L-arginine biosensor (ARG-Select) to obtain increased L-arginine producers among a large number of mutant strains. This biosensor was constructed by ArgR protein and argC promoter, and could provide the strain with the output of bacterial growth via the reporter gene sacB; strains with high L-arginine production could survive in 10% sucrose screening. To extend the screening limitation of 10% sucrose, the sensitivity of ArgR protein to L-arginine was decreased. Corynebacterium crenatum SYPA5-5 and its systems pathway engineered strain Cc6 were chosen as the original strains. This biosensor was employed, and L-arginine hyperproducing mutants were screened. Finally, the HArg1 and DArg36 mutants of C. crenatum SYPA5-5 and Cc6 could produce 56.7 and 95.5 g L–1 of L-arginine, respectively, which represent increases of 35.0 and 13.5%. These results demonstrate that the transcription factor-based biosensor could be applied in high yield strains selection as an effective high-throughput screening method.The high yield mutants require a high-throughput screening method to obtain them quickly. Here, we developed an  Corynebacterium glutamicum AR1 strain, removal of the regulatory repressors of the arginine operon, optimization of the NADPH level, and disruption of the L-glutamate exporter allowed the strain to produce 92.5 g L–1 of L-arginine in a 5-L bioreactor -based system with the output of reporter genes, including fluorescence intensity or antibiotic resistance  was obtained by UV and EMS mutagenesis, and it could produce L-arginine (30 g L–1) in shake-flask fermentation under optimal culture conditions  and incubated for 30 min. Total RNA was purified using a MasterPureTM RNA purification kit . The first strand cDNAs were synthesized by HiScript®II Q RT SuperMix  using total RNA (0.5 μg). For RT-qPCR, the 16S rRNA was chosen as the endogenous control. The reaction mixture was prepared in a qPCR tube and consisted of 10 μL of 2× ChamQ Universal SYBR qPCR Master Mix, 0.4 μL of forward and reverse primers (10 μM each) respectively, 1.0 μL of cDNA, and 8.2 μL of ddH2O. The RT-qPCR was performed on a Bio-Rad CFX96 Manager PCR system  using the following parameters: 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 10 s and 60°C for 30 s and extension at 72°C for 20 s. The 2Ct–ΔΔ method was applied to analyze the data, which were normalized to the transcription level of 16S rRNA.Strains were cultivated at 30°C and 180 rpm and induced by 0.05 mM IPTG when the ODCorynebacterium sp. strains were first cultivated at 30°C in broth medium (BHI) with 50 μg mL–1 of kanamycin or ampicillin added. The preliminary screening agar medium was L-arginine production fermentation medium 2SO4 40 g L–1, KH2PO4 1.5 g L–1, KCl 1 g L–1, MgSO4.7H2O 0.5 g L–1, FeSO4.7H2O 0.02 g L–1, and MnSO4.H2O 0.02 g L–1) containing 10% sucrose and 0.05 mM IPTG. Three days later, the mutant colonies were inoculated into 24-deep-well plates with 3 mL of fermentation medium without sucrose, and they were cultivated for 48 h until the cell’s concentration reached an OD600 = 3.5–4.0. For shake-flask fermentation, mutant strains were activated on fermentation agar plates for 24 h and inoculated in 10 mL of seed medium (BHI). Then, 1.5 mL of seed culture was transferred into 30 mL of fermentation medium 2SO4 40 g L–1, KH2PO4 1.5 g L–1, KCl 1 g L–1, MgSO4.7H2O 0.5 g L–1, FeSO4.7H2O 0.02 g L–1, MnSO4.H2O 0.02 g L–1, and CaCO3 20 g L–1) in 250-mL shake flasks and cultivated at 220 rpm for 96 h. Subsequently, the yield of L-arginine was measured. Repeated fermentation of mutant strains was required to ensure genetic stability.Both 600 = 0.2–0.3. Mutagenesis was performed using the ARTP mutation breeding system . First, 10 μL of cell culture was placed on a piece of metal slide and put on the minidisc. The main parameters included the input power (100 W), ventilation flow rate (10 L min–1), and the distance between the sample plate and ion generator (2 mm).Before treating with atmospheric and room temperature plasma (ARTP) mutagenesis, cells were cultivated to the mid and late stages of logarithmic growth. The cells were washed twice with sterile saline and diluted to an ODCells were treated by an ion beam for 30 s. Then, the sheet iron with 10 μL of mutagenized cells was placed into 990 μL of sterile saline. Finally, the cells were diluted and spread on the fermentation medium plates containing 10% sucrose. After incubation at 30°C for 3 days, the numbers of control colonies (A) and surviving colonies (B) were counted and the lethality rates were calculated with the following equation: (A − B)/A × 100%.gfp were cultivated for 6 h in 1.0 mL of fermentation medium containing 50 μg mL–1 of kanamycin or ampicillin for cells’ recovery. Afterward, 100 μL of the cell culture was incubated in 10 mL of fresh medium and induced by IPTG until the OD600 reached 0.5. Then, the cells were washed twice with phosphate buffer (pH 7.4) and diluted to OD600 = 0.1.Before cells were analyzed by flow cytometry, 10 μL of mutagenized cells with the reporter gene of C. crenatum SYPA5-5 cells were used to exclude the background for cell sorting. Two gates with different fluorescence intensity were circled out based on pre-analysis of mutagenized cells, and four-way purity was used as the precision mode for cell sorting with a threshold rate of up to 6,000 events s–1. Data were analyzed using FlowJo v10.0.7 analysis software .Fluorescence sorting of cells was performed by a FACSAria II flow cytometer  using an excitation wavelength of 488 ± 20 nm and an emission wavelength of 520 ± 20 nm at a sample pressure of 70 psi. The forward-scatter characteristics (FSC) and side-scatter characteristics (SSC) were detected with a 488-nm laser. Non-fluorescent cells of –1, NaCl 10 g L–1, peptone 10 g L–1, yeast extract 5 g L–1, agar 20 g L–1, corn steep liquor 25 g L–1, pH 7.0) for 24 h. Single colonies were inoculated into seed medium 2SO4 10 g L–1, pH 7.0) and cultivated at 30°C and 180 rpm for 24 h until the OD600 = 16–18. The seed culture was then transferred into 1.5 L of fermentation medium 2SO4 30 g L–1, KH2PO4 2 g L–1, MgSO4.7H2O 0.5 g L–1, urea 1 g L–1, molasses 10 g L–1, pH 7.0) at 1:100 vol/vol. The feed medium  was fed into the fermenters when the concentration of glucose was less than 20 g L–1. The pH was controlled at 7.0 by 50% NH3.H2O, and the agitation speed was 600 rpm.Fed-batch fermentation was carried out in 5-L fermenters . Strains were first activated on an agar plate . The reaction was incubated at 30°C for 30 min, and the absorbance was measured at 520 nm.The production of c method . This me–1 NaHCO3 and 150 μL of 1% DNFB-acetonitrile. Then, the mixture was heated at 60°C in darkness for 60 min. L-Arginine production was detected by using an HPLC system, and the separation was achieved by using a platisil 5 μm ODS, 250 × 4.6 mm C18 column. The mobile phase consisted of 0.1 mol L–1 NaAc (pH 6.4)-acetonitrile (86:14), with a flow rate of 1.0 mL min–1, injection volume of 10 μL, column temperature of 30°C, and detection wavelength of 360 nm.For the shake-flask and fed-batch fermentation, cell culture was centrifuged at 8,000 rpm for 1 min, and 1 mL of culture supernatant was treated with 1 mL of 0.5 mol L1 using the structure of Mycobacterium tuberculosis (PDB: 6A2Q) as a template. The binding of the L-arginine molecule to the ArgR protein was included in the structure. The model was assembled primarily as a tool to predict mutation sites. Model observation, image processing, and correlation analyses regarding the binding pocket of ArgR protein were performed by PyMOL program , and the ARG-Select biosensor system exhibits a sensitive response to different L-arginine concentrations.Finally, RT-qPCR was performed on the recombinant ARG-Select biosensor to analyze the change in reporter gene expression at different arginine . Howevergfp-based biosensor was used to further verify the relationship between fluorescence intensity and L-arginine production  or lower (P2) than the control. Subsequently, cells in the two gates were collected and spread on agar plate with 50 μg mL–1 of kanamycin or ampicillin added. We randomly selected 200 strains from each gate and determined the L-arginine production in 24-deep-well plates for 48 h. Cells in P1 showed strong fluorescence and could produce 1.91 g L–1 of L-arginine on average; the L-arginine production of cells in P2 showed low fluorescence, and the cells could produce 2.48 g L–1 of L-arginine. The difference of L-arginine production between P1 and P2 cells was statistically significant (P < 0.01). Most of the weak cells could not be further identified and isolated by FACS. This result indicated that the fluorescence in a gfp-based biosensor system was capable of predicting the L-arginine yield of strains. The obvious difference of the L-arginine production between the strains in the two gates demonstrated the ability of the ARG-Select biosensor.To examine the effectiveness of the ARG-Select biosensor, the oduction . The strCorynebacterium crenatum SYPA5-5 is a mutant strain that could produce 30 g L–1L-arginine in our study. Previously, it was reported that a negative mutation (C → T) of argR occurred in C. crenatum SYPA5-5 that led to obviously increased expression of the argC–argH cluster  grew on the plate, and they were randomly picked with 200 small colonies (colony diameter ≤0.80 mm) to determine the L-arginine production in 24-deep-well plates. An obvious difference in L-arginine production was found between large and small colonies  and five small  colonies were cultivated to obtain the growth curves (600) was measured every 24 h. As expected, different growth rates were shown between the large and small colonies, and strains with high L-arginine production showed a fast growth rate  will be applied to increase the L-arginine production, which is achieved by further mutation and screening.In this study, the increased All datasets generated for this study are included in the article/supplementary material.MX and PL performed the experiments and wrote the manuscript. JC and AP helped in the analysis design and data interpretation. ZR designed the experiments and reviewed the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Background: Jaktinib is a novel selective janus kinase 1/2 inhibitor. The phase I first-in-human study evaluated the tolerance and pharmacokinetics of jaktinib in healthy Chinese subjects.Methods: A randomized, double-blind, placebo-controlled study were designed. A total of 126 healthy subjects were enrolled into the single ascending dose, multiple ascending dose and food effect study. Safety endpoints included adverse events, abnormal vital signs, 12-lead ECGs, abdominal ultrasound, chest x-ray, physical examination and clinical laboratory tests. Blood, urine and feces samples were collected at predetermined time points for pharmacokinetic analysis of jaktinib, the metabolites ZG0244 and ZG0245, which are formed by oxidation or hydrolysis metabolic pathway, respectively.Results: Jaktinib was absorbed with a median time to peak plasma concentration of 1.25–3.5 h and was eliminated with a half-life of 2.952–9.040 h. Linear pharmacokinetic characteristic was presented over the dose range from 