A relationship between hypovitaminosis D and infection with HIV and HCV has been established in the scientific literature. Studies comparing these illnesses to other risk factors for development of hypovitaminosis D, such as being of Middle Eastern origin, have been lacking. The goals of this study were: (a) to document vitamin D levels in groups of individuals at high risk of developing its deficiency, (b) analyze the data collected to numerically determine which group had the lowest average vitamin D levels, and (c) discuss the impact of the findings and offer possible explanations.This retrospective observational study involved reviewing medical charts and documenting recent vitamin D levels. Our subgroups were: (a) individuals infected with HIV, (b) individuals infected with HCV, (c) individuals co‐infected with HIV/HCV, and (d) people of Middle Eastern origin. The gathered data was subsequently subjected to statistical analysis.People of Middle Eastern origin were found more likely to be vitamin D deficient as compared to those infected with HIV, HCV, or co‐infected with both HIV and HCV.This suggests that genetic and environmental factors unique to otherwise healthy Middle Eastern people are more detrimental, in terms of developing hypovitaminosis D, than being chronically infected with the aforementioned illnesses. Relatively recently, vitamin D has also been shown to have immunomodulatory effects; its receptors have been found on monocytes, macrophages, as well as B and T lymphocytes Holick, . SynthesThe Endocrine Society (ENDO) and the International Osteoporosis Foundation (IOF) recommends that levels of 25(OH)D be at or above 75 nmol/L  HIV infection, (b) HCV infection, (c) HIV‐HCV co‐infection, and (d) Middle Eastern origin. By setting out to accomplish this task, the hope was to highlight the relative contribution of each of these factors in developing vitamin D deficiency and thereby facilitate discourse about the course of action moving forward. Knowing which factor is most dominant will enable discussion on policy change considerations where applicable, such as those related to vitamin D screening, and also it will spark enthusiasm for research exploring that factor further.2This retrospective observational study looked at vitamin D levels in people attending an infectious diseases clinic in Vancouver, BC. The medical charts of 266 patients were anonymized and reviewed to tabulate vitamin D levels. Individuals were divided into the following four categories: (a) infected with HIV, (b) infected with HCV, (c) co‐infected with HIV and HCV, and (d) individuals of Middle Eastern background without HIV or HCV. The respective number of people in each category was as follows: (a) 26, (b) 46, (c) 38, and (d) 156.When determining vitamin D levels, the bone marker 25‐hydroxyvitamin D was used. The total amount of 25‐hydroxyvitamin D is represented by the sum of 25‐hydroxylated vitamin D2 and vitamin D3 species. The values indicating low, normal, high, and toxic were determined by local lab services. Consequently, we defined vitamin D levels <75 nmol/L as low, 75–149 nmol/L as normal, 150–200 nmol/L as high, and above 200 nmol/L as toxic. The collected data was then subjected to statistical analysis; the software used for this purpose was Microsoft Excel version 14.0.7015.1000. The average vitamin D level was calculated for people in each category; the corresponding confidence interval and p value was also computed. The resultant values for the HIV, HCV, and co‐infected subgroups were then compared against the Middle Eastern subgroup.3n = 26; CI = 54.58–75.03; p = <0.05) in the HIV subgroup, 72.83 nmol/L  in the HCV subgroup, 65.18 nmol/L  in the co‐infected subgroup, and 43.02 nmol/L  in the Middle Eastern subgroup. These findings are presented in Table Our results showed that people of Middle Eastern background had, on average, lower levels of vitamin D than those infected with HIV, HCV, or those co‐infected with both HIV and HCV. It was found that 7.7% of HIV, 6.5% of HCV, and 2.6% of co‐infected individuals were vitamin D deficient, as compared to 35.3% of Middle Eastern individuals. The mean value of vitamin D in each of our subgroups was as follows: 64.81 nmol/L . It was found that the vitamin D level of Turkish women with Western‐style clothing was on average 56 nmol/L; for those who wore hijab it was 32 nmol/L, whereas for those who wore the niqab, it was only 9 nmol/L  review as per Tri‐Council Policy Statement 2: Ethical Conduct for Research involving Humans (TCPS 2) 2014, Article 2.4.The authors declare that there is no conflict of interest regarding the publication of this paper.


Background: Erectile dysfunction (ED) is a disorder that is frequently observed in people with chronic kidney disease who undergo hemodialysis (HD). In the context of evidence-based medicine, we aimed to investigate the effect of low-dose tadalafil on sexual function in patients undergoing HD.Methods: The medical records of 30 males (aged 29–65 years) with end-stage renal disease (ESRD) on a HD program, and who had received 5 mg tadalafil twice weekly, were retrospectively evaluated. Changes in erectile and ejaculatory function were evaluated using the International Erectile Function Index questionnaire, the Erection Hardness Scale (EHS), and the Male Sexual Health Questionnaire (MSHQ).Results: The mean age of the patients was 47.6 ± 10.1 years, their mean body mass index was 24.3 ± 4.2 kg/m2, their mean hemoglobin was 11.9 ± 0.9 g/dL, and their mean creatinine clearance was 5.8 ± 1.1 mL/min. At the third month of treatment, 36.6% of the patients had no ED, 40% had mild ED, 10% had mild-to-moderate ED, and 13.3% had moderate ED. The mean MSHQ scores (p < .05) and the mean EHS scores (p = .001) were significantly improved. There was no significant difference between Beck's Depression Inventory scores (p > .05), but Hamilton anxiety rate scores decreased significantly (p = .001). The quality-of-life score improved throughout the study period (p < .05).Conclusions: Tadalafil therapy is an effective therapeutic option in patients with ESRD who undergo HD, not only for the treatment of ED, but also for ejaculatory function, with acceptable adverse effects. The delicate balance between detumesence and erection is maintained by harmonious interaction of the sympathetic and parasympathetic nervous systems. Alfa-1 adrenergic stimulation, which is mediated by various mediators, leads to penile smooth-muscle contraction in the flaccid phase of the penis .Erectile dysfunction (ED) is a disorder that is frequently observed with increasing prevalence in people with end-stage renal disease (ESRD) who undergo a hemodialysis (HD) program . SeveralESRD has become a global problem, and its incidence is increasing . The chrSeveral studies have reported that ED prevalence is as high as 80% in ESRD people on an HD program than in the normal population . In addiIn the present study, we aimed to investigate the effect of 5 mg tadalafil, administered twice-weekly for three months, on sexual function in people with ESRD undergoing HD, using the International Index of Erectile Function questionnaire (IIEF)  and the This study was designed as a retrospective clinical trial. After obtaining approval from the Ethics Committee of Ondokuz Mayis University, Samsun, Turkey, all the patients were informed of the study for ethical reasons, and their written consents were obtained. The required number of patients was determined using a power analysis. The medical records of 30 males (aged 29–65 years) with ESRD and on HD who had previously consulted our clinic regarding sexual dysfunction, and had received 5 mg tadalafil twice weekly, were retrospectively evaluated . At the The questionnaires were interviewed face to face with patients in one session. As needed, quantitative variables were repeated throughout the study period. Changes in erectile function were evaluated using the short form of the Turkish validated IIEF , which cThe psychological state of the patients was assessed as minimal, mild, moderate or severe depression, using the Beck’s Depression Inventory (BDI) ,22 and tn (%) and/or median (min–max). The intragroup data were compared using the Wilcoxon signed rank test for non-normal data. Comparisons between data were performed using a paired-sample test. Pearson’s chi-square and Fisher’s exact test were used for comparisons of percentages. A p values of less than 0.05 was considered statistically significant.The statistical analysis was carried out using Statistical Package for the Social Sciences for Windows software, version 21.0  and the Shapiro–Wilk test was used to analyze the normal distribution of the quantitative data. The results are given as mean ± standard deviation, 2, and their mean hemoglobin was 11.9 ± 0.9 g/dL. The mean HD duration was 78.4 ± 27 months (24–120), the mean creatinine clearance was 5.8 ± 1.1 mL/min (4–8), and the frequency of HD was three sessions per week. The mean total serum testosterone level was 7.2 ± 1 (4.4–8.9) mmol/L, and only four patients were in the normal limits. The mean prolactin level was 5.9 ± 2.4 (3–12) ng/mL, and 11 patients were hypercholesterolemic and hypertriglyceridemic (36.6%)  .At the beginning of tadalafil treatment, 10 of the patients had mild ED (33.3%), six had mild-to-moderate ED (20%), and 14 had moderate ED (46.6%); at the first month of treatment, 15 patients had mild ED (50%), five had mild-to-moderate ED (16.6%), and eight had moderate ED (28.6%). At the third month of treatment, 11 patients had no ED (36.6%), 12 had mild ED (40%), three had mild-to-moderate (10%), and four had moderate ED (13.3%). Overall, the ED rate was 100%, 93.3% and 66.6% at pretreatment, at the first treatment month, and after the third month of treatment, respectively .p = .05), the third month of treatment score was significantly increased compared to the pretreatment value (p = .001)  .p < .05). The mean EHS [p = .001).The mean MSHQ123 scores at pretreatment and at the first and third month of treatment were 9.9 ± 2.4, 10.6 ± 2.6 and 11.4 ± 0.7; the mean MSHQejD scores were 3.1 ± 0.9, 3.6 ± 0.7 and 4.0 ± 0.7, respectively. The mean MSHQ123 and mean MSHQejD scores were significantly increased at the first and third months of treatment compared to pretreatment values (mean EHS  scores ap > .05). Four of the patients had borderline clinical depression (13.3%), and one had moderate depression (3.3%) throughout the treatment period. The mean pretreatment, and the first- and third-month treatment HARS scores were 19.3 ± 4.2, 12.5 ± 3.1 and 10.5 ± 2.6, respectively. The first month and third month of treatment mean HARS scores were statistically decreased compared to the mean pretreatment score (p = .001) (p = .04) and third month (p = .02) of treatment.The mean BDI scores at pretreatment and at the first- and third-month treatment periods were 2.9 ± 5.4, 2.5 ± 2.2 and 2.9 ± 2.3, respectively, but there was no statistically significant difference ( = .001) . The quaTmax in healthy men, and metabolism is completed in the liver, almost 36% of an oral dose is excreted in the urine [Prior to the phosphodiesterase type-5 (PDE5) inhibitor era, ED treatment consisted of conservative options, including dialysis, correction of anemia using erythropoietin, testosterone supplementation, vacuum devices, penile or intraurethral vasoactive injections or surgical procedures, such as penile prosthesis and vascular surgery . Althoughe urine . To datehe urine . while Jhe urine . Our stuMany studies have reported that patients have discontinued tadalafil treatment due to adverse effects, or the lack of drug efficacy. Although none of the patients in the present study stopped the therapy due to minor side effects, 28 (93.3%) stated that they would not be able to continue the therapy for a long time because tadalafil was not covered by their insurance. It has previously been reported that diabetes mellitus and hypertension are the comorbidities most commonly associated with ED in HD patients . AlthougOf the patients in the present study, 26 were hypoandrogenic due to abovementioned reasons, in accordance with previous studies, but none were hyperprolactinemic . AnotherA limited number of previous studies have investigated ejaculatory disorders in ESRD. In those studies, ejaculation disorders were reported in 28–51.5% of HD patients . EjaculaPsychogenic causes are another etiological reason in ESRD patients compared to age-matched counterparts without renal failure . AccordiIn the present study, none of the patients showed depression according, to the BDI which was stable throughout the 3-month period. In a review, Chilcot et al. reported that the prevalence of depression was 8.1–71.4% in different studies . HoweverThe short duration of therapy due to treatment costs, the low number of patients studied, and an absence of evaluation of the patients' partners.Tadalafil therapy is an effective therapeutic option in people with ESRD who undergo HD. Tadalafil 5 mg twice weekly not only improved ED, but also improved ejaculatory function, with acceptable adverse effects. Correctable conditions, such as hypertension and hyperlipidemia, which promote endothelial dysfunction, may influence treatment success rate, and a systematic approach is obligatory for improved outcomes.


