The PCR-LFS method exhibits high specificity at an annealing temperature of 62 °C and does not cross-react with other animal sources. This strategy is also highly sensitive, being able to detect 0.1% of chicken in artificial adulterated meat. The results of the test strips can be observed with the naked eye within 5 min, and this result is consistent with the electrophoresis result, demonstrating its high accuracy. Moreover, the detection system has already been successfully used to detect chicken in commercial samples. Hence, this PCR-LFS strategy provides a potential tool to verify the authenticity of chicken.The aim of this study was to develop an accurate, easy-to-use, and cost-effective method for the detection of chicken adulteration based on polymerase chain reaction (PCR) and lateral flow strip (LFS). We compared six DNA extraction methods, namely the cetyltrimethylammonium bromide (CTAB) method, salt method, urea method, SDS method, guanidine isothiocyanate method, and commercial kit method. The chicken cytb gene was used as a target to design specific primers. The specificity and sensitivity of the PCR-LFS system were tested using a self-assembled lateral flow measurement sensor. The results showed that the DNA concentration obtained by salt methods is up to 533 ± 84 ng µL Meat products are an important source of nutrition for the human body. As people’s living conditions and quality of life increase, the demand for meat products is increasing. However, food fraud is rampant, and food-safety issues have become increasingly concerning. Just like the horsemeat fiasco in Europe in 2013, unscrupulous merchants sold horsemeat disguised as beef . The aboAt present, methods of meat composition identification are mainly based on protein and nucleic acid analysis . ProteinThe colloidal gold lateral flow sensor is a new immunolabelling technique. The test is achieved by the specific binding of antigens and antibodies with colloidal gold as the tracer . It is s2CO3, NaHCO3, sodium citrate (C6H5Na3O7), guanidine isothiocyanate (C2H6N4S), sodium N-dodecanoylsalcosinate (C15H10NO4Na), β-mercaptoethanol, alcohol, isoamyl alcohol, and isopropanol were purchased from Sangon Biotech . HAuCl4 was purchased from Sinopharm . Bovine serum albumin (BSA), sucrose, and alginate were obtained from Meilunbio Biotech . Absorbent pads (CH27) and sample pads (SB06) were purchased from KinBio Biotech . The gold absorbent pad (GL0194), PVC backing plate (DB-6), and nitrocellulose membrane (Millipore 135) were obtained from Jiyi Biotech . PCR Master Max and the anti-5-FAM antibody were obtained from BBI Biotech . Streptavidin and biotin-BSA were obtained from Solarbio Biotech .Ethylene diamine tetraacetic acid (EDTA), cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), Tris-HCl, NaCl, urea, NaSeven fresh meat samples  and 48 processed meat samples  were acquired from local markets in Ningbo, China. The purchased meat samples were rinsed with distilled water, ground in a meat grinder, and stored in sealed bags at −20 °C. To simulate chicken adulteration, we mixed different proportions of chicken meat with beef, lamb, and ostrich meat , and wer260/A280 and A260/A230 to determine the purity of the DNA.Six different methods were used to extract DNA from the chicken samples, and three replicate experiments were carried out for each method. DNA is extracted in five steps: sample lysis, organic solvent extraction, nucleic acid precipitation, wash the precipitate, and dissolve DNA A. The DN−1). Then, the mixture was centrifuged for 5 min at 13,000× g, and the supernatant was transferred to a new tube with 400 µL of trichloromethane-isoamyl alcohol . Thereafter, the solution was centrifuged for another 5 min at 13,000× g, and the supernatant was mixed with 400 µL of isopropanol. The mixture was kept at room temperature for 45 min and then centrifuged at 13,000× g for 5 min. The precipitant was washed with ethanol/water solution . The ethanol was discarded after centrifugation, and the residual ethanol was evaporated at ambient temperature. Finally, the precipitated DNA was dissolved in TE buffer.The CTAB method was performed according to the literature, with slight modifications . Thirty −1). Then 400 µL of 6 M NaCl was added and mixed upside down. The mixture was centrifuged at 13,000× g for 5 min, and the supernatant was mixed with 500 µL of isopropanol. Thereafter, the solution was centrifuged for another 5 min at 13,000× g, and the precipitate was washed with ethanol/water solution . The ethanol was discarded after centrifugation, and the residual ethanol was evaporated at an ambient temperature. Finally, the precipitated DNA was dissolved in TE buffer.The salt method was performed according to the literature, with slight modifications . Thirty −1). Then, 50 µL of 5 M NaCl and 500 µL of phenol/chloroform/isoamyl alcohol  were added to the purified DNA. The mixture was centrifuged at 13,000× g for 5 min, and the supernatant was removed in 500 µL of isopropanol. Thereafter, the solution was centrifuged for another 5 min at 13,000× g, and the precipitate was washed with ethanol/water solution . The ethanol was discarded after centrifugation, and the residual ethanol was evaporated at an ambient temperature. Finally, the precipitated DNA was dissolved in TE buffer.The urea method was carried out according to the literature, with slight modifications . Thirty −1). An equivalent amount of Tris-saturated phenol was added and mixed upside down. Then, the mixture was centrifuged for 5 min at 13000× g. Extraction was carried out with an equal volume of chloroform/isoamyl alcohol . Thereafter, the solution was centrifuged for another 5 min at 13,000× g, and the supernatant was mixed with 500 µL of isopropanol. The mixture was kept at −20 °C for 2 h and then centrifuged at 13,000× g for 5 min. The precipitate was washed with ethanol/water solution . Subsequently, ethanol was discarded after centrifugation, and the residual ethanol was evaporated at an ambient temperature. Finally, the precipitated DNA was dissolved in TE buffer.The SDS method was performed according to the literature, with slight modifications . Thirty 2H6N4S, 25 mM C6H5Na3O7, 20 mM C15H10NO4Na, 100 mM β-mercaptoethanol, 20 mM CBS buffer). and incubated at 60 °C for 20 min after adding 2 µL of Proteinase K (20 mg mL−1). Then, 300 µL of chloroform/isoamyl alcohol (24:1) and 300 µL of Tris-saturated phenol were added to the purified DNA. Then, the mixture was centrifuged for 5 min at 13,000× g, and the supernatant was mixed with 500 µL of chloroform/isoamyl alcohol . Thereafter, the solution was centrifuged for another 5 min 13,000× g, and the supernatant was mixed with 500 µL of isopropanol. Centrifuge at 13,000× g for 5 min, and the precipitate was washed by ethanol/water solution . Ethanol was discarded after centrifugation, and the residual ethanol was evaporated at an ambient temperature. Finally, the precipitated DNA was dissolved in TE buffer.The guanidine isothiocyanate method was performed according to the literature with slight modifications . Thirty The specific operation process of the Tiangen method was performed according to the TIANamp Genomic DNA Kit manual . Take 30 mg of animal tissue in 200 µL of GA buffer and shake it to suspension. Add 20 µL proteinase K and place at 56 °C for 2 h until the tissue is lysed. After adding 200 µL of buffer GB, mix well and leave at 70 °C for 10 min. Then, 200 µL of anhydrous ethanol was added and mixed well. Transfer the solution from the previous step into the adsorption column CB3 (column in a collection tube), centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. Add 600 µL of rinse solution PW to the adsorption column CB3, centrifuge at 12,000 rpm for 30 s, and discard the waste liquid. Repeat the previous step again. Centrifuge for another 2 min at 12,000 rpm to remove the residual rinse solution, and leave the sample at room temperature for a few minutes to thoroughly dry the residual rinse solution on the adsorbent material. Finally, transfer the adsorption column to a clean centrifuge tube, drop 100 µL of TE buffer on the adsorption membrane, placed at room temperature for 5 min, centrifuged, and collected the DNA solution.https://www.ncbi.nlm.nih.gov/tools/primer-blast (accessed on 25 July 2022)) was further used to assess primer specificity. The forward primer (FR) was 5′-Biotin-CATGACCCAAATCCTCACCG-3′ , where the 5′ end was modified with biotin; the reverse primer (PR) was 5′-Fam-TCCTTGTAGAGGTAGGAGCCGTA-3′ , where the 5′ end was grafted with Fam. The above primers were all produced by Hangjing Biotech Co., Ltd. .A partial sequence of the chicken cytb gene was downloaded from GenBank (Accession No.: L07520.1). Primer Premier 6 was then used to design specific primers. During the design process, extreme caution was exercised to avoid the creation of complicated secondary structures between primers, which would probably result in false-positive test results. The nucleic acid alignment program “BLAST” (Available online: −1), 1 µL of reverse primer (10 µmol L−1), 1 µL of template DNA, 10 µL of PCR Master Mix, and 12 µL of ddH2O. The PCR amplification program was as follows: pre-denaturation was first carried out at 95 °C for 5 min, followed by denaturation at 95 °C for 30 s, annealing was performed at 62 °C for 30 s and, extension at 72 °C for 30 s (35 cycles). ddH2O was used instead of DNA as a negative control. Amplificants were kept at 4 °C before gel electrophoresis analysis (140 V for 30 min).The PCR amplification system consisted of 1 µL of forward primer (10 µmol L−1) were added per ml of colloidal gold solution to determine the optimal amount for protein labeling on AuNPs. The AuNPs-SA composites were stored at 4 °C before further use. The colloidal gold particles were characterized by UV spectrophotometry and transmission electron microscopy (TEM).Colloidal gold particles (AuNPs) were synthesized using the conventional trisodium citrate reduction method . One mil−1 of anti-5-Fam antibody and 1 mg mL−1 of BSA-biotin were sprayed separately on the nitrocellulose membrane as the test line and the control line, respectively. Finally, all the above-mentioned pads were sticked together and cut into 4 mm in width. The obtained test strips were placed in a sealed container with a desiccant and stored at room temperature before further use.The test strip was composed of five parts: the sample pad, gold absorption pad, nitrocellulose membrane, absorbent pad, and PVC backing plate. The gold absorbent pads were sprayed with the pretreatment solution  and dried at 40 °C for 2 h . The preThe mechanism for detecting chicken adulteration using the PCR-LFS strategy is shown in 2O as the negative control.The genomic DNA of chicken, duck, pig, beef, lamb, horse, and ostrich was extracted using the salt method and used to determine the specificity of the PCR-LFS system. The sample DNA was amplified using four different annealing temperatures , with ddHThree different types of meat adulteration were simulated to test the sensitivity of the PCR-LFS method. To replicate real-life chicken adulteration, different percentages of chicken  were mixed with beef, lamb, and a mixture of beef, lamb, and ostrich.p < 0.05 being a significant difference. The Malvern particle size analyzer was used to measure the diameter of nanoparticles, histograms of the size distribution were plotted using Origin, and the histograms were fitted using a Gaussian equation.DNA concentration and purity were determined using NanoDrop 2000, and the mean and standard deviation were calculated using Microsoft Excel software . Origin software  was used to plot histograms to analyze the relationship between different DNA extraction methods (x) and concentration (y). Significant differences in the DNA concentrations extracted by the different methods were analyzed by a one-way analysis of variance (ANOVA) using GraphPad Prism 8 , with −1, 533 ± 84 ng µL−1, 314 ± 13 ng µL−1, 251 ± 58 ng µL−1, 241 ± 75 ng µL−1, and 289 ± 11 ng µL−1, respectively  between 450 nm and 600 nm (max) = 0.4271 × (particle diameter) + 514.56, the preliminary calculation