  in 2% of FCS in 1 M PBS, followed by incubation for 30 minutes with a biotinylated secondary anti‐rabbit antibody, and another 30‐minute incubation with an avidine/biotine complex (Thermo Fisher Scientific Life Sciences). Antigen visualization was performed using 25 mg of 3,3′‐diaminobenzidine tetrahydrochloride (Sigma‐Aldrich) in 50 ml of Tris‐HCl 0.05 M, pH 7.6, with 12.5 μl of H2O2, and stopped at the desired time. Sections were then mounted on superfrost slides and thionin countercoloration was performed.After 13 weeks (including the development of the lesion and consequent treatment), the animals were killed with sodium pentobarbital  equipped with a digital camera , and with the help of Visiomorph software , the boundaries of the SNc area were drawn. The delineation of this region was performed through identification of anatomic standard reference points and with the help of a rat brain atlas + cells in the SNc area were counted in both hemispheres. Data are presented as the percentage of remaining TH+ cells in the injected side, compared with control side.To ensure a representative sampling among all the animals when quantifying the SNc TH‐positive cells, six identical TH‐labeled sections spanning the entire mesenphalon were chosen, including all the portions of the SNc. Using a bright‐field microscope  and analyzed for gray intensity after calibrating the software program. This was done using the “optical density step tablet” to determine the optical density (O.D.) of the selected sections and performed according to program instructions. This method provided a gross estimation of Parkinsonian pathology on lesioned side. From this, striatum O.D. values were determined in both hemispheres using a 1.1‐mm2 rectangular grid, encompassing the injection sites, as determined by anatomical references and rat brain atlas For estimating TH immunoreactive striatal fibers, total immunoreactivity of all TH fibers was measured by densitometry, as described by Febbraro et al. http://www.gelifesciences.com) and 100 μg of protein per sample was subjected to liquid digestion with trypsin (2 μg of trypsin per sample) overnight at 37°C http://www.agilent.com) before liquid chromatography‐tandem mass spectrometry (LC‐MS/MS).Three biological replicates of hMSC CM were processed as previously described https://sciex.com) by performing both information‐dependent acquisition (IDA) and SWATH on the same sample. Peptides were resolved by liquid chromatography  on a Halo Fused‐Core C18 reverse phase column  at 5 μl/minute using an acetonitrile gradient in 0.1% formic acid .LC‐MS/MS analysis was performed as previously detailed Protein identification from the IDA experiments was obtained by searching against the human and bovine species from UniProt database using the ProteinPilot software . An independent false discovery rate (FDR) analysis using the target‐decoy approach provided with ProteinPilot software was used to assess the quality of the identifications, and positive identifications were considered when identified proteins and peptides reached a 5% local FDR http://pantherdb.org).Additionally, a specific library of precursor masses and fragment ions created from a pool of hMSC samples, described by Teixiera et al. https://www.luminexcorp.com), and each factor concentration was calculated or obtained using Bioplex Manager version 6.1 software , and expressed as picograms per milliliter.The hMSC CM was first concentrated using a 5‐kDa cutoff filter  according to manufacturer's guidelines, as previously described t test. For RotaRod and staircase tests, upon 6‐OHDA and hMSC CM or Neurobasal‐A medium injections, analysis of variance repeated measures followed by post hoc Bonferroni for multiple comparisons was performed using the SPSS statistics program . Data are presented as mean ± SEM. The significance value was set at p < .05.Statistical evaluation of the animal apomorphine behavioral test injection (after 6‐OHDA) was performed using Student's t = 5.898; p < .001;  = 16.72; p < .001; η2partial = 0.411;  = 50.81; p < .001; η2partial = 0.637; t = 6.66; left‐side t = 6.81; p < .001; To further evaluate the functional integrity of the animal dopaminergic system after 6‐OHDA injection , the apoTo evaluate the effects of the hMSC CM  in 6‐OHDA‐injected animals, animal motor performances were assessed at 1, 4, and 7 weeks after CM injection during the RotaRod and staircase tests. = 31.58; p < .001; η2partial = 0.733) but no effect for the factor time  = 0.727; p = .477; η2partial = 0.031) and no interaction between these factors  = 2.24; p = .087; η2partial = 0.163). Comparing the CM‐injected animals with the untreated group , post hoc testing revealed that the motor coordination of the former group significantly improved after hMSC CM injection  = 48.33; p < .001; η2partial = 0.775), for the factor time  = 18.01; p < .001; η2partial = 0.391), and the interaction between these factors  = 6.17; p < .001; η2partial = 