25 to 400 mg. No obvious accumulation was observed after multiple doses for 10 days. Administration after a high-fat breakfast significantly increased the absorption of jaktinib. The accumulated fraction of jaktinib and the determined metabolites excreted in urine and feces was 19.478%. Jaktinib was well tolerated in all single dose cohorts. In multiple dose cohorts, 200 mg q24 h method was evaluated as maximally tolerated dose. Neutropenia, diarrhea, dizziness and headache were the most frequently reported treatment related adverse events. No deaths, serious or Grade ≥4 adverse events was developed.Conclusion: Jaktinib was well tolerated when single dose ranging from 25 to 400 mg and multiple dose up to 200 mg q24 h. The safety and pharmacokinetic characteristics support the next trial in myelofibrosis patients. It is amia (ET) . The agemia (ET) . The conmia (ET) . AllogenIn 2005, the relationship between Janus kinase (JAK) mutations and MF was unveiled. The JAK2 V617 F mutation has been identified in 50–60% of patients with MF. RuxolitJaktinib is a newly developed selective JAK1/2 inhibitor with 50% inhibitory concentration (IC50) of 0.1 μM on cells transformed by selected oncogenic mutants of tyrosine kinases. It has the similar inhibition potency to momelotinib and ruxolitinib. Preclinical toxicology studies were conducted in both Wistar rats and Beagle dogs with oral administration of jaktinib. The no observed adverse effect level (NOAEL) were 18.3 mg/kg/day and 30 mg/kg/day in Beagle dogs and Wistar rats, respectively. Based on the NOAEL dose and a 10-fold safety factor, the recommended phase I starting dose was 25 mg/day. This was a first-in-human study to investigate the tolerance, safety, pharmacokinetics and food effect of jaktinib in healthy Chinese volunteers.2, and a total body weight of male and female subjects was no less than 50 and 45 kg, respectively. Other eligibility criteria included no history of cardiac, hepatic, renal, gastrointestinal, or neurologic diseases. All subjects were required to practice birth control and have no plan to conceive during the next 6 months.Eligible volunteers were healthy male and female Chinese aged 18–45 years with body mass index of 18–28 kg/mExclusion criteria included smoking more than five cigarettes per day within the past 3 months; any clinically significant laboratory test or 12-lead ECG abnormality; history of drug abuse and/or alcoholism; intake of any other drugs, vitamins, or herbal medicine within 14 days or intake of any drugs known to influence the activity of drug metabolizing enzymes  within 28 days before the first dose of the trial medication; pregnant or breastfeeding; multiple food and drug allergies.The study was approved by the Ethics Committee of the First Hospital of Jilin University, and conducted in accordance with the ethical principles originating in the Declaration of Helsinki version 2013 and in compliance with International Conference on Harmonization Good Clinical Practice Guidelines. All volunteers provided written informed consent.ClinicalTrials.gov identifier: NCT03314402) from October 2017 to March 2018. Jaktinib hydrochloride 50 mg film-coated tablets were produced and supplied by Suzhou Zelgen Biopharmaceuticals Co., Ltd., China. The study comprised three parts: single ascending dose (SAD), multiple ascending dose (MAD) and food effect.This was a phase I, randomized, double-blind, placebo-controlled, dose-escalation study conducted at First Hospital of Jilin University, China . Among these cohorts, 25 mg was bisected by the 50 mg specification under good manufacturing practices (GMP) condition and provided the certificate of analysis. In each cohort, eight healthy volunteers were randomized (3:1) to receive jaktinib or placebo with a similar proportion of male and female. Jaktinib or placebo was administrated after an overnight fast.MAD study was conducted after evaluating the tolerance of the single dose 100 mg cohort. In MAD part, volunteers were designed into five cohorts . In each cohort, 10 volunteers were randomized (8:2) to receive jaktinib or placebo in a close proportion of male and female and jakitinib or placebo was administrated for 10 days under fasting condition ranging from 100 mg q24h to 150 mg q12h. The MAD part was initiated after the safety and tolerance evaluation from SAD 100 mg cohorts. Progression to higher MAD doses was allowed after review the safety data of the prior MAD cohorts.Food effect part was a two-period crossover study and conducted after finishing both SAD and MAD parts. 12 volunteers were randomized (1:1) with a similar proportion of male and female to receive their first dose of jaktinib 200 mg under fasted (group A) or fed (group B) conditions (period 1). After a washout period of 5 days, volunteers received their second dose of jaktinib 200 mg under the other condition . For group A, the urine and feces samples for pharmacokinetic (PK) analysis were collected in the first period.Dose-limiting toxicities (DLTs) were defined according to the NCI CTCAE v.4.03 as any occurrence of the following drug-related adverse events (AEs): more than one third of volunteers suffered Grade ≥4 drug-related hematological toxicities or Grade ≥3 drug-related nonhematological toxicities; one drug-related serious adverse events (SAEs).The primary objectives of the study were to evaluate the safety and tolerance, determine the DLT and maximal tolerated dose (MTD) of jaktinib. The secondary objectives were to explore the PK profile and the food effect on PK profile of jaktinib.2EDTA anticoagulant at 0 (pre-dose), 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 14, 24 and 36 h post-dosing in SAD cohorts. For MAD cohorts, blood samples were collected at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8 and 12 h on day 1; 0 h on day 2, 3, 4, 6 and 7; 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, 36 and 48 h on day 10. In food effect cohort, blood samples were collected at 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, 36, 48, 72, 96 and 120 h in period one of group A; 0, 0.25, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 24, 36 and 48 h in period two of group A and the two periods of group B. Blood samples were centrifuged at 3,000 rpm for 10 min at 2–8 °C and stored at -80 °C until liquid chromatography-mass spectrophotometry (LC-MS/MS) analysis. Urine and feces samples were collected in period one of group A of food effect cohort with the intervals 0, 0–6, 6–12, 12–24, 24–48, 48–72, 72–96, 96–120 and 0–120 h, respectively.Blood samples (4 ml) for pharmacokinetic analysis of jaktinib, main metabolites ZG0244 (similar JAK1/2 inhibitory activity to jaktinib) and ZG0245 (no JAK1/2 inhibitory activity) were collected in tubes containing Kmax), time to peak plasma concentration (Tmax), area under the plasma concentration–time curve from time 0 to 12 or 24 h , AUC from time 0 to the last timepoint with a quantifiable concentration (AUClast), AUC from time 0 to infinity (AUC0-∞), terminal elimination half-life (t1/2), clearance (CL/F), apparent volume of distribution (Vz/F) and the parameters at steady state in MAD cohorts: C,max,ss, Cmin,ss, Tmax,ss, AUC0-24,ss, AUClast,ss, AUC0-∞,ss, CL/Fss and Vz/Fss. Urine and feces PK parameter was accumulated excretion recovery ratio from time zero to 120 h (Fe0–120h).Plasma pharmacokinetic data were analyzed by standard non-compartmental methods using WinNonlin version 7.0 (Certara USA Inc.) and the pharmacokinetic parameters included peak plasma concentration  and 2, 6, 24, 48 h after administration in SAD cohorts; 0, 2, 6 h on day 1, 0 h on days 2, 3, 4, 7, 0, 2, 6, 24, 48 h after the last dose in MAD cohorts; 0, 2, 6, 24, 48, 72, 120 h in period 1 and 2, 6, 24, 48 h in period two in food effect cohort.No concomitant medications were allowed during the study except for the treatments of AEs. Potent inhibitors and inducers of hepatic metabolic enzymes were prohibited from 4 weeks before administration to throughout the study and closely monitoring should be given when combining CYP2C9 substrates with narrow range of treatment.max and AUC parameters to log-transformed dose, was used to investigate dose proportionality. Food effect pharmacokinetic parameters were analyzed using the mixed effect model with period, sequence and treatment as fixed effects and subjects as the random effect. Logarithmic transformation of Cmax, AUC0-∞ and AUClast were performed to calculate the 90% confidence intervals (CIs) of the geometric mean ratio (GMR) between fasting and fed administration. If the calculated 90% CI values fall entirely within the range of 0.80–1.25, it is indicative of no significant food effect.Statistical analysis was performed using SAS software, version 9.4 . Descriptive statistics were used to summarize continuous variables as cases, means with standard deviations, medians, maximum and minimum. For categorical variables, frequencies and percentages were used. A regression power model, relating log-transformed CA total of 126 healthy volunteers were enrolled into the study; 64, 50 and 12 volunteers enrolled in the SAD, MAD and food effect part, respectively. The baseline demographic characteristics of the enrolled cases are presented in max 1.25–3.5 h, which is independent of dose. The t1/2 were 2.952–9.040 h and didn't show a proportional increase with increasing dosages of administration. The jaktinib AUC0-∞ and Cmax increased dependent on dose, the increase trend was basically in line with the dose proportionality. The correlative analysis of dosages and pharmacokinetic properties were analyzed using WinNonlin linear mixed effects model and the slopes (90% CI) were 1.15 (0.96–1.34) and 1.00 (0.86–1.15) for ln AUC0-∞ and ln Cmax, close to 1, suggesting a clear linear pharmacokinetic characteristic within the dose range of jaktinib 25–400 mg. The CL/F was 76.212–150.190 L/h with a large Vz/F 533.561–1033.554 L, suggesting a wide distribution in the body. For metabolites ZG0244 and ZG0245, the Tmax were 2–4 and 4–8 h with the t1/2 3.042–8.511 and 8.08–24.549 h, respectively. The metabolic rate were 0.789–1.325 and 0.279–0.584 , decreased as the dose increasing. The mean jaktinib, metabolites ZG0244 and ZG0245 plasma concentration-time profiles after SAD are shown in ac was calculated using AUC0-24 and AUC0-24,ss, and for q12h, it was AUC0-12 and AUC0-12,ss. The Rac were 0.98 , 1.16 , 1.24 , 0.94 , 1.38  for 100 mg q24h, 150 mg q24h, 100 mg q12h, 200 mg q24h and 150 mg q12h cohorts, respectively, showing no obvious accumulation. For metabolites ZG0244 and ZG0245, the Rac was 0.668–1.150 and 2.252–5.286, respectively. No accumulation was observed for ZG0244 and there was moderate accumulation for ZG0245. max of 2.6-fold and AUC0-∞ of 2.28-fold.The inter-individual variation of main PK parameters decreased in the fed state. The results of mixed effect model analysis showed no calculated 90% CI values of AUC0-t, AUC0-∞ and Cmax fell entirely within the range of 0.80–1.25 (max was significantly prolonged from 2 h in the fasted state to 4 h in the fed state (p < 0.05). The metabolites ZG0244 and ZG0245 showed a similar food effect PK characteristics to jaktinib. In the fed state, the Cmax of 1.56-fold and AUC0-∞ of 1.93-fold