BPA) with Ni(II) salt yielded [(BPA)NiCl2(H2O)] (NiBPA). The Ni(II) in NiBPA bound to a BPA ligand, two chloride, and one aqua ligands. Because most medications inhibit biological processes by binding to a specific protein, the stopped-flow technique was used to investigate DNA/protein binding in-vitro, and a mechanism was proposed. NiBPA binds to DNA/protein more strongly than BPA via a static quenching mechanism. Using the stopped-flow technique, a mechanism was proposed. BSA interacts with BPA via a fast reversible step followed by a slow irreversible step, whereas NiBPA interacts via two reversible steps. DNA, on the other hand, binds to BPA and NiBPA via the same mechanism through two reversible steps. Although BSA interacts with NiBPA much faster, NiBPA has a much higher affinity for DNA (2077 M) than BSA (30.3 M). Compared to NiBPA, BPA was found to form a more stable BSA complex. When BPA and NiBPA bind to DNA, the Ni(II) center was found to influence the rate but not the mechanism, whereas, for BSA, the Ni(II) center was found to change both the mechanism and the rate. Additionally, NiBPA exhibited significant cytotoxicity and antibacterial activity, which is consistent with the binding constants but not the kinetic stability. This shows that in our situation, biological activity is significantly more influenced by binding constants than by kinetic stability. Due to its selectivity and cytotoxic activity, complex NiBPA is anticipated to be used in medicine.Reaction of bis(2-picolyl)amine ( Metal complexes with the ability to bind and react with specific DNA locations under physiological conditions are of great interest in the development of anticancer drugs . By attaPolypyridyl ligands are very interesting in biological and materials science ,27,28,29BSA is the most extensively researched serum albumin due to its structural similarity to human serum albumin (HSA). This finding emphasizes the importance of studying the interaction behavior of metal complexes with BSA while evaluating their anticancer properties. Our journey with detailed mechanistic studies on DNA/protein binding recently began with the challenge of exploring this type of interaction via related systems while also attempting to answer the question, is the metal center important for binding? ,43,44,45BPA) in medicine, we describe the synthesis and characterization of [(BPA)NiCl2(H2O)] (NiBPA). Furthermore, a detailed kinetic investigation of the interaction of BPA and its Ni(II) complex (NiBPA) with ct-DNA/protein has been performed using UV/vis, fluorescence, and the stopped-flow technique, and a mechanism was reported. To evaluate the antibacterial activities of both compounds, Escherichia coli, Salmonella choleraesuis, Staphylococcus aureus, and Proteus mirabilis bacteria strains were chosen. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the compounds against these bacteria were tested in vitro. Additionally, the cytotoxicity against HepG2 human liver cancer cells is reported, and the results are correlated with the binding constants. Based on these intriguing findings, and because of the role and activity of nickel and its complexes in biological systems , as wellBPA, which was used without further purification. The IR spectra were captured using a Bruker Alpha-Atunated FT-IR Spectrophotometer in the 400–4000 cm−1 range. Shimadzu spectrophotometer UV-240 was used to obtain electronic absorption spectra in a methanolic solution. At room temperature, magnetic moments were measured using Gouy’s method.All of the chemicals used were of the highest analytical grade. Merck Chemical Company provided the ligand 2  in ethanol (20 mL) was heated, and a solution of BPA  in ethanol (20 mL) was added. The mixture was heated for two hours. After cooling to room temperature, 40 mL of n-hexane was added, and the resulting precipitate was isolated by filtering to give NiBPA: Yield: 75% . Elemental Analysis calculated for C12H15Cl2N3NiO; C, 41.55; H, 4.36; N, 12.11; Found: C, 41.22; H, 4.10; N, 12.03. IR : ν(NH): 3164, 1517. ν(C=N)met: 1678, ν(M-N=C): 721, ν(M–N): 492. Λm = 20 Ω−1mol−1cm2.At 60 °C, a solution of Ni(II)ClBPA and NiBPA . BPA and NiBPA were dissolved in DMSO and diluted in phosphate buffer (pH 7.2) (DMSO percent by v/v does not exceed 3%). Intrinsic fluorescence was measured with the ct-DNA solution at the excitation wavelength , and emission spectra were recorded at 550–700 nm after aliquot addition of BPA and NiBPA.Electronic absorption measurements were performed with a constant concentration of ctDNA and increasing concentrations of The excitation and emission wavelengths of BSA at 280 nm were monitored in protein binding studies using BSA (0.5 M) solution prepared in Tris-HCl buffer (pH 7.5), and the solution was stored in a dark place at 4 °C.BPA and NiBPA under pseudo-first-order conditions. For each experimental condition, all recorded rate constants were taken into account, from which the mean values of at least three independent kinetic runs were calculated.The kinetics of protein and DNA binding was measured using an Applied KinetAsyst SF-61DX2 stopped-flow instrument. At 25 °C, equal volumes of compounds against protein and DNA solutions were rapidly mixed in the stopped-flow instrument. The binding reactions were studied using a 10-fold excess of the ligand BPA and one for human liver cancer (HepG2) cell lines. MTT can be converted into formazan by living cells with an active metabolism, but this ability is lost in dying cells. Therefore, in this study, the MTT assay was used to determine how many viable cells there were. Before the investigated compounds were added, cells were cultured in 96-well flat-bottomed microtiter plates at a density of 1 × 104 cells/well (100 l/well) for 70 to 80 percent confluent cultures, and the cultures were then incubated at 37 °C and 5 percent CO2 for 24 h. The compounds were used in a range of concentrations , and the cells were cultured for 24 h. Then, 10 l of the 12 mM MTT stock solution (5 mg/mL MTT in sterile PBS) was added to each well at the conclusion of the incubation period. After that, the plate was incubated at 37 °C for 4 h. The purple formazan crystal that formed at the bottom of the wells was removed, and 100 l of DMSO was used to dissolve it for 20 min. There was also a negative control that involved adding 10 mL of the MTT stock solution to 100 mL of medium on its own. On an ELISA reader, the absorbance at 570 nm was read . The percentage of cells inhibited was calculated using:T is the optical density of the test sample, To is the optical density at time zero, and C is the optical density of the control.Methyl thiazolyl tetrazolium (MTT) colorimetry  was used2(H2O)] (NiBPA) was produced in good yield by treating hydrated NiCl2 with one equivalent of BPA in boiling ethanol at 60 °C. IR and UV-vis spectroscopies, as well as elemental analysis, were used to characterize complex NiBPA. NiBPA, which shows an intense peak at λmax = 284 nm, indicating the inter ligand π→π* transition in the BPA ligand. A less intense band at 477 nm, on the other hand, is attributed to the nickel dπ→π* MLCT transition. At 688 nm, a significant low-energy absorption band assigned for the d–d transition was seen. This may be assigned for the 3A2g(F) to 3T1g(F)(v2) transition and support the octahedral geometry of the nickel complex  and thus leads to upward curvy plots of 0/II versus [Q] at higher [Q]. ormation ,54 accorqK) was calculated using the Stern–Volmer and Scatchard equation and plotted as 0/I vs.I [Q] to better understand the quenching process (Kbin is the compound’s BSA binding constant and n is the number of binding sites. From the log (0 − II)/I vs. log [Q] plot (Kbin) and the number of binding sites (n) have been calculated  for BPA and NiBPA strongly supported the existence of a single binding site in BSA. The qK and binK values for these compounds also indicated that NiBPA interacts with BSA more strongly than the BPA under investigation.The quenching constant  and NiBPA (0.52 × 105 M−1) are similar to those of well-known groove binding rather than classical intercalation, implying that NiBPA interacts with ct-DNA much more strongly than BPA under investigation.Absorption titration information was depicted in BPA and propose a mechanism in order to obtain such information. The kinetic traces were recorded at 265 nm within 100 s (BPA) and 5 s (NiBPA), respectively, to capture the interaction process with BSA. The absorbance of NiBPA increased sharply in the first 1 s of the reaction with BSA and then gradually decreased over the next 5 s. In contrast, the interaction of BPA with BSA shows a distinct pattern, with a gradual increase in absorption. In the case of ct-DNA interaction, the kinetic traces were recorded at 290 nm, and both BPA and NiBPA followed a similar pattern since kinetic traces for BPA and NiBPA were collected within 100 s, covering the entire interaction process. After a sharp decrease in the first 5 s, the absorbance gradually increased over the next 100 s. Over the concentration range of BPA and NiBPA, the study of kinetic traces performed under pseudo-first-order conditions may be fitted to two kinetic phases  and dissociates forty times faster (−1k = 7.3 ± 0.3 s−1). This means that the binding affinity of BSA to BPA, a1K, 1/k−1k, of 73.5 M−1 is about ten times higher than that of NiBPA (7.3 M−1) and that the equilibrium dissociation constants of BPA, d1K, −1/k1k, is 13.6 × 10−3 M and about ten times slower than that of NiBPA (136 × 10−3 M). This means that the formed BPA-BSA is far more stable than NiBPA-BSA.The results, presented in −1 for NiBPA was detected in the second reaction step, but an irreversible reaction with a 2K = 0.69 M−1 with BPA was observed. The second phase is primarily concerned with the BSA condensation process, in addition to the electrostatic interaction. As shown in BPA (−k2 ≈ 0) suggests that the newly formed BPA-BSA complex is kinetically more stable in solution than NiBPA-BSA.A reversible reaction with a coordination affinity of 0.69 Mobs1k) followed by a slow first-order isomerization process (rate constant obs2k). Both obs1k and obs2k increased linearly with BPA and NiBPA concentration  [bindG.The difference between the free energy, G, of BSA molecules alone in the solution and when bound together is related to the equilibrium dissociation constant, tion (4)  gives thT, R represents the absolute temperature and the universal gas coefficient, respectively. The ΔbindG values, calculated using Equation (4), are −10.6 and −5.1 kJ mol−1 for the first step binding of BSA to BPA and NiBPA, respectively. The negative sign for G indicates spontaneous binding to both BPA and NiBPA, and the greater negative value of BPA compared to NiBPA indicates that binding with BPA is very favorable. Both compounds interact with ct-DNA through a similar mechanism that is reversible in both reaction steps  and dissociates from the binary complex with (−1k = 0.36 ± 0.1 s−1). This means that the DNA binding affinity of 1, Ka1,1k/−1k is slightly higher (53.6 M−1) than that of BPA (40.0 M−1) and that the equilibrium dissociation constant of NiBPA, Kd1, k−1/k1 is ×10−3 M, which is slightly slower (25.6 × 10−3 M). Because of the presence of a Ni(II) center, NiBPA has a higher affinity and a lower dissociation constant than BPA, indicating that the formed 1–DNA is much more stable than BPA–DNA. Gbind values of −9.1 kJ mol−1 (BPA) and −9.7 kJ mol−1 (NiBPA) indicate that this step is spontaneous for both compounds. The data from the second reaction step, which included internal DNA rearrangement as well as electrostatic interaction and isomerization reaction, followed the same pattern as the first; a reversible reaction was observed for both BPA and NiBPA. The DNA affinity for BPA (Ka2 = 2k/−2k) is 47.5 M−1, which is slightly higher than NiBPA (39 M−1), and the equilibrium dissociation constant for BDA (Kd2 = k−2/k2) is 2.1 × 10−2 M, which is slightly slower than NiBPA (2.6 × 10−3 M). bindG values for this phase are −9.4 kJ mol−1 (BPA) and −9.0 kJ mol−1 (NiBPA), indicating that this step is spontaneous for both compounds.While on steps . The inid,K from the individual equilibrium dissociation constants, d1K and d2K, as well as the overall association constants, aK.Equation (5) shows how to calculate the overall equilibrium dissociation constant, NiBPA (2077 M) is higher than that of BPA (1899). The overall reaction bindG values are −18.7 kJ mol−1 (BPA) and −18.9 (NiBPA) kJ mol−1, indicating that the reaction is spontaneous for both compounds.The data in BPA and NiBPA were tested against human liver cancer (HepG2) and normal liver (Wi38) cell lines at various concentrations by MTT assay. The percentages of cell viability in the presence of BPA and NiBPA with different concentration levels  are depicted in BPA and NiBPA can be measured by the IC50 values and were found to be 100.0 ± 1.0 and 75.0 ± 0.5 μg/mL, respectively. From this data, one can conclude the following. Firstly, both complexes were cytotoxic active, but the activity was less than that for the normal drug . Secondly, the Ni(II) complex NiBPA exhibits higher cytotoxicity compared to BPA. The complexation of BPA to nickel center in complex NiBPA may be the main factor for the better cytotoxicity of NiBPA. The cytotoxic selectivity of both compounds was investigated by measuring their effect on the cell viability of normal liver (Wi38) cell lines. In the case of the normal cells, the IC50 values suppressed by BPA and its Ni(II) complex NiBPA are 237.2 ± 1.3 and 120.0 ± 0.5 μg/mL, respectively. The IC50 values of the normal cells are much higher than that of the cancer cells, indicating that both compounds can be effective and selective toward cancer cells. Thus, complex NiBPA is the most active, indicating that nickel ion coordination was crucial for cytotoxicity and that complexation improved the anticancer activity. The positive charge in NiBPA due to the neutral BPA ligand may increase cytotoxicity. Additionally, these findings are in agreement with the affinities and binding constants of every compound that was studied for interaction with BSA.The interesting findings from BSA binding studies inspired us to use the MTT assay to test all compounds’ anticancer activity. In vitro cytotoxic activity of BPA and its nickel complex NiBPA were tested in vitro for antibacterial activity against Gram-positive (S. aureus and S epidermidis) and Gram-negative (P. mirabilis and E. coli) bacteria. BPA and NiBPA as the diameter of the growth-inhibition region in millimeters. NiBPA exhibited strong antibacterial activity, and its antibacterial activity was much greater than that of BPA, but it was less than that of the standard drug (TOB). Second, NiBPA demonstrated greater antimicrobial activity against Gram-negative bacteria than Gram-positive bacteria. This is explained by the metal ion’s polarity and cationic charge, as NiBPA has a cationic property with two positive charges. Gram-negative bacteria were more sensitive to NiBPA than Gram-positive bacteria due to differences in cell surface characteristics; Gram-negative bacteria had a higher negative charge on their cell surface than Gram-positive bacteria [NiBPA was stronger than that of Gram-positive bacteria due to the higher negative charge on the cell surface, despite the presence of an outer membrane in Gram-negative bacteria  complex NiBPA. In comparison to BPA, complex NiBPA binds strongly to DNA/protein via a static quenching mechanism. A mechanism for the interaction was proposed using stopped-flow techniques. BSA interacts with BPA and NiBPA via two different mechanisms. BPA’s interaction is characterized by fast reversible followed by slow irreversible steps, whereas NiBPA’s interaction is characterized by two reversible steps. The binding parameters for the first step of BPA  and NiBPA  and for the second step of BPA (K2 = 0.82 M−1) and 1  were determined. Overall binding parameters for NiBPA . BPA and its Ni(II) complex NiBPA bind to DNA in a similar way, with two reversible steps: fast second-order binding followed by slow first-order isomerization. The binding parameters for the first step of BPA  and NiBPA  and for the second step of BPA  and NiBPA  were determined. Overall binding parameters for BPA  and NiBPA  were also determined and showed that the relative reactivity is approximately (BPA)/(NiBPA) = 9/10. The significantly negative G values support a spontaneous binding reaction to both BPA and its Ni(II) complex NiBPA. The following points can be summarized by comparing the results of the interactions of BPA and NiBPA with DNA with those of BSA. BSA interacts with BPA and NiBPA via two different mechanisms: (i) A fast reversible followed by slow irreversible steps characterized the interaction of BPA, whereas two reversible steps are observed with NiBPA.; (ii) BPA and NiBPA interact with DNA via a similar mechanism via two reversible steps; (iii) Complex NiBPA was found to have much higher affinity to DNA (2077 M) than BSA (30.3 M), despite the fact that BSA interacts with this, which is explained by the fact that the fast association of NiBPA with BSA is followed by a fast dissociation reaction; (iv) BPA formed a more stable BSA complex than its Ni(II) complex. This is also understandable because they both bind via different mechanisms, with the second binding step for BPA being irreversible and the second binding step for NiBPA being reversible; (v) Interestingly, when BPA and NiBPA bind to both DNA, the Ni(II) center was found to affect the rate but not the mechanism. In the case of BSA interaction with BPA and its Ni(II) complex, the Ni(II) center was found to change both the mechanism and the rate. Additionally, NiBPA exhibited significant cytotoxicity against the human liver cancer HepG2 cell as well as antibacterial activity, which is consistent with the binding constants but not the kinetic stability. This shows that in our situation, cytotoxic and antibacterial activities are significantly more influenced by binding constants than by kinetic stability. Due to its selectivity and cytotoxic activity, the complex NiBPA is anticipated to be used in medicine.New Ni(II) complexes containing bispicolylamine have been described and characterized in this study. Because almost all pharmaceuticals work by interfering with biological functions by binding to a specific protein or DNA, in vitro DNA/protein binding of


Even though hot melt extrusion (HME) is a commonly applied process in the pharmaceutical area, determination of the optimal process parameters is demanding. The goal of this study was to find a rational approach for predetermining suitable extrusion parameters, with a focus on material temperature and throughput. A two-step optimization procedure, called scale-independent optimization strategy (SIOS), was applied and developed further, including the use of an autogenic extrusion mode. Three different polymers  were considered, and different optimal process parameters were assessed. The maximum barrel load was dependent on the polymers’ bulk density and the extruder size. The melt temperature was influenced by the screw speed and the rheological behavior of the polymer. The melt viscosity depended mainly on the screw speed and was self-adjusted in the autogenic extrusion. A new approach, called SIOS 2.0, was suggested for calculating the extrusion process parameters  based on the material data and a few extrusion experiments. A major challenge in today’s pharmaceutical research is the poor solubility of newly developed drugs ,2. Even A common manufacturing technique for ASDs is hot melt extrusion (HME) ,9. CorotThe critical process parameters are directly adjustable by the operator. In contrast, the dependent critical process parameters are not directly selectable but are related to the critical process parameters, as well as to the critical material attributes . Recently, the scale-independent optimization strategy (SIOS) was proposed as a method to tailor an optimized extrusion process . AccordiSFL) ,13, are ion (1)) .(1)SFL=The SIOS is a two-step efficiency optimization procedure for reaching an optimized operating point, which aims for a high throughput and a low power consumption while producing a homogeneous cylindrical strand (coherent extrudate). During the first step (point A to B), the screw speed is decreased stepwise at a constant throughput until the barrel is completely filled (point B). This point can be detected from a developed backlog in the feeding zone. Thereafter, a slightly higher screw speed is used (point C). In the second step (point C to D), the throughput and the screw speed are increased simultaneously to increase the efficiency of the process at a constant barrel load until degradation is observed (point D). Operating the extruder without external barrel heating or cooling is often called autogenic or adiabatic extrusion. It is used to increase the robustness and scalability of the process, since the surface-to-volume ratio of the process unit (barrel and screw) becomes irrelevant . These tThe motor power (sumption . In adia˙loss=0) . Thus, tIn this work, a theoretical approach was developed for predetermining the optimal process parameters to produce a coherent extrudate at a maximum throughput and defined melt temperature. Therefore, autogenic extrusion was added to the scale-independent optimization strategy. The approach was applied to different polymers, and its applicability to industrial processes was investigated. A mathematical model for the barrel load and the melt temperature was developed, with the aim of predicting the optimized operating point in accordance with the SIOS.Three pharmaceutical polymers commonly used in hot melt extrusion were utilized in this study ,17,18. TA loss-in-weight feeder  was used for dosing the material in the extruder. The extrusion experiments were carried out in a corotating twin-screw extruder , containing modular screw elements with a 28.3 mm diameter and a length of 32 D . A heateThe screw and barrel design was similar to that which was used in previous investigations . For allFor starting the extrusion, a barrel temperature profile was set. Barrels 1 and 2 were cooled to 20 °C and barrel 3 was heated to 80 °C. The subsequent barrel elements were set to the material manufacturers’ recommended temperatures for extruding the excipients . During the first optimization step, the screw speed was decreased stepwise (from 200 to 20 rpm) at a constant throughput (3 kg/h). For further processing, the barrel temperature control in barrels 1–3 remained activated to avoid sticking in the feeding zone. Temperature control was disabled in barrels 4–8 to perform autogenic extrusion, which is an extension of the SIOS. In the second optimization step, the throughput was increased stepwise from 3 kg/h up to 42 kg/h. The screw speed was increased accordingly to keep the specific feed load constant (Equation (1)). The temperatures used for rheology measurements were close to the extrusion temperatures.Oscillation rheology measurements covered the high shear rate range relevant for extrusion.Dried polymers were utilized due to the plasticizing effect of the water on the polymers.Sample preparation via “MeltPrep” technology was preferred to minimize the air bubbles in the molten polymer.Repetitive measurements were made to enable confidence in the data.The rheological data for all three polymers were taken from the literature. The datasets were chosen based on five criteria: The literature data were refitted to the Carreau model (Equation (3))  which waR) and the temperature (T). A material-specific activation energy (The Carreau model describes the dynamic viscosity (approach . The tem as well .The process conditions in extrusion were chosen in accordance with the SIOS . In thisThese experiments applied the SIOS for the first time to two other polymers (SOL and bBMA) which are commonly used in the hot melt extrusion of amorphous solid dispersions ,25,26,27The specific feed load is a dimensionless number that represents the load of the extrusion barrel. Different maximum specific feed loads  in order to save time and resources by conducting preliminary experiments to determine the powder slip.In the autogenic extrusion , C to D According to Hagen–Poiseuille law  ) .