Aspergillus fumigatus EFBL was purified to their electrophoretic homogeneity by gel-filtration and ion-exchange chromatography, with overall purification folds 2.8 and yield 9.43%. The apparent molecular subunit structure of the purified A. fumigatus amidase was 50 kDa, with highest activity at reaction temperature of 40 °C and pH of 7.5 The enzyme displayed a significant thermal stability as revealed from the value of T1/2 (13.37 h), and thermal denaturation rate (Kr 0.832 × 10–3 min) at 50 °C, with metalloproteinic identity. The purified enzyme had a significant activity for acrylamide degradation in various food products such as meat, cookies, potato chips, and bread as revealed from the HPLC analysis and LC–MS analysis. So, with the purified amidase, the acrylamide in the food products was degraded by about 95% to acrylic acid, ensuring the possibility of using this enzyme in abolishing the toxic acrylamide in the foods products. This is the first report exploring the potency of A. fumigatus amidase for an actual degradation of acrylamide in foods efficiently. Further biochemical analyses are ongoing to assess the affinity of this enzyme for selective hydrolyses of acrylamide in foods, without affecting the beneficial stereochemical related compounds.Acrylamide is the major by-product of the Maillard reactions in foods with the overheating processes of L-asparagine-rich foods with reducing sugars that usually allied with neurotoxicity and carcinogenicity. Several approaches have been used to prevent the formation of acrylamide, however, degrading the already formed acrylamide in foods remains unequivocal. Acrylamide hydrolyzing enzyme “amidohydrolase” is one of the most promising enzymes for acrylamide degradation in foods. So, amidohydrolase “amidase” from thermotolerant The online version contains supplementary material available at 10.1007/s00284-023-03544-1. Acrylamide is one of the major by-products of Maillard reactions in foods during the processing, due to the reactivity of L-asparagine with the reducing sugars by overheating at 120 °C for at least 25 min , thus acRalstonia eutropha, Bacillus licheniformis, Enterobacter aerogenes, Stenotrophomonas acidaminiphila, and Moraxella osloensis  w–28 w24–2assessed .The experimental fungal isolates were identified based on their macroscopic and microscopic features by growing on PDA and Czapek-Dox media, according to the reference keys –32. The 425 nm, and the concentration of ammonia was calculated from the inference of authentic concentrations of ammonium sulfate [The most potent acrylamide-decomposing fungal isolate was grown on potato dextrose broth medium with 0.5% acrylamide for 10 days at 30 °C, then the fungal biomass was collected by filtration, and then intracellular crude proteins were extracted , 37–39.  sulfate , 24, 25. sulfate , regardiAspergillus fumigatus was grown on potato dextrose broth medium amended with 0.5% acrylamide, incubated at the desired conditions, the fungal pellets were collected, and then washed by potassium phosphate buffer. The fungal biomass was pulverized in liquid nitrogen and the intracellular crude proteins were extracted [xtracted , 39, 41.xtracted , 37, 38 xtracted –35, 39. xtracted . The mosxtracted –38. The xtracted , normaliA. fumigatus amidase such as reaction temperature, reaction pH, and thermal stability were investigated [2+, Fe3+, Ca2+, Hg2+, Fe3+, Al3+, Zn2+, Na+, Cu2+) for 2 h at 4 °C at final concentration 1 mM and then assessing the residual enzymatic activity. Different amino acid analogues such as 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB), hydroxylamine, guanidine thiocyanate, iodoacetate, 3-methyl-2-benzo-thiazolinone hydrazone (MBTH), and phenylmethylsulfonyl fluoride (PMSF) were incubated with the enzyme at 1 mM for 2 h at 4 °C and then measured their residual enzyme activity.The biochemical properties of the purified stigated . The reastigated , 43–45. 203 nm [203.The functionality of amidase to degrade acrylamide in different food products mainly meat, bread, cookies, and potato chips was determined . One gra203 nm , compareThe prepared homogenates of the food products were defatted with hexane, evaporated, and the concentration of acrylamide and its degradation by-products were determined . The acrAspergillus fumigatus EFBL has been deposited to the GenBank with accession # MW737636.1 and at Assiut University Mycological Center, Egypt, with deposition # AUMC14078.The experiments were performed in triplicates, and the results were expressed by mean ± STDV. The statistical analysis was assessed using one-way ANOVA  test, and the means were compared with Duncan’s test at 0.05 level.Aspergillus fumigatus EFBL (MW737636.1), A. flavus (MT951414.1), A. niger, and A. awomari exhibited the highest reliable growth on acrylamide-containing PDA media , unlike the noticeable tolerance of acrylamide toxicity displayed by A. fumigatus. At 0.5% acrylamide, the growth of A. flavus, A. awomari, and A. niger was reduced by about 80–90%, comparing to acrylamide-free media. However, a noticeable tolerance to acrylamide toxicity has been observed by A. fumigatus, as revealed from their visual growth and biomass, and at 0.5% acrylamide the fungal growth was reduced by about 5% comparing to control. Remarkably, the deep blue-colored conidial pigmentation of A. fumigatus EFBL was subsequently faded with the higher acrylamide concentration. The morphological feature of the potent acrylamide-hydrolyzing fungal isolate “Aspergillus fumigatus” is shown in Fig. S2. The morphological identity of the A. fumigatus was confirmed from the ITS sequence that has been deposited on GenBank with accession number MW737636.1. The tolerance of A. fumigatus EFBL to acrylamide reveals their possessing to powerful detoxifying system of acrylamide-degrading enzymes “amidases” that counteract the acrylamide toxicity by converting it to acrylic acid and ammonia.Twenty-five fungal isolates were preliminary screened for acrylamide degradation by growing on modified potato dextrose agar with 0.2% acrylamide. A visual fluctuation was observed on the ability of the tested fungi to grow on acrylamide-containing PDA media (Data not shown). Among the experimented fungi, four isolates, namely dia Fig. . The morAspergillus fumigatus was grown on the PDB medium containing acrylamide (0.5%) for 8 days at 30 °C, then the fungal pellets were collected, washed and their intracellular proteins were extracted, and further purified by ion-exchange and gel-filtration chromatography. The overall purification profile of amidase from A. fumigatus EFBL is shown in Table A. fumigatus amidase was assessed by incubating the mixture at 10, 20, 30, 40, 50, and 60 °C and then measuring the enzyme activity by the standard assay. A gradual increase in the activity of A. fumigatus amidase was observed with the temperature, till maximum activity at 30–40 °C, with a noticeable reduction in the enzyme activity by about 20%, comparing to control (35 °C)  of the purified A. fumigatus amidase was 250, 75.7, 29.9, and 16.3 h, at 4, 20, 30, and 40 °C, respectively, while the thermal denaturation rate (Kr) of amidase was 0.002 × 10–3, 0.15 × 10–3, 0.245 × 10–3, and 0.499 × 10–3 min, at 4, 20, 30, and 40 °C, respectively. Obviously, the thermal structural denaturation rate of the enzyme was increased with the higher temperature and with pre-incubation time. The influence of reaction pH (2–10) on the activity of purified A. fumigatus amidase was assessed. A remarkable increase in the activity of amidase with the reaction pH, till maximum activity at pH 7.0, is followed by a decrease in the enzyme activity with higher pHs (pH 10) , i.e., by about nine-fold reduction to acrylamide upon enzyme treatment. However, the acrylic acid concentration of bread samples was increased to 34.0 μg/g in bread treated with enzyme, ensuring the enzyme functionality . The half-life time (T1/2) of the purified A. fumigatus acrylamidase was 250, 75.7, 29.9, and 16.3 h, at 4, 20, 30, and 40 °C, respectively. The thermal denaturation rate (Kr) of amidase at 4 and 40 °C was 0.002 × 10–3 and 0.499 × 10–3 min. A noticeable reduction to the enzyme activity was observed at highly acidic and alkaline pHs. Similar biochemical properties of acrylamidase were reported [2+, Cu2+, and Ba2+, followed by monovalent cations K+ and Na+ at 1 mM. The enzyme activity was reduced by ~ 30% in response to PMSF, MBTH, and DTNB, ensuring the implantation of surface thiols amino acids on the catalytic sites of enzyme [A. fumigatus was assessed in the experimental mice, with no signs of toxicity, based on the tested biochemical and hematological parameters [ terreus  and Cuprccus sp.  have the44.5 kDa , 19. Therameters .A. fumigatus acrylamidase in acrylamide degradation in food products such as meat, bread, cookies, and potato chips was assessed. The acrylamide content in the tested food products was extremely reduced upon implementation of amidase, comparing to control. The acrylamide content in meat was reduced by 3.4 folds in response to the enzyme, ensuring the efficient functionality of enzyme in foods applications. The acrylamide content of cookies was dramatically reduced by about 20 folds, upon the enzymatic treatment. The dramatic reduction of acrylamide contents in the food products ensures the functionality of amidase in degradation of acrylamide into ammonia and acrylic acids that could be a co-supportive approach with L-asparaginase as a pre-acrylamide formation control. The purified acrylamidase from S. acidaminiphila had a significant activity in degradation of authentic acrylamide into ammonia and acrylic acid [Lactobacillus acidophilus has the ability to produce acrylamidase [R. eutropha, X. maltophilia, Pseudomonas sp., E. aerogenes, and V. boronicumulans were reported to be acrylamidase producers [A. fumigatus acrylamidase, ensuring the efficiency of enzyme in food applications. Similarly, bioconversion of authentic acrylamide into acrylic acid upon treatment with acrylamidase from S. acidaminiphila, was reported [A. fumigatus acrylamidase in food applications, negating the possibility of toxic metabolites that might be released from using of native microorganisms.The activity of the purified lic acid , 22, 24.lamidase , 47. Conroducers , 15, 16 roducers , 24. Thereported , 49. TheA. fumigatus EFBL to its molecular homogeneity by gel-filtration and ion-exchange chromatography and their overall biochemical properties were studied. The acrylamide-degrading activity of the purified enzyme has been assessed in various food products. The acrylamide contents of the processed foods such as meat, cookies, potato chips, and bread were strongly reduced by ~ 90% in response to the enzyme treatment, comparing to control food samples, as revealed from the HPLC analysis. Upon addition of the purified amidase to the tested food products, the acrylamide was completely degraded to acrylic acid as revealed from the LC/MS profile, ensuring the functionality of this enzyme in food applications. Interestingly, this is the first report exploring the potency of amidase to hydrolyze the acrylamide compound in food products, authenticating their efficiency, selectivity to the acrylamide compound, and resistance to the other chemicals compounds that might inhibit their activity in foods. However, further biochemical analyses are required to assess the affinity of this enzyme for selectively hydrolyze acrylamide in foods, without affecting amino acids, and beneficial compounds that might be stereochemically similar to acrylamide.Acrylamide amidase was purified from Supplementary file1 (PPTX 3649 kb)Below is the link to the electronic supplementary material.


