To compare the von Mises stresses of the internal fixation devices among different short segment pedicle screw fixation techniques to treat thoracic 12 vertebral fractures, especially the mono-segment pedicle screw fixation and intermediate unilateral pedicle screw fixation techniques.Finite element methods were utilised to investigate the biomechanical comparison of the four posterior short segment pedicle screw fixation techniques .The range of motion (ROM) in flexion, axial rotation, and lateral bending was the smallest in the I6+2 fixation model, followed by the I5+2 and S4+2 fixation models, but lateral bending was the largest in the M4+2 fixation model. The maximal stress of the upper pedicle screw is larger than the lower pedicle screw in S4+2 and M4+2. The largest maximal von Mises stress was observed in the upper pedicle screw in the S4+2 and M4+2 fixation models and in the lower pedicle screw in the I6+2 and I5+2 fixation models. The values of the largest maximal von Mises stress of the pedicle screws and rods during all states of motion were 263.1 MPa and 304.5 MPa in the S4+2 fixation model, 291.6 MPa and 340.5 MPa in the M4+2 fixation model, 182.9 MPa and 263.2 MPa in the I6+2 fixation model, and 269.3 MPa and 383.7 MPa in the I5+2 fixation model, respectively. Comparing the stress between different spinal loadings, the maximal von Mises stress of the implants were observed in flexion in all implanted models.Additional bilateral pedicle screws at the level of the fracture to SPSF may result in a stiffer construct and less von Mises stress for pedicle screws and rods. The largest maximal von Mises stress of the pedicle screws during all states of motion were observed in the mono-segment pedicle screw fixation technique. Approximately 90% of spinal fractures are found in the thoracolumbar segment, and burst fractures comprise 10%–20% of such injuries In this study, we investigated the biomechanical comparison of the four posterior short segment pedicle screw fixation techniques  used in thoracolumbar burst fractures. We developed a finite element model of the intact thoracolumbar junction from T9 to L3 with thoracic 12 vertebral fracture, and various spinal fusion models were developed using the intact model. The range of motion (ROM), von Mises stress, and stress nephogram of the pedicle screws and rods were evaluated.The software Mimics 10.0 , Rapidform 2006 , and Abaqus 6.9  were used. A computed tomography scanner  was used to collect raw data in DICOM format with a scan slice of 0.625 mm.A finite element model of the T9-L3 spine, which included seven vertebras and six discs, was reconstructed. Geometrical details of T9-L3 spine vertebras were obtained from 64 spiral computed tomography (CT) images of a 40 years old healthy male (65kg and 175cm) without a history of spine injury, osteoporosis and radiographic evidence of degenerative sign. The CT images were scanned and imported into Mimics 10.0 . The surface model was then exported into Rapidform 2006  to generate and enhance the quality of the solid model. Eventually, it was imported to Abaqus 6.9  for meshing and analysis. Each vertebral body consisted of cortical bone and cancellous bone, and each vertebral disc was composed of nucleus pulposus, annulus fibrosus, and endplates. Posterior elements were built separately from the vertebral bodies. Based on Boolean operation, the lower half of the T12 segment was resected, and the structure of the posterior part was reserved to establish a finite element model of an unstable thoracolumbar fracture Fig. 1.Four fixation models were established using the unstable thoracolumbar fracture: the S4+2 model, M4+2 model, I6+2 model, and I5+2 model. In the current study, S4+2 means traditional short-segment pedicle screw fixation, M4+2 means mono-segment pedicle screw fixation, I6+2 means intermediate bilateral pedicle screw fixation, I5+2 means intermediate unilateral pedicle screw fixation. The implants and the vertebral structure were precisely engaged. The diameter of the screw was 6 mm, and the total length of the screw was 45 mm. The fixation models are shown in The inferior endplate of L5 was constrained in all degrees of freedom. A pure moment of 10 Nm combined with a pre-compressive load of 150 N To validate the rationalities of the models, including model simplification, material properties, boundary conditions, and loads, a moment of 10 Nm and a compressive load of 150 N were applied to the reference point. These loading conditions are adopted from the biomechanical experiments and published finite element analyses The range of motion (ROM) of T11-L2, von Mises stress, and stress nephogram of the pedicle screws and rods of the four fixation finite element models under six loading  conditions were analysed. No statistical analysis was performed in the manuscript because only one subject was modeled.Compared with the intact normal spine model, the four fixation models showed a decreased range of motion (ROM) in all states of motion, as shown in The largest maximal von Mises stress of pedicle screws all happened in flexion among all of the fixation models during all states of motion Fig. 3. The largest maximal von Mises stress of rods all happened in flexion among all of the fixation models during all states of motion Fig. 4.  and demonstrate that the stress was concentrated at the screw root under flexion conditions.The results of the pedicle screw stress distribution are shown in There are several posterior surgical techniques currently employed to treat thoracolumbar burst fractures, and a large number of biomechanical studies suggest that reinforcement with a fracture-level screw combination can help to improve the biomechanical stability The finite element model constructed in this study was based on data collected from a spinal CT scan of the patient's thoracolumbar region. The geometric structure of the thoracolumbar vertebrae was generated using medical three-dimensional reconstruction Mimics software. The geometric surface data were preprocessed through smoothing and grid reduction and optimised by Rapidform software to accurately represent the geometry of all models and to minimise the analytic error due to the loss of geometric data. The vertebrae were made up of a solid volume (cancellous bone) and a layer of shell  with a thickness of 0.4 mm. The intervertebral disc was constructed as a continuum structure that occupied the intervertebral space and was partitioned into annulus fibrosus and nucleus pulposus in approximately 6-to-4 ratio In the current study, we attempted to re-create a burst fracture model where the inferior half of T12 and the intervertebral disc between T12 and L1 were completely resected. The construct is highly artificial as depicted, with absence of significant osseous and discal anatomy. We think the spinal fracture model we have built is an extreme case which is similar to A3.1 type that can provide almost little support to the upper vertebrae, and is severe instability, and then we compare the von Mises stresses of the internal fixation devices among different short segment pedicle screw fixation techniques through the extreme spinal fracture model with severe instability. As is known to all, the most common pattern of burst fracture is fracture between the pedicles in the upper half of the body. The spinal fracture model we have built is more severe instability than the most common pattern of burst fracture, we chose the extreme case and carry out our biomechanical study, the conclusions draw from the study on the extreme spinal fracture model with more severe instability can also provide references for clinical treatment of A3.1 type spinal burst fractures.Compared with the intact normal spine model, the four fixation models showed a decreased range of motion (ROM) in all states of motion. The ROM in flexion, axial rotation, and lateral bending was the smallest in the I6+2 fixation model, followed by the I5+2 and S4+2 fixation models, but the ROM was largest in the M4+2 fixation model. In axial rotation, the ROM could be ranked in the following order from small to large: I6+2, I5+2, S4+2, and M4+2. The results were consistent with previous biomechanical studies that suggested that reinforcement with fracture-level screw combinations can help to improve biomechanical stability In the current study, the largest maximal von Mises stress of pedicle screws all happened in flexion among all of the fixation models during all states of motion. The value of largest maximal von Mises stress of pedicle screws during all states of motion was largest in the M4+2 fixation model. As shown in the stress distribution results, a maximum level of pedicle screw stress was apparent at the root of the screws under all loading conditions. In clinical practice, the majority of screw breaks occur at this site. These findings were also consistent with the stress features of pedicle screws described by Kim The value of the largest maximal von Mises stress on rods during all states of motion was the largest in the I5+2 fixation model. The value of the largest maximal von Mises stress of the pedicle screws and rods during nearly all states of motion was smallest in the I6+2 fixation model. The pedicle screws at the fracture site carry some loading stress, thus relieving stress from internal fixation. Therefore, this stress distribution may reduce the possibility of broken or loosened screws. Flexion, extension, right/left lateral bending, and right/left axial rotation tests were conducted to measure the loading forces on the screws. In the current study, we found that additional bilateral pedicle screws at the level of the fracture may result in a stiffer construct and less von Mises stress on pedicle screws compared with traditional short-segment 4 pedicle screw fixation. Index-level pedicle screw fixation to the SPSF decreased the von Mises stress of the pedicle screws; the von Mises stress of the internal fixation was scattered. With pedicle screw fixation at the level of the fracture, the T12 vertebral screw carried a portion of the loading stress. The intermediate screw is thought to function as a push point with an anterior vector, thus creating a lordotic force. The intermediate screw also provides improved “three point fixation”, thus decreasing the cantilever and parallelogram effects and correcting junction kyphosis.There were a few limitations to this study. The most common pattern of burst fracture is fracture between the pedicles in the upper half of the body. In the current study, we attempted to re-create a burst fracture model where only the inferior half of T12 was resected. In fact the fracture model re-created by the authors was not the most common clinical scenarios. We acknowledge that our model for simulating the fracture is done of for ease of computer simulation, and not a direct reflection of the reality of burst fractures. That's because the fracture model in the current study can be easily created through finite element analysis method and ensure the repeatability of the study, the type of the burst fracture in the current study is similar to A 3.1  according to Magerl's classification In theoretically, the spine should be a symmetric construct and the ROM and implant stress should be similar in left/right bending or rotation. However, we kept the reference model  in its natural configuration of the subject to respect the fact that it was not as symmetry as expected. Despite that, in term of the ROM in the symmetric fixed models , the differences between left and right lateral bending and rotation were less than 5.2% . In termAdditional bilateral pedicle screws at the level of the fracture may result in a stiffer construct and less von Mises stress on pedicle screws and rods compared with SPSF. Index-level pedicle screw fixation to the SPSF decreased the von Mises stress of the pedicle screws, and the von Mises stress of the internal fixation was scattered, thus reducing screw fatigue and breakage. The largest maximal von Mises stress of the pedicle screws during all states of motion were observed in the mono-segment pedicle screw fixation technique. The conclusions draw from the study can provide references for clinical treatment of A3.1 type spinal burst fractures, but we can't motivate surgeons to make clinical and surgical decision for a more broad spectrum of burst fractures.


