Hepatozoon spp. are tick-borne parasites causing subclinical to clinical disease in wild and domestic animals. Aim of this study was to determine Hepatozoon prevalence and species distribution among wild mammals and ticks in Europe.Samples of wild mammals and ticks, originating from Austria, Bosnia and Herzegovina, Croatia, Belgium and the Netherlands, were tested with PCR to amplify a ~ 670-bp fragment of the small subunit ribosomal RNA gene.Hepatozooncanis was detected in samples of 178 animals , H.martis in 125 , H.sciuri in 13 , Hepatozoon sp. in 47  and H.ayorgbor in 4 . Regarding origin, 2.9% (6/208) tested positive from Austria, 2.8% (1/36) from Bosnia and Herzegovina, 14.6% (173/1186) from Croatia and 13.9% (190/1371) from Belgium/the Netherlands. Of the 754 ticks collected, 0.0% (0/35) Hyalomma sp., 16.0% (4/25) Dermacentor spp., 0.0% (0/23) Haemaphysalis spp., 5.3% (24/50) Ixodes and 1.4% (3/221) Rhipicephalus spp. tested positive for Hepatozoon , most often H.canis (n = 22).Of the 2801 mammal samples that were used for this study, 370 (13.2%) tested positive. Hepatozooncanis is most present in mammals  and ticks, followed by H.martis, which was found merely in stone martens and pine martens. None of the rodent-associated Hepatozoon spp. were detected in the ticks, suggesting the possible implication of other arthropod species or non-vectorial routes in the transmission cycle of the hemoprotozoans in rodents. Our findings of H.canis in ticks other than R.sanguineus add to the observation that other ticks are also involved in the life cycle of Hepatozoon. Now that presence of Hepatozoon has been demonstrated in red foxes, gray wolves, mustelids and rodents from the Netherlands and/or Belgium, veterinary clinicians should be aware of the possibility of spill-over to domestic animals, such as dogs.The online version contains supplementary material available at 10.1186/s13071-022-05626-8. Hepatozoon spp. (Adeleorina: Hepatozoidae) are apicomplexan vector-borne blood parasites with a complex life cycle [Hepatozoon transmission is not achieved by arthropod blood feeding on the vertebrate host, but with the infection taking place when the intermediate host ingests the definitive host. Also, transmission routes other than vector-borne have been described. For some Hepatozoon species, such as H.americanum (canids), H.sipedon (reptiles), H.caimani (caiman crocodiles) and H.ayorgbor (snakes), transmission can take place via predation of prey [H.canis (canids) and Hepatozoon sp. of garter snakes (Thamnophiselegans), vertical transmission has been described [fe cycle , 2. Vertfe cycle –6. Unlik of prey –12 or, iescribed –16.Hepatozoon in wild animals such as canids [Hepatozoon in wild animals is mainly spillover to domestic animals. Hepatozoon infection in dogs and cats is known to cause subclinical to severe disease, which can be worsened by co-infection with, e.g., bacteria or other hemoparasites as well [The pathogenicity of s canids , 18, fels canids  and musts canids  seems tos canids . The imp as well , 22–31.Hepatozoon infections. Several Hepatozoon species have been described in European wild and domestic mammals with these methods, with most studies focusing on carnivores [H.silvestris and/or H.felis in European wild cats (Felissilvestrissilvestris) in Bosnia and Herzegovina [H.martis has been detected in mustelids from Bosnia and Herzegovina and Croatia [H.ursi in brown bears (Ursusarctos) from Turkey [Hepatozoon sp. in pine martens from Scotland, UK [Vulpesvulpes) [Canislupus) [Canisaureus) [H.canis can be high. In contrast, prevalence of H.canis in domestic dogs [Hepatozoon, finding H.canis in camels from Saudi-Arabia [Hepatozoon sp. in ticks collected from cattle in Pakistan [Hepatozoon in wild Lagomorpha, except for a Spanish study in which no Hepatozoon was detected in European hares (Lepuseuropaeus) [Until a decade ago, histological methods prevailed in the characterization of blood parasites, with few exceptions –36. Sincrnivores , 39, e.gzegovina , 40, Spazegovina  and Hungzegovina , and in zegovina , 43. Als Croatia , Hungary Croatia  and Spai Croatia  and in w Croatia , 45, H.m Turkey  and Hepaland, UK . In wildvulpes) , 47, 48,slupus) , 50 and aureus) , prevaletic dogs , 53 and i-Arabia  and in ti-Arabia  and Romai-Arabia  and HepaPakistan . To our ropaeus) .Hepatozoon has been reported in Europe in Finland, Estonia and western Russia [Hepatozoon could be identified to species level, in bank voles (Myodes = Clethrionomysglareolus) H.erhardovae was often detected [H.sciuri in red squirrels (Sciurivulgaris) [H.griseisciuri in gray squirrels (Sciuriscarolinensis) [H.lavieri in common voles  [In rodents, n Russia , Lithuann Russia , Poland n Russia –62, Hungn Russia , the Czen Russia , Slovakin Russia , Great Bn Russia –69, Germn Russia , 71, Ausn Russia , Turkey n Russia  and Spain Russia . In rodedetected , 71, 72 ulgaris) , H.grisarvalis) .Hepatozoon in European ticks are also scarcely reported. It has long been believed that Rhipicephalussanguineus sensu lato is the only known vector of H.canis in Europe and of many other Hepatozoon species [Hepatozoon sp. in other tick species raise questions about their vectorial role. Those findings include H.canis in Ixodesricinus, I.canisuga, I.hexagonus and Dermacentorreticulatus ticks feeding on foxes in Germany [Haemaphysalisconcinna ticks collected from a dog in Poland [I.ricinus) from goats  in Romania [H.canis positive questing I.ricinus ticks were found in Slovakia and the Czech Republic [R.turanicus collected from an infected fox in Italy [Data about  species , 74, 75. Germany , in Haemn Poland  and in t Romania . Also, HRepublic  and in ain Italy .Hepatozoon among wild mammals and ticks in Europe. For this, we investigated a wide range of ungulates, carnivores and small mammals and ticks collected from animals and vegetation from five European countries. Animals and ticks were tested for the presence of Hepatozoon spp. using PCR and sequencing methods.Here, we aim to gain more knowledge on species distribution and prevalence of Canislupus) one or multiple samples were collected after a complete necropsy  using DNA blood and tissue kit  according to the manufacturer’s instructions.For extraction of individual ticks’ DNA NucleoSpin® DNA Insect (Macherey Nagel) was used. The forward primer HepF 5’-ATACATGAGCAAAATCTCAAC-3’ and the reverse primer HepR 5’-CTTATTATTCCATGCTGCAG-3’ were used to amplify a fragment of ~ 670 bp of the 18S rRNA gene .H.canis confirmed with sequencing from earlier studies) and negative (water from GoTaq G2 Mastermix) controls including extraction controls were used in all amplifications. The amplification product was analyzed using capillary electrophoresis on the QIAexcel system . For the purpose of further DNA sequencing, amplified PCR product was purified using ExoSAP-IT-PCR Clean-Up Reagent, according to the manufacturer’s instructions . Sequencing in both directions was performed by Macrogen Europe with the same primers used for PCRs. The sequences were assembled using the SeqMan Pro software edited with EditSeq of the Lasergene software  and compared with available sequences using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cg) system analysis.PCR reaction mixtures of 20 µl were prepared containing 10 µl G2 GOTaq mastermix , 7.2 µl DNase/RNase-Free distilled water (Promega), 0.4 µl 10 pmol/µl of each primer and 2 µl of sample. Positive  clustering analysis using the tree builder tool incorporated in Geneious Prime (HKY). Hepatozoon sequences generated in this study were deposited in the NCBI GenBank® database under the accession numbers MH656727-MH656732 and KT274177-KT274186.The 18S rRNA sequences of Hepatozoon with PCR. The mammals were order Artiodactyla (n = 1233), further divided in the Families Bovidae (n = 181), Suidae (n = 289) and Cervidae (n = 763); order Carnivora (n = 865), further divided in the Families Canidae (n = 336), Ursidae (n = 79), Mustelidae (n = 446) and Procyonidae (n = 4); order Eulipotyphia (n = 1), of which only the family Erinaceidae (n = 1); order Lagomorpha (n = 171), of which only the family Leporidae (n = 171) and order Rodentia (n = 531), further divided into the families Sciuridae (n = 53), Cricetidae (n = 167) and Muridae (n = 311). Of these 2801 animals, 36 originated from Bosnia and Herzegovina, 1186 from Croatia, 208 from Austria and 1371 from Belgium/the Netherlands. From Austria and Bosnia and Herzegovina, no Eulipotyphia, Lagomorpha or Rodentia were tested, and from the Netherlands/Belgium, no Eulipotyphia and Lagomorpha. Precise numbers of each mammal species and origin are given in Table For this study, 2801 mammals and 754 ticks were tested for the presence of la n = 123, furthen = 376, collected from the environment n = 91). Table Hepatozoon DNA in their spleen samples.In total 754 ticks of 15 tick species  mammal samples tested positive for Hepatozoon. The Carnivora showed highest prevalence (34.8%), followed by Rodentia (12.1%). Artiodactyla and Lagomorpha showed the lowest Hepatozoon prevalence . Within the Artiodactyla, only chamois (Bovidae) and roe deer (Cervidae) tested positive. The only animal that was tested within the order Eulipotyphia  tested positive. Within the Carnivora, the four families showed differences in prevalence: the Canidae showed the highest prevalence (49.7%), and within that family especially the golden jackals (80.8%), followed by gray wolves  and red foxes . Within the Mustelidae, pine martens were most frequently infected, followed by stone martens and European polecats. In contrast, all samples of the Families Ursidae and Procyonidae of the Carnivora tested negative. Within the Rodentia, the Muridae showed much lower prevalence (3.2%) than Sciuridae (24.5%) and Cricetidae (24.6%).See Tables n = 1186) and the Netherlands/Belgium (n = 1371) showed higher prevalence  than animals from Austria (n = 208) and Bosnia (n = 36) . The chamois and roe deer (Artiodactyla) that tested positive, were all from Austria. It is interesting to point out the difference in prevalence of Hepatozoon among bank voles (Croatia 81.8% and the Netherlands/Belgium 10.5%) and wood mice (Croatia 14.6% and the Netherlands/Belgium 0.0%).Regarding origin, the mammals from Croatia , followed by Ixodes (5.3%) and Rhipicephalus (1.4%). None of the ticks of the genera Hyalomma (n = 35) and Haemaphysalis (n = 23) and none of the ticks from the Netherlands (n = 287) tested positive. Ticks that were collected from animals  tested positive more often than ticks collected from the environment .Overall, 31 (4.1%) of the 754 collected ticks tested positive for on Table . Ticks oHepatozoon species were detected in the tested mammals and ticks. Also, Hepatozoon that could not be further specified to species level (Hepatozoon sp.) was found. Within all tested animals, H.canis was most prevalent . Interestingly, H.canis isolate MH656730 was most prevalent in mammals  from Austria, Bosnia, Croatia and the Netherlands/Belgium, while H.canis isolate MH656729 was most often detected in ticks , and isolated solely from Croatian mammals , Mustelidae (badger), Erinaceaidae (European hedgehog) and Leporidae (European hare), while