Patients and Methods. PD patients who were recruited from January 2016 to November 2017  and evaluated again at a 2-year ± 30-day follow-up (V2) from 35 centers in Spain from the COPPADIS cohort were included in this longitudinal prospective study. Subjects were classified as with or without drooling according to item 19 of the NMSS  at V0, V1 (1-year ± 15 days), and V2 for patients and at V0 and V2 for controls.  Drooling in Parkinson's disease (PD) is frequent but often goes underrecognized. Our aim was to examine the prevalence of drooling in a PD cohort and compare it with a control group. Specifically, we identified factors associated with drooling and conducted subanalyses in a subgroup of very early PD patients. p < 0.0001), 43.7% (264/604) at V1, and 48.2% (242/502) at V2 (3.2% (4/124) in controls; p < 0.0001), with a period prevalence of 63.6% (306/481). Being older , being male , having greater nonmotor symptom (NMS) burden at the baseline , and having a greater increase in the NMS burden from V0 to V2  were identified as independent predictors of drooling after the 2-year follow-up. Similar results were observed in the group of patients with ≤2 years since symptom onset, with a cumulative prevalence of 64.6% and a higher score on the UPDRS-III at V0  as a predictor of drooling at V2.  The frequency of drooling in PD patients was 40.1% (277/691) at V0 (2.4% (5/201) in controls;  Drooling is frequent in PD patients even at the initial onset of the disease and is associated with a greater motor severity and NMS burden. Sialorrhea, commonly referred to as drooling, is defined as excessive saliva beyond the margin of the lip. Drooling can be a complication of Parkinson's disease (PD) and is one of the most prevalent complaints of patients, but it is often underrecognized and undertreated . A wide Although many studies have analyzed the frequency of drooling in PD, there is less information about its prevalence and associated factors in early PD patients and how it impacts QoL and change over time. Some studies have reported a prevalence of about 20% in de novo and untreated PD patients and that prevalence increases in the long term , 28. Ourhttps://bmcneurol.biomedcentral.com/articles/10.1186/s12883-016-0548-9 [PD patients who were recruited from January 2016 to November 2017  and evaluated again at a 2-year ± 30-day follow-up (V2) from 35 centers in Spain from the COPPADIS cohort  were inc6-0548-9 .Information on sociodemographic aspects, factors related to PD, comorbidity, and treatment were collected. Motor status, nonmotor symptoms (NMS), QoL, and disability were assessed at V0 and at V2 using different validated scales: Hoehn and Yahr (H&Y), UPDRS-III and UPDRS-IV, Freezing of Gait Questionnaire (FOGQ)), Parkinson's Disease Cognitive Rating Scale (PD-CRS), Nonmotor Symptoms Scale (NMSS), Beck Depression Inventory-II (BDI-II), Parkinson's disease sleep scale (PDSS), Neuropsychiatric Inventory (NPI), Questionnaire for impulsive-compulsive disorders in Parkinson's Disease-Rating Scale (QUIP-RS), visual analog scale-pain (VAS-Pain), Visual Analog Fatigue Scale (VAFS)), the 39-item Parkinson's Disease Questionnaire (PDQ-39), the EUROHIS-QOL 8-item index (EUROHIS-QOL8), and ADLS  . In patiV0+V1+V2, from 0 to 36) were calculated. Patients reporting drooling during the three visits were defined as patients with “persistent drooling.” The same method was used to define dysphagia (item 20 of the NMSS) [Subjects were classified as with or without drooling according to item 19 of the NMSS . This ithe NMSS)  and hypohe NMSS) .t-test, Mann–Whitney U test, chi-square test, or Fisher test were used as appropriate (distribution for variables was verified by one-sample Kolmogorov–Smirnov test).Data were processed using SPSS 20.0 for Windows. For comparisons between PD patients in the control group and PD patients with and without drooling, the Student's V0+V1+V2 score as the dependent variable), respectively. Variables with univariate associations with p values <0.20 were included in a multivariable model, and a backward selection process was used to remove variables individually until all remaining variables were significant at the 0.10 level. For exploring the association between drooling and QoL, linear regression models were used with PDQ-39SI  and EUROHIS-QOL8  as dependent variables. The total domain scores of the PDQ-39 were expressed as a percentage of the corresponding maximum possible score, and a summary index was obtained as an average of the domain scores (PDQ-39SI). The effect was controlled by age, gender, disease duration, LEDD, comorbidities  and drooling severity (NMSS-Droolingan drugs ), motor an drugs .p value was considered significant for all analyses when it was <0.05.For PD patients, analyses were conducted in the entire cohort and in the subgroup of patients with ≤2 years of disease duration since symptoms' onset (PD ≤ 2 y) at the baseline. The For this study, we received approval from the Comité de Ética de la Investigación Clínica de Galicia in Spain . Written informed consents from all participants in this study were obtained. COPPADIS-2015 was classified by the AEMPS (Agencia Española del Medicamento y Productos Sanitarios) as a postauthorization prospective follow-up study with the code COH-PAK-2014-01.p < 0.0001) in the control group than in PD patients (2.4% at V0 and 3.2% at V2 in controls). In the patients  with assessments carried out during all visits , 63.6% (306/481) of them reported drooling at least once during the study . Specifically, 18.9% (91/481) in only one visit, 19.1% (92/481) in two out of the three visits, and 25.6% (123/481) in all three visits   and 206 patients in the control group  were considered valid for the analysis. The frequency of drooling in PD patients was 40.1% (277/691) at V0; 43.7% (264/604) at V1; 48.2% (242/502) at V2 . At V0 arooling) . In the rooling) .V0+V1+V2 score was higher in patients with persistent drooling (p < 0.0001): drooling in one visit, 1.95 ± 1.67 (N = 91); drooling in two out of the three visits, 4.22 ± 2.95 (N = 92); persistent drooling, 10.36 ± 6.12 (N = 123). Drooling was more frequent in patients with dysphagia than in those without dysphagia: 60% (96/160) vs. 34.1% (181/531) (p < 0.0001) at V0; 57.9% (99/171) vs. 38.1% (165/433) (p < 0.0001) at V1; 55.9% (76/136) vs. 45.4% (60/166/366) (p = 0.023) at V2 , at V1 , at V2 , and after considering all visits together . Drooling was also more frequent in patients with hypomimia than in those without hypomimia at V0 , at V1 , and at V2  , at V1 , at V2 , and after considering all visits . Similar results were observed in the PD ≤ 2 y group, with significant correlations between drooling burden and dysphagia burden  and between drooling burden and hypomimia burden  after considering the sum of the burden of all visits during the follow-up. Drooling was more frequent in patients with dysphagia at V0 and at V1 and with hypomimia at V2 than in those patients with these symptoms in the PD ≤ 2 y group  at V2 . Droolin= 0.002) . A signi y group . Regardip < 0.0001), older age , and a higher LEDD   . Patient 0.0001) , and aut 0.0001) . In the  0.0001) . Compare 0.0001) .p = 0.019), to be male , to have a higher score on the UPDRS-III  and the NMSS , and to have dysphagia  were independent factors associated with drooling at the baseline (p = 0.001). Like as seen with baseline predictions, being older , being male , having a greater NMS burden at the baseline , and having a greater increase in the NMS burden from V0 to V2  were identified as independent predictors of drooling after the 2-year follow-up (p < 0.0001). Moreover, to have drooling at the baseline multiplied by 6 , the probability of drooling at V2 after adjustment must be receiving anticholinergic drugs and the other covariates of the model. In the PD ≤ 2 y group, a higher UPDRS-III score at V0 was the only predictor of drooling at V2 identified  (p = 0.011). Finally, to have a greater NMS burden at the baseline  and a greater increase in the NMS burden from V0 to V2  were the most significant factors associated with drooling severely at V2 in the entire cohort, whereas to have at the baseline, a greater score on the UPDRS-III  and the NMSS  were in the PD ≤ 2 y group  . Specifi y group . Similarβ = 0.180; 95% CI, 2.928–6.992; p < 0.0001) and at V2  and also with a worse global QoL (EUROHIS-QOL8 as dependent variable) at V0  and at V2 . In the PD ≤ 2 y group, drooling was associated with a worse health-related QoL at V0  and at V2  and with a worse global QoL at V0  as well. However, after adjustment to covariates defined in the methods, the association between drooling and both health-related and global QoL at V0 and at V2 was not significant, not even when persisting drooling or the NMSS-DroolingV0+V1+V2 score was considered in the model. A correlation was observed between the NMSS-DroolingV0+V1+V2 score and the score on both PDQ-39SI and EUROHIS-QOL8 at V2 in the entire cohort ; EUROHIS-QOL8, r = −0.222 (p < 0.0001)) and in the PD ≤ 2 y group ; EUROHIS-QOL8, r = −0.304 (p = 0.001)). QoL at V2 was worse in patients with persistent drooling in both the entire cohort ; EUROHIS-QOL8, 3.64 ± 0.51 vs. 3.8 ± 0.59 (p < 0.005)) and in the PD ≤ 2 y group ; EUROHIS-QOL8, 3.54 ± 0.53 vs. 3.88 ± 0.57 (p = 0.006)). Finally, by domains, drooling was an independent factor associated with a worse “Activities of daily living”  and “Communication”  at V0 in the entire cohort.With regard to QoL, drooling was associated with a worse health-related QoL (PDQ-39SI as the dependent variable) at V0 (The present study represents one of the largest cohorts of PD patients in whom the prevalence of drooling was reported using a validated global NMS scale. We observed that drooling was common in PD patients, clearly much more frequent than in the control group, and was associated with the male gender, older age, and a greater motor and nonmotor severity. In addition, patients with drooling had a worse global and health-related QoL, although the effect of drooling on QoL was not significant after adjusting to other covariates. Importantly, we observed that drooling was also a very frequent symptom at the beginning of the disease, as seen in the very early PD patients, suggesting the clinical importance of asking for the presence of drooling at the beginning of the patient's follow-up.About 2 out of every 3 patients from the Spanish cohort COPPADIS reported drooling over a 2-year follow-up. This cumulative prevalence is in line with the previously published data . HoweverIn addition to male gender, many other variables have been associated with drooling in PD patients such as dysphagia , 42, dysThe present study has some important limitations. Drooling was considered based on an answer to a simple clinical question from the NMSS and not after using a specific scale , 41. HowIn conclusion, this study observes a high prevalence of drooling in PD patients, clearly much more so than in control subjects, and that this feature is frequent even at the first stages of the disease as well. Dysphagia is associated with drooling, and a higher motor score and a greater NMS burden are predictors of drooling. PD patients with drooling have a worse QoL, and drooling is also an independent factor associated with communication problems. Thus, drooling screening and therapeutic options should be considered in clinical practice.