0.306). Comparing the CM‐injected animals with the 6‐OHDA group, post hoc analysis revealed that the injection of hMSC CM led to a significant improvement of the success rate of eaten pellets  = 34.06; p < .001; η2partial = 0.709), the factor time  = 3.22; p = .029; η2partial = 0.103), and the interaction between these factors  = 3.13; p = .009; η2partial = 0.183). Post hoc testing of the effects of the hMSCs CM revealed an increased success rate of eaten pellets (in the affected side) when compared with the 6‐OHDA group  = 213.34; p < .001; η2partial = 0.949). Injection of the hMSC CM appeared to play a role in the survival of DA neurons: There was a significantly higher number of TH‐positive cells observed in the SNc (CM: 25.36% ± 5.45%) when compared with the 6‐OHDA group (5.08% ± 1.60%)  = 350.69; p < .001; η2partial = 0.997) in which the injection of hMSC CM was able to increase the TH expression levels when compared with the 6‐OHDA group  when compared with the sham group, indicating a decline in the functional integrity of the DA system. In addition, we have also verified that the animals’ motor coordination was affected  on motor coordination (as seen in the RotaRod test), we observed that its injection was able to improve the motor performance of the CM‐injected animals when compared with the 6‐OHDA group . Interes+ neurons and fibers , we observed an improvement in animal behavior when compared with the 6‐OHDA group. We also found that the injection of the hMSC secretome was able to increase the densities of TH‐positive cells, a fact that most likely explains the improved behavioral performance of the CM‐injected animals. In addition to the important neuroregulatory molecules present in the hMSC CM , the presence of PEDF might also have a role in its outcomes because it has been described as an important neurotrophic and neuroprotective molecule in PD. Overall, our results suggest strongly that the use of the hMSC secretome may be a new and important tool for the treatment of PD because the secretome is able to modulate the DA neuronal survival and animal behavior.F.G.T.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; M.M.C. and A.J.R.: data analysis and interpretation; K.M.P.: data analysis and interpretation, manuscript writing; B.M.‐P.: collection and/or assembly of data; S.A. and B.M.: collection and/or assembly of data, data analysis and interpretation; L.A.B.: provision of study material, data analysis and interpretation, manuscript writing; N.S.: data analysis and interpretation, manuscript writing, final approval of manuscript; A.J.S.: financial support, provision of study material, data analysis and interpretation, manuscript writing, final approval of manuscript.The authors indicated no potential conflicts of interest.


Magnolia officinalis, exerted anticancer effects through a variety of mechanisms on multiple types of cancers. In this study, the molecular mechanisms of honokiol in suppressing the human oral squamous cell carcinoma (OSCC) cells were evaluated. Treatment of two OSCC cell lines with honokiol resulted in reducing the cell proliferation and arresting the cell cycle at G1 stage which was correlated with the down‐regulation of Cdk2 and Cdk4 and the up‐regulation of cell cycle suppressors, p21 and p27. In addition, the caspase‐dependent programmed cell death was substantially detected, and the autophagy was induced as the autophagosome formation and autophagic flux proceeded. Modulation of autophagy by autophagic inducer, rapamycin or inhibitors, 3‐MA or bafilomycin, potentiated the honokiol‐mediated anti‐OSCC effects where honokiol exerted multiple actions in suppression of MAPK pathway and regulation of Akt/mTOR or AMPK pathways. As compared to clinical therapeutic agent, 5‐FU, honokiol exhibited more potent activity against OSCC cells and synergistically enhanced the cytotoxic effect of 5‐FU. Furthermore, orally administrated honokiol exerted effective antitumour activity in vivo in OSCC‐xenografted mice. Thus, this study revealed that honokiol could be a promising candidate in preventing human OSCCs.Honokiol, an active natural product derived from  Oral squamous cell carcinoma (OSCC) is one of the most common neoplasia worldwide In vitro and in vivo studies suggest that honokiol has multiple anticancer actions against numerous solid and haematological cancers including human hepatoma, myelogenous leukaemia, lung adenocarcinoma, breast cancer, ovarian cancer, prostate cancer and gastrointestinal cancer Honokiol, a biphenolic phytochemical, is one of dominant biphenolic compounds isolated from Magnolia plant species. It has been used in Traditional Chinese Medicine (TCM) for the treatment of various biological properties including antidepressant, anti‐emetic, anti‐oxidative, antithrombotic, anti‐angiogenesis, anti‐anxiolytic, anti‐inflammatory and antitumour activities in vitro, HSC‐3 and HSC‐4, through the induction of apoptosis Recently, honokiol alone (in