P < 0.05). Light microscope observation shows that HJJPD could control the degree of lipid denaturation of the mouse liver tissue to a great extent. RT-qPCR results show that the mRNA expression levels of peroxisome proliferative activated receptor gamma (PPARG), tumor necrosis factor-α (TNF-α), antiserine/threonine protein kinase 1 (AKT1), and prostaglandin-endoperoxide synthase (PTGS2) in the liver tissues of the three HJJPD groups  were significantly lower than those in the model group (P < 0.05). HJJPD can exert its effect by inhibiting hepatic steatosis and related mRNA expression and decreasing the levels of other liver-related indexes. This study suggested that HJJPD exerted its effect on NAFLD by modulating multitargets with multicompounds through multipathways. It also demonstrated that the network pharmacology-based approach might provide insights for understanding the interrelationship between complex diseases and interventions of HJJPD.This paper was designed to predict the mechanisms of the active components of Huaji Jianpi Decoction (HJJPD) against nonalcoholic fatty liver disease (NAFLD) based on network pharmacology-combined animal experiments. The candidate compounds of HJJPD and its relative targets were obtained from TCMSP and PharmMapper web server, and the intersection genes for NAFLD were discerned using OMIM, GeneCards, and DisGeNET. Then, the target protein-protein interaction (PPI) and component-target-pathway networks were constructed. Moreover, gene function annotation (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to study the potential signaling pathways associated with HJJPD's effect on NAFLD. Molecular docking simulation was preformed to validate the binding affinity between potential core components and key targets. Eventually, the candidate targets, the possible pathway, and the mechanism of HJJPD were predicted by the network pharmacology-based strategy, followed by experimental validation in the NAFLD mice model treated with HJJPD. A total of 55 candidate compounds and 36 corresponding genes were identified from HJJPD that are associated with activity against NAFLD, and then the network of them was constructed. Inflammatory response and lipid metabolism-related signaling pathways were identified as the critical signaling pathways mediating the therapeutic effect of the active bioactive ingredients on NAFLD. Compared with the model group, the liver wet weight, liver/body ratio, the levels of total cholesterol (TC), triglyceride (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and high-density lipoprotein (HDL) in serum in the HJJPD low-dose (17.52 g/kg·d), medium-dose (35.04 g/kg·d), and high-dose (70.07 g/kg·d) groups significantly decreased ( With the globalization of obesity and relative metabolic disorders, excess nutrition leads to the prevalence of nonalcoholic fatty liver disease (NAFLD) worldwide . There aTraditional Chinese medicine (TCM) has been widely applied for the regulation of the physiological function of the human body with the advantage of holistic concept and differentiation treatment , 8. ManyAtractylodes macrocephala (Baizhu), Astragalus memeranaceus (Fisch.) Bge. Var. Mongholicus (Bge.) Hsiao (Huangqi), Pinellia ternata (Thunb.) Breit. (Banxia), Citrus reticulata Blanco (Chenpi), Wolfiporia cocos (F. A. Wolf) Ryvarden & Gilb. (Fuling), Coix lacryma-jobi L. var mayuen (ROman.) Stapf (Yiyi Ren), Atractylodes Lancea (Thunb.) DC. (Cangzhu), Citrus aurantium L. (Zhishi), Alisma plantago-aquatica Linn. (Zexie), Ligusticum chuanxiong hort (Chuanxiong), and Nelumbo nucifera Gaertn (Heye) [G. uralensis, C. pinnatifida, and C. reticulata, as the anti-inflammatory and lipid-lowering drug, have remarkable effect in the treatment of liver diseases and NAFLD [C.pinnatifida was a common therapeutic drug that was added in the TCM prescription for the treatment of NAFLD and liver cirrhosis. Hence, HJJPD may be the superior therapeutic strategy for NAFLD according to TCM experience and scientific research. Presently, the molecular mechanisms underlying the efficacy of HJJPD remain uncertain. It is quite meaningful to further study the chemical composition and pharmacology of HJJPD for the treatment of NAFLD.Network pharmacology is a thriving interdisciplinary technology that is related to Chinese traditional medicine, chemical informatics, computer science, and bioinformatics, and it is an efficient and powerful tool for studying the bioactive ingredients and mechanism of Chinese traditional medicine. Huaji Jianpi Decoction (HJJPD), as a folk prescription, was derived from the famous TCM prescription of Zhizhu pill, Liujunzi Detection, and Jiaosanxian, composed of n (Heye) . As per n (Heye) . The maind NAFLD . EspeciaHJJPDs are composed of a variety of TCMs, and they are characterized by multiple components, targets, and pathways, which is of great difficulty for studying the chemical composition and mechanism of HJJPDs . Network80 SPF male C57BL/6N mice aged 3 weeks and weighing 20 ± 2.0 g were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.  and treated in accordance with the Guide for the Care and Use of Laboratory Animals. The experimental site is the third level scientific research laboratory of traditional Chinese medicine pharmacology, College of Traditional Chinese Medicine, Hebei University (No : TCM-09-315). All animal experiments were reviewed and approved by the Animal Care and Use Committee of Renmin Hospital of Hebei University (IACUC-2018043).Atractylodes macrocephala (Baizhu), Astragalus memeranaceus (Fisch.) Bge. Var. Mongholicus (Bge.) Hsiao (Huangqi), Pinellia ternata (Thunb.) Breit. (Banxia), Citrus reticulata Blanco (Chenpi), Wolfiporia cocos (F.A. Wolf) Ryvarden & Gilb. (Fuling), Coix lacryma-jobi L.var.mayuen (ROman.) Stapf (Yiyi Ren), Atractylodes Lancea (Thunb.) DC. (Cangzhu), Citrus aurantium L. (Zhishi), Alisma plantago-aquatica Linn. (Zexie), Ligusticum chuanxiong hort (Chuanxiong), and Nelumbo nucifera Gaertn (Heye) were obtained from Hebei Anguo medicine market  and identified by Professor Keli Chen of Hubei University of Chinese Medicine. All herbals met the requirements of the Chinese Pharmacopoeia (2020 edition), which have been preserved in the College of Traditional Chinese Medicine, Hebei University (No: TCM-09-315).Trizol reagent kit, total RNA extraction reagent, and DNA marker were purchased from Aidelai Biotechnology Co., Ltd. . HJJPD consisted of Baizhu (30 g), Huangqi (30 g), Banxia (10 g), Chenpi (10 g), Fuling (30 g), Yiyi Ren (30 g), Cangzhu (15 g), Zhishi (15 g), Zexie (10 g), Chuanxiong (10 g), and Heye (10 g). The above medicinal materials are fully mixed and then made into a powder sample using a plant powder extractor . The sample was extracted thrice using traditional Chinese medicine extractor  for 1 h. Following extraction, the samples were centrifuged at 5000 g for 15 min at 25°C using a type centrifuge  to obtain the extracts, which were concentrated using a vacuum rotary evaporator  at 40°C, and the above concentrated extracts were freeze-dried by vacuum freeze dryer , yielding 20.4 g of water extract.http://www.gene2newdrug.com/) and Traditional Chinese Medicine System Pharmacology database and Analysis Platform  on the basis of the rule of five . Meanwhile, the main compounds of herbs were also retained, which has been extensively studied by reported pieces of research [http://www.swissadme.ch/), and target proteins (probability > 0.1) were selected. The target genes for NAFLD were collected from Gene Cards database (https://www.genecards.org/).The active compounds of HJJPD and protein targets of the collected active compounds were obtained from Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine , and the collected target genes and active components were obtained for subsequent network analysis as visualized using Cytoscape 3.7.2 software. Cytoscape software  was applied to visualize the relationship between active compounds and diseases of NAFLD. The core active ingredients were identified in the treatment of NAFLD. The candidate genes were imported into the STRING database (https://string.db.org/) for the construction of PPI network, and the visual networks were established by Cytoscape software . The key genes were selected according to the degree value.The target genes related to NAFLD and active components of HJJPD were obtained by the online Venn map platform (http://metascape.org/gp/index.html). The bubble map of GO and KEGG enrichment results was established using R Studio. The visualization and integration of enrichment results were carried out using Enrichlot and ggplot2 R package. The above steps were completed by R software 3.6.2.The functional annotation of GO and KEGG pathways, including the relevant biological processes (BP), cellular components (CC), molecular functions (MF), and signal pathways of potential anti-NAFLD targets, were performed using the Metascape platform (∗mol format and target protein in ∗PDB format were obtained from software Chem office. According to Homo sapiens and refinement resolution (1.0∼2.0 Å), the target proteins in ∗PDB format were selected from the RSCB PDB database (https://www.rcsb.org/). Then, the ligands and water molecules in the target protein were removed using software Pymol 2.5, and then it was stored in ∗pdbqt format. Lastly, Autodock Vina software was used for molecular docking between active compounds and target proteins [A molecular docking simulation was conducted to assess the binding energy of the core compounds with the key targets. Autodock Vina 1.5.6 software developed by Olson's research group in Scripps Research Institute was adopted to assess molecular docking . Top 10 proteins , 17. TheThe mice in the normal and model groups  were fed normal and high-fat diet, respectively. After 10 weeks, 50 obese mice were randomly divided into 5 groups, including the model group, positive control group, and low-, medium-, and high-dose HJJPD-treated groups. The positive control group was given 0.005 g/mL obeticholic acid by gavage for 6 weeks, and normal saline was given to the control group. Low-, medium-, and high-dose HJJPD-treated groups were given 17.52 g/kg·d, 35.04 g/kg·d, and 70.07 g/kg·d of HJJPD, respectively. Meanwhile, the body weight was recorded at 0, 4, 8, 12, 16, 20, and 24 weeks.All blood samples were collected from the mice orbit after an overnight fast at 24 weeks, and they were centrifuged  to collect the supernatant for subsequent experiments. Mice were sacrificed and dissected. Then, the liver tissues were collected and measured for the