Mentha sp. leaves with improved antioxidant properties and, for the first time, with optimal antimicrobial activity. Among the solvents evaluated, water was selected as the extractant in order to develop a green procedure and also for its improved bioactive properties . MAE operating conditions were optimized by means of a 3-level factorial experimental design , and further applied to the extraction of bioactives from 6 different Mentha species. A comparative LC-Q MS and LC-QToF MS analysis of these MAE extracts was carried out for the first time in a single study, allowing the characterization of up to 40 phenolics and the quantitation of the most abundant. Antioxidant, antimicrobial  and antifungal  activities of MAE extracts depended on the Mentha species considered. In conclusion, the new MAE method developed here is shown as a green and efficient approach to provide multifunctional Mentha sp. extracts with an added value as natural food preservatives.A microwave-assisted extraction (MAE) procedure has been optimized to simultaneously provide multifunctional extracts of  In the last few years, there has been an increased interest in searching for new functional ingredients of natural origin that provide foods with added health benefits ,2,3. In Mentha Linneo belonging to the Lamiaceae family comprises about 19 species of aromatic plants widely distributed through temperate areas of Europe, Asia, Australia, Africa and North America −). Data acquisition and processing were performed using Agilent Mass Hunter Workstation Acquisition Rev. B.02.00 software.The instrument employed for these analyses was an Agilent 1200 Series LC system coupled to a 6520 quadrupole-time of flight (QToF) mass spectrometer (Agilent Technologies), using an ESI interface working in the negative-ion mode. The electrospray voltage was set at 4.5 kV, the fragmentor voltage at 150 V and the drying gas temperature at 300 °C. Nitrogen (99.5% purity) was used as the nebulizer (207 kPa) and drying gas (6 L min−1. The mobile phase was a binary mixture of solvent A (water with 0.1% acetic acid) and B (acetonitrile with 0.1% acetic acid), according to the following gradient: 0 min: 5% B; 20–25 min: 36% B; 35 min: 90% B; 36–50 min: 5% B. The injection volume was 5 µL, and the column temperature was maintained at 25 °C.Chromatographic separations were carried out on a reverse-phase Luna C18 analytical column   operating at a flow rate of 0.4 mL min−1 range. Goodness of fit for these calibration curves and reproducibility of the method were previously assured. Results were expressed as milligrams per gram of dry sample.Quantitation of phenolics was performed in triplicate using external standard calibration curves within the 0.01–100 µg·mL−1 Na2CO3  were added, and the samples were vortexed briefly. The Eppendorfs were then allowed to stand in the dark for 20 min at room temperature. Following this, the samples were centrifuged at 13,000 r.p.m. for 3 min, and their absorbance was read (n = 3) at 750 nm. The same procedure was repeated with aqueous solutions of gallic acid in the 10–100 mg L−1 concentration range to build up a calibration curve. Results were expressed as gallic acid equivalents (GAE) (mg mL−1 or mg·g−1 dry sample).The TPC of MAE extracts was determined using the Folin-Ciocalteu (2N) reagent and gallic acid as standard (both from Sigma-Aldrich), according to the method optimized by Singelton et al. , with slMentha sp. extracts was measured (n = 3) in terms of hydrogen donating or free radical scavenging ability using the stable DPPH radical method [The antioxidant activity of l method . AliquotI (%) = ((controlA − sampleA)/controlA) × 100, where controlA is the absorbance of the control reaction , and sampleA is the absorbance of the test sample. The concentration of extract providing 50% inhibition (IC50) was estimated from the curve plotting of I (%) against the extract concentration. Antioxidant reagent Trolox (Sigma-Aldrich) was used as positive control. Results were also expressed as mg of Trolox equivalents (TE) g−1 of dry sample or as mg of TE mL−1 of extract.The inhibition percent of the free radical was estimated using the following equation: Mentha sp. were tested against Gram (+) bacteria , Gram (−) bacteria (Escherichia coli (CECT 515) and Salmonella typhimurium (CECT 4594)) and against the yeast Candida albicans (ATCC 32354). These activities were determined by the agar disk diffusion assay according to Palá-Paúl et al. [C. albicans. Once the incubation was complete, the diameters of the growth inhibition halos and the bacteriostatic effect (growth rate inhibition) were measured in centimeters (including the diameter of discs). Measurements were carried out in duplicate.The antibacterial and antifungal activity of MAE extracts of l et al. , with slp < 0.05) of differences was evaluated by analysis of variance .Data were subjected to statistical analysis by using Statistica 7.0 software . Significance . Moreover, the aqueous MAE extract was the only one showing antimicrobial activity against Staphylococcus aureus and Escherichia coli bacteria and against the yeast Candida albicans. Regardless of the solvent considered, no extract was active against Salmonella typhimurium. The greatest inhibition halo of bacterial growth provided by this aqueous extract was for S. aureus (1.7 ± 0.3 cm)  . TherefoM. rotundifolia using different water volumes (12 and 15 mL), but identical temperature and time conditions , were compared. As no significant (p < 0.05) differences were observed in the antioxidant activity of both experiments (7.1 mg TE mL−1), 12 mL of water were selected as optimal to minimize the volume of extractant required.In order to select the optimal s/V ratio, results from the extraction of 1 g of TPCR, DPPHR and AMR). The antioxidant activity measured in terms of Trolox equivalents (TE) varied between 6.1 and 8.9 mg mL−1, and the TPC between 4.5 and 5.8 mg·GAE mL−1. In general, the highest values of both responses were obtained at high temperatures (100 °C), while at 50 °C, the extracts presented low TPC concentrations, as well as low antioxidant activity. Regarding the antimicrobial activity (AMR), extracts obtained at a low temperature (50 °C) showed no activity against S. aureus, whereas those subjected to MAE at 75–100 °C gave rise to growth inhibition halos in the 1.1–1.7 cm range .The optimization of the extraction temperature and time was subsequently carried out by means of a 3-level factorial experimental design. DPPHR and TPCR. Regarding the extraction time, TPCR presented its optimal value (5.91 mg·GAE mL−1) for extractions of 30 min, while the maximum response for DPPHR (9.203 mg TE mL−1) was obtained for 5 min extractions. T and T·t were the most significant (p < 0.05) variables for TPCR, and the equation of the model was TPCR = 5.024 + 0.0033·T − 0.0552·t + 0.00074·T·t (R2 = 91%). As for antioxidant activity, the only significant (p < 0.05) variable was T, and the equation explaining a percentage high enough of the variability of this response was DPPHR = 3.446 + 0.0615·T + 0.104·t − 0.0018·T·t (R2 = 76%). For antimicrobial activity, high temperatures (93 °C) and intermediate extraction times (19 min) provided the optimal conditions to maximize the growth inhibition halo against S. aureus by MAE extracts (AMR = 1.61 cm). The most significant (p < 0.05) factors in this individual optimization were T and T2, and the equation of the model was AMR = −5.967 + 0.163·T − 0.00088·T2 (R2 = 94%). Finally, when a multiple response (DR = 0.85) that simultaneously maximized DPPHR, TPCR and AMR was considered, 100 °C and 14.7 min were selected as the optimal operating parameters to provide multifunctional extracts of M. rotundifolia. Under these conditions, the calculated and experimental responses were very similar, with only DPPHR showing a higher dispersion (32%)  . First, on (32%) .M. rotundifolia [O-glucoside were extracted in cycles C1 and C2 . Percentages lower than 7% of selected phenolics were also extracted in the third extraction cycle. Because of the longer processing time and higher dilution associated with mixing different extraction cycles, a single MAE cycle was considered as a trade-off to recover most of the target bioactives while keeping the required drying step to a minimum for better extract preservation. Therefore, optimal MAE conditions to obtain multifunctional extracts from M. rotundifolia leaves were 100 °C, 14.7 min, 1 g of dry leaves/12 mL of water and 1 extraction cycle.Under the previously optimized temperature and time conditions, the number of cycles was also evaluated for selected phenolics previously described as major components in ndifolia ,37,38. WMentha species. The compounds present in these extracts were identified by using LC-QToF MS. −) and characteristic MS/MS fragments. A total of 40 compounds were identified, 21 of which were unequivocally identified using commercial standards. Whereas some compounds, such as hesperetin, chlorogenic and rosmarinic acids, catechin and gallocatechin isomer, etc., were detected in all the species, most of the compounds were found to be characteristic for some of them. Thus, for example, a higher number of low-molecular-weight compounds  were present in the extracts of M. longifolia and M. cervina, whereas in the M. spicata extract, compounds of higher molecular weight, such as eriocitrin, diosmin, rutin, etc., were detected. Although many of these compounds have already been described in leaf extracts of some Mentha species  and trimeric (lithospermic acid) forms are the most characteristic phenolic acids of the genus Mentha sp. [M. pulegium. Different isomers of lithospermic acid were also detected mainly in M. spicata and M. suaveolens. Chlorogenic acid, obtained by condensation of caffeic and quinic acids, and its isomers were also detected irrespective of the Mentha species, while other derivatives, such as caftaric acid or the different isomers of salvianolic acid, were only detected in some species. In addition to caffeic acid derivatives, other acids, such as gentisic acid, cinnamic acid, etc., were also detected in the analyzed extracts.The previously optimized MAE method was applied to obtain multifunctional aqueous extracts from leaves of different  spicata , in M. v cervina , in M. rpulegium ), the idivatives , for whiivatives . Althougntha sp. . In thisMentha leaves and, among them, luteolin and its derivatives [O-glucoside and luteolin-7-O-rutinoside were detected in almost all the samples analyzed in this study, although with different abundances. However, apigenin, a flavone previously described in some Mentha species, such as M. arvensis [M. piperita [M. spicata [M. spicata and M. longifolia. Flavonols, such as kaempferol, were also detected in M. pulegium, M. longifolia, M. rotundifolia and M. suaveolens.Regarding flavonoids, flavones are the most characteristic of ivatives . Luteoliarvensis  or M. pipiperita , or flav spicata ,38,45, w−1 of dry sample) of the main phenolic compounds of MAE extracts from different Mentha species was carried out by HPLC-MS in SIM mode , while M. suaveolens had the highest phenolic concentration (14.30 mg·g−1 of dry sample).Quantitative analysis  and p-coumaric acid (0.15 mg·g−1 of dry sample) and intermediate levels of rosmarinic acid (1.7 mg·g−1 of dry sample), similar to those present in the M. rotundifolia extract. These compounds have also been previously detected by Aldogman et al. [Mentha suaveolens L. leaves from Saudi Arabia at different levels. Low concentrations of luteolin and caffeic acid, characteristic of M. suaveolens ethanolic extracts [−1 of dry sample).Among the different n et al.  in Menthextracts , were alM. spicata presented the highest concentration of rosmarinic acid (4.4 mg·g−1 of dry sample), as previously found by Dorman et al. [−1 of dry sample), chlorogenic acid (0.020 mg·g−1 of dry sample) and salvianolic acid B (0.66 mg·g−1 of dry sample) were also detected in this sample at concentrations higher than those of other Mentha species; these compounds were also reported by Rita et al. [M. spicata infusions (concentrations ranging from 1.6 to 7.8 μg·mL−1 extract).n et al.  and Ritan et al. . Caffeica et al.  in M. spM. cervina extracts, concentrations of 4-hydroxybenzoic acid, syringic acid and