Salivary lactate has been suggested as a non-invasive anaerobic biomarker in sports medicine for decades, yet has not been widely applied until now. This study aimed to explore possible issues related to its application and suggest directions for future method improvement.n = 20). The samples were collected via chewing a cotton ball for one minute and centrifuging it afterwards. The evaluation included variation with mouth rinse times, consistency at different sampling times, change after treadmill or cycle ergometer trainings, and association with blood lactate. Sample sizes were calculated prior to the study. One-way analysis of variance (ANOVA), intra-class correlation coefficients (ICC) and relative standard deviation (RSD) were used to evaluate data variances. Pearson correlation was applied to show correlation between salivary and blood lactate. Effect sizes and power were calculated following ANOVA and correlation analyses.A liquid chromatography–mass spectrometry (LC-MS) method for the determination of salivary lactate was developed, validated and applied on saliva samples collected from a group of professional sprinters , and stabilized after mouth rinsing for three times. The concentrations at resting state across three weeks were consistent at group level , but varied greatly individually . Salivary lactate level significantly increased after treadmill and cycle ergometer trainings , and displayed positive correlation with blood lactate concentration . Significant difference between male and female participants was observed in none of the tests conducted in this study.The RSD of the LC-MS method was 19.70%. Salivary lactate concentration was affected by mouth rinse times before sampling (ANOVA Salivary lactate was found to be a potential anaerobic biomarker. However, reproducible methods for sample collection and analysis, as well as more knowledge on the secretion mechanism and pattern of salivary lactate are required to make it a practical anaerobic biomarker. However, blood sampling test is an invasive test, which brings pain, stress, and risk of infection . Despite these promising data generated decades ago, salivary lactate is still not widely applied in sports medicine nowadays. In this study, we aimed to explore what might have prevented salivary lactate from being used in practice.Saliva is a type of non-invasive biological sample that can be easily collected. Positive correlation between salivary lactate and blood lactate , as wellSpecifically, factors that potentially affect the analysis of salivary lactate, including analytical method, mouth rinses before sampling, and data consistency at different sampling days were evaluated. Elevation of salivary lactate during and after two types of short-time high-intensity trainings, and its correlation with blood lactate were also verified.A customized liquid chromatography–mass spectrometry (LC–MS) method was developed and validated for the analysis of salivary lactate. This method was chosen for its high throughput. In fact, we also simultaneously measured 23 other potential salivary biomarkers of physical activities, including creatinine, uric acid, urea, cortisol, testosterone, choline, 5-hydroxyindole acetic acid, homovanillic acid and 15 essential amino acids together with lactate, with the hope of discovering new salivary biomarkers in sports. However, these 23 compounds were either undetected or unchanged after the exercise training, and hence the data were not shown in this report.The participants were 20 professional sprinters, including eleven females and nine males. Six of them were 100-meter runners, three were 200-meter runners and eleven were 400-meter runners. They had been engaged in training for three years or longer at a training center where the study was conducted. Verbal informed consent from the athletes and their coaches were obtained prior to sampling. All the participants fulfilled the following criteria: (1) they were in good health, without sport injuries or bleeding wounds in the mouths, nor underwent dental surgery in the last three months; (2) they did not smoke, drink alcohol, or participate in strenuous physical activities 24 h prior to the study; and (3) they did not eat food or chew gum two hours prior to sampling. The study was conformed to the Declaration of Helsinki and approved by the ethics committee of the Stomatological Hospital of Chongqing Medical University, with the approval number of 2021(061).To obtain a saliva sample, a medical cotton ball weighing approximately 0.3 g was chewed for one minute, and spitted into a 10 ml centrifuge tube. The tube was immediately sealed and temporarily stored in an ice box for no more than 2 h before transported to the laboratory and stored at −20 °C.The evaluation was consisted of five parts: (1) validation of method accuracy and precision, (2) the influence of mouth rinses, (3) consistency at resting state, (4) variation during training, and (5) correlation with blood lactate. Accordingly, the participants were asked to provide saliva samples as follows: (1) at resting state, before and after one, two and three times of mouth rinses with 10 ml of drinking water ; (2) at resting state, before breakfast (approximately 7 am) in Monday mornings of three consecutive weeks and after three times of mouth rinses ; and (3) five minutes before, immediately after and five minutes after acute high-intensity treadmill training with increasing load or cycle ergometer trainings  after three times of mouth rinses.As many professional sprinters in the training center as possible were recruited into this study. Sample sizes required were calculated based on the effect sizes of previous similar studies , the sigThe treadmill and cycle ergometer trainings started at approximately 9 am on two Thursday mornings with a one-month interval. The participants provided their information including sex, age, height and body weight, and sat quietly for 20 min prior to the trainings. The acute high-intensity treadmill training with increasing load was conducted with a Metalyzer 3B  spiroergometry system and a RUN 7410 treadmill  preheated for 30 min. The training included five-minute warm-up at 8 km/h and 0° grade, 8 min load-increasing stage with increasing speed and slope every minute based on the participants’ individual capacities, and three-minute recovery stage at 5–7 km/h, 0° grade. The participants were equipped with heart rate monitors and accompanied by a team doctor. They were encouraged to complete the training but allowed to stop anytime when exhausted. The exhaustion criteria were set as the heart rate remaining at no less than 180 beats/min for two minutes, and the rate of perceived exertion (RPE) scale  reachingPrior to analysis, the cotton ball soaked with saliva was defrosted at room temperature and transferred into a 4 ml Eppendorf tube with a small hole drilled at the bottom. The 4 ml tube was then put into a clean 10 ml Eppendorf tube whose cap was cut off. The inner diameter of the 10 ml Eppendorf tube was just between the outer diameters of the body and edge of the 4 ml Eppendorf tube, enabling the 4 ml tube to be stuck at the top of the 10 ml tube. After that, the 10 ml tube was centrifuged with an Eppendorf Centrifuge 5920 R  at 4 °C and 3,000 rpm for 3 min, so that the saliva in the cotton ball was collected in the 10 ml Eppendorf tube. 100 µl of the saliva sample was transfer into a 1.5 ml Eppendorf tube, and vortex-mixed with 20 µl of 1 µg/ml internal standard 2-Cl-phenylalanine. Then, 1 ml of acetonitrile was added and the mixture vortexed again. After that, the 1.5 ml centrifuge tube was centrifuged with an Eppendorf Centrifuge 5427 R  at 4 °C and 10,000 rpm for 10 min. The supernatant was transferred to a new 1.5 ml Eppendorf tube, dried with a Labconco CentriVap vacuum dryer  at 40 °C, and then reconstituted in 100 µl of 5% methanol. The solution was centrifuged again at 4 °C, 10,000 rpm for 5 min and 80 µl of the supernatant was transfer to an LC-MS vial for injection. Calibration samples were prepared in the same way  as the saliva samples. A pool sample was prepared by mixing 100 µl aliquots of all the test samples and used in method validation.The LC-MS analysis was performed using an Agilent 1290-6495C Ultra High Performance Liquid Chromatography-Triple Quadrupole Tandem Mass Spectrometry . LC separation was performed using a Waters HSS T3  column with a Waters ACQUITY UPLC HSS T3 VanGuard  pre-column. Mobile phase A was aqueous solution of 0.1% formic acid, and mobile phase B was acetonitrile. The gradients were: 0–0.5 min: 5% B, 0.5–1 min: 5–50% B, 1–7.5 min: 50% B, 7.5–8 min: 50–5% B, 8–10 min: 5% B. The flow rate was 0.3 ml/min, the injection volume was 2 µl, and the column temperature was 40 °C. The ion source parameters for the mass spectrometry were: Gas Temp: 200 °C, Gas Flow: 14 L/min, Nebulizer: 35 psi, Sheath Gas Temp: 300 °C, Sheath Gas flow: 11 L/min, Capillary: −3000 V, Nozzle Voltage: 500 V, iFunnel H: −150 V, L: −60 V. Multiple reaction monitoring (MRM) mode was used to analyze lactate and the internal standard 2-Cl-phenylalanine. Q1(m/z), Q3(m/z) and CV(eV) were set as 89.0, 43.2 and 13, respectively, for lactate; and 198.0, 181.0 and 9, respectively, for 2-Cl-phenylalanine. Lactate standard was purchased from Bidepharm , and 2-Cl-phenylalanine was from MedChemExpress .The method was validated for accuracy and precision. Accuracy was determined by adding 10 µl/ml of lactate into a pool sample and calculating the recovery, which was the ratio of measured concentration increase to the theoretical concentration increase (10 µl/ml). Precision was determined by analyzing seven 100 µl sub-aliquots of the pool sample twice, and calculating the relative standard deviation (RSD) of the fourteen measured concentrations.P value for each comparison with family-wise significance and confidence level set at 0.05 was reported. Intra-class correlation coefficients (ICC) and RSD of multiple sampling times were used to evaluate data variances of each participant. Pearson correlation coefficient (r) was calculated for salivary lactate and blood lactate of the same participants at the same time points. Effect sizes and power were calculated following ANOVA and correlation analyses. ANOVA, RSD and Pearson correlation were calculated using GraphPad Prism 8 for mac , while ICC was calculated with IBM SPSS Statistics software, version 20.0.0 . Effect sizes of ANOVA (η2) were calculated from ANOVA F values, using the equation η2 = F × (k−1) / [F × (k−1) + (n−k)] (β) and sample sizes of all analyses were calculated with R software and package “pwr” (Each set of data is expressed as mean ± standard deviation (SD). Paired one-way analysis of variance (ANOVA) and Tukey’ post-hoc multiple comparisons test were used to compare data among different time points of the same group of participants, and multiplicity adjusted + (n−k)] , while t+ (n−k)]  and pack+ (n−k)] . Power (ge “pwr” .η2) was 0.40, power (1–β) was 0.99, ICC was 0.23, and mean RSD of four sampling was 55.30%. The average concentration decrease with the increasing times of mouth rinses. After three times of mouth rinses, the salivary lactate concentration was significantly lower than that after one rinse, so three times of mouth rinses prior to sampling was applied in the following evaluations.It was calculated that the method accuracy was 95.9% and the RSD was 19.7%. Using this method, it was found that the times of mouth rinses  significant affected salivary lactate levels . ANOVA pvia analyzing samples collected once a week  = 0.03, power (1–β) = 0.20). However, marked individual variance was observed, which was shown by an ICC of 0.22 and a mean RSD of 56.16%.We then investigated whether the salivary lactate concentration was consistent at different sampling days e a week . ANOVA sp value was 0.0002, effect size (η2) was 0.46, and power (1–β) was 0.9999 for treadmill trainings; while ANOVA p value was 0.0019, effect size (η2) was 0.40, and power (1–β) was 0.9993 for cycle ergometer trainings. There was no significant difference between immediately and 5 min after training.Next, we verified whether salivary lactate increased after training. Despite large inter-individual variances, it was clear that the salivary lactate levels significantly increased after the treadmill  and cyclOne participant missed the blood lactate test, and another participant missed one time point of the blood lactate test on the day of cycle ergometer training. Consequently, 14 out of the 16 participants underwent the blood lactate before and immediately after the cycle ergometer training, and one out of the 16 participants only underwent blood lactate test at one time point, which provided a total of 14 × 2 + 1 = 29 pairs of blood-salivary lactate data. The 29 pairs of salivary-blood lactate data showed significant and positive correlation . The 95%In this study, we evaluated whether salivary lactate can be used as an anaerobic biomarker. Significant increase of salivary lactate during and after two forms of short-time high-intensity exercises , as wellLactobacillus produce lactate (Firstly, the production of saliva lactate was influenced by multiple factors. Even with the same sampling procedures, at the same time of day and day of week, saliva samples were found to vary, with the RSD as high as 56.16% . It shou lactate , which cSecondly, saliva collection methods may affect lactate concentration. Saliva is secreted at the rate of approximately 0.5 ml/min by three major pairs of salivary glands and a number of minor mucous glands, and the flow rate may increase with stimuli . Thus, sFinally, the precision of the analytical method was not satisfactory. In this study, an LC-MS method with an RSD of 19.70% was developed to analyze salivary lactate. In contrast, enzymatic methods for salivary lactate measurements normally produce an RSD of approximately 2% or less . This maTo summarize, salivary lactate is a promising biomarker for the evaluation of anaerobic capacities, but reliable sampling and analytical methods, together with comprehensive knowledge on the mechanism and pattern of salivary lactate secretion, are crucial elements to make it practical.


