Exploring the Light-Emitting Agents in Renilla Luciferases by an Effective QM/MM Approach.

Bioluminescence is a fascinating natural phenomenon, wherein organisms produce light through specific biochemical reactions. Among these organisms, Renilla luciferase (RLuc) derived from the sea pansy Renilla reniformis is notable for its blue light emission and has potential applications in bioluminescent tagging. Our study focuses on RLuc8, a variant of RLuc with eight amino acid substitutions. Recent studies have shown that the luminescent emitter coelenteramide can adopt multiple protonation states, which may be influenced by nearby residues at the enzyme's active site, demonstrating a complex interplay between protein structure and bioluminescence. Herein, using the quantum mechanical consistent force field method and the semimacroscopic protein dipole-Langevin dipole method with linear response approximation, we show that the phenolate state of coelenteramide in RLuc8 is the primary light-emitting species in agreement with experimental results. Our calculations also suggest that the proton transfer (PT) from neutral coelenteramide to Asp162 plays a crucial role in the bioluminescence process. Additionally, we reproduced the observed emission maximum for the amide anion in RLuc8-D120A and the pyrazine anion in the presence of a Na+ counterion in RLuc8-D162A, suggesting that these are the primary emitters. Furthermore, our calculations on the neutral emitter in the engineered AncFT-D160A enzyme, structurally akin to RLuc8-D162A but with a considerably blue-shifted emission peak, aligned with the observed data, possibly explaining the variance in emission peaks. Overall, this study demonstrates an effective approach to investigate chromophores' bimolecular states while incorporating the PT process in emission spectra calculations, contributing valuable insights for future studies of PT in photoproteins.

[3D printing navigation template assisted pedicle screw placement for the treatment of typeâ ¡old odontoid fractures].

OBJECTIVE: To compare the safety and clinical efficacy of freehand and 3D printing navigation template assisted screw placement in patients with old odontoid fractures of typeⅡ. METHODS: Total of 38 patients with old odontoid fractures of typeⅡwere treated from November 2018 to December 2022, all of which presented as chronic neck pain. According to the different methods of screw insertion into the pedicle, the patients were divided into a navigation template group and a freehand group. In the navigation template group, there were 17 patients including 9 males and 8 females with an average age of (51.30±13.20) years old, disease duration was (22.18±7.59) months. In the freehand group, there 21 patients including 7 males and 14 females with an average age of (49.46±11.92) years old, disease duration was (19.52±9.17) months. The intraoperative blood loss, operation time, and postoperative drainage output were recorded and compared between two groups. The accuracy of screw placement was evaluated by CT scan. Before operation and 1 year after operation, cervical pain was assessed by visual analogue scale(VAS), neurological changes were evaluated by the Japanese Orthopaedic Association (JOA) score, and the degree of spinal cord injury was assessed by the American Spinal Injury Association (ASIA) injury scale. RESULTS: All patients were followed up for (25.31±1.21) months. The operation time of template group (112.00±20.48) min had significantly shorter than that of the freehand group(124.29±15.24) min(P<0.05), while there were no significant differences between two groups in terms of intraoperative blood loss, postoperative drainage, and hospital stay(P>0.05). At 1 year after operation, in template group and freehand group, the VAS [(2.88±0.86), (2.90±0.83)] and JOA [(14.94±1.82), (14.62±2.19)] improved with preoperative [VAS(4.71±0.92), (4.86±0.79) and JOA (12.18±2.30), (11.95±2.31)](P<0.05), with no significant difference between two groups (P>0.05). No significant improvement was observed in ASIA grading in either group at 1 year after operation(P>0.05), and there was no significant difference between two groups(P>0.05). The template group had significantly better accuracy of screw placement in the pedicle of the axis than the freehand group (P<0.05), while no significant difference was observed between two groups in the accuracy of screw placement in the pedicle of the atlas (P>0.05). CONCLUSION: In the treatment of typeⅡold odontoid fractures with posterior pedicle screw fixation, 3D printing navigation template screw placement can significantly shorten the operation time, achieve similar clinical efficacy as free-hand screw placement, and significantly improve the accuracy of screw placement in the pedicle of the axis.