rosmarinic acid were found in the range of 3–4 mg·g−1 of dry sample. Salvianolic acid A and sagerinic acid were also present at moderate levels in this MAE extract (0.4 mg·g−1 dry sample). The presence of these compounds (except for syringic acid) has been previously reported in the literature in infusions and hydroalcoholic extracts of this species [As for  species ,28,53.Mentha species, MAE extracts of M. longifolia were characterized by low concentrations of rosmarinic acid (0.22 mg·g−1 of dry sample) and high concentrations of 4-hydroxybenzoic acid (3.7 mg·g−1 of dry sample). On the contrary, Krzyzanowska et al. [−1 of dry sample on average) in M. longifolia collected in Poland and in Czechia, respectively, and extremely low concentrations of 4-hydroxybenzoic acid (0.83 µg g−1 of dry sample). Similarly, Bahadori et al. [−1 extract) and sinapic acid (7132 μg·g−1 extract) as the most abundant compounds in infusions from M. longifolia collected in Iran, but 4-hydroxybenzoic acid was not detected. The differences described above may be associated with a number of factors related to the sample analyzed  or the methodology followed for bioactive extraction , among others.In contrast to other a et al.  and Ćavaa et al.  reportedi et al.  identifiM. pulegium were 4-hydroxybenzoic acid and rosmarinic acid , followed by quinic acid (0.092 mg·g−1 of dry sample), sagerinic acid (0.026 mg·g−1 of dry sample) and salvianolic acid A (0.019 mg·g−1 of dry sample). These results are in agreement with the composition previously described by Brahmi et al. [M. pulegium extracts, compounds not detected or detected at trace levels in the MAE extracts analyzed in this work. This discrepancy is probably due to the fact that these phenolic compounds arise from glycosides that were released during a sample hydrolysis process that was not followed in our study.The major phenolic compounds found in the MAE extract of i et al.  for ethaMentha species, the phenolic profile of the M. rotundifolia MAE extract was characterized by a high content of rosmarinic acid (2.5 mg·g−1 of dry sample), as well as high concentrations of syringic acid and salvianolic acid A . There is scarce data of individual phenolics present in M. rotundifolia extracts in the literature. In a study by Yaiha et al. [M. rotundifolia leaves from different geographical areas of Tunisia, rosmarinic acid was also found as one of the most abundant phenolics (ranging between 6.53 and 116.15 mg·g−1 extract). However, salvianolic acid A and syringic acid were not detected. These authors also reported a significant qualitative and quantitative phenolic variability between the studied samples, probably due to the wide diversity of ecological factors characterizing the sites where samples had been collected.Compared to the other a et al.  on the cMentha species under study are shown in M. spicata and M. rotundifolia extracts showed significantly higher TPC values  than the other Mentha species, with M. pulegium extract showing the lowest value (18.7 mg·GAE·g−1 of dry sample). A wide variability in TPC has also been described in previous studies for extracts of different Mentha species. Thus, in a recent study by Ćavar Zeljković et al. [M. suaveolens presented the highest TPC (58.93 mg·GAE·g−1), while M. cervina extracts showed the lowest (14.81 mg·GAE·g−1).The total phenolic content and antioxidant activity (determined by the DPPH method) of MAE extracts from the different ć et al. , methanoMentha, Salvia, Sideritis, etc.) have been described to possess antioxidant activity. However, whereas the number of papers dealing with the TPC and antioxidant activity of Mentha sp. essential oils and extracts  is high [Mentha species (Mentha piperita) [As reported in the literature , essenti is high ,26,56, oiperita) ,25.−1 of dry sample) than alcoholic (ethanol and methanol) extracts obtained by conventional extraction from different Mentha species collected in Algeria  [Mentha viridis and Mentha pulegium: 16.5 and 8.4 mg·GAE·g−1 dry sample, respectively) [M. piperita by MAE using methylene chloride as solvent (40.49 mg·GAE·g−1) [M. piperita from Serbia, obtained using 40–80% ethanol as extractant (64–113 mg·GAE·g−1 of dry sample) [Mentha species from Romania (51–98 mg·GAE·g−1 of dry sample). Similar to the results described here, this last study found the highest concentrations of total phenolic compounds in M. rotundifolia, followed by M. spicata and M. suaveolens [In general, the aqueous MAE extracts obtained in this work showed higher TPC values (18.7–48.6 mg·GAE·g sample)  and in Gctively) , and simGAE·g−1) . However sample) , and thoaveolens .M. spicata (120 mg·TE·g−1 dry sample), followed by M. suaveolens (69 mg·TE·g−1 dry sample); no significant differences were found in the remaining samples evaluated (49–57 mg·TE·g−1 of dry sample). These results are in agreement with those of Pavlic et al. [−1 of dry sample), and are higher than those obtained from this Mentha specie using methylene chloride as MAE extractant (19.79–69.15 mg·TE·g−1) [The highest ability for DPPH free radical scavenging was found in c et al. , who rep·TE·g−1) .IC50 values for aqueous extracts obtained by infusion and by SLE from M. cervina [M. spicata [−1) than those of the aqueous MAE extracts obtained in this work from the same mint species . A similar antioxidant activity  has been previously reported for ethanolic extracts obtained at room temperature, for 24 h, from leaves of M. spicata, M. pulegium and M. rotundifolia collected in Algeria [IC50 values  have also been previously reported by Moldovan et al. [Mentha species. In agreement with previous references [Mentha species, harvesting locations and efficiency for bioactive extraction of all these methodologies could be responsible for the variations observed regarding both the TPC content and antioxidant activity of Mentha extracts.The  cervina , and by  spicata , were hi species . Compare Algeria . Slightln et al.  for 70% ferences ,45,52,57M. spicata), no significant correlations were found between the results of these two assays. Therefore, it is likely that the type and concentration of phenolic compounds, as well as the presence of other compounds with antioxidant activity in MAE extracts, could be responsible for this lack of correlation. Similar results have been previously described by other authors in studies on the antioxidant activity of different types of samples [Although some similarities between the TPC and DPPH results were observed  and Gram (−)  bacteria and of the yeast Candida albicans in the presence of the MAE extracts of the different Mentha species. Both the bactericidal and bacteriostatic effect of each extract were considered for the different strains. It is known that the antimicrobial and antifungal activities depend on the Mentha species considered [Mentha species evaluated, except for M. rotundifolia, presented moderate bactericidal activity against E. coli , the M. cervina, M. suaveolens and M. pulegium extracts were the only ones that showed an additional bacteriostatic effect .nsidered ,45. Althnsidered ,12, the Mentha species other than M. suaveolens were also active against S. aureus . Similarly to previous references [M. rotundifolia and M. spicata extracts were found to be the most active in inhibiting the growth of this microorganism. In addition, M. pulegium and M. cervina were the only samples showing a bacteriostatic effect against S. aureus, with no significant differences in the activity determined for both extracts . Regarding S. typhimurium, all Mentha sp. extracts assayed failed to inhibit the growth of this Gram (−) bacteria. M. cervina was the only species that was active against C. albicans .ferences , M. rotuMentha species [M. pulegium and M. cervina showing the widest inhibitory spectrum. As for different extraction techniques, the MAE aqueous extracts obtained in this work compared favorably with respect to Soxhlet methanolic extracts of M. longifolia [M. longifolia and M. pulegium [M. spicata, M. suaveolens and M. rotundifolia [Although to a lesser extent than essential oils reported in the literature for several  species ,12, MAE ngifolia  and withpulegium , and werndifolia .Mentha species . In agreement with the variable phenolic composition determined by LC-Q MS and LC-QToF MS, the bioactivity of MAE extracts was found to be highly dependent on the Mentha sp. considered. This contribution, in which MAE extracts from six different Mentha species were obtained under identical operating conditions, represents an advantage over previous references, in which the variety of extraction techniques, bioactivity assays, etc. applied to a single or very limited number of Mentha species did not allow the proper comparison of this effect. Moreover, this study evaluates the antimicrobial  activity of Mentha MAE extracts for the first time, thereby extending the multifunctionality of these antioxidant-rich extracts and the number of potential applications  in the food industry, among others.In conclusion, a fast, efficient and environmentally friendly MAE method (using water as the solvent) has been fully optimized, considering a multiple response based on antioxidant capacity and, for the first time, antimicrobial activity, to obtain multifunctional extracts from six different


We aimed to determine a method to identify normal cerebrospinal fluid (CSF) glucose levels by examining the correlation between blood and CSF glucose levels in patients with normal and abnormal glucose metabolism.One hundred ninety-five patients were divided into two groups according to their glucose metabolism. The glucose levels were obtained from CSF and fingertip blood at 6, 5, 4, 3, 2, 1, and 0  h before lumbar puncture. SPSS 22.0 software was used for the statistical analysis.In both the normal and abnormal glucose metabolism groups, CSF glucose levels increased with blood glucose levels at 6, 5, 4, 3, 2, 1, and 0  h before lumbar puncture. In the normal glucose metabolism group, the CSF/blood glucose ratio range was 0.35–0.95 at 0–6  h before lumbar puncture, and the CSF/average blood glucose ratio range was 0.43–0.74. In the abnormal glucose metabolism group, the CSF/blood glucose ratio range was 0.25–1.2 at 0–6  h before lumbar puncture, and the CSF/average blood glucose ratio range was 0.33–0.78.The CSF glucose level is influenced by the blood glucose level 6  h before lumbar puncture. In patients with normal glucose metabolism, direct measurement of the CSF glucose level can be used to determine whether the CSF level is normal. However, in patients with abnormal or unclear glucose metabolism, the CSF/average blood glucose ratio should be used to determine whether the CSF glucose level is normal. Cerebrospinal fluid (CSF) examination is important for the diagnosis of central nervous system (CNS) diseases. A decline in the CSF glucose level is an important indicator for diagnosis of CNS infection and carcinoma. The CSF glucose level is influenced by the serum glucose level. Fluctuations in blood glucose parallel changes in the CSF glucose , 2. Low We collected clinical data from 195 patients in our department between April 2018 and December 2022. All patients were adults who underwent CSF examination for routine diagnostic and treatment purposes. We collected the patients with demyelinating disease of CNS, autoimmune encephalitis, peripheral neuropathy, primary headache, metabolic encephalopathy, degenerative disease, idiopathic intracranial hypertension, motor neuron disease and the patient whose primary disease had been cured, and patients without neurological diseases . The patients with CSF consumption diseases, such as CNS infection, CNS tumor, prion disease, cerebral infarction, SAH and so on, were excluded. All patients signed an informed consent form to participate in the study.We determined the CSF glucose and the fingertip blood glucose levels at 6 (6Pre), 5 (5Pre), 4 (4Pre), 3 (3Pre), 2 (2Pre), 1 (1Pre), and 0 h  before lumbar puncture and calculated the average fingertip blood glucose levels at each of these times (Ave). The glucose metabolism state was determined according to the 2018 American Diabetes Association diagnostic criteria  and the abnormal glucose metabolism group (106 patients). The abnormal glucose metabolism group included patients with