Campomanesia adamantium (Myrtaceae) and tested for cross‐amplification in the related species C. sessiliflora.A novel set of nuclear microsatellite markers was developed and characterized for C. sessiliflora; more than 55.5% of the microsatellite loci amplified, confirming the use of these microsatellite markers in a related species.Forty‐one primer pairs were designed for simple sequence repeat loci, of which 36 successfully amplified and were polymorphic. The number of alleles ranged from two to 14, with an average of 8.14 alleles per locus. Additionally, cross‐amplification was tested in C. adamantium and a closely related species, which will aid in future conservation efforts.We developed a set of microsatellite markers that will be useful for future studies of genetic diversity and population structure of  Campomanesia adamantium (Cambess.) O. Berg (Myrtaceae) should be highlighted because of its environmental significance. This species can resist flooding and has been used for spoiled‐area reforestation projects  markers –derived simple sequence repeat (SSR) markers from Eucalyptus L'Hér. (Myrtaceae)  Mattos, a species native to Brazil with medicinal and economic value.Therefore, the goal of this study was to isolate and characterize microsatellite markers for C. adamantium  were collected to develop the microsatellite markers, and 45 samples from three natural populations were collected to validate the microsatellite markers for this species  and ligation of fragments with double‐stranded adapters 5′‐CTCTTGCTTACGCGTGGACTA‐3′ and 5′‐TAGTCCACGCGTAAGCAAGAGCACA‐3′. The enrichment was based on hybridization capture using (CT)8 and (GT)8 biotin‐linked probes and streptavidin‐coated magnetic beads . Microsatellite‐enriched DNA fragments were amplified by PCR, cloned into a pGEM‐T Easy Vector (Promega Corporation), and then inserted into Escherichia coli XL1‐Blue competent cells (Promega Corporation) using an electroporation technique. Positive clones were selected via a culture medium containing ampicillin and β‐galactosidase.Genomic DNA of e et al. . The isoNinety‐six colonies were selected using blue/white screening, and the sequencing was performed on an automated ABI 3500xL Genetic Analyzer  with T7 and SP6 primers and the BigDye Terminator version 3.1 Cycle Sequencing Kit (Perkin Elmer–Applied Biosystems). Microsatellite sequences were identified in 41 clones with good quality sequences for primer design using Primer3Plus software . In addition, for markers CAMP01, CAMP03, CAMP04, CAMP08, CAMP13, CAMP17, CAMP24, CAMP25, CAMP28, and CAMP36, the genotyping was performed with n ≥ 45, using additional individuals sampled from Crispim et al.  (Fermentas). PCR cycling conditions consisted of an initial denaturation at 94°C for 5 min; followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min; and a final extension cycle at 72°C for 15 min. Amplified products were verified visually on 2% agarose gels. Polymorphism evaluation and genotyping were performed using electrophoresis on a 7% polyacrylamide gel stained with silver nitrate  and compared them with the data of transferable markers  from the research by Crispim et al.  and observed heterozygosity (Ho), and the polymorphism information content. We used the statistical environment software R  within each population using the same package. Confidence intervals were obtained with 10,000 bootstrap replicates. The test for null allele presence was performed using MICRO‐CHECKER  values using an excluding null alleles (ENA) correction. After accounting for null allele frequencies, loci with frequencies of ≥0.2 were considered potentially problematic for the calculations.Statistical analyses were performed using GenAlEx version 6.5 software ; the number of alleles ranged from two to 14, with an average of 8.14 per locus. The Ho and He ranged from 0.00 to 0.91 (average 0.52) and from 0.09 to 0.89 (average 0.78), respectively. When we tested the previously mentioned set of 10 markers  using an increased number of individuals (n ≥ 45), the number of alleles ranged from 11 to 23, with an average of 17.10. The level of Ho and He ranged from 0.24 to 0.72 (average 0.51) and from 0.81 to 0.92 (average 0.87), respectively  in C. adamantium were detected for 11 loci in the Dourados population, 15 loci in the Bonito population, and 10 loci in the Cerro Corá population .The test for null alleles indicated significant results in some loci in the populations Table . The mosC. sessiliflora, 20 microsatellite loci cross‐amplified (Table P < 0.05) from HWE were verified in three loci.In ed Table . The numC. adamantium. The microsatellite loci described in this study successfully cross‐amplified in C. sessiliflora, suggesting that these markers could be used to support genetic conservation and breeding programs for C. adamantium and other species in the genus.We developed a panel of microsatellite markers that will be helpful for future studies of genetic diversity and population structure of B.A.C., A.B., and M.C.V designed the project. T.O.C. collected the samples. B.A.C., A.A.V., T.G.D., J.S.F., and M.M.B performed the experiments. B.A.C., T.G.D., A.B., M.M.B., and M.I.Z. wrote the manuscript.


Neospora caninum is an obligate intracellular parasite, which causes significant economic losses in the cattle industry. However, the immune mechanism of the parasite–host interaction is not yet fully understood. Extracellular vesicles (EVs) have emerged as a ubiquitous mechanism by which almost all cells, especially immune and tumor cells, participate in intercellular communications. Although studies have indicated that EVs secreted by Toxoplasma gondii or Trypanosoma brucei promote exchanges of biological molecules important for the host–parasite interplay, however, EVs and their biological activities in N. caninum is not clear. Here, we used multiple methods, including electron microscopy, nanoparticle tracking analysis, RT-PCR, immunofluorescence, western blot, proteomics, and cytokine analyses, to examine the properties of N. caninum EVs. We found that N. caninum produced EVs that are similar to mammalian exosomes, which generally range from 30 to 150 nm in diameter. It was shown that N. caninum EVs could remarkably increase the production of pro-inflammatory cytokines IL-12p40, TNF-α, IL-1β, IL-6, and IFN-γ by wild-type (WT) mouse bone marrow-derived macrophages (BMDMs) whereas the secretion of IL-12p40, TNF-α, and IFN-γ was very strongly downregulated in TLR2−/− mouse BMDMs. The levels of IL-6 were not affected, but the secretion of IL-10 was upregulated. We found that the phosphorylation levels of P38, ERK, and JNK were significantly reduced in the TLR2−/− cells compared with those in WT mouse BMDMs and that treatment with chemical inhibiters of P38, ERK, and JNK resulted in upregulation of IL-6, IL-12p40, and IL-10 production. Together, these results demonstrated that N. caninum EVs could be rapidly internalized to deliver proteins to the host cells and modulate the host cell immune responses through MAPK signaling pathway in a TLR2-dependent manner. Our study is the first to reveal potential roles for N. caninum EVs in host communication and immune response in parasite–host interactions. Neospora caninum is an obligate intracellular parasite which naturally takes dog and cattle as hosts  in response to IFN-γ  . EVs are evasion . Researce burden , 19. Morto IFN-γ . Mice innd IL-10 , suggestN. caninum and defined EVs-associated proteins. These EVs could be rapidly internalized thereby delivering their contents to the host cells. Furthermore, it was demonstrated that these N. caninum EVs could activate TLR2 in mouse bone marrow-derived macrophages (BMDMs) and that they had regulatory effects on the secretion of inflammatory cytokines through the TLR2 and MAPK signaling pathway, which indicated a potential role in parasite–host interactions.In the present report, we characterized the physical properties of EVs released by −/−) mice were obtained from the Model Animal Research Center of Nanjing University . All mice were housed under specific-pathogen-free conditions in the National Experimental Teaching Demonstration Center of Jilin University . The food and water provided were sterile.Female C57BL/6 mice (6–8 weeks old) were purchased from the Changsheng Experimental Animal Center , and TLR2-deficient (TLR2Neospora caninum tachyzoites (Nc-1) were maintained by serial passage in VERO cells in RPMI-1640 medium, supplemented with 2% fetal bovine serum (FBS) , 2 mM l-glutamine, 100 U penicillin, and 100 µg of streptomycin (P/S)  in a 5% CO2 atmosphere at 37°C. Free N. caninum were obtained from the cell cultures as described previously  but without P/S.eviously . Briefly−/− mice were euthanized and immersed in 75% ethanol; the femurs and tibiae were removed, briefly sterilized in 75% ethanol, immersed in PBS + P/S for 5 min, and placed in RPMI-1640 + P/S. This medium was used to wash out the marrow cavity plugs, and the bone marrow cells were resuspended in 1640 supplemented with 25% L929-cell conditioned medium as a source of granulocyte/macrophage colony-stimulating factor with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, plated onto 100 mm dishes, and cultured at 37°C in a 5% CO2 atmosphere. The culture medium was changed 4 days post isolation. After 7 days, the adherent cells were removed and analyzed by flow cytometry. The positive phenotype was confirmed on the basis of having CD11b expression over 95%, and the cells were retained for further experimental procedures.Bone marrow-derived macrophages were isolated using the previously described protocols . BrieflyNeospora caninum EVs were purified as previously described with a slight modification to the protocol , followed by further ultracentrifugation  at 100,000 × g for 70 min at 4°C to spin down the expected N. caninum EVs, which were then resuspended in 7 ml PBS. The EVs-rich fraction was washed twice with PBS, then resuspended in 200 µl PBS for further processing.protocol . After 2Neospora caninum lysate antigen (NLA) and excretory secretory antigens (ESAs) were prepared as previously described with a slight modification  with protease inhibitors  and subjected to ultrasound (60 H/30 s) on ice. After centrifugation at 10,000 × g for 30 min at 4°C, the NLA was collected and filtered using 0.22 µm membranes. For the preparation of ESAs, approximately 2 × 108 tachyzoites/ml were incubated at 37°C for 3 h in 2 ml serum-free RPMI-1640 containing P/S. After centrifugation for 10 min at 1,000 × g, the ESA-containing supernatant was filtered and collected for further processing.fication . For theThe collected proteins were either stored at −80°C or directly used in additional experiments. The pellets were resuspended in protein loading buffer and stored at −20°C or immediately analyzed by western blot. All protein concentrations were measured using the BCA Protein Assay Kit .N. caninum EVs, NLA, and ESAs, the ToxinSensor™ chromogenic LAL Endotoxin Assay Kit  was used according to the manufacturer’s instructions. Briefly, 