It is of interest to know whether an environmental factor (E) can attenuate or exacerbate the adverse influence of a candidate gene. To this end, we here evaluate the performance of “genetic risk score” (GRS) approaches to detect “gene-environment interactions” (G × E). In the first stage, a GRS is calculated according to the genotypes of variants in a candidate gene. In the second stage, we test whether E can significantly modify this GRS effect. This two-stage procedure can not only provide a p-value for a G × E test but also guide inferences on how E modifies the adverse effect of a gene. With systematic simulations, we compared several ways to construct a GRS. If E exacerbates the adverse influence of a gene, GRS formed by the elastic net (ENET) or the least absolute shrinkage and selection operator (LASSO) is recommended. However, the performance of ENET or LASSO will be compromised if E attenuates the adverse influence of a gene, and using the ridge regression (RIDGE) can be more powerful in this situation. Applying RIDGE to 18,424 subjects in the Taiwan Biobank, we showed that performing regular exercise can attenuate the adverse influence of the FTO gene on four obesity measures: BMI (p = 0.0009), body fat percentage (p = 0.0031), waist circumference (p = 0.0052), and hip circumference (p = 0.0001). As another example, we used RIDGE and found the FGF5 gene has a stronger effect on blood pressure in Han Chinese with a higher waist-to-hip ratio [p = 0.0013 for diastolic blood pressure (DBP) and p = 0.0027 for systolic blood pressure (SBP)]. This study provides an evaluation on the GRS approaches, which is important to infer whether E attenuates or exacerbates the adverse influence of a candidate gene.Some candidate genes have been robustly reported to be associated with complex traits, such as the  Althougnalyses) . Most evity risk . This mefibroblast growth factor 5 (FGF5) gene is associated with blood pressure levels in Han Chinese  attenuates or exacerbates the effects of these risk variants. However, most G × E methods provide only eraction . In conteraction . ApproprTo address this issue, GRS approaches using internal weights have been proposed for pathway-based G × E studies  and GWASIn this study, we described how to use GRS approaches to infer whether E attenuates or exacerbates the adverse influence of a gene . Moreover, we compared GRS approaches with the “Set-Based gene-EnviRonment InterAction test” (SBERIA) , the “inL SNPs in a gene or a pathway. Let g[⋅] be the link function, Y_i be the phenotype that can be either continuous or binary, Gij be the number of minor alleles  at the j-th SNP , E_i be the environmental factor, Xi be the vector of non-genetic covariates such as the age and sex, and the subscript “i” represents data for the i-th subject . In addition to the additive model , Gij can also be coded according to the dominant or recessive genetic model.Usually, a GRS combines information of multiple nearly independent SNPs across the genome , 2020. HGij later and first regress Y_i on Xi by a linear model or a logistic regression model, as follows:Some GRS approaches involve SNP selection and then aggregate the information of selected SNPs. Therefore, we leave Y_i) or Y_i) be the predicted mean of Y_i under model (1). Therefore, the covariate-adjusted phenotype for the i-th subject is Let Gi1,⋯,GiL, as follows:Subsequently, we regress 0...βL] be the vector of regression coefficients in model (2). The ordinary least squares (OLS) estimate of β is as follows:Let β′=, and n-length vector of covariate-adjusted phenotypes. Because of linkage disequilibrium (LD), the SNPs in a gene are usually highly correlated with each other. In this situation, where l2-norm penalty, as follows:Ridge regression (RIDGE)  is used The regularization parameter λ controls the amount of shrinkage. When λ is close to 0, l1-norm penalty (Least absolute shrinkage and selection operator (LASSO) was later proposed to estimate regression coefficients by minimizing the residual sum of squares and an  penalty  as follojs to 0 . Because equation (5) selects SNPs that are marginally associated with the covariate-adjusted phenotype (E_i and Gij×Ei interaction term have not been included in Eq. (5). If βj is shrunk to 0, the j-th SNP is regarded as unassociated with the covariate-adjusted phenotype and will not be used for the construction of a GRS.The regularization parameter λ controls the amount of shrinkage. Moreover, variable selection can be performed by shrinking some βl1-norm and an l2-norm, as follows:ENET  strikes The regularization parameter λ controls the amount of shrinkage, whereas α is a penalty weight ranging from 0 (RIDGE) to 1 (LASSO). As suggested by Similar to genetic studies using penalized regression approaches , we usedn. GWASs  and pathn. GWASs  have reci-th subject is constructed by Gij is the number of minor alleles  at the j-th SNPof the i-th subject. A positive GRS′. In contrast, a negative GRS′. The GRS′ is then transformed into a Z-score that represents how many standard deviations the GRS′ is from the mean. The standardized GRS Z-score is denoted as GRSi. A larger GRS is always associated with an increased phenotype.RIDGE, LASSO, and ENET are all techniques for regression models that suffer from multicollinearity. Therefore, the pruning stage to remove SNPs with LD is not required here. After obtaining Afterwards, we fit the following generalized linear model (GLM):G > 0 because a larger GRS is always associated with an increased phenotype. Adding a constraint (γG > 0) is expected to improve power, although for simplicity we here perform a GLM without this constraint. By testing H0:γInt=0vs.H1:γInt≠0, we evaluate whether G × E exists. For continuous traits, each 1 standard deviation (SD) increase in GRS is associated with a where γ1,⋯,βL (Eqs 4–6) and then to test H0:γInt=0vs.H1:γInt≠0 (model 7) without data splitting. Theoretically, The whole data can be used to estimate βst power . Empiricst power , pathwayst power , and GWAst power  have preThe abovementioned GRS approaches with some form of penalized regression  were compared with the SBERIA , iSKAT , and ADASBERIA is the first gene-based G × E test based on the GRS concept. The phenotype is first regressed on each SNP separately, while adjusting for covariates such as sex, age, and ancestry principal components (PCs), as follows:j-th SNP and p_j be the p-value of testing H0:βj≠0vs.H1:βj≠0. The GRS of the i-th subject is constructed by I(pj < 0.1) is an indicator variable with a value of 1 if pj < 0.1 and 0 otherwise, Z-score is denoted as GRSi. Then, the following GLM is fitted:Let H0:γInt=0vs.H1:γInt≠0, we can evaluate whether G × E exists , whereas ENET and LASSO fit a multivariate model incorporating L SNPs simultaneously (Eqs 5 and 6). Second, in the model for testing GRS × E, SBERIA includes the main effects of all the L SNPs (GGRSi in Eq. 7).There are two fundamental differences between SBERIA and ENET (or LASSO). First, in the marginal-association filtering stage, SBERIA fits We compared the abovementioned GRS tests with iSKAT  and ADABIntj is the interaction effect between the j-th SNP and E. Assuming δIntjs follow a distribution with a mean of 0 and a variance of τ, the null hypothesis of all δIntjs=0 can be reduced to τ=0. The iSKAT is a score statistic for testing the variance component, i.e., H0:τ=0 vs. H1:τ > 0. The statistic can be referred to as Eq. (6) in where δj-th SNP :In ADABF, we consider the following model for the H0:δIntj=0 vs. H1:δIntj≠0 for the j-th SNP . A Bayes factor (BF) was calculated for each SNP × E, where BF=Pr(Data|H1)/Pr(Data|H0). A larger BF indicates that the relative evidence in favor of H_1 (SNP × E interaction exists) is stronger. Because the number of SNPs exhibiting interactions with E varies gene by gene, ADABF exhaustively searches for the evidence of G × E interaction by considering the largest BF, combining the largest 2 BFs, combining the largest 3 BFs,…, to aggregating all the L BFs in the gene. The significance of G × E interaction is then determined by the efficient sequential resampling procedure  were drawn for simulations, including 48, 95, and 242 SNPs, respectively. It is common to see multiple trait-associated SNPs to be included in the same gene (P (E = 1) = 0.2 and P (E = 1) = 0.5, respectively. For continuous exposures, E was randomly selected from the normal distribution with a mean of 0 and a standard deviation of 0.5.To reflect the real LD structures of the human genome, we used the genotypes of 18,424 TWB subjects as our simulation material. Three genes , βIntd is the effect size of interaction between the d-th trait-associated SNP and E , D is the number of trait-associated SNPs that also exhibit interactions with E (D = 4 or 2 in our simulation), and εi is the random error term following the standard normal distribution.where βi-th subject was simulated as follows:The binary trait of the 0 was log(0.1/0.9)=−2.2 or log(0.4/0.6)=−0.4. Yi=1 represents that the i-th subject was diseased whereas Yi=0 indicates that he/she was non-diseased. This setting corresponds to a disease prevalence of 10% or 40%. A disease prevalence of 40% is also a reasonable setting for some complex diseases. For example, the worldwide prevalence of hypertension among adults aged ≥25 years was ∼40% , were uniformly sampled from 0.04 to 0.08 for continuous traits or uniformly sampled from log(1.05) to log(1.15) for binary traits. This simulation setting for binary trait (odds ratios uniformly sampled from 1.05 to 1.15) concurs with broad GWAS findings, where most odds ratios < 1.5 and many odds ratios < 1.2 =0.2624 for binary traits, respectively. When evaluating type I error rates, data have to be generated from the null hypothesis of no SNP×E interactions, and therefore we specified all βIntds=0 . When assessing power, the magnitudes of SNP×E interaction effects, |βIntd|s , were uniformly sampled from 0.04 to 0.08 for continuous traits or uniformly sampled from log(1.05) to log(1.15) for binary traits. To not favor GRS-based approaches that construct GRSs with SNPs marginal effects, the sampling of |βIntd|s  was independent of the sampling of |βGd|s . Power was evaluated under 14 simulation scenarios listed in Unlike small effect sizes for SNPs, the effect size of E was assumed to be larger, because some E is critical to traits . The magnitude of E, |βYi=1 in binary traits. Scenarios 1, 3, 6, 8, 10, and 13 indicate that a larger E exacerbates the adverse effect of a candidate gene, whereas scenarios 2, 4, 7, 9, 11, and 14 present that a larger E attenuates the adverse effect of a candidate gene. Scenarios 5 and 12 are cross-over situations, representing that a larger E exacerbates the adverse effect of 50% of trait-associated SNPs while attenuating the adverse effect of the remaining 50% of trait-associated SNPs.Scenarios 1–7 describe that a larger E is associated with an increase in trait, whereas scenarios 8–14 represent that a larger E is associated with a decrease in trait. Suppose that a higher trait is linked to a less healthy situation, e.g., The TWB aims to build a research database that integrates the genomic profiles and lifestyle patterns of residents aged 30–70 years in Taiwan . Communip-values < 5.7 × 10–7  have to be set at 0 in order to evaluate type I error rates. The main effects of