liver index and subsequent histological analysis. The liver index (g/g) was calculated usingAspartate aminotransferase (AST), alanine aminotransferase (ALT), the total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were analyzed by HITACHI 7600 automatic biochemical analyzer .3 pieces on ice, fixed in 2.5% glutaraldehyde solution for 2 h, washed with 0.1 M PBS for 3 times, fixed with 1% osmium acid for 1 h, washed with 0.1 M PBS for 3 times, dehydrated with alcohol  and acetone gradient, and subjected to Epon812 penetration. Semithin sections (0.5–2 nm) were performed on the embedded mass of liver tissue, and toluidine blue staining was performed. Morphological changes of liver in mice were observed under the optical microscope.The liver tissues were dissected and taken, cut into 1 mmα, AKT1, and PTGS2) were analyzed by RT-qPCR. The RNA extraction of the liver tissue was performed according to the operation guidance in the kit instructions. The nucleic acid protein analyzer detects the quality and concentration of total RNA. The A260/A280 ratio is approximately 2.0, and the RNA content is calculated by test kit. 2.0 μg of total RNA was taken and cDNA was synthesized by reverse transcription  was applied for comparisons among three groups, and post hoc analyses were carried out with the Newman–Keuls multiple comparison test. Statistical significance was considered at P < 0.05 .All experiments were repeated at least three times, and the experimental results are shown as the mean ± SD. Student's b, poricoic acid a, alisol a monoacetate, kumatakenin, baicalin, etc. Table S1 shows the material basis of HJJPD. 4 compounds were overlapped across different herbs, including (+)-Eudesma-4 (15), 7 (11)-Dien-8-One in Baizhu and Cangzhu, 3-O-trans ferulylquinic acid in Chenpi and Chuanxiong, and Nobiletin, Tangeretin in Chenpi and Fuling. The top 10 compounds by the degree value  with chemical structures, molecular weight (Mw), the number of hydrogen bond donors, hydrogen bond acceptors, rotatable bonds, and lipid-water partition coefficient are as shown in HJJPD consists of Baizhu (30 g), Huangqi (30 g), Banxia (10 g), Chenpi (10 g), Fuling (30 g), Yiyi Ren (30 g), Cangzhu (15 g), Zhishi (15 g), Zexie (10 g), Chuanxiong (10 g), and Heye (10 g). HJJPD was extracted using traditional Chinese medicine extractor, and the yield of the extract was 17%. NAFLD, as the most common comorbidity, was the attractive research target for TCM. Baizhu, Fuling, and Huangqin had been reported as the potential TCM for improving NAFLD . AccordiNAFLD was searched as the key word in the Genecard database . ConsequP < 0.05). P < 0.05). The GO functional and KEGG enrichment analyses were performed to study the crucial biological processes of HJJPD in the treatment of NAFLD. As per the GO analysis results, 36 genes were enriched in 508 GO entries that consisted of 2401 biological progress (BP), 9 cellular components (CC), and 25 molecular functions (MF) (β (Table S3), PPARG, PTGS2, ESR1, LPL, PPARA, and HMGCR. These targets play an important role in the PPI network and KEGG signaling pathway, and they have a significant impact on glucose and lipid metabolism disorders and inflammation . Binding energy ≤ −5.0 kJ/mol was used as the basis for screening hypoglycemic activity. The results show that the binding energy of all active ingredients to protein was less than −5 kJ/mol, which further indicated that the active ingredients had strong binding activity to protein, expect Poricoic acide A [α, and the highest binding affinity (-11.2 kJ/mol) was observed in Atractylol III and AKT1 , indicating that HJJPD can inhibit the increase of liver weight to some extent [P < 0.05). In addition, the liver/body weight ratio of mice in the HJJPD treatment group was significantly lower than that in the model group (P < 0.05). Obesity is an important risk factor for NAFLD, and the effects of HJPD on mice body and liver weight are shown in e extent . The livBlood biochemical index can provide the information of various ions, sugars, lipids, proteins, enzymes, hormones, and various metabolites, which is conductive to the determination of treatment effect. α, AKT1, PPARG, and PTGS2) predicted by network pharmacology were estimated by RT-qPCR . Compared with the model group, the mRNA expression levels of PPARG, TNF-α, AKT1, and PTGS2 in the HJJPD-treated groups and the positive control group were significantly lower than those in the model group (P < 0.05). These results suggest that HJJPD has protective effects on NAFLD by inhibiting inflammatory factors, glucose, and lipid metabolism.To explore the effect of HJJPD on NAFLD, relevant signaling pathways . Molecular docking is an important method to realize structure-based drug design and screening by studying the interaction between ligand molecules and receptor molecules, predicting their affinity and binding mode [α, and the highest binding affinity (−11.2 kJ/mol) was observed in Atractylol III and AKT1  and top 10 target proteins . These results are consistent with those reported by Wang et al.  and Zhu α, AKT1, and PTGS2 for validation purposes. The experiment results show that HJJPD  could obviously inhibit the mRNA expression levels of PPARG, TNF-α, AKT1, and PTGS2 in the liver and adipose tissues, indicating that HJJPD can regulate the inflammatory response and lipid metabolism and reduce the role of hepatic steatosis [Subsequently, NAFLD mice induced by HDF are applied to further ascertain the function of HJJPD in the amelioration of NAFLD by the validation experiment. When the synthesis and secretion of TC in hepatocytes are damaged and cannot be balanced, lipids rapidly deposit in hepatocytes and cannot be excreted, leading to hepatocyte disease and nonalcoholic fatty liver . A largeteatosis . These eα, AKT1, and PTGS2 in the liver tissues. All in all, HJJPD could ameliorate HFD-induced NAFLD by regulating the inflammatory response and lipid metabolism and decreasing levels of liver-related indexes and the role of hepatic steatosis. This study provides a scientific basis for the further analysis of the clinical application of CCT in NAFLD. However, the further downstream mechanism of HJJPD treatment still needs to be investigated. Additionally, to develop more inexpensive and safer clinical applications, we need to analyze the active components of HJJPD and explore how HJJPD treats NAFLD using different signaling pathways related to inflammation and lipid metabolism.Taken together, the present study puts forward for the first time a comprehensive strategy that combines pharmacological experiments and network pharmacology methods to explore the material basis of HJJPD and its possible mechanisms of the active components of HJJPD against NAFLD. Through active ingredient screening, target prediction, PPI network construction, KEGG pathway, GO biological process analysis, and experimental verification, HJJPD protective mechanism for NAFLD was clarified. It has been demonstrated that inflammatory response- and lipid metabolism- related signaling pathways were identified as the critical signaling pathways mediating the therapeutic effect of the active bioactive ingredients on NAFLD. HJJPD  could significantly decrease the liver wet weight, liver/body ratio, and the levels of TC, TG, AST, ALT, and HDL in serum, and reduce the mRNA expression levels of PPARG, TNF-


Leishmania infantum in South American coatis inhabiting two forest fragments in Campo Grande, Mato Grosso do Sul, Midwest region of Brazil, an endemic area of human and canine visceral leishmaniasis (VL).The aim of the present study was to investigate the occurrence of Parque Estadual do Prosa” (PEP) and in the residential area “Vila da Base Aérea” (VBA) from March 2018 to April 2019. As a longitudinal study that include up to six recaptures of the same individual, a total of 190 capture events were obtained. Blood, bone marrow and skin samples were obtained for parasitological (axenic culture), serological  and molecular diagnostic assays .A total of 110 South American coatis were sampled in the conservation unit “L. infantum was found in 33 individuals, six in PEP and 27 in VBA. Furthermore, L. infantum was detected by molecular analysis in 16 individuals, seven from PEP and nine from VBA. We also isolated L. infantum from bone marrow of one individual and detected a single positive skin sample in molecular assay from other individual, both from VBA.Seropositivity for L. infantum in this and other potential L. infantum reservoirs that inhabit urbanized regions endemic to VL.An overall infection rate of 36.4% (40/110) was observed, significantly higher in the VBA (49.1%) than in the PEP (21.6%), probably because VBA presents: (i) a large number of resident dogs and chickens that would be attracting sandflies; (ii) a denser population of this wild mammal species; and (iii) physical barriers and a lack of functional connectivity in the surroundings, preventing these animals to disperse out. We conclude that South American coati populations living in urban forest fragments of Campo Grande are affected by the epidemiological scenario of VL, known to involve dogs, vectors and humans. We highlight the importance of investigate the parasitism by  Leishmania infantum is the etiological agent of visceral leishmaniasis (VL), one of the most important parasitic diseases worldwide. Brazil ranks the top five countries with the highest number of human cases and the highest fatality rate  to urban environments   (L. infantum reservoir host (L. infantum seems to be less competent to infect vectors than dogs (L. infantum in intact skin of a captive bush dog (Speothos venaticus) (Chrysocyon brachyurus) was also very low (Wild carnivore species have been reported parasitized by n thous) , althougvetulus) . Indeed,oir host , as atteoir host . Nevethehan dogs . Apart fnaticus) , althougnaticus) . The travery low . As thesvery low .Nasua nasua; hereafter “coati”) is a carnivore species well adapted to urban forest fragments in some regions of Brazil , the capital of Mato Grosso do Sul (MS) state, Midwest Brazil, is considered an endemic area for human and canine VL . In thistic cats  and wildtic cats . Moreoves region , indicatsmission . Since, Parque Estadual do Prosa” (PEP)  and “Vila da Base Aérea” (VBA) , located in the urban area of CG/MS  and riparian forest, and is located in the sanitary district of “Prosa”  that receives and keeps several wild animals from all over the MS state.The PEP is a conservation unit of 135 hectares composed of Lagoa”, is a complex of 197 hectares divided into a