H.canis MH656730 was detected in carnivores, Canidae , Mustelidae (badger), but also in Artiodactyla Cervidae (roe deer). Of the 38 ticks that were collected from Hepatozoon-negative foxes, in 16 (42.1%) ticks H.canis MH656729 was detected (Table H. martis (MH656728) was most prevalent . In Sciuridae (squirrels), only H. sciuri (MH656732) was detected. In Rodentia, Hepatozoon sp. vole isolate (MH656731) was detected in bank voles (Cricetidae) from the Netherlands and Croatia and H.ayorgbor (EF157822) in yellow necked mice and wood mice  wolves of which different numbers of organs (range 1 to 9) were screened to assess the distribution of H.canis, 71  had one or more positive organ(s)  or two (n = 34) organs were tested. Three and four organs were tested in six wolves each. Six organs were tested in four wolves, eight organs in one wolf and finally nine organs in two wolves. The organs that were most tested were spleen (n = 64), lymph node (n = 36) and skeletal muscle (n = 20). Bone marrow (n = 3) and blood (n = 1) were tested the least and brain (n = 7), myocardium (n = 10), lungs (n = 12), kidneys (n = 12) and liver (n = 13) in between.Of the 119 , followed by samples of lungs , lymph nodes  and bone marrow . The seven brain samples and one blood sample tested negative. Also, myocardium , liver , kidney  and skeletal muscle  samples tested positive.Hepatozoon prevalence and species diversity. Hepatozoon was detected in mammals from all five countries, with differences in prevalence. Hepatozoon prevalence of mammals in Austria and Bosnia and Herzogovina was ~ 3% compared to a prevalence in Croatia and Belgium/the Netherlands of 14–15%. The main reason for this difference in prevalence seems to be sampling bias. The animal species with the highest prevalence  from Austria. Also, in Austrian roe deer (C.capreolus), H.canis was detected. Broader host specificity is known for H.martis and H.canis, although the species were detected in other Carnivora, Canidae and Mustelidae [H.canis in the spleen samples of roe deer and chamois therefore represents an unexpected finding. Although prevalence was low, current detection could suggest lack of host specificity as seen in other tick-borne apicomplexans, e.g. Theileriacapreoli infecting gray wolves [In this study, 670-bp fragments of the 18S rRNA gene were used for phylogenetic analysis and species determination. Even though for this purpose amplifications of longer fragments  or next-H.canis , H.feliH.felis  and the .martis , H.sciu.sciuri  and H.aayorgbor . Surprisstelidae . The prey wolves .Hepatozoon species that are associated with Carnivores were detected . This is not surprising, since Hepatozoon transmission is known to take place by carnivores (intermediate hosts) ingesting infected ticks (definitive hosts) [H.canis not only in R.sanguineus, but also in D.reticulatus, I.hexagonus, I.ricinus, I.canisuga and I.ventalloi, which adds to the observations that other tick species than R.sanguineus may also be definitive tick hosts in the life cycle of H.canis [Hepatozoon since merely detection of the parasites in a tick is insufficient to designate the tick species as a vector.In the 31 positive (4.1%) ticks, only e hosts) . We founH.canis , 94. MorHepatozoon species could be identified: H.sciuri (in squirrels) and H.ayorgbor (in A.flavicollis and A.sylvaticus). The Hepatozoon sp. detected in bank voles (M.glareolus) from Croatia and the Netherlands could not be identified to species level. All Hepatozoon spp. sequences of bank voles from this study were (nearly) identical to each other and to Hepatozoon sequences from voles in GenBank®  is the first reported in European mammals. In the life cycle of H.ayorgbor, snakes can be infected via predation of rodents, with rodents serving as paratenic or intermediate hosts and mosquitoes as definitive invertebrate hosts [H.ayorgbor was detected and of which the sequence from GenBank® (MZ475989) was used in our sequence analysis was truly infected or merely contaminated via blood feeding on an infected host is therefore not clear. Hepatozoonayorgbor-like sequence was detected in three spleen samples of great gerbils in northwestern China [H.ayorgbor in the blood and liver of a ball python (Pythonregius) fed with tissues of mice experimentally infected with H.ayorgbor [H.ayorgbor- or H.ayorgbor-related genotypes.The species that were detected in rodents were not detected in the tested ticks. This could mean that ticks are not involved in the life cycle of these  mammals ) and/or te hosts , but finte hosts  suggest rn China , sharingnk® MZ47589 was usayorgbor . TogetheHepatozoon in carnivores and rodents from the Netherlands and Belgium are the first reported, but our study is also the first performed regarding detection of Hepatozoon in those countries. The species that were identified in mammals from the Netherlands and Belgium were H.canis in gray wolves and red foxes, H.martis in stone martens, pine martens and European polecats, and H.sciuri in squirrels. Even though a high prevalence of Hepatozoon was found in wild carnivores from the Netherlands/Belgium, to our knowledge, in The Netherlands no autochthonous Hepatozoon spillover from wild to domestic carnivores has been reported so far. No Hepatozoon was detected in the investigated Dutch and Belgian ticks. This could be because transmission of H.canis in for example foxes takes place via ticks in fox burrows  or via canisuga , which mle Table .The results of the investigated gray wolves show that spleen samples are most likely to test positive in case of a positive animal, which is in accordance with other reports .Hepatozoon is widely present in wild mammals and ticks originating from several countries in West, Central and Southeast Europe. Presence of Hepatozoon was confirmed in ticks other than the ‘usual suspect’ R.sanguineus. Besides confirming presence of Hepatozoon in wild mammals and ticks in countries in which Hepatozoon was previously detected, presence of this tick-borne parasite in the Netherlands/Belgium was demonstrated for the first time, even though circulation in ticks could not be confirmed. Since spillover from wildlife to domestic animals in countries where Hepatozoon is endemic occurs, veterinary clinicians in the Netherlands/Belgium should be aware of the presence of this tick-borne disease.Our results show that Additional file 1: Table S1. Overall prevalence of Hepatozoon and species in tested animal species and among countries.Additional file 2: Table S2. Hepatozoonayorgbor isolates from Croatian small wild rodents compared to the python isolate EF157822. Table S3. Hepatozoon sp. isolates infecting Croatian small wild rodents compared to Spanish Myodesglareolus isolates AY600625 and AY600626.Additional file 3: Table S4. Distribution of H.canis in organs of gray wolves.


The decline in motor function associated with chronic ankle instability (CAI) can be assessed using Functional Performance tests. Ankle muscular strength, endurance and range of motion (ROM) has been assessed in previous studies but functional activities such as sprinting and change of direction are less well studied in athletes with CAI. Hence the aim of this study was to determine how sprint, change of direction, ankle isometric strength, endurance and ROM measures may be associated with discriminate athletes with and without CAI.n = 53 or no CAI: n = 53) provided informed consent to participate in this study. Participants performed three functional performance tests, ) and change of direction test. Range of motion for dorsiflexion was measured using weight bearing lunge test and inversion, eversion and plantarflexion using Saunders® digital inclinometer. Strength was assessed using Baseline® hand-held dynamometer for plantarflexors, dorsiflexors, invertors and evertors. Muscular endurance was assessed by single heel raise test and Modified single heel raise test. Between-group comparisons utilised Student’s t-test and Mann-Whitney U-tests, with a number of unique variable and multivariable binomial logistic regression performed to determine which performance measures may discriminate participants with CAI.One hundred and six participants (CAI: p < 0.008). While several measures of ROM (plantarflexion and dorsiflexion), strength (inversion and eversion) and both muscular endurance tests were significantly associated with CAI in the univariable analysis, the strongest association was the functional performance tests, especially MICDOT time (odds ratio (95% CI): 0.06 [0.02–0.17], sensitivity 94.3%, specificity 88.7%). Multivariable regression analyses indicated that performance across the functional performance tests were more strongly associated with CAI than any ankle ROM, muscular strength or endurance test. Further, the inclusion of the best ankle range of motion, strength or muscular endurance tests did not significantly improve upon the association of the MICDOT with CAI.The CAI participants performed significantly worse in the three functional performance tests as well as multiple measures of ankle ROM, isometric strength and muscular endurance (Chronic ankle instability in athletic populations appears to be highly associated with declines in functional performance and to a somewhat lesser extent, ankle range of motion, strength and muscle endurance measures. This may suggest that optimal rehabilitation for athletes with CAI may require a greater focus on improving sprinting speed and change of direction ability in the mid to latter stages of rehabilitation, with regular assessments of these functional performance tests necessary to guide the progression and overload of this training. One of the most frequent musculoskeletal injuries among athletes is lateral ankle sprain (LAS), accounting for 25% to 30% of injuries . Out of n = 48–82), mixed populations , and the between-study differences in the potential predictor variables assessed  and self-reported measures of function. The assessment of objective and subjective ankle function is vital to tracking rehabilitation milestones and is commonly assessed in the CAI literature. A recent meta-analysis indicates some, but definitely, not all of these muscular strength assessments are associated with CAI . Furtherassessed . TherefoFunctional performance tests (FPTs), when performed together with ankle muscular strength, endurance, and ROM tests, may provide a more comprehensive assessment of performance deficits than ankle function tests alone . Some coThe reductions in general physical and specific training in athletes with CAI are likely to negatively affect their performance ability in sprinting and change of direction (COD) tasks . In thesThis case-control study included 106 participants (53 with and 53 without CAI) between the ages of 18–30 years. The sample size for the study was calculated using G*power software for a multiple linear regression test. For a medium effect size and 13 predictor variables, considering a 1 percent margin of error and 90% study’s statistical power, the total sample size required for this study is 99 (50 per group). The sample size was achieved, whereby we had 53 participants in each group . Using a convenience sampling approach, participants