comparison with 5‐FU) has been shown to inhibit the cell growth of two OSCC cell lines 2. In addition, SAS cells were transfected with siRNA  to block the expression of ATG5. Lipofectamine 2000  was mixed into serum‐free DMEM containing siRNA or scrambled RNA.Two OSCC cell lines (OC2 and OCSL), derived from males with habits of drinking, smoking and betel quid chewing in Taiwan 3 cells/100 μl) were plated into 96‐well tissue culture plates and grown in the above‐mentioned medium. The cells were treated with medium only (containing 0.01% DMSO) or medium containing honokiol at 5, 10, 20, 30, 40, 50 and 60 μM. After cultivation for 24, 48 and 72 hr, the number of metabolically active cells was determined by cell counting kit‐8 (CCK‐8) assay . The final results were analysed by statistical methods in three independent studies.Honokiol was purchased from BioVision . Cells  and incubated at room temperature in the dark for 30 min. DNA content was analysed using FACScan  with ModFit LT 3.3 software. In addition, the cell cycle markers, such as cyclin D1, cyclin E, cdk4, cdk2, p21 and p27, were determined by Western blot with the antibodies .g for 10 min. The cell pellets were resuspended in 100 μl of staining solution (2 μl annexin V‐FITC and 2 μl PI in 100 μl binding buffer) and incubated for 15 min. at room temperature in darkness. Annexin V or PI fluorescent intensities were analysed by FACScan , and 10,000 cells were evaluated in each sample.After treatment of the cells, annexin V staining (Sigma‐Aldrich) was performed to detect apoptotic cells. The cells were washed with PBS twice and centrifuged at 1500 × 3VO4, 1 mM PMSF, 1% NP‐40)with cocktail of protease inhibitors, and the protein concentration in the supernatant was determined by Bio‐Rad protein assay kit . The cell lysate (50 μg) was subjected to the sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE) analysis, and the separated proteins were electrically transferred to a PVDF membrane . The membrane was probed with sequential additions of primary and matched secondary antibodies, and the signal was developed with enhanced chemiluminescence (ECL) substrate and acquired by BioSpectrum 800 Imaging System . The antibodies used were as follows: anti‐LC3 , anti‐Akt , anti‐phosphorylated‐Akt (Santa Cruz Biotechnology), anti‐mTOR , anti‐phosphorylated‐mTOR , anti‐phosphorylated‐p70S6 K , anti‐p62 (Abgent), anticyclin D1 , anti‐Cdk4 , anti‐p27 , anticyclin E , anti‐Cdk2 , anti‐p21 , anti‐caspase‐8 , anti‐caspase‐3 , anti‐PARP , anti‐Bcl‐xl , anti‐Bid (Epitomics), anti‐caspase‐9 , anti‐GAPDH  and secondary antibodies, antimouse IgG Ab and anti‐rabbit‐IgG Ab(GeneTex).Cells were cultured in 10‐cm culture dishes and treated with or without compounds. The DMSO was used as negative control. The cell lysate was prepared using lysis buffer  were seeded on a slide and cultured for 24 hr. The cells were treated with DMSO, rapamycin or honokiol, and then fixed in methanol for 20 min. The slides were incubated for 30 min. in 0.1% Triton X‐100 in phosphate‐buffered saline. Anti‐LC3 polyclonal antibody  was added on the slide and left overnight at 4°C. The fluorescent change in the cells was captured and analysed by high‐content image analyzer, BD pathway 435 . The percentage of cells with LC3 puncta formation and the average number of LC3 puncta per cell were analysed by BD Attovision software (BD Biosciences).Cells (1 × 106 cells/mice) were separately injected subcutaneously (s.c.) into the right flank of the nude mice, but only the SAS xenograft nude mice model was successfully established. The experiment protocol was conducted as follows: Six‐week‐old male nude mice were randomized into three groups (three mice/group in each experiment); 2 × 106 cells of SAS were injected s.c. at day 0. After 10 days post‐injection, and the tumour was growth at less 1 mm3, mice were treated with DMSO or honokiol. Control group mice were orally fed with DMSO, and the experimental group mice were orally fed with honokiol 5 mg/kg or 15 mg/kg at days 1, 4, 7, 10, 13, 16, 19 and 22, Tumour size was measured at above days. Tumour volume was estimated by the equation . Closing the experiment on day 35, the nude mice were killed, and the tumour was harvested and weighed. Two independent experiments were conducted. The experimental protocol was complied with Taiwan's Animal Protection Act and was approved by the Laboratory Animal Care and Use Committee of the National Chiayi University .The 6‐week‐old male nude mice (BALB/cAnN.Cg‐Foxn1nu/CrlNarl) were purchased from the National Laboratory Animal Center. Mice were maintained in the animal facility in the Department of Life Science, National Dong Hwa University, Hualien, Taiwan. OC2, OCSL and SAS cells  at 48 hr and at higher dose (i.e. 40 μM for OC2 and 30 μM for OCSL)  did not increase the population of annexin VTo clarify the ability of honokiol‐induced cell death in human OSCC cells extensively, autophagy was examined in