The limits of detection (S/N = 3) of five QAs ranged from 0.03 to 0.133 ng·mL−1. The intra-day and inter-day relative standard deviations of the five QAs with the spiked concentration of 50 ng·mL−1 were 2.8–4.0 and 3.0–8.8, respectively. The recoveries of five QAs in the honey and pork samples were 81.6–119.7%, which proved that the proposed method has great potential for the efficient extraction and determination of QAs in complex samples.The extraction of quinolone antibiotics (QAs) is crucial for the environment and human health. In this work, polyacrylonitrile (PAN)/covalent organic framework TpPa–1 nanofiber was prepared by an electrospinning technique and used as an adsorbent for dispersive solid-phase extraction (dSPE) of five QAs in the honey and pork. The morphology and structure of the adsorbent were characterized, and the extraction and desorption conditions for the targeted analytes were optimized. Under the optimal conditions, a sensitive method was developed by using PAN/TpPa–1 nanofiber as an adsorbent coupled with high-performance liquid chromatography (HPLC) for five QAs detection. It offered good linearity in the ranges of 0.5–200 ng·mL As natural or synthetic compounds, antibiotics have been widely used in human and veterinary medicine and can inhibit the proliferation of bacteria . QuinoloTo date, a variety of detection methods, such as fluorescence , surfaceIn recent years, some sample pretreatment techniques, such as conventional liquid–liquid extraction (LLE) ,18, soliCOFs, as a new kind of porous organic material, are constructed by the connection of organic monomers via strong covalent bonds, which have attracted a wide range of attention of researchers . Due to In this work, PAN/TpPa–1 nanofiber membrane was prepared by doping COFs into PAN polymer solution and subsequently electrospinning. The PAN/TpPa–1 nanofiber was used as a dSPE adsorbent for the extraction of five QAs from animal-derived foods. The main parameters influencing the extraction efficiency of QAs, such as the amount of PAN/TpPa–1, desorption solvent, desorption time, extraction time, ionic strength of sample solution, pH of sample solution, and the analytical performance of the monitoring QAs, were evaluated. A sensitive method was developed by using PAN/TpPa–1-based dSPE coupled with HPLC and a UV detector and was successfully applied to the analysis of five QAs in honey and pork samples.P-phenylenediamine (Pa–1), mesitylene, and polyacrylonitrile  were purchased from Shanghai Macklin Biochemical Co., Ltd. . Pefloxacin (PEF), norfloxacin (NOR), enrofloxacin (ENR), and orbifloxacin (ORB) were obtained from Qingdao Tenglong Weibo Technology Co., Ltd. . Sarafloxacin (SAR) was obtained from Shanghai Yuanye Bio-Technology Co., Ltd. . Methanol, ammonia , formic acid, acetonitrile 1,4-dioxane, and N, N-dimethylformamide (DMF) were purchased from Tianjin Concord Technology Co., Ltd. . Honey and pork samples were obtained from a local supermarket in Tianjin. Ultrapure water used for all experiments was purchased from Watsons Water .The 1,3,5-Triformylphloroglucinol (Tp) was obtained from Yanshen Technology Co., Ltd. . −1. The X-ray diffraction (XRD) pattern was recorded in the range of 2θ from 2 to 35° with an X-ray diffractometer . The tube voltage was 40 kV, the tube current was 150 mA, and the scanning speed was 15 deg/min. The surface area and pore size were calculated by nitrogen adsorption and desorption isotherms at 77 K, according to the Brunauer–Emmett–Teller (BET) method . The thermogravimetric analysis (TGA) was performed on a TG209C . The temperature range was from 25 °C to 800 °C, and the heating rate was 10 °C/min.All nanofibers obtained in this work were fabricated by an NS Lab NanoSpider . Scanning electron microscopy (SEM) images were obtained with a SU3500 . All samples needed sputtering before observation, and the operating voltage was 30 kV. The FT-IR spectra of materials were obtained by FT-IR spectrometer  in the range of 3500–500 cmv/v), and the volumetric flow rate was set at 0.5 mL·min−1. The injection volume is 20 μL, and the detection wavelength was 277 nm for analytes. Five QAs were separated with the elution order of PEF, NOR, ENR, ORB, and SAR.The HPLC measurement was carried out by an LC-20AT  HPLC system equipped with an SPD-20A UV/vis detector. An Inert Sustain C18 Column  was used for separating five QAs. The mobile phase was water (containing 0.3% formic acid)–methanol  were added into a glass bottle. The mixture was sonicated for 30 s to obtain a homogenous dispersion, and 2.0 mL of 3 M aqueous acetic acid was added. The mixture was transferred into a 50-mL Teflon-lined reactor. The reactor was sealed and then heated at 120 °C for 72 h. The product was washed with acetone and anhydrous ethanol and then dried at 80 °C under vacuum overnight, and a deep red-colored powder was collected.TpPa–1 was prepared according to the reported literature with slight modifications . TypicalAs shown in A 10.0 g honey sample was vortexed with 30 mL of acetonitrile for 2 min. Then, the mixture was sonicated for 30 min. After that, the sample was centrifuged at 6000 r/min for 7 min, and the sediment was repeatedly extracted with 30 mL of acetonitrile once. The above extraction steps were repeated for five groups. The supernatants obtained were combined to evaporate dry with a rotary evaporator, and the residues were dissolved with 5 mL of acetonitrile. After being filtered by a 0.22-µm filter membrane, the obtained solution was diluted to 50 mL with ultrapure water and stored in a refrigerator at 4 °C until use. The pork was cut into small pieces. A 10.0 g pork sample was vortexed with 30 mL of 1% acetic acid-acetonitrile for 5 min. Then, the sample was centrifuged at 3500 r/min for 5 min. The supernatant was collected, and the residues were extracted again. The above extraction steps were repeated for five groups. The supernatant obtained was combined. Then, 30 mL of n-hexane was added into the solutions mentioned above. The solution was vortexed for 1 min and immediately centrifuged at 3500 r/min for 5 min. The n-hexane layer was discarded, and the acetonitrile layer was collected. Afterwards, 10 mL of isopropanol was placed in the collected acetonitrile layer with adequate mixing. The above solution was evaporated until dry on a rotary evaporator, and the residues were dissolved in 5 mL of acetonitrile. The next step is similar to the honey sample. v/v) by shaking for 15 min. After being filtered by a 0.22-µm membrane, the eluent was concentrated until dry using nitrogen. Finally, the residues were redissolved with 50 µL of eluent for HPLC analysis. The dSPE procedure is shown in The dSPE procedure utilizing the PAN/TpPa–1 as an adsorbent was as follows. First, 10.0 mg of PAN/TpPa–1 was placed in a 50-mL centrifugal tube and activated with 2.0 mL of methanol and 2.0 mL of water for 1 min in turn. Then, 20 mL of the sample solution was loaded into the centrifugal tube, and the mixture was shaken for 40 min. Thereafter, the PAN/TpPa–1 was washed with 2.0 mL of water, and the adsorbed target analytes were eluted with 1.0 mL of 2.0% ammonia-methanol  and merged with the peak of the C=C stretching band at 1582 cm−1, which was due to the strong hydrogen bond in the keto structure. The peaks at 1256 cm−1 come from C–N stretching. This indicated that TpPa–1 was successfully synthesized and the tautomerism of TpPa–1 tended to form a keto structure instead of an enol structure. The FT-IR of Tp, Pa–1, and COF TpPa–1 is displayed in 2θ), corresponding to the (100) reflecting plane, and the minor peaks appear at ~8.1° and ~27° (2θ), attributable to the (200) and (001) planes, respectively. At the higher ~27° (2θ), it was mainly due to π–π stacking between COFs layers. This indicated that TpPa–1 has good crystallinity, which was consistent with the simulation results. The unit cell was constructed with a hexagonal model P6/M space group, and the unit cell parameters were a = b = 22.82 Å and c = 3.34 Å. After TpPa–1 was doped into PAN, there were no obvious peaks at ~4.7°, ~8.1°, and ~27° (2θ), and the XRD pattern was basically consistent with PAN, which may be due to the low content of TpPa–1 doped into PAN.X-ray diffraction (XRD) analysis was used to study the crystal structures of TpPa–1, PAN, and PAN/TpPa–1 nanofiber. It can be seen in 2 adsorption–desorption isotherm of TpPa–1 increased sharply when P/P0 was very low because the interaction between adsorbent and adsorbate was enhanced in narrow micropores, which led to micropore filling at extremely low relative pressures. However, the saturated pressure was reached, and the adsorbate condensed led to the curve rising. This was a typical type I adsorption isotherm of microporous materials, indicating that TpPa–1 was mainly composed of micropores with a pore diameter of about 1.7 nm and a specific surface area of 778.4 m2·g−1. Therefore, TpPa–1 is very suitable for the adsorption of five QAs whose sizes were between 1.24 nm and 1.55 nm  method. As shown in  1.55 nm . As showThe thermogravimetric analysis (TGA) of the materials is shown in The amount of adsorbent is of great significance to the dSPE procedure. In this work, 4 mg, 7 mg, 10 mg, and 13 mg were selected to investigate the influence of the amount of adsorbent on the extraction of QAs. With the increase in adsorbent mass from 4 mg to 10 mg, the peak areas of the chromatogram for five QAs were greatly improved. When the adsorbent mass increased from 10 mg to 13 mg, the peak areas of the targets did not significantly increase; so, the amount of PAN/TpPa–1 nanofiber selected for further research was 10 mg A.Methanol, acetonitrile, ethyl acetate, and acetone were selected as eluents to investigate the effect of desorption solvent. The results showed that the target analytes were hardly eluted under the condition of these eluents. Considering that QAs are a kind of acid–base amphoteric compound, it may be necessary to add an appropriate acid or alkali to the eluant to improve the elution efficiency. Therefore, the desorption effects of 2% formic acid-methanol, 1% ammonia-methanol, 2% ammonia-methanol, and 4% ammonia-methanol were investigated, and the results are shown in During the process of dSPE, the extraction time is important. In this work, the effect of extraction time on the dSPE process was investigated. As shown in −1 . The LODs (S/N = 3) and LOQs (S/N = 10) were in the range of 0.03–0.133 ng·mL−1 and 0.086–0.288 ng·mL−1, respectively. The intra-day RSD and inter-day RSD were 2.8–4.3% and 3.0–8.8%, respectively, which showed the method has good precision.Under the optimal conditions, the validity of this method was proved by linear range, limits of detection (LODs), and limits of quantitation (LOQs). As shown in −1) were investigated  as real samples and three spiked concentrations (5, 50, and 100 ng·mLstigated . Five QATo evaluate the performance of this method, it was compared with other methods for adsorbing QAs . ThroughIn this work, a novel PAN/TpPa–1 nanofiber was synthesized by a facile electrospinning method and applied as a dSPE adsorbent for extraction of five trace QAs in honey and pork, combined with HPLC–UV detection. The prepared PAN/TpPa–1 material exhibited a good extraction effect towards QAs probably due to π–π interactions and hydrogen-bond formation. The established dSPE/HPLC–UV method exhibited a wide linear range, low LODs, and satisfactory recoveries. Furthermore, the PAN/TpPa–1 nanofiber possessed good reusability and easy operation. In addition, the results for the extraction of five QAs provide important reference information and an experimental basis. Therefore, the dSPE/HPLC–UV method based on PAN/TpPa–1 had excellent extraction performance for QAs and has application potential for the determination of QAs in complex samples.