100 µl of standards, samples, and LAL reagent water were carefully dispensed into different endotoxin-free vials, then 100 µl LAL lysate was added and mixed well by swirling gently, incubated at 37°C for 30 min. After proper incubation, 100 µl of chromogenic substrate was added to each of the reactions and incubated at 37°C for 6 min. The reactions were terminated by adding 500 µl each of the three color stabilizer solutions and gently swirled to mix well. The endotoxin levels were then measured by spectrophotometry at 545 nm. Distilled water was used as blank to adjust the photometer to zero absorbance.To assessed the level of endotoxin in N. caninum EVs can enter into BMDM, N. caninum EVs were labeled with the PKH67 Green Fluorescent Cell Linker Kit  according to the manufacturer’s protocol with minor modifications. Fifty micrograms of N. caninum EVs were diluted in 100 µl PBS and added to 1 ml Diluent C, then 4 µl of PKH67 dye was added and incubated for 4 min in darkness, after which 1 ml 1% BSA/PBS was added to bind excess dye. The labeled EVs were washed at 100,000 × g for 1 h, and the pellet was diluted in 100 µl PBS in an opaque tube at 4°C and used for the internalization experiments. PBS was treated in parallel. An aliquot of 25 µg PKH67-labeled N. caninum EVs was added to BMDMs growing on glass coverslips in 24-well plates. At 0, 2, 4, 6, 8, and 12 h after treatment, the cells were washed twice with PBS, then dried at room temperature (RT) and fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.25% Triton X-100 in PBS for 10 min and washed. F-actin was stained with TRITC Phalloidin , and the nuclei were stained with DAPI . We utilized N. caninum polyclonal antibodies against 14-3-3, HSP70, and enolase for confocal microscopy. The mouse polyclonal antibodies against Nc14-3-3, NcHSP70, and NcEnolase were prepared according to previously described protocols  according to the manufacturer’s protocol. Ten 8-week-old BALB/C mice were subcutaneously immunized with the obtained proteins, which were emulsified with Freund’s complete/incomplete adjuvant , and finally the corresponding mouse polyclonal antibodies were generated. For immunolocalization, the protocol was similar to the method described above except that N. caninum EVs were not labeled, and at 3 and 6 h after treatment, the cells were washed twice with PBS, then dried at RT and fixed in 4% paraformaldehyde for 10 min. They were then permeabilized with 0.25% Triton X-100 in PBS for 10 min, blocked with 3% BSA in PBS (PBS-BSA) for 2 h at RT, and incubated with a 1:100 dilution of the 14-3-3, HSP70, or enolase antibody overnight at 4°C. Finally, they were washed and incubated with Alexa Fluor-conjugated secondary antibody  for 1 h at RT. The coverslips were mounted using DAPI before analysis on an Olympus FV1000 Laser Scanning Confocal microscope (Japan).To examine whether rotocols . Briefly6 BMDMs were seeded in 6-well culture plates and incubated for 12 h with 50 µg/ml purified N. caninum EVs or Pam3Cys-Ser-(Lys) 4 (Pam3CSK4)  -Ct (control). Every sample was tested in triplicate, and the data used for the final analysis were from three independent experiments.On day 7 after isolation, 3 × 100 µg/ml) . RNA was0 µg/ml) . All RNAN. caninum EVs were resuspended in PBS and applied to a carbon-coated copper grid and incubated for 1 min at RT. After removal of the liquid using filter paper, 20 µl of 3% phosphotungstic acid was used to negatively stain the grid for 5 min at RT. The grid was dried for 15 min and then examined using transmission electron microscopy . For ultrastructural analysis of the secreted EVs, N. caninum tachyzoites were harvested after 6 h incubation. The pellet was then fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer overnight. After washing with the same buffer, the samples were post-fixed with osmium tetroxide and dehydrated in a graded acetone series prior to embedding in epoxy resin. Ultrathin sections (70–80 nm) were cut from the resin blocks using a Reichert-Jung Ultracut E Ultramicrotome. Formvar grids covered with isolated vesicles or with ultrathin sections were examined using TEM.The enriched N. caninum tachyzoites were cultured in 24-well plates  containing glass cover slips that had been pre-coated with 0.1 mg/ml poly-l-lysine  and washed three times with distilled water. After a 6-h incubation, the cells were fixed with 4% glutaraldehyde for 24 h, washed with PBS and post-fixed in 1.0% osmium tetroxide. Then, the samples were dehydrated with a series of 30, 50, 70, 80, 90, 95, and 100% alcohol solutions, critical-point dried, sputter-coated with gold, and visualized with a Hitachi S-3400N scanning electron microscope .For SEM, N. caninum EVs were characterized for size distribution and quantitated using a ZetaView PMX 110 instrument  and its corresponding software, ZetaView 8.02.28.The Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed at Shanghai Omicsspace Biotech Co., Ltd. . Briefly, the proteins were precipitated with 15% trichloroacetic acid/acetone and digested with trypsin at a final concentration of 2 ng/ml. After incubation at 37°C for 18 h, the reactions were quenched by the addition of formic acid to a final concentration of 1% prior to the LC-MS/MS analysis. The buffers used for chromatography were 0.2% formic acid (buffer A) and 100% acetonitrile/0.2% formic acid (buffer B). The Thermo Scientific analytical column was equilibrated with buffer A before the samples were loaded from the autosampler to the EASY trap column  and then separated. The peptides were loaded onto the column and eluted with a three-slope gradient. Buffer B first increased from 5 to 28% in 40 min and then from 28 to 90% in 2 min, then remained at 90% for 18 min. The digested product was analyzed using an Orbitrap-Elite mass spectrometer  after separation using capillary HPLC and desalting. The analysis time was 60 min.N. caninum database  with 20 ppm peptide mass tolerance and 0.1 Da fragment mass tolerance with allowance for two missed cleavages in the trypsin digests. Carbamidomethylation was specified in both search engines as a fixed modification, and oxidation of methionine residues was specified in Mascot as a variable modification. The identification of a protein was accepted if its peptides had FDR ≤0.01 (high confidence) and it contained at least two identified peptides. Annotations with gene ontology and Kyoto Encyclopedia of Genes and Genomes for identified proteins were also performed.The MS/MS data were searched against the NCBI database with the Mascot software version 2.3 (Matrix Science). Mascot was set up to search the N. caninum protein was prepared as follows: free N. caninum tachyzoites were resuspended in CelLytic™ M lysis reagent with Benzonase  and incubated at RT for 5 min; then, an equal volume of a solution of 2% SDS and 1 mM EDTA was added. For N. caninum EVs, the pellets were resuspended in ice-cold cell lysis buffer supplemented with a proteinase inhibitor mixture and a phosphatase inhibitor . Thirty micrograms of each protein sample were separated on a 12% SDS-PAGE gel, and the proteins were then transferred to PVDF membranes . After a 2-h blocking step in TBS-0.05% Tween 20 (TBST) containing 5% skim milk, the membranes were incubated at 4°C overnight with the following antibodies: polyclonal mouse antibodies anti-14-3-3 (1/100), anti-enolase (1/100), and anti-HSP70 (1/100); primary rabbit antibodies anti-p-P38 , anti-total P38 , anti-p-ERK1/2 , anti-total ERK1/2 , anti-p-JNK , and anti-total JNK  ; and sera from N. caninum-infected or healthy mice (1/200) (prepared in our lab). For all secondary antibody incubations, HRP-conjugated goat anti-mouse or goat anti-rabbit antibodies  were used at a 1:2,000 dilution. The membranes were visualized using an ECL Western Blot Detection System .The 5 cells/well and treated with 50 µg/ml purified N. caninum EVs or Pam3CSK4 (10 µg/ml) for 8, 12, and 24 h. In some experiments, the BMDMs were pretreated for 2 h with a P38 inhibitor , 1 h with an ERK inhibitor , 1 h with a JNK inhibitor  or with PBS at 37°C before the treatment with N. caninum EVs. After 8, 12, or 24 h of stimulation, all supernatants were collected for ELISA assays, and Cytokine ELISA Ready-SET-Go kits  were used to determine IL-12p40, TNF-α, IFN-γ, IL-10, IL-1β, and IL-6 levels following the manufacturer’s instructions. The assays were read at 450 nm and the OD values obtained were converted to pg/ml by interpolation of the standard curve.For the detection of inflammatory cytokines, BMDMs were seeded in 24-well culture plates at a density of 6 × 10P < 0.05, **P < 0.01, and ***P < 0.001.Data are analysis by one-way analysis of variance using SPSS 19.0 software  and expressed as the mean ± SEM. The graphs were generated in GraphPad Prism 7.00. All experiments were performed three times with three technical replicates. Significance is shown as *N. caninum produces EVs, vesicles were isolated from parasite growth media through a series of ultracentrifugation steps, similar to that described for isolating exosomes from the parasitic protozoan Leishmania  were detected with at least two unique peptides -type, in which IL-12, IFN-γ, and NO are sequentially produced by the cells of the immune system  and N. caninum-derived polypeptides  on MAPK activation in BMDMs. The results showed that all of these proteins could induce the phosphorylation of p38, ERK, and JNK after treatment for 30 or 60 min  with PBS or N. caninum EVs for 60 min at 37°C. The phosphorylation levels of p38, ERK, and JNK were significantly reduced in the TLR2−/− cells, which demonstrated that N. caninum EVs induced phosphorylation of P38, ERK, and JNK via TLR2  and significantly increased production of IL-6, IL-12, and IL-10 when BMDMs were separately pretreated with P38, ERK, and JNK inhibitors. In conclusion, the secretion of inflammatory cytokines in BMDMs induced by N. caninum EVs was regulated through the MAPK signaling pathway.To address the mechanism of the N. caninum could modulate the cytokines expression of BMDMs by triggering TLR2 and MAPK signaling pathways, but the exact mechanism was not yet fully understood. In addition, the role of EVs in regulating the immune response in vivo is presently unknown. Our current knowledge of N. caninum EVs biogenesis did not allow for specific inhibition of the production or release of EVs. Direct evaluation of the importance of EVs during an in vivo infection is therefore currently not possible. In summary, our results suggested that N. caninum EVs are actively involved in the host–parasite interactions during the N. caninum infection. It will be important to elucidate the biological activities of these EVs in vivo in future studies.We demonstrated here that EVs released by All animal experimental procedures were performed in strict accordance with the Animal Welfare and Research Ethics Committee at Jilin University (IACUC Permit Number: 20160612).SL, PG, XiZ, and JL drafted the main manuscript and performed the data analysis; SL, LT, CZ, and XuZ planned and performed the experiments; SL, XW, XL, PG, and JL were responsible for the experimental design; and JY, ZY, JL, and XiZ were responsible for guiding and supporting the experiments and revising the manuscript. All authors read and approved the final manuscript.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.


