Agastache rugosa are used as a food material and traditional medicine in Asia. A 50% ethanol extract exhibited potent xanthine oxidase (XO) inhibitory activity (IC50 = 32.4 µg/mL). To investigate the major components responsible for this effect, seven known compounds were identified from A. rugosa; among these, salvianolic acid B (2) was isolated from this plant for the first time. Moreover, acacetin (7) exhibited the most potent inhibitory activity with an IC50 value of 0.58 µM, lower than that of allopurinol (IC50 = 4.2 µM), which is commonly used as a XO inhibitor. Comparative activity screening revealed that the C6-bonded monosaccharides (3) or sugars substituted with acetyl or malonyl groups (4–6) are critical for XO inhibition when converted to aglycone (7). The most potent inhibitor (7) in the A. rugosa extract (ARE) exhibited mixed-type inhibition kinetics and reversible inhibition toward XO. Furthermore, the hydrolysis of ARE almost converted to an inhibitor (7), which displayed the highest efficacy; UPLC-qTof MS revealed an increased content, up to five times more compared with that before treatment. This study will contribute to the enhancement in the industrial value of ARE hydrolysates as a functional ingredient and natural drug toward the management of hyperuricemia and treatment of gout.The aerial parts of  Agastache rugosa (Fisch. and C.A.Mey.) Kuntze.The main causes of hyperuricemia are the over-production of uric acid, insufficient excretion in the urine, or a combination of both ,2. When A. rugosa is a perennial plant belonging to the Lamiales order Lamiaceae family; it is known to form a colony and has high growth power even in barren mountain fields − . 1H NMR and 13C NMR data, see Salvianolic acid B (2). Amorphous white powder; [α]20D +94.0 ; λmax (MeOH) 254, 287, 329 nm; HR-ESI-MS, m/z 717.1453 [M–H]− . 1H NMR  δH 3.10-2.95 , 4.40 , 5.11–5.20 , 5.83 , 6.29 , 6.46 , 6.57-6.65 , 6.76 , 7.17 , 7.68 . 13C NMR  δC 172.4 (C-9″), 170.1 (C-10″), 168.1 (C-9‴), 166.9 (C-10‴), 148.9 (C-8), 146.5 (C-7), 146.4 , 145.9 , 145.1 , 133.7 (C-1′), 129.2 (C-1″), 128.7 (C-1‴), 126.2 (C-9), 124.7 (C-4), 144.1 (C-12‴), 121.9 , 118.3 , 117.5 , 116.3 , 113.2 (C-2′), 88.8 (C-2), 74.4 (C-8″), 74.4 (C-8″), 57.2 (C-3), 37.8 (C-7‴), and 37.2 (C-7″).Tilianin (3). White-yellowish powder; λmax (MeOH) 267, 332 nm; HR-ESI-MS, m/z 447.1286 [M+H]+ . 1H NMR and 13C NMR data, see Acacetin 7-O--β-D-glucoside (4). Yellowish amorphous powder; λmax (MeOH) 267, 332 nm; HR-ESI-MS, m/z 533.1293 [M+H]+ . 1H NMR and 13C NMR data, see Acacetin 7-O-(2″-O-acetyl)-β-D-glucoside (5). Yellowish crystalline powder; λmax (MeOH) 267, 332 nm; HR-ESI-MS, m/z 489.1394 [M+H]+ . 1H NMR and 13C NMR data, see Acacetin 7-O--β-D-glucoside (6). Pale-brown amorphous powder; λmax (MeOH) 267, 332 nm; HR-ESI-MS, m/z 575.1399 [M+H]+ . 1H NMR and 13C NMR data, see Acacetin (7). White-yellowish powder; λmax (MeOH) 267, 332 nm; HR-ESI-MS, m/z 285.0755 [M+H]+ . 1H NMR and 13C NMR data, see 2O containing 0.1% FA; B: ACN containing 0.1% FA). The linear gradient elution was optimized as follows: 0 min, 10% B; 0–1 min, 10% B; 1–8 min, 10–40% B; 8–11 min, 40–90% B; 11–11.3 min, 90–100% B; 11.3–13.3 min, 100% B; 13.3–13.4 min, 100–10% B; and 13.4–15 min, return to initial elution condition of 10% B. The qTof MS was operated in two different ion modes (negative and positive) under the following conditions: cone voltage 30 V, capillary voltage 2.5 kV, source temperature 110 °C, and desolvation temperature 400 °C. A sprayer with a calibration (lock MS) standard solution of leucine-enkephalin  was used. The UV chromatograms were obtained using Empower 2, and full scan MS data and MS2 spectra were collected using the MassLynx software.The identification of the main component peaks was performed by a UPLC-coupled PDA detector. An amount of 2.0 μL of each sample was injected into a BEH C18 column  at a flow rate of 0.4 mL (per min) and eluted using a chromatographic gradient of 2 mobile phases (A: H1–7) for evaluation of inhibitory effects were dissolved in DMSO at 4 mM and further diluted at various concentrations. First, 125 µL of 100 mM sodium pyrophosphate buffer , 30 µL of 0.1 units XO enzyme, 5 µL of test samples, and 40 µL of the substrate (0.5 mM xanthine) were mixed. The reaction mixture (200 µL) in a 300 µL well plate was incubated at 37 °C and the UV absorbance at 295 nm was screened every 1 min for 15 min. The inhibition mechanism and constants  of a binding site for the inhibitor to a free enzyme (KI) or an enzyme–substrate complex (KIS) were determined using the Lineweaver–Burk and Dixon plots + ion at m/z 285.0755  in the ESI-HR-MS analysis using qTof MS. The 13C-NMR data enabled carbons corresponding to seven C–C double bonds and one carbonyl group to be identified, thus accounting for 8 of the 11 degrees of unsaturation. The extra three degrees of unsaturation were ascribed to three aromatic rings. In the 1H-NMR spectrum, the presence of seven aromatic protons indicated a trisubstituted typical flavonoid skeleton. Four proton values for ring B protons appeared in the form of two sets of doublets (J = 8.8 Hz) at δ 8.02  and 7.10  with their corresponding carbons at 128.3 and 114.6 ppm, respectively. Thus, 7 was identified as acacetin.As a result of separation and purification through repeated CC, seven XO inhibitory phenolic compounds  and their derivatives because they induce a very strong effect on the XO inhibitory activity according to our study.an brain . Further1–7 were evaluated for their inhibitory effects on XO activity and their inhibitory mechanism was derived through kinetic analysis. Phenolic acid derivatives (1 and 2), flavone glycosides , and flavone aglycone (7) had a concentration-dependent effect (50: >25 μM) except for acacetin (7), a flavonoid aglycone (All isolated compounds t effect A. Nile eaglycone .7 exhibited considerably higher inhibitory activity (75.5%) at the same concentration of 5 μM compared with apigenin (67.2%) and allopurinol (51.9%), which were used as positive controls. Acacetin (7) (IC50 = 0.58 µM), which is more effective than apigenin (IC50 = 0.87 µM) and is known to have the highest inhibitory activity among flavonoids, was revealed for the first time. It appears that inhibition was higher when (7) has a methoxy group instead of a hydroxyl group at the 4′-position. These results are in agreement with those reported by Liu, in which some flavonoids have higher inhibitory activity when they are substituted with methoxy groups −; 2, m/z 717.1453 [M–H]−; 3, m/z 447.1286 [M+H]+; 4, m/z 533.1293 [M+H]+; 5, m/z 489.1394 [M+H]+; 6, m/z 575.1399 [M+H]+; 7, m/z 285.0755 [M+H]+). The standard curve demonstrated a highly validated correlation coefficient (r2 > 0.998) and was linear and reproducible. As a result of the quantitative analysis, it was confirmed that the contents of 1–7 in the extracted 50% EtOH were 4.05 mg/g, 0.06 mg/g, 9.04 mg/g, 5.12 mg/g, 0.37 mg/g, 0.45 mg/g, and 1.96 mg/g per dry sample, respectively. tR), UV absorption maxima (λmax), and MS spectral data, including molecular ion and elemental composition of the peaks (1–7) [7), which has the highest inhibitory activity on XO, is accumulated as a monosaccharide glycoside (3) or mostly substituted with units of malonyl-glc (4), acetyl-glc (5), or acetyl-malonyl-glc (6). Thus, to hydrolyze glycoside compounds (3–6) to aglycone 7  and the content increased by more than 5 times compared with that before treatment (1.96 mg/g per dry sample). According to Kim et al., the acacetin (7) content increased to 3 mg/g per dry sample level when ARE was treated with lactic acid bacteria [7 content caused by bacterial treatment indicates that the selective hydrolysis of only the glucose moiety with the β form or its complete conversion to an aglycone is difficult. In addition, regarding the industrial use of the hydrolysis extract of 50% ARE, more research is needed involving acid hydrolysis in addition to treatment with enzymes or bacteria because acid hydrolysis is more competitive in terms of manufacturing cost.To increase the industrial applicability of the ARE, the extraction conditions were optimized and the content of potent XO inhibitor (ks (1–7) ,20. A PDlycone 7 B, 50% AR7) with a low micromolar inhibition (IC50 = 0.58 μM) against XO. The evaluation of the detailed kinetic analysis using double-reciprocal plots revealed that 7 exhibited a mixed-type inhibition (Ki = 0.61 μM). The index/active ingredient information was provided, and rapid analysis methods for standardization and quality control of the ARE were conducted. In addition, this work presents the possibility of developing a material that maximizes the active ingredient through hydrolysis; we expect that the ARE can be industrially used as a natural drug for the management of hyperuricemia and treatment of gout.The ARE exhibited a significant inhibitory effect on XO in vitro. Importantly, it was revealed for the first time that the most potent inhibitor was acacetin (


