Medial patellofemoral ligament (MPFL) reconstruction combined with tibial tubercle osteotomy (TTO) and lateral retinacular release (LRR) is one of the main treatment methods for patellar instability. So far, few studies have evaluated the clinical effectiveness and assessed potential risk factors for recurrent patellar instability.To report the clinical outcomes of MPFL reconstruction combined with TTO and LRR at least three years after operation and to identify potential risk factors for recurrent patellar instability.A retrospective analysis of medical records for patients treated with MPFL, TTO and LRR from 2013 to 2017 was performed. Preoperative assessment for imaging examination included trochlear dysplasia according to Dejour classification, patella alta with the Caton-Deschamps index (CDI), tibial tubercle–trochlear groove distance. Postoperative assessment for knee function included Kujala, IKDC and Tegner scores. Failure rate which was defined by a postoperative dislocation was also reported.P = 0.006), Tegner  and Kujala . Age (p = 0.011) and trochlear dysplasia (p = 0.016) were considered to be two failure factors for MPFL combined with TTO and LRR.A total of 108 knees in 98 patients were included in the study. The mean age at operation was 19.2 ± 6.1 years , and the mean follow-up was 61.3 ± 15.4 months . All patients included had trochlear dysplasia , and 67% had patellar alta. The mean postoperative scores of Tegner, Kujala and IKDC were 5.3 ± 1.3 (2–8), 90.5 ± 15.5 (24–100) and 72.7 ± 12.1 (26–86). Postoperative dislocation happened in 6 patients (5.6%). Female gender was a risk factor for lower IKDC . Female gender was a risk factor for worse postoperative outcomes. Preoperative failure risk factors in this study were age and trochlear dysplasia.Level IV; Case series Recurrent patellar instability is a common problem, which could cause anterior knee pain, limit the motion of the knee joint, and increase the risk of patellar osteoarthritis. The stability of patellofemoral joint is maintained by the joint action of bony anatomical structure and soft tissue on patella. A few studies have proved that the surgical procedures—MPFL alone or MPFL combined with TTO—can get good results. However few studies described the good outcome of MPFL reconstruction combined with LRR and TTO, information about multiple risk factors for poor outcomes of the combined treatment is rare, and no articles reported risk factors for failure of the combined treatment.As a surgical method, MPFL combined with TTO and LRR would be a reliable choice with a low failure rate. Female gender was a risk factor for worse postoperative outcomes. Preoperative failure risk factors in this study were age and trochlear dysplasia.Recurrent patellar instability is a common problem, which could cause patellofemoral pain, limit the motion of the knee joint, and increase the risk of patellofemoral osteoarthritis . The staThe medial patellofemoral ligament (MPFL) is the most important soft tissue to keep patellar from lateral displacement from zero to thirty of knee flexion. It has been proved that the injury of MPFL happens during all lateral patellar dislocations , 3. WithTibial tuberosity to trochlear groove (TT–TG) distance is an index for measuring the lateralization of the tibial tuberosity . Except So far, numerous studies described the good outcome of MPFL reconstruction alone or MPFL reconstruction with tibial tubercle osteotomy (TTO) , 13, butThis study received institutional review board approval. All procedures were performed in accordance with relevant guidelines.normal-Aactual; Fig. The patients included in the study had at least 2 patellar dislocations. One experienced surgeon (JL) performed MPFL reconstruction combined with LRR and TTO treatment from 2013 to 2017. The indications for TTO were CDI ≥ 1.3 , gender, and follow-up time. Preoperative radiographs were reviewed for patellar alta and trochlear dysplasia. Patellar alta was defined using a Caton-Deschamps Index (CDI) of more than 1.3 based on lateral radiographs. Trochlear dysplasia was defined using Dejour classification based on computed tomography (CT) slices. Preoperative TT-TG distance was defined based on CT slices. Measurements were performed by 1 musculoskeletal radiologist. Postoperative indicators included postoperative re-dislocation besides Tegner, IKDC and Kujala scores. The shortest postoperative follow-up time was three years.All surgical procedures were performed by one senior surgeon, who used the same technique. The patient was placed in supine position with a tourniquet placed on the thigh root. An autogenous hamstring tendon graft was then harvested and prepared. A 12-cm longitudinal incision was made on the medial side of patella. The patella was pushed toward the medial side. If the patella moved less than one fourth of the patella, the lateral patellar retinaculum would be partially severed for the release. The range of release was determined by the patella movement. A continuous incision which went through the capsule and lateral retinaculum ranging from, and including the vastus lateralis tendon to the joint line, 1 cm lateral to the patella was usually made. After the lateral retinaculum was released, the periosteum was dissected with osteotome at the medial or /and inferior part of tibial tubercle. Then wedge-shaped osteotomy was done at the medial or /and inferior part of tibial tubercle.The area of the bone mass was about 3.0 * 1.0 cm. After the wedge-shaped osteotomy, another wedge-shaped osteotomy was done at the tibial tubercle. The area of the bone mass with patellar tendon was about 3.0 * 1.0 cm. Then the position of the two bone mass was exchanged. The bone mass with patellar tendon was fixed with two 4.5 * 40 mm absorbable screws. After the transferring of the tibial tubercle, two parallel bone tunnels with a diameter of 4.5 mm were drilled in the medial side of the patella, and another tunnel with a diameter of 6.0 mm was drilled on the site between the adductor tubercle and the proximal part of the superficial medial collateral ligament. The grafts were placed into the bone tunnel guided by a trans-femoral pin. Appropriate graft tension was obtained by cycling the knee from full extension to full flexion. The graft was then secured within the tunnel with a diameter of 6-mm interference screw and fixed at 30° of knee flexion. The detail was seen in Figs. 4A quality post-operative rehabilitation program was essential to having a successful outcome from a patellar stabilization procedure. In early stage, the goals of rehabilitation would initially focus on protection for healing, mobility and range of motion. Twenty-four hours after operation, ankle pump and straight-leg raise started with the knee joint fixed in extension position for two weeks. Then the knee flexion exercise started. At week 6, the knee flexed to 120°. The patients returned to daily activities around the third month.All calculations were made with IBM SPSS 25.0. Statistical significance was set at P < 0.05. Number of observed values, mean and SD, and minimum and maximum were reported for quantitative data. Number of observed values and number and percentage of patients per class were reported for qualitative data. Wilcoxon sign ranked sums was used for evaluating continuous variables and χ-square analysis was used for evaluating categorical variables.A total of 108 knees in 98 patients were included in the study. Of these, 10 patients (10.2%) had bilateral reconstructions. In the follow-up, 15 patients (13.3%) were lost. The mean age at operation was 19.2 ± 6.1 years , and the mean follow-up was 61.3 ± 15.4 months . All patients included had trochlear dysplasia , and 67% had patellar alta. The mean CDI was 1.2 ; mean TT-TG distance, 22 mm ; All data details were summarized in Table p = 0.587; Tegner: p = 0.542; Kujala: p = 0.542). Patients with patellar height ≥ 1.3 had mean Kujala, Tegner and IKDC scores of 92.5, 5.4 and 75 as compared with 89.4, 5.3 and 71.5 for those with patellar height < 1.3 . Patients with TT-TG ≥ 20 mm had mean Kujala, Tegner and IKDC scores of 91.5, 5.4 and 73.6 as compared with 87.6, 5.1 and 70.4 for those with TT-TG < 20 mm . Patients with age ≥ 18 had mean Kujala, Tegner and IKDC scores of 86.4, 5.1 and 69.8 as compared with 94.3, 5.6 and 75.5 for those with age < 18 .Patients with BMI ≥ 25 had mean Kujala, Tegner and IKDC scores of 86.4, 5.1 and 69.8 as compared with 94.3, 5.6 and 75.5 for those with age < 25 .Female patients had mean Kujala, Tegner and IKDC scores of 88.2, 5.1 and 70.7 as compared with 96.6, 6.0 and 78.1 for male patients .The results showed that increasing trochlear dysplasia, increasing patellar height,, increasing TT-TG distance, BMI and age did not have any significant impact in the improvement of their Kujala, Tegner and IKDC scores, but Female gender as risk factor had a negative impact for IKDC, Tegner and Kujala scores. All data details were summarized in Fig. Six knees 5.6%) had postoperative dislocation, 4 with medial subluxation, 4 with lateral subluxation and 4 with obvious knee pain (VAS ≥ 4), 2 with operative incision disruption, 2 with fat liquefaction. All of the TTOs were clinically and radiographically healed by 3 months. The mean scores for Kujala, Tegner and IKDC were 90.5 ± 15.5 , 5.3 ± 1.3  and 72.7 ± 12.1 , respectively. Patients with grade-A trochlear dysplasia had mean Kujala, Tegner and IKDC scores of 93.6, 5.5 and 72.8. Patients with grade-B trochlear dysplasia had mean Kujala, Tegner and IKDC scores of 93.3, 5.8 and 76.4. Patients with grade-C trochlear dysplasia had mean Kujala, Tegner and IKDC scores of 87.5, 5.1 and 70. Patients with grade-D trochlear dysplasia had mean Kujala, Tegner and IKDC scores of 89.8, 5.3 and 74.2. There were no significant differences in postoperative outcomes for different grades of trochlear dysplasia (IKDC: .6% had pp = 0.016), and between age < 18 and age ≥ 18 (p = 0.011). The result showed that the failure rate was significantly associated with trochlear dysplasia and age. However, there were no significant differences in the failure rates between CDI ≥ 1.3 and CDI < 1.3 (p = 0.088), between females and males (p = 0.669), between BMI < 25 and BMI ≥ 25 (p = 1.000) and between TT-TG < 20 and TT-TG ≥ 20 (p = 0.336). The result showed that the failure rate was not significantly associated with patellar height, sex, BMI and TT-TG distance. All data details were summarized in Table The χ-square analysis indicated that there were significant differences in the failure rates among different grades of trochlear dysplasia  were lost to follow-up despite attempts to contact them by telephone, which might cause bias risk. Third, among the patients contacted for the follow-up, no patients completed the preoperative assessment of the function of knee joint using related scales, which make us unable to evaluate the difference between preoperative and postoperative. Nevertheless, this was the first study about the function of combined treatment  at least three years after surgery and preoperative risk factors.As a surgical method, MPFL combined with TTO and LRR would be a reliable choice with a low failure rate (5.6%). Female gender was a risk factor for worse postoperative outcomes. Preoperative failure risk factors in this study were age and trochlear dysplasia.