BPAP) and its selectively recognise Fe(III) and Fe(II) ions with visible, naked eye colour changes and fluorometric selectivity towards Zn2+ and Cd2+ ions in aqueous medium.Currently considerable research both in life and in environmental sciences is dedicated to chemosensors able to detect metals of biological interest such as zinc and iron or other toxic and carcinogenic, as cadmium, mercury, chromium, lead. Recently, a new chemosensor strategy of “single chemosensor for multiple metals” has emerged. For this scope, many fluorescent sensors for Cd(II) and Zn(II) have been designed and synthetized, as ligand systems or in polymeric matrices This article is submitted as a companion paper to Caruso et al. (2018)  Specifications Table•BPAP along a and c axes.The data show some molecular structure of •BPAP and its zinc complex (lengths and angles).The data report relevant structural data of •The data report Job׳s plot analysis for the binding Zn(II) and Cd(II) with ligand system.•1H NMR and 13C NMR of BPAP and BPAP metal complexes are reported.1BPAP) and its selectively recognise Fe(III) and Fe(II) ions with visible, naked eye colour changes and fluorometric selectivity towards Zn2+ and Cd2+ ions in aqueous medium.The data presented in this article are related to the research article entitled “A real-time tripodal colorimetric/fluorescence sensor for multiple target metal ions” The following data are a necessary support for the identification of materials and properties of the ligand system and its complexes.2α radiation, λ = 0.71073 Å, CCD rotation images, thick slices, φ and ω scans to fill asymmetric unit). Semiempirical absorption corrections  and 1582070 (Zn-BPAP). These data can be obtained free of charge from www.ccdc.cam.ac.uk/data_request/cif.All crystal data were deposited at Cambridge Crystallographic Data Centre with assigned number CCDC 1582069 (1H and 13C NMR are reported in 1H NMR spectrum of Zn-BPAP is shown.NMR spectra were recorded in DMSO using a Bruker Spectrometer operating at 400 MHz. For BPAP, both 2+ and Cd2+ (BPAP in ethanol. Volumes of 3.00, 2.75, 2.50, 2.00, 1.50, 1.00, 0.50, 0.25 and 0 mL of the solution of ligand were taken and transferred to vials and volumes of 0, 0.25, 0.50, 1.00, 1.50, 2.00, 2.50, 2.75, 3.00 mL of metal ion added, each vial having a total volume of 3.0 mL. Fluorescence spectra were recorded at room temperature after shaking each vial for a few seconds.Job׳s plot measurement of Znand Cd2+  has been


Participants were presented with four colored shapes sequentially and asked to recall the color of one probed item following a delay. They were informed that the first item was more valuable  or as valuable as the other items , and that this item would be tested more frequently  or as frequently  as the other items. Probe value and probe frequency boosts were observed at the first position, though both were accompanied by costs to other items. Probe value and probe frequency boosts were additive, suggesting the manipulations yield independent effects. Further supporting this, experiment 2 revealed that probe frequency boosts are not reliant on executive resources, directly contrasting with previous findings regarding probe value. Taken together, these outcomes suggest there may be several ways in which attention can be directed in working memory.In visual working memory tasks, memory for an item is enhanced if participants are told that the item is relatively more valuable than others presented within the same trial. Experiment 1 explored whether these probe value boosts (termed  The most commonly employed paradigm is retro‐cueing, in which a cue is presented immediately following an array of to‐be‐remembered items. This cue typically informs participants which item will, or is most likely to, be tested at retrieval. Retro‐cues improve memory for the cued item,The reward associated with an item can also be increased to encourage participants to direct attention toward it.In research using the probe value paradigm, memory for the more valuable item has been tested as frequently as memory for the other items.These research questions were investigated in the current set of experiments. Experiment 1 orthogonally manipulated probe value and probe frequency, targeting both manipulations at the first item. Experiment 2 explored probe frequency effects further, examining whether boosts are reliant on executive resources during encoding and maintenance, as probe value effects appear to be.Experiment 1 orthogonally manipulated probe value  or not ) and probe frequency  or not ). Both manipulations were targeted at the first item. Based on previous findings, it was predicted that significant probe value boosts would be observed at the first SP, whereby memory for this item would be higher in the differential probe value condition than the equal probe value condition.M = 20.42; SD = 1.15; nine males). Participants were native English speakers with no known learning difficulties, normal or corrected‐to‐normal vision, and no color blindness. Participants were undergraduate students who were reimbursed for their time with course credits. The experiment was approved by the School of Psychology Ethics Committee at the University of Leeds (UK).Forty‐four young adults took part  with a color from a pool of six options . No shape or color was repeated within the same trial. All stimuli subtended a visual angle of approximately 1.5°, based on a viewing distance of 50 cm. Shapes were presented on a white background at one of eight points around a 2° imaginary circle positioned at the center of the screen. Locations were selected randomly, with the constraint that no location could be used more than once per trial. The test cue was an outline of one of the stimuli presented during the encoding phase. This was displayed in the center of the screen. In the equal probe frequency condition, the first SP was tested as often as other items (25% of the time). In the differential probe frequency condition, the first SP was assessed 70% of the time, whereas the other items were each probed 10% of the time.The study employed a 2 × 2 × 4 within subject design, manipulating probe value , probe frequency , and SP (1–4). Participants completed four blocks of 40 trials; one for each combination of probe value and probe frequency conditions. The order of probe frequency blocks and the order of probe value blocks within the probe frequency conditions were counterbalanced. In the equal probe frequency conditions, each SP was tested 10 times. In the differential probe frequency conditions, the first SP was tested 28 times and the other SPs were each tested four times. The SPs tested were randomly distributed within the blocks. At the start of each block, participants completed four practice trials to familiarize themselves with the condition.Each condition commenced with the provision of written instructions. In the differential probe value conditions, participants were told that correct recall of the first item would earn them four points, whereas correct recall of the other items would earn them one point. In the equal probe value conditions, they were told each item was worth one point. The points were part of a notional reward system: the number of points accrued was never tallied and no actual rewards were given. During the instructions, participants were also informed about the probe frequency manipulation. In the equal probe frequency conditions, they were told that all items would be tested the same number of times. In the differential probe frequency condition, they were told that the first item would be tested more often than the other items.The experimental paradigm used is displayed in Figure The dependent variable was accuracy, determined by the proportion of trials where participants responded correctly. Findings are first reported as a function of SP. Further planned analysis was then conducted at SP1, as this is the position at which the manipulations were targeted.10 values describe how many times more likely the alternative hypothesis is to the null hypothesis, whereas BF01 describes the ratio of how likely the null hypothesis is compared with the alternative hypothesis.In experiment 1 and the subsequent experiments, data were analyzed using frequentist and Bayes Factor (BF) methods. BF analysis assesses the strength of evidence for the alternative hypothesis relative to the null hypothesis, and also provides a test of equivalence between conditions.M = 0.52, SE = 0.02; Equal M = 0.53, SE = 0.02;  = 0.38, P = 0.54, MSE = 0.038, ηp2 < 0.01, ηG2 < 0.01; BF01 = 10.09) or probe frequency  = 2.57, P = 0.12, MSE = 0.063, ηp2 = 0.056, ηG2 < 0.01; BF01 = 2.01), demonstrating that neither manipulation affected overall performance on the task. A main effect of SP emerged  = 40.07, P < 0.001, MSE = 0.064, ηp2 = 0.48, ηG2 = 0.17; BF10 > 1000). Pairwise comparisons (corrected using Bonferroni–Holm) revealed significant differences between SP1  and SP2 , SP1 and SP3 , SP1 and SP4 , SP2 and SP4 (P < 0.001), and SP3 and SP4 (P < 0.001). Significant interactions emerged between probe value and SP  = 25.01, P < 0.001, MSE = 0.036, ηp2 = 0.37, ηG2 = 0.069; BF10 > 1000), and probe frequency and SP  = 19.15, P < 0.001, MSE = 0.042, ηp2 = 0.31, ηG2 = 0.063; BF10 > 1000), indicating that the effects of probe value and probe frequency differed depending upon the SP tested. No interactions emerged between probe value and probe frequency  = 0.11, P = 0.74, MSE = 0.032, ηp2 < 0.01, ηG2 < 0.01; BF01 = 8.34) or probe value, probe frequency, and SP  = 0.25, P = 0.83, MSE = 0.037, ηp2 < 0.01, ηG2 < 0.01; BF01 = 27.22). These outcomes were corroborated by BF analysis, which revealed that the most likely model included a main effect of SP, as well as interactions between probe value and SP, and probe frequency and SP (BF10 > 1000 relative to the null model with random effects of participant only).Mean accuracy as a function of probe value, probe frequency, and SP is displayed in Figure t‐tests (corrected using Bonferroni–Holm) were conducted. Mean proportion correct as a function of probe value and SP is displayed in Table t(43) = 8.85, P < 0.001, BF10 > 1000, d = 1.33). The pattern was reversed at SP4, with participants exhibiting higher accuracy in the equal probe value condition (t(43) = –4.08, P < 0.001, BF10 = 130.6, d = –0.61). No significant differences between probe value conditions were found at SP2 (t(43) = –1.71, P = 0.19, BF01 = 1.64, d = –0.26) or SP3 (t(43) = –1.38, P = 0.19, BF01 = 2.56, d = –0.21). In summary, this indicates that increasing the value of the first item boosted performance at SP1, had no significant effect on performance at SP2 and SP3, and negatively affected performance at SP4.To