Multi–site imaging consortiums strive to increase participant numbers by pooling data across sites, but scanner related differences can bias results. This study combines data from three research MRI centers, including three different scanner models from two vendors, to examine non–harmonized T1–weighted brain imaging protocols in two cohorts. First, 23 human traveling phantoms were scanned twice each at all three sites  to quantify within–participant variability of brain volumes , and to calculate site–specific correction factors for each structure. Sample size calculations were used to determine the number of traveling phantoms needed to achieve effect sizes for observed differences to help guide future studies. Next, cross–sectional lifespan volume trajectories were examined in 856 healthy participants (5—91 years of age) scanned at these sites. Cross–sectional trajectories of volume versus age for each structure were then compared before and after application of traveling phantom based site–specific correction factors, as well as correction using the open–source method ComBat. Although small systematic differences between sites were observed in the traveling phantom analysis, correction for site using either method had little impact on the lifespan trajectories. Only white matter had small but significant differences in the intercept parameter after ComBat correction (but not traveling phantom based correction), while no other fits differed. This suggests that age–related changes over the lifespan outweigh systematic differences between scanners for volumetric analysis. This work will help guide pooling of multisite datasets as well as meta–analyses of data from non–harmonized protocols. Multi–site imaging consortiums are becoming an increasingly common strategy to increase power through large participant numbers and to improve generalizability of patient studies. These collaborations have advanced our understanding of numerous disorders, e.g., Alzheimer's disease (ADNI) , adolescMany studies attempt to prospectively minimize this variability by harmonizing scan protocols across sites . This reduces but does not eliminate variability, which inevitably still stems from hardware and software differences  . Within–This study examines within–participant reliability of brain volumes across 23 traveling human phantoms, scanned twice at each of three sites , for a total of 138 scans. These data are used to (i) quantify systematic differences in brain volumes between three sites, (ii) determine the number of traveling phantoms needed to detect these site–specific effects; and (iii) determine if correcting for these differences (using two methods) changes the lifespan trajectory of brain volumes assessed in a cross–sectional cohort of 856 healthy individuals (5—91 years of age) scanned across these three sites. Traveling phantom based corrections of lifespan data is compared to correction using the open–source data harmonization method ComBat , in orden = 534, Site 2: ACH n = 52, Site 3: FMC n = 270; This study includes two datasets: (i) a “traveling phantom” cohort and ii) a self–reported healthy lifespan cohort. The traveling phantom dataset consists of 23 healthy adults  who were scanned twice at each at the University of Alberta (UofA), Alberta Children's Hospital (ACH), and Foothills Medical Center (FMC) for a total of 138 scans . The hea3, TE/TR/TI = 2.4/1800/900 ms, 3:39 min at Site 1 – UofA; (ii) 3T GE MR750W with 32 channel coil, axial IR–SPGR, 0.8 mm isotropic, TE/TR/TI = 3.2/8.2/600 ms, 5:38 min at Site 2 – ACH; and (iii) 3T GE MR750 with a 12–channel coil, sagittal IR–SPGR, 1 mm isotropic, TE/TR/TI = 2.6/6.3/650 ms, 5:44 min at Site 3— FMC  3T Siemens Prisma with 64 channel coil, sagittal MPRAGE, 0.87 × 0.87 × 0.85 mme 3— FMC . Table 2All images  were processed in volBrain  to yieldn = 23) was chosen to well exceed the typical number used in past literature, e.g., n = 2—10  using partial eta squared values from RM–ANOVA to determine the number of traveling human phantoms needed to detect observed effects of site on volume measurements for each structure.The percent difference between consecutive within–participant scans at each site was calculated as a measure of the test-retest reliability of each scanner. Reliability of volume measurements across sites was assessed with intraclass correlation coefficients (ICC) and Repeated Measures ANOVA (with pairwise comparisons between sites), after averaging each participants' consecutive scans within a site. Coefficient of variation (CV) within participants across all three sites, and between participants at each site were also calculated. Given that the number of traveling human phantoms included in this study .Site–specific traveling phantom–based correction factors (TP–correction) were calculated by using the participant mean volume for each site divided by the grand mean for that participant . This percent difference was then averaged across all 23 participants per structure for each site and used to correct the absolute volumes of the healthy development cohort for systematic site difference using a ratio multiplier. For example, if one site was found to have an average of 2% higher volumes for a given structure compared to the grand mean, then the individual volumes for that structure for all scans from that site were multiplied by 1/1.02. As an alternative to traveling–phantom based correction, raw lifespan data was also corrected using ComBat  which emZ statistics. In addition, correlations between volumes given by TP–correction vs. ComBat were tested with Pearson's correlations. For the purposes of this manuscript, volumes of males and females were combined into a single fit vs. age.Lifespan volume trajectories in the main dataset were estimated with linear, quadratic, cubic and exponential curves; significant curves with the lowest AIC values were considered the best fit , calculan = 267, age 5—90 with the same age distribution as the total sample) in addition to all of the scans from Site 2 ACH (n = 52) and Site 3 FMC (n = 270) for a total of 589 participants. Z–tests were used to test for differences in fit parameters between raw data, TP–corrected and ComBat corrected data in this subset. Note that ComBat correction was run separately in this cohort given that ComBat cannot be run ad–hoc on single subjects.Given that 62% of the lifespan sample was scanned at UofA (thus dominating the fits), a post–hoc analysis was performed to repeat curve fitting in a subset of participants that included only half of the sample from Site 1 UofA  was <1% for all structures at all sites, except lateral ventricle volume (1%) and white matter volume (1.6%) at Site 3. Within–participant CVs across all three sites ranged from 1.2—4.5% across structures . In all The first analyses here are on the uncorrected raw volumes vs. age   along with the full sample from the other two sites revealed that with the exception of the caudate and putamen, all structures fit the same trajectories in the subsample (n = 589) as in the full sample (n = 856)  . Differe– 0.998)  which lin = 856) . The caun = 856) . In thisn = 856) .This study demonstrated small systematic variations of brain volume (within–participant differences of 1—5%) across three 3T MRI scanners (two vendors – Siemens and GE with two different models), measured in 23 healthy adult traveling phantoms. ICC values ranged from 0.873 (white matter) to 0.992 , suggesting excellent overall consistency between scanners. These values are on par or higher than ICC values reported in past volumetric traveling phantom studies , 5, 19. via automated detection of structural boundaries). Image quality differences may also vary by site based on scanning procedures, an observation that may be particularly true for younger and older participants where motion may be more common , pulse sequence software and image contrast, and analysis pipelines . Absolute volumes can be assessed with calibrated resolution phantoms such as used in ADNI , howeverThese results hold promise for the feasibility of multi–site meta–analysis studies of volume based on automated segmentation of 3D T1–weighted images at 3T to advance our understanding of brain development, aging and disease through pooling of very large participant numbers.