Video-assisted thoracoscopic surgery for Andersson lesion in ankylosing spondylitis: A case report and literature review.

RATIONALE: Andersson lesion (AL), a phenomenon initially described by Andersson nearly 80 years ago, has been the subject of extensive research and various treatment modalities. The ongoing debate surrounding the need for anterior surgery in AL cases has spurred numerous proposed approaches. Despite the demonstrated efficacy of anterior surgery in achieving fusion and stabilization, its implementation is associated with prolonged operation time and heightened intraoperative bleeding. PATIENT CONCERNS: A 32-year-old male patient presented at our hospital in February 2019 with a 2-month history of bilateral lower extremity weakness and sensory disturbances. These symptoms were exacerbated by a recent fall. DIAGNOSIS: AL conbined with ankylosing spondylitis. INTERVENTIONS: A 1-stage posterior fixation and decompression procedure was performed to ensure spinal stability, minimize deformities, and reduce surgical trauma. To achieve these goals, a 2-stage approach was employed, which included video-assisted thoracoscope-guided vertebrectomy, spinal canal decompression, and bone graft fusion. OUTCOMES: No recurrences of significant pain, limb numbness, or other symptoms were reported, ultimately leading to an improved quality of life for the patient. LESSONS: We utilized video-assisted thoracoscopic surgery technology for anterior bone graft fusion in a patient with AL to minimize the trauma of secondary surgery. However, the 3-year follow-up showed insufficient bony fusion at the fracture site. Nevertheless, the patient maintained spinal stability with posterior internal fixation and no significant kyphosis or symptoms. Thus, standalone posterior fixation may suffice for favorable clinical outcomes in patients with AL.

Treatment of unstable C1 semi-ring fractures with direct C1 pedicle screw fixation using a navigational template: A case report and literature review.

RATIONALE: Because of the risk of C1 to C2 instability, which would reduce the mobility of the occipito-atlanto-axis articulation, unstable C1 semi-ring fractures are typically treated with C1 to C2 or C0 to C2 fusion. The vertebral artery and spinal cord are at risk of harm during the installation of C1 pedicle screws. There is a need for a method that can maintain the occipito-atlanto-axis articulation's mobility and increase the safety of C1 pedicle screw fixation, particularly for surgeons who have less experience inserting C1 pedicle screws freehand. PATIENT CONCERNS: A 45-year-old man who had suffered a severe fall from a height of 2.5 m presented with pain in his cervical spine. Magnetic resonance imaging and computed tomography were used to diagnose unstable atlas fractures. DIAGNOSIS: According to radiographic studies, the patient had a unilateral anterior and posterior arch fracture (semi-ring fracture, Landells type II), as well as fractures and transverse ligament avulsion at the attachment site. INTERVENTIONS: We fixed the C1 directly with a pedicle screw using a navigational template. OUTCOMES: Both during and after the operation, there were no connected complications. Imaging at 12 months after surgery demonstrated that the fracture had united. The average visual analog scale score decreased from 8 before surgery to 2. LESSONS: In particular for surgeons with less experience placing freehand C1 pedicle screws, direct C1 pedicle screw fixation with the aid of a navigational template was a good option because it can preserve the mobility of the occipito-atlanto-axis articulation and improve the safety of C1 pedicle screw.

Biomechanical Properties of Novel Lateral Hole Pedicle Screws and Solid Pedicle Screws: A Comparative Study in the Beagle Dogs.