Keratin 5 (K5) is present in the basal layer of a stratified squamous keratinized and non-keratinized epithelium. K5 and K14 have been demonstrated in the mucosa and tumors of the oral cavity, oropharynx, hypopharynx and larynx, and in the mitotic active basal cells of a stratified squamous epithelium. The aim of the present study was to assess K5 expression in squamocellular carcinoma with various localizations in the head and neck. A total of 13 biopsy fragments were included from patients diagnosed with squamocellular carcinoma of the larynx area (n=2), pharynx (n=2), hard palate (n=1), tongue (n=2), submandibular (n=1), lip (n=1), gingival sulcus (n=1), nasal pyramid (n=1), maxilla (n=1) and zygomatic (n=1). The immunohistochemical staining for K5 was evaluated according to the following score criteria: 0 (0% positive cells); 1 (<10% positive cells); 2 (10–30% positive cells); and 3 (>30% positive cells). K5 expression was observed in all squamocellular carcinomas included in the present study with scores between 1 and 3. For well- and moderately-differentiated histopathological types, a maximum score of 3 was recorded for all of the cases, not including the laryngeal area, which presented a score of 2. The following scores were identified in the regions of the poorly differentiated carcinomas: Jaw, 3; gingival sulcus, 2; and tongue and submandibular area, 1. These observations may aid with an improved stratification of head and neck squamocellular carcinoma, thus improving the diagnosis and treatment strategies for this type of cancer. Cytokeratins (CKs) are proteins of the intermediary filaments of keratin, situated intracytoplasmically and present in the cytoskeleton of all epithelial cells. The term CK was initially used at the end of the 1970s . There aSquamocellular carcinomas, independently from their origin, are characterized by the expression of the Ks of the stratified epithelia  and overexpression of K6 and K16 in hyperproliferating strata . The expUsing these data as a stratification method, the aim of the present study was to identify the K5 expression features in the squamocellular carcinoma that were located in various regions of the oral and maxillofacial area in order to improve the diagnostic accuracy.A total of 13 biopsy fragments were evaluated, which were obtained from patients that had been diagnosed with squamocellular carcinoma in the following areas; larynx (n=2), pharynx (n=2), hard palate (n=1), tongue (n=2), submandibular (n=1), lip (n=1), gingival sulcus (n=1), nasal pyramid (n=1), maxilla (n=1) and zygomatic (n=1). Patients provided written informed consent.The biopsy fragments were placed into buffered formalin (10%) for 48 h and subsequently paraffin was added. All stages of the immunohistochemical technique were facilitated by the use of an automated immunohistochemistry instrument , according to the manufacturer’s instructions and using a primary monoclonal mouse antibody specific for CK5 . Following dehydration in pure alcohol, the sections were placed in benzene to replace ethanol prior to embedding in paraffin and subsequently fitted using Canada balsam . The microscopic evaluation was performed with a Nikon Eclipse E600 microscope  and the images were obtained using the Laboratory Universal Computer Image Analysis G system . The immunohistochemistry for K5 in the tumor cells was evaluated according to the following scores: 0 (0% CK5-positive cells), 1 (<10% CK5-positive cells), 2 (10–30% CK5-positive cells) and 3 (>30% CK5-positive cells).The morphological labeling indicated the presence of seven cases of well-differentiated squamocellular carcinoma , two were moderately differentiated  and four were poorly differentiated . In certain cases, at the level of the larynx, scores of 2 were observed in addition to the presence of two distribution models, which were as follows: i) Homogeneous distribution, all the cells in the tumor area were positive for CK5 labeling; and ii) heterogeneous distribution, the CK5-positive cells were prevalent at the periphery of the tumor areas. However, the distribution of the immunohistochemical staining score was always 2 .For the second case that originated from the larynx, the score was 3, the distribution was homogeneous and a prevalent cytoplasmic pattern was demonstrated .The well-differentiated squamocellular carcinoma cases, originating from the pharynx, exhibited a CK5 expression score of 3 with a homogeneous distribution pattern and intense immunohistochemical staining . All of Moderately-differentiated squamocellular carcinoma cases originating from the lip and hard palate demonstrated a homogeneous distribution of CK5 in all the cells of the tumor area with a score of 3.With regard to the intensity of the immunohistochemical staining and the associated expression pattern, the following were observed: i) Maximal intensity and a cytoplasmic pattern in the isolated cells, in addition to a mixed pattern in the carcinomas located in the lip . ii) HetFor all the four cases that were included in the poorly-differentiated grade carcinoma category located in the tongue, submandibular area, maxillary and gingival sulcus, a marked reduction in the scores for the CK5 immunohistochemistry was observed; for squamocellular carcinoma originating from the tongue, a score of 1 was observed . The celThe squamocellular carcinoma case originating from the submandibular area presented isolated regions with positive cells in the tumor area  as well as cytoplasmic expression . For theThe squamocellular carcinoma that originated from the gingival sulcus area preserved the same mixed aspect with positive and negative cells present at the level of the tumor areas. In this case, the intensity of the reaction was moderate  and the expression patterns were cytoplasmic or mixed  and 5.et al (et al (Expression of CK5 and CK14 was found to be specific for mucosal areas and for tumors that originated from the oral cavity, oropharynx, hypopharynx and larynx, and in the basal actively mitotic cells of the squamous stratified epithelia . Velluciet al  and Marll (et al  observedet al (et al observed that only 14.2% of the non-squamocellular carcinomas expressed CK5 and CK6, with distribution in a reduced number of tumor cells. In the present study, all the cases of squamocellular carcinomas originating from the oral and maxillofacial areas expressed CK5 in the tumor cells, with scores ranging from 1 to 3. Whereas, the variation between the intensity scores for CK5/6 and p63 was relatively small in a previous study (et al (et al (Kaufmann et al  demonstrus study . Crook ey (et al  highlighl (et al  reportedet al (According to Chung et al  specificIn conclusion, CK5 was expressed in all of the types of squamocellular carcinoma that were included in the present study, with scores varying from 1 to 3 and a higher expression observed in the poorly-differentiated carcinomas as follows: A score of 3 for those originating from the jaw, 2 for the one originating from the gingival sulcus, and 1 for carcinomas of the tongue and submandibular area. The expression was present for well- and moderately-differentiated histopathological grades with a maximal score of 3 for all of the cases, with the exception of one carcinoma of the larynx where the score was 2. The present study confirms the role of CK5 in the definition of the differentiation of squamocellular carcinoma of head and neck revealing a differential expression depending on the anatomic site of the primary tumour. On these bases, additional studies on a larger series of patients are required.