investigate the interaction between probe value and SP, a series of paired sample t‐tests (corrected using Bonferroni–Holm) were also conducted to investigate the interaction between probe frequency and SP. Mean proportion correct as a function of probe frequency and SP is displayed in Table t(43) = 6.05, P < 0.001, BF10 > 1000, d = 0.91). The opposite pattern of results was observed at SP3 and SP4, with participants exhibiting significantly higher accuracy in the equal probe frequency condition (SP3: t(43) = –2.47, P = 0.035, BF10 = 2.45, d = –0.37; SP4: t(43) = –4.51, P < 0.001, BF10 = 450.47, d = –0.68). No effect of probe frequency emerged at SP2 (t(43) = –0.87, P = 0.39, BF01 = 4.35, d = –0.13). To summarize, this demonstrates that increasing the likelihood of the first item being assessed enhanced accuracy at SP1, had no significant effect at SP2, and reduced accuracy at SP3 and SP4.A series of paired sample F = 78.28, P < 0.001, MSE = 0.021, ηp2 = 0.65, ηG2 = 0.21; BF10 > 1000), with participants exhibiting higher accuracy in the differential probe value condition. There was also a significant main effect of probe frequency  = 36.57, P < 0.001, MSE = 0.029, ηp2 = 0.46, ηG2 = 0.15; BF10 > 1000), with participants exhibiting higher accuracy in the differential probe frequency condition. No significant interaction emerged between probe value and probe frequency  = 0.17, P = 0.69, MSE = 0.015, ηp2 < 0.01, ηG2 < 0.01; BF01 = 4.22), suggesting that probe value boosts do not differ depending on probe frequency and that the manipulations have independent effects. BF analysis supported these conclusions, with the most likely model including main effects of probe value and probe frequency (BF10 > 1000 relative to the null model with random effects of participant only).As both manipulations were targeted at SP1, further analysis was conducted at this SP to explore whether an interaction emerged between probe value and probe frequency. A 2  × 2 (Probe frequency) within‐subjects ANOVA revealed a significant main effect of probe value . This indicates that probe value effects are not affected by the frequency with which the more valuable item is assessed at retrieval. Perhaps more importantly, this indicates that probe value and probe frequency are independent in their impacts on WM, and that they might encourage individuals to direct attention in different ways. Evidence for the latter is preliminary, however, warranting additional research to explore this possibility further.Previous research has suggested that probe value effects are reduced if participants engage in an attentionally demanding concurrent task during encoding and maintenance.et al.Hu To the best of our knowledge, research to date has not explored whether probe frequency boosts in WM also rely on executive control. However, a series of studies have demonstrated that individuals reliably encode frequency information during memory taskset al.To allow comparisons with the probe value literature, the methodology used was similar to that used by Hu M = 22.11, SD = 3.58; 10 males). Participants were either paid or given course credit. The experiment was approved by the School of Psychology Ethics Committee at the University of Leeds (UK).Twenty‐four young adults participated , load  and SP (1–4). Participants completed four blocks of 40 trials; one for each combination of probe frequency and load. The order of probe frequency blocks and the order of load blocks within the probe frequency conditions were counterbalanced. The SPs tested were randomly distributed within the blocks.et al.Participants were told that correctly recalling the color of the shape tested would gain them one point. The probe frequency instructions were the same as in experiment 1. The experimental paradigm was also identical to experiment 1, except that participants were presented with a number between 20 and 99 at the start of the trial as opposed to the word “la.” In the low load conditions, participants were asked to repeat the number until retrieval. In the high load conditions, participants were asked to count upwards in steps of two from the number until the retrieval phase . This load manipulation is identical to that employed by Hu The dependent variable was accuracy, determined by mean proportion correct. Findings are first reported across SPs, followed by further planned analysis at SP1.M = 0.48, SE = 0.03; Equal M = 0.50, SE = 0.02; F = 0.39, P = 0.54, MSE = 0.056, ηp2 = 0.016, ηG2 < 0.01; BF01 = 7.25), indicating that increasing the likelihood of the first item being assessed did not affect overall performance on the task. There was, however, a main effect of load  = 77.86, P < 0.001, MSE = 0.023, ηp2 = 0.77, ηG2 = 0.081; BF10 > 1000), with higher accuracy in the low load condition  relative to the high load condition . There was also a significant main effect of SP  = 10.48, P < 0.001, MSE = 0.081, ηp2 = 0.31, ηG2 = 0.11; BF10 > 1000), with pairwise comparisons (corrected using Bonferroni–Holm) revealing significant differences at SP1  and SP2 , SP2 and SP4  and SP3  and SP4 (P < 0.001). A significant interaction emerged between probe frequency and SP  = 17.79, P < 0.001, MSE = 0.052, ηp2 = 0.44, ηG2 = 0.12; BF10 > 1000), indicating that the effects of probe frequency differed depending on the SP tested. In contrast, there was no interaction between load and SP  = 1.31, P = 0.28, MSE = 0.037, ηp2 = 0.054, ηG2 < 0.01; BF01 = 10.96). There were also no interactions between probe frequency and load  < 0.01, P = 0.96, MSE = 0.034, ηp2 < 0.01, ηG2 < 0.01; BF01 = 6.51), and probe frequency, load and SP  = 0.27, P = 0.85, MSE = 0.033, ηp2 = 0.012, ηG2 < 0.01; BF01 = 14.2). These findings were corroborated by BF analysis, which revealed that the most likely model included main effects of load and SP, as well as an interaction between probe frequency and SP (BF10 > 1000 relative to the null model with random effects of participant only).Mean proportion correct as a function of probe frequency, SP, and load is displayed in Figure t‐tests (corrected using Bonferroni–Holm) were conducted. Accuracy as a function of probe frequency and SP is displayed in Table t(23) = 6.63, P < 0.001, BF10 > 1000, d = 1.35). This pattern was reversed at SP3 (t(23) = –2.78, P = 0.021, BF10 = 4.63, d = –0.57) and SP4 (t(23) = –3.65, P = 0.004, BF10 = 27.09, d = –0.74), with participants exhibiting higher accuracy in the equal probe frequency condition. No significant effect of probe frequency emerged at SP2 (t(23) = 0.76, P = 0.46, BF01 = 3.57, d = 0.15). To summarize, these outcomes indicate that increasing the likelihood of the first item being tested enhanced memory at SP1, had no significant effect at SP2, and impaired memory at SP3 and SP4.To investigate the interaction between probe frequency and SP, a series of F = 43.89, P < 0.001, MSE = 0.03, ηp2 = 0.66, ηG2 = 0.29; BF10 > 1000), with participants exhibiting higher accuracy in the differential probe frequency condition relative to the equal probe frequency condition. There was also a main effect of load  = 18.66, P < 0.001, MSE = 0.023, ηp2 = 0.45, ηG2 = 0.12; BF10 = 435.73), with participant exhibiting higher accuracy in the low load condition. There was, however, no significant interaction between probe frequency and load  = 0.24, P = 0.63, MSE = 0.016, ηp2 = 0.01, ηG2 < 0.01; BF01 = 3.33), suggesting that the probe frequency boosts observed were not affected by load. BF analysis revealed that the most likely model included main effects of probe frequency and load (BF10 > 1000 relative to the null model with random effects of participant only).As in experiment 1, further planned analysis was conducted at SP1 to explore whether an interaction emerged between probe frequency and load. A 2 (Probe frequency) × 2 (Load) within‐subjects ANOVA revealed a main effect of probe frequency . This replicates the outcomes from experiment 1, further demonstrating that the direction of attention can negatively affect items that are not focused on.A pair of experiments explored how attention can be directed in visual WM. Of particular interest was whether probe value boosts are affected by the probability with which the more valuable item is tested at retrieval  or whether these manipulations yield independent effects. In experiment 1, probe value and probe frequency boosts were observed, although the two effects were additive. This demonstrates that probe value boosts are not affected by probe frequency and that the manipulations have independent effects on performance. This latter finding was further supported by experiment 2, which indicated that probe frequency boosts are not reliant on executive resources during encoding and maintenance, unlike probe value effects.Such findings might be taken as evidence that the manipulations involve distinct underlying mechanisms. But how might the boosts emerge? Probe value effects are thought to reflect the more valuable item being retained in the FoA for longer periods of time or more frequently relative to less valuable items.Alternatively, the probe value and probe frequency manipulations may involve the same underlying mechanism. Experiment 1 revealed additive effects when probe value and probe frequency were employed together, which we interpret as indicating the operation of independent underlying mechanisms. However, these outcomes might be expected if the manipulations involve the same mechanism, but neither fully saturate it. Instead of involving distinct mechanisms, the probe value and probe frequency manipulations might therefore activate the same mechanism but in different ways. Experiment 2 would then indicate that activation of this mechanism is somewhat automatic when probe frequency is increased, but under more strategic control when probe value is manipulated. To delineate between these possibilities, it would be useful for additional research to further explore how the probe value and probe frequency manipulations differ and the cognitive mechanisms involved in both.Regardless of the outcomes of this further research, the current findings have important implications for the relationship between WM and attention, suggesting that not all forms of attentional direction are functionally equivalent. Researchers should therefore avoid assumptions that different attentional manipulations encourage participants to direct attention in the same way, as this could result in inaccurate or erroneous conclusions. These findings may also have important practical implications, indicating that individuals can direct attention to more important information in WM if they have prior knowledge regarding value or goal relevance. This might be particularly useful for everyday tasks that rely on WM, such as learning,In summary, these experiments suggest that the manipulation of probe value and probe frequency encourages participants to direct attention in different ways. Although probe value effects appear to depend on executive resources during encoding and maintenance,The authors declare no competing interests.


