GENESIS 2.1: High-Performance Molecular Dynamics Software for Enhanced Sampling and Free-Energy Calculations for Atomistic, Coarse-Grained, and Quantum Mechanics/Molecular Mechanics Models.

GENeralized-Ensemble SImulation System (GENESIS) is a molecular dynamics (MD) software developed to simulate the conformational dynamics of a single biomolecule, as well as molecular interactions in large biomolecular assemblies and between multiple biomolecules in cellular environments. To achieve the latter purpose, the earlier versions of GENESIS emphasized high performance in atomistic MD simulations on massively parallel supercomputers, with or without graphics processing units (GPUs). Here, we implemented multiscale MD simulations that include atomistic, coarse-grained, and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. They demonstrate high performance and are integrated with enhanced conformational sampling algorithms and free-energy calculations without using external programs except for the QM programs. In this article, we review new functions, molecular models, and other essential features in GENESIS version 2.1 and discuss ongoing developments for future releases.

Learning QM/MM potential using equivariant multiscale model.

The machine learning (ML) method emerges as an efficient and precise surrogate model for high-level electronic structure theory. Its application has been limited to closed chemical systems without considering external potentials from the surrounding environment. To address this limitation and incorporate the influence of external potentials, polarization effects, and long-range interactions between a chemical system and its environment, the first two terms of the Taylor expansion of an electrostatic operator have been used as extra input to the existing ML model to represent the electrostatic environments. However, high-order electrostatic interaction is often essential to account for external potentials from the environment. The existing models based only on invariant features cannot capture significant distribution patterns of the external potentials. Here, we propose a novel ML model that includes high-order terms of the Taylor expansion of an electrostatic operator and uses an equivariant model, which can generate a high-order tensor covariant with rotations as a base model. Therefore, we can use the multipole-expansion equation to derive a useful representation by accounting for polarization and intermolecular interaction. Moreover, to deal with long-range interactions, we follow the same strategy adopted to derive long-range interactions between a target system and its environment media. Our model achieves higher prediction accuracy and transferability among various environment media with these modifications.

Full-endoscopic spine surgery in oldest old patients aged over 90 years:A case report.

BACKGROUND: Transforaminal full-endoscopic spine surgery (FESS) is the least invasive spinal surgery and can be performed under local anesthesia. In Japan, the population is rapidly aging and the number of spinal surgeries performed in the elderly is also increasing. OBJECT: In this report, we describe 3 patients aged 90 years or older in whom we performed FESS under local anesthesia. CASE: The first case was a 90-year-old man who presented with severe leg pain. He had multiple medical comorbidities and was unsuitable for general anesthesia. We performed FESS. After surgery, the leg pain resolved with full recovery of muscle strength. He was discharged with no perioperative complications. The second case was a 90-year-old man who presented with severe leg pain. MRI showed a herniated nucleus pulposus and foraminal stenosis at L4/5. We performed FESS. The leg pain improved immediately after surgery. The third case was a 91-year-old woman in whom we diagnosed left L5 radiculopathy due to foraminal stenosis at L5/S1. After surgery, her leg pain was relieved. CONCLUSION: FESS is a good surgical procedure for elderly patients who are in a poor general condition because it is minimally invasive and can be performed under local anesthesia with early mobilization. J. Med. Invest. 71 : 169-173, February, 2024.

Similarity scores of vibrational spectra reveal the atomistic structure of pentapeptides in multiple basins.

Vibrational spectroscopy combined with theoretical calculations is a powerful tool for analyzing the interaction and conformation of peptides at the atomistic level. Nonetheless, identifying the structure becomes increasingly difficult as the peptide size grows large. One example is acetyl-SIVSF-N-methylamide, a capped pentapeptide, whose atomistic structure has remained unknown since its first observation [T. Sekiguchi, M. Tamura, H. Oba, P. Çarçarbal, R. R. Lozada-Garcia, A. Zehnacker-Rentien, G. Grégoire, S. Ishiuchi and M. Fujii, Angew. Chem., Int. Ed., 2018, 57, 5626-5629]. Here, we propose a novel conformational search method, which exploits the structure-spectrum correlation using a similarity score that measures the agreement of theoretical and experimental spectra. Surprisingly, the two conformers have distinctly different energy and geometry. The second conformer is 25 kJ mol-1 higher in energy than the other, lowest-energy conformer. The result implies that there are multiple pathways in the early stage of the folding process: one to the global minimum and the other to a different basin. Once such a structure is established, the second conformer is unlikely to overcome the barrier to produce the most stable structure due to a vastly different hydrogen bond network of the backbone. Our proposed method can characterize the lowest-energy conformer and kinetically trapped, high-energy conformers of complex biomolecules.