P < .05. Thirty patients  aged 14 to 60 years old (33.4 ± 11.9) were enrolled in this study. The mean overall basal quality of life score was 3.41 ± 1.22. After 11 weeks, the mean RQLQ score was 1.74 ± 1.09 , and all the domain scores improved . Each of the 28 individual item scores on the RQLQ showed clinical  and statistical (P < .05) improvements. DLE might be a beneficial adjuvant treatment for AR. Our results provide preliminary data for future research.Allergic rhinitis (AR) has considerable impact on the general health of individuals. Therefore, treatment trials should include an evaluation of quality of life. We aimed to determine changes in the quality of life of moderate/severe AR patients treated with standard treatment in addition to dialyzable leukocyte extract (DLE), a peptide-based immunomodulator. In a prospective, non-controlled trial, DLE was added to the standard treatment regimen for patients with moderate/severe AR. DLE was administered orally at 2 mg per day for 5 days, followed by 4 mg per week for 5 weeks, and then 2 mg per week for 5 weeks. The primary endpoints were overall improved Standardized Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) scores, domain scores, and individual item scores of 0.5 points or higher. Statistical significance was defined as Clinical trials registration ID: NCT02506998 The incidence rate in Latin America is approximately 7%; in Mexican children, it averages at 15.4% (7.1–28%) with variations, depending on region and degree of urbanization. Disease control in primary care settings is poor and correlates with disease perception.Allergic rhinitis (AR) is a common disease worldwide.,6 Moreover, its effect on comorbidities such as asthma reflects the need for good treatment options. The impact of AR on sleep, daily activities, and productivity has been assessed in multiple studies, suggesting that quality of life (QoL) evaluation is an important aspect to consider in clinical trials.,9Although not life-threatening, AR considerably affects general well-being, absenteeism, work, and school performance.,11 Favoring Th1/Treg can thus improve allergic symptoms. Dialyzable leukocyte extract (DLE) is a complex drug made up of a mixture of peptides under 10 kDa in size, whose primary component is ubiquitin. The proposed mechanism of action of DLE is immune modulation, which is initiated by ubiquitin activating its receptor C-X-C chemokine receptor type 4 in the stomach and via the vagus nerve, affecting innate and adaptive signaling pathways and thereby favoring the T-helper (Th1) immune regulatory response.,15 With the use of DLE, the production of certain cytokines is clinically modified, including a decrease in tumor necrosis factor alpha and interleukin 6, which drive an immune-regulatory response.–16 The administration of DLE improves the clinical response in allergies and other immune-mediated diseases.–19The T-helper cell balance in allergic diseases is skewed toward a Th2/Th17 response, and it has been proposed that Th1/Treg cytokines play a negative regulatory role in Th2 inflammatory predominance.This study aimed to determine changes in QoL of patients with moderate/severe AR, treated with standard treatment in addition to DLE.,20 Patients were required to have at least 6 weeks of standard AR treatment with adequately prescribed antihistamines, leukotriene inhibitors, intranasal steroids, intranasal/ophthalmic sodium cromoglycate, and/or immunotherapy in varying combinations before entering the protocol. Confirmation of a type 1 hypersensitivity response through previous positive skin or in vitro allergen testing was required. Asymptomatic patients, those with severe chronic diseases, and those with immunodeficiency during enrollment were excluded. Subjects with incomplete visits were excluded from the analysis. No data substitutions were made.We included 30 patients aged 14 to 60 years with a confirmed diagnosis of moderate/severe AR. The disease was confirmed, and the severity was classified based on the Allergic Rhinitis and its Impact on Asthma guidelines.®, National Polytechnic Institute, Mexico) was added. DLE dose and duration of treatment were based on previous recommendations by Berrón-Pérez et al, allowing enough time for a measurable effect to occur as with other allergy medications. The initial DLE dose was 2 mg orally per day for 5 days, followed by 2 mg twice a week for 5 weeks, and then 2 mg per week for 5 weeks. Clinical history was reviewed during each visit , physical examination was performed, and the Spanish (Mexico) version of the self-administered Standardized Rhinoconjunctivitis Quality of Life Questionnaire (RQLQ) was administered. For patients aged 14 to 17 years, an RQLQ score ≥ 12 was used.This was a prospective, open-label, non-controlled trial. The standard treatment was continued without any modifications, and DLE  to 6 (very bothersome). The total score was calculated from the average score of all items. The scores from each of the 7 domains  were obtained from the average score of the items in each domain. The scores for each item were also obtained. The primary endpoint was a change from baseline in the RQLQ overall scores, domain scores, and individual item scores of at least 0.5, minimal important difference (MID).All patients who underwent DLE signed an informed consent form as part of the IC 13-001 approved by the University Ethics Committee. Patients younger than 18 years also provided oral assent.t test and one-way analysis of variance were used to determine the differences between the groups and for before and after comparison. Differences were considered statistically significant at P < .05, and a ≥0.5-point response on the RQLQ was considered clinically significant. Each item in the RQLQ questionnaire was analyzed.Statistical analysis was performed using GraphPad Prism software version 6.0f  and SPSS version 21 . The normality of the data was checked for skewness and kurtosis, and was confirmed using the Shapiro–Wilk normality test. Dispersion and central tendency statistics were used to describe variables. A 2-tailed The sample size was calculated using G*power 3 (Heinrich Heine Universität Duesseldorf) according to the following parameters: Type I error 0.05, power 0.8, and effect size 0.43, based on previous observations. Patients whose overall symptoms worsened during the study were considered to have AE. The intention-to-treat population, consisting of all subjects who received at least 1 dose of DLE, was used for safety analysis.The presence of any adverse events (AE) associated with the medication was assessed as required by the Mexican Official Standard Norm.Thirty patients  between the ages of 14 and 60 years (33.4 ± 11.9) were followed up for 11 weeks  were taking antihistamines, 13 (43.3%) were taking nasal corticosteroids, 5 (16.7%) were taking montelukast, 5 (16.7%) were taking sublingual or subcutaneous immunotherapy, and 1 patient (3.3%) had ophthalmic sodium cromoglycate . After 1 week of DLE use, 14 (46.7%) patients had significantly improved QoL scores (≥0.5). After 6 weeks, the mean RQLQ overall score was 2.05 ± 0.83 , and 25 (83.3%) patients had significantly improved QoL scores (≥0.5). After 11 weeks, the mean RQLQ overall score was 1.74 ± 1.09 , and 26 (86.7%) patients had significantly improved QoL scores (≥0.5)  Total Score” and Table S2, Supplemental Digital Content, http://links.lww.com/MD/J221 “Follow-up RQLQ(S)”).The median basal overall RQLQ score before DLE was 3.41 ± 1.22. One week after the addition of DLE, the mean RQLQ overall score was 2.89 ± 1.27 , or the amount of AR medication taken Table .P < .001, 95% CI 0.91–2.15. MID was ≥0.5 in 24 (80%) patients.After 11 weeks of DLE treatment, the mean daily activity score improved from 3.62 ± 1.3 to 2.09 ± 1.25, P < .001, 95% CI 0.9–2.26. MID was ≥0.5 in 23 (76.7%) patients.The mean sleep symptom score improved from 3.1 ± 1.51 to 1.52 ± 1.33, P = .001, 95% CI 0.51–1.82. MID was ≥0.5 in 19 (63.3%) patients.Clinical improvement was seen in non-hay fever symptoms, and the mean score improved from 2.77 ± 1.67 to 1.6 ± 1.25, P < .001, 95% CI 1.55–2.85. MID was ≥0.5 in 25 (83.3%) patients.Practical problems mean score improved from 4.43 ± 1.46 to 2.23 ± 1.4, P < .001, 95% CI 1.36–2.67. MID was ≥0.5 in 24 (80%) patients.The mean nasal symptom score improved from 4.1 ± 1.5 to 2.08 ± 1.24, P < .001, 95% CI 1.05–2.17. MID was ≥0.5 in 20 (66.7%) patients.The mean score for ocular symptoms improved from 3.2 ± 1.66 to 1.6 ± 1.32, P < .001, 95% CI 1.23–2.55. MID was ≥0.5 in 24 (80%) patients.Emotional symptoms improved from 3.48 ± 1.52 to 1.59 ± 1.11, http://links.lww.com/MD/J222 “Domain Scores”).The scores for each domain before and after 11 weeks of adjuvant DLE are shown in Figure http://links.lww.com/MD/J223 “Items Scores” show the scores for the remaining individual items.Each individual item in the RQLQ questionnaire showed clinical (MID ≥ 0.5) and statistical improvements with the use of DLE , not including immunotherapy. A third medication (leukotriene antagonist) may benefit pediatric patients with AR, and some may occasionally use local vasoconstrictors. found that ocular symptoms had the highest impact on QoL, followed by nasal obstruction and pruritus, to a lesser degree.All 7 domains  in the RQLQ questionnaire showed statistical and clinical improvements during DLE treatment. Each of the 28 individual items in the questionnaire also showed statistical and clinical improvement. Bousquet et al The items in the questionnaire with the most improvement were sneezing, nasal congestion, pruritus  and nasal secretion . This suggests that DLE can be beneficial for improving the main symptoms of AR. As expected, due to the psychometric qualities of the questionnaire, the individual item score improvement was consistent with the domain score and overall RQLQ score improvement. This allows us to conclude that there was a consistent and general improvement in the QoL of AR patients who had DLE added to their treatment.The main symptoms of AR are sneezing, nasal congestion, pruritus, and nasal secretion.Despite the DLE dose reduction in each phase, we observed a significant improvement in the RQLQ scores over time, suggesting that DLE dosing once a week may be adequate for most patients. However, we cannot exclude the possibility that a higher initial dose is beneficial. Additional studies comparing the efficacies of different doses are required to resolve this issue. Three patients (10%) in this study presented with non-serious AE that correlated with the type of adverse event reported by Homberg et al Interestingly, the symptom that least improved was headaches, which is consistent with the finding that DLE may cause headaches.The most common AE associated with the use of DLE are headache, increased disease symptoms, rash, and fatigue, which are present in less than 2% of DLE users.,32 It is helpful to guide treatment options and responses; however, the scores are based on the patient’s subjective point of view and personality traits. Nevertheless, DLE appears to be beneficial regardless of basal treatment. Additional studies are necessary to evaluate subgroups of patients with similar traits. Evaluation of symptom scores, visual analog scales, and Th1/Th2/Th17/Treg cytokine profiles is required to fully determine the effectiveness of DLE treatment for AR.The limitations of this study include the small number of subjects, lack of a control group, and the fact that only QoL was evaluated. Due to the sample size and lack of a control group, no definite conclusions can be made regarding efficacy of DLE until larger, controlled, multicenter studies involving different populations are done. Nonetheless, this study provides encouraging results in favor of continuing the study of DLE as a possible treatment option for AR. QoL is one of the many tools available for assessing patients with AR.DLE may be beneficial as adjuvant treatment for AR. Our results provide preliminary data for future research. Interestingly, the patients showed additional improvement with increased treatment time, and dose reduction did not have a worsening effect.The authors wish to thank Elizabeth Juniper and her team for allowing us to use the RQLQ questionnaire.Conceptualization: Toni Angela Homberg, María Carmen Jiménez-Martínez.Data curation: Toni Angela Homberg, Ivan Lara, Consuelo Andaluz.Formal analysis: Pedro Martín Hernández-Martínez, Toni Angela Homberg, María C. JIménez-Martínez.Funding acquisition: Sonia Mayra Pérez-Tapia.Investigation: Toni Angela Homberg, Ivan Lara, Consuelo Andaluz, Edgar Cervantes-Trujano.Methodology: Ivan Lara, Consuelo Andaluz, Edgar Cervantes-Trujano, Pedro Martín Hernández-Martínez, María Carmen Jiménez-Martínez.Project administration: Toni Angela Homberg, María Carmen Jiménez-Martínez.Supervision: Sonia Mayra Pérez-Tapia, María Carmen Jiménez-Martínez.Validation: Pedro Martín Hernández-Martínez, María Carmen Jiménez-Martínez.Visualization: María Carmen Jiménez-Martínez.Writing – original draft: Toni Angela Homberg.Writing – review & editing: Sonia Mayra Pérez-Tapia, María Carmen Jiménez-Martínez.