were recruited and 53 matched participants each were divided into two groups between December 2019 and February 2021. These participants were involved in university-level sports, including football (34%), basketball (17.9%), track and field (17.9%), badminton (17%), and volleyball (13.2%). The study was approved by the institutional ethics committee (IEC), KMC Mangalore (KMC MLR 11-19/584). Potential participants read and provided their written informed consent before participation. The study was conducted following the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement.Participants were divided into two groups, those with and without CAI, based on several criteria. Based on the inclusion criteria stated by International Ankle Consortium , athletei.e., 30-m sprint test, MICODT, and change of direction test. All the FPTs were performed in random order. Each test was performed three times, and the best time of the three trials was recorded. The participant was given a rest interval of 30 s after each trial and 1 min after three trials of each FPT.Participants performed 10 min of a self-selected warm-up prior to performing the assessments. Assessments of ankle ROM were performed first, followed by muscular strength, muscular endurance tests and finally, FPTs. Participants were provided with a demonstration and verbal instructions prior to each test, with familiarization and two submaximal trials also provided for the FPTs, The three FPTs were performed on a natural grass terrain, and participants wore running shoes without spikes. In the 30-m sprint test, the participant starts from a stand start and, on the researcher’s command, sprints for 30 m to the finish line, with timing assessed by trained researchers using a stopwatch. This sprinting speed assessment by a stopwatch has been reported to have excellent reliability (ICC = 0.95–0.97) , a resulRange of motion was assessed in a random order for ankle dorsiflexion (DF), plantarflexion (PF), inversion (INV), and eversion (EVE) using a Saunders® digital Inclinometer . Ankle DF was assessed using Weight Bearing Lunge Test (WBLT), where the participants performed the test with a digital inclinometer placed 15 cm below the tibial tuberosity . The WBLIsometric muscle strength was assessed randomly for ankle plantarflexors, dorsiflexors, invertors, and evertors using a Baseline® digital push-pull dynamometer . Isometric strength for ankle dorsiflexors, invertors, and evertors was assessed in the supine position and plantarflexors in the prone position, with force applied using a dynamometer at the metatarsal heads. Each participant was instructed to perform three maximal voluntary isometric contractions for each muscle group, each held for 3–5 s. All the assessments were performed supine with the participant’s hips and knees extended and the lower limb stabilized proximal to the ankle joint. These assessments have been reported to have excellent inter-rater reliability (ICC = 0.77 to 0.88) . The aveThe single heel raise test (SHRT) and modified single heel raise test (MSHRT) were used to assess muscular endurance . For theShapiro-Wilk tests identified normal distribution for all three FPTs and non-normal distribution for most of the ankle ROM, strength, and muscular endurance variables. Thus, between-group comparisons were conducted using Student’s t-tests for the three FPTs while the Mann-Whitney U-test for the remaining outcomes. Cohen’s D for the between group differences was determined by calculating the mean difference between two groups, and dividing it by the pooled standard deviation using Microsoft Excel.p < 0.05 was considered statistically significant.Univariable and multivariable binomial logistic regression were used to gain insight into the odds of the participants having CAI based on the FPT and ankle function tests performed in this study. Specifically, univariable binomial linear regressions were performed on each of the FPT, ROM, isometric strength, and muscular endurance measures . The firi.e., FPT, ROM, isometric strength, and muscular endurance outcomes. No significant differences were in sex, age, height, body mass, and body mass index (BMI). A significantly reduced CAIT score was observed for CAI compared to non-CAI participants.A summary of the FPT, ROM isometric muscle strength, and endurance outcomes for both groups is provided in A summary of the multivariable binomial logistic regressions determining the association between CAI and each of the four categories  is presented in One of the novel results of the current study was that significant functional performance deficits, as measured in the 30-m sprint, MICODT, and COD tests were identified in athletes with CAI compared to those without CAI. The question then emerges about how such functional performance deficits may be reduced in athletes with CAI. One approach might be to determine how characteristics such as functional performance in sprinting and change of direction task and more ankle joint specific tests of ROM, strength, and muscular endurance may be associated with CAI. Identifying such characteristics may then inform the primary focus of rehabilitation programs and return to play guidelines for athletes with CAI.A series of univariable and multivariable binomial logistic regressions were performed to achieve this aim. The univariable binomial logistic regression results indicated that the test most associated with CAI was the MICDOT. The association between the MICDOT and CAI appears more excellent than the results of any other similar studies, including the posterior talar glide test  or a varA series of multivariable binomial linear regression analyses were also performed to identify the combination of most highly associated tests with CAI. The first of these regression analyses was performed within each of the four categories of functional and ankle assessments. The FPTs had the strongest associations to CAI, with the lowest associations observed for the muscular endurance tests. The final multivariable binomial linear regression involved the inclusion of the most highly associated variable from each of the four categories of assessments. While this model, which included the MICDOT, dorsiflexion ROM, eversion strength, and single heel raise muscular endurance, was highly associated with CAI, such results were not substantially improved from that of the univariable binomial analysis involving the MICDOT in isolation. Combining the functional performance tests had the highest sensitivity at 84.9%  and, theIt is, however, unlikely that most athletes in the early-mid stages of rehabilitation from CAI would be able to safely perform high-intensity change of direction tasks as required in the MICDOT. In this stage of their rehabilitation, it might be prudent for the athlete to focus on improving aspects of ankle function that are most associated with CAI. Results of the current study indicated that participants with CAI typically had numerous significant reductions in ankle ROM, isometric strength, and muscular endurance compared to those without CAI, with all of these muscle function characteristics potentially influential in determining their functional performance. Specifically, participants with CAI had significantly reduced plantarflexion and dorsiflexion ROM, inversion and eversion strength, and muscular endurance as assessed in two different single leg heel raise tests. These findings are consistent with previous research, whereby deficits in a variety of measures of ankle function have been observed in individuals with CAI .Interestingly, in our study, participants with CAI showed significantly reduced plantarflexor performance in the two single leg muscular endurance tests but failed to demonstrate any significant differences in isometric plantarflexor muscular strength. Further, univariable binomial regression analyses indicated that of the four muscular strength and two muscular endurance tests, only plantarflexion and dorsiflexion strength were not significantly associated with CAI. Such results may have considerable clinical relevance to the assessments and exercise prescription required for athletes with CAI.Concerning the most appropriate ankle joint assessments for athletes with CAI, it is unclear what the relative differences in the predictive ability of the isometric dynamometry of the plantarflexors and dorsiflexors compared to simple, single leg muscular endurance tests represent. While our results suggest muscular endurance may be more critical than isometric strength, it may also be that the greater multiplanar ankle control required in the single-leg muscle endurance tests is more reflective of the challenges imposed on the ankle during high-intensity movements such as change of direction and sprinting . Thus, fExercise prescription applications of these results also suggest a focus on increasing ankle eversion (and perhaps inversion) strength, and single-leg calf raise performance should be emphasized. A recent training study involving 54 athletes with a recurrent lateral ankle sprain provides longitudinal support for this view. These athletes completed either 6 weeks of resistance training  or balance training  . At the i.e., young adult athletic populations with moderate levels of chronic ankle instability.One of the limitations of our study was that our case-control design does not allow us to predict the development of CAI; instead, it allows us to gain insight into factors associated with athletes who already have CAI. For studies wishing to determine what factors may predict the development of CAI, a well-designed prospective cohort study would provide insight into the potential predictive power of the FPTs and ankle function tests. Another potential limitation of the study was the potential for some fatigue effects during the testing session. However, pilot testing and participant feedback suggested such fatigue effects were minimal. In addition, we recorded the strength measures in absolute terms rather than normalizing to body mass which may be considered another limitation. The study results may also be limited to individuals with similar characteristics to those of the participants, Athletes with CAI demonstrated significantly slower sprinting and COD times, ankle ROM, isometric strength, and muscular endurance, which were indicative of their reduced functional performance compared to non-CAI participants. Logistic regression results indicated that performance in the MICODT was most predictive of CAI, with a specificity of 94.3% and sensitivity of 84.9%. Further, ROM of the plantarflexion and dorsiflexors, eversion isometric strength, and the number of repetitions performed in the SHRT were also predictive of CAI. These results indicate that athletes with CAI may initially need to focus their rehabilitation on improving plantarflexion and dorsiflexion ROM, eversion isometric strength, single leg calf muscular endurance, and focus in the later stages on improving their change of direction ability before they safely return to play.10.7717/peerj.13390/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.13390/supp-2Supplemental Information 2Click here for additional data file.