Purpose: To evaluate the safety, tolerability, pharmacokinetics and immunogenicity of DS002 injection, an anti-nerve growth factor (anti-NGF) monoclonal antibody for treating pain conditions, in healthy Chinese subjects.Methods: This study was a single-center, randomized, double-blind, single-dose escalation, placebo-controlled design (CTR20210155). A total of 53 healthy subjects, 27 male and 26 female, were enrolled in this study, and one subject withdrew from the study before administration. Seven dose groups were set up, which were 0.5 mg, 1.0 mg, 2.0 mg, 4.0 mg, 7.0 mg, 12.0 mg and 20.0 mg, respectively. The drug was administered by single subcutaneous injection. Four subjects were enrolled in the first dose group (0.5 mg) received DS002. Other dose groups enrolled eight subjects each, six of whom received DS002 while the other two received a placebo. Safety, tolerability, pharmacokinetic parameters and immunogenicity of DS002 were assessed.Results: DS002 was well tolerated; all adverse events were Grade 1–2, and did not reach the termination standard of dose increment within the range of 0.5–20.0 mg. Adverse event rates were generally similar across treatments. After a single subcutaneous injection, the median Tmax in different dose groups ranged 167.77–337.38 h; mean t1/2 ranged 176.80–294.23 h, the volume of distribution (Vz) ranged 5265.42–7212.00 ml, and the clearance rate (CL) ranged 12.69–24.75 ml/h. In the dose range of 0.5–20.0 mg, Cmax ranged from 51.83 ± 22.74 ng/ml to 2048.86 ± 564.78 ng/ml, AUC0-t ranged from 20615.16 ± 5698.28 h·ng/mL to 1669608.11 ± 387246.36 h·ng/mL, and AUC0-inf ranged from 21852.45 ± 5920.21 h·ng/mL to 1673504.66 ± 389106.13 h·ng/mL. They all increased with dose escalation, and Cmax and AUC0-t did not have a significant dose-linear relationship, whilst AUC0-t was not dose-dependent at all. anti-drug antibody test results of each group of all subjects in this trial were negative.Conclusion: DS002 showed satisfactory safety within the dose range of 0.5 mg–20.0 mg. The absorption and metabolism of DS002 were slow, it exhibited a low volume of distribution and the clearance rate was low. These data suggest that DS002, by blocking nerve growth factor, is expected to become a novel, safe and non-addictive treatment for pain conditions. Chronic pain affects more than 30% of people in the world. The individual and societal burden of chronic pain are substantial . In the The nerve growth factor (NGF) belongs to the neurotrophic factor family. It was originally extracted from mouse submandibular gland and snake venom, and exists in almost all vertebrates . NGF exiAnti-NGF antibody drugs include Pfizer/Lilly’s Tanezumab and Regeneron/Tiva’s Fasinumab, both of which have achieved significant efficacy in the treatment of moderate to severe osteoarthritis and chronic lower back pain. However, both drugs reported severe adverse reactions in joints, including osteonecrosis and rapidly progressive osteoarthritis (RPOA), . TherefoIn vitro studies have shown that DS002 can successfully block the binding of NGF to TrkA receptors. In pre-clinical studies, DS002 treatment demostrated potent analgesic effects in several pain models including OA pain, chemotherapy-induced pain, and cancer pain. Based on the preclinical repeated toxicity test and efficacy test, as well as the clinical trial of drugs at the same target  was approved by the ethics committee of Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, and carried out according to the Declaration of Helsinki (2013 version), the Guideline For Good Clinical Practice issued by the International Council for Harmonisation (ICH), the Good Clinical Practice (GCP) issued by the National Medical Products Administration (NMPA) and relevant regulations. Written informed consent was obtained from all subjects.The overall study was a single-center, randomized, double-blind, single-dose escalation, placebo-controlled phase I clinical trial. A total of 53 Chinese healthy subjects were enrolled in this study, including 27 males and 26 females, and 52 subjects completed this clinical trial.The dose-escalation design was used to evaluate the safety, tolerability, pharmacokinetics and immunogenicity of DS002 after a single administration. Seven dose groups were set up, including 0.5, 1.0, 2.0, 4.0, 7.0, 12.0 and 20.0 mg, not per kg, respectively. DS002 was administered by subcutaneous injection. Four subjects were enrolled in the first dose group (0.5 mg), and each subject received a singledose of DS002 subcutaneously. In the 2nd to 7th dose groups, eight subjects were enrolled in each group, among whom 6 received DS002 and two subjects received placebo. A series of blood samples were collected before and after DS002 administration to evaluate the PK parameters and immunogenicity of DS002 in healthy subjects. Subjects were clinically followed up on the 4th, 8th, 15th, 22nd, 29th, 43rd, 57th, 85th and 113th days, biological samples were collected and safety and tolerability were assesseded. Subjects were followed up by telephone on the 120th day, which was the end of each dose group. . The nexDuring hospitalization in the research center, the subjects were managed with a unified diet and rest protocol. On the day before DS002 administration, the subjects stayed in the Phase I trials unit, took a unified dinner, and then fasted overnight for at least 10 h. The next morning, they ate a small amount of standard breakfast and then received DS002 injection subcutaneously. After injection, they ate the standard meals. Within 48 h prior to enrollment and during the whole trial period, it was forbidden to take any food and drink rich in caffeine or xanthine , smoking and drinking alcohol-containing products were also not allowed. Strenuous exercise  were avoided during the whole trial period. Subjects were told to take proper contraception from the start of the study period to 6 months after DS002 administration.This study was a double-blind trial design (except for the first dose group). Blindness was maintained for the generation of random numbers, the coding of drugs used in the trial, the drug matched to each subject, the recording and evaluation of test results, the monitoring of the test process and data management. On the day of DS002 administration, the investigator checked the information of all subjects to ensure that they met the inclusion criteria of the trial, and then administered according to the drug number matched with the random number of the subject. All relevant personnel remained blind during the clinical study.PK blood sampling time points: for determination of the concentration of DS002, blood samples were collected at 17 time points in each dose group: within 1 h before DS002 administration, and 6 h, 8 h, 12 h, D2 (24 h), D2 (36 h), D3 (48 h), D3 (60 h), D4 (72 h), D8 (168 h), D15 (336 h), D22 (504 h), D29 (672 h), D43 , D57 , D85 (2016 h), and D113 (2688 h) after administration. About 3 ml of venous blood was collected at each time point .Immunogenicity (ADA) blood sampling time points: blood samples were collected at 6 time points in each dose group, including within 1 h before administration, D8 (168 h), D15 (336 h), D29 (672 h), D57  and D113 (2688 h) after administration, and about 3 ml of venous blood was collected at each time point .2, the male body weight was ≥50 kg and female ≥45 kg. Effective contraception should be used during the trial and continued for 6 months after administration. Subjects fully understood the content, objectives and characteristics of the study and could complete the study as planned, voluntarily participated in the study and signed written informed consent.Subjects were healthy volunteers aged 18–45 years, including both males and females (not pregnant or breastfeeding). The body mass index (BMI) of the subjects was 19.0–26.0 kg/mSubjects were excluded if they met one of the following: Those who had an allergic history or allergic constitution in the past; Those who had a history of bone or joint diseases, peripheral or autonomic neuropathy, spontaneous bleeding of unknown causes and (or) disease or medical history of continuous bleeding after trauma, abnormal thyroid function or thyroid hormone, or malignant tumor; Smokers, alcoholics or drug abusers. Subjects were not allowed to take any prescription drugs, over-the-counter drugs and natural health products within 14 days before screening.The primary objective of this study was to evaluate the safety and tolerability of DS002 as a single administration in healthy subjects. The secondary objective was to evaluate the pharmacokinetic parameters and immunogenicity of DS002 in healthy subjects after a single administration.The safety indicators of this study included the incidence of adverse event  and serious adverse event (SAE); Laboratory examinations; Neurological examination; Injection site reaction; 12-lead electrocardiogram (ECG); Abdominal B ultrasound; Vital signs; Physical examination.max, Tmax, AUC0-t, AUC0-inf, t1/2, MRT, λz; Immunogenicity index: ADA after single