SNPs, |βGd|s , and the environmental factor, |βE|, have been described in section “Simulation study.” Based on 10,000 replications for each scenario, To evaluate type I error rates, continuous traits and binary traits were simulated according to models (12) and (13), respectively. Scenarios 1 and 8 in H0:γInt=0  and correctly specifying the sign of γInt (γInt can be found from models 7 and 9). The iSKAT . P (E = 1) = 0.2. If E exacerbates the adverse effect of a gene, ENET and LASSO outperform the other methods . If E attenuates the adverse effect of a gene, RIDGE is more powerful . Moreover, ADABF was the optimal test under the cross-over scenario (scenarios 5 and 12).We compared the power of the 4 GRS-based tests and 2 G × E methods: iSKAT  and ADABhe iSKAT  and ADABhe iSKAT  methods Among the 4 GRS-based tests, ENET, LASSO, and SBERIA first select SNPs marginally associated with the phenotype. ENET and LASSO select SNPs according to the multivariate ENET regression (Eq. 6) and multivariate LASSO regression (Eq. 5), respectively. SBERIA selects SNPs based on regressions considering one SNP at a time (Eq. 8). The performance of these three methods to detect G × E depends on their ability to find the true trait-associated SNPs. p-value threshold of 0.1, in the filtering stage it usually selects more SNPs than ENET and LASSO do. Therefore, as shown in Gid×Ei cannot be included; therefore, a βIntd in the opposite direction to βGd will weaken the magnitude of the marginal effect of the d-th trait-associated SNP. In contrast, a βIntd in the same direction to βGd will strengthen the magnitude of the marginal effect of the d-th trait-associated SNP. Therefore, for each method, the ability to pinpoint true trait-associated SNPs is inferior in attenuation scenarios than in exacerbation scenarios  among all rejections of H0:γInt=0 . With 1,000 simulation replications under each scenario, P (E = 1) = 0.2. The true γInt is positive under scenarios 1, 3, 6, 8, 10, and 13; negative under scenarios 2, 4, 7, 9, 11, and 14. The cross-over scenarios (5 and 12) were not considered here, because the true sign of γInt was unclear when E = 1 (or a larger continuous E) exacerbates the adverse effect of 50% of trait-associated SNPs but attenuates the adverse effect of the remaining 50% of trait-associated SNPs.We then investigated the percentage of sign-misspecifications for each GRS-based method. In the presence of G × E, we calculated the percentage of wrongly specifying the sign of γInt were usually correctly specified except scenarios 4 and 11. SBERIA generally makes more mistakes in indicating the true direction for γInt, because it filters SNPs by considering only one SNP at a time (Eq. 8). P (E = 1) = 0.5 and a continuous E, respectively. Each method under P (E = 1) = 0.5 was slightly more powerful than that under P (E = 1) = 0.2, but the comparisons across methods were similar to those shown in P (Y = 1) = 0.4 than under P (Y = 1) = 0.1. Concerning different probabilities of being exposed, each method was more powerful under P (E = 1) = 0.5 than under P (E = 1) = 0.2. Nonetheless, the comparisons across methods were similar to those described for continuous traits.The performance of each method for binary-trait simulations can be found in Ints were in the opposite direction to βGs. Therefore, detecting G × E and correctly specifying the sign for γInt were more challenging under scenarios 4 and 11.Regarding the 14 simulation scenarios listed in Regarding the computation time –S29, SBEFTO gene is obesity-associated and has been replicated by many studies , body fat percentage (BFP), waist circumference (WC), hip circumference (HC), and the waist-to-hip ratio (WHR).The  studies . It span studies , 50 kb i studies . A totalRegular physical exercise was defined as engaging in 30 min of “exercise” three times a week. “Exercise” indicated leisure-time activities such as jogging, yoga, mountain climbing, or playing basketball. According to the TWB questionnaire, activities during work were not counted in “exercise.” Among the 18,424 subjects, 7,652 (41.5%) reported performing regular exercise, while 10,764 reported no regular exercise. A total of eight subjects did not respond to this question. Covariates adjusted in all models included sex, age (in years), educational attainment, drinking status, smoking status, and the first 10 ancestry PCs.FTO gene on BMI, BFP, WC, and HC. The other three GRS-based tests  provided significant results for three obesity measures, respectively. All FTO on obesity measures.FTO. Take BMI-associated SNPs as examples. A total of 12 out of the 242 SNPs reached the genome-wide significance  when testing H0:βj=0vs.H1:βj≠ 0 in the following model:Genetic risk score-based tests are more powerful than iSKAT and ADABF because performing regular exercise generally blunted the effects of the trait-increasing alleles in Y_i is BMI, Gij is the number of minor alleles  at the j-th SNP , and Xi is the vector of covariates of the i-th subject . All Where Ei=1 if yes; Ei=0 if no) and the SNP × exercise interaction were included in the 12 models, as follows:When exercise . This indicated that the minor alleles of the 12 SNPs were associated with increased BMIs, but their effects were blunted by performing regular exercise. As mentioned above, a βIntj in the opposite direction to βj weakens the magnitude of marginal effect of this SNP, and therefore FTO gene harbors more BMI-associated SNPs than our simulation scenario 9, and these SNPs interact with E in the same direction. Therefore, GRS-based tests are more powerful than iSKAT and ADABF.all 2 lower BMI in exercisers than in non-exercisers . Each 1 SD increase in WC-GRS was associated with a 0.3854 cm lower WC in exercisers than in non-exercisers (p = 0.0052). Each 1 SD increase in HC-GRS was associated with a 0.3868 cm lower HC in exercisers than in non-exercisers (p = 0.0001). As shown by 2 (the length of orange bar in P = 1.1×10−20). This association was stronger in non-exercisers than in exercisers ; in exercisers, each 1 SD increase in BMI-GRS was associated with a 0.1382 kg/m2 (the length of blue bar in P = 1.4×10−4).According to RIDGE, each 1 SD increase in BMI-GRS was associated with a 0.1743 kg/mFTO gene on both WC and HC, but not on WHR because it is a ratio of WC to HC. This negative result for WHR is in line with the result from polygenic analyses  (Performing regular exercises is associated with an attenuation of the adverse influence of the  genome) .FGF5 gene is associated with blood pressure levels in Han Chinese , drinking status, smoking status, and the first 10 ancestry PCs. As described above, a total of 18,424 unrelated TWB subjects were included in our analysis, where 9,093 were males and 9,331 were females.In addition to binary exposures, we also provide an example for continuous exposures. The  Chinese . FGF5 sped tests , our anaed tests . TherefoFGF5 × WHR interaction on DBP and SBP (PInt < 0.05). Positive FGF5.FTO and FGF5, respectively).The above two examples demonstrate using GRS-based methods to infer whether E attenuates or exacerbates the adverse influence of a candidate gene   . SearchiL SNPs in L respective regressions (Eq. 8). RIDGE has the advantage of accommodating LD among SNPs and providing regression coefficients that lead to minimum MSE. Although RIDGE has been used to address LD in genetic association analysis .In this work, we described how to use GRS approaches to infer whether E attenuates or exacerbates the adverse effect of a gene. GRS approaches can not only provide a In the cross-over situations (simulation scenarios 5 and 12), where E exacerbates the adverse effects of some SNPs but attenuates the adverse effects of others, GRS approaches were underpowered and ADABF became the most useful. None of the six methods could outperform the others across all situations. In this work, we performed simulations to explore the relative performances of these six tests in detecting G × E and correctly indicating the interaction direction.FTO × exercise interaction on five obesity measures  measures ; (2) FGFe levels . The γIn−6). When testing for many genes simultaneously, even true positive G × Es would have difficulty in standing out among all the noise .Individual-level Taiwan Biobank data are available upon application to Taiwan Biobank . The patients/participants provided their written informed consent to participate in this study.W-YL developed the methods and the analysis tools, designed and performed the simulation studies, analyzed the TWB data, and wrote the manuscript. Y-SL contributed to the programming for the simulations and data analyses. C-CC, Y-LL, S-JT, and P-HK contributed to the writing of the manuscript. P-HK, Y-LL, and S-JT provided the TWB data. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The suitability of a PAS polypeptide as a precipitant to grow protein crystals with high X-ray diffraction quality has been demonstrated, adding this novel class of biosynthetic polymers to the toolset for protein crystallography as an alternative to polyethylene glycol. l-proline, l-alanine and/or l-serine. PAS polymers are strongly hydrophilic and highly soluble in water, where they exhibit a natively disordered conformation without any detectable secondary or tertiary structure, similar to polyethylene glycol (PEG), which constitutes the most widely applied precipitant for protein crystallization to date. To investigate the potential of PAS polymers for structural studies by X-ray crystallography, two proteins that were successfully crystallized using PEG in the past, hen egg-white lysozyme and the Fragaria × ananassaO-methyltransferase, were subjected to crystallization screens with a 200-residue PAS polypeptide. The PAS polymer was applied as a precipitant using a vapor-diffusion setup that allowed individual optimization of the precipitant concentration in the droplet in the reservoir. As a result, crystals of both proteins showing high diffraction quality were obtained using the PAS precipitant. The genetic definition and precise macromolecular composition of PAS polymers, both in sequence and in length, distinguish them from all natural and synthetic polymers that have been utilized for protein crystallization so far, including PEG, and facilitate their adaptation for future applications. Thus, PAS polymers offer potential as novel precipitants for biomolecular crystallography.Proline/alanine-rich sequence (PAS) polypeptides represent a novel class of biosynthetic polymers comprising repetitive sequences of the small proteinogenic amino acids  Apart from sample quality  was the first such polymer to be introduced into protein crystallization, enabling the X-ray structure determination of deoxyhemoglobin A from crystals grown in a PEG-containing buffer  represent a novel class of biodegradable biopolymers that are currently under development as a biological alternative to PEG for extension of the plasma half-life of therapeutic proteins  and the 2.