of the colloidal gold particle diameter was 18 nm, and the results of this particle size were consistent with previous studies [Gold nanoparticles have been selected as signal molecules for flow measurement sensors due to their high stability, ease of synthesis, strong colorimetric signal, and high protein adsorption capacity . The cold 600 nm A. Accord studies . TransmiBefore obtaining AuNPs-SA conjugates, we need to select the optimal amount of streptavidin to label the AuNPs. Accurate amounts of protein labeling ensure the stability of immune complexes. The results showed that the tubes exhibited aggregation from red to blue when the amount of colloidal gold-labeled protein was 5 µg, 10 µg and 15 µg per ml. The colloidal gold solution remains red when the protein labeling amount is 20 µg and 25 µg A. InsuffThe specificity of the PCR-LFS system was assessed using seven different meat samples . According to the test results, the non-specific products were present in lanes 2 and 6 when the annealing temperature was 58 °C A, which w/w) of chicken in beef and lamb, and the color of the test strip becomes lighter as the adulteration ratio decreases. However, gel electrophoresis detected only 1% of the chickens in adulterated meat  of chicken in beef, lamb and ostrich. In addition, the system has a high application value, as the PCR amplicons can be used directly in the test strip, and the results can be visualized by the naked eye within 5 min. In summary, the developed PCR-LFS strategy can be applied to the detection of meat adulteration in resource-limited areas.This study proposed and developed an accurate, simple, and cost-effective detection strategy for chicken adulteration based on the PCR-LFS system. The inexpensive, environmentally friendly salt method can be used as an alternative to commercial kits to efficiently extract DNA at high concentrations and purity. High specificity of this system at an annealing temperature of 60 °C, without primer secondary structure and non-specific products. The system with good sensitivity to detect 0.1% (


In patients with SpaceOAR, average rectal wall doses decreased to 39.0, 36.9, 33.5, and 23.9 Gy to the volume of 0.5, 1, 2, and 5 cm3, respectively (p < 0.01). In PSI plans, rectal wall doses were on average 78.5, 60.9, 41.8, and 14.8 Gy to the volume of 0.5, 1, 2, and 5 cm3, respectively, in patients without spacer. In contrast, the doses decreased to 34.5, 28.4, 20.6 (p < 0.01), and 8.5 Gy (p < 0.05) to rectal wall volume of 0.5, 1, 2, and 5 cm3, respectively, in patient with SpaceOAR. To demonstrate rectal sum dose sparing, dose‐biological effective dose (BED) calculation was accomplished in those patients who showed >60% overlap of rectal volumetric doses between VMAT and PSI. In patients with SpaceOAR, average BEDsum was decreased up to 34%, which was 90.1, 78.9, 65.9, and 40.8 Gy to rectal volume of 0.5, 1, 2, and 5 cm3, respectively, in comparison to 137.4, 116.7, 93.0, and 50.2 Gy to the volume of 0.5, 1, 2, and 5 cm3, respectively, in those with no spacer. Our result suggested a significant reduction of rectal doses in those patients who underwent a combination of VMAT and LDR with hydrogel spacer placement.This study aimed to evaluate rectal dose reduction in prostate cancer patients who underwent a combination of volumetric modulated arc therapy (VMAT) and low‐dose‐rate (LDR) brachytherapy with insertion of hydrogel spacer (SpaceOAR). For this study, 35 patients receiving hydrogel spacer and 30 patients receiving no spacer were retrospectively enrolled. Patient was treated to doses of 45 Gy to the primary tumor site and nodal regions over 25 fractions using VMAT and 100 Gy to the prostate using prostate seed implant (PSI). In VMAT plans of patients with no spacer, mean doses of rectal wall were 43.6, 42.4, 40.1, and 28.8 Gy to the volume of 0.5, 1, 2, and 5 cm Radiation dose escalation results in improved clinical outcomes, but it also increases risk of rectal toxicity.Several studies have shown significant dosimetric impact by insertion of hydrogel spacer on rectum, and consequently, lower risk of rectal toxicity was observed during and after radiation treatment.22.1In this retrospective study, 65 patients with prostate cancer including 35 patients receiving hydrogel spacer (SpaceOAR) and 30 patients receiving no spacer were enrolled. Selection of the patients was based on the following criteria: (1) patient was treated to doses of 45 Gy over 25 fractions using volumetric modulated arc therapy (VMAT) and a boost 100 Gy using Palladium‐103 (Pd‐103) seed implant; (2) prostate plus lymph nodes in pelvic regions were included in the treatment.2.2Insertion of hydrogel spacer has been described in detail by other studies.2.345 according to the prescriptions. Normal tissue structures, including rectum, bladder, small bowel, large bowel, and femur heads, were contoured as organ‐at‐risk (OAR). Rectum was outlined as a solid organ, and rectal wall was defined as 0.4 cm thickness inside the outer contour of rectum. All contours underwent departmental peer review prior to planning in Eclipse .Non‐contrast computed tomography (CT) simulation was performed at 0.3 cm intervals as per our standard procedure, approximately 2 weeks after placement of SpaceOAR. Co‐registration of the CT and magnetic resonance imaging (MRI) was performed for verification of the spacer, delineation of target and normal tissue structures using Velocity . Target volumes of the prostate and high‐risk nodal region were outlined, known as PTV2.43 and heterogeneity correction applied. Two weeks after VMAT, patient underwent LDR brachytherapy that was delivered using Pd‐103 seed implant.The EBRT portion of therapy was delivered using conventional fractionation (45 Gy in 25 fractions). Two‐arc VMAT plan was generated using 6MV photons. The anisotropic analytical algorithm (AAA) was used as dose calculation model with a grid size of 0.25 × 0.25 × 0.25 cm3, were denoted as D0.5cc, D1cc, D2cc, and D5cc. Dose coverage of PTV45 was showed in maximum pixel dose (Dmax), relative dose D95, and conformity index (CI) in VMAT plan. CI was defined as percent volume of target PTV covered by 100% of the prescribed dose. Dose CI above 70% had been previously considered acceptable in the literature.D90, V100, and V150. Statistical significance of dosimetric outcomes between two‐group patients was analyzed using an unpaired Student's t‐test (Microsoft Excel 365).Both VMAT and PSI plans were exported in DICOM‐RT format to Velocity for examination of target dose coverage and OAR dose sparing. Rectal wall dose was evaluated using the VMAT structure set after completing both rigid and deformable image registration between PSI image and VMAT image datasets. Briefly, the CT images for PSI plan were aligned with the images for VMAT plan by using mutual information‐based rigid registration, and the rectal volumetric dose was examined. To further limit the impact of rectal wall positioning uncertainties between images acquired on different days, B‐spline deformable image registration was applied in each patient. In Velocity, a region of interest was generated by expanding the PTV and rectum by 1 cm in three dimensions. Structure‐based deformable registration was performed using the 1 cm expanded volume around the PTV and rectum. Percent difference of the rectal doses evaluated under two types of image registration was presented in Table 2.5i.e. D0.5cc, D1cc, D2cc, and D5cc, was converted to a structure, and then intersection of the dose structures was measured in absolute volume (cm3). Using Velocity, we were able to analyze percent overlap in each pair of the volumetric doses obtained from VMAT and PSI in each patient. Although deformable image registration reduced some alignment uncertainty in the rectal wall, complete overlap of the volumetric doses was unlikely to occur, especially in the small volume dose, i.e. D0.5cc. To obtain rectal sum dose, the patient who had greater than 60% overlapping in D0.5cc, D1cc, D2cc, and D5cc would be favored in selection in BEDsum calculation.Percent overlap of the rectal volumetric doses was examined between VMAT and PSI plans in each patient in Velocity. Briefly, each volumetric dose, 2.6n treatment fractions, dose d per fraction was completed by using Equation (1):α and β were the radiosensitivity parameters for the linear‐quadratic responses, respectively. The quotient α/β was the dose at which linear and quadratic terms contribute equally to biological response. Cell proliferation for rectum, as a normal tissue, was not taken into account in treatment period of prostate cancer patient.The linear‐quadratic model has been widely accepted in describing cell response to radiation.D was the dose delivered to the full decay, 0 = D * λD was defined as initial dose rate, λ was decay constant, and μ was the sublethal repair coefficient. Equation (rT) was significantly longer for late responding normal tissues. In this study, dose‐BED conversion was completed for rectal wall doses using the parameters according to AAPM report TG‐137,D was the rectal dose delivered to the full decay . λ was Pd‐103 decay constant (λ = 0.0408 day−1). The rectal repair coefficient μ was 43.3217day−1 (μ = ln2/Tr), where rT was sublethal repair half time (r T= 0.016 day). Rectal α/β ratio was 4 Gy .For rectal dose‐BED conversion in PSI plan, the equivalent BED calculation, derived by Zhang BED=D1+D33.145 coverage was examined in relative dose D95, maximum pixel doses (Dmax), and CI. Dose D95 on each PTV45 was adequate to satisfy D95 > 42.8 Gy that was 95% of the prescription, as required. The average D95 was 44.3 Gy in patients with SpaceOAR and 44.2 Gy in those without spacer. Maximum pixel dose was an average of 48.3 and 48.5 Gy in patients having SpacerOAR and those having no spacer, respectively, which satisfied the maximum dose less than 110% prescription dose objective. The average CI for PTV45 was 92.1% in patients with SpaceOAR and 91.8% in those without spacer. A significant difference of PTV45 coverage was not observed between the two groups of patients (p > 0.05). In the PSI plan, target coverage was assessed with D90, V100, and V150. The average D90 was 100.9 and 103.5 Gy in patients with and without the insertion, respectively. Average V150 was 57.8% in patients with SpaceOAR and 63.8% in patients with no spacer. On average, V100 was found to be 88.9% in patient having SpaceOAR and 91.8% in those having no spacer. There was no statistically significant difference in D90, V100, and V150 found between the two groups (p > 0.05).The target dose coverage in both VMAT and PSI plans were evaluated in each patient and did not appear to be significantly affected by the use of a hydrogel spacer Table . In the 3.23 and an average distance between prostate and rectum was about 1 (± 0.2) cm. An average volume of rectal wall was 13.1 cm3 with range from 8.8 to 21.2 cm3 in patients with SpaceOAR, and 12.6 cm3 in range of 8.7–17.2 cm3 in those with no spacer. There was no significant difference observed between the two groups.An interspace created by insertion of hydrogel spacer was outlined as SpaceOAR Figure . An averD0.5cc, D1cc, D2cc, and D5cc) were evaluated in the VMAT structural set in Velocity. Percent difference in the rectal wall doses measured under rigid and deformable image registrations is depicted in Table Combined volumetric doses of the rectal wall , on average of 39.0, 36.9, 33.5, and 23.9 Gy to rectal volume of 0.5, 1, 2, and 5 cm3, respectively. In PSI plans, on average, rectal wall doses in patients without the spacer were 78.5, 60.9, 41.8, and 14.8 Gy to the volume of 0.5, 1, 2, and 5 cm3, respectively. Significant decreases of the doses were observed in patients with SpaceOAR, which were on average 34.5, 28.4, 20.6 (p < 0.01), and 8.5 Gy (p < 0.05) to rectal wall volume of 0.5, 1, 2, and 5 cm3, respectively.Rectal wall doses were significantly decreased in both VMAT Table  and PSI 3.3sum), it was necessary to examine whether the volumetric doses overlapped in VMAT and PSI. Using Velocity, we calculated percent intersection of the dose volumes in VMAT and PSI plans  between