Tedizolid is an oxazolidinone anti-MRSA drug with included in the National Health Insurance Drug Price List in 2018. The effect of hemodialysis on tedizolid phosphate concentrations has been reported; pre-dialysis concentrations decreased by 10% compared to post- dialysis concentrations. However, the material of the dialysis membrane remains unknown. In addition, there have been no reports on the effects of continuous hemodiafiltration. In this study, we investigated the effects of continuous hemodiafiltration on tedizolid using two types of dialysis membranes made of different materials.The adsorption of tedizolid, linezolid, and vancomycin to two different dialysis membranes was investigated, and the clearance of each drug was calculated by experiments using an in vitro continuous hemodiafiltration model.The adsorption of tedizolid, linezolid, and vancomycin on the dialysis membranes was examined, and no adsorption was observed. Experimental results from the continuous hemodiafiltration model showed that linezolid and vancomycin concentrations decreased over time: after two hours, the respective decreases were 26.48 ± 7.14% and 28.51 ± 2.32% for polysulfone membranes, respectively. The decrease was 23.57 ± 4.95% and 28.73 ± 5.13% for the polymethylmethacrylate membranes, respectively. These results suggested that linezolid and vancomycin were eliminated by continuous hemodiafiltration. In contrast, tedizolid phosphate and tedizolid concentrations decreased slightly in the polysulfone and polymethylmethacrylate membranes. The decrease in concentrations were 2.10 ± 0.77% and 2.97 ± 0.60% for the polysulfone membranes, respectively. For the polymethylmethacrylate membranes, the decrease in concentration were 2.01 ± 0.88% and 1.73 ± 0.27%, respectively.These results suggested that tedizolid should not be considered for dose control during continuous hemodiafiltration. Patients with severe infections or sepsis caused by methicillin-resistant Staphylococcus aureus (MRSA) often develop complications resulting from acute renal dysfunction or changes in other organ functions. In such cases, hemodiafiltration (HDF), particularly continuous hemodiafiltration (CHDF), which has less impact on blood flow, is widely used in intensive care units. In patients using HDF or CHDF, the optimal dose of antimicrobial agents, including anti-MRSA agents, should be determined based on the patient's renal function and the estimated efficiency of drug clearance by the dialysis membrane , 2. WhenSivextro® (tedizolid phosphate) is a novel oxazolidinone antimicrobial agent indicated for MRSA infections, which was listed in the National Health Insurance Drug Price List in May 2018. It is similar to the oxazolidinone antimicrobial agent, linezolid (LZD), which binds to the 50S subunit of the ribosome and inhibits the formation of the 70S initiation complex, thereby inhibiting bacterial protein synthesis and growth. Tedizolid phosphate is a prodrug that is converted in vivo into its active form, tedizolid (TZD), which exhibits antimicrobial activity. Regarding the effect of hemodialysis on TZD, the interview form reported a 10% decrease in TZD concentration before dialysis compared with after dialysis. However, the material of the dialysis membrane remains unknown. In addition, there have been no reports on the effects of CHDF. Some drugs are lost in HDF and CHDF by diffusion and ultrafiltration and also by adsorption to the filtration membrane –6. Anti-The aim of this study was designed to compare the adsorption of tedizolid phosphate and TZD on two types of dialysis membranes with different membrane structures, mainly used in CHDF with other anti-MRSA agents (LZD and VCM) and to investigate the potential for a CHDF-induced decrease in TZD concentration through in vitro experiments.Tedizolid phosphate standard samples were purchased from MedChemExpress , and TZD was purchased from Nacalai Tesque Corporation . Sivextro (tedizolid phosphate) and sodium acetate were purchased from MSD Corporation  and Nacalai Tesque Corporation, respectively. LZD, VCM, acetonitrile, and methanol were purchased from the FUJIFILM Wako Pure Chemical Corporation . Bovine serum albumin (BSA) was added to saline as pseudo-blood and was purchased from Iwai Chemical Corporation. A syringe filter  was used for sample solution pretreatment. A saline solution  was purchased from Otsuka Pharmaceutical Corporation .max) of each drug at clinical administration.The adsorption rates of tedizolid phosphate, TZD, LZD, and VCM onto two types of dialysis membranes were investigated Table . The expThe hollow fibers  of each dialysis membrane were soaked in solutions of tedizolid phosphate, TZD, LZD, and VCM, stirred for 1 h at room temperature, and collected. To 0.2 ml of this sample, an equal volume of methanol was added (0.2 ml), and the proteins were removed by centrifugation . Protein-free samples were assayed by high- performance liquid chromatography (HPLC).b) of 100 ml/min. The flow rate of the dialysate (Qd) was set to 450 ml/hr, and the flow rates of the substitution solution (Qs) and ultrafiltrate (Qf) were set to 200 ml/hr. These flow rates were set based on the actual clinical flow rates. The pseudo-blood containing the drug solution was circulated in the circuit to equalize the concentration of the drug solution, and the experiment was then started. Samples were collected from the inlet (Cin), outlet (Cout), and filtrate (Cf) of the dialyzer at 0, 0.5, 1, and 2 h.CHDF was performed using an in vitro model with a blood purification system  Fig. . Saline The elimination rates of tedizolid phosphate, TZD, LZD, and VCM were calculated as follows:0: Initial drug concentrationCn: Drug concentration at the time of samplingCCHDF) was calculated using the following equation:The clearance of each drug in the in vitro CHDF . The analyses of tedizolid phosphate and TZD were performed in accordance with previous reports –9. The mP < 0.05.Data are expressed as the mean ± SE. One-way analysis of variance (ANOVA) was applied to nonreplicated measurements. Repeated-measures ANOVA followed by the Tukey–Kramer test was applied for repeated or serial determinations. Statistical significance was set at The elimination rate of tedizolid phosphate onto the PS and PMMA membranes after 1 h significantly increased for both membranes compared with that of the control Fig. . HoweverP < 0.01). The elimination rates of tedizolid phosphate, TZD, LZD and VCM under BSA-free conditions were 12.31 ± 0.29%, 9.75 ± 1.97%, 25.30 ± 5.28% and 27.39 ± 3.04% for the PMMA membrane and 6.58 ± 0.86%, 9.88 ± 1.40%, 19.82 ± 2.22% and 27.14 ± 2.87% for the PS membrane, respectively.The elimination rates after 2 h of tedizolid phosphate, TZD, LZD, and VCM were 1.48 ± 0.38% to 2.01 ± 0.88%, 0.88 ± 0.63% to 1.73 ± 0.27%, 15.36 ± 1.23% to 23.57 ± 4.95%, and 26.30 ± 4. 78% to 28.73 ± 5.13% for the PMMA membrane and 1.86 ± 0.76% to 2.10 ± 0.77%, 0.91 ± 0.38% to 2.97 ± 0.60%, 21.01 ± 0.98% to 26.48 ± 7.14%, and 25.56 ± 4.36% to 28.51 ± 2.32% for the PS f membrane .Concentrations after 2 h of tedizolid phosphate, TZD, LZD, and VCM filtrates ranged from 2.68 ± 1.12% to 3.40 ± 0.26%, 1.06 ± 0.01% to 1.09 ± 0.01%, 18.21 ± 0.92% to 20.69 ± 2.74%, and 18.43 ± 1.19% to 19.73 ± 2.08% were detected in the PMMA membrane, 2.59 ± 1.36% to 3.21 ± 1.18%, 0.71 ± 0.01% to 0.72 ± 0.01%, 15.09 ± 2.26% to 15.93 ± 1.75%, and 17.90 ± 0.91% to 18.88 ± 1.88% were detected in the PS membrane .The CLSerum albumin concentrations tend to be lower in critically ill patients than in healthy subjects, which may increase the apparent volume of drug distribution and clearance due to high protein-binding rates. In addition, the disposition, metabolism, and excretion of drugs are non-physiological in patients with renal failure. For example, teicoplanin has a high protein-binding rate and is known from previous reports to adsorb on PMMA and PS membranes, affecting clearance . These aThis study compared the affinities of tedizolid phosphate and TZD with those of other anti-methicillin-resistant Staphylococcus aureus agents (LZD and VCM) on two different types of dialysis membranes with different structures, mainly used in CHDF. The PS membrane used in the experiments is the most commonly used membrane in CHDF; PS membranes have an asymmetric structure and are excellent for water removal and filtration. Hypercytokinemia is also observed in septic acute kidney injury, and dialysis membranes with adsorption properties such as PMMA and AN69ST membranes are used. In this study, symmetrically structured PMMA membrane was used as membranes with adsorptive properties, and in vitro experiments were performed to investigate the differences in TZD removal rates in dialysis membranes with different membrane structures.The PS membrane used in this experiment has an asymmetric structure with a thin active layer on the surface that affects solute permeability and has a high solute elimination capacity from molecular weight substances to β2-microglobulin (β2-MG) with no albumin leakage , 16. HomFirst, experiments were conducted to compare the affinity of the dialysis membrane hollow fibers added to a saline solution to the dialysis membranes. The results showed that TZD, LZD, and VCM were comparable to the control; however, tedizolid phosphate was significantly eliminated compared to the control.CHDF were examined using an in vitro CHDF model. The results showed that tedizolid phosphate and TZD showed a slight decrease in concentration compared with LZD and VCM. Although there was a significant difference in the elimination rate of tedizolid phosphate compared with that of the control in the adsorption to the dialysis membrane experiment, the elimination rate was lower in the in vitro CHDF model. This difference may be due to the fact that, in the adsorption experiment, TZD made contact with both the inside and outside of the dialysis membrane, whereas in the in vitro CHDF model, TZD made contact with the inner surface of the membrane only. The filtrate concentrations of LZD and VCM, which showed a significant decrease, were similar to the elimination rates of LZD and VCM. The CLCHDF of VCM in PMMA obtained in this study was close to the value reported previously (1.35 L/hr), which may reflect the in vivo conditions [The elimination rates of tedizolid phosphate, TZD, LZD, VCM, filtrate concentration, and CLnditions . These rHowever, the present experiments were performed in vitro with dialysis membranes and pseudo-blood  and may not fully represent the clinical situation. In addition, other dialysis membranes besides the one used in this study have been used in actual clinical practice. Elucidating these phenomena in a clinical setting would be meaningful, especially in terms of TZD administration and drug selection for hemodialysis patients.With the increase in the number of long-term hemodialysis patients and elderly hemodialysis patients, the number of hemodialysis patients requiring antimicrobial agents is increasing; however, because most antimicrobial agents are non-dialyzable, their extracorporeal elimination during hemodialysis treatment is not a major problem. The TZD interview form reported a 10% change in concentration after hemodialysis; however, the dialysis membrane material was unknown.In this study, we investigated the elimination of the dialysis membranes (PS and PMMA) used in CHDF. In addition, TZD has different pharmacokinetics in oral and intravenous administration. Experiments have proven that TZD is not easily affected by CHDF, providing a new indicator of the actual situation in the medical field.


