Skeletonema marinoi, Cyclotella cryptica) and one eustigmatophyte (Nannochloropsis oceanica) were grown alone or in combination. Concentrations of ten vitamins , carotenoids and polyphenols, together with total flavonoids, sterols, lipids, proteins and carbohydrates, were compared. Moreover, antioxidant capacity and chemopreventive potential in terms inhibiting four human tumor-derived and normal cell lines proliferation were evaluated. Co-cultivation can engender biomass with emergent properties regarding bioactivity or bioactive chemical profile, depending on the combined species. The high vitamin content of C. cryptica or N. oceanica further enhanced (until 10% more) when co-cultivated, explaining the two-fold increase of the antioxidant capacity of the combined C. cryptica and N. oceanica biomass. Differently, the chemopreventive activity was valuably enhanced when coupling the two diatoms C. cryptica and S. marinoi. The results obtained in this pilot study promote microalgal co-cultivation as a valuable strategy aiming to boost their application in eco-sustainable biotechnology.Microalgal biotechnology is gaining importance. However, key issues in the pipeline from species selection towards large biomass production still require improvements to maximize the yield and lower the microalgal production costs. This study explores a co-cultivation strategy to improve the bioactive compounds richness of the harvested microalgal biomass. Based on their biotechnological potential, two diatoms ( Spirulina, Chlorella, Dunaliella, or Haematococcus:  × 100, where A0 = absorbance of DPPH+ and A1= absorbance of sample.An aliquot of 50 mg of dried algal powder was used to test the ability of the extract to scavenge 2,2-diphenyl-1-picrylhydrazyl . Microalgal dried biomass was re-suspended in 500 μL of methanol and shacked vigorously for 2 min. The solution was sonicated for 1 min with a micro tip at 20% output on ice  and then the tubes were centrifuged at 3900× + radical. Microalgal dry biomass was re-suspended in 500 μL of methanol and shacked vigorously for 2 min. The solution was sonicated for 1 min with a micro tip at 20% output on ice (S-250A Branson Ultrasonic) and centrifuged at 3900× g for 15 min at 4 °C. then, an aliquot of 10 μL of sample was mixed with 990 μL of an ABTS+ solution and kept for 6 min at 30 °C. The ABTS+ solution was prepared as follow: ABTS  was weighed and dissolved in water, with a final concentration of 7 mM. Potassium persulfate  powder was weighed and dissolved in water with a final concentration of 2.45 mM. To form the ABTS+ radical, an equal volume of ABTS and potassium persulfate solution was mixed and stored in darkness at room temperature for 12 h. The ABTS+ solution was then diluted with methanol to reach an absorbance of 0.70 ± 0.02 AU, measured in a UV-VIS spectrophotometer  at 734 nm. After incubation of the sample with ABTS+ solution, the absorbance was read at 734 nm using a UV-VIS spectrophotometer . The scavenging assay was done in triplicate and the results were calculated according to the equation: ABTS+ % Inhibition = [(A0 − A1)/A0] × 100, where A0= absorbance of ABTS+ and A1= absorbance of sample.An aliquot of 50 mg of dried algal powder was used to test the ability of the extract to scavenge ABTSg for 10 min). Phenol (0.2 mL) was added to 0.4 mL of sample, then 1 mL of concentrated sulfuric acid was added. After 30 min at room temperature, the absorbance was read at 490 nm using a UV-VIS spectrophotometer . The total carbohydrates concentration was quantified referring to a calibration curve using glucose as standard.Total carbohydrate concentration was determined on 30 mg of dried algal powder with the phenol-sulfuric acid method, slightly modified from . The drig for 8 min. The absorbance of an aliquot of 1.5 µL of the supernatant was read in triplicate in a NanoDrop 1000 UV-Vis spectrophotometer at 280 nm and 260 nm to estimate the protein concentration [Dried microalgal powder (15 mg) was re-suspended in 500 µL of RIPA Lysis and Extraction Buffer  and sonicated for 90 s  with a micro tip at 20% output on ice (S-250A Branson Ultrasonic). Samples were then centrifuged at 13,000× ntration .g for 10 min. After discarding the upper layer, the lower layer was transferred into a fresh tube and the pellets were re-extracted in a solution of chloroform (2 mL) and methanol (2 mL). The extract was passed through a layer of anhydrous sodium sulfate using Whatman No. 1 filter paper in a funnel and then dried by rotary evaporation at 4 °C and weighted. The dried total lipid content was used for total sterol concentration (TSC) estimation [For total lipid concentration estimation , 100 mg timation . The lipv/v. After 4 min, 600 μL of Na2CO3 (75 g L−1) were added to the mixture and left 2 h at room temperature. Then, absorbance was read at 765 nm using a UV-VIS spectrophotometer  and the concentrations were estimated using gallic acid (GA) as standard [3) colorimetric method [3 2% was mixed to 600 μL of sample pre-diluted 1:2 v/v in methanol 80% v/v. The mixture was left for 1 h at room temperature. Then, absorbance was read at 410 nm using a UV-VIS spectrophotometer  and the concentrations were estimated using quercetin (Q) as standard.The total phenolic content (TPC) and total flavonoid content (TFC) of the microalgal biomass were estimated in aliquots of 30 mg of dried powder. An aliquot of 150 μL of the extract was mixed with 750 µL of Folin-Ciocalteu’s phenol reagent, pre-diluted in distilled water 1:10 standard . TFC wasc method  as follov/v) and B, absolute methanol with a flow rate set up at 1.7 mL min−1. During the 12 min elution, the gradient between the solvents was programmed as follow: 75% A (0 min), 50% A (1 min), 0% A (8 min), 0% A (11 min), 75% A (12 min). Pigments were detected at 440 nm using a Hewlett Packard photodiode array detector model DAD series 1100, which provides the 400–700 nm spectrum for each detected pigment. Pigments were identified based on their retention time and absorption spectrum, while pure standards from the D.H.I. Water & Environment  allowed their quantification.Pigment analysis was done following the protocol in . Fifteeng for 8 min. Vitamins A, B1, B2, B6, B12, C, D2, D3, E and H were quantified performing a competitive ELISA assay in 96 well plate : 5 µL of each microalgal extract, 45 µL of coating buffer solution  and 50 µL of primary antibody solution  were added in each well and incubated at 37 °C for 1 h. At the end of the second incubation, 50 µL of TMB substrate 1×  were added in each well and incubated at room temperature for 15 min. The absorbance was read at 450 nm with the reference measurement at 620 nm, using a Microplate Reader: Infinite® M1000 PRO . The vitamin concentration (in ng mg−1 DW) was quantified referring to calibration curve using pure vitamins as standard.Dried microalgal powder (15 mg) was re-suspended in 500 µL of RIPA Lysis and Extraction Buffer  and sonicated for 90 s  with a micro tip at 20% output on ice (S-250A Branson Ultrasonic). The samples were then centrifuged at 13,000× g for 5 min and the supernatants were filtered on a 0.2 µm polyamide membrane. The supernatant was then injected in a Hewlett Packard series 1100 HPLC  equipped with a 50 µL loop and a reversed-phase column . The mobile phase, as described in [−1. Chromatograms were acquired at three different wavelengths . Phenolic compounds were identified based on their retention time and absorption spectrum (registered between 220 and 450 nm) and with comparison with nineteen pure standards  allowing the quantification of these compounds. Due to experimental problems, the phenolic compounds analysis provided reliable results only for the mono-cultivated S.m. and C.c.An aliquot of around 130 mg was used to determine the phenolic compounds profile of the dried microalgal powder. Extraction was done in 2 mL of 75% aqueous methanol, with BHT  to preserve polyphenol stability. Extraction was performed with ultrasounds for 2 min followed by a 30 min lasting ultrasonic bath. Then, the mixture was kept for 30 min at 4 °C. Extracts were then centrifuged at 1200× ribed in , consistv/v) fetal bovine serum (FBS), 100 units mL−1 penicillin, 100 units mL−1 streptomycin and 2 mM of L-glutamine, in a 5% CO2 atmosphere at 37 °C. The PC3 cell line was grown in RPMI 1640 supplemented with 10% (v/v) FBS, 100 units mL−1 penicillin, 100 units mL−1 streptomycin and 2 mM of L-glutamine, in a 5% CO2 atmosphere at 37 °C. The A2058 cell line was grown in DMEM/F12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12), supplemented with 10% FBS, 100 units mL−1 penicillin and 100 units mL−1 streptomycin, in a 5% CO2 atmosphere at 37 °C. The HT-29 cell line was grown in McCoy’s 5a Medium Modified supplemented with 10% (v/v) FBS, 100 units mL−1 penicillin and 100 units mL−1 streptomycin, in a 5% CO2 atmosphere at 37 °C. Human cell lines were seeded in 96 well plate , with an initial concentration of 2 × 103 cells well−1 and kept overnight for attachment. The four human cell lines were then incubated with microalgal extracts at three concentrations  for 48 h. Microalgal extracts were prepared as follow: Microalgal dried biomass was re-suspended in 500 μL of methanol and shacked vigorously for 2 min. The