military operational area and a residential area surrounded by a dense vegetation that includes riparian forest and veredas (a plant formation with hydromorphic soil located in the vicinity of river springs) (Dasypus novemcinctus), Azara’s agouti (Dasyprocta azarae), Brazilian guinea pig (Cavia aperea) and howler-monkey (Alouatta caraya), and other species such as the coati and Capybara (Hydrochoerus hydrochaeris) are abundant.The VBA, located in the sanitary district of “®, Brazil) baited with 15 to 25 grams of bacon. All the captured coatis were sedated with an association of tiletamine hydrochloride and zolazepam hydrochloride , tagged with subcutaneous transponders  and numbered colored ear-tags . Blood samples were collected through femoral venipuncture using 0.25 x 0.8 mm needles (BD Vacutainer®) and deposited in clot activator tubes to obtain the serum. Bone marrow (BM) samples were collected from manubrium sterni of animals > 2.5 kg using 0.40 x 1.2 mm hypodermic needles and 10 mL syringes, and then placed in tubes containing Ethylenediamine Tetraacetic Acid (EDTA). These procedures were performed after appropriate asepsis of the collection site using bactericide soap, iodized ethanol and ethanol 70%. Ear skin fragments (0.4 to 30 mg) were obtained with sterilized material and stored in 99% ethanol . After total recovery from anesthesia, the animals were released at the capture site.The coatis were captured and randomly recaptured in box traps , the Instituto de Meio Ambiente de Mato Grosso do Sul , and Air force cooperation agreement (No.01/GAP-CG/2018). The present study was approved by the Ethics Committee for Animal Use of Universidade Católica Dom Bosco, Campo Grande, MS (license number 001/2017).The field procedures were conducted in accordance with the L. infantum IgG was performed through the Enzyme-Linked Immunosorbent Assay (ELISA)  , diluted at 1:70,000, and each microtiter polystyrene plate contained positive and negative control samples in duplicates. The cut-off point was established by the mean Optical Density (OD) of the negative control ± three standard deviations and the gray range adopted was 20% above the cut-off value. Serum samples were also submitted to a Dual Path Platform chromatographic immunoassay  for rapid detection of K26/K39 specific antibodies against L. infantum    (clioc.fiocruz.br) of Instituto Oswaldo Cruz and characterized by them using isoenzyme electrophoresis of G6PDH and 6PGDH  with Schneider’s insect medium overlay supplemented with 10% fetal bovine serum. The tubes were incubated at 26°C – 28°C and weekly examined for 30 days. After the exponential growth phase of the flagellates, the positive culture was cryopreserved at – 196°C under liquid nitrogen. The promastigote forms of the isolated culture were deposited in the Collection of nd 6PGDH .® Blood and Tissue Kit . The DNA was eluted in 50 µL elution buffer and stored at -20°C until molecular analysis. The concentration and quality were assessed through spectrophotometry .Genomic DNA was extracted from 200 µL of the BM samples through the phenol-chloroform method according to Leishmania spp. according to ®, United States) at ACTGene Molecular Analysis biotechnology company (https://actgene.com.br/). Negative samples at HSP70 PCR were submitted to L. infantum-specific kDNA PCR (MC1/MC2) according to ®, United States), using positive controls of L. infantum (LHV14 strain) and L. amazonensis (IFLA/BR/1967/PH8 strain) and negative controls (ultrapure water). The PCR products were visualized on 1.5% agarose , stained with Gel Red Nucleic Acid Stain .The BM and skin samples were firstly screened through Polymerase Chain Reaction (PCR) using a pair of primers (L1/L2) directed to the kinetoplastid DNA (kDNA) minicircle molecules of ® CVL), the Cohen’s Kappa test was performed with a 95% confidence interval and interpreted as follows: poor agreement (k = 0), slight agreement (0.20 ≤ k ≥ 0), fair agreement (0.40 ≤ k ≥ 0.21), moderate agreement (0.6 ≤ k ≥ 0:41), substantial agreement (0.80 ≤ k ≥ 0.61), and almost perfect agreement (1.0 ≤ k ≥ 0.81). To check a possible influence of the studied areas and sex on L. infantum infection, a Chi-squared test (p < 0.05) was applied considering the individuals. We considered infection by L. infantum as positivity in any of the parasitological or molecular tests and/or positivity in both serological assays. Both Cohen’s Kappa and Chi-squared tests were performed through the R software  in 80 occasions Figure 4L. infantum, indicating an overall seroprevalence of 30% (33/110). In PEP, the seroprevalence rate was 11.8% (6/51), corresponding to two males and four females, while in VBA the seroprevalence was estimated in 45.8% (27/59), corresponding to 11 males and 16 females. Despite the discordance observed in 19 samples (16 ELISA+/DPP- and three ELISA-/DPP+), the Cohen’s kappa test attested a substantial agreement between ELISA and DPP . Only one serum sample of a non-recaptured female coati from VBA, that was seropositive to L. infantum, presented seropositivity for T. cruzi, indicating a mixed infection.A total of 65 samples from 33 individuals were seropositive (ELISA+/DPP+) to L. infantum from one BM sample derived from a seropositive male captured in VBA (ID 11528)  Figure 5Leishmania sp. Posteriorly, L. infantum infection was confirmed in 16 individuals (nine by HSP70 sequencing and seven by MC1/MC2 PCR), seven from PEP  and nine from VBA . The results of parasitological and molecular assays performed in the coatis are presented in L. infantum in molecular tests, four were classified as seropositive and 15 were seronegative. Only one skin sample was positive in kDNA PCR and L. infantum infection was confirmed by MC1/MC2 PCR. The same individual  was seropositive and also positive in BM molecular assay (with L. infantum infection confirmed by HSP70 sequencing).The screening kDNA PCR in BM samples showed an infection rate of 41.2% (35/85) for L. donovani HSP70 gene obtained from samples of humans from Ethiopia and China (accession numbers: FN669773.1 and JX970996.1 respectively). The obtained sequences were deposited in the GenBank database under the following accession numbers: ON805836 to ON805844 . These 40 individuals correspond to 24 positive by serological tests , seven positive by molecular tests, and nine positive by both tests. We observed through Chi-squared test that infection was significantly higher in VBA  than in PEP  . Our analysis did not show influence of sex in L. infantum infection .Together, the parasitological, serology and molecular tests showed a L. infantum infection patterns , we observed distinct patterns Table 1.L. infantum, 19 individuals were recaptured during the study at least on one occasion. Eleven individuals remained positive by at least one diagnostic test throughout their recaptures . Our results demonstrate that this wild carnivore species is somehow affected by the L. infantum epidemiological scenario at the studied area. Indeed, (i) the endemicity of VL in humans and dogs at CG  abpis s.l. , and , indicating a spatial influence. This finding may be related to the geographical localization of the studied areas, as well as differences in land use. For instance, VBA is a residential area with many dogs, the main domestic reservoir host of L. infantum, and chickens, which although refractory to Leishmania infection, are amplifiers and attractors of sandfly populations  may be related to several factors including the absence or low abundance of competent reservoirs, a higher richness of mammal species than VBA, and the CRAS, that is located in this conserved area. Although the coatis have access to the adjacent areas n effect . In addiL. infantum in an ear skin fragment of an individual that was also seropositive and presented L. infantum DNA in BM sample (ID 10826), indicates that L. infantum is capable to colonize intact skin of coatis. In view of the importance for L. infantum transmission, domestic dogs are the main reservoir species mainly because they can present high parasitic loads in the skin, favoring the infection of the vectors , as it is more sensitive and highly specific for L. donovani s.l., improved the detectability of molecular diagnostics. Concerning the recaptures, molecular negativity observed in the first recaptures of the individuals 10320 and 10322, and in the second recapture of the individual 10323  that was seropositive in the capture and seronegative in the three subsequent recaptures  remained positive in molecular test in consecutive captures, indicating that individuals may be capable of sustain BM parasitism for at least five months. At this point, the remaining question is whether these animals are capable of sustain high parasite loads in the skin and act as source of infection to sand flies.In contrast, we found evidence of active transmission of Leishmania species besides L. infantum in the sampled coatis, since (i) 54.3% (19/35) of the infections detected by kDNA PCR were not confirmed by L. infantum-specific targets, and (ii) we found 16 samples ELISA positive and DPP negative that were also negative in the serological tests performed to detect other common trypanosomatids that infects wild mammals: T. cruzi. Geographical overlap among Leishmania species is reported  the high population densities of this mammal host in different urban forest fragments, (ii) their proximity to domiciliated areas and contact with human and dogs, (iii) the ability to maintain viable parasites in bone marrow, as demonstrated by L. infantum isolation and (iv) the possibility to maintain parasites in no lesioned ear skin, as demonstrated by L. infantum DNA detection. Given these findings, we highlight the importance to investigate the pattern of L. infantum infection in coatis inhabiting urban forest fragments to define their role as L. infantum reservoirs in endemic areas for VL.Coatis living in urban forested fragments at CG are affected by the epidemiological scenario of VL transmission that evolves dogs, humans and The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article.The animal study was reviewed and approved by Ethics Committee for Animal Use of Universidade Católica Dom Bosco, Campo Grande, MS (license number 001/2017).GCdeM, WTGB, ALRR and HMH wrote the manuscript. GCdeM, WTGB, ACR and WOdeA collected biological samples. SCdasCX, FMA and ARdaS contributed in diagnostic tests execution. FMS performed statistical analysis. CEdeO, GEdeOP, SCdasCX, FMA, GBdeA, AMJ, ALRR and HMH critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.