−1 in 10 mM sodium acetate pH 4.5. FaOMT was purified as described elsewhere  and concentrated to 14.4 mg ml−1 in 150 mM NaCl, 20 mM HEPES–NaOH pH 7.5, 2 mM β-mercaptoethanol, 0.02%(w/v) sodium azide with the addition of 2 mMS-adenosylmethionine (SAM). Both enzymes were known to crystallize in the presence of PEG 3350 as a precipitant: in 20%(w/v) PEG 3350, 0.2 M NaCl, 0.1 M sodium acetate pH 4.5 for HEL  PEG 3350, 0.2 M MgCl2, 0.1 M HEPES–NaOH pH 7.0 for FaOMT–SAM. In the present study, PEG 3350 was substituted by suitable concentrations of the Pro/Ala polymer using the same buffer conditions.HEL was purchased from AppliChem  and dissolved at 30 mg mlEscherichia coli and purified to homogeneity according to published procedures  sodium azide to yield a 50%(w/v) stock solution.The recombinant monodisperse 200-residue Pro/Ala polymer (PA200) was produced in id Fig. 1. PA200 ww/v) PA200 solution with 1 µl protein solution to yield initial PA200 concentrations of up to 25%(w/v). Crystallization of HEL and FaOMT was performed by hanging-drop vapor diffusion in Crystalgen SuperClear plates  by mixing 1 µl crystallization solution (precipitant plus buffer) with 1 µl protein solution. The crystallization solution consisted of 6.25 or 12.5%(w/v) PA200 with 0.2 M NaCl, 0.1 M sodium acetate pH 4.5 or with 0.2 M MgCl2, 0.1 M HEPES–NaOH pH 7.0 for HEL or FaOMT, respectively. In order to keep the consumption of the Pro/Ala polymer low, the 1 ml reservoir solutions contained stepwise increasing concentrations of PEG 3350 instead of PA200  PEG 3350 (instead of PA200) in the crystallization droplet  PA200, 20%(v/v) ethylene glycol, 0.2 M NaCl, 0.1 M sodium acetate pH 4.5 for the HEL crystals and 12.5%(w/v) PA200, 20%(v/v) ethylene glycol, 0.2 M MgCl2, 0.1 M HEPES–NaOH pH 7.0 for the FaOMT crystals. X-ray diffraction data were collected on beamline 14.2 of BESSY at the Helmholtz-Zentrum Berlin  as search models. Model building and refinement were performed with Coot , corresponding to 31 mM. The molecular mass of PA200 (16 225 Da) is 4.8-fold higher than the reference PEG polymer PEG 3350 applied in this study, whereas its length in a fully extended conformation exceeds that of PEG 3350 by a factor of just 2.6. While the number of hydrogen-bond acceptors per monomer unit (carbonyl groups in PA200 versus ether O atoms in PEG 3350) is identical for PA200 and PEG 3350, 65% of the monomer units in the Pro/Ala polymer, i.e. all of the alanine residues with their amide groups, also act as hydrogen-bond donors .In contrast to the polydisperse synthetic or semisynthetic organic polymers used for protein crystallization to date, in particular PEG . As it is difficult to estimate the concentration of PEG 3350 that is equivalent in terms of hygroscopy to a certain percentage of PA200, protein droplets with identical PA200 contents were equilibrated against varying concentrations of PEG 3350 as described below  to yield a crystallization droplet. This droplet is then placed next to a larger volume of the precipitant solution, termed the reservoir, and is equilibrated in a sealed reaction vessel. Thereby, all solutes in the droplet are concentrated by way of vapor diffusion, considering that the initial precipitant/buffer concentration in the droplet is only 50% of that in the reservoir, until the supersaturation of the protein in the droplet is sufficient for nucleation and, ideally, crystal growth  containing 6.25 or 12.5%(w/v) PA200 to yield starting concentrations of the biosynthetic polymer of 3.125 or 6.25%(w/v) . These droplets were equilibrated against six different reservoir solutions containing 10, 15, 20, 25, 30 or 35%(w/v) PEG 3350  PA200 were observed at ≥30 and ≥25%(w/v) PEG 3350, respectively , and again equilibrated against reservoir solutions containing 10–35%(w/v) PEG 3350  PEG 3350, 50 mM citrate buffer pH 4.0; 1.45 Å resolution] and FaOMT , with r.m.s.d. values of 0.28 and 0.50 Å for 129 and 352 equivalent Cα positions, respectively.To demonstrate that the single crystals grown in the presence of PA200 were suitable for X-ray diffraction experiments, data sets were collected at the BESSY synchrotron source for both HEL and FaOMT and refined to resolutions of 1.2 and 2.1 Å, respectively. Both data sets showed reasonable statistics Table 1. After m4.et al., 2013Taken together, we have demonstrated the utility of a PAS polypeptide as a precipitant to grow protein crystals, adding this novel class of biopolymers to the toolset for protein crystallography. Variations in length and PAS sequence as well as amino-acid composition, as described previously (Breibeck & Skerra, 2018hen egg-white lysozyme, 6yjwPDB reference: O-methyltransferase, 6yjxPDB reference:


This study aimed to investigate the risk of acute kidney injury (AKI) in hospitalized patients based on admission serum ionized calcium levels. This is a cohort study of all hospitalized adult patients, from January 2009 to December 2013 at a tertiary referral hospital, who had available serum ionized calcium at the time of admission. We excluded patients who had end-stage kidney disease or AKI at admission. We stratified admission serum ionized calcium into 6 groups; ≤ 4.39, 4.40–4.59, 4.60–4.79, 4.80–4.99, 5.00–5.19, and ≥ 5.20 mg/dL. We used serum creatinine criterion of KDIGO definition for diagnosis of AKI. We performed logistic regression analysis to assess the risk of in-hospital AKI occurrence based on admission serum ionized calcium, using serum ionized calcium of 5.00–5.19 mg/dL as the reference group. We studied a total of 25,844 hospitalized patients. Of these, 3,294 (12.7%) developed AKI in hospital, and 622 (2.4%) had AKI stage 2 or 3. We observed a U-shaped association between admission serum ionized calcium and in-hospital AKI, with nadir in-hospital AKI was in serum ionized calcium of 5.00–5.19 mg/dL. After adjustment for confounders, low serum ionized calcium of 4.40–4.59, ≤ 4.39 mg/dL and elevated serum ionized calcium ≥ 5.20 mg/dL were associated with increased risk of AKI with odds ratio of 1.33 (95% CI 1.14–1.56), 1.45 (95% CI 1.21–1.74), and 1.26 (95% CI 1.04–1.54), respectively. Both hypocalcemia, and hypercalcemia at the time of admission were associated with an increased risk of hospital-acquired AKI. Importantly, AKI is independently associated with in-hospital mortality and de novo or progression of chronic kidney disease6. Early detection and management for AKI and its complications are needed to decrease its dire clinical impacts. Calcium homeostasis is commonly disturbed as a consequence of AKI7. However, whether calcium, specifically ionized calcium plays a role in the development of AKI is unclear.Acute kidney injury (AKI) is a common complication among hospitalized patients with approximate incidence of 20%11. From the kidney standpoint, AKI in the setting of hypercalcemia can arise via several mechanisms such as volume depletion from polyuria (decreased collecting duct cells response to vasopressin) and direct alterations of intravascular tone14. Besides, persistent hypercalcemia can lead to calcium deposits in the kidneys or nephrocalcinosis15. Previous studies have shown associations between electrolyte disturbances and a higher incidence of AKI among hospitalized patients, specifically an imbalance in total calcium, phosphate, and calcium-phosphorus product17. These abnormalities seem to directly or indirectly correlate with serum ionized calcium18. Therefore, serum ionized calcium might be a useful marker to predict AKI and might also be independently associated with AKI. However, data on the impact of serum ionized calcium on incidence of AKI among hospitalized patients is limited.Ionized calcium is an essential mineral required for many physiologic functions in the body, from cellular function, intracellular messenger transduction, hormonal activity, to cardiac function and neuronal activityOur study aimed to assess the association between admission serum ionized calcium and risk of in-hospital AKI among hospitalized patients retrieving data from an electronic medical record system.This historical cohort study, conducted at Mayo Clinic, Rochester, MN, USA, included all hospitalized adult patients from January 1st, 2009 to December 31st, 2013 who had available serum ionized calcium within 24 hours of hospital admission. We excluded individuals with end-stage kidney disease (ESKD), patients whose AKI was present at the time of admission, and those whose serum creatinine was not available during hospitalization. For patients with recurrent admissions, only the first hospital admission during the study period was analyzed. Mayo Clinic Institutional Review Board reviewed and approved this project (IRB number 15-000024) and waived informed consent due to the minimal risk nature of this study. The study was conducted in accordance with the relevant guidelines and regulations.19. The Charlson Comorbidity Index was calculated to assess comorbidity burden at the time of admission20. Principal diagnoses were grouped based on admission ICD-9 codes. ESKD was identified using ICD-9 diagnosis code or eGFR of ≤ 15 mL/min/1.73 m2.Institutional electronic medical record system was used to automated retrieval of data collection for clinical characteristics, demographic information, and laboratory data. The predictor of interest was the admission serum ionized calcium, defined as the first serum ionized calcium measured within 24 hours of hospital admission. Estimated glomerular filtration rate (eGFR) was calculated based on age, sex, race, and baseline serum creatinine, using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation21. Severe AKI was considered as AKI KDIGO stage 2 or 3. The most recent outpatient serum creatinine prior to the admission was regarded as baseline serum creatinine. If outpatient baseline serum creatinine was absent, the Modification of Diet in Renal Disease equation was used to back-estimate baseline serum creatinine level, assuming normal baseline GFR of 75 mL/min/1.73 m2, as suggested by the guideline21.The outcome of interest was the occurrence of acute kidney injury (AKI) within 7 days of hospital admission. AKI was diagnosed by the KDIGO serum creatinine criterion, which was defined as an absolute increase in serum creatinine of ≥ 0.3 mg/dL within 48 h or ≥ 1.5 times baseline within 7 days after admission dateVariables were summarized as mean ± standard deviation (SD) for continuous variables and as number with percentage for categorical variables. ANOVA and Chi-squared test was used to compare clinical characteristics and outcomes among admission serum ionized calcium groups, as appropriate. Based on its distribution, admission serum ionized calcium was categorized into six groups; ≤ 4.39, 4.40–4.59, 4.60–4.79, 4.80–4.99, 5.00–5.19, and ≥ 5.20 mg/dL. Restricted cubic spline with 5 knots based on serum ionized calcium was fitted to visualize the possible non-linear relationship between serum ionized calcium at admission and risk of in-hospital AKI. Then, multivariate logistic regression analysis was performed to obtain adjusted odds ratio (OR) of in-hospital AKI occurrence based on admission serum ionized calcium groups, using serum ionized calcium of 5.00–5.20 mg/dL as the reference group because AKI least occurred in the hospital when admission serum ionized calcium was within this range. The adjusting variables were priori-defined and included age, sex, race, baseline eGFR, Charlson Comorbidity Index, comorbidity conditions, admission type, admission service, principal diagnosis, medications, the use for vasopressor and mechanical ventilator at hospital admission, admission serum phosphate, serum magnesium, and serum albumin. P-value was two-tailed with the value of < 0.05 considered statistically significant. All analyses were performed using JMP statistical software . The restricted cubic spline was constructed using STATA .2. The mean admission serum ionized calcium was 4.8 ± 0.3 mg/dL. The distribution of admission serum ionized calcium was as follows: 8% in serum ionized calcium of ≤ 4.39 mg/dL, 13% in 4.40–4.59 mg/dL, 31% in 4.60–4.79 mg/dL, 29% in 4.80–4.99 mg/dL, 13% in 5.00–5.19 mg/dL, and 6% in ≥ 5.20 mg/dL. Table Figure Of 25,844 patients studied, 3,294 12.7%) developed AKI in hospital; 10.3% in stage 1, 1.3% in stage 2, and 1.1% in stage 3. 