was observed for ZG0244 and it was both 1.94-fold for ZG0245. The median Tmax was extended by 2 and 1.5 h, respectively. .80–1.25 , it is iFor metabolic transformation study, jaktinb and ZG0244 were detected in urine samples and in feces samples, jaktinib, ZG0244, ZG0245, ZG0243 and ZG0262 were detected. The 0–120 h accumulated excretion recovery ratio was 19.478% in urine and feces .Overall, jaktinib was well tolerated in all the SAD and MAD cohorts except for 150 mg q12 h cohort. Treatment-related AEs (TRAEs) were reported in 18 (28.1%) and 37 (74.0%) volunteers in SAD and MAD cohorts. In the food effect cohort, 3 (25.0%) and 5 (41.7%)volunteers reported treatment-related AEs in the fasted and fed state. The most frequently reported treatment-related AEs were neutropenia, diarrhea, dizziness and headache. No deaths, SAEs and Grade ≥4 AEs were reported in all the cohorts.In the SAD part, most reported TRAEs were Grade 1 except one Grade 3 Varicella, which was reported five days after administration and the subject recovered after the treatment of second-generation cephalosporin cefuroxime axetil. Headache (10.4%), positive urine erythrocyte (8.3%) were the most frequently reported AEs (≥5%) for volunteers who received jaktinib, and 0%, 6.3% for volunteers who received placebo. No dose-dependent TRAEs were observed .In the MAD part, most reported TRAEs were Grade 1 or 2. All the Grade 3 AEs were neutropenia with 1 (12.5%) in the 100 mg q12h cohort and 5 (62.5%) in the 150 mg q12h cohort. The volunteers suffered Grade 3 neutropenia stopped jaktinib administration immediately and recovered to normal without additional treatment. Neutropenia (27.5%), diarrhea (22.5%), dizziness (17.5%) and leukopenia (12.5%) were the most frequently reported TRAEs (≥10%) in jaktinib group showed a greater proportion than placebo group . No obvious dose-dependent TRAEs were observed .In the food effect part, all TRAEs were mild to moderate in severity. A higher incidence of TRAEs were observed in the fed state but the difference was not statistically significant .This phase 1 study evaluated the safety, tolerability, PK characteristics of SAD, MAD and food effect of jaktinib, in healthy Chinese volunteers. The baseline demographic characteristics were similar across all the cohorts.0-∞) 0.789–1.325 and 0.279–0.584 respectively after SAD of 25–400 mg jaktinib. ZG0244 appeared in plasma almost simultaneously with jaktinib and revealed a similar PK characteristics. For ZG0245, it appeared slowly in plasma and the Tmax was about several hours longer than jaktinib and ZG0244. There was a trend of dose-proportional increase in exposure to jaktinib with no obviously differences observed for t1/2.After single administration, jaktinib was rapidly absorbed in plasma with high inter-individual variations in PK parameters. ZG0244 was the most abundant metabolite, followed by ZG0245, with the MR  than in 100 mg q12h (807.632 h·ng/ml), this may attribute to a higher variable coefficient (CV) of ZG0245, 118.7% in 100 mg q12h and 103.5% in 150 mg q12h, respectively. High-fat meals increased the exposure of jaktinib and metabolites and delayed the time to peak concentration. Administration without food is recommended for the future studies based on the following considerations: firstly, the concentrations in plasma after administrations with high-fat food were very high and additional study for food impact with low-fat meal need to be conducted; And secondly, for agents of long-term use, it is difficult to administrate after high-fat meal, because some MF patients can't tolerated the high-fat meals, especially for patients of q12h administration. Considering these differences in diet and disease state of MF patients, administrations without food can achieve a relatively consistent exposure to obtain favorable effect. Feces was the main way of excretion and the unmetabolized jaktinib was the main detected substance in the excrements, which showed potential of further test in patients with renal insufficiency.In MAD cohorts, visual inspection of trough concentration data, steady-state plasma concentration were reached at approximately 4 days for jaktinib. The TIn the SAD part, most AEs were mild in severity. One volunteer at dose 250 mg experienced Grade 3 varicella, which was considered related to the pharmacological mechanism of JAK inhibitor. Virus infection (herpes zoster) was also observed in the RESPONSE trial of ruxolitinib with the incidence rates 4.7% and 0% in ruxolitinib and control groups, respectively . HeadachIn the MAD part, neutropenia, diarrhea and dizziness were the most common AEs. These AEs have been also observed in ruxolitinib and momelotinib clinical trials . The incIn conclusion, the current study showed that single oral administration of jaktinib 25–400 mg and multiple doses up to 200 mg q24h were well tolerated with no DLTs observed. The accumulated safety and PK data in healthy subjects support further evaluation in patient clinical trials for the treatment of MF.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Ethics Committee of the First Hospital of Jilin University, China. The patients/participants provided their written informed consent to participate in this study.JL and YD wrote the article. YD, JL and BL designed the research. JL, BL, HY, XZ and HW performed the clinical trial. JL, XZ and YD analyzed the data.The work was funded by Suzhou Zelgen Biopharmaceuticals Co., Ltd. The work was also financially supported by the National Major Scientific and Technological Special Project for “Significant New Drug Development” during the Thirteenth Five-Year Plan Period of China (Project Nos. 2017ZX09304004 and 2017ZX09101001-002-004), the National Natural Science Foundation of China (Project No. 81602897).BL and HY are employees of Suzhou Zelgen Biopharmaceuticals Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Eimeria tenella AMA1 (EtAMA1) is critical for sporozoite invasion of host cells. Here, we show that EtAMA1 is a microneme protein secreted by sporozoites, confirming previous results. Individual and combined treatment with antibodies of EtAMA1 and its interacting proteins, E. tenella rhoptry neck protein 2 (EtRON2) and Eimeria-specific protein (EtESP), elicited significant anti-invasion effects on the parasite in a concentration-dependent manner. The overexpression of EtAMA1 in DF-1 cells showed a significant increase of sporozoite invasion. Isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS were used to screen differentially expressed proteins (DEPs) in DF-1 cells transiently transfected with EtAMA1. In total, 3953 distinct nonredundant proteins were identiﬁed and 163 of these were found to be differentially expressed, including 91 upregulated proteins and 72 downregulated proteins. The DEPs were mainly localized within the cytoplasm and were involved in protein binding and poly(A)-RNA binding. KEEG analyses suggested that the key pathways that the DEPs belonged to included melanogenesis, spliceosomes, tight junctions, and the FoxO and MAPK signaling pathways. The data in this study not only provide a comprehensive dataset for the overall protein changes caused by EtAMA1 expression, but also shed light on EtAMA1’s potential molecular mechanisms during Eimeria infections.Apical membrane antigen 1 (AMA1) is a type I integral membrane protein that is highly conserved in apicomplexan parasites. Previous studies have shown that  Parasites of the phylum Apicomplexa are responsible for some of the most devastating and burdensome diseases to humans and animals worldwide, including malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis . Host-ceToxoplasma and Plasmodium detailed the composition of the MJ complex, which mainly includes rhoptry neck proteins (RONs) and apical membrane antigen1 (AMA1) [P. knowlesi nearly 40 years ago [Toxoplasma tachyzoites [Plasmodium merozoites [Neospora tachyzoites [Babesia merozoites [Previous reports on 1 (AMA1) , 3, 7. M1 (AMA1) . Of all ears ago , AMA1 isears ago . Numerouhyzoites , Plasmodrozoites , Neosporhyzoites , and Babrozoites  to theirEimeria tenella is widely considered to be the most economically relevant and well-known of the seven Eimeria species that cause coccidiosis in chickens [Eimeria tenella has a complex life cycle that includes two major asexual developmental stages, including sporozoites and the merozoites [E. tenella life-cycle stages found that EtAMA1 was detected only in sporozoites [chickens . Eimeriarozoites . The exprozoites . Proteomrozoites . Jiang erozoites . Speciﬁcrozoites , 23, 29.E. tenella rhoptry neck protein 2 (EtRON2), microneme protein 2 (EtMIC2), and an Eimeria-specific protein (EtESP) (GenBank accession No. XM_013373193) [E. tenella putative cystathionine beta-synthase, four conserved hypothetical proteins (one in the serine/threonine protein kinase family), and seven unknown proteins, but other putative proteins must be further identified [Eimeria host cell invasion.EtAMA1 can interact with 3373193) , 44. Othentified . Thus, tAll animal procedures were approved by the Animal Ethics Committee of the Shanghai Veterinary Institute, Chinese Academy of Agricultural Science. Experiments were conducted in accordance with animal ethics guidelines and approved protocols.E. tenella was maintained and propagated by regular in vivo passage through 2-week-old Yellow chickens reared under specific pathogen-free conditions [The Shanghai strain of nditions . Unsporunditions . The chiEtAMA1, E. tenella microneme 2 protein (EtMIC2) was used as the experimental control [6 fresh sporozoites were resuspended in 100 μL PBS or complete medium and incubated for 2 h at 4 °C or at 41 °C. Then 5 μM, 10 μM, or 20 μM of staurosporine , a protein kinase inhibitor known to speciﬁcally inhibit microneme secretion [g (Eppendorf Centrifuge 5810R). Supernatants containing excretory-secretory antigens (ESAs) were harvested and EtAMA1 and EtMIC2 secretion was analyzed by western blotting with anti-rEtAMA1 or anti-rEtMIC2 antibodies, respectively [To test the secretion of  control , 43. A tecretion , 19, disectively , 43.E. tenella, the purified sporozoites were transferred to glass slides and air-dried. Sporozoites were fixed in 2% paraformaldehyde in PBS, air-dried, and permeabilized with 1% Triton X-100 in PBS for 15 min. The slides were blocked with 2% (w/v) bovine serum albumin in PBS for 2 h at 37 °C and incubated with polyclonal anti-rEtAMA1 mouse serum, and anti-rEtESP or anti-rEtRON2 rabbit serum [To identify co-localization of EtAMA1 and its interaction proteins, EtRON2 and EtESP, in it serum  diluted E. tenella sporozoites. The ani-rEtAMA1, ani-rEtRON2, and ani-rEtESP antibodies were purified using protein A + G agarose (Beyotime), according to the manufacturer’s instructions. DF-1 cells (2 × 105 cells per well) were cultivated in 24-well plates  in CM for 12 h at 37 °C and 5% CO2. Freshly purified sporozoites were marked using carboxyfluorescein diacetate succinimidyl ester , according to the manufacturer’s instructions. The labelled sporozoites were preincubated at 37 °C for 2 h with the purified anti-rEtAMA1, rEtESP or rEtRON2 IgG, or combinations of anti-rEtAMA1 and rEtESP IgGs (with a ratio of 1:1), or anti-rEtAMA1 and rEtRON2 IgGs (with a ratio of 1:1) at concentrations of 100, 200, 400, or 600 μg/mL [2 with a sporozoite/cell ratio of 3:1. The cells were washed, trypsinized, harvested, and analyzed using a flow cytometer . The infected cells, non-infected cells and free sporozoites were gated using the software RXP for subsequent delineation and counting of the infected (containing the labeled sporozoites) and non-infected (fluorescence-free) cells. Three replicates were performed for all the tests, and a total of three tests were conducted. The percentage of infected cells in the presence or absence of inhibitory antibodies was calculated as previously described [A neutralization assay with antibodies was used to assess the potential involvement of EtAMA1 and its interaction proteins, EtRON2 and EtESP, in host cell invasion by 00 μg/mL . The samescribed .LN610018.1) was amplified using the primers: 5′–GCGAATTCATGGGGCCACCCCCGTCTTTGGCAAAAAC–3′ and 5′–GCAGA TCTGCACTTGG TCTCCCAGTCG–3′ containing EcoRI sand BgIII restriction sites (underlined). The PCR products were ligated into the pcDNA3.1(+) vector and designated as pcDNA3.1(+)-EtAMA1. The constructed plasmid was identified by sequencing and purified using a Qiagen Plasmid Giga Kit (Qiagen). The plasmid concentration was determined by spectrophotometry at 260 nm.The extracellular domain I of EtAMA1 (GenBank accession No. EtAMA1 or pcDNA3.1(+) using Lipofectamine® (Invitrogen). Six hours later, the DNA-transfection reagent mixture was replaced by opti-MEM. At 48 h post-transfection, the expression of EtAMA1 in transfected DF-1 cells was confirmed by indirect fluorescent antibody testing (IFA) -g et al. ). The prEtAMA1 or pcDNA3.1(+) transfected DF-1 cells were invaded by fluorescently-labeled E. tenella sporozoites with CFSE at a ratio of three sporozoites per cell for 8 h at 41 °C, and then subjected to flow cytometry to determine the invasion rate. The sporozoite-infected un-treated DF-1 cells were the control group.The pcDNA3.1(+)-EtAMA1-transfected and empty vector pcDNA3.1(+)-transfected DF-1 cells were seeded into T25 culture ﬂasks and cultured in a 5% CO2 incubator at 37 °C for 48 h. Each group was treated with three independent biological replicates. The transfected cells were washed three times with chilled PBS, collected with cell scrapers, and homogenized in lysis buffer , then incubated for 3 min in boiling water and sonicated twice on ice, and clariﬁed by centrifugation at 16,000 ×g at 25 °C for 10 min. Protein content was determined using the BCA protein assay reagent (Beyotime). Protein digestion, iTRAQ labeling, peptide fractionation using strong cation exchange (SCX) chromatography, and liquid chromatography (LC)-electrospray ionization tandem MS (MS/MS) analyses with Q Exactive were conducted as previously described [The pcDNA3.1(+)-escribed , 42.Gallus gallus database  and the decoy database. For identiﬁcation, the following options were used. Peptide mass tolerance = 20 ppm, MS/MS tolerance = 0.1 Da, enzyme = trypsin, Missed cleavage = 2, fixed modiﬁcation: carbamidomethyl (C), iTRAQ8plex (K), iTRAQ8plex(N-term), variable modiﬁcation: oxidation (M), and FDR ≤ 0.01. To define up- and downregulated proteins, the levels for significantly differentially expressed proteins (DEPs) were set at a fold change > 1.2 and < 0.83, respectively, with a p-value < 0.05 [MS/MS spectra were searched using the Mascot 2.2 engine against the UniProt e < 0.05 , 47.https://www.blast2go.com/), was used to annotate DEPs to create histograms of GO annotations. For pathway analysis, the DEPs were mapped to the terms in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database using the KAAS program (http://www.genome.jp/kaas-bin/kaas_main).The Gene Ontology (GO) program, Blast2GO  and GraphPad Prism version 6.0 .p < 0.05 and < 0.01, respectively.The significance of differences in sporozoite invasion rates or inhibitory percentages between the groups was evaluated by one-way analysis of variance (ANOVA) and the mean values were compared using Duncan’s multiple range test. Differences were considered significant and extremely significant if To determine whether EtAMA1 is a secreted protein, sporozoites were incubated in PBS and CM at 4 °C or 41 °C, respectively. Western blotting showed that EtAMA1 was secreted when sporozoites were incubated at 41 °C in CM, and EtMIC2 was secreted in PBS and CM at 4 °C or 41 °C . TherefoTo demonstrate that EtAMA1 secretion was dependent on the micronemal pathway, staurosporine was used as this protein kinase inhibitor speciﬁcally inhibits micronemal secretion , 19. In The locations of EtAMA1 and its interacting proteins, EtESP and EtRON2, were detected in sporozoites incubated in culture medium. IFAs showed that EtAMA1, EtESP and EtRON2 were distributed to the apical end of sporozoites  and 2b, p < 0.001) (p < 0.05)  . When co < 0.05) .EtAMA1 was transiently transfected into the DF-1 cells and its expression was confirmed by IFA. Intense green ﬂuorescence was detected in DF-1 cells transfected with pcDNA3.1(+)-EtAMA1 and none was detected in cells transfected with pcDNA3.1(+) (EtAMA1 plasmid was similar to that of the control groups (EtAMA1 plasmid was significantly higher than that of cells expressing the pcDNA3.1-(+) plasmid (p < 0.05), and extremely significantly higher than that of the normal (un-treated) cells (p < 0.01) -NA3.1(+) ; thus, dl groups . Sporozo < 0.01) .Supplementary Table S1). This included 91 upregulated proteins and 72 downregulated proteins. The top ﬁve upregulated proteins were the glucagon like peptide 2 receptor, the protein transport protein Sec61 subunit beta, keratin , Wnt-11, and heat shock protein 25. The top ﬁve downregulated proteins were the coiled-coil domain-containing protein 186, the pleckstrin homology domain-containing family O member 1, the 40S ribosomal protein S24, the signal transducer and activator of transcription, and an uncharacterized protein. Collectively, these results indicated that EtAMA1-transfection induced a distinct proteomic proﬁle in DF-1 cells, which caused the host cells to sharply alter their related proteins in response.After processing the MS/MS spectra using Mascot software, 16,385 unique peptides were mapped to 3953 proteins from DF-1 cells. A total of 163 proteins were found to be signiﬁcantly differentially expressed in DF-1 cells in response to transient transfection with EtAMA1 , the negative regulation of biological processes (13%), and single organism cellular processes (12%) . AdditioToxoplasma gondii and the secretion of EtMIC3 [The full-length cDNA of EtAMA1 was 2349 bp, with a 1608 bp open reading frame encoding a protein of 535 amino acids. Analysis of the amino acid sequences revealed that EtAMA1 is a type I integral membrane protein with a transmembrane domain, a signal peptide, an ectodomain, a cytoplasmic C terminal domain, and an AMA1 superfamily domain . EtAMA1 f EtMIC3 . These rf EtMIC3 .E. tenella had shown that EtAMA1 transcription was stage-speciﬁc [E. tenella sporozoite invasion in host cells [E. tenella sporozoite invasion was also regulated by the overexpression of EtAMA1, invasion experiments were performed with EtAMA1-transfected DF-1 cells. The results showed that the sporozoite invasion of EtAMA1-transfected DF-1 cells was significantly higher than that of the control group, thus indicating that the overexpression of EtAMA1 could promote sporozoite invasion.Comparison of RNAseq datasets from unsporulated oocysts, sporulated oocysts, sporozoites, 2nd-generation merozoites, and the gametocyte lifecycle stages of -speciﬁc , 39. The-speciﬁc . Immunoﬂ-speciﬁc , 29. Blost cells , 23, 29.T. gondii and Plasmodium, AMA1 interacts directly with rhoptry neck protein 2 (RON2), which is secreted from the parasite rhoptries and specifically localizes to the MJ. The RON2-AMA1 interaction is a critical step in MJ-dependent invasion of host cells by apicomplexan parasites [E. tenella sporozoites, and a total of 14 putative interaction proteins were obtained [E. tenella rhoptry neck protein 2 (EtRON2), microneme protein 2 (EtMIC2), and Eimeria-specific protein (EtESP) interacted with EtAMA1 [Previous studies showed that in arasites , 37. To obtained . Some puh EtAMA1 , 44. In h EtAMA1 .EtAMA1 plays an important role in sporozoite invasion, but its molecular mechanism remains unknown. To investigate the effect of EtAMA1 on host cells, the proteomic changes of transfected DF-1 cells were investigated using iTRAQ. iTRAQ has become a powerful quantitative proteomic method with advantages over traditional proteomic techniques. Such advantages include higher throughput, increased sensitivity, and greater accuracy. iTRAQ has been used successfully to explore host-pathogen interactions for viruses , 40, bacPrevious reports showed that transiently-expressed heterologous proteins could significantly change cell proteomes, and in particular, proteins involved in different pathways , 36. In Eimeria infections, changes in the cytoskeleton of the host cells affect cell adhesion and migration, and may play an important role in the invasion and development of Eimeria spp. In the present study, several cytoskeleton-related proteins, including TPM1, alpha-actinin-1 (ACTN1), myosin regulatory light chain interacting protein, septin-2, and keratin were significantly upregulated by the transfection of EtAMA1. Such upregulations may impact cytoskeletal organization, and cell migration and movement tendency. These observations clearly indicated that changes in the cytoskeleton of cells affect cell adhesion and migration, which may play an important role in the invasion and development of Eimeria spp.The cytoskeleton is a cellular scaffolding contained within the cytoplasm. It maintains cell shape, provides mechanical strength, directs locomotion, regulates chromosome separation during mitosis and meiosis, and regulates the intracellular transport of organelles in cells . In the Eimeria host-cell invasion.In conclusion, the present study revealed that EtAMA1 was secreted by micronemes. Both rEtAMA1-specific antibodies and its interacting proteins, EtESP, EtRON2, inhibited parasite invasion, and the overexpression of EtAMA1 in cells promoted sporozoite invasion, thus suggesting that EtAMA1 was involved in host-cell sporozoite invasion. The proteomic changes in EtAMA1-trancfected DF-1 cells were analyzed using an iTRAQ-based proteomic method. Our analyses of the DEPs were comprehensive, but further studies are necessary to understand the functions of the identified proteins regulated by EtAMA1 in DF-1 cells. Such studies will be important to understand the precise functions and molecular mechanisms of EtAMA1 during https://www.parasite-journal.org/10.1051/parasite/2020068/olmSupplementary material is available at Table S1. Detailed list of proteins of DF-1 cells transfected transiently with EtAMA1.Detailed list of 163 proteins differentially expressed in DF-1 cells transfected transiently with EtAMA1.This work was supported by grants from the National Science Foundation of China (Nos. 31672551 and 31572266), the National Key R&D program of China (No. 2017YFD0500400), and the National Parasitic Resources Center (No. NPRC-2019-194-30).The authors declare that they have no competing interests.