diabetes, impaired fasting blood glucose, and impaired glucose tolerance. Patients with a history of abnormal glucose metabolism and those who completed a glucose tolerance test within 1 year were directly included in groups. Those with no clear glucose metabolism status were required to undergo a 75-g glucose tolerance test.In the normal glucose metabolism group and the abnormal glucose metabolism group, the patients were divided into three groups depend on their disease: (1) CNS diseases group, (2) disease of peripheral nervous system group, and (3) others group. Others group including patients with motor neuron disease, patient whose primary disease had been cured and patients without neurological diseases.In the normal glucose metabolism group and the abnormal glucose metabolism group, the patients were divided into two groups according to the use of corticosteroids.p ≤ 0.05 was considered significant. The independent-sample T test was used to analyze the effect of age on CSF glucose and the age differences between the normal glucose metabolism group and the abnormal glucose metabolism group. The bivariate correlation analysis method was used to analyze the correlation between CSF glucose and blood glucose levels at each time point and age. Non-parametric test was used to analyze the gender, diagnosis, use of corticosteroids differences between the normal glucose metabolism group and the abnormal glucose metabolism group.SPSS 22.0 software was used to analyze the data, and a value of The blood glucose and CSF glucose in the normal glucose metabolism group and the abnormal glucose metabolism groupIn the normal glucose metabolism group, the blood glucose level in the 6Pre, 5Pre, 4Pre, 3Pre, 2Pre, 1Pre, and 0Pre groups were 5.51 ± 0.77 mmol/L, 5.57 ± 0.81 mmol/L, 5.54 ± 0.82 mmol/L, 5.86 ± 1.23 mmol/L, 5.99 ± 1.07 mmol/L, 6.50 ± 0.96 mmol/L, 6.41 ± 1.02 mmol/L, respectively. The CSF level was 3.48 ± 0.43 mmol/L.Differences of age, gender, diagnosis and medication between the normal glucose metabolism group and the abnormal glucose metabolism groupIn the abnormal glucose metabolism group, the blood glucose level in the 6Pre, 5Pre, 4Pre, 3Pre, 2Pre, 1Pre, and 0Pre groups were 7.93 ± 2.66 mmol/L, 7.92 ± 2.51 mmol/L, 7.86 ± 2.58 mmol/L, 7.99 ± 2.61 mmol/L, 8.27 ± 2.67 mmol/L, 8.75 ± 2.95 mmol/L, 8.64 ± 2.83 mmol/L, respectively. The CSF level was 4.39 ± 1.15 mmol/L.F = 1.688, p = 0.195).In the normal glucose metabolism group, the patients average age was 34.37 ± 12.74 years old. In the abnormal glucose metabolism group, the average age was 44.37 ± 14.66 years old. There was no difference in age between the two groups (p = 0.962).There were 49 males in the 89 patients of normal glucose metabolism group, 58 males in the 106 patients of abnormal glucose metabolism group. There was no difference in gender between the two groups (p = 0.391).In the normal glucose metabolism group, there 50 patients in the CNS disease group, 13 patients in the disease of peripheral nervous system group, 26 patients in the others group. In the abnormal glucose metabolism group, there 53 patients in the CNS disease group,17 patients in the disease of peripheral nervous system group, 36 patients in the others group. There was no difference in diagnosis between the two groups (p = 0.088).Correlation between CSF glucose and age and genderThere were 47 patients use corticosteroids in the normal glucose metabolism group, and 43 patients use corticosteroids in the abnormal glucose metabolism group. There was no difference in the use of corticosteroids between the two groups (p = 0.373) or gender .In the normal glucose metabolism group, we found no relationship between CSF glucose and age  or gender .Correlation between CSF and blood glucose levels at each time point before lumbar punctureIn the abnormal glucose metabolism group, we found no relationship between CSF glucose and age   .p < 0.05)  .CSF glucIn the normal glucose metabolism group, the CSF/blood glucose ratio range was 0.35–0.95 in the 0–6Pre groups, and the CSF/average blood glucose ratio was 0.43–0.74. The distribution of the CSF glucose/blood glucose ratio in each group and the average value showed little variation .In the abnormal glucose metabolism group, the CSF/blood glucose ratio range was 0.25–1.2 in the 0–6Pre groups, and the CSF/average blood glucose ratio range was 0.33–0.78. The distribution of the CSF/blood glucose ratio across the groups showed greater variation than that in the normal glucose metabolism group .10.Glucose is the main energy-providing substance in the CSF. The CSF glucose level declines when energy-consuming diseases are present. Therefore, the CSF glucose level is important in the diagnosis of and evaluation of therapy for infectious CNS diseases, as well as for distinguishing between bacterial and non-bacterial infections . StandarThe glucose levels of diabetic patients are usually higher than those of people with normal glucose metabolism; therefore, the CSF glucose level should exhibit a similar trend. However, to date, no studies examining normal CSF glucose levels have provided a standard for diabetic patients. Some studies did not consider glucose metabolism , 10, whiHuman CSF is circulated every 4–6 h. The CSF collected at a lumbar puncture is produced 4–6 h before the puncture, and the production of glucose in the CSF is related to the blood glucose level. Therefore, in this study, we measured blood glucose levels at different times within 6 h prior to lumbar puncture to analyze the correlation between the CSF glucose and blood glucose levels. The result showed that, in both the normal and abnormal glucose metabolism groups, the CSF glucose level increased with the blood glucose level at 6, 5, 4, 3, 2, 1, and 0 h before lumbar puncture. This study confirmed that the CSF glucose level was influenced by the blood glucose level within 6 h before lumbar puncture, instead of only at the time of puncture. The glucose level in the CSF is closely related to the blood glucose level at 0–6 h before lumbar puncture. This is consistent with previous animal experiments performed by our research group, indicating that the blood glucose level should be monitored in patients 6 h before lumbar puncture .In 2016, a study demonstrated that the blood glucose level increases immediately after food intake, and a period occurred during which the blood glucose level was increased but the CSF glucose level was still low  for the publication of any potentially identifiable images or data included in this article.QT and XX collected the data, performed the statistical analysis, and wrote the manuscript. JZ took full responsibility for the whole study and revised the manuscript. MH, YM, LW, XW, HW, and SY helped collect and interpret the data. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To measure proximal ulna dorsal angulation (PUDA) and olecranon tip-to-apex distance (TTA) in pediatric population to aid surgeons with data for proximal ulna fractures fixation.Retrospective review of the hospital radiographic database. All elbow radiographs were identified and after implementing exclusion criteria, included were 95 patients aged 0–10; 53 patients aged 11–14; and 53 patients aged 15–18. PUDA was defined as the angle between lines placed on the “flat spot” of the olecranon and the dorsal edge of the ulnar shaft and TTA as the distance between the tip of the olecranon to the apex of angulation. Two evaluators performed measurements independently.r = − 0.56, p < 0.001), while TTA was positively correlated with age . Reliability levels of 0.81–1 or 0.61–0.80 were achieved for most of intra- and inter-rater reliabilities besides two levels of 0.41–60 and one of 0.21–0.40.In age group 0–10, mean PUDA was 7.53°, range 3.8–13.7, 95% CI 7.16–7.91, while mean TTA was 22.04 mm, range 8.8–50.5, 95% CI 19.92–24.17. In age group 11–14, mean PUDA was 4.99°, range 2.5–9.3, 95% CI (4.61–5.37), while mean TTA was 37.41 mm, range 16.5–66.6, 95% CI (34.91–39.90). In age group 15–18, mean PUDA was 5.18°, range 2.9–8.1, 95% CI (4.75–5.61), while mean TTA was 43.79 mm, range 24.5–79.4, 95% CI (41.38–46.19). PUDA was negatively correlated with age (The main study finding is that in most cases mean age-group values may serve as a template for proximal ulna fixation. There are some cases in which X-ray of contralateral elbow may provide surgeon with a better template.II. Proximal ulna dorsal angulation (PUDA) is the intersection angle of lines drawn along the dorsal flat surface of the olecranon and the dorsal prominence of the ulnar shaft. This angle typically measures typically between 4.3 and 8.5° in adults , 22, 27.While multiple authors measured PUDA , 22, 27 The study was approved by institutional Ethical Committee, decision number RNN/230/19/KE from 9th April 2019. Due to retrospective-database nature of the study Ethical Committee did not demand collecting individual informed consent.The study was designed as retrospective chart review of consecutive Radiology Department patients at the Central Teaching Hospital of the Medical University of Lodz, Poland. Sample size analysis was not performed, but all lateral elbow radiographs available in hospital database of patients 0–18 years old were identified. Exclusion criteria included radiographs with fractures around the elbow and radiographs with invalid lateral projection . Valid lExamples of measurements from age groups: (1) 0–10 years old; (2) 11–14 years old; (3) 15–18 years old. Proximal Ulna Dorsal Angulation (PUDA) was defined as angle of intersection of lines A and B. Line A is tangent to the dorsal edge of the ulnar shaft and line B is tangent to the “flat spot” of the olecranon. Olecranon Tip-To-Apex distance (TTA) was defined as the distance measured on the line tangent to dorsal flat surface of the olecranon, between proximal tip of olecranon and place of proximal ulna angulation.Two evaluators interpreted the radiographs independently. The first examination was performed as a trial run and the radiographs were reexamined by each evaluator at 1- and 2-week intervals after the first examination. The trial measurement was not included in outcome analysis. Radiographs were examined in a random order and results of previous measurements were blinded to avoid recall bias.t- test or U Mann–Whitney test, accordingly to the data distribution [p < 0.05.The values of second and third examination were analyzed statistically using Statistica 13.1 software. Afterwards, intra-, and inter- reliability measurements were assessed using the Krippendorff alpha test. The following interpretation of reliabilities was utilized: poor < 0.20; fair 0.21–0.40; moderate 0.41–0.60; good 0.61–0.80; very good 0.81–1 . Final vribution . StatistAfter the process of selection presented on the Flowchart in the Fig. p < 0.001) and it was negatively correlated with age   Fig. .Table 2Fp < 0.001 and 11–14 vs. 15–18, p < 0.001) and it was positively correlated with age  ; however, this difference was not statistically significant when analyzed in age subgroups  than in boys  [It is difficult to directly compare the results of this study with the literature, as while there was a study assessing PUDA and TTA in children 0–12 years old , and theeported) . Howevereported) . Due to eported) . As to Peported) , 24–27. eported) , 24, 25.As to associations between PUDA, TTA and gender, the results of this study remain in partial agreement with the literature. In this study the mean PUDA value was higher in girls when all patients were accounted into analysis. However, no significant differences were observed in subgroup analysis. In the study of Han et al. concerning children 0–12 years old, no gender differences in any of analyzed morphological variables were found . AuthorsThis study holds some limitations. First, it was retrospective in design. However, all previous studies measuring PUDA or TTA were also performed either on cadavers or retrospectively , 22, 27.This study provided original values of PUDA and TTA in various children and adolescents age groups. The main study finding is that while as shown by 95%CIs, in most cases mean age-group values may serve as a template for proximal ulna fixation, there are some cases in which X-ray of contralateral elbow may provide surgeon with a better template.