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Health Research Ethics Boards of the University of Alberta and University of Calgary. Written informed consent to participate in this study was provided by the participant, or their parent/legal guardian for participants under the age of 18.ST contributed to study design, data collection, statistical analysis, created figures, and wrote the manuscript. ES, JR, CM, and MB carried out data collection and processing. CM, CL, RF, DE, and CB designed the study, oversaw all aspects of data collection and analysis, and edited the manuscript. All authors contributed to the article and approved the submitted version.Operating funds for the University of Alberta cohort were provided by the University of Alberta Hospital Foundation, Women's and Children's Health Research Institute, and Canadian Institutes for Health Research. Data collection at the Alberta Children's Hospital was funded by the Natural Sciences and Engineering Research Council. Data collection at the Foothills Medical Center was supported by Canadian Institutes of Health Research and the Seaman Family MR Research Center. Salary support was provided by the Canada Research Chairs Program (CB and CL) and the Hopewell Professorship (RF).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to characterize the microbial community which colonizes these niches and to determine the influence of the pre-treatment of raw-domestic wastewater on these communities. This was achieved through a detailed sampling campaign of two OWTSs. At each site, the STU areas were split whereby half received effluent directly from septic tanks, and half received more highly treated effluents from packaged aerobic treatment systems . Effluents from the RBC had a higher level of pre-treatment [~90% Total Organic Carbon (TOC) removal], compared to the media filter . A total of 92 samples were obtained from both STU locations and characterized by 16S rRNA gene sequencing analysis. The fully treated effluent from the RBC resulted in greater microbial community richness and diversity within the STUs compared to the STUs receiving partially treated effluents. The microbial community structure found within the STU receiving fully treated effluents was significantly different from its septic tank, primary effluent counterpart. Moreover, the distance along each STU appears to have a greater impact on the community structure than the depth in each STU. Our findings highlight the spatial variability of diversity, Phylum- and Genus-level taxa, and functional groups within the STUs, which supports the assumption that specialized biomes develop around the application of effluents under different degrees of treatment and distance from the source. This research indicates that the application of pre-treated effluents infers significant changes in the microbial community structure, which in turn has important implications for the functionality of the STU, and consequently the potential risks to public health and the environment.The growth of microbial mats or “ A typical on-site wastewater treatment system (OWTS) uses a septic tank to provide primary treatment and a limited amount of anaerobic digestion. Further treatment occurs as effluent percolates through the soil-stone matrix of the soil treatment unit (STU) which can vary in their configuration according to site specific design requirements Gill, . The undMore recently, there has been a proliferation of packaged treatment systems that provide additional (secondary) treatment to the effluent before being discharged to the STU. Regardless of using a secondary treatment unit, the STU remains a crucial component of domestic wastewater treatment, particularly, the development of the biomat or “clogging layer,” which grows over the base of the STUs correlated to the level of organic and nutrient loading Bouma, . This laE. coli, enterococci, and bacteriophages as surrogates for human enteric viruses , bulk organics , and fecal indicator bacteria such as Several factors impact the microbial community structure within a wastewater treatment system, including influent composition, environmental conditions, system processes, plant configuration, and operational parameters  , and four percolation trenches, two of which were fed directly from the septic tank system as the primary effluent (PE), and the other two percolation trenches were fed with the secondary treated effluent (SE) from packaged treatment systems, as shown in At selected locations, the soil was excavated from the surface down into the percolation trenches to the gravel subsoil interface at the base of the trenches. Core samples were then taken with a 25.4-mm stainless steel corer. At each site and for each system, a single sample was taken in each of the three different depths from this infiltrative interface  , for eacThe microbial community was assessed by next-generation amplicon sequencing of the 16S rRNA in a paired end mode. DNA extracts were sequenced with an Illumina MiSeq platform  using the primer set F515/R806 targeting 294 bp of the V4 region of the bacterial 16S rRNA gene, as described by Kozich et al. .Pair end reads were converted into exact amplicon sequence variant libraries produced using the DADA2 pipeline package with the R program  was assessed by computing the total number of Operational Taxonomic Units (OTUs), abundance-based coverage estimator (ACE) , nitrite-N (NO2-N), and ammonium–N (NH4-N) and phosphorus as ortho-phosphate (PO4-P) using a Konelab 20i chemistry analyser . Assessing the spatial distribution of the volumetric water content (VWC) and long-term changes in water retention within the STUs was achieved by using a network of 80 and 92 soil moisture sensors , which have been installed during the construction of Sites A and B, respectively. Sensors were installed by auguring a 10-cm diameter hole to a desired depth, with sensors positioned into undisturbed subsoil at a required depth below the STU. Control sensors were installed outside of the STU area at the corresponding depths to those below the STU. Calibrations were performed according to the manufacturer's instructions using site-specific subsoils retrieved from test holes excavated prior to the construction of the sites. All sensor data were collected every hour and stored on a CR1000 data logger with two AM15/32 multiplexers .Both research sites were fitted with automated weather stations  measuring air temperature, relative humidity, atmospheric pressure, net radiation, wind speed and direction, and rainfall. Hydraulic loadings were determined using calibrated tipping buckets (described previously). A network of suction lysimeters  was installed at each site spaced longitudinally along each trench and at three different depths beneath the infiltrative surface down to 50 cm depth in the soil. For sample collection, a suction of 50 kPa was applied using a vacuum-pressure hand pump and samples were collected 24 h later. The effluent and porewater samples extracted from lysimeters were stored on ice for <6 h of transport to be analyzed in the environmental engineering laboratory at Trinity College Dublin. The organic loadings of the PE and SE fed into the STUs were determined as chemical oxygen demand (COD) using dichromate digestion test kits  and the total organic carbon (TOC) was determined using a Shimadzu TOC-V analyzer . Nitrogen species were analyzed as nitrate-nitrogen  under accession number Sites A and B received a mean annual rainfall of 928.6 and 972.4 mm, at mean temperatures of 10.0 and 10.4°C, with low values of −7.2 and −7.4°C and high values of 30.8 and 30.3°C, respectively, over the study period. At the time of sampling in August 2018, the total monthly precipitation was 41.9 and 17.6 mm, at mean temperatures of 15.7 and 15.6°C for Sites A and B, respectively. Detailed trend graphs of total monthly precipitation, monthly air temperatures, and monthly actual evapotranspiration for both sites can be found in the The average