OBJECTIVE: Although pedicle screws are widely used to reconstruct the stability of the spine, screw loosening is a common complication after spine surgery. The main objective of this study was to investigate whether the application of the hollow lateral hole structure had the potential to improve the stability of the pedicle screw by comparing the biomechanical properties of the novel lateral hole pedicle screws (LHPSs) with those of the solid pedicle screws (SPSs) in beagle dogs. METHODS: The cancellous bone of the distal femur, proximal femur, distal tibia, and proximal tibia were chosen as implantation sites in beagle dogs. In each of 12 dogs, four LHPSs, and four SPSs were implanted into both lower limbs. At 1, 2, and 3 months after surgery, four dogs were randomly sampled and sacrificed. The LHPS group and SPS group were subdivided into four subgroups according to the length of their duration of implantation (0, 1, 2, 3 months). The biomechanical properties of both pedicle screws were evaluated by pull-out and the cyclic bending tests. RESULTS: The results of the study showed that no significant difference was found between LHPSs (276.62 ± 50.11 N) and SPSs (282.47 ± 42.98 N) in pull-out tests at time 0 (P > 0.05). At the same time point after implantations, LHPSs exhibited significantly higher maximal pullout strength than SPSs (month 1: 360.51 ± 25.63 vs 325.87 ± 28.11 N; month 2: 416.59 ± 23.78 vs 362.12 ± 29.27 N; month 3: 447.05 ± 38.26 vs 376.63 ± 32.36 N) (P < 0.05). Moreover, compared with SPSs, LHPSs withstood more loading cycles (month 2: 592 ± 21 vs 534 ± 48 times; month 3: 596 ± 10 vs 543 ± 59 times), and exhibiting less displacement before loosening at month 2 (1.70 ± 0.17 vs 1.96 ± 0.10 mm) and 3 (1.69 ± 0.19 vs 1.92 ± 0.14 mm) (P < 0.05), but no significant difference in time 0 and month 1 (P > 0.05). CONCLUSIONS: The pedicle screw with the hollow lateral hole structure could allow bone to grow into the inner architecture, which improved biomechanical properties by extending the contact area between screw and bone tissue after implantation into the cancellous bone. It indicated that LHPS could reduce loosening of the pedicle screws in long term after surgery.

Elucidation of the α-Ketoamide Inhibition Mechanism: Revealing the Critical Role of the Electrostatic Reorganization Effect of Asp17 in the Active Site of the 20S Proteasome.

The 20S proteasome is an attractive drug target for the development of anticancer agents because it plays an important role in cellular protein degradation. It has a threonine residue that can act as a nucleophile to attack inhibitors with an electrophilic warhead, forming a covalent adduct. Fundamental understanding of the reaction mechanism between covalent inhibitors and the proteasome may assist the design and refinement of compounds with the desired activity. In this study, we investigated the covalent inhibition mechanism of an α-keto phenylamide inhibitor of the proteasome. We calculated the noncovalent binding free energy using the PDLD/S-LRA/ß method and the reaction free energy through the empirical valence bond method (EVB). Several possible reaction pathways were explored. Subsequently, we validated the calculated activation and reaction free energies of the most plausible pathways by performing kinetic experiments. Furthermore, the effects of different ionization states of Asp17 on the activation energy at each step were also discussed. The results revealed that the ionization states of Asp17 remarkably affect the activation energies and there is an electrostatic reorganization of Asp17 during the course of the reaction. Our results demonstrate the critical electrostatic effect of Asp17 in the active site of the 20S proteasome.

Realistic simulations of proton transport along the gramicidin channel: demonstrating the importance of solvation effects.

The nature of proton transduction (PTR) through a file of water molecules, along the gramicidin A (gA) channel, has long been considered as being highly relevant to PTR in biological systems. Previous attempts to model this process implied that the so-called Grotthuss mechanism and the corresponding orientation of the water file plays a major role. The present work reexamines the PTR in gA by combining a fully microscopic empirical valence bond (EVB) model and a recently developed simplified EVB-based model with Langevin dynamics (LD) simulations. The full model is used first to evaluate the free energy profile for a stepwise PTR process. The corresponding results are then used to construct the effective potential of the simplified EVB. This later model is then used in Langevin dynamics simulations, taking into account the correct physics of possible concerted motions and the effect of the solvent reorganization. The simulations reproduce the observed experimental trend and lead to a picture that is quite different from that assumed previously. It is found that the PTR in gA is controlled by the change in solvation energy of the transferred proton along the channel axis. Although the time dependent electrostatic fluctuations of the channel and water dipoles play their usual role in modulating the proton-transfer process (Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 444), the PTR rate is mainly determined by the free energy profile. Furthermore, the energetics of the reorientation of the unprotonated water file do not appear to provide a consistent way of assessing the activation barrier for the PTR process. It seems to us that in the case of gA, and probably other systems with significant electrostatic barriers for the transfer of the proton charge, the PTR rate is controlled by the electrostatic barrier. This finding has clear consequences with regards to PTR processes in biological systems.