Scomber japonicus) meat in a refrigerator for 20 days. The inhibition of crude urease activity from Vibrio parahaemolyticus using HHP treatment was also investigated. The mackerel meat storage experiment demonstrated that production of trimethylamine (TMA) and volatile basic nitrogen (VBN), the main components of fishy smell, was significantly reduced on the 20th day of storage after the HHP treatment compared to the untreated mackerels. The results demonstrated that the increased ammonia nitrogen rates in the 2000, 3000, and 4000 bar, HHP-treated groups decreased by 23.8%, 23.8%, and 31.0%, respectively, compared to the untreated groups. The enzyme activity of crude urease was significantly reduced in the HHP-treated group compared to that in the untreated group. Measurement of the volatile organic compounds (VOCs) in mackerel meat during storage indicated that the content of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavor due to spoilage, were significantly reduced by HHP treatment. Collectively, our results suggested that HHP treatment would be useful for inhibiting the activity of urease, thereby reducing the fishy smells from fish and shellfish.In this study, the physicochemical changes related to fishy smell were determined by storing high hydrostatic pressure (HHP)-treated mackerel ( Fishes start losing freshness after death, and the content of TMA, ammonia, and VBN, which are the substances that cause fishy odor because of the activation of microorganisms, gradually increases. Thus, in the absence of appropriate processing mechanisms, the time by activating for consumption, is limited .High hydrostatic pressure (HHP), from the isostatic pressing process long used for ceramic materials, is now being applied to the food industry. Variations of the HHP process depend on the pressure-transmitting fluid (water or gas), temperature, and the specific product. The applied pressure and treatment time will depend on the type of product being processed and the expected final product. In general, for enzyme inactivation, a pressure higher than the pressure used for microorganism inactivation should be used .e.g., UV irradiation, low-temperature osmotic dehydration, radiation, etc.) [HHP treatment technology to improve food storage and preference is one of the technologies that can provide safer, high-quality, and value-added food than other non-thermal treatment technologies  .Fish spoilage occurs because of the presence of endogenous enzymes and spoilage microorganisms. It leads to rapid quality deterioration of fresh fish during handling and storage and limited shelf life of the product . HHP tre5Consequently, the application of HHP treatment technology has been increasing recently, and various effects of HHP treatment on fish have been reported depending on different factors, such as the nature of the species and its size and chemical composition , 9.Scomber japonicus) [Products studied by introducing HHP treatment technology include fruits, salad dressings, yogurt, juice, and processed products using rice . In addiponicus) .Sardinops sagax), Jack mackerel (Trachurus japonicus), and Pacific saury (Cololabis saira), are four fishes with external blue coloring. Mackerel is rich in lipids and proteins and contains a large number of nucleic acids, which facilitate cell division and replication, and large amounts of taurine, which is known to be effective in preventing stroke. In particular, the content of polyunsaturated fatty acids, such as EPA and DHA, is very high in mackerel [Mackerel, along with sardine  was purchased from a local market (Korea). The skin, gill, and viscera of the mackerel were aseptically removed on a clean bench. The meat was rinsed with sterile distilled water, placed in a sterile blender and ground with 0.05% ascorbic acid at 1,000 rpm for 30 s in an ice box.Mackerel  were used and cultured in lysogeny broth (LB) (pH 5.5) supplemented with 2% NaCl, 0.5% glucose, and 0.2% urea. After adding 2–3% of the pre-culture solution to the LB medium, it was incubated at 37°C for 6–7 h, followed by centrifugation at 6,992 ×g for 10 min at 4°C. The precipitate was washed twice with saline solution and washed with 20 mM phosphate buffer (pH 7.0). The cells were crushed using an ultrasonic crusher, centrifuged at 15,729 ×g for 10 min at 4°C, and the supernatant was used as crude urease enzyme.Crude urease enzyme was extracted from m et al. . V. paraThe mackerel meat and crude urease enzyme were vacuum-packed and placed on ice to minimize any temperature effects prior to HHP treatment. The samples were processed in a pressure chamber  with a volume of 215 L, and with tap water as the pressure transfer fluid. The pressure time was approximately 18-20 s per 1000 bar, and the pressure release time was less than 35 s. The initial water temperature was 19°C, and the increase in temperature due to adiabatic heating was 2°C per 1000 bar. The treatment groups were high-pressure treated in batches at 2000, 3000, and 4000 bar for 3 min, but the control group was not treated. Compression and decompression times were not included in the treatment. After treatment, the samples were stored at 4°C for 20 days, and TMA, VBN, pH, color values, volatile organic compounds (VOCs), and ammonia nitrogen were measured at 10-day intervals.Canavalia ensiformis) urease .Urease enzyme activity was measured using the method described by Weatherburn . Brieflyg for 10 min, and then filtered. The filtrate (4 ml) was mixed with 1 ml of 20% formaldehyde, 10 ml of anhydrous toluene, and 3 ml of saturated K2CO3 solution followed by vortexing for 1 min. The mixed sample was allowed to stand for 5 min, and the separated toluene supernatant was dehydrated by adding Na2SO4 for 1 min. The dehydrated supernatant was reacted with 0.02% picric acid (1:1 ratio), and the absorbance was measured at 410 nm.The TMA content of HHP-treated mackerel meat was measured by modifying the AOAC  method . HHP-tre2SO4 was added into the inner well of the Conway unit. Vaseline gel was applied at the contact part of the cover plate and the outer well. The cover plate was closed to approximately 2/3, and 1 ml of saturated K2CO3 solution was added to the right side of the outer well. The cover plate was then rapidly closed and fixed by a clip. The Conway unit was shaken to mix the outer well solution equally and incubated at 25°C for 1 h. The cover plate was opened, and one drop of the Brunswik indicator was added to the inner well. This was titrated by 0.01 N NaOH using a micro burette until the color changed to green. The titration was repeated twice.The VBN content was measured by modifying Conway's method of the Korean Food Standards Codex . HHP-treet al. [The ammonia nitrogen content was measured according to the method described by Kim et al. . HHP-treThe color values of mackerel meat treated with HHP were measured as lightness (L*), yellowness (b*), and redness (a*) using a colorimeter . The measurement was repeated at least five times to obtain an average value. The values of the standard color plate used were L * = 98.98, a * = 0.21, and b * = -0.28.n = 5) .HHP-treated (3 g) or untreated mackerel meat was added to 30 ml of distilled water and homogenized with a homogenizer for 2 min at 10,000 rpm. The pH of the homogenized samples was measured at room temperature using a pH meter . Statistical evaluation was carried out using analysis of variance with SAS software , according to Duncan's multiple range test (p < 0.05).Data were expressed as the mean ± SEM (Trimethylamine oxide (TMAO) in fish and shellfish is reduced to TMA by bacteria or enzymes after death, causing a fishy smell, and through this, the degree of spoilage is measured , 29. TMAIn this study, the changes in TMA levels during storage at 4°C in HHP-treated or untreated mackerel meat are reported in et al. [These results were similar to those of Gou et al. , whereinet al. , 29. TheWhen fresh mackerel is spoiled, the quantity of VBN contained in trace amounts in the meat increases. As fish decay progresses, proteins are decomposed into low-molecular substances, such as peptides, amino acids, and peptones, and the content of VBN increases . In addiIn this study, HHP-treated or untreated mackerel samples were stored at 4°C for 20 days, and the changes in VBN content were measured according to storage days . On day et al. [In general, when the VBN content is more than 50 mg/100 g, it is considered complete spoilage . The VBNet al. , who treThe color change of mackerel meat by HHP is shown in et al. [et al. [Citrus junos or Prunus mume immersion solution and HHP in mackerel. Choi et al. [This result is similar to the study by Cruz-Romero et al.  which sh [et al.  reportedi et al.  also repi et al. . TherefoThe pH value of fresh fish after death is usually around 5.5-6.5 , and theIn this study, we found that the pH values of the untreated group and the 2000 bar-treated group significantly increased from 5.49 to 6.47 on day 0, and 5.59 to 6.25 on day 20 . Howeveret al. [Shewanella putrefaciens, which is considered to be the major TMA-producing bacteria in seafood. It is widely known that an increase in the pH value of fish muscle leads to the accumulation of undesirable alkaline compounds, such as ammonia and TMA, which are primarily derived from microbial activity [These results were similar to those reported by Reyes et al. , which sactivity .As a result of measuring the ammonia nitrogen content during the storage period of 0–20 days , the ammIn aquatic organisms, various products are produced as final metabolites of nitrogen compounds, like ammonia. In general, when the freshness of fish decreases, a severe ammonia odor occurs because the urea contained in muscles is decomposed by urease secreted by bacteria to produce ammonia . When foTherefore, the urease activity secreted by microorganisms present in fish can be inhibited by high hydrostatic treatment, thereby suppressing the fishy smell.V. parahaemolyticus treated with HHP is shown in The activity of the crude urease enzyme extracted from The results of this study demonstrated that the urease activity was inhibited by HHP treatment, and it can be assumed that HHP treatment will be useful for the inhibition of fishy smell by inhibiting urease activity.To evaluate the change in the contents of VOCs in HHP-treated mackerel meat during storage for 20 days, a total of 38 VOCs were detected, including 14 aldehydes, 5 alcohols, 4 ketones and 15 other compounds (data not shown). The contents of ethanol, 2-butanone, 3-methylbutanal, and trans-2-pentenal, which are known to cause off-flavor due to fish spoilage, are shown in Ethanol is a common volatile compound generated by the microbial decay of fish -45. Lerket al.[During storage for 20 days, the contents of 2-butanone in the untreated group and the groups treated at 2000, 3000, and 4000 bar increased by 209.18%, 128.51%, 69.19%, and 109.28%, respectively. The results indicated that the increase in the rate of 2-butanone content by 2000, 3000, and 4000 bar HHP treatment was decreased by 38.56%, 66.92%, and 47.75%, respectively, compared to the untreated group. Subsequently, Jonsdottir 3-methylbutanal content was detected in any group on day 0. The content of 3-methylbutanal in the groups treated with 2000, 3000, and 4000 bar pressure decreased by 89.33%, 84.46%, and 88.0 3%, respectively, compared to the untreated group on the 10th day of storage and by 88.34%, 89.82%, and 91.58%, respectively on the 20th day of storage.Previous studies have reported that the production of various short-chain alcohols, aldehydes, and ketones, such as ethanol, 3-methylbutanal, and 2-butanone, is a result of metabolic activity during fish spoilage by microorganisms -50. In aTherefore, HHP treatment is believed to delay the spoilage of fish by microorganisms, thereby reducing off-flavor, and our study suggested the possibility of inhibiting the action of urease by reducing the microorganisms.