quality of the effluent from the septic tanks and packaged treatment plants that were feeding the percolation trenches is shown in The two systems at Sites A and B have been in operation for ~35 and 29 months, respectively, when the soil samples from the percolation area were taken. An intensive research study has characterized the performance of the OSWTSs in terms of the three-dimensional attenuation of contaminants as they passed down through the soil, and the effluent quality of the upstream treatment units (septic tanks and secondary treatment units)—as detailed by Knappe et al. . The in-3 cm−3 above the baseline value over a period of 30 days. The biomat position has extended horizontally for 15 m from the inlet for PE-STUs at Sites A and B. Site A exhibited a faster growth rate, with the biomat reaching 15 m in 10 months, and Site B reaching that length in 13 months. Growth was more arrested at the SE-STU with the biomat horizontally extending by 7.5 m and 10 m for Sites A and B after 3 years in operation, respectively. Vertical growth of the biofilm appears to be limited to approximately 5 cm from the infiltrative surface.Research performed by Knappe et al.  revealedAcidobacteriota, Actinobacteriota, and Proteobacteria , and for Site B, Acidobacteriota, Proteobacteria, and Chloroflexi  as described in Proteobacteria, Acidobacteria, and Chloroflexi for PE Site A , PE Site B , and SE Site A , respectively. In Site B SE-STU, Proteobacteria, Firmicutes, and Bacteroidetes were the dominant phyla with abundances of 39.29 ± 1.75, 19.89 ± 4.49, and 9.84 ± 1.56, respectively. Gammabacteria consisted of the majority abundances of Proteobacteria across all STUs  to 220,139 (max). Read depth was rarefied to the approximate average value of 35,000 reads per sample. The most abundant phyla in control subsoils for Site A were all STUs .Acidobacteriota (Site A: 3.6 ± 0.54 and Site B: 2.7 ± 0.42), Actinobacteria (Site A: 1.15 ± 0.77 and Site B: 0.51 ± 0.16), and Chloroflexi (Site A: 2.02 ± 2.08 and Site B1: 29 ± 0.09) as shown in The top 10 most abundant species-level taxa were selected for and compared across compartments within the systems at both sites . The conMetanosarcina sp. and Thauera sp. genus were also high with abundances of 3.1 and 5.98, respectively. In the SE fed STUs, Mycobacterium sp. (Site A: 0.78 ± 0.12 and Site B: 0.39 ± 0.06) was the most abundant species within both sites, followed by Pirellula sp. (1.08 ± 0.3) in Site A and Bacillus (0.8 ± 0.21) in Site B. The community profiles for topsoil samples did not vary largely between the samples at both sites and effluent types. At Site A, the phylum Acidobacteriota and Actinobacterium abundances were 22.03 ± 5.84 and 12.58 ± 6.41 in the PE-TT, respectively, and abundances in the SE-TT were 17.89 ± 0.69 and 11.14 ± 2.01, respectively. At Site B, the Acidobacteriota and Actinobacterium abundances were 17.27 ± 2.53 and 10.67 ± 1.45 in the PE-TT, respectively, and abundances in the SE-TT were 15 ± 7.89 and 15.71 ± 9.41, respectively.In PE effluent samples, Firmicutes was the most abundant genus, with abundances of 0.91 and 3.33 for Sites A and B, respectively. In the Site B PE samples, Actinobacteriota species sequences at 1 m along the PE fed trench were higher by an average of 2% with relative read abundance when compared to the control subsoil sample  along the STU compared to the relative sequence read abundances found within the control subsoil. In the PE-STU, at Site A, there was an average drop of 3% in the read abundance relative species belonging to the Acidobacteriota phylum at 1 m along the trench and an average of 1% at a 12-m sampling point. The parallel SE fed STU showed a 1% reduction across the sampled length of the trench. There was an average drop of 1–2% of Firmicute Bacillales and Verrumicrobiota Chtoniobacterales species sequences in both PE and SE-STUs.The greatest variance within the microbial community structure was determined from the STU base samples; changes in genus abundances were observed with respect to the distance from the inlet to the trenches and the relative read abundance within the subsoil control sample. At Site A, l sample . For theMethanosarcina sp., 3–4% in Synergistota species, and 9% in Proteobacteria Thauera sp. within the SE-STU relative to the abundances found within the control subsoil, as shown in Methanosarcina sp., Romboutsia sp., and Thauera sp.In Site B, there was an increase of 2% for Firmicute species sampled at 5–12 m from the head of the trench. The highest increase, in Site B, is observed for the Firmicute species at 2 and 7% for the PE and SE fed STUs, respectively. There were 3-10% increases in p = 0.03, Wilcoxon test), but there was no overall significant difference in diversity . There was no significant difference in the diversity or richness between the control soil samples . The Site A SE-STU was significantly more rich in species  and diverse  than that of Site B. Statistical analysis of alpha diversity and species richness  and species richness (Chao1) between all compartments across both sites, Site A was significantly richer in species . There were significant differences in the microbial community composition between the topsoil control samples and the subsoil samples , and statistically significant differences between the STUs of the same effluent type when compared between the sites . Permutational analysis of variance applying distance matrices results indicated that there was no significant difference between the control subsoil communities across sites  group which was determined from the existing literature. Relative read abundance was measured and compared across sites, STUs, and environmental compartments , as shown in The STUs dosed with Primary effluents, Denitrifying bacteria, Polyphosphate-Accumulating Organisms (PAO), Nitrite Oxidizing Bacteria (NOB), Methanogens, Glycogen Accumulating Organisms, Ammonia Oxidizing Bacteria (AOB), AMO and Acetogen functional groups showed higher relative abundances when compared to the control subsoil sample . There wFor the SE fed STU samples, all functional groups  showed an increase in relative read abundance with respect to the control subsoil sample. The only notable reductions were the Denitrifiers and NOB functional groups in Site B, with reductions of 0.58–77 and 0.47–0.73 respectively, lower than the abundances found within the subsoil control samples . In the sat values measured at Site A. Variations in richness and species diversity in PE-STUs were less significant and can be attributed to soil pore size and average loading rates which has also been known to impact the growth rate of the biofilm . Two research sites were selected due to their proximity of each other, being in the same climate region and having the same the soil type. Both sites are classified as typical Luvisol soils, averaging at pH 8 with Little known variance  and both sites also employ similar land management practices   bacteria e et al.  who confe et al. . The pree et al. . These re et al. . Increase et al. . Howevere et al. . Our stue et al. . This suThis study presents the first direct microbial comparative analysis between STUs for on-site wastewater treatment systems receiving domestic effluents with different levels of pre-treatment. This analysis has been conducted in STUs which have already been successfully characterized for soil clogging, under the same environmental, hydrological, and subsoil conditions.The microbial community richness and diversity within the STU system were significantly affected by the level of pre-treatment of wastewater. This outcome appears to follow the productivity diversity relationship theory that, over time, the addition of organics to these systems results in low diversity communities. The effect is not linear and the addition of a steady flow of lower concentrations of organics and nutrients may be more selective for copiotrophic bacteria than for the raw effluent.The STU receiving the fully pre-treated effluent contained the highest relative abundance of Denitrifying bacteria, however the increases in functional richness may not indicate the attenuating capacity of the system. Attenuation appears to be linked to the extent of the coverage of the STU by the biomat. This suggests that the increases in functional richness within the STU are secondary to bioclogging, as metabolic rates could be limited by hydraulic conductivity.The community structure or beta diversity of the STUs was significantly impacted by the level of pre-treatment and the horizontal distance downstream of the inlet. Depth did not significantly impact community structure in any of the STUs.This study has effectively profiled two developed and stunted biomats within the STUS of two separate research sites. More temporal data are required to assess the development of these communities in the field. In doing so, it will be possible to establish the critical points of transition for the STUs, profiling communities at different stages of the growth of the biomat which will provide researchers with in-depth understanding of the biological clogging process and how to manage it.PRJNA794316.