administration of DS002.PK indexes: PK parameters of DS002 after a single administration, including but not limited to CElectrochemiluminescence Immunoassay (ECLIA) methods for the analysis of the pharmacokinetics (PK) and anti-drug antibody (ADA) of DS002 in serum were established.For PK samples, the MSD plate was pre-coated with huNGF-his, and after adding the sample, it was incubated to form an antigen-antibody complex. After incubation, the unbound DS002 was washed away with a washing solution. Detection antibody Sulfo-Anti-DS002 antibody was then added and incubated; after incubation, unbound antibody was washed off with washing solution; finally the reading buffer was added, and MSD  was used to collect the signal. The data were analyzed and processed with Watson LIMS (V7.4.1 and V7.6.1). The calibration ranges of DS002 were 2.50 ng/ml∼500.00 ng/ml. The quality control samples  were determined with an accuracy of 89.0%–114.3%, the precision of 17.6% (CV) for inter-assay and accuracy of 104.0%–106.1%, precision of 19.8% (CV) for intra-assay. There was no matrix effect for human serum. No hook effect was observed for samples up to 5000.00 ng/ml. The sample can be diluted up to 1,250 fold with a total CV(%) of 5.5%.The ADA samples were measured by screening assay, confirmatory assay, and titer assay. The method is based on the multivalency of antibodies and the specific binding of antigen-antibody. First, each control sample and the test sample were diluted by 1:20 times of acidification. Then each diluted sample solution was added to TrkA-hFc working solution for purification to remove interferences. The purified solution was then added with Biotin-DS002 and Suflo-DS002 Master Mixture (with or without drug), incubating on a dilution plate to allow a bridging reaction between the drug and the anti-drug antibody to form a bridging complex. After the reaction was completed, it was transferred to the MSD plate for incubation. After incubation, the plate was washed and then the reading buffer was added, and the signal was read on the MSD. For the screening assay, the HPC samples (5000 ng/ml) were determined with a precision of 4.4% (CV) for inter-assay and 11.9% (CV) for intra-assay. The LPC samples (16 ng/ml) were determined with a precision of 14% (CV) for inter-assay and 10.8% (CV) for intra-assay. For the confirmatory assay, the HPC samples (5000 ng/ml) were determined with a precision of 0.1% (CV) for inter-assay and 0.1% (CV) for intra-assay. The LPC samples (16 ng/ml) were determined with a precision of 25.9% (CV) for inter-assay and 18.3% (CV) for intra-assay. There was no matrix effect for human serum. No hook effect was observed for samples up to 41000.00 ng/ml.® (Version 8.0 and above) were used for statistical analysis. The classified variables were summarized by frequency and percentage. Continuous variables were summarized by sample size, mean, standard deviation, median, minimum and maximum. For some continuous variables, the coefficient of variation or the geometric coefficient of variation, geometric mean, lower quartile and upper quartile were also counted. The number of subjects enrolled in each dose group and the situation of dropped cases were summarized. A descriptive statistical analysis on baseline characteristics of the enrolled cases were made. Safety and PK data were summarized using appropriate statistical tables and diagrams.Unless otherwise specified, SAS (version 9.4 and above) and Phoenix™ WinNonlin Full analysis set (FAS): all randomized subjects. Safety analysis set (SS): all subjects who have received the study drug at least once and have at least one safety evaluation index. PK concentration and parameter analysis set (PKCS and PKPS): all subjects who have used the study drug and have at least one PK concentration or parameter data. Immunogenicity analysis set (IS): all subjects who have received the study drug at least once and have at least one post-baseline immunogenicity evaluation data.Based on the PKCS, the concentration data were tabulated and described by dose groups. The drug concentration-time curves (linear and semi-logarithmic) were drawn according to the planned blood collection time.® (version 8.0 or above) was adopted. PK parameters were calculated according to the actual blood collection time, and PK parameters were listed and described statistically. A power model was used to analyze the linear relationship between main PK parameters  and dose.Based on PKPS, a Non-Compartmental Analysis by Phoenix™ WinNonlinThe study was conducted from 22 February 2021 to 24 May 2022. In this trial, a total of 53 healthy Chinese subjects in seven dose groups were included in the FAS, and 52 were included in the SS (one subject withdrew from the study before administration), including 40 subjects receiving DS002, four in the 0.5 mg dose group, and six in other dose groups; There were 12 subjects receiving placebo. All 52 subjects completed the study . The demAt least one adverse event (AE) occurred in 36 of 40 (90%) healthy subjects who received DS002 and 12 of 12 (100%) subjects who received placebo. Forty-five subjects (86.54%) had adverse reactions (ARs). 12 (25.00%) subjects of DS002 had AEs of grade 2 . No grade 3 or grade 4 AEs were reported. The TEAEs of subjects are shown in Among the 40 subjects injected DS002, the AEs with an incidence rate of more than 5% include the following: upper respiratory tract infection ; decreased triiodothyronine, and elevated serum uric acid ; elevated amylase ; elevated serum thyrotropin ; decreased white blood cell count ; arthralgia ; positive urine red blood cell, positive urine occult blood, elevated triglycerides, elevated alanine aminotransferase, prolonged QTc interval of ECG, hypokalemia, and decreased free thyroxine ; increased neutrophil count, abnormal ECG T wave, increased white blood cell count, elevated free thyroxine, elevated γ-glutamyltransferase, elevated aspartate aminotransferase, elevated serum calcium and elevated free triiodothyronine .In the placebo group, 12 subjects (100.00%) had AEs. The incidence rates from high to low were elevated amylase ; elevated serum thyrotropin, decreased leukocyte count, and increased neutrophil count ; decreased triiodothyronine, abnormal ECG T wave, and positive urine protein ; all (100.00%) had ARs. Of which one subject (8.33%) had 2 times Grade 2 AE.The incidences of AEs and ARs in each dose group showed an overall upward trend with dose escalation, and the incidence of AEs and ARs between DS002 and placebo subjects was similar. One case of Grade 2 AE (elevated amylase without symptom) was reported in the 12.0 mg dose group, whilst the rest of elevated amylase AEs were all Grade 1. No serious adverse event (SAE) was reported in subjects who received DS002 injection. None of the subjects died or dropped out to TEAE.In brief, after a single dose of DS002, none of the dose groups reached the termination standard of dose escalation, and the safety and tolerability profiles of DS002 were satisfied.All 40 subjects receiving DS002 were included in the PK analysis. The PK parameters of the DS002 and placebo groups are shown in _% Extrap of DS002 in the dose range of 0.5 mg–20 mg was less than 5.81%  and one subject in the placebo group. The symptoms were mild without any discomfort such as activity disorder, and were all resolved without intervention. It is worth noting that no severe bone and joint events or peripheral neuropathy/paresthesia were found. No rapid progression of osteoarthritis (RPOA) was observed even at high doses, which was the most prominent concern when the US Food and Drug Administration shelved the development plan of similar anti-NGF drugs. Collectively, the safety data of DS002 indicate that it may serve as provide a new therapeutic strategy for the target of analgesic treatment.1/2 of tanezumab is about 21 day .In adults, NGF and its interaction with TrkA have been found to play a vital role in the nociception and plasticity of the nervous system under pain conditions. Therefore, various monoclonal antibody therapies targeting this pathway have been studied to treat chronic pain. Although the Food and Drug Administration once shelved the development plan for an anti-NGF monoclonal antibody due to safety concerns, this target still has great clinical value and application prospect. Currently, no anti-NGF monoclonal antibody has been approved for clinical use, but three anti-NGF drugs are under development: tanezumab, fulranumab and fasinumab . Tanezumt 21 day , while tIn conclusion, the absorption and metabolism of a single subcutaneous injection of DS002 in healthy subjects were slow, it exhibited a low volume of distribution, and the clearance rate was low. Single subcutaneous injection of DS002 was well tolerated and non-immunogenic within the dose range of 0.5 mg–20.0 mg. The anti-NFG antibody represented by DS002 shows promise as a novel, safe and non-addictive approach for pain treatment. This first-in-human study supports further development of DS002 as a potential analgesic treatment.