With a view to explaining the structure-affinity properties of the ligands of the protein FKBP12, we characterized a binding intermediate state between this protein and a high-affinity ligand. Indeed, the nature and extent of the intermolecular contacts developed in such a species may play a role on its stability and, hence, on the overall association rate. To find the binding intermediate, a molecular simulation protocol was used to unbind the ligand by gradually decreasing the biasing forces introduced. The intermediate was subsequently refined with 17 independent stochastic boundary molecular dynamics simulations that provide a consistent picture of the intermediate state. In this state, the core region of the ligand remains stable, notably because of the two anchoring oxygen atoms that correspond to recurrent motifs found in all FKBP12 ligand core structures. Besides, the non-core regions participate in numerous transient intermolecular and intramolecular contacts. The dynamic aspect of most of the contacts seems important both for the ligand to retain at least a part of its configurational entropy and for avoiding a trapped state along the binding pathway. Since the transient and anchoring contacts contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant  This protein is a target in the treatment of transplant rejection. For example, the two immunosuppresive exogenous ligands FK506 and rapamycin can bind tightly to FKBP12 with an inhibition constant of 0.6 and 0.3 nM, respectively 8 and 308, shown in 308 is also reported in FKBP12 is a 12 kDa enzyme found mainly in the cytosol that catalyzes the peptidylprolyl 8 shown in 308  would not account for the ten-fold difference in affinity. Holt et al.8, which would not satisfactorily explain the large binding free energy difference. To unravel the factors responsible for the binding free energy difference of various FKBP12 ligands, the ligand dynamics was also examined by undertaking molecular simulations et al.8. Their study underlies the fact that the binding free energy is dominated by the contribution of hydrophobic contacts but also by the entropic penalty due to the loss of translational, rotational, and conformational degrees of freedom of the ligands. Actually, the hydrophobic contacts seem not to be a primary determinant of the ligand affinity, as demonstrated by the experimental work of Yang et al.8. In this study, an engineered hole has been created inside the protein binding pocket to fit various hydrophobic moieties of the FKBP12 ligands with the aim of enhancing their binding properties. Unexpectedly, the binding affinity of these ligands are unrelated to the extent of complementarity between the protein and its ligand, i.e. to the number of hydrophobic contacts. The above theoretical and experimental results thus reveal that the ligand entropy contributes significantly to the binding free energy, which proves to be most awkward in attempts at scoring fixed FKBP12-ligand complexes or at deriving structure-affinity relationships of the ligands.The development of FKBP12 inhibitors represents a major interest for extending the potential of many therapeutic treatments. It is difficult, however, to derive structure-activity relationships for the FKBP12 ligands since highly diverse structures exhibit excellent binding properties. Indeed, the known nanomolar affinity ligands of FKBP12 differ not only by the structure of the central region of the ligand, the core, but also by the peripheral non-core moieties 308, which displays a rather different core structure as compared to 8  The same protocol as in our previous study was used and is hereafter summarized For the LD simulations, the system was initially energy-minimized and then heated from 0 to 300 K in steps of 25 K (for 600 ps); different random seeds for the distribution of initial velocities led to six independent simulations. The heating run was followed by an equilibration divided in two periods of 500 ps each. For the heating and for the first equilibration periods, the sets C1, C2, C3, and C4 of NOE restraints were used . After the second equilibration period that used the sets C1, C2, and C3 of NOE restraints, the six LD production runs were conducted with the same sets C1, C2, and C3 of NOE restraints for a total simulation time of 53 ns . The setTo compare the results obtained with the LD simulations, the bound state was also simulated by using six independent stochastic boundary molecular dynamics (SBD) simulations For the SBD simulations, the system was then heated from 0 to 300 K using a step of 10 K by assigning velocities every picosecond (for 30 ps) and using three different random seeds. During the heating, the sets C1, C2, C4, and C5 of NOE restraints were used. The heating run was followed by two successive equilibration runs of 300 and 150 ps, with the atomic velocities rescaled. The sets C1, C2, C4, and C5 of NOE restraints were applied during the first equilibration run; however, all the restraints were removed during both the second equilibration and the production runs. The sets C1, C2, and C4 were used for the same reasons as for the LD simulations; the set C5 was used to achieve agreement with the experimental position of the ligand in the binding pocket  to validate the force field and solvation parameters that are also used in the subsequent step of unbinding simulations, and (ii) to obtain initial structures for the unbinding simulations. Implicit solvent simulations are required for the unbinding step as has been recommended for unfolding or unbinding events The same unbinding and refinement protocols as in our previous work were followed α atom of Glu5 decreases (dRC); otherwise, no external perturbation is applied, and the increased distance dRC is taken as the new reaction coordinate for the next time step. The BMD method allows sampling the unbinding pathway under quasi-equilibrium conditions for an implicit solvation model, as previously shown et al. have shown that the BMD simulations cause the least perturbation in a system As the unbinding is a long-extended process RC and of dCM; the latter represents the distance between the center of mass of the binding pocket and each of the four centers of mass of the four ligand moieties . This procedure was also followed by Curcio et al. in their study of forced unbinding of fluorescein from anti-fluorescein antibody FITC-E2 iBu, Tol, and Ethe) with the Cα atoms aligned on those of the X-ray structure of the bound state.In the initial BMD simulations . However, most of the metastable states found in step 1 have a lifetime that is lower than 150 ps. In this case, the last structure obtained in the portion of the simulations that corresponds to constant mean values of dRC, dCM, and RMSDs was chosen for the step 2 whose purpose is to increase as much as possible the lifetime of these metastable states. Indeed, during the unbinding in step 1, the intermolecular distances was increased and the number of intermolecular contacts was decreased, thereby reducing the intermolecular forces. Hence, in step 2, since a smaller value of the perturbative force is needed, the simulations were started by gradually decreasing the value of RC, dCM, and RMSDs were then obtained in the step 2b. If the lifetime of such a metastable state is higher than 150 ps, then it is used in the clustering step 3. If not, the simulations describe a metastable state that is too short and, to increase its lifetime, the last structure in the portion of the simulations that corresponds to constant mean values of dRC, dCM, and RMSDs was used as starting structure in the step 2a with a lower value of RC, dCM, and RMSDs for the protein and for the ligand. The simulations with a lifetime greater than 150 ps were added to the pool of states analyzed in the step 3.From the unbinding simulations  to further probe the stability of the binding intermediate located using LD simulations and (ii) to refine the results since explicit solvent model is known to provide a more realistic description of a system. For the SBD simulations, the same protocol as for the bound state was used, except that the water molecules were here added to the selected structures in a sphere of 22 Å in diameter centered on the binding site. This refinement protocol was performed by 17 independent SBD simulations with no biasing forces or restraints on the system for a total run of 25 ns of production In this section, the results for the intermediate IS308 are presented. A snapshot of IS308 is shown in α atoms aligned on those of the X-ray structure of the bound state. In the first type, hereafter named A1, all the Cα atoms are aligned on their reference position in the crystal. In the second, named A2, all the Cα atoms are aligned on their reference position except those of a given group of atoms G; this allows examining the displacement of the group G with respect to the rest of the protein. The α atoms of all the residues, except those that are in the 80s loop, is 1.11 Å. On the other hand, for the 80s loop, the value calculated by A2 indicates a global distortion of 4.01 Å in IS308 and of 2.15 Å in the SBD simulations of the complexed state CS308, the latter value being similar to the average value calculated for all the Cα atoms in CS308 . For IS308, a detailed analysis of the various sub-regions in the 80s loop indicates that the segment Ala84-Pro93 undergoes a large displacement and contributes mainly to the distortion of this loop (RMSD of 4.57 Å obtained by A2); in particular, the largest displacement inside this residue range is found for the tip of the loop, Gly89-Ile90 . This feature is similar to what was found in the binding intermediate for the ligand 8, IS8 (4.5 Å for the residue range 88–93 and a displacement of 5.5 Å for Gly89) The structure of the protein in the intermediate state IS308 was analyzed by performing two types of RMSD calculations with the CiBu, Tol, and Ethe); we checked that they remained constant during the course of each simulation as well as consistent among the set of simulations. To measure the deviations from the X-ray structure, RMSD calculations for the heavy atoms of each ligand moiety were also performed with the alignment A1. The average values of iPe in 8). In particular, the Tol moiety in 308 and its counterpart iPe in 8 both interact with the mobile side chain of His87, which is at the entrance of the binding pocket, to form many transient contacts (vide infra). Another similarity between these intermediates concerns the oxygen atoms O4 in IS308 and O3 in IS8 that both points to the small side cavity defined by the residues 82, 87, 90, and 91 of the distorted 80s loop . It is also noteworthy in iBu  when one compares the two ligand positions in the bound states In the intermediate IS308, we found that Tyr82 of FKBP12 makes the highest number of intermolecular contacts (12.9 vs. 11.3 for His87). As can be seen in iBu and all the atoms of the Tol cycle. In IS308, but also in the free state, two of these intramolecular contacts are formed . In 308 to FKBP12. The set of molecular dynamics simulations indicate consistent results for all the structural characteristics such as the structure of the protein, the position of the ligand or the type of intermolecular contacts. The stability of the ligand at the entrance of the binding pocket is due to the numerous transient contacts as well as to a few permanent contacts. The transient nature of the majority of the contacts formed could help to avoid a trapped intermediate state along the complexation pathway.We characterized an intermediate state along the binding pathway of ligand iBu), favors transient contacts between interchangeable atoms of the ligand and some protein residues. Recently, the importance of this type of transient contacts was shown in a study on the contributions in the configurational entropy of the side chains of residues during the formation of the complex PKA/AKAP The mobility due to torsions or to rotational oscillations of the peripheral moieties, along with their peculiar geometry . On the basis of these observations, a binding mechanism between the ligand 308 or 8 and a rigid protein FKBP12 that would occur without any binding intermediate would be prevented by a large steric hindrance for the entrance into the binding pocket. The results gathered from the docking of the ligand FK506 to a rigid protein FKBP12 308/8 share an analogous binding mode with nearly superimposable binding regions. In this rigid docking study, the author failed to identify a geometry close to the experimentally reported structure of FKBP12-FK506 because of unfavorable steric overlaps. Interestingly, this author has found that the relaxation of the protein in the 80s loop region helps to accommodate the ligand in the binding pocket. Hence, during