Phytophthora species cause destructive plant diseases worldwide. All Phytophthora species, except for one, are listed as plant quarantine organisms in Japan. The exception, Phytophthora nicotianae is considered to be a domestic species. The injurious pests Phytophthora ramorum, Phytophthora lateralis, and Phytophthora kernoviae are invasive pathogens that cause tree mortality worldwide, mainly in the United States and the United Kingdom. To effectively control Phytophthora diseases, we established detection methods that utilize the loop-mediated isothermal amplification (LAMP) of the genus Phytophthora and the four species P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae. LAMP primers for P. ramorum, P. lateralis, and P. kernoviae were newly designed in the present study. Our multiplex assay includes the detection of plant DNA as an internal control. When the optimum ratio between plant and pathogen primers was used in multiplex LAMP assays, 1 pg to 100 fg of pathogen DNA was detected with similar sensitivity to that in simplex LAMP assays. The detection of plant DNA in the absence of pathogens enables us to check for and avoid undesirable negative results caused by enzyme inactivation or the contamination of amplification inhibitors from plant tissues. The total time from sample collection to results is approximately 120 min, and, thus, our multiplex LAMP assay may be used as an accurate and time-saving detection method for Phytophthora pathogens. Phytophthora species cause destructive diseases in forest trees and nursery plants  or dead-end (non-sporulating) hosts. In forests in California and Oregon in the United States (US), P. ramorum causes trunk cankers on susceptible oak species (Quercus spp.) without sporulation, but does not cause leaf or twig symptoms  infected with P. ramorum shows leaf blight followed by the appearance of abundant sporangia on the leaves (P. ramorum infected tanoak (Lithocarpus densiflorus) in North America (Larix kaempferi) in the United Kingdom (UK)  . Tree spP. ramorum was initially detected in the UK in 2002 . In addition to the UK  and occasionally Pacific yew (Taxus brevifolia) . DNA were extracted from mycelial colonies using the PrepManTM Ultra Sample Preparation Reagent (Thermo Fisher Scientific). A small amount of the mycelial mat was collected by scraping with an inoculating needle, transferred to a 1.5-mL Eppendorf tube containing 100 μL of 50% PrepMan Ultra Reagent, and incubated at 100°C for 10 min. After a 3-min incubation at room temperature, the sample was centrifuged at 21,880×g for 3 min. The supernatant (approximately 80 μL) was transferred to a new 1.5-mL tube and the total DNA concentration was measured using the QuantiFluor® dsDNA System (Promega) with a Qubit®2.0 Fluorometer (Invitrogen). The concentration was adjusted to 100 pg μL–1 with sterilized deionized water (SDW) and stored at 4°C until used.The isolates used in the present study are listed in P. ramorum, P. lateralis, and P. kernoviae were designed using Primer Explorer V5 software . The primers for P. ramorum and P. lateralis were based on the cytochrome c oxidase subunit I (cox1) gene of the isolates CBS 101553  and CPHST BL 42 , respectively . The supernatant was transferred to a 1.5-mL Eppendorf tube and centrifuged under the same conditions. The supernatant was transferred to a new 1.5-mL tube and diluted 20 times with SDW. Samples were stored at 4°C until used as templates.The plant samples used in the present study are listed in TM PCR Inhibitor Removal Kit (Zymo Research). The non-diluted DNA extract (50 or 100 μL) was purified according to the manufacturer’s protocol. Samples were stored at 4°C until used as templates.When weak or no detection results were obtained, extracted DNA was subjected to additional purification using the OneStepPhytophthora isolates used in the present study are listed in P. nicotianae and P. capsici were prepared as previously described , Japanese andromeda (Pieris japonica ssp. japonica), and common camellia (Camellia japonica) were also used in this experiment. Isolates of P. ramorum, P. lateralis, P. kernoviae, Phytophthora foliorum, and Phytophthora hibernalis were used to inoculate the leaves. P. foliorum and P. hibernalis are phylogenetically closely related to P. ramorum and P. lateralis; all four species belong to subclade 8c , primer mixture(s), and a DNA template: 1 μL (default) or 2 μL (for DNA extracted from plants). The 1× primer mixture for each pathogen contained 0.2 μM each of the F3 and B3 primers (0.05 μM for P. nicotianae) and 1.6 μM each of the FIP and BIP primers. The 1× mixture for the previously designed plant LAMP primers (®II (Optigene) with the following conditions: pre-heat at 65 or 68°C for 5 min; amplification at 65 or 68°C for 60 min; and an annealing curve analysis at 98 to 80°C, ramping at 0.05°C s–1. The temperatures of the pre-heat and amplification steps were optimized at 65°C for all primer sets, except Phytophthora genus-specific primers, which were optimized at 68°C. Therefore, multiplex assays were performed at 65°C for all assays, except those that included the Phytophthora genus-specific primers, which were performed at 68°C. The format of the raw data (.gen) was changed to a text file using Genie Explorer version 2.0.6.3 software (Optigene), and amplification curves and annealing curves were then created using Microsoft Excel. The sensitivity of each LAMP assay was assessed with at least three experiments. The specificity and detectability of each assay was evaluated at least twice for each DNA sample.The LAMP reaction was conducted in a total volume of 25 μL containing 1× Isothermal Master Mix , 4 mg mLP. ramorum, P. lateralis, and P. kernoviae, we designed LAMP primer sets  of Phytophthora, 12 species (12 isolates) of Pythium, one species of Phytopythium, and one isolate each of the following soil-borne pathogens: Aphanomyces cochlioides, Fusarium oxysporum f. sp. fragariae, Plasmodiophora brassicae, Rhizoctonia solani, Saprolegnia parasitica, Sclerotinia sclerotiorum, and Verticillium albo-atrum , 450, and ATCC 201856 ; and P. kernoviae Pk-1, 2654, and P1571 were tested. The specificity of each amplification was confirmed with a single peak in the annealing curve analysis at approximately 82°C for P. ramorum , P. lateralis (isolates WPC P3361 and ATCC 201856), and P. kernoviae (isolates Pk-1 and P1571) were 10, 100, and 10 fg, respectively. Representative data are shown in P. ramorum isolate CBS 101553, P. lateralis isolate 450, and P. kernoviae isolate 2654 (data not shown).To test the detection limits of the LAMP primers for  ramorum D, 83°C fateralis E, and 86ernoviae F. The deThe optimum primer ratios for multiplex LAMP assays designed to detect pathogens in plant samples were selected. “Optimum” indicates the concentration of plant primers that may be used without disturbing pathogen detection, while functioning as an internal control for non-infected plants. We used the 1× concentration of the pathogen primer mixes combined with different concentrations of the plant primer mix, and tested these mixtures using reaction temperatures of 65 or 68°C in the pre-heat and amplification steps.P. ramorum, P. lateralis, P. kernoviae, and P. nicotianae, we used the P. ramorum primers in reactions performed at 65°C. The P. ramorum species-specific primer set (at the 1× concentration) was mixed with different concentrations of the modified plant primer set , and then tested in assays with mycelial DNA extracted from P. ramorum Pr-1 and/or plant DNA extracted from the leaves of Rhododendron sp. (P. ramorum) DNA only (1 pg), and 4) a mixture of plant DNA (71.5 ng) and pathogen DNA (1 pg). In reactions containing the 0.15× or 0.1× plant primers with both types of DNA, pathogen DNA (peak at approximately 82°C) and plant DNA (peak at approximately 84°C) were both simultaneously detected .To select the optimum ratio in the species-specific multiplex LAMP detection of dron sp. . Four sadetected A and B. detected C and D. detected C, implyidetected D. Based Phytophthora species, the amplification reaction was performed at 68°C. We used Phytophthora genus-specific primers (1×) combined with different concentrations of the modified plant primers  and assayed with the DNA samples described above. In reactions containing the 0.2× plant primer and both types of DNA, pathogen DNA (peak at approximately 86°C) and plant DNA (peak at approximately 84°C) were simultaneously detected , and P. nicotianae  and Japanese andromeda (Pieris japonica ssp. japonica) were inoculated with P. ramorum, P. lateralis, P. kernoviae, P. foliorum, and P. hibernalis. The detached leaves of common camellia (Camellia japonica), which were inoculated with P. ramorum, P. lateralis, and P. kernoviae, were examined. Tomato (Solanum lycopersicum) and eggplant (Solanum melongena) fruits were inoculated with P. capsici and P. nicotianae. On days three to five post-inoculation, extracted DNA from the infected tissues were tested for the detection of the pathogen using multiplex LAMP assays. Target pathogen DNA with its specific primer set was detected as a single peak at the same temperatures as those described above for the sensitivity assays , P. lateralis (42 isolates), and P. kernoviae (22 isolates) from the NCBI nucleotide database (https://www.ncbi.nlm.nih.gov/nucleotide/) to confirm the conservation of the species-specific sequences in the designed primers. In addition, the primer sets were tested with mycelial DNA from the isolates of P. ramorum (30 isolates), P. lateralis (4 isolates), and P. kernoviae (9 isolates) that had originated from different countries and/or host plants. All the isolates tested were detected by each species-specific primer. Therefore, intra-species sequence variations among the isolates examined did not affect our species-specific detection.To detect important pathogenic species of the genus Pittosporum tobira, were more difficult to detect , the annealing peak of pathogen DNA was indistinct or formed dual peaks with plant DNA  is important when screening plant samples for infecting pathogens because it shows that negative results are not due to poor DNA extraction or other errors. The simultaneous amplification of the pathogen and plant DNA by multiplex LAMP is preferred, but is very difficult. In natural samples, the relative amounts of DNA originating from pathogens and plants markedly vary depending on the severity of the infection. In LAMP assays, the strong amplification of more abundant DNA may overwhelm the amplification of less abundant DNA . Furtherlant DNA . This relant DNA .Phytophthora disease management. The genus Phytophthora has a large number of host plants. Based on database searches in 2019, mainly from the USDA Agricultural Research Service, approximately 2,600 host plant species of 956 genera belonging to 184 families have been reported to host Phytophthora species worldwide. Moreover, previous studies suggested that global warming enhances disease development and affects the distribution of Phytophthora pathogens  Multiplex LAMP Detection of the Genus https://doi.org/10.1264/jsme2.ME21019Supplementary Material 1Supplementary Material 2Supplementary Material 3