Bombax ceiba L.  was rightly called the “silent doctor” in the past as every part of it had medicinal value. For centuries, humans have used this plant according to the traditional medicinal systems of China, Ayurveda, and tribal communities. Recently, with an emerging interest in herbals, attention has been paid to scientifically validating medicinal claims for the treatment of diabetes using secondary metabolites of B. ceiba L. flowers. In the present study, specific secondary metabolites from the flowers of B. ceiba L. were isolated in good yield using the solvent extraction methodology, and their in vitro anti-diabetic efficacy was examined. Extraction efficiency of each solvent for secondary metabolites was found in following order: water > ethanol> methanol > chloroform > petroleum ether. Quantitative analysis of secondary metabolites showed 120.33 ± 2.33 mg/gm polyphenols, 60.77 ± 1.02 mg/g flavonoids, 60.26 ± 1.20 mg/g glycosaponins, 0.167 ± 0.02 mg/g polysaccharides for water extract; 91.00 ± 1.00 mg/g polyphenols, 9.22 ± 1.02 mg/g flavonoids, 43.90 ± 0.30 mg/g glycosaponins, 0.090 ± 0.03 mg/g poly saccharides for ethanol extract; 52.00 ± 2.64 mg/g polyphenols, 35.22 ± 0.38 mg/g flavonoids, 72.26 ± 1.05 mg/g glycosaponins, 0.147 ± 0.01 mg/g polysaccharides for methanol extract; 11.33 ± 0.58 mg/g polyphenols, 23.66 ± 1.76 mg/g flavonoids, 32.8 ± 0.75 mg/g glycosaponins, 0.013 ± 0.02 mg/g polysaccharides for chloroform extract; and 3.33 ± 1.53 mg/g polyphenols, 1.89 ± 1.39 mg/g flavonoids, 21.67 ± 1.24 mg/g glycosaponins, 0.005 ± 0.01 mg/g polysaccharides for petroleum ether extract. Glucose uptake by yeast cells increased 70.38 ± 2.17% by water extract. Bombax ceiba L., such as flowers, stem bark and leaves, have proven to be hypoglycaemic in animal models, and root powder was proven to be anti-diabetic for type 2 patients in 2008 [Bombax ceiba L. For example, demulcent and tonic uses are associated with the roots, gum and bark [Bombax ceiba L. for intestinal worms [Many parts of  in 2008 ,2,3,4,5. in 2008 ,7,8,9. O in 2008 ,13,14,15and bark . The baral worms ,18,19.Bombax ceiba L.: scopolamine, protocatechuic acid, esculetin, isomangiferin, mangiferin, isovitexin, vitexin, rutin, chlorogenic acid, methyl chlorogenate, vanillic acid, quercetin, fraxetin [Various researchers have found a variety of useful compounds in the flowers of fraxetin , palmitifraxetin , , locally famous as the “sumbal tree”, was selected. Considering the importance of this plant, this study attempts to extract the secondary metabolites from the flowers of Bombax ceiba L. using a solvent extraction strategy. Several solvents are employed in this study for the extraction of secondary metabolites in cold and hot conditions. FTIR and UV-vis spectroscopic analysis of the extracts was performed to analyse the specific functional group in the metabolites. Subsequently, a quantitative determination of the secondary metabolites, including polyphenols, flavonoids, glycosaponins, and polysaccharides, was carried out, and an in vitro model of anti-diabetic potential was optimized for all of the flower extracts.The above discussion shows the magnitude of traditional value found in Bombax ceiba L.), locally famous as the “sumbal tree”, was selected. The fully blossomed flowers of Bombax ceiba L. were collected in March from the University College of Pharmacy, University of the Punjab, Lahore, Pakistan, from different trees in the front garden. The flowers were cleaned and shade-dried for 30 days. Further flowers were dried in an Anton Paar oven at 40 °C for three days. Dried flowers were sieved, pulverized and stored in air-tight containers [In the present study, the red silk cotton tree  first, then chloroform (61–62 °C), and lastly methanol (64–65 °C). For this purpose, 430 g of dry floral powder was packed in a filter paper bag of appropriate capacity, and approximately 3.5 L of petroleum ether was added. The temperature of the apparatus was kept below the boiling point of the solvent. All the solvents were evaporated using a rotary evaporator. The extracts were concentrated to nearly 100 mL of thick fluid, and for further removal of leftover solvent, the liquid extracts were dried in the oven at 50 °C. The extracts were weighed repeatedly to observe whether a constant mass was achieved. When the remaining mass became constant, it showed that there is no more solvent to be evaporated; then, the semi-solid or solid masses were removed from the oven. The extracts were weighed, and the % yield was calculated regarding initial weight. The extracts were stored at room temperature in air-tight bottles and away from direct exposure to sunlight.Twenty-five grams of dry floral powder with 150 mL of ethanol in a 1000 mL beaker was allowed to be stirred. The beaker was covered with aluminium foil to avoid loss of solvent. After 1 h, the solvent was decanted and filtered. Another 150 mL of ethanol was added to the same powder and stirred over the magnetic plate for 1 h. The solvent was again collected. Another 150 mL of ethanol was added, and the filtration procedure was repeated. The three ethanol filtrates were combined in the same place. After ethanol filtrates, the left over powder material was mixed with 150 mL distilled water, stirred for 1 h and filtered. All the solvents were evaporated using a rotary evaporator. The extracts were concentrated to nearly 100 mL of thick fluid, and for further removal of leftover solvent, the liquid extracts were dried in the oven at 70 °C. The extracts were weighed repeatedly to observe whether a constant mass was achieved. When the remaining mass became constant, it showed that there is no more solvent to be evaporated; then, the semi-solid or solid masses were removed from the oven. The extracts were weighed, and the % yield was calculated regarding initial weight. The extracts were stored at room temperature in air-tight bottles and away from direct exposure to sunlight.Sr. No.SamplePhysical CharacteristicsSign of Instability1.PowderLight brown and amorphousNo Apparent Change2.PBCYellowish green semi-solid with petroleum smellNo Apparent Change3.CBCGreenish semi-solid with characteristic smellNo Apparent Change4.MBCDark brown semi-solid with no smellNo Apparent Change5.EBCLight brown semi-solid with no smellNo Apparent Change6.WBCDark brown semi-solid with no smellSemisolid to SolidOrganoleptic properties of extracts, i.e., colour, smell, consistency and physical state, were noted and observed throughout experimentation to ascertain physical stability.2CO3 and then add methanol to make the final volume 3 mL. For extracts, take 200 µL from extract stock solution, 200 µL FC reagent and 1 mL of 15% Na2CO3 and make 3 mL volume with methanol. For the blank, 200 µL of methanol in place of the extract is used. Allow standing for 90 min in the incubator at room temperature. Absorbance is measured at 760 nm. Draw the calibration curve for the standard. The concentration of total phenolics in extracts is read through the standard curve and expressed as mg of Gallic acid per gram of extract. The procedure is repeated for each extract separately and readings are taken in triplicate.Total polyphenols were found using the Singleton (1965) method, in which 1 mg/mL solution in water or methanol was prepared for Gallic acid and extracts. These were standard stock solutions of Gallic acid and standard solutions of extract, respectively. The procedure was as follows. Take 10 µL from Gallic acid solution, add 990 µL distilled water. Take 20, 40, 80, 120 µL from gallic acid stock solution separately and make up each dilution to 1000 µL with distilled water. Take 200 µL from each dilution and add to it 200 µL FC reagent and 1 mL of 15% Na3)3, 100 µL of 1 M CH3COOH and 4.6 mL of distilled water were added. The same reagents were added to 200 µL of extract. The samples were allowed to stand for 45 min at 25 °C. The absorbance was measured by a spectrophotometer at 415 nm. The standard curve was drawn for quercetin, and the results were calculated. The procedure was repeated for each extract separately, and readings were taken in triplicate.Quercetin was used as a standard antioxidant. A stock solution of 1 mg/mL quercetin was diluted to 10 µg/mL, 20 µg/mL, 40 µg/mL, 80 µg/mL and 120 µg/mL using analytical grade methanol as a solvent. Various extracts were prepared with methanol of 1 mg/mL strength. To 200 µg/mL of each strength of quercetin, 100 µL of 10% Al was dissolved in 7 mL of 80% hot ethanol was vortexed for 2 min and centrifuged for 10 min at 2700 rpm. Extracts were washed repeatedly until the supernatant did not give colour with the anthrone reagent. Residue was collected and dried from the water bath. Then, 25% HCl and distilled water were mixed in equal quantities (1:1). The collected residue was extracted with 10 mL of 1:1 HCl and distilled water for twenty minutes at 0 °C. The tube was subjected to 2700 rpm for 10 min in centrifuge, and the supernatant was collected in a 100 mL volumetric flask, with the volume made up with distilled water. A hundred microlitres of diluted supernatant was taken in the test tube, and 0.9 mL of distilled water and 4 mL anthrone were added and heated in a boiling water bath for 10 min; the reaction mixture was rapidly cooled, and absorbance was measured at 630 nm against the blank. The 20, 40, 60, 80, 100, and 200 µg/mL glucose solutions were used to draw the standard curve. Glucose concentration in extract was found from the standard curve and multiplied by 0.9 to obtain the total polysaccharides. The procedure was repeated for each extract separately, and readings were taken in triplicate.Saccharomyces cerevisiae yeast strain was washed three times and again by centrifugation  in normal saline until the supernatant became clear. Supernatants were discarded every time, and the remaining washed yeast was used to make a 10% v/v suspension in ice-cold water. A 100 µg/mL concentration of all extracts was prepared and added to a 10 mM glucose solution. Reaction mixtures were incubated at 37 °C for ten minutes, and then 100 µL of yeast suspension was added in all test tubes. Tubes were vortexed to allow homogeneous mixing and incubated further for one hour at 60 °C. After incubation, the reaction mixture was centrifuged . Glucose was estimated by the anthrone method in the supernatant.The method of Bhutkar and Bhise (2013) was adopted with some modifications. Commercially available max was recorded for each against the respective methanol blank. A Fourier transform infrared spectrometer, Perkin- Elemer, USA, was used. For sample preparation, one milligram of dry floral powder was taken in a pestle and mortar to reduce its size further. Then, 100 mg of KBr was taken in a tarred crucible until constant weight was achieved and then mixed with dry floral powder in a mortar to achieve thorough mixing. A disc was prepared by compression in a hydraulic press machine, i.e., KBr disc. The spectrum was scanned in range of 4000–400 cm−1.An ultraviolet spectrophotometer, UV-2550, with an operating system UV probe 2.21, Agilent Technologies, Waldbronn, Germany, was used. All extracts obtained after hot and cold extraction were subjected to ultraviolet visible spectroscopy prior to experimentation. Stock solutions of 1 mg/mL strength were prepared for all extracts in distilled water or water miscible solvents (For example Methanol and DMSO). The stock solutions