This study investigated the effects of cow’s milk on recovery from repeated simulated team games (STGs) in females. Twenty female team-sport athletes completed an STG circuit . Measures of muscle function, soreness and tiredness, symptoms of stress and serum markers of muscle damage and oxidative stress were determined pre- and 24 h, 48 h, 72 h and 96 h following the circuit. At 48 h, a second STG was completed. Sprint performance , lap time, countermovement jump (CMJ), heart rate and RPE were recorded during each STG. Immediately following each STG, participants consumed either 500 mL of cow’s milk (MILK) or 500 mL of an energy-matched carbohydrate (CHO) solution. Compared to CHO, MILK had a beneficial effect in attenuating losses in peak torque for knee extension (60°/s)  = 0.26 to 0.28) knee flexion (60°/s) . A benefit for MILK was observed for 5 m sprint , 10 m sprint  and symptoms of stress . Mostly unclear outcomes for other variables were observed. For STG variables, trivial  and unclear  outcomes were recorded. In conclusion, the consumption of 500 mL of milk attenuated losses in muscle function and perceptions of stress following repeated simulated team-sports games. However, further investigation is warranted to determine whether MILK can influence subsequent team-sport performance. Participation in a single team-sport match results in fatigue, muscle damage and oxidative stress in males ,2 and feMinimizing muscle damage and enhancing recovery between training sessions or games could be beneficial for maintaining performance in subsequent bouts. Recent research has shown that the consumption of cow’s milk following muscle damaging exercise can attenuate decreases in muscle functional capacity in males ,10 and fGiven the benefits of cow’s milk for enhanced recovery following muscle damaging protocols, and with the short recovery periods experienced by athletes between team-sport training sessions and games, it makes the expectation tenable that post-exercise consumption of cow’s milk may combat the negative effects of muscle damage and fatigue, thus accelerating the rate of recovery between matches. Accordingly, this study aims to examine the effect of cow’s milk on recovery from a simulated team game and a second simulated team game following a 48 h recovery period. Additionally, the effect of cow’s milk on ‘in-game’ measures in the second simulated game is explored.Twenty female team-sport athletes , from the amateur sports of camogie and ladies Gaelic football, participated in this investigation. All participants were training for and competing in their chosen sport at the time of data collection. Eligibility was assessed with a screening questionnaire, and volunteers were excluded from the study if they met any of the following criteria: current or recent use of nutritional supplements, intolerance to dairy or lactose products, injury in previous 3 months, surgery in previous 6 months, known coronary disease, uncontrolled metabolic disorder or respiratory disease, pregnancy and/or post-partum. Following verbal and written briefings written informed consent was provided. Ethics approval was gained from the Ethics Committees of Middlesex University and Institute of Technology Carlow, where data collection took place.Participants visited the laboratory on seven occasions. The first visit was comprised of study briefing and familiarization with all variables. Baseline values for all measures were determined at the second visit, following which participants were divided into two groups, cow’s milk (MILK) and an energy-matched carbohydrate (CHO) solution, matched according to player position and peak torque of the dominant leg knee extensors (60°/s). Data collection took place at the same time each day following 24 h abstinence from alcohol, caffeine and exercise.Participants completed all measures following an overnight fast and a standardized snack. No more than 5 days post-baseline measures, participants completed an exercise protocol comprising a circuit with simulated team-sport games (STG1). Participants returned to the laboratory at 24 h and 48 h post-exercise to repeat baseline measures. Upon completion of measures 48 h post-exercise, participants repeated the circuit (STG2) returning again to repeat baseline measures 72 h and 96 h post STG1. Participants were asked to refrain from exercise for the duration of the experimental period, and from treating symptoms of muscle damage.Participants completed a modified version of a validated protocol, described in , designeThirty minutes prior to commencing each STG, each participant consumed a snack . Immediately following the STG, participants were provided with either 500 mL of cow’s milk  or 500 mL of an energy-matched carbohydrate solution, which was consumed within 30 min. Participants were not blinded to the beverage received. Macronutrient composition (per 500 mL) of the cow’s milk was as follows: Energy 910 kJ/215 kcal, Protein 17.0 g, Carbohydrate 25.5 g, and Fat 5.0 g. The energy-matched carbohydrate solution  consisted of glucose mixed with water and a flavoured cordial. Each participant completed a food diary for 24 h prior to testing and for the subsequent 4 days. Participants were provided with a weighing scale and measuring jug and were instructed to follow their usual eating habits. Analysis  indicated that there were no differences in energy, carbohydrate, protein or fat intake between groups.−1). Protein Carbonyls (PCs) were determined using a commercially available kit  utilising a methodology based on that described by Levine et al. [Each day, a blood sample was collected by venepuncture from a forearm vein in serum separator (SST) tubes. A sample was also collected 2 h post-completion of the STG. Samples were centrifuged, aliqouted and stored at −70 °C for later analysis. Serum creatine kinase (CK) and high sensitivity C-reactive protein (hsCRP) were determined singly using high-sensitivity procedures . Lipid hydroperoxides (LOOHs) were determined singly using the ferrous oxidation of xylenol orange method (FOX 1), using a modification of previous methods ,19. Absoe et al. .Muscle soreness during the completion of a squat to approximately 90° knee flexion, and during isokinetic testing, was measured on a visual analogue scale (VAS), with subjects rating soreness on a scale of 0 (no soreness) to 10 (as bad as it could be). A similar VAS was used to measure muscle tiredness.Symptoms of stress were monitored using the Daily Analysis of Life Demands (DALDA) questionnaire . The queTo determine peak torque (Nm), participants completed three dominant-leg maximal effort knee extension and flexion repetitions at 60°/s and 180°/s, with 60 s recovery between speeds, on a Biodex System3 Isokinetic dynamometer . Rate of force development (RFD) was determined over 100–200 ms of an isometric contraction . BrieflyData analysis was carried out by making probabilistic magnitude-based inferences about the true values of the effect of intervention on outcomes by expressing the uncertainty as 90% confidence limits (CLs) . Within-Post-STG1 analysis of within-group effects revealed decreases in peak torque, CMJ, RSI, sprint performance and RFD, increases in CK and hsCRP, and soreness and tiredness. Minimal effects were seen on oxidative stress markers. The impact of STG2 on muscle function was minimal, with mostly trivial or unclear outcomes, though there was a trivial decrease in sprint performance. STG2 resulted in increased CK, hsCRP for CHO, and LOOHs for MILK. Mean effects, ±90% CI, with qualitative inferences and effect size are presented in Baseline (B) peak torque values for knee extension at 60°/s for MILK and CHO were 156.7 ± 27.3 Nm and 163.0 ± 14.9 Nm respectively. There was a benefit for MILK at B-24 h (−5.9 ± 6.8% vs. −10.0 ± 5.8%) and B-48 h (−4.5 ± 8.6% vs. −9.3 ± 7.8%). B-72 h effects were unclear and B-96 h effects were trivial. For 60°/s leg flexion baseline values for MILK and CHO were 81.4 ± 9.3 Nm and 81.7 ± 11.2 Nm. A benefit of MILK was observed at B-24 h (−4.2 ± 13.7% vs. −11.3 ± 7.8%), B-48 h (−4.7 ± 12.4% vs. −14.1 ± 13.5%) and B-72 h (−2.2 ± 13.8% vs. −10.6 ± 15.8%). See Baseline values for knee extension at 180°/s were 106.5 ± 19.8 Nm and 113.0 ± 11.9 Nm for MILK and CHO respectively. Outcomes were trivial  and unclear . For knee flexion at 180°/s, baseline values for MILK and CHO were 67.8 ± 11.7 Nm and 66.9 ± 11.3 Nm. A benefit for MILK was seen at B-48 h (−3.0 ± 2.8% vs. −8.9 ± 6.3%). Other comparisons were unclear.−1 and 448.1 ± 151.9 Nm·s−1. Unclear outcomes were observed B-24 h, B-72 h and B-96 h, with a trivial outcome at B-48 h. Baseline CMJ values for MILK and CHO were 25.8 ± 4.5 cm and 27.7 ± 10.6 cm. Comparisons at all timepoints were unclear.Baseline values for RFD were 429.2 ±125.2 Nm·s−1 and 113.6 ± 18.3 cm·s−1 for MILK and CHO. Comparisons at B-24 h and B-48 h were unclear. Comparisons from B-72 h (−17.3 ± 10.4% vs. −7.2 ± 24.9%) and B-96 h (−9.5 ± 10.8% vs. −0.3 ± 25.2%) revealed a possible small harmful effect of milk.RSI values at baseline were 105.8 ± 17.2 cm·sBaseline values for 5 m sprint performance were 1.17 ± 0.10 s and 1.18 ± 0.04 s for MILK and CHO. A benefit of MILK was seen at B-24 h (−1.4 ± 3.0% vs. −3.1 ± 6.3%), B-48 h (−1.5 ± 4.1% vs. −3.9 ± 7.0%) and at B-72 h (−2.3 ± 4.4% vs. −5.0 ± 9.5%). Comparisons at B-96 h were unclear. See All comparisons of soreness during a squat were unclear. For soreness during isokinetic testing, a possible benefit was seen for MILK at B-24 h (51.0 ± 7.4 vs. 65.5 ± 13.8%). Other comparisons were unclear. For tiredness, a trivial outcome was noted at B–24 h with a benefit for MILK at B-72 h (49.0 ± 17.9 vs. 60.0 ± 19.4%). Other comparisons were unclear.For DALDA B, a benefit of MILK was observed at B-24 h (11.6 ± 8.9% vs. 21.2 ± 11.3%), B-48 h (17.2 ± 14.4% vs. 32.0 ± 11.8%) and B-72 h (16.8 ± 15.5% vs. 29.6 ± 14.6%), with an unclear outcome at B-96 h. See Baseline CK values were 196.2 ± 130.0 and 188.6 ± 125.0 U/L for MILK and CHO respectively. Unclear outcomes were found all timepoints. See Mean effects for all measures of muscle function, soreness and tiredness, symptoms of stress, CK, hsCRP, LOOHs and PCs can be seen in Changes in STG variables from STG1 to STG2 were observed. Analysis of within-group effects revealed a decrease in 5 m sprint performance, but a likely increase in 15 m sprint performance for both MILK and CHO. Trivial effects were seen for CMJ, lap time and HR for MILK and CHO. HR was likely lower in STG2 for CHO, but the outcome was trivial for MILK. Changes can be seen in Comparisons of MILK vs. CHO for 5 m sprint performance a, 15 m sThis study is the first to examine the effect of cow’s milk on recovery from repeated simulated team-sport games in females. The principal findings were that cow’s milk had a positive effect on attenuating losses in peak torque, sprint performance and symptoms of stress, and on increases in some measures of muscle soreness and tiredness following STG1. Cow’s milk did not benefit any variable within STG2. STG2 did not lead to a secondary decrease in muscle function , soreness, tiredness or perceptions of stress. There was a post-STG2 increase in CK and in hsCRP (CHO) and LOOHs (MILK).The consumption of cow’s milk had a beneficial effect on muscle function, specifically peak torque and sprint performance (5 and 10 m). Similar benefits have been noted when cow’s milk was consumed following repeated sprinting and jumping in females . This beThe effect of cow’s milk on measures of soreness and tiredness was primarily unclear, though a likely benefit for MILK was noted for tiredness from baseline-72 h. Given that soreness is a subjective measure and that this investigation employed an independent groups design, it is difficult to compare conditions. Cow’s milk had beneficial effects on perceptions of the symptoms of stress, as previously observed , with paComparison of hsCRP data indicates a likely harmful effect of MILK over the 48 h post STG1, though an unclear outcome following STG2. This early benefit for CHO is a result of a decrease in hsCRP levels from baseline to 2 h and baseline to 48 h, while the MILK group showed increases during this time. It has been shown that carbohydrate consumption attenuates increases in inflammatory markers following intermittent exercise . HoweverIn accordance with previous research with female athletes ,36, the Interestingly, despite cow’s milk benefiting muscle function measures over the recovery period, a comparison of sprint performance and CMJ from STG1 to STG2 revealed no clear benefit of MILK. Of relevance, Mohr et al.  highlighSurprisingly, STG2 had little impact on measures of muscle function, soreness, tiredness or perceptions of stress. Differences from 48–72 h and 48–96 h were typically trivial or unclear with some measures  continuing to return towards baseline values despite the demands of STG2, the exception being sprint performance. Nonetheless, performance of all muscle function variables remained below baseline at 96 h, which has implications for coaches and athletes in terms of training programmes and competition fixtures. The two-fold increase in the pattern of CK was as expected, with an increase observed following STG1, returning towards baseline before a second increase following STG2. This is similar to the pattern observed following repeated match play ,8, thougLimitations of this study include the small sample size and the use of an independent groups design. The latter, however, was chosen to reduce the impact of a Repeated Bout Effect whereby participation in an exercise bout with a high eccentric load results in reduced muscle damage in the next subsequent bout of exercise . DespiteIn conclusion, the present study clearly demonstrates that the consumption of cow’s milk following two simulated team games separated by 48 h can attenuate losses in peak torque, sprint performance and symptoms of stress compared to an energy-matched carbohydrate drink in female athletes, with benefits also for some measures of soreness and tiredness. From a practical perspective, this study indicates that cow’s milk may serve to enhance the recovery of female athletes following team-sport participation. No benefit of cow’s milk was observed for sprint performance, jump height or lap times within the second STG protocol, which questions how transferable isolated measures of recovery are to real match performances. Further research is necessary to elucidate whether cow’s milk can affect within-game performance variables when recovery periods are limited.