(9)γ˙diAt this point, it is worth mentioning that the die shear stress ). However, the shape of this function is quite similar for the three polymers, which was unexpected. Apparently, each screw speed of the extrusion screw leads to the same die viscosity, regardless of the material. Moreover, a hyperbolic trend was observed in the data and was used to model the data with Equation (10).When considering the die viscosity as a function of screw speed , right, Here, n is the screw speed, which correlates to the shear rate in the extruder screw (compare Equation (7)). The correlation parameter  model using die shear rate and melt temperature. Ideally, these two viscosities should be the same, but there are several limitations to using a production scale extruder as a capillary rheometer, such as the in-feed behavior until laminar flow is reached. However, a correlation between these two viscosities was found , left—alThe two viscosities were correlated to the screw speed in a double logarithmic approach, which is quite common in the field of rheology .(11)lgηIn this way, the measured die viscosity , and the die viscosity can be converted to an extruder-independent viscosity Determine the following material characteristics:2.free cross-sectional area of the screw (screw diameter (screw pitch in the feeding section (die radius (die length (Determine the following extruder parameters:3.n). Calculate the slip , while high speeds increase the material consumption.Choose a reasonable screw speed  and set the mass flow for this specific speed using Equation (5) at Wait for the steady state of the extrusion process at these conditions, at which point, homogeneous, coherent (commonly transparent) extrudate strands are obtained.Turn off the barrel temperature control to allow autogenic extrusion. However, continue cooling the feeding section and heating the die to the manufacturer’s recommended process temperature for the material .Measure the melt temperature  after steady state is reached , which may take several minutes. Note the screw speed, mass flow rate, melt temperature and die pressure.Move to other process conditions. If the power consumption is reasonably low , the screw speed and the mass flow rate should be lowered by the same ratio . If the power consumption is relatively high, the higher screw speed and mass flow rate should be increased by the same ratio .Measure the melt temperature and die pressure after reaching steady state. Note the corresponding mass flow rate and screw speed.Fit the model parameters  with fewer experiments (SIOS 2.0). A stepwise procedure is presented as follows:The predictive power of this new concept was evaluated using the same experimental data that were presented before. However, the data set was reevaluated in a crossover design. The extrusion performance of one polymer was predicted by the two other polymers and subsequently compared to the experimental data . The melt temperatures of PVPVA and SOL were predicted well using the new model. There were systematic deviations at low screw speeds, which might be attributed to incomplete thermal equilibration. However, the desired range for any production process will be at a high speed and high throughput. The predicted melt temperature of bBMA is systematically lower than the measured values. The extrusion behavior of this polymer is less related to the rheological data found in the literature. However, temperature differences of 5K at the desired process conditions seem to be acceptable. The prediction of the mass flow rate reflects the differences in slip between the polymers. Since deviations were of less than two percent, no relevant differences between the model and experiment were recognized. These results demonstrate the predictive power of the new modelling concept SIOS 2.0. Direct process parameters, such as screw speed and mass flow rate, can be chosen, and dependent process parameters, such as specific feed load and melt temperature, can be predicted. Since this concept is valid for polymers with different structures, it is likely that this concept would be valid for more similar materials, such as polymers and their corresponding amorphous solid dispersion formulations.In this work, the scale-independent optimization strategy (SIOS), according to Wesholowski et al. , was extDifferences between the polymers in terms of the maximum barrel load and melt temperature were attributed to the critical material attributes. The maximum barrel load was found to be dependent on the polymer bulk density and the process-related slip. The melt temperature was related to polymer rheology, and it adjusted automatically based on the screw speed. Two mathematical models were developed to predict the throughput, as well as the melt temperature, based on extruder dimensions, material properties and several extrusion experiments. Thus, the existing SIOS was extended to SIOS 2.0, reducing the time and experimental effort.


To confirm these results, in vitro kinase assays and two-dimensional phosphopeptide analyses were performed with tau441 phosphomutants confirming Alzheimer’s disease-associated residues Ser68/Thr71 and Ser289 as CK1δ-specific phosphorylation sites. Treatment of differentiated human neural progenitor cells with PF-670462 and Western blot analysis identified Ser214 as CK1δ-targeted phosphorylation site. The use of an in vitro tau aggregation assay demonstrated a possible role of CK1δ in tau aggregation. Results obtained in this study highlight the potential of CK1δ to be a promising target in the treatment of Alzheimer’s disease.A main pathological event in Alzheimer’s disease is the generation of neurofibrillary tangles originating from hyperphosphorylated and subsequently aggregated tau proteins. Previous reports demonstrated the critical involvement of members of the protein kinase family CK1 in the pathogenesis of Alzheimer’s disease by hyperphosphorylation of tau. However, precise mechanisms and effects of CK1-mediated tau phosphorylation are still not fully understood. In this study, we analyzed recombinant tau441 phosphorylated by CK1δ  Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by an irreversible process of changes involving specific neurons of the neocortex, hippocampus, and other regions of the brain, leading to a cognitive impairment followed by a mental and functional decline. Generally, AD is responsible for more than 80% of dementia cases in elderly people worldwide . One of Tau proteins belong to the family of microtubule-associated proteins (MAPs)  and occuin vitro and in vivo substrates (reviewed in in vitro by using mass spectrometry (MS). MS analysis revealed 33 CK1δ-specific tau phosphorylation sites, while previously detected phosphorylation of Ser202/Thr205 and Ser396/Ser404 could not be confirmed  family. Members of the CK1 family are highly conserved serine/threonine-specific, ubiquitously expressed protein kinases. So far, seven different CK1 isoforms  and their splice variants were identified in mammals. CK1 is able to recognize canonical as well as noncanonical consensus sequences within a substrate resulting in over 150 different onfirmed . In bothonfirmed . Becausein vitro by using different techniques including MS, in vitro kinase assays and two-dimensional phosphopeptide analysis. The results were further supported by a cell-based assay and Western blot analysis using phospho-specific antibodies. Additionally, we demonstrated that CK1δ co-localized with tau in neuronal cells and that CK1δ-mediated phosphorylation led to an increased in vitro tau aggregation. With our data, we provide a comprehensive analysis of CK1δ-mediated site-specific tau phosphorylation and confirm a functional influence of CK1δ on tau aggregation.Tau hyperphosphorylation and tau-phosphorylating kinases have become attractive targets in the treatment of AD. Therefore, the characterization of tau hyperphosphorylation by tau-targeting kinases, such as CK1, is necessary to understand the pathophysiological mechanisms and to provide better therapeutical approaches addressing tau pathology. Here, we characterized the contribution of CK1δ to AD-associated tau phosphorylation sites 1-155 and tau441243-441) and phosphorylation site mutants of full-length tau441  were created by using inverse PCR and the PCR primer pairs as indicated in 156-242, Gibson Assembly® was performed according to manufacturer’s instructions . Sanger DNA sequencing  confirmed successful introduction of mutations.The codon-optimized bacterial expression vector pET28a(+)tau441 encoding for N-terminal 6xHis-tagged human microtubule-associated protein tau (MAPT) isoform 4 (tau441) was synthesized by Biomatik . Plasmids encoding for tau441 fragments . Protein production was conducted in 450 ml lysogeny broth (LB) medium supplemented with 15 μg/ml kanamycin and induced with 0.5 mM IPTG at an OD600 of 0.6 AU for 3 h at 30°C. Bacteria were harvested by centrifugation for 10 min at 3,200 g and 4°C. Bacteria were lysed in lysis buffer composed of 50 mM sodium phosphate buffer (pH 7.0), 350 mM NaCl, 15 mM imidazole, 0.5% NP-40, 10% glycerol, 1 mM benzamidine, 0.25 g/L aprotinin and 10 mg lysozyme. Cell lysates were cleared by centrifugation for 20 min at 15,500 g and 4°C. According to Barghorn et al.  (50% (v/v) in PBS) and incubated at 4°C rotating overnight. Bound proteins were washed (50 mM sodium phosphate buffer (pH 7.0), 350 mM NaCl, 15 mM imidazole, 10% glycerol, 0.25 g/L aprotinin) and eluted stepwise  followed by dialysis using PD-10 desalting columns . Production of GST-tagged and 6xHis-tagged kinases was carried out as described previously , 2 µCi [γ-32P]-ATP (only for radiometric determination), 300 nM CK1δ and 1 µg  substrate. As substrates either tau441, tau441 fragments or tau441 phosphorylation site mutants were used. Reaction was carried out for 30 min at 30°C in triplicates. Proteins were separated by SDS-PAGE on 10% gels followed by Coomassie blue staining. Radioactively labeled substrate bands were visualized on dried gels by autoradiography. For quantification of phosphorylated products, radioactively labeled substrate bands were excised from dried gels and phosphate incorporation was determined by Cherenkov counting . In vitro phosphorylated tau441 (wild type), fragments and phosphorylation site mutants were analyzed by two-dimensional phosphopeptide analysis using standard protocols described previously . Protein bands of interest were incubated with 5% (w/v) polyvinylpyrrolidone (in 10 mM acetic acid) at 37°C for 30 min, washed with 50 mM ammonium bicarbonate buffer and digested with 10 µg TPCK-trypsin. Digested proteins were further oxidized with performic acid on ice for 2 h. Radioactively labeled phosphopeptides were separated on cellulose TLC plates  by electrophoresis at pH 1.9 (containing 6% (v/v) formic acid, 1.25% (v/v) acetic acid and 0.25% (v/v) pyridine in dH2O) followed by ascending chromatographic separation in buffer (composed of 37.5% (v/v) n-butanol, 7.5% (v/v) acetic acid and 25% (v/v) pyridine in dH2O). Radioactively labeled phosphopeptides were visualized by autoradiography.The reaction was performed in a total volume of 15 µl containing the kinase buffer  in 100 mM Tris/HCl (pH 6.8) was induced with freshly prepared 150 µM arachidonic acid (10 mM in ethanol) as described previously  at intervals of 1.5 min within 30 min. Data were displayed and fit to one-phase association