The selective assembly of Kv subunits into one of four subfamilies of tetrameric, voltage-gated potassium channels is mediated by the T1 tetramerization domain. Here, it was found that unlike the other Kv T1 domains that have been studied to date, the human Kv2.1 T1 domain forms a pentamer, and that zinc binding and electrostatics contribute to the stability of the proteins. The Kv family of voltage-gated potassium channels regulate neuronal excitability. The biophysical characteristics of Kv channels can be matched to the needs of different neurons by forming homotetrameric or heterotetrameric channels within one of four subfamilies. The cytoplasmic tetramerization (T1) domain plays a major role in dictating the compatibility of different Kv subunits. The only Kv subfamily lacking a representative structure of the T1 domain is the Kv2 family. Here, X-ray crystallography was used to solve the structure of the human Kv2.1 T1 domain. The structure is similar to those of other T1 domains, but surprisingly formed a pentamer instead of a tetramer. In solution the Kv2.1 T1 domain also formed a pentamer, as determined by inline SEC–MALS–SAXS and negative-stain electron microscopy. The Kv2.1 T1–T1 interface involves electrostatic interactions, including a salt bridge formed by the negative charges in a previously described CDD motif, and inter-subunit coordination of zinc. It is shown that zinc binding is important for stability. In conclusion, the Kv2.1 T1 domain behaves differently from the other Kv T1 domains, which may reflect the versatility of Kv2.1, which can assemble with the regulatory KvS subunits and scaffold ER–plasma membrane contacts. The cells were harvested by centrifugation and stored at −80°C. The harvested cells were resuspended in lysis buffer  and supplemented with DNase I and protease-inhibitor cocktail (Roche). The cells were disrupted using an Emulsiflex C3 (Avestin) to release the protein. The His-tagged fusion protein was purified using an Ni–NTA column (Qiagen) and the elution fraction was incubated with TEV protease for 15–18 h at 4°C to cleave the N-terminal His tag. The cleaved protein was further purified using a HiLoad 16/600 Superdex 200 pg gel-filtration column  in 20 mM Tris–HCl pH 8.0, 150 mM NaCl, 5% glycerol, 0.01% Triton X-100, 1 mM DTT. The purified protein was concentrated using Amicon Ultra centrifugal filter devices . The protein samples for dynamic light-scattering (DLS) and circular-dichroism (CD) measurements were dialyzed into phosphate buffer  at 4°C overnight before use.Human Kv2.1 residues 29–147, corresponding to the T1 domain, were cloned using NdeI and BamHI sites in a modified pET-28a vector (Novagen) to express a fusion protein with an N-terminal hexahistidine (6×His) affinity tag followed by a Tobacco etch virus (TEV) protease cleavage site (ENLYFQG). Transformed 2.2.−1 Kv2.1 T1 with the reservoir solution in a 1:1 ratio. Initial crystals were obtained at 291 K in 0.2 M magnesium chloride, 15% PEG 400, 0.1 M sodium HEPES pH 7.5. The crystals were flash-cooled in liquid nitrogen using 30% PEG 400 as a cryoprotectant (see PDB entry 7spd). Crystals of Kv2.1 T1 prepared by supplementing the size-exclusion buffer with 50 µM zinc sulfate were obtained in 0.2 M magnesium chloride hexahydrate, 0.1 M Tris pH 8.5, 3.4 M 1,6-hexanediol and were flash-cooled in liquid nitrogen without additional cryoprotection (see PDB entry 7re5). X-ray diffraction data were collected at 100 K on beamline 4.2.2 at the Advanced Light Source (ALS), Berkeley, California, USA.Kv2.1 T1 was crystallized using the hanging-drop vapor-diffusion method by mixing 8 mg ml2.3.XDS . The buried surface area was determined using the PISA server . The elution trajectory was redirected into the SAXS sample-flow cell. Scattering data were collected every 1 s using 0.5 s exposure on a PILATUS3 1M pixel detector (Dectris) covering a q range of 0.0045 < q < 0.35 Å−1 . The BioXTAS RAW software was used to collect the SAXS data  and the radius of gyration (Rg) were calculated from the Guinier fit. The normalized Kratky plot, the pair distance distribution plot P(r) and the Porod volume were calculated using GNOM embedded in BioXTAS RAW. Low-resolution ab initio bead-based models of proteins were constructed from the experimental data using GASBOR  between the simulated and experimental scattering curves.Following data reduction and buffer subtraction, the SAXS data were further analyzed using the 2.6.M Kv2.1 T1 was added to a 1 mm path-length quartz cuvette . The CD ellipticities (θ) were measured from 200 to 260 nm with a 1 nm increment, a scanning speed of 50 nm min−1 and a data-integration time of 2 s with a standard sensitivity. Buffers were used for baseline measurements. The final ellipticities were recorded as an average of four baseline-corrected scans. The ellipticities (θ) were used to calculate the mean residue ellipticity using the formula [θ] = θ/cnl, where c is the concentration of the protein in moles, n is the number of residues and l is the path length of the cuvette. To examine the effect of Zn2+ on the stability of Kv2.1 T1, 1 mM EDTA was added to Kv2.1 T1 samples and incubated at 4°C for 12 h, and the far-UV CD spectra were then measured in the same way as for the samples in the absence of EDTA. To detect the thermal unfolding, denaturation experiments were carried out using an automated 1°C incremental temperature ramp in the interval 20–95°C, with a 30 s equilibration time at each measurement step. The thermal unfolding profiles of the samples were characterized using the mean residue ellipticity minimum at 222 nm (θ222) to determine Tm by fitting the Boltzmann sigmoid equation using Prism (GraphPad).Far-UV CD spectra of Kv2.1 T1 were acquired using a J-815 spectrometer (JASCO) connected to a Peltier temperature controller. All experiments were performed in duplicate. For each measurement, 300 µl sample consisting of 20 µ2.7.−1. Before measurements, the sample was incubated at 10°C for 3 min. The DLS measurements were averaged from five acquisitions of 1 s each. To examine the effect of Zn2+ on the stability of Kv2.1 T1, 1 mM EDTA was added to Kv2.1 T1 and incubated at 4°C for 12 h and the size was then measured by DLS and compared with that of samples without adding any EDTA. The DLS data were analyzed using Wyatt software and were fitted by the linear intersection method to determine the Tonset values.Thermal ramp stability measurements were made by DLS in a plastic cuvette with 1 mm path length at various temperatures ranging from 10 to 80°C, with a ramp rate of 1°C min2.8.−1 was added to a glow-discharged carbon-coated copper grid (Electron Microscopy Science). The grids were stained in 2%(w/v) uranyl acetate . The grids were blotted with filter paper (Whatman) to absorb residual solution between each step.For negative staining, 3 µl protein solution at 0.01 mg mlcryoSPARC to determine the oligomeric state of Kv2.1 T1  were submitted to the  al. 2022. Briefly3.3.1.E. coli BL21 (DE3) cells and purified by Ni–NTA chromatography followed by TEV cleavage to remove the N-terminal His tag and finally Superdex 200 chromatography. Crystals of Kv2.1 T1 belonged to space group P41212. Initial phase estimates were obtained by molecular replacement using a Kv3.1 T1-domain monomer (PDB entry 3kvt) as a search model, and the resulting Kv2.1 T1 structure was refined at a resolution of 2.5 Å (PDB entry 7re5). Electron densities for residues 29–133 were identified; the missing C-terminal residues (134–147) may be disordered. Surprisingly, five protein molecules were present in the asymmetric unit of the Kv2.1 T1 crystals, forming a pentameric ring, as shown from the C-terminal (top) and side views . Diffraction data and refinement statistics are presented in Table 1X5CX20CC motif. The Kv2.1 T1 pentameric rings were stacked as dimers in the crystal and a closer examination of the interface between rings revealed a second metal ion coordinated by residues introduced by the N-terminal NdeI cloning site. Representative electron densities and verification of the metal as Zn2+ by X-ray fluorescence spectroscopy are shown in Supplementary Fig. S1. We also obtained a lower resolution (2.7 Å) structure in the alternative space group C2221, which was also a pentamer but with partial zinc occupancy . The core BTB fold of the Kv2.1 T1 monomers is similar to the monomers of the pentameric T1-like domain from KCTD5  and the tetrameric Kv4.2 T1 domain . The five Kv2.1 T1 subunits are rotated from the central axis of the ring by 70.5°, 71.3°, 71.5°, 73.2° and 73.5°, which is more open than the arrangement in KCTD5 . Overall, the pentameric ring of Kv2.1 T1 subunits is less compact than that for KCTD5.The T1 domain of voltage-gated potassium channel Kv2.1  was expressed in D5 Fig. 1c and thin Fig. 1d. The fa). Electrostatics make a particular contribution to the N-terminal portion of the T1 domain, with a positively charged bulge fitting into a negatively charged concave area on the adjacent subunit. The residues constituting the negatively charged bulge are highly conserved across the T1 family. However, the residues constituting the matching positive patch are less conserved and thus may contribute to the selectivity of T1-domain assembly .The surface of the interface between adjacent Kv2.1 T1 subunits is characterized by complementary charges Fig. 2a. Electly Fig. 2b.2+ binding and CDD motifs  that assemble with Kv2 proteins. This motif, occupied by Zn2+, is at the C-terminal end of the T1 domain, which in the context of the full-length protein would be the end closest to the transmembrane domains. In Kv2.1 it is formed by His105, Cys132 and Cys133, with the third cysteine, Cys111, provided by the adjacent subunit . The CDD motif is found in a loop between α3 and β3 at the other end of the subunit interface from zinc . Asp74 and Asp75 of this motif are in the interface and form a salt bridge with Arg31 and Arg32. The arginines are only conserved in Kv2.1 and Kv2.2. Together, these two motifs are well positioned to provide stability to higher-ordered assemblies of Kv2.1 T1 domains. Functional Kv channels are tetramers, and we asked whether these two motifs could still be involved in stabilizing either homotetrameric or heterotetrameric assemblies of Kv2.1 T1 by examining the interface of models generated using YASARA or AlphaFold2. It is likely that Zn2+ binding increases the stability of all forms, while the CDD motif should make Kv2.1 homodimers or homotetramers more compact and potentially more stable .Two motifs that have previously been implicated in selective higher-order assembly of T1 domains are the Zn3.2.a). The MALS data are most consistent with a pentameric assembly of Kv2.1 T1. From the SAXS data, it was determined that the Kv2.1 T1 protein was monodisperse (linear in a Guinier plot) and globular (with a Gaussian peak in a Kratky plot) . The experimental scattering curve of Kv2.1 T1 was fitted to a tetrameric homology model of Kv2.1 T1 generated using YASARA , but the fit was very poor, with χ2 = 27. Fitting the experimental scattering curve to the pentameric crystal structure resulted in a good fit with χ2 = 1.7 . To aid in visualization, the calculated