Growth impairment is a common manifestation in Noonan syndrome (NS). Recombinant human GH (rhGH) treatment has been shown to increase growth and adult height (AH) in a few studies. We aimed to evaluate the growth trajectory towards the AH, and the effects of rhGH treatment in a large cohort of NS children.PTPN11 mutations, 94 who reached AH, were recruited. Auxological data were collected at 2, 5, and 10 years, at pubertal onset, at AH. Sixty-eight NS subjects affected with GH deficiency (GHD) were treated with rhGH at a mean dose of 0.24 mg/kg per week until AH achievement.Retrospective, multicenter, cohort study including subjects with genetic diagnosis of NS. A total of 228 NS patients, 154 with p<0.001), while no significant differences were found between HSDS measurements at 2, 5, and 10 years, at pubertal onset, and at AH. HSDS increased from −3.10 ± 0.84 to −2.31 ± 0.99 during rhGH treatment, with a total height gain of 0.79 ± 0.74, and no significant difference between untreated and treated NS at AH.ANOVA analysis showed a significant difference between birth length and height standard deviation scores (HSDS) at the different key ages (PTPN11 gene and in the less common genotypes.rhGH treatment at the standard dose used for children with GH idiopathic deficiency is effective in improving growth and AH in NS with GHD. Further studies are needed to assess genotype-specific response to rhGH treatment in the different pathogenic variants of  Noonan syndrome (NS) is a multisystem disorder characterized by facial and skeletal dysmorphisms, short stature, congenital heart diseases, organ dysfunction, and mild-to-moderate developmental/learning delay , 2. NS cRandomized studies in NS patients who reached AH have not been published so far, and available data are difficult to compare due to the heterogeneous treatment protocols, sample size, as well as different cohort selection criteria and limited data on AH.versus untreated NS subjects.The aims of this multicenter retrospective study were to evaluate the growth trajectory towards the AH, and the effects of rhGH treatment in a large cohort of Italian NS children, by comparing rhGH-treated 2. Bone age (BA) was determined using the Greulich and Pyle method and evaluated for each key age recorded on standard datasheets.Seven Italian Pediatric Endocrinology Centers participated in this retrospective study. Inclusion criteria were molecular diagnosis of NS and at least three height measurements among the key ages recorded on standard datasheets , in order to evaluate growth trajectory. For subjects who had undergone rhGH therapy, height measurements expressed as height SDS (HSDS), were evaluated at the beginning of treatment, after the first and the second year, and at the end of treatment. Anthropometric measurements were compared with the standard growth charts for the Italian population , 28 and Patients with height (H) ≤ −3 SDS, or H ≤ −2 SDS and growth velocity (GV) ≤ −1 SDS, or severe reduction in GV (≤ −2 SDS/year) were tested for GHD by pharmacological stimulation tests, as indicated by the Italian Drug Agency , note number 39. GHD was defined as a peak of GH value less than 10 ng/ml or 8 ng/ml, according to revised criteria starting from 2015, in two GH stimulating tests.Pubertal stages were assessed with the Tanner method . GV cm/. AH was The study was approved by the Comitato Etico Indipendente, Azienda Ospedaliero-Universitaria “Consorziale Policlinico,” Bari, Italy. Children’s parents or guardians gave written informed consent.t-test) was used for normally distributed data; Mann–Whitney U test was used for not normally distributed data. ANOVA was applied to compare means between more than two groups. The level of significance was set in all cases at p<0.05. The normal distribution of the measured variables was assessed by Shapiro-Wilk test, whereas the homogeneity of variances across groups was verified using Levene’s test. Variables with non-normal distribution were expressed as medians and interquartile ranges (IQRs) and were compared using Kruskal–Wallis test. In case of significant results, pairwise comparisons using Tukey test or Dunn’s method for parametric or non-parametric ANOVA analysis were performed, respectively. Friedman test (ANOVA on rank for repeated measurements) was used to evaluate the effect of rhGH therapy on height measurements during the treatment period. Multiple regression analysis, using the forward stepwise approach, was used to study the effect of some variables on the outcome, represented by the total statural gain ∆HSDS (height at the end of therapy − height at baseline). Normality test (p>0.05), equal variance test (p>0.05), and Durbin-Watson test  were verified before performing multiple regression analysis, to assess normal distribution of population, variance homogeneity, and autocorrelation in the residuals, respectively. Statistical analyses were performed using SigmaPlot® Software, version 12 for Windows.Numerical data considered in the study were expressed as mean ± SDS. Mean HSDS for NS population was compared with those of the normal population  and thosde novo mutations. Among them, 154 (67.5%) had PTPN11 mutations, while 74 (32.5%) had the following gene mutations: 21 SOS1, 16 SHOC2, 9 RAF1, 8 KRAS, 5 BRAF, 6 LZTR1, 3 RIT1, 2 SOS2, 1 MEK, 1 NRAS, 1 CBL, and 1 MAP2K2. Most of the patients  were born at term; 32 (14%) were born before 37 weeks. Overall, 160 patients (70.2%) had cardiac malformations, and 18 (11.2%) underwent surgery. Data on AH were obtained from 94 patients (41.2%)  were collected. All cases were  (41.2%) Figure 1p=0.008) and had a bigger head circumference (p= 0.001) at birth, while BW was similar to healthy neonates (NS patients were significantly shorter (neonates Table 1.p<0.001), while no significant differences were found between HSDS measurements at 2, 5, and 10 years, at pubertal onset, and at AH, as shown in ANOVA analysis among length/height measurements showed a significant difference between BL SDS and HSDS at the different key ages  and PTPN11−, the latter including patients with mutation in the other NS-related genes. BL did not differ between the two groups, while PTPN11+ subjects had a lower BW than PTPN11− . At the other crucial time points, no significant difference was found between the two genotype groups.Patients were divided into two groups according to genotype: At the onset of puberty, NS females had a mean age of 12.1 ± 2.3 years, with a mean BA of 11.3 ± 1.7 years, while male patients had a mean age of 12.1 ± 1.3 years, with a mean BA of 10.8 ± 1.3 years. The frequency of delayed puberty was 45% in females and 10% in males.PTPN11+ and PTPN11− subjects.In females, HSDS decreased at puberty onset  and increased during the pubertal spurt, reaching an AH SDS of −2.02 ± 1.09 (−0.04 ± 0.94 Ranke standard). In boys, a steadier growth trend with a HSDS of −2.14 ± 0.87 (0.02 ± 0.96 Ranke standard) at pubertal onset and −2.08 ± 0.96 SDS (0.23 ± 0.85 Ranke standard) at AH was observed. According to genotype, no significant difference was found between height at the start of puberty in PTPN11+, while the other 20 subjects (29%) showed different genotypes . At the beginning of treatment, the mean age was 7.2 ± 3.5 years (range: 0.9–14.1 years). The mean duration of rhGH therapy was 6.3 ± 4.0 years, and the mean dose of rhGH administered was 0.24 mg/kg per week. HSDS evaluated at the beginning of treatment, after the first and the second year, and at the end of treatment is shown in Among rhGH-treated NS subjects, 48 (71%) were versus −0.83 ± 1.46, respectively; p=0.917). There was no significant difference in age and weight between male and female patients at the beginning of treatment, but female patients were significantly shorter  than male patients  (p=0.001).HSDS was also evaluated in male and female patients Table 2.p>0.05). The trend of HSDS during the treatment period is shown in p<0.001) (p=0.011). BA increased from 5.80 ± 3.81 years to 6.59 ± 3.76 years after one year, and to 8.36 ± 3.70 years after two years of treatment (p=0.012). The BA/chronologic age ratio increased from 0.72 ± 0.19 at the start of treatment to 0.77 ± 0.16 after one year, and to 0.84 ± 0.15 after 2 years of treatment (p=0.021). AH was reached by 41 out of the 68 NS subjects (60.3%) who had undergone rhGH treatment, at the mean age of 19.42 ± 1.38 years, and the mean value was −2.24 ± 0.89 SDS (0.09 ± 0.79 Ranke standard).Multiple comparisons performed using Tukey test showed that there was an increase in HSDS mean values during rhGH treatment with the best results obtained in the first year Table 2.p=0.024), the 5-year-olds groups (p<0.001), the pubertal onset groups (p <0.001), and a slightly significant difference between the 10-year-olds groups (p=0.043). On the contrary, no significant difference was found between the AH groups (p=0.123) (p<0.001) (The comparison of H measurements between the rhGH treated and untreated NS subjects showed a significant difference between the 2-year-olds groups (p=0.123) Table 3.PTPN11+ subset and to male sex patients’ subgroup. Taking into account pubertal age of NS patients, two different kinds of multiple regression analysis were performed.To assess which parameters affected ∆HSDS during rhGH therapy, a multiple regression analysis was performed. ∆HSDS was set as the outcome (dependent variable), while chronological age, IGF-I, rhGH dose, baseline HSDS, type of mutation, TH, and sex were considered as independent variables. To perform this analysis, qualitative variables such as type of mutation and sex were transformed into dummy variables . In particular, value 1 was assigned to 2 = 0.431, p<0.001R), IGF-I , and type of mutation . These results showed that patients with female sex, PTPN11+, and lower IGF-I values were more likely to achieve a wider ∆HSDS, hence a higher height at the end of therapy.2 = 0.281R, p=0.008) and sex . These results suggested that males and patients PTPN11+ reached a height at the end of therapy closer to their TH values . In contrast, a significantly greater increase in HSDS has been observed with 0.066 vs 0.033 mg/kg/day of rhGH without adverse effects  rhGH treatment in GH deficient NS subjects is effective in improving height gain and AH, according to Ranke standard; (ii) the greatest loss in height SDS occurs within the first 2 years of life in NS subjects, thus it is recommended to start rhGH treatment early; (iii) GH deficiency alone is insufficient to explain the cause of short stature in NS subjects.More studies are required to investigate which genotype or molecular alteration produces a good response to the treatment in order to more accurately select the candidates for rhGH therapy.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by Comitato Etico Indipendente, Azienda Ospedaliero-Universitaria “Consorziale Policlinico,” Bari, Italy. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.AL and MF conceptualized and designed the study, drafted the initial manuscript, and reviewed and revised the manuscript. VD’A performed statistical analysis. MCh, MD, AG, SE, AM, GP, ES, AS, and SV collected data and rewieved the manuscript. MCa, MM, LM, MW, and GF coordinated and supervised data collection and critically reviewed the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version. All authors agree to be accountable for all aspects of the work.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.