Moringa oleifera. The four UAE factors, solvent ratio (A), solvent–solid ratio (B), extraction temperature (C), and extraction time (D), were optimized using response surface methodology (RSM). A Box–Behnken design was used for the experimental design. The optimal conditions were found to be a 50% v/v solvent ratio, a 30% v/w solvent–solid ratio, 35 °C extraction temperature, and 45 min extraction time. The experimental value of extraction yield (R1), TFC (R2), TPC (R3), and DPP IV enzyme inhibitory activity (R4)  agreed with those predicted by RSM models , thus demonstrating the appropriateness of the model used and the ability of the RSM to optimize the extraction conditions. Excellent DPP IV enzyme inhibitory activity was exhibited by M. oleifera compared with the standard, sitagliptin. While the modeled equation fits the data, the t-test is not significant, suggesting that the experimental values agree with those predicted by the RSM–BBDThis study optimized ultrasound-assisted extraction conditions to maximize the extraction yield, total flavonoid content (TFC), total phenolic content (TPC), and DPP IV enzyme inhibitory activity from  M. oleifera leaves are myricetin, quercetin, and kaempferol, which are present in concentrations of 5.8, 0.207, and 7.57 mg/g, respectively [M. oleifera leaf extract is a potential instrument for insulin resistance in diabetes mellitus. The presence of the flavonoid activity of Moringa leaves (M. oleifera) encouraged the development of optimized extraction methods. To achieve this, it is necessary to choose the right method in the extraction process to obtain an optimal compound content. When analyzing the chemical components present in a plant, the choice of extraction process must be taken into account [A key source of bioactive compounds for developing new anti-diabetic medications for insulin resistance is now natural products. Recently, more and more natural components have been described as having antidiabetic properties in insulin resistance, and many attempts have been made to elucidate the mechanisms by which these occur . Recent ectively ,4. In drectively . M. olei account .There are two extraction techniques, namely conventional and non-conventional. In conventional extraction, compounds are extracted from the plant matrix, employing diffusion and mass transfer principles after the plant matrix has been homogenized and submerged in one or more solvents. Although this approach is fairly straightforward, it has disadvantages, such as low efficiency because of the high solvent requirements . At the R2 = 0.99) and is expressed in QE, varying between 51.86–77.00 mg/g in QE. The TPC was determined from the regression equation of the calibration curve  and is expressed in GAE, varying between 70.06–100.98 mg/g in GAE [One of the aims of this research is to optimize the extraction method of UAE using the Response Surface Methodology (RSM) approach. BBD is a type of RSM-independent design that is recommended for optimization processes with three or more independent variables . Box–Behg in GAE . Researcg in GAE M. oleifera constituents. However, the application of response surface methodology (RSM) for the optimization of ultrasound-assisted extraction (UAE) of Moringa oleifera with respect to the extraction yield, the content of bioactive compounds, and biological effects in vitro has not previously been undertaken. On this basis, we aimed to study the effects of different UAE extraction variables  on extraction yield (R1), TFC (R2), TPC (R3), and DPP IV enzyme inhibitory activity (R4).Several studies have previously been conducted for the extraction and quantification of The ranges for the four independent extraction variables, namely, solvent ratio (A), solvent–solid ratio (B), temperature (C), and time (D), at levels  for extraction parameter optimization by the BBD method were selected based on observations from the single-factor experiments  Table 1Table 1.M. oleifera. In R1, R2, R3, and R4. The results were fitted into Equation (1) :Y is the expected matter of D, 0β is the intersection of the equations, iβ is the linear coefficient, iiβ is the squared coefficient, ijβ is the coefficient of the cross outcome, and iX and jX are the coded independent variables. The F-test is used to determine a polynomial equation’s relevance. Each coefficient in the polynomial equation’s statistical significance is evaluated using the p-value [R1–R5):All independent variables were employed with the BBD to investigate the effects on the extraction yield, TFC, TPC, and DPP IV enzyme inhibitory activity in  p-value ,25 to gew/w), TFC (51.86–77.00 mg QE/g extract), TPC (70.06–101.48 mg GAE/g extract), and DPP IV enzyme inhibitory activity (42.86–90.42% inhibition).R2 of 0.9691, 0.9864, 0.9317, and 0.9323 was found to be the best-fit models for the analysis of R1, R2, R3, and R4, respectively. In R1, R2, R3, and R4.For the BBD-based experimental design, a quadratic model with an R2/predicted R2 values for R1, R2, R3, and R4  were found to be close to showing a correlation between adjusted and predicted values. Furthermore, while differences between squared and predicted R values should be less than 0.2 for every dependent variable , the signal-to-noise ratio can be used, which should be greater than 4 to fit the model. In this experiment, the signal-to-noise ratio for R1, R2, R3, and R4 were 7.4695, 8.9156, 8.3776, and 15.8775, respectively; all the values were more than 4, indicating that the models were fitted correctly and suggesting that the proposed model can be used to navigate the design space. The ANOVA  results for the quadratic models of R1, R2, R3, and R4 are listed.The adjusted R2, R3, and R4, the F-values were found to be 6.69, 4.24, and 11.81, respectively, implying that each quadratic model was significant and that there was only a 0.11, 0.83, and 0.01% chance, respectively, that these F-values could occur due the noise. The p-values for the proposed quadratic model of all dependent variables were found to be very low (<0.05), suggesting that the models developed for the analysis of all the variables were significant. Lack of fit F-values of 1.02, 0.45, 0.27, and 0.29 for R1, R2, R3, and R4, respectively, imply that the lack of fit is not significant relative to the pure error; thus, it is appropriate to fit the model and predict the response. There is a 59.30, 83.97, 93.96, and 92.91% chance that a lack of fit F-values this large could occur due to noise for the variable yields, TFC, TPC, and DPP IV enzyme inhibitory activity, respectively.The model F-values for yield were 5.69, implying that the deal is significant. There is only a 0.23% chance that an F-value this large occurs due to noise. Similarly, for p < 0.05) and to affect R1, R2, R3, and R4. The quadratic effect of the solvent ratio (A) exhibited significance and affected R4. The effect on the solvent–solid ratio (B) indicated significance and affected R2 and R4. Temperature (C) showed a significant effect on R1 and R4. Time (D) exhibited a significant effect and affected only R3.The influences of the individual independent variables A, B, C, and D are listed in , the solvent ratio, solvent–solid ratio, temperature, and time were interpreted in the range of , , , and , respectively. The confidence interval for each response was 95% in the mentioned ranges on the plots.As shown in Figure 2R2 value above 0.9. The R values obtained indicated that more than 99% of the response variables (R1–R4) could be described by the RSM model. The high value of R2 for each response indicated that the BBD design fitted well into the quadratic polynomial models that were developed. These results confirmed the predictability of the models in determining the optimum conditions needed to obtain the highest extraction yield, TFC, TPC, and DPP IV enzyme inhibitory activity , solvent–solid ratio (50% v/w), temperature (35°), and time (45 min) could produce the optimum extraction yield, TFC, TPC, and DPP IV enzyme inhibitory activity . Optimized conditions for the simultaneous maximum extraction yield, TFC, TPC, and DPP IV enzyme inhibitory activity were determined. In the BBD analysis, the optimized conditions using the solvent ratio , glass beakers , measuring cups , ultrasonicator , oven, Whatman filter paper no. 42, rotary vacuum evaporator , analytical balance , micropipettes , micropipette tips , 1.5 and 2 mL microtubes , 96-well microplates , and a multi-mode microplate reader .Reagents used were 96% ethanol , 10% aluminum chloride , 1 M sodium acetate , Folin–Ciocalteu reagent , sodium carbonate , quercetin , gallic acid , dimethyl sulfoxide (DMSO) , and a dipeptidyl peptidase IV (DPP IV) inhibitor screening kit consisting of assay buffer (25 μL), enzymes (100 μL), substrate (200 μL) and sitagliptin (50 μL) .M. oleifera) obtained from the Research Institute for Medicinal Plants and Spices , Bogor, Indonesia. The age of the harvested Moringa leaves (M. oleifera) was not lower than 30 days. The wet sorting process was carried out manually by separating impurities or other foreign materials prior to the washing process by removing unnecessary parts. The Moringa leaves were dried in an oven at 40 °C until a moisture content of less than 10% was obtained. Dry sorting was performed manually by separating foreign objects such as plant parts that were not needed and other impurities that were still present and left in the dried simplicia. The dried sample was powdered to a particle size of 40 mesh. The drying and grinding procedures were performed for one batch of samples, and these procedures were not replicated.The material employed in this study was dried simplicia of Moringa leaves  using the UAE method, which was preceded by immersion of the sample in a solvent at a certain solvent ratio. The extraction time is calculated when the temperature for each run reaches a predetermined temperature. The extract obtained was filtered using a Whatman® brand membrane filter holder (300 mL capacity) Cat. No. 1960-004, then the filtrate was concentrated with a rotary evaporator until a thick extract was obtained. The percentage yield of the extract was calculated after obtaining a constant viscous extract weight to the weight of the dry powder of M. oleifera by using the following equation: In general, chemical compounds such as flavonoids, which are known to have biologically relevant properties ,9, can bM. oleifera extract. Quercetin was utilized as a reference substance. A 10 µL volume of 10% aluminum chloride was measured and put into a 1.5 mL microtube, to which was added 50 µL of each of the 27 samples of 1 mg/mL thick extract of M. oleifera leaves, 150 µL of 96% ethanol, and 10 µL of sodium acetate 1 M. Each of these mixtures was put into a 96-well microplate and shaken in a multi-mode microplate reader for 1 min. These were then incubated at 37 °C protected from light for 40 min and the absorbance was read on a multi-mode microplate reader at λ 415 nm [A multi-mode microplate reader was used to measure the total flavonoid content of the λ 415 nm . Total fλ 415 nm ,30.M. oleifera extract was measured. Gallic acid was employed as a reference ingredient. The thick extract of M. oleifera leaves of 1 mg/mL from 27 samples was measured by putting as much as 12.5 µL each into a 96-well microplate, to which was added 50 µL of Folin–Ciocalteu reagent. Then, the microplates were placed in the shaker for 1 min in a multi-mode microplate reader and incubated for 4 min protected from light. A volume of 37.5 µL Na2CO3 was added, and the samples were shaken for 1 min, incubated for 2 h protected from light, and their absorbance read on a multi-mode microplate reader at λ 765 nm [With a multi-mode microplate reader and the Folin–Ciocalteu method, the total phenolic content of λ 765 nm . The totλ 765 nm ,30.M. oleifera leaves consisting of DPP IV assay buffer (49 µL), DPP IV enzyme (1 µL) that was given a shaker treatment for 1 min and incubated for 10 min at 37 °C protected from light, to which was then added DPP IV assay buffer (23 µL) and DPP IV substrate (2 µL) and the mixture shaken for 1 min. To each of the 27 samples (25 µL) was added the viscous extract that had been