were diluted to prepare working solutions of 100 µg/mL strength. Scans were taken in a UV-Vis range of 200–800 nm, and λA schematic presentation of the present study is given in Bombax ceiba L. flowers, multiple extraction solvents, including petroleum ether, chloroform, methanol, ethanol and water, were applied for the extraction of secondary metabolites from the subject flowers. After extraction in multiple solvents, the percentage yield of secondary metabolites in each extract was determined and is given in A schematic presentation of the current research work is helpful to understand the direction and objective of this study. It is clear from Organoleptic characteristics were observed and recorded multiple times with naked-eye observations. Methanol and water extracts were sticky and semi-solid, while water extract turned into hard solid material as more and more water evaporated with time. Petroleum ether extract had a characteristic strong smell that was greater than the chloroform extract. Tools such as the smell and colour of extracts can be used to indicate any instability due to light, temperature, humidity and other factors. The details of organoleptic characteristics are given in −1, carbonyl group stretching peaks at 1742 and 1711 cm−1 and C-H stretching peaks at 2852 cm−1, 2921 cm−1 and 3007 cm−1. No peak is visible in the range of 3200–3500 cm−1, indicating the absence of -OH and N-H groups. The absence of -OH and N-H groups shows that hydroxyl, phenolic and amide functional groups are not present in the PBC extract. The FTIR of PBC is given in −1 indicate the presence of the –CH group. The stretching peaks at 1726 cm−1 identify the carbonyl group present in the extract. The peaks in the range of <1000 cm−1 can be ascribed to the bending vibrations of C-H and aromatic rings. A wide band in the range of 3200–3500 cm−1 indicates the presence of a hydroxyl or phenolic functional group in the structures of the CBC-based extract  was adopted with some modifications. The detail of this method is described in the experimental section. A comparison of secondary metabolites and anti-diabetic activity of the extracts is given in Bombax ceiba L. flowers was achieved using a simple and easy approach. The hot solvent extraction gave better performance as compared to the cold one. All the extractions were analysed by the FTIR and UV-vis spectra to obtain comparative quantitative estimation of secondary metabolites  in each extract. Quantitative analysis of secondary metabolites showed 120.33 ± 2.33 mg/gm polyphenols, 60.77 ± 1.02 mg/g flavonoids, 60.26 ± 1.20 mg/g glycosaponins, 0.167 ± 0.02 mg/g polysaccharides for water extract; 91.00 ± 1.00 mg/g polyphenols, 9.22 ± 1.02 mg/g flavonoids, 43.90 ± 0.30 mg/g glycosaponins, 0.090 ± 0.03 mg/g poly saccharides for ethanol extract; 52.00 ± 2.64 mg/g polyphenols, 35.22 ± 0.38 mg/g flavonoids, 72.26 ± 1.05 mg/g glycosaponins, 0.147 ± 0.01 mg/g polysaccharides for methanol extract; 11.33 ± 0.58 mg/g polyphenols, 23.66 ± 1.76 mg/g flavonoids, 32.8 ± 0.75 mg/g glycosaponins, 0.013 ± 0.02 mg/g polysaccharides for chloroform extract; and 3.33 ± 1.53 mg/g polyphenols, 1.89 ± 1.39 mg/g flavonoids, 21.67 ± 1.24 mg/g glycosaponins, 0.005 ± 0.01 mg/g polysaccharides for petroleum ether extract. Glucose uptake by yeast cells was increased 70.38 ± 2.17% by the water extract, while glucose uptake by yeast cells was less for other extracts. The quantitative determination of secondary metabolites showed that extraction with hot water gives the maximum extraction of secondary metabolites such as polyphenols, flavonoids, glycosaponins and polysaccharides. In vitro testing results show that water extracts of Bombax ceiba L. flowers increase glucose uptake by yeast cells by 70.38 ± 2.17%. Therefore, it can be used for the synthesis of anti-diabetic drugs in liquid or solid form. It would be a herbal medication for diabetic patients, without any side effects, and thus would also be economical.In conclusion, successful solvent extraction of


Mentha piperita L.) available in Poland. For this purpose, we applied ICP-MS-based methodology for content analysis  and also the specific health risk assessment for a comprehensive assessment. Our strategy was based on weekly intake (µg/L of infusion/week) considering weekly tea consumption. Then, based on the weekly consumption of tea per adult, individuals were compared to the Temporary Tolerated Weekly Intake (PTWI) designated by the Joint FAO Expert Committee on Food Additives (JECFA), and the weekly consumption per body weight (µg/L of infusion/week/bw) was estimated. Daily exposure to Co in the tested products is in a range of 0.093–0.238 µg/day. In comparison, the established PDE  for Co by the ICH guideline (the ICH guideline Q3D (R1), 2019) is 50 µg/day. For lithium, PDE is approximately 560 µg/day and, in our study, the estimated daily exposure for Li in investigated products is in a range of 0.226–2.41 µg/day. Our studies found also low levels of Cs (in a range of 0.0598–0.195 µg/L), Mo (in a range of 0.663–3.261 µg/L), and Sr (0.223–65.842 µg/L) in infusions. For Molybdenum, the established PDE is approximately 3400 µg/day. There are no documents about Sr and Co in the Joint FAO/WHO Expert Committee on Food Additives (JECFA) database and the ICH guideline Q3D (R1). In the case of V, the established PDE is 120 µg/day, and the level of V in analyzed infusions is in a range of 0.284–0.702 µg/L. Silver and gold were present only in a few samples, and the estimated daily exposure for Ag is approximately 0.04575 µg/day for product A and approximately 0.1885 µg/day for product O, except for Au, which is in a range of 0.000356–0.114 µg/L. The estimated daily exposure for Ag is 167 µg/day and, for Au, it is in a range of 8.9 × 105–0.0285 µg/day. It can be concluded that levels of all investigated elements  in the daily dose should not threaten the consumer’s health after consuming mint tea infusions.The purpose of our studies is the evaluation of the health risks of the novel elemental impurities  in mint tea infusions ( For centuries, plant extracts were used by people for different health purposes in different forms, e.g., tinctures, mixtures, and also teas . Teas, i12; however, no particular biological function of cobalt in the human body has been identified [90Sr)are extremely dangerous for bones [133Cs, and is also slightly inclusive of the other eleven main radioactive isotopes that can be noxious to humans [Mentha piperita L.) available in Polish markets;Health risk assessment of new and traditional EI  for the first time in mint tea infusions (EI profile in mint tea infusions (µg/L of infusion) as raw results;Estimation of the weekly intake (µg/L of infusion/week) established on weekly tea consumption (based on );Estimation of the weekly intake per body weight (µg/L of infusion/week/bw) based on weekly tea consumption per individual (~70 kg bw) in comparison to the Provisional Tolerable Weekly Intake (PTWI) appointed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA); andIndividual health risk assessment for elements without PTWI values.A complex problem for the environment and human health are novel elemental impurities (NEIs). NEIs are not a typical EI  resulting from current environmental exposure. One of the hottest topics related to NEIs are metallic nanoparticles ; however, more is needed to know about their environmental fate, levels, and effects . Excessientified . Co compentified . Excessientified . Toxicitentified . On the entified . In the entified . High coentified . The extor bones . Anotheror bones . Howeveror bones . Cesium o humans . Cesium o humans . The jusn = 17) were purchased in stores in Poland in five cities  from June 2021 to September 2021. The analysed samples were in various forms, such as raw materials (leaf-like/needle-like) and tea bag packages . No additional steps in sample preparation were required. The samples were not subjected to further preparatory steps. They have been applied in our studies based on consumer conditions (tea infusion). The characteristics of the analyzed mint tea samples analyzed are shown in Samples of mint tea  containing Ag, Au, Co, Cs, Li, Mo, Se, Sr, and V were utilized as an internal standard. −1, respectively. All experimental conditions are summarized in The determination of investigated elements  was performed by the methodology based on the ICP-MS technique ). We used the simultaneous multielement detection mode. The plasma excitation power was 1150 W; the gas flow rates for plasma gas, carrier gas, and makeup gas were 15.0, 1.1, and 1.0 L minA tea infusion process was performed based on the information described in The determination of the elements was based on contemporary multi-element detection of Ag, Au, Co, Cs, Li, Mo, Se, Sr, and V by an Elan DRC-e Perkin Elmer (US) spectrometer. The determination of elements was based on simultaneous multielement detection of the investigated elements by the Elan DRC-e Perkin Elmer (US) spectrometer. When the infusion process was complete, the obtained solutions were decanted and cooled to room temperature until analyzed by ICP-MS. The workflow of our study is shown in n = 5) with concentrations of Ag, Au, Cs, and Sr were 0.0, 1.0, 2.0, 5.0, and 10.0 µg/L, and for Co, Li, Mo, and V, they were 0.0, 2.0, 4.0, 10.0, and 20.0 µg/L. Additionally, for Se, the concentrations were 0.0, 10.0, 20.0, 50.0, and 100.0 µg/L. Then, solutions of the predetermined concentrations were applied for the calibration procedure. The analysis was precise and accurate, as indicated by the values of the correlation coefficient (0.995 < R < 0.999). The determination of the investigated elements in the analyzed samples was performed using the calibration curves . The working solutions (The tea infusion (µg/L of infusion) using elemental impurity profile, the half violin plots, and the descriptive statistics ;The assessment of weekly intake (µg/L of infusion/week) established on weekly tea consumption , as seeAn estimation of the health hazard of investigated EI in mint teas from Polish markets involves three steps:3.Based on the weekly tea consumption per individual (at about 70 kg/bw) in comparison to the PTWI. The weekly intake was evaluated depending on body weight (µg/L of infusion/week/bw), as seen in Equation (2):Excel macros  were applied for initial data collection. All data were expressed as means of five independent replicates with the standard error (the mean ± standard error). Descriptive statistics were made using the Origin 2021 Pro program (licensed by Jagiellonian University): minimum, maximum, and mean. Moreover, utilizing the same program, EI profiles were created as half-violin charts for each element. Each stage of the health risk assessment approach was calculated for self-programmed macros in Excel  and the Origin 2021 Pro program (licensed by Jagiellonian University).To ensure better transparency and more precise analysis of each EI separately , the plots as a violin with the box were prepared . Additio−4 µg/L in sample G). It should be underlined that the highest levels of EI were observed for Sr in five samples: E (50.711 ± 0.87 µg/L), F (64.270 ± 0.98 µg/L), I (62.103 ± 1.12 µg/L), O (65.841 ± 0.94 µg/L), and Q (63.263 ± 1.11 µg/L). These observations are not correlated with tea-related factors . On the other hand, Sr levels were relatively low in other samples (in a range of 0.223 