exponential model using Prism 8 .Tau441 was phosphorylated eviously . Tau agg® VM from Merck Millipore, Darmstadt, Germany) were expanded and maintained in proliferation medium  supplemented with 2% (v/v) B-27 neural supplement , 2 μg/μl heparin , 20 ng/ml human basic fibroblast growth factor (bFGF) , 20 ng/ml human epidermal growth factor (EGF) , and 100 U/ml penicillin-streptomycin solution ) as described previously (6 naïve hNPCs were seeded onto Matrigel-coated glass slides in a 6-well with differentiation medium (proliferation medium without growth factors) and differentiated for 2 weeks.Immortalized human neural progenitor cells (hNPCs)  or DMSO as vehicle control for 24 h and subsequently lysed.To generate a cell culture system for modeling the AD pathology, hNPCs were stably transfected with lentiviral DNA constructs pCSCW-APPSL-IRES-GFP and pCSCW-PSEN1(ΔE9)-IRES-mCherry, which encode full-length human APP695 with K670N/M671L/V717I (Swedish and London mutation) and GFP or human presenilin 1 with a deletion in exon 9 (PSEN1 (ΔE9)) and mCherry. The lentiviral DNA constructs were kindly provided by Prof. Dr. Doo Kim . Lentiviral transduction and the subsequent enrichment of high-expressing transduced hNPCs via fluorescence-activated cell sorting (FACS) was performed according to 3VO4, 1 mM NaF containing fresh phosphatase inhibitor cocktail  and cOmpleteTM protease inhibitor cocktail . Cell lysates were cleared by centrifugation at 10,000 x g for 10 min at 4°C. 10 µg of cleared lysates were separated on 10% (v/v) gels in SDS-PAGE and transferred to a 0.2 µm PVDF membrane . Membranes were blocked in 5% (w/v) BSA in TBST for 1 h and incubated with anti-tau antibody , anti-pSer202/pThr205-tau antibody , anti-pSer214-tau antibody , anti-pSer416-tau antibody  and anti-β-actin antibody  overnight. Immunocomplexes were detected using a secondary antibody (horseradish-peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibody, 1:10,000). Immunoreactivity was detected by enhanced chemiluminescence using the Fusion FX imaging system . Signal intensities were quantitatively determined by using ImageJ .Transduced hNPCs were washed with 20 mM Tris-HCl (pH 7.6) buffer containing 140 mM NaCl. Cell lysates used for Western blot analyses were prepared in RIPA lysis buffer composed of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 1 mM EDTA, 2 mM PMSF, 2 mM PNT, 1 mM Na2) at 4°C for 20 min followed by permeabilization with 0.3% (v/v) Triton-X 100 in 1x PEM buffer at RT for 5 min. Cells were washed briefly with 1x PEM and blocked with 5% (w/v) BSA in 1x PEM at RT for 30 min. Thereafter, cells were incubated with anti-tau antibody  at 4°C overnight. Cells were washed three times with 1x PEM and subsequently incubated with Alexa Fluor 488 anti-mouse antibody  at RT for 1 h. Then, cells were washed three times with 1x PEM. Cultures were blocked again in 5% (w/v) BSA in 1x PEM at RT for 30 min, washed three times with 1x PEM and stained with anti-CK1δ antibody  or anti-MAP2 antibody . After washing the 2D grown cells, they were incubated with Alexa Fluor 647 anti-goat antibody  or Alexa Fluor 633 anti-rabbit antibody  at RT for 1 h. Then, cells were washed three times with 1x PEM and stained with 0.1 μg/ml DAPI  at RT for 5 min. Washed cells were finally mounted with ProLongTM Glass Antifade Mountant . Images were captured using the Leica SP8 confocal microscope  at ×63 magnification.After differentiation and inhibitor treatment, cells were washed briefly with 1x PBS and fixed with 4% (v/v) paraformaldehyde in 1x PEM buffer (80 mM PIPES (pH 6.8), 1 mM EGTA, 5 mM MgClCo-localization analysis was carried out using R  and RStudio . The required packages include imager  and colop values ≤0.05 (shown as * for p ≤ 0.05 and ns for not significant) were considered to be statistically significant.Results are presented as the mean of experiments at least performed in triplicates. Evaluation and statistical analysis of the results were performed using Prism 8 . Statistical significance was tested by using the nonparametric Mann-Whitney U test. Hyperphosphorylation of tau by various kinases plays an important role in the pathogenesis of AD. Previously, several studies demonstrated a potential role of CK1 in the hyperphosphorylation of tau that could be linked to the development of AD. The present study intends to demonstrate a specific involvement of CK1 in tau hyperphosphorylation and aggregation.E. coli, purified via immobilized metal affinity chromatography and subsequently phosphorylated by human recombinant CK1δ in vitro. LC-MS/MS data generated from in vitro phosphorylated and trypsin-digested tau441 revealed 91.8% and 94.1% sequence coverage of non-phosphorylated tau441 and CK1δ-phosphorylated tau441, respectively, based on the amino acid sequence of the longest human tau isoform tau441 (UniProt: P10636-8). Additionally, MS analysis did not detect any phosphorylation of non-phosphorylated tau441. In total, we detected phosphopeptides corresponding to ten different phosphorylation sites  contains several putative target sites for CK1-mediated phosphorylation . Initialon sites ; Table 1Most of the phosphorylation sites, which could be detected in our study, were recently identified by 1-155, tau441156-242 and tau441243-441). Each of them contains a specific domain of tau441  and corresponding phosphomutants tau441ediction , one phoe tau441 . Additioin vitro . No new 2A+T427A .in vitro kinase assay. However, CK1δ-specific inhibition by PF-670462 did not affect tau phosphorylation at Ser202/Thr205 and Ser416  revealed that CK1δ prefers to phosphorylate tau441 within the MT-binding and C-terminal domain (aa 244-441) as well as the proline-rich domain (aa 197-244), which is in line with the findings of several other studies, demonstrating that most of the known AD-associated phosphorylation sites are located within the central region (172-251) and the C-terminal region (aa 368-441)  .in silico approaches and MS analysis showed that CK1δ specifically phosphorylates the AD-associated phosphorylation sites Ser68, Thr71, Ser198, Ser214, Ser289, Thr414/Ser416 and Thr427  and vice versa. In vitro experiments based on in vitro kinase assays confirmed that the AD-associated phosphorylation sites Ser68 and/or Thr71 and Ser289 on tau441 are targeted by CK1δ. Although Ser68 and/or Thr71 were not detected by MS analysis, in vitro kinase assays and phosphopeptide analysis clearly identified these phosphorylation sites as CK1δ-specific highlighting the potential of these methods. The failure to detect these phosphorylation sites by MS analysis could have been due to difficulties in separation, the loss of phosphorylation at these residues during sample preparation or reduced peptide lengths peptides, that is probably caused by peptide precipitation and unpolymerized acrylamide cross-linked to proteins interfering tryptic digestion, that leads to inefficient tryptic peptide digestion and insufficient membrane detachment  or experimentally identified . Results obtained from our aggregation experiments demonstrated a potential role of CK1δ in tau aggregation, which has already been shown for several other kinases including TTBK1 and Fyn (CK1δ phosphorylates tau at specific AD-associated residues  from AD . To dire and Fyn . In a bi and Fyn . TTBK1 i and Fyn .in vitro aggregation of tau. Therefore, our findings clearly support the assumption that CK1δ has an essential role in tau hyperphosphorylation and the pathogenesis of AD.In summary, we clearly identified AD-associated tau phosphorylation sites, which can be targeted by CK1δ. Furthermore, we demonstrated that CK1δ co-localizes with tau in differentiated neuronal cells. For the first time, we provide experimental proof that CK1δ-mediated phosphorylation plays an important role at least in the


Scyliorhinus stellaris) maintained under human care. Median (RI) in major leukocyte types were similar to other Carcharhinid sharks as lymphocytes were the predominant leukocyte with 38.0 (28.2–53.5)%, followed by coarse eosinophilic granulocytes with 20.0 (12.2–31.7)%, fine eosinophilic granulocytes with 6.0 (1.2–12.8) %, monocytes with 2.0 (0.0–6.0)%, and neutrophils with 2.0 (0.0–6.0)%. Nursehound Shark produced granulated thrombocytes, which were classified as granulocytes and represented 28.5 (12.4–39.7)% of all leukocytes. Median (RI) manual red blood cell and white blood cell counts were 177.50 (132.50–210.00) x 109 cells/l and 8.26 (5.24–14.23) x 109 cells/l, respectively. Median (RI) plasma chemistry values showed alkaline phosphatase 7.7 (4.2–13.0) U/l, aspartate aminotransferase 7.6 (3.3–17.1) U/l, blood urea nitrogen 281.6 (261.2–305.0) mmol/l, calcium 3.97 (3.59–4.47) mmol/l, total cholesterol 2.04 (1.02–3.91) mmol/l, chloride 233.0 (215.2–259.0) mmol/l, iron 3.79 (1.74–6.93) μmol/l, glucose 0.87 (0.47–1.44 mmol/l), potassium 3.8 (2.9–4.6) mmol/l, sodium 243.0 (227.7–271.0) mmol/l, phosphorus 1.58 (1.13–2.10) mmol/l, total protein 24.0 (20.0–35.0) g/l, and triglycerides 0.97 (0.49–3.35) mmol/l. Creatine kinase, gamma glutamyl transferase, and lactate dehydrogenase levels were below the instrument reading range.Studies determining baseline hematological reference intervals (RI) in elasmobranchs are very limited. In this study, blood samples were collected from 94 clinically healthy Nursehound Shark ( Population-based reference values (RV) represent the most widely used tool for the interpretation of individual clinical laboratory test results and are typically reported as reference intervals (RI) comprising 90–95% of a healthy reference population , 2. AfteBlood collection should be included in the routine health evaluation of elasmobranchs, as underlying disease diagnosis in fish can be challenging and blood work adds important information for their precise health status determination , 10, 11.Scyliorhinus stellaris) of the order Carcharhiniformes is a medium-sized oviparous catshark found in the Northeast Atlantic Ocean and the Mediterranean Sea (Scyliorhinus canicula) (The Nursehound Shark (nean Sea . It is cnean Sea . Althouganicula) . In addiin situ studies with free-ranging Nursehound Shark.The objective of this study was to document the main hematological and plasma chemical RV for the Nursehound Shark, as well as possible differences which could be associated with sex and developmental stage, using a population of healthy individuals maintained under human care. This information will allow a more accurate interpretation of blood analytical values and improve the quality of the health evaluations performed in this species. Additionally, it will provide a better understanding and comparison of hematology and plasma chemistry with other studied elasmobranch species. It will also allow for further comparison to All procedures were approved by the Animal Care and Welfare Committee at Oceanogràfic of Valencia and the Generalitat Valenciana under the project reference OCE-22-19.www.oceanografic.org), Finisterrae Aquarium , Bioparc Gijón Aquarium ; Museo Marítimo del Cantábrico , and Sevilla Aquarium .This study was designed as a prospective experimental trial, arranged as a collaboration between five different Spanish aquaria which housed Nursehound Shark: Oceanogràfic Aquarium of Valencia   or subadult  , to examine possible differences in their analytical values. It was not possible to accurately classify animals as sexually mature/immature nor determine if adult females were carrying eggs. Sampled animals were housed in five different aquaria in Spain. We collected blood samples from 94 clinically healthy Nursehound Shark . We determined gender based on the presence of claspers  . We deteSqualus acanthias) when samples were collected after feeding , squid (Loligo spp.), herring (Clupea spp.), or mackerel (Scomber spp.) supplemented with potassium iodide capsules and Akwavit® fish eating animal tablets . Nursehound Shark housed at