dissolved in DPP IV assay buffer until each mixture had a content of 1000 ppm/well [The inhibitory activity of the DPP IV enzyme was determined in vitro using a DPP IV inhibitor screening kit and a multi-mode microplate reader for the 5 test groups, with each group consisting of 3 wells on a 96-well multi-plate. The group divisions were as follows: Group I was a positive control consisting of DPP IV assay buffer (49 µL) and DPP IV enzyme (1 µL) that was shaker-treated for 1 min and incubated for 10 min at 37 °C protected from light, to which was then added DPP IV assay buffer (23 µL) and DPP IV substrate (2 µL). This was shaken for 1 min, and, finally, sitagliptin (2.5 µL dissolved in 22.5 µL DPP IV assay buffer) was added. Group II was the negative control consisting of DPP IV assay buffer (49 µL), DPP IV enzyme (1 µL) that was treated with a shaker for 1 min and incubated for 10 min at 37 °C protected from light, to which was then added DPP IV assay buffer (23 µL) and DPP IV substrate (2 µL). Group III was a blank with DMSO consisting of DPP IV assay buffer (49 µL) that was treated with a shaker for 1 min and incubated for 10 min at 37 °C protected from light, to which was then added DPP IV assay buffer (23 µL) and DPP IV substrate (2 µL). DMSO (1 µL) was added and given a shaker treatment for 1 min and incubated for 10 min at 37 °C protected from light. Group IV was a blank without DMSO consisting of DPP IV assay buffer (49 µL) that was treated with a shaker for 1 min and incubated for 10 min at 37 °C protected from light, to which was then added DPP IV assay buffer (23 µL) and DPP IV substrate (2µL). Group V was a concentrated extract of ppm/well . Then thv/v), solvent–solid ratio , temperature , and time .The extraction variables  were selected based on the observation of single-factor effects, which were used to optimize all extraction variables using the BBD method to obtain maximum values for the total extraction yield, TFC, TPC, and the insulin-resistant antidiabetic activity assay. The impact of single factors on the total extraction yield, TFC, TPC, and the insulin-resistant antidiabetic activity assay was evaluated by varying the solvent ratio  Box–Behnken design  was used for the optimization of the independent variables solvent ratio (A), solvent–solid ratio (B), temperature (C), and time (D), at low (−1), medium (0), and high levels (+1) (R1), TFC (R2), TPC (R3), and DPP IV enzyme inhibitory activity (R4). Three-dimensional contour plots were made to deduce the independent variable’s effects on R1, R2, R3, and R4, and the “biggest-is-best” principle was used for each variable to obtain the optimum outcome, with p-values ≤ 0.05 considered to be significant. A final confirmation experiment (n = 4) was performed using optimized values for model validation.A 4-factorial (4els (+1) . A totalM. oleifera leaf extraction and allows a better understanding of the relationship between independent variables and response variables. The model was verified statistically with ANOVA. Under the optimal conditions, the actual values were in good agreement with the predicted values, as the RSE values for the optimum conditions were less than 0.2%. The quadratic effect of the solvent ratio (A) exhibited significance and affected R4. The solvent–solid ratio (B) exhibited significant effects on R2 and R4. Temperature (C) exhibited significant effects and affected R1 and R4. Time (D) exhibited a significant effect on R3 and R4. DPP IV enzyme inhibitory activity was influenced by all independent variables (p < 0.05). The optimization conditions were as follows: solvent ratio of50% v/v, solvent–solid ratio of30% v/w, temperature 35 °C, and time 45 min. Under these conditions, the experimental and model predicted yield value, TFC, TPC, and DPP IV enzyme inhibitory activity . Therefore, in this study, M. oleifera leaf extraction was successfully optimized.This study demonstrated that RSM is an effective tool for optimizing the conditions of


Staphylococcus aureus (MIC of 1.563 mg/mL) and Escherichia coli (MIC of 3.125 mg/mL). Findings revealed that, in comparison to 0.02% potassium sorbate, blueberries treated with a high concentration of UPPP in a dipping treatment displayed superior quality maintenance after 7 days of storage at 4°C. Importantly, analysis of the various metrics showed that treatment with UPPP was significantly better compared to blueberries treated with 0.02% potassium sorbate. For example, the decay rate, weight loss, and total number of colonies of blueberries treated with 0.02% potassium sorbate were 55.56, 3.48%, and 4.24 ± 0.07 log CFU/mL, whereas the values of the above indexes for blueberries treated with 25 mg/mL of UPPP were 22.22, 3.09%, and 3.10 ± 0.17 log CFU/mL, respectively. Conversely, blueberries that were not dipped in any preservative displayed signs of deterioration as early as the 3rd day of the storage period, highlighting the potential of UPPP as a valuable method for preserving fruits and vegetables. Therefore, UPPP holds great promise as an innovative natural food preservative, effectively enhancing food safety, quality, and extending shelf-life.Proanthocyanidins (PCs) extracted from ume have many well-known functional properties. The aim of this study was to explore a novel natural food preservative using ume plum pulp proanthocyanidins (UPPP). The crude product of PCs from ume plum was obtained by using ethanol as extraction solvent and ultrasonic-assisted extraction, and then the pure product of UPPP was obtained by purification with AB-8 resin. The bacteriostatic ability of UPPP and the freshness preservation effect on blueberry were analyzed. The results showed that UPPP had a high inhibitory effect on  As a dual-use species of medicine and food, ume has received widespread attention from both China and foreign countries, and can be eaten raw or used to make preserved fruit, jam or sour plum soup, which contains a large number of organic acids, flavonoids, lipids and other components (Ume is processed from the dried nearly ripe fruit of mponents . Ume hasmponents . Modern mponents .3 and prodelphinidin C exhibited the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) on E. coli in the comparison with the other 12 phenolics from Quercus ilex leaves  activity, superoxide dismutase (SOD) activity, and colony counts were determined in an attempt to unravel the potential of UPPP as a natural food preservative especially for blueberry preservation. This study was expected to provide valuable insights into the potential application of UPPP as natural food preservatives. The findings would contribute to enhancing food safety, quality, and shelf life of blueberries and aid in the development of safer and more effective preservation technologies. In addition, it was also conducive to the active development of new resources such as PCs, which would ultimately maximize the utilization of ume resources.2.2.1.Staphylococcus aureus , Escherichia coli  gene, partial cds.), Aspergillus niger , POD kit, SOD kit , Handy Plate® Aerobic Count Plate .Ume , sterile TTC solution , 2.2.The optimal extraction conditions of UPPP were obtained by single-factor and response surface experiments. An appropriate amount of degreased ume powder was weighed, sieved to 0.25 mm, and UPPP was extracted with an ultrasonic extractor  with 49% ethanol in the ratio of 15:1 at the ultrasonic power of 155 W. The extraction time was set to 48 min, and the extraction temperature was 45°C. The crude extract was centrifuged for 10 min , the supernatant was transferred, and 1/4 of the supernatant volume was evaporated by rotation to obtain the concentrated liquid. The concentrated extract was diluted to a certain concentration and purified by AB-8 macroporous adsorption resin.During the purification process, the pre-treated AB-8 macroporous resin was loaded into a 30 mm × 300 mm column. The pH value was adjusted to 4, and then the concentrated extract solution obtained above was diluted to a concentration of 1.0 mg/mL and loaded into the chromatographic column. The loading flow rate was 1.25 mL/min and the loading volume was 2 BV. After loading, the sample was allowed to stand for 4 h, followed by washing with distilled water until the effluent was colorless to remove soluble proteins, sugars, and pigments. Subsequently, elution was carried out using 60% ethanol eluent, with a flow rate of 1.25 mL/min and an elution volume of 4 BV. The eluate was collected and subjected to rotary evaporation and freeze-dried at −40°C using a vacuum freeze dryer  and stored away from light to obtain the purified UPPP .The above experimental steps were repeated three times. The extraction, separation and purification process of UPPP is shown in 2.3.The PCs standard  was prepared into concentration gradient solutions of 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL and 6 mg/mL, and the absorbance value was determined according to the vanillin hydrochloride method , and 502c: concentration of UPPP (mg/mL); v: sample volume (mL); d: dilution multiple; m: mass of skimmed ume powder (g).The purity of PCs in UPPP was calculated according to the following equation:2.4.−1 to 400 cm−1 , thoroughly mixed in an onyx mortar, pressed, and scanned with a FTIR spectrometer  in the range of 4,000 cm400 cm−1 .2.5.To identify the presence of PCs in the purified products, the procedure described in the literature  was foll2.6.2.6.1.8 CFU mL−1) and, then, tenfold diluted twice  and, then, tenfold diluted twice  and fungi (A. niger) by using the agar well diffusion method  were punched in the medium, and then 90 μL of UPPP and sterile water (blank control) were added to the medium wells, respectively. 100 μL of fungal spore solution (concentration of 1 × 106 CFU/mL) was added to PDA medium, and holes were punched in the medium (6 mm in diameter), then 90 μL of UPPP and sterile water (blank control) were added to the medium wells, respectively, and incubated at 37°C  was added to each well, then the 96-well plates were incubated at 37°C in a thermostatic microbiological incubator for 24 h. The bacterial growth of each well was observed and analyzed, and the lowest concentration with no bacterial growth and no OD increase was defined as MIC value  was added to each well and incubated at 100 rpm/min in a thermostatic air-bath shaker  at 37°C. The OD value at 600 nm was measured and recorded every 2 h to plot the growth curve. Three biological replicates were performed for each group, and the bacterial growth curves were expressed as the mean value of turbidity, with absorbance corresponding to the time interval.Bacterial growth curve analysis was performed according to the  E. coli . The ana2.6.4.S. aureus by scanning electron microscopy according to a previously reported method (S. aureus (108 CFU/mL) was treated with the MIC concentration of UPPP at 37°C for 2 h. The bacteria were centrifuged at 6000 rpm for 10 min to precipitate and washed three times with PBS. The precipitated cells were fixed with 2.5% glutaraldehyde at 4°C for 24 h and then dehydrated with a series of different concentrations  of ethanol for 10 min. Finally, the dehydrated samples were coated with gold and examined by SEM . A control experiment was performed without UPPP treatment.To confirm and validate the results observed with light microscopy, we examined the cellular structure of d method  with sli2.7.Blueberries of uniform size, color, and ripeness, free from mechanical damage, pests and diseases were selected and randomly divided into three groups of nine each. The blueberries were washed with sterile water, dried in aseptic operation at 25°C, immersed in different preservation solutions  for 15 min, and then, after the surface of the blueberries was dried, they were packed in special fruit packing boxes made of PET, and then stored in a refrigerator at 4°C for 7 days, and data such as weight and color change were measured every 24 h. Distilled water was used instead of the preservation solution as a control.2.7.1.The decay rate of blueberries was detected using a modified version of a reported method . The fru2.7.2.The change in weight of blueberries for each day was measured using the weighing