honokiol‐treated OSCC cells. Although the basal level expression pattern of LC3 (the marker of autophagy) in OC2 and OCSL cells under normal condition was different, that is OC2 cells showing endogenous LC3‐II expression, the expression levels of LC3‐II post‐honokiol treatment in OC2 and OCSL were comparable to those of rapamycin treatment in each cell line Fig. A and D. The effect of honokiol on growth inhibition of OSCC cells revealed that OCSL cells are more sensitive than OC2 cells to the treatment. This might be partly due to the fact that signalling transduction in these two cell lines was different. Upon honokiol treatment, the expression levels of survival signalling  in OCSL cells were notably down‐regulated, while these molecules at least p‐ERK and p‐JNK were up‐regulated in OC2 cells Fig. A. In addThe effective treatments for patients with oral cancer of stages III and IV are generally surgical excision and radiation combined with chemotherapy using 5‐FU and cisplatin. Here, treatment with honokiol or 5‐FU showed a similar antiproliferation effect on OC2 cells Fig. A. Howevein vivo, a xenograft nude mice model was established. As OC2 and OCSL cells failed to grow in nude mice, a xenograft with another OSCC cells, SAS cell, was used to evaluate the antitumour effect of honokiol in vivo. The antitumour effects of honokiol on SAS cells were evaluated. It was shown that honokiol suppressed SAS cell growth in a dosage‐dependent manner and genetically knock‐down of Atg5 rendered the cells less sensitive to honokiol treatment , STAT3, epidermal growth factor receptor (EGFR), c‐Met, c‐FLIP, bone morphogenetic protein 7 (BMP7), p53, RhoA, cell survival signalling and cell cycle mediators Autophagy is a catabolic process involving cellular recycling and is believed to help in survival and longevity of cancer cells by buffering metabolic stress. Inhibition of autophagy in an environment of nutrient deprivation can induce cell death. However, enhancement of autophagy can also be a beneficial tool in the fight against cancers Importantly, honokiol shows better antiproliferation effect than 5‐FU and has a synergistic effect with 5‐FU in the treatment of human OSCC cells Fig. . In the et al. have identified a side population (SP) cells harbouring stem cell‐like properties in the eight human OSCC cell lines in vivo; however, SAS cells display tumorigenic capability in xenograft mice model. It was suggested that SP cells might be the absence or few in the population of OC2 and OCSL cells. In addition, preclinical researches of honokiol have showed wide‐ranging biological and clinically relevant effects on tumour treatment, without appreciable toxicity in animal models et al. had revealed that no significant abnormalities are observed in rats, including body weight, mean daily food intake, haematological values, serum biochemical values and tissue pathologic changes, in administration with high dosage of honokiol (80 mg/kg) in vivo.Huang in vivo. In this study, honokiol has shown the promising antitumour activity without exacerbating significant toxicities with regard to the body weight loss and the liver and kidney histological examinations, which was in line with other observations, previously In the present study, we found that both OC2 and OCSL cells cannot establish tumours in nude mice; hence, SAS cells, another OSCC cell line, were used to evaluate the antitumour benefit of honokiol We have read the Journal's policy, and the authors confirm that there is no conflict of interests.CHK, KJH and YRL conceived and designed the experiments; CHK, SHC, CYL and YRL analysed the data; CHK, KJH and YRL wrote the first draft of the manuscript; CHK, KJH, SHC, CYL and YRL read and confirm the criteria for authorship.Fig. S1 Honokiol inhibited the growth of human normal cells. Hs68 cells were incubated with various concentration of honokiol, and the cell viability was determined by CCK‐8 analysis. The data present as the mean ± S.D. of three independent experimentsClick here for additional data file.Fig. S2 (A) The OC2 cell morphology and the autophagy induced by honokiol combined with autophagy agonist, rapamycin, and antagonists, 3‐MA and bafilomycin. The morphologic changes were observed under a microscope. (B) OC2 cell incubated with DMSO, 3‐MA, bafilomycin, rapamycin and honokiol, and then the cell lysates were collected for western blotting of LC3‐II and p62 proteins. GAPDH was used as an internal controlClick here for additional data file. Click here for additional data file.Fig. S3 (A) The OCSL cell morphology and the autophagy induced by honokiol combined with autophagy agonist. The morphologic changes were observed under a microscope. (B) OCSL cell incubated with DMSO, 3‐MA, bafilomycin, rapamycin and honokiol, and then the cell lysates were collected for western blotting of LC3‐II and p62 proteins. GAPDH was used as an internal controlClick here for additional data file. Click here for additional data file.