The role of germline genetic factors in determining survival from cutaneous melanoma (CM) is not well understood.To perform a genome-wide association study (GWAS) meta-analysis of melanoma-specific survival (MSS), and test whether a CM-susceptibility polygenic risk score (PRS) is associated with MSS.We conducted two Cox proportional-hazard GWAS of MSS using data from the Melanoma Institute Australia, a high ultraviolet (UV) radiation setting  and UK Biobank , and combined them in a fixed-effects meta-analysis. Significant (P < 5 × 10–8) results were investigated in the Leeds Melanoma Cohort . We also developed a CM-susceptibility PRS using a large independent GWAS meta-analysis . The PRS was tested for an association with MSS in the MIA and UKB cohorts.Two loci were significantly associated with MSS in the meta-analysis of MIA and UKB with lead SNPs rs41309643  on chromosome 1, and rs75682113  on chromosome 7. While neither SNP replicated in the LMC, rs75682113 was significantly associated in the combined discovery and replication sets. After adjusting for age at diagnosis, sex and the first ten principal components, a one standard deviation increase in the CM-susceptibility PRS was associated with improved MSS in the discovery meta-analysis . However, this was only driven by the high UV setting cohort .We found two loci potentially associated with MSS. Increased genetic susceptibility to develop CM is associated with improved MSS in a high UV setting.The online version contains supplementary material available at 10.1186/s12967-022-03613-2. Cutaneous melanoma (CM) is the third most common skin cancer and is responsible for over 1,300 deaths in Australia annually . While sCM-susceptibility is driven by host factors including skin pigmentation and number of naevi, as well as environmental factors, most importantly exposure to ultraviolet radiation –9. GermlAlthough there are well known prognostic factors for melanoma-specific survival (MSS) including primary tumour thickness, ulceration, mitotic rate, melanoma type, anatomical site and the stage of the tumour at diagnosis , 12, theA powerful approach to test whether germline genetic risk for a given disease or trait (e.g. risk for CM) influences another trait (e.g. MSS) is to combine individual genetic effects in a polygenic risk score (PRS). Death from all causes has been associated with the joint effect of PRSs associated with risk of a range of diseases  or associated with mortality risk factors  , suggestTo explore these two questions, we first aimed to identify germline genetic factors that influence MSS by performing a large-scale GWAS of MSS. Following this we assessed whether a PRS for CM-susceptibility (referred to as PRS_susceptibility) was associated with MSS.Samples for this cohort were derived from the Melanoma Institute Australia (MIA) Biospecimen Bank (protocol HREC/10/RPAH/530) and patient information from the MIA Research Database (protocol HREC/11/RPAH/444). With written, informed consent, patients with histo-pathological confirmed CM cases managed at MIA, Sydney, Australia were identified from this Biospecimen Bank and Database. Participants’ clinical and biospecimen data were captured and prospectively collected follow- up for outcomes including death due to melanoma. MIA study protocols were approved by the Sydney Local Health District Ethics Review Committee, Royal Prince Alfred Hospital, Camperdown, Australia. Participants were genotyped in phases using the Oncoarray in 2014 and 2016, and the Global Screening Array in 2018 .−10 for patients with melanoma or less than 5 × 10−4 in CM-free individuals in Landi et al., [–6, or a low GenTrain score (< 0.6) [Full details of the GWAS data cleaning quality control for both MIA datasets have been previously reported , 21. Bri (< 0.6) .For this study, the primary endpoint was death from melanoma which was ascertained through MIA clinical records and linkage to Australian Cancer Registries , electoral rolls, and the Birth and Death Register. This analysis was restricted to 5,672 participants of European ancestry diagnosed with CM. For participants with multiple CM, the first primary CM was used to define the start point. MSS survival time (in years) was defined as the duration between the date of diagnosis of the (first) primary CM, and the date of death due to melanoma. Patients were censored on the last day of follow-up or when they died of non-melanoma causes.UK Biobank (UKB) is a large population-based cohort of approximately 500,000 adult participants (40–70 years at recruitment) recruited with informed consent from the United Kingdom between 2006 and 2010. Participants were followed up for disease outcomes including death from melanoma. Details on participant recruitment, phenotype measurement and genotyping have been published elsewhere , 24. In The Leeds Melanoma Cohort (LMC) is a population-based cohort of 2,184 participants diagnosed with incident melanoma between September 2000 and December 2012 and residing in Yorkshire and the North of England . DetailsMelanoma diagnoses were clinico-histopathologically confirmed through data linkage with the Northern and Yorkshire Cancer Registry and Information Service. Samples were genotyped using the Infinium HumanOmniExpressExome array . After genetic quality control procedures , this present study was restricted to 1,947 participants with genetic and phenotype data, and consent. Ethical approval for research involving the LMC was obtained from the Northern and Yorkshire Research Ethics Committee, and all participants provided written informed consent.–4 or missingness > 0.03 were excluded, as were any individuals with call rates < 0.97, identified as first degree relatives and/or European outliers by principal components analysis. Samples were imputed using the Haplotype Reference Consortium panel at the University of Michigan imputation server [SNPs with MAF < 0.03, control Hardy–Weinberg equilibrium (HWE) P < 10n server  and varisurvival package [First, we conducted two GWAS of MSS in the MIA cohort (5762 patients with melanoma and 800 melanoma-specific deaths) and in UKB cohort (5220 melanoma patients and 241 melanoma-specific deaths). Using Cox proportional-hazard modelling, hazard ratios (HRs) were computed using PLINK 1.9  and the  package . In both2) were computed. Lead genome-wide significant (P < 5 × 10–8) SNPs independent at linkage disequilibrium (LD) r2 < 0.1 were identified using FUMA v1.3.6a (https://fuma.ctglab.nl/) [Next, we conducted a meta-analysis for both GWAS  using a fixed-effects inverse-variance weighted model in METAL . In addilab.nl/) .Lead SNPs were tested for replication in the LMC . The replication p-value threshold was set to 0.05. Next, we conducted a fixed- and random- effects inverse-variance meta-analysis of the lead SNPs from all three sets  using METAL . For theAs the three MSS GWAS cohorts contributed to the discovery CM-susceptibility GWAS meta-analysis , and oveWith the exception of the self-reported 23andMe, Inc. dataset, all CM cases were histopathologically confirmed; previous work has shown that 23andMe cases are very similar to the confirmed cases: the susceptibility loci show very similar effects in the self-reported and confirmed CM cases . Each stOnly SNPs with an imputation quality score > 0.5 were included, and a fixed-effects inverse variance weighted meta-analysis of log odds ratios (ORs) was performed using PLINK 1.9 . Next, w–6), a low GenTrain score (< 0.6), or a low MAF (< 0.01). Cleaned genotype data were imputed to the haplotype reference consortium (v1) panel using the University of Michigan imputation server [The QSkin Sun and Health Study (QSkin) cohort is a population-based cohort comprising over 43,000 adult participants recruited from Queensland, Australia. Detailed information on participant recruitment, phenotype measurement, genotyping and quality control measures have been published elsewhere , 33. In n server .The Human Research Ethics Committee of QIMR Berghofer Medical Research Institute, Brisbane, Australia approved the study protocol and all participants provided written informed consent. We selected 16,708 participants  of European ancestry. CM cases were ascertained through data linkage with the Queensland Cancer Registry as well as assessing histopathology reports from pathology laboratories in Queensland.We used the CM-susceptibility GWAS data (generated above) and an LD reference panel of 2,000 unrelated individuals of European ancestry from UKB, to generate 30 PRS_susceptibility models at 1 megabase (Mb), 2 Mb, 3 Mb, 4 Mb and 5 Mb of LD radii each with varying fractions of causal SNPs i.e. 1 (F0), 0.1 (F1), 0.01 (F2), 0.001 (F3), 0.0001 (F4), and 0.00002 (F5). For this analysis we used LDpred, a Bayesian method that utilises all SNPs in the discovery GWAS (here CM-susceptibility GWAS), and their LD information, to derive LD-adjusted effect estimates (log ORs) for the trait (here CM-susceptibility) .2 [Next, we used the QSkin validation cohort to select the optimally performing PRS. Next, for each model we computed scores for 16,708 individuals  in the QSkin Cohort using the LDpred-adjusted effect sizes (log ORs) and the imputed allelic dosages using PLINK 1.9 [2  to selec2 . Model psurvival package in R [The best performing PRS_susceptibility model was applied to the MIA and UKB cohorts using imputed allelic dosages and PLINK 1.9. The PRS was normalised to have a mean of 0 and an SD of 1 and tested for association with MSS in a Cox proportional hazard model adjusted for age at diagnosis, sex and the first ten PCs using the age in R . We furtage in R . We thenP), naevus count (PRSN) and telomere length (PRSTL) and tested whether they were associated with MSS . The MIA survival GWAS, CM risk GWAS meta-analysis, and LD panel  were filtered to a common set of SNPs, and then pruned to LD independence (LD r2 < 0.01) in plink v1.9b6.26, leaving 102,286 SNPs. As per Dudbridge et al., 2019, the log(HR) effect sizes from the MIA and UKB survival GWAS were regressed onto the log(OR) effect sizes from the CM risk GWAS meta-analysis, and SCIMEX (1000 simulations) was used to adjust the regression slope for regression dilution to calculate the correction factor, and its variance, which was used to adjust the survival GWAS results. The impact of index-bias on the association between polygenic risk for melanoma, and survival was estimated as done in Howe et al. [-8 and independent (LD r2 <  0.01) were selected, and the inverse-variance weighted method  was useThis analysis was restricted to 5,762 melanoma patients in the MIA cohort, 5,220 in the UKB cohort, and 1,947 in the LMC. Summary data on mean age at diagnosis, sex, duration of follow up and the number of melanoma-specific deaths are presented in Table –8) loci (Table –8) on chromosome 1 (1q42.13) and rs75682113 (P = 1.07 × 10–8) on chromosome 7 (7p14.1)  with high heterogeneity metrics  (formerly ADCK3) gene in blood. COQ8A is induced by p53 in response to DNA damage and inhibition of COQ8A counteracts p53-induced apoptosis [Succinyl-Coa:Glutarate-Coa Transferase (SUGCT) gene. This SNP has not been reported as an eQTL for any genes. Independent variants in the SUGCT gene have been associated with glutaric aciduria type 3 disease susceptibility [Rs41309643 on chromosome 1 is an intron of the poptosis . rs756822 of 7.02%  and a 5 Mb LD radius performed best, with a Nagelkerke’s R and a 5 –5). However, the association between the PRS_susceptibility and MSS was highly heterogeneous across the two studies , with the finding mainly driven by the high UV setting cohort . Although not statistically significant, the magnitude and direction for the random effects model was also consistent with the fixed-effects results . The inverse association between polygenic susceptibility to melanoma and MSS persisted after excluding genomic regions associated with naevus count  and pigmentation . The association between polygenic risk for melanoma and MSS was not replicated (P > 0.05) in the LMC; however, the directions of the effect estimates were consistent , even after excluding naevus and pigmentation loci . Sensitivity analyses showed that the skin colour PRS was also associated with improved MSS , while the naevus count PRS also provided suggestive evidence  . In addition, the association was consistent even after adjusting for age, sex, 10 PCs, AJCC 2010 Stage, and primary tumour thickness  . After excluding the first two years of follow-up (following diagnosis), there was no evidence of lead-time bias  . We applied the index-event bias proposed in Dudbridge et al. [In the MIA cohort the PRS_susceptibility remained associated with improved survival after excluding participants with melanoma 5) Table . There w5) Table . In a st5) Table a and pri5) Table b. The PRe et al.  to the M–8) loci for MSS at 1q42.13 and 7p14.1. While the two loci did not formally replicate in an independent cohort, the confidence intervals (particularly for rs75682113) in the replication set overlap the estimate from the discovery