surface envelope for Kv2.1 T1 was superimposed with cartoons for the tetrameric homology model or pentameric crystal structure . The pentameric structure best fits the SAXS data. We also used negative-stain electron microscopy (EM) to investigate the oligomeric state of Kv2.1 T1. Top views of ring-shaped particles for Kv2.1 T1 could be seen in the raw electron micrographs, and after 2D class averaging the rings could be more clearly seen to be pentamers  as an independent method to determine the stoichiometry of Kv2.1 T1 complexes in solution. The molecular weight of the Kv2.1 T1 monomer is 14.6 kDa, so the theoretical mass of a tetramer is 58.4 kDa, while that of a pentamer is 73 kDa. The absolute molecular mass of Kv2.1 T1 measured by MALS was 71 ± 1 kDa Fig. 4a. The M.7 Fig. 4b. To aire Fig. 4c. The prs Fig. 5. In conc3.3.2+ in Kv3 and Kv4 T1 domains has been determined to be critical for the transition from monomers to tetramers as well as for protein stability  and assessed the effect that this had on the protein using circular-dichroism (CD) and dynamic light-scattering (DLS) thermal unfolding experiments. The CD spectra calculated for Kv1.1, Kv3.1 and Kv4.1 T1 domains determined using PDB2CD .The role of Zn2+ Fig. 6a.b). The fraction of unfolded protein (fU) as a function of temperature exhibited two transitions. The α-helical signal began to diminish at 40°C and unfolding increased until ∼60°C, and at ∼80°C the amount of unfolded protein again increased. In the presence of EDTA the curve was shifted to the left and the differences between the two phases were less striking. To calculate the melting temperature , the fraction of unfolded protein (fU) as a function of temperature from 10 to 70 °C (to encompass the first transition) was fitted by a sigmoidal curve. The Tm of Kv2.1 T1 was 50 ± 1°C and decreased to 38 ± 1°C in the presence of EDTA. Our results imply that Kv2.1 T1 undergoes a large conformational change at lower temperature in the absence of bound Zn2+. Using DLS as a complementary approach, we monitored the hydrodynamic radius as a function of temperature (10–80°C). The onset transition temperature  of Kv2.1 T1, defined by a linear transition plot, was 42.5 ± 0.5°C. Addition of EDTA reduced the Tonset to 29.5 ± 0.5°C . These data support the CD experiments and we conclude that Zn2+ binding increases the stability of the protein.The thermal unfolding experiments were monitored by CD at 222 nm at 1°C intervals from 20 to 95°C Fig. 6b. The f°C Fig. 6c. These4.Here, we report the crystal structure of the T1 domain of Kv2.1. This T1 domain is very similar to previously described T1 domains from other Kv subfamilies, except that it forms a pentamer. All of the previously reported structures of isolated Kv T1 domains are tetramers, which is consistent with the tetrameric assembly of full-length Kv subunits into functional channels. Our X-ray structural data are supported by solution analysis of the Kv2.1 T1 domain using SEC–MALS–SAXS and EM imaging.et al., 2005et al., 2019et al., 2017In the larger family of BTB folds (of which the T1 domain is one class), there is a variety of stoichiometries from monomers to hexamers  channels, and several KChIP proteins serve a similar role to Kvβ for Kv4 channels  appear to have lost the ability to bind zinc since the histidine in the Het al., 2022In conclusion, we provide the first structural analysis of the human Kv2.1 T1 domain. Since structures of Kv1, Kv3 and Kv4 T1 domains have been solved, we started this work with the expectation of providing a representative T1 domain for the missing channel-forming Kv subfamily. We were consequently surprised to find that the Kv2.1 T1 domain behaves differently from the others in the stoichiometry that we observed. Future work will need to determine how the Kv2.1 T1 domain is arranged in the context of the full-length channel. While this manuscript was under review, an abstract was published indicating that a cryo-EM structure of full-length Kv2.1 will soon be available for just such an analysis (Fernández-Mariño Kv2.1 tetramerization domain, 7re5PDB reference: 7spdPDB reference: 10.1107/S205979832200568X/jv5009sup1.pdfSupplementary Figures and Table. DOI: https://doi.org/10.1101/2021.11.09.467962A preprint of this article is available from bioRxiv.:


The AAA + ATPase p97 is an essential unfoldase/segragase involved in a multitude of cellular processes. It functions as a molecular machine critical for protein homeostasis, homotypic membrane fusion events and organelle biogenesis during mitosis in which it acts in concert with cofactors p47 and p37. Cofactors assist p97 in extracting and unfolding protein substrates through ATP hydrolysis. In contrast to other p97ʼs cofactors, p37 uniquely increases the ATPase activity of p97. Disease-causing mutations in p97, including mutations that cause neurodegenerative diseases, increase cofactor association with its N-domain, ATPase activity and improper substrate processing. Upregulation of p97 has also been observed in various cancers. This study aims towards the characterization of the protein–protein interaction between p97 and p37 at the atomic level. We defined the interacting residues in p97 and p37. The knowledge will facilitate the design of unique small molecules inhibiting this interaction with insights into cancer therapy and drug design.The homology model of human p37 UBX domain was built from the X-ray crystal structure of p47 C-terminus from rat  as a template and assessed by model validation analysis. According to the HDOCK, HAWKDOCK, MM-GBSA binding free energy calculations and Arpeggio, we found that there are several hydrophobic and two hydrogen-bonding interactions between p37 UBX and p97 N-D1 domain. Residues of p37 UBX predicted to be involved in the interactions with p97 N-D1 domain interface are highly conserved among UBX cofactors.This study provides a reliable structural insight into the p37-p97 complex binding sites at the atomic level though molecular docking coupled with molecular dynamics simulation. This can guide the rational design of small molecule drugs for inhibiting mutant p97 activity. Homotypic membrane fusion events occur to reassemble organelles such as the Golgi apparatus and the endoplasmic reticulum (ER) after mitosis . One essMutations in p97 are associated with neurodegenerative conditions collectively referred to as multisystem proteinopathy (MSP1) –10. ThesThe interaction between p97 and its cofactors p37 and p47 have been shown to be required for organelle biogenesis. p47 is required for the reassembly of Golgi fragments and for ER network formation –14. ThisBoth p37 and p47 share similar domains and motifs. They both have a C-terminal UBX (ubiquitin regulatory X) domain which allows binding to the p97 N-terminal . They alUBX domain-containing cofactors bind to p97 through docking the UBX domain to the hydrophobic groove between Nn and Nc . A high-Currently, there is no structural characterisation of p37 or any of its domains. As previously described, p37 is involved in alternative pathways for membrane fusion and organelle biogenesis. Structural analysis of p97-p37 binding is necessary to understand its mechanism in regulating p97 activity and role in these cellular processes. To discover insights into the mechanism of p97-p37 binding, this study utilised homology modelling to predict structures of p37 UBX domain and protein–protein docking to simulate the binding of the p37 UBX to the p97 N-D1domain.Homology modelling software utilises proteins with previously determined structures to predict proteins of unknown function. Servers such as PHYRE2 and I-TASSER were used to predict the structure of the p37 UBX domain –29.In this project, HDOCK and HAWKDOCK were applied as a prediction docking server to characterising the binding of p37 UBX and p97 N-D1 domain. Furthermore, the key residues for PPIs are highlighted by the MM/GBSA program based on the binding free energy  . The mCSHomology modelling was performed to predict the secondary and tertiary structure of the human p37 cofactor (Uniprot ID: Q14CS0) using SWISS-MODEL, PHYRE 2 and I-TASSER servers , 28, 35.Model Validation was applied to validate the accuracy of predicted tertiary structures and detect the most accurate candidate model among them. The predicted models were assessed by the quality assessment tools Qualitative Model Energy Analysis (QMEAN) , and theHDOCK and HAWKDOCK were applied to conduct molecular docking based on blind docking for detection of possible binding sites between UBX domain of human p37(Uniprot ID: Q14CS0) and N-D1 domains of human p97(Uniprot ID: P55072) to find favourable protein–protein complex pose. , 37. At The top docked model of the p97 N-D1 domain in complex with p37 UBX domain obtained from molecular docking and the MM/GBSA program was further analyzed using molecular dynamics simulation to ensure the conformational stability of the p97 N-D1—p37 UBX complex in a solvated model system over time of simulation following methodology described by others , 40, 41.The secondary structure of human p37 (Uniprot ID: Q14CS0) was predicted using homology modelling via three different prediction servers including PHYRE2, SWISS-MODEL and I-TASSER. The N-terminal domain of p37 from residues 1 to 136 was predicted as a disordered region. The SEP domain of human p47  and the C terminus from rat p47  were chosen as templates for the SEP and UBX domain of human p37, respectively. Although many regions of p37 obtained from these servers were similar, the secondary structure of p37 predicted by PHYRE2 was more consistent with those of templates (data not shown). According to the PHYRE2 prediction server, the p37 SEP domain is comprised of two ⍺-helices  and three β-strands . Moreover, the p37 UBX domain is composed of two ⍺-helices  and four β-strands .Since p37 binds to the p97 N domain through the UBX domain , 45, theQMEAN server is a composite scoring function that estimates global (entire structure) and local (residue) quality of the predicted model . The quaIn addition, the overall root mean square deviation (RMSD) score for the predicted p37 UBX domain via PHYRE 2 and I-TASSER based on the template (1S3S) were 0.51 and 3.42, respectively . After calculating the distance between carbon α of the interacting residues, HAWKDOCK server was used to predict the best matching binding mode of p37 UBX to p97 N-D1 domain based on different restraints. Finally, the best docked pose was detected according to the HAWKDOCK score (-3647.93) and MM/GBSA, a functionality of HawkDock sever, with the free binding energy of -41.21 kcal/mol  of each trajectory frame to the initial conformation. Time series and histograms of the RMSD are presented in Fig. To ensure that the binding interaction between p37 UBX and p97 N-D1 is stable throughout the MD simulation, we counted the number of H-bonds formed between p37 UBX and p97 N-D1 at each step of the simulation. Figure In this work, we studied the protein–protein interaction between p97 and the p37 cofactor via computational methods, which is essential for understanding the assembly of p97 and p37 in processes such as membrane fusion and organelle biogenesis. Since there is no structural