In this study, we studied the therapeutic effectiveness of percutaneous drainage with antibiotics and the need for an interval appendectomy for treating appendiceal abscess in children with a research-oriented dataset released by the Bureau of National Health Insurance in Taiwan through the Collaboration Center for Health Information Application (CCHIA).We identified 1225 patients under 18 years of age who had non-surgical treatment for an appendiceal abscess between 2007 and 2012 in a Taiwan CCHIA dataset. The treatment included percutaneous drainage with antibiotics or antibiotics alone. We also analyzed data of patient’s baseline characteristics, outcomes of percutaneous drainage, and indicating factors for performing an interval appendectomy.p < 0.05), a significantly smaller chance of receiving an interval appendectomy (p < 0.05), and significantly fewer postoperative complications after the interval appendectomy (p < 0.05) than those without percutaneous drainage treatment. Older children (13 ~ 18 years) patients were found to have a significantly smaller need to receive an interval appendectomy than those who were ≤ 6 years of age (odd ratio (OR) = 2.071, 95 % confidence interval (CI) = 1.34–3.19, p < 0.01), and those who were 7 ~ 12 years old . In addition, those treated with percutaneous drainage were significantly less indicated to receive an interval appendectomy later . In addition, those with recurrent appendicitis had a significantly increased incidence of receiving an interval appendectomy later .Totally, 6190 children had an appendiceal abscess, an 1225 patients received non-operative treatment. Of 1225 patients, 150 patients received treatment with percutaneous drainage and antibiotics, 78 had recurrent appendicitis, 185 went on to receive an interval appendectomy, and 10 had postoperative complications after the interval appendectomy. We found that patients treated with percutaneous drainage and antibiotics had a significantly lower rate of recurrent appendicitis (In this study, we used nationwide data to demonstrate therapeutic effectiveness of percutaneous drainage and antibiotics was more beneficial than only antibiotics in treating patients with an appendiceal abscess. We also found three factors that were significantly associated with receiving an interval appendectomy: recurrent appendicitis, being aged ≤ 13 years, and treatment with antibiotics only. Appendicitis is a common pediatric surgical emergency. Even with the use of ultrasonagraphy and computed tomography (CT), perforation rates in children have been reported to be 30 % over the last 30 years, and some perforated patients presented themselves with an appendiceal abscess –3.Appendiceal abscess can immediately be treated by surgery or by non-surgical management, consisting of treatment with parenteral antibiotics alone, or treatment with antibiotics and ultrasound- or CT-guided drainage, followed by an interval appendectomy , 5. But In this study, we used dataset from Taiwan’s National Health Insurance (NHI) to analyze the effectiveness of percutaneous drainage therapy, and to determine the possible indicating factors for performing an interval appendectomy.This study used data from the Taiwan National Health Insurance (NHI) program, which the government initiated in 1995, and which covers 99 % of the population of 23 million people. The Bureau of the NHI (BNHI) in Taiwan has released a research-oriented database through the Collaboration Center for Health Information Application (CCHIA). In 1999, the BNHI began to release all claims data in electronic form, to allow researchers to trace almost all uses of medical services for all people in Taiwan, including all children with appendicitis.We extracted datasets between 2007 and 2012 from the NHI research database (NHIRD) released by the BNHI through the CCHIA. The study protocol was approved by the Taipei Medical University Joint Institutional Review Board without the need to obtain signatures of study participants, because the NHIRD consists of de-linked secondary data.International Classification of Disease, Ninth Revision, Clinical Modification (ICD-9-CM) codes were available since 2007.We enrolled 1225 pediatric patients (≤18 years of age) with a diagnosis of an appendiceal abscess who received non-surgical treatment between January 2007 and December 2012, because the Inpatient claims, which included records of all hospitalizations, provided a substantial amount of information. We linked study participants to the inpatient claims data to identify appendiceal abscess (540.9), percutaneous drainage (470.22C) and postoperative complications include an intra-abdominal abscess  and postoperative bowel obstruction  based on ICD-9-CM codes.Patients were divided into three age groups: ≤ 6, 7 ~ 12, and 13 ~ 18 years old. The definition of an interval appendectomy is a patient who had initial medical therapy with intravenous antibiotics or possibly drainage followed by an elective appendectomy. The definition of recurrent appendicitis is a patient who has appendicitis again after initial non-surgical treatment without an