VMAT and PSI in individual patient, for example, we proposed that 60% or more overlapping would be sufficient in analysis of rectal sum doses (BEDsum). There were 11 of 35 patients with SpaceOAR and 12 of 30 patients with no spacer showing greater than 60% overlap in all examined doses, who were selected in BEDsum calculation.The rectal dose‐BED conversion was carried out in each patient. To further combine the BED to sum dose . BEDsum_5cc decreased by 18%, but it was still not statistically significant (p = 0.051), possibly due to the limited sample size.In those 12 patients with no spacer, average BED4sum) in patients who underwent a combination of EBRT and LDR therapy with hydrogel spacer, and showed decreases of rectal BEDsum_0.5cc, BEDsum_1cc, and BEDsum_2cc by up to 34% in comparison with those patients who did not have spacer placement.This study assessed dosimetric impact by hydrogel spacer insertion on rectal sum dose using BED calculation in prostate cancer patients who underwent EBRT and LDR combination therapy. It has been well documented that late rectal toxicity is correlated to the volume of the anterior rectal wall that receives the highest radiation dose.sum. First, due to changes in bladder and rectal filling, changes in the position of the anterior rectal wall were inevitable. This led to uncertainties in rectal dose volume, that is, D0.5cc, D1cc, D2cc, and D5cc, between VMAT and PSI CT image sets that were acquired on different days. As this could impact rectal BED summation, we used deformable image registration to mitigate this concern. Certainly, the higher percentage of the volumetric dose intersection between VMAT and PSI was seen, the more reasonable estimation of rectal sum dose would be. We showed that greater than 90% overlap could be seen in relatively large volumetric doses, such as D5cc, in both groups of patients, but 100% overlap was unlikely. Meanwhile, it was feasible to see greater than 60% overlap in the volumetric doses D0.5cc in some patients. Determining the overlap threshold for proper dose summation could be challenged. Here, we proposed that intersection volume 0.3 cm3 or more occurred in the examined dose volume 0.5 cm3 (D0.5cc), i.e. 60% overlapping, would be considered a substantially impressive volume in affecting rectal BEDsum_0.5cc. Therefore, rectal BED calculation was carried out in those patients who showed 60% or more overlaps of the volumetric doses between VMAT and PSI. We believe that the BEDsum could be reasonably useful in estimating rectal sum dose, even in the condition of 100% dose intersection between VMAT and PSI, as the worst‐case scenario. Furthermore, image registration could be another limitation affecting measurement of the dose overlapping. After considering rigid and deformable image registration in Velocity, we compared the rectal doses in each patient using both techniques and decided to utilize deformable image registration because it has been reported that deformable image registration can be used for response evaluation and cumulative dose estimation.sum calculation and correlation with rectal toxicity during and after the treatment, an attempt to develop planning guidelines that incorporate summed dosimetry. Furthermore, a novel method using voxel‐by‐voxel dose summation to improve the accuracy of combined modality dosimetry has been developed in our institutesum could potentially yield new planning guidelines.The current work has a few limitations in demonstration of rectal BED5sum calculation would provide valuable information in assessment of dosimetric impact by insertion of hydrogel spacer on rectal sum dose sparing in prostate cancer patients who underwent a combination of EBRT and LDR therapy. Statistically significant dosimetric advantages in rectal wall sparing were observed in favor of patients with hydrogel spacer, indicating up to 34% reduction of rectal wall volumetric dose compared with patients treated without spacer. This trend held for combined dosimetry as well as EBRT and LDR components.We concluded that rectal BEDThe authors declare no conflict of interest for this work.Project design, data collection, EBRT planning, data analysis, and manuscript writing: Honglai Zhang. Performing prostate implant and treatment planning, post‐implant dosimetry, and data collection: Lin Wang. Performing prostate implant and treatment planning, post‐implant dosimetry, data analysis, and manuscript revision: Adam C. Riegel. EBRT treatment planning: Jeffrey Antone. Project development, performing prostate implant, and manuscript revision: Louis Potters. Project development, performing prostate implant, and manuscript revision: Lucille Lee. Project design, data analysis, and manuscript writing: Yijian Cao


The chromatographic separation was performed on an ACE Excel 2 Phenyl column (50 × 2.1 mm). Mass quantification was carried out on the multiple reaction monitoring of the transitions of m/z 473.1→338.1, 480.1→338.1, 356.0→324.0, and 362.2→326.2 for clevidipine, clevidipine-d7, H152/81, and H152/81-13C-d3. The validated method gave an excellent linearity over a concentration range of 0.1–30 ng/ml for clevidipine and 2–600 ng/ml for H152/81. Other fully validated content such as accuracy, precision, extraction recovery, matrix effect, and stability were also investigated and showed satisfactory results. It was strongly recommended that whole blood is the first choice for clinical bioanalysis. Using whole blood for sample analysis can reduce the whole blood collection volume (1 ml vs. 4 ml) and shorten the time from sample collection to storage to 5 min, and there is no centrifugation process and precooling in the ice water bath, which can further reduce the instability caused by exposure. The method was successfully applied to a bioequivalence study of clevidipine butyrate-injectable emulsion.Clevidipine is an ultrashort-acting dihydropyridine calcium antagonist, which can control blood pressure accurately. It is necessary to develop a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method to quantitate clevidipine and its active metabolite H152/81 for clinical pharmacokinetic study and therapeutic drug monitoring. Liquid–liquid extraction was used for sample preparation, and clevidipine-d Nowadays, various factors such as smoking, medications, non-compliance, and poorly controlled chronic hypertension result in an acute rise in blood pressure, which leads to multiple damage and complications, such as heart failure, stroke, myocardial infarction, and renal dysfunction . Acute sClevidipine, in the category of arterial vasodilators, is an ultrashort-acting dihydropyridine L-type calcium channel antagonist . The reaIn earlier years, the quantitation of clevidipine and H152/81 has been reported by HPLC with UV or fluorescence detection methods and capillary gas chromatography–mass spectrometry . HoweverYujia Zhang et al. developed and fully validated a simultaneous determination method of clevidipine and H152/81 in human plasma, and the method exhibited good linearity over the concentration ranges of 0.100–40.0 ng/ml for clevidipine and 5.00–400 ng/ml for H152/81with NaF and ascorbic acid used as esterase inhibitor and antioxidant, respectively .In this study, a rapid, simple, and more reliable LC-MS/MS method was established and validated for simultaneous determination of clevidipine and H152/81 in human whole blood. Based on the result, there was a conclusion that the method provided higher specificity, sensitivity, and short analysis time. The method using whole blood for sample analysis can reduce the whole blood volume and shorten the time from collection to storage to 5 min. The proposed method was successfully applied to a bioequivalence study of 8 mg/h (16 ml/h) clevidipine butyrate-injectable emulsion by intravenous administration, which lasted 30 min in healthy Chinese volunteers under fed conditions.7  was finally used for the preparation of calibration standard samples and quality control (QC) samples.Ten percent(w/v) SDS solution containing 0.05 M ascorbic acid was added to fresh K7/H152/81-13C-d3. All solutions were stored at −20°C.Stock solutions (1 mg/ml) of analytes and internal standards were generated by dissolving in DMSO. The standard working solutions of analytes were prepared by serially diluting stock solutions of acetonitrile–water . In a similar process, the QC working solutions were prepared with an independent stock solution of analytes. The IS working solutions were prepared by diluting stock solutions of acetonitrile–water  at concentrations of 20/200 ng/ml for clevidipine–dCalibration standards and QC samples were prepared by mixing working solutions with stabilized blood to obtain calibration standards, and concentrations of the calibration standards were 0.1/2, 0.2/4, 0.5/10, 2/40, 5/100, 15/300, 27/540, and 30/600 ng/ml for clevidipine/H152/81, and the QC samples  were 0.1/2, 0.3/6, 1.5/30, 8/160, and 24/480 ng/ml for clevidipine/H152/81.4AC and 0.025% acetic acid and injected into the LC-MS system.Liquid–liquid extraction (LLE) was used to extract clevidipine and H152/81 from human blood. Aliquots of 25 μl IS working solution, 50 μl blood sample, and 50 μl of 0.1% formic acid were added to 1.5-ml polypropylene tubes precooled in an ice bath. After vortex-mixing for 1 min, 500 μl of MTBE was added to each sample and vigorously vortexed for ten min. Subsequently, the samples were centrifuged at 10,000 g for five min at 4°C. Then, 200 μl of the supernatants were transferred into a clean 96-well plate and evaporated under nitrogen at room temperature. Finally, the residues of the sample were reconstituted in 200 μl of methanol–water  containing 2 mM NH+ mass spectrometer (MS) from Sciex . Chromatographic separation was performed on an ACE Excel 2 Phenyl column (50 × 2.1 mm id) maintained at 40°C with 2 mM ammonium acetate in water (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.6 ml/min and injection volume of 20 μl. The gradients were as follows: 0–2.00 min, 30–35% B; 2.00–4.00 min, 35–65% B; 4.00–4.01 min, 65–95% B; 4.01–4.70 min, 95% B; 4.70–4.71 min, 95–30% B; and 4.71–5.50 min, 30% B. Mass quantification was operated in the positive electrospray ionization (ESI) mode. By using the multiple reaction monitoring (MRM) mode, the optimized transitions were m/z 473.1→338.1 for clevidipine, m/z 480.1→338.1 for clevidipine-d7, m/z 356.0→324.0 for H152/81, and m/z 362.2→326.2 for H152/81-13C-d3. The optimal parameters are shown in The LC-MS/MS system consists of a Shimadzu UPLC system  connected with an AB SCIEX Triple Quad 6500The assay method validation was performed according to the bioanalytical method validation guidelines . A full 2. The acceptance criteria were that 75% (at least six) non-negative calibrators should be with a relative deviation (RE) within ±15% at each concentration (within ± 20% at LLOQ) and the calculation curves involved a coefficient of determination (r2) ≥ 0.99.The calculation curves contained eight different concentrations at two repeats and were prepared in the biological samples by spiking human whole blood with standard solutions and validated in at least three batches. The range of calculation curves was 0.1–30 ng/ml for clevidipine and 2.0–600 ng/ml for H152/81. The calculation curves were produced by plotting the concentration of the standards against the analyte to IS peak area ratios using least square linear regressions with a weighting factor of 1/xThe selectivity of the bioanalytical method included the interference between human blank whole blood and analytes. Six individual human blank whole blood samples with and without analytes and ISs were compared to investigate the potential interfering peaks at the retention time. The responses of analytes in the blank should not exceed 20% of LLOQ and 5% of the average IS responses of the calibrators and QCs, respectively.Five concentration levels of QC samples  with six duplicates repeated in three independent batches were used to verify the accuracy and precision of intra- and inter-batch of the method. The accuracy was expressed as the RE with nominal concentration, and the acceptance