information regarding p37 and its interaction with p97, this study aimed to provide the first structural insights prior to experimental methods, which often involve lengthy optimisation and analysis. Like p47, this cofactor is involved in Golgi and ER biogenesis and plays a pivotal role in their maintenance during interphase and their reassembly at the end of mitosis .p37 is composed of two domains including SEP and UBX which have high similarity to those of p47. We characterized the secondary structure of p37 via homology modelling to determine the secondary structure features of the SEP and UBX domains. p37 belongs to the largest family of p97 cofactors named UBX proteins that are involved in the interaction with p97 N domain through their UBX domain. In addition, this domain contains interacting residues that are conserved among members of this family. Therefore, the tertiary structure of the p37 UBX domain was predicted based on homology modelling via three powerful and prevalent predication servers. Model validation analysis showed that the tertiary structure predicted by PHYRE2 was the most accurate. In this model, the p37 UBX domain was modelled based on the C-terminus of p47 from rat as a template, with 100.0% confidence and 64% identity.Despite the fact that both p47 and p37 participate in Golgi and ER biogenesis and need VCIP135 for their function, there are some sharp differences in their structures and roles. The main structural difference between p37 and p47 is the lack of the UBA domain in the N-terminus of p37, which in p47 binds to monoubiquitinated substrates in the presence of p97. The amino acid region 69–92 found in p47 is also absent in p37 , 47. Morpose) as well as assessment of the binding affinity [As there is no solved structure of p37 in complex with p97, we applied protein docking to model the interaction between p37 UBX and p97 N-D1 at the atomic level and characterize their interacting key residues to elucidate fundamental biochemical processes. The docking is based on two steps including the prediction of the ligand conformation, position, and orientation in terms of its receptor  calculated by the HAWKDOCK algorithm score and the MM/GBSA program, respectively Fig. . AccordiD values of ∼20 nM and 29 nM, respectively [Meanwhile, mCSM was applied to confirm that key residues involved in the interaction between p37 UBX and p97 N domain were essential to stabilising the complex. mCSM predicts the impact of mutations on protein–protein interactions and protein complex stability by determining free energy differences between wild-type and mutant residues . We founectively . InteresThe protein–protein interactions for complex having minimum binding energy (stronger binding) were further assessed by molecular dynamics simulations analysis. MD simulations studies were performed up to 100 ns through GENESIS to analyse dynamics and structure of complex. The analysis of RMSD and RMSF indicated the conformational stability of the predicted p97 N-D1 complexed with p37 UBX in the solvated model system throughout the simulation. Meanwhile, The H-bonds analysis confirmed a stable interaction between p37 UBX and p97 N-D1 during the simulation.This study provides the first structural insights into the p37-p97 complex based on homology modelling, protein–protein docking and molecular dynamics that identified key residues in the protein–protein interaction between p37 and p97. Our conclusion showed that how human p37 can interact with p97 and recruit it for the membrane fusion mechanism. Intriguingly, many interacting residues in p37 cofactor are conserved in p47, indicating that the significance of these conserved residues in interaction with p97. The results of our work on binding interactions between the p37 and p97 would be useful for subsequent studies, including understanding the mechanisms of membrane fusion events and identifying therapeutic targets for human disease within this system.


L* a* b* value was not statistically different in each variety of flour. Fourier transform infrared spectroscopy (FTIR) analysis was used for the biochemical change in flour. The PCA and cluster analysis results revealed that cassava flour from Pirun 6 was different from Pirun 2 and Pirun 4. After that, the test using selected cassava flour from Pirun 6 to test the physical properties and sensory attributes of gluten-free muffins compared with wheat flour found that gluten-free muffins were overall better than basic muffins.In Thailand new edible cassava varieties have been developed to be used in the food industry. The aim of this research was to analyze the difference between flour from three cassava varieties and to evaluate the suitability and quality of flour for gluten-free muffins. The physico-chemical properties of flour from three varieties were studied. The results showed the moisture content of flour was between 10.65 ± 0.01 and 10.85 ± 0.45%. Total protein content was highly significant with a difference of 1.97 ± 0.00%, 2.15 ± 0.01%, and 2.18 ± 0.01%, respectively. Moreover, ash and fat in each flour were highly significant. Amylose content was 19.93 ± 0.47%, and the viscosity was 6286.00 ± 1.52 mPa.s. The color of flour values of  Manihot esculenta) is one of the most important economic crops in Thailand with an annual production of around 25 million tons. It is the fifth edible plant, following rice, potato, maize, and wheat [Cassava  from field trials grown in organic systems were obtained from Pak Chong district, Nakhon Ratchasima province, Thailand. The cassava was planted in April 2021  and harvested in December 2021 . The experimental areas were thoroughly plowed two times and high beds of approximately 45 cm were made. Then, planted was completed vertically with 1 × 1 m spacing with a drip irrigation system. The weeds were managed at 1, 2, and 3 months after planting. Harvest was undertaken at 8 months old to produce flour in the next step.® FD115, Neckarsulm, Baden-Württemberg, Germany) at 60 °C for 48 h, and then ground until fine to obtain powder granules 9–20 microns using a beater  for analysis.The samples were produced using 50 kg of freshly harvested cassava tubers from each of the varieties that were peeled to remove the outer skin. Then they were soaked in brine and washed to remove stains and dirt. After that, they were cut into 1 mm pieces, then baked  Co., Ltd. Bangkok, Thailand using an in-house method based on AOAC Official Method 994.12 (2000). The nitrogen content of the samples was determined by the Micro-Kjeldahl method (conversion factor of 5.95) to convert to crude protein. The weight difference methods were used to determine moisture and ash content, while crude fat was determined using the AOAC procedure with petroleum ether as the solvent. The carbohydrate content was calculated by the percentage of difference as Equation (1) :%CHO = 1The cassava flour samples (100 g) for total cyanide content were determined using the pyridine/pyrazolone method . AcrylamL*a* b* color space.The color of cassava flour was determined using a Colorimeter . Results were expressed in the CIE −1 and scanned 64 times in the area of 4000–400 cm−1. The spectra were corrected with FTIR spectrometer . Spectral equipment control was carried out by OPUS 7.2 software  at the Synchrotron Light Research Institute (SLRI), Thailand.Cassava flour samples were analyzed to compare the differences of cassava flour from each cassava variety by FTIR. The resolution of flour samples was 4 cmDehydrated cassava flour samples were on a carbon tape attached to a piece of aluminum stubs , coated with 35 nm of gold-aluminum, and then observed under a Scanning electron microscope  at an accelerating voltage of 20 kV and photographed at a magnification of 8000×.All ingredients were weighed according to muffin-making formulations. Using 2 eggs mixed with 100 g of sugar and 1/4 teaspoon (tsp) of salt, the eggs were beaten until they turned pale yellow. Then 125 mg of fresh milk, 60 mg of oil, and 1 tsp of vanilla flavor were added and mixed well. After that, 150 g of cassava flour or all-purpose flour and baking powder 1 ½ tsp were added. The mixture was poured into a muffin cup and baked at 170 °C for 20 min in an electric multideck baking oven . A gluten-free muffin from cassava flour was compared with a basic muffin from all-purpose flour in the same proportion.Fifteen weight measurements of gluten-free muffin and all-purpose flour muffin /samples were taken for each formulation before and after baking. Losses were calculated as the following Equation (2):L*a* b* color space. The reported color of the muffin was the average color obtained from the measurement of the top and bottom surfaces of 3 dots on each side with 15 pieces per replication of each formulation.The color of the gluten-free muffin and the all-purpose flour muffin were measured with a colorimeter . Results were reported as color values in the CIE The firmness of the gluten-free muffin and the all-purpose flour muffin were measured with Acquisition Rate (PRS) PRS probe surfaces . The probe was pressed onto the sample at a velocity of 1 mm/s for a distance of 50% of the sample height. Then the probe was moved back up at a speed of 5 mm/s and the droplet traveled for 30 s. After that, the probe was pressed on the sample again at the same speed.Thirty panelists determined the sensory characteristics of the gluten-free muffin and the all-purpose flour muffin. Sensory attributes including color, taste, flavor, texture, and overall acceptance were evaluated using a 9-point hedonic scale, ranging from 1 (dislike extremely) to 9 (like extremely) ,20.p ≤ 0.05.The experiments of proximate analysis, total cyanide content, acrylamide, amylose, gluten content, viscosity, bake loss, color of muffin, firmness of muffin, and sensory test were conducted in triplicate. Data reported are averages of three determinations, by dissolved one-way analysis of variance (ANOVA) using SPSS version 14. The New Duncan’s Multiple Range Test (DMRT) differentiated treatments at The proximate composition of the cassava flour is shown in The results of amylose content, acrylamide, cyanide content, gluten content, and viscosity of cassava flour revealed that amylose content at 19.93 ± 0.470%, and acrylamide and gluten content were not found in flour. The cassava flour’s cyanide content at 2.93 ± 0.16 mg/kg dry basis did not exceed the Codex Alimentarius limit (10 mg/kg of dry weight) considered harmless to consumers. The viscosity of the flour was 6286.00 ± 1.52 mPa.s .L*, a* and b*) were non significantly different between samples. The characteristics of lightness L* value were between 97.65 ± 0.65 and 99.46 ± 0.40 which represented the whiteness of the flour. The average a* value (red–green degree) of flour samples was between 1.01 ± 0.34 and 1.75 ± 0.12. The flour derived from cassava varieties Pirun 4 had the highest redness. However, the three cassava varieties showed low redness in the flour. The b* averaged (yellow–blue degree) between 14.73 ± 0.04 and 15.76 ± 1.20, indicating a slight yellowness of the flour. The overall color characteristics of each cassava flour are shown in Three varieties of cassava flour samples were