167 (0.6%) patients required renal replacement therapy in the hospital. The incidence of hospital-acquired AKI was 16.5% in patients with admission serum ionized calcium of ≤ 4.39, 14.8% in 4.40–4.59, 12.5% in 4.60–4.79, 11.6% in 4.80–4.99, 10.4% in 5.00–5.19, and 15.0% in ≥ 5.20 mg/dL (Table 2.7% deveThe subgroup analysis based on admission type showed that, among patients with elective hospital admission, only decreased serum ionized calcium ≤ 4.59 mg/dL was significantly associated with increased risk of hospital-acquired AKI, whereas, among patients with urgent/emergent admissions, both decreased serum ionized calcium of ≤ 4.79 mg/dL and elevated serum ionized calcium of ≥ 5.20 mg/dL were significantly associated with increased risk of hospital-acquired AKI Table . HoweverThe subgroup analysis based on admission service showed that among patients admitted in medical service, only decreased serum ionized calcium ≤ 4.59 mg/dL was significantly associated with increased risk of hospital-acquired AKI, whereas, among patients admitted in surgical service, both decreased serum ionized calcium of ≤ 4.59 mg/dL and elevated serum ionized calcium of ≥ 5.20 mg/dL were significantly associated with increased risk of hospital-acquired AKI Table . HoweverThe findings of our study showed an independent association between admission level of serum ionized calcium and the risk of in-hospital AKI with a U-curve association. Ionized calcium ≤ 4.59 mg/dL or ≥ 5.20 mg/dL was significantly associated with higher occurrence of in-hospital AKI. For subgroup analysis of the risk of in-hospital severe AKI, our results suggested that ionized calcium ≤ 4.79 mg/dL or ≥ 5.20 mg/dL was significantly associated with higher risk of severe AKI.22. However, total calcium measurements have considerable limitations in the identification of actual calcium abnormalities, such as its dependency on serum albumin level10. Our study is the largest cohort to demonstrate the U-curve association between serum ionized calcium and risk of in-hospital AKI. We postulated that abnormality in calcium affects vascular tone, including renal vessels. The imbalance between renal vasoconstriction and vasodilatation, in turn, could result in AKI. Hypercalcemia interferes with the kidneys’ ability to concentrate urine leading to volume depletion with predisposition to AKI15. Nonetheless, it could be the degree of severity of the disease that explains this association. Low serum ionized calcium might be related to severity of the illness or sepsis25. The inflammatory cytokines during the illness impair PTH secretion from parathyroid gland along with resistance to PTH at the end-organs26. Furthermore, calcitriol production is also suppressed during this severe illness27. These result in low serum ionized calcium. At the same time, severity of the disease is also associated with higher risk of AKI. Among critically ill patients with AKI, recent study demonstrated that low serum ionized calcium levels were independent predictors of all-cause mortality8. Further studies are needed to understand more about the mechanistic pathways of this association.Previous studies have demonstrated the AKI risk among patients with various serum calcium levels6, future studies are needed to assess whether the incorporation of ionized calcium levels in a prognostic model at the time of hospital admission will help identify high-risk patients for AKI during hospitalization. Besides, further trials to investigate the benefit of protocol for correction of abnormal serum ionized calcium levels on the risk of AKI in hospitalized patients may be necessary.Our study has several limitations. First, the design of the study is observational and retrospective. Hence, causal relationship cannot be established. In addition, some critical information, such as the causes of serum ionized calcium derangements, urine output, and urine electrolytes, were not available or incomplete in our database and, therefore, we were not able to report them. There might be some residual confounders that we unaccounted for when we analyzed the association. Second, we had only single level of serum ionized calcium at the admission so we could not explore whether the time course of changes in serum ionized calcium might influentially affect the risk of AKI. Third, as serum ionized calcium levels at hospital admission were the predictor of interest, only patients with available admission serum ionized calcium were included. However, serum ionized calcium levels were measured in a limited proportion of hospitalized patients at the time of hospital admission Figure . As showTo summarize, admission ionized calcium either ≤ 4.59 mg/dL or ≥ 5.20 mg/dL is significantly associated with a higher risk of in-hospital AKI while admission serum ionized calcium of either ≤ 4.79 mg/dL or ≥ 5.20 mg/dL is significantly associated with higher risk of in-hospital severe AKI.Supplementary Information.


The percent of farms in Meru and Nyandarua with nutrient contents below critical levels were 66% and 20% for N, 46% and 85% for P, 67% and 31% for S, 9% and 51% for Cu, and 87% and 80% for B, respectively. Low tissue nutrient concentrations were observed for N, P, K, and S irrespective of the sites. Soil pH correlated strongly with majority of the analyzed soil and tissue nutrients. These results affirm the need to design integrative soil fertility management strategies to correct the impoverished soil fertility status in the study area.Inherent low soil fertility remains a hindrance to potato production in Kenya and continues to pose a threat to food security. A study was conducted in Nyandarua and Meru counties to assess the soil fertility status in smallholder potato farms. Soil and plant tissue samples were collected and analysed for selected nutrients  from 198 farms. Critical nutrient levels were used to assess the sufficiency levels of nutrients for potato growth. Soils in the sampled farms were weakly to strongly acidic (pH-CaCl Solanum tuberosum L.) in sub-Saharan Africa is greatly constrained by the impoverished soil fertility caused mainly by poor soil nutrient management strategies2. Potato is a heavy feeder crop with regard to the primary nutrients . For instance, to attain tuber yield of 48 tons ha−1, potato tubers remove 47.6 kg N, 24 kg P, 103.4 kg K and 5 kg S, while the haulm requires 31.8 kg N, 8.2 kg P, 47.6 kg K and 3.2 kg S3. These nutrient amounts can only be supplied through fertilizer application, a strategy that may be beyond the means of the resource constrained smallholder farmers5. Nitrogen supply influences tuber bulking rate and the time of tuber growth6, K plays an important role in increasing tuber yield, size and quality7, while P enhances root development, tuber set and promotes tuber maturity3. Sulphur is an integral component in proteins and activates many enzymes regulating potato growth. Soil pH, SOM, Ca, Mg, Fe, Zn, Mn, Mo, Ni, and B are also essential for potato growth and development8.The productivity of potato crop 36. Calcium chloride (0.0125 M) was used to extract soil mineral N and S37.Soils were sampled from each target farms in zigzag pattern and at an interval of approximately 10 m. On average, ten (10) soil replicates were taken within rows and inter rows of each farm with a 15 mm diameter soil auger at 0–30 cm depth. The samples were mixed into a composite for each farm and a sub sample of 100 g fresh weight was taken to the laboratory and frozen at 4 th leaf from the top of the growing tip). This leaf was considered the critical part for tissue nutrient analyses because younger or older tissue have varying nutrient concentrations which can lead to erroneous interpretations16. All leaflets were put into khaki paper bags embedded with silica gel to absorb the moisture. Foliar samples were oven dried at 60 °C, ground using Wiley mill, wet digested, and analysed for mineral nitrogen calorimetrically using Skalar, and for Ca, P, S, K, Mg, B, Zn, S and Cu using ICP-OES after dissolving the ashes  in dilute HCl38.Tissue samples were extracted at mid flowering stage from the youngest fully expanded potato leaves  analysis.Descriptive statistical analyses including means, range and standard deviations were used to explore the data using STATA software and graphs plotted in MS Excel. Nutrient sufficiency ranges  Table  were use−1 for these areas  and Nyandarua (95%) Fig. . This feeas Fig. . General2) ranged from 3.9 to 6.2, with an overall mean of 5.0 ± 0.5, with 86% of sampled farms having values below the critical level of 5.5  and Nyandarua (5.6 ± 5.3). Calcium and Mg were generally optimal in Meru and Nyandarua. Ca-Mg ratio was higher in Meru (3.63 ± 0.88) compared to Nyandarua (1.98 ± 0.77).In Nyandarua, the soil pH CaCl ranged f−1 in Meru and 47.39 ± 4.98 g kg−1 in Nyandarua , soil P and negatively correlated to soil Cu r = −0.40) , K (r = 0.47), Ca (r = 0.79), Mg r = 0.69) and B (r = 0.74) and negatively to Cu (r = −0.59) in Meru , P (0.46), K (0.7), Ca (0.77), Mg (0.57) and B (0.75). Similarly, Cu was negatively correlated to all the nutrients and significantly with pH (−0.46), K (−0.37), Ca (−0.41) and B (−0.42). Boron was positively correlated to all analysed nutrients apart from the correlation with Cu which was negative.Factor analysis for soil chemical properties in Meru retained 7 principal factors with the first three factors accounting for 91% proportion of the variance. Zinc and S were the most unique soil chemical properties in Meru  influenced the first factor. The higher the bases  the higher the soil pH. This would thus reinforce that soil pH is a major factor to consider for sustainable nutrient management72. Similar relations have elsewhere been reported74. Nutrient interaction in the soil and within the plant also affects crop nutrient uptake75.The low soil nutrient contents measured for N, P, K, S and B were reflected in the tissue nutrient concentrations which were consistently lower indicating that nutrient uptake by potato crop was influenced by soil nutrient concentrations. The correlations were however not very strong which could be explained by other confounding factors influencing nutrient uptake such as crop variety, farm management, climatic factors and crop health statusThis study shows that N, P, K, S and B are the key limiting nutrients to potato production in the major potato growing areas of Kenya. These nutrients need to be integrated in nutrient management programs for major potato growing areas of Kenya. Soil pH was found to be low in all the sampled farms and was the major factor that influenced nutrient uptake by potato crop. Improved cropping systems and soil management practices are required to adjust the soil pH for enhanced soil fertility in the study areas.