Finisterrae Aquarium were fed once daily, 3 days per week, with pieces of thawed hake, mackerel, and prawn (Penaus spp.) supplemented with Vetregard® immunostimulants , Vit-amin C and Mazuri® Shark & Ray tablets . Shark housed at Bioparc Gijón Aquarium were fed once daily, 3 days per week, with pieces of thawed hake, squid, capelin , and sprat (Sprattus spp.); feed trays were supplemented with spirulina and Calfostonic® powder . Finally, shark housed at Museo Marítimo del Cantábrico were fed once daily, 4 days per week with pieces of thawed mackerel, Humboldt squid (Dosidicus gigas), and pollock (Pollachius spp.). All aquaria participating in the study maintained a similar feeding intake rate for their Nursehound Shark, ranging from 5 to 10% body weight per week.Environmental conditions varied slightly between aquaria included in the study. In all aquaria, shark were maintained in tanks provided with life support systems which included mechanical filtration (through sand filters), protein skimmers, biological filtration, and disinfection through UV. In all aquaria, ammonia was maintained under 0.01 ppm, nitrite under 0.05 ppm, and nitrate under 100 ppm. Holding systems in Finisterrae Aquarium, Bioparc Gijón Aquarium, and Museo Marítimo del Cantábrico were filled with natural sea water, filtered from the Cantabrian Sea , while Oceanogràfic Aquarium was supplied with natural seawater filtered from the coast of Valencia (Mediterranean Sea) and Sevilla Aquarium was supplied with water filtered from the coast of Cadiz, Spain (Atlantic Ocean). Water temperature, pH, and salinity in the holding systems of Oceanogràfic Aquarium were 18°C, 8.0 and 35.0 ‰, respectively; 18.5°C, 7.9, and 35.2 ‰ in Finisterrae Aquarium; 13°C, 8.0, and 35.0 ‰ in Bioparc Gijón Aquarium; 19°C, 7.8 and 35.6 ‰ in Museo Marítimo del Cantábrico; 18°C, 8.1 and 34.5 ‰ in Sevilla Aquarium. Diet also varied slightly between the different aquaria: Nursehound Shark housed at Oceanogràfic and Sevilla Aquariums were fed once daily, 3 days per week, with pieces of thawed hake , which we determined using microhematocrit tubes, centrifuged at 1,372 g  for 6 min at room temperature 24°C) in a portable centrifuge . We determined plasma total solids (TS) using an analog refractometer . Next, we collected 10 μl of blood and mixed with them with 490 microliter Natt-Herricks solution  to perform RBC and total granulocyte manual counts using a Neubauer improved counting chamber . We performed blood smears using lithium-heparin blood samples, previously homogenized, left to dry at ambient temperature (24°C), and stained using a Diff-Quick stain . We also performed leukocyte differential counts using a portable optical microscope  equipped with an IS E-Plan achromat objective 100 x and immersion oil. We inspected each smear to exclude the presence of platelet and leukocyte aggregates. Leukocyte differentials consisted of counting a minimum of 100 WBC per animal and sample, and the further calculation of cell percentages. Scyliorhinid shark leukocytes have been studied under electron microscopy in previous studies and have been classified into four types according to the structure of their granules °C in a p, 24.After hematology analysis, we centrifuged lithium heparin tubes at 448 g  for 5 min to obtain plasma using the same centrifuge. We collected plasma, transferred it to 1.5 ml Eppendorf tubes, which were frozen at −18°C until processing. We Processed all samples within a maximum of 30 days from collection at the laboratory located at the veterinary clinic of Oceanogràfic Aquarium using a Beckman Coulter® Chemistry Analyzer through spectrophotometry and potentiometry . Plasma chemistry profiles included alkaline phosphatase (ALP) , aspartate aminotransferase (AST) , blood urea nitrogen (BUN) (Urease/GLDH method), calcium (Ca) (oCPC method), total cholesterol (CHO-POD method), chloride (Cl) (ISE indirect), creatine kinase (CK) (IFCC method), gamma-glutamyl transferase (GGT) (IFCC method), glucose (Gluc) (HK G6P-DH method), iron (Fe) (TPTZ method), lactate dehydrogenase (LDH) (IFCC method), sodium (Na) (ISE indirect), phosphorus , potassium (K) (ISE indirect), total proteins (TP) (Biuret method), and triglycerides (Trig) (GPO-POD method).p-value of 0.2 as a cut off value instead of 0.05, following the previously established recommendations for RI determination using small sample sizes  were calculated using a bootstrap method. Because all obtained data were not normally distributed, we used nonparametric statistics, and we report summary statistics as median values throughout the manuscript. We also provide mean values, SD, maximum, and minimum values for every analyte in the tables presented in the results section of this manuscript. We evaluated differences between sexes  and developmental stage (adult vs. subadult) for morphometrical, hematological, and biochemical analytes using a Mann-Whitney U test. We evaluated differences between aquaria and subgroups  using a Kruskal-Wallis test and Dunn's post hoc tests with Bonferroni corrections. In addition, we performed a Spearman's rank-order correlation, and calculated the coefficient of determination (R2) for size  and the rest of the hematological and plasma chemical parameters. We performed statistical analyses using the statistical software package RStudio® . We set statistical significance at p < 0.05. We have expressed results using the Standard International System of Units.We established reference intervals following the recommended guidelines published by the American Society of Veterinary Clinical Pathology (ASCVP), using the RefVal adv. 2.0 Excel addin and the MedCalc® statistical software , 9. We ile sizes . If p ≥ p = 0.817, 0.607, and 0.522, respectively; Mann-Whitney U test). When comparing across subgroups , differences were also not significant .Median weight was 2.40 kg (range = 0.70–7.45 kg), median TL was 79.0 cm (range = 51.0–113.0 cm), median SVL was 38.0 cm (range = 18.0–56.0 cm). A comparison for morphological measurements between female and male sharks is provided in the p > 0.05; Mann-Whitney U test), and we did not detect significant differences when comparing the hematological values obtained from the different aquaria participating in the study . Furthermore, we did not detectstatistically significant differences in hematological parameters between developmental stages (adult vs. subadult)  and across the different subgroups  . Main peripheral blood cells found in the Nursehound Shark are presented in Descriptive statistics for hematology analytes are shown in p < 0.05; Mann-Whitney U test). Plasma total cholesterol was also significantly higher in adult males (median 2.36 mmol/l) when compared to adult females (median 1.57 mmol/l) , but comparison between subadult males (median 2.12 mmol/l) and subadult females (median 1.86 mmol/l) showed no significant differences , thus revealing that differences in cholesterol between sexes were stronger in adult animals. No statistically significant differences were detected when comparing total cholesterol levels between adult males vs. subadult males  nor between adult females vs. subadult females . Except total cholesterol, no statistically significant differences in any other parameter were detected between sexes. Variations in plasma chemical values between aquaria were only statistically significant for ALP . Correlation between size  and biochemical parameters was low (R2 < 0.150). Despite this low correlation, statistically significant differences were found for GGT between adult and subadult shark .Descriptive statistics for blood chemistry are presented in Aquaria allow for the achievement of safe, efficient, and economic study of elasmobranchs, as well as procurement of important information and expansion of knowledge in many areas of elasmobranch biology and medicine , 28.Chiloscyllium plagiosum) (0.13 l/l), aquarium-maintained Smooth Dogfish (Mustelus canis) (0.24 l/l), and free-ranging Bonnethead Shark (Sphyrna tiburo) (0.22 l/l) (Dasyatis americana) (56 g/l) and the aquarium-maintained Spotted Eagle Ray (Aetobatus narinari) (57 g/l) (9 erythrocytes/l) were similar to those obtained in other non-obligate ram-ventilating benthic elasmobranchs such as the Winter Skate (Leucoraja ocellata) (200.0 x 109 erythrocytes/l), and lower than those reported in pelagic elasmobranch species such as the Cownose Ray (Rhinoptera bonasus) (511.2 x 109 erythrocytes/l) and other obligate ram-ventilating Carcharhinid sharks such as the Sandbar Shark (Carcharhinus plumbeus) (402.0 x 109 erythrocytes/l) and the Blacktip Shark (Carcharhinus limbatus) (476.0 x 109 erythrocytes/l) (Median PCV (0.17 l/l) values were within ranges of those reported in previous studies with elasmobranchs, which show typically low values in healthy elasmobranchs and important variations between species (aquarium-maintained Bamboo Shark (.22 l/l) , 29, 30.(57 g/l) , 16, 31.(57 g/l) . Median cytes/l) –34. Captcytes/l) .Torpedo spp.), and the Port Jackson Shark (Heterodontus portusjacksoni) (Rhincodon typus), where FEG have been reported as the most frequent peripheral granulocyte (Ginglymostoma cirratum) have demonstrated phagocytic activity in vitro, and despite immature granulocytes are described as a normal finding in blood smears from clinically healthy elasmobranchs, a left-shift in leukocyte differential counts has been also described in elasmobranchs suffering from infectious and inflammatory pathologies  , 38, 39.nulocyte , 37. Granulocyte , 35. Simnulocyte , 37. Basnulocyte , 13, 30.hologies .Carcharhinus melanopterus) (13.7 x109 cells/l) (Carcharias taurus) and the Tiger shark  (16.3 (4.5–40.5) x 109 cells/l)  were kept within the previously reported values for other elasmobranch species such as the aquarium-maintained Whale Shark (5.1 x 109 cells/l) and the Blacktip Reef Shark (cells/l) , 39, 41.cells/l) , 43. Howcells/l) , 44. Thep < 0.05, Dunn's test, Bonferroni-adjusted). Variations were not significant when compared to Oceanogràfic of Valencia (median 7.8 U/l) and Sevilla Aquarium (median 7.9 U/l). Alkaline phosphatase activity is involved in the mineralization of both bony and cartilaginous skeletons (Carcharhinus leucas) (median 2 U/l), the free-ranging Bonnethead Shark (median 5 U/l), the free-ranging Grey Nurse Shark (median 20 U/l), and the aquarium-maintained Cownose Ray (median 34 U/l)  compared to Museo Marítimo del Cantábrico (5.6 U/l) and Finisterrae Aquarium (median 7.3 U/l)  , 34, 42.median 7. U/l and  34 U/l) , 36, 43.p < 0.05; Mann-Whitney U test). This enzyme, which is present in cell membranes, contributes to maintain intracellular homeostasis and is a physiological biomarker of hepatobiliary disfunction, increasing in sharks with bile accumulation (Prionacea glauca) (0–2 U/l) and the free-ranging Scalloped Hammerhead (Sphyrna lewini) (0–4 U/l) (Median GGT levels were significantly higher in shark classified as adults (0.7 U/l) compared to subadults (0.4 U/l)  .Rhizoprionodon terraenovae) (22.5 U/l) and Bonnethead Shark (39.0 U/l), though very close to those of wild-caught Spiny Dogfish (7.0 U/l) and aquarium maintained Smooth Dogfish (10.6 U/l) . Reported plasma CK values vary greatly between the different studies involving elasmobranchs; median CK was 80.5 U/l in wild-caught southern stingrays, values ranged from 18 to 735 U/l in wild-caught Bonnethead Sharks, low levels (2–13 U/l) were described in Smooth Dogfish under human care, and CK levels under the limit of detection (<4 U/l) were also reported in aquarium-maintained Cownose Rays , 30, 34.0.6 U/l) , 30. In 0.6 U/l) , 30, 34.0.6 U/l) . The men0.6 U/l) . Former 0.6 U/l) , 49. Due0.6 U/l) . Tonic iaenovae) .5 U/l anaenovae) .5 U/l anrhinidae , 51. As aenovae) .5 U/l anSlight non-statistically significant variations in glucose values were detected across aquaria as median levels were higher in Oceanogràfic Aquarium (0.91 mmol/l) than in Finisterrae