In this report, surface-enhanced Raman scattering (SERS) technology was used to achieve rapid detection and identification of clenbuterol hydrochloride (CB) residues. First, the effects of several different organic solvents on the extraction efficiency were compared, and it was found that clenbuterol in pork had a better enhancement effect using ethyl acetate as an extraction agent. Then, SERS signals of clenbuterol in different solvents were compared, and it was found that clenbuterol had a better enhancement effect in an aqueous solution. Therefore, water was chosen as the solvent for clenbuterol detection. Next, enhancement effect was compared using different concentration of sodium chloride solution as the aggregating compound. Finally, pork samples with different clenbuterol content  were prepared for quantitative analysis. The SERS spectra of samples were collected with 0.5 mol/L of NaCl solution as aggregating compound and gold colloid as an enhanced substrate. Multiple scattering correction (MSC) and automatic Whittaker filter (AWF) were used for preprocessing, and the fluorescence background contained in the original Raman spectra was removed. A unary linear regression model was established between SERS intensity at 1472 cm-1 and clenbuterol content in pork samples. The model had a better linear relationship with a correlation coefficient R2 of 0.99 and a root mean square error of 0.263 µg/g. This method can be used for rapid screening of pork containing clenbuterol in the market.Clenbuterol (CB) is a synthetic  Clenbutlalcohol . Feedinglalcohol ,6. Many lalcohol . TherefoThere are many ways to detect CB, including high performance liquid chromatography (HPLC) , capilla4–106 times. In recent years, SERS technology has achieved significant progress in detection for trace analysis of prohibited or restricted chemicals, such as food additives [Surface-enhanced Raman spectroscopy (SERS) is a novel analytical tool providing ultrasensitive detection based on the enhanced local electromagnetic field near the nanostructured noble metal surfaces, which allows trace level detection in combination with detailed qualitative information, giving characteristic responses for each molecule that are true fingerprints of a certain compound ,16. SERSdditives , pesticidditives , and antdditives .Sample preparation has always been the main bottleneck of chemical residue detection in food. In particular, Raman spectra contain strong background signals and irrelevant characteristic bands because livestock and poultry meat contain more complex substances such as protein, fat, and pigment, when target substance in livestock and poultry meat are detected using SERS. The fluorescence background and characteristic bands produced by irrelevant components will greatly disturb the Raman signals of the target substance, and even mask the Raman signal of the target substance.18 sorbent, PSA sorbent, enhanced matrix removal-lipid kit, MgSO4, and other reagents to remove the matrix are often used in the purification process, which is expensive and time consuming. Therefore, the development of a simple and effective sample pretreatment process can greatly improve time and efficiency.The pre-processing steps of clenbuterol detection using liquid chromatography with tandem mass spectrometry (LC-MS) are as follows: extraction, centrifugation, degreasing, filtration, concentration, and purification . These sIn this report, the effects of several different organic solvents on the extraction results were explored. Then, comparing clenbuterol signals in several different solvents, ultrapure water was chosen as the solvent for clenbuterol detection. Finally, the unary linear regression model between clenbuterol content in pork and SERS intensity was established under optimal detection conditions. 4∙4H2O) was purchased from Shenyang Jinke Reagent Co., LTD . The pure product of clenbuterol hydrochloride  was received from Beijing Shengda-Huijie Technology Co., LTD . Sodium citrate (C6H5Na3O7) and sodium chloride were obtained from Shanghai Macklin Technology Co., LTD . Ethyl acetate (C4H8O2), acetone, methanol, and acetonitrile were purchased from Beijing Chemical Plant. All reagents were dissolved and diluted with ultrapure water. The fresh pig muscle tissues were bought at Beijing Xing-fu Supermarket. All glassware was cleaned with aqua regia before the test.Chloroauric acid tetrahydrate  was obtained from Mettler Toledo Measurement Technology Co. Ltd. . Vortex mixing (QL-901) was received from Haimen Qilin Bell Instrument Manufacturing Co., Ltd. . Constant temperature magnetic stirrer (88-1) and centrifuge (TDZ5-WS) were purchased from Changzhou Guohua Electric Co., Ltd.  and Hunan Xiang-xi Instrument Equipment Co., Ltd. , respectively.The Raman spectra acquisition system used in this paper consists of a Raman spectrometer ), a 785 nm laser, optical fiber and power. For spectra acquisition, the integral time was set to 4 s and laser power was measured to at 100 mW. A UV/vis Spectrophotometer 756S was used to collect UV/vis absorption spectra of gold colloid in the 1190–1100 nm spectral range. The TEM image of gold colloid was obtained by a Transmission electron microscopy .Gold colloid was synthesized by reducing chloroauric acid tetrahydrate with sodium citrate . The proA total of 10 mg of clenbuterol was added to 100 mL of ultrapure water to obtain 100 µg/mL of clenbuterol aqueous solution. The 100 µg/mL of clenbuterol aqueous solution was diluted to 6 different concentrations of 1, 3, 5, 7, 9, and 10 µg/mL by ultrapure water. Tissue samples (2 ± 0.01 g) were ground, weighed, and added to a 15 mL plastic centrifuge tube. Then, 200 µL of 10 µg/mL clenbuterol solution was added. The mixture was vortexed and mixed for 1 min. Thus, a standard sample of pork containing clenbuterol (10 µg/g) was obtained. Likewise, pork samples containing clenbuterol  were prepared by adding 200 µL of clenbuterol aqueous solutions with different concentrations. The clenbuterol content of the prepared pork samples was calculated by mass ratio of the actual added clenbuterol and pork mass. Three pork samples were prepared for each concentration.The pre-processing steps of samples before detection are shown in As shown in R2) and root mean square error of correction (RMSEC) [Affected by various factors such as system fluctuations, changes in the external environment, and the fluorescence background generated by impurities, the obtained spectra signals contained other irrelevant information and fluorescence interference in addition to the information of the target substance. Therefore, the fluorescence background of the Raman spectra is removed using automatic Whitacre fitting algorithm (AWF), and multiple scattering correction (MSC) was used to eliminate baseline drift. The original and pretreated SERS spectra of clenbuterol in pork are shown in  (RMSEC) . If the  (RMSEC) . MATLAB UV/vis absorption spectra were used to characterize the optical properties of the colloidal solution, shown in −1 were attributed to clenbuterol. The in-plane deformation of the phenyl ring coupled to C-C-Cl deformations band is at 382 cm−1; the band at 647 cm-1 is attributed to out-of-plane ring deformation; the band at 787 cm−1 can be assigned to stretching vibration of C-Cl coupled to in-plane stretching ring; the band at 1259 cm-1 is linked to the stretching of C-N coupled to in-plane ring deformations; the band centered at 1472 cm−1 is assigned to the in-plane stretching vibrations coupled with the anilines C-N stretching vibration [−1 can be assigned to the C = C stretching band of the aromatic ring [−1 in different solvents, from which we can see that clenbuterol has the best enhancement performance in aqueous solution. This may be due to clenbuterol in organic solvent being difficult to bind to gold colloid due to the competitive adsorption of organic solvent molecules. Subsequently, ultrapure water was used to redissolve the extracted clenbuterol.In this report, SERS spectra of clenbuterol (10 µg/mL) in four different solvents of water, acetonitrile, acetone and methanol were compared. From ibration ; and thetic ring . Figure 2SO4 was added to absorb excess moisture when methanol was used as an extractant. Since clenbuterol has the best enhancement in aqueous solution, ultrapure water was chosen to redissolve the residue of the concentrated extract. As shown in −1 of clenbuterol can be observed, which have the same bands as the clenbuterol aqueous solution. The band at 1027 cm−1 observed on curves a and b was caused by fat in pork. As can be seen from curves c, d, e, and f, when acetonitrile and methanol were used as extraction agents, the SERS spectra of clenbuterol in the pork had similar bands to the control group, so they could not be used to determine whether it contained clenbuterol or not. Therefore, ethyl acetate has a better extraction effect on clenbuterol from pork.CB is a weakly alkaline compound (pKa = 9.6). When pH > pKa, CB is soluble in organic solvents. Otherwise, the CB is soluble in water when pH < pKa . When etThe enhancement performance will be influenced by the charge properties and binding degree between metal nanoparticles and different molecules. The charge balance is upset when electrolytes are added, which causes particles to coalesce . The con−1 using NaCl solution with different concentrations as aggregating compounds are shown in −1 first increased and then decreased, which may be due to the gradual aggregation of uniformly dispersed gold nanoparticles. Since clenbuterol is attached to the gold nanoparticles, when the gold nanoparticles gradually gather, the quantity of the target substance at the “hot spot” gradually increases. With the aggregation of gold nanoparticles, agglomeration precipitation will occur, which is not conducive to the enhancement of Raman signals of target molecules. As can be seen from As shown in −1 are shown in −1 were 6.9%, 6.2%, 6.0%, 4.6%, 5.1%, and 5.0%, respectively. The results show that this method has a better repeatability and stability.A total of 20 samples of pork contenting clenbuterol (10 µg/g) were selected to verify the repeatability of the proposed method. SERS spectra of pork containing clenbuterol (10 µg/g) are shown in Pork samples with different clenbuterol content  were prepared for quantitative analysis. Ethyl acetate and 10% sodium carbonate solutions were selected as extractants, and the pork samples were treated using the pre-treatment method described in −1 was selected for data analysis. As is shown in −1 is as follows:−1.Since the original spectra collected had different fluorescence background signals, MSC was used to eliminate baseline drift, and AWF was used to remove the baseline. The characteristic band intensity of clenbuterol at 1472 cm−1 of spectra were interpolated into the linear equation to verify the prediction performance of the model. As is shown in R2 = 0.99) and lower root mean square error (RMSEC = 0.263 µg/g).The peak intensities at 1472 cmIn this report, SERS technology was used to detect clenbuterol in pork. Compared with the traditional detection method, this method has the advantages of simple operation, short detection time and fast detection speed, which can be used for the crude screening of pork containing clenbuterol in the market. However, the detection limit is 1 µg/g, which is relatively high. A better enhancement substrate is needed to reduce the detection limit in the future. In addition, with the rapid hardware update of Raman system and the gradual deepening of stoichiometry, the detection limit will be gradually reduced.−1 and clenbuterol content in pork samples. The established model had a better linear relationship, with a correlation coefficient R2 of 0.99 and a root mean square error of 0.263 µg/g. This method can be used for rapid crude screening of pork containing clenbuterol in the market.In this report, different extraction agents were compared, and it was found that clenbuterol in pork had a better enhancement effect using ethyl acetate as an extraction agent. Furthermore, the clenbuterol signals in several different solvents were compared, and it was found that clenbuterol had a better enhancement effect in aqueous solution. Therefore, water was chosen as the solvent for clenbuterol detection. Then, NaCl with different concentrations as aggregating compounds were compared, and the results showed the enhancement effect was best using 0.5 M NaCl as the aggregating compound. Finally, pork samples with different clenbuterol content  were prepared for quantitative analysis. The SERS spectra of samples were collected with 0.5 M of NaCl solution as the aggregating compound and gold colloid as an enhanced substrate. MSC and AWF were used to eliminate baseline drift and remove the fluorescence background contained in the original Raman spectra, respectively. A unary linear regression model was established between SERS intensity at 1472 cm