criterion was that the RE should be within ±15% of the target concentration (within ± 20% at LLOQ). Precision was demonstrated as the relative standard deviation (RSD), which should be ≤15% at each level (≤20% at LLOQ).To investigate the recovery of analytes and ISs, six replicates from spiked blood samples at three concentration levels  were used to assess the extraction process, by calculating the mean peak areas required from QC samples to that of the post-extraction of blank spiked blood QC solutions. The matrix effect of the analytes and ISs was evaluated by comparing the responses of the post-extraction six different blank human spiked blood samples with those of the neat solutions at three QC concentration levels .Dilution integrity was evaluated by DQC samples  with fivefold dilution to determinate the influence of the accuracy and precision of the dilution procedure. The mean accuracy of the dilution QCs should be within ±15% of the nominal concentration, and the precision should not exceed 15%.Carryover was investigated by injecting an extracted blank matrix immediately after the upper limit of quantitation (ULOQ) sample. The acceptance criteria were that the peak area at the retention time of analyte in the blank sample should be <20% of the analyte peak area in the LLOQ sample and <5% of the average peak area of IS.The stability of analytes in the matrix was evaluated by the spiked QC samples (LQC and HQC) with six replicates under the following conditions: room temperature with an ice bath for one h, four freeze–thaw cycles at −80°C, long term at −20°C and −80°C for 6 and 34 days, and the autosampler stability at a setup temperature (8°C) for 146 h. All the samples were analyzed with the freshly prepared calibration curves. The stability of stock and working solutions of analytes and ISs was calculated by comparing the peak area of the stock and working solution storage at −20°C for 35 days with that of freshly prepared solutions.2. The design of the study was a single-center, open-label, randomized, two-period, two-treatment, and a single-dose, crossover bioequivalence study for a test  and a reference  formulation under fed conditions with a cleaning period of seven days between the two formulations. The protocol and informed consent form were approved by the Ethics Committee of Beijing Chao-yang Hospital, Capital Medical University (BCYH-CMU), with the ethical approval number 2017-drug-8, and all volunteers provided with informed written consent in BCYH-CMU. The whole blood samples (1 ml each) were collected from the forearm vein within 30 min before administration, and 10, 20, 30, 32, 34, 36, 38, 42, 50 min, 1, 1.5, 2.5, 4.5, 8.5, 12.5, 24.5, and 48.5 h after administration, and were then put into a K2EDTA blood collection tube, which was preloaded with 1 ml stabilizer ((10% w/v) SDS aqueous solution containing 0.05 M ascorbic acid). The samples were placed slightly upside down 8–10 times directly after collection and stored below −60°C (the refrigerator temperature was set at −70 ± 10°C) until analysis. The pharmacokinetic parameters of clevidipine and H152/81 were evaluated by non-compartmental analysis using Phoenix™ WinNonlin® software version 8.0, and other data were summarized and analyzed using SAS version 9.4 software.The validated method was successfully applied to detect the whole blood concentration of clevidipine and H152/81 after an intravenous administration of 8 mg/h (16 ml/h) which lasted 30 min in 24 healthy Chinese volunteers in the age from 20 to 45 years and a body mass index (BMI) from 19.1 to 25.4 kg/mmax and the elimination phase.Incurred sample reanalysis (ISR) was performed to verify the reproducibility of the method. In this study, ISR samples include 85 samples for clevidipine and 111 samples for H152/81, of which concentrations were near C+ ions at m/z 473.1, 480.1, and [M + H]+ ions at m/z 356.0, 362.2 were chosen as the precursor ions for clevidipine, clevidipine-d7, H152/81, and H152/81-13C-d3, respectively. Due to the existence of chlorine, the precursor ion of H152/81 had a serious isotopic interference to the precursor ion at m/z 360 of H152/81-13C-d3. Selecting the isotope precursor ion at m/z 362.2 of H152/81-13C-d3 can avoid the isotope interference. The product ion mass spectra of analytes and ISs were optimized under collision-induced dissociation, and the most stable fragment ions were selected at m/z 338.1, 338.1, 324.0, and 326.2 for clevidipine, clevidipine-d7, H152/81, and H152/81-13C-d3, respectively  which achieved excellent resolution and satisfactory sensitivity was finally selected. Some solvents such as methanol, acetonitrile, water, formic acid, and ammonium acetate were considered for the mobile phase. In the aspect of the elution effect, acetonitrile was much stronger than methanol. Meanwhile, using acetonitrile as the mobile phase could yield lower background noise. The addition of 2 mM ammonium acetate in water could enhance the peak shape of analytes and ISs. Finally, acetonitrile and water containing 2 mM ammonium acetate as mobile phases with a gradient elution at 0.6 ml/min of flow rate provided symmetric peaks, optimal retention time, and higher sensitivity to the analytes and ISs.7 and H152/81-13C-d3 were chosen as the internal standards in this study.As we know, the stable isotope-labeled standard is the best choice as the internal standard due to the similarities in mass and chromatographic behaviors such as retention action, ionization, and extraction efficiency with the analytes. Therefore, using a stable isotope-labeled standard was suitable for improving both the precision and the trueness of methodology in the LC-MS/MS analysis. Thus, clevidipine-d ml vs. 4 ml) and shorten the time from sample collection to storage to 5 min, and there is no centrifugation process and precooling in the ice water bath, which can further reduce the instability caused by exposure. Therefore, we strongly recommend that the whole blood is the first choice for clinical bioanalysis.Due to the wide presence of esterase, which is located in the membrane or the cytosol of the red blood cells, caused the instability and fast degradation of clevidipine and its metabolites throughout the sample collection, processing, and before sample extraction. It is recommended that the concentration of analytes in whole blood, rather than plasma, is more accurate in quantification . CompareAs we all know, clevidipine is unstable and can be rapidly hydrolyzed by esterase in whole blood due to its ester group. SDS, a common esterase inhibitor, was demonstrated to prevent clevidipine from hydrolysis distinctly in whole blood, particularly at low temperature . In addiCompared with protein precipitation, the LLE method displayed fewer interference components, better signal-to-noise ratio, and a higher extraction recovery for sample preparation. Regular different extraction solvents and different numbers of pH modifiers were assessed to improve the LLE procedure in this study. Compared with ethyl acetate, the recovery of MTBE remained at the same high level, and the matrix interference caused by emulsification was also significantly reduced. Eventually, MTBE was chosen as the optimum LLE agent and 0.1% FA was utilized to obtain high and stable extraction efficiency.The calculation curve contained eight different concentrations and showed excellent linearity in the concentration range of 0.1–30 ng/ml for clevidipine and 2.0–600 ng/ml for H152/81. The correlation coefficients of clevidipine and H152/81 were better than 0.9951 in all the validation batches. The LLOQ of clevidipine and H152/81 was 0.1 ng/ml and 2.0 ng/ml  respectively, which were sufficient for the quantification of clevidipine and H152/81 in the human whole blood sample study.7, and H152/81-13C-d3 was 3.79, 1.20, 3.78, and 1.19 min, respectively. There was no distinct interference at the retention times of analytes and ISs in the human blank whole blood, which indicated that the method displayed superior selectivity.The selectivity of this method was evaluated by comparing the human blank whole blood samples collected from six different sources with the corresponding spiked samples. As The intra- and inter-batch accuracies were within the range of -2.5 to 4.7% and the intra- and inter-batch precisions were less than 6.1% and 7.3% with six duplicates repeated in three independent batches . These r7 and H152/81-13C-d3, respectively. The matrix effects for clevidipine were 114, 117, and 115% at the concentrations of 0.3, 8.0, and 24 ng/ml, respectively, and those for H152/81 were 98.8, 97.8, and 101% at the concentrations of 6, 160, and 480 ng/ml, respectively. These results indicated that no matrix effect for both analytes and ISs were seen in the method in which the recovery was consistent and reproducible.The extraction recoveries in low-, medium-, and high-QC samples were 80.3, 83.4, and 80.4% for clevidipine and 76.8, 78.6, and 80.6% for H152/81, respectively, while the extraction recoveries of ISs were 82.5 and 79.0% for clevidipine-dThe accuracy of the DQC samples ranged from 101.5 to 105.6% for clevidipine and 96.7 to 105.3% for H152/81, and the precision of clevidipine and H152/81 was 1.5% and 2.9%, respectively, indicating that there was no significant effect on dilution to quantify analytes before sample analysis.The peak area at the retention time of the analytes and ISs in matrix blank samples injected after the ULOQ samples had no obvious interference, which was within ±20% of the analyte peak area in the LLOQ samples and within ±5% of the average peak area of ISs.The stock and working solutions of the analytes were stable with storage at −20°C for 34 days. The results of the stability of clevidipine and H152/81 in human blood  showed tmax, AUC0−t, AUC0−∞, and their 90% confidence intervals ranged from 97.57 to 119.86% for clevidipine and 97.62 to 106.46% for H152/81, which were within the acceptance criterion of 80–125%, showing the bioequivalence of the test formulation with the reference product in terms of rate and extent of absorption. Meanwhile, there was no significant difference appeared in the variance analysis between the period and sequence.In this study, because of the occurrence of adverse events, 23 of the 24 subjects in the reference group and 21 of the 24 subjects in the test group were eventually incorporated into the pharmacokinetic analysis, while 20 of the 24 subjects were finally brought into the bioequivalence study. The reasons for the abscission of three subjects were the changes of the ST-T segment of the ECG before the second administration, the decrease in plasma fibrinogen before the cleaning period, the increase in the heart rate and blood pressure, which appeared six min after the first administration. The average blood concentration–time semi-logarithmic curves of clevidipine and H152/81 obtained for test and reference formulations are displayed in The percent change of incurred samples was within ±20% that compared with the same initial samples. The ISR result indicated the perfect reproducibility and reliability of this method.To the best of our knowledge, this is the first time that a rapid and sensitive LC-MS/MS method was developed and validated for the simultaneous quantitation of clevidipine and its active metabolite H152/81 in human whole blood for bioequivalence study. It is strongly recommended that whole blood is the first choice for clinical bioanalysis. Collecting whole blood samples greatly reduces the blood sample collection volume and the time from collection to storage, and there is no centrifugation process and precooling in the ice water bath, which can further reduce the instability caused by exposure. The method with the LLE sample preparation process minimized the strong matrix effect caused by endogenous substances and had a lower LLOQ (0.1 ng/ml for clevidipine and 2.0 ng/ml for H152/81). Moreover, using SDS and ascorbic acid can effectively reduce the hydrolysis and oxidation of clevidipine and H152/81 during analysis. Eventually, the method was successfully applied to the bioequivalence study in healthy Chinese volunteers.