appendectomy. Excluded from the study were patients for whom the initial non-surgical treatment failed or who had recurrent appendicitis and received immediate surgery.t-test. We also present categorical variables as ratios, and analyzed differences in non-parametric variables between groups with Chi-square test. A multivariate logistic regression analysis was used to identify independent factors including factors of different age groups, gender, the use of antibiotics, the use of percutaneous drainage, and recurrent or non-recurrent appendicitis. The relationship between potential variables and the necessity for an interval appendectomy was assessed by odds ratios (ORs) and related 95 % confidence intervals (CIs).We performed a univariate analysis between different age group and treatment methods. We present continuous variables as the mean ± standard deviation (SD) and analyzed differences in these parametric variables between groups with Student’s p values were < 0.05.We used the Statistical Analytic System software version 9.3  to analyze the data in this study. Differences between groups were considered significant if Totally, 6190 children had an appendiceal abscess, and non-surgical treatment was used in 1,225 patients. Table p < 0.05). Table p < 0.05), a significantly smaller chance of receiving an interval appendectomy (p < 0.05), and a significantly smaller chance of postoperative complications (p < 0.05) after an interval appendectomy than those without percutaneous drainage treatment.Of the 1225 children presenting with an appendiceal abscess had non-surgical treatment. 150 (2.2 %) patients were treated by percutaneous drainage with antibiotics, and the remaining 1075 (97.8 %) were only given antibiotics without percutaneous drainage. The average time of hospital stay for patients treated with percutaneous drainage (at 12.8 ± 8.9 days) was longer than that without drainage (9.7 ± 6.4 days) (p < 0.05), older (13 ~ 18 years) patients had a significantly smaller need to receive an interval appendectomy than those who were ≤ 6 years, , and those who were 7–12 years old , and patients with recurrent appendicitis were significantly likely to have an increased incidence of receiving an interval appendectomy .In our study Table , 185 15 showed tTo the best of our knowledge, this is the first study to use a large nationwide database to study the effectiveness of percutaneous drainage therapy, and to determine the indicating factors for receiving an interval appendectomy in patients with appendiceal abscess.Some recent reports mentioned that treatment of acute appendicitis with abscess formation or phlegmon with non-surgical management in children followed by an elective appendectomy has become well-recognized and accepted –15. Non-p < 0.05), but also had a significantly lower rate of recurrent appendicitis (p < 0.05) and had a significantly smaller chance of having postoperative complications (p < 0.05) after an interval appendectomy. The average time of hospital stay (12.8 ± 8.9 days) was longer for patients treated with percutaneous drainage than those without drainage (9.7 ± 6.4 days) but with antibiotics alone (p < 0.05). Based on these data, we suggest that the therapeutic effectiveness of percutaneous drainage may be due to patients’ hospitalization until drainage was completely stopped.Previous reports revealed that percutaneous drainage with the use of antibiotics is more efficient than treatment with antibiotics alone to successfully and completely treat appendiceal abscess without an interval appendectomy , 12. We p < 0.05) and were significantly older (13 ~ 18 years) (p < 0.01). But if the pediatric patients had recurrent appendicitis, they had a significantly greater need to receive an interval appendectomy .There are few reports dealing with indicating factors for performing an interval appendectomy. The presence of an appendicolith, increased blood C-reactive protein levels, elevated percent bands of white blood cells, and partial small bowel obstruction on admission are reported to be associated with an increased risk of recurrent appendicitis, and thus, such patients should receive an interval appendectomy –18. In tReaders are warned against over-interpreting our study data because it has two major limitations. First, with a population-based database, we had no other available clinical data including patients’ descriptions of clinical presentation, laboratory data, severity of appendiceal abscess, or pathologic confirmation of an appendiceal abscess. Second, we were unsure whether the staff surgeon decided to treat patients with non-surgical treatment, the use of percutaneous drainage, and performing an interval appendectomy.In this our study, we used nationwide data in Taiwan to demonstrate that the therapeutic effectiveness of percutaneous drainage and antibiotics was more beneficial than antibiotics alone in treating patients with appendiceal abscess. We also found three factors significantly associated with receiving an interval appendectomy: recurrent appendicitis, being aged less than 13 years, and treatment with antibiotics only.