the binding process of FK506 or related ligands such as 308 or 8, a displacement of the 80s loop would facilitate the binding to FKBP12. This hypothesis is further supported by a comparison between solution structures of unliganded FKBP12 and a crystal structure of FKBP12-FK506 iPe moiety of the ligand have to fit simultaneously into the main pocket and the small side-cavity, respectively.Another important feature of the intermediate states IS308 and IS8 is the distortion of the 80s loop. In the respective complexed state, the ligand 308.The formation and the stability of the binding intermediates IS308 and IS8 result from the three following contributions: (i) an entropic penalty due to the steric hindrance with the 80s loop that is reduced owing to its displacement, (ii) an enthalpic gain due to the numerous transient and few permanent interactions in the intermediate, and (iii) the above-discussed configurational entropy that increases with the number of transient contacts and that is hypothesized to be large since most of the numerous contacts made in IS are transient. Since these three energetic considerations contribute to increasing the stability of the intermediate, as a corollary, the dissociation rate constant onstant   and 2. H8 or C2SN7 in 308 that is still solvated in IS/TS and that only barely interacts with the protein (308 and by O2 and O3 in 8). Hence, if the above condition is satisfied, our model of IS would come closest to explaining both the two recurrent oxygen atoms and the prevalence of hydrophobic counterparts in various FKBP12 ligands as substituents for C2SN7, as these structural features are linked to hydrogen bond formation and desolvation that can drive the intermediate to the final complex. Under the above-defined condition, these forces would therefore increase A relationship between the characteristics of the binding intermediate and their incidence on the association rate constant  protein ; in CS, α , allows the transient contacts to be interchangeable , mainly by the atoms of the ligand core. Besides, our analysis of the binding intermediate in terms of contacts throws some light on the role played by the peripheral moieties in this binding step: Ethe and Tol are major transient contacts makers. The transient nature of these interactions seems crucial for the affinity of binding since it has been shown, in the context of the protein-protein interactions, that their quantity determines the affinity We characterized a binding intermediate state IS308 between the protein FKBP12 and a high-affinity ligand 8As the peripheral groups are mainly involved in transient interactions, we found that O1 and O5 are of particular importance for the stability of the ligand in IS308 since they act as two anchors, forming permanent contacts with Ile56 and Ile90. Interestingly, the same result was found in the case of the dissimilar ligand S1 TextDefinition of the sets C1, C2 and C5 of NOE restraints.(PDF)Click here for additional data file.S1 TableSummary of the NOE restraints that were used to perform the MD simulations. The use of these restraints resulted with a good agreement with the experimental structure . See the definition of the sets of restraints C3 and C4 in (PDF)Click here for additional data file.S2 TableSet of NOE restraints C3. The values of R(PDF)Click here for additional data file.S3 TableSet of NOE restraints C4. The values of R(PDF)Click here for additional data file.S4 TableAverage distances between the respective center of mass of the four ligand moieties and that of the binding pocket. The average (PDF)Click here for additional data file.S5 TableRMSDs of the ligand and of its four moieties from the respective X-ray structural positions. The average values (in Å) calculated from the subset of LD simulations that passed the acceptance criteria (see text) are given along with the ensemble averages (only the heavy atoms were considered). The alignment was based upon all the protein Cα atoms. For comparison, the ensemble average values for CS308, calculated from the SBD simulations of the complexed state, are also reported.(PDF)Click here for additional data file.S6 TableTime average and ensemble average distances (Å) for the most persistent contacts between the protein and the ligand. A persistent contact was considered whenever the corresponding contact frequency was higher than 50% in at least one simulation. The reported fluctuation range (Å) represents the minimum and the maximum of the fluctuations in the simulation set.(PDF)Click here for additional data file.


To determine inter-session and intra/inter-individual variations of the attenuations of aortic blood/myocardium with MDCT in the context of calcium scoring. To evaluate whether these variations are dependent on patients’ characteristics.Fifty-four volunteers were evaluated with calcium scoring non-enhanced CT. We measured attenuations  and standard deviations  of the blood in the ascending aorta and of the myocardium of left ventricle. Every volunteer was examined twice to study the inter-session variation. The fat pad thickness at the sternum and noise (SD of air) were measured too. These values were correlated with the measured aortic/ventricular attenuations and their SDs (Pearson). Historically fixed thresholds (90 and 130 HU) were tested against different models based on attenuations of blood/ventricle.The mean attenuation was 46HU  with mean SD 23HU for the blood, and 39HU (10-82HU) with mean SD 18 HU for the myocardium. The attenuation/SD of the blood were significantly higher than those of the myocardium (p<0.01). The inter-session variation was not significant. There was a poor correlation between SD of aortic blood/ventricle with fat thickness/noise. Based on existing models, 90 HU threshold offers a confidence interval of approximately 95% and 130 HU more than 99%.Historical thresholds offer high confidence intervals for exclusion of aortic blood/myocardium and by the way for detecting calcifications. Nevertheless, considering the large variations of blood/myocardium CT values and the influence of patient’s characteristics, a better approach might be an adaptive threshold. The quantification of coronary calcifications is now a recognized method for stratifying the risk for future cardiovascular events –3. Descr2) with a minimum attenuation value of 130 HU [Despite new advances, the principle of the calcification scores, including the volume and the mass measurements, remains unchanged and is still based on the detection of calcified regions of interest in the vessel wall. By design, each region of interest was defined as a region satisfying two conditions: a minimum of 3 adjacent pixels  used to detect calcifications, given the values and variations of blood and myocardium.This work was approved by the Commission of ethics and clinical research of the University of Lausanne. Written informed consent was obtained from all the participants.Fifty-four healthy participants with no known cardio-vascular condition were included in the study (31 men and 23 women), with a mean age of 60±12 years. Inclusion criteria: volunteers older than 18 years without the following health conditions screened during a pre-examination visit: presence of angina, active or past history of smoking ≤ 5 years, any medication for diabetes, dyslipidemia, arterial hypertension, erectile dysfunction. Technical exclusion criteria after the examination were as followed: streak artifacts due to metallic structures and motion artifacts due to breathing or arrhythmia. No volunteer was excluded.Data were acquired on a 64-detector VCT system . Acquisitions were obtained without injection of contrast medium. All CT data were obtained with prospective ECG-gating at 75% of the R-R cycle with 120 kV and 200 mA. For each volunteer, the acquisitions were repeated twice (session 1 followed by session 2) and reconstructed with a slice thickness of 1.25 mm and with a slice thickness of 2.5 mm (54 X 2 X 2 = 216 data sets). FOV was 25 cm, and matrix was 512 X 512.The analysis was performed on an Advantage Window workstation version 4.3 .2), and (b) the attenuation and SD of the medio-lateral free wall of the left ventricle (ROI of 10 mm2). At the same level, the pre-sternal fat pad thickness and the standard deviation of the attenuation of the air (noise) were also measured (ROI of 1 cm2)  Figs .The mean SD in all volunteers represents the intra-individual variation. The range of values between volunteers represents the inter-individual variation. The difference of values between sessions represents the inter-session variation.The significance of differences between measurements in one session and between measurements in two sessions were studied with a Student t-test (p<0.05). The effect of thicker slices (1.25 mm to 2.5 mm) on noise and SD of blood and myocardium was also evaluated with the t-test.The correlation between the SDs, the fat thickness, and the noise were analyzed with a Pearson test. When the r correlation coefficient is between -1.0 and -0.7, there is a strong negative correlation. When r is between -0.7 and -0.3, the correlation is weak and negative. With a coefficient between -0.3 and 0.3, there is little or no correlation. From 0.3 to 0.7, the r coefficient shows the presence of a weak positive correlation. When the coefficient is between 0.7 and 1.0, there is a strong positive coefficient.aorta = HUaorta + nSDaorta, and thresholdventricle = HUventricle + nSDventricle, with n = 1, 2, 3, or 4. The hypothesis is that a higher n will increase the confidence interval and will allow a better discrimination of calcifications, expecting that calcifications have higher attenuations. By definition, 1 SD represents a confidence interval of 68.2%, 2 SD 95.4%, 3 SD 99.6%, and 4 SD 99.8%. Each of these models was compared with the historical thresholds.To evaluate the impact of the attenuations of the blood and of the myocardium on the adequacy of theoretical fixed thresholds , two models were used as follows: thresholdDistributions of individual aortic and ventricular attenuations measured in the first session are presented in Figs In the first session, the mean attenuation of the blood in the ascending aorta was 46 HU (from 17 HU to 84 HU) with a mean SD of 23 HU. In the same session, the mean attenuation of the lateral wall of the left ventricle was 38 HU (from 16 HU to 62 HU) with a mean SD of 18 HU. The attenuation of the blood in the ascending aorta was significantly higher than the attenuation of the ventricle (p < 0.01). The standard deviation of the attenuation of the blood in the ascending aorta was also significantly higher than the SD of the attenuation of the left ventricle (p < 0.01).In the second session, the mean attenuation of the blood in the ascending aorta was 46 HU (from 23 HU to 81 HU) with a mean SD of 23 HU. In the same session, the mean attenuation of the lateral wall of the left ventricle was 39 HU (from 10 HU to 82 HU) with a mean SD of 19HU. The attenuation of the blood in the ascending aorta was significantly higher than the attenuation of the ventricle (p < 0.01). The standard deviation of the attenuation of the blood in the ascending aorta was also significantly higher than the SD of the attenuation of the left ventricle (p < 0.01).The intersession comparison showed that there was no difference in the attenuation of the blood in the ascending aorta and in the attenuation of the ventricle between the two sessions . Neither the SD of the attenuation of the blood in the ascending aorta nor the SD of the attenuation of the ventricle was different between the two sessions .The averaging effect of doubling the slice thickness (from 1.25 mm to 2.5 mm) significantly reduced the image noise , the SD of the attenuation of the aortic blood , and the SD of the attenuation of the ventricular wall . On the other hand, the measurements of the attenuation of the aortic blood and of the ventricle wall were not different when doubling the slice thickness .There was a weak positive correlation between the SD of the attenuation of the aortic blood and the fat thickness, between the SD of the attenuation of the ventricular wall and the fat thickness, between the SD of the attenuation of the aortic blood and the noise, and between the SD of the attenuation of the ventricular wall and the noise . There waorta + n SDaorta), there was a large variation. As an example, for n = 3, the threshold values vary from 59.8 HU to 183.4 HU (mean = 113.4 HU). The model based on the attenuation of the ventricle (HUventricle + n SDventricle) also showed a large variation: for n = 3, the thresholds vary from 49.8 HU to 193.8 HU (mean 93.1 HU). When we calculated the model based on the individual attenuation of the aortic blood plus n SD Click here for additional data file.