Lactobacillus spp. that dominate the healthy vaginal microbiome contributes to a low vaginal pH (3.5–4.5). During bacterial vaginosis (BV), strict and facultative anaerobes including Gardnerella vaginalis become predominant, leading to an increase in the vaginal pH (> 4.5). BV enhances the risk of obstetrical complications, acquisition of sexually transmitted infections, and cervical cancer. Factors critical for the maintenance of the healthy vaginal microbiome or the transition to the BV microbiome are not well defined. Vaginal pH may affect glycogen metabolism by the vaginal microflora, thus influencing the shift in the vaginal microbiome.Glycogen metabolism by L. jensenii 62G, L. gasseri 63 AM, and L. crispatus JV-V01, and G. vaginalis JCP8151A at specific initial pH conditions for 30 d. L. jensenii at all three starting pH levels , G. vaginalis at pH 4.5 and 5.0, and L. gasseri at pH 5.0 exhibited the long-term stationary phase when grown in MSVF. L. gasseri at pH 4.5 and L. crispatus at pH 5.0 displayed an extended lag phase over 30 d suggesting inefficient glycogen metabolism. Glycogen was essential for the growth of L. jensenii, L. crispatus, and G. vaginalis; only L. gasseri was able to survive in MSVF without glycogen, and only at pH 5.0, where it used glucose. All four species were able to survive for 15 d in MSVF with half the glycogen content but only at specific starting pH levels – pH 4.5 and 5.0 for L. jensenii, L. gasseri, and G. vaginalis and pH 5.0 for L. crispatus.The medium simulating vaginal fluid (MSVF) supported growth of G. vaginalis JCP8151A by affecting their ability to metabolize glycogen. Further, we found that L. jensenii 62G is capable of glycogen metabolism over a broader pH range (4.0–5.0) while L. crispatus JV-V01 glycogen utilization is pH sensitive . Finally, our results showed that G. vaginalis JCP8151A can colonize the vaginal tract for an extended period as long as the pH remains at 4.5 or above.These results suggest that variations in the vaginal pH critically influence the colonization of the vaginal tract by lactobacilli and The online version contains supplementary material available at 10.1186/s12866-023-02916-8. Lactobacillus spp. – L. jensenii, L. crispatus, L. gasseri, and L. iners . Ma. MaL. je1A Table . Despiterispatus , we retrG. vaginalis ranging from pH 4.7 to 5.9, and CST V from 4.3 to 5.1 . Pr. PrG. vaed Table .L. crispatus and L. acidophilus, respectively, has been shown to be repressed when the cells are grown on glucose but not when maltose or maltotriose is present  , Lactobacillus gasseri 63 AM  obtained from the American Type Culture Collection  were used in this study. The standard laboratory media New York City III broth (NYCB) (ATCC Medium 1685 formulation [https://www.atcc.org/]) and de Man, Rogosa, and Sharpe broth (MRSB)  were used for G. vaginalis and the lactobacilli, respectively, as well as medium simulating vaginal fluid (MSVF) for all strains [ strains , 31, 38.G. vaginalis or the different Lactobacillus spp. were inoculated into NYCB or MRSB, respectively, and incubated at 37℃ under 5% CO2 for 48 h. One-mL aliquots of the 48 h cultures were centrifuged and the pellets were washed three times in the medium to be used for the growth curve experiments  and resuspended in the same medium. The washed bacterial cells were then used to adjust fresh media to a starting OD600 of 0.02 to yield an initial inoculum of 104 CFU/mL in 10 µL of culture for subsequent experiments.Frozen stocks of 2 in a humid chamber for designated time periods. Samples were collected at 1-, 2-, or 5-d intervals depending on the species tested and the medium used to assess the viability and pH of the cultures. At specified time points, 60 µL of culture from individual wells were diluted tenfold and 10-µL aliquots were spotted in triplicate on chocolate agar  or MRS agar (Oxoid™) for G. vaginalis or Lactobacillus spp., respectively, to determine the number of CFU/mL. A 400-µL aliquot was used to determine the pH level of the respective cultures at each time point.Cultures were grown in MSVF, which closely mimic the vaginal fluid, for 30 dpi to reflect the 30-d cycle of conditions within the vagina; that is, the average menstrual cycle for women of child-bearing age, the group in which most BV occurs, is 28 d (21–35 d) , 83. As 4 CFU/mL of each of the strains and incubated for up to 15 d. At 5-d intervals, the CFU/mL (viability) were determined, and the pH levels were measured.The MSVF medium contains 10 g/L of glycogen, 10 g/L of glucose and 0.25 g/L of mucin . To asse4 CFU/mL of L. gasseri for MSVF_0Gly0Glu and MSVF_0Gly0Glu0Muc or 104 CFU/mL of L. jensenii or G. vaginalis for MSVF_0Glu. The numbers of CFU/mL were determined MSVF_0Gly0Glu and MSVF_0Gly0Glu0Muc and at 5-d intervals over 15 d for MSVF_0Glu.Additional MSVF was prepared without glycogen or glucose (MSVF_0Gly0Glu), without glycogen, glucose, or mucin (MSVF_0Gly0Glu0Muc), and without glucose (MSVF_0Glu); these media were adjusted to pH 5.0. Microtiter plates were prepared and inoculated with 10L. jensenii or G. vaginalis was grown in MSVF at pH 5.0 for 6 d and 12 d, a predetermined concentration of carbenicillin sufficient to inhibit growth  was added to the cultures and incubation was continued for an additional 24 h  at which time the numbers of CFU/mL were assessed.To determine whether the bacteria in the prolonged stationary phase were metabolically active, the carboxypenicillin carbenicillin  was added to the media. L. jensenii or G. vaginalis, the strains were grown in grown in MSVF at pH 5.0 for 6 d and 12 d and a concentrated solution of glycogen in water was added to the cultures to a total of 2.5 mg/mL. Incubation continued for an additional 24 h and the numbers of CFU/mL were assessed at 7 d or 13 d, respectively.To determine if glycogen limitation was affecting the growth of n = 3). The CFU data were routinely log transformed prior to graphing and statistical analysis. Two-way ANOVA with Tukey’s multiple comparisons posttest or Šídák’s multiple comparisons posttest (as recommended by the program) was used to determine significance between time points in the growth curve analyses both for CFU and pH. One-way ANOVA with Dunnett’s multiple comparisons posttest was used to compare each treatment to fMSVF or appropriate control; one-way ANOVA with Tukey’s multiple comparisons posttest was used to compare differences among the fractions . Two-tailed unpaired t tests were used to compare individual pairs. Statistical significance is shown as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns, no significant difference.GraphPad Prism version 9.4.0 (673)  was used for all statistical analyses. Data represent the means ± SEM of three independent experiments for each group (Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2Supplementary Material 3Supplementary Material 4


Bacillus thuringiensis-based insecticide formulations are largely recognized as among the most effective, ecologically safe, and long-lasting methods of managing insect pests. New B. thuringiensis strains with high mosquito control effectiveness were isolated, identified, genetically defined, and physiologically characterized. Eight B. thuringiensis strains were identified and shown to carry endotoxin-producing genes. Using a scanning electron microscope, results revealed typical crystal forms of various shapes in B. thuringiensis strains. Fourteen cry and cyt genes were found in the strains examined. Although the genome of the B. thuringiensis A4 strain had twelve cry and cyt genes, not all of them were expressed, and only a few protein profiles were observed. The larvicidal activity of the eight B. thuringiensis strains was found to be positive (LC50: 1.4–28.5 g/ml and LC95: 15.3-130.3 g/ml). Bioassays in a laboratory environment demonstrated that preparations containing B. thuringiensis spores and crystals were particularly active to mosquito larvae and adults. These new findings show that the novel preparation containing B. thuringiensis A4 spores and crystals mixture might be used to control larval and adult mosquitoes in a sustainable and ecologically friendly manner.Mosquitoes of many species are key disease vectors, killing millions of people each year. The online version contains supplementary material available at 10.1186/s12866-023-02842-9. Mosquitoes are recognized as one of the most important groups of insects in transmitting many major infectious illnesses, such as malaria, filariasis, dengue, Rift Valley and yellow fevers, chikungunya and Zika fevers, and Japanese encephalitis, which cause millions of deaths each year , 2. The Culex pipiens pallens has had a high incidence in Shandong province and constitutes a potential vector of different diseases including the West Nile virus in China. Unfortunately, this species has developed insecticide resistance debilitating effective control [Furthermore, the widespread and intense use of chemical pesticides for mosquito control has resulted in a number of environmental and human health issues, including the disruption of natural-biological control mechanisms, the development of mosquito resistance, and unfavorable impacts on beneficial species , 5. Spec control .B. thuringiensis spore and crystal mixture is among the few alternatives to conventional chemical compounds for vector control. This has been isolated from soil, aquatic habitats like sewage, dead insects, herbivore dung, stored grains, phylloplane, and woods [The formulation based on nd woods . Becausend woods . This band woods , 9.B. thuringiensis usually produces insecticidal proteins and δ-endotoxins [cry and two cyt δ-endotoxin genes with insecticidal activity [B. thuringiensis strains can vary between countries and regions. For that reason, many B. thuringiensis strains were isolated and characterized around the world with the main objective of finding novel active cry genes to control resistant insects. The parasporal crystal form is an indication of Cry proteins content; hence, it is used as the first criterion of the classification of B. thuringiensis isolates [cry and cyt genes can predict the insecticidal activity of a novel isolate. Due to the complexity of the δ-endotoxins action, it is also necessary to assess the frequency of the genes involved in larval mortality to understand the mode of action of these proteins in larval intestinal cells [cry and cyt genes allows for high toxic activity, causing larval death within 24 h after exposure [During the sporulation stage, dotoxins , 11. Hofactivity . The genisolates –15. The al cells –18. The exposure .B. thuringiensis genes encoding proteins with demonstrated anti-dipteran activity such as cry1, cry2, cry4, cry10, cry11, cry19, cry20, cry24, cry27, cry30, cry39, cry44, cry47, cry50, cry54, cry56, mpp60, tpp80, cyt1, and cyt2 genes [Aedes, Anopheles, and Culex, respectively [Additionally, there are many ectively –26.B. thuringiensis protoxin activation of these protoxins into toxic forms, and the toxins bind to receptors on the intestinal epithelium in this active state in different groups (orders) of insects [On the other hand, the initiation of larval toxicity occurs soon after the bacterial crystals are ingested, which are later solubilized in the alkaline pH of the larval midgut, releasing the protoxins. Different proteases are involved in  insects –30. This insects .B. thuringiensis-based products are extensively utilized, and several formulations have been designed. Suspension concentrates, wettable powders, and grained formulations are the most common commercial products [B. thuringiensis formulations using ice granules have been utilized [B. thuringiensis, manufactured by Valent BioSciences Corporation was evaluated on field condition in Cabo Verde, with a mortality rate ranging from 43.1 to 90.9% [products . Formulautilized . A dispeto 90.9% . Recentlto 90.9% .B. thuringiensis subsp. israelensis crystals through enemas into the midguts of Aedes aegypti