cohorts. Confirmation of these two loci will require replication in larger cohorts. rs75682113 is particularly promising as it was genome-wide significant (P < 5 × 10–8) in our meta-analysis of the discovery and replication samples.In this study, we performed the largest GWAS for MSS to date using data from Australia and the United Kingdom and potentially have identified two independent, novel, genome-wide significant  indicate that the effect sizes may not be consistent across the three studies, with a very strong result in the MIA cohort (a high UV setting) and weaker associations in the UK samples  and other effects. Firstly, the MIA and UKB meta-analysis results did not replicate in the LMC. Secondly, the high heterogeneity metrics e.g. I indicateAnalyses of outcomes such as survival necessitate the inclusion of people based on having the index disease, which can introduce an index event bias . This caIn a more detailed analysis in the MIA cohort, our study suggests that this inverse association is consistent even after further adjusting for (and testing for interaction with) strong predictors of MSS like tumour stage and primary tumour thickness at diagnosis. The stratified analysis shows that the association is not modified by primary tumour thickness or stage. Thus, if replicated in additional cohorts, a CM-susceptibility PRS is potentially an independent prognostic factor for MSS.To our knowledge, while no prior study has examined the association of a CM susceptibility PRS and survival outcome, similar inverse relationships have been found in other cancers e.g. higher breast cancer PRSs and better breast cancer prognosis/characteristics , 51. AlsThe mechanisms underlying this inverse association are unclear. Particularly for MSS, it could be that a higher genetic risk for CM leads to thin melanomas or slow-growing melanomas that are less lethal , 55, 56,As noted in our study above, higher nevus counts may be associated with a lower chance of dying from melanoma . It is pAnother possible mechanism could be via gene-environment interaction, where those at highest genetic risk of CM benefit more from treatment (e.g. immunotherapy), as it is the case for those at high genetic risk for coronary artery disease (CAD) and treatment benefits from PCSK9 inhibitors in the FOURIER and ODYSSEY OUTCOMES trials , 59.PRS_susceptibility for profiling and monitoring patients for melanoma outcomes following diagnosis during the “melanoma follow-up care program” [This study presents new insights that highlight the potential clinical utility of program” , 61. In In a GWAS meta-analysis of MSS, we identified two novel loci potentially associated with survival from cutaneous melanoma, both of which contain candidate genes linked to tumour progression; however, replication in large independent cohorts is required. In line with observations in other cancers and complex diseases, increased germline genetic susceptibility for CM was strongly but heterogeneously associated with improved MSS especially in a high UV setting. If validated, a PRS_susceptibility could be used to predict melanoma outcomes after diagnosis and profile patients for personalised care.Additional file 1: Figure 1. Manhattan plot for the MSS GWAS meta-analysis between MIA and UKB cohorts. Figure 2. Skin colour polygenic risk score model performance in the validation cohort (QSkin). The x-axis represents the different melanoma polygenic risk score (PRS) models of varying fractions of causal SNPs (i.e. 1 (F0), 0.1 (F1), 0.01 (F2),0.001(F3),0.0001 (F4) and 0.00002 (F5)) at the different radii of the linkage disequilibrium (LD) (i.e. 2 megabase (mb) and 5 mb). The y-axis represents Nagelkerke’s R2 (%) for each of the 12 PRS models. The horizontal dashed black line highlights the optimal model (F1-2mb) (i.e. with the highest Nagelkerke’s R2). Figure 3. The association between polygenic risk scores and the risk of melanoma in QSkin. Figure 4. The association by quartile of polygenic risk for melanoma susceptibility and melanoma specific survival in the MIA cohort. Figure 5. Association of standalone skin colour and naevus PRSs and melanoma specific survival in MIA and UKB.Additional file 2: Table 1. Cohorts used for the cutaneous melanoma susceptibility discovery meta-analysis GWAS. Table 2. Per study melanoma specific survival  GWAS results. Table 3. Pigmentation loci excluded from the cutaneous melanoma susceptibility polygenic risk score. Table 4. Naevi loci excluded from the cutaneous melanoma susceptibility polygenic risk score. Table 5. Naevous count SNPs included in the naevus polygenic risk score.


The purpose of the present study  was to evaluate the preemptive analgesic effects of pregabalin combined with celecoxib in total knee arthroplasty (TKA). n = 36), celecoxib group (n = 38), pregabalin group (n = 38), and combination group (n = 37). Each group was given the corresponding preemptive analgesia regimen at 12 and 2 hours before surgery. The pain score at rest and upon movement, cumulative dosage of sufentanil, knee range of motion (ROM), high-sensitivityC-reactive protein (hs-CRP) level, and adverse effects were evaluated after TKA to compare the effects of the preemptive analgesia regimens among the four groups.  From January 2019 to June 2021, we enrolled 149 patients who underwent TKA and divided them into four groups: the placebo group (P < 0.05). The cumulative dose of sufentanil within 48 hours after surgery was lowest in the combined group among the four groups (P < 0.05). Hs-CRP, ROM, and postoperative nausea and vomiting (PONV) were within 72 hours after surgery significantly improved in the combination group compared with those of the three other groups (P < 0.05).  The pain scores upon movement were significantly lower in the combination group than in the other three groups at 6, 12, 24, and 48 hours after surgery (ChiCTR2100041595. The preemptive analgesia regimen of pregabalin combined with celecoxib had positive effects on improving acute pain and reducing the cumulative dose of opioids after TKA. This trial is registered with  Total knee arthroplasty (TKA), a surgical method for the treatment of terminal stage knee osteoarthritis, can effectively relieve pain and restore knee function. However, patients experience moderate to severe pain after TKA. Acute pain after TKA can have a negative effect on enhanced recovery after surgery among patients . If acutThe mechanism of postoperative pain is complicated. In the past, it was believed that postoperative pain after TKA was mainly nociceptive pain caused by surgical trauma and tissue inflammation . Howeverγ-aminobutyric acid (GABA). Different from the mechanism of traditional analgesics, it has obvious anti-central and peripheral pain sensitization effects through inhibiting the voltage-dependent calcium channels of the peripheral and central nervous systems [The use of oral analgesics in advance is a convenient and effective method for preemptive analgesia after TKA. Celecoxib or other cyclooxygenase-2 (COX-2) inhibitors selectively inhibit the activity of COX-2, reduce prostaglandin production and inflammatory reactions, and achieve effective control of pain and anti-peripheral pain sensitization effects. In particular, it has a positive effect on reducing nociceptive pain mediated by inflammatory mechanisms after TKA , 12. Man systems . Therefo systems , 18. TheThe purpose of our study was to evaluate the effectiveness of pregabalin combined with celecoxib as TKA preemptive analgesia by compared the pain score, cumulative dosage of opioids, high-sensitivity C-reactive protein (hs-CRP) level, and adverse effects after TKA. We hypothesised that the effectiveness of pregabalin combined with celecoxib as TKA preemptive analgesia would lead to satisfactory clinical outcomes.http://www.chictr.org.cn . All patients signed written informed consent forms.The present study was a double-blind randomized clinical trial and was conducted in the affiliated hospital of our university. The study was approved by the ethics committee of the affiliated hospital of our university and conducted in accordance with the Declaration of Helsinki. In addition, the study was retrospectively registered at From January 2019 to June 2021, 201 patients who underwent TKA were subjected to a strict eligibility review. The inclusion criteria were as follows: patients who received elective, initial, and single TKA. The exclusion criteria were as follows: (1) patients with an American Society of Anesthesiologists (ASA) classification of grade 4 or higher, (2) patients with a history of liver and kidney insufficiency, severe cardiopulmonary disease, severe digestive tract disease, and mental illness, (3) patients with an allergy to celecoxib or pregabalin or to anesthetic drugs, (4) patients who had taken celecoxib or pregabalin within 2 weeks before surgery, and (5) patients with cognitive impairment. A total of 160 patients were finally included in the study.μg/kg + ondansetron 0.3 mg/mL + 0.9% sodium chloride injection, which were diluted to 100 mL. The first loading dose was 3 mL; there was no background dose. The additional dose of self-controlled analgesia was 1 mL each time, the lock-in time was 15 min, and the use of PCIA lasted for 48 hours. After the surgery, if the patients had a visual analog scale (VAS) score >4, 10 mg morphine was given as remedial analgesia, and none of the patients underwent a nerve block. All patients were managed with a standardized TKA accelerated rehabilitation program, and immediately after the operation, they were instructed to perform lower limb quadriceps muscle contraction exercises, lower limb ankle pump exercises, and active and passive knee flexion activities.Pregabalin and celecoxib are produced by Pfizer in the United States under the trade names Lyrica and Celebrex, respectively. Pregabalin, celecoxib, and the placebo were packaged in the same opaque packaging by the University Hospital Health Research Center pharmacy. The placebo capsule contained a mixture of 50% cellulose and 50% starch. An assistant from the Clinical Research Center used a random number table to divide the patients into four groups: the control group, celecoxib group, pregabalin group, and combination group. The patient group assignments were sealed in envelopes. Anesthesiologists who did not participate in the study opened the envelope 12 hours before the surgery, distributed drugs according to the patient group assignment, and distributed the same drugs again 2 hours before the surgery. The researchers involved did not know the patient group assignment during the surgery or data analysis. The medication program was as follows: placebo group: 200 mg + 150 mg placebo; pregabalin group: 150 mg pregabalin + 200 mg placebo; celecoxib group: 200 mg celecoxib + 150 mg placebo; and pregabalin combined with celecoxib group: 150 mg pregabalin + 200 mg celecoxib. Each group was given the same medication program again 2 hours before surgery. The patients were closely monitored for adverse effects after each administration of the medicine. All patients were given general anesthesia by senior anesthesiologists in our hospital. All operations were performed by two senior doctors with more than ten years of experience in TKA. Periarticular infiltration of the cocktail ingredient (0.5% ropivacaine + 10 mg/ml triamcinolone acetonide acetate + 0.1% epinephrine hydrochloride) was performed before closed the incision. Afterwards, the four groups of patients received patient-controlled intravenous analgesia (PCIA) immediately after the laryngeal mask was removed. The analgesic formula was sufentanil 2 The main observation indicators included the following:(1) VAS pain score at rest and upon movement at 6, 12, 24, and 48 hours after the surgery ; (2) cumulative dose of opioids within 48 hours ; (3) the hs-CRP level before and after the surgery; (4) time of first successful straight leg elevation: the standard is that the patient can maintain a flexed ankle joint and straight leg raise at least 40 cm above the bed for more than 4 s; (5) maximum knee flexion range of motion (ROM) at 24, 48, and 72 hours after surgery; (6) time to first remedial analgesia after surgery; and (7) complications including postoperative nausea and vomiting (PONV), dizziness, gastrointestinal hemorrhage, urinary retention, deep vein thrombosis (DVT), and severity of sedation.P < 0.05 indicated that the difference was statistically significant.Power analysis was performed based on the data from a previous study  to calcuA total of 160 patients were enrolled in the study, 7 patients discontinued PCIA because of severe vomiting reaction to sufentanil. 