Thermopower waves are a recently developed energy conversion concept utilizing dynamic temperature and chemical potential gradients to harvest electrical energy while the combustion wave propagates along the hybrid layers of nanomaterials and chemical fuels. The intrinsic properties of the core nanomaterials and chemical fuels in the hybrid composites can broadly affect the energy generation, as well as the combustion process, of thermopower waves. So far, most research has focused on the application of new core nanomaterials to enhance energy generation. In this study, we demonstrate that the alignment of core nanomaterials can significantly influence a number of aspects of the thermopower waves, while the nanomaterials involved are identical carbon nanotubes (CNTs). Diversely structured, large-area CNT/fuel composites of one-dimensional aligned CNT arrays (1D CNT arrays), randomly oriented CNT films (2D CNT films), and randomly aggregated bulk CNT clusters (3D CNT clusters) were fabricated to evaluate the energy generation, as well as the propagation of the thermal wave, from thermopower waves. The more the core nanostructures were aligned, the less inversion of temperature gradients and the less cross-propagation of multiple thermopower waves occurred. These characteristics of the aligned structures prevented the cancellation of charge carrier movements among the core nanomaterials and produced the relative enhancement of the energy generation and the specific power with a single-polarity voltage signal. Understanding this effect of structure on energy generation from thermopower waves can help in the design of optimized hybrid composites of nanomaterials and fuels, especially designs based on the internal alignment of the materials. More generally, we believe that this work provides clues to the process of chemical to thermal to electrical energy conversion inside/outside hybrid nanostructured materials. Chemical combustion technologies deliver a high energy density, and the output can be directly converted into a number of types of mechanical and thermal energy. When combustion is integrated with moving parts, electrical energy can be produced. Most research at the micro/nanoscale has focused on the conversion of chemical energy into mechanical or thermal energy by combustion because the mechanical apparatus required for the chemical-to-electrical energy conversion are difficult to be integrated at the micro/nanoscale. There have been serious efforts to utilize combustion as the main energy source for various micro/nanoscopic applications, such as microthrusters ,2, micro2Te3 films and Bi2Te3-Sb2Te3 have been used as the core thermoelectric materials for generating thermopower waves, and the maximum output voltages reached 150 and 200 mV, respectively [2Te3 increased the peak output voltage [2[2 film prepared using MnO2 powders provided 1.8 V. Although the core thermoelectric materials play an important role in the generation of thermopower waves, other factors which implement the thermopower waves, such as the core material alignment and the chemical fuel composition, can also affect the fundamental phenomena and response.For high-Seebeck-coefficient materials in the low-temperature regime, Biectively . The ser voltage  by up to voltage ,14 and MIn this work, we explored the effects that different arrangements of nanostructures, composed of networks of CNTs, have on the different aspects of the energy generation, temperature gradient, and reaction velocity, as well as mutual correlation in thermopower waves. A one-dimensional aligned CNT array (1D CNT array), a randomly oriented CNT film (2D CNT film), and a randomly aggregated bulk CNT cluster (3D CNT cluster) were fabricated to be the core materials of the thermopower wave devices, and layers of chemical fuel were deposited on the CNT surfaces using a wet impregnation process. During the combustion of the chemical fuel, the temperature gradient at the beginning position and at the ending position in the 1D CNT array and 2D CNT film was 500°C to 700°C, and preheating along the CNTs increased the temperatures of the CNTs prior to the reaction. Due to cooling behind the reacted region and the thermal wave propagation, the temperature gradient was inverted in thermopower waves; this resulted in an inverted output voltage signal, which was normally observed in the 2D CNT film and 3D CNT cluster. The output voltages and specific powers of the 1D CNT arrays showed the highest values among the three types of CNT-based nanostructures due to the effective energy transfer in the alignment direction without any inversion of the temperature gradient. Moreover, the energy generation from thermopower waves depends on the structural characteristics of the CNT network, regardless of the total mass of the CNT/fuel composites. Our experimental investigation of the effects of the structure of large-area CNT/fuel composites revealed that the alignment and arrangement of nanostructured networks significantly changes the output voltage, peak specific power, and temperature distribution as well as the reaction propagation velocity. These results suggest that miniaturized sources of thermopower waves have the capability to maintain or enhance the energy generation, because the overall performance does not depend on the total mass of the specific system. Thus, one of these sources can be used as a scalable power source. This work indicates specific strategies for obtaining the ideal alignment of nanostructured materials for the optimized generation of thermopower waves.To investigate the effect of the structure of the core nanomaterials, three different types of CNT networks: a 1D CNT array, a 2D CNT film, and a 3D CNT cluster, were designed and evaluated as the core materials for thermopower wave generation. Schematic representations of these three types of CNT networks and the corresponding hybrid composites with the chemical fuels are shown in Figure 2O3 and 1-nm-thick Fe layers on a silicon wafer was conducted by e-beam evaporation. Ethylene was the carbon source, and Joule heating by tungsten at the entrance of the quartz tube promoted the decomposition of the carbon. Argon was used as the carrier gas, and hydrogen formed the nanoparticles on the Fe layer, the roots of the MWCNTs. After the TCVD process, a freestanding VACNT array was obtained by separating the CNT layer from the silicon wafer. The length of the VACNT array was 3 to 5 mm, while the cross-sectional area of the 1D CNTs was controlled by the post-processing treatment physically, without any chemical treatment. Figure Vertically aligned CNTs (VACNTs) were synthesized by the thermal chemical vapor deposition (TCVD) method . The depCNTs were dispersed in deionized (DI) water with surfactants (2 wt.% sodium dodecyl sulfate or sodium cholate) by homogenization for 1 h at 6,500 rpm. After a cuphorn sonicator was used to disperse the CNTs for 10 min, density gradient ultracentrifugation was applied to remove the CNT bundles . The decRandomly aggregated bulk CNT clusters were fabricated, starting with the above method used for CNT purification ,20. NitrTwo different combinations of combustible fuels were used to fabricate the CNT/fuel composites. The first mixture was picric acid and sodium azide. Picric acid was selected as the main chemical fuel due to its large enthalpy of combustion  , and sodThe thermopower wave measurement was conducted using three main pieces of equipment: an oscilloscope , a high-speed CCD camera system , and two optical pyrometers  at specific positions during thermopower wave propagation. The reaction front of the thermal waves passed the first and second optical pyrometers, in turn, at the ignition position and the ending position of the propagation, respectively. It was assumed that the time at which the temperature reached its maximum value indicated the time of the passing of the reaction front during combustion. The as-prepared 1D, 2D, and 3D CNT/fuel composites were fixed on silicon wafers, and copper films were used as electrodes. Silver paste was used as an adhesive to join the ends of the CNT structures and the copper electrodes. Combustion was initiated by tungsten Joule heating at the leading edge of the CNT/fuel structures. Because the tungsten element only contacted the chemical fuel layer, and not the CNTs, there was no electrical disturbance from Joule heating.Typical combustion events in the 1D, 2D, and 3D CNT/fuel composites are shown in Figure A comparison of reaction front velocities revealed which structure was able to transfer its reaction most efficiently. The 3D CNT/fuel composite generated essentially random combustion behavior, lacking any clear reaction front, so it was not possible to determine the velocity of the chemical reaction. The propagation of reaction had been randomly distributed inside and outside of the 3D CNT random networks. However, the 1D and 2D CNT/fuel composites revealed clear reaction fronts of the thermopower wave, and the reaction velocities were determined from the high-speed microscopic images. Especially, in the 2D CNT films, the reaction was propagated as fan shapes from the starting position on the surface of the films, similar to getting wetted, and the reaction front on the films were possible to be measured by a high-speed microscopic setup. The average reaction velocity in the 1D CNT/fuel composite was much faster than that in the 2D CNT/fuel composite, as shown in Figure The different combustion modes depending on the particular structures produced diverse voltage signals that were synchronized with the thermal distributions from the thermopower waves, as shown in Figure 2)3C6H2ONa) and hydrogen azide (H-N3) in DI water when the H+ and Na+ ions are exchanged because the picric acid is more acidic than hydrazoic acid (HN3). In this condition, the π-stacked benzene structure can be constructed by replacing the hydroxide group, which induces the hydrodynamic alignment of fuel mixtures. The polarity difference between picric acid  and the 2,4,6-trinitro sodium phenoxide induces continuous benzene stacking, which leads to the formation of the one-dimensional cylindrical structures of the fuel mixtures, like the unique fuel alignment in Figure The marked difference in the absolute magnitude in energy generation between the picric acid/sodium azide and the nitrocellulose/sodium azide cases was due to the reconstruction of the chemical structures in fuel mixtures. Figure The temperature distributions in the 1D, 2D, and 3D CNT structures also support the finding that the total energy generation and specific power were maximized in the 1D CNT/fuel composite. To amplify the energy generation, the charge carrier movement should proceed in one direction without being cancelled by motion in the opposite direction. As shown in Figure In summary, we explored the effect of the structure of large-area CNT/fuel composites on the energy generation from thermopower waves. The different types of CNT structures - 1D CNT arrays, 2D CNT films, and 3D CNT clusters - were fabricated as the core of the CNT/fuel composites. The thermopower waves in the three different nanostructured composites were characterized by the propagation of the combustion front, the real-time voltage signal, the total energy generation, and the specific power. The 1D CNT/fuel composite produced a voltage signal with a single polarity, which was driven by the unidirectional temperature gradient, and this physical characteristic resulted in relatively large energy generation per unit mass and specific power. The voltage signal of the 2D CNT/fuel composite showed a dynamic change in polarity during the propagation of the thermopower waves. The inversion of the temperature gradients between the starting time and the ending time caused this polarity change, and the change was associated with the cancellation of some of the effective charge carrier movements and a loss of energy during the conversion process in comparison with the 1D CNT structures. The voltage signal in the 3D CNT/fuel composite was highly irregular, and there were heavy losses in energy conversion owing to the randomly propagating reaction throughout the CNT network. Understanding this structural effect on energy generation from thermopower waves can help tune device designs to optimize the hybrid composites of nanomaterials and fuels, specifically, tuning based on the effective alignment of the nanomaterials. More generally, we believe that this work provides clues to understanding the chemical to thermal to electrical energy conversion inside/outside nanostructured materials.The authors declare that they have no competing interests.HH, TY, KL, and WC took the tasks of fabrication of carbon nanotube/fuel composites in multiple dimensions, measurements of thermopower waves, overall data collection, and draft writing. JU, HK, JH, and WK provided the dispersed carbon nanotubes in solution phase as well as carbon nanotube networks in various alignments. All authors read and approved the final manuscript.