method. Three trials were repeated for each group of treatments. The rate of weight loss was expressed as a percentage of weight loss relative to the initial weight. The following formula was used to calculate the weight loss rate of blueberry:0 and Wn are the weight of blueberry at 0 day and nth days, respectively.Where W2.7.3.The hardness of blueberry was measured using a mass spectrometer  with a probe diameter of 50 mm, a contact pressure of 20 gf, and a compression deformation rate of 40% using the TPA test method . Three trials were repeated for each group of treatments.2.7.4.Blueberry is susceptible to browning during storage, and the color change can be reflected by the total color difference ΔE value. During storage, color parameters  of blueberries from different treatment groups were measured daily using a colorimeter . Each set of experiments was repeated three times. The ΔE of blueberry was calculated as follows:∗, Δa∗, and Δb∗ are the change of blueberry at 0 day to 7 days. L∗ represents the black and white value, a∗ represents the red and green value, b∗ represents the yellow and blue value.Where ΔL2.7.5.Blueberries of different treatment groups were wrapped with gauze, squeezed out the juice and dripped in the center of the detection groove of the saccharimeter , and left to stand for 1 min, then pressed the start button to start the detection, and the number shown on the display was the soluble solids of the blueberries. Each set of experiments was repeated three times.2.7.6.The blueberries of different treatment groups were taken out from the storage environment, put into homogenization bags and added with the same quality of deionized water, and put into a beat homogenizer to homogenize at the speed of 10 for 90s to get the blueberry homogenate.POD activity (U/g FW): Blueberries from day 0 and day 7 of storage were prepared as homogenates which were measured according to the steps described in the POD activity kit  to calculate the POD activity of the blueberries. Three biological replicates were performed.SOD activity (U/g FW): Homogenized blueberries from day 0 and day 7 of storage were measured as described in the SOD activity kit  to calculate the SOD activity of blueberries. Three biological replicates were performed.2.7.7.In each of the different treatment groups, 2.5 g of blueberries were placed in a sterile homogenization bag containing 22.5 mL of PBS buffer, and the homogenizer was agitated for 2 min to make a 1:10 homogenate of blueberries. Then, 1 mL of blueberry homogenate was injected into a test tube containing 9 mL of PBS buffer and shaken well to make a 1:100 sample homogenate, and so on to prepare a 10-fold series of diluted blueberry homogenate. A sterilized pipette was used to suck 1 mL of blueberry homogenate into the center of the colony test slice, and left to stand for 5 min, and the transparent side was upward after the Plate Count Agar (PCA) medium solidified, and then placed in a 37°C constant temperature incubator to observe the colonies and count them after 48 h of incubation . Each seC: number of colonies on the colony test piece (CFU); D: dilution multiple; V: the volume of blueberry homogenate used for testing (mL).2.8.p < 0.05 were considered statistically significant.All experiments were performed three times using freshly prepared samples, and results were reported as the mean and standard deviation of these measurements. Statistical analysis was performed using Origin 2021, Graphpad Prism 8 and SPSS. To compare differences between multiple groups, analysis of variance (ANOVA) was performed on the data. 3.3.1.The ultrasonic extraction yielded UPPP content of 70.05 ± 0.12 mg/g, and separation and purification with macroporous resin AB-8 resulted in UPPP purity of 87.56%. The sample was then freeze-dried, resulting in a brown powder.3.2.−1, which indicated that the mass fraction of the structural unit procyanidin was higher than that of prodelphinidins in UPPP, and it was thus clear that the structural unit of UPPP was mainly procyanidin. The PCs dominated by prodelphinidins have an absorption peak near 730 cm−1, while the PCs dominated by procyanidin have an absorption peak near 770 cm−1 – 780 cm−1, which is attributed to their higher out-of-plane deformation vibration frequency than that of the PCs dominated by prodelphinidins, and the infrared absorption peak in UPPP at 779 cm−1 also confirms that the main structural unit of UPPP is procyanidin ₂ or vanillin . The abo3.4.3.4.1.S. aureus, and E. coli, but not on A. niger. The results of the bacterial zone of inhibition diameters were shown in S. aureus were 20.41 ± 1.34 mm and for E. coli were 12.97 ± 0.05 mm. The experiments proved that UPPP has good bacterial inhibition ability.The inhibition ability of UPPP was determined by punching method, and it was shown in 3.4.2.S. aureus, 3.125 mg/mL for E. coli, in contrast, at a mass concentration of 25 mg/mL of UPPP, there was no inhibitory effect on A. niger. It can be seen that UPPP has a good inhibitory effect on bacteria, especially S. aureus.The MIC of UPPP was determined as shown in 3.4.3.S. aureus in the control group reached the exponential growth phase after 4 h and did not enter the stable phase after 12 h. After treatment with 0.125 MIC, 0.25 MIC and 0.5 MIC of UPPP, S. aureus entered the exponential growth phase after 4 h and none of them reached the stable phase after 12 h. The absorbance of 1 MIC did not change in 12 h, indicating that S. aureus The absorbance of 1 MIC did not change at 12 h, indicating that S. aureus was very sensitive to 1 MIC concentration; different concentrations of UPPP had inhibitory effect on S. aureus at 12 h.As shown in E. coli treated with Control, 0.25 MIC, and 0.125 MIC entered the exponential growth phase after 4 h of incubation and reached the stationary phase at 6 h. The growth curve of E. coli treated with 0.5 MIC of UPPP lagged behind the control group by 2 h and entered the stabilization period at 8 h. Furthermore, 1 MIC completely inhibited the growth of E. coli within 12 h.The results in 3.4.4.S. aureus bacterial cells was observed by using scanning electron microscope and the results were shown in S. aureus cultured under normal conditions with complete cellular morphology, the bacterial cell surface was complete and smooth overall presenting round spherical cells, the bacterial body was full and tightly structured. S. aureus was treated with MIC concentration of UPPP, the bacterial surface showed varying degrees of deformation, unclear outline, surface collapse, cell membrane depression, breakage, and the appearance of aberrant cells, which indicated that the cell wall and cell membrane structure of S. aureus were damaged by UPPP, and the incompleteness of the cell wall and cell membrane structure led to the leakage of cellular contents, which caused the bacterium to lose its integrity, resulting in bacterial suppressive effect.The effect of UPPP on the morphology of 3.5.3.5.1.p < 0.05). In conclusion, the treatment group can reduce the decay rate of blueberries during storage, and 25 mg/mL UPPP has the best effect on delaying the decay of blueberries.The results of the decay rate of blueberry stored for 7 days are shown in 3.5.2.p < 0.05) in the weight loss rate when compared with the Control group, proving that the UPPP treatment was effective in reducing the weight loss of blueberry, with the concentration of 25 mg/mL resulting in the lowest rate of weight loss of blueberry.The results of the 7-day experiment on weight loss rate of UPPP-treated blueberry is shown in 3.5.3.p < 0.05), and the UPPP treatment group with a concentration of 25 mg/mL had the least change in hardness before and after storage, which was the most effective in maintaining the hardness of blueberry. The hardness of blueberry was maintained optimum.The 7-day experimental results of hardness of UPPP-treated blueberry is shown in 3.5.4.* value (brightness), a* value , and b* value . The experimental results are shown in * and b* values of the blueberry and Control groups treated with 0.02% potassium sorbate, UPPP at concentrations of 5 mg/mL and 25 mg/mL, decreased, and the a* values increased, reflecting a decrease in brightness and an increase in surface color saturation. ∆E could reflect the overall change in color of blueberry during storage. There was a general trend of increasing ∆E during storage, which was consistent with the normal metabolism of blueberries. Compared with day 0, the ∆E of the control, 0.02% potassium sorbate-treated, 5 mg/mL UPPP-treated, and 25 mg/mL UPPP-treated groups were 4.59 ± 0.24, 3.81 ± 0.31, 2.79 ± 0.36, and 2.21 ± 0.13, which were significantly different (p < 0.05) in all the blueberry-treated groups when compared to the control group, and the UPPP treatment groups were all <0.02% potassium sorbate, indicating that the UPPP treatment was able to maintain the color change of blueberries.The color change of UPPP-treated blueberry was assessed by measuring the L3.5.5.p < 0.05) difference in the rate of decrease of soluble solids when compared with the Control group, implying that different concentrations of UPPP had an effect on the soluble solids of blueberry. Among them, UPPP at a concentration of 5 mg/mL was effective in reducing the consumption of soluble solids.Soluble solids is an important indicator of fruit ripening and senescence, and the results of 7-day experiments of soluble solids and soluble solids decline rate of UPPP-treated blueberries were shown in 3.5.6.p < 0.05) in POD activity between the treated and control groups after 7 days of storage, suggesting that UPPP was able to better limit the membrane peroxidation process, thus maintaining the integrity of blueberry cells.p < 0.05). At the end of the storage period, the SOD activities of the control, 0.02% potassium sorbate-treated, 5 mg/mL UPPP-treated, and 25 mg/mL UPPP-treated groups were 48.04 ± 1.32 U/g FW, 58.13 ± 2.50 U/g FW, 61.83 ± 1.17 U/g FW, and 69.20 ± 2.20 U/g FW, respectively, indicating that 25 mg/mL UPPP was important in maintaining the activity of reactive oxygen scavenging enzymes, preventing the accumulation of reactive oxygen species and the occurrence of membrane lipid peroxidation, delaying senescence and inhibiting fruit softening in blueberry. In conclusion, UPPP inhibited browning of blueberry by inducing POD activity and SOD activity to alleviate peroxidation.As shown in 3.5.7.p < 0.05). By comparing the total number of colonies of blueberries treated with different concentrations of UPPP and 0.02% potassium sorbate, it was found that UPPP treatment was the most effective, especially with a concentration of 25 mg/mL. The results showed that the different concentrations of UPPP had strong antibacterial activity, which could significantly inhibit the total number of colonies of blueberries during the storage process, and thus reduced the microbial infestation of blueberries and the rate of rotting.As shown in 4.S. aureus , but poorer antibacterial effect against E. coli (12.97 ± 0.05 mm). The physiological and biochemical indexes of blueberry stored at 4°C for 7 d after treatment were determined. The results showed that the decay rate, weight loss rate, hardness, color change, soluble solids decline rate and total colony number of blueberry in the UPPP-treated group were lower than those in the blank control group and the 0.02% potassium sorbate group; and the POD activity and SOD activity were higher than those in the blank control group and the 0.02% potassium sorbate group. In conclusion, UPPP is a natural food preservative, which can significantly increase the shelf life of blueberry, providing a scientific basis for the application of UPPP in the preservation of blueberry.In this paper, we investigated the antibacterial properties of UPPP and its preservation effect on blueberry. UPPP showed the best antibacterial effect against The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.LL: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing. HQ: Writing – review & editing. YuL: Writing – review & editing. YiL: Writing – review & editing. LW: Writing – review & editing. WZ: Writing – review & editing. FM: Writing – review & editing.