Saccharomyces cerevisiae ASNase II coded by the ASP3 gene was biosynthesized by recombinant Pichia pastoris MUTs under the control of the AOX1 promoter, using different cultivation strategies. In particular, we applied multistage fed-batch cultivation divided in four distinct phases to produce ASNase II and determine the fermentation parameters, namely specific growth rate, biomass yield, and enzyme activity. Cultivation of recombinant P. pastoris under favorable conditions in a modified defined medium ensured a dry biomass concentration of 31 gdcw.L−1 during glycerol batch phase, corresponding to a biomass yield of 0.77 gdcw.g−1. After 12 h of glycerol feeding under limiting conditions, cell concentration achieved 65 gdcw.L−1 while ethanol concentration was very low. During the phase of methanol induction, biomass concentration achieved 91 gdcw.L−1, periplasmic specific enzyme activity 37.1 U.g−1, overall enzyme volumetric productivity 31 U.L−1.h−1, while the specific growth rate fell to 0.039 h−1. Our results showed that the best strategy employed for the ASNase II production was using glycerol fed-batch phase with pseudo exponential feeding plus induction with continuous methanol feeding.L-Asparaginase (ASNase) is used in the treatment of acute lymphoblastic leukemia, being produced and commercialized only from bacterial sources. Alternative  Escherichia coli or unmodified from E. coli and Erwinia chrysanthemi have been approved for ALL treatment  is the enzyme that catalyzes the conversion of L-asparagine to aspartic acid and ammonia. It is used as a fundamental drug in protocols for the treatment of acute lymphoblastic leukemia (ALL). Up to now, only bacterial enzymes either modified (PEGylated) from l Jones, . AlthougPichia pastoris has been widely used for the expression of more than 1,000 heterologous proteins  promoter in high cell density cultures is often carried out according to a multi-stage fermentation protocol  was first cloned into the pET22b vector  for C-terminal histidine tail insertion. This construct was used as template to amplify the insert ASP3_26-362 + his using the following primers: forward AGCGGGCCTAGGGAAGAGAAGAATTCTTC by inserting the restriction site Avr II and reverse AAGGAAAAAAGCGGCCGCGGATCTCAGTG by inserting the NotI site. This insert was then digested with the Avr II and Not I enzymes and cloned into the pPIC9K vector  at these restriction sites. The constructs were confirmed by sequencing. The correct clone was linearized with the Sal I restriction enzyme and transformed into the P. pastoris strain KM71 (arg4 his4 AOX1::ARG4) (Invitrogen) by electroporation. Transformants were selected for geneticin resistance (G418 at concentrations increasing from 0.25 to 4 mg/mL). The clone with resistance to 4 mg/mL had its genomic DNA extracted with PureLink® Genomic DNA Mini kit . Integration of the pPIC9K + ASP3_26-362 + his construct was confirmed by PCR using the primers flanking the insertion site in the genome at the AOX locus (forward GACTGGTTCCAATTGACAAGC and reverse GCAAATGGCATTCTGACATCC). For more details about the steps of plasmid construction, gene sequence, and vector diagram, please see the The −1 yeast extract, 20 g.L−1 peptone, 100 mM potassium phosphate, pH 6.0, 3.4 g.L−1 yeast nitrogen base medium, 10 g.L−1 (NH4)2SO4, 4 mg.L−1 biotin, and 10 g.L−1 glycerol]. The culture was incubated in an orbital shaker  at 30°C and 250 rpm until an optical density >30 was reached. Cells were then stored at −80°C in BMGY enriched with 20% (v/v) glycerol in 1.5-mL microtubes. To prepare the pre-inoculum, cells were thawed and inoculated in a 250-mL Erlenmeyer flask containing 50 mL of BMGY medium under the same conditions as above.To produce a working cell bank, the yeast strain was reactivated in yeast peptone dextrose (YPD) agar under incubation for 24 h at 30°C. The activated cells were transferred to a 250-mL Erlenmeyer flask containing 100 mL of buffered glycerol complex medium (BMGY) [composition: 10 g.LBiomass specific growth rate (μ) was estimated from the experimental data of biomass concentration by the equation  on consumed substrate was defined as:The yield of biomass  or modified salt fermentation medium (SFMm)] were incubated as above until glycerol depletion. A 50-mL aliquot of fermented broth was centrifuged at 3,320 g and 4°C for 10 min and resuspended in 250-mL Erlenmeyer flasks containing 50 mL of buffered methanol complex medium (BMMY) having the same composition as BMGY except for 30 mL.L−1 methanol instead of 10 g.L−1 glycerol. The ASNase II induction phase was carried out at 20°C and 250 rpm on the same medium as that used for the growth phase, by two pulse additions of 30 mL.L−1 of pure methanol at the start and after 24 h. The induction phase was ended after 48 h.Cultures were carried out using a previously-prepared pre-inoculum where the frozen suspension was reactivated. After inoculation with a dry cell weight (dcw) concentration of about 1 g−1 of 85% (v/v) H3PO4, 0.93 g.L−1 CaSO4, 18.2 g.L−1 K2SO4, 14.9 g.L−1 MgSO2O, 4.13 g.L−1 KOH, 13–20 g.L−1 (NH4)2SO4, and 4.35 mL.L−1Pichia trace metal solution (PTM) , while SFMm contained 12 g.L−1 KH2PO4, 4.7 g.L−1 MgSO2O, 0.36 g.L−1 CaCl2O, 13–20 g.L−1 (NH4)2SO4, and 4.35 ml.L−1 PTM (pH adjusted to 5.0 with NaOH).BSMm contained 26.7 mL.Lx1), methanol concentration (x2) and induction time (x3) were selected as the independent variables, while periplasmic specific ASNase II activity (U.g−1) and biomass concentration (gdcw.L−1) as the responses  for x2, and 48, 72, and 96 h for x3, giving a total of 11 runs including two repetitions of the central point to estimate the pure error. The results were analyzed by means of Statistica software, version 10 .The induction phase was investigated through experiments carried out according to a Box–Behnken design  by means of a moisture analyzer, model MOC63u unibloc . The volume of cell suspension ensuring an initial biomass concentration of 1 gdcw.L−1 was centrifuged at 3,320 g, and the pellet resuspended in BSMm in a 3 L bioreactor, model Bioflo 115 .The inoculum was prepared by growing a stock culture of recombinant −1 glycerol and 13 g.L−1 (NH4)2SO4. The bioreactor was operated at 30°C during cell growth and starvation phases, and pH was monitored and controlled automatically at 5.0 with NaOH. During the induction phase, temperature was reduced at 20°C, and medium pH was maintained at 6.0.Cultures were performed on BSMm plus 40 g.LPichia pastoris in Shake Flasks, 33−1 Fractional Factorial Design for the Induction Phase in Shake Flasks, and Pichia pastoris Cultivation in Bioreactor were selected based on the work of Ferrara et al.  on cell growth and ethanol production, three different kLa values, selected on previous screening of conditions (results not shown), were tested in bioreactor, namely 84 h−1 ; 115 h−1 ; and 160 h−1 , in batch mode until glycerol depletion.To evaluate the influence of oxygen mass transfer coefficient  vs. time (h) for different aeration rates and agitation conditions, being C the dissolved oxygen concentration at time t and CS that at saturation (mg.L−1).od Wise,  by bubbl−1.h−1 plus PTM solution at 15 mLPTM.L4)2SO4 at 0.325 g(NH4)2SO4.gkLa = 160 h−1.The bioreactor was operated in batch mode for 20 h with an initial medium volume of 1.5 L, after which fed-batch culture was performed with glycerol as carbon source for further biomass growth. During 50% (w/v) glycerol fed-batch phase, the specific feeding flow rate was varied in the range 5.6–12.6 mL.Lrglycerol, gglycerol.L−1.h−1) was calculated from the bioreactor mass balance by the equation , t the time (h), tf the time when starting the feed (h), and μset the specific growth rate set for the fed-batch phase (h−1) defined as in Equation (1).where YFi, L.h−1) after the time interval Δt (h) was calculated as:To ensure an exponential feeding profile during the growth phase, the volumetric feeding rate (where:Vi and Vi−1 the volumes of medium in the bioreactor (L) after and before Δt, Cglycerol the glycerol concentration in the feed solution (g.L−1), Fi−1 the volumetric feeding rate of glycerol solution before Δt, and rglycerol, i the glycerol volumetric consumption rate after Δt (gglycerol.L−1.h−1).being −1 plus 15 mLPTM.LkLa = 160 h−1.After a 1–2 h starvation period following the above high cell density culture, methanol fed-batch phase was performed feeding a 100% methanol solution at specific flow rates in the range 3.75–5.25 mL.hBiomass dry weight was quantified by measuring the optical density at 600 nm using a microplate reader, model Spectramax® plus 384 . The weight was measured again, and the dry weight was calculated as the difference between the final and initial values.600) vs. dry cell weight concentration (X), expressed in gdcw.L−1:Cell concentration in the samples was then calculated according to the following standard curve of optical density at 600 nm . Centrifuged samples (10 μL) were mixed with 1.0 mL of triglycerides assay kit and incubated at 37°C for 10 min. The optical density was read with the above microplate reader at 505 nm using distilled water as a blank. To prepare a standard curve, dilute solutions of pure glycerol  in the defined BSMm medium were used.