Transforaminal Full-Endoscopic Ventral Facetectomy: Midterm Results and Factors Associated with Poor Surgical Outcomes.

BACKGROUND: Full-endoscopic spine surgery (FESS) is a well-established procedure for herniated nucleus pulposus. It is a minimally invasive surgery that can be performed under local anesthesia through only an 8-mm skin incision. With improvements in surgical equipment such as high-speed drills, the indications for FESS have expanded to include lumbar spinal stenosis (LSS). We perform a transforaminal full-endoscopic ventral facetectomy (TF-FEVF) for unilateral nerve root-type lateral recess stenosis (LRS) using a transforaminal approach under local anesthesia.The aim of this study was to examine the postoperative results of TF-FEVF for LRS and to identify factors associated with poor surgical outcomes. 85 patients who underwent TF-FEVF for LRS under local anesthesia. Clinical outcomes were determined by visual analog scale (VAS) and the modified MacNab criteria. Evaluation was performed using magnetic resonance imaging (MRI), computed tomography (CT), and flexion-extension radiographs. METHODS: This study involved 85 patients (47 males and 38 females) who underwent TF-FEVF for LRS. The mean age was 70.5 years and the mean follow-up duration was 14.8 months. Data were collected on sex, age, level of operation, diagnosis, history of spine surgery at the same level, and duration of follow-up. The diagnosis was categorized as LSS with or without disk bulging. Clinical evaluation was performed using the VAS and modified MacNab criteria. MRI was used to evaluate the degree of disk degeneration, vertebral endplate degeneration, disk height, thickening of the ligamentum flavum, and stenosis. Bony stenosis was evaluated using CT. Sagittal translation and sagittal angulation were also measured by flexion-extension radiographs, and the Cobb angle was measured using a standing front view radiograph. All variables were compared between patients with excellent/good outcomes (E/G group) and those with fair/poor outcomes (F/P group) using the modified MacNab criteria. RESULTS: Postoperative VAS showed that leg pain decreased from 59.0 ± 28.6 preoperatively to 17.9 ± 27.2 at the final follow-up (p < 0.01) and that lower back pain also decreased from 60.7 ± 26.6 preoperatively to 27.3 ± 28.6 at final follow-up (p < 0.01). According to the modified MacNab criteria, the results during the final follow-up were excellent in 39 cases, good in 21 cases, fair in 13 cases, and poor in 12 cases. There were no significant differences in sex, age, diagnosis, history of spine surgery, and duration of follow-up periods between the 60 cases (70.6%) in the E/G group and the 25 cases (29.4%) in the F/P group. Imaging evaluation revealed statistically significant differences between the E/G group and the F/P group in intervertebral angle flexion (3.2 vs. 0.4 degrees; p < 0.05), sagittal angulation (4.3 vs. 8.1 degrees; p < 0.05), slip in flexion (0.9 vs. 2.8 mm; p < 0.05), sagittal translation (0.7 vs. 1.6 mm; p < 0.05), and Cobb angle (-0.5 vs. -1.9 degrees; p < 0.05). CONCLUSION: Midterm results of TF-FEVF were generally favorable; factors contributing to good or poor TF-FEVF outcomes were large sagittal angulation, large sagittal translation, and concave side.

Transforming Growth Factor-ß Induces Interleukin-6 Secretion from Human Ligamentum Flavum-Derived Cells through Partial Activation of p38 and p44/42 Mitogen-Activated Protein Kinases.