solution was sonicated for 1 min with a micro tip at 20% output on ice (S-250A Branson Ultrasonic) and then the tubes were centrifuged at 3900× g for 15 min at 4 °C. The supernatants were then dried by rotary evaporation at 37 °C and the dried methanolic extracts were resuspended in DMSO (0.1%) and human cell culture medium. At the end of the 48 h incubation, the MTT -2,5-diphenyltetrazolium Bromide, Applichem, Darmstadt, Germany) viability assay was performed. Cells were incubated with 10 μL (5 mg mL−1) of MTT for 3 h in a 5% CO2 atmosphere at 37 °C. The formazan crystals (produced by viable cells) were dissolved with 100 μL of isopropyl alcohol. The absorbance was then recorded on a microplate reader at a wavelength of 570 nm Infinite® M1000 PRO . The results were represented as a percent of viable cells estimated as the ratio between the absorbance of each sample  and the absorbance of the control (untreated cells).Four human cell lines were used to perform the MTT assay: prostate cancer , melanoma , colon-rectal adenocarcinoma  and normal prostate epithelium . The cell lines A2058 and HT29 represent primary tumor cells, while PC3 cell line derived from prostate cancer metastasis in bone marrow in a disease state IV. PNT2 cell line was established by immortalization of normal adult prostatic epithelial cells by transfection with a plasmid containing SV40 genome with a defective replication origin. The PNT2 cell line was grown in RPMI 1640 supplemented with 10% (® Software (2021). Mean comparison was carried out using the Student’s t-test. For viability assay, two-way ANOVA was used for the assessment of variance within and between the control and treated groups. Dunnett’s method was applied to compare the mean from treated cells with the control mean (untreated cells) by using GraphPad® Software (2021). A confidence interval at 95% for the difference between the two means was estimated.For each data measured on the triplicate cultivations, mean and standard deviation were estimated using GraphPadCoupling two species together did not inhibit growth of the co-cultivated species, both in 50 mL flasks  and in 2p < 0.05; p < 0.05). This result was confirmed by the significant enhancement of the DPPH scavenging ability for the biomass C.c. + N.o. compared to the other biomasses  and N.o. . The same trend was found for the carbohydrate content, two and four folds higher in C.c. than in S.m. (p < 0.01) and N.o. (p < 0.001), respectively (p > 0.05), with N.o. accounting for the greatest lipid contribution . Conversely, the lipid content decreased when N.o. was coupled with S.m. or C.c. compared to N.o. alone (39 or 52%).The macromolecular composition differed among the three species d–f. Protectively d–f. Lipiion 84%, d–f. Co-cion 84%, d–f. The p < 0.05) and the lowest TFC or TCC (p < 0.05) , carotenoids , polyphenols  and flavonoids  differed among the biomass obtained from the three cultivated species g–i. N.o. < 0.05) g–i.p < 0.01). Indeed, the common pigments in the three species, chlorophyll a and β-carotene, were significantly higher in C.c. biomass than in the two other species . TFC was also higher in C.c. biomass (p < 0.01) (C.c. exhibited a higher TCC than the two other species ( < 0.01) g–i, whil < 0.01) .p < 0.05, p < 0.05, −1 DW, respectively), while absent in mono-cultivated N.o. Conversely, no difference in diatoxanthin  nor in diadinoxanthin  between co-cultivation and mono-cultivation was revealed . However, the de-epoxidation state (DES = Dt/(Dt + Dd); [p < 0.05), both in mono- and co-cultures.Coupling N.o. with diatoms provided the biomass with the highest TSC (t + Dd); ) was sig−1 DW  and D2 were the most concentrated in all harvested biomasses from mono-cultivation  and only with S.m. for B2, B6, H, D2, E and A (at least p < 0.05). The lowest vitamin concentration  compared to C.c. alone. Generally, this co-cultivation enhanced the vitamin content (p < 0.01).The co-cultivation of C.c. + N.o. (the two species with the higher vitamin concentration reported from mono-culture) increased the concentration of the vitamins A, E, C, B content j–l, the p < 0.0001) at the highest concentration (100 µg mL−1) on both cancer and normal cell lines. The two lower concentrations (1 and 10 µg mL−1) exhibited different effects on the human cell lines. At the lowest concentration (1 µg mL−1), S.m. extract induced an antiproliferative effect on the colon-rectal adenocarcinoma cell line , with a cell viability lowering at 77% (p < 0.0005), while it did not affect normal prostate epithelium cell line (PNT2) viability. Differently, the lowest concentration (1 µg mL−1) of C.c. extract decreased the prostate cancer cell line  viability to 75% (p < 0.001) without compromising the PNT2 normal cell viability. As for C.c., N.o. extract (1 µg mL−1) displayed an antiproliferative effect on the PC3 cell line without affecting the PNT2 cell growth (p < 0.0001).The extracts from mono-cultures  displayep < 0.0001) (−1) of this extract decreased the viability of all cell lines (p < 0.0001), although no effect was reported with the two other concentrations . S.m. + N.o. extract inhibited the human cell line A2058 viability at a concentration of 100 µg ml−1 , although the antiproliferative effect against the other cell lines was lower compared to S.m. or N.o. extracts from mono-cultivation.Co-cultivation modified the antiproliferative capacity of the biomass extracts . At the  0.0001) d compare 0.0001) a. Moreov 0.0001) d. Regard1, H and B12 [This study provides a broad integration of biochemical properties and bioactivities of biomass harvested from microalgae cultivated alone and in co-culture, also comparing their growth performance. The findings highlight the benefits of microalgal co-cultivation as a tool to produce enriched biomass with emergent biological properties, enhancing biotechnological interests, without increasing cultivation costs. This might represent a way to improve the microalgal role as a resource for human-health-applied biotechnology. The main advantage offered by the co-cultivation strategy is its capacity to enhance the bioactivity interest of the biomass, with an enrichment in vitamins and other bioactive molecules and/or in improving the bioactive properties of the biomass. The augmentation of the vitamins content is extremely appealing, being these compounds crucial for human health ,44,45. A and B12 , their c and B12 ), sugges and B12 . This feSkeletonema marinoi and Cyclotella cryptica and the Eustigmatophyceae Nannochloropsis oceanica, are able to grow in couple without generating competition-related growth inhibition. Competition for resources, light or nutrients, does not significantly affect the co-existence of the species, as it occurs in situ, with the co-occurrence of many species in a small habitat , probabparadox” ,46,47. Tparadox” ). Indeedparadox” .Nannochloropsis [Cyclotella cryptica further increases the overall vitamin content together with the high phytosterols and carotenoids concentration. The increase in all vitamins reaches at least 10%, except for the vitamins C and H (5 and 2% lower), while carotenoids increase by 97% and sterols by 8%. The enrichment of the biochemical profile of N.o. + C.c. positively modulates the bioactive property of this mix, increasing its antioxidant capacity. Yet, the antiproliferative activity of N.o. + C.c. extract is enhanced when added at 100 µg mL−1 to the three human cancer cell lines, compared to the extracts from other cultures. Thishighlights the improved beneficial chemopreventive activities of the couple N.o. + C.c.. Vitamins or carotenoids—both enhanced in N.o. + C.c.—activities against cancer cells are well documented [12 or B6, among the others, are known to interfere with intracellular viral replication [The results obtained in our study confirms the vitamin richness of loropsis . Couplincumented ,51,52. Icumented ,54. Indecumented , the latcumented . The riclication ,58. Thislication , withoutlication ,61,62.C. cryptica and N. oceanica is enhanced by 10% when these two species are coupled, further increasing the antioxidant capacity of the biomass . The antioxidant property enhancement leads to a significant increase in the antiproliferative capacity against the three cancer cell lines tested. The synergy of the two diatoms C. cryptica and S. marinoi generates a biomass with improved chemopreventive activity, especially against the proliferation of the aggressive cancer prostate PC3 cells.This pioneer study opens a new route to produce microalgal biomass with an increased bioactive compounds richness, e.g., vitamins, or/and an improved the bioactive property. Coupling two species with alternative biochemical or nutraceutical profile can improve the yield of vitamins and the antioxidant or chemopreventive properties of the biomass. The high vitamin content detected in These results are worthy of interest, since the bioactivity benefit from co-cultivation does not add any cost compared to mono-algal cultivation. This study experimentally proposes a new microalgal cultivation strategy aiming to boost their application in eco-sustainable biotechnology and paves the way for further investigations to better define its strengths and limits towards potential up-scaling application.