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI BioProject—JK and LG devised the study design. Fieldwork was performed by JK, CS, CB, and LG. Lab work was performed by AC and JK and coordinated by TC and LG. Data analysis was performed by AC under the guidance of JK, CB, MA, and LG. The first draft was written by AC. All authors contributed to the revision of the manuscript and approved the submission of the manuscript.The funding for the study was provided by the Irish Research Council (IRC) as a Starting Laureate Award (Grant Number IRCLA/2017/246). The funding source had no role in the design and execution of this study, analysis, and interpretation of the data, or decision to submit results.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


N-arylpyrrole and TZD. The resultant derivatives at different concentrations were screened against key enzymes of glucose metabolism and glucose utilisation in the liver (HEP-G2) cell line. Additionally, peroxisome proliferator-activated receptor-γ activation was performed through docking studies. Docking of these molecules against PPAR-γ predicted strong binding, similar to that of rosiglitazone. Hence, TZDD2 was able to increase glucose uptake in the liver cells as compared to the control. The enzymatic inhibition assays showed a relative inhibition activity; with all four derivatives exhibiting ≥ 50% inhibition activity in the α-amylase inhibition assay and a concentration dependent activity in the α-glucosidase inhibition assay. All four derivatives exhibited ≥30% inhibition in the aldose reductase inhibition assay, except TZDD1 at 10 µg/mL. Interestingly, TZDD3 showed a decreasing inhibition activity. In the dipeptidyl peptidase–4 inhibition assay, TZDD2 and TZDD4 exhibited ≥20% inhibition activity.Diabetes mellitus (DM) and related complications continue to exert a significant burden on health care systems globally. Although conventional pharmacological therapies are beneficial in the management of this metabolic condition, it is still necessary to seek novel potential molecules for its management. On this basis, we have synthesised and evaluated the anti-diabetic properties of four novel thiazolidinedione (TZD)-derivatives. The TZD derivatives were synthesised through the pharmacophore hybridisation strategy based on  Diabetes mellitus prevalence continues to show an upward trajectory, exerting a serious cost burden on healthcare systems all over the world ,2,3,4,5.Type 2 diabetes treatment options are increasingly expensive, not readily available, and frequently have numerous side-effects such as hypoglycemia, diarrhoea, weight gain, and abnormal liver function ,6. HenceN-heterocyclic amines as an appendant on the N-3 of the TZD scaffold since several compounds containing these structural scaffolds are known to exert biological activity. Earlier studies have shown that TZD-derived hybrids with an N-functionalized side chain on the N-3 nitrogen TZD ring display improved activity, and such modifications exhibit low toxicity towards primary cultured non-malignant human hepatocytes +, calcd. for C22H24N3O3S, 410.1538.A yellow solid ; m.p. 256–258 °C; Rf  0.25; IR νmax/cm−1: 3381 (OH), 1720 , 1655 ,1587 , 1498 (C=C); δH : 7.87 , 7.52–7.44 , 7.18 , 6.20 , 4.28 , 2.14 , 2.02 ; δC : 168.5, 167.3, 166.1, 137.5, 136.9, 132.3, 129.7 (2C), 129.0, 128.9, 128.0 (2C), 115.7, 112.7, 105.5, 44.3, 12.9, 11.2; HRMS: m/z (ESI) found 357.0913 [M + H]+, calcd. for C18H17N2O4S, 357.0909.A yellow solid ; m.p. 118–120 °C; RThe α-amylase inhibitory activity was determined using the method described by the Worthington Enzyme Manual with slight modifications ,29. The Acontrol − Asample)/Acontrol) × 100%, where Acontrol was the absorbance of the control (without the inhibitor); Asample was the absorbance in the presence of TZD derivatives.Inhibition (%) = . The Ki values were calculated with GraphPad Prism 9.2.0. (332) by plotting the reciprocal of maximum velocity (1/Vmax) (y-axis) against the derivative concentrations (x-axis). The types of inhibition parameters were all calculated with GraphPad Prism 9.2.0. Briefly, 30 µL of α-glucosidase enzyme (0.5 IU/mL) was dissolved in the 0.02 M phosphate buffer (pH 6.9), and then was pre-incubated at 37 °C with the above-mentioned derivative (50 µL) in a 96-well plate, for 5 min. In sequence, pNPG  was added and incubated in the reaction mixture at 37 °C for 30 min.The mode of inhibition of the biological enzyme α-glucosidase, by the most potent derivative (TZDD3) in this study, was determined using the Michaelis-Menten and Lineweaver-Burk plots to determine the kinetic constants, as well as plots . The inhThe aldose reductase (AR) inhibition assay was performed according to the method described by Kazeem et al. with minor modifications ,32. All control − ∆Abssample)/∆Abscontrol] × 100%, where ∆Abs is the change is absorbance.% Inhibition =  × 100%, where ∆Abs is the change is absorbance.% Inhibition = [ product information (Sigma-Aldrich.com) with minor modifications to the volumes to suit the 96-well plate assay.3 flask under tissue culture conditions. Upon confluency (80%), cells were trypsinized and seeded into the 96-well plates. Upon confluency, the cell preparations were exposed to different concentrations  of TZDDs, and a media glucose reading was taken before incubation (time = 0). Thereafter, the plates containing cells and treatments were incubated for 24 h under tissue culture conditions. The non-treated cells and insulin treated cells served as absolute and positive controls, respectively. After a 24 h incubation, the medium glucose was taken using a glucometer [HEPG-2 liver cells were cultured in MEME supplemented with pen/strep (1%) and FBS (10%) in a 75 cmucometer . The perGlucose uptake (%) = (T24/T0) × 100To determine the cell viability, the acid phosphatase assay method was followed . BrieflyCell viability (%) = (Ab compound/Ab control) × 100https://www.rcsb.org/) [The 3D crystal structure of peroxisome proliferator-activated receptor gamma (PPAR-γ) was downloaded from the Protein Data Bank (PDB) (sb.org/) . The prohttps://proteins.plus/) and the Protein-Ligand Interaction Profiler (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) to determine the binding conformations [The grid boxes included the entire binding site of this protein and provided enough space for the ligand’s translational and rotational walk. For each ligand, a docking experiment consisting of 50 stimulations was performed, and the analysis was based on binding free energies and root mean square deviation (RMSD) values. The ligand molecules were then ranked in order of increasing docking energies. Results were analysed using binding energy as well as ProteinPlus analysis tools software (rmations . No furtp < 0.05 level. Each experiment was carried out in triplicates and repeated twice. Statistical analysis of results from each experiment was conducted using GraphPad Prism 9.2.0. Further statistical analysis was performed using a one-way ANOVA followed by a Tukey-Kramer post hoc test used to test the significance between a test compound and the absolute control. The statistical analysis was performed using absorbance or fluorescence readings before any normalization. The statistical significance was acknowledged at the Taken together, our compounds show moderate activity in several targets for diabetes management. These observations may suggest that TZZDs can exert antidiabetic activity by inhibiting or activating various key targets in DM management, which could be of paramount therapeutic value. For these reasons, further investigations, including in vivo studies, are critical moving forward to gain holistic therapeutic insights.