Coarse particulate matter (PM10–2.5) exposure has not been extensively studied, particularly for long-term exposure, and the biological mechanisms remain uncertain.Exposure to particulate matter air pollution has been associated with cardiovascular disease (CVD) morbidity and mortality; however, most studies have focused on fine particulate matter  enrolled in the longitudinal Study of Women’s Health Across the Nation (SWAN) at six study sites from 1999 to 2004. Residential locations and the USEPA air monitoring network measurements were used to assign exposure to one-year PM10–2.5, as well as co-pollutants. Linear mixed-effects regression models were used to describe the association between PM10–2.5 exposure and markers, including demographic, health and other covariates.We examined the association between ambient concentrations of PM3) increase in one-year PM10–2.5 exposure was associated with a 5.5%  increase in levels of plasminogen activator inhibitor-1 (PAI-1) and 4.1%  increase in high-sensitivity C-creative Protein (hs-CRP). Stratified analyses suggested that the association with PAI-1 was particularly strong in some subgroups, including women who were peri-menopausal, were less educated, had a body mass index lower than 25, and reported low alcohol consumption. The association between PM10–2.5 and PAI-1 remained unchanged with adjustment for PM2.5, ozone, nitrogen dioxide, and carbon monoxide.Each interquartile (4 μg/m10–2.5 exposure may be associated with changes in coagulation independently from PM2.5, and thus, contribute to CVD risk in midlife women.Long-term PMThe online version contains supplementary material available at 10.1186/s12940-020-00663-1. Cardiovascular disease (CVD) is the leading cause of death for men and women in the US . Risk fa2.5 and PM10, respectively) .Analyses were performed in SAS 9.4 . All tests were two-sided and After applying the exclusions described above, 1694 women with 5982 observations during the study period were available for analyses. Approximately 68% of the women had three or more clinic visits between 1999 and 2004. The race/ethnicity distribution by study site for this population reflected the SWAN sampling strategy discussed previously. Over 50% of women in Chicago, Los Angeles, and Oakland had completed college, while in Detroit, Newark, and Pittsburgh, approximately 27% of the women had completed college. Most women in the study were overweight (25 < BMI ≤ 30) or obese (BMI ≥ 30), with the exception of women from the Oakland and Los Angeles sites. Additionally, most women in this study were neither current smokers nor high alcohol consumers (2+ servings/week or > 0.3 servings/day) , and had a slightly larger sample size (N = 5466) than the one-year exposure (N = 5175), because PM2.5 measurements started from 1998 which limited the ability to calculate prior one-year exposure for some early visits. Los Angeles site had the highest levels of one-year PM10–2.5 exposure at 17.5 ± 2.3 μg/m3, while Pittsburgh had the lowest levels at 8.5 ± 3.8 μg/m3. Due to the limited availability of PM10 monitors, however, Los Angeles only had a small number of visits with co-located PM10 and PM2.5 monitors, and thus matched PM10–2.5 data. Distributions of one-year average exposure of co-pollutants, including PM2.5, ozone, CO, NO2 and SO2 exposure, can be found in Table SThe average prior one-year PM3) increase in the prior one-year exposure of PM10–2.5 was associated with a 5.5%  increase in PAI-1 level. This association was consistent for the six-month exposure window, with a 4.0%  increase in PAI-1 per 4 μg/m3 increase in PM10–2.5. The inflammation marker, hs-CRP, was also associated with PM10–2.5, with a 4.1%  and a 6.0%  increase in hs-CRP per 4 μg/m3 increase in prior one-year and six-month PM10–2.5 exposure, respectively. Associations with other markers were not observed.We observed that an interquartile  in women with a BMI <  25, 7.5%  in peri-menopausal women, 7.6%  in women with equivalent or less than a high school education, and 7.5%  in women who reported low alcohol consumption. Additionally, women who reported low alcohol consumption had an 8.8%  increase in hs-CRP, and women with equivalent or less than a high school education had 2.4%  decrease in factor VIIc, per 4 μg/m3 increase in PM10–2.5. The stratification results for the other inflammatory and hemostatic markers were not statistically significant.Potential effect modifiers were evaluated for the one-year PMks Table . For a 410–2.5, given the availability of co-pollutant data. For most sites, 70–98% of serum samples had matched co-pollutant exposure, except for ozone in Detroit (0.5%)  and Newark (48%), NO2 in Detroit (57%), Chicago (65%), and Newark (65%), and SO2 in Oakland (53%) (Table S10–2.5 exposure appeared to be moderately correlated with one-year PM2.5 (r = 0.29) and NO2 (r = 0.30). The interquartile ranges for the prior one-year average exposures of co-pollutants was 3 μg/m3 for PM2.5, 0.007 ppm for ozone, 0.3 ppm for CO, 5 ppb for NO2, and 3.0 ppb for SO2.The co-pollutant models were conducted in subsets of observations compared with the single pollutant models of PM2.5, NO2, and CO were positively associated with PAI-1 [10–2.5, PM2.5, CO, and NO2 were each still positively associated with PAI-1 levels, and the associations were stronger for PM2.5, CO, and NO2 than PM10–2.5. In contrast, O3 had a negative association with PAI-1. Nevertheless, these co-pollutants did not confound the association between PM10–2.5 and PAI-1  increase per 4 μg/m3 increase in PM10–2.5. Similarly, PAI-1 increased by 4.8%  per 4 μg/m3 increase in PM10–2.5 after adjustment for co-exposure to CO, and by 4.3%  per 4 μg/m3 increase in PM10–2.5 independent of exposure to NO2. Confounding by SO2 cannot be evaluated, because in the subset of data with matched SO2 measurements, PM10–2.5 showed no association with PAI-1 regardless of including SO2 or not. The availability of SO2 measurements may have introduced bias for testing the association between PM10–2.5 and PAI-1.Our previous studies have found that one-year exposures to PMth PAI-1 , 19. In 10–2.5 exposure levels were positively associated with the levels of PAI-1, and marginally positively associated with hs-CRP. Elevated PAI-1 increases the risk of thrombosis by inhibiting tPA activity, which initiates the fibrinolysis processes that break down excess blood clotting. Therefore, the associations we found suggested that elevated long-term exposure to coarse particles may increase the risk of thrombosis.This study provided new evidence for associations between long-term exposure to coarse particles and CVD markers. Specifically, we found that prior one-year PM10–2.5 with CVD markers. Chen and Schwarz observed increased white blood cell count, a potential inflammatory marker, was associated with one-year local PM10 levels (ranging 14.6–78.5 μg/m3) in the US [10 (ranging 11.0–36.1 μg/m3) [10–2.5 exposures between 2000 and 2006 varied by individuals, with a mean of 5.0 ± 1.7 μg/m3, and found positive, though not statistically significant, associations between long-term PM10–2.5 concentrations and inflammation and coagulation markers [10–2.5 contributed most to inflammation and coagulation effects, respectively, which were robust to the adjustment for PM2.5. As an indicator of abrasive brake wear, copper is expected in substantial amount in PM10–2.5 at most of the SWAN sites, which are major metropolitan areas with intense traffic flow, particularly in Los Angeles [10–2.5 coagulation among different locations and populations.As mentioned earlier, very few studies were conducted on the associations of long-term exposure to PMn the US . In cont1 μg/m3) . Inconsi markers . The aut Angeles . Our stu2.5 and ambient gases with inflammatory and hemostatic markers in the SWAN cohort. One-year exposure to PM2.5 was associated with increases in hs-CRP  and PAI-1  per 10 μg/m3 increase in PM2.5 concentrations [2 were also positively associated with PAI-1, though not statistically significant [10–2.5 was similar to that from PM2.5, PM10–2.5 exposure was weakly correlated with PM2.5, regardless of whether long-term  or short-term  exposure. The two-pollutant analyses confirmed the associations with PM2.5 and ambient gases, and further demonstrated that PM10–2.5 created additional burden on inflammation and coagulation. The previous studies did not control for PM10–2.5, so some of the associations observed may have been due to confounding by PM10–2.5.Our previous studies have identified associations between exposures to PMtrations . One-yeaI-1  pe10–2.5 cause health effects through mechanisms different from those of PM2.5. However, it has been observed that coarse PM of low density, such as soil particles, have deposited deeper in the lung as well [2.5 and PM10–2.5 affected nasal and bronchial epithelial cells and immune response differently, but both increased release of IL-6 in bronchial epithelial cells [10–2.5 sourced from the Los Angeles Basin for 1 month, and found that PM10–2.5 was the only particles contributing to this process [10–2.5 may have stronger associations with inflammation and coagulation than PM2.5 [Considering the variations in chemical composition and the deposition locations within the lungs associated with their sizes, we expect that PM as well , suggestal cells . Ljubimo process . A few tan PM2.5 –34.10–2.5 particles travel deeper in the lung and cause systemic inflammation and oxidative stress similar to that from PM2.5. Generated by the endothelium, PAI-1 is activated by chronic inflammatory conditions and endothelium injury to inhibit fibrinolysis, the process that degrades blood clots; in other words, PAI-1 allows blood clot formation and shortens bleeding time. Another possible pathway is that PM10–2.5 induces respiratory tract inflammation. Inflammatory cytokines produced in the respiratory tract can potentially enter the circulatory system where they can stimulate the liver to release coagulation factors that can alter hemostasis [10–2.5. Given the marginal/unstable association with hs-CRP and absent relationship with fibrinogen and factor VIIc, we cannot conclude whether inflammation and thrombosis were from the same biological process, or differentiate whether such coagulation reflected vascular inflammation or deep vein thrombosis. Future studies are warranted to explore the physiological consequences and mechanisms involved with chronic exposure to PM10–2.5, especially to the specific chemical species of PM10–2.5.Furthermore, risks derived from short-term and long-term exposure may be generated through different mechanisms. Coagulation activation, indicated by the increase of PAI-1 level, is a typical risk that has been associated with long-term exposure . Two potmostasis . This co10–2.5 exposure appeared particularly strong in some subgroups, including women with a BMI <  25, women who were peri-menopausal, women with equivalent or less than a high school education, and women who reported low alcohol consumption. That is to say, apart from toxicity of chemicals, physiological condition, i.e., BMI and menopausal status, and socioeconomic background, i.e., education, could influence vulnerability to external exposures. For women, the menopausal transition is a complex and multifaceted process that involves multiple organ systems and genetic variability where physiological impacts can manifest differently among individual women [Our findings also suggest that the associations between PAI-1 and one-year PMal women . Being i10–2.5 exposure. Second, because residential history has been maintained for each SWAN participant, we were able to use ambient monitoring data, along with residential history, to assign exposure levels to participants. Finally, substantial demographic and health information collected in SWAN allowed us to examine the impact of effect modifiers, including time-varying menopausal status for this midlife population.This study had several strengths. First, SWAN is a large, multi-site, longitudinal study, which provided coverage of a wide range of ambient exposure levels and particle compositions associated with local sources. Also, with the same participants followed longitudinally and the same sampling/analytical approaches for measurement of biomarkers consistently used, our data provided a unique opportunity to examine health effects associated with long-term PM10–2.5 exposure need to be noted when interpreting our results. PM10–2.5 was not routinely measured directly, its level was either calculated through the ambient monitor concentrations of PM10 and PM2.5 as we did, or obtained by modeling. Some studies used estimations from land use regression models, which were based on limited cross-sectional measurements to simulate long-term spatial variation [10–2.5 exposures of the Multi-Ethnic Study of Atherosclerosis cohort were estimated using land use regression model, ranging from 3.8 ± 1.3 μg/m3 in Winston-Salem, North Carolina to 5.6 ± 1.2 μg/m3 in Chicago, Illinois [10–2.5 concentrations were not commonly estimated through satellite data in the USA, as PM2.5 has been the primary focus. We acknowledge that PM10–2.5 concentration tends to be more heterogeneous across space than PM2.5, resulting in great uncertainty for PM10–2.5 estimations through nearby stationary monitors within a 20 km buffer. The assigned exposure may be overestimated or underestimated depending on whether there are sources nearby. We assume that the exposure assignment error tends to be non-differential [10–2.5 concentrations using data obtained from co-located PM10 and PM2.5 monitors to avoid potential bias. However, PM10 monitoring had been greatly reduced , which limited the ability to calculate PM10–2.5 data. This was a particular concern for the Los Angeles site, where only 24% of women had matched PM10–2.5 measurements. The mean average one-year PM10–2.5 exposure in our study ranged from 8.5 ± 3.8 μg/ m3 to 17.5 ± 2.3 μg/m3 at different sites, which aligned with the levels reported in the literature.Meanwhile, the limitations of estimating PMariation , 16. Theerential . Additio10–2.5 compositions by study location may influence the associations with biomarkers, but composition information was not available to use in our study.A few other limitations also need to be considered. First, as a common issue in cohort studies, loss of follow-up was also observed in SWAN, which reduced statistical power. In our dataset, 35% of participants had completed one or two visits only, including almost all participants at Newark site and half of the participants at Los Angeles site, which was equivalent to 20% of visits. We were not able to run the regression model within these subjects because of the small sample size; however, sensitivity tests excluding these participants confirmed the associations we observed