In conclusion, this study indicates that the amounts of nutrients removed by modern soybean varieties were greater relative to the historical values recorded in Brazil, excluding Ca and S. Nonetheless, in the middle to long term (10 years), a significant impact of plant breeding on grain nutrient concentration was recorded only for N and Mg. The difference in nutrient removal patterns between Brazil and other countries indicates an integrated effect of management, genotype, and environment on nutrient removal. These findings provide guidance for optimal nutrient management and specific information for plant breeding programs to understand nutrient variability.Knowing the nutrient removal by soybean grain harvest in different varieties, locations, and over time is essential to correctly adjust agronomic recommendations, update farmers’ practices, and increase nutrient use efficiency. A field-based research trial was carried out to assess macronutrients  removed in grain by modern soybean varieties from southern Brazil introduced between 2007 and 2016. We examined changes between our set of modern varieties and a dataset of historical values encompassing a wide range of varieties introduced before 2007. Moreover, we undertook a synthesis analysis using scientific literature published after 2007 to investigate nutrient removal by grain among modern Brazilian soybeans and a dataset that included field trials from Argentina, United States, and India. There were no yield gains across the years for modern soybean varieties introduced among 2007 and 2016 in Brazil, although the grain N and Mg concentrations decreased. Modern Brazilian soybeans increased nutrient removal compared with that by soybeans historically planted in Brazil, with 11.1, 26.9, 45.0, and 31.6% more N, P, K, and Mg removed, respectively. Our results indicated that soybean growing in Brazil removed 4.3% less N relative to the values reported in the literature dataset, whereas K removal was 21.4% greater. A significant difference was also recorded for high-yield soybean varieties, and Brazilian varieties removed 11.8% less N and 8.6% more K than varieties in the literature dataset. No differences were found among locations for P removal, averaging 4.9 kg Mg Glycine max (L.) Merrill] is a major leguminous crop grown throughout the world. Processed soybean grain is the world’s largest source of protein for animals, and beans’ abundant stored lipids are also widely used as oilseed (Soybean [ oilseed  and as a oilseed . There i oilseed . However oilseed . Brazil –1 year–1) over the past 4 decades .–1.We used a randomized complete block design in the field with four replications and a set of 12 modern soybean varieties used in southern Brazil. The soybean varieties were grown in the 2017/2018 crop season at four different locations in the State of Paraná: Floresta (23°35′23″ S and 52°04′21″ W), Maringá (23°02′21″ S and 52°04′56″ W), Cambé (23°15′46″ S and 51°14′62″ W), and Apucarana (23°33′39″ S and 51°22′85″ W). The plots consisted of six rows that were 5 m long and had 0.45-m spacing and sown at a rate of 266,000 seeds haTo assess nutrient removal by soybean at harvest, we used 12 well-planted varieties in southern Brazil. The soybean varieties studied here were introduced between 2007 and 2016. The term “modern” was used following grouping adopted in a previous study , and morThe soils of the experimental areas were classified as typical Oxisols except for Maringá, which was classified as a typical Ultisol (USDA Soil Taxonomy). The climate was classified as subtropical humid mesothermal Cfa , charactBradyrhizobium japonicum at a concentration of 5.0 × 109 viable cells per mL g–1 and a seed rate of 2 mL kg–1. All the experimental areas were fertilized based on soil fertility characteristics  to obtain the yield results. Grain nutrient concentration and nutrient content were also expressed on a dry basis .2SO4 and subsequent distillation using the micro-Kjeldahl method. To obtain the total nutrient content for P, sulfur (S), calcium (Ca), Mg, and K of the soybean grains, digestion by nitric-perchloric (4:1) solution was performed. Total S was determined by applying the turbidimetry method with barium sulfate. Ca and Mg concentrations were determined using an Agilent® Microwave Plasma Atomic Emission Spectrometer (MP-AES). Total K and P were determined by using a flame photometer and metavanadate colorimetry, respectively. All the analyses were performed according to Soybean grain samples were individually ground to a powder in a Wiley mill with a stainless-steel blade and passed through a 40-mesh sieve. A total of 1,000 mg of milled grain was used to determine the exported nutrients. The N concentration was determined by means of complete digestion in concentrated HTo examine the validity of our findings, we compared our data with the literature. We compiled soybean studies established from 2007 to 2019 from field trials dealing with management practices, genotypes, and environmental effects. The year 2007 was chosen as the starting point considering that the soybean varieties in our study were introduced on dates ranging from 2007 to 2016. All the articles that reported studies of grain yield, grain moisture, and N, P, and K removal by grain harvest were included. The entire resultant database consisted of 313 plot-specific data from 16 datasets . The numThe search keywords used were “soybean,” “grain yield,” “nutrient uptake,” “nutrient removal,” “nitrogen,” “phosphorus,” and “potassium.” We searched the Web of Science Core Collection, Scopus, Springer Link, and Google Scholar citation databases. Grain yield and nutrient grain content were all adjusted to a dry basis.Most studies in the literature reported no data dispersion in terms of the variance, standard deviation, or standard error of their averages, making it difficult to perform a meta-analysis . Therefo–1) as a function of yield (Mg ha–1) in the current study. The grain nutrient content was calculated by multiplying the nutrient percentage by the yield on a dry basis. The nutrient values used in the historical model were 4.7, 0.45, 1.4, 0.23, 0.18, and 0.47 g 100 g–1 for N, P, K, Ca, Mg, and S, respectively.To describe the differences in grain nutrient removal between the modern varieties grown in our field trial and the historical values for varieties from southern Brazil introduced before 2007, a theoretical model for nutrient removal by grain harvest was estimated based on historical values for this region. A similar approach was taken by ® software. The pie chart showing relationships among nutrient concentrations was built using the CORRPLOT package in R software  was found. In accordance with previous studies (σ2 or R2) for the yield and grain nutrient relationships among models was determined by linear regression between the residuals of the grain nutrient concentration and grain nutrient content  in our experimental dataset, y is the overall mean of the yield or nutrient removal for the dataset of interest , and n is the number of observations of the present study (n = 192).where Σ is the sum, –1; Changes in nutrient removal were plotted against changes in grain yield only compared to the literature, as the amount of nutrient removal estimated for historical values was calculated using the yield of our study. In addition, to investigate the nutritional change patterns of high-yield soybean varieties , averaging 3.5 Mg ha–1 , with a  Mg ha–1 . A sligh Mg ha–1 . The obsectively . The amoectively . The ave–1) performed in this study. Our results indicated that N × P (r = 0.37∗∗∗), N × S (r = 0.21∗∗∗), K × Ca (r = 0.40∗∗∗), K × Mg (r = 0.37∗∗∗), Ca × Mg (r = 0.41∗∗), P × Mg, P × S, and S × Mg (r = 0.18∗∗∗) had positive correlations, whereas N × K (r = –0.37∗∗∗) and P × Ca (r = –0.41∗∗∗) were negatively correlated. Correlations among P × K, N × Ca, N × Mg, S × K, and S × Ca were not significantly different, indicating zero interaction.p < 0.05) among all the datasets (–1 grain), which included studies from Argentina, India, and the United States. The slope between accumulated N in grain and yield for our study (45.6 kg N Mg–1 grain) was greater than that of the model built from historical values, which included regional varieties introduced before 2007 (41 kg N Mg–1 grain). The box-and-whisker plots of the nutrients removed were created to display the distribution for each dataset, which also included a separate analysis for data points classified as high yield (>4 Mg ha–1). Details of the descriptive analysis are available in –1, respectively , resulting in P removal of 4.95 kg Mg–1 grain . Considering all the yield data, the total P removed in the study, historical, and literature datasets was 19, 15.9, and 16.4 kg ha–1, respectively  among the three datasets (–1 grain), followed by the literature dataset (17.9 kg K Mg–1 grain) and the model predicted using historical values (14.2 kg K Mg–1 grain). Overall, the total K removed from the study, historical, and literature datasets was 73, 50, and 59 kg ha–1, respectively  than that derived from historical values (2.3 kg Ca Mg–1 grain). The slope of Mg removal to yield was greater for this study than for the estimated model derived from the historical dataset (p < 0.05). The slopes were 2.37 and 1.80 kg Mg–1 grain for the data from this study and the historical dataset, respectively (p < 0.05) between datasets. Our study showed a lower slope (2.63 kg S Mg–1 grain) relative to the slope of the model built from historical values (4.7 kg S Mg–1 grain). The distributions of Ca, Mg, and S removal for all the yield data  was ∼25% above the average rain-fed on-farm soybean yields in Brazil  is consistent with the long-term studies by –1 year–1. Concerning Mg grain concentration, although previous studies also found a wide range of concentrations (0.17–0.34 g 100 g–1) due to varietal differences  N and more K (21.4%) than soybean varieties in the literature dataset, and more relevant differences were observed in high-yield soybean for N (–11.8%). No differences were found among locations for P removal. These results revealed significant variability in N and K removal due to location effect and suggest that soybean growing in Brazil can meet high-yield with less N relative to the other locations studied, such as Argentina, India, and the United States. Additional studies are needed to further elucidate how soil properties, management, genotype, and environment affect nutrient removal among different locations.The original contributions presented in the study are included in the article/ME, LL, RS, and SO designed the experiments and wrote the manuscript. MB, TI, and AB edited the manuscript. ME, LL, RS, and MB performed the experiments. ME and SO analyzed the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Information theory is a unifying mathematical theory to measure information content, which is key for research in cryptography, statistical physics, and quantum computing ,2,3. A cm random variables Y and Z, defined as:H the Shannon entropy [Nga et al. study a system of  entropy . The autThe general entropy correlation model proposed by the authors rely on two claims, both incorrect:Claim 1.In Equation (13) and Section 2.2.2, Nga et al. claim that higher-order correlations are bounded by pairwise correlations:Claim 2.In Equations (16) and (20), Nga et al. use Claim 1 to prove that, for any subset of m variable, its joint entropy with We propose two examples for Proposition 1.Consider the four i.i.d. discrete random variables Proof. As In our second example, we obtain Proposition 2.Consider three discrete random variables Proof. We have Overall, the two new inequalities derived by Nga et al. for the joint entropy Finally, deriving constraints and bounds on joint entropies is a computationally difficult task and an active field of research ,9,10,11.eorem 1, ). For ineorem 1, )) lower em 2.34, ):(2)H of 1,,3, by EqSimilar bounds can be obtained for quations  and 3)  m>3 usineorem 6, ). This georem 6, . Taken t