Haemonchus contortus eggs. With increased trade in animals within and between countries and continents, it has become important to correctly identify H. contortus eggs in fecal samples. To validate the outcome of diagnostic tests, sheep feces (n = 38) were collected from naturally infected flocks in Sweden. Subsamples were analyzed with (a) McMaster egg counting; (b) differential counting of eggs after staining with peanut agglutinin (PNA); (c) detection of DNA following amplification by real-time quantitative polymerase chain reaction (qPCR); and (d) loop-mediated isothermal amplification (LAMP). Differences between similar tests (microscopic and molecular) and SD (±SD) were analyzed with Bland–Altman plots and Spearman rank correlation. Strongylid egg counts ranged from 200 to 12,100 eggs per gram (epg) . Microscopy showed presence of H. contortus eggs in 27 (73%) unstained samples and in 28 (76%) samples stained with PNA, whereas 29 samples (78%) tested positive in LAMP and 34 (91%) in qPCR analysis. The cycle threshold (Ct) values with LAMP ranged between 13 and 38 (mean ± SD = 21 ± 7), and those in qPCR between 25 and 49 (mean ± SD = 33 ± 6). In the LAMP and qPCR analyses, seven (19%) and three (8%) samples, respectively, had a cycle threshold (Ct) >35, whereas no reactions were observed in eight (22%) and three (8%) samples, respectively. There was good agreement between the diagnostic tests based on microscopic examination and DNA detection, although the molecular tests were more sensitive. The bias between the microscopy methods (−4.2 ± 11) was smaller than for the molecular tests (−9.8 ± 10). The observed ranking in terms of test sensitivity was: McMaster counting by conventional microscopy < PNA < LAMP < qPCR. In conclusion, H. contortus can be identified by McMaster counting, without major mistakes regarding false positive results. However, molecular methods provide the capacity to diagnose H. contortus eggs with increased accuracy. This is essential when animals are investigated in quarantine or in studies evaluating anthelmintic treatment efficacy. These methods could also be applied to fecal samples from wildlife to investigate nematode transmission between wildlife and livestock.We compared four methods for identification of  Haemonchus contortus is a cosmopolitan blood-feeding parasite in the abomasum of ruminants. It is recognized as one of the greatest health problems in small ruminants, leading to reduced production and loss of income for farmers throughout the world  used for restocking purposes  demonstrated low levels of Haemonchus  methods based on examination of parasite DNA . The litH. contortus eggs based either on microscopy or molecular methods. The scientific objective was to determine the presence of H. contortus by comparing results and evaluating the performance of each assay relative to the gold standard of conventional microscopy. This was considered essential to check the reliability and test performance of egg count data on H. contortus produced by a veterinary diagnostic laboratory in Sweden.The aim of this study was to compare four diagnostic assays for detection of H. contortus and their individual egg counts after McMaster examination, as described below. The underlying intention was to have access to four sample groups: one without H. contortus (Negative group) and three additional groups with the parasite plus low, medium, and high egg counts.Sheep feces were collected at a commercial laboratory in Sweden receiving ≈6,000 diagnostic samples on an annual basis. In total, 38 egg-positive samples originating from 17 naturally infected flocks were monitored for strongylid parasites. Individual samples of fresh feces were analyzed within 48 h of sampling by four different methods, of which two were based on detection of parasite eggs by microscopic examination and the remaining two by analysis with two different molecular diagnostic tools. All 38 samples contained strongylid eggs but were selected on the basis of both declared absence or presence of Feces samples were collected by licensed veterinarians from the rectum of sheep between June and July 2014, as part of routine diagnostic screening, and no ethical permission was required under Swedish legislation .H. contortus eggs were determined based on their characteristic shape, dark brown blastomeres, and body dimensions . For PNA staining, eggs in 3 g feces were enriched by flotation with saturated NaCl, pipetted into new tubes, subjected to repeat centrifugation at 300 g for 5 min and washed several times in phosphate-buffered saline (PBS). In the staining process, the final pellet of parasite eggs was resuspended in 1 mL of PNA-FITC  and then incubated in dissolved lectin suspension for ≈1 h at room temperature. Before microscopic examination, egg samples were washed twice with PBS as described earlier and then transferred to a glass slide with 3 mL fluorescent mounting fluid  and covered with a coverslip. Nematode eggs were viewed in the dark and lectin-binding intensity was recorded with an Olympus BH2-RFC fluorescence microscope. Three slides were produced for each sample, from which eggs were counted both under bright field illumination and when viewed with fluorescence.Conventional egg counting was first performed with the McMaster method and then, after flotation and staining of eggs with peanut agglutinin (PNA), essentially as described by Jurasek et al. . McMasteHaemonchus DNA was performed following extraction and amplification either by real-time qPCR or by loop-mediated isothermal amplification (LAMP). The DNA extraction step of the total amount of eggs in 3 g feces was based on the Nucleospin Tissue Kit (Macherey-Nagel). In brief, flotated eggs were washed and transferred into Eppendorf