The MS detection was performed in positive ion mode using electrospray ionization (ESI) source. This method showed good linearity over the range of 1~500 ng/mL (R2 > 0.999), and the lower limit of quantitation (LLOQ) was 1.0 ng/mL. The intraday precision and interday precision were both within 8.5%, whereas the accuracies were in the range of -2.6%–6.0%. The average recoveries of galgravin in rat plasma were between 92.3% and 99.3%. Moreover, galgravin was stable throughout storage and processing with all RSDs below 12.1%. After the successful application of this optimized method, the oral bioavailability of galgravin was determined to be 8.5%. This study will be helpful to the future research and development of galgravin.This paper presents a new high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method with a rapid analysis of 6 min to determine the concentration of galgravin in rat plasma so as to study its pharmacokinetic features and bioavailability in vivo. Schisandrin was selected as the internal standard (IS). After extracting the analyte from plasma samples with ethyl acetate, methanol-H Piper wallichii [Tridax procumbens [Acorus tatarinowii Schott [Schisandra propinqua [Lignans have attracted extensive attention of researchers owing to their wide bioactivities including antioxidant, antitumor, and hepatoprotective effects, as well as antileishmanial and antimalarial activities in recent years –4. Galgrallichii , leaves ocumbens , rhizomei Schott , and theropinqua . Modern ropinqua , 10. In ropinqua , 12. Galropinqua . Evidencropinqua .1H and 13C NMR to identify the structure of this compound, the researchers once used vacuum liquid chromatography, semipreparative HPLC system [Currently, there are few literature reports on galgravin. In addition to using C system , and supC system  during tC system , 16.Although galgravin has a variety of pharmacological activities, there is no literature on its pharmacokinetics in vivo. It is widely known that HPLC-MS/MS method is much useful in the pharmacokinetic study of quantifying the main components of drugs because of its high selectivity and sensitivity. A new and efficient HPLC-MS/MS analysis method was established in this research and first successfully applied to investigate the pharmacokinetics and bioavailability of galgravin in two different ways of administration (intragastric and intravenous dosing). The study would provide some references for further research and application of this compound.Chromatographic grade methanol and acetonitrile were obtained from Thermo Fisher Scientific Co. Ltd. (Canada). Both ethyl acetate  and formic acid  were also of HPLC grade. Ultrapure water was prepared by a Milli-Q water purification system . Galgravin (purity ≥ 98%) and schisandrin (purity ≥ 99%) were purchased from Chengdu Dexter Biotechnology Co. Ltd. The chemical structures of galgravin and IS are presented in The separation and detection of the analyte were carried out using the LC-MS/MS system, which comprises mainly of an Agilent-1200 high-performance liquid chromatography system  and an Agilent-6430 triple quadrupole tandem mass spectrometer .μm, 4.6 × 50 mm) with column temperature of 25°C and a run time of 6 min. The mobile phase system was composed of methanol and 0.1% formic acid water . 5 μL sample was injected into HPLC-MS/MS system under the flow rate of 0.3 mL/min.The chromatographic separation was achieved by using a Waters XBridge™ BEH C18 column (2.5 The positive ESI with multiple reaction monitoring (MRM) mode was applied for mass detection. The specific parameters of galgravin and IS are shown in μL) for preparation of serial CS solutions, yielding final concentrations of 1, 2, 5, 10, 25, 50, 100, 250, and 500 ng/mL for galgravin and 40 ng/mL for IS. The concentrations of QC samples for method validation were 2, 50, and 400 ng/mL, respectively.A certain amount of galgravin and IS reference material were precisely weighed and dissolved with methanol to prepare respective stock solution at concentration of 1 mg/mL. Then, the stock solution of galgravin was diluted serially with methanol to prepare the working solutions over the concentration range of 5-2500 ng/mL. And IS working solution was at 200 ng/mL. All solutions were stored in a refrigerator at -20°C. Appropriate amount of working solution was added into blank rat plasma , the peak area ratio of galgravin to IS as dependent variable (y), and 1/x as weight factor. Then, the correlation coefficient (R2) was used to evaluate the linearity. The sensitivity of the method was reflected by LLOQ with signal-to-noise ratio (SNR) greater than 10. At this concentration, not only should the relative error (RE) be within ±20% but also the relative standard deviation (RSD) was less than 20%.The calibration curve was constructed at nine concentration levels , and regression equation was calculated with the concentration of galgravin as independent variable (n = 6) were prepared and analyzed continuously for 3 days to investigate intra- and interday precision and accuracy. The precision and accuracy are expressed by RSD and RE values, respectively. Meanwhile, the acceptance criteria were RSD ≤ 15% and RE within ±15%.QC samples at three concentrations  were as follows: short-term stability (2 h at room temperature), freeze-thaw stability (three freeze-thaw cycles), postpreparative stability (12 h in autosampler), and long-term stability (1 week at -80°C).μL) were obtained from the orbital fossa vein into heparinized centrifuge tubes at prodose, 0.03, 0.08, 0.17, 0.5, 0.75, 1, 2, 4, 6, 8, 10, 12, 24, and 36 h after administration. The other group was injected with galgravin solution via tail vein. The dosage was 2 mg/kg, and blood samples were collected at prodose, 0.03, 0.08, 0.17, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 12 h. After centrifugation at 3,000 × g for 10 min, the plasma samples were immediately separated, followed by transfer into clean tubes and storage at −80°C. The animal studies were approved by the Animal Ethics Committee of Tianjin University Traditional Chinese Medicine (TCM-LAEC20200046).Twelve male Sprague-Dawley rats  were randomly divided into two groups and fed in suitable environment . After fasting overnight, the first group of rats received a single gavage administration of galgravin suspension (0.5% CMC-Na as solvent) at 20 mg/kg, and serial blood samples  using the following formula: F (%) = (AUCi.g. × Dosei.v.)/(AUCi.v. × Dosei.g.) × 100.The plasma concentration of galgravin was quantitatively calculated using MassHunter Workstation software (version B.09.00). DAS Software  was applied to evaluate the exact pharmacokinetic parameters. The area under the concentration-time curve (AUC) is essential for the calculation of oral absolute bioavailability , ZORBAX Eclipse C18 column , and X Bridge™ BEH C18 column  were investigated during the method development. The results showed that the peak shape of the analyte obtained by CORTECS™ C18 column  was asymmetric compared to X Bridge™ BEH C18 column , and the chromatographic response significantly reduced using ZORBAX Eclipse C18 column  with longer analysis time compared to X Bridge™ BEH C18 column . Therefore, the X Bridge™ BEH C18 column  was selected finally. Meanwhile, the influence of different mobile phase combinations  or added buffers  on the chromatographic behavior of the target compound was investigated. Methanol and 0.1% formic acid water were selected because of the symmetric peak shape, appropriate retention time, and detection sensitivity. The optimal column temperature was 25°C. The major MS spectrometry parameters were optimized individually to obtain better responses. Results showed that the signal response of galgravin and IS in positive ion mode was higher in contrast to the negative mode. In addition, the optimal ion transition pairs for quantitation were at m/z 373.3 ⟶ 235.2 for galgravin and m/z 433.2 ⟶ 384.3 for IS in MRM mode.In this study, CORTECS™ C18 column (2.7 Three extraction methods were investigated to find an appropriate preparation method of plasma sample in this study. It was found that liquid-liquid extraction (LLE) with ethyl acetate provided better recovery and process efficiency of galgravin compared to protein precipitation with methanol and acetonitrile. Hence, LLE was selected to extract the analyte from biological samples.The typical MRM spectra of blank plasma (a), blank plasma spiked with galgravin and IS (b), and plasma sample after gavage (c) are displayed in y = 0.020756x + 0.005814 (R2 > 0.999) by weighted least squares linear regression. And the LLOQ was established as low as 1.0 ng/mL, which indicated that the method has high sensitivity.Good linearity was attained in the range of 1-500 ng/mL. The typical regression equation of calibration curve was The results of intra- and interday precision and accuracy are shown in As listed in The novel analytical method was proved to be successful to determine the plasma samples obtained from rats that received intragastric and intravenous administrations of galgravin. Cmax, 48.42 ± 37.66 ng/mL) at 2.08 h (Tmax) and declined with the T1/2 of 3.99 h after a single oral administration to rats (20 mg/kg), suggesting that galgravin had a moderate speed of absorption and metabolism in blood circulatory system after oral administration. However, Cmax,oral was much lower than Cmax,i.v., which means that the absorption of galgravin was not very well. The AUCt)(0 −  (314.25 ± 179.84 h ng/mL) was close to the AUC(0 − ∞) (315.75 ± 179.70 h ng/mL), which indicated that the monitoring time of this study was reasonable. After intravenous injection of galgravin solution at 2 mg/kg, the Cmax of 332.80 ± 63.59 ng/mL was achieved at 0.03 h without absorption process; then, plasma concentration of galgravin decreased exponentially. Short MRT (1.27 ± 0.19 h) and T1/2 (1.34 ± 0.21 h) suggested fast elimination tendency of galgravin. The AUCt)(0 −  and AUC(0 − ∞) were 369.56 ± 66.06 h ng/mL and 371.36 ± 66.59 h ng/mL, respectively. Finally, the calculated oral bioavailability of galgravin was 8.5%.The concentration of galgravin in plasma reached its peak (Based on the above results, the pharmacokinetic characteristics of galgravin may affect its clinical application in the future. Its insufficient oral absorption could be compensated by increasing the administered dose, and the longer retention time in the body may keep the drug in effect for a longer period of time. By comparison, intravenous administration shows the advantage of a faster onset of action and would be beneficial for rapid clinical control of the condition.In the study, an efficient and sensitive analysis method of HPLC-MS/MS was developed and optimized to determine galgravin in plasma after single-dose oral and intravenous administration to rats. The described method had been proved to be successful in studying the bioavailability of galgravin. The absolute bioavailability of 8.5% indicated oral malabsorption of galgravin. This paper provides the first pharmacokinetic and bioavailability study of galgravin, which might be helpful for its further research and applications.