Scylla paramamosain), new exoskeleton, gills, and appendages are formed after a molting, which contributes to a 40 to 90% increase in body weight. However, little is currently known about the associations between molting and the dynamic changes of microbiota and physiological characteristics in mud crabs. In this study, the effects of molting on changes of the microbiome, immune response, and digestive enzyme activities in mud crabs were investigated. The results showed dynamic changes in the abundances and community compositions of crab-associated microbiota harboring the gills, subcuticular epidermis, hepatopancreas, midgut, and hemolymph during molting. Renewed microbiota was observed in the gills and midgut of crabs at the postmolt stages, which seems to be related to the formation of a new exoskeleton after the molting. A significant positive correlation between the expression of two antimicrobial peptide (AMP) genes (SpALF5 and SpCrustin) and the relative abundance of two predominant microorganisms  in hemolymph was observed in the whole molt cycle, suggesting that AMPs play a role in modulating hemolymph microbiota. Furthermore, digestive enzymes might play a vital role in the changes of microbiota harboring the hepatopancreas and midgut, which provide suitable conditions for restoring and reconstructing host-microbiome homeostasis during molting. In conclusion, this study confirms that molting affects host-associated microbiota and further sheds light on the effects on the immune response and the digestive systems as well.Molting is a crucial lifelong process in the growth, development, and reproduction of crustaceans. In mud crab  and the expression of AMP genes (SpALF5 and SpCrustin) was observed in the hemolymph of crabs during the whole molt cycle, suggesting the modulation of hemolymph microbes by AMPs. Furthermore, the digestive enzymes were found to participate in the regulation of microbiota in hepatopancreas and midgut, consequently providing a suitable condition for the restoration and reconstruction of host-microbiome homeostasis during the molting. This study confirms that molting affects the microbial communities and concomitantly influences the immune and digestive systems in mud crabs. This is also the first time the homeostasis of the host and microbiome, and the associations between molting and physiological characteristics in crustaceans, have been revealed. Scylla paramamosain) is a commercially important aquaculture species on the coast of southeast China, with its annual production reported to be above 160,000 tons in 2019 . , 9. Scylin 2019 –, 11. Thein 2019 –.Vibrio, Aeromonas, and Hematodinium, in the disease crabs compared with the healthy individuals  and 0, 3, 6, and 15 days postmolt. Changes in immunological parameters  and activity of digestive enzymes  were correspondingly investigated in the hemolymph and hepatopancreas of mud crabs. The distinct patterns of the host’s physiological characteristics and microbiota were observed at pre- and postmolting.P < 0.05; P < 0.05; Changes in the morphometric characteristics of crabs, including bodyweight, carapace length, and width, were measured at pre (Ds)- and postmolt (0 to 15 days) stages. The Ds crabs are those with a crack on their exoskeleton  and moltP < 0.05) (P < 0.05) (P > 0.05)  was higher in the hepatopancreas of crabs at 0 to 6 days than those at Ds  and F. T < 0.05)  and F. A < 0.05) . However > 0.05)  and F.6 cells/ml (15 days) (6 cells/ml) , and this value reduced to the basal level at 3 days and 15 days (P < 0.05), which remained at a low value of 0.13 mmol/liter at 15 days (The average number of hemocytes at the intermolt stage was 3.9 × 1015 days) . A signi 15 days . The tot 15 days .SpALF5 and SpCrustin, significantly increased by 3.3-fold and 8.5-fold at 0 days compared to Ds  and decreased to the basal level from 3 days to 15 days  Shannon, (B) Chao1, (C) observed species, (D) PD whole tree. G, gills. Se, subcuticular epidermis. Mi, midgut. Hemo, hemolymph. H, hepatopancreas. Sw, seawater. Ds, double-shelled crab. The numbers 0d, 3d, 6d, and 15d represent the sampled crabs at different days after a molt. The white dot indicates the median, and the box covers between the 25% and 75% quantiles. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00917-21.2FIG S2FIG S2, TIF file, 0.2 MB.Venn diagrams showing the shared bacterial groups in a specific crab tissue in a molt cycle (at genus level). G, gill. Se, subcuticular epidermis. Mi, midgut. Hemo, hemolymph. H, hepatopancreas. Ds, double-shelled crab. The numbers of 0, 3, 6, and 15 represent the sampled crabs at different days after a molt. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 8 cells/g), and the lowest was at 0 days (3.1 × 106 cells/g). There was a gradual increase from 0 days to 15 days, with the highest density of 3.4 × 107 cells/g at 15 days  (P < 0.05), whereas the relative abundances of Halomonas and Devosia significantly increased from Ds to 0 days (P < 0.05) before decreasing from 3 days to 15 days , subcuticular epidermis (B), midgut (C), hemolymph (D), and hepatopancreas (E) at the genus level. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 5 cells/g) and 6 days (6.5 × 105 cells/g) (6 cells/g) was found at 15 days, followed by 3 days (2.8 × 106 cells/g) and Ds (2.1 × 106 cells/g). The microbial diversity reached the highest level at Ds and maintained at the similar lower values between 0 days and 15 days  . The hig 15 days . In tota 15 days . Twenty-t 0 days B.7 cells/g; 3 days, 4.2 × 108 cells/g; 6 days, 3.6 × 108 cells/g; and 15 days, 4.2 × 108 cells/g), although this was lower than that at Ds (6.1 × 108 cells/g) (Rhodobacteraceae (at Ds), Vibrionales (at 3 days), and Micrococcaceae, Actinomycetales, and Actinobacteria (at 6 days)  and Photobacterium , were significantly decreased from Ds to 0 days and rapidly recovered at 15 days (P < 0.05). Fifty-three bacterial taxa were newly identified at 0 days, but 40 of them (such as Bradyrhizobium and Xanthobacter) disappeared in the postmolt stages  . A total 6 days) . The relt stages C. During10.1128/mSystems.00917-21.3FIG S3FIG S3, TIF file, 0.2 MB.LEfSe identifies the most differentially abundant bacterial groups in a molt cycle, including (A) gill, (B) subcuticular epidermis, (C) midgut, (D) hemolymph, and (E) hepatopancreas. The linear discriminant analysis (LDA) scores are computed based on their relative abundances in each sample. G, gill. Se, subcuticular epidermis. Mi, midgut. Hemo, hemolymph. H, hepatopancreas. Ds, double-shelled crab. The numbers 0, 3, 6, and 15 represent the crabs on different days after a molt. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 4 cells/ml at Ds, 1.8 × 104 cells/ml at 0 days, 2.2 × 104 cells/ml at 3 days, 1.3 × 104 cells/ml at 6 days, and 1.6 × 104 cells/ml at 15 days , Devosia , Photobacterium , and Shewanella  significantly decreased from Ds to 0 days. Some bacterial genera, such as Paracoccus, Sphingobacterium, Serratia, Arcobacter, Deinococcus, Alteromonas, and Phaeobacter, were detected throughout the molting cycle except for 3 days postmolt. Some bacterial taxa, such as unclassified Phyllobacteriaceae, Escherichia, Acinetobacter, Pseudomonas, Flavobacterium, Delftia, and Marinomonas, were significantly increased or newly detected after the molt (from Ds to 0 days)  D. The reHalomonas, Acinetobacter, Nesterenkonia, and Devosia) changed their relative abundances throughout the molt cycle  to 28.86% (at 0 days) or 28.86% (at 0 days) to 78.71% (at 3 days) (P < 0.05) (Pseudomonadaceae and Devosia showed a significant increase at 0 days compared with those at Ds (P < 0.05) (Sphingomonas (at Ds), Moraxellaceae (at 0 days), Halomonas (at 0 days), Devosia (at 0 days), Clostridiales (at 3 days), unclassified Caulobacteraceae (at 6 days), and Oceanospirillales (at 15 days)  E. The un < 0.05) E. Seven 15 days) . The rel10.1128/mSystems.00917-21.10TABLE S4Table S4, XLSX file, 2.0 MB.Microbial community compositions in different crab tissues in a molt cycle. Detailed values for Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the This study shows that various tissues of mud crabs harbored distinct relative abundance and communities of microorganisms, which confirms the hypothesis that host-associated microorganisms are tissue specific . BecauseFlavobacterium, Pseudoalteromonas, and Vibrio, decreased after molting, suggesting that molting plays an essential role in removing pathogenic bacteria from the gills of mud crabs. In mud crab, gills are responsible for gaseous exchange, osmolyte transport, nitrogenous excretion, acid-base balance, volume regulation between the body and external environment, and protection against infection and also act as a potential hematopoietic organ  but recovered at 3 days (4.2 × 108 cells/g) to reach levels that are similar to those at 15 days (4.2 × 108 cells/g) and Ds (6.1 × 108 cells/g), which could be attributed to the observation that crabs cease feeding during molting  in the midgut was observed, which might come from the surrounding seawater and feed  and environmental factors (foods and seawater), which finally maintain the homeostasis between crabs and their associated microbiota .SpALF5 and SpCrustin), phagocytosis (SpLAMP and SpRab5), agglutination (SpCTL-B), and melanization (SpproPO) in the hemolymph of mud crabs at 0 days compared with