µg/L–0.309 µg/L).All investigated elemental impurities were present in all analyzed mint tea infusions, except silver and gold. Ag was present only in two samples . Au was present in five samples  at shallow levels (in a range of 0.000356–0.114 µg/L). Four EI were characterized by levels below 1 µg/L . Additionally, four EI was characterized by levels below 10 µg/L . The relatively high and variable levels of EI were observed for Cr and Sr (Cr in a range of 7.317–23.451 µg/L and Sr in a range of 0.223–65.842 µg/L). The lower level was noted for Au  compared with only a few samples (n = 6), based on the literature [The analysis and comparison of our data  with the literature values  terature .For the health risk assessment of investigated EI, the second step is the assessment of EI weekly exposure in comparison with the weekly intake of mint tea (based on consumption scenarios). It is difficult because there are a lot of consumption scenarios. The worst-case (WC) scenario is usually applied in toxicological risk assessment, i.e., in this case, the highest possible frequency of tea consumption per week. Based on the assumptions of , that thThe last step in the appropriate health risk assessment of investigated elements in mint tea infusions is the assessment of weekly intake based on weight and weekly tea consumption. For this purpose, the values of the weekly intake for each EI in investigated samples  were calAs = 0.015 mg/kg bw/week [Cd = 7.0 µg/kg bw/week [Pb = 0.025 mg/kg bw/week [Se = 66.0 µg/kg bw/week [An appropriate baseline  for the final step in the applied health risk assessment is the comparison of the obtained estimation of weekly intake depending on body weight with the values of the provisional tolerable weekly intake. This widely applied parameter in assessing limitations for redundant elements in the diet is issued by JECFA ,27. The  bw/week , PTWICd  bw/week , PTWIPb  bw/week , and sel bw/week ). For thThe results in Due to the lack of established PTWI values for NEIs , the individual health risk assessment, including all available information, has been described below.Given the rare application of Ag and in the absence of knowledge of the exact nature of this element in food, specification and any toxicological reference values were not prepared by JECFA since 1978  until noThe established permitted daily exposure  for Ag by the ICH guideline Q3D (R1) on elemental impurities  is 167 µ−5–0.0285 µg/day. In comparison to the established PDE  for Au by the ICH guideline Q3D (R1) [As was the case with Ag, any toxicological reference values were not prepared for Au by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) since 1978  until noQ3D (R1)  of 134 µ12; however, no particular biological function of this element in the human body has been identified [As was mentioned in the introduction, Co is an essential element for humans because it is an integral component of Vitamin Bentified . There ientified  of 50 µg133Cs. It is also slightly inclusive of the other eleven major radioactive isotopes, which can be noxious for humans [137Cs with various components of tea leaves fermented from Chernobyl-contaminated green tea; however, there are no values for Cs level in infusion). Hence, it is impossible to compare the obtained results, but this element’s low levels indicate no severe problems for human health.Cesium is an interesting but underestimated element because natural Cs mainly exist as r humans . Our stuLithium is a common element in the environment but is more often applied  as a pharmaceutical in treating mania, bipolar disorder, and recurrent unipolar depression rather than in industry. In general, there is a deficiency of studies related to this element about existence in herbs or herbs infusions. The established PDE  for Li by the ICH guideline Q3D (R1)  is basedMo is an interesting element because the range between toxicity and deficiency in animals is narrow; therefore, careful control of Mo in animal diets is essential ,34. From90Sr) are highly hazardous for bones [Sr is an extraordinary element because it has never been shown to be essential, but radioactive isotopes  compared to only a few samples (n = 6) from the literature [The level of investigated EI in all of the investigated mint tea infusions occurs but at a relatively low level. The obtained elemental profiles in mint tea infusions indicated the presence of Co (0.372–0.952 µg/L), Cs (0.078–0.186 µg/L), Li (0.904–9.641 µg/L), Mo (0.663–2.970 µg/L), Se (0.622–3.417 µg/L), Sr (0.223–65.842 µg/L), and V (0.039–0.702 µg/L) in all investigated samples. There was a lack of Ag impurities (15/17) and Au impurities (12/17) in most investigated samples. Comparing the obtained results with the literature values  indicateterature .The health risk assessment for Se in the analyzed products indicated no health hazard to consumers for weekly exposure. Due to the lack of established PTWI values for other investigated EI , the individual health risk assessment, including all available information, has been made. The obtained results indicated that EI levels in daily doses should not represent any health risk to the recipient after consuming mint tea infusions from products accessible in Polish markets.Our designed health risk assessment strategy for investigated EI provides pioneer data  may be worthwhile for additional investigators and manufacturers. Additionally, a well-design health risk assessment methodology will be valuable and essential for public health and environmental studies. Because these environmental investigations are scarce, it would be valuable to carry out a broader study considering other mint tea infusions in different countries.


Lumbar puncture (LP) is a common and relatively safe neurological procedure. It can be complicated by post-dural puncture headache (PDPH) after both diagnostic and therapeutic procedures. The aim of this study is to identify the incidence, risk factors and clinical characterization of PDPH in the inpatient setting of the main tertiary neurology hospital in Kuwait.We conducted a prospective observational cohort study that included patients who were admitted to neurology department at Ibn Sina hospital, Kuwait, from January 1, 2019 to December 31, 2020, on whom, LP was performed for diagnostic and/or therapeutic reasons. Multivariate logistic regression analysis was performed to evaluate the association between PDPH and different clinical parameters.p = 0.001), female gender (p = 0 .001), low BMI (p < 0 .001), pre-LP headache (p = 0.001), history of previous PDPH (p = 0.001), and number of LP attempts (p < 0.001). PDPH was statistically significantly higher in patients with optic neuritis (p = 0.009), and cerebral venous thrombosis (p = 0.007), and lower in patients with peripheral neuropathy (p = 0.011) and spinal muscular atrophy (p = 0.042).A total of 285 patients were included; 225 females (78.9%), mean age of 32.9 ± 11.7 years. PDPH was reported by 84 patients (29.5%), with mean headache onset of 1.7 ± 0.8 days, and mean duration of 2.4 ± 2.1 days. The commonest headache type was dull aching in 49 patients (58.3%). Headache severity was mild to moderate in 64 patients (76.2%), with mean NRS of 4.1 ± 0.9. Most PDPH (99.3%) resolved with conservative medical management, with only 2 patients (0.7%) requiring epidural blood patch. In multivariate logistic regression model, there was a statistically significant correlation between development of PDPH and young age (Findings from clinical practice in the main tertiary neurology hospital in Kuwait were in line with literature findings. Younger age, female gender, lower BMI, pre-procedural headache, previous history of PDPH, and number of LP attempts were found to be independent risk factors for developing PDPH. To our knowledge, this study represents the first comprehensive description of PDPH in a population from the Arabian Gulf Region. The claThis particular type of headache has been an area of interest to physicians from different specialties, and several clinical studies have attempted to identify procedure- and non-procedure-related risk factors in the literature. Several modifiable and non-modifiable independent risk factors for PDPH have been documented in both anesthesiology and neurology studies. Class I and II evidence regarding the development of PDPH in literature included; female gender, age between 20 and 50 years, lower body mass index (BMI), previous history of PDPH, larger needle diameter, use of cutting needles, perpendicular insertion of the needle bevel to the long axis of the spine, and pregnancy –5. As a The exact mechanism of headache in PDPH is still uncertain, and several theories have been proposed. The most common theory is downward stretch of pain sensitive structures when patients assume an upright position, secondary to cerebrospinal fluid (CSF) volume loss . Other tIn this study, we aimed to evaluate the incidence, risk factors and clinical characteristics of PDPH in the inpatient setting of the main tertiary neurology hospital in Kuwait. To our knowledge, this work represents the first comprehensive description of PDPH from a population of the Arabian Gulf Region.We conducted a prospective observational cohort study that included all patients who were admitted to the neurology department at Ibn Sina hospital, Kuwait, from January 1, 2019 to December 31, 2020, on whom LP was performed for diagnostic and/or therapeutic reasons. Patients who agreed to participate were included consecutively upon admission to hospital, following their voluntary and informed consent. The study was approved by the Scientific Research Committee of Department of Neurology, Ibn Sina hospital, Kuwait.Demographic data included age, gender, body weight, body height, and BMI. All patients underwent complete history taking, detailed neurological examination, routine laboratory investigations  and brain imaging (magnetic resonance imaging or computed tomography) prior to LP.22-gauge, atraumatic, non-cutting spinal needle. LPs were performed in either left lateral recumbent or sitting positions. All patients received local injection of lidocaine 1% or 2%, and the procedure was performed in a midline approach, at either L4–L5, or L3–L4 intervertebral space levels. As a standard of care, LP technique included inserting the needle bevel parallel to the dural fibers, passively withdrawing CSF, and replacing the stylet before withdrawing the spinal needle. CSF opening and closing pressures were measured using manometer in lateral recumbent position with the legs relaxed and partially extended, regardless of initial position of the procedure. Routine CSF analysis including levels of protein, glucose, red blood cells (RBCs), and white blood cells (WBCs) was done in all cases.As per department protocol, all the procedures were standardized using a In the postprocedural period, all patients were instructed to rest in bed for 4 h and they could walk afterwards if their clinical condition permitted. PDPH was assessed on the day of the procedure, upon discharge, and on day 7 and day 14 after LP, either in-person or by telephone, by the study investigators . PDPH was diagnosed according to the ICHD-3 diagnostic criteria .Headache-related information included: presence of pre-procedural headache, history of previous PDPH, headache onset, type, duration, and severity. Headache severity was evaluated using Numerical