Aquarium (0.83 mmol/l), Bioparc Gijón Aquarium (0.80 mmol/l), Museo Marítimo del Cantábrico (0.85 mmol/l), and Sevilla Aquarium (0.86 mmol/l). Previous studies have determined that glucose can decrease by 5–7% per hour in unprocessed blood samples due to glycolysis . Variatip > 0.05; Kruskal-Wallis, Bonferroni-adjusted), likely due to the similar environmental and management conditions and equal sampling protocols.Electrolyte values  obtained in this study were similar to those previously reported in other elasmobranchs maintained under human care such as the Smooth Dogfish, the Cownose Ray, and the Southern Stingray , 30, 34.p < 0.01). Important variations in cholesterol and lipoproteins have been described according to species, season, and reproductive status in elasmobranchs, also showing higher cholesterol levels in male Small-spotted Catshark when compared to females , but the difference between subadult males and subadult females was not significant . Similar to cholesterol, variations in plasma triglyceride levels have been reported in elasmobranchs related to sexual maturation, reproduction, and food availability  when compared to females (median 1.71 mmol/l) . Correlation coefficient between weight and triglyceride levels, as well as between triglyceride levels and total cholesterol levels was very low . Caution should be taken when interpreting cholesterol levels in fish, as this molecule is a precursor of steroidal hormones, an increased values have been reported after stressful stimuli such as aerial exposure or anesthesia for procedures over 5 min .Study design: PM-E and DG-P. Data acquisition: PM-E, CR-S, TA-A, and MV-T. Data processing: PM-E and CS. Preparation of the manuscript: PM-E. Revision of the manuscript: DG-P, CR-S, and TE. All authors have read and agreed to the published version of the manuscript.Funding for this study was provided by the Complutense University of Madrid Predoctoral Contract  and the Fundación Oceanogràfic (Under the project reference number OCE-22-19).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The triple pelvic osteotomy is an established surgical method with multiple modifications regarding surgical technique and choice of implant. The stability of the osteotomy is affected by numerous factors, and among these, the three-dimensional implant configuration is a scientifically less explored aspect.We used a finite element model of a hemi-pelvis with a standardized triple osteotomy to calculate relative flexibility for loads in all translational degrees of freedom for five different implant configurations. Two of the configurations used entry points only feasible when implant removal was not necessary.The stability of the osteotomy improved with an increased distance between the implants in the plane of the osteotomy as well as for a more perpendicular angle relative to the osteotomy plane. The implant configurations with more entry points available made this easier to adhere to.The use of bioabsorbable implants may provide better opportunities for optimal implant constructs which can, to a certain degree, compensate for the lesser mechanical stiffness of bioabsorbable polymers as compared to metal implants.The online version contains supplementary material available at 10.1186/s13018-022-03169-3. The triple pelvic osteotomy (TPO) is a re-directional osteotomy normally used in children over the age of 6–8 years old when the symphysis has become too stiff to allow a Salter osteotomy. The procedure is also an alternative to periacetabular osteotomies (PAO) in adults. Numerous modifications of the TPO have been proposed over the years with the Steel, Tönnis, and Carlioz modifications being the most well-known –3.As in a Salter osteotomy, the TPO entails a cut of the ilium, and in addition also osteotomies of the ischial and pubic bone. Since the entire acetabulum is mobilized, the TPO is inherently more unstable than most other pelvic osteotomies. Numerous different methods of fixation have been used for the iliac osteotomy, including K-wires, Steinmann pins, and screws and plates –6. RecenThe research regarding pelvic osteotomies has mainly been focusing on modifications of the osteotomies or type of implant used rather than how the positioning of the implants affects the stability of the osteotomy. The classic papers presenting the surgical method of a TPO or Salter osteotomy go into little detail regarding the exact placement of the implants. Salter simply states that “a stout Kirschner wire is inserted across the osteotomy site, through the graft and into the distal fragment posterior to the acetabulum in order to prevent any subsequent shift of the graft or of the distal fragment” . Tönnis . who performed a cadaver-based study comparing the stability of three different screw configurations for a Tönnis triple osteotomy and a PAO [Traditionally, the implants have been inserted from the iliac crest, in or close to the harvest site of the bone graft. The use of bioabsorbable screws, negating the need for implant removal, enables the surgeon to place the screws from any location that can be reached from the surgical approach . Even thnd a PAO . In thisi.e., entry points, directions, and implant lengths), virtual models of mechanical systems can be used. The finite element (FE) method is a mathematical model to numerically solve partial differential equations that describe mechanical systems in fields such as structural analysis, heat transfer, and fluid flow [. performed an extensive review on FE analysis of acetabulum pressure related to surgery of developmental dysplasia of the hip [To further evaluate the stability of an osteotomy for different implant configurations  to the implant beam elements. These nodes are highlighted as white dots in Fig. The implants were modeled using beam elements with a circular cross section of 4.5 mm in diameter and using an isotropic linear elastic material model with a Young’s modulus of 5 000 MPa.*Contact Pair, Surafce_To_Surface, with friction coefficient 0.2), as suggestedTo model the boundary conditions, the ilium was constrained in all translational and rotational DOF at the SI joint surface, see Fig. Pelvis loading was applied at the center of the femoral head, for one direction (X/Y/Z translation or X/Y/Z rotation) at a time, using prescribed unit displacements. The load, 1 mm translation or 1 degree rotation, was distributed over the lunate surface of acetabulum  1–6).Finally, to be easily comparable to the work by Yassir et al., stability of the virtual osteotomy was evaluated in terms of mechanical flexibilityFor the present simulation, five implant placement configurations, corresponding to five different FE models, were chosen to represent different clinically feasible fixation solutions of the osteotomy Table . In confFigure Averaging the relative translational flexibility in Fig. The FE method has been proven to be useful for postoperative modeling of different orthopedic applications and has been used to evaluate different fixation methods for PAO , 27, 28.In this study, we present a FE analysis of how screw placements affect the stability of a standardized TPO. Model E, with the most entry points available, clearly had the best results from a stability point of view. Model C, with a distally placed screw, also showed high stability, but placing a screw from the acetabular fragment has drawbacks. Firstly, extraction of a screw (or pin) from this entry point can be challenging, secondly the antero-lateral corner superior to the acetabulum is small and can potentially break, and thirdly implants in this location may also interfere with hip abduction. In the other models with all screws placed from the superior fragment , the option to place the screws from more entry points (D) outperformed the models with less entry points available (A and B).Adamczyk et al. used an in vitro model to compare the stability of bioabsorbable PLLA and metaIn the present study, we have not used physiological loads but rather focused on a generic prescribed displacement of 1 mm or 1 degree in a given direction. The displacements for different fixation methods are in the same order of magnitude as found by Yassir et al.   and/or a postero-medial dislocation of the distal fragment (X− in our model). A rotation around the X-axis affects the ante/retroversion of the acetabulum with both being undesirable but an increased retroversion even less so. A displacement in both X− and Y− negates the correction achieved and means that the distal fragment may lose the hinge of cortical bone in the infero-medial corner of the osteotomy.Due to the complexity of the three-dimensional shape of the pelvis, the theoretically possible entry points and subsequent angles and lengths of an implant are innumerable and the modeling had to be restricted to study only a few clinically feasible solutions. Our study does not present reference values but intend to serve as a guide for surgical planning of TPO by outlining factors affecting stability and support. Moreover, our study was performed with a singular type of implant, corresponding to a 4.5-mm resorbable 85L/15G PLGA screw. Compared to commonly used 2-mm-steel K-wires, the 4.5-mm bioabsorbable screw has 60% less bending stiffness. Thus, for loading directions where the implants are mainly subjected to bending forces, e.g., in the X- and Y-directions, the joint overall flexibility should be comparable. For loading directions where the implants are mainly subjected to axial loads, (Z-), a 2-mm K-wire is almost 10 times stiffer than a 4.5-mm bioabsorbable screw, but this is of less clinical relevance. Also, lacking threads, the K-wire cannot fully utilize its axial stiffness, so the pull-out strength of a 4.5-mm bioabsorbable screw remains superior. Depending on the specific type of implant, the absolute magnitude of the results will vary to some extent, but the relative stability/flexibility between different implant configurations is expected to be consistent.Although the study demonstrated the comparison of different fixation systems, the modeling had its limitations. Similar to other established finite element models of the pelvis , the impUsing a similar motivation, we have not performed any mesh converges study. Since flexibility (or stiffness) relates to the displacement while stress/strain relates to the derivative of the displacement, structural flexibility will converge much faster than stress/strain. Therefore, mesh convergence studies are of greater importance for stress or strain analyses. A relevant example is presented in the ABAQUS (2016) user guideline, where even a very coarse mesh consisting of just 14 elements gives a stiffness prediction within 3% of the accurate result, while the stress at the critical area is not converged even using the finest mesh consisting of 1800 elements.Moreover, the individual shape of the pelvis may vary greatly, affected by sex, age, and as shaped by the underlying condition necessitating surgery. The shape of the pelvis should not, however, greatly affect the relative stability between the screw configurations. Nevertheless, additional studies are warranted to evaluate the applicability of our findings in clinical practice. While virtual models cannot completely replace experimental testing, they provide a valuable tool that is rapidly evolving.In a FE model of a TPO, different implant placement configurations had a substantial impact on the stability of the osteotomy. As predicted, a greater spread of the implants over the osteotomy and configurations more perpendicular to the osteotomy improved the stability. The findings suggest that bioabsorbable screws in TPO, enabling a higher degree of freedom when selecting entry points, provide superior preoperative stability as compared to TPO with only traditional entry points.Additional file 1: Appendix 1. Tabulation of all results from the FE simulations.