2 patients asked to withdraw from the study and 2 patients were suspended surgery. Consequently, 149 patients were included in the final data analysis. The CONSORT study participant flow diagram is shown in P > 0.05)  .P < 0.05). The pain scores at rest in the combination group were significantly lower than those in the celecoxib group at 6 hours after surgery (P=0.018 < 0.05) (P < 0.05)  = 37.88, P < 0.001). Then, a simple effects model was used for further analysis. In terms of a time effect, we observed that the pain score on movement in the combined group showed a significantly continuous downward trend over time within 48 hours after surgery (P < 0.05) (µg) was significantly lower than those of the other three groups within 48 hours after surgery (P < 0.05) (P < 0.05).As shown in  < 0.05) . The pai < 0.05) . There w < 0.05) . The cum < 0.05) . The firP=0.386). The hs-CRP level was significantly lower in the combination group than in the other three groups at 24, 48, and 72 hours after surgery (P < 0.05). The hs-CRP level was significantly lower in the celecoxib group than that in the pregabalin and placebo groups at 24, 48, and 72 hours after surgery (P < 0.05) (P < 0.05) (P < 0.05). As shown in P < 0.05).As shown in  < 0.05) . ROM inc < 0.05) . The timX), and the cumulative dose of sufentanil within 48 hours was the dependent variable (Y). The regression equation   was the independent variable (< 0.001)  suggestePerioperative pain management is key to the smooth implementation of enhanced recovery after surgery (ERAS), which has rapidly become a popular concept in the field of orthopedics. Although the combined application of various postoperative analgesia methods can improve the postoperative acute pain of TKA patients, many still suffer from moderate or even severe pain , 20.Preemptive analgesia is an important part of multimodal analgesia, and its value is increasingly recognized by clinicians and researchers. Some studies have found that preemptive analgesia can further improve postoperative pain in TKA patients –10. HoweIn the present study, we observed that the combination group had better improvement in pain scores at rest and upon movement than the other three groups. The pain scores at rest in the combined group were always low level within 48 hours, and unlike the placebo group, there were no large fluctuations in pain scores, which indicated that the analgesic effect of the combination regimen was stable and relatively durable. However, the pain upon movement was more significant than that at rest because all of the patients need to exercise as soon as possible after TKA to enhance recovery. In the study of Lee et al. , patientCurrently, analgesia after TKA mainly involves a variety of opioids, such as sufentanil, tramadol, and morphine. Opioids mainly act on opioid receptors in the pain center of the brain to inhibit the generation and amplification of pain signals. However, opioids have significant side effects, such as nausea and vomiting, drug dependence, urinary retention, excessive sedation, and respiratory depression. The majority of TKA patients are elderly patients, and the incidence of opioid side effects is significantly higher in elderly patients than in young and middle-aged patients . One of The key core of preemptive analgesia is to reduce the degree of peripheral and central pain sensitization and to improve the pain threshold of patients in advance . Lower lOur study has some limitations. First, our research was limited to within 72 hours after surgery, so we cannot be sure whether control of acute postoperative pain can promote long-term knee function recovery and reduce the possibility of long-term chronic pain. Second, we did not accurately estimate sample size, which may affect the efficacy of the present study. Third, according to the relevant study , the minLast, in terms of postoperative complications, our sample size was too small to comprehensively and objectively evaluate the safety of analgesia programs.The preemptive analgesia regimen of pregabalin combined with celecoxib had positive effects on improving acute pain and reducing the cumulative dose of opioids after TKA.


Three experiments (N = 112) examined how aesthetic appeal influences performance in a classic visual search task. In each experiment, participants completed 320 visual search trials, with icons varying in rated aesthetic appeal and either visual complexity  than unappealing targets across all experiments, and compared to neutral distractors, appealing distractors slowed search RT down. These findings are the first to show that an object’s aesthetic appeal influences visual search performance.Aesthetic appeal of a visual image can influence performance in time-critical tasks, even if it is irrelevant to the task. This series of experiments examined whether aesthetic appeal can act as an object attribute that guides visual search. If appeal enhances the salience of the ents N = 12 examin Recruitment primarily took place via the Psychology Department’s participant pool, with additional students sourced via posters across the University’s campus. The study was approved by the College of Human & Health Sciences, Swansea University, Research Ethics Committee.Forty undergraduate and postgraduate Swansea University students, 34 females and eight males, took part in the study in exchange for participant pool credits. The usual sample size for visual search experiments manipulating variables within-participants is between ten and 25 participants. For current study G*Power (3.1) analysis suggested a minimum of 18 to obtain a medium effect size (.06) for the key Appeal and Set Size interaction. Here we recruited a little over double the required participants ensuring a sufficient number of subjective icon ratings. Specifically, as participants would be asked to rate icons in terms of attractiveness, we needed to avoid missing values for some categories for some participants. Participant ages ranged from 19 to 35 years  appear in Table The visual search task was based on a 2 (Target Presence: present vs. absent) × 4  × 2 (Complexity: complex vs. simple) × 2  repeated-measures design yielding 32 experimental conditions. There were ten trials per condition trials per condition, yielding 320 trials per participant. The dependent measure was RT.Participants were tested individually in a quiet well-lit laboratory. Participants first completed the rating task followed by a computer-based visual search task. For the rating task, each participant was presented with a booklet of 60 icons which included instructions and examples. Participants rated each icon by circling a Likert scale response from 1  to 5 . The booklets started on different pages for each participant to avoid any potential order effects.Following the ratings task participants carried out the visual search task. Trial presentation and recording of responses was controlled via PsyScope  was reported for all significant effects  of participants used the full scale of appeal ratings. No participants used fewer than 3 points on the Likert scale. Appeal ratings per icon type are shown in Table For the visual search task, errors (4.29%) were removed from the data and analysed separately. Correct responses that were ±3 SDs from the mean per participant per condition  were classed as outliers, removed from the data, and not analysed further.In this set of analyses, appealing and unappealing icons were determined based on ratings provided by participants in McDougall and Reppa . SeparatA 4  × 2 (Complexity: complex versus simple) × 2  repeated-measures ANOVA, with appeal determined based on the ratings obtained by McDougall and Reppa , was cart(39) = 5.10, p < .001, d = .81. The same difference was observed between simple appealing and unappealing icons, but the effect size was smaller, t(39) = 3.87, p = .003, d = .61. The Set Size × Complexity interaction was also significant with shallower slopes for simple compared to complex icons . There were no other significant interactions.The main effect of Set Size was significant with RT increasing as set size increased. The main effects of Complexity and of Appeal were also significant. There was also a significant Complexity × Appeal interaction: pairwise comparisons confirmed that search RT was faster for complex appealing compared to complex unappealing icons, target-present trials were submitted to a 2 (Complexity: complex vs. simple) ×  repeated-measures ANOVA. There was only a significant main effect of Complexity with steeper slopes for complex (18 ms/item) compared to simple icons (14 ms/item). The main effect of Appeal was not significant and neither was the Complexity × Appeal interaction.The RT by set size slopes for appeal interacted with visual complexity to boost search times when the target was present in the array – search RT was faster when targets were appealing compared to unappealing icons with this difference being larger for unappealing complex icons. In contrast, search efficiency was influenced by complexity only, with shallower slopes for visually simple compared to visually complex icons.Overall, icon target-absent RT showed a significant main effect of Set Size with increasing RT as the number of distractors increased. The main effects of Complexity and Appeal were significant as was Complexity × Appeal interaction. Pairwise comparisons to examine the interaction, showed that target-absent RT was overall faster when looking for complex appealing compared to unappealing targets, t(39) = 4.73, p < .001, d = .75, while there was no difference in search RT between simple appealing and simple unappealing target, t(39) = .29, p = .78, d = .05.A repeated-measures ANOVA for F = 7.75, p < .001, ηp2 = .17, reflecting steeper RT slopes for complex compared to simple targets . The Set Size × Appeal interaction was not significant but the significant three-way interaction was significant. The three-way interaction was driven by the interaction between Complexity and Appeal on search slopes .There was also a significant interaction between Set Size and Complexity, target-absent trials were submitted to a 2 (Complexity: complex vs. simple) ×  ANOVA and reveal a significant main effect of Complexity but not Appeal. The Complexity × Appeal interaction was significant: pairwise comparisons confirmed that search slopes when looking for complex targets were shallower when they were appealing (53 ms/item) compared to when they were unappealing (64 ms/item), t(39) = 2.60, p < .001, d = .41, with no significant difference between simple appealing and simple unappealing slopes (50 and 43 ms/item respectively), t(39) = 1.95, p = .07, d = .25.Search slopes for appeal interacted with visual complexity to influence overall search RT: search was terminated later for unappealing compared to appealing targets, with this difference being larger for complex compared to simple target icons. Search efficiency was also determined by the interaction between visual complexity and appeal, with shallower search slopes when searching for appealing targets, especially when they were complex.Overall, for target-absent trials Appeal was coded separately for each participant depending on their own ratings of the target icons. That means that an icon rated as high in appeal for one participant may have been rated as neutral or unappealing by another. As before separate analyses were carried out for target-present and target-absent trials. The results of these analyses are shown in Table A 4  × 2 (Complexity: complex versus simple) × 3  repeated-measures ANOVA, with appeal determined based on each participant’s subjective appeal ratings, was carried out on correct RT. The main effect of Set Size was significant with reaction times increasing as set size increased. The main effects of Complexity and Appeal were significant but, this time, the Complexity × Appeal interaction was not significant. The Set Size × Complexity interaction was significant revealing shallower slopes for simple compared to complex icons . There were no other significant interactions.target-present trials were submitted to a 2 (Complexity: complex vs. simple) ×  repeated-measures ANOVA. There was only a significant main effect of Complexity with steeper slopes for complex (18 ms/item) compared to simple icons (9 ms/item). The main effect of Appeal was not significant and neither was the Complexity × Appeal interaction.The RT by set size slopes for icon appeal and visual complexity independently boosted search times when the target was present in the array – search RT was faster when targets were appealing compared to when unappealing, and faster when they were simple compared to visually complex. In contrast to the pattern of results when appeal was based on pre-existing ratings, when appeal was determined subjectively, there was no longer an interaction between appeal and complexity.Overall, both Search efficiency was only influenced by complexity, with shallower slopes for visually simple compared to visually complex icons. Nevertheless, the slopes for simple appealing and simple neutral icons were less than 10 ms per item which is in the ‘quite efficient’ search range.target-absent RT showed a significant main effect of Set Size, with increasing RT as the number of distractors increased, as well as Complexity and Appeal. The Complexity × Appeal interaction was not significant. There