those at Ds, which indicates the role of molting in activating host immune response. Antimicrobial peptides (AMPs) are essential immune molecules that protect hosts against exogenous microorganisms. Hemocytes of healthy Pacific oysters could express basal levels of two AMPs (defensins and proline-rich peptides), which indicates that hemolymph microorganisms stimulate the generation of these AMPs to boost host immunity  correlated positively and significantly with the relative abundances of Halomonas and Shewanella during molting , two phagocytosis-related genes, SpLAMP and SpRab5, coupled with the increased ROS level, indicates an immune response by the crabs to invasion by the exogenous bacteria or proliferation of indigenous bacteria. This observation is probably explained by the important role of hemolymph in the host immune response. The microbiota colonizing the hemolymph are tightly controlled by host-microbiome homeostasis immunity  are capable of synthesizing digestive enzymes   –62. Here amylase . The resp shells . Methylohitinase . The bac molting , which i molting –67. CollIn conclusion, this study confirms the hypothesis that molting affects the microbiota in the tissues of mud crabs. Renewed microbiota was generated in gills and midgut after a molt, and dynamic changes of crab-associated microbiota are observed in a molt cycle. This study suggests that the molting would further alter the immune response and the digestive systems in mud crab as well. Future studies using metagenomics, metatranscriptomics, and/or culturomics are needed to identify the potential functions of the host-associated microorganisms during molting in mud crabs.The mud crabs used in this study were obtained from a local aquaculture farm in Xiamen, Fujian, China. The animals were processed according to the Regulations for the Administration of Affairs Concerning Experimental Animals established by Guangdong Provincial Department of Science and Technology on animal use and care. The experiments were approved by the Institutional Animal Care and Use Committee of Shantou University.Ruditapes philippinarum, Cipangopaludina cahayensis, and Sinonovacula constricta at a total amount of 10 to ∼20 g per day. During the culturing period, the average temperature (24.6°C), salinity (20.9 ‰), pH (7.9), and nitrate (35.4 mg/liter) and ammonium concentrations (0.05 mg/liter) in the seawater were relatively stable , which were healthy with no signs of disease, had complete appendages, and no material attachments on their body surfaces, were used. Before the experiments, crabs were cultured in a “crab house” aquaculture system equipped with a continuous recirculating seawater system. The seawater used was drawn from nearby natural seawater, which was then passed through a filter system containing activated carbon and silica sand, with the seawater’s daily exchange capacity maintained at 600 to 800% in volume. Crabs were individually kept in the units of the crab house (40 by 25 by 18 cm) to avoid fighting or being killed during molting. The crab house was cleaned once daily to remove crab feces and residual feeds. Crabs were fed daily with a mixture of able see . In this10.1128/mSystems.00917-21.4FIG S4FIG S4, TIF file, 2.1 MB.Crab molting and environmental conditions during the sampling period. The black vertical bar at the bottom indicates the number of molted crabs at a certain date. The orange bar indicates the sampling date of the double-shelled crabs. The temperature is measured once every hour. The salinity is measured once every day, and two sudden drops of the seawater salinity were due to rainfall events (October 10 and October 24). Average T., Max T., and Min T. are the average, highest, and lowest temperatures in a day. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSystems.00917-21.5FIG S5FIG S5, TIF file, 2.2 MB.Concentrations of nitrate, ammonium, and pH of the culturing seawater during the sampling period. Three replicate samples are measured for each point. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The body weight, carapace length, and width of each crab were measured at the beginning (when the crabs were introduced to the crab house) and end (the time of sample collection) of each experiment. The hepatopancreas index (HI) was calculated according to the formula hepatopancreas index (hi) = total hepatopancreas weight/bodyweight.2). Pieces of food  used for feeding the mud crabs were collected and cut into small pieces (<0.2 cm2). Tissue samples were quickly weighed, and 0.1 g of each sample was preserved in 500 μl of 1× phosphate-buffered saline (PBS)-ethanol  before being stored at −20°C until further analyses. One liter of culturing seawater was filtered through a sterile filter with a 0.22-μm mesh membrane  using a vacuum pump, and the microbial cells from the culturing seawater were collected by direct filtering of the water. Half of the membranes were stored at −20°C for DNA extraction, and the other half were fixed with sterile formalin solution for 2 h and stored at 4°C for cell counting.The sampling procedure was performed in a sterile laminar flow hood, with the surface of each crab disinfected and wiped three times with 75% (vol/vol) ethanol. Hemolymph was withdrawn via the arthropodal membrane  using a 1-ml disposable syringe into a tube containing precooled sterile anticoagulant buffer  at a ratio of 1:1. Two hundred microliters of the anticoagulant-hemolymph mixture was fixed with 4 ml of sterile formalin solution  for 2 h and stored at 4°C for cell enumeration (three replicates). The gills, subcuticular epidermis (a thin tissue that attached to the inner part of the shell), and midgut were collected into 2-ml sterile tubes with separate flame-sterilized scissors, which were then cut into pieces  for 20 min. After staining, the dye was removed and the membrane was immersed in 25 μl of 10% glycerin. Cells were counted at ×1,000 magnification using a fluorescence microscope  with a blue filter as described elsewhere , 69. EacThe total microbial DNA of hemolymph, gills, subcuticular epidermis, midgut, hepatopancreas, and crab foods were extracted using the PowerFecal DNA isolation kit  according to the manufacturer’s protocols. The hypervariable V3-V4 regions of the bacterial 16S rRNA genes were amplified by PCR using primers 341F and 806R . PCRs we10.1128/mSystems.00917-21.7TABLE S1Table S1, XLSX file, 0.01 MB.PCR primers used in this study. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the g for 20 min at 4°C to isolate hemocytes, followed by washing with 1× PBS and resuspended with 200 μl of 1× PBS containing 10 μM DCFH-DA. The mixture was incubated (protected from light) for 20 min at 37°C, after which the fluorescence intensity was measured at 488 and 525 nm using an Infinite 200 PRO NanoQuant microplate reader . Total antioxidant capacity was determined using a commercial test kit (number A015-2-1) according to the manufacturer's instructions . The optical density was measured at 405 nm using a microplate reader.Approximately 20 μl of hemolymph was placed in a hemocytometer for total hemocyte count using an inverted phase-contrast microscope . All hemocytes in both the top and bottom fields (1 by 1 mm) of the hemocytometer were counted. One milliliter of diluted hemolymph was used to measure ROS levels with a reactive oxygen species assay kit . Briefly, hemolymph was centrifuged at 800 × g for 10 min at 4°C. The supernatant was used to measure enzymatic activities of lipase, amylase, and trypsin using commercial test kits   according to the manufacturer's instructions. An increase of 0.001 per min is defined as one active unit (U). Chitinase activity was measured using an enzyme-linked immunosorbent assay kit  according to the manufacturer's instructions.The hepatopancreas of each crab was weighed and homogenized with 1× PBS before being centrifuged at 3,000 × SpALF5 and SpCrustin), phagocytosis (SpLAMP and SpRab5), agglutination (SpCTL-B), and melanization (SpproPO) . First-strand cDNAs were synthesized using the PrimeScript RT reagent kit with gDNA Eraser  according to the manufacturer’s instructions. The quantitative real-time PCR (RT-qPCR) analysis used the SYBR Premix Ex Taq II kit   by following the manufacturer’s instructions on a LightCycler 480 . The RT-qPCR total reaction volume was 20 μl containing 10 μl of SYBR Premix Ex Taq II, 2 μl of the 4-fold diluted cDNA, 0.8 μl (10 μM) each of the forward and reverse primers, and 6.4 μl of ultrapure water. The primers used for the six immune-related genes  are in The expression of six immune-related genes representing the four major types of innate immunity in mud crab were measured, including those of antimicrobial response (SpproPO) , 70–74. The raw sequence data analysis used a previously described procedure , 30. Aft10.1128/mSystems.00917-21.8TABLE S2Table S2, XLSX file, 0.01 MB.Number of bacterial 16S rRNA gene sequences used in this study. Download Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P value of <0.05. The LDA effect size (LEfSe) analysis was performed with an LDA score threshold of >2.0.The statistical significance between two samples of crab physiology, hepatopancreas digestive enzymes, hemolymph conditions, expression of immune-related genes, and microbial abundances was analyzed using the Mann-Whitney U test (SPSS 22.0). Metastat analysis with Fisher’s exact test was performed in R software (version 3.2.0)  to deterPRJNA640924.The 16S rRNA gene sequences of this study were deposited in the Sequence Read Archive of the National Center for Biotechnology Information (NCBI) under the accession number 10.1128/mSystems.00917-21.6FIG S6R. philippinarum, C. cahayensis, and S. constricta. Download FIG S6, TIF file, 0.5 MB.Microbial community compositions of the culturing seawater (A) and crab foods (B). The crab foods are the mixture of three animals, including Copyright © 2021 Zhang et al.2021Zhang et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the