This study thus indicated that skating on synthetic ice alters the skating patterns such that the on-ice distinctive features can no longer differentiate between low- and high-calibre skating joint angles. This wearable technology has the potential to help skating coaches keep track of the players’ progress by assessing the skaters’ performance, wheresoever.Hockey skating objective assessment can help coaches detect players’ performance drop early and avoid fatigue-induced injuries. This study aimed to calculate and experimentally validate the 3D angles of lower limb joints of hockey skaters obtained by inertial measurement units and explore the effectiveness of the on-ice distinctive features measured using these wearable sensors in differentiating low- and high-calibre skaters. Twelve able-bodied individuals, six high-calibre and six low-calibre skaters, were recruited to skate forward on a synthetic ice surface. Five IMUs were placed on their dominant leg and pelvis. The 3D lower-limb joint angles were obtained by IMUs and experimentally validated against those obtained by a motion capture system with a maximum root mean square error of 5 deg. Additionally, among twelve joint angle-based distinctive features identified in other on-ice studies, only three were significantly different ( One of the key components of ice hockey players’ skills is skating ,2. Like Wearable technologies such as GPS and accelerometers measure essential kinematics in team sports ,16,17,18Biomechanical variances between high- and low-calibre players’ skating have been emphasized to understand the relationship between skating biomechanics and players’ performance. Previous research has highlighted substantial differences between groups of players with different skill levels in ice experiments ,9,19,24.While most public ice rinks were closed due to COVID-19, synthetic ice showed to be an alternative for skaters to exercise and be prepared for competitions and assists coaches in monitoring their players remotely. Stidwill et al.  showed tThe objectives of this study are to: (1) calculate the 3D angles of lower limb joints of participants skating using IMUs, (2) experimentally validate the accuracy of the obtained angles against those measured by a motion capture system, and (3) experimentally investigate if the kinematic features of lower limb joints during skating on synthetic ice measured by this wearable system can differentiate low- and high-calibre skaters. The outcome of this technology will be an optimal set of wearable IMU sensors ready to be used in on-ice and on-synthetic-ice experiments to measure the 3D kinematics of the skaters.Twelve able-bodied individuals were recruited to participate in this study. By a K-means clustering algorithm based on the participant’s years of ice skating experience, they are clustered into two groups of six high-calibre skaters  and six low-calibre skaters . All participants were healthy individuals and could skate on synthetic ice without trouble. They were informed of the experimental procedures and gave informed written consent before the test. This study was approved by the University of Alberta’s research ethics board (Pro00092821).2 synthetic ice panels. Four IMUs composed of accelerometers, gyroscopes, and magnetometers  [2, angular velocity: ±2000 deg/s, and magnetic field: ±1.9 Gauss, plate size: 10 × 7 cm) were placed on the pelvis, thighs, shanks, and hockey skates on the dominant legs of the participants without constraining their movements  , full-scovements . As the ovements . The parIMU readouts, including the tri-axial gyroscope, accelerometer, and magnetometer measurements, are used to obtain the sensor orientations using sensor fusion algorithms ,31,32. TThe RMS errors between IMU-based and camera recordings-based angles for each trial of all participants were calculated;The average of the RMS error was calculated over all trials of each participant;The computed average value for each participant is presented as boxplots for each 3D joint angle.Finally, the Root mean square (RMS) between the 3D joint angles calculated from the IMUs’ readout and the angles calculated from the reference system were obtained:In the next step, using these validated angles and the on-ice distinctive parameters, a supervised classification analysis was developed to differentiate low- and high-calibre skaters’ profiles using the on-ice distinctive features.p-value was lower than or equal to 0.05, the feature was chosen to classify high- and low-calibre skaters in this study. Then, a K-nearest neighbour (KNN) model was used to classify high- and low-calibre skaters  were randomly selected and added to the testing set, and the other participants’ data (75% of the data) were added to the training set;A KNN classification was trained for k = 1 to k = 15, and the sensitivity, specificity, precision, and accuracy of these classifiers were calculated for the testing set;Steps (1–3) were repeated 12 times so that each participant was added to the testing set three times;The average of all obtained sensitivity, specificity, precision, and accuracy for these repeated measures was calculated for each k.First, we experimentally investigated if the distinctive features measured by this wearable IMU system would be different between low- and high-calibre skaters on synthetic ice. All the temporal events of skating were obtained based on the camera recordings as the reference system in order to avoid mixing errors due to sensor orientation estimation and temporal event detection. Then, a Friedman’s test was used to verify whether the distinctive features obtained from the literature studies  was used to optimize the feature space dimensions used in the cross-validation analysis. The best three components obtained from the PCA on on-ice distinctive features were selected as the new feature space of the KNN classification, and steps (1–5) were repeated .Sixty measurement trials from the twelve participants, five trials for each, were obtained ; one comp-value < 0.05): (i) ankle dorsiflexion range, (ii) hip adduction angle in push-off instant, and (iii) knee flexion angle in a push-off instant, referred to hereafter as selected features. When the KNN classification with k varying from 1 to 15 was used based on these three selected features to differentiate high-calibre skaters from low-calibre skaters, the classifiers’ sensitivity, specificity, precision, and accuracy ranged from 46% to 67%, 61% to 86%, 47% to 67%, and 59% to 75%, respectively  was comparable with those reported in gait analysis . TheLack of familiarity with skating on synthetic ice may have affected the skating patterns of the participants since most of the study’s participants, even high-calibre ones, had not had much experience with synthetic ice skating prior to the skating sessions.Skating on a shorter distance—here, a 14 m-distance—requires faster acceleration and deceleration than real ice skating , which cThe reported surface-skates blades friction coefficient of the synthetic surfaces (0.27–0.37) is higher than the reported ice skating surfaces (0.002–0.007) ,39,40,41The 3D joint angles of the lower limbs of low- and high-calibre skater groups showed significant differences in several distinctive features . SecondThe first step toward improving hockey players’ efficiency in ice hockey matches and training is accurate performance assessments, and wearable technology helps hockey coaches do this assessment in a less intrusive way. In this study, 3D joint angles of ice hockey skaters were obtained using an IMU-based wearable technology and experimentally validated their accuracy against a camera-based motion capture system. Further, a supervised learning algorithm was developed to classify low- and high-calibre skaters’ kinematics using on-ice calibre-based distinctive features. We discovered that skating on synthetic ice alters the skaters’ skating patterns such that the on-ice distinctive features could not differentiate low- and high-calibre skaters on synthetic ice with high accuracy and sensitivity. Characterizing the biomechanics of skating on synthetic ice and comparing it with on-ice skating biomechanics is important since it is an alternative to on-ice skating. The next step of this technology development would be using it on ice to analyse the player performance on ice experiments. Using the output of this technology, skating coaches and trainers can keep track of the skaters’ progress and improve their efficiency by assessing their skating performance during training sessions and matches, even remotely.