This study aimed to assess the diagnostic performance of circulating tumor DNA (ctDNA) in hepatocellular carcinoma (HCC).We enrolled all relevant studies published up to 5 January 2022. Three primary subgroups were investigated: qualitative or quantitative ctDNA analyses, combined alpha-fetoprotein (AFP), and ctDNA assay. In addition to the three primary subgroups, we also evaluated the diagnostic value of methylated SEPTIN9 (mSEPT9), which has been studied extensively in the diagnosis of hepatocellular carcinoma. After a search based on four primary databases, we used a bivariate linear mixed model to analyze the pooled sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR). We also plotted hierarchical summary receiver operating characteristics (HSROC) and utilized lambda as well as the area under the curve (AUC) to create summary receiver operating characteristic (SROC) curves to estimate the diagnostic value of ctDNA.A total of 59 qualified articles with 9,766 subjects were incorporated into our meta-analysis. The integrated SEN, SPE, and DOR in the qualitative studies were 0.50 (95% CI [0.43–0.56]), 0.90 (95% CI [0.86–0.93]), and 8.72 (95% CI [6.18–12.32]), respectively, yielding an AUC of 0.78 and lambda of 1.93 (95% CI [1.56–2.33]). For quantitative studies, the corresponding values were 0.69 (95% CI [0.63–0.74]), 0.84 (95% CI [0.77–0.89]), 11.88 (95% CI [7.78–18.12]), 0.81, and 2.32 (95% CI [1.96–2.69]), respectively. Six studies were included to evaluate the SETP9 methylation, which yielded an AUC of 0.86, a SEN of 0.80 (95% CI [0.71–0.87]), and a SPE of 0.77 (95% CI [0.68–0.85]). Likewise, ctDNA concentration yielded an AUC of 0.73, with a SEN of 0.63 (95% CI [0.56–0.70]) and a SPE of 0.86 (95% CI [0.74–0.93]). AFP combined with ctDNA assay resulted in an AUC of 0.89, with a SEN of 0.82 (95% CI [0.77–0.86]) and a SPE of 0.84 (95% CI [0.76–0.90]).This study shows that circulating tumor DNA, particularly mSEPT9, shows promising diagnostic potential in HCC; however, it is not enough to diagnose HCC independently, and ctDNA combined with conventional assays such as AFP can effectively improve diagnostic performance. Among all primary liver cancers, hepatocellular carcinoma (HCC) accounts for >80% of primary liver cancers worldwide  and tissue biopsy for tumor diagnosis and monitoring . Liquid In 1977, Two authors (Jiajie Li and Yanqing Lv) independently conducted a comprehensive search for relevant articles in PubMed, Web of Science, Embase, and Cochrane Library. The query terms were as follows: “circulating tumor DNA” OR “circulating DNA” OR “ctDNA” OR “plasma DNA” OR “serum” DNA” OR “blood DNA” and “Liver Neoplasms” OR “Neoplasms, Hepatic” OR “Neoplasms, Liver” OR “Liver Neoplasm” OR “Neoplasm, Liver” OR “Hepatic Neoplasms” OR “Hepatic Neoplasm” OR “Neoplasm, Hepatic” OR “Cancer of Liver” OR “Hepatocellular Cancer” OR “Cancers, Hepatocellular” OR “Hepatocellular Cancers” OR “Hepatic Cancer” OR “Cancer, Hepatic” OR “Cancers, Hepatic” OR “Hepatic Cancers” OR “Liver Cancer” OR “Cancer, Liver” OR “Cancers, Liver” OR “Liver Cancers” OR “Cancer of the Liver” OR “Cancer, Hepatocellular” AND “diagnosis” OR “sensitivity” OR “specificity” OR “accuracy”. The language of all the articles was limited to English and the experiment target was limited to human beings. We also manually screened references from included articles and related reviews in order to expand the search.e.g., SEN, SPE, true positives [TP], false positives [FP], true negatives [FN], and false negatives [FN]); (c) CtDNA markers were used for the first diagnosis of HCC, not for the diagnosis of recurrence and metastasis; (d) controls were cancer-free adults; and (e) articles were published in English and experiment sources were human beings. The main exclusion criteria were as follows: (a) reviews, conference abstracts, meta-analysis, editorials, letters, reply, case report, commentary, short survey, notes, research highlight, and duplicate publications; (b) the sample size of studies was less than 10; (c) we failed to obtain the full text; (d) overlapping publications that included the same population and gene; and (e) there were multiple genes or gene models.Studies that met the following criteria were eligible for inclusion: (a) diagnostic accuracy of ctDNA was evaluated in plasma or serum; (b) sufficient data were acquired or could be calculated from the raw data , the quality of studies was assessed and rated by two authors independently, and the risk of bias and applicability concerns was categorized as low, unclear, or high. If the answer to all the iconic questions in a range was “yes”, then the risk of bias can be assessed as low, if the answer to any of the information questions is “no”, then the risk of bias was judged as “high”, and when there was not enough information, we defined it as unclear risk. Divergences were discussed together to reach a consensus and if an agreement was unable to be met, Huifan Ji made a judgement.P value were used to identify the presence of the threshold effect. The threshold effect was considered to exist when the P value was lower than 0.05. If heterogeneity resulted from the non-threshold effect, I2 and χ2 were used to evaluate heterogeneity between the enrolled articles. I2 >50% or P < 0.05 for χ2 suggested significant heterogeneity , meta-disc 1.4, to perform this diagnostic meta-analysis. The pooled sensitivity and specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and corresponding 95% confidence interval (95% CI) were calculated using a bivariate generalized linear mixed model to evaluate the test accuracy. Simultaneously, we plotted hierarchical summary receiver operating characteristics (HSROC) and utilized lambda as well as the area under the curve (AUC) which created by summary receiver operating characteristic (SROC) curves to estimate the diagnostic value of ctDNA. The logit estimates of SEN, SPE, and respective variances were used to construct the HSROC curves. Fagan’s nomogram was applied to interpret the clinical utility of ctDNA for HCC . The Speogeneity . Subgrouogeneity , and trin = 92). All the included studies included quantitative analysis to measure ctDNA concentration and single-gene methylation concentration (n = 19) (n = 40) (n = 31) (n = 31) did not clearly state the study design. Among those factors known at the time of collection, sampling time was either before treatment or surgery (n = 19), and samples were obtained from plasma (n = 15), serum (n = 24), or plasma/serum (n = 1). There were 23 articles with sample sizes ≥100, while the remaining sample sizes<100.(n = 19)  and qual(n = 40) . Of thes(n = 40) . In the (n = 40)  , while (n = 40)  . Other (n = 31) , five in(n = 31) , three i(n = 31)  , and on(n = 31) . A totaln = 15). Eight studies evaluated the performance of ctDNA concentration as a diagnostic indicator, 10 studies chose single gene methylation concentration, and one selected hTERT concentration. As for study design, the majority of them were not described clearly (n = 16), but two were prospective studies, and one was a retrospective study. Nine studies had a known time of collection before treatment and surgery. Samples were obtained from plasma (n = 9) and serum (n = 10). Twelve publications had sample sizes ≥100 and the rest had sample sizes smaller than 100. The assay methods used to measure the concentrations of ctDNA were real-time quantitative polymerase chain reaction (RT-qPCR) (n = 5), droplet digital PCR DNA (Dd-PCR) (n = 3), quantitative methylation specific PCR (Q-MSP) (n = 4), quantitative PCR (QPCR) (n = 4), ultraviolet transilluminator (n = 1), and Qubit dsDNA (n = 1). For ctDNA mixed with the AFP subgroup, 18 papers were studied with 1,790 HCC patients and 1,614 non-cancer participants.In the quantitative analysis subgroup, there were a total of 1,446 HCC patients and 1,835 non-cancer control participants . Patients with liver cirrhosis, HCV infection, and HBV infection were chosen as the control group in nine publications  , and onAs shown in 2 statistic: 94.01%) and a SPE of 0.90  (2 statistic:92.21%), NLR was 0.56 , DOR was 8.72 , and the AUC for the SROC curve was 0.78 (95%CI [0.74–0.81]). The HSROC graph was plotted and the value of beta was 0.23 (95% CI [−0.05–0.56]), z value was 1.61, and p value was 0.106, which indicated that the graph was symmetrical. Lambda was 1.93 (95% CI [1.56–2.33]), which suggested a moderate level of diagnostic value  and 0.84 , respectively (2 statistic: 88.24%) and NLR was 0.37 . The pooled DOR was 11.87  and the AUC for the SROC curve was 0.81 (95% CI [0.77–0.84]). In addition, the value of beta was 0.62 (95% CI [0.17–1.07]), z statistic was 2.68, and p value was 0.007, indicating that the HSROC was asymmetric. Lambda was 2.32 (95%CI [1.96–2.69]), which indicated a moderate level of diagnostic value  . In addiic value . The Speectively . The PLRic value . The Spe2 statistic: 85.81%) and a SPE of 0.84  (2 statistic:92.31), 0.22 , 23.63 , respectively. The results obtained by the HSROC model showed that the value of beta was 0.56 (95% CI [−0.03–1.15]), z statistic was 1.87, and p value was 0.062. The estimate for the “Lambda” and its 95% confidence interval was 3.24 (95% CI [2.63–3.85]), suggesting a high level of diagnostic value  . The comic value .2 statistic:85.27%), 0.77 , (2 statistic:74.47%), 0.25 , 14.06 , 0.86, and 2.64 (95% CI [1.73–3.55]), (2 statistic:1.38%), 0.86  (2 statistic:57.20%), 0.42 , 10.86 , 0.73, and 2.01 (95% CI [1.48–2.55]) , , 3.57 (9–3.55]), , respect:83.85%) , 4.61 (98–2.55]) , respectvs. Africa), sample source (plasma vs. serum), control type , sample size (≥100 vs. <100), publication year (2000–2010 vs. 2011–2021), assay methods  and America (n = 3) was not sufficient enough to perform subgroup analysis. The qualitative analysis of the ctDNA subgroup based on different regions revealed that the sampling area from Asia showed better diagnostic performance  than the area in Africa . Another subgroup analysis associated with years suggested that studies from 2000–2010  had SEN and SPE values of 0.39 and 0.98, respectively, and had lower sensitivity but greater specificity than studies from 2011–2021 , which had SEN and SPE values of 0.54 and 0.86, respectively. As for sample source, when we compared the samples collected from plasma, the sample source from serum did not show a great difference with a DOR of 5.77 vs 10.61 and an AUC of 0.76 vs. 0.77, which were quite different from the results in the quantitative subgroup. Also, studies with a sample size of ≥100 cases  were not drastically different when compared with studies with a sample size of <100 cases . Additionally, the single-gene mutation subgroup  had rather low SEN and higher SPE when compared with the single-gene methylation subgroup . Similarly, the quantitative analysis of the ctDNA subgroup showed a SEN of 0.65 and a SPE of 0.92, and sampling from plasma  achieved a greater diagnostic value compared to the subgroup that sampled from serum , which had a SEN of 0.71 and SPE of 0.76. Likewise, subgroup analyses related to control type showed that studies had satisfactory diagnostic value in discriminating HCC patients from healthy volunteers  compared with those using benign liver disorder patients. In terms of assay methods, research utilizing RT-PCR detective methods  revealed superior diagnostic accuracy in discriminating HCC patients from the control group with a SEN of 0.75 and SPE of 0.88 compared with research using other detective methods  with a SEN of 0.68 and SPE of 0.83, respectively.Subgroup analysis was applied based on different covariates to explore the potential sources of heterogeneity: region (Asia  studies), and sinWe also performed a multivariable meta-regression to further explore the