Test–retest reliability for baseline conductivity was evaluated using the intraclass correlation coefficient. The baseline and post-exercise conductivity values (mean ± standard deviation) [S/m] of muscles, cartilages, and nerves were 1.73 ± 0.40 and 1.82 ± 0.50 (p = 0.048), 2.29 ± 0.47 and 2.51 ± 0.37 (p = 0.006), and 2.35 ± 0.57 and 2.36 ± 0.57 (p = 0.927), respectively. Intraclass correlation coefficient for the baseline conductivity of muscles, cartilages, and nerves were 0.89, 0.67, and 0.89, respectively. In conclusion, in vivo conductivity measurement of musculoskeletal tissues is feasible using MR-EPT. Conductivity of muscles and cartilages significantly changed with an overall increase after exercise.This study aimed to investigate whether in vivo MR-electrical properties tomography (MR-EPT) is feasible in musculoskeletal tissues by evaluating the conductivity of muscle, cartilage, and peripheral nerve around the knee joint, and to explore whether these measurements change after exercise. This prospective study was approved by the institutional review board. On February 2020, ten healthy volunteers provided written informed consent and underwent MRI of the right knee using a three-dimensional balanced steady-state free precession (bSSFP) sequence. To test the effect of loading, the subjects performed 60 squatting exercises after baseline MRI, immediately followed by post-exercise MRI with the same sequences. After reconstruction of conductivity map based on the bSSFP sequence, conductivity of muscles, cartilages, and nerves were measured. Measurements between the baseline and post-exercise MRI were compared using the paired  In biological tissues, it is closely related to water content, intracellular and extracellular fluid volumes, ion concentration, and the cellular membrane permeability, thus has been considered to be a fundamental biophysical property3. As several experimental studies have shown that conductivity can reflect pathologic status as well as tissue type7, it has emerged as a potentially important tissue biomarker for clinical diagnostics9.Electrical conductivity is an intrinsic property of a material that represents the capability to transfer electrical current inside the medium7, the first non-invasive in vivo assessment of conductivity was made using the electrical impedance tomography (EIT)10. Among multiple efforts to improve non-invasive conductivity imaging, MR-EIT provided higher spatial resolution and eliminated the need to solve the ill-posed inverse problem in EIT11. However, as it required injection of electrical current into the body, safety concerns were raised9. Another technique called magnetic induction tomography did not involve direct contact with the subject, nevertheless, this non-MRI-based method exhibited poor spatial resolution12.While previous investigations mostly relied on ex vivo measurements13, another non-invasive approach termed “MR-electrical properties tomography (MR-EPT)” has been introduced by Katscher et al.14. They reconstructed tissue conductivity from the measured B1+ field generated by the radiofrequency (RF) coil. It is considered to be advantageous regarding practicability, as it can be acquired using standard MRI system with a regular coil and does not apply externally mounted electrodes or current8. With increasing interest, there has been continuous progress in the research of MR-EPT to validate it15, prove its feasibility16, and estimate specific absorption rate (SAR)17. Whilst several clinical studies have investigated MR-EPT in various organs including breast19, brain20, and liver21, the number of studies that utilized MR-EPT in musculoskeletal tissues has been limited22.Based on the concept of deriving conductivity from the measured MR signalsThus, the purpose of this study was to investigate whether in vivo imaging is feasible in musculoskeletal tissues using MR-EPT by evaluating the conductivity of muscle, cartilage, and peripheral nerves around the knee joint. Under the hypothesis that conductivity could reflect physiologic changes, this study also sought to explore whether the conductivity of musculoskeletal tissues changes after exercise, and to interpret their changes with reference to other quantitative measurements including T2, T2* relaxation times (RT) and diffusion tensor imaging (DTI) parameters.This prospective study was approved by the institutional review board  and registered at cris.nih.go.kr (KCT0004839). After being advised of the purpose of the study, all subjects gave written informed consent. The study was conducted in accordance with the declaration of Helsinki.22, the mean and standard deviation of muscle conductivity using MR-EPT has been reported to be 0.93 S/m (Siemens per meter = Ω−1 m−1) and 0.26 S/m, respectively. The sample size (n) needed to construct a 95% confidence interval with a margin of error of 0.17 S/m was prospectively calculated using the following equation: n = 1.962 × SD2/d2, where SD and d are the standard deviation of muscle conductivity and the margin of error, respectively. Therefore, the sample size was calculated to be nine. Assuming dropout rate to be 10%, ten subjects were required.According to a previous studyTen healthy adults were recruited who had body mass index between 18.5 and 25.0, without any discomfort in their lower extremities, previous history of internal derangement, surgery in the right knee, or contraindication to MRI such as claustrophobia.8. Both magnitude and phase images were obtained in the axial plane. To evaluate the test–retest reliability of conductivity, all volunteers underwent two iterations of the bSSFP sequence. Thereafter, T2 map (T2-weighted multi-echo spin-echo sequence), T2* map (T2*-weighted multi-echo fast-field echo sequence with ∆B0 correction), and DTI (single-shot echo-planar imaging) in the axial planes were acquired. T2 and T2* maps were computed using a pixel-by-pixel basis, mono-exponential, non-negative least-square fitting algorithm. For DTI, diffusion gradients were applied in six directions and diffusion encoding was done with monopolar gradient pulses. Detailed MRI parameters are summarized in Table All volunteers underwent baseline imaging using a 3.0-T MRI scanner  and a 16-channel knee coil. For conductivity assessment, a three-dimensional (3D) balanced steady-state free precession (bSSFP)  sequence was chosen; this sequence was selected due to its relatively benign off-resonance behavior and robustness against unwanted phase contribution from flow, motion, and eddy current23. Standing with weight equally distributed and the feet separated shoulder width apart, they were asked to stand in a position of knee flexion at 90° bilaterally. Keeping the knees not moving past the toes, they held this position for 3 s, followed by repositioning to stand upright. They performed 60 repetitions under supervision by an observer to ensure identical exercise among the volunteers. After exercising, the volunteers were immediately repositioned on the MR table for post-exercise imaging using the same sequences and protocols as the baseline imaging, including two iterations of the bSSFP sequence. The first and second post-exercise bSSFP sequences were acquired 2 and 6 min after exercise cessation in all volunteers, respectively. Volunteers’ skin was marked under the guidance of integrated laser to maintain identical imaging position between the baseline and post-exercise imaging.To test the effect of loading, the volunteers performed squatting exercise outside the MRI scanner after baseline imaging as described in a previous literatureB1 magnitude) was used:ω = Larmor frequency, Δ = Laplacian operator, μ = magnetic permeability of tissue , and Φ+  = RF transmit phase estimated as half of the measured transceive phase Φ± 8. Care has to be taken that Φ± does not show any phase wraps when applying this division by two. However, since the total phase span across the field of view has been roughly 60° for the experiments in this study, no dedicated phase unwrapping was needed. The second derivative required for Δ was performed numerically by fitting a local parabola to a number N of voxels (“kernel”) on each side of the respective target voxel of Φ±. A maximum number of N was chosen as Nmax = 10 and reduced as soon as the bSSFP magnitude of a kernel voxel deviates more than 10% from the target voxel, providing a suitable trade-off between too low signal-to-noise ratio  and decreased image resolution (for too large N) and avoids the typical boundary artifacts of EPT. As the Laplacian operator inevitably leads to noise amplification, a bilateral median filter was applied to the conductivity resulting from those equations, again applying the locally shaped kernel as done for the differentiation step. This approach has successfully been applied in previous studies for brain tumor conductivity24 and similarly also for breast tumor conductivity25.After data acquisition, all images were transferred to an independent workstation for analysis with manufacturer-supplied software . The conductivity maps were reconstructed from the phase images of the 3D bSSFP sequence. To convert the phase information to conductivity (σ), the following equation , semimembranosus muscles (SM), and biceps femoris muscle, including both long and short heads (BF) were selected because they had sufficient cross-sectional area. The ROIs were placed on each muscle so that the boundaries were within 1–2 mm from the muscle fascia.Cartilages: Axial planes that revealed the maximum thickness of the patellar and trochlear cartilages were selected for evaluation. The ROIs were placed to cover the entire cartilage, encompassing both deep and superficial cartilage, and medial and lateral facets; joint fluid or subchondral bone were carefully avoided.Nerve: At the same axial plane as that for muscle analysis, the ROIs were carefully drawn to fall within the visualized boundary of the tibial nerve, excluding the perineural vessels.Two board-certified radiologists  analyzed the MRIs. Among T2-weighted images with six different echo times, one was selected and regarded as anatomical reference that best differentiated subchondral bone, articular cartilage, and joint fluid from the cartilage. Using the manufacturer-supplied software , conductivities of muscles, cartilages, and nerves were measured by placing regions of interests (ROIs) on the magnitude image of the 3D bSSFP sequence using freehand drawing tools according to the following criteria  was used for image processing and ROI placement on the T2, T2* maps, and DTI using the same criteria and anatomical reference images. The mean T2 and T2* RTs, fractional anisotropy (FA), and mean diffusivity (MD) of the same muscles, cartilages, and nerves were measured at the corresponding areas.t-test was used to explore if the post-exercise values varied from the baseline imaging. Test–retest reliability between conductivities obtained from the 1st and 2nd baseline bSSFP sequences by reader I and interobserver agreement between the two readers were assessed using the intraclass correlation coefficient (ICC). An ICC of 1.0 was considered to represent perfect agreement; 0.81–0.99, almost perfect agreement; 0.61–0.80, substantial agreement; 0.41–0.60, moderate agreement; 0.21–0.40, fair agreement; and 0.20 or less, slight agreement26. All statistical analyses were performed using MedCalc version 19.0.7 . p values less than 0.05 