An anterior cruciate ligament (ACL) reconstruction is a common treatment for patients with ACL rupture that aims to regain pre-injury knee stability and kinematics. During the ACL reconstruction, one method to fix the graft is the use of an interference screw (IS). The IS should provide initial stability and secure the graft during the healing period. In recent years, magnesium has emerged as an alternative material to permanent metal and polymer ISs. In addition, differences in designs, such as the shape of the IS, can influence the fixation performance of the IS. Therefore, in this biomechanical experiment, two different screw designs with two ligament materials were compared in an insertion and a pull-out test at a rate of 1 mm/s. The screw designs were a conventional polymer screw and a magnesium screw. Porcine tendon and nylon rope were used as ligament materials. All tests were performed in polyurethane foam blocks with 15 PCF density . As a result, both screw designs required an insertion torque of less than 3 Nm. There was a significant difference between the porcine and nylon rope in pull-out tests for each screw design. The magnesium screw had the highest pull-out force at 412.14 ± 50.00 N for porcine tendon and 707.38 ± 21.81 N for nylon rope. There were no significant differences in tunnel widening (narrow–wide ratio) between each ligament material. The magnesium screw showed the lowest narrow–wide tunnel ratio, implying a better ability to compress the graft to the tunnel. In conclusion, a more optimized magnesium IS design resulted in better graft fixation and an improved ACL reconstruction outcome. The anterior cruciate ligament (ACL) is one of the most important ligaments in the knee, influencing stability and kinematics. It is the most commonly injured ligament in the knee . The ACLOne factor that affects the ability to return to sport is the surgical technique . The typA meta-analysis comparing bioabsorbable and metallic IS fixation in ACL reconstruction in ten studies with a total of 790 patients found no statistical differences in infection rate, arthrometer testing, pivot-shift testing, International Knee Documentation Committee final score, and Lysholm score . Apart fSince the biomechanical testing of fixation by interference screws has shown fixation failures ,16,17, tAn initial evaluation of the screw insertion and fixation performance can provide an impression of the screw implementation and is, therefore, a good indication of a well-designed IS. The testing method for ISs is adapted from the guideline for the in vitro testing of the cruciate ligaments and ligament reconstruction  and the In addition to the pull-out test as initial evaluation, observation of the tunnel widening (the change in shape of the tunnel) can be performed. Since the size of the IS combined with the graft is normally larger than the diameter of the tunnel, the compression occurring in the fixation can cause the tunnel to widen. The observation of the tunnel widening should imply how the screw functions towards the fixation .Therefore, in this study, a magnesium IS design, see The experiment consisted of two parts, the insertion and the pull-out test, which were carried out one after the other for all samples. A bioasorbable magnesium IS (n = 10) was used for comparison with a conventional polymer IS (n = 10). A total of three 15 PCF polyurethane foam blocks  were selected to represent the bone material. Porcine flexor tendon and nylon rope were selected to represent the ACL graft for the experiment. Porcine tendon is a common material for representing ACL material. Furthermore, since this was an initial evaluation of the ISs, nylon rope was used as an additional material, aiming to eliminate any possible inconsistency in porcine tendons, such as those due to graft size, ligament stretch, deformation, or damage during the test, that could directly effect the full performance (mainly focusing on the fixation performance) of the screw. The rope was considered rigid and was not subjected to large strains. Therefore, this nylon rope set-up did not represent the actual implementation condition of the screws but determined the performance of the screw with the least influence on the non-focused parameter.The test set-ups were adapted, as mentioned previously, from the in vitro ligament testing  and the The experiment consisted of two IS designs (polymer IS and magnesium IS) tested with a porcine tendon and a nylon rope as an ACL replacement alternative, creating four groups for the test set-up. Within each group, the tests were performed five times. All ISs had a diameter of 9 mm and were 30 mm in length. The pitch and pitch depth of each screw design were measured using a Vernier caliper . The pitch was measured at 2.5 mm in both screw designs. The pitching depth for the polymer and magnesium screws was 1 mm and 0.825 mm, respectively. In A porcine flexor tendon and a 6 × 9 mm width elliptical-shaped nylon rope were selected as the ACL material for the test. Porcine feet were ordered from a local butcher. Therefore, no ethical approval was required. The porcine flexor tendon was harvested and stored in phosphate-buffered saline (PBS) and frozen at −16 °C. The ligaments were thawed to room temperature before the experiment. A total of ten porcine tendons with a size averaging 7.86 ± 0.63 mm in width and ten nylon ropes were prepared.3 blocks. In each block, three holes were drilled. Tunnels of 9 mm and 10 mm diameter were drilled for the porcine tendon and nylon rope test, respectively, as these tunnel sizes proved suitable in the initial tests. Each tunnel diameter was measured and recorded.The 15 PCF solid rigid polyurethane foam blocks were cut into seven smaller 11 × 3.5 × 4 cmBefore the experiment, the ligament clamp was connected to the testing machine with a 2.5 kN load cell . Additionally, at the beginning of each test, a ligament specimen (either porcine tendon or nylon rope) was fixed to a ligament clamp for the insertion and pull-out test.The screw insertion was performed by fixing the bone block to a 3D-printed screw insertion platform, preventing the bone block from moving . After the ligament material was fixed to the ligament clamp, the free end was pulled through the bone block tunnel (insertion tunnel) from the back to the open side of the tunnel. The ligament was pulled, and the screw was inserted to fix the ligament to the tunnel. The insertion torque was measured using a digital torque screwdriver  to verify that the insertion torque met the target of less than 3 Nm insertion torque. After the insertion was performed, the foam block with implemented IS was removed and then placed in a block holder .The pull-out test was performed by placing the implemented IS in a block holder with the tunnel axially aligned to the pulling direction, as shown in Ligament material slippage: when the ligament slips along the tunnel where the screw remains intact in the tunnel;Screw loosening: when the screw loses its interaction with the tunnel wall and moves out or within the tunnel;Screw breakage: when the material of the screw cannot withstand the load and breaks/fails.The pull-out test was performed at a rate of 1 mm/s until the machine reached a set travel limit of 96 mm. The first peak force was recorded as the maximum pull-out force. The modes of failure, such as ligament material slippage, screw loosening, and screw breakage, were observed. The determination of the mode of failure was as follows:After the pull-out test, the screw and the ligament were removed from the tunnel. The tunnel was measured at the insertion site. Since the tunnel was widened to an eliptical shape, the measurement consisted of the narrow (minor axis) and wide (major axis) sides of the tunnel. t-test. The variance of each set-up was checked to determine an equal or unequal variance t-test. Statistical significance was determined at p-values < 0.05.Statistical analysis was conducted for the mean and standard deviation. Each test set-up was analyzed using a two-tailed, two-sample p < 0.05). Moreover, for both ACL materials, the pull-out force of the magnesium screw design was significantly higher than that of the polymer screw design (p < 0.05).The insertion torque of both screw designs and ligament materials satisfied the goal of not exceeding 3 Nm insertion torque. The modes of failure were all caused by ligament material slippage in all experiments. In addition to the pull-out force from the experiment, the tunnel widening pattern, described as a narrow–wide ratio, was observed. p = 0.49), while the polymer screw showed a significant difference between the two ligament materials (p < 0.05). When comparing the tunnel widening pattern between the screw designs, there was a significant difference in the porcine tendon test (p < 0.05), and in the nylon rope test (p = 0.05). The tunnel widening ratio allowed the results of the two different tunnel diameter to be compared. The narrow–wide ratio of each test set-up is discussed in the next section.Biodegradable screws have been developed as an alternative to metal . The advBefore the test, all screw designs were visually investigated. The diameter, length, and pitch of both screws were the same except for a shallower pitch depth of the magnesium screw compared to the polymer screw. One finding was that the finishing surface of the polymer screw was smoother than the surface finish