evaluated in a colorimeter , it was found that the color values  and loading plot analyzed the differences between the three varieties of cassava flour found that the three varieties were clearly different, as shown in from PC3 B,C.It was found that the cassava flour varieties Pirun 2 and Pirun 4 were similar in the cluster analysis for FTIR but were a different group compared with Pirun 6 . The resMicrostructural analysis of flour samples using a scanning electron microscope (SEM) reveals that the morphology of different varieties of flour distribution of granules were found evenly. They were spherical and dome-shaped, ranging from 9 to 20 µm in size . There wFrom the physico-chemical properties of three varieties of cassava flour, it was found that all of the flours were not different in terms of physico-chemical properties. Therefore, flour from cassava variety Pirun 6 was selected for use in the next experiments, because there was low cyanide and fat content, and high ash, protein, and viscosity values.a*) values than the basic muffin formula. However, the gluten-free muffins containing cassava flour were a yellow color (b*) with no significant differences when compared with the basic muffins (A basic muffin formula as compared with gluten-free muffins containing cassava flour was tested for baking loss (%), firmness (N), and color. The results found that gluten-free muffins had a higher bake weight loss than the basic muffin at 13.13 ± 0.56%, while the firmness values were not significantly different between the two muffin formulations . The col muffins .The results of the sensory characteristics of the basic muffin formula and gluten-free muffins containing cassava flour showed that the texture appearance, color, taste, and crunchiness were not different. However, tasters rated the smell, wallowing sensation, and overall liking of gluten-free muffins containing cassava flour over basic muffins , indicatL*, a* and b*) were non-significantly different between samples. The characteristics of lightness L* value were between 97.65 ± 0.65 and 99.46 ± 0.40 which represented the whiteness of the flour. The average a* value (red-green degree) of flour samples was between 1.01 ± 0.34–1.75 ± 0.12. The b* averaged (yellow–blue degree) between 14.73 ± 0.04 and 15.76 ± 1.20 indicated a slight yellowness of the flour. Chisenga et al. [−1 representing –C-H (CH2) stretching. The peak of the protein of flour from cassava varieties Pirun 6 was higher than Pirun 2 and Pirun 4 at wave number 1638 cm−1 representing amide I (-C=O) stretching, respectively. FTIR results showed proximate compositions of protein and fat content in flour from cassava varieties Pirun 6. The flour from cassava varieties Pirun 2, Pirun 4, and Pirun 6 quality traits varied among the cassava varieties, and even within the same shape, these physico-chemical components were still not consistent, and the source of variation was due to differences in flour fiber, ash, fat, protein, total cyanide, and peak viscosity. Therefore, it could be said that the differences in chemical components would be a characteristic of each variety [a*) values than the basic muffin formula. This is because cassava flour affects the formation of color in gluten-free muffins such as the Maillard reaction (reaction between carbohydrates with protein in the product) and caramelization (the sugar contained in the product is exposed to heat) at the time of baking [In Thailand, the local varieties of edible cassava used in the food industry have many variations in the nutrient quality of the cassava root depending on the cassava variety used. A study of the physico-chemical properties of flour from three varieties showed that the moisture content of three cultivars of cassava flour was between 10.56 ± 0.01 and 10.85 ± 0.45%. Hasmadi et al.  reporteda et al. . The clu variety . Microst variety . This is variety . The tes variety . The colf baking . From thEdible cassava of the three varieties—Pirun 2, Pirun 4, and Pirun 6—eventually have the potential to be applied in the food industry. Several analyses of cassava flour’s physico-chemical suitability and quality of flour for gluten-free muffins  showed positive results. The results found that different varieties influenced the proximate compositions of cassava flour, and significant differences were observed in ash, fat, protein, total cyanide, and peak viscosity. Significant differences were also observed for the spectral characteristics of the flour from cassava variety Pirun 6 showed chemical components  such as lipids, proteins, etc., by the FTIR technique. The physical characteristics of gluten-free muffins showed that the characteristics and color values of muffins using cassava flour had no significant difference when compared with muffins using wheat flour. The sensory evaluation of gluten-free muffins using cassava flour compared with wheat flour was acceptable by the sensory panelists. These studies may contribute to the development of gluten-free products based on cassava flour, aiming to substitute for wheat flour.


Mid-infrared metamaterial absorbers have many applications in the field of infrared detection, infrared thermal energy utilization, radiation refrigeration, invisible camouflage, etc. In this study, we designed an ultra-broadband mid-infrared metamaterial absorber based on multi-sized resonators. The structure of the absorber consisted of a gold substrate and nine resonators. The simulated results showed that the absorptivity of the absorber was higher than 90% in the 8.33–15.09 μm waveband with an average absorptivity of 95.17%. The energy distributions of the electric and magnetic fields were introduced to investigate the physics of broadband absorption. Moreover, we combined the multi-layer structure with the plane random arrangement structure to achieve a balance between thickness and width. Our study further illustrates the potential application of multi-sized resonators in metamaterial absorbers to realize high absorptivity and ultra-broadband to improve the performance of devices applied in infrared detection, radiation refrigeration, and other fields. As a composite with an artificial structure, metamaterials have unique properties that many natural materials do not have, such as a negative dielectric constant , negativSince the first perfect metamaterial absorber (MA) was presented by Landy  in 2008,In this paper, we present an ultra-broadband mid-infrared metamaterial absorber with nine resonators placed on a gold substrate with the resonators arranged as a 3 × 3 five-layer unit cell. The absorptivity of this MA is greater than 90% in the 8.33–15.09 μm waveband with an average absorptivity of 95.17%. Our study illustrates the potential application of multi-sized resonators in metamaterial absorbers to realize higher absorptivity and ultra-broadband. The proposed MA has potential application value in the fields of infrared detection, infrared thermal energy utilization, radiation refrigeration, invisible camouflage, etc.2O3, Au, and SiO2 placed on a square gold substrate, and we use the subscript w to indicate the sequence number of the resonators. Moreover, without considering the z-direction, we set the geometric center of the substrate as the coordinate origin; thus, the coordinates of resonator 1–9 center are , , , , , , , , and , respectively. The characteristics of the materials applied in the MA were the relative permittivity and loss tangent of Al2O3 and SiO2, and the conductivity of gold; the values of which are 2.28; 0.04, 3.9, and 0.025; and 4.56 × 107 S/m, respectively. Additionally, we list the dimensions of the MA structural parameters in In max, while the boundary condition in the Zmin direction was set as grounding (Et = 0).We carried out all the simulations using CST microwave studio 2020 software. We chose the frequency domain analyzer as the simulation solver to obtain the absorptivity of the MA with the simulation and calculation. The mesh type was tetrahedral. Further, in the x and y directions, we set the conditions of the boundary as the unit cell. Set as open add space, the condition of the boundary was applied in the direction of ZTo calculate the absorptivity of the proposed MA, we used the equation The absorptivity of the MA is shown in Investigating how the incidence and polarization affect the absorptivity, we further illustrated the performance of the proposed MA, and the results are displayed in p becomes larger, and the minimum value of p is 12 μm due to the structure of the MA.We further simulated how the changes in the structure parameters affect the absorptivity, and the result is shown in To investigate the distribution of the electric and magnetic fields, we analyzed the absorption mechanism of the MA. We concluded that most of the energy distributed in the gold substrate and middle gold layer, and the remaining minor electric field energy and magnetic field energy distributed in the up and down, and left and right sides of the resonator in the top gold layer, respectively. Not only the resonators but also the coupling of several resonators with different sizes contributed to the generation of the resonant peaks. Additionally, we present a comparison between the proposed MA and those reported in several previous works in 2O3, and SiO2 to absorb mid-infrared waves. Then, we changed the size of each resonator to expand the bandwidth because resonators with different sizes have different operation wavelengths and the relationship between them is positive. We further rearranged the resonators to change their distances, which created variation in the coupling condition between the resonators. Therefore, the performance of our MA was improved. By conducting simulations and calculations, we ensured the final design of the proposed MA. The results showed that the absorptivity of the proposed MA in the 8.33–15.09 μm waveband was higher than 90%, with an average absorptivity of 95.17%, and the absorption bandwidth was about 6.76 μm. The proposed MA has promising application value in the fields of infrared detection, infrared thermal energy utilization, radiation refrigeration, invisible camouflage, etc. In summary, we proposed a metamaterial absorber characterized by ultra-broadband absorption in the mid-infrared range based on multi-sized resonators. Initially, we designed a 3 × 3 five-layer structure with Au, Al