For the C and H groups, an equivalent volume of normal saline was administered. During heating, the respiratory frequency and rectal temperature were measured and recorded once every 30 min. After 2.5 h of heating, the wet/dry weight (W/D) ratio of the lung tissues of the rats was measured. Additionally, the cellular morphologies of the lung tissues were observed under light and electron microscopes. The respiratory frequencies and lung tissue W/D ratios of the rats in the various hyperthermia groups were significantly higher than those of the rats in the C group . The respiratory frequencies and lung tissue W/D values of the HU and HU1 groups were significantly lower than those of the H group . Under the light microscope, the bronchial surrounding tissues of the HU and HU1 groups were loose, and the majority of the pulmonary alveolar structures were normal; the H and HU2 groups presented a number of changes, including pulmonary interstitial hyperemia, alveolar epithelial swelling and emphysema. Under the electron microscope, it was observed in the type II epithelial cells of the pulmonary alveoli of the H group that the mitochondria were swollen, the cell ridges were shortened, the microvilli were thin and increased, and the alveolar wall was thickened. Also, an increased number of infiltrating neutrophils were visible. In addition, the type II epithelial cells of the HU2 group also presented these changes to different extents and the changes in the HU and HU1 groups were the mildest. These results indicate that the early application of UTI relieves edema and the extent of cell injury of the lung tissue in rats with systemic hyperthermia.Hyperthermia not only directly induces cell injury of body tissues, but also causes the body to release large amounts of inflammatory mediators and cells with extensive biological activities to induce a systemic inflammatory response and immune dysfunction. Thus, hyperthermia causes systemic inflammatory response syndrome, aggravating injuries to various organs. This study aimed to observe the effects of ulinastatin (UTI) administered at different time points on the cellular morphologies of the lung tissues of rats with systemic hyperthermia. A total of 40 male Sprague Dawley rats were randomly divided into five groups: The normal control group (C group), the hyperthermia group without medication (H group), the hyperthermia and UTI pre-treatment group (HU group), the group treated with UTI at 1 h after hyperthermia (HU1 group), and the group treated with UTI at 2 h after hyperthermia (HU2 group). The systemic hyperthermia rat model was established in a heating chamber with a biological oxygen supply. For the HU, HU1 and HU2 groups, UTI (5×10 Hyperthermia may be defined as overheating of the body, including heatstroke caused by an over-high ambient temperature, or as an abnormally high body temperature . The potUlinastatin (UTI) has been used in the process of successfully treating a case of malignant hyperthermia (MH) . UTI is The purpose of the present study was to observe the cellular morphological changes of the lung tissue in rats with hyperthermia and the effects of intervention with UTI administration at different time points on cellular morphology. These observations were conducted to explore the mechanism of action by which UTI treats fatal hyperthermia in the clinic and the importance of the time of application.4 U/kg UTI was administered to the rats prior to heating), the HU1 group (5×104 U/kg UTI was administered after 1 h of heating), and the HU2 group (5×104 U/kg UTI was administered after 2 h of heating). UTI was provided by Tianpu Biochemical Pharmaceutical Co. Ltd. . This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health . The animal use protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Nanfang Hospital, Southern Medical University.A total of 40 specific pathogen-free Sprague Dawley male rats with body weights ranging from 180 to 220 g were provided by the Experimental Animal Center of Southern Medical University  and randomly divided into five groups, with eight rats in each group. The groups were as follows: The C group , the H group , the HU group  was administered at the initiation of heating, and after 1 h and 2 h of heating, respectively. For the other two groups, an equivalent volume of normal saline was administered at the beginning of the experiment. After 2.5 h of heating, all rats were removed from the heating chamber and treated as subsequently described. The time at the start of the experiment was expressed as T0. During the experiment, the respiratory frequency and rectal temperature of the rats were measured and recorded once every 30 min. A total of six recordings at different time points during the experiment were conducted and they were expressed as T0–T150, respectively.The rats were anesthetized with 3% pentobarbital  by intraperitoneal injection and placed into a heating chamber with a biological oxygen supply . Also, the previously examined rectal temperature was used as the basic value. Subsequently, the rats in all groups other than the C group were heated in the heating chamber at 35°C and a relative humidity of 60%. For the HU, HU1 and HU2 groups, 5×10After removal of the rats from the heating chamber, the rat thoracic cavity of each group (n=8) was opened to remove the left upper lung tissue before perfusion with paraformaldehyde. Water and blood staining on the lung tissue surface was absorbed with filter paper, and the tissue was weighed in a weighing disk (wet weight), dried in a constant temperature oven at 80°C and weighed again (dry weight). Subsequently, the W/D ratio was calculated.Following the opening of the thoracic cavity, buffer solution containing 4% paraformaldehyde was rapidly perfused via the right ventricle. The left lower lung tissue was removed, soaked and fixed in 4% paraformaldehyde for 24 h, conventionally dehydrated with alcohol, embedded with paraffin wax and sectioned into ultrathin slices. The slices were stained with hematoxylin and eosin and were then observed for pathological changes under a Nikon microscope  and photographed.3. The tissue sections were soaked in 2% glutaraldehyde (this step was completed within 1 min), fixed with 2% osmic acid, dehydrated with gradient alcohol, embedded with epoxy resin and sectioned into ultrathin slices. The slices were stained with uranium acetate and lead citrate, and were observed and photographed under a H-7500 Transmission Electron Microscope .The thoracic cavities of three rats from each group were opened to remove the right upper lung tissue, and each tissue sample was torn into small sections with a size of ~1 mmThe data were analyzed using SPSS statistical software, version 13.0 , and the experimental data were expressed as the mean ± standard deviation. One-way analysis of variance was used for comparisons of the mean value of various parameter indicators among the groups. P<0.05 was considered to indicate a statistically significant difference.No significant difference in the body weight, basic rectal temperature and rectal temperatures of the rats at various time points were identified among the groups  with the exception of the respiratory rate of group H1 at 30 and 60 min. In addition, the respiratory frequencies and lung tissue W/D ratios of the HU and HU1 groups were significantly lower than those of the H group . Between the HU2 and H groups, no significant differences in the respiratory frequency and lung tissue W/D values were identified P>0.05,  and 2.In the C group, the bronchial pulmonary alveoli tissues of the animals were integrated; the bronchial wall did not present hyperemia and edema; the pulmonary alveoli did not present shrinkage or dilation; and no clear exudation in the alveolar space was observed . CompareUnder the electron microscope, it was observed in the C group that the pulmonary alveoli were integrated; the nuclear membrane was complete; the nuclear chromatin was uniform; and mitochondria, rough endoplasmic reticula, Golgi apparatus and lysosomes and lamellated bodies arranged in concentric circles or in parallel were present in the cytoplasm of the type II epithelial cells . The chaet al (2+ ATP enzyme activity and Ca2+ content of myocardial cell mitochondria caused mitochondrial function disorders (et al (et al (As there are numerous methods of causing an over-high body temperature, there are a number of different damage mechanisms of the body associated with hyperthermia –10. Prevet al  observedisorders . This iss (et al  investigl (et al  hypothesUTI is a trypsin inhibitor isolated and purified from human urine. UTI inhibits the activities of a variety of types of proteins, carbohydrates and lipid hydrolases in the body, scavenges oxygen free radicals, relieves local tissue peroxidation, inhibits the synthesis of excess superoxide and myocardial depressant factor, and reduces the excessive release of inflammatory cytokines, resulting in improved microcirculation and immune regulation. UTI has been widely used for treating clinical critical diseases, including severe acute pancreatitis and disseminated intravascular coagulation –13. A nuDuring surgery, there are numerous factors that may induce fatal hyperthermia –9, incluTo the best of our knowledge, there are no studies on the applications and trials of UTI in rescuing patients with fatal hyperthermia. The results of the present study show that early intervention with UTI relieves the extent of hyperthermia-induced lung tissue cell damage in rats with hyperthermia and has a certain protective effect on the lung. This study provides an experimental basis for the reasonable application of UTI in cases of intraoperative fatal hyperthermia in the clinic.