Although the foot is involved in load-bearing and shock absorption, foot pressure (FP), ground contact area (CA), and gait cycle (GC) in flatfeet (FF) have not been examined in detail. We aimed to analyze the influence of FF on FP, CA, and GC. We included 20 and 21 women with FF and normal feet (NF), respectively. A Footscan plantar pressure plate  was used to analyze FP, CA, and GC. FP was applied to the unit area of 10 compartments. GC analysis was performed using phase-time measurements by dividing the GC into four phases. In the analysis, FP and CA were compared between the FF and NF groups. A comparison of GC was similarly performed between the two groups.The data provided in this article will be useful when designing studies on the effect of foot shape on FP, CA, and GC during gait. They can be used to evaluate the effects of foot shape on gait disturbance.•These data will be beneficial for researchers in measuring the plantar pressure distribution during walking.•These data can be used to compare the sole pressure distribution and walking cycle before and after exercise and when using the insole.1The dataset and supplementary files provide relevant details on the plantar pressure distribution and walking cycle, and the data are presented in the tables and figures.The data obtained from participants with flat and healthy feet are presented in The raw data are described [Mendeley Data], and all data for the maximum plantar pressure (MaxP), CA, and phase-time are available. Foot pressure (FP) was defined as the MaxP, and the MaxP and contact area (CA) were measured in the following 10 compartments: Toe1 (T1), Toe2–5 (T2–5), Metatarsal 1 (M1), Metatarsal 2 (M2), Metatarsal 3 (M3), Metatarsal 4 (M4), Metatarsal 5 (M5), Midfoot (MF), Heel medial (HM), and Heel lateral (HL) .Participants in the FF and NF groups walked and stepped on the RsScan device, which was placed 5 m away from them, with one foot.2 during walking for participants in the FF and NF groups.t-test was performed for MaxP, for participants in the FF and NF groups, in 10 compartments: T1, T2–5, M1, M2, M3, M4, M5, MF, HM, and HL. In T1, the FF group showed a significantly higher MaxP than the NF group.A t-test was performed for CA, for participants in the FF and NF groups, in 10 compartments: T1, T2–5, M1, M2, M3, M4, M5, MF, HM, and HL. In MF, the FF group showed a significantly higher CA than the NF group.A The values of initial contact phase (ICP), forefoot contact phase (FFCP), foot flat phase (FFP), and forefoot push-off phase (FFPOP) were 49.7 ± 22.6 ms, 30.3 ± 16.4 ms, 314.4 ± 42.9 ms, and 287.4 ± 38.3 ms, respectively, in the FF group and 50.9 ± 13.2 ms, 30.2 ± 13.3 ms, 304.7 ± 51.8 ms, and 285.4 ± 52.9 ms, respectively, in the NF group. No significant differences were observed in phase-time measurements between the FF and NF groups.22.1Twenty women with FF ] years; height, 1.58 [0.03 SD] m; body mass, 53.8 [7.5 SD] kg; foot length, 23.2 [0.7 SD] cm) and 21 women with NF  were used to acquire these data. The data collected were not case-controlled.2.2The arch index (AI) The AI has received considerable scientific attention and has allowed researchers and clinicians to classify static arch structures as either high (≤0.21), low (≥0.26), or normal (0.21−0.26) 2; Resolution: 8 bits. The participants were instructed to walk barefoot and step on the RsScan on only one foot for a total of three sets. MaxP areas of the foot were measured during gait at T1, T2–5, M1, M2, M3, M4, M5, MF, HM, and HL and was applied to the unit area of 10 foot regions. Similarly, the pressure applied to the CA was measured. Phase-time measurements of the walking time (ms) were obtained by dividing the walking cycle into ICP, FFCP, FFP, and FFPOP. The values (%) were obtained by dividing each phase time by one GC. ICP was defined as the period from the first heel contact to the first metatarsal contact, and FCP was defined as the period from the first metatarsal contact to the contact of all (M1~M5) metatarsals. While FFP was defined as the period from the contact of all metatarsals until the heel  left the plate, FFPOP was defined as the period from the last heel contact to the last foot contact.RsScan was used for the measurement. The specifications of RsScan are as follows: Dimensions: 578 × 418 × 12 mm; Effective measuring range: 488 × 325 mm; Number of sensors: 4096; Sensor size: 7.62 × 5.08 mm; Measurement pressure range: 1–127 N/cmICP and FFCP correspond to the loading response, FFP corresponds to the mid stance, and FFPOP corresponds to the terminal stance and pre swing t-test, and Welch's t-test were performed on normally distributed data, and the Mann–Whitney U test was conducted on abnormally distributed data. A p-value <0.05 was considered statistically significant.In the analysis, MaxP and CA were compared between the low and normal arch groups, and the time required for ICP, FFCP, FFP, and FFPOP was similarly compared between the two groups. F-test, Student's The ethics committee of Hokkaido Bunkyo University approved all study protocols, and each participant provided written informed consent prior to enrollment (number 02008).Yuichi Takata: Conceptualization, Methodology; Shinji Matsuoka: Statistical analysis; Genta Asano: Software; Ryoki Kawamura: Investigation; Kazushi Kimura, Shigenori Miyamoto: Supervision; Yuichi Takata, Hiroshi Hashida: Writing- Reviewing and Editing.The authors declare that they have no known competing financial interests or personal relationships, which have or could be perceived to have influenced the work reported in this article.


Syzygium aromaticum seeds, as well as investigating its biological activities, insecticide effect, and allelopathic properties. The extraction yield was about 14.3 and 7.14% for grounded and ungrounded seeds, respectively. The GC-MS analysis allowed the identification of 17 heterogeneous compounds, including eugenol (68.7-87.4%), as major compound, cyperene (20.5-7.2%), phenethyl isovalerate (6.4-3.6%), and cis-thujopsene (1.9-0.8%), respectively, for grounded and ungrounded seeds. Concerning the antibacterial activity, the diameter of the inhibition zone reached 35 mm when the essential oil extracted from grounded seeds was applied against Escherichia coli. Regarding the antioxidant activity via the DPPH radical scavenging test, the IC50 varied from 1.2 ± 0.1 to 2.8 ± 0.5 μg/mL. With respect to reducing power, the efficient concentration EC50 ranged from 32 to 50 μg/mL. The essential oil exhibited also an allelopathic effect against seeds of Hyoscyamus niger, as well as an insecticide effect against Sitophilus oryzae with a DL50 value of 252.4 μL/L air. These findings enhance the use of this spice as a natural food preservative and encourage its use in several fields, including pharmaceutical, cosmetics, agriculture, and therapy, that could be a strategic way to guarantee the consumer's health.This study is aimed at identifying the chemical composition of the essential oil extracted from the  Over the last decades, the use of aromatic plants for traditional medicinal purposes has gained a great interest. Thus, the Arab and Egyptian civilizations have attributed their use to religious recommendations . ThereafNowadays, modern medicine uses the therapeutic properties of essential oils and their constituents. Actually, many volatile compounds are common ingredients of pharmaceutical preparations. Thymol is used in dental care for its antiseptic properties, and eugenol is recommended due to its analgesic effect . In an aSyzygium aromaticum, also called clove, is a dried flower bud, which belongs to the Myrtaceae family. This species is indigenous to the Maluku islands in Indonesia, and it is the most important and second valuable spice in world trade [ld trade . Clove bld trade , 8.Several researchers have reported the antibacterial activity of the essential oil from clove buds against different food-borne pathogens , 10. HowSyzygium aromaticum (S. aromaticum) (synonym: Eugenia cariophylata), commonly known as clove, belongs to the Myrtaceae family, subfamily of Myrtoideae and Syzygiae tribe. It is a median-sized tree (8-12 m) native from the Maluku islands in east Indonesia [According to the US National Plant Germplasm System, ndonesia . It is a2SO4 and stored in amber-colored bottles at 4°C for subsequent analysis. The Na2SO4 was removed by filtration of the EO. The extraction yield was calculated as follows:Both grounded and ungrounded buds were subjected, separately, to hydrodistillation during 6 h using a Clevenger-type apparatus. The raw material was directly submerged in 500 mL distilled water in a round bottom flask (1 L capacity) containing a few pumice stones to regulate boiling. The entire extraction method was operated at atmospheric pressure. This technique consists of boiling the mixture, then condensing the vapors that are released using a water condenser. The condensation of this vapor mixture causes its separation into an organic phase, called essential oil, containing the majority of the odorous compounds, and an aqueous phase, called aromatic water, which contains very few odorous compounds. As the density of clove bud essential oil (1.062) at 20°C was superior to the water density (1.00), the separation of the organic and aqueous phases was a little bit difficult. In fact, the organic phase was below the aqueous one. For this purpose, the distillate was placed in a decanter. Salt-saturated water was then added. After shaking and degassing, the mixture was decanted. This step, called release, allows the separation of the essential oil from the aqueous phase, since the essential oil is less soluble in salt water than in water. The obtained essential oil was then recovered with the help of the decanter. After that, it was dried with NaS. aromaticum bud essential oil used the procedures of Ben Hassine et al. [μm). The analysis was performed using the following temperature program: temperature rise from 60°C to 260°C with a gradient of 5°C/min and 15 min isotherm at 260°C. A second gradient was applied to 340°C at 40°C/min. The injection volume was 1 μL in 1 : 10 split mode. The carrier gas was helium with a flow rate of 1 mL/min. Injector temperature was held at 200°C. The mass spectrometer (Varian Saturn GC/MS/MS 4D) was adjusted for an emission current of 10 μA and electron multiplier voltage between 1.400 and 1.500 V. Trap and transfer line temperatures were 220°C and 250°C, respectively. Mass scanning was from 40 to 650 amu. The identification of the oil constituents was based on a comparison of their retention indices (RI) relative to C5-C24 n-alkanes with those of the literature or with those of authentic compounds available in our laboratory. Further identification was made by matching their recorded mass spectra with those stored in NIST 08 and other published mass spectra. The percentage composition of the essential oil was calculated by the normalization method from the GC peak areas, assuming identical mass response factor for all compounds.The identification of volatile compounds from e et al. . Analyti• solution (0.2 mM). After 30 min of incubation at room temperature, the absorbance was measured at 520 nm. The DPPH• inhibition percentage (IP) was expressed as follows: IP(%) = ((Ablank − Asample)/Ablank)∗100. Here, Ablank is the absorbance of the control containing only the solvent and DPPH• solution. Radical scavenging activity was expressed in terms of IC50 (μg/mL), and BHT was used as standard. IC50 represents the concentration at which clove EO exerts half of its maximal DPPH• inhibitory effect. It indicates how much EO is needed to inhibit the radical by half, thus providing a measure of potency. A lower value of IC50 indicates more potent inhibitors. All measurements were replicated three times.Spectrophotometric measurements were assessed according to the method of Ben Hassine et al. . A volumS. aromaticum essential oil was mixed with 2.5 mL of sodium phosphate buffer  and 2.5 mL of 1% K3Fe (CN)6. The whole mixture was incubated in a water bath at 50°C for 20 min. Then, 2.5 mL of a 10% TCA solution was added to the mixture, which was centrifuged at 6500 rpm for 10 min. The supernatant (2.5 mL) was then mixed with 2.5 mL of distilled water and 0.5 mL of a 0.1% FeCl3 solution. The intensity of the blue-green color was measured at 700 nm, and BHT was used as a positive control. The IC50% value (μg/mL) is the effective concentration at which the absorbance was 0.5 for reducing antioxidant power and was obtained by interpolation from linear regression analysis. All measurements were replicated three times.Experiments were done according to Kammoun El Euch et al. . One mL Staphylococcus aureus ATCC 6538, Listeria monocytogenes FMCC B-128, and Bacillus cereus ATCC 168) and two Gram-negative types  were used. A suspension of the tested microorganism (0.1 mL of 108 cells per mL) was spread on nutrient agar. The discs have a 6 mm diameter, and against each microbial strain, one dose each of 1, 5, and 10 μL