adult. The susceptibility from different mosquito species was evaluated after oral administration of solubilized B. thuringiensis [B. thuringiensis was supplied in a saline and sucrose solution. Also, the ingestion of B. thuringiensis or Cry toxins in an attractive toxic sugar baits had a high mortality in different mosquito adults [Besides, evidence about the biological activity on mosquito adults was initially reported by Klowden et al. , who intngiensis . A high o adults –40.B. thuringiensis has been isolated and investigated in a variety of ecologies since its discovery by Ishiwata in 1902. However, insecticides based on B. thuringiensis have not completely replaced dangerous chemical pesticides, and many ecologies remain unknown [B. thuringiensis isolates with naturally enhanced toxicity for biocontrol of mosquito larvae and adults.Until now, various alternatives have been used to controlling mosquitoes , 42. How unknown . TherefoB. thuringiensis isolation. One gram of each soil sample was suspended in 10 ml of Luria-Bertani (LB) broth medium . The mixture was then incubated in a shaker incubator at 30 °C for 4 h. After incubation, the samples were heated at 80 °C for 15 min. From each of these samples, 100 µl was spread on nutrient agar medium (yeast extract (3 g/L), peptone (5 g/L), sodium chloride (5 g/L), and agar (15 g/L))  plates and incubated for 72 h at 30 °C. After that, each colony was grown on nutrient medium (yeast extract (3 g/L), peptone (5 g/L), and sodium chloride (5 g/L)) (Sangon-Biotech) plates supplemented with 100 µg/ml of penicillin G  and incubated at 30 °C for 96 h. B. thuringiensis-like colonies, white, large, and nearly circular with fine irregular margins and glossy, less glossy, or rough were selected.Soil samples were collected in sterile plastic tubes from three different districts in Rizhao, Shandong, China, People’s Republic of China  was suspended in 100 µl sterile distilled water in an Eppendorf tube and boiled in a water bath for 10 min. B. thuringiensis suspensions were then immediately cool-shocked at -20°C. This heat-shock process was repeated three times to allow complete cell lysis before centrifugation at 3,500× g for 10 min at 4°C. The resultant supernatant containing the crude DNA was used for PCR amplification [16S rRNA gene was used for molecular identification. A fragment of the 16S rRNA gene (1.4 kb) was amplified by polymerase chain reaction (PCR) using the forward primer 27F 5’-AGAGTTTGATCCTGGCTCAG-3’, reverse primer 1492R 5’-GGTTACCTTGTTACGACTT-3’, and the SanTaq Plus PCR Master Mix . The amplification process was conducted in a Thermal Cycler T100 machine  using a Taq PCR Master Mix Kit . The PCR reaction was as follows: 95oC for 10 min; 35 cycles of 95oC for 30 s, 55oC for 1 min, and 72oC for 1.5 min; and a final extension at 72oC for 10 min. The purified PCR fragment was sequenced using an ABI 3730 DNA sequencer . The 16 S rRNA gene fragment sequence  was identified using BLASTN homology searches [B. thuringiensis strains identified were examined for the presence of parasporal inclusion bodies by light microscope. The bacteria were prepared as films on slides and heat fixed, then stained with Coomassie Brilliant Blue. Slides were observed with bright-field microscopy using a 100× oil immersion objective. Subsequently, crystals and spores morphologies were analyzed with Scanning Electron Microscopy (SEM) . A mixture of spores and crystals in sterile distilled was placed on SEM sample holder. Samples were dried at 37oC in oven, metalized and placed in vacuum chamber of SEM to capture images.A colony taken from a 24-hour culture of each fication . The 16Ssearches  from theB. thuringiensis strains  obtained according to the method mentioned above was used for PCR amplification [The supernatant containing the crude DNA from fication , using tB. thuringiensis (A4) culture with spore and crystal mixture was centrifuged, and the pellet was washed thrice with 1 M NaCl. The pellet was then washed 3x with distilled water. The spore-crystal pellet was resuspended in 50 mM NaOH and incubated at room temperature for 1 h to solubilize the crystal proteins. The purified crystal proteins were combined with 2x boiling buffer  at the ratio of 2:1, respectively. The samples were then boiled for 5 min along with a broad-range protein marker. The samples were loaded onto an SDS-PAGE gel with a 10% separating gel and 3% stacking gel. The electrophoresis was run at 100 V for 2 h. The gels were stained with a staining solution containing 0.025% Coomassie Brilliant Blue R250. Destaining was performed overnight with a solution containing ethanol, glacial acetic acid, and water at the ratio of 5:7:88, respectively. Additionally, the Bradford protein assay was used to measure the concentration of total protein [The  protein . Five diB. thuringiensis culture  preserved in glycerol was added to 40 ml of the LB medium in an Erlenmeyer flask. The B. thuringiensis fermentation was conducted in an incubator shaker at 37 °C and 120 rpm for 48 h. Subsequently, the culture was transferred to a fermenter with 40 L of medium for B. thuringiensis fermentation. The medium composition was the following: glucose, 10 g/L; corn powder, 10 g/L; soybean powder, 5 g/L; yeast extract, 1.5 g/L; peptone, 0.5 g/L; KH2PO4, 2.5 g/L; CaCO3, 0.5 g/L; and MgSO4•7H2O, 0.25 g/L; at a pH of 7.5 [A volume of 40 µl of H of 7.5 . Batch fLarvae assay: The fermentations of A2, A4, A8, A10, B6, C2, C4 and C5 strains were used in the experiment. The bioassays were developed according to the guidelines for the laboratory testing of mosquito larvicide from the World Health Organization [Culex pipiens pallens species was used in the experiments due the incidence and prevalence in the Shandong province [B. thuringiensis powder with a concentration of 7.5 × 1013 colony forming units (CFU) per gram and 30 mg/g of total proteins was used in the experiments. The CFU was determined as follows: the B. thuringiensis dry powder (0.1 g) was dissolved in 10 ml of sterile water. The solution was diluted tenfold gradient and place on the nutrient agar medium. Three plates were used for each dilution. The plates were incubated overnight at 37 ℃. Finally, the colonies were counted in each plate (the effective range of colonies was between 30 and 300). The average value was multiplied for the dilution and calculate the concentration of the original dry powder solution. On the other hand, the different concentrations were prepared by serial dilution for each isolate. The control was prepared following the same procedure but without B. thuringiensis. The experiment was conducted in five repetitions. The bioassays were evaluated at 24 h after the application of B. thuringiensis, and the live and dead larvae were counted. The LC50 and LC95 were determined by a Probit analysis run in the POLO PLUS [nization  with modOLO PLUS  program,OLO PLUS   + odorant solution (10% v:v).Treatment A: mixture of water + B. thuringiensis A4 spore-crystal mixture .Treatment B: mixture of water + Treatment C: mixture of water + odorant solution (10% v:v).Treatment D: water.A total of 85 adult mosquitoes of the same age were used in each treatment. The mosquito diet (10 ml) was replaced without spore-crystal mixture every day. The experiments were developed at 37 °C and 70% relative humidity. The bioassays were monitored every 24 h for two week, and the live and dead adult mosquitoes were counted. .B. thuringiensis. The proportion of colonies of B. thuringiensis was low at 5.7% of the total  corresponded to B. thuringiensis. Smooth spherical shape, spherical shape with an undulated surface, spherical but deflated balloon shape, and spherical with one concave side and a pointy-edged shape were identified  were tested for positive amplification of the cry and cyt genes using the previously described specific primers. The most frequent genes of the new B. thuringiensis isolates analyzed were cry1, cry11Ba, cry4Aa, cry10Aa, cry11Aa, cry10, and cry11    and odorant natural compounds displayed significantly high mortality of mosquito adults (68%) compared with the control treatments . The bioactivity of preparation from isolate A4 showed an interesting low LC50 and LC95 concentration, compared with previous works where the concentrations of preparation of the new B. thuringiensis isolates were higher, including the reference B. thuringiensis subsp. israelensis H14 strain [B. thuringiensis isolates had a lower LC50 concentration against Culex pipiens [Aedes aegypti larvae, respectively [B. thuringiensis, but this should not be taken as a final criterion, without a real-time comparison with reference isolates or similar isolates. Although, the behavior of the reference isolates may vary in relation to the mosquito species and the experimental conditions of the region. This is the reason for the continuous search for native B. thuringiensis strains to be used in the specific regions where they were previously isolated.Preparation of spore-crystal mixture from local he world , 71, 72. pipiens  or Aedesectively . The comB. thuringiensis as a mosquito larvicide is widely known, previous research on its biological effects on adult mosquitos has shown interesting and contradictory results [B. thuringiensis and natural odorant caused significant mortality of mosquito adults after ingestion. These odorant baits are one alternative strategy to attract mosquitoes. Also, the probable presence of attractive toxic sugar in the natural compounds can have an additive effect on mortality. Both attractive toxic sugar baits and attractant compound present in fruits have been shown to be effective against several important mosquito vectors [B. thuringiensis and natural odorants on wild mosquito species populations.The use of  results , 74–76.  vectors , 78. TheB. thuringiensis isolates from Rizhao soil are promising for the biological control of C. pipiens pallens larvae, which can be evidenced by the biological activity in larvae caused by the Cry and Cyt toxins, making these isolates highly toxic and enabling them to be safely used as a biotechnological tool for the manufacture of biological larvicide with different combinations of toxins from those currently used. We believe that the discovery of the A4 strain may prove crucial for future bioinsecticide production against mosquito vectors. This is due to its greatly enhanced mosquitocidal activity against C. pipiens pallens, combined with its possible economic and environmental advantages. In the trials, a comparison with B. thuringiensis subsp. israelensis isolates would have been useful. The objective was to isolate and compare novel native isolates under laboratory conditions. However, these results only reach a candidate product. Assays on a small, medium, and large scale will be required in natural conditions using a final product formulated. In the future, a comparison of the final product formulated with other commercial pesticides would be required.The new preparations of spore-crystal mixture of Below is the link to the electronic supplementary material.Supplementary Figure 1. Map of Rizhao showing the locations of the sites from which the soil samples were collected (stars). Stars indicate sites from which toxic Bacillus thuringiensis was isolated. Supplementary Figure 2. Schematic representation of spore-crystal mixture from B. thuringiensis and natural odorant production to the control of larvae and adult mosquitoes. Supplementary Figure 3. Schematic representation of the evaluation of biological activity preparation of spore-crystal mixture from B. thuringiensis against larvae (A) and adult (B) mosquitoes under controlled conditions. Supplementary Figure 4. Original full-length SDS-PAGE gels of spore-crystal mixture from local B. thuringiensis strains. Supplementary Figure 5. Evaluation of LC50 of spore-crystal mixture from B. thuringiensis strain A4 using Probit, arithmetic, and logarithmic analyses. Strain A4 and one replication.