Rating Scale (NRS). Patients self-reported the severity of headache on a scale from (0 to 10); scores from 1 to 3 were interpreted as mild headache, scores from 4 to 7 were considered moderate, and scores from 8 to 10 were considered as severe headache. All patients who developed PDPH were offered medical treatment, and were advised to visit their treating physician in case of severe and/or persistent headaches.p < 0.1 in univariate analysis. Significance of the obtained results was set at p ≤ 0.05 level.Data were analyzed using IBM SPSS software package version 25.0 . Qualitative data were described using number and percent. The Kolmogorov–Smirnov test was used to verify the normality of distribution. Quantitative data were described using range (minimum and maximum), mean, standard deviation, and median. Chi-square test (Fisher or Monte Carlo), and student t-test were used to compare two groups for normally distributed quantitative variables. Mann–Whitney test was used to compare between two groups for not normally distributed quantitative variables. All of the variables that were statistically significant in univariate logistic regression model were entered into multivariate logistic regression model to explore the factors that are independently associated with development of PDPH. The multivariate model contained variables that were associated with 2). Of them, 139 patients (48.8%) had idiopathic intracranial hypertension (IIH), 90 (31.6%) had demyelinating diseases, 26 (9.1%) had peripheral neuropathy (PN), 8 (2.8%) had optic neuritis (ON), 5 (1.8%) had dementia, 4 (1.4%) had cerebral venous thrombosis (CVT), 3 (1.1%) had autoimmune encephalitis, and only 1 patient (0.4%) had CNS infection. Nine patients (3.2%) had spinal muscular atrophy (SMA) and underwent intrathecal treatment with nusinersen (Spinraza ®) 12 mg/5 ml after withdrawing 5 ml of CSF.A total of 285 consecutive procedures were performed in our study during the assigned period , mean age of 32.9 ± 11.7 years, and mean BMI of 33.5 ± 37.6 kg/mPDPH was reported by 84 patients (29.5%). Mean headache onset was 1.7 ± 0.8 days, with 85.7% having headache onset in the first 2 days. Mean headache duration was 2.4 ± 2.1 days. The commonest headache type was dull aching in 49 patients (58.3%%), followed by throbbing in 25 (29.8%), and other types in 10 patients (11.9%). Headache severity was mild in 34 (40.5%), moderate in 30 (35.7%), and severe in 20 patients (23.8%). Mean pain intensity using NRS was 4.1 ± 0.9. Headache complaint prior to LP was reported by 212 patients (74.4%), while history of previous PDPH was reported by 24 patients (8.4%).All patients with PDPH were offered medical treatment , with only 2 patients (0.7%) with persistent headaches needed an epidural blood patch.As regards to LP, it was performed in left lateral position in 241 patients (84.6%), and in sitting position in 44 patients (15.4%). L4–L5 intervertebral space level was used in 216 patients (75.8%), while L3–L4 intervertebral space level was used in 69 patients (24.2%). Median number of LP attempts was 2, ranging from (1 – 6). LP was performed by a registrar in 189 patients (66.3%), senior registrar in 79 (27.7%), assistant registrar in 13 (4.6%), and consultant in 4 patients (1.4%). Intravenous fluids were given after LP for only 34 patients (11.9%).2O, mean closing pressure was 174.9 ± 40.3 mm H2O, and mean amount of withdrawn CSF was 8.4 ± 4.3 ml. Mean CSF protein was 296.6 ± 113.2 mg/mL, mean CSF glucose was 4.1 ± 0.8 mmol/L, mean CSF RBCs was 31.5 ± 142.6, and mean CSF WBCs was 4.5 ± 5.7 cells/µL. The demographic and clinical characteristics of our cohort are shown in Table As regards to CSF, mean opening pressure was 272 ± 145.6 mm Hp < 0.001), female gender (p = 0 0.006), low BMI , pre-LP headache (p < 0.001), history of previous PDPH (p < 0.001), low levels of RBCs in CSF , and number of LP attempts . As regards to clinical diagnosis; a statically significant association with developing PDPH was observed in patients with ON (p = 0.009), and CVT (p = 0.007), while patients with PN (p = 0.011) and SMA (p = 0.042) had less PDPH, which was statistically significant. Other parameters were not found to be statistically significant in our cohort. Correlation findings are summarized in Table There was a statistically significant association between development of PDPH and younger age , female gender , low BMI , pre-LP headache , history of previous PDPH , and number of LP attempts  remained statistically significantly associated with PDPH. However, RBCs in CSF , and the different clinical diagnoses in our cohort were not found to be statistically significant in the multivariate model. The detailed results of univariate and multivariate analysis are shown in Table In multivariate logistic regression, younger age . However, as regards to CSF pressure, there was no statistically significant difference between patients with normal and high CSF pressure at a cutoff value of > 25 cmH2O .Furthermore, analysis of risk factors showed that the incidence of PDPH in patients with a BMI < 30.0 kg/m (39.3%) was higher than in obese patients with a BMI ≥ 30.0 kg/m (60.7%) , with less female participants (61.7%) than our study. Another reason could be the nature of our prospective study, where we evaluate patients after LP and specifically ask if they have headache. Many PDPH studies are retrospective, thus only patients who report headaches are included.The majority of headaches (85.7%) started within the first 2 days of the procedure, and lasted for average of 2.4 days. This is similar to findings in the literature, where around 66% of PDPH start within the first 48 h of LP, and nearly 90% within the first 72 h . HoweverThe commonest headache type was dull aching in 58.3%, followed by throbbing in 29.8% of patients. In a review of literature , the comMoreover, PDPH was self-limited in the majority of patients, with only 2 patients (0.7%) requiring a blood patch. These values compare favorably to published studies of PDPH. In a study by de Almeida and colleagues , PDPH waThe current study supports some earlier findings for PDPH risk factors in the literature, including young age, female gender, low BMI, headache before LP, previous history of PDPH, and number of attempted punctures , 20.Young age and female gender are the most important well-documented risk factors for development of PDPH , 7, 21. Female gender presented a risk for PDPH 8-times greater than that of males, which was found to be higher than reported numbers in the literature. Amorim and colleagues  reportedThe possible underlying etiologies for age and gender differences in PDPH include; differences in pain perception, differences in elasticity of the dura, psychosocial factors, and hormonal effects on the reactivity of cerebral blood vessels , 25.2, the percentage of PDPH was 45%, in comparison to patients with BMI greater than 30 kg/m2where the incidence was 25%, in a study of 99 patients who had accidental dural puncture. Another study by Peralta and colleagues [2, and 39% if the BMI was greater than 31.5 kg/m2 (P = 0.0004).Lower BMI in our study was found to be an independent risk factor for PDPH, similar to other earlier reports in literature. Faure and colleagues , reportelleagues , which eFurthermore, morbid obesity was found to decrease the incidence of PDPH in several reports . The proIn our cohort, the majority of patients had headache prior to LP, and more than 70% of them developed PDPH, with a statistical significance. This was similar to the findings by Kuntz and colleagues , who fouA previous history of PDPH was estimated to have 2–3 times higher risk for developing a new attack of PDPH in several reports, compared to patients who never developed PDPH before , 30. AmoHigher number of LP attempts increased the risk of PDPH in our study. It has been shown that PDPH is more common if two verified punctures into the subarachnoid space are made . A prospIn this study, PDPH was statistically significantly higher in patients with ON and CVT, and lower in patients with PN and SMA, although this association was not confirmed in the multivariate regression analysis. This could be attributed to other clinical characteristics in those patients, such as the presence of headache prior to LP, or the predominance of female gender in patients with CVT and ON, in our cohort.Several studies in the literature reported variable effects of different clinical diagnoses on the development of PDPH. Similar to our findings, dementia , 34 and In our study, CSF opening pressure, closing pressure, CSF volumes or constituents, were not associated with the development of PDPH, similar to several previous reports , 38, 39.p > 0.05). One can argue that this lack of correlation is attributed to the lesser degrees of variability in the technical performance of the LPs, as all neurologists in the department have more than 3 years of experience, under close supervision and rigorous training.Moreover, the lack of association of LP location, position, duration of rest after LP, and post-LP fluids and PDPH in our cohort, was in line with findings in the literature , 19, 41.In literature, more than 85% of PDPH resolves with conservative measures, or without any specific treatment . DespiteThis study has some limitations. First, the results were obtained from a single-center, and the sample size was not calculated according to a predefined outcome. However, we included all consecutive eligible patients admitted to the largest neurology tertiary hospital in Kuwait, during the study period, which also minimized the possibility of selection bias. Few studies in the literature present a uniform data set regarding LPs and PDPH, as most studies use different techniques and protocols. Moreover, our findings came in line with published data on PDPH, which further validates our sample, and confirms that our cohort is representative to the population of patients undergoing LP in neurology departments.Secondly, given our department standardized protocol for LPs, we could not evaluate the impact of certain procedural factors such as; needle type, needle size, bevel direction, and non-replacement of the stylet, on the development of PDPH. Moreover, LPs in the study were performed by physicians of varying experience, which could have affected the results. However, all of them have received vigorous training for the procedure, and we aimed to assess this variation on the primary outcome, as this represents the real-life clinical situation in most hospitals.Our findings from clinical practice in the main tertiary neurology hospital in Kuwait were in line with literature data regarding PDPH. To our knowledge, this study represents the first comprehensive description of PDPH from the Arabian Gulf Region.In the majority of patients, headache started within the first 2 days of the procedure, lasted for average of 2.4 days, was dull aching or throbbing in nature, and of mild to moderate severity. Independent risk factors included younger age, female gender, lower BMI, pre-procedural headache, previous history of PDPH, and number of LP attempts. Identification of risk factors is important in predicting PDPH, and preventing this painful adverse event should be the main goal of neurologists dealing with this population. These results should be validated in further multicenter studies with larger cohorts to confirm our findings.