of the screw made of magnesium due to the material surface treatment.The first assessment of this study was the assessment of the insertion torque. The insertion torque limit was set at 3 Nm based on the literature, which states that (1) insertion torque ranges between 0.5 and 3.5 Nm , (2) in The evaluation of the IS performance focused on the investigation of the surface condition and the shape of the screw. In the pull-out test, the magnesium IS showed significantly higher pull-out force than the polymer IS in both porcine tendon and nylon rope cases. This means that, in the same fixation condition , the magnesium IS provides better initial fixation strength compared to the polymer IS. However, this conclusion cannot be applied when comparing the porcine tendon and nylon rope of the same IS because of the difference in the fixation condition . One observation is that, for the polymer IS, the pull-out force increased by 216 N when using nylon rope, while, for the magnesium IS, it increased by 238 N. The 20 N difference could imply an effect of the material surface condition since the nylon rope set-up created more friction with the IS. Therefore, the surface condition reflects more in this set-up than the more slippery porcine tendon set-up. For this case, it could be implied that the shape of the IS influences the fixation strength more than the surface condition.Regarding the evaluation of the shape of the IS, it was found that, in the literature, there was no significant difference in pull-out behavior for ISs with the same material with different pitches and pitch depths of 1 mm and 0.3 mm, respectively . The pitDaneshvarhashjin et al.  investigThe foam block used for the testing complied to the ASTM F1839 standard. A 9 mm tunnel and screw diameter was an optimum set-up for the IS implementation since the graft slippage is the lowest when the screw and tunnel have the same diameter . HoweverOne additional observation is the tunnel widening pattern after the insertion. The insertion results of the magnesium IS show that the nylon rope had a smaller deviation, and there was no significant difference in tunnel widening compared to porcine tendons. The tunnel widening pattern showed the interaction of the bone–screw–ligament. The ratio of approximately 0.92 in the magnesium IS shows that the IS compressed the ligament directly to the tunnel wall, resulting in an elliptical tunnel. There was no significant difference in the tunnel widening ratio between the porcine tendon and nylon rope in the magnesium IS cases. This means that, for magnesium ISs, nylon rope can give the same material response as porcine tendon. For the polymer IS, the tunnel widening ratio was higher than that in the magnesium IS cases, which means the tunnel after the insertion was more rounded. In this case, more investigation into the optimum ligament compression influencing the fixational strength should have been conducted.Despite all efforts, drawbacks still exist in this study. The experiment aimed to compare different IS designs. Since many fixation methods exist, the experimental set-up represented a monofixation with a single band graft. The result is not to be applied to all graft and fixation methods . Due to the current set-up, the foam block had to be moved to the block holder. This required more steps during the test. Fixing the bone block in one position may help to smoothen the experimental procedure and reduce the risk of possible errors. As modes of failures, only ligament material slippage, screw loosening, and screw breakage were observed. Additional observation of the nature of graft failure, including transverse rupture or constriction of the graft (and the position of the rupture), could possibly give a more detailed view of the modes of failure. Tunnel widening was only observed at the entry point of the tunnel, not for the overall length of the tunnel. Thus, the tunnel widening did not correctly imitate any tunnel widening after ACL reconstruction or during the rehabilitation process. An additional limitation is that the exact dimension of the screw was not determined but was visually observed. It was shown that parameters such as the body slope had a higher influence on the pull-out force than others such as screw depth and pitch. However, to determine the exact design parameters that influence the fixation performance, further studies have to be conducted.Reconstruction of the ACL aims to approximate the mechanical and biological properties of the original ligament. The adequate fixation of the artificial tissue is, therefore, important. It can be concluded that a combination of parameters in screw design, such as the material, surface condition, length, pitch, pitch depth, and slope design, influences the performance of the IS. An optimized design improves fixation and leads to a better ACL reconstruction outcome. Problems such as graft fiber transverse rupture and graft necking can be minimized. Lastly, since the magnesium screw had a promising outcome, it can be further investigated in a different test set-up and protocol such as by a mechanical dynamic test and various implantation approaches. Biodegradable screws should provide sufficient mechanical fixation until adequate biological fixation has been achieved and should then be degraded completely .


Multisite phosphorylation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, is required for receptor activation by natriuretic peptides (NPs) because alanine substitutions for the first four GC-A phosphorylation sites produce an enzyme that cannot be stimulated by NPs. In contrast, single Glu substitutions for the first six chemically identified GC-A phosphorylation sites to mimic the negative charge of phosphate produced an enzyme that is activated by NPs but had an elevated Michaelis constant (Km), resulting in low activity. Here, we show that vicinal (double adjacent) Glu substitutions for the same sites to mimic the two negative charges of phosphate produced a near wild type (WT) enzyme with a low Km. Unlike the enzyme with single glutamate substitutions, the vicinally substituted enzyme did not require the functionally identified Ser-473-Glu substitution to achieve WT-like activity. Importantly, the negative charge associated with either phosphorylation or glutamate substitutions was required for allosteric activation of GC-A by ATP. We conclude that vicinal Glu substitutions are better phosphomimetics than single Glu substitutions and that phosphorylation is required for allosteric activation of GC-A in the absence and presence of NP. Finally, we suggest that the putative functionally identified phosphorylation sites, Ser-473 in GC-A and Ser-489 in GC-B, are not phosphorylation sites at all. Atrial natriuretic peptide (NP) and B-type NP reduce blood pressure, blood volume, heart size and metabolism by activating guanylyl cyclase (GC)-A . C-type The kinase homology domain of GC-A, as well as the same domains in GC-B, GC-C, and GC-E, bind to and are allosterically regulated by ATP , which i32PO4 labeled GC-A protein isolated from transfected 293 cells  and unlabeled NPs were from Sigma . Protease inhibitor tablets were from Roche, Inc. .Ala or Glu substitutions for single or multiple phosphorylation sites were generated on the CMV3-GC-A plasmid as previously described .Human embryonic kidney 293T cells were transiently transfected with 5 μg of pCMV3-GC-A plasmids containing single or multiple phosphorylation site mutations by the HEPES-calcium-phosphate precipitation method as previously described .g for 15 min at 4°C. The supernatant was aspirated and the membrane pellet was resuspended with 0.5 ml PIB and centrifuged again at 20,000g for 15 min at 4°C. The supernatant was aspirated, and the pellet was resuspended in PIB to yield a protein concentration between 1 and 3 mg/ml. Crude membranes were assayed for GC activity without freezing.Cells cultured on 10 cm plates were placed in serum-free media for 4 h before membranes were prepared. Membranes were harvested at 4°C by washing the plates twice with phosphate buffered saline, scraping the cells off the plates in 0.6 ml phosphatase inhibitor buffer PIB; . Cells w2, and a nucleotide regenerating system as reported  for 15 min. This wash was repeated two times for a total of three washes. The gels were then rinsed in water, scanned with a 532 nm laser, and imaged with FUJI FLA 5000 software as previously described . After ip-values. Unpaired t-tests were used to analyze single substrate GC assays. A significant difference in all tests was where p < 0.05. Horizontal bars above and below individual data points represent the standard error of the mean. When no vertical bars are shown, they are within the symbol itself.Substrate-velocity curves were analyzed by nonlinear regression with an allosteric sigmoidal model in Prism 7 to determine the Vmax, Km, and Hill coefficients. A Michaelis–Menten model was used to determine Vmax and Km in the presence of ATP. “Km is represented by K half in some figures.” For each pair of enzymes and each model parameter, we compared the full model fit against a null model in which that parameter was set to be equal. Because of this, we used the extra sum of squares F test to generate 50) and enzymatic (Vmax and Km) characteristics to phosphorylated WT-GC-A , The Fund for Science, and The Hormone Receptor Fund.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.