were considered to indicate statistical significance.Paired The subjects constituted of five men and five women volunteers, ranging in age from 24 to 30 years (26.8 ± 1.8 years). The patellar and trochlear cartilages in two volunteers, were excluded on second baseline imaging due to severe artifacts. The duration of exercise ranged from 3 min 56 s to 7 min 51 s (6 min 11 s ± 1 min 17 s).Because there was almost perfect interobserver agreement on measurements of conductivity in muscles, cartilages, and nerves, data obtained by one of the readers were used. The result of the test–retest reliability of the baseline conductivity was almost perfect in all muscles and the tibial nerve. Cartilages tended to show lower test–retest reliability, which were regarded as substantial Table .Table 2TThe range of baseline conductivity was as follows: all muscles, 1.03–2.58 S/m; VM muscle, 1.03–1.95 S/m; SM muscle, 1.15–2.48 S/m; BF muscle, 1.45–2.58 S/m; all cartilages, 1.12–2.98 S/m; patellar cartilage, 1.11–2.80 S/m; trochlear cartilage, 1.51–2.98 S/m; tibial nerve, 1.47–3.00 S/m.p = 0.048), + 9.05 ± 11.07% (p = 0.029), and + 9.49 ± 13.69% (p = 0.006), respectively [mean ± standard deviation], with an overall increase from the baseline; conductivity of the VM muscle, patellar, and trochlear cartilages tended to increase on the second post-exercise imaging, which changed by + 8.59 ± 16.12% (p = 0.126), + 8.84 ± 4.13% (p = 0.061), and + 10.12 ± 4.71% (p = 0.060), respectively , + 19.89 ± 8.37% (p < 0.001), + 5.98 ± 6.11% (p = 0.013), and + 14.89 ± 4.42% (p < 0.001), respectively, with an overall increase compared to the baseline. In contrast, the BF muscle showed overall post-exercise decrease of the T2 RT, T2* RT, and MD, which changed by − 3.82 ± 4.39% (p = 0.022), − 3.79 ± 4.66% (p = 0.030), and − 3.59 ± 2.93% (p = 0.004) from the baseline, respectively. Regarding all muscles, overall post-exercise increase was observed in the T2* RT and FA, which changed by + 4.34 ± 11.97% (p = 0.018) and + 3.52 ± 6.72% (p = 0.012) from the baseline, respectively. No other significant differences in the T2 RT, T2* RT, FA, and MD were noted between the baseline and post-exercise imaging (Table After exercise, the T2 RT, T2* RT, FA, and MD of the VM muscle changed by + 17.54 ± 11.10% (This study measured in vivo conductivity of muscles, cartilages, and nerves around the knee joint in normal volunteers using MR-EPT and showed its feasibility. Additionally, this study revealed that post-exercise conductivity significantly changed with a tendency to increase in muscles and cartilages at 6 min after exercise cessation.29, probably due to various species and study conditions. In human subjects, muscle conductivity has been reported to be 0.62 S/m30 and 0.93 S/m22. In ex vivo studies, conductivity of cartilages and nerves were reported to be 0.51–1.14 S/m31 and 0.39 S/m29, respectively. Though different experimental conditions make direct comparison difficult, the conductivity values of our study were higher than those of previous investigations.In previous animal studies, results of muscle conductivity assessment observed a large variation ranging between 0.56 and 1.05 S/m20, maps in this study were suspected to suffer from B1 inhomogeneity. The bSSFP sequence is known to have relative robustness against B0 inhomogeneity, as long as banding artifacts from signal voids are avoided, and in fact B0 inhomogeneities are too small to produce those banding artifacts in the knee images of this study; yet, conductivity reconstruction is vulnerable to B1 inhomogeneity8. Conductivity reconstruction as applied in this study is based on B1 phase only, i.e., based on the assumption of a constant B1 magnitude. It has been reported that a violation of this assumption leads to an artificial increase of reconstructed conductivity, contributing to the observed high conductivity values32. A phantom simulation as described in the B1 magnitude to exclude this artificial conductivity increase. Nevertheless, as B1 inhomogeneity is similar for all scans in this study, it can be expected that the observed exercise-induced increase of conductivity is not affected by this issue. Furthermore, this study adopted the so-called “transceive phase assumption”, estimating the absolute B1+ phase Φ+ to be half of the transceive phase Φ± as the B1+ phase is not directly measurable by standard MR sequences33. Even though it has been widely used, violation against this assumption is thought to lower the accuracy of conductivity evaluation. Phase error has been reported to be larger at the peripheral regions indicating that the merit of this assumption deteriorates34. In addition, geometric asymmetry may lead to unequal contributions of the transmission and reception process in the total transceive phase33. It can also be prone to inaccuracies when imaging subjects with asymmetrical electrical properties35. In this context, substantial phase error might have been produced in this study. As extremities are hardly scanned in the isocenter area and are inherently asymmetric with right- or left-sided predilection, this phase error could be regarded as one of the major problems to be resolved for implementing MR-EPT in musculoskeletal imaging. Given that the previous study22 from which we estimated the sample size examined pelvic region, wide range of conductivity with relatively large SD in our study also might be explained by aforementioned reasons. Despite of previous study that validated MR-EPT15, our study suggests that it may be vulnerable to reconstruction error in musculoskeletal regions requiring further validation.Compared to the conductivity maps in previous studies on MR-EPT37. Osmotic shift, water accumulation, increase in volume of intracellular space of perfused muscle and intracellular acidification increases T2 RT40, whereas blood inflow exceeding oxygen extraction fraction increases T2* RT41. For DTI parameters, tightening of muscle fibers and changes in intra- and extracellular volume, pseudodiffusion due to increased blood volume are suggested to increase FA and MD, respectively42. In contrast, the post-exercise T2, T2* RT, and MD of the BF muscle decreased in this study, contrary to the VM muscle. As squatting elicits greater activity of quadriceps relative to other muscles43, decreased T2, T2* RT, and MD may imply compensatory vasoconstriction in the BF muscle.The post-exercise T2 and T2* RTs, FA, and MD significantly increased in the VM muscle, which was comparable to the findings of previous studies44, increased conductivity could not be explained by increased water content as T2 RT were decreased in the BF muscle and not changed in the cartilages. Thus, it could be reasonable to presume that increased conductivity may reflect increased tissue sodium concentration, which probably occurs several minutes after the exercise cessation rather than immediate post-exercise period. However, considering that the duration of exercise varied between the volunteers and that there was a difference in the pattern of change in conductivity after exercise as shown in Fig. On the other hand, conductivity tended to increase in both VM and BF muscles and cartilages on the second post-exercise MRI compared to the baseline. While conductivity is affected by both water content and tissue sodium concentration46. Whereas sodium MRI is still challenging due to limitations in hardware and software, MR-EPT is advantageous as it uses standard MR scanners. Considering usefulness of sodium MRI in evaluating early cartilage degeneration47, it could be reasonable to consider cartilage imaging as one of the promising applications of MR-EPT in the musculoskeletal regions. However, conductivity of the SM muscle did not show tendency of post-exercise increase. Although we presumed that no significant physiologic change occurred in the SM muscle after exercise considering all the other parameters were also not changed, the basis of this result remains unclear. Furthermore, the fact that the differences between baseline and post-exercise conductivity in muscles and cartilages were significant only when they were grouped except for the BF muscle requires future investigation to elucidate its true significance. In addition to the technical issues regarding B1 inhomogeneity, relatively low test–retest reliability in the cartilage possibly due to their small size and curved shape is another hurdle that should be overcome.Our result showing changes of conductivity in muscle is partially comparable to those of previous studies reporting signal intensity increase of muscle after exercise using sodium MRI48, change of temperature after exercise may have affected our study results.This study had several limitations. First, the MR-EPT technique was not validated in the musculoskeletal regions. Validation using a knee-sized phantom and conductivity probe could help proving its reliability in musculoskeletal imaging. Second, substantial inhomogeneity was noted in the conductivity maps with regions of increased conductivity, as described above. Further optimization would be mandatory, which may provide conductivity values closer to the values reported in previous investigations. Third, the time intervals between exercise cessation and acquisition of each sequence for quantitative measurements were variable. The duration of exercise was also variable among the volunteers, which also may have influenced the study results particularly in post-exercise changes of conductivity. Fourth, misalignment of knee joints between the baseline and post-exercise scans could have been possible. Intelligent software which automatically plans the scanning geometries would be helpful, in addition to our effort using skin marking and integrated laser. Finally, temperature measurements of target tissues were not performed, assuming them to be 37 °C for both baseline and post-exercise MRI. As temperature coefficient of conductivity variation is about 2%/°CIn conclusion, in vivo conductivity measurement was feasible in musculoskeletal tissues using MR-EPT around the knee joint. Although technological hurdles should be overcome that might result in artificial increase of conductivity values and relatively poor reproducibility of cartilage conductivity, significant post-exercise change in conductivity may suggest its potential as a biomarker to reflect physiologic status of musculoskeletal tissues. Further clinical studies with validation based on this initial experience of MR-EPT would be mandatory to elucidate true significance of conductivity in musculoskeletal imaging and to expand its applications particularly to indirect assessment of tissue sodium concentration.Supplementary Information.