Previous work on temporally sparse multifocal methods suggests that the results are correlated with disability and progression in people with multiple sclerosis (PwMS). Here, we assess the diagnostic power of three cortically mediated sparse multifocal pupillographic objective perimetry (mfPOP) methods that quantified response-delay and light-sensitivity at up to 44 regions of both visual fields concurrently.g). Linear models examined significance. Concurrent testing of both eyes permitted assessment of between-eye asymmetries.One high-spatial-resolution mfPOP method, P129, and two rapid medium-resolution methods, W12 and W20, were tested on 44 PwMS and controls. W12 and W20 took 82 s to test both visual fields concurrently, providing response delay and sensitivity at each field location, while P129 took 7 min. Diagnostic power was assessed using areas under the receiver operating characteristic (AUROC) curves and effect-size  in relapsing–remitting MS, and 96.5% ± 2.30% in progressive MS. Performance increased with increasing disability scores, with even moderate EDSS 2 to 4.5 PwMS producing AUROCs of 82.1 to 89.8%, Hedge’s Overall, the 82-s tests (W12 and W20) performed better than P129. The results suggest that mfPOP assesses a correlate of disease severity rather than a history of inflammation, and that it may be useful in the clinical management of PwMS. They have been shown to have diagnostic power related to the overall functional status of people with MS (PwMS), particularly if so-called temporally sparse stimuli are employed [Measures of regional or global brain atrophy are not well-correlated with progression of disability in multiple sclerosis (MS) . Loss ofemployed . Multifoemployed , 6. Respemployed , as suppemployed . Thus, aemployed –11, it hemployed , migrainemployed , epilepsemployed , concussemployed  and multemployed . With reemployed , which wemployed .Our original cross-sectional study of 85 PwMS indicated that mfPOP results were highly correlated with clinical disease severity . As has One consideration is that a more rapid, spatially coarser, assessment of the quadrants of the inner and outer visual fields may be sufficient for use in neurological testing. With this in mind, new mfPOP algorithms have recently been developed with total test durations of just 82 s . This stThis study was approved by the ACT Health Human Research Ethics Committee (ETH 7.07.667) and Australian National University Human Ethics Committee (2010/194) and conformed to the Declaration of Helsinki. Informed written consent was obtained from all participants.Forty-four people with MS were studied, 31 of whom were female. All PwMS had been diagnosed by a neurologist based upon clinical and laboratory findings. Thirty-one had relapsing–remitting MS (RRMS) and the other 13 had progressive MS. Their ages were 60.7 ± 10.1 years (mean ± SD), range 36 to 78 years. They were part of a cohort of 85 persons who had been studied 10 years earlier , 19. ExpThe disease status of PwMS was classified in three different ways: clinical type of MS, EDSS score, and any history of ON. Possible clinical types were relapsing–remitting MS (RRMS), secondary progressive MS (SPMS) or primary progressive MS (PPMS). For some analyses, EDSS scores were divided into 3 groups, each with approximately equal numbers: ≤ 2.5; 2.5 to 4.5; and In addition to mfPOP testing, all participants underwent ocular examination that included best corrected visual acuity (log-MAR), slit-lamp examination, posterior pole and retinal nerve fibre layer optical coherence tomography , and Matrix or HFA 24–2 automated perimetry . A prototype of the FDA-cleared ObjectiveFIELD Analyser (OFA)  was used for the mfPOP testing.The standard mfPOP stimulus is a 44-region/eye, 7-min test referred to as P129. It has previously been reported to have high signal-to-noise ratios , reprodu2 in P129, from 49 to 156 cd/m2 in W12, and from 53 to 167 cd/m2 in W20. The stimulus backgrounds were yellow at 10 cd/m2 in all three.Figure Results of P129 for PwMS were compared to pre-existing normative data obtained from 115 individuals in the same age range (57.8 ± 12.8 year). We also tested 40 age-matched normal controls (60.2 ± 12.5 years) for comparisons using W20 and W12.g . The accepted cutoffs for effect-sizes for d and g are: small 0.2, medium 0.5, large 0.8, very large 1.2, and huge 2.0. Similar AUROCs and g-values were also derived for different sub-groups of interest.Pupillary responses to stimulation at each test region from each participant generated a mean time-to-peak constriction (‘delay’), along with a standardised peak constriction amplitude which was converted to decibels (‘sensitivity’). The response estimation and related multifocal analysis methods have been given previously , 25. Diatotal deviations (TDs), i.e. the differences from the normative values for each individual at each visual-field location.Per-region deviations, or Pattern deviations (PDs). These provide within-individual controlled data by subtracting a response level corresponding to the overall ‘normal’ of that field. To do this, the 86th percentile of the TD data for each visual field was subtracted from TD measurements for all visual-field locations in any given test.Between-eye asymmetry (Asymm). This involves calculating the difference between pairs of test results arising from anatomically equivalent regions of the two eyes, e.g. superior temporal left eye cf. superior temporal right eye. This helps control for inter-individual variability. Although the subject of many scientific studies, Asymm has not yet been incorporated into commercial perimeters, probably because existing commercial perimeters test one eye at a time resulting in potential confounding by test order. This confounder does not apply to OFA which tests all regions of both visual fields concurrently.Our AUROC analysis methods have been published previously , 23, 26.To investigate how many regions/field might provide good diagnostic power, the measures of interest  from each visual field stimulus location were sorted from worst  to best . Initially, the single worst stimulus location from every participant (both PwMS and controls) was used to generate an AUROC. Thereafter, the means of the worst 2 locations in each field were included in the AUROC analysis. This process was repeated for the means of the worst 3, then 4, and up to 12 regions. Change in AUROC as a function of N-worst locations provides information about the distribution of visual field losses , 23, 26.g looking at the 4- and 12-worst region delays of each individual’s visual fields, looking at PDs and Asymms, for each of the three mfPOP tests, broken down by diagnostic classification, i.e. RRMS  while those of the progressive group were mostly in the in the ‘huge’ range.Of the 44 PwMS, 31 were classified as RRMS, 2 as PPMS and 11 as SPMS. Owing to the small numbers, the two progressive classifications were collapsed for further analysis. The overall average EDSS score was 3.81 ± 2.00 (mean ± SD) and the scores for the RRMS and progressive MS groups were 2.8 ± 1.32 (mean ± SD) and 6.2 ± 1.03, respectively. Twenty-three out of 31 (74%) RRMS participants and 7/13 (54%) participants with progressive disease had a history of ON. Table g of 1.11 (‘large’). A similar analysis for W20 yielded an AUROC of 87.3% ± 3.79% and a Hedges’ g of 1.45 (‘very large’). For P129 the values were 81.7% ± 4.10% and 1.52 (‘very large’). Thus, the pooled values are intermediate between those in Table In all analyses using delays, Asymm performed better than PD. W20 performed better than W12 or P129 in the RRMS group, but it performed less well in those with progressive MS. Combining RRMS and progressive groups and comparing them to controls for the 4 worst regions, Asymm data for W12 yielded an AUROC of 87.0% ± 3.85% and a Hedges’ g values were ≥ 1.12 (‘very large’). For W12, the moderately severe EDSS 2.5 to 4.5 group produced an %AUROC of 89.8 ± 4.74%.Table p = 4.00E − 13 for the EDSS 2.5 to 4.5 group. Given the means of the 4-worst regions and the cyclopean data, there was 1 data point per subject.Table g values for delays were generally larger in participants who had not had a history of ON. This was also true for the AUROC results for N = 12, but not N = 4.Table Looking at sensitivity as opposed to the previous analyses, which all utilised delay, analysis of TDs yielded AUROCs of > 85% for PwMS with progressive disease. The result was consistent with the fact that these individuals were likely to have higher EDSS scores and so have experienced more neural degeneration. For individuals with less marked disability, however, sensitivity provided little useful diagnostic power compared to delay.g > 2.0, ‘huge’). AUROCs were greater in participants with higher EDSS scores, but were 89.8 ± 4.74 for W12 for those PwMS in the moderate disease severity group who had scores of 2.5 to 4.5. This resulted in very high significance levels  in a population of people with multiple sclerosis (PwMS) using three different per-region measures . The results showed that analysis of delay (time-to-peak) of pupil response was superior to analysis of sensitivity (amplitude). Analysis of interocular asymmetry (Asymm) performed better than pattern deviations (PD), and both performed much better than total deviations (TD). The 82-s mfPOP versions yielded AUROCs of 83.0 to 86.6% in RRMS participants, and 93.0% to 96.5% in those with progressive disease  performed as well, or better than the longer test (P129) on almost all measures. W12 and W20 each had some advantages. W12 generated somewhat higher AUROC scores in the progressive group, but W20 performed better in the relapsing–remitting group. There was little difference in the performance of W12 and W20 when comparing different EDSS groups or whether or not there was a history of ON. Overall, the findings of this study, combined with the independent finding that mfPOP results are predictive of development of progressive disease 10 years later , suggestg scores, strongly suggest the results are robust. Much current MS research is directed towards improving biomarkers that can assist in diagnosis, monitoring and prognosis of the disease. Multiple clinical, image based, and blood- or CSF-based biomarkers already exist [The number of individuals in this study was small, and it is important to validate the findings in a larger, prospective group. However, the consistently high AUROC values, along with the ‘very large’ to ‘huge’ Hedges’ dy exist , but thedy exist ). mfPOP The newer mfPOP algorithms allow objective, non-invasive assessment of PwMS in 82 s. The diagnostic power is high, meaning that mfPOP has potential as a clinically useful marker of disease in MS. Similar performance for eyes with and without a history of optic neuritis strongly suggests the test is assessing something related to disease severity/progression rather than just a history of inflammation. This is consistent with the finding that an older version of mfPOP has demonstrated ability to predict disease progression over 10 years .


RSC Advances article due to the following instances of matched/similar images that have been identified that weaken this article, which occurred due to honest human errors.I, the undersigned author, hereby wholly retract this Following the previous publication of a correction to correct errors in Fig. 1, 3, 7 and 9, further instances of duplicating images have been identified within the corrected Fig. 3 and 7, as well as additional errors in the original article, that undermine this article.In Fig. 3 of the correction notice:S. enterica MTCC-3219 2 μg ml−1 10−10 is identical to the panel for S. typhirmurium MTCC-3224 4 μg ml−1 10−5 after one year of storage, to the panel 1 h treated with SBT@AgNPs in Fig. 7, and to the panels E. coli MTCC No. 62 6 μg ml−1 and 8 μg ml−1 in Fig. 5 of ref. The panel for freshly prepared S. typhirmurium MTCC-3224 6 μg ml−1 10−10 is identical to the panel for S. typhirmurium MTCC-3224 6 μg ml−1 10−10 after one year of storage and to the panel 0.5 h treated with SBT@AgNPs in Fig. 7.The panel for freshly prepared S. typhirmurium MTCC-3224 4 μg ml−1 10−5 is identical to the panel for freshly prepared S. enterica MTCC-3219 6 μg ml−1 10−10.The panel for freshly prepared S. typhirmurium MTCC-3224 2 μg ml−1 10−10 is identical to the panel for S. typhirmurium MTCC-3224 6 μg ml−1 10−5 after one year of storage.The panel for freshly prepared S. typhirmurium MTCC-3224 4 μg ml−1 10−10 is identical to the panel for S. typhirmurium MTCC-3224 2 μg ml−1 10−10 after one year of storage.The panel for freshly prepared S. enterica MTCC-3219 4 μg ml−1 10−5 after one year of storage is identical to the panel for S. enterica MTCC-3219 6 μg ml−1 10−10 after one year of storage.The panel for In Fig. 3a, a′, c and c′ of the original article, the error bars are erroneous.S. typhirmurium are identical.In Fig. 4 of the original article, both the panels for Thiyagarajan Kalaiyarasan and Vijay K. Bharti responded to all enquiries and submitted data related to the above concerns. However, to avoid any future ambiguity to the readers, the article is retracted.Vijay K. Bharti and O. P. Chaurasia were informed about the retraction of the article but have not responded.Signed: Kalaiyarasan ThiyagarajanDate: 1/6/2022RSC AdvancesRetraction endorsed by Laura Fisher, Executive Editor,