Xenopus laevis to determine conserved or specific features in the amphibian diencephalon. From early embryonic stages to the beginning of the larval period, the expression patterns of 22 markers were analyzed by means of combined In situ hybridization (ISH) and immunohistochemical techniques. The early genoarchitecture observed in the diencephalon allowed us to discern the boundaries of the thalamus with the prethalamus, pretectum, and epithalamus. Common molecular features were observed in the thalamic prepatterning among vertebrates in which Wnt3a, Fez, Pax6 and Xiro1 expression were of particular importance in Xenopus. The formation of the zona limitans intrathalamica was observed, as in other vertebrates, by the progressive expression of Shh. The largely conserved expressions of Nkx2.2 in the rostral thalamic domain vs. Gbx2 and Ngn2 (among others) in the caudal domain strongly suggest the role of Shh as morphogen in the amphibian thalamus. All these data showed that the molecular characteristics observed during preparttening and patterning in the thalamus of the anuran Xenopus (anamniote) share many features with those described during thalamic development in amniotes (common patterns in tetrapods) but also with zebrafish, strengthening the idea of a basic organization of this diencephalic region across vertebrates.Previous developmental studies of the thalamus  have defined the molecular basis for the acquisition of the thalamic competence (preparttening), the subsequent formation of the secondary organizer in the zona limitans intrathalamica, and the early specification of two anteroposterior domains  in response to inducing activities and that are shared in birds and mammals. In the present study we have analyzed the embryonic development of the thalamus in the anuran  The thalamus is a pivotal forebrain structure that serves as relay station for sensory information (except olfaction) to the overlaying cortex in mammals Jones, . Thus, iAnatomically, the thalamus represents the largest part of the diencephalon. According to the current prosomeric model of forebrain development , also known as mid-diencephalic organizer , that are distinguishable by molecular markers , and Pax7  and other anamniotes. Interestingly, the analysis of the genoarchitecture has been revealed as a powerful tool in the identification the regions in the amphibian brain that are homologous to those with similar genetic features in other vertebrate groups  and Spain , after approval from the Complutense University to conduct the experiments described. For the present study, a total of 371 embryonic 0/63/EU aXenopus laevis embryos were obtained by human chorionic gonadotropin (HCG)-induced egg-laying followed by in vitro fertilization. Adult frogs were purchased from the CNRS colony . Embryos were selected and staged according to Nieuwkoop and Faber (N-morpholino]) propanesulfonic acid [MOPS](Sigma-Aldrich), 20 mM EGTA (Sigma-Aldrich), and 10 mM magnesium sulfate in 4% formaldehyde, adjusted to pH 7.4.) fixative solution overnight at 4°C. They were then processed in toto for In situ hybridization (ISH) or Immunohistochemistry (IHC) and finally sectioned on a freezing microtome.nd Faber  into embThe embryos were processed for ISH with several digoxigenin-UTP-labeled probes and with fluorescein-UTP-labeled (Shh) antisense riboprobes Table . The plain toto  for antisense and sense control probes. The embryos were processed Selected embryos were fixed and processed in toto, for ISH with digoxigenin-UTP-labeled antisense riboprobes , where each of those was combined with fluorescein-UTP-labeled antisense riboprobes of Shh in hybridization buffer at 55°C overnight. Shh was the first probe to reveal following the same steps that in simple ISH using an AP-coupled anti-fluorescein antibody , by NBT/BCIP and was observed in blue color. After that, the embryos were treated with Glycine 0.1M-HCl-0.1%Tween (pH 2.2) to break the AP unions, and were incubated with an AP-coupled anti-DIG antibody . AP staining was developed with INT (2-[4-iodophenyl]-3-[4-nitrophenyl]-5-phenyltetrazolium chloride)/BCIP solution  and was observed in orange color.ISH. Antibody cocktails were used as follows: (1) Incubation at 4°C for 72 h in a mixture of mouse anti-Pax7, mouse anti-Nkx2.2 or mouse anti-Isl1  (Table Different two-step protocols for bright field or immunohistofluorescence (IHF) were conducted after ]) Table  after hyin toto embryos as follows: (1) First incubation was conducted for 72 h at 4°C in the dilution of a mixture of primary antibodies .Western blot analysis with the Pax7 antibody detected a single band at the same molecular weight as the major product detected in rat brain extract (about 55 kDa) . The specificity of the Nkx2.2 antibody has been confirmed by an absence of labeling in Nkx2.2−/− mice . The DNA region of NK2 transcription factor of chick was cloned by polymerase chain reaction into the Xenopus laevis . The antibody was isolated from antiserum by immunospecific methods of purification, and antigen-specific affinity isolation removed essentially all rabbit serum proteins, including immunoglobulins that do not specifically bind to GABA. In previous studies, the same antibody has been used in different species, including The labeled sections were analyzed with and Olympus BX51 microscope equipped for fluorescence with appropriate filter combinations. Selected sections were photographed using a digital camera (Olympus DP70). Contrast and brightness of the photomicrographs were adjusted in Adobe PhotoShop CS4  and figures were mounted in Canvas 11 .Xenopus distinct parts of the diencephalon, including the epithalamus, arise  and obtain precise spatial gene expression maps in the developing thalamus and adjacent regions. We analyzed the embryonic development starting at stage 29/30, when [3H]-thymidine studies indicated that in Xenopus embryos changes in morphology as the longitudinal axis is straightened, and the progressive differences in the diencephalic organization can be inferred by the labeling of two main markers, Shh and Gbx2  the brain of  Figures . Thus, tIn early embryos, the expression of Shh was observed predominantly in the ventral neural tube but, at levels of the diencephalon, the Shh expression extended dorsally in the basal part of the forming Zli Figures . The extIn the diencephalic alar plate of embryos before stage 33, Pax6 expression occupied the alar domains, sharply highlighting the p1-mesencephalic boundary Figure . SubsequInterestingly, Wnt3a expression in the dorsal part of the developing thalamus was also noted at early stages Figure  and its Other markers of the thalamic region during early embryonic stages included the transcription factor Tcf4, whose expression extended from the p3/p2 boundary (marked by the Zli) to the mesencephalon Figure . At stagThe rostral boundary of the embryonic thalamus at the dorsal part of the brain was also assessed by the expression of Fez , which strongly labeled the dorsalmost part of p3 in contrast to the total lack of expression in p2, thus highlighting the p3/p2 boundary Figures . TherefoStarting by stage 30, the homeobox gene Nkx2.2 was expressed adjacent to the Shh expresión domain, forming a narrow band along the neural tube and in the neural epithelium flanking the developing Zli. The progressive dorsalward ascent of the Shh expression in the Zli was accompanied by a corresponding progressive modification of the longitudinal band that expressed Nkx2.2, which was pushed upward by the Zli as it formed Figures .The embryonic thalamus early formed two molecularly distinct subdivisions: the small rostral thalamus (r-Th) showing the expression of Nkx2.2, and the larger caudal thalamus (c-Th) that expressed Gbx2, Ngn2 and Lhx9, among others, already at early stages.Xenopus from these early stages. Other markers, such as Emx1, were clearly located in basal part of p2 (p2b) partially overlapping the Nkx2.2 domain, and continued caudally in the alar p1 and mesencephalon . Dorsal to the c-Th develops the epithalamus  that is molecularly distinct. Expression of Gbx2, which was located in postmitotic cells in the mz in a small region close to the basal boundary in the early thalamus Figures , extendeAt earlier stages, Gli3 expression occupied a narrow zone above the Zli and it was seen later in the dorsal aspect of the c-Th and pretectum, both in the vz and mz Figures , whereasBy stage 37, Ngn2 expression was detectable in the vz at the Zli and in the vz and deep part of the mz of the c-Th Figures , in contXenopus embryos at stage 40 (about 70 h postfertilization) are characterized by the opening of the mouth and strikingly increase of body length (6.3–6.8 mm). The morphology of the forebrain also changes notably from previous stages and a fast progressive enlargement of the secondary prosencephalon  takes place  served to highlight the thalamic boundaries Figure . HoweverThis period starts at the time the embryos begin spontaneous swimming, and continues for about 3 days, when the yolk is completely absorbed. It ends when the animals initiate to feed independently, which marks the beginning of the long larval period stage 45). During the late embryonic stages, the brain changed considerably, the Zli extended more dorsally, penetrating most of the alar territory between p2-p3. In addition, the alar part of p2, including the thalamus and the epithalamus, increased in size and complexity Figures  and 5.. During At these stages, the Nkx2.2 expressing region (including the r-Th) increased in size and the intensity of the immunoreaction was higher than at previous stages Figures . MarkersIn the region of the r-Th, labeled for Nkx2.2, overlapping expressions of Lhx1 Figures , Tcf4 F and, onlRegarding the Gli transcription factors, Gli1 labeled primarily the rostrodorsal part of the thalamus, even partially overlapping the Nkx2.2 expressing zone Figures , whereasXenopus laevis was examined morphologically by using combinations of markers revealed by ISH or IHC. In general, one of the markers in each combination corresponded to a well-characterized reference probe such as the Zli marker Shh , the formation of the Zli, and the early specification of thalamic domains (patterning). All the data will be interpreted according to the current prosomeric model  and, at very early developmental stages, the posterior limit of the Pax6 expression coincides with that of Tcf4  and essentially, Shh .It is currently accepted that Shh released from the organizer in the Zli regulates regionalization and patterning of the thalamus, as has been demonstrated elegantly in zebrafish  and caudal  liminal domains, previously referred to as rostral and caudal shell domains, with respect to the Zli, reflecting precisely the situation reported for amniotes  that apparently drive the progressive and gradient-dependent downregulation of Pax6 in the thalamus, first in the r-Th and later in the c-Th  and the nuclei of the habenula. It has been generally considered in relation to the thalamus, although with significant developmental differences, and functional characteristics in the mature brain. Because many of the markers used here to characterize the developing thalamus were also observed in the epithalamus we include a few comments on its early genoarchitecture.Xenopus acquires its competence by the expressions of similar combinations of markers   study focuses on a model organism  and compares gene expression patterns and molecular profiles that define early brain structures. This is based on the notion that evolutionary close relatives  share fundamental features of their gene networks during development. In addition, the “same” genomic traits (genomic sequence homology) are rather frequent in related species.The aim of our study was to analyze the molecular underpinnings of the development of the thalamus in The molecular specification of the rostral and caudal domains in the embryonic thalamus proper was described in great detail in the mouse embryo (Vue et al., Xenopus thalamus we have described and correlated one to another the expression of 22 markers, which serves to highlight that prepatterning and patterning features are largely shared among tetrapods. Furthermore, recent research in the zebrafish diencephalon identified numerous neurological structures and ontogenetic processes as highly conserved across vertebrates (Scholpp and Lumsden, In this scenario, in the present analysis of the All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. AG and RM devised the study. SB performed most of the experiments. The three authors contributed to the data analysis. SB and RM led the figure preparation and wrote the majority of the article, further completed and edited by AG. All authors approved the article.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.