The dependence of IC50 on the substrate concentration allows us to distinguish between non-competitive inhibition  and oxy-tyrosinase (Eox), 50 value.Tyrosinase is the enzyme involved in melanization and is also responsible for the browning of fruits and vegetables. Control of its activity can be carried out using inhibitors, which is interesting in terms of quantitatively understanding the action of these regulators. In the study of the inhibition of the diphenolase activity of tyrosinase, it is intriguing to know the strength and type of inhibition. The strength is indicated by the value of the inhibition constant(s), and the type can be, in a first approximation: competitive, non-competitive, uncompetitive and mixed. In this work, it is proposed to calculate the degree of inhibition ( Possible types of inhibition:In case B, there is no ambiguity between the types of inhibition. It would be a non-competitive inhibitor with f IC50NC . The conStep 6. Solution of the ambiguity between competitive inhibition and mixed type (1). Two o KI1app , in the nd KI2appB. These Step 7. Solution of the ambiguity between uncompetitive and mixed type (2) inhibition. Mushroom tyrosinase (3130 U/mg) was purchased from Merck Life  and purified as previously described . ProteinBenzoate, cinnamate and L-dopa were purchased from Merck Life . Stock solutions of substrates were prepared in 0.15 mM phosphoric acid to prevent auto-oxidation.Absorption was recorded in a visible-ultraviolet PerkinElmer Lambda 35-spectrophotometer , online interfaced with a compatible laptop. The temperature was maintained at 25 °C. Kinetics assays were also carried out with the above instruments by measuring the appearance of the products in the reaction medium. The activity on L-dopa was measured at 475 nm .Initial rate  or constants (mixed). When the substrate concentration is varied, keeping the inhibitor concentration fixed, the non-linear regression analysis of the corresponding equation provides the values of Non-linear regression analysis  of the iThe REFERASS computer program was used to obtain the rate equations of these mechanisms in the presence of inhibitor or of an alternative substrate .Agaricus bisporus. Input protein structure for docking was prepared by adding all hydrogen atoms and removing water molecules. The met and oxy forms of tyrosinase were built as previously described [Molecular docking of the ligands was studied in the active site of mushroom tyrosinase. Their chemical structures were built from chemical structures obtained from the PubChem Substance and Compound database  Table S. The molescribed . Gasteigescribed ,63. Autoescribed . Other described . PyMOL 2escribed . Dockingescribed .50 parameter and its quantitative relationships with the apparent inhibition constants are highlighted. Thus, from the IC50 value, the inhibition constant for a competitive, non-competitive, or uncompetitive inhibitor can be determined when the inhibition mechanism is confirmed. In the case of mixed inhibition, the The study of tyrosinase inhibition is very important due to the multiple applications it can have in pathological processes of pigmentation, such as the browning of fruits and vegetables. The importance of the IC


Despite no significant difference in anteroposterior and mediolateral torso kinematics, vector magnitudes were significantly greater  when the stance was performed off-ice. The increased vertical acceleration observed when goalies performed the movement off-ice could be due to a failure to maintain adequate posture without the support of the external load. The results of this study may help inform off-ice training interventions for ice hockey goalkeeping.In ice hockey, the butterfly style/stance is a technique distinguished by the goalkeepers  dropping to their knees to block attempts to score. Although this goalie style has been around for many years, comparisons between on and off-ice attire has not been undertaken. Therefore, this preliminary study compared differences in torso acceleration and energy expenditure by way of the Metabolic Equivalent of Task (MET) during off-ice and on-ice butterfly stances/saves. Seven participants each performed 8 on-ice butterfly saves/stances whilst wearing full hockey attire followed by 8 off-ice butterfly stances without wearing full hockey attire whilst torso acceleration was collected. The off-ice movement significantly increased vertical torso acceleration ( Ice hockey is a high intensity sport that requires versatile biomechanical demands for athletes. Successful performance in ice hockey necessitates intervals of maximal physical strength and power ,2 and enAn ice hockey goalkeeper is usually positioned in a designated area in front of the net to cover the goal. The goalkeeper then applies different save techniques in order to prevent the opponent from scoring. Led by Patrick Roy, a then new technique brought the game to its knees by introducing a more effective way of goalkeeping . This teDuring a regular National Hockey League (NHL) game, a goalkeeper makes about 45 saves in a full or half butterfly position  and dropThe testing of on-ice and off-ice parameters is fundamental to determine the specific performance of ice hockey players. In preceding studies, off-ice performance  were analyzed to determine the relationship to hockey playing ,13. BecaAccording to the scientific literature, the total game time for ice hockey players, excluding goalkeepers, is approximately 15 to 25 min, depending on the position of the player ,14. In c1 min1. Despite the prevalence of the MET, use of the MET metric in ice hockey is minimal. Such information could be useful for coaches when comparing off-ice training to on-ice requirements given the evidence that off-ice training may have a motor transfer effect to on-ice agility [Physical activity refers to bodily movements produced by skeletal muscles that expends energy . The amo agility . Existing research has primarily examined hip kinematics during the butterfly save  in an atA common method used to facilitate a post-activation potentiation response is performing sport specific movement patterns with added resistance that provides additional resistance to movement. An ice hockey goalkeeper can expect their on-ice attire to weigh approximately between 14–20 kg (dry weight)  which maIn recent years, new technologies such as inertial sensors based on Micro Electro Mechanical Systems (MEMS) have been developed, which have low production costs, are small in size and have the ability to measure kinematics over large periods of time ,23,24. Mn = 7) semi-professional male ice hockey players participated in this preliminary study  having been approved by the University Research Ethics Committee (HREC H21114). All of the ice hockey goalkeepers included in the study were training at least once per week for a duration of two hours or more that included on and off-ice activity. Additionally, all of the goalkeepers were playing competitively (defined as an on-ice game against opposition whereby points are gained for the winning team) at least once per week. The participants had at least two years of both on and off ice training including butterfly technique training. Goalkeepers were excluded from the study if they had any injury (acute or chronic) or illness at the time of the test that prevented them from exerting maximum effort. Only players  over the age of 18 were asked to participate in this study. Before participation in the study, written informed consent was obtained from all of the goalkeepers. Seven  or Ice Hockey Victoria (IHV). None of the goalkeepers reported any current or ongoing neuromuscular diseases or musculoskeletal injuries specific to the ankle, knee, or hip joints, and no participant reported taking any dietary or performance supplements that might be expected to affect performance during the study. All the participants were fully accustomed with the procedures used in this research and were informed that they could withdraw from the study at any time without penalty.This was a randomised and crossover study that was performed in an on-ice and off-ice environment. The study design consisted of two parts that included participants randomly drawn into either an on-ice or an off-ice group. Therefore, four of the goalkeepers performed the butterfly stance on-ice while the remaining three goalkeepers performed the stance off-ice. Once completed, the participants changed groups in order to complete the protocol in either an on-ice or an off-ice environment.For the on-ice component of the study, the goalkeepers were required to complete 8 on-ice deep blocking butterfly stances while wearing characteristic on-ice goalkeeping attire. On-ice hockey apparel consisted of helmet, shoulder pads, padded gloves, padded shorts, shin guards, elbow pads, chest protector, athletic cup groin protector and hockey skates. After an acoustic countdown  participants performed the butterfly technique. For the off-ice component of the study, the goalkeepers were once more required to complete 8 deep blocking butterfly stances while wearing part hockey attire that included shin guards and padded shorts. Thus, data was obtained on-ice (with attire) and off-ice (without full attire) . A duratAerobic metabolism is required for goalkeepers to recover between short and high intensity periods. This is to maintain an alert and ready position for long periods of time, and to make numerous saves during short intense duration . This shThe off-ice tests were performed in a modified indoor setting complete with gymnasium landing mats. The modified gymnasium was 5 × 4 m. The use of landing mats was necessary in order to limit potential injuries during the off-ice tests as participants would have been required to land directly onto solid flooring. To further reduce the risk of injury off the ice, the participants retained lower shin guards and padded shorts whilst performing the butterfly stance to enable safe landing. All participants performed the off-ice tests without footwear. The synthetic rink was 6 × 4 m and was made from ultra-high molecular weight polyethylene (UHMWP) sheets . Each sheet was 13 mm in height (thickness) and assembled into interlocking pieces. The testing procedure used a standardised sequence in that the day started by measuring anthropometric  and personal data . All of the participants were measured using traditional digital scales  with and without hockey attire in order to obtain the weighted mean. A Sportline 240 Econosport stopwatch  was used to monitor time which was manually recorded by the principal author. To limit any outside factors that could potentially change the motion of the participant, data was collected in one day with both tests conducted from 0900–1200. The tests were performed during ambient conditions . The participants were cooled with two vertical standing pedestal fans. Advances in small and precise accelerometers provide real-time detection of motion . Thus, aAlthough primarily a physics term, the centre of mass  is a theoretical point and an indicator of a body’s position relative to the centralized point of mass. Although the relative position of the CoM will depend on the gender and the location of the limbs, when in the anatomical position the CoM and centre of gravity lies approximately anterior to the second sacral vertebra ,27. Spec2. The device was secured to the participants by double sided tape between the L5 and S1 spinous process [The quantification of torso motion can be inferred by the relative amount that the CoM accelerates. This acceleration is made by data output that captures acceleration magnitude in three orthogonal axes where vertical , mediolateral  and anteroposterior  are aligned with anatomical axis and planes of motion. As acceleration is the derivative of velocity and velocity is the derivative of position with respect to time, trunk acceleration can be presented as meters per second per second, or m/s process ,29 [Figu process .2, was determined over the 8 butterfly stances. Thus, the numeric of counts and