of pure EO was applied to each disc, respectively, and separately, by injecting and placing on the inoculated plates. Penicillin (P), chloramphenicol (C), gentamicin (GM), and erythromycin 500 (E) were used as standard antibiotics to determine the sensitivity of the bacterial species tested. These plates, after staying at 4°C for 2 h to allow the diffusion of volatile compounds inside the Petri dishes, were incubated for 48 h at 37°C. Antimicrobial activity was evaluated by measuring the inhibition zone diameter (IZD) (mm) against the tested microorganism. The strain's sensitivity was evaluated according to IZD (Ф) as follows: (-) 6 mm < Ф < 10 mm: the strain is insensitive to the EO action; (+) 10 mm ≤ Ф < 15 mm: the strain is sensitive; (+ +) 15 mm ≤ Ф < 20 mm: the strain is very sensitive; (+ + +) Ф > 20 mm: the strain is extremely sensitive to the EO action.The antimicrobial activity of clove buds essential oil was achieved according to the disc diffusion method . Three tIn light of previous results, tests for the allelopathic and insecticidal effects were carried out with the grounded clove bud essential oil, since it was responsible for the highest biological activity results.H. niger germination was tested following the method adopted by Zahed et al. with slight modifications [μL/mL) were used. Essential oil was diluted in 1‰ MeOH. Distilled water served as the control. Whatman No. 2 filter paper was placed in 9 cm diameter Petri dishes moistened with 5 mL of EO solutions. Five replications of 25 seeds were used for each treatment. To prevent evaporation, Petri dishes were sealed with parafilm and placed in an incubator at 25°C in the dark. After 10 days, the number of germinated seeds was recorded and the percentage of germination was calculated using the following formula:n is the number of germinated seeds in the test solution, and N is the total number of seeds. Seeds showing radicle emergence (2 mm) were recorded as germinated.The allelopathic effects of clove EO on ications . Five co(1) Insect Rearing. Sitophilus oryzae adults were obtained from cultures initiated in the Laboratory of Materials, Molecules, and Applications at IPEST, La Marsa, Tunisia. Corn (Zea mays) seeds were used as a host. Insects were reared in darkness at 25°C in a relative humidity (R.H.) of 65 ± 5% and 16/8 (L/D) photoperiod. The old adult insects (7-10 days) were used for fumigant assay.(2) Fumigant Assay. Fumigant toxicity assay of S. aromaticum essential oil was evaluated using the method described by Bachrouch et al. [S. oryzae adults (1–7 days old). When no leg or antennal movements were observed, insects were considered dead. To evaluate the insects' mortality, amounts of 2.5, 5, 7.5, and 10 μL EO from clove grounded buds, corresponding to concentrations of 41.7, 83.3, 125, and 166.7 μL/L air, respectively, were prepared. Control insects were kept under the same conditions without any EO, and each dose was replicated three times. The number of dead and alive insects in each bottle was counted 24 h after initial exposure. The mortality was evaluated by direct hourly observation of the insects. Probit analysis [50 and LC95 values after 24 h of treatment .h et al. . Two-cenanalysis  was usedP < 0.05. LC50 and LC95 were calculated by Probit analysis using IBM SPSS statistics version 19 (2010). Data were calculated for significance by ANOVA  using SPSS 20.1 . Statistical differences between the essential oils were estimated by Tukey's test. Student's t-test analysis was used to compare between essential oil extraction yields. Multiple range tests were conducted at 5% significance level.All data were expressed as means ± standard deviations of triplicate measurements. The confidence limits were set at S. aromaticum essential oil from Algeria. They obtained an extraction yield of 11.6%. Similarly, Kapadiya and Desai [Obtained results showed that grounded samples (particle size = 0.6 mm) present the highest extraction yield. While the ungrounded clove buds (particle size between 1.5 and 2 cm) exhibited an EO extraction yield of 7.1 ± 0.8%, the grounded ones showed an extraction yield two times higher than the first EO and reached the value of 14.3 ± 0.6% . These rnd Desai  have found Desai . The decnd Desai . In addind Desai .β-caryophyllene (11.5%) was one of the major components [β-cariofilen, α-humulen, β-pinene, limonene, farnesol, benzaldehyde 2-heptanone, and ethylanoate were the most common compounds [cis-thujopsene, and phenyl isovalerate, it would be preferable to ground the clove buds to improve their contents in the obtained essential oil.The volatile compounds of the different essential oils (grounded and ungrounded) of clove buds were analyzed by GC-MS . A compamponents . This laompounds . Accordiompounds . This vaompounds . The parompounds . Kapadiyompounds  also indompounds  which isβ-caryophyllene, and eugenyl acetate, which are known to inhibit reactive oxidative species by scavenging free radicals [50 = 1.2 ± 0.1 μg/mL) was about two times greater than that of ungrounded ones (IC50 = 2.8 ± 0.5 μg/mL). These results were very significant when compared to BHT (IC50 = 35.5 ± 0.57 μg/mL). These findings were in perfect agreement with Gülçin et al. [• scavenging activity of clove bud EO was more important than various synthetic antioxidants, namely, BHT, α-tocopherol, and Trolox. This important activity could be related to the high content of eugenol according to many scientific reports proving that eugenol is a potent chelator with values of IC50, ranging from 4.4 to 130.5 μg/mL [50 value (32 ± 1.3 μg/mL) than theEC50value of intact buds (50 ± 4.5 μg/mL) with eugenol amount of 87.39%.Several researchers have reported the efficacy of employing DPPH radical and FRAP methods to evaluate the antioxidant potential by estimating the radical scavenging activity , 24. Comradicals .  Figuren et al.  who repo.5 μg/mL , 27. Res50 was equal to 25.8 ± 0.2 μg/mL in the same experimental conditions. This finding could be related to the loss and the decomposition of clove buds during the grinding process, and thus to a reduction of the amount of volatile chemical compounds. It is important to note that there are no previous citations concerning the FRAP test on clove buds EO. These results were obtained after three measurements for both clove batches (ungrounded and grounded).The BHT EC+ and Gram− bacteria with IZD ranging between 17.2 ± 4.5 and 35.5 ± 2.6 mm. Statistical analysis showed a significant difference between EO from grounded and ungrounded clove buds. These findings were in perfect agreement with previous reports which elucidated important MIC values for L. monocytogenes (0.05 mg/mL) and S. enteritidis (0.1 mg/mL) [in vitro study that the MIC values of clove oil and eugenol to Bacillus cereus were 25 and 15 μg/mL, and their MBC values were 40 and 25 μg/mL, respectively, whereas the half maximal inhibitory concentration (IC50) of clove oil against this important food source gram-positive pathogen was 5.02 μg/mL, and that of eugenol was 4.05 μg/mL [S. aromaticum EO containing 56.06% eugenol showed a strong inhibitory effect up to a concentration of 0.30 mg/mL against pathogenic bacteria  [Syzygium aromaticum revealed promising growth inhibitory activity with MICs of 100 and 400 μg/mL, respectively, against S. aureus and E. coli. Abdullah et al. recorded an IZD against S. aureus of 13 ± 2.2 and 22 ± 1.6 mm at 1.3 and 10% concentration, respectively, [E. coli is the most sensitive recording the highest IZD of about 28.7, 34, and 35.5 mm, respectively, to 1, 5, and 10 μL applied doses. Besides, these results conventionally indicated that the increase of bioactive inhibitory power was closely correlated to EO type (from grounded or ungrounded buds) and concentration  . This ac05 μg/mL . Radünz 05 μg/mL  demonstr aureus) , Syzygiuctively, . Howeverctively, . Besidesntration . All the acetate , 37. The acetate . All thelus spp. , 40. Thelus spp. , rainbowlus spp. , ground lus spp. , cooked lus spp. . These fH. niger germination rate under different clove buds EO concentrations. Noticeably better results were demonstrated for the essential oil (30 μL) in comparison with the control seeds. Indeed, clove buds EO achieved complete inhibition of H. niger germination during the whole experiment period; however, only 91% was recorded for the control seeds. Controls prepared with a methanol/water (1‰) mixture did not affect H. niger germination in comparison with controls in water. In addition, obtained results showed the dose-dependent manner of the allelopathic effect. These findings were in line with those reported by Amparo-Blazquez [S. aromaticum EO caused an important inhibition of seedling growth against broccoli, lambsquarters, and redroot pigweed. The mechanism of germination inhibition is not well known, but some studies reported that a lack of electrolyte, cell destruction, and membrane permeability caused by EO could be implicated [Blazquez , which iplicated . An inhiplicated . EOs cauplicated . These fplicated . Kegley plicated .S. oryzae was significantly susceptible to the EO after 24 h of exposure. Probit analysis showed that 50% of mortality was achieved at 252.4 μL/L air concentration, when the confidence limits were about 95%. LD95 was estimated at 426.2 μL/L air. Cardiet et al. [S. oryzae with LC90 observed at 355 μL/L. According to Devi and Devi [S. oryzae was susceptible to the toxic effect of clove oil resulting in 92% mortality by 21 days. Vendan et al. [S. oryzae recorded at a concentration of 400 μL/L air of clove oil and during 72 h of exposure time. Mishra et al. [S. aromaticum killed the adults of S. oryzae by fumigant action. They also mentioned that the fumigant toxicity was proportional to exposure time and concentration. The authors indicated that S. aromaticum EO had stronger toxicity in comparison to A. marmelos EO against S. oryzae. For that reason, Jairoce et al. [50 value obtained for clove oil after 24 h exposure against C. chinensis adults was about 0.3 μg/mL. The efficacy of clove oil is due to its major components, such as phenylpropanoids like carvacrol, thymol, eugenol, and cinnamaldehyde. The authors indicated that clove EO may be developed as possible natural fumigants or repellents to control the pulse beetle.The insecticidal activity was examined using the paper filter contact method. and Devi , S. oryza et al.  reportede et al.  recommenS. aromaticum essential oil. The assessment of the grinding effect on clove buds EO was performed initially. It was demonstrated that the grounded buds gave higher essential oil yield than intact ones, with respective values of 14.3 and 7.1%. Chemical composition analysis identified particularly four major compounds, respectively, as mentioned below for unground and ground clove buds EOs: eugenol (87.4-68.7%), cyperene (7.2-20.5%), phenethyl isovalerate (3.6-6.4%), and cis-thujopsene (0.8-1.9%). α-Patchoulene was present at 0.2% in the chemical composition of grounded clove bud EO. For the antibacterial activity, the best inhibition zone diameter was observed against E. coli(35.5 ± 2.6 mm), when the grounded clove buds EO was applied. This important antimicrobial activity enhances the potential use of essential oil as a food preservative. Regarding the antioxidant power, both tested EOs were more potent radical DPPH scavengers  than α-tocopherol, as they exhibited noticeable ferric-reducing power . The significant antioxidant power encourages the application of clove buds essential oil in the composition of several drugs as a natural remedy. Furthermore, our results highlighted the potent herbicidal effect of S. aromaticum oil on seed germination of H. niger, as well as its allelopathic effect. These findings promote the use of clove buds essential oil as a synthetic pesticide alternative to preserve maize, wheat, or other grass seeds during their storage period.The main objectives of this study were the chemical and biological evaluations of