Athletes with a history of ankle sprain that do not suffer from ankle instability (copers) exhibit similar balance and neuromuscular control compared to healthy athletes.Athletes with chronic ankle instability show a deficit in neuromuscular control and balance, which may increase the risk of subsequent injuries.Athletes with chronic ankle instability are advised to use neuromuscular training in their rehabilitation program to improve balance and neuromuscular performance to reduce the risk of injury.More than 70% of people with ankle sprain experience chronic ankle instability. However, some people are well adapted to this damage (copers) and do not suffer from chronic ankle instability (CAI). This cross-sectional study involved 34 female athletes, who were classified into three groups  and tested on a Biodex Balance System. Surface electromyography (EMG) and balance scores were monitored. The coper and healthy group exhibited higher medial gastrocnemius (MG) EMG activity during unstable balance conditions. The rectus abdominus (RA) in the coper group and rectus femoris (RF) in the healthy group showed greater EMG activity compared to CAI during unstable conditions. During stable conditions, the coper group showed greater RA EMG activity compared to CAI, as well as higher tibialis anterior (TA) EMG activity compared to the healthy group. Additionally, balance error scores were higher in the CAI group than those in the healthy group under unstable conditions. In conclusion, decreased EMG activity of the MG, RF, and RA in CAI athletes may contribute to impaired balance in these individuals. The increased EMG activity of the MG, TA, and RA in copers might result in more trunk and ankle stability. Ankle sprains are one of the most prevalent musculoskeletal injuries, with athletes in the United States reporting ankle sprain accounting for 15% of total injuries  that leaCompared to healthy individuals, patients with a history of ankle sprain during single-leg stance demonstrate a dominant postural control strategy from the hip rather than the ankle . ResearcIn the case of copers, they showed a comparable postural control strategy to healthy individuals during open and closed eyes in a single-leg stance test ,11,15; hPrevious research has shown that during various balance tasks, people with CAI exhibited atypical levels of muscle activation than copers ,22,23,24Thus, it was hypothesized that the muscles involved in maintaining balance are different between individuals with CAI, compared to the copers group, and this can lead to balance disorders in these individuals. Secondly, it was also hypothesized that muscle activity of these individuals can differ between stable and unstable surfaces while balancing on them. Another hypothesis contends that individuals with CAI have reduced ankle muscle activity due to injury, and are more likely to activate the proximal muscles to compensate for this deficiency. By examining these cases, we can have a better understanding of copers for adapting to injury, which will be important in improving balance control and problems in people with CAI. Therefore, the purpose of this study was to investigate the balance and neuromuscular activity of the muscles involved in postural control strategies, such as tibialis anterior (TA), medial gastrocnemius (MG), rectus femoris (RF), biceps femoris (BF), erector spinae (ES), and rectus abdominis (RA), during a single-leg stance between CAI, copers, and healthy athletes.A sample of thirty-four female athletes were allocated to three groups: CAI, copers, and healthy athletes . The samFive minutes of self-paced walking was completed as a warmup. All participants wore sports clothes and shoes during warm-up, and short tops and shorts during measurements. After the warmup, participants were familiarized with the balance tests at two levels of difficulty (level 3: unstable and level 12: stable) on the Biodex Balance System (BBS) system. All participants were given a practice session using the same testing protocol, as a familiarization with the balance platform. For the CAI and coper groups, all measurements were taken from the injured limb, and for healthy athletes (control group) from the dominant leg, which was defined as the leg used to kick a soccer ball . AthleteBalance scores was recorded at two levels of stability (level 3: unstable and level 12: stable) using the BBS athlete single-leg test. If the postural stability was not met during the test, the results from the related trial were nullified and the participant repeated the trial once more. The average scores of the anterior–posterior and medial–lateral indices from three trials were used for statistical analysis. The BBS measures the amount of body tilt around each axis in dynamic and closed-chain conditions, provides stability indices, and calculates the stability indices of anterior–posterior, medial–lateral stability, and the overall stability index . Lower values indicate better balance of the individual.EMG  activity from TA, MG, RF, BF, ES, and RA were recorded at a sampling frequency of 1000 Hz  = 110 dB, input impedance = 10 Gohm, Gain: 305). The skin was prepared by removing excess hair, abrading, and cleaning with an alcohol swab. The electrodes  were placed on the TA, MG, RF, BF, ES, and RA muscles following SENIAM guidelines, as illustrated in The Brown–Forsythe F-test was used to compare the difference in height, weight and body mass index (BMI) between the three groups due to unequal sample sizes between groups. Non-parametric Kruskal–Wallis H tests were used for EMG activity for each muscle and BBS scores at both level 3 and level 12 of BBS. If a significant effect was found, a post hoc Mann–Whitney U test was carried out to identify the differences among groups . Furthermore, Wilcoxon signed ranks tests were used to compare EMG activity for each muscle and BBS scores between two difficulty levels of the BBS. Alpha level was set to 0.05 for Kruskal–Wallis H tests. A Bonferroni correction on the alpha level was calculated for Mann–Whitney U and Wilcoxon signed ranks tests to reduce type I error. Effect sizes were calculated for the non-parametric tests (r) in which the values were considered small (0.1–0.29), medium (0.3–0.49), and large (>0.5) . All stap > 0.05).MG activity was higher in the coper (↑133%) and healthy athletes (↑116%) groups compared to the CAI group. RA activity in the coper group was elevated compared to the CAI group (↑77%). RF activity was higher in the healthy athletes (↑148%) compared to the CAI group . No signThe coper group showed higher (↑68%) RA EMG activity compared to the CAI group. Additionally, TA muscle had greater EMG activity in the coper group (↑105%) compared to the healthy athletes. RF EMG activity was greater in healthy athletes (↑166%) compared to the CAI group .p > 0.05).EMG muscle activity of the TA in the CAI (↑76%) and healthy (↑64%) groups; BF in the CAI (↑39%) group; RF in the CAI (↑46%) and coper (↑47%) groups; ES in the CAI (↑54%) and healthy (↑11%) groups; and RA in the CAI (↑6%) and coper (↑12%) groups had significantly higher EMG activity from level 12 (stable) compared to level 3 (unstable) BBS . The MG (2) = 7.346, p = 0.025) medial–lateral balance error scores (↑142%) compared to the healthy group . The overall balance error score was greater (H(2) = 8.051, p = 0.018) in CAI (↑106%) compared to the healthy athletes .The CAI group showed increased (H(2) = 7.341, p = 0.025) compared (↑44%) to the healthy athletes . No further difference was observed between the CAI, coper, and healthy groups (p > 0.05).The copers achieved overall higher balance error scores , CAI (↑124), and healthy (↑60%) groups in the unstable condition. Greater values were found for anterior–posterior scores for copers (↑42%) and CAI (↑49%), as well as overall balance for copers (↑102%) and CAI (↑82%) in the unstable condition . The primary findings of this study were that individuals with CAI showed a large magnitude (effect size) decrease in EMG activity for proximal muscle groups (MG) and distal (RF and RA) to the ankle during a single-leg stance. A secondary finding was that both medial–lateral and overall balance during unstable conditions demonstrated significant, large magnitude, higher  indices in the CAI group than the healthy controls.Previous studies showed a decrease in ankle, knee, and hip EMG activity in people with CAI during functional balance tasks  ,22. SimiIn addition to the muscles supporting the ankle joint, muscles distal from the ankle  showed decreased EMG activity in people with CAI. A previous study partially supports these findings, reporting that patients with CAI demonstrated a decreased hip and ankle muscle activity during performance of the Star Excursion Balance test (SEBT) . The altAnother finding was that no significant difference in balance indices or EMG activity in unstable conditions were found between healthy controls and copers. Very little work has been done on the BBS; however, other results support the findings of this study . RegardiWith regards to balance performance, two studies found no significant difference in the performance scores during a SEBT between copers and healthy individuals ,41. For The results of this study demonstrated that the EMG activity of the CAI group was lower than the other two groups during single-leg stance. Individuals with CAI presented a decrease in EMG activity of muscles both distal (RF and RA) and proximal (MG) to the ankle during single-leg stance in the unstable condition; however, it is difficult to conjecture with certainty which strategy in these individuals is more likely to contribute to maintaining the balance with a single-leg stance. Moreover, people with CAI had balance deficits in the medial–lateral direction. Reduced muscle activity in individuals with CAI could be one of the reasons for the balance deficits in these individuals. Increased EMG activity of the MG, TA, and RA in copers might contribute to compensatory mechanisms, which results in greater trunk and ankle stability.


An analogous population of GATA3-expressing Tregs was also identified in human melanoma tumors. Our study provides insights into the role of TSLP in programming a protumoral immune microenvironment in cutaneous melanoma.Malignant melanoma is a major public health issue displaying frequent resistance to targeted therapy and immunotherapy. A major challenge lies in better understanding how melanoma cells evade immune elimination and how tumor growth and metastasis is facilitated by the tumor microenvironment. Here, we show that expression of the cytokine thymic stromal lymphopoietin (TSLP) by epidermal keratinocytes is induced by cutaneous melanoma in both mice and humans. Using genetically engineered models of melanoma and tumor cell grafting combined with TSLP-KO or overexpression, we defined a crosstalk between melanoma cells, keratinocytes, and immune cells in establishing a tumor-promoting microenvironment. Keratinocyte-derived TSLP is induced by signals derived from melanoma cells and subsequently acts via immune cells to promote melanoma progression and metastasis. Furthermore, we show that TSLP signals through TSLP receptor–expressing (TSLPR-expressing) DCs to play an unrecognized role in promoting GATA3  Cutaneous malignant melanoma is the most aggressive of human skin cancers, causing the majority (75%) of skin cancer–related deaths with an incidence of 15–25 per 100,000 . Immune Keratinocytes, the major cellular component in skin, actively participate in regulating skin and systemic immune responses, but their role in shaping the melanoma TME has been largely ignored. Thymic stromal lymphopoietin (TSLP), a pro-Th2 cytokine predominantly expressed by epithelial cells and keratinocytes, was initially recognized in driving pathogenesis of atopic diseases, including atopic dermatitis, asthma, and allergic march , 6, and V600E expression with or without ablation of the tumor suppressor Pten gene in melanocytes  treatment induces oncogenic Brafanocytes , 16, nampatients , activatpatients  and, in patients . Using tpatients  with itspatients –21 in BrTyr:Cre-ERT2(Tg/0)BrafLSL–V600E/+Ptenlox/lox mice by breeding Tyr:Cre-ERT2(tg/0)BrafLSL–V600E/+ , in agreement with previous reports BrafLSL–V600E/+Ptenlox/lox mice)  . RNA in ox mice) . These r–/–Tslp background (named Braf/Pten/Tslp–/–) by breeding the Tyr:Cre-ERT2BrafLSL–V600E/+Ptenlox/lox mice with –/–Tslp mice : BrafLSL–V600E/+ (Braf mice), appearing 