raw acceleration data are presented. The ActiGraph accelerometer records activity counts in a given time interval, for example, half-minute or minute intervals. Thus, observations are expressed as activity counts per 60 s. Counts are a consequence of summating post-filtered accelerometer values (raw data at 100 Hz) into epoch “chunks.” The value of the counts will vary based on the frequency and intensity of the raw acceleration relative to the goalkeeper’s movement.The ActiGraph device was controlled wirelessly from the principal author via a typical Hewlett Packard PC using the ActiLife software program . Only the local acceleration components of the participants were analysed. For repeatability, each participant was assigned a device which was subsequently used for both tests. For each test, the mean torso activity counts and raw acceleration magnitude, the latter expressed in m/s1 value for the on-ice and off-ice butterfly stances. This method for establishing counts/min1 ensured the activity monitor and metabolic measurements were synchronized for each second of data collection. Triaxial acceleration data collected from the ActiGraph was converted to activity counts through a process called actigraphy, which results in an interpretable and objective measure of physical activity . The acc1 (corresponding to 1 MET). The MET results are presented for the on and off-ice butterfly stances with direct comparisons with the Compendium code that was used and the corresponding MET value.For comparison with the Compendium of Physical Activities , the MET2 + y2 + z2. Analysis was performed in the time domain with the mean of each epoch analysed. Prior to both tests, the sensor was initialized according to manufacturer instructions. Concerning the magnitude of triaxial acceleration, this was a result of summing post-filtered accelerometer values (raw data at 100 Hz) into epoch “chunks” via an ActiLife GT3X file and an AGD file. From here, raw data was converted into a CSV file and manually filtered into epochs based on the frequency and intensity of raw acceleration. The vector magnitude incorporated the vertical, anteroposterior and lateral axes and refers to the length and size of the vector. Thus, vector magnitude was calculated where VM = √ xThe normality of appropriate data sets was confirmed by the Shapiro–Wilks test using the Analyse-it statistical package . The assumptions of normality, linearity, and homogeneity were satisfied. Means and standard deviation (±SD) were analysed for the local acceleration components. Despite the small sample size of participants, parametric tests were deemed appropriate given the assumption of a normal distribution. In contrast, most nonparametric tests use some way of ranking the measurements of the distribution , which wt-test was used to assess differences between on-ice (Test 1) and off-ice (Test 2) with a modified Cohen’s d calculated to assess effect sizes and interpreted according to established guidelines where 0.1–0.3 , greater than 0.3–0.5 (moderate), greater than 0.5–0.7 (large), greater than 0.7–0.9 (very large) and greater than 0.9 (extremely large) [t-test was used to compare temporal raw magnitudes of acceleration with the MET values. Regression analysis was performed to analyse the relationship between accelerometer counts and the magnitude of triaxial torso temporal accelerations with the MET. The rationale for this approach was to describe and estimate the association between the magnitude of temporal torso accelerations to the MET in both on and off-ice environments. Therefore, a coefficient of determination (r2) was performed along with two predictive equations that represented on and off-ice environments. Post-hoc processing was completed using raw accelerometry data to assess individual differences in the magnitude of torso acceleration between off-ice and on-ice butterfly stances. A range of ActiGraph counts were considered as cut-points for defining intensity levels. The method of selecting an optimal cut-point was based on two criteria: (1) correct identification of the target or higher intensity activities ; and (2) correct exclusion of lower intensity activities . In both tests, means were compared using a priori of 0.05.The student’s y large) . A paireConcerning the magnitude of torso acceleration, post-hoc processing revelated that inter individual differences were greater when the butterfly stance was performed off-ice as evident by the wider dispersion of ±SD. Significant differences and extremely large effects were detected in the vertical and mediolateral channels .t-tests (Results in the paired t-tests  indicatep < 0.001). Overall, the MET for the vector magnitudes for the on-ice and off-ice butterfly stances did not significantly differ to the Compendium values.The measured MET and the Compendium standard MET are presented in r2 value for a line of best fit was 0.92 . The best fit line produced the following equation:The combination of temporal magnitudes of torso acceleration for the on-ice butterfly technique resulted in a combined intensity value. In this case, the magnitude of acceleration was summed together. The r2 value for a line of best fit was 0.49 . In this instance the line of best fit produced the following equation:Equally, the grouping of temporal magnitudes of torso acceleration for the off-ice butterfly technique was summated. The The present study aimed to compare differences in torso acceleration and the MET during the butterfly stance with and without attire in off and on-ice environments using a triaxial accelerometer. We hypothesized that significant differences would occur in on-ice and off-ice performance in vertical and anteroposterior magnitudes of torso acceleration, as well as a low relationship between the MET in on-ice and off-ice environments. Our study used a randomized and crossover design methodology to understand the differences in longitudinal, mediolateral, and anteroposterior torso accelerations and the associated levels of energy expenditure. Moreover, data capture occurred in-the-field using unobtrusive sensor technology. p = 0.0097, d > 1). This partially confirms our hypothesis that significant differences would occur in vertical acceleration of the torso. The impact of the butterfly stance was assessed through off and on-ice measures that involved performance of a characteristic butterfly stance with and without typical full goalkeeping attire that is worn during on-ice practice and competition. The results from the current study demonstrated that the magnitude of vertical acceleration of the torso was significantly greater when the goalkeepers performed the butterfly save off-ice and without full goalkeeping attire  attains a steady level which can be maintained for a long period of time. In contrast, anaerobic power and capacity are two factors that are well known to be of critical importance in hockey performance [The results of the current study demonstrated that the MET was greater when the butterfly stance was performed off-ice. In particular and relative to the Compendium code, the activity for competitive ice hockey has a MET of 10 (code 15362). Despite our MET values being marginally higher compared to the Compendium, overall results remain consistent with others. For instance, our results are consistent with Bassett et al.  who founformance .The goalkeeper’s actions are characterized by fast, repetitive, and explosive movements of short duration with periods of lower intensity actions or rest . A potenAbdominal endurance is fundamental because it allows for good stability in order for players to perform fast velocity actions . In our n = 7) of semi-professional goalkeepers. Of these goalkeepers, a wide dispersion of age ranges was seen (36 ± 10 years), suggesting variable aerobic, anaerobic, and kinematical differences between the participants. Normally, professional male goalkeepers are significantly older than amateurs, which can be explained in that biological maturity is a predictor utilized during hockey camp selections to determine which players will access higher playing levels [Our preliminary study included a small sample size  to on-ice skating acceleration . Whilst Given the small number of participants in this study, the results cannot be generalised or inferred to a wider population. Another limitation is that the participants consisted of semi-professional ice hockey goalkeepers and not elite nor professional players. This might potentially lead to biased comparisons because results from this study are not specifically comparable to goalkeepers from the NHL. However, in the current literature comparisons between on-ice and off-ice kinematical capacity assessments in goalkeepers are lacking. Goalkeepers were required to perform off-ice stances in a modified gymnasium setting. This included the use of landing mats to limit potential injuries as participants would have been required to land directly onto solid flooring. Although the landing mat may have provided cushioning when goalkeepers dropped to their knees during the save, this is representative of a typical training situation. Therefore, future research should apply specific criterion for defining the experimental protocol in order to correctly categorise the ability elite, semi-professional, recreational and junior goalkeepers. Testing protocols should also reflect the demands of on-ice competition experienced by the experimental population. Additionally, a continued focus on current strength and conditioning off-ice data when training with and without the addition of equipment will provide a more in-depth understanding of the ever-changing demands of performing the butterfly stance that will in-turn shape the direction of future research. For example, training off-ice with different weighted loads could assist in retainment of movement that matches on-ice energy expenditure, as measured by activity counts. In summary, the results gained from this study could serve as benchmark values for strength and conditioning specialists when they work with goalkeepers of different performance levels. In summary, the present study explored the effects of a goalkeeper’s ice hockey attire related to kinematical aspects of the butterfly technique and the relative difference between activity counts and the MET. The off-ice butterfly stance resulted in greater vector magnitudes predominately due to larger extents of vertical acceleration. Coaches should be encouraged to teach goalkeepers’ how to perform the butterfly technique prior to on-ice training. Drawing on the results of this study, strategic interventions could aim at improving postural stability through development of refined torso and CoM movement control. The increased vertical acceleration observed when the goalkeeper’s performed the movement off-ice could be due to a failure to maintain adequate posture without the support of the external load. The results of this study may help inform off-ice training interventions for ice hockey goalkeeping. The results from this preliminary study found that the off-ice butterfly stance resulted in greater vector magnitudes predominately due to larger extents of vertical acceleration. This was reflected in the differences between the MET given that a greater MET occurred off-ice compared to on-ice. This was observed despite participants not wearing upper body attire during the off-ice movement. Determining the relationship between the MET, the magnitude of torso acceleration and the on-ice and off-ice test, it may be possible to establish a range of kinematic and physiological responses that are desirable to determine the physical performance of ice hockey goalkeepers. However, caution is needed when extending our results to elite ice hockey goalkeepers. Continued research should aim at refining and developing new accelerometry procedures and field-based testing methods. This will enable practitioners to identify goalkeepers susceptible to increased and undesired METs and greater magnitude of torso acceleration in order to improve specific training strategies.