source of heterogeneity . The resBased on Fagan’s nomogram, when we set the pretest probability to 25%, ctDNA increased the probability of a positive value to 62%, while there was a 16% probability that it ignored HCC patients with a negative test . Similarp = 0.86), while a coefficient of 11.80  indicated a study bias among studies that used a qualitative methodological approach. Trim and fill analysis (p = 0.00) for the mixed model before we filled in the missing studies and the outcome altered to 0.668   . Also, wHCC patients can benefit from early-stage diagnosis, but the damage and cost of tissue biopsy is not widely accepted by many patients with no or mild symptoms. Therefore, an increasing number of novel and available biomarkers for the early detection and diagnosis of HCC have been widely studied. Due to the development of next generation sequencing and other detective methods, many gene models  and genee.g., mSEPT9 methylation) has better diagnostic value than AFP.The pooled SEN and SPE values based on 67 studies in the qualitative subgroup were 0.50 and 0.90, respectively, while the quantitative analysis group yielded higher SEN and SPE values of 0.69 and 0.84, respectively. The superior diagnostic performance of the quantitative analysis compared to the qualitative subgroup may be due to the low detection rate of some genes in the earlier period  , low divs 7.06, and 59.26 vs 9.45, respectively. The plasma’s DOR was 20.54 in the quantitative ctDNA assay, and the serum’s was only 8.00. In addition, serum samples generally yield more ccfDNA, but the additional material is derived from leukocyte lysis during clotting, which dilutes the ctDNA content (n>50) and is not a specific biomarker for HCC, suggesting that it is not sufficient for clinical use. The DOR value of the AFP and ctDNA combined assay was 23.62, while the DOR of AFP was 10.64 at the cut-off value of 20–100 ng/ml in More importantly, we also used DOR as a single indicator to evaluate the diagnostic performance of the enrolled publications. Generally, a DOR value of >10 is considered good discriminatory performance. In this meta-analysis, the DOR values for the quantitative and qualitative ctDNA assay to distinguish HCC cases from control subjects were 11.78 and 8.72, respectively, indicating that quantitative assays showed a better performance than qualitative assays. The pooled DOR values for the quantitative and qualitative analysis of ctDNA to distinguish HCC from healthy volunteers and benign patients was 30.87  content . The DORLRs are indicators that reflect the authenticity of SEN and SPE. Although the results of AUC and DOR suggested a high level of accuracy, our pooled PLR and NLR results were less than satisfactory. In our qualitative meta-analysis, the PLR was 4.89 and the NLR was 0.56. Our results indicated that HCC patients had approximately four to five times greater chance of a TP than the control group according to the positive test results. The NLR was 0.56, which revealed that ctDNA-negative participants may have a 56% possibility of verifying HCC. Likewise, the pooled PLR and NLR of the quantitative analysis were 4.36 and 0.37, respectively. These results were quite similar to previous studies  . The poThere was no published bias in the quantitative analysis and AFP-ctDNA combined group according to the asymmetric Deek’s funnel plot test. However, there were some concerns about publication bias in the qualitative analysis subgroup. Results may be biased because positive results are more likely to be published. However, the results were robust after trim and fill analysis was utilized. SEN analysis in the mSEPT9 and ctDNA subgroup was robust , indicatDuring our study, we noticed genetic deviations such as mutations in TP53 , CTNNB1 The major limitations of this meta-analysis are as follows: First, in order to enroll all the studies that met the inclusion and exclusion standards as closely as possible, we took into consideration studies that included many genes with low SEN and SPE, but may have underrated the diagnostic performance of ctDNA. Moreover, the number of publications included in the ctDNA concentration subgroup and mSEPT9 subgroup were relatively small, and we need larger cohort studies to support our results. Third, most enrolled studies did not clearly point out the study type and many studies were case-control studies, which reduced the persuasiveness of the article. Also, many studies failed to provide information about some covariates such as vascular invasion, tumor staging, number of metastases, etiology, average age of participants, and tumor size. Further, the detection of samples lacks a standardized detective technology process, which may be source of heterogeneity. Finally, enrolled papers were limited to English, which may have generated some bias. Therefore, large-scale prospective studies using standardized detective technology and processes are needed in the future to support the conclusions of this meta-analysis.In summary, this meta-analysis showed that quantitative and qualitative subgroups had a medium to high level of diagnostic value, and the quantitative analysis showed better diagnostic value. We also specifically analyzed ctDNA level and mSEPT9 assay, which have potential to be applied as effective novel biomarkers for HCC in clinical work. It is worth noting that the mSEPT9 assay showed a satisfactory result in a study published in 2018 . The com10.7717/peerj.14303/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14303/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14303/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14303/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14303/supp-5Supplemental Information 5(A) The qualitative detection subgroup. (B) The quantitative detection subgroupClick here for additional data file.10.7717/peerj.14303/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.14303/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.14303/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.14303/supp-9Supplemental Information 9Click here for additional data file.


LOXHD1 gene (DFNB77), encoding lipoxygenase homology domain 1, is a rare hearing loss gene found in several populations. To evaluate the importance of LOXHD1 variants in Chinese patients with NSHL, we performed genetic analysis on LOXHD1 in 2,901 sporadic Chinese patients to identify the aspect and frequency of LOXHD1 causative variants. Next-generation sequencing using a custom gene panel of HL was conducted on 2,641 unrelated patients and whole-exome sequencing on the remaining 260 patients. A total of 33 likely causative variants were identified in 21 patients, including 20 novel variants and 13 previously reported pathogenic variants. Each of the 20 novel variants was evaluated according to ACMG criteria. These findings showed that causative variants in LOXHD1 were found in about 0.72%  of Chinese NSHL patients. This study is by far the largest number of novel variants identified in this gene expanding the range of pathogenic variants in LOXHD1, and suggests that variants in this gene occur relatively commonly in Chinese NSHL patients. This extensive investigation of LOXHD1 in Chinese NSHL patients proposed six recurrent LOXHD1 variants. These findings may assist in both molecular diagnosis and genetic counseling.Non-syndromic hearing loss (NSHL) is a common neurosensory disease with an extreme genetic heterogeneity which has been linked to variants in over 120 genes. The  Autosomal recessive non-syndromic hearing loss (ARNSHL) accounts for ∼80% of cases of inherited HL. The two most common genes linked to ARNSHL are GJB2 and SLC26A4, observed in multiple populations. In 2009, biallelic variants in LOXHD1  was identified as the cause of ARNSHL, DFNB77 (MIM #613079) (Hearing loss (HL) is common and is known to affect approximately one to three out of 1,000 newborns . HL show#613079) .LOXHD1 usually present with heterogeneous hearing phenotypes. Although LOXHD1 pathogenic variants tend to be linked to late-onset HL with an onset age of older than 10 years old (yo)  , onset mudiogram .LOXHD1 genetic load in NSHL. In relatively larger cohorts with more than 100 patients, the prevalence of LOXHD1-associated HL in United States HL patients was found to be 0.71%   while in 2019, Maekawa et al. identified 28 affected individuals with LOXHD1 variants among 8,074 Japanese HL patients (0.35%) , considering only late-onset, down-sloping HL  , 1.5% in8/1,119) , and 0.98/1,119) . In 2015 (0.35%) . A reporHL cases . In 2021oping HL .LOXHD1 have been identified to be related with HL. To date, over one hundred pathogenic variants in LOXHD1 have been identified. Although a few sporadic cases of LOXHD1-associated HL have been described in Chinese patients , 107 pathogenic variants of patients , little The DNA of 2,901 sporadic NSHL patients belonging to the Han Chinese population was analyzed. The participants were recruited from the Departments of Otolaryngology Head and Neck Surgery, PLA General Hospital, between June 2015 and August 2021, and ranged in age from 6 months to 28 years. All participants presented with bilateral, congenital or late-onset, mild to profound NSHL (age of onset between 0 and 10 years) with no other obvious systemic disorders. Other explanations for HL, such as infection, malformation, or tumors, were ruled out.The clinical data acquired at the time of assessment included medical history (by questionnaire), temporal bone computed tomography (CT), otoscopy, pure-tone audiometric examination (if over the age of 6 years), auditory steady-state response , acoustic immittance, auditory brainstem responses, and distortion product otoacoustic emission.HL was assessed using pure-tone hearing thresholds or ASSR response thresholds . Hearing was scored using the American Speech-Language-Hearing Association (ASHA) guidelines as normal (threshold ≤25 dB), mild HL (threshold: 25.1–40 dB), moderate HL (threshold: 40.1–55 dB), moderately severe HL (threshold: 55.1–70 dB), severe HL (threshold: 70.1–90 dB), and profound HL . The progression of HL was indicated by a 15 dB increase in the hearing or response threshold in one or both ears from one audiogram to the next increased hearing or response threshold. The hearing frequencies were categorized as low (125–500 Hz), mid (1–2 kHz), or high (4–8 kHz). Prelingual HL was defined as that occurring before the development of speech , while postlingual loss occurred after speech development . EvaluatThe audiograms were described according to shape, specifically, down-sloping , flat , U-shaped (≥10 dB difference between the poorest mid-frequency [1–2 kHz] threshold and thresholds at higher and lower frequencies), ascending (≥15 dB difference between the 250–500 Hz and the [better] 4–8 kHz thresholds), residual (limited hearing at only a few frequencies), complete deafness (no hearing at maximum outputs regardless of frequency), and unclassified (none of the above).de novo) or autosomal recessive inheritance patterns, only variants de novo in study participants or variants homozygous or compound heterozygous in study participants and meanwhile heterozygous in their parents were picked as candidate. The variants were comprehensive interpreted using VarSome , as per instructions. Whole-exome capture was applied in 260 patients. A custom capture panel  encompassing 3,156 regions of approximately 850 kb target size was constructed to cover the exons and flanking intronic sequences (∼50 bp) for 159 deafness genes  was appl VarSome . Manual  VarSome . The finhttps://www.ncbi.nlm.nih.gov/clinvar/) under the accession numbers SCV002011797-SCV002011798.The data, including phenotypes and observed variants, have been submitted to ClinVar  carrying