Permanent deformation is one of the dominant asphalt concrete damages. Significant progress has been made to realistically predict the damage. In the last decade, the mechanistic approach has been the focus of research, and the fundamental theories of viscoelasticity, viscoplasticity, continuum mechanics, and micromechanics are applied to develop the material laws (constitutive equations). This paper reviews the advancement of permanent deformation models including analogical, microstructural, and continuum-based methods. Pavement analysis using the nonlinear damage approach (PANDA) is the most comprehensive and theoretically sound approach that is available in the literature. The model coupled different damages and other phenomena . The anisotropic microstructure approach can be incorporated into the PANDA approach for a more realistic prediction. Moreover, the interaction of fatigue and permanent deformation is the gap that is lacking in the literature. The mechanistic approaches have the capacity to couple these damages for unified asphalt concrete damage prediction. HIn a cyclic creep-recovery test, material hardening grows, and the hysteresis loops shift horizontally due to the accumulation of irrecoverable viscoplastic strain (open loops). On the other hand, in the idealized fatigue test (either stress- or strain-controlled), cyclic load is applied that causes stiffness reduction and a phase angle increment with a negligible viscoplastic strain . In simultaneous fatigue-permanent deformation damage, the material hardening contributes to initiate fatigue cracking at intermediate temperatures, and the fatigue cracks accelerate the microstructure change and contribute to viscoplastic deformation. The viscous flow creates a plastic zone (the crack initiation point), and fatigue cracking can evolve without plastic flow (aggregate movement and re-orientation). That means the simultaneous occurrence of the two damages cannot be ignored as they are interdependent. Thus, the hysteresis loops can be modified by superposing the two loops a to accoOne of the limitations for interaction damage modeling is the lack of integrated testing protocol for permanent deformation and fatigue. Some attempts were made, such as Indirect Tension (IDT) testing for deformation and fracture and applying a haversine loading waveform in fatigue tests ,105. TheThe main objective in the design and service life estimation of asphalt concrete is to estimate the number of cycles . Some progresses are being made to transition from empirical to mechanistic-empirical design methods. The primary motivation of this literature study was to explore the advancement of asphalt concrete permanent deformation characterization, constitutive modeling, and application. The current state of research is focused on the formulation of a mechanistic method that applies the fundamental theories of mechanics and materials to predict permanent deformation damage. In the last decade, promising advancements have been made in the development of comprehensive and coupled permanent deformation modeling. Pavement analysis using the nonlinear damage approach  constitutive model is one of the notable progresses that is gaining wide acceptance (as the next generation mechanistic pavement design approach). Another promising area of progress is the microstructural approach aided with 3D digital image analysis equipment and finite element modeling. This digital technology-based permanent deformation modeling is also the future prospect for accurate permanent deformation modeling and prediction of different asphalt mixtures. The mechanistic methods offer unlimited potential to expand the modeling parameters, and different damages and phenomena such as aging, healing, moisture damage, pre-crack, etc., can be coupled to unify damage prediction. The drawbacks of mechanistic models are sophistication, requiring extensive testing, and the calibration of several modeling variables. For example, the viscoelastic–viscoplastic–viscodamage model requires more than 21 model variables to be optimized using at least two different experiments and several test repetitions at different temperatures stresses and strains. The improvement of the latest models from the classic viscoplastic strain hardening model is the consideration of cyclic hardening and relaxation mechanisms and the viscodamage of asphalt concrete. Although the mechanistic method is theoretically appealing, the calibration cost and rigorous equations can be considered the limitations. The micromechanics approach considers the evolution of permanent deformation related to changes in the microstructure of asphalt concrete constituents (aggregates and mastic). Thus, the micromechanics method is regarded as the most realistic way of modeling heterogeneous materials such as asphalt concrete. Based on the extensive literature study, the permanent deformation prediction and modeling approaches can be categorized into four aspects, as shown in A review study was conducted to explore the state of the art on permanent deformation prediction from the 1960s’ pure empirical to latest mechanistic (from 2011) methods. The review study revealed that the latest constitutive models integrate and couple different theories, i.e., continuum mechanics (1958), nonlinear viscoelastic (1969), viscoplasticity (1971), and viscodamage (2011), along with the crucial time–temperature superposition principle. Such coupling techniques offered advantages of integrating different asphalt concrete damages and opened the possibility of a unified asphalt damage model in the future. The PANDA model is one of the most comprehensive permanent deformation modeling approaches available in the literature . The calibration and/or validation tests are reliant upon the conventional creep or creep-recovery tests in either confined or unconfined modes. The computation and experiment cost of mechanistic methods are the limitations. The practical application of the mechanistic models is very limited at the moment. Moreover, the fatigue–permanent deformation (rutting) interaction is often ignored in the existing (studied) literature. It is inferred that both damages can evolve simultaneously as the same load caused both damages. The mechanistic approach has the potential to couple the two predominant pavement damages. From the extensive study, it can be synthesized that a unified permanent deformation damage model can be developed by integrating continuum damage and microstructure approaches and coupling fatigue, moisture, healing, aging, and other physical and chemical phenomena in asphalt concrete.The mechanistic method is “universally” applicable to predict damage regardless of climatic conditions, stress state, or material type. This characteristic presents wide, open research questions, for example, (1) developing a unified pavement damage performance prediction model, (2) a coupled model for fatigue and rutting damages using continuum mechanics and viscoelastic and viscoplastic theories, (3) considering (coupling) different strains such as shear and axial strains in permanent deformation prediction models, and (4) developing simplified (unified) asphalt concrete test methods to characterized different damages simultaneously . The authors of this paper are conducting research on the simultaneous creep–fatigue damage evolution in a sequential manner.


Based on the results of the research, recommendations were made regarding forging elongation technology and the geometry of working tools in order to ensure the closure of metallurgical discontinuities during hot elongation operations of Zr-1% Nb alloy ingots.In this article, we present research results on the closing conditions of internal metallurgical discontinuities during the hot elongation operation of a Zr-1%Nb alloy ingot using physical and numerical modeling. Research on the influence of thermal and deformation parameters of elongation operations on the rheological behavior of a Zr-1% Nb alloy was conducted using the Gleeble 3800 metallurgical process simulator. Modeling of the influence of thermal–mechanical parameters of hot elongation operations in combinations of rhombic and flat anvils on the closure of metallurgical discontinuities was performed with the help of the FORGE Zirconium-based alloys are widely used for the production of structural parts operating under extreme conditions, including fuel rods for nuclear reactor cores ,2,3. ZirA Zr-1%Nb M5) alloy is a basic alloy used for the production of fuel rod parts  for PWR reactors [ alloy isManufacturing technology for the production of round Zr-1%Nb alloy parts includes the double vacuum–arc remelting of ingots, hot ingot forging, hot billet pressing with or without piercing, several cycles of cold pilger rolling for semi-finished products, and the intermediate and final heat treatment of finished products . In the We conducted a literature analysis ,14,15,16According to ,3,19, thIn this paper, we present guidelines for the development of forging elongation technology during the forging of zirconium alloy ingots in order to ensure the welding of internal metallurgical discontinuities in ingots. Ingots were obtained through a process of double vacuum melting in an arc furnace. In order to achieve the purpose of the work, we proposed that forge elongation operations be carried out under hot conditions using flat and rhombic anvils.In order to develop technological guidelines, numerical tests of the ingot forging process were carried out using three different anvil compositions, including two shaped anvils.®NxT 2.1. program based on MES, which allows for the tracking of changes in hydrostatic pressure distributions and deformation intensity in plastically processed metal during any stage of the elongation operation [The elongation operation was modeled using the Forgeperation . The disDuring model tests of forging elongation operations, the results of plastometric tests of a given Zr alloy were used, based on which, graphs of the dependence of the stress on the deformation of the actual Zr alloy were developed, and according to which, the coefficients of the flow stress function were selected.Based on the analysis of the results of numerical research, guidelines for forging elongation technology were developed in order to weld internal metallurgical discontinuities with flat and rhombic anvils.The test results reported herein can contribute to the improvement of the structural and mechanical properties of Zr alloy rods, supporting the appropriate selection of anvil shapes.The purpose of the plastometric tests was to determine the coefficients of the deformation resistance function, taking into account the influence of temperature and strain rate. The determination of these coefficients is necessary for numerical calculations using the finite element method. We cannot to carry out relevant modeling without these data.The material used for the research was an M5 alloy with the following chemical composition: Zr–1.1%Nb–0.05%Fe—0.6%O.p of the M5 alloy in the conditions of multi-transition hot forging, an experimental diagram was developed ; in addition, it is the most favorable deformation state to describe the rheological properties of a material during plastic deformation .p−ε using the generalizing relationship—functions of Henzel A., Spittel T. [p—flow stress, T—temperature of the deformed material, ε—actual deformation, έ—strain rate, A, m1–m9—function coefficients.In order to make practical use of the results of plastometric tests of the M5 zirconium alloy, an approximation of flow curves was carried out σittel T. :σp = A·e®NxT 2.1 ”Rheology Database” program at the Faculty of Production Engineering and Materials Technology of the Czestochowa University of Technology.The approximation of the test results was carried out according to the method presented in  in the FThe paper analyzed the elongation operation of zirconium alloy rods for three anvil compositions, including rhombic anvil compositions and rhombic flat anvil compositions. The numerical modeling of the elongation operation was carried out in order to analyze the possibility of welding metallurgical discontinuities in zirconium alloy rods for individual rhombic and flat anvil compositions. Each of the anvil compositions was characterized by a different geometric surface of the deformation basin. The variable geometric surface of the deformation basin resulted in different directions and reversals of the pressure and friction forces in the deformed material in the deformation zone, which had an impact on the welding mechanism of the modeled metallurgical discontinuities inside the deformed rods. The constant parameters of the elongation operation were: forging start temperature, relative crush value, and application angle value, while for individual operations, the type of composition used in the incubators was changed.