Angiostrongylus cantonensis, severe damage has been observed in the hippocampus. This component has been considered to have associations with spatial learning and memory in humans and vertebrates. This study was designed to determine the impairments in behavior, learning, and memory in BALB/c and C57BL/6 mice heavily infected with the parasite.Parasitic infections may cause significant effects on behavior, learning, and memory of the host. In the brain of mice heavily infected with A. cantonensis. After infection, daily changes in weight and dietary consumption, worm recoveries and survival rates were determined. The forced swimming test, open field test, and Morris water maze test were employed to evaluate depression- and anxiety-like behavior as well as impairments in spatial learning and memory, respectively.Each mouse was inoculated with 50 third-stage larvae of The worm recovery rate in the BALB/c mice was significantly lower than that of C57BL/6 mice from day 14 post-infection. The survival rate in infected BALB/c mice decreased to 0% by day 25 whereas those with swim-training survived three more days. On day 42, the C57BL/6 mice had a survival rate of 85.7% in the swimming group and 70% in the non-swimming group. Significant differences were found in weight between infected and non-infected BALB/c and C57BL/6 mice from day 13 and day 12, respectively with corresponding changes in their dietary consumption. Depression-like behavior was found in the infected BALB/c mice but not in C57BL/6 mice. However, anxiety-like behavior was found to occur only in C57BL/6 mice. Impaired spatial learning and memory were also found in the two strains of mice which occurred from day 14 post-infection.A. cantonensis causes depression, anxiety, and impairments in spatial learning and memory in heavily infected mice. Moreover, significantly higher severity was observed in the Th-2 dominant BALB/c mice.Results of this study indicate that  Heligmosomoides polygyrus and Eimeria vermiformis were respectively observed to have poor spatial learning and a reduced avoidance of cat odor [Strongyloides ratti did not show deficits in learning [Toxocara canis [Toxoplasma gondii [T. gondii in specific forebrain areas of rats has been attributed to attenuation of predator odor aversion and changes in anxiety-related behavior [T. gondii and anxiety and cognitive disorders has also been reported [Parasitic infections have been reported to have significant effects on behavior, learning, and memory of the host. Mice infected with cat odor , 2. Howelearning . Althougra canis  and Toxoa gondii –7 have bbehavior . Associareported , 9.Angiostrongylus cantonensis is a neurotropic nematode causing eosinophilic meningitis and/or eosinophilic meningoencephalitis in humans. The infection is due to ingestion of raw or uncooked intermediate hosts (snails or slugs), paratenic hosts , or vegetables contaminated with the infective third-stage larvae (L3) [vae (L3) , 11. Aftvae (L3) . Severe vae (L3) . Using tvae (L3) .A. cantonensis causes significant pathological changes in the brain of infected mice especially in the hippocampus [Lactobacillus helveticus to rats with ammonium acetate induced chronic hyperammonemia has been reported to elevate cognitive functions [Streptococcus pneumoniae not only prevented apoptosis and reduced damages in hippocampus, but also improved spatial memory and learning [A. cantonensis infection causes damage to the hippocampus, these changes may lead to impairments in the behavior, learning, and memory of the host. In this study, we employed the force swimming test to evaluate the level of depression, the open field test to assess anxiety, and the Morris water maze test to determine changes in learning and memory in heavily infected BALB/c and C57BL/6 mice with A. cantonensis.In our previous studies, we have reported that pocampus  and diffpocampus . Rats wipocampus . Moreovepocampus , 19. Daiunctions . Treatmelearning . In addilearning . Since AA. cantonensis has been maintained in our laboratory since 1980. Its life-cycle is maintained through Biomphalaria glabrata snails and Sprague-Dawley (SD) rats [d) and C57BL/6 (H-2b) mice were used for experimental studies. Animals were purchased from the National Laboratory Animal Center  and BioLASCO Taiwan Co., Ltd. . These animals were reared in the Laboratory Animal Center of Chang-Gung University. They were kept in plastic cages and provided with food and water ad libitum. All procedures were reviewed and approved by the Institutional Animal Care and Use Committee of Chang Gung University .A Taiwan strain of SD) rats . The ratA. cantonensis were removed from the standing precipitations using a medical dropper and counted under a dissecting microscope. Each mouse was inoculated with 50 L3 by stomach intubation.On day 21 post-infection, tissues of the snail hosts were removed and homogenized after shell crushing. The tissue samples were digested with 0.6% (w/v) pepsin-HCl (pH 2–3) for at 37 °C for 1 h . L3 of AMice undertaking the tests have to exercise in the training and testing stages, as it was a requirement to determine whether exercise affected the status of infection. There were two controls groups (10 BALB/c mice and 10 C57BL/6 mice) and two corresponding infected groups with equal numbers. Throughout the experimental studies, each mouse was weighed daily. In addition, the daily dietary consumption was determined by recording the intake of food and drinking water.A. cantonensis infection. There were 10 mice in each of the control and infected groups. The test was carried out on days 7, 14 and 21 post-infection. The tested mouse is placed in a transparent cylindrical tank (45 cm height × 10 cm radius) and filled with water (25 ± 1 °C) at a level of 15 cm from the bottom for 6 min. The duration that the mouse does not swim in the last 4 min is recorded [The forced swim test is a behavioral test used for the evaluation on the level of depression after recorded .A. cantonensis infected mice. There were 10 mice in each of the control and infected groups. The test was carried out on day 9 post-infection. The open field apparatus consisted of a square tank (40 × 40 cm) with walls 40 cm high made of gray polyvinyl chloride plastic board. Mice were transported to the testing room and left in their home cages for 1 h. The animal was placed in the middle of the tank and allowed to move freely. Speed and time of the locomotor activity was monitored for 1 h. Tendency to anxiety was defined as time in the middle less than time on the edge [The open field test was used to assess anxiety-like behavior in the edge .A. cantonensis on spatial learning and memory. There were 10 mice in each of the control and infected groups. The mice were trained on days 3–6, 10–13 and 17–20 post-infection, and tests performed on days 7, 14 and 21 days post-infection. The custom-made maze was a circular pool 120 cm in diameter and 40 cm in height, filled with water at 25 ± 1 °C and made opaque by adding milk. In the acquisition session, each mouse was given 4 trials per day and 4 days of training in total to find a hidden platform located 1.5 cm below the water surface. Each mouse was placed into the pool, facing the wall, with a different starting point for each trial that the direct route to the platform differed each time. The time required by the mouse to find and stand on the platform was recorded for up to 90 s. The mouse was allowed to stay on the platform for 30 s. It was then removed from the maze and placed into its cage. For the mouse that found the platform within 90 s, the animal was placed on the platform for 30 s. The inter-trial interval was at least 30 min. In the probe session, on day 5, the platform was removed from the pool. The mouse was tested in a probe trial for 60 s. Mouse swimming tracts were recorded using a TopScan automated tracking system  [The Morris water maze test was used to assess the effect of ia, USA) .The infected mice were sacrificed by inhalation of 3% (v/v) isoflurane . Brains were removed from the cranial cavity. Worm recovery rates were then determined by counting the number worms under a dissecting microscope.P < 0.05 was considered to be statistically significant.Statistical analyses were performed using Microsoft Excel 2010 for Windows . Data were expressed as the mean ± standard deviation (SD). Differences between groups were analyzed by Student’s t-test. t(4) = 0.000169, P < 0.001) whereas that of C57BL/6 mice decreased only on day 21 post-infection (t(4) = 0.001452, P < 0.01). In addition, the rate in the BALB/c mice was significantly lower than that of C57BL/6 mice on days 14 (t(4) = 0.000331, P < 0.001) and 21 (t(4) = 0.006658, P < 0.01) . The survival rate in BALB/c mice without undertaking the water maze test (the non-swimming group) rapidly decreased to 0% by day 25 whereas those undertaking the test (the swimming group) survived three more days. Among the C57BL/6 mice, the survival rate only became 70% on day 42 in the non-swimming group and that in the swimming group remained 85.7% on the same day. The mortality rate in the swimming group decreased from 30% to 14%  = 0.320876, P > 0.05). From day 14, the weight of the infected group significantly decreased (t(9) = 0.006903, P < 0.01)  = 0.011617, P < 0.05)  = 0.031012, P < 0.05)  = 0.326197, P > 0.05) and the weight of the infected mice significantly decreased from day 12 (t(9) = 0.037645, P < 0.05)  = 0.113181, P > 0.05). From day 13, food intake in the infected group significantly decreased (t(9) = 0.004678, P < 0.01)  = 0.692658, P > 0.05). However, food intake in the infected mice significantly decreased from day 13 (t(9) = 0.004806, P < 0.01)  = 0.794676, P > 0.05). From day 12, the water intake in the infected group significantly decreased (t(9) = 0.002576, P < 0.01)  = 0.204049, P > 0.05); however there was a significant decrease of food intake in the infected mice from day 14 (t(9) = 0.019749, P < 0.05)  = 0.018582, P < 0.05)  = 0.158094, P > 0.05). Moreover, the immobility time in the infected mice was found to be significantly shorter than the uninfected mice on day 14 (t(9) = 0.013031, P < 0.05) and day 21 (t(9) = 0.00011, P < 0.001), indicating an increase in activity and no occurrence of depression  = 0.024914, P < 0.05)  = 0.029771, P < 0.05)  Fig. a. No sigion Fig. b. Althou05) Fig. a and a lA. cantonensis, BALB/c and C57BL/6 mice were continuously trained in the water maze for 4 days to observe their spatial learning ability. Before day 7, no significant difference in escape latency to the platform was found in infected and uninfected BALB/c mice (t(9) = 0.561855, P > 0.05)  = 0.000049, P < 0.001)  = 0.803257, P > 0.05)  = 0.013565, P < 0.05)  = 0.002013, P < 0.01)  = 0.016921, P < 0.05), 14 (t(9) = 0.015133, P < 0.05), and 21 (t(9) = 0.000047, P < 0.001). In the infected C57BL/6 mice, this phenomena only occurred on days 7 (t(9) = 0.03045, P < 0.05) and 21 (t(9) = 0.004776, P < 0.01) (Fig. A. cantonensis infection.After removing the water maze platform, infected BALB/c mice stayed in the target quadrant of the original platform for a significantly shorter time than the uninfected group (t = 0.0020t = 0.0020t = 0.0020A. cantonensis. However, this suggestion requires further experiments.Mice undertaking the water maze test were required to swim for four days in the training stage. The tested infected BALB/c mice may survive after day 28 and C57BL/6 mice had a high survival rate of 87% Fig. . It has A. cantonensis, and pathologic changes were not limited to the meninges but also in the cerebral parenchyma especially in the hippocampus [In our previous study, eosinophilic meningitis was found to occur from day 12 post-infection in ICR mice infected with pocampus . Hippocapocampus  and the pocampus . In addipocampus  or persipocampus  may alsopocampus .A. cantonensis decreased on day 21 (Fig. In this study, we demonstrated that the recovery rate of worms in the two strains of mice infected with  21 Fig. . This fi 21 Fig.  and wate 21 Fig.  intake. A. cantonensis may cause mechanical damage to the brain of the host. The water maze test also demonstrated that the infected BALB/c mice required a longer time to find the hidden platform and had a shorter residence time at this position than their C57BL/6 counterpart (Fig. By using the forced swimming test, we demonstrated that infected BALB/c mice developed depression on day 21. However, infected C57BL/6 mice had a significantly shorter immobility time (Fig. art Fig. , showingChanges in the immune response have been associated with spatial memory. It has been shown that SCID BALB/c mice have a lower spatial memory. However, their spatial memory and learning abilities was significantly improved after injection of T cells . A waterA. cantonensis is able to cause cognitive impairments in heavily infected mice. The infection may cause depression, anxiety, and impairments in spatial learning and memory in heavily infected mice. Moreover, significantly higher severity was observed in the Th-2 dominant BALB/c mice.In conclusion,


To the editor,Surgical site infection (SSI) is a major complication of spinal surgery involving implants. Although immunosuppressive effects of anesthesia including opioids have been implicated , whetherWe retrospectively reviewed 127 patients who underwent surgery for scoliosis at Kagoshima University Hospital from January 2012 to December 2017. In all patients, anesthesia was induced with propofol, remifentanil, and rocuronium. Anesthesia was maintained with continuous intravenous target-controlled infusion of propofol with an effect-site concentration of 2.5 to 4.0 μg/mL for patients ≥ 16 years old or continuous infusion at a rate of 4.0 to 6.0 mg/kg/h for patients < 16 years old to maintain the bispectral index at 40 to 60. During surgery, remifentanil was administered at 0.2 to 1.0 μg/kg/min. Rocuronium was not used to measure motor evoked potentials during the operation. After the end of surgery, sedation with dexmedetomidine (0.4–0.7 g/kg/h) and propofol (3.0–4.0 g/kg/h) was continued until arrival in the intensive care unit.P = 0.026]. The immunological parameters such as the neutrophil count, lymphocyte count, and N/L ratio were not different between patients with and without SSI immediately after the end of anesthesia although an elevated N/L ratio is reportedly correlated with the incidence of SSI after posterior lumbar instrumentation surgery . This is consistent with a previous report showing that the CRP level on POD 7 is a reliable marker for SSI following instrumented spinal fusion [P = 0.015], possibly because of postoperative pain due to infection.SSI occurred in 7 of 127 patients (5.5%). The onset of SSI differed among the patients, ranging from postoperative day (POD) 8 to POD 77. The total use of remifentanil was not different between patients with and without SSI. The preoperative C-reactive protein (CRP) level was significantly higher in patients with than without SSI [median (interquartile range), 0.5 0.09–0.63) vs. 0.03 (0.02–0.15) mg/dL, respectively;  vs. 0.03In conclusion, immunological parameters immediately after the end of anesthesia were not different between patients with and without SSI. The correlation between perioperative opioid use and the incidence of SSI was not clear. The parameter directly reflecting the onset of SSI might be CRP level after POD7.