thirty-three likely causative he PLATs ; Table 1in cis in his father whose hearing was normal. The variant c.5888delG in LOXHD1 leading to a premature termination codon was coded as “PVS1+PS1+PM2+PP3+PP5” and listed as “pathogenic” as mentioned above, while c.1262G > A leading to a single amino acid substitution was coded as “PM2+BP1+BP4” and listed as of “uncertain significance”. Therefore, we presumed c.5888delG was the possible causative variant in patient No.19. Patient No.1 showed two heterozygous variants (c.2641G > A and c.1420G > T) in LOXHD1, in addition to two compound heterozygous variants (c.1256A > T and c.1255A > C) in HARS1. Given that the two HARS1 variants were coded as “c.1256A > T: PM2+PP3” and “c.5557C > G: PM2+BP4” and listed as “Uncertain significance” according to the ACMG criteria with the same population frequency of 0.00109 in East Asia, and a lack of reports of their possible pathogenic significance, we assumed HARS1 was not the causative gene in patient No.1. Similarly, in patient No.8, in addition to the compound heterozygous variants (c.2438T > A and c.2635C > T) in LOXHD1, variants (c.3658G > A and c.5557C > G) in MYO15A were identified, with the parents as carriers. There are no reported pathogenic associations of these MYO15A variants, and the ACMG codes are “c.3658G > A: PP2+BS1+BS2+BP4” and “c.5557C > G: PM1+PM2+PP2+BP4”; thus, they were listed as variants of benign and uncertain significance, respectively. MYO15A was, therefore, not considered to be the causative gene in this family. Nevertheless, as it is possible that these variants may have influenced HL, their relevance cannot be discounted altogether and further investigation should be undertaken. In patient No.7, compound heterozygous variants (c.611–2A > T and c.1846T > C) in LOXHD1 were identified in the affected proband and c.611–2A > T was confirmed as inherited from his mother; the c.1846T > C variant could not be classified as de novo or not as the father’s blood sample was not available. A similar situation was seen in patient No.17, where the parents’ blood samples were not available.The candidate variants selected from the sequencing data are shown in LOXHD1 identified in this study, a total of six variants were recurrent, including four reported  and two novel (c.1420G > T and c.6413G > A) variants. Among the reported variants, c.5888delG was detected in three unrelated patients in the heterozygous state  and in one patient in the homozygous state (No.19), and c.611-2A > T was detected in three unrelated patients in the heterozygous state . Both c.5888delG and c.611-2A > T were defined as “pathogenic” according to the ACMG criteria. The frequency of c.5888delG in the Chinese NSHL population was estimated as 0.0862% , which is four times higher than the value of 0.000183 for the East Asian population recorded in the gnomAD Exomes database. The frequency of c.611-2A > T in the Chinese NSHL population was estimated as 0.0517%  and it was not found in the gnomAD exomes or gnomAD genomes databases. The other four variants, c.2641G > A, c.4247G > A, c.1420G > T, and c.6413G > A, were detected in two unrelated patients separately in the heterozygous state and their frequencies in the Chinese NSHL population were estimated as 0.0345% . The variants c.2641G > A, c.4247G > A and c.1420G > T were defined as “likely pathogenic”, “pathogenic”, and “pathogenic”, respectively, according to the ACMG criteria and their frequencies in the East Asian population were not found in either the gnomAD exomes database or the gnomAD genomes database. Although c.6413G > A was classified as a variant of “uncertain significance” according to the ACMG criteria, it is noteworthy that the variant frequency is recorded as 0.033 in the East Asian population in the gnomAD exomes database, which indicated that this variant is likely to be benign.Of all the variants in LOXHD1 compound heterozygous/homozygous variants, including age of onset, visiting age, audiogram classification, HL progression, and vestibular symptoms. The age of onset varied from newborn to 10 years, with most participants showing evidence of congenital HL between the ages of 0–3 years . There were no vestibular symptoms present in any of the participants.By evaluating of the types of HL among the 15 probands with available audiograms, relationships between genotype and phenotype could be determined . The HL Of the 11 patients with follow-up information, four complained of progressive HL while seven gave feedback of stable hearing status. Patient No.4, the only patient with multiple hearing examination results, diagnosed with bilateral symmetric severe hearing loss at 1 yo, showed progressive HL on the left ear and a relatively stable hearing status after 2 years of follow-up ; Table 2LOXHD1 variants were associated with ∼0.72%  of sporadic NSHL cases in a patient cohort without apparent disorders in other systems. All the LOXHD1 variant–positive patients were under 30 years old and 90.5% (19/21) had HL of congenital onset. In total, 13 reported pathogenic variants and 20 novel variants of LOXHD1 were identified including six recurrent variants.It was observed that pathogenic Loxhd1 homologous to human LOXHD1 was first cloned by Grillet et al., in 2009 and was found to be expressed in the cochlear and vestibular hair cells of young mice and in hair cells along the length of the stereocilia in adult mice . The LOXHD1 protein is highly evolutionarily conserved, and consists of 15 PLAT domains  or 16 PLAT domains  and the relevant versions used have been adjusted to transcripts variant 1 (NM_144612) in this study. The assumed function of the PLAT domain is to facilitate access to the plasma membrane in a Ca2+-dependent manner  and bilateral symmetric down-sloping audiogram configurations  that were in accordance with previous reports  of NSHL in Chinese patients, an incidence similar to that reported in the American population   variants in LOXHD1 are nonsense  . In orde8/1,119) -Type1. O8/1,119) -Type2. Bnonsense .LOXHD1. LOXHD1 variants filtered using the Human Gene Mutation Database (HGMD) Professional version (release 2021.02) and appraised through literature review to date, including the 20 novel variants identified in this study. The findings have also expanded the variant spectrum of LOXHD1 in the Chinese deafness population from 28 to 51. LOXHD1 variants seen in different populations on the schematic diagram of the protein domain structure. Among all the 13 reported variants, c.5888delG (p.G1963Afs*136), c.611–2A > T, c.3268C > T (p.R1090W), c.4247G > A (p.W1416*) and c.4212+5G > A (NM_144612.6) have only been reported in Chinese HL patients (Our findings expand the spectrum of variants seen in patients , c.1270+patients , and thepatients . NotablyLOXHD1 were not detected in this study; these should be investigated in further studies.This study has two minor limitations. First, most patients in our cohort did not present with multiple audiograms, leading to difficulties in estimating HL progression. We, therefore, had to rely on parental observation. Second, copy number variations in LOXHD1 variants in 2,901 Chinese patients with NSHL and investigated the clinical and genetic features of 21 patients. Our findings indicate that variants in LOXHD1 represent a comparatively common cause of NSHL in the Chinese population. It is hoped that these findings will assist with diagnosis, management, and genetic counseling of NSHL patients, as well as suggesting directions for therapeutic development.In conclusion, we evaluated the importance of


Brassicaceae edible plants are rich in bioactive compounds and promote health benefits. However, there is less interest in expanding knowledge about the Brassica cultivars to date. In particular, underutilized species and local cultivars could constitute a source of agrodiversity in adapting to the territory with likely higher contents of nutraceutical compounds. In this context, Bróquil  is a traditional Brassicaceae crop grown in the Spanish region of Aragón. Currently, it is cultivated mainly in family orchards for autoconsumption and, in minority, in small farms for local markets. This study evaluates a collection of 13 bróquil landraces from the Spanish Vegetable Genebank of the Agrifood Research and Technology Center of Zaragoza (BGHZ-CITA), describing their mineral contents, bioactive compounds, and antioxidant activities, including a broccoli commercial variety “Parthenon” as the control. The study reports data on the health-promoting nutrients and antioxidants of bróquil for the first time. Under our experimental conditions, we found that bróquil has a great variability for these compounds that showed on average similar or higher levels than the broccoli control. The different bróquil landraces also revealed variability in both intraccessions and interaccessions due to the lack of a formal breeding selection. Despite this variability, we highlight accession HB5 that corresponds to Headed Bróquil BGHZ6685. In particular, we can stand out its antioxidant activity of 87.07 ± 0.81%I, total phenolic content of 13.21 ± 0.53 mg GAE g−1 dw, total flavonoid content of 14.50 ± 1.29 mg QE g−1 dw, total glucosinolate content of 43.70 ± 1.09 mg SnE g−1 dw, and vitamin C content of 7.21 ± 0.13 mg AA g−1 dw. Regarding bróquil mineral composition, K was the highest macroelement (22.66–33.62 mg g−1 dw), followed by Ca, P, and S whose values were relatively lower compared to K. Mg and Na showed the lowest values. Among the microelements evaluated , iron was the most abundant detected, higher in all bróquil accessions than in broccoli, except for one accession. Therefore, the results reported for bróquil landraces show promising nutritional quality. This could lead to an increase in agrobiodiversity and contribute to a more diversified and healthy diet. Vegetable crops represent a fundamental ingredient in the human diet due to their high nutritional value and bioactive content and could serve toward improving food security and nutritional quality . HoweverBrassicaceae edible plants due to their richness in bioactive compounds and health benefits  is a traditional winter cole crop that has been cultivated in the Huesca Province of the Aragón region, Northeast Spain, since ancient times  were used for this study: six headed bróquil accessions (HB) and seven green bróquil accessions (GB) . Seeds of 13 landraces of bróquil (ons (GB) . The comThe assay was carried out in the experimental fields of CPIFP Montearagón  Huesca, Spain, during the fall-winter of 2019–2020 using a randomized complete block design with three replications. Each replication consisted of 14 plants.