V) channel subtypes, including NaV1.7, are promising targets for the treatment of neurological diseases, such as chronic pain. Cone snail-derived µ-conotoxins are small, potent NaV channel inhibitors which represent potential drug leads. Of the 22 µ-conotoxins characterised so far, only a small number, including KIIIA and CnIIIC, have shown inhibition against human NaV1.7. We have recently identified a novel µ-conotoxin, SxIIIC, from Conus striolatus. Here we present the isolation of native peptide, chemical synthesis, characterisation of human NaV channel activity by whole-cell patch-clamp electrophysiology and analysis of the NMR solution structure. SxIIIC displays a unique NaV channel selectivity profile (1.4 > 1.3 > 1.1 ≈ 1.6 ≈ 1.7 > 1.2 >> 1.5 ≈ 1.8) when compared to other µ-conotoxins and represents one of the most potent human NaV1.7 putative pore blockers (IC50 152.2 ± 21.8 nM) to date. NMR analysis reveals the structure of SxIIIC includes the characteristic α-helix seen in other µ-conotoxins. Future investigations into structure-activity relationships of SxIIIC are expected to provide insights into residues important for NaV channel pore blocker selectivity and subsequently important for chronic pain drug development.Voltage-gated sodium (Na V) channels are highly conserved pore-forming proteins permeable to sodium ions and are important for the initiation and propagation of action potentials . χ1. χ126]. y scores . The finThe NMR solution structure of SxIIIC is composed of a series of turns followed by a C-terminal α-helix between residues Arg13 and Asp17 d. MediumV1.7, along with NaV1.1, 1.3, 1.6, 1.8, and 1.9, plays an important role in pain signalling and has shown promise as a drug target for pain therapies. Toxins isolated from highly venomous creatures have evolved to potently and specifically target NaV channels and offer a great starting point as drug leads. However, a major limitation in their success has been the high sequence homology of the NaV channel pore, which could lead to undesirable side effects caused by off-target inhibition. As most toxins modulators that target the therapeutically relevant subtype NaV1.7 act as gating modifiers and bind to the voltage-sensing domain, they offer limited insights into the channel pore interactions. Despite µ-conotoxins NaV1.7 pore blockers being less potent than gating modifiers, they may be used to further our understanding of potency and selectivity for NaV channel. In this body of work, we have isolated a novel µ-conotoxin, SxIIIC, from C. striolatus, characterised the selectivity profile against hNaV channel subtypes and determined the NMR solution structure.NaC. striolatus transcriptome [V channel subtypes by automated whole-cell patch-clamp electrophysiology. SxIIIC displayed potent inhibition of hNaV1.4 (IC50 15.11 ± 10.7 nM) which is characteristic of most other µ-conotoxins, and may provide an evolutionary advantage as NaV1.4 is expressed exclusively in skeletal muscle and cone snail venom acts rapidly to immobilise prey [V1.4, synthetic SxIIIC was significantly less potent against hNaV1.2 (IC50 363.8 ± 53.8 nM). This was surprising as many other µ-conotoxins preferentially target NaV1.4 and NaV1.2 [V1.3, as mentioned earlier [V1.7 is consistent with that of the native material, which inhibited NaV1.7 expressed in SH-SY5Y cells. Biological activity of the alternate folding arrangements remains to be determined.We first used activity-guided fractionation to isolate native SxIIIC from crude venom and subsequently determined the peptide sequence by Edman degradation. This sequence was identical to STRIO_41, recently reported from a criptome . The unucriptome . When weise prey . Interesd NaV1.2 , with th earlier . HoweverV1.7, potent inhibition of this isoform is nonetheless unusual for the µ-conotoxins. Only two µ-conotoxins have reported nanomolar activity against hNaV1.7 isoforms: KIIIA and CnIIIC (485 nM) [V1.7 with an IC50 of 147 nM [50 of 363 nM which is approximately two-fold less active than SxIIIC. At hNaV1.7, the wash-off rate of SxIIIC was markedly slower than that of KIIIA assessed under identical experimental conditions. Notably, previous reports of a more readily reversible KIIIA [V1.7. Notably, the protocols used to compute IC50 values at each subtype do not directly permit calculation of on-rates, although these appeared comparatively slower at NaV1.4 and NaV1.7  . In 2011f 147 nM . Howeverf 147 nM , KIIIA wle KIIIA  could bele KIIIA , and we d NaV1.7 . FurtherV channel subtypes, particularly within the pore region, these findings may indicate subtle differences that could be exploited to further our understanding of potent NaV1.7 inhibition. Compared to KIIIA, the N-terminal tail of SxIIIC is extended by two residues, while the peptide sequence between the second and third cysteines (referred to as loop 1) is extended by four residues. It is possible that the extended loops of SxIIIC permit more interactions with NaV channels than KIIIA, and we thus sought to determine the NMR structure of SxIIIC by 2D homonuclear NMR for comparison to other μ-conotoxins and to provide a foundation for future structure-activity relationship studies. SxIIIC structure is composed of a C-terminal α-helix and is stabilised by three disulfide bonds. A marginally shorter α-helix seen in the final structure compared to the earlier predicted structure is likely explained by additional restraints applied during refinement.Given the high sequence homology between µ-conotoxins d and theV channels provide insights into the molecular basis of µ-conotoxin binding [V channel inhibition, including Lys7, Trp8, Arg10, Asp11, and His12 (KIIIA numbering) [V1.2 structure  and skeletal subtypes (NaV1.4) and may have influenced the observation of decreased potency at NaV1.2 [V1.7 (IC50 1.3 ± 0.23 µM) [V channel inhibition and selectivity.Recent landmark cryo-EM studies of hNa binding ,43. The  binding ,35,38. Rmbering) ,45. SxIImbering) . It is ltructure , residuet NaV1.2 . Interes0.23 µM) . FurtherV1.7 inhibitors, acting as a presumptive pore blockers, and presents as a promising peptide for NaV channel inhibitor development.In conclusion, we discovered a novel µ-conotoxin SxIIIC, produced the peptide synthetically and evaluated its pharmacological and structure characteristics. To date, SxIIIC is one of the most potent µ-conotoxin hNa


As sodium level in diabetic ketoacidosis (DKA) and hyperglycemic hyperosmolar state (HHS) is usually low, normal, or slightly elevated, severe hypernatremia with DKA and/or HHS is rare. Case 1 was a 14-year-old boy, presenting with typical laboratory test values and symptoms consistent with DKA and HHS. His corrected sodium level, 172 mEq/L, might have occurred as a result of consuming 6 L/day of highly carbonated, carbohydrate- and sodium-rich drinks during the week preceding the diagnosis. This patient developed right lung artery thrombosis, which did not require treatment. Case 2 was a 10-year-old girl, presenting with typical laboratory test values and symptoms of DKA and HHS. Her corrected sodium level, 175 mEq/L, might have occurred as a result of large electrolyte-free water loss associated with osmotic diuresis. These two cases of patients presenting with DKA-HHS and severe hypernatremia are the first to be reported in Japan. Diabetic ketoacidosis (DKA) and hyperglycemic hyperosmolar state (HHS) are complications that present in the acute phase of diabetes mellitus (DM). They are associated with increased risk of mortality -2. DKA pHyperglycemic hyperosmolar state is defined based on the following laboratory values: 1) BG > 600 mg/dL, 2) venous pH > 7.25 and/or arterial pH > 7.30, 3) HCO3- > 15 mmol/L, 4) mild ketonuria or absent to mild ketonemia, 5) plasma osmolarity > 320 mOsm/kg, and 6) altered level of consciousness  or seizures [To date, few cases of pediatric DKA-HHS with severe hypernatremia have been only reported, as DKA tends to present with hyponatremia or eunatremia, while HHS tends to present with hyponatremia, eunatremia, or mild hypernatremia due to sodium chloride excretion . In 2014Case 1Case 1 was a 14-year-old Japanese boy who had been prescribed methylphenidate hydrochloride for attention deficit hyperactivity disorder (ADHD). He was born at 40 weeks of gestational age, with a birth weight of 2690 g (-1.2 SD) and birth length of 46.4 cm (-1.7 SD). He had a height of 172.0 cm (+1.35 SD), body weight of 63.0 kg (+1.0 SD), and body mass index (BMI) of 21.3 kg/m2 (+0.6 SD). His body weight was 72.0 kg a few weeks before admission. There was no family history of DM. One month before the diagnosis of DM, the patient had polydipsia and thirst. In the week preceding the diagnosis, he consumed over 6 L/day of a sports drink  and 2.5 L/day of milk . He presented with generalized fatigue and received 0.5 L/day intravenous drip (DIV) of 0.9% saline with 2.6% dextrose  continuously for two days for thirst, vomiting, and fatigue. However, his symptoms did not improve and therefore, was referred to our hospital. He was restless but able to maintain conversation. His vital signs are shown in Table We administered 0.58%-0.9% saline and insulin at 0.05 U/kg/h via DIV. Finally, his sodium level returned to the normal range after 112 hours from admission Figure . On admiOne year postadmission, the patient has a height of 172.8 cm (+0.9 SD), body weight of 80.7 kg (+2.2 SD), and BMI of 27 kg/m2 (+1.7 SD). His treatment consists of 24 units/day of insulin degludec and 35 units/day of insulin aspart (0.73 units/body weight/day). He was diagnosed with T1DM, complicated with DKA-HHS and severe hypernatremia due to soft drink ketosis.Case 2Case 2 was a 10-year-old Japanese girl, with no significant past medical history. She was born at 39 weeks of gestational age, with a birth weight of 3202 g (+1.1 SD) and birth length of 49.5 cm (+0.4 SD). She had a height of 148.6 cm (+1.8 SD), body weight of 26.2 kg (-0.9 SD), and BMI of 11.9 kg/m2 (-3.6 SD). Her body weight was 31.1 kg one month prior to presentation There was no family history of DM. She was referred to our hospital after presenting with generalized fatigue, polydipsia, and polyuria. She was able to maintain conversation but was moderately restless in general. Her vital signs are shown in Table We administered 0.58%-0.9% saline and insulin 0.05 U/kg/h via DIV. Finally, her sodium level returned to the normal range after 78 hours from admission Figure . She didSix months postadmission, the patient is 146.4 cm (+1.2 SD) tall, with a body weight of 33.4 kg (-0.6 SD) and BMI of 13.6 kg/m2 (-2.1 SD). Her treatment consists of 10 units/day of insulin degludec and 19 units/day of insulin lispro (0.87 units/body weight/day). She was diagnosed with T1DM, complicated with DKA-HHS and severe hypernatremia.To the best of our knowledge, this is the first report of two pediatric cases of DKA-HHS with severe hypernatremia, including a case with complications of asymptomatic rhabdomyolysis, thrombosis of right pulmonary artery, pneumomediastinum, and subcutaneous emphysema simultaneously, in Japan.Diabetic ketoacidosis-hyperglycemic hyperosmolar state occurs in 13.8% of pediatric hyperglycemic emergencies . It is nHyperglycemic hyperosmolar state tends to present with hyponatremia, eunatremia, or slight hypernatremia, as HHS is induced by dilution of extracellular fluid and/or dehydration of osmotic diuresis. DKA may present with mild hypernatremia due to severe dehydration. However, exact mechanism of severe hypernatremia is unknown . The mecAmong the severe complications associated with DKA is brain edema. Slow correction of hyperglycemia and hypernatremia lowers the risk of brain edema . In pediThe present cases were difficult to diagnose. Case 1 was diagnosed with T2DM because insulin-related antibodies were all negative, and 24 hours urine C-peptide did not show absence of insulin. However, he was not obese and required insulin therapy (0.73 units/body weight/day), indicating that he may have slowly progressive T1DM. Case 2 was diagnosed with T1DM because the IA-2 antibody was weakly positive. She was also not obese and required insulin therapy (0.87 units/body weight/day). However, 24 hours urine C-peptide showed presence of insulin. She may still have T2DM, but further observation is necessary.We report the first two Japanese pediatric cases of DKA-HHS with severe hypernatremia. DKA-HHS results in more complications than only DKA or HHS. The findings from these cases should be considered to address the possibility of pediatric patients developing rarer complications of diabetes, including rhabdomyolysis, thrombosis, pneumomediastinum, and subcutaneous emphysema. We believe that careful fluid therapy in DKA-HHS prevents severe life-threatening complications such as brain edema or death, although several complications except for the life-threatening ones may occur.