Duodenal samples were collected when the rabbits were aged 49, 56, 63 and 70 days. The mean villus height, crypt depth and small intestine length were measured. Significant interactions (P<0.001) between group and age were identified in the villi height and crypt depths. The maximum mean villus height was found in the R2 group in 56-day-old rabbits (643.14 μm), while the minimum was found in the ADL group in 49-day-old rabbits (460.29 μm). The longest (P<0.001) small intestine was measured in the R1 group in 63-day-old rabbits (347.60 cm), while the shortest was measured in the ADL group in 49-day-old rabbits (263.60 cm). The models show that villus height, crypt depth and the length of the small intestine change with the intensity of feed restriction and age.The objective of this study was to investigate the effect of seven days of feed restriction (between days 42 and 49) on the morphology of the small intestine in experimental rabbit models. Sixty weaned Hyplus rabbits (35 days old) were included in the experiment and split into three groups of 20 rabbits. The first control group (n=20) received feed  The effect of feed restriction has been studied in numerous animal models –4, but tRabbit models are very specific. The rabbit is a herbivorous species with functional and morphological specificities of its digestive tract due to the quality of feed ingested. Information on the outcomes of the length and percentage quantity of restriction is important for veterinary practice and clinical medicine, and could be useful for other specific practical purposes. It is necessary to determine the effects of different lengths and intensities of feed restriction, as well as the effect of restriction at different ages, and to verify that there are no pathological effects on the morphology of the small intestine. This can be investigated by observing the changes that occur in intestinal morphology. However, the absorption of nutrients depends on the digestive tract, and it can be assumed that quantitative restriction of feed intake can change the morphology of the small intestine mucosa . It wellThis study was approved by the Ethics Committee of the Institute of Animal Science  and the Central Commission for Animal Welfare of the Ministry of Agriculture of the Czech Republic  and carried out according to the guidelines for applied nutrition experiments in rabbits .ad libitum  during the experiment. Rabbits from the R1 and R2 groups were on feed restriction  between 42 and 49 days of age. Animals from both feed restriction groups were fed ad libitum prior to and following feed restriction. Rabbits were fed by a commercial type of feed mixture with 165 g crude protein, 183 g crude fiber, 38 g crude fat and 17.1 MJ gross energy/kg (as-fed basis) (Sixty Hyplus broiler rabbits (PS 19 × PS 40) were kept in the rabbit building of the Institute of Animal Science under controlled environmental conditions  and housed in cages (50×40×42.5 cm). Three experimental groups  were formed. Control rabbits were fed d basis) . ClinicaFor small intestine analysis, five rabbits per group were sacrificed at the ages of 49, 56, 63 and 70 days. Samples of small intestine were obtained by the necropsy method using standard procedures. The entire small intestine from pylorus to ileocaecal junction was first measured. One representative sample was subsequently taken from each animal, 20 cm under the pylorus, and the samples were placed in a 4% Bouin solution  for histological analysis. All the samples were collected within 30 min after sacrifice. The samples were fixed for one week.The samples were processed in accordance with standard histological methods. Slices with a 5-μm thickness were cut from each sample and stained with haematoxylin and eosin . Standard slides  were used for histological purposes. The prepared samples were evaluated under a Nikon Eclipse E600 light microscope . Villus height and crypt depth were measured using the NIS-Elements version 3.0 software (Nikon Corporation Instruments Company). The height of each villus was measured from the top of the villus to the crypt transition, and the crypt depth was defined as the invagination between two villi. The heights of 40 villi and the depths of 40 crypts were measured per animal.The data were examined by two-way analysis of variance with group and age interactions using the Statistical Analysis System (SAS) 2003 software . P<0.05 was considered to indicate a statistically significant difference.No clinical signs of digestive problems occurred during the experiment. No significant differences were identified among the groups with regard to live weight. The data collected throughout the experiment are presented in At the beginning of the experiment the weights of rabbits in the three groups had approximately the same values: ADL, 1,678 g; R1, 1,676 g and R2, 1,751 g. At the end of the experiment the weights of the rabbits had increased: ADL, 2,892 g; R1, 2,650 g and R2, 2,791 g. No significant interaction between group and age was identified.It was found that the length of the small intestine significantly (P<0.001) increased with age; however, the level of restriction did not significantly affect this parameter. The longest small intestine was measured in the R1 group in 63-day-old rabbits (347.60 cm) and the shortest in the ADL group in 49-day-old rabbits (263.60 cm).The data for villus height show interactions between the feeding regime and the age of rabbits. In general, villus height significantly (P<0.001) increased with rabbit age. However, differences were observed between the ADL and restricted rabbits. In the ADL rabbits, the villus height regularly increased with age. In the R1 group, the mean villus height at the age of 49 days, immediately subsequent to restriction, was significantly higher than that in the ADL group. In the following week, the mean villus height in the R1 group was shorter than that in the other two groups. The maximum mean villus height was observed in the R2 group at 56 days; this was significantly different from the heights in the other two groups at the same age.As in the case of villus height, a significant interaction (P<0.001) was detected between age and group for crypt depth. It was found that the ADL group showed a reduction in crypt depth when the depths at the beginning and end of the experiment were compared (from 128.36 to 116.64 μm). Conversely, in the R1 and R2 groups, the crypts were significantly (P<0.001) deeper at 70 days of age in comparison with the depths at the beginning of the experiment .The physiological functions of the gastrointestinal tract are dependent on the nutrition and quantity of feed. Trying to establish the feed conversion ratio is currently one of the main tasks of all animal breeding sectors. There is a search for the optimal dose of feed that would ensure good yield and also reduce health risk factors.In rabbits, it is still not known how long a restrictive diet should be applied for and what the effects of restriction for a shorter period of time are on the morphology of the small intestine. Previous studies have shown that starvation is not compatible with a healthy life as it exerts numerous negative effects on the body, although it is common in nature ,19. The The present study revealed that, following one week of feed restriction in the 49-day-old rabbits, mean villus height was greater than that in the ADL group. However, at the end of the experiment, the mean villus height in the R1 and R2 groups was lower than that in the ADL group. It is possible that, in the absence of nutrients, the villous relief increased, and resulted in higher resorption activity. An increase in resorption may also have been effected by a greater small intestine length. With a gradual adaptation to normal conditions in the realimentation period, the heights of the villi then returned to levels comparable to those of the ADL group. In a previous study, which evaluated the morphometric changes in the small intestine of nursing piglets caused by 60% restriction, it was stated that this restriction resulted in a significantly reduced thickness of the mucosa and villus height compared with the control group . In otheet al (et al (Martignon et al  stated tl (et al , which fl (et al ,30. It cl (et al  the lengl (et al . Some of