The residues of the four pesticides in flour were significantly lower than those in wheat, ranging from 38.68% to 98.04%. Chlorpyrifos and omethoate levels showed a slight decrease during the kneading and fermentation process, and further decreases of 2.46%–29.51% for chlorpyrifos and 14.22%–71.11% for omethoate were found in CSB; however, most of the groups of cypermethrin and deltamethrin showed various degrees of increases in kneaded and fermented dough and steamed bread compared with flour. The mechanism of this increase is unknown and needs further research.To investigate the fractioning of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during wheat milling and the fate of four pesticides during Chinese steamed bread (CSB) processing, wheat samples, which were sprayed twice with chlorpyrifos, omethoate, cypermethrin, and deltamethrin at three levels of concentrations during the grain‐filling stage, were milled, and wheat flour was processed to CSB. The residues of four pesticides in the milling products, kneaded dough, fermented dough, and CSB were determined with GC‐MS/MS. The concentrations of chlorpyrifos, omethoate, cypermethrin, and deltamethrin in bran were 1.46–1.57, 1.85–2.13, 1.27–1.86, and 1.63–2.33 times higher than those in wheat, respectively, while the residues of the four pesticides in shorts decreased approximately 27.97% to 57.02% for chlorpyrifos, 6.22% to 44.77% for cypermethrin, and 13.13% to 61.15% for deltamethrin compared with the residues in wheat ( Milling process decreased chlorpyrifos, omethoate, cypermethrin, and deltamethrin levels in flour. Cypermethrin and deltamethrin levels showed various degrees of increases during Chinese steamed bread processing. Changes of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during Chinese steamed bread making process were firstly detailed reported. Many kinds of pesticides are used yearly to control pests and diseases that threaten wheat yield and quality. Hence, pesticides play a major role in controlling pests and diseases, improving crop yield and quality, and ensuring the quality and safety of agricultural products  of wheat have been established by many countries and organizations, such as the European Union (EU), Codex Alimentarius Commission (CAC), and USA. In EU, Australia, USA, Japan, and CAC, the MRLs of chlorpyrifos in wheat are 0.05, 0.1, 0.5, 0.5, and 0.5 mg/kg, respectively; the MRLs of cypermethrin in wheat are 2, 0.2, 0.2, 0.2, and 0.2 mg/kg, respectively; and the MRLs of deltamethrin in wheat are 2, 2, 1, 2, and 2 mg/kg, respectively. The MRLs of omethoate in wheat are 0.01, 0.05, and 0.1 mg/kg in the European Union (EU), Australia, and Japan, respectively , a traditional staple food, has been consumed for nearly 2,000 years in China, representing more than 40% of Chinese wheat consumption  were obtained from Agro‐Environment Protection Institute, Ministry of Agriculture . The commercial pesticide chlorpyrifos (45% emulsifiable concentrate [EC]) was purchased from the Jiangsu Jiahui Chemical Co., Ltd.; the commercial pesticide omethoate (40% EC) was purchased from Shandong Dongtai Agrochemicals Co., Ltd.; the commercial pesticide cypermethrin (45% EC) was purchased from Shandong Zhongshi Pharmaceutical Co., Ltd.; and the commercial pesticide deltamethrin (25 g/L EC) was purchased from Bayer CropScience Co., Ltd. Organic solvents, including acetonitrile, formic acid, and n‐hexane, used for both sample extraction and analysis, were of HPLC grade and purchased from Thermo Fisher Scientific Corporation. UPLC‐grade acetone was purchased from MREDA Technology, Inc. Ultrapure water was obtained from a Milli‐Q system (Millipore).2.2The standard stock solutions of chlorpyrifos, omethoate, cypermethrin, and deltamethrin (100 mg/L) were diluted with acetone and stored at −20°C. Working standard solutions of chlorpyrifos, omethoate, cypermethrin, and deltamethrin  were prepared by diluting the stock solution. Correspondingly, matrix‐matched standard solutions of chlorpyrifos, omethoate, cypermethrin, and deltamethrin  were prepared by diluting the working matrix standard solutions. These solutions were stored in the darkness at 4°C, and the working standard solutions were not observed to degrade over 3 months.2.3A wheat field at Shunyi Farm, located in the northeast of Beijing, China , was divided into control plot, which was sprayed with water, and treatment plots, which were sprayed with 4 kinds of pesticides at 3 concentrations. Plot #1 was used as the control. Plots #2‐#5 were sprayed with commercial pesticides chlorpyrifos 45% EC, omethoate 40% EC, deltamethrin 25 g/L EC, and cypermethrin 45% EC at the recommended dosage . Plots #6‐#9 were sprayed with the 4 pesticides at twofold the recommended dosage and plots #10‐#13 at ten‐fold the recommended dosage. Each plot was sprayed twice at 14 and 7 d before harvest. Plots were randomly arranged, and each treatment was replicated 3 times. Wheat samples were harvested, placed in polyethylene bags and stored in a −40°C freezer.2.4All wheat samples were wetted to 16.5% moisture content and left overnight before milling. Milling was carried out by a Chopin CD1 Laboratory Mill  with two breaks and one stripping system. Three fractions, including bran, shorts, and flour, as well as wheat grain, were obtained and submitted for analyses of pesticide residues.2.5Chinese streamed bread was prepared according to the Chinese Standard . The basic formula for making CSB consisted of 100 g of milled flour, 48 ml of distilled warm water (38°C), and 1 g of active dry yeast. The ingredients were mixed evenly by hand for 3 min, and then, dough was fermented in a fermentation cabinet  for 1 hr. After that, it was kneaded and shaped manually for 5 min into a round dough piece with a smooth surface. After resting at room temperature for 15 min, the dough was put in a steamer when the water was boiling and was steamed (100°C) for 20 min, followed by cooling at room temperature for 40–60 min. Three replicate samples at every stage  were taken. All representative samples were stored at −20°C until analysis.2.6v/v) for 30 min using an automatic shaker. Afterward, 4 g of magnesium sulfate (MgSO4), 1 g of sodium chloride (NaCl), 1 g of sodium citrate dihydrate, and 0.5 g of sodium hydrogen citrate sesquihydrate were added and shaken vigorously for 2 min, and the sample was centrifuged for 5 min at 6,037 g. Then, a clean‐up, dispersive, solid‐phase extraction (d‐SPE) was conducted by adding 5 ml of the supernatant phase to a 15‐ml centrifuge tube that contained 900 mg of MgSO4, 150 of mg PSA, and 150 of mg C18. The sample was immediately vortexed for 1 min and centrifuged for 5 min at 6,037 g, and then, 2 ml of the supernatant‐cleaned extract was evaporated to dryness in a nitrogen evaporator with a water bath at 60°C. The dry residue was then dissolved in 1 ml of acetone, followed by filtering through a 0.22‐μm nylon syringe filter (Jinteng). After that, it was ready for analysis.The extraction and cleaning up procedure was carried out following the QuEChERS method. Five grams of homogenized sample were weighted in a 50‐mL polypropylene centrifuge tube and then extracted with 20 ml of acetonitrile , that was coupled to a triple quadrupole mass spectrometer (Bruker 300 MS), operating in the electron impact ionization (EI) mode. The instrument conditions are as follows: the column was initially maintained at 60°C, and then, the temperature was increased to 250°C at a rate of 20°C/min and finally increased to 300°C at a rate of 5°C/min and maintained for 4.5 min. The total run time was 24 min. Argon collision gas flow was maintained at 1.8 m Torr pressure. Helium (purity > 99.999%) was employed as a carrier gas at 1.2 ml/min, and injections were carried out in split/splitless mode at 250°C using 1‐μL injection volumes.2.8Recovery experiments were conducted by spiking untreated wheat samples at five different levels of 0.02, 0.05, 0.1, 0.2, and 0.5 mg/kg with intermediate pesticide working solutions  in acetone. Triplicates of each concentration were analyzed. The limits of detection (LODs) and limits of quantitation (LOQs) for the four pesticides were assessed at a signal‐to‐noise (S/N) ratio of 3 and 10, respectively.2.9p < .05, and data were reported as the mean value ± the standard deviation (SD) of five replicates.Statistical analysis was performed using the PSAW Statistic 19.0 (SPSS) statistical software package. All data were subjected to a one‐way analysis of variance (one‐way ANOVA). Homogeneity of variance was confirmed before ANOVA, and differences among the means were analyzed by Duncan's multiple range test. Data were shown and analyzed as micrograms per kilogram of matrix (µg/kg) on a dry matter basis. The differences were regarded as significant when 1/C2, where C1 and C2 are postprocessing and preprocessing pesticide residue levels (mg/kg), respectively.The processing factor (PF) during different wheat milling fractions and CSB processing is calculated as follows: PF = C33.1R2), which show the correlation between the concentrations of pesticide residues and the detective areas in different wheat matrices, were higher than 99.10%, demonstrating that the methods were sensitive and selective , in which the concentrations of the four pesticides in bran were significantly enhanced (p < .05), and in flour, they were significantly lower than in wheat (p < .05) (Table p < .05); however, the omethoate showed a different trend with a marked increase in shorts compared with in wheat (p < .05) in the ten‐fold treatment group due to its strong systemic conductibility. Further degradation of four pesticide levels was found in flour, with ranges of an 85.45%–89.67% decrease in chlorpyrifos, 85.68%–98.04% decrease in omethoate, 38.68%–83.69% decrease in cypermethrin, and 63.10%–93.65% decrease in deltamethrin. Additionally, the PF values of chlorpyrifos, cypermethrin, and deltamethrin were <1 in shorts and PF values of four pesticide residues decreased to 0.02–0.61 in flour  Table . The con5) Table . MeanwhiHigh concentrations of pesticides accumulated in the bran fraction may be related to the strength of fixation of residues on the bran coat. The thicker of the bran layers and higher yield of bran may have led to more residues accumulated on the bran coat. The transfer of pesticide residues to bran, shorts, and flour fractions can also be attributed to a translocation phenomenon generated by mechanical power during the milling process. Residues are not completely absorbed into the bran layer and can still be easily removed and transferred to the other layers (shorts and flours) through amplified friction, crushing, rolling, or sieving steps during the milling process  compared with flour, while chlorpyrifos concentration showed a slight decrease during the kneading process. This could be contributed to the fact that the insufficient mixing of the flour caused inconsistent concentrations among the samples. Samples from the recommended dosage of omethoate and deltamethrin were too little to quantify because of their lower initial levels in flour. Additionally, in this study, the increased pesticide concentrations after kneading were mostly found in the recommended dosage and twofold of the recommend dosage groups. Hence, it is suggested that the enhanced matrix interference resulted in increased recovery of pesticides in the low level, and this was in accordance with the results that are shown in Table In general, it is accepted that most processing techniques, such as cleaning, peeling, milling, extruding, cooking, baking, canning, and juicing, might reduce the pesticide residues in food  . Obviously, pyrethroid groups showed a completely opposite trend compared with organophosphorus groups during fermentation, which might be partly governed by their chemical structure. Mao et al. , during the fermentation of sweet orange peel, whereas no significant changes were observed in pyrethroid groups such as cypermethrin, deltamethrin, and bifenthrin during the fermentation process, indicating that the stability of pesticides might significantly affect the degradation rate of pesticides during fermentation. Furthermore, the observed increases in cypermethrin and deltamethrin levels in fermented dough are also likely due to a concentration effect or affinity for the lipid moiety of pesticides  Figure . Similar) Figure , in whic) Figure  reportedp < .05), increasing 1.16‐fold to 1.43‐fold compared to the flour, except for a decrease presented in the ten‐fold treatment group (loss of 16.41% for cypermethrin and 21.69% for deltamethrin)  compared with the initial levels in flour Figure , who spi4p < .05), with omethoate showing a different trend with a marked increase in the ten‐fold treatment group in shorts compared with that in wheat (p < .05). Decreases in the four pesticides ranging from 38.68% to 98.04% were observed in flour.This study investigated the fractioning and fate of chlorpyrifos, omethoate, cypermethrin, and deltamethrin during wheat milling and Chinese steamed bread processing. The levels of chlorpyrifos, omethoate, cypermethrin, and deltamethrin in bran were 1.46–1.57, 1.85–2.13, 1.27–1.86, and 1.63–2.33 times higher than those in wheat, respectively, while reductions of 27.97%–57.02% for chlorpyrifos, 6.22%–44.77% for cypermethrin, and 13.13%–61.15% for deltamethrin were observed in shorts compared with those in wheat . Although the concentrations of cypermethrin and deltamethrin in flour did not exceed the MRLs of China (0.2 and 0.5 mg/kg), most of the cypermethrin and deltamethrin groups showed various degrees of increases in kneaded and fermented dough and CSB compared with flour due to a concentration effect, thermal and hydrolytic stability and a strong affinity for the lipid moiety of cypermethrin and deltamethrin, which might also endanger human food safety. The research results provide further information for wheat‐based food risk assessment in terms of pesticide residues.There are no conflicts of interest.This study does not involve any human or animal testing.