Rhynchanthus beesianus W. W. Smith, an edible, medicinal, and ornamental plant, is mainly cultivated in China and Myanmar. The essential oil (EO) from R. beesianus rhizome has been used as an aromatic stomachic in China. The chemical composition and biological activities of EO from R. beesianus rhizome were reported for the first time. Based on gas chromatography with flame ionization or mass selective detection (GC-FID/MS) results, the major constituents of EO were 1,8-cineole (47.6%), borneol (15.0%), methyleugenol (11.2%), and bornyl formate (7.6%). For bioactivities, EO showed a significant antibacterial activity against Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, and Proteus vulgaris with the diameter of the inhibition zone (DIZ) (8.66–10.56 mm), minimal inhibitory concentration (MIC) (3.13–6.25 mg/mL), and minimal bactericidal concentration (MBC) (6.25–12.5 mg/mL). Moreover, EO (128 μg/mL) significantly inhibited the production of proinflammatory mediators nitric oxide (NO) (92.73 ± 1.50%) and cytokines tumor necrosis factor-α (TNF-α) (20.29 ± 0.17%) and interleukin-6 (IL-6) (61.08 ± 0.13%) in lipopolysaccharide (LPS)-induced RAW264.7 macrophages without any cytotoxic effect. Moreover, EO exhibited significant acetylcholinesterase (AChE) inhibitory activity (the concentration of the sample that affords a 50% inhibition in the assay (IC50) = 1.03 ± 0.18 mg/mL) and moderate α-glucosidase inhibition effect (IC50 = 11.60 ± 0.25 mg/mL). Thus, the EO could be regarded as a bioactive natural product and has a high exploitation potential in the cosmetics and pharmaceutical industries. Essential oils are volatile complex compounds with a strong odor that are formed by aromatic plants . About 3The Zingiberaceae family comprises approximately 52 genera and 1600 species and is mainly distributed in the tropical and subtropical regions . Many spRhynchanthus J. D. Hooker, a small genus in the family Zingiberaceae, has approximately four species and is mainly distributed in Myanmar, Indonesia, and Southern China -2,5 diphenyl tetrazolium bromide (MTT) assay. Compared with the untreated control cells, R. beesianus EO showed no significant cytotoxicity on L929 and RAW264.7 cells at 16−128 μg/mL (α in LPS-induced RAW264.7 macrophages at doses of 64 μg/mL (2731.02 ± 1.80 pg/mL) and 128 μg/mL (2451.02 ± 3.52 pg/mL). The maximum inhibition rate of EO (20.29 ± 0.17% at 128 μg/mL) was comparable to that of DXM (21.34 ± 0.20% at 20 μg/mL). Additionally, compared with the LPS group (974.28 ± 4.15 pg/mL), EO effectively inhibited the secretion of IL-6 in RAW264.7 cells induced by LPS at doses of 16 μg/mL (803.81 ± 28.82 pg/mL), 32 μg/mL (738.81 ± 5.87 pg/mL), 64 μg/mL (550.48 ± 8.30 pg/mL), and 128 μg/mL (379.17 ± 0.31 pg/mL). In particular, the inhibitory ratios of EO at 64 μg/mL (43.50 ± 1.09%) and 128 μg/mL (61.08 ± 0.13%) were exceeded that of DXM (21.90 ± 0.14% at 20 μg/mL) (The anti-inflammatory activity of the EO from 28 μg/mL . Therefo28 μg/mL A. The ac0 μg/mL) D. α and IL-6) play key roles in the development of the inflammation process, and compounds that inhibit their overproduction may be good candidates in the treatment of many inflammation-related diseases [R. beesianus EO, reduced LPS-induced inflammation by inhibiting TNF-α and IL-6 [α-terpineol, and α-pinene, have been demonstrated in the previous reports [R. beesianus EO. These results suggest that the R. beesianus EO can be used as a new source of natural anti-inflammatory molecules in the cosmetics and pharmaceutical industries.The proinflammatory mediator (NO) and cytokines  and butyrylcholinesterase (BChE) were investigated and the results are shown in The inhibitory effect of 50 = 11.60 ± 0.25 mg/mL). The inhibitory activities of 1,8-cineole and methyleugenol against α-glucosidase were reported in the previous studies [R. beesianus EO could be a natural source of potent α-glucosidase inhibitors.α-Glucosidase inhibitors, such as acarbose, have been used to treat type 2 diabetes by reducing post-meal blood glucose and insulin levels . Pre-dia studies ,39. Henc50 = 53.71 ± 4.89 mg/mL). Tyrosinase is a key enzyme which involved the catalysis of the only rate-limiting step in mammalian melanogenesis, so tyrosinase inhibitors are used to treat hyperpigmentation in the human skin ,41. As s50 value of 1.03 ± 0.18 mg/mL. However, EO showed a weak inhibitory effect on BChE (IC50 = 104.22 ± 11.61 mg/mL). According to the previous studies, 1,8-cineole and α-pinene showed a strong AChE inhibitory activity and were found to be uncompetitive reversible AChE inhibitors [α-terpineol has been demonstrated in the previous studies [R. beesianus EO can be used as a new source of AChE inhibitor in the pharmaceutical industry.The inhibition of cholinesterase can reduce the decomposition of acetylcholine to enhance cholinergic neurotransmission, which has become an effective treatment strategy for Alzheimer’s disease . EO exerhibitors ,43,44. A studies . TherefoThe fresh R. beesianus rhizome was collected from Guangxi Province of China in July 2019. The identity of this species was confirmed by Prof. Guoxiong Hu of Guizhou University. The voucher specimen (Voucher No: RB-20190711) was deposited at National & Local Joint Engineering Research Center for the Exploition of Homology Resources of Southwest Medicine and Food, Guizhou University.w/w). EO was dried over anhydrous Na2SO4, filtered, and stored at 4 °C until further analysis.The fresh, finely chopped rhizomes (2.0 kg) were subjected to hydrodistillation for 4 h using a Clevenger-type apparatus to obtain the essential oil  and EI mode (70 eV). The interface temperature and ion source temperature were 280 °C and 230 °C, respectively. The relative percentage of chemical constituents was determined by the peak area. The retention index (RI) was determined by referring to a series of n-alkanes (C8–C21). The constituents of the EO were identified by comparison of their retention index and mass spectrum with those listed in NIST 2017 and Wiley 275 databases. The EO was analyzed by an Agilent 6890 gas chromatograph (GC) equipped with a flame ionization detector (FID). Capillary column: HP-5MS . The injection volume was 1 μL and split injection was used (split ratio 1:20). The flow rate of carrier gas helium was set at 1 mL/min. The following GC oven temperature was used: kept at 70 °C (2 min), 2 °C per min to 180 °C (55 min), 10 °C per min to 310 °C (13 min), and held at 310 °C (4 min). The GC-MS analysis was carried out using an Agilent 6890 gas chromatograph equipped with an Agilent 5975C mass selective detector . GC column and parameters were the same as in GC-FID. The mass spectra operated in the mass range  was evenly spread on the Mueller–Hinton agar medium. The filter paper discs (diameter 6 mm) containing EO or streptomycin solution (20 μL) were added and the diameter of inhibition zone (DIZ) was recorded after 24 h incubation at 37 °C.The diameter of the inhibition zone (DIZ) was assayed according to our previously published disc agar diffusion method . Briefly5 CFU/mL. The 96-well plates were incubated for 24 h at 37 °C. Then, 20 μL of resazurin solution (0.1 mg/mL) was added to each well and incubated for 2 h in the dark at 37 °C. The MIC was determined as the minimum sample concentration without color change. For the determination of MBC value, the sample (10 μL) from the wells without color change was subcultured in a Mueller–Hinton agar plate for 24 h at 37 °C. MBC was defined as the minimum sample concentration without bacterial growth.The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values were evaluated by the microplate dilution method . Briefly2 atmosphere in Dulbecco’s modified eagle medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 medium, respectively. Both media were supplemented with 2 mM glutamine, 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin. The cytotoxic activity was determined by the MTT assay with slight modification [4 cells per well and incubated for 24 h. Subsequently, 100 μL of the diluted EO solution was added and cultured for 24 h. Then, the MTT  solution  was added. After 4 h of incubation, the supernate was discarded and 150 μL of DMSO was added to each well to dissolve formazan crystal. The absorbance was measured using a Varioskan Lux Multimode microplate reader  at 490 nm to evaluate the cell viability and proliferation inhibitory ratio.Murine macrophages RAW264.7 and murine fibroblast cells L929 were maintained at 37 °C with 5% COfication . The cel4 cells per well and allowed to grow for 24 h. After removing the medium, the cells were incubated with 100 μL of two-fold serially diluted EO solution for 2 h. Then, 100μL lipopolysaccharide (LPS) was added to the final concentration of 1 μg/mL and incubated for another 24 h. Morphological changes of the treated RAW264.7 cells were recorded using an inverted microscope . Subsequently, the supernatants were collected and centrifuged. The accumulation amount of NO in the supernatant was detected using a colorimetric NO detection kit following the manufacturer’s instructions . The secretion of IL-6 and TNF-α was determined using respective ELISA determination kits according to the manufacturer’s instructions . Dexamethasone (DXM) (20 μg/mL) was used as a positive control.The RAW264.7 cell suspensions (100 μL) were added to each well at the density of 2 × 102CO3 solution , and finally the absorbance was measured at 405 nm. Acarbose was used as a positive reference. The α-glucosidase inhibitory effect was expressed using IC50 values.The α-glucosidase inhibitory activity was assayed according to the method previously described with slight modification . EO solu50 values.The inhibitory activity of tyrosinase was evaluated according to the method with L-tyrosine as a substrate . EO solu50 values.The inhibitory effects of cholinesterase including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were assayed according to Ellman’s method with marginal modification . EO soluP < 0.05).All experiments were performed independently at least three times, and the data were expressed as the mean ± standard deviation (SD). SPSS software (version 19.0) was used for statistical analysis. The significant difference between the two groups was compared by one-way analysis of variance (ANOVA) using Tukey’s multiple range tests  and cytokines (TNF-α and IL-6) in LPS-induced RAW264.7 cells without any cytotoxic effect. Furthermore, EO revealed a moderate α-glucosidase inhibition effect and significant AChE inhibitory activity. Thus, the R. beesianus EO could be regarded as a source of bioactive products with high exploitation potential in the cosmetic and pharmaceutical industries.To our knowledge, the chemical composition, antibacterial, anti-inflammatory, and enzyme inhibitory activities of the essential oil from