The findings of this study provide evidence of how the level of physical fitness (neuromuscular and aerobic) relates to physical performance parameters recorded in official competitions.The aim of this study was to analyze the relationships between the level of physical fitness and the physical performance parameters recorded by GPS in official FIFA matches of the Chilean women’s senior national football team in the period 2018–2020. Twenty-six female field players (age (mean ± SD) 26.8 ± 3.3 years, height 157.8 ± 21.5 cm, weight 58.9 ± 4.9 kg) participated in the study. Physical fitness variables were assessed: muscular strength, countermovement jump (CMJ), speed, agility and aerobic fitness. Physical performance variables were recorded by GPS in 26 official FIFA matches. The most notable associations with significant statistical significance ( Football is the most played sport in the world, and although it has traditionally been played mainly by men, it is now played by both men and women ,2. The p−1, 3735 m; 7–13 km·h−1, 3856 m; 13–19 km·h−1, 2168 m; 19–23 km·h−1, 495 m; and >23 km·h−1, 180 m [For example, the distance covered at different speeds at the Women’s World Cup in France 2019 has been recorded. The average total distance covered was 10,435 m, distributed in the following speed ranges: 0–7 km·h(r = −0.30 to 0.78) [There is no consensus in the literature on the relationship between the fitness level and performance in official matches. On the one hand, the aerobic fitness field test (level 1 recovery Yo-Yo) and neuromuscular performance tests have been found to be associated with physical performance variables obtained in matches such as total distance covered, distance covered at a high intensity and distance covered at a very high intensity ,12. Linkto 0.78) . In thisto 0.78) . In contto 0.78) ,14. TherThis is a quantitative, cross-sectional, descriptive, correlational and descriptive research work.n = 26; goalkeepers were not included). It was conducted in official FIFA and France 2019 World Cup matches in the period 2018–2020. The age (mean ± SD) was 26.8 ± 3.3 years, height was 157.8 ± 21.5 cm, weight was 58.9 ± 4.9 kg, muscle percentage was 48.28 ± 2.08% and fat percentage was 23.21 ± 2.03%. All the participants had played in a senior category in different leagues or professional competitions at the national level in different countries . They had 7 ± 2 years of experience in official competitions. At the time of the evaluations, the Chilean national team was ranked 36th out of 155 according to the FIFA women’s world ranking.Female outfield players from the Chilean national football team participated in this study . Day 2, running speed, time to run 10 m (T10) and time to run 30 m (T30). Day 3, countermovement jump (CMJ) and Illinois agility time (IAT). Day 4, Yo-Yo intermittent recovery test level 1 (YYIRL1).These tests are commonly performed by women’s football teams ,18,19,20This test has been validated to assess muscle strength levels in lower limbs in football players ,22. BefoThis test is regularly performed to measure the lower extremity power of female football players ,23. PrioThis test is frequently used to assess acceleration and maximal running speed capacity in football players ,25 Figu. Prior tThis test is often used to assess agility and changes of direction in football players ,27. PrioThis test is commonly used to assess intermittent endurance capacity in football players ,31,32,33−1 in meters (HSR), number of sprints at >18 km·h−1 (N°S), peak speed in km·h−1 (PS), meters per minute (M/M) and number of accelerations >2 m·s−2 (N°AC) were recorded. These data are often used by coaches and sport scientists to monitor loads and observe performance in training and official matches in women’s football [The total distance (TD) in meters, high-speed run >18 km·hfootball ,37,38,39The study was conducted according to the guidelines of the Declaration of Helsinki  and approved by the Institutional Ethics Committee of University Hospitals Virgen Macarena and Virgen del Rocío from Seville, Spain . In addition, all the participants had a medical examination prior to the start of the season. All the participants underwent the tests without any injury or physical discomfort, and informed consent was obtained from all subjects involved in the study.r), according to normality, to study the level of association between physical fitness and physical performance variables in official matches. The r values were interpreted as trivial (0.00–0.09), small (0.10–0.29), moderate (0.30–0.49), large (0.50–0.69), very large (0.70–0.89), almost perfect (0.90–0.99) and perfect (1.0) [p ≤ 0.05. Statistical analysis was carried out using SPSS IBM software, Version 22 .For the descriptive analysis, the mean and standard deviation per playing position of the variables analyzed were calculated. The intraclass correlation coefficient (ICC) and coefficient of variation (CV) were found to analyze the relative and absolute reliability of variables that were measured more than twice. The Shapiro–Wilk test was performed to analyze whether the variables follow a normal distribution. Finally, correlations were calculated using Pearson or Spearman (ct (1.0) . The 95%The aim of this study was to analyze the relationships between the level of physical fitness and physical performance parameters recorded in official FIFA matches of the Chilean women’s senior national football team in the period 2018–2020.r = −0.398), and RS, T10, T30, CMJ and IAT were observed to be statistically significantly associated with N°AC . These results are in agreement with those reported by Rago et al. [r = 0.40) and with N°AC (r = 0.57). Similar associations were found between 5-m sprints and the N°AC in small-sided games and in regulation dimensions . These results might suggest that players who are able to produce a lot of force per unit time in the explosive tests of sprinting (5 m sprint) and jumping (CMJ) will be able to perform more high-intensity accelerations in training and in football matches. The findings of Rago et al. [Significant relationships were found between neuromuscular performance-oriented fitness variables and physical performance indicators in official matches : a signio et al. , who invo et al.  coincideo et al. , and it o et al. . Therefor = 0.490, 0.390, 0.491 and 0.589, respectively). These results are in agreement with those reported by Rago et al. [r = 0.47) and full-size football (r = 0.57) and also to HSR in full-size football (r = 0.46). On the other hand, a negative correlation was observed with N°AC . Something similar was reported by Aquino et al. [r = 0.76). Krustrup et al. [r = 0.56) and HSR (0.76). These findings suggest that the YYIRL1 test has a high sensitivity to the ability to run longer distances and at a higher intensity in official football matches, irrespective of the level or sex of the football players. For example, young football players who performed better on the YYIRL1 test covered a greater total distance (r = 0.65) and ran more at higher intensities (r = 0.77) [−1; R2 = 42%) and sprints  [R2 = 28%), high-intensity running  and total high-intensity activities . In professional female football players, YYIRL1 scores were related to total distance covered  and distance covered in high-intensity running during matches  [On the other hand, the aerobic performance-oriented fitness variable YYIRL1  showed ro et al.  who inveo et al.  studied p et al. , who ass = 0.77) . Specifi2 = 57%) . In male2 = 57%)  reported2 = 69%) .Finally, the findings presented in our study demonstrate strong associations between YYIRL1 and distances covered in official matches. Furthermore, YYIRL1 is one of the few field tests that demonstrates a similarity in the external load represented by distance run per minute (M/M) in official matches. For example, taking our records, the mean duration of YYIRL1 is 12 min and the mean distance covered is 1436 m. Therefore, the estimated work rate is 118 M/M , and thaThe main findings of this study provide evidence on how the level of physical fitness (neuromuscular and aerobic) is related to physical performance parameters recorded in official matches using GPS devices. On the one hand, neuromuscular fitness indicators could be good predictors of N°AC in official matches, particularly performance in the T10 and CMJ tests, explaining more variance in the number of high-intensity accelerations in official competitions. On the other hand, the intermittent endurance capacity obtained in the YYIRL1 test is a good predictor of performance in total distance covered (TD) and distances covered at a high speed in elite women’s football matches. These findings therefore suggest that analyzing the level of physical fitness by means of field tests could provide relevant information to select players with a greater aptitude for elite competition.From the results obtained, we can conclude that there is a moderate relationship between 1RM and TD, and RS, T10, T30, CMJ and IAT exhibit moderate relationships with N°AC. On the one hand, neuromuscular fitness indicators could explain the performance of N°AC in official matches and in particular of the performance in T10 and CMJ tests, according to a greater variance in the number of high-intensity accelerations in official competitions. In addition, the intermittent endurance capacity obtained in the YYIRL1 test may be a good parameter of performance in total distance run (TD) and distances run at high intensity in elite women’s soccer matches. These results suggest, therefore, that the analysis of the level of physical fitness through field tests can be a useful indicator to identify those players who present a better sporting form to compete in high performance; however, by obtaining moderate relationships, they do not allow prediction of performance, considering also that field tests cannot be used as predictors of performance due to the complex and multifactorial nature of the competition , but theField evaluations represent an interesting approach to assess the physical fitness of athletes and thus identify which ones are in a better sporting shape to withstand the demands of high-performance competition. The results of our study demonstrate that fitness levels (neuromuscular and aerobic) are associated with measures of competitive performance.