®NxT 2.1, a product of Transvalor Solution, was used to model the elongation operation in three anvil compositions in order to achieve the welding of metallurgical discontinuities during the deformation of the zirconium alloy rod. This program allows for the thermomechanical simulation of, among others, plastic processing. A detailed description of temperature, energy, strain, and deformation functions, as well as thermomechanical and friction laws used during the calculations, can be found in [®NxT 2.1. is based on the finite element method (FEM). In the paper, to simulate the elongation operation, a thermo-viscoplastic model of the deformed body, which is based on the theory of large plastic deformations, was used. To generate the grid of finite elements, tetrahedral elements with a base of triangles were used. In the model input created in this way, the number of nodes equal to the range of 8900 ÷ 9500 was assumed for the simulation, while the number of tetrahedral elements was in the range of 40,700 ÷ 42,000. The value of the coefficient of friction between the anvil surface and the deformed rod was adopted in accordance with the Coulomb law, and it was μ = 0.3. It was assumed that the heat transfer coefficient between the anvils and the material was λ = 10,000 W/m2K, while the heat transfer coefficient between the metal and the environment was equal to λ = 10 W/m2K. The ambient temperature was assumed to be 25 °C, while the anvil temperature was assumed to be 350 °C. The initial temperature of the model rod was 950 °C. In all the forging transitions, 35% relative compaction was adopted. The feed rate of the upper anvil was v = 8 mm/s, while the lower anvil was assumed to be stationary.A commercial computer program on the PC FORGEfound in . For thefound in , which i®NxT 2.1. The inference of discontinuous welding is based on the values of hydrostatic pressure and the temperature of the bar being elongated. Therefore, in the elongation operation, the goal is to achieve the maximum possible values of hydrostatic pressure within the existing defects, as well as to maintain a high temperature close to the temperature at the beginning of forging. During the welding process of the discontinuity, on the right and left sides of the discontinuity, there are medium stresses with large values; these are generally negative medium stresses (there is high hydrostatic pressure). On the other hand, at the top and bottom of the welded discontinuity, unfavorable positive medium stresses with very high values are formed (there is no hydrostatic pressure) [The diffusion model is not included in FORGEressure) .Zirconium alloy rods with diameters of 100 mm and lengths of l = 50 mm were deformed in each of the three anvil compositions in four forging transitions.The shape and dimensions of the anvils used in the work are presented in For the assembly of rhombic anvils, they were treated in a manner analogous to the forging scheme described above . Shape aThe shape, dimensions, and arrangement of the modeled metallurgical discontinuities on the front surface of the model zirconium alloy rod are presented in The model input for the elongation operation was a cylinder with a diameter of 100 mm and a length of 50 mm, in which nine metallurgical discontinuities with an input length of 50 mm were modeled; the first axial one had a diameter of 10 mm, and the eight remaining discontinuities with diameters of 1 mm were distributed around the perimeter of the model input at a distance of 25 mm from its axis. The input modeled in this way was a reflection of the ingot obtained after the process of double vacuum melting in an arc furnace. On the other hand, artificially modeled holes were used to simulate internal casting discontinuities such as middle porosity and casting voids. A detailed description of this type of defect can be found in the literature ,29,30,31® program, which is a product of Autodesk. Metallurgical discontinuities were drawn as rollers inside the model input. Using the “difference” tool in AutoCad, metallurgical discontinuities were understood by the program as holes in the model input. The model input drawing with artificially simulated metallurgical discontinuities was exported to FORGE®NxT 2.1 by the file format “*.stl”. The same actions were carried out for all anvil compositions. In the FORGE®NxT 2.1 program, using the “stlmeshing” and “volumemeshing” tools, a 2D triangular mesh was applied to the input and artificially modeled discontinuities, followed by a 3D quadrilateral mesh. In addition, in order to properly simulate the closure of discontinuities and their correct formation, the built-in “FoldsDetection”, “SelfContact”, and “Damage” tools were used. Thanks to the use of the presented tools, during the FEM simulation, in the areas of welded discontinuities, there were no computational errors or degeneration of the grid. When welding discontinuities, the nodal points of the quadrilateral elements in close contact connect with each other, and thus, the modeled metallurgical discontinuity is eliminated. Thanks to such an adopted modeling methodology, there were no errors in the form of the penetration of the nodes of tetrahedral elements around the closed discontinuities.The model input and anvils were designed in the commercial AutoCad Mechanical 2009®NxT 2.1 to RinoCeros 3.0®; then, the grid of tetrahedral elements was separated in this program, and the elements of the rod outline were removed. The next step was to merge the previously broken grid of tetrahedral elements . In the last step, using the “physical parameters” tool in RinoCeros 3.0®, the total volume of discontinuities for individual elongation operations was obtained, which is shown in Based on the results of numerical simulations of the welding process of metallurgical discontinuities during the operation of elongating Zr alloy rods in flat and rhombic anvils, the volume values of the remaining unwelded discontinuities were determined. The procedure was as follows: after the simulation of the elongation operation was completed, a file with a deformed rod datum in the “*.stl” format was exported in FORGEAfter the elongation operations of the rods in three anvil assemblies, the hydrostatic pressure values at the nodal points of the tetrahedrons constituting the contour of unheated discontinuities were determined as the arithmetic mean of the occurring stresses. An analogy was made for the deformation intensity values. However, all the results presented in the paper are burdened with a certain error resulting from numerical errors and generalizations used in retrieving the data needed to analyze the results obtained.The authors analyzed the distributions of strain intensity and hydrostatic pressure only after the first and second forging passes, since in the third forging pass, all of the metallurgical discontinuities were closed for all anvil geometry options.έ from 0.5 s−1 to 5.0 s−1, obtained using the Gleeble 3800 plastometer. Data in p decreased about 2.5 times. An increase in the strain rate from 0.5 s−1 to 5.0 s−1 led to an increase in the value of the flow stress σp. At T = 770 °C, this increase was about 29%, while at T = 850 °C, this increase was about 36%, and at T = 950 °C, it was 43%. Thus, the effects of increasing strain rate on flow stress σp with increasing test temperature were accelerated. Such an effect could have a connection with the decrease in the plastic deformation thermal effect with regard to the test temperature acceleration [leration .−1 over the entire tested temperature range; the flow curves σp−ε reached the maximum stress value for a given deformation value, whereby as the temperature increased, the maximum flow stress value σp moved in the direction of smaller actual deformation values.έ = 0.5 s−1 and έ = 5.0 s−1 were of a different nature. The maximum value of flow stress σp at the strain rate equal to έ = 0.5 s−1 was obtained for the value of actual deformation equal to ε = 0.12, and with a further increase in deformation, a decrease in the value of flow stress σp was observed. For the strain rate έ = 5 s−1, the value of flow stress σp increased to the value of actual deformation ε = 0.3, after exceeding which, the plateau effect was observed on the curve.The data presented in This difference may have been due to the fact that as the strain rate increases, the process of dynamic softening is delayed. It is possible that dynamic recrystallization is inhibited and the softening of the metal proceeds in accordance with the mechanism of dynamic polygonization. In addition, at a temperature of T = 862 °C, a polymorphic transformation took place in the zirconium, when the crystalline network changed from hexagonal A3 to a regular spatially centered network A2 .