was a significant interaction between Set Size and Complexity reflecting steeper RT slopes for complex compared to simple targets . No other interactions were significant.A repeated-measures ANOVA for target-absent trials were submitted to a 2 (Complexity: complex vs. simple) ×  ANOVA, showed only a significant main effect of Complexity with steeper slopes when looking for complex (56 ms/item) compared to simple (46 ms/item) targets. The main effect of Appeal was not significant and neither was Complexity × Appeal interaction.Search slopes for appeal and visual complexity influenced overall search RT: search was terminated later for unappealing compared to appealing targets, and for complex compared to simple targets. Search efficiency was only determined by visual complexity, with steeper search slopes when looking for complex targets.Overall, for target-absent trials both Experiment Visual complexity significantly slowed down search times and led to more inefficient searches. Previous work using search tasks without manipulating search array size has found a negative impact of visual complexity icons and symbols on performance  were recruited in exchange of participant pool credits. Participants had normal or corrected-to-normal vision and were naïve to the aim of the experiment. The study was approved by the College of Human & Health Sciences, Swansea University, Research Ethics Committee.The same apparatus was used as in Experiment The design and procedure were identical to that of Experiment Error trials accounted for 5.50% of all trials with 1.2% false alarms  and 4.2% misses . All error trials were removed from the calculation of correct mean response times (RT) and analysed separately. RT that was ±3SDs from the mean per participant and per condition (1.4%) were removed from the correct RT data and not analysed further. Cell means for correct RT in target-present and target-absent trials for Experiment target-present trials only revealed a significant main effect of Set Size with increasing RT with larger set sizes. The main effect of Complexity was significant with simple icons being found faster than complex icons. The main effect of Appeal was also significant with appealing icons found significantly faster than unappealing icons. The Complexity × Appeal interaction was significant. Pairwise comparisons to examine the Complexity × Appeal interaction showed longer search RT for complex unappealing compared to complex appealing targets  = 4.69, p < .001, d = .92), but a nonsignificant difference between simple appealing and unappealing targets  = 1.46, p = .16, d = .29). There were no other significant interactions.A repeated-measures ANOVA on target-present trials were submitted to a 2 (Complexity: complex vs. simple) ×  repeated-measures ANOVA. Neither the main effect of Appeal, nor Complexity were significant, and neither was their interaction.Search slopes for visual complexity and appeal influenced search RT when the target was present in the array, with faster searches when the target was simple and when the targets were appealing. Search efficiency was influenced neither by visual complexity nor by appeal.Overall, both target-absent RT showed a significant main effect of Set Size with increasing RT as the number of distractors increased. The main effect of Complexity was significant as was the main effect of Appeal. The Complexity × Appeal interaction was significant. Pairwise comparisons to examine the interaction, showed that target-absent RT was overall faster when looking for complex appealing targets, compared to unappealing targets  = 5.67, p < .001, d = 1.11) and while there was no difference in search RT between simple appealing and unappealing target  = 1.38, p = .18, d =.27). The only other significant interaction was between Set Size and Complexity, reflecting steeper RT slopes for complex compared to simple targets . Neither the Set Size × Appeal nor the three-way interaction were significant.A repeated-measures ANOVA for target-absent trials were submitted to a 2 (Complexity: complex vs. simple) ×  ANOVA, showed a significant main effect of Complexity, with steeper slopes for complex (60 ms/item) compared to simple (50 ms/item) target icons. Neither the main effect of Appeal nor the interaction were significant.Search slopes for appeal interacted with visual complexity to influence overall search RT: search was terminated later for unappealing compared to appealing targets, with this difference being larger for complex compared to simple target icons. Search efficiency was determined only by visual complexity, with steeper search slopes when searching for complex compared to simple targets.Overall, Experiment In Experiment M = 25.09, SD = 10.45) took part in Experiment Twenty-six new participants, 17 females and nine males, with ages ranging from 19 to 55 years  and correct trials with responses that were ±3 SDs from the mean per participant per condition (1.6%) were removed from the data and were not analysed further. One participant’s data was removed from the calculation of mean RT as their overall RT exceeded 3 standard deviations from the group mean. Mean correct response times (ms) for the remaining 25 participants per condition are shown in Fig. .42% and target-present trials only, revealed a significant main effect of Set Size and a significant main effect of Complexity. The main effect of Appeal was no longer significant but there was a significant Complexity × Appeal interaction. Pairwise comparisons showed no difference in RT between simple appealing and unappealing target icons, t(24) = 1.82, p = .08, but search times were faster for complex appealing compared to complex unappealing icons, t(24) = 2.51, p < .01, d = .50. The interaction between Complexity × Set Size was also significant with steeper search slopes for complex compared to simple target icons . There were no other significant interactions, including the theoretically relevant Set Size by Appeal interaction.A repeated-measures ANOVA on correct RT of target-present trials were submitted to a 2 (Complexity: complex vs. simple) ×  repeated-measures ANOVA. The main effect of Complexity was significant with steeper slopes for complex (28 ms/item) compared to simple targets (19 ms/item). Neither the main effect of Appeal nor the interaction were significant.Search slopes for target-absent trials, showed a significant main effect of Set Size significant main effect of Complexity as well as a significant Set Size × Complexity interaction reflecting steeper search slopes for complex targets . The main effect of Appeal was not significant. The Complexity × Appeal interaction was significant, with faster search termination RTs for complex appealing targets compared to unappealing counterparts (t(24) = 2.57, p = .001, d = .51), but no difference in search RT between simple appealing and unappealing targets [t(24) = 0.13, p = .90). None of the other interactions were significant.A repeated-measures ANOVA on correct RT of target-absent trials were submitted to a 2 (Complexity: complex vs. simple) ×  ANOVA. Only the main effect of Complexity was significant with steeper slopes for complex (80 ms/item) compared to simple targets (63 ms/item). Neither the main effect of Appeal, nor the interaction were significant.Search slopes for search RT and search efficiency for target-present and target-absent trials were only influenced by the visual complexity of the target icon. Target appeal no longer facilitated search or search termination times.In summary, when distractor icons were appealing, Experiments steeper slopes for complex unappealing compared to complex appealing targets, t(49) = 2.02, p = .04, d = .28, but no such significant difference between simple appealing and unappealing targets, t(49) = .52, p = .60. Target Appeal did not have a significant main effect on search slopes but it interacted with Distractor Appeal. Pairwise comparisons confirmed that appealing targets yielded flatter slopes when distractors were neutral in appeal, (17 ms/item) compared to when distractors were appealing (25 ms/item), t(49) = 3.07, p = .003, d = .86. Unappealing targets yielded similar slopes irrespective of whether they appeared among neutral or appealing distractors , t(49) = .41, p = .68, d = .11. The Distractor Appeal × Complexity interaction was not significant and neither was the three-way interaction.A mixed 2 (Complexity: complex vs. simple) × 2  × 2  ANOVA was carried out on search slopes for target-present trials, with Distractor type as the between-participants variable. There was a significant main effect of Complexity steeper slopes for complex compared to simple targets, and of Distractor Appeal with steeper slopes when distractors were appealing compared to when they were of medium appeal. Target appeal interacted with Complexity, with In summary, when the target was present, distractor appeal influenced search slopes overall, with more inefficient searches when distractors were of high compared to neutral appeal. Distractor appeal also interacted with target complexity to yield steeper slopes for complex target icons when they were surrounded by appealing compared neutral appeal distractors. Critically, distractor appeal interacted with target appeal, with steeper slopes for appealing targets amongst appealing distractors compared to neutral appeal distractors.target-absent trials, showed a significant main effect of Distractor Appeal with steeper search slopes when search was among appealing distractors (71 ms/item) compared to neutral distractors (55 ms/item). Target Complexity was also significant on target-absent slopes with steeper slopes for complex (70 ms/item) compared to simple targets (56 ms/item). There were no significant interactions.The same ANOVA on Experiment Taken together the findings from Experiments Search slopes were steeper when distractors were appealing, compared to when they were neutral in appeal, and that was the case both for target-present and target-absent trials. This suggests that appealing ‘crowds’ are searched slower than crowds of neutral appeal. The slower search among appealing distractors, may be due to the difficulty in disengaging from appealing distractors  and with normal or corrected-to-normal vision, took part in Experiment Twenty new undergraduate Swansea University students , with ages ranging from 19 to 39 years  × 4  × 2 (Concreteness: concrete vs. abstract) × 2  repeated-measures design was used, yielding 32 within-participant conditions. There were ten trials per condition yielding a total of 320 trials per participant. The dependent measure was RT.The procedure of the visual search task was identical to that in Experiment Error rates were low accounting for about 6.2% of all trials . Error trials were removed from the correct RT analyses, prior to estimating the mean RT per condition. Correct trials with responses that were ±3 SDs from the mean per participant per condition (0.33%) were excluded from the analyses. Correct mean RT per condition for target-present and target-absent trials appear in Fig. target-present trials. The main effect of Set Size was significant with RT increasing with larger set sizes. The main effect of Concreteness was significant faster search termination times for concrete icons compared to abstract icons. The main effect of Appeal was also significant with faster search termination times for appealing icons compared to unappealing icons. None of the interactions were significant.A 4  × 2 (Concreteness: concrete vs. abstract) × 2  repeated-measures ANOVA was carried out on correct RT of A 2 (Concreteness: concrete vs. abstract) × 2  repeated-measures ANOVA on search slopes, showed no significant main effect of Concreteness no significant main effect of Appeal and no significant interaction.appeal and concreteness independently influenced search RT - it took less time to find the target if it was appealing than unappealing, and when it was concrete, as opposed to abstract. Neither appeal nor concreteness however influenced search efficiency when the target was present.In summary, when the target was present, target-absent trials. This showed a significant main effect of Set Size with RT increasing with larger set sizes. The main effect of Concreteness was significant with faster termination times for concrete compared to abstract target icons. The main effect of Appeal was also significant with faster termination times when looking for appealing compared to unappealing targets. There were no significant interactions.A 4  × 2 (Concreteness: concrete vs. abstract) × 2  repeated-measures ANOVA was carried out on correct RT of p < .05), but not when they were concrete (p > .05).A 2 (Concreteness: concrete vs. abstract) × 2  repeated-measures ANOVA on search slopes for target-absent trials, showed a significant main effect of Concreteness. There was no significant main effect of Appeal but the interaction was significant simple effects analysis confirmed that slopes were steeper in unappealing compared