STUDY DESIGN: This experimental study was performed using human ligamentum flavum-derived cells (HFCs). PURPOSE: To investigate the intracellular signaling mechanism of interleukin-6 (IL-6) secretion in transforming growth factor-ß (TGF- ß)-stimulated HFCs. OVERVIEW OF LITERATURE: Lumbar spinal stenosis (LSS) is a prevalent disease among the elderly, characterized by debilitating pain in the lower extremities. Although the number of patients with LSS has increased in recent years, the underlying pathomechanism remains unclear. Clinical examinations typically rely on magnetic resonance imaging to diagnose patients, revealing ligamentum flavum hypertrophy. Some studies have suggested an association between ligamentum flavum hypertrophy and inflammation/fibrosis, and expression of TGF-ß and IL-6 has been observed in surgically obtained ligamentum flavum samples. However, direct evidence linking TGF-ß and IL-6 expression in HFCs is lacking. METHODS: HFCs were obtained from patients with LSS who had undergone decompression surgery. The cells were stimulated with TGF-ß and pretreated with either the p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 or the p44/42 MAP kinase inhibitor FR180204. IL-6 secretion in the cell culture medium and IL-6 messenger RNA (mRNA) expression levels were analyzed using an enzyme-linked immunoassay and real-time polymerase chain reaction, respectively. RESULTS: TGF-ß administration resulted in a dose- and time-dependent stimulation of IL-6 release. Treatment with SB203580 and FR180204 markedly suppressed TGF-ß-induced IL-6 secretion from HFCs. Moreover, these inhibitors suppressed IL-6 mRNA expression in response to TGF-ß stimulation. CONCLUSIONS: Our findings indicate that TGF-ß induces IL-6 protein secretion and gene expression in HFCs through the activation of p38 or p44/42 MAP kinases. These results suggest a potential association between IL-6-mediated inflammatory response and tissue hypertrophy in LSS, and we provide insights into molecular targets for therapeutic interventions targeting LSS-related inflammation through our analysis of the MAP kinase pathway using HFCs.

Allosteric regulation of ß-reaction stage I in tryptophan synthase upon the α-ligand binding.

Tryptophan synthase (TRPS) is a bifunctional enzyme consisting of α- and ß-subunits that catalyzes the last two steps of L-tryptophan (L-Trp) biosynthesis. The first stage of the reaction at the ß-subunit is called ß-reaction stage I, which converts the ß-ligand from an internal aldimine [E(Ain)] to an α-aminoacrylate [E(A-A)] intermediate. The activity is known to increase 3-10-fold upon the binding of 3-indole-D-glycerol-3'-phosphate (IGP) at the α-subunit. The effect of α-ligand binding on ß-reaction stage I at the distal ß-active site is not well understood despite the abundant structural information available for TRPS. Here, we investigate the ß-reaction stage I by carrying out minimum-energy pathway searches based on a hybrid quantum mechanics/molecular mechanics (QM/MM) model. The free-energy differences along the pathway are also examined using QM/MM umbrella sampling simulations with QM calculations at the B3LYP-D3/aug-cc-pVDZ level of theory. Our simulations suggest that the sidechain orientation of ßD305 near the ß-ligand likely plays an essential role in the allosteric regulation: a hydrogen bond is formed between ßD305 and the ß-ligand in the absence of the α-ligand, prohibiting a smooth rotation of the hydroxyl group in the quinonoid intermediate, whereas the dihedral angle rotates smoothly after the hydrogen bond is switched from ßD305-ß-ligand to ßD305-ßR141. This switch could occur upon the IGP-binding at the α-subunit, as evidenced by the existing TRPS crystal structures.

Advantages of Revision Transforaminal Full-Endoscopic Spine Surgery in Patients who have Previously Undergone Posterior Spine Surgery.

BACKGROUND: Revision lumbar spine surgery via a posterior approach is more challenging than primary surgery because of epidural or perineural scar tissue. It demands more extensive removal of the posterior structures to confirm intact bony landmarks and could cause iatrogenic instability; therefore, fusion surgery is often added. However, adjacent segment disease after fusion surgery could be a problem, and further exposure of the posterior muscles could result in multiple operated back syndrome. To address these problems, we now perform transforaminal full-endoscopic spine surgery (TF-FES) as revision surgery in patients who have previously undergone posterior lumbar surgery. There have been several reports on the advantages of TF-FES, which include feasibility of local anesthesia, minimal invasiveness to posterior structures, and less scar tissue with fewer adhesions. In this study, we aim to assess the clinical outcomes of revision TF-FES and its advantages. METHODS: We evaluated 48 consecutive patients with a history of posterior lumbar spine surgery who underwent revision TF-FES (at 60 levels) under local anesthesia. Intraoperative blood loss, operating time, and complication rate were evaluated. Postoperative outcomes were assessed using the modified Macnab criteria and visual analog scale (VAS) scores for leg pain, back pain, and leg numbness. We also compared the outcome of revision FES with that of primary FES. RESULTS: Mean operating time was 70.5 ± 14.4 (52-106) minutes. Blood loss was unmeasurable. The clinical outcomes were rated as excellent at 16 levels (26.7%), good at 28 (46.7%), fair at 10 (16.7%), and poor at 6 (10.0%). The mean preoperative VAS score was 6.0