−1, and n-propanol used as internal standard. Injections for desorption of standards from needle trap device were made by splitless mode for 3 min at injection port temperature of 280°C, then the needle trap device was removed from the injector, and the system was switched into the split mode. The column oven temperature was held at 40°C for 5 min, ramped to 150°C at the rate of 10°C.min−1 and held at the final temperature for 5 min.Ethanol and methanol concentrations were determined by a gas chromatograph, model 6890N , equipped with a fused-silica capillary column Poraplot Q (10 m × 320 μm × 5 μm), and a split-splitless injector. The carrier gas was helium at a pressure of 60 kPa in the injection port. The detector temperature was maintained at 280°C. The gas flow rate was set at 0.8 mL.ming at 4°C for 10 min, and cells were resuspended in 0.9 mL of 20 mM Tris-HCl buffer (pH 6.8), 0.2 mL of 100 mM asparagine, and 0.2 mL of 1.0 M hydroxylamine (pH 7.0)  were added, and cell suspension centrifuged at 7,000 g for 10 min. Supernatant optical density was read at 500 nm with the same equipment as above  based on asparagine hydroxylaminolysis, i.e., conversion of asparagine into β-aspartohydroxamate and ammonia. Samples (1.0 mL) of fermentation cultures were centrifuged at 3,320 TM Standards .Samples of culture medium were assayed by SDS-PAGE in 12% polyacryamide gel (1.5 mm) to check the extracellular enzyme production and the purity degree  stock solution.Protein concentration in the culture medium was determined using the Bicinchoninic Acid (BCA) Protein Assay kit . Briefly, the protein sample (20 μL) was added to a 96 well plate containing 200 μL of the prepared BCA working reagent solution and incubated for 30 min at 37°C. Then, the optical density was read at 562 nm. Total protein concentration was obtained by a standard curve of several dilutions of a 1,000 mg.mLP. pastoris is usually performed in complex BMGY medium, whose composition, however, is not optimized; therefore, modifications have been proposed   and k is the number of steps to reduce the experimental design . However, in the presence of many factors, it is not advisable to adopt a complete design from the beginning, but it is rather preferable to start with a fractional design trying to identify the actually significant factors. Another advantage of using an experimental design is that it allows simultaneously studying more factors with fewer experiments compared with several parametric variations. Such a reduction of the set of experiments can be described mathematically as 3e flasks . Such a e flasks .To describe the behavior of the system, second-order polynomial models were used to correlate independent variables with responses according to the equation:Y is the response, β0 is the interception coefficient, βi are the linear terms, βii are the quadratic terms, βij are the interaction terms, and xi and xj are the coded levels of the independent variables.where p < 0.05). It can be seen that the positive quadratic effect of temperature was by far the strongest one on this response, followed by the negative quadratic effect of induction time, while all the others were not statistically significant or very weak.P. pastoris MUTs using methanol concentration below 3% (v/v) and lower temperatures (10–20°C).It is possible to observe in the response surface plot of −1) within the selected ranges of independent variables a temperature of 20°C, a methanol concentration of 3% (v/v) and an induction time of 48 h.On the other hand, consistently with the observations of the Pareto chart, albeit only a few significant, the induction time exerted a negative effect on the expression level, therefore induction times as shorter as possible (48 h) should be used to maximize it . TherefoP. pastoris in bioreactor are usually carried out on chemically-defined high-salt concentration broth consisting of a modified basal salt medium (BSMm) enriched with a Pichia trace metal (PTM) solution 2SO4 at concentration in the range 13–20 g.L−1, and the pH controlled by addition of NaOH  about 7% higher than that obtained in SFMm (0.143 h−1). Based on these considerations, the better production results obtained in the former medium suggested us to select it for bioreactor experiments.Cell growth profiles in shake flasks using such BSMm and modified salt fermentation medium (SFMm) are shown in S. cerevisiae ASNase II periplasmic activity was fully inactivated at very low pH values (below 3.2)  gave similar results to the others, suggesting no nitrogen limitation, it was selected for subsequent runs to decrease reagent costs.There was no ASNase II activity at the end of the induction phase because of strong acidity (pH ≤ 3.3) and low buffering capacity of both media. This result agrees with the observation that dcw.L−1 and a glycerol fed-batch phase (GFP) during which glycerol was fed in limiting level and cell density was >50 gdcw.L−1. As a result, fermentations in bioreactor were carried out in three steps, namely GBP, GFP, and ASNase II production during methanol fed-batch phase (MFP), with only a short glycerol starvation period before starting MFP in BSMm.To avoid a long oxygen limitation period in the bioreactor 2SO4 and 40 g.L−1 glycerol under kLa of 84, 110, and 160 h−1 and controlling the pH at 5.0 to favor salt solubility  (0.73 and 0.77 g.g−1) (kLa (84 h−1), despite the almost coincident Yx/s values, μ and cell productivity were 42–48% and 24–28% lower, respectively, due to reduction of the oxygen level. These μ values fall in the typical range for P. pastoris grown on glycerol as the sole carbon source  , while a Körner,  and are The yields obtained in bioreactor were appreciably higher when compared to those obtained in shake flasks on the same medium (results not shown).max.To achieve high cell densities, while simultaneously avoiding ethanol formation, GFP was carried out based on GBP growth and biomass yields. According to D'Anjou and Daugulis , ethanolglycerol.L−1.h−1 was calculated for our system by Equation (3) at kLa = 160 h−1, being μset = 0.072 h−1, and Yx/s = 0.77 g.gms) was < < μ during the exponential growth, μ and Yx/s were constant, no oxygen limitation occurred during GFP, sampling volume, water evaporation and biomass dilution effect were negligible because of a volume variation <10% 2SO4.g−1). Using the μ and Yx/s values from batch fermentations and assuming a dry cell concentration of 28 gdcw.L−1 and an initial volume of 1.5 L, the flow rate pattern summarized in To avoid excess ammonium inhibition  were substantially lower than that in the batch phase (0.219 h−1) just because glycerol was fed at growth-limiting level.dcw.L−1 at the start of constant feed and 65.8 gdcw.L−1 at the end of the 12 h period. On the other hand, the pseudo-exponential feed started after the batch period when cell density was 24.7 gdcw.L−1 and stopped after 12 h, reaching about the same cell concentration (68.3 gdcw.L−1) as the constant feed. At the end of fermentations, ethanol level was very low (< 0.2 gethanol.L−1) and similar for the two feeding patterns.Cell concentration achieved 27.7 gP. pastoris using methanol as the sole carbon source at high cell density. To ensure glycerol and ethanol depletion, the culture was submitted to a 1–2 h starvation period between GFP and induction phase. The same agitation speed and aeration rate as in the other phases (700 rpm and 1.0 vvm) were adopted, feeding pure methanol plus 12 mLPTM.Lmethanol.LThe objective of this part of the work was to induce ASNase II production by recombinant −1 and lasted 20 h, GFP was performed with exponential feed rate for 12 h, and, after the starvation period, glycerol and ethanol were totally consumed, while cell concentration reached 70 gdcw.L−1.The profiles of cell growth, periplasmic specific ASNase II activity, and glycerol concentration in the medium during fermentation are illustrated in dcw.L−1.After 34 h of fermentation, i.e., at the end of glycerol starvation period, the culture was induced for 34 h. The growth rate was low during the induction phase, at the end ASNase II specific activity (25.4 U.g−1) in GFP.The use of methanol pulses was reported to be a simple and effective strategy to produce recombinant proteins , glycerol and ethanol were absent, and cell concentration reached 61.0 gdcw.L−1 before the induction phase.The profiles of cell, glycerol, and methanol concentrations as well as periplasmic specific ASNase II activity are illustrated in dcw.L−1. After the first methanol pulse, a 2-h adaptation period was observed during which methanol was not consumed. Multiple pulses led to significant variations of μ that ranged from only 0.006 h−1 in the first pulse to 0.039 h−1 in the last one, with an average value throughout the whole induction phase of 0.033 h−1. After every methanol pulses, dry biomass concentration slightly decreased, and dissolved oxygen level dropped to < 10%, confirming methanol toxicity and the high oxygen requirement of methanol metabolism , which confirms the important role of oxygen in proteins induction and of high cell density in gene expression  may have been due to an interaction of S. cerevisiae cell wall with some unidentified domain of ASNase II. Therefore, in the present case, it is possible that the enzyme secreted to the extracellular environment by P. pastoris may have undergone some modification to an inactive form. Likewise, Martínez et al.  under continuous methanol feeding was lower than that observed with pulses, cell concentration at the end of the 73-h induction period almost the same (89.4 gdcw.L−1), and specific ASNase II activity  more than one order of magnitude higher than that (265 U.L−1) reported by Ferrara et al.  methanol pulses every 24 h for a 48 h period in shake culture. In BSMm medium, biomass-to-glycerol yield was about 53% higher than in defined medium.kLa conditions, the specific growth rate was 0.21 h−1 and biomass yield 0.77 g.gdcw.L−1, respectively), although biomass yields (0.55 and 0.51 g.g−1). The adoption of a starvation period before methanol feeding avoided any AOX repression due to the presence of ethanol or residual glycerol from glycerol fed-batch phase. The use of continuous methanol feeding for ASNase II induction allowed optimal control of methanol concentration in the medium, avoiding any oxygen limitation. Under these conditions, ASNase II was expressed at the highest levels. The results of this work demonstrate that not only methanol concentration but also oxygen availability in the induction phase are crucial for high recombinant protein production under the AOX promoter regulation.In glycerol batch fermentations under optimum P. pastoris system for ASNase expression. AC, AL, GM, LF, and AP conceived and designed the study, interpreted the results, and drafted the manuscript. All authors revised and approved the final manuscript.DR, OP-P, KT-O, JF-S, and IS-M performed bioreactor and flask fermentations and analysis in BMGY, BSMm, and SFMm media, acquired the data, performed data calculations, standardization and assay of periplasmic specific ASNase II activity, interpreted the results and drafted the initial manuscript. MP and TB designed and constructed the recombinant 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.