to appealing target trials when targets were abstract  × 2  repeated-measures ANOVA on search slopes, showed no significant main effect of Familiarity, and only a marginally significant effect of Appeal and no significant interaction.appeal and familiarity independently influenced search RT – it took less time to find the target if it was appealing than unappealing, and when it was concrete, as opposed to abstract. As was the case with concreteness and appeal, neither appeal nor familiarity however influenced search efficiency when the target was present.In summary, when the target was present, target-absent trials. This showed a significant main effect of Set Size with RT increasing with larger set sizes. The main effect of Familiarity was significant with faster termination times for familiar compared to unfamiliar target icons. The main effect of Appeal was not significant, and there were no significant interactions.A 4  × 2 (Familiarity: familiar vs. unfamiliar) × 2  repeated-measures ANOVA was carried out on correct RT of p = .09)A 2 (Familiarity: familiar vs. unfamiliar) × 2  repeated-measures ANOVA on search slopes for target-absent trials, showed than neither of the two main effects were significant. The interaction was only marginally significant , even when it is not task relevant. However, unlike visual complexity it did not influence search efficiency – search slopes were in the ‘quite inefficient’ range for both abstract and concrete icons and there was no search asymmetry between the two conditions. Therefore, even though concreteness can speed search up it only does so after the item is attended to. Separate analyses showed that the effects of familiarity on visual search are similar to those of concreteness. This is not surprising given their high correlation: concrete icons rely on the use of visual metaphors with the real world, often using familiar objects to represent mean producing a close relationship between the two icon characteristics.Critically, as in the previous experiments, icon appeal moderated search performance: search was faster when looking for an appealing target, compared to unappealing targets. This finding corroborates previous evidence from a localisation task, showing that appealing icons are localised faster than unappealing icons Search efficiency was determined by visual complexity, with icons rated as visually simple guiding search more efficiently than those rated as visually complex.(ii)not blind to beauty – to borrow a term from Gerritsen et al. There was no evidence that appeal was pre-attentively processed and guided search.(iv)distractors were appealing, the search advantage for appealing targets was reduced, and it was not replaced by advantage for unappealing targets.When (v)distractors took longer than neutral distractors to be rejected - as evidenced by the slower search times and less efficient searches among appealing compared to neutral distractors. Those findings are discussed in turn.Appealing Attractiveness of objects, people, or interfaces is key to much of our everyday life decisions and behaviours, with significant socio-economic impact . Second, the procedure used was clearly sensitive to changes in stimuli – the pattern of results changed when we used appealing distractors. This suggests that our participants were sensitive to the appeal of stimuli on the screen. Of course, the absence of evidence for pre-attentive processing does not rule out the possibility that visual appeal is an important object dimension. The aesthetic appeal of a stimulus lies along a continuum, with most stimuli lies somewhere along it. Lack of efficient searches may be due either to distractors never being at zero level of appeal, or to the targets not being sufficiently high in appeal.The icons used in the current study, and their properties, are published elsewhere , and when set sizes were large (Exps. 1 and 2a). Both findings corroborate previous evidence showing conditional effects of appeal on performance with icons and with websites (e.g., Moshagen et al., appeal may increase motivation which in turn can speed up search performance. The current findings lend further support to this notion – we find unconditional effects of appeal on performance for stimuli we personally like and thus find more rewarding (e.g., Della Libera & Chelazzi, Obtaining subjective ratings of appeal in Experiment Other work has shown that stimuli that are associated with rewards can become visually salient and accordingly enhance performance in visual cognition tasks (e.g., Hickey et al., perceived to be more usable (e.g., Kurosu & Kashimura, become more usable because users make more of an effort with them (e.g., Wiedenbeck, are more usable. This finding is potentially relevant to different types of stimuli and user experience, where optimising performance could have considerable costs. People can be sensitive to performance costs as small as 150 ms (e.g., Gray & Boehm-Davis, The current findings are important in the field of HCI because they go beyond the evidence showing that more aesthetically appealing interfaces are In conclusion, the current findings show that visual aesthetic appeal, as a stimulus characteristic, even when task irrelevant, can lead to efficient performance in visual search tasks. Future work needs to examine the mechanism with which appeal can influence performance, be it via positive affect or some other emotional or cognitive process.


Ideal root canal shaping might be more challenging particularly in cases with severely curved canals or complex anatomical variations. Glide path preparation is suggested as a critical step to achieve ideal canal preparation. The present study is aimed at evaluating transportation at different levels of the canal following glide path preparation by five different path finders. #10 and #15, 0.02), (2) One G , (3) PathFile , (4) GPS , and (5) K file  (Control group). The first four groups were NiTi rotary instruments, while the last group was a stainless steel hand file. The aforementioned files were used after canal negotiation by a #10 stainless steel hand file. Before- and after-preparation photos were taken and were superimposed in Adobe PhotoShop CC 2019. Transportation measurements were conducted in Digimizer. Absolute canal transportation was calculated at 10 cross-sections. Intergroup and intragroup data analysis were conducted using one-way and repeated measures ANOVA tests, respectively, in SPSS 26.0. The significance level was set to 0.05.  The study was conducted on 100 S-shaped canal simulator blocks. Glide path was prepared in five groups including (1) Scout RaCe (p < 0.001), rotary groups were not statistically different. In the coronal third, K files led to significantly more transportation compared to Scout RaCe and PathFile (p < 0.05).  Although K file led to significantly more transportation in the apical and middle thirds ( Within the limitations of the present study, regardless of the recruited rotary system, glide path preparation using NiTi rotary instruments leads to less canal transportation compared to stainless steel hand files. An ideal root canal preparation is essential in order to facilitate irrigation, disinfection, and proper obturation of the canal, thus preventing or eliminating apical periodontitis . MeanwhiA glide path is defined as a smooth radicular tunnel extending from the canal orifice to the physiologic terminus . CreatinGlide path creation can be carried out with either precurved stainless steel K files or NiTi rotary path finder instruments . K filesStudies have shown that glide path creation with NiTi instruments is faster and causes fewer procedural errors than K files . It has One G  is a 0.03 tapered NiTi glide path file with a tip size of 0.14 mm. It is suggested to be used with a torque limit of 1.2 Ncm and a speed of 250–400 rpm. PathFile (PF)  NiTi rotary instruments are used for creating an initial glide path mechanically. The system consists of 3 path finder instruments with ISO 13, 16, and 19 tip sizes, a 0.02 taper, and a square cross section. PathFile is suggested to be used with a torque limit of 5 and 6 Ncm and a speed of 300 rpm. Scout RaCe  rotary pathfinding instruments are 3 instruments, with ISO 10, 15, and 20 tip sizes and a 0.02 constant taper, manufactured from conventional NiTi. Scout RaCe is suggested to be used with a torque limit of 1.5 Ncm and a speed of 800 rpm. PathFile and Scout RaCe instruments have 4 cutting edges with a square cross section. The GPS files  are NiTi rotary path files with a tip size of 0.15 mm and a 0.03 constant taper. It is used with continuous rotation, a torque limit of 1.5 Ncm, and a speed of 300–500 rpm.K-files  are hand files manufactured from twisted square stainless steel blanks. They are the standard instruments used for root canal preparation. Because of the sharp and mirror-like finished edges with high ductility, these instruments have excellent working characteristics. K files are used with a watch-winding hand motion and can also be reciprocated.There are a couple of studies investigating the deviation of the canal during glide path preparation , 15, 16,#10 and #15, 0.02), (2) One G , (3) PathFile , (4) GPS , (5) K file  (Control). The first four groups were NiTi rotary instruments, while the last group was a stainless steel hand file. Canal simulator endo-blocks were used in the present study to standardize the methodology and control confounding variables [#10 stainless steel K-file  and then rotary instruments were introduced into the canal with gentle strokes till the working length was reached. Glide path preparation was carried out in the first group by Scout RaCe #10 and #15; in the second group by One G file  as a single file; in the third group by PathFile #13 and #16; in the fourth group by GPS ; and in the fifth group by #15 K-file . K-files were introduced into the canal with watch-winding motion and circumferential motion, subsequently. Rotary files were used in order to prepare a glide path according to the manufacturer's catalogue , assigned to 5 groups (20 blocks were assigned to each group): (1) Scout RaCe  is presented in G > GPS > Scout RaCe, Middle third: GPS > One G > Pathfile > Scout RaCe, Coronal third, One G > GPS > PathFile > Scout RaCe.NiTi path finder files mean transportation at each level was observed as follows: Apical third, PathFile > One p < 0.001). Pairwise comparisons between NiTi groups did not show any significant differences among them in the apical and middle thirds of the canal. In the coronal third, Scout RaCe, and PathFile were associated with less canal transportation when compared to the control group . However, GPS and One G were not significantly different from the control group regarding coronal transportation . Pairwise comparisons between NiTi groups did not show any significant differences among them in the coronal third of the canal.In the apical and middle thirds of the canal, all of the path finders showed significantly less canal transportation compared to the control group (K file) (p > 0.05) (p=0.001). PathFile deviated the coronal third of the canal significantly less than the apical (p=0.008) and the middle (p < 0.001) thirds of the canal. K file also showed the same pattern as the PathFile (p < 0.001).Intragroup comparisons revealed that canal deviation caused by Scout RaCe and One G was not significantly different throughout the canal; i.e., the apical, middle, and coronal transportation values in each group were not statistically different ( > 0.05) . GPS shoThe present study has focused on the transportation caused by five path finder files, including One G, PathFile, GPS, Scout RaCe, and K file, on different levels of an S-shaped canal.The major determinants of canal transportation mentioned by previous authors are canal anatomy, instrument design, instrument alloy, and instrumentation technique . On the As apical transportation may compromise the apical seal of the obturation, it might be of great concern in terms of treatment outcome . Among tConsidering the main objectives of cleaning and shaping by Schilder  and the ex vivo models. Further in vivo studies are also required to investigate the clinical significance of the observed differences among the path finding instruments; i.e., whether the difference among the path finding instruments in terms of canal transportation affects treatment outcomes significantly.The present study has recruited endo-blocks as S-shaped canal simulators. Thus the results must be interpreted with caution. It is suggested for future studies to investigate canal transportation values associated with different path-finding instruments on Within the limitations of the present study, glide path preparation using K files results in significantly more transportation at different levels of the canal; while none of the NiTi rotary path finding instruments investigated in the present study, including One G, Scout RaCe, GPS, and PathFile, outperformed the others with regard to canal transportation.