Aim is to identify if age, sex, type of posterolateral approach (mini vs standard), surgical time and time from surgery to drainage removal were independent risk factors for heterotopic ossifications after total hip arthroplasty.Patients who underwent a THA with posterolateral approach during a 15 years period were included. The exclusion criteria were absence of X-rays follow-up or HO prophylaxis protocol adoption. The following data were collected: age, sex, type of approach , surgical time, time from surgery to drainage removal. Two orthopedic surgeons independently reviewed the 2 years follow-up X-rays and classified the HO according to Brooker classification. Severe HO was defined if HO were classified as major than grade 2. Correlation between severe HO and risk factor has been tested with multivariable analysis.p = 0.001). Patients older than 65 years showed higher severe HO rate . Standard posterolateral approach was performed in 75.4% and severe HO rate was 32.8% versus 27.1% in those treated with the minimally invasive approach (p = 0.067).About 1225 patients were included: mean age of 63.8 years, 504 were men. HO were found in 67.6%. Men showed higher severe HO rate than woman . In 47.4% of patients, the drainage was removed in the first post-operative day, in this group severe HO rate was significantly lower than the others: 24.8 versus 36.2% (p = 0.001).In 75.6% of cases surgery lasted less than 90 min and this group showed a severe HO rate in 29.1%, while patient with longer surgical time showed a rate of 35.7% (Male sex, age older than 65 years, surgical time longer than 90 min and delayed drainage removal are risk factors for severe HO. Patients with one or more of those risk factors should be identified as good candidates for HO prophylaxis. Total hip arthroplasty (THA), described as “the operation of the century”  for its Several risk factors for HO after a THA are described in the literature. Among these, there are patient related factors  , 11 and Aim of the study is to identify if age, sex, type of postero-lateral approach (mini vs extended), surgical time and time from surgery to drainage removal were independent risk factors for HO formation after THA.This retrospective study was conducted in a single large teaching hospital, study protocol was approved by the local committee (2018/2022), trial number 038.815 .Patients were included if they underwent a THA with postero-lateral approach during a 15 years period. The exclusion criteria were absence of X-rays follow-up longer than 2 years, HO prophylaxis protocol adoption or simultaneous bilateral THA.The following data were collected: age at surgery, sex, type of approach , surgical time, time to drainage removal. Cut-off values were determined: 65 years for age, 90 min for surgery duration and 24 h for postoperative drainage removal.t test or the Mann–Whitney test for continuous outcomes. The Kolmogorov–Smirnov test was used to determine whether data were normally distributed. The relationship between severe HO and study characteristics was assessed with multivariate linear regression models. p values lower than 0.05 were considered statistically significant. All analyses were performed using Stata version 12 .Two orthopedic surgeons reviewed the two-year follow-up X-rays and classified the HO (if present) according to the Brooker classification system : grade 0During the study period, 1859 patients underwent a THA with postero-lateral approach. In 589 cases no x-rays follow-up longer than 2 years was retrievable, 27 underwent an HO prophylaxis and 18 underwent a simultaneous bilateral THA, therefore 1225 patients were included in our study.Mean age of 63.8 years (SD 13), 721 were women (58.9%) and 504 men (41.1%).p = 0.001).HO were found in 67.6% of cases. According to the Brooker classification system, 32.3% of patients were classified as grade 1, 16.5% as grade 2, 11.4% as grade 3 and 7.4% as grade 4 (Table p = 0.002) was found between the first and the second group, respectively with a rate of 30.3% and 39.9%.Among the 1225 patients, 48.6% of them was 65 years old or younger, while 51.4% was older. A significant difference in severe HO prevalence (p = 0.067).In 75.4% of cases a classic postero-lateral approach was performed, while in 24.6% of cases a minimally invasive approach was used. Patients treated with the classic postero-lateral approach showed severe HO rate of 32.8% versus 27.1% found in those treated with the minimally invasive approach (p = 0.033).Regarding surgical time, in 75.6% of cases surgery lasted 90 min or less, while in 24.4% of cases it lasted longer. In the first group the severe HO rate was 29.1%, while in the second group it was 35.7%, with a statistically significant difference (p = 0.001).In 47.4% of patients the drainage was removed before 24 h from surgery, in 52.6% after 24 h. In the second group severe HO rate was significantly higher: respectively 24.8 versus 36.2% ) after THA , 16. RadIn our study we found an overall HO incidence of 67.6% similarly to other authors who reported an incidence up to 90% , 21. In The presence of HO has an important impact on the clinical outcome in total hip replacement. Literature reported that 15% of HO patients suffers from pain and limited range of motion of the involved hip . Eggli eIn our study, a statistically significant correlation between male sex and severe HO was demonstrated and this result is consistent with the literature: male sex is widely considered a strong risk factor for clinical relevant HO after THA implant , 24, 25.About the age, there is not a complete concordance between authors: Zhu et al.  and EgglThe degree of soft tissue trauma during surgery and surgery duration are demonstrated to be important risk factors for HO development , 27, 28.Surgical time is also considered as an indirect index of tissue damage: a long lasting surgery is usually due to a more complex treatment which implies a wider tissue trauma. In fact, we found a statistically significant higher rate in HO ≥ 2 between the patients that underwent surgery longer than 90 min.To the best of our knowledge, just only one study focused on the intra-articular drainage as a potential risk factor: Di Benedetto  analyzedOur study presents several limits. Its retrospective single center nature may limit our conclusions. Furthermore, we used the Brooker classification system and the latter is based on a single anteroposterior X-ray of the pelvis and may under or overestimate the HO extension but it is the most widely diffused , 30. EveMale sex, age older than 65 years, surgical time longer than 90 min and delayed drainage removal are risk factor for severe HO development. Patients with one or more of those risk factors should be identified as good candidates for HO prophylactic treatment.


Aerosols have a severe impact on the Earth's climate, human health, and ecosystem. To understand the impacts of aerosols on climate, human health, and the ecosystem we must need to understand the variability of aerosols and their optical properties. Therefore, we used Aqua-MODIS retrieved aerosol optical depth (AOD) (550 nm) and Angstrom exponent (AE) (440/870) data to analyze the Spatio-temporal seasonal variability of aerosols and their relationship with different meteorological parameters over Pakistan from 2002 to 2021. High (>0.5) AOD values were observed during the summer season and low (<0.8) in the spring season. AE values were observed to be high (>1) in the northern regions of Pakistan indicating the dominance of fine mode particles during the winter season. Moreover, AOD showed a positive correlation with Relative Humidity (RH), Evapotranspiration, Wind speed (WS), and Temperature. On the other hand, it showed a negative correlation with Soil moisture (SM), Normalized difference vegetation index (NDVI), and precipitation over Pakistan. Therefore, considering the outcomes of this study will help policymakers to understand the spatiotemporal variability of aerosols and their seasonal correlation with different meteorological parameters. AerosThe weather conditions and the emission of aerosols from local sources make up the concentration of aerosols in a particular region. The increased number of aerosols in Asia is due to an increase in traffic, urbanization, and industrialization . The divTariq et al.  analyzedAngstrom exponent (AE) is a parameter that provides information about the size of aerosol particles by utilizing AOD . AE equaSingh et al.  studied Densely vegetative areas have a significant impact on wind speed (WS) and adhere to the movement of pollutants over a long distance from the ground . Sakar aPreviously conducted studies have investigated the spatiotemporal distribution of aerosols emitted from anthropogenic activities over Pakistan . Accordi22.1Pakistan is a densely populated country having a population of 225 million. It is located between latitudes 24°–37° N and longitude 61°–76° E. There lies the Himalaya and Karakoram ranges in the northern areas of Pakistan . Similarly, for AE, we have used MODIS-Aqua with deep blue Angstrom Exponent for land (0.412–0.47 μm) monthly product (MYD08_M3v6.1) with 1° × 1° spatial resolution from the same website. Version 6 of AOD and AE is the latest and most improved product that provides improved cloud masking and atmospheric profile algorithms.In 1999, MODIS sensor onboard the Aqua satellite was launched . It has 2.2.2https://giovanni.gsfc.nasa.gov/giovanni/). Details of all the datasets are given in Data for a meteorological and topological parameter was obtained from Atmospheric Infrared Sounder (AIRS) for Relative Humidity (RH) at 925 hPa. Similarly, data for PR, NDVI, surface wind speed (SWS), and Atmospheric temperature (AT) was obtained from the Tropical Rainfall Measuring Mission (TRMM), MODIS sensor onboard the Terra satellite and the Modern-Era Retrospective analysis for Research and Applications, version 2 (MERRA-2) Model respectively. Data for SM and evapotranspiration was collected from Global Land Data Assimilation System (GLDAS). All the datasets are collected from the website Giovanni NASA , spring , summer , and autumn . These datasets were utilized to observe the spatial variability of AOD and AE over different seasons among all provinces of Pakistan.Furthermore, seasonal datasets were also downloaded for NDVI, SM, evapotranspiration, and other meteorological parameters. Data was downloaded in the form of TIFF (tagged image file format) and later on, imported into the ArcGIS software.ArcGIS software was used to carry out all the analysis. After downloading the data, the resolutions of the data sets were equalized by using a resampling tool in the data management category of ArcGIS. Finally, correlation maps were exported from the ArcGIS software, and the results were compared.33.1By contrast, AE distribution among provinces of Pakistan was in such a way that the approximate values of 1.06, 1.15, 0.89, and 0.82 of AE are observed in the eastern, northern, southern, and western regions of Pakistan respectively. Variation of AE was high in Punjab and Khyber Pakhtunkhwa (approximately 1.06 and 1.15), indicating that the fine mode aerosol particles increase in these regions. The dominance of fine-mode aerosols in Khyber Pakhtunkhwa and Punjab indicated an increase in biomass burning and related anthropogenic activities, especially in winter. Ali et al.  analyzed3.1.13.1.2The seasonal spatial distribution of AE from 2002 to 2022 is shown in 3.1.33.1.4According to 