tubes and incubated overnight at 56°C with proteinase K in lysis buffer (0.20 mg/mL) while being subjected to gentle shaking on a PCMT thermo-shaker (Grant-bio). DNA was then extracted according to the manufacturer’s instructions. Aliquots of the same DNA were examined with both methods, as described below.Detection of Haemonchus primers targeting the internal transcribed spacer region (ITS2) of the ribosomal RNA gene array were used. The primer sequences employed were Hc forward 5′ GTT ACA ATT TCA TAA CAT CAC GT 3′ and Hc reverse 5′ TTT ACA GTT TGC AGA ACT TA 3′ (H. contortus), was determined for identical technical duplicates in a Rotor-Gene 3000 (Corbett). Cycling conditions were: 95°C for 15 min followed by 45 cycles of 94°C for 15 s, 50°C for 30 s, and 72°C for 30 s. The results were analyzed using Rotor-Gene 6.1.90 software (Corbett).In the qPCR analysis, species-specific CT TA 3′ . The qPCegion ITS of the rThe LAMP assay was carried out “blind” using prototype pelleted LAMP reagents (V6.21) designed by MAST Group Ltd. , adapting the assay described by Melville et al. . In briep < 0.05.Graphs were prepared and differences between groups were tested with the Kruskal–Wallis non-parametric test followed by Dunn’s multiple comparison in GraphPad Prism6. This software was also used to compare test performance with Bland–Altman plots and Spearman rank correlation. All statistical tests were considered significant at n = 38) contained strongylid eggs, in numbers ranging from 200 to 12,100 epg  . The remaining 28 H. contortus-containing samples were divided into three groups based on their FEC level: Low , Medium , and High . The mean epg ± SD in these groups was: 328 ± 82 in Low, 720 ± 180 in Medium, and 3,600 ± 3,673 in High, and the proportion of H. contortus eggs was 69 ± 34, 85 ± 17, and 90 ± 8%, respectively  between the groups.McMaster fecal egg counts (FECs) showed that all samples tested  identified as being H. contortus-positive. A total of 79% (73/92), 85% (355/417), and 91% (486/535) of the eggs in the Low, Medium, and High groups tested PNA-positive when examined with fluorescence   were PNA-positive in one of the 10 samples deemed to be % were PNH. contortus by McMaster and after lectin staining with PNA are shown in Figure Differences in the proportion of eggs identified as p = 0.0011) between the sample groups. The value was 46 ± 7.5 (mean ± SD) for samples in the Negative group, 37 ± 9.0 in Low, 30 ± 2.0 in Medium, and 30 ± 2.9 in High  between the sample groups. The value was 34 ± 4.6 (mean ± SD) in the Negative group, 22 ± 5.0 in Low, 17 ± 1.8 in Medium, and 17 ± 1.2 in High  had isothermal (Ct) values >35, and eight (22%) samples had no reaction. In the qPCR analysis, three (8%) samples had a cycle threshold (Ct) >35, and only three (8%) showed no reaction. Those samples where no increase was observed were replaced by a value of 50 for graphical reasons. With LAMP, the equivalent of Ct values ranged between 25 and 50 and differed significantly   Figure . The agr) Figure B.H. contortus than conventional microscopy.In this study, there was generally good agreement between the four different diagnostic methods compared. The two molecular methods were more sensitive than the two based on microscopy, but the discrepancy between the microscopy methods (−4.2 ± 11) was less than for the molecular tests (−9.8 ± 10). However, both molecular diagnostic tests, and especially qPCR, were more sensitive in detection of H. contortus eggs, have been developed, further validated and refined  were positive compared with samples examined by LAMP (n = 29). This illustrates that qPCR is superior to LAMP, although it is an open question whether the Ct values are directly comparable between these tests. Another study on H. contortus eggs isolated from ovine samples has reported 10-fold higher sensitivity of results obtained by fluorescent LAMP compared with conventional PCR (r = 0.7), it is difficult to make direct comparisons between Ct values obtained with these molecular methods, as the thresholds obviously differ between the methods (Figure H. contortus-negative and Low groups. Furthermore, the Ct values obtained with qPCR were in general lower than those obtained with LAMP.On comparing the outcomes of the molecular testing, it was evident that more samples examined by qPCR (onal PCR . Althougonal PCR  than in onal PCR . AlthougIt was beyond the scope of this study to investigate whether it is practical to set up and offer these techniques in a smaller diagnostic setting receiving routine samples from veterinarians. Still, LAMP in particular seems to provide a viable option, even in a small diagnostic laboratory, because it can generate highly sensitive and reliable results in less than an hour even in a resource-limited situation. By contrast, qPCR requires more sophisticated equipment, such as a real-time PCR thermal cycler.H. contortus in sheep with the McMaster method at flock level, without making major mistakes with false positives. However, both PNA and especially the two molecular methods were obviously more sensitive than microscopy. Although this increased sensitivity is not always required for high-throughput diagnostic laboratories, it is vital for certain purposes, for example, when it important to avoid the spread of isolates of H. contortus, especially if they are resistant to anthelmintics.From this study, it can be concluded that in most situations it is possible to identify Conceptualization and funding acquisition: JH. Acquisition of data: SL and LM. Analysis and interpretation of data and drafting of manuscript: JH and PS. Critical revision: all the authors.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. The reviewer BP and handling editor declared their shared affiliation.