Each interval was run with minute-to-minute pace variation around average speed. CR was measured over the last 2 min. The coefficient of variation (CV) of running speed was calculated to quantify pace variations: ±0.0 m∙s−1 (CV = 0%), ±0.04 m∙s−1 (CV = 1.4%), ±0.13 m∙s−1(CV = 4.2%), and ±0.22 m∙s−1(CV = 7%). No differences in CR, HR, or blood lactate (BLa) were found amongst the variations in running pace. Rating of perceived exertion (RPE) was significantly higher only in the 7% CV condition. The results support earlier studies with short term (3s) pace variations, that pace variation within the limits often seen in competitive races did not affect CR when measured at running speeds below VT.The aerobic cost of running (CR), an important determinant of running performance, is usually measured during constant speed running. However, constant speed does not adequately reflect the nature of human locomotion, particularly competitive races, which include stochastic variations in pace. Studies in non-athletic individuals suggest that stochastic variations in running velocity produce little change in CR. This study was designed to evaluate whether variations in running speed influence CR in trained runners. Twenty competitive runners (12 m, VO Fractional utilization is of established importance within competitive runners [2 (particularly in elite runners) [2 to represent energy cost in sub-elite runners. As defining features of human performance, world record (WR) performances have been of interest to physiologists. A number of physiological determinants have emerged as indicators of performance. These determinants include maximal aerobic capacity, fractional utilization of aerobic capacity, lactate threshold, and the aerobic cost of running (CR) . The VO2udinally  or unchaudinally , althougudinally . Given t runners  and is p runners ,8. The C runners . Althoug runners . Studies runners , which m runners . Recent  runners . However runners  have sho runners  was very runners ,4,5. Accrunners) , we haveThe subjects in this study were 20 competitive runners  recruited from a NCAA Division III track team. All runners had an extensive running background and were fit and healthy at the time of testing . Approval was obtained from the university human subjects committee  and each subject provided written informed consent prior to participation. Descriptive characteristics of the subjects are provided in 2max and ventilatory threshold (VT). The treadmill grade was constant at 1% to account for the lack of wind resistance in the laboratory [−1) to maximal running in increments of 0.21 m·s−1 during each minute. At least 48 h later, each subject completed Trial 2 consisting of four, 6-minute running intervals with 2 min of walking at 1.33 m·s−1 between intervals. The average velocity of each run was set to require ~90% VT, to ensure that VO2 was likely to be in steady state. One interval was completed at a constant speed (control) while the other three intervals were run with the pace altered above and below the average speed each minute by 0.044 m·s−1, 0.134 m·s−1 or 0.223 m·s−1. For example, if the subject’s average speed was determined as 3.35 m·s−1 at ~90% VT, the constant-paced interval was run at 3.35 m·s−1 for 6 min. The varied pace intervals were run at 3.31/3.39, 3.22/3.48, and 3.13/3.57 m·s−1, respectively. Each of the varied pace intervals were 6 min in duration with velocity change occurring every minute. The order of sets was counterbalanced to account for thermal stress and fatigue. Within each set, the slower segment was run first. The VO2 during the last 2 min of each 6-minute set was accepted as CR.Each subject performed two trials. Each trial was performed after a normal regeneration day (40–60 min of easy running) within the coach’s periodization scheme. Trial 1 was an incremental treadmill test to determine VOboratory . The spe2 was measured using open circuit spirometry with a metabolic cart . Calibration procedures were standard, a 3-L syringe to calibrate the pneumotach and a reference gas (~16% O2 and ~5% CO2) and room air to calibrate the gas analyzers. Heart rate was monitored using radio-telemetry . Both VO2 and HR were recorded as the average over the last 2 min of each set. Blood lactate was measured in fingertip blood during walking following each 6-minute interval using dry chemistry . Ratings of perceived exertion (RPEs) were made using the Category-Ratio (0–10) Borg Scale [2 required to run 1 km (mL·kg−1·km−1). CR of each varied pace run was compared with the CR of the constant pace run. VT was estimated using both the v-slope and ventilatory equivalent method [For all tests, VOt method .p-value of < 0.05 was considered statistically significant. The Stata statistical software, version 141  was used for statistical analysis. A preliminary analysis was run with gender as a co-variate. It was not significant relative to changes in CR by the CV of running speed. Accordingly, the primary analysis was based on the pooled results of male and female subjects. The difference in CR, and other outcome measures, expressed in relation to the coefficient of variation (CV) of running speed within each run was evaluated using repeated measures ANOVA. Post-hoc tests were done using the Tukey Test. A −1, 4.2% for 0.134, and 7.0% for 0.223 m·s−1. The physiological responses to the varied and constant pace intervals are presented graphically in −1·km−1), HR  or blood lactate , for the control, 1.4, 4.2 and 7.0% CV of velocity, respectively. A significant difference (p = 0.0246) was found in RPE  with a pace variation at 7.0% CV.There were no significant differences between the varied and constant pace intervals for CR  coincided, in this sample of sub-elite runners, with a CV of 1.4 ± 0.1, 4.2 ± 0.4, and 7.0 ± 0.7 % of average velocity, respectively. A run with no changes in velocity (0% CV) served as a control. This magnitude of CV was of the same magnitude as often reported in races when runners are improving their best performances [The primary purpose of this study was to investigate how variations in pace around a constant average speed affected CR. The study reflected the nature of competitive races in which runners typically change their speed for tactical purposes.  or due tormances . It was ormances , who stuWhile the ability of this group of runners was far below the ability of elite runners, who might be capable of WR, and the relative speed chosen (<VT) was below competition velocity in this population, the results suggest that variations in running velocity, per se, do not inherently change the CR. Future studies, perhaps using more accomplished runners, or technology not dependent on the limits of pulmonary gas exchange might amplify our results.−1 km−1) was comparatively high compared to that observed in more elite runners [2max within this sample of competitive sub-elite, runners (best performance was 85–90% of WR velocity) it seems reasonable to suggest that the CR was a large determinant of the performance level of these subjects. Other data from our laboratory have noted a normal range of variation in CR, suggesting the internal validity of the present data [The absolute value for CR , suggesting that large variations in pace might have a deleterious effect. However, Veneman et al.  recently2 kinetics, which are known to produce near steady state values for VO2 during interval training, seem a likely explanation for the observed lack of variation in CR.Minetti et al.  have arg2 might more reasonably represent the CR than during events where the CR probably exceeds VO2max. However, in highly competitive events, there are often large stochastic variations in momentary running velocity [CR is a potentially important determinant of success in the performance of competitive runners. Given the tendency for recent WR, at least in shorter events, to be run with smaller values for CV of velocity , it woulvelocity . Althougvelocity ,4,5,8, dSupporting earlier findings in non-competitive runners , variati


Ziziphora clinopodioides Lam (CoNPs) and assessment of their cytotoxicity, antioxidant, antifungal, antibacterial, and cutaneous wound healing properties. The synthesized CoNPs were characterized using different techniques including UV–Vis., FT-IR spectroscopy, X‐ray diffraction (XRD), energy dispersive X-ray spectrometry (EDS), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). According to the XRD analysis, 28.19 nm was measured for the crystal size of NPs. TEM and SEM images exhibited a uniform spherical morphology and average diameters of 29.08 nm for the biosynthesized nanoparticles. Agar diffusion tests were done to determine the antibacterial and antifungal characteristics. Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum fungicidal concentration (MFC) were specified by macro-broth dilution assay. CoNPs indicated higher antibacterial and antifungal effects than many standard antibiotics (p ≤ 0.01). Also, CoNPs prevented the growth of all bacteria at 2–4 mg/mL concentrations and removed them at 2–8 mg/mL concentrations (p ≤ 0.01). In the case of antifungal effects of CoNPs, they inhibited the growth of all fungi at 1–4 mg/mL concentrations and destroyed them at 2–16 mg/mL concentrations (p ≤ 0.01). The synthesized CoNPs had great cell viability dose-dependently and indicated this method was nontoxic. DPPH free radical scavenging test was done to assess the antioxidant potentials, which revealed similar antioxidant potentials for CoNPs and butylated hydroxytoluene. In vivo experiment, after creating the cutaneous wound, the rats were randomly divided into six groups: untreated control, treatment with Eucerin basal ointment, treatment with 3% tetracycline ointment, treatment with 0.2% Co(NO3)2 ointment, treatment with 0.2% Z. clinopodioides ointment, and treatment with 0.2% CoNPs ointment. These groups were treated for 10 days. For histopathological and biochemical analysis of the healing trend, a 3 × 3 cm section was prepared from all dermal thicknesses at day 10. Use of CoNPs ointment in the treatment groups substantially raised (p ≤ 0.01) the wound contracture, hydroxyl proline, hexosamine, hexuronic acid, fibrocyte, and fibrocytes/fibroblast rate and remarkably decreased (p ≤ 0.01) the wound area, total cells, neutrophil, and lymphocyte compared to other groups. In conclusion, CoNPs can be used as a medical supplement owing to their non-cytotoxic, antioxidant, antibacterial, antifungal, and cutaneous wound healing effects. Additionally, the novel nanoparticles (Co(NO3)2 and CoNPs) were good inhibitors of the α-glycosidase, and cholinesterase enzymes.The aim of the experiment was a green synthesis of cobalt nanoparticles from the aqueous extract of  Nanotechnology are based on nanoparticles, particles with a 3D structure and a size of 1–100 nm. These materials are available in various sizes and shapes such as crystal, spherical, needle, and rod forms1. Different physical, chemical, and biological methods are used to produce nanoparticles. Use of physical methods requires high temperature, pressure, and cost. On the other hand, most chemical methods use chemicals that are toxic and hazardous to the environment and biological systems. Another problem of using this method is the production of toxic products2. Hence, there is an increasing need to discover a highly efficient and inexpensive method free of toxins and environmental damage. The biological method is one of the methods that is increasingly being used for the production of nanoparticles2. There is a bulky list of sources used for the biological production of metal nanoparticles, including plants and plant extracts3. Recently, plants have been increasingly used for the