3.1.5Temperature is also one of the major natural factors affecting the scattering of pollutants in the atmosphere. It can be seen in 3.1.6According to 3.1.7According to 3.1.8−2) during all seasons in Baluchistan, Khyber Pakhtunkhwa, and some areas of Sindh. The lowest range of SM content (40-60 kgm−2) is found majorly in Punjab and Sindh. Similarly, the highest SM content (280 kgm−2) is found to be in northern Kashmir.According to 3.1.9−0.07), Sindh (3.9 × 10−0.07), and southern eastern Punjab (6.7 × 10−0.06). However, a moderate evapotranspiration rate is observed in northern Punjab (1.5 × 10−0.05) during winter. In summer, a high rate of evapotranspiration (4.7 × 10−0.05) is found in northern Punjab, all over Khyber Pakhtunkhwa, and in southern Kashmir. Similarly, in the case of Kashmir, a high rate of evapotranspiration (4.7 × 10−0.05) is found during the spring and autumn seasons. A moderate range (3.5 × 10−0.06) of evapotranspiration is found in Punjab, Khyber Pakhtunkhwa, and in some cities of Sindh during the autumn and spring seasons. Similarly, the lowest value (3.9 × 10−0.07) is found in Baluchistan during the autumn and spring seasons.According to 3.2The correlation between AOD and AE derived from Aqua-MODIS over Pakistan has been shown in During the spring season, a very weak positive correlation has been observed in northern Khyber Pakhtunkhwa, Kashmir, and Punjab. These areas have an AOD value of ∼0.7 with the presence of coarse mode particles indicated by a low value of AE (0.6) , Fig. 5 During the spring season, a weak positive correlation between NDVI and AOD can be observed in Khyber Pakhtunkhwa. According to According to During the spring season, a strong positive correlation is observed in southern Punjab, almost all the areas of Sindh, and a few areas of Baluchistan. The aerosol burden is found to be moderate (0.7) in respective areas in Punjab, Sindh, and Baluchistan. Similarly, WS is also moderate (greater than 6 km/h). An extremely weak negative correlation is found in northern Punjab. In spring, the aerosol burden is found to be moderate because frequent dust storm events occur in which wind helps to transport the aerosols from Thar, Thal, and surrounding arid areas to Sindh, Baluchistan, and Punjab ,67. The During the autumn season S positivFrom A weak negative correlation is found to be in northern Khyber Pakhtunkhwa. Aerosols are found to be moderate in concentration (0.6) but the temperature is also low to moderate (180 k) , Fig. 8.During the summer season, a weak positive correlation is found in western Baluchistan, Sindh, southeastern Punjab, and Khyber Pakhtunkhwa. According to During the spring season, a strong positive correlation between AOD and temperature is found in Sindh, Baluchistan, and southern Khyber Pakhtunkhwa. Similarly, a negative correlation is found in Khyber Pakhtunkhwa and Kashmir. During the autumn season, a strong positive correlation is found all over Baluchistan, and Khyber Pakhtunkhwa which includes Chagai, Zhob, Quetta, Khuzdar, Peshawar, Mardan, and Kohistan. AOD is lowest during the spring, winter, and autumn seasons in above mentioned regions because of weak vertical mixing in the atmosphere and the absence of strong connective winds in the low temperatures .A negative correlation is observed in southern western Punjab and Gilgit Baltistan. Punjab has high AOD due to the harvesting season of rice in which rice husk contributes to atmospheric pollution in the atmosphere followed by the burning of left-over rice residues . All theAccording to During summers, a positive correlation is found in northwestern Punjab (0.8) and in Khyber Pakhtunkhwa (0.9). A moderate (40%) amount of RH is present in the above regions with low AOD (0.6) in Khyber Pakhtunkhwa and moderate to high AOD (greater than 0.8) in Punjab. A negative correlation is found in some areas of Punjab (−0.6), Sindh (−0.6), and Gilgit Baltistan (−0.7). However, the cities of Srinagar, Muzaffarabad, and Gilgit have a low aerosol burden (0.3) and moderate RH (50%) , Fig. 9.During spring, a strong positive correlation is found in south-eastern Sindh (∼1) and Kashmir (∼0.7). According to A negative correlation is found in southern Khyber Pakhtunkhwa (−0.7), and Baluchistan (−0.8). The RH is low (30%) in these areas along with a low AOD (0.3). During autumn, a strong positive correlation is found in a few areas of Khyber Pakhtunkhwa (1), northern western Punjab (0.8), eastern Baluchistan (0.7), and all over Sindh (greater than 0.8). Areas of Peshawar and Khuzdar show relatively low aerosol burden (0.2) with low to moderate (35%) RH. However, in Sindh and Punjab, the aerosol burden is high (greater than 0.9) and RH is low (<20%). A negative correlation is found in northwestern Baluchistan (∼-1) so, according to All these results show that AOD and RH show a direct relation in the summer and winter seasons. However, in autumn and spring, AOD and RH show an indirect relationship between them. It can be seen in In summer, a weak positive correlation (0.3) is found in the northern area of Punjab and the eastern area of Khyber Pakhtunkhwa. According to A negative correlation (less than 0.07) is found in a few areas of Punjab, Sindh, and Kashmir. High aerosol burden (1) with low precipitation (0.02 mm/day) is found in Sindh and Punjab. However, in Muzaffarabad, as well as in Srinagar, the aerosol burden is low (0.4), and precipitation (0.2 mm/day) is found to be high to moderate. Precipitation is high in northern Pakistan because it receives winds laden with moisture and rainfall that washes out the aerosols present in the atmosphere .During the spring season, a weak positive correlation is found in a few areas of northeastern Baluchistan, northern Sindh, and Kashmir. These areas have a low PR (0.02 mm/day) and low aerosol burden (0.6) in spring , Fig. 10During autumn, a strong positive correlation (greater than 0.4) is found in Sindh and Baluchistan. In Sindh, aerosol burden (0.8) is found to be high, whereas precipitation (0.02 mm/day) is found to be low. Similarly, a negative correlation (0.04) is found in Baluchistan and eastern Punjab S. Signif−2) in Baluchistan and Khyber Pakhtunkhwa and very low (40 kgm−2) in Sindh and Punjab. SM is directly linked with precipitation. A decrease in precipitation in Punjab and Sindh during winter ultimately shows a decrease in SM whereas the northern area of Baluchistan, Kashmir, and Khyber Pakhtunkhwa have moderate SM due to low temperature that prevents evapotranspiration and high rainfall that add moisture to the soil. Increased AOD in Punjab and Sindh is due to the low moisture content in the soil [It can be seen in the soil .−2) in Khyber Pakhtunkhwa and Srinagar and lowest in Punjab (40 kgm−2). During summers SM content in Punjab is low due to the arid climate followed by hot temperatures (above 40 °C) that cause excessive evaporation thus losing moisture from the soil that holds up aerosol [During summers, a positive correlation can be observed in the northern Punjab (0.6) and eastern Khyber Pakhtunkhwa (0.7). Whereas a negative correlation can be observed in southern Punjab (−0.6), northern Khyber Pakhtunkhwa (−0.5), and Srinagar (−0.9). According to  aerosol .−2) and AOD (0.2) both are low in Baluchistan and northern Khyber Pakhtunkhwa. However, AOD is moderate (0.8) in Sindh and Punjab and SM is very low (40 kgm−2). SM is low in Baluchistan due to late summer droughts that stress the SM by increasing the evapotranspiration process [During spring, a weak positive (0.3) correlation can be observed in western Baluchistan and northern Khyber Pakhtunkhwa. Whereas a strong negative correlation (−0.9) can be observed in Sindh and the southern Punjab area. According to  process . Zamin e process  conducteDuring autumn, a strong positive correlation (greater than 0.6) can be observed in Sindh and Baluchistan. Whereas a negative correlation (−0.7) can be observed in western Punjab and the area of Kashmir. According to −0.07 kgm−2s−1) in Baluchistan, Khyber Pakhtunkhwa, and Sindh. Only Punjab has a high value (4.7 × 10−0.05 kgm−2s−1) for evapotranspiration. Punjab faces high AOD and evapotranspiration due to high temperatures compared to the rest of the provinces. The studies of Shahriar [It can be seen in Shahriar  and KaurShahriar  have rep−0.05 kgm−2s−1) and low in southern Punjab (6.7 × 10−0.06 kgm−2s−1). Rainfall was high over Punjab in monsoon but over a few years, it has moved to northern and western Khyber Pakhtunkhwa due to changes in climatic patterns. A decrease in rainfall in Punjab increases the evapotranspiration rate [During the summer season, a strong positive correlation (0.9) can be observed in western Punjab and southern Khyber Pakhtunkhwa. Whereas a negative correlation can be observed in Kashmir (−0.6) and southern Punjab (−0.3) S. Accordion rate .−0.06 kgm−2s−1) in Baluchistan and Sindh but high (4.7 × 10−0.05 kgm−2s−1) in northern Punjab and Khyber Pakhtunkhwa. Similarly, −0.05 kgm−2s−1) in Baluchistan, Punjab, and Sindh. Khan and Hasan [During the spring season, a positive correlation can be observed in Kashmir (0.6) and western Khyber Pakhtunkhwa (0.7), whereas a negative correlation (−0.5) can be observed in northern Punjab, Baluchistan, Khyber Pakhtunkhwa, and Sindh. According to nd Hasan  reported4Environmental pollution is one of the major issues in developing countries like Pakistan. Therefore, in this study, we analyzed aerosol optical properties and their relationship with SM, evapotranspiration, NDVI, and other meteorological parameters over Pakistan from 2002 to 2021. High AOD values observed in Sindh (0.48) and Punjab (0.55) are associated with desert dust and anthropogenic activities within the region. High AE values observed in Punjab (1.06) and Khyber Pakhtunkhwa (1.15) indicate the dominance of fine-mode particles. Furthermore, NDVI was observed to be high (0.8) in Kashmir and Khyber Pakhtunkhwa during winter. Whereas, in Baluchistan (10–12 km/h) and Sindh (12 km/h), WS was observed to be high during the winter and spring season. The correlation of AOD with WS, temperature, SM, and evapotranspiration over Pakistan showed a direct relation whereas, NDVI and precipitation showed an inverse relation throughout the year. Furthermore, during the summer and winter seasons, AOD showed a direct relation to RH. Significant seasonal changes in AOD measurements demand further research into the area's meteorological pollution source location and transportation. Different methods like afforestation, efficient use of fuel, use of public transport, restriction on the use of certain industrial plants, etc. can be used to reduce air pollution.Data will be made available on request.Not required.Not Applicable.Not Applicable.The work did not receive any funding.Uzma Basharat: Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing – original draft. Salman Tariq: Conceptualization, Data curation, Formal analysis, Methodology, Software, Writing – original draft. Muhammad Nawaz Chaudhry: Conceptualization, Data curation, Formal analysis, Investigation, Software, Writing – original draft. Muhammad Khan: Conceptualization, Data curation, Formal analysis, Investigation, Software, Writing – original draft. Ephraim Bonah Agyekum: Formal analysis, Investigation, Methodology, Writing – review & editing. Wulfran Fendzi Mbasso: Formal analysis, Investigation, Methodology, Writing – review & editing. Salah Kamel: Formal analysis, Investigation, Methodology, Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.