−1  during one elongation operation. It is worth mentioning that during the execution of a single elongation operation using only one anvil assembly, it is not possible to obtain differentiated metal flow kinematics in the entire volume of the deformed rod.From the analysis of the fourth transitions, it follows that in the majority of the cross-sectional area of the forged rod, a favorable and even hydrostatic pressure of 25 MPa was obtained.In each of the forging elongation operations of ingots after the casting process of metallic alloys, carried out in order to weld metallurgical discontinuities such as middle porosity and metallurgical voids, in the area of the discontinuities, especially in axial areas, it should be sought to obtain the highest value of deformation intensity due to the relatively large areas of the surfaces of these discontinuities. However, in production practice, when carrying out the rod-lengthening operation, it is difficult to introduce a deformation state characterized by high deformation values, especially compressive strains in the axial zones of the forging, as well as in other local zones of the deformed bar, where, as a rule, there are also small discontinuities. In order to achieve the intended character of deformations, favoring the welding of discontinuities, the authors proposed that forging elongation operations should be carried out in rhombic anvils, thanks to which, it is possible to control the direction, return and value of the friction force vectors and pressure forces acting in the deformation zone and to select the appropriate values of the main technological parameters of the elongation operation.In the first forging transition in the axial zone of the deformed rod, the deformation intensity values ranged from 1.00 to 1.50 and did not lead to the complete welding of the axial discontinuity by the angle of 90° and reimplementation of the 35% crumple; the deformation intensity values were the same, and the axial discontinuity was completely welded, which is visible in the second transition. On the other hand, in the case of discontinuities located at a distance of 25 mm from the axis of the deformed rod, after the first forging transition, two discontinuities remained under the direct action of the upper rhombic anvil (first transition). After the second forging transition, one discontinuity remained along the x-axis direction on the right side of the deformed rod. It happened because both in the first forging transition and the second, in these areas, there was a deformation intensity with small values ranging from 0.01 to 0.15. Little or negligible intensification of deformations in the area of discontinuities during forging operations does not contribute to the welding of discontinuities.The analysis of the data of the third transition shows that after the third forging transition with 35% crumple, after the rod was heated to 950 °C and rotated by 90°, all discontinuities were welded. This was the result of a higher value of the deformation intensity in the areas where there were previously unwelded discontinuities. The values of the deformation intensity ranged from 0.45 to 0.57 and differed from the values occurring after two forging transitions.Based on the data of fourth transition, it can be concluded that after the fourth and last forging transition, there was still a favorable distribution of deformation intensity favoring the welding of discontinuities; in the central area, the value of the deformation intensity ranged from 0.90 to 1.50, while in areas distant from the rod axis, it was within the limits of 0.45–0.90.The data of first transition show that in the axial zone of the deformed rod, the deformation intensity value was 3.25, but this did not result in welding the discontinuity in the rod axis. The two discontinuities originally 25 mm away from the axis of the forged rod on both sides along the x-axis were also not completely welded, because the value of the deformation intensity was 0.14. This was due to the free movement of the material towards the surface that was not limited by tools, where tensile deformations were mostly present. Only the discontinuities under the direct action of the surface of flat anvils, where compressive deformations prevailed, were completely welded.By analyzing the data of the second transition, it can be concluded that in the axial zone of the deformed rod after the second forging transition , a high intensity of deformation was maintained, which ultimately resulted in welding the axial discontinuity; the value of the deformation intensity in this area was 3.25. However, also after the second forging transition, in the areas of the deformed rod under the influence of the working surfaces of the flat anvils, the intensity of the deformation was relatively low, which resulted in the remaining discontinuities not being completely welded. The value of the deformation intensity in this area was still equal to 0.14.On the basis of the analysis of the distribution of the deformation intensity values presented in figures of the third transition, it can be seen that all discontinuities were welded.The values of the deformation intensity in the central area of the deformed rod were within 2–3, while in the area distant from the rod axis, they ranged in the range of 0.87–1.06. The exceptions were the areas located right next to the contact surface of the material with the anvil and the areas not limited by the working surfaces of the anvils along the x-axis. The value of the deformation intensity was small there, amounting to 0.14.The data in the figures of the fourth transition show that the distribution of the deformation intensity value after the fourth transition did not change compared to the state occurring after the third forging transition. In the central part of the rod, the values of the deformation intensity were recorded in the range 1.46–3.25, while in the area distant from the rod axis, the values were within the range of 0.87–1.06.Analyzing the data first transition, it can be concluded that the largest axial discontinuity was welded after the first forging transition carried out in rhombic anvils. The value of the deformation intensity in this area ranged from 1.77 to 2.28, while the discontinuities located under the tops of the anvil working surfaces remained unwelded. The values of the deformation intensity in these areas ranged between 0.15 and 0.36. It follows that the intensification of deformations was too small to lead to welding of the discontinuities in the deformed zirconium alloy rod. The use of rhombic anvils, due to the shape of their working surfaces, affected the favorable distribution of the intensity of deformations in the axial areas of the deformed rod. In the areas under the anvil tips, the value of the deformation intensity was insufficient for the welding of metallurgical discontinuities. The pressure exerted on the forging by the rhombic anvils was almost entirely exerted in the direction of the axis of the forged rod.The data of the second transition show that in the axial zone of the deformed rod, after rotation by an angle of 90°, the deformation intensity distribution favorable for the process of welding the discontinuities was maintained. The values of the deformation intensity ranged between 1.24 and 2.28. After both the first and the second forging transition, the remaining discontinuities were not welded due to the unfavorable distribution of the deformation intensity, the values of which were small and ranged from 0.15 to 0.36. The material flowed freely along the x-axis into spaces not limited by the working surfaces of rhombic anvils. Tensile deformations occurred in these areas, which adversely affected the process of welding the discontinuities.In the third transition, after replacing rhombic anvils with flat anvils and heating the rod to the initial forging temperature and rotating it by 90°, the remaining discontinuities were welded. In the central area of the deformed rod, the deformation intensity values ranged from 1.08 to 2.64, while in the remaining areas, the deformation intensity values ranged from 0.38 to 1.08.After the fourth and last forging transition, the distribution of the deformation intensity values in the metal was very similar to that in the third transition. The deformation intensity values, especially in the central part of the deformed rod, were very favorable and ranged from 1.08 to 2.64, while in other areas, except for small areas at the contact of the deformed material with the tool, the deformation intensity values fluctuated within 0.38–1.08.By analyzing the data in 3, in relation to the initial value of 4239 mm3.The data in 3 were obtained, already in the second forging transition, after turning the deformed bar by 90° and resetting the 35% relative crumple, the value of the total volume of unwelded discontinuities significantly decreased and ranged from 66.40 to 82.80 mm3. The research shows that rhombic anvils can also be used to weld metallurgical discontinuities. Here, in contrast to the rhombic flat and flat anvils, after the first forging transition, a reduction in the total volume of discontinuities to the value of 136.50 mm3 was noted. However, after the second forging transition, the area of unwelded discontinuities decreased to the value of 124.33 mm3. The total, the initial value of the volume of all modeled discontinuities was equal to 4239 mm3.By analyzing the data in The data in 3 to 66.40 mm3. The arithmetic mean values of the deformation intensity calculated for the rod elongation operation in the remaining anvil compositions had much lower values.The analysis of the data in Summing up, on the basis of the results shown in The use of figured (cut-out) anvils in the hot forging process significantly affects the welding of internal metallurgical discontinuities in local zones of deformed zirconium alloy forging, depending on the geometry of the anvils used.To weld metallurgical discontinuities, it is necessary to carry out forging with the maximum possible values of relative reduction up to 40%.The implementation of forging with significant reductions contributes to the formation of favorable compressive deformations in the local areas of the forging, which create conditions for welding metallurgical discontinuities.When forging a zirconium alloy ingot using flat and rhomboid flat anvils, high values of hydrostatic pressure are noted, which contributes to a significant reduction in the total volume of metallurgical defects.The axial discontinuity of the largest size was welded using rhombic anvils in the first forging pass.When using all variants of anvils, the welding of metallurgical discontinuities was noted in the third forging pass.As a result of the research, it was found that the complete welding of metallurgical discontinuities is achieved by using figured (cut-out) anvils in the first passes of forging and in subsequent passes of forging flat anvils.The authors intend to carry out the verification of the obtained modeling results under laboratory conditions in the near future.Based on the analysis of the results of the conducted research, the following conclusions were drawn:


Cyclin-dependent kinase 5 (Cdk5) is a crucial regulator of neuronal signal transduction. Cdk5 activity is implicated in various neuropsychiatric and neurodegenerative conditions such as stress, anxiety, depression, addiction, Alzheimer’s disease, and Parkinson’s disease. While constitutive Cdk5 knockout is perinatally lethal, conditional knockout mice display resilience to stress-induction, enhanced cognition, neuroprotection from stroke and head trauma, and ameliorated neurodegeneration. Thus, Cdk5 represents a prime target for treatment in a spectrum of neurological and neuropsychiatric conditions. While intracranial infusions or treatment of acutely dissected brain tissue with compounds that inhibit Cdk5 have allowed the study of kinase function and corroborated conditional knockout findings, potent brain-penetrant systemically deliverable Cdk5 inhibitors are extremely limited, and no Cdk5 inhibitor has been approved to treat any neuropsychiatric or degenerative diseases to date. Here, we screened aminopyrazole-based analogs as potential Cdk5 inhibitors and identified a novel analog, 25–106, as a uniquely brain-penetrant anti-Cdk5 drug. We characterize the pharmacokinetic and dynamic responses of 25–106 in mice and functionally validate the effects of Cdk5 inhibition on open field and tail-suspension behaviors. Altogether, 25–106 represents a promising preclinical Cdk5 inhibitor that can be systemically administered with significant potential as a neurological/neuropsychiatric therapeutic. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase predominantly expressed in mature neurons . Unlike 2+ homeostasis. Increased intracellular Ca2+ can activate the protease calpain, which then cleaves Cdk5’s physiological coactivator p35 into a truncated aberrant coactivator p25 and a p10 fragment , Parkinson’s disease (PD), ischemia, and brain injury . This isfragment . The p25fragment . Furtherfragment . These cfragment . For thefragment . Elevatefragment . Deletiofragment . Thus, CTherapeutic infusion of preclinical inhibitors of Cdk5 into the brain has been successfully shown to ameliorate neurological disease phenotypes in mice. However, none of these compounds has proven effective in clinical trials . A majorin vivo and displays high potency for inhibition in vitro. Furthermore, this inhibitor modulates behavioral phenotypes linked to Cdk5 through previous characterizations of conditional knockout mice. These studies further advance our understanding of Cdk5’s role in neuropsychiatric behavior, bring forth a new pharmacological tool to assess Cdk5 activity, and suggest a promising new preclinical drug.Here, by screening a small panel of aminopyrazole analogs, we identify analog, 25–106, that can be delivered systemically, penetrate the blood–brain barrier, and pharmacologically inhibit Cdk5/p35 activity in the brain. These data demonstrate that 25–106 inhibits Cdk5 activity in vivo, ex vivo, and behavioral studies. This age range was selected to be consistent with previous studies of conditional Cdk5 knockout mice , coupling constant in hertz (Hz), and integration. The purity of the final compounds was determined by analytical HPLC and was found to be ≥95% pure. HPLC was performed on a Waters Alliance 2,690 system equipped with a Waters 2,996 photodiode array detector and an auto-sampler under the following conditions: column, Phenomenex Luna-2 RP-C18 ; solvent A, H2O containing 0.1% formic acid (FA); solvent B, CH3CN containing 0.1% FA; gradient, 60% B to 100% B over 8 min followed by 100% B over 6 min; injection volume, 25 μL; flow rate, 1 ml/min retention times and purity data for each target compound are provided in the Experimental Section. The purified compound was further confirmed by MS analysis using a Quattro Micro triple–quadrupole mass spectrometer using an electron spray ionization (ESI) technique and a TOF mass analyzer.All reagents were purchased from commercial sources and were used without further purification. Flash chromatography was carried out on silica gel (200–400 mesh). Thin-layer chromatography (TLC) was run on pre-coated ANALTECH uniplate and observed under UV light at 254 nM and with a basic potassium permanganate dip. Column chromatography was performed with silica gel . 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded in chloroform-d or DMSO-Synthesis of 2- acetyl chloride . To a so2Cl2, washed with brine, and dried with MgSO4. The crude product was purified using silica gel column chromatography .Synthesis of tert-butyl 3-amino-5-cyclobutyl-1H-pyrazole-1-carboxylate (2) . 3-amino2. The reaction mixture was cooled to 0°C and freshly prepared 2-acetyl chloride  dichloromethane was added dropwise at 0°C. Following addition, the reaction temperature was allowed to rise to room temperature and, the mixture was stirred for an additional 3 h. The crude mixture was washed with brine, extracted with dichloromethane, dried with MgSO4, and concentrated. The crude mixture was purified using silica gel column chromatography ; 1H NMR  δ 10.31 , 6.87 , 6.57 , 3.91 , 3.87 , 3.72 , 3.55 , 2.41–2.14 , 2.12–1.85 , 1.72–1.56 .Synthesis of tert-butyl 5-cyclobutyl-3-[2-acetamido]-1H-pyrazole-1-carboxylate (3) . Compounin vacuo, and the resulting solid was recrystallized to yield a colorless solid . Purity was assessed by HPLC (>98%); 1H NMR  δ 12.04 , 10.41 , 6.64 , 6.31 , 3.76 , 3.63 , 3.51 , 3.48–3.37 , 2.35–2.18 , 2.18–2.02 , 1.95 , 1.89–1.73 ; 13C NMR  δ 168.1, 153.1, 147.8, 147.5, 136.7, 132.2, 107.0, 93.8, 60.4, 56.3, 43.3, 31.6, 29.4, 18.6; MS calculated for C18H23N3O4 m/z 345.16, found mass: 346.14.Synthesis of N-(5-cyclobutyl-1H-pyrazol-3-yl)-2-acetamide (25–106). To a stirred solution of tert-butyl 5-cyclobutyl-3-[2-acetamido]-1H-pyrazole-1-carboxylate  in dichloromethane (5 ml) at 0°C, trifluoroacetic acid (1 ml) was added dropwise, and the reaction mixture was stirred at room temperature for 3 h. After completion, the reaction mixture was concentrated 2) linear regression analysis of the 25–106/IS peak area ratio vs. the 25–106 concentration from 1.0–80.0 ng/ml (ng/g for tissue) for the low curve and 80.0–10,000 ng/ml (ng/g for tissue) for the high curve. The concentrations of incurred and quality control samples were calculated with the same regression analysis, and the results were reported in ng/mL (plasma) or ng/g (tissue) of 25–106.A quantitative determination of 25–106 in mouse plasma and tissue homogenate was accomplished by the use of protein precipitation and high-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). 5- amiloride (HMA) was used as the internal standard (IS). 25–106 and IS were typically extracted from 50 µL of mouse plasma or tissue homogenate using protein precipitation with methanol. The extracts were analyzed by reverse-phase chromatography using an ACE Excel 3 C18-PFP column under isocratic conditions at a flow rate of 500 μL/min. The column temperature was maintained at 40°C. The mobile phase A consisted of 0.1% formic acid in water, and the mobile phase B was acetonitrile. A triple–quadrupole mass spectrometer  equipped with TurboV IonSpray operating in positive-ion mode was used. Column effluents were analyzed by multiple reaction monitoring (MRM). The precursor/product transitions are 346.1 to 137.1 m/z for 25–106 and 312.37 to 129.5 m/z for IS. The calibration curve was fit using weighted  crystal structure was retrieved from the Protein Data Bank and analyzed using Maestro version 12.3.013 (Schrödinger Inc.). The protein preparation was performed using Protein Preparation Wizard in which missing hydrogen atoms, side chains, and loops were added, followed by grid generation, ligand preparation, and docking. The protein structures were minimized using the OPLS3e force field to optimize hydrogen bonding networks and converging heavy atoms to an RMSD of 0.3 Å. The prepared protein structure was subjected to SiteMap for binding site analysis as implemented in Schrödinger Suite 2020–1. At least 15 site points per reported site were required. A more restricted definition of hydrophobicity together with a standard grid was used. Site maps at 4 Å or more from the nearest site points were cropped. The structures of 25–106 and roscovitine were subjected to Lig Prep to generate conformers and possible protonation at pH of 7 ± 2, which serves as an input for the docking process. The receptor grid was generated using the receptor grid generation tool in Maestro (Schrödinger Inc.). GLIDE XP was used to perform all the dockings with the van der Waals radii of nonpolar atoms for each of the ligands being scaled by a factor of 0.8 and the partial charge cutoff of 0.15. The docked poses from GLIDE XP were analyzed using Maestro version 12.3.013 (Schrödinger Inc.).3, 1.25 mM KH2PO4, 1.5 mM MgSO4, 10 mM d-glucose, and 1.5 mM CaCl2). The brains were subsequently sectioned into normal Kreb’s solution at 350 µ thickness. The brain slices were transferred to a 30°C solution of normal Kreb’s containing 10 µM deaminase for a 1-h recovery period. The sections were then incubated in normal Kreb’s solution with and without the indicated compounds across the dose and time ranges indicated. The sections were rapidly snap-frozen in dry ice to terminate treatment regimes.Brain slice pharmacology was performed as described . BrieflyImmunoblotting was performed as previously described . Mouse bIn vitro phosphorylation reactions were conducted as dose-response kinase inhibition assays with three highly homologous CDKs, namely, Cdk2/CyclinE (1.5 nM), CDK5/p25 (0.35 nM), and Cdk9/CyclinT1 (9 nM). Briefly, the abovementioned kinases were incubated with ATP (30 μM), the corresponding substrate (20 μM), and an increasing concentration of 25–106 (0–5 μM) for 30 min. The kinase activity of the abovementioned reactions was graphed against the concentration of 25–106 to determine the IC50 and Ki values for each kinase.The OFT was performed as described . With thThe tail suspension test was conducted as previously described . The micThe physicochemical properties of five FDA-approved CNS drugs   Ghose e, were esvia immunoblotting the phosphorylation of established Cdk5 sites, phospho-Thr75 DARPP-32 and phospho-Ser1116 NR2B , and brain striatal lysates from various time points were subjected to quantitative immunoblot analysis for phosho-Ser549 Synapsin I levels. Three compounds, 20–107, 20–132, and 20–143, had no effect on Cdk5 activity compared to that of vehicle alone (data not shown). In contrast, 25–106 significantly reduced phospho-Ser549 synapsin I levels in the striatum 2 h after I.V. injection (Each of the four compounds tested in brain slices was further screened for their ability to penetrate the blood–brain barrier (BBB) and inhibit Cdk5-dependent activity njection . Moreovein vitro selectivity of 25–106, we conducted a dose–response kinase inhibition assay with three highly homologous Cdks, Cdk2/CyclinE, Cdk5/p25, and Cdk9/CyclinT1  detection method to assess the distribution, concentration, and elimination of 25–106 throughout several organ systems. For these studies, 5- amiloride (HMA) was used as an internal standard. Mass spectra product transitions were observed as 25–106 displayed a prominent peak of the parent compound at 346.6 m/z, with the product transition for 25–106 displaying a larger peak at 137.9 m/z , elimination rate constant (Ke), half-life (T1/2), last measurable concentration (Clast), 24-h area-under-curve (AUC24h), distribution volume (V), and clearance (CL), were calculated  (R2 = 0.9454)  . The CL  0.9454) . Of noteinjected .max results of 101.13, 118.25, 376.88, and 668.75 ng/g for 10, 50, 100, and 200 mg/kg injections, respectively (1/2 of 25–106 in the brain remained relatively constant (excluding the lowest dose of 10 mg/kg), with an average half-life of 6.23, 5.03, and 4.39 h for 50, 100, and 200 mg/kg injections, respectively , reaching Cectively ; Table 3ectively ; Table 3max, Clast, and AUC could be determined for these samples  activity and protein kinase A (PKA) signaling (Previous studies have demonstrated striatal Cdk5 knockout or inhibition results in reduced anxiety-like behaviors in mice ignaling . Impaireignaling . Here, wignaling . Therefoignaling . Since tignaling . In addiignaling . Cdk5 acignaling .As perhaps the first robust systemic inhibitor, 25–106 represents an exciting and expandable and translatable pharmacological tool to study the function of Cdk5 activity in wild-type animals. Achieving systemic applicability may be considered a step forward toward the testing of Cdk5 inhibitors to treat neuropsychiatric and neurodegenerative diseases. This provides a promising landscape for future studies to assess the effects of brain-permeable Cdk5 inhibitors to combat stress, anxiety, depression, addiction, cancer, and neurodegeneration.