synthesis of nanoparticles. In general, photosynthesis of nanoparticles by plants has many advantages. In photosynthesis of nanoparticles by plant extracts, water is used as a solvent, which is free of risk4. Biosynthesis of nanoparticles by plant extracts is very easy and does not require specific conditions needed in physical and chemical methods. Plant extracts have a higher reduction potential than microbial culture media, thereby requiring less time for the formation of nanoparticles.5 The contamination created by biosynthesis of nanoparticles by plant extracts is less than other methods and is approximately zero. Therefore, the biosynthesis of nanoparticles by plant extracts has fewer environmental and is more environmentally friendly5. However, the production speed, quality, and other characteristics of the nanoparticles produced by plant extracts depend on factors such as nature of plant extract, the concentration of the extract, salt concentration, pH, temperature, and duration of the reaction7. In previous studies indicated that metal nanoparticles of plant extract have strong potentials in the treatment of bacterial, fungal, and skin diseases10.Nanotechnology and nanoscience are the study and application of extremely small things and can be used across all the other science fields, such as biology, chemistry, physics, materials science, and engineering11. The basic principles of cutaneous wound healing are the minimization of tissue damage, adequate blood supply, oxygenation, proper diet, humid environment to create anatomic integrity, and function of the affected site12. The cutaneous wound healing process includes accumulation of platelets, coagulation, inflammatory response to damage, changing the underlying materials, angiogenesis, and re-epithelization12. Inflammation is a normal phenomenon in the wound healing process and is important for the elimination of the contaminating microorganisms. Prolonged inflammation occurs in the absence of effective decontamination. When microbial cleaning is incomplete, bacteria and endotoxins can prolong the pro-inflammatory cytokines as well as the inflammatory phase13. Subcutaneous cells begin to make collagen following injury and regenerate the epithelial cells. Hence, it is therapeutically important to discover medicines to accelerate the regeneration of dermis and epidermis against skin injuries14. In recent years, the use of chemicals has encouraged the researchers to conduct many studies on the use of traditional and herbal medicines. These studies have revealed that natural drugs are the only treatment modality in some cases, and the compounds available in them have been used in pharmaceutical industries15. Since no definitive drug has been introduced for wound healing, it is necessary to perform studies on the effects of herbal drugs and their metal nanoparticles on cutaneous wound healing15.Cutaneous wound healing is a dynamic, complex, and regular response to impairment that requires the interaction of different types of cells, structural proteins, growth factors, and proteinases19. They have been the foundation for inhibition and treatment of experimental a24. One of the most important herbal medicines that are widely used is Ziziphora clinopodioides Lam. It belongs to Ziziphora genus and Lamiaceae family with the Persian name of kakuti-e kuhi is a perennial plant. In Iran, several hundred species in 49 genera of the Lamiaceae family are scattered26. Z. clinopodioides is used in Iranian traditional medicine for treatment of gastrointestinal disorders, common cold, and inflammations is a member of Labiatae family25. Various properties such as antifungal27, antibacterial28, anti-inflammatory29, and antioxidant30 have been revealed as the effects of this plant. It has chemical components including flavonoids, α and β pinen, terpenoides, thymol, piperitenone, sis-isopulegone, pulegone, and cineol32. According to these compounds, it can be having notable therapeutical effects against various diseases. Due to our ongoing interest on the biosynthesis of metal nanoparticles and heterogeneous catalysts, we wish to report for the first time, green synthesis, detailed morphological, structural, and its catalytic applications of CoNPs synthesized by Z. clinopodioides leaves. CoNPs have synthesized with plant extract having the ecofriendly polyphenol which acts as a reducing agent and a capping agent. Also, considering the therapeutical effects of Z. clinopodioides, we made an attempt to study the cytotoxicity, antioxidant, antibacterial, antifungal, and cutaneous wound healing effects of CoNPs.Iran has experimental plants that are widely distributed throughout the country, particularly in Kermanshah province, west of Iran All materials were obtained from Sigma Aldrich chemicals.Z. clinopodioides was collected from Kermanshah city in the west of Iran  distilled water, heated at 45 °C, and stirred for 2 h. It was then kept at ambient temperature for 24 h. Next, the extract was filtered with Whatman paper #2. The primary extract was fed into a vacuum distillation apparatus (a rotary machine with a vacuum pump), and the solvent was evaporated at 40 °C for 1 h, yielding the condensed extract. To prepare the powder of the extract, the condensed solution was put in the oven for 48 h at 40 °C, and the obtained substance was lyophilized35.After complete drying of37. Firstly, 2.5 g of plant extract was dissolved in 62.5 mL of deionized/distilled water, then 30 mL of Co(NO3)2·6H2O with a concentration of 30 mM was added to the solution. The mixture was refluxed for 90 min at 60 °C. Then 5 mL of NaOH 2% was slowly added to the mixture during the reaction time. The color of the solution was changed to brown color. In the next step, the mixture was centrifuged at 6,000 rpm for 15 min. Finally, the residue was dried in an oven for 3 h at 50 °C. The obtained brown powder was kept in a vial for chemical characterization and biological activities.Biosynthesis of cobalt nanoparticles was carried out according to the previous studies with some modification−1 (KBr disc); MIRA3TESCAN-XMU was used to report the FE-SEM Images and EDS result. The XRD pattern of CoNPs was recorded in the 2θ range of 20°–80° by a GNR EXPLORER instrument at a voltage of 40 kV, a current of 30 mA, and Cu-Kα radiation (1.5406 Å). The average crystal size of CoNPs was calculated using X‐ray diffraction according to the Debye–Scherrer equationDifferent techniques were used to characterize the synthesized CoNPs. The methods include UV–Vis., FT-IR spectroscopy; XRD, SEM, EDS, and TEM techniques. Different parameters of the nanoparticle, such as shape, particle size, fractal dimensions, crystallinity and surface area are obtained by these techniques. The UV–Vis. spectra were obtained by a PhotonixAr 2015 UV–Vis. Spectrophotometer (200–800 nm); The FT-IR spectra were recorded using a Shimadzu FT-IR 8400 in the range of 400–4,000 cm2. After cell density reached 80%, the sample was exposed to 1% of EDTA-trypsin solution. After 3 min incubation at 37 °C along with 5% CO2 in the cell culture incubator and observing the cells detached from the plate floor, the sample was centrifuged for 5 min at 5,000 rpm and the cell deposition was trypsinized by adding the culture medium. Then, the cell suspensions were counted by Neobar slide after trypan blue staining, and cell toxicity test was done by MTT assay. For this reason, 10,000 HUVEC cells along with 200 µL complete culture medium were added to each 98-plate culture plate. To achieve cells with single layer density, the plate was incubated again at 37 °C along with 5% CO2. After 80% of cell growth was achieved, the culture medium was removed and the surface of the cells was irrigated with FBS, and 100 µL double concentration culture medium was added afterward. Then, 100 µL Co(NO3)2, Z. clinopodioides, and CoNPs solution soluble in PBS were added to the well 1 (1000 µg/mL). After mixing Co(NO3)2, Z. clinopodioides, and CoNPs in the culture medium, 100 µL of the first well was removed and added to the second well. Next, 100 µL of the second well was removed and added to well 3. This process was continued up to well 11 so that half of the Co(NO3)2, Z. clinopodioides, and CoNPs were added to each well. Well 12 only contained the cell and single concentration complete culture medium and remained as control. The plate was incubated at 37 °C for 24 h at the presence of 5% CO2, after which cell toxicity was determined by tetrazolium staining. After that, 10 µL of tetrazolium stain (5 mg/mL) was added to the wells, including the control, and the plate was incubated at 37 °C for 2 h at the presence of 5% CO2. Then, the stain was removed from the wells and 100 µL of DMSO was added to the wells. The plate was wrapped in an aluminum foil and shaken for 20 min in a shaker. Finally, cell viability was recorded by an ELISA reader at a wavelength of 570 nm according to the following formula38:Human umbilical vein endothelial cells (HUVECs) was used to investigate the efficacy of silver nanoparticles in the culture medium. To this end, the cell line was placed in T25 flasks along with complete culture medium, including DMEM (Dulbecco's Modified Eagle Medium), 10% decamplmaneh fetal bovine serum, and 1% penicillin–streptomycin solution and incubated at 37 °C along with 5% CO3)2, Z. clinopodioides, and CoNPs were mixed with 2 mL 0.004% DPPH solution. The control solution contained 2 mL DPPH and 2 mL ethanol. The solutions were kept in darkness at room temperature for 30 min. Then, the absorption rate of the samples was measured at 517 nm by the following formula compared to the control sample39:To determine the trapping potential of DPPH, different concentrations of the Co and Streptococcus pneumonia (ATCC No. 49619) were procured as lyophilized from Iranian Research Organization for Science and Technology. Also, four fungal species, namely Candida albicans (PFCC No. 89-1000), Candida glabrata (PFCC No. 164-665), Candida krusei (PFCC No. 52951), Candida guilliermondii (PFCC No. 88-1947), and four bacterial species, namely Pseudomonas aeruginosa (ATCC No. 27853), Escherichia coli O157:H7 (ATCC No. 25922), Bacillus subtilis (ATCC No. 6633), and Staphylococcus aureus (ATCC No. 25923) were procured as lyophilized from Pasteur Institute of Iran.3)2, Z. clinopodioides, and CoNPs were added to the wells and disks. In this study, distilled water was negative control and antifungal  and antibacterial  antibiotics were positive controls. The zone of growth inhibition was recorded after 24 h of incubation at 37 °C40.Agar disk-diffusion and well-diffusion methods were used to analyze the antifungal and antibacterial activities. To this end, the prepared microbial suspension with 0.5 McFarland turbidity standard was cultured onto Mueller Hinton Agar and Sabouraud Dextrose Agar in completely sterile conditions. In the well diffusion method, 6-mm wells were created by a Pasteur pipette on the culture medium with constant distances. In the disk diffusion method, 6-mm blank disks were used on agar culture medium. Then, 60 µL of different dilutions of Co(NO3)2, Z. clinopodioides, and CoNPs were added to macro broth tubes, following which 60 µL fungal and bacterial suspensions were added and incubated for 24 h at 37 °C. Then, the concentration with minimum dilution and no turbidity was considered MIC40.Macro broth dilution method was used to determine Minimum Inhibitory Concentration (MIC). Different dilutions of Co and minimum fungicidal concentration (MFC), 60 µL MIC and three preceding chambers were cultured on Muller Hinton Agar and Sabouraud Dextrose Agar, respectively. After 24 h incubation at 37 °C, the minimum concentration with no fungal and bacterial