−2 in outdoor cultivation. Lines of the drip irrigation system were implanted following the plant row. No mineral fertilizers were applied. Previous crops were legumes. No signs of aphids, lepidoptera, fungi, viruses, other plant insect attacks, or infection were detected, and thus, no phytosanitary treatment was necessary.The seeds of 13 bróquil and commercial broccoli were sown in foam trays filled with a mixture of peat moss and vermiculite (1:1 volume). Soil preparation, planting, and other agronomic practices were carried out uniformly following the standard bróquil growth practices. The seedlings were transplanted at the third true leaf stage on the 12th of September with a 60 cm distance between the plants and a row-to-row distance of 60 cm, equivalent to a crop density of 2.77 plants mThree plants per replication were harvested at the commercial maturity stage and transported immediately to the laboratory. Broccoli was ready to harvest when the heads are well formed and green but still in the compact bud in the same way as bróquil but yellowish. Before harvesting, the edible parts were separated, washed, chopped, and the moisture content was determined using an oven at 105°C for 24 h. For each field replication, 100 g was prepared and frozen for later lyophilization. The freeze-dried samples were milled in a ball mill. The samples were stored in airtight plastic bags in a freezer at −18°C until analysis. The analyses were performed in triplicate, and the results were expressed based on dry weight for each sample.The extract from freeze-dried bróquil samples was prepared by following the procedure described by Mawlong et al.  with sli2CO3) was added. An intense blue color developed after 30 min in the dark at ambient temperature and its absorbance was measured at 760 nm. Gallic acid was used as a standard for calibration at different concentrations (2–20 mg L−1). The results were expressed as mg of gallic acid equivalents per gram of the dry weight sample (mg GAE g−1 dw).For TPC determination, the Folin–Ciocalteu method was used with slight modifications . To 0.5 3) were added. After 5 min of incubation, 0.3 ml of 10% aluminum chloride (AlCl3) was added. Before 1 min, 2 ml of 1 M sodium hydroxide (NaOH) was added. Finally, the volume was made up to 10 ml with distilled water and homogenized. An orange-yellowish color was developed and the absorbance was measured at 510 nm. Quercetin was used as a standard for calibration at different concentrations (4–30 mg L−1). The results were expressed as mg of the quercetin equivalents per gram of the dry weight sample (mg QE g−1 dw).For TFC determination, the aluminum chloride method was used . To 0.5 2PdCl4) were added. After incubation at 20°C for 1 h, absorbance was measured at 425 nm. Sinigrin was used as a standard for calibration at different concentrations (10–90 mg L−1). The results were expressed as mg of the sinigrin equivalents per gram of the dry weight sample (mg SnE g−1 dw).For TGLC determination, the tetrachloropalladate method was used . To 0.5 2SO4 was added. To 0.1 ml of this mix, 0.9 ml of 15% (m/v) DCPIP was added and the absorbance was measured immediately after mixing for 15 s at 515 nm. Ascorbic acid was used as a standard for calibration at different concentrations (10–140 mg L−1). The results were expressed in mg ascorbic acid per gram of the dry weight sample (mg AA g−1 dw).The total content of vitamin C [the sum of ascorbic acid (AA) and dehydroascorbic acid (DHAA)] was determined by the 2,6-dichloroindophenol (DCIP) spectrophotometric method  and the −1) and the absorbance was measured at 510 nm after keeping it at 10 min at ambient temperature. A blank control was prepared and measured with 0.2 ml of 80% methanol and 1 ml of DPPH. The lower absorbance of the reaction mixture after a 10-min reaction indicated higher free radical scavenging activity. The results were expressed as percentage inhibition (%I) using the following equation: %I = ((Ac - As) / Ac) × 100.Samples were prepared as described previously for determining the activities of the bioactive compounds. For the determination of antioxidant activity (AA), the DPPH  radical method was used . This meAc is the absorbance of the control (blank) and As is the absorbance in the presence of the bróquil methanolic extract.where −1 of dry weight sample), (mg TE g−1 dw), according to previous studies using the following equation: y = 0.0164x – 0.4719 (R2 = 0.9924), where x is the %I.Besides, AA is expressed as an activity equivalent of Trolox  were added and made up to a volume of 10 ml with distilled water and shaken. After 10 min, the absorbance was measured at 420 nm. Sodium sulfate (Na2SO4) was used as a standard for calibration at different concentrations of sulfate (5–40 mg L−1). The results were expressed in mg sulfur per gram of the dry weight sample. The concentration of P was determined by the colorimetric procedure using acid ammonium molybdate. To 0.03 ml of the solution sample, 3 ml of distilled water was added and the pH was adjusted to 3–10 with 1 M NaOH. Then, 0.2 ml of ascorbic acid and 0.4 ml of a mixed solution  were added and made up to 10 ml with distilled water and shaken. After 10 min, the absorbance of the molybdenum blue complex was measured at 880 nm. Potassium acid phosphate (K2HPO4) was used as a standard for calibration at different concentrations of phosphate (0.1–1.0 mg L−1). The results were expressed in mg phosphorus per gram of the dry weight sample.For the determination of P and S, spectrophotometric methods based on the standard methods for water analysis  were usepost-hoc Tukey-b test to construct homogeneous groups. The differences between individual means were deemed to be significant at a p-value of < 0.05. All analyses were performed using the SPSS statistical package .The results were analyzed from the mean of determinations for duplicate samples prepared for each genotype. Data were expressed as the mean ± standard deviation (SD). The mean was compared using the one-way analysis of variance (ANOVA) followed by a The phytochemical analysis of bróquil included total phenolic, flavonoid, and glucosinolate contents and vitamin C. The results presented were obtained from the analysis of dried and subsequently freeze-dried plant material. The moisture range was 86.3–91.2% for headed bróquil, 83.0–89.8% for green bróquil, and 87.5% for broccoli.−1 dw (HB5). Minimum values (4.69 mg g−1) were obtained for broccoli. The plant material showed significant differences between accessions  to 13.21 mg GAE gcessions . Data co−1 dw in broccoli to 14.50 mg QE g−1 dw in HB5  and significant differences were detected between all accessions  to 48.51 mg SnE gcessions . Three h−1 (HB3). Significant differences were detected between accessions (Vitamin C content ranged from 4.59 (HB2) to 8.45 mg AA gcessions . Four ac−1 dw. No significant differences were detected between headed bróquil accessions and green bróquil ones. Notably, the high standard deviation among the accessions evidences the heterogeneity of these landraces regarding the antioxidant activity. Except for GB4, bróquil accessions did not show statistically significant differences regarding antioxidant activity when compared to broccoli.Low variability was detected among bróquil accessions in terms of antioxidant activity . Data raThe present study revealed that the average ash content in bróquil samples vary from 7.02 to 9.24 g per 100 g of dry matter in samples GB4 and HB3, respectively, while in broccoli, the ash content was 7.14 g per 100 g of dry matter. All macronutrients and microelements showed significant differences among the bróquil accessions and broccoli control , 3.−1 for GB7 and HB4, respectively. The broccoli control presented an intermediate value of 25.64 mg g−1 and was included in the homogeneous group with a lower potassium content. In contrast, bróquil accessions HB2, HB3, HB4, and GB1 have higher potassium values. The following macroelements were phosphorus, calcium, and sulfur, with average values ranging from 6 mg g−1 in all cases. Magnesium and sodium were the macroelements with the lowest contents, with values below 2 mg g−1. Green and headed bróquil did not show differences according to their macroelement content. The broccoli variety, used as the control, had high values of magnesium (1.89 mg g−1), thus establishing a homogeneous group with HB3 and GB1 and highlighting its high sodium content of 1.10 mg g−1, since the bróquil accessions presented values between 0.20 and 0.40 mg g−1.The main ash component was potassium ranging from 22.66 to 33.62 mg g−1 was significantly lower than the values obtained for bróquil accessions, except for GB6. The iron content of bróquil accessions ranged from 0.135 (GB3 and HB2) to 0.106 mg g−1 (GB6). The maximum and minimum contents of manganese were obtained for bróquil accessions, especially GB3 (0.033 mg g−1) and HB2 (0.019 mg g−1), while broccoli showed an intermediate value of 0.023 mg g−1. The higher values for zinc were obtained from the broccoli (0.058 mg g−1) and the bróquil accessions HB3, HB4, HB6, GB2, and GB3. The minimum zinc content obtained was 0.022 mg g−1 from the HB1 accession.Broccoli's iron content of 0.088 mg gBrassica landrace maintained in a small area of the Spanish Aragón region. The work constitutes an approach to the bioactive properties of this type of Brassicas performed for the first time since no previous bibliographical references exist. Consequently, the potential beneficial effects of bróquil may only contrast the B. oleracea typologies. Brassica species and, in particular, broccoli are reputed for their nutritional value. They are a source of phytochemicals  and diverse nutrients   , 25, 26.−1 dw which is higher than broccoli (4.7 mg GAE g−1 dw). These results range from 3.7 to 12.1, 7.5 to 4.6, and 6.4 mg GAE g−1 dw for commercial broccoli, Italian broccoli landraces, and broccoli as reported by Koh et al.  was higher than that obtained for broccoli (4.3 mg QE g−1 dw), which suggests a higher nutritional as well as nutraceutical value of bróquil cultivars. Naguid et al. . Six bróquil accessions showed similar TGLC values while seven bróquil accessions, including both headed and green types, showed significantly higher values than broccoli. Bhandari et al. .The variability reported in this study clearly shows that the contents of macronutrients and micronutrients in bróquil accessions depend on the genotype. The content of macronutrients in bróquil landraces follows the sequence: K > Ca > P > S > Mg > Na. The content of micronutrients is arranged as follows: Fe > Mn > Zn. Among the macronutrients, the content of K was the highest, followed by those of Ca, P, and S whose values were relatively lower compared to K. The level of Mg was relatively lower compared to the others. We can also highlight the lower amount of Na obtained for all bróquil accessions. A normal amount of K in a typical diet of healthy people is about 2–5.9 ge 782 mg . Transfo−1 fresh weight . The bioavailability of calcium (Ca) in Brassicas is compared to milk  which comprises all the diversity for this Spanish local edible brassica. The results describe their mineral content, bioactive compounds, and antioxidant activity. No differences were detected between green and headed bróquil; however, a great variability within each typology was observed, due to the lack of a formal breeding selection. The results show promising contents of total phenolic compounds, flavonoids, glucosinolates, and vitamin C. The antioxidant activity value of bróquil, obtained under our experimental conditions, is similar to or higher than broccoli. Moreover, bróquil contributes to daily mineral intake because the results show high quantities of potassium, calcium, phosphorus, sulfur, and iron. The variability found also makes these landraces an important resource for genetic improvement, especially regarding the HB5 accession, which could be considered a promising candidate for the bróquil-headed type due to the high values for most of the parameters assessed. In conclusion, our results revealed interesting antioxidant properties and mineral composition for this traditional crop, which reinforce the nutritional value of bróquil, enriched in bioactive compounds that promote healthy nutrition.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CMo: investigation, data curation, formal analysis, funding acquisition, writing—original draft, supervision, methodology, and writing—review and editing. RZ: investigation, data curation, formal analysis, writing original draft, supervision, methodology, and writing—review and editing. SL: investigation, data curation, formal analysis, and writing the original draft. CMa: investigation, data curation, writing original draft, supervision, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.