Introduction: In clinical practice, warfarin is often combined with Compound Danshen dripping pill (CDDP) for the treatment of cardiovascular diseases. However, warfarin has a narrow therapeutic index, wide interindividual variability (genetic and non-genetic factors), and is susceptible to drug-drug interactions. Our previous study indicated that CDDP might interact with warfarin in individuals with the epoxide hydrolase gene  A/A subtype. We sought to clarify the interaction between CDDP and warfarin associated with EPHX1 in a comprehensive and accurate manner.Methods: Here, EPHX1 A and EPHX1 G cell lines were established. Expression of microsomal epoxide hydrolase (mEH), vitamin K epoxide reductase (VKOR), and vitamin K-dependent clotting factors  was measured by western blotting upon incubation with CDDP and warfarin. mEH activity was evaluated by measuring the transformation of epoxyeicosatrienoic acids into dihydroxyeicosatrienoic acids. Then, healthy volunteers (HVs) with the EPHX1 A/A genotype were recruited and administered warfarin and CDDP to investigate the pharmacokinetics and pharmacodynamics of warfarin.Results: CDDP combined with warfarin could decrease expression of mEH and VKOR, and increase protein expression of FII, FVII, FIX, and FX, in EPHX1 A cells. CDDP could slightly influence the pharmacokinetics/pharmacodynamics of warfarin in HVs with the EPHX1 A/A genotype.Discussion: Rational combination of CDDP and warfarin was safe with no risk of bleeding, but the therapeutic management is also needed. The clinical study is posted in the China Clinical Trial Registry (ChiCTR190002434). Use of herbs and herbal products is widespread and increasing steadily worldwide . Sales oHowever, the increasing use of medicinal plants has attracted growing concern with regard to their efficacy and safety if combined with conventional medicines. Herbal plants contain multiple phytochemicals that act upon different targets and pathways. Hence, there is a risk of interactions if another drug is administered simultaneously or successively . TherefoWarfarin has a narrow therapeutic index, wide variability in efficacy between individuals, and can interact with other drugs . It actsIn China, the most prevalent herbal medicine used against cardiovascular diseases is Compound Danshen dripping pill (CDDP). It was first launched by the China Food and Drug Administration in 1995, and has been approved by the Australian Therapeutic Goods Administration. It is undergoing a phase-III clinical trial overseen by the US Food and Drug Administration .In clinical practice, warfarin often combines with CDDP, and many scholars have reported on this interaction . Howeverin vivo.Recently, we recruited patients with coronary heart diseases and atrial fibrillation to investigate the interaction between CDDP and warfarin . CDDP diThus, we wished to clarify the interaction between CDDP and warfarin associated with EPHX1 in a comprehensive and accurate manner. Hence, transfected EPHX1 A cells and transfected EPHX1 G cells were adopted to explore the interaction between CDDP and warfarin, and the result was verified using HVs with the EPHX1 A/A genotype.2 at 37°C. EPHX1 A cells or EPHX1 G cells were prepared as shown in Human hepatocarcinoma (HepG2) cells were purchased from the Chinese Academy of Medical Sciences & Peking Union Medical College . They were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in an atmosphere of 5% COTransfected cells were cultured in medium containing puromycin (2 μg/mL) to select stably transfected cells. Subsequently, a microscope  equipped with a bandpass filter of 488 nm was used to capture images of intracellular green fluorescence. Then, we used a flow cytometer  to measure the proportion of intracellular green fluorescence from the green fluorescent protein that integrated with transfected cells. Finally, western blotting was applied to measure mEH expression in transfected cells.2 at 37°C. Then, the cells were centrifugated and the supernatant was retained. Finally, the blue-purple crystals were dissolved using dimethyl sulfoxide (150 µL) with agitation for 10 min. The absorbance of each well was determined at 490 nm. Cell viability . Warfarin was obtained from Orion Corporation . Transfected cells were plated evenly on a 96-well cell-culture plate with ∼8,000 cells/well. They were cultured for 24 h at 37°C. Then, they were treated with CDDP or warfarin at 0.0625–8.00 mg/mL for 24 h or 72 h. Subsequently, to each well was added 10 µL of 3--2,5-diphenyltetrazolium bromide solution (5 mg/mL), and cells were allowed to incubate for an additional 4 h in an atmosphere of 5% COiability  was calcWe wished to measure expression of mEH, VKOR, FII, FVII, FIX, and FX. Whole-cell extracts were prepared using radio immunoprecipitation assay buffer  incubated on ice for 30 min. Cell lysates were centrifuged . The protein concentration of the supernatant was determined by a bicinchoninic acid protein assay kit . Lysates (10 µg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 10% gels. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes . PVDF membranes were blocked with 10% non-fat dried milk at room temperature (Solarbio) for 1.5 h and incubated overnight at 4°C with antibody directed against mEH , VKOR, FII, and FIX , FVII , FX , and glyceraldehyde 3-phosphate dehydrogenase  in TBS-T buffer (Tris (20 mM), pH 7.6, NaCl (100 mM), 0.1% Tween-20) for immunoblotting. After incubation for 2 h at room temperature with secondary anti-rabbit immunoglobulin-G antibody conjugated with horseradish peroxidase , proteins were visualized by an electrochemiluminescence detection system. Western-blotting signals were quantified using ImageJ 1.6.0.20  and were normalized to GAPDH expression.mEH catalyzes the hydrolysis of lipophilic epoxides to their corresponding dihydrodiols . Free epThe concentration of 14,15-EET and 14,15-DHET was measured by liquid chromatography tandem-mass spectrometry (LC-MS/MS). The separation and detection of 14,15-EET (319.2→219.2) and 14,15-DHET (337.2→207.0) was undertaken in an Acquity ultra-performance liquid chromatography (UPLC) system  with a Triple Quad 5500 MS/MS detector . Detailed information and method validation are shown in Transfected EPHX1 A cells, transfected EPHX1 G cells, and transfected BL cells were seeded 1 day before treatment with a substrate or inhibitor. Then, the cell-culture medium was replaced with complete medium containing CDDP (0.5 mg/mL) and warfarin (0.125 mg/mL) (CDDP + War group), or warfarin (0.125 mg/mL) (War group), or valpramide (100 mg/mL) . After incubation with 14,15-EET, 60 µL of the cell culture medium was harvested at 0, 0.25, 0.5, 1, 1.5, 2, and 3 h after addition of 14,15-EET. Subsequently, to the harvested cell-culture medium was added 3 µL of chloramphenicol  and extracted with ethyl acetate (180 µL) with vortex-mixing for 3 min and centrifugation . The supernatant was evaporated under a stream of nitrogen and re-dissolved by 50% methanol/water (60.0 µL), then 2.00 µL was injected into the LC-MS/MS system.This was an open-label, three-period, controlled before-and-after trial . It was conducted at the Second Affiliated Hospital within Tianjin University of Traditional Chinese Medicine . The protocol was approved by the ethics committees of the Second Affiliated Hospital within Tianjin University of Traditional Chinese Medicine. Written informed consent was provided by all participants. This trial was in accordance with the Declaration of Helsinki 1964 and its later amendments.Men and women aged 18–45 years participated in this trial if they were healthy and the EPHX1 A/A genotype was confirmed. The key exclusion criteria were coagulation disorders or allergies to drugs.Our trial comprised three periods. In the first period, warfarin  was taken once a day. After 3 days, individuals entered the second period, in which CDDP  was administered for 5 days. Finally, participants were treated with a combination of warfarin  and CDDP  in the third period.During the first and third period, 15 blood samples (2-mL each time) were collected for the concentration analysis of warfarin at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12, 24, 36, 48, and 72 h after administration. Eight blood samples were collected for the detection of coagulation indices before as well as 12, 24, 30, 36, 48, 54, and 72 h after administration. Moreover, blood samples were taken from participants once a day before CDDP administration for assays of coagulation indices to ensure safety.max), time to reach Cmax (tmax), the area under the curve from time 0 h–144 h (AUC0–144), the area under the curve from time 0 to infinity (AUC0–∞), half-life (t1/2), volume of distribution (Vd), and clearance (CL).The plasma concentration of S-warfarin and R-warfarin was measured by LC-MS/MS, as described previously . The phaIn addition, the coagulation indices of prothrombin time (PT), INR, thrombin time (TT), activated prothrombin time (APTT), and fibrinogen level were detected according to the manufacturer instructions of assay kits in the clinical laboratory of the Second Affiliated Hospital within Tianjin University of Traditional Chinese Medicine.in vitro.Blood samples of the trough concentration and peak concentration of warfarin were used to measure the content of mEH, VKOR, FII, FVII, FIX, FX, and FIIa using the respective human ELISA kits. mEH and VKOR kits were from Lanso . FII and FX kits were from Cusabio . FVII and FIX kits were from Ruifan . The FIIa kit was from Abcam. FIIa is also called thrombin and not exist p-values (two-sided) were calculated and paired t-tests were used for statistical analyses if the data passed the normality (Shapiro–Wilk) test, or the Wilcoxon matched-pairs test if the distribution was not normal. Results are the mean ± SD for continuous variables with a normal distribution. p < 0.05 was considered significant.Statistical analyses were carried out using SPSS 22.0 . Graphs were plotted using Prism 8.0 . ANOVA was used to compare the results of transfected EPHX1 A cells or transfected EPHX1 G cells by western blotting. For HVs with the EPHX1 A/A genotype, in vitro (p < 0.05) (Transfected EPHX1 A cells and transfected EPHX1 G cells were established to explore differences between EPHX1 A and EPHX1 G subtypes in vitro . Subsequin vitro . The effin vitro . Western < 0.05) . These rFor the control group, mEH expression  in transp < 0.05) compared with that in the War group in transfected EPHX1 A cells. Protein expression of FII, FVII, FIX, and FX p < 0.05) in transfected EPHX1 A cells, and there was no significant difference between the two groups for transfected EPHX1 G cells and transfected BL cells (p > 0.05). Thus, various concentrations of CDDP  combined with warfarin (0.125 mg/mL) were incubated in transfected EPHX1 A cells to assess the interaction with CDDP. Expression of mEH and FIX was significantly different (p < 0.05), whereas the other indicators did not show a significant difference between the three concentrations of CDDP (Protein expression of mEH and VKOR  was decr of CDDP . Expressp < 0.05)  was chosen and metabolic efficiency was calculated. The curves for the metabolic efficiency of 14,15-EET in transfected EPHX1 A cells and transfected EPHX1 G cells were higher than that for transfected BL cells ( < 0.05) .p < 0.05) (p < 0.05)  . When co < 0.05) . These fFrom March 2019 to September 2019, 15 HVs with the EPHX1 A/A genotype were selected from the Second Affiliated Hospital within Tianjin University of Traditional Chinese Medicine. Eight HVs were enrolled and seven  completed our clinical trial .A concentration-time curve was plotted . The plap < 0.05) in the AUC, Cmax, CL, and Vd between the first period and third period, whereas t1/2 and Tmax did not show a significant difference  and peak concentration (Cmax) time points of warfarin as measured by ELISAs.The content of mEH, VKOR, FVII, FIX, FX, FII, and FIIa in HVs with the EPHX1 A/A genotype was measured. Samples were chosen from the trough concentration . These results suggested that CDDP combined with warfarin might affect the content of mEH, VKOR, FVII, FIX, FX, FII, and FIIa in HVs with the EPHX1 A/A genotype compared with those taking only warfarin.The content of all indices did not show a significant difference between Car group . Thus, CIn recent years, formulations based on traditional Chinese medicine (TCM) have been used widely in clinical therapy and are, in general, considered safe . Howeverin vitro results were verified in HVs with the EPHX1 A/A genotype.Warfarin and CDDP are often used in combination for treatment of cardiovascular diseases . PrevioumEH was first purified from rabbit liver  and thenIn humans, the EPHX1 (SNP: rs2292566) gene includes A/A, A/G, and A/A subtypes. A genetic polymorphism is a potential determinant of mEH activity, and contributes to differences among individuals . The difCDDP combined with warfarin in EPHX1 A cells could reduce expression of mEH and VKOR, inhibit the enzyme activity of mEH , increasd and CL between the two periods after this adjustment (Warfarin combined with CDDP impacted the blood concentrations and pharmacokinetic parameters of warfarin in HVs with the EPHX1 A/A genotype, which were increased by ∼30%. The trough concentration of warfarin in the third period  suggestejustment .The INR is used routinely to monitor the level of anticoagulation elicited by warfarin . AccordiThere are some limitations in this study. The dose and ratio of warfarin and CDDP in cell experiment were not inconsistent with the clinical dose. For the cell experiment, the concentration of warfarin and CDDP were determined by the MTT assay  and the CDDP combined with warfarin might reduce expression of mEH and VKOR, and increase protein expression of FII, FVII, FIX, and FX, in EPHX1 A cells. In HVs with the EPHX1 A/A genotype, CDDP could influence the pharmacokinetics/pharmacodynamics of warfarin slightly, but the combination is safe with no risk of bleeding, but this is still a warning signal for patients with EPHX1 A/A.