These findings provided the experimental evidence that N-ZH with favorable anticancer activity can be used as a therapeutic agent and strategy for lung cancer treatment in future clinical trials.Lung cancer is one of the most common cancers in the world with a high mortality rate. Zein is a protein compound whose protein isolate is not useful and whose protein hydrolysis produces biological activity. By encapsulating this bioactive compound inside the nanoparticles (NPs), it causes itself to reach the tumor site and destroy it rapidly. In this study, the effects of zein hydrolysate (ZH) and nano-liposomal ZH (N-ZH) were investigated on the human A549 cell line. Western blotting and cell cycle analyses showed that ZH and N-ZH caused cytotoxicity. They induced apoptosis via cell cycle arrest at the G0 phase, as well as significant increases in pro-apoptotic genes, such as Bax, caspase-3, -8, -9, and p53, accompanied with significant decreases in the anti-apoptotic marker Bcl-2. Based on the results, the cytotoxic and anticancer effects of N-ZH were higher than those of free ZH. In conclusion, liposomes improved the performance of ZH and dramatically reduced the IC The results show that 1.8 million people are diagnosed with lung cancer each year and 1.6 million people die from the disease , alcalase enzyme, lecithin, Tween 80, cholesterol, ethanol, RPMI, RNase A, and yellow tetrazolium 3--2,5 diphenyl tetrazolium bromide (MTT) was purchased from Sigma-Aldrich . Fetal bovine serum and DMSO were provided by Thermo , PBS, trypsin/EDTA, propidium iodide (PI), Triton X100, and DCFDA/H2DCFDA Cellular ROS Assay Kit were obtained from Abcam . Penicillin, streptomycin, and Tris-HCl buffer were purchased from GeneDirex, Inc. . Annexin V-FITC , Bax, Bcl-2, caspase-3, -8, -9, and p53 antibodies were purchased from Santa Cruz Biotechnology .2.2.w/v) was hydrolyzed with alcalase at pH 9 in a 50 °C shaker incubator for 4 h. The enzyme-to-substrate mass ratio was 2.5% w/v. During hydrolysis, the pH of zein suspension was maintained at 9.0 by adding 1 M NaOH. After 4 h of hydrolysis, the pH of the hydrolysate was reduced to 7.0 by adding 1 M HCl, and the enzyme was inactivated by heating the mixture at 85 °C for 10 min. Next, the suspension was centrifuged at 5000 × g for 10 min. The hydrolysate was collected and freeze-dried for further analysis . Using a rotary evaporator at 40 °C, the solvent was removed from the mixture, and a thin layer was prepared. The resulting film was hydrated by 5 mL of water containing 10 mg of ZH and 10 mL of 1X PBS solution. To reduce the size of the liposomes, the formulation was placed in an ice bath for 10 min  with an ultrasound probe  at 80% power  and encapsulation efficiency (EE) percentages were determined by measuring the concentration of unloaded ZH in liposomes. Briefly, 2 mL of each sample was placed into the Amicon (Ultra-4 100 k–a 30 kDa) filter and centrifuged at 4000 rpm for 10 min. Next, by UV-vis spectrophotometry , the concentration of unloaded ZH was determined at 278 nm. EE was calculated using 2.5.Nanoliposome size and ZP were measured at room temperature using dynamic light scattering and a Malvern zeta sizer . All samples were diluted 10 times with distilled water, and all measurements were performed in triplicate.2.6.4 cells in each well) were incubated in 96-well plates for 24 h. After 24 h, the cells were treated with ZH and N-ZH at concentrations of 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024 μg/mL for 48 h. The cells were then incubated with MTT (5 mg/mL in PBS) for 4 h at 37 °C. The supernatant was discarded, and the cells were incubated with 200 μL of DMSO for 20 min, and finally measured at 570 nm using an ELISA Reader  were incubated for 24 h. The cells were incubated with ZH and N-ZH suspension at the IC50 dose for 48 h. The cells were then removed from the wells and washed twice with PBS. For every 100 μL of cell sample, 10 μL of 50 mg/μL PI solution and 5 μL of 25 mg/μL FITC-conjugated Annexin V solution were added, and the cells were kept in darkness for 20 min. Samples were analyzed by flow cytometry on a MACS Quant instrument.  and flow cytometry. In summary, A549 cells (102.7.2.6) treated with ZH and N-ZH were washed with PBS and fixed for 24 h at −20 °C in 70% cold ethanol. After 24 h, the cells were washed with PBS and incubated with 10–50 μg/mL RNase A and 20 μg/mL PI at 37 °C in darkness for 20 min. Finally, they were analyzed by flow cytometry  were treated with the IC50 dose of ZH and N-ZH for 48 h. Afterward, the cells were washed with PBS and incubated with 2-7-dichlorodihydrofluorescein diacetate (DCFH-DA) for 40 min at 37 °C. The cells were subsequently washed twice with PBS. ROS levels in the cells were measured by flow cytometry . The percentage of apoptotic cells was calculated by the Image-Pro Plus 6.0 software  were cultured in a 12-well plate. Next, using a plastic pipette tip, a thin scratch was created in the cell layer, followed by treatment with N-ZH and ZH at the IC50 dose. At 0, 12, and 24 h, photographs of the cells were taken  for 1 day. After that, Bax, Bcl-2, caspase-3, 8, 9, and p53 antibodies were mixed with the blocking solution, and the membrane was further incubated for 16 to 18 h. After initial staining of the membrane with secondary rabbit antibody with concentration (1: 1000) for all primary antibodies for one hour and 15 min at room temperature. The GAPDH antibody was used as a loading control . 3.3.1.The liposome size was calculated to be 43.44 ± 11.8, with an −2.25 ± 0.37 ZP and 0.184 PDI. Moreover, the EE and DL of the liposome formulation were calculated as 94.6 ± 3.1 and 9.46 ± 0.31, respectively.3.2.50 value of N-ZH and ZH was calculated to be 61 and 864 μg/mL, respectively, against A549 cells after 48 h of treatment. These results suggested that ZH and N-ZH were cytotoxic to A549 human cancer cells, with N-ZH showing 14 times higher cytotoxicity than ZH (The IC than ZH .3.3.3.3.1.p < .0001) increased Annexin V upregulation. N-ZH inhibited cell proliferation, and but promoted cell apoptosis, as indicated by the increased fluorescence intensity of Annexin V (Apoptosis in N-ZH- and ZH-treated cells was examined by assessing the binding of fluorescent Annexin V. Exposure to N-ZH and ZH promoted the staining of A549 cells with fluorescent Annexin V . Specifinnexin V .3.3.2.p < .001). The rate of cells in the Sub-G1 phase was significantly increased among N-ZH-treated cells compared to that among control cells (*p < .001).N-ZH led to cell-cycle arrest in A549 cells. As shown in 3.3.3.ROS have previously been shown to mediate intracellular signaling cascades and stimulate programmed cell death pathways .The migration of A549 cells was investigated using the scratch method. As shown in 3.3.6.p < .0001), caspase-9 (****p < .0001), caspase-3 (****p < .0001), and p53 (****p < .0001) increased, whereas the ratio of Bcl-2 to Bax (***p < .001) decreased. These data suggested that N-ZH induced apoptosis in A549 cells via the mitochondrial and external pathways  and the external pathway Elmore, . Therefop < .0001) improvement in the apoptosis-inducing effect of N-ZH at much lower concentrations compared to ZH. Hsieh et al. analyzed the antitumor activity of soy protein extract on rat fibroblasts and reported antitumor activity. Other studies have reported the anticancer activity of turnip flour-derived peptides . Cancer cell migration was also inhibited in a previous study (Nyiramana et al., The antitumor activity of ZH was determined via an MTT assay. Compared to egg white protein hydrolysate (Moon et al., in vivo. Liposomes allow efficient delivery of encapsulated compounds to the target sites while minimizing systemic toxicity. In the present study, the liposomes were in the appropriate diameter size range. The ideal characteristics of liposome systems include a size below 100 nm, low PDI, and good dispersion. Liposomal EE and DL were higher for ZH, indicating good encapsulation efficiency and good ZH loading within liposomes. Compared to nanoparticles in a previous study (Hong et al., in vivo (Torchilin, Liposomes play a significant role in enhancing drug delivery in many areas of biomedicine. Liposomes can stabilize therapeutic compounds, overcome cellular and tissue absorption barriers, and elevate the distribution of compounds at the target sites Taken together, the findings of this study showed that ZH significantly increased A549 cell proliferation by increasing the expression of genes effective in inducing internal and external apoptosis including caspases-3, -8, -9 and P53 and the important Bcl-2 family and stopping It suppresses the cell cycle in phase S . It also5.In this study, we found that ZH exposure activated cell apoptosis pathways. ZH-loaded liposomes were successfully synthesized in this study. These liposomes improved the anticancer effect of ZH, inducing apoptosis even at low doses. These findings show that N-ZH can inhibit the survival and migration of cancer cells.