To determine the agreement of measurements between color light-emitting diode corneal topography (Cassini) and Scheimpflug camera keratometry (Pentacam HR).The current retrospective study investigated 117 right eyes of 117 healthy patients before cataract surgery from June 2017 to July 2017. Steep K, flat K, mean K, astigmatism, and axis for both anterior and posterior corneal surface were measured using the two devices. The measured values were converted into J vectors such as J0 and J45. The mean difference for those measurement values were compared between the two instruments, and the agreement was evaluated using the Bland-Altman plot I.P = 0.004]) and astigmatism (0.90D [0.58 to 1.30] and 0.70D [0.40 to 1.30] by Cassini and Pentacam [P = 0.002]) on the anterior corneal surface and flat K (− 6.21D [− 6.39 to − 6.07] and − 6.30D [− 6.5 to − 6.10] by Cassini and Pentacam [P < 0.001]), mean K (− 6.39D [− 6.54 to − 6.25] and − 6.40D [− 6.60 to − 6.30] by Cassini and Pentacam [P = 0.019]), and astigmatism (0.33D [0.22 to 0.47] and 0.30D [0.15 to 0.40] by Cassini and Pentacam [P = 0.002]) on the posterior corneal surface. The mean difference (= Cassini – Pentacam) with 95% limit of agreement for mean K and astigmatism of the anterior corneal surface were 0.082D (− 0.60 to 0.76) and 0.11D (− 0.73 to 0.95) for measurements obtained by the two instruments, respectively. Regarding keratometric values from the posterior corneal surface, the mean differences for flat K, mean K, and astigmatism were − 0.081D (− 0.42 to 0.26), − 0.030D (− 0.32 to 0.26), and 0.067D (− 0.33 to 0.46), respectively. Intraclass correlation coefficients for steep K, flat K, mean K, and vector J0 were higher than 0.9 in the anterior cornea. Positive correlation in steep K, flat K, mean K, astigmatism, and J0 was found between two devices in both anterior and posterior cornea (P < 0.001).There were statistically significant differences in mean K (44.21D [43.34 to 45.34] and 44.30D [43.30 to 45.10] by Cassini and Pentacam [Corneal refractive power and astigmatism tend to be higher when measured using Cassini than Pentacam HR in both anterior and posterior cornea. The two different devices might not be used interchangeably.RESI0439KC17.Retrospectively registered. Registration number:  In cataract surgery, refractive power of the cornea plays an important role in the calculation of power in artificial intraocular lens (IOL) insertion. Accurate measurement of the corneal refractive power and astigmatism are crucial factors in intraoperative correction of astigmatism as well as for improvement of the patient’s vision and satisfaction with treatment , 2. BothAmong devices used for anterior segment biometry, Pentacam HR , a type of rotational Scheimpflug camera, and the recently developed Cassini  –7. The cThis study aims to comparatively analyze the refractive power, astigmatism, and axis of the anterior and posterior cornea and assess whether the two devices can be used interchangeably.RESI0439). Eyes with preoperative pathologies that could affect corneal astigmatism, such as pterygium, severe corneal turbidity, corneal pathology due to severe dry eye, and diseases of the eyelid, orbit, and conjunctiva were excluded from the analysis.A retrospective study was conducted on 117 eyes of 117 patients undergoing normal cataract surgery in our center’s Ophthalmology Department from June 7, 2017 to July 27, 2017. The stage of cataract for them ranged from 1 to 4 by Pentacam nucleus staging system. Right eye was selected from each subject. The study was conducted in adherence to the Declaration of Helsinki as well as under the approval of the Medical Research Ethics Board of the Catholic University of Korea  and Scheimpflug camera keratometry  in that order, at an interval of under 10 min. Keratometry of their eyes was performed before applying any kind of eye drops. We performed the procedure after guiding the subject to fix their chin and forehead on the equipment’s test bed and to observe the internal fixation target in the test equipment while sitting in front of the test equipment. In order to minimize errors in measurement due to changes in the tear layer, patients were encouraged to blink sufficiently before each measurement. The same investigator took three measurements, and the average of the three values was used in subsequent analyses to compare each test result. Both Cassini and Pentacam HR provided qualitative information, and only those with “OK” or “measurement successful” results were included in the analysis.Cassini reconstructs the specular reflections from 679 color-LEDs by a point-to-point method, thereby instantly analyzing and measuring the shapes of the anterior and posterior cornea, the corneal curvature, lower-order aberration, and higher-order aberration , 10. TheCassini and Pentacam HR were both used to measure the steep K, flat K, mean K, astigmatism, and axis of the anterior and posterior sides of a central 3-mm corneal area (simulated keratometry). J0 and J45, which are Jackson cross-cylinder (JCC) values, were used for the analysis of astigmatism through power vector analysis . The magP values of less than 0.05 were deemed statistically significant.SPSS software  and R software  were used for statistical analyses. The paired t-test was used to compare the average measured values between the two devices. Intraclass correlation coefficient (ICC) was calculated to evaluate the degree of agreement between the two devices. Pearson correlation was used to obtain the correlation coefficient between the measurement methods and the scatter plots were produced using the LOWESS curve. The agreement between the values measured using the two devices was analyzed by the Bland-Altman plot and is expressed as 95% limits of agreement. Mean patient age was 65.1 ± 11.5 years (range 22–93). Thirty-four subjects (29%) were men, and 57 eyes (49%) were right eyes. The median astigmatism values for both anterior, posterior, and total cornea were larger in Cassini than in Pentacam HR (Table P = 0.057) for steep K, 0.01D  for flat K, 0.08D  for mean K, 3.88°  for axis, − 0.04D  for vector J0, 0.00D  for J45, and 0.11D  for astigmatism (Table P = 0.791) for steep K, 0.08D  for flat K, 0.03D  for mean K, 4.25°  for axis, − 0.04D  for vector J0, − 0.01D  for J45, and 0.07D  for astigmatism. In the posterior cornea, flat K, mean K, vector J0, and astigmatism showed statistically significant differences.The difference (= Cassini - Pentacam HR) in average values of the anterior cornea was 0.08D ; however, in the posterior cornea, J45 showed no significant correlation . In the present study, the average values measured in the anterior cornea by the Cassini and Pentacam HR was 44.34 ± 1.35D and 44.25 ± 1.34D, respectively and in the posterior cornea was 6.43 ± 0.23D and 6.43 ± 0.24D for mean K. The average corneal curvature and the average astigmatism size measured by the two devices showed small differences of maximum 0.43D and 0.54D in the anterior cornea and maximum 0.18D and 0.27D in the posterior cornea.Comparison of measurements using Cassini and Pentacam HR indicated significant differences in the average refractive power of the cornea and astigmatism in both the anterior and the posterior cornea. Pentacam HR reconstructs the images of the anterior segment into a 3-dimensional structure and allows for the visualization of the anterior and posterior cornea, as well as measurement of astigmatism in certain areas of the cornea. In a study on 493 eyes, Ho et al.  reportedThe two devices may not be used interchangeably. In the present study, the results of Pearson correlation analysis showed high correlation between the corneal refractive powers of the anterior and posterior cornea ; however, based on the 95% agreement limit of − 0.60~0.76D and − 0.26~0.32D in the anterior and posterior cornea, respectively. This might be caused that the two devices have different methods of measuring the corneal curvature and the accuracy of measurement or the calibration of each device affects the values. Though both devices measure the same 3-mm central corneal area, Pentacam HR requires a longer duration of time compared with Cassini; therefore, factors involved in patient cooperation, such as eye stability, compensatory saccadic movement, and consistent eye opening, would affect the test results.The limitation of this study is that measurement error may have occurred, since two investigators were involved in the measurement and in addition, inter-reader agreement could not be determined. Moreover, the study was conducted only on normal corneas, and difference due to the innate characteristics of each device was not considered. In the present retrospective study, we calculated the required sample size using G Power for a power of 0.8. The number of required samples was 128. However, the number of samples was as low as 117 people. Additional studies that compensate for the above limitation and those with repeated measurements are required for results that are more accurate.In conclusion, both two devices are useful in measuring keratometry, but the two devices might be non-interchangeable.