2 months after Tam injection or topical 4-HT treatment with melanomas barely observed within 6 months, as previously reported  mice that lack mature T, B, NK, and innate lymphoid cells (ILCs) . Calculation of tumor cell areas on REs and LEs showed that the RE/LE ratio was much higher in WT mice compared with –/–Tslp mice  grafted B16F10 melanoma cells into ears of WT C57BL/6J mice or immunodeficient NOD.s (ILCs) . We treaa growth . MC903 p–/– mice , demonst–/– mice . TogetheCrlf2 gene with CD11c-Cre transgenic mice (Tg/0/Crlf2L2/L2 mice (named as Crlf2CD11c–/–) together with their CD11c-Cre0/0/Crlf2L2/L2 CT littermates were i.d. grafted with B16F10 cells, followed by MC903 treatment on RE and EtOH on LE, as shown in Crlf2CD11c–/– mice  cell responses in mice , 27 and nic mice . CD11c-C–/– mice , suggestOn the other hand, we investigated whether TSLP was induced by signals derived from melanoma cells or from immune cells. ELISA measurement showed that TSLP protein levels were elevated in B16F10-grafted WT or NSG ears in presence of melanomas . TSLP inTogether, the above data suggest that melanoma-derived signals induced TSLP expression in keratinocytes that, in turn, signals through DCs expressing TSLPR to trigger immune cascades promoting melanoma growth and metastasis.Tslp–/– mice and draining dorsal melanomas of Braf/Pten/Tslp+/+ and Braf/Pten/Tslp–/– mice. Expression of Th2-type cytokines IL-4, IL-5, and IL-13 and chemokines CCL17 and CCL22 was upregulated in Braf/Pten/Tslp+/+ mice compared with WT mice, but it was diminished in Braf/Pten/Tslp–/– mice, in agreement with the recognized role of TSLP in promoting Th2 response . In addition, highest levels of IL-10 were observed in GATA3+ Tregs, followed by GATA3– Tregs and GATA3+ Th2 cells, although levels remained weak for all populations  or EtOH (on LE) as described in + Tregs were significantly reduced in Crlf2CD11c–/– mice, in both frequency and cell number . Gating CD4+ T cells with ST2 and Foxp3 (+Foxp3+ (ST2+ Tregs) indeed exhibited GATA3 and OX40 expression compared with ST2–Foxp3+ (ST2– Tregs)  . Moreove–/– ILNs , in a ma at different ratios from 1:8 to 1:1 (Treg/Tresp). Under these conditions, ST2+CD25+ Tregs suppressed CD8 Tresp proliferation and, strikingly, much more potently than ST2–CD25+ Tregs  toward a reduction in GATA3+ Th2 cells  Tregs at Braf/Pten cutaneous tumor sites.We next examined whether GATA3ed genes , as obse of TSLP . Flow cy+ tumors . Among Ch2 cells . Gating – tumors . Note al– tumors . Togethe+ Tregs accumulated in MC903-treated Braf mouse ears. Upon MC903 treatment, Braf/Tslp+/+ ears showed an increase in the frequency of ST2+ Tregs, which was diminished in Braf/Tslp–/– ears  were analyzed by IHC using a previously validated anti–human TSLP antibody (n = 2), compound nevi (n = 6), or intradermal nevi (n = 7)  of the CD4+ T cells , TNFRSF9 (encoding 4-1BB), TNFRSF18 (encoding GITR), CCR8, IKZF2 (encoding Helios), CTLA4, ICOS, and TIGIT (+ Tregs identified in mouse melanoma (IL1RL1 (encoding ST2), NRP1, and LAG3 was very low, whereas although PDCD1 (encoding PD-1) was expressed in C2, it was most strongly expressed in C4 and did not appear to be a signature gene for C2  or Th17  cells, except that C1 showed a highest level for Th1 cytokine gene IFNG , TNFRSF9, TNFRSF18, CCR8, IKZF2 (Helios), CTLA4, ICOS, and TIGIT.Together, these analyses of the single-cell expression of CD4+Foxp3+) cells expressing a signature of surface markers including ST2, CCR8, ICOS, PD-1, CTLA-4, and OX40 and exhibiting a potent suppressor activity on CD8+ cytotoxic T lymphocyte (CTL) proliferation and IFN-γ production. Together, our data define a potentially novel pathway by which TSLP promotes melanoma growth and metastasis via regulation of the tumor-associated immunosuppressive microenvironment . Also, most of these previous studies focused on TSLP-Th2 mechanisms in cancer through the implication of Th2 cytokines IL-4 and IL-13 , 46 — asd humans –37. Durid humans  reporteda et al. , suggesta et al.  that wer+ (ST2+) Tregs exhibit a highly suppressive activity for CD8+ CTLs, representing a plausible pathway for the protumoral function of TSLP. In agreement with this, a higher CD8/CD4 ratio was observed in Braf/Pten tumors following TSLP ablation  and CD11b+Ly6CintLy6G+ myeloid cells (PMN-MDSCs) compared with Braf/Pten mice, accompanied by reduced serum levels of S100A8/A9  Treg axis reported here differs from that reported by Obata-Ninomiya et al.  or immune checkpoint therapies (targeting immune cells) are required. Of particular note, the recent approval of tezepelumab (anti-TSLP antibody) to treat asthma in humans provides the possibility to translate our mouse studies to human melanoma. Moreover, it will be important to assess whether TSLP expression or/and serum levels could serve as prognostic factors for cutaneous melanoma patients, as recently reported in gastric cancer  from formalin-fixed paraffin-embedded (FFPE) tissue from primary melanoma and nevi were retrospectively retrieved from the department of dermatology of Strasbourg University Hospital   .4 cells) were i.d. injected into mouse ears. Tumor growth was monitored by digital photography of the skin and analyzed using ImageJ (NIH) software. The Lu1205 cell line derived from human melanoma patients (2 × 104) was i.d. injected into ears of NSG mice.Mouse melanoma B16F10 cell line was provided by Lionel Larue  B16F10 cells  was dissolved in 100% ethanol and topically applied on mouse ears (2 nmol in 25 μL) as described before . For thet test with Welch’s correction or the 2-tailed Mann-Whitney U rank-sum nonparametric test, depending on results from the Kolmogorov-Smirnov test for normality. Comparison of more than 2 groups was performed by ordinary 1-way ANOVA, followed by Tukey’s post hoc test. Data are presented as mean ± SEM (for Student’s t test or 1-way ANOVA), or with median (for Mann-Whitney U rank-sum nonparametric test). Two-tailed Fisher’s exact test was used to compare percentages of TSLP+ samples from the examined in situ melanoma or invasive melanoma biopsies  and Union (2010/63/UE) and the French National Committee (87/848) guidelines and policies, and with approval of the National Ethics Committee.Crlf2-floxed mice. EF performed ex vivo culture study and prepared mouse cohorts. AB and DL provided human melanoma FFPE biopsies and analyzed IHC data. WY, BG, DC, AB, LD, ID, and ML analyzed and interpreted data. WY, BG, DC, AB, DL, ID, and ML wrote and revised the manuscript. ML directed the study and supervised the work.WY, BG, DC, AB, ID, and ML conceived and designed the study. WY BG, and DC conducted most of experiments and acquired data, and the chronological order of their contribution to this work was used in assigning the authorship order among co–first authors. AB contributed to IHC and multiplex IF studies with human melanoma biopsies. DC and P. Meyer performed in vitro Treg functional study. DC, P. Meyer, and GD performed scRNA-Seq data analyses. CH, P. Meyer, P. Marschall, EF, JS, and MG contributed to flow cytometry analyses. CH, EF, and PH contributed to qPCR analyses. PL contributed to IHC analyses of mouse melanoma samples. GM initiated and contributed to Braf/TSLP and Braf/Pten/TSLP mouse breeding and cohort generation. MCB contributed to design and generation of


This research was focused to examine the potential targets, action network, and mechanism of urolithin A (UA) in spinal cord injury (SCI) management exploiting the network pharmacology (NP).  We used the SwissTargetPrediction, PharmMapper, and TargetNet databases to obtain UA action targets. We searched the OMIM, GeneCards, CTD, and DrugBank databases to screen selected target genes for SCI treatment. The intersection of target genes between the UA and SCI databases was obtained by constructing Venn diagrams, which led to the identification of common druggable targets for the disease. The relationship network of the targets was built with Cytoscape 3.7.2, and the protein interaction network was analyzed with the STRING platform. The protein-protein interaction (PPI) network can be built on the STRING database. Gene Ontology (GO) function and KEGG pathway analyses of target intersections were completed with the DAVID 6.8 database. We constructed preliminary network targets for actions underlying UA-SCI interactions. Using the AutoDock software, we examined the molecular docking interactions between UA and its target proteins and further verified the mechanism of the action of UA. κB1 as possible therapeutic targets. Enrichment analysis revealed that the PI3K-AKT, VEGF, and TNF signaling pathways could be critical for the NP analysis. Molecular docking indicated that UA had a strong affinity for docked proteins .  We obtained 318 UA drug targets and 1492 SCI disease targets. We identified a total of 118 common UA-SCI targets. Based on the PPI analysis, we identified MAPK1, SRC, AKT1, HRAS, MAPK8, HSP90AA1, MAPK14, JAK2, ESR1, and NF- We employed an NP approach to validate and predict the underlying mechanisms associated with UA therapy for SCI. An additional purpose of this study was to provide a theoretical basis for further experimental studies on UA's potential in SCI treatment. Spinal cord injury (SCI) causes the most severe spine-related complications and can often result in functional limb impairment below the level of the injured segment . SCI notin vitro and in vivo disease models. With its potent antioxidant and anti-inflammatory effects, EA can counteract diverse types of neurotoxic and neurodegenerative conditions [The blood-spinal cord barrier (BSCB) is functionally similar to the blood-brain barrier (BBB) that separates the spinal cord from plasma and significantly interferes with the entry of therapeutic drugs into the spinal cord . Importanditions . HoweverNetwork pharmacology (NP) exploits network topology mapping and bioinformatics to analyze the complex relationship between drugs, drug targets, affected signaling pathways, and diseases. In this context, the NP approach can improve the efficiency of target-oriented drug findings, thus significantly reducing the therapeutic development costs as well as providing new ideas for studying the drug-disease interaction mechanisms in clinical research . Here, whttps://www.swisstargetprediction.ch/) was applied to query UA's targets and names of relevant target genes. Then, the PharmMapper database (https://www.lilab-ecust.cn/pharmmapper/submitfile.html) was applied to restrict the species to humans, as well as the TargetNet database (https://targetnet.scbdd.com/) to identify potential target genes. Furthermore, we employed the UniProt database (https://www.uniprot.org/uploadlists/) to query genes corresponding to potential target proteins in screening for the active ingredient. Our results were derived from targeting UA through Excel searching and sorting. We used the search term “Spinal cord injury” in databases such as OMIM (https://www.omim.org/), GeneCard (https://www.genecards.org/), CTD (https://ctdbase.org/), and DrugBank (https://www.DrugBank.ca).UA is a chemically simple compound. The SwissTargetPrediction database (https://bioinfogp.cnb.csic.es/tools/venny/index.html) tool to map SCI-related targets and active ingredient targets in the form of Venn diagrams. Next, shared targets between the UA and SCI were noted as the potential target set for UA in SCI treatment.After merging and compiling the database search results, duplicate targets were removed. Intersections of the predicted target for the active component of UA and the retrieved results of SCI-related target genes were determined, and common targets were screened out as probable action targets of UA in SCI. We applied the Venny 2.1 (https://string-db.org/) was utilized to identify potential therapeutic targets of UA related to SCI pathology within the PPI network, setting the species type to “Homo sapiens” and a high confidence score (0.9) [https://cytoscape.org) was used to visually output the PPI network.PPI networks are constructed from protein targets. that interact among themselves to execute certain biological processes such as cell signaling, regulation of gene expressions, metabolic pathways like energy production, and regulation of cell cycle checkpoints . The STRre (0.9) . Followihttps://david.ncifcrf.gov/), was utilized for statistical analysis and graphical displays. This database has also been exploited for the development of online packages/tools such as Bioinformatics (https://www.bioinformatics.com.cn/) and OmicShare (https://www.omicshare.com/tools/) for mapping of GO-annotated and KEGG pathway enriched factors. We used these online platforms  to examine the biological processes (BP), molecular functions (MF), cellular components (CC), and signaling pathways linked to UA in SCI treatment.The DAVID 6.8, an open-source database (https://sankeymatic.com/) was plotted to visualize the “hubs in UA-bioactive compounds-top 10 hub genes.” Selected critical bioactive chemical compounds designated for low molecular weight ligands, and their respective great receptor proteins were subjected to the molecular docking assay. The original document of latent bioactive compounds related to UA may be publicly accessed through the PubChem compounds database (https://pubchem.ncbi.nlm.nih.gov/) and that of the top 10 hub genes (PDB format) through the RCSB Protein Data Bank (https://www.rcsb.org/). After performing the initial analysis with the AutoDock tool [https://pymol.org/2/) for the docking assay [After determining UA-related targets and active compounds, we next screened for active compounds that could modulate the functions of those top 10 hub genes. A Sankey diagram  for UA in SCI treatment.A PPI network within the STRING database was constructed by entering previously identified 118 potential targets for UA in relation to SCI pathology, and setting the species to “sapiens” , which isapiens” , where iP < 0.05) (P=0.05). A total of 148 pathways were found to be enriched in this analysis. The top 20 pathways thus identified, including the P13K-AKT, MAPK, TNF, VEGF, and HIF-1 signaling pathways, suggest that the pathways targeted by UA may modulate inflammatory responses and inhibit cellular apoptosis in SCI patients , cellular components (CC), and molecular function (MF), which showed the enrichment of 123 GO terms associated with targets of UA against SCI. We then screened the top 20 GO terms ( < 0.05) . Furtherpatients κB1, CASP3, and BCL-2. Furthermore, this finding indicated the reliability of using the NP approach in parallel to the molecular docking for the target identification. The docking results showed that the minimum binding energy between UA and the ligand-binding sites in the receptor proteins MAPK1, AKT1, JAK2, NF-κB1, CASP3, and BCL-2 was less than −5.0 kcal mol−1 (−1), suggesting a tight binding interaction of UA with the target proteins.Molecular docking analysis further confirmed the pathway prediction results revealing the existence of substantial interactions between the active ingredients of UA and their respective hub genes. Exploring the underlying mechanism of UA in SCI management might delineate the mechanistic regulation of targets such as MAPK1, AKT1, JAK2, NF-al mol−1 . MoreoveSCI is characterized by an abnormal change in the structure and function of the spinal cord and is directly or indirectly caused by a variety of pathogenic factors. This often leads to impairment or loss of sensory, motor, and neurological functions of the limbs below the injured segment . SCI is MAPK1, SPC, AKT1, HRAS, MAPK8, and NF-κB1 for UA in the SCI treatment. These six target genes may critically modulate the UA-based treatment of SCI. Previous studies have shown that traumatic SCI leads to the activation of NF-κB and MAPK pathways [κB and MAPK signaling was neuroprotection against the SCI. Abdelazeem et al. [κB as well as MAPK signaling pathways in bone marrow stromal cells by inhibiting the generation of reactive oxygen species. Others have also shown that UA could inhibit the NF-κB nuclear translocation by blocking IKBα phosphorylation, thereby modulating the activity of NF-κB under the diseased condition [Using the STRING interaction network database, we constructed a target interaction network representing UA effects on the therapy of SCI. All together, 118 core targets were screened and selected. The NP analysis and molecular docking results predicted several key target genes, including pathways . Thus, bpathways  and Xiaopathways  found thm et al.  reportedondition . The MAPondition . UA playondition  has demoα, IL-6, and similar cytokines. Once the central nervous system (CNS) is injured, the JAK/STAT3 pathway participates in gliosis and scar formation by regulating the differentiation of neural progenitor cells (NPCs) and activation of astrocytes in SCI tissues [AKT was shown to have a central role in the key target network for UA in the treatment of SCI. AKT is a serine-threonine kinase related to protein kinase C (PKC) , which i tissues , 33. Zhe tissues  have sho tissues  has furt tissues . Endopla tissues . Li et a tissues  reported tissues . SCI is  tissues  have fouα increases immediately in response to CNS damage (including SCI), skin, muscle, and bone injuries [α inhibits the differentiation of BMSCs into NPCs and significantly restored neurological function in SCI rats. Han et al. [α, IL-1β, and IL-6, promote axonal regeneration, and help in locomotor function improvement. Furthermore, glial scar formation and cavities were reduced in the transplanted rats.The KEGG pathway analysis explored that the major pathways to participate in SCI pathology contain the PI3K-AKT, VEGF, and TNF signaling pathways. PI3K-AKT is critical in neuronal regeneration and inhibition of neuronal apoptosis. This pathway also regulates a variety of cellular responses, such as cell cycle, stress, and apoptosis . Two keyinjuries . Peng etinjuries  have foun et al.  reportedMAPK1, AKT1, MAPK8, MAPK14, NF-κB1, and CASP3, were the hub genes. We also identified critical UA pathways associated with SCI treatment, including the PI3K-AKT, VEGF, and TNF signaling pathways. Here, we employed the NP approach to study the mechanism of UA for treating SCI and to provide a basis for future studies. Our predicted disease targets for UA components closely matched with those in the existing literature, indicating that the use of a combination of NP and molecular docking to study the treatment mechanism could be useful and accurate. However, this study was mainly grounded in bioinformatics, and further studies with animal models and/or cell experiments are needed to verify the main regulatory targets of UA in SCI therapy.In summary, this study utilized the NP methodology to explore the role and mechanism of UA for treating SCI. The results of this study suggest that UA may regulate inflammation and inhibit apoptosis through multiple pathways, contributing to improved functioning of damaged nerves in SCI. There were 118 therapeutic targets in UA-SCI target screening, among which


The dysregulation of host signaling pathways plays a critical role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and viral pathogenesis. While a number of viral proteins that can block type I IFN signaling have been identified, a comprehensive analysis of SARS-CoV-2 proteins in the regulation of other signaling pathways that can be critical for viral infection and its pathophysiology is still lacking. Here, we screened the effect of 21 SARS-CoV-2 proteins on 10 different host signaling pathways, namely, Wnt, p53, TGFβ, c-Myc, Hypoxia, Hippo, AP-1, Notch, Oct4/Sox2, and NF-κB, using a luciferase reporter assay. As a result, we identified several SARS-CoV-2 proteins that could act as activators or inhibitors for distinct signaling pathways in the context of overexpression in HEK293T cells. We also provided evidence for p53 being an intrinsic host restriction factor of SARS-CoV-2. We found that the overexpression of p53 is capable of reducing virus production, while the main viral protease nsp5 can repress the transcriptional activity of p53, which depends on the protease function of nsp5. Taken together, our results provide a foundation for future studies, which can explore how the dysregulation of specific signaling pathways by SARS-CoV-2 proteins can control viral infection and pathogenesis. SARS-CoV-2, the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, is an enveloped, 30 kb positive-sense single-stranded RNA virus that belongs to the genus Betacoronavirus in the family Coronaviridae . AlthougViruses are known to be able to effectively subvert immune response pathways and dysregulate cell signaling pathways to create a cellular milieu permissive for viral replication. Importantly, the viral modulation of host signaling pathways can change the activity of transcription factors downstream in the signaling cascade, resulting in an altered host gene transcriptome. In the case of SARS-CoV-2, it is still poorly understood which host signaling pathways are usurped by the virus, which can promote infection and viral pathogenesis. This study aimed to fill this knowledge gap by screening the effect of each of the SARS-CoV-2 proteins on 10 different host signaling pathways, many of which are known to be critical in the regulation of viral infections.Cell lines 293T, HEK293 (ATCC), and Vero E6 (ATCC) were maintained in DMEM medium supplemented with 10% FBS and penicillin/streptomycin (P/S). The luciferase reporter plasmids of signaling pathways were purchased from Addgene . These plasmids were the following: Wnt signaling , p53 signaling , TGF-beta signaling , c-Myc signaling , Hypoxia signaling , Hippo signaling , AP-1 signaling , Notch signaling , OCT4/SOX2 , and NF-kB signaling . The p21 promoter luciferase plasmid was made by cloning 2.4 kb of DNA sequence upstream of the transcription start site of p21 gene into pGL3 luciferase plasmid. The V5-p53 expression vector was purchased from Addgene (#22945). The vectors expressing C-terminally 2xStrep epitope-tagged SARS-CoV-2 proteins were purchased from Addgene and were used in a study by Gordon DE et al. [p-value) was determined through a two-tailed Student’s t test, and p < 0.05 was considered statistically significant.293T and HEK293 were transfected with the luciferase reporter plasmids, along with gene expression plasmids, using the transfection reagent polyethylenimine (PEI 25K) . At 48 h post-transfection, the cells were collected in a lysis buffer (0.5% Triton-×100 diluted in 1X DPBS). Cell lysates were mixed with ONE-Glo luciferase substrate , and the luciferase activity was measured using a Promega GloMax-Multi Detection System. All luciferase assays were carried out in triplicate. The luciferase reporter data show the average of the three independent luciferase experiments. Significance . Following a 72 h incubation at 37 °C and 5% CO2, cells were fixed and stained in a crystal violet solution . Following a 30 min inactivation timepoint, cell monolayers were rinsed with PBS and plaques were enumerated.V5-p53 was expressed in Vero E6 cells by using lentiviral transduction. Two days after lentiviral transduction, the cells were trypsinized, and 250,000 cells were seeded in each well of a 6-well dish. The next day, the cells were infected with the clinical isolate UF-1 of SARS-CoV-2  at a mulTo investigate the effect of SARS-CoV-2 proteins on host signaling pathways, we performed a luciferase reporter screen utilizing reporter plasmids, in which the promoters of luciferase gene were responsive to distinct signaling pathways, such as Wnt, p53, TGFβ, c-Myc, Hypoxia, Hippo, AP-1, Notch, Oct4/Sox2, and NF-κB A. We co-The results of the signaling pathway screen are summarized in Importantly, we found multiple viral proteins that have the potential to control the TGFβ, AP-1, and NF-κB host signaling pathways, which are major regulators of the immune response pathways and are dysregulated during SARS-CoV-2 infection in patients. In fact, a recent study showed that orf3a, M, and orf7a of SARS-CoV-2 are NF-κB activators, and orf7a-mediated NF-κB activation triggers an elevated expression of cytokines and chemokines, which are frequently increased in severely affected COVID-19 patients . In agreThe role of p53 in the regulation of the cell cycle, DNA repair, apoptosis, and cellular stress responses is well-characterized . In addiPrevious studies have shown that p53 can have both antiviral and proviral activities ,31. To iIn summary, we identified several coronavirus proteins that can control various cellular signaling pathways, which have not been explored in previous studies. Our results provide a foundation for future studies, which will determine how the coronavirus proteins that we identified in our screen can control distinct host signaling pathways during SARS-CoV-2 infection, and how these novel virus–host interactions can contribute to the progression of SARS-CoV-2 infection and COVID-19-associated multiple organ failure. One of our novel findings is the identification of p53 as a component of the host intrinsic antiviral immunity that restricts SARS-CoV-2 replication. Importantly, we found several viral proteins that can dysregulate the transcriptional activity of p53. Because p53 is a pleiotropic host factor, the modulation of p53 activity by SARS-CoV-2 impacts many different cellular processes that can contribute to the diverse pathology of COVID-19.