growth was considered MBC and MFC, respectively. All tests were done in tripletAll animal procedures were approved by standards of Kermanshah Payame Noor University (No. 01/Z/G 1395/12/01) on Humane Care and Use of Laboratory Animals, in accordance with the Research Ethics Committee of the Ministry of Health and Medical Education in Iran , based on the Helsinki Protocol . A total of 60 male rats of the same race with the weight of 220 ± 5 g were used in this study. The rats were kept in individual cages at 22 ± 2 °C, in 12:12 h dark–light cycle, and with free access to water and food. The rats were anesthetized by intramuscular administration of 40 mg/kg ketamine. After induction of anesthesia, the hair between the two scapulae was shaven, and 3 × 3 cm of the area was disinfected with 70% ethanol. A wound (2 × 2 cm) was made by a scalpel, which involved the removal of all cutaneous layers. The depth of the wound included dermis and hypodermis 2 ointment, treatment with 0.2% Z. clinopodioides ointment, and treatment with 0.2% CoNPs ointment. The ointment was applied to the wound bed for 10 consequent days.After creating the cutaneous wound, the rats were randomly divided into six groups: untreated control, treatment with Eucerin basal ointment, treatment with 3% tetracycline ointment, treatment with 0.2% Co was adjusted similar to the work of TAO et al.34. As mentioned earlier, the absorption values were determined at 405 nm34.As previously revealed, the inhibition effect of new nanoparticles (CoNPs) on pain and BChE activities was specified according to Ellman's spectrophotometric methodP ≤ 0.01).The obtained results were fed into SPSS-22 software and analyzed by one-way ANOVA, followed by Duncan post-hoc test  was used to recognize surface morphology and size of CoNPs indicated the formation of homogeneous cobalt NPs with an average diameter size of 29.07 nm. Figure 47. CoNPs was also biosynthesized using methanolic extracts of Conocarpus erectus and Nerium indicum. The size of particles was estimated in the range of 20–60 nm37. The average particle size of CoNPs, which was produced using aqueous extracts of Raphanus sativus, was reported 80 nm36. The particle size ranging from 20 to 50 nm was reported for of cobalt nanoparticles that were biosynthesized using Moringa oleifera extract48.TEM micrograph showed the surface morphology of synthesized nanoparticles Fig. . The parThe EDS analysis of CoNPs is shown in Fig. E. coli and S. typhimurium (p ≤ 0.01). CoNPs prevented B. subtilis, S. pneumonia/S. aureus/P. aeruginosa/C. glabrata/C. guilliermondii/C. krusei, and E. coli/S. typhimurium/C. albicans growth at 1, 2, and 4 mg/mL concentrations, respectively and destroyed B. subtilis/S. pneumonia/S. aureus/P. aeruginosa/C. krusei, E. coli/C. guilliermondii/C. glabrata/C. albicans, and S. typhimurium at 2, 4, and 8 mg/mL concentrations, respectively. Thus, the findings showed strong antifungal and antibacterial properties of CoNPs against all of the tested fungi and bacteria. Moreover, CoNPs had the highest antibacterial and antifungal effects on B. subtilis and C. krusei, respectively (p ≤ 0.01). In agreement with our experiment, in study of Hemmati et al.5 showed that metal nanoparticles had strong antibacterial activities against Gram-negative bacteria include Proteus mirabilis (ATCC No. 25933), Shigella flexneri (ATCC No. 12022), Listeria monocytogenes (ATCC No. 13932), Klebsiella pneumonia (ATCC No. 9997), Pseudomonas aeruginosa (ATCC No. 27853), Escherichia coli O157:H7 (ATCC No. 25922), and Salmonella typhimurium (ATCC No. 14028) and Gram-positive bacteria include Enterococcus faecalis (ATCC No. 29212), Bacillus subtilis (ATCC No. 6633), Streptococcus pyogenes (ATCC No. 10403), Staphylococcus saprophyticus (ATCC No. 49453), Staphylococcus epidermidis (ATCC No. 12228), Staphylococcus aureus (ATCC No. 25923), and Streptococcus pneumonia (ATCC No. 49619)5.Analysis of results in this research Tables , 4, 5, 6p ≤ 0.01) amended the above parameters at day 10 compared to the other groups  the number of fibrocyte, the concentration of hydroxyproline, hexuronic acid, and hexosamine and fibrocyte/fibroblast ratio at day 10 compared to the other groups  the number of these cells at day 10 compared to the other groups  indicated that CoNPs ointment regulated significantly  indicated impressive prevention similar to BHT. The IC50 of Z. clinopodioides, BHT, and CoNPs were 448, 342, and 342 µg/mL, respectively 2, Z. clinopodioides, and CoNPs were examined by MTT test for 48 h regarding cytotoxic effects on HUVEC cells. The absorbance rate was determined at 570 nm, which indicated extraordinary viability on HUVEC line even up to 1000 μg/mL for Co(NO3)2, Z. clinopodioides, and CoNPs 2 showed Ki value of 0.42 ± 0.11 mM. The IC50 values of TAC as positive control for AChE and new nanoparticles were examined in the following order: Co(NO3)2  < CoNPs  < TAC . The IC50 values of TAC as positive control for BChE and new nanoparticles are in the following order: Co(NO3)2  < CoNPs  < TAC . In addition, new nanoparticles effectively inhibited BChE with values of 1.18 ± 0.17 and 1.91 ± 0.38 mM Ki, respectively. At the same time, all of these new nanoparticles synthesized had nearly close inhibition profiles. The most active Co(NO3)2 effectively inhibited BChE, with Ki value of 1.18 ± 0.17 mM. Indeed, recording novel inhibitors targeting AChE has still been of significant interest to the researchers. Additionally, it is recorded that selective BChE inhibitors can circumvent classical cholinergic toxicity. Hence, the development of novel selective BChE inhibitor compounds can provide additional benefits in the therapy of AD61.All of novel nanoparticles (CoNPs) had remarkably higher AChE inhibitory effect than of control AChE inhibitor compound such as Tacrine. Indeed, the Ki values of novel nanoparticles and standard compound (tacrine) are summarized in Table 2: 0.9371) < Co(NO3)2  < ACR . Anti-diabetic drugs that are used in clinical practice, such as acarbose, voglibose and miglitol, competitively inhibit α-glucosidase in brush border of small intestine which subsequently interrupt hydrolysis of carbohydrate and improve postprandial hyperglycemia62.For enzyme glycosidase, new nanoparticles (CoNPs) have IC50 values of 15.86 and 11.26 µM and Ki values of 18.51 ± 2.73 and 15.70 ± 3.10 mM (Table Z. clinopodioides aqueous extract, in which no chemical substance was used. Due to the existing major problems in the physical and chemical methods for producing nanoparticles, there is a need to easy, low-cost, and non-toxic procedures. FT-IR, UV–Vis spectroscopy, EDS, and FESEM techniques were used to characterize CoNPs synthesized. The synthesized CoNPs have great antioxidant, antifungal, antibacterial, and cutaneous wound healing potentials. Also, the absence of any notable toxicity is another advantage that was evaluated and confirmed by the recent study. After confirming in the clinical trial sutides, this formulation can be used for the treatment of several types of cutaneous wounds in humans.The recent research indicated an ecofriendly, clean and useful method to synthesize cobalt nanoparticles using


Eosinophilic arthritis is characterized by arthritis, eosinophilia, normal laboratory findings, unresponsiveness to nonsteroidal anti‐inflammatory drugs and favorable response to corticosteroids. We diagnosed a female patient with this rare disease. Eosinophilic arthritis is characterized by arthritis, eosinophilia, normal laboratory findings, unresponsiveness to nonsteroidal anti‐inflammatory drugs and favorable response to corticosteroids. We diagnosed a female patient with this rare disease. We diagnosed a female patient with eosinophilic arthritis and treated her using a corticosteroid. Her symptoms were relieved after several days. It is important for physicians to be aware of this rare disease.3 is considered as elevated. Peripheral eosinophilia is characterized as follows: mild, 500‐1500 cells/mm3; moderate, 1500‐5000 cells/mm3; and severe, >5000 cells/mm3. The common causes of eosinophilia include allergic or atopic disorders and parasitic infections. In our case, the patient complained of edema and pain in her wrists and ankles and presented moderate eosinophilia. However, she had normal laboratory findings and was unresponsive to nonsteroidal anti‐inflammatory drugs (NSAIDs). Therefore, we diagnosed her with eosinophilic arthritis, which is quite rare.Eosinophils are granulocytes derived from progenitor cells of monocytes, macrophages, and basophils. They are components of the innate immune system, and eosinophils have various functions, including defense against parasites and intracellular bacteria and modulation of immediate hypersensitivity reactions.23; erythrocytes, 4.99 million cells/mm3; and platelets, 253 000 cells/mm3. Her hemoglobin was 13.6 g/dL, and hematocrit was 41.7%. The eosinophil level was 15.5% (1767 cells/mm3). Her blood biochemical analysis results were as follows: total protein, 7.6 g/dL; serum albumin, 4.5 g/dL; Na, 138 mEq/L; K, 4.3 mEq/L; Cl, 104 mEq/L; serum creatinine, 0.66 mg/dL; estimated glomerular filtration rate, 85.1 mL/min; aspartate aminotransferase, 20 IU/L; alanine transaminase, 20 IU/L; blood sugar, 104 mg/dL; thyroid‐stimulating hormone, 1.23 μU/mL; free triiodothyroxine, 2.63 pg/mL; free thyroxine, 0.94 ng/dL; and one‐hour erythrocyte sedimentation, 8 mm. An immunological examination revealed normal levels of rheumatoid factor, antistreptolysin O, antistreptokinase, anticyclic citrullinated peptide antibodies, antideoxyribonucleic antibodies, antiribonucleic acid antibodies, perinuclear antineutrophil cytoplasmic antibodies, cytoplasmic antineutrophil cytoplasmic antibodies, and immunoglobulin E. We treated her with a NSAID but her symptoms were not relieved. Her WBC and eosinophil levels increased to 11 900 cells/mm3 and 19.4% (2308 cells/mm3), respectively. Therefore, we diagnosed her with eosinophilic arthritis and treated her with prednisolone (30 mg/day). Within several days, her edema and pain were relieved . Prednisolone was gradually tapered and ceased two months later, no occurrence of relapse for at least one year.A 30‐year‐old Japanese woman presented to an orthopedist after experiencing edema and pain on her wrists and ankles for a week. She was prescribed with NSAID, which did not relieve her symptoms. This resulted in her visiting our hospital. She had been healthy and had not used any ordinary drug or supplement. Additionally, she had no history of allergies and parasitosis. However, her mother suffered from hyperthyroidism according to her family history. A physical examination confirmed edema in both her wrists and ankles, with the edema being more severe on the right side Figure . Pitting33), normal laboratory findings, unresponsiveness to NSAIDs, and favorable response to corticosteroids. The pathogenesis of this disease was unknown and could have resulted from a reaction to some unknown agents or allergens.We report the rare case of a 30‐year‐old Japanese woman with eosinophilic arthritis. Polyarthritis with peripheral blood eosinophilia is associated with several diseases. Peripheral blood eosinophilia is common among patients with concomitant allergies or helminthic infections.4We report the case of a 30‐year‐old Japanese woman presenting with edema and pain in her wrists and ankles, who was diagnosed with eosinophilic arthritis. The etiology of this disease is unknown, and it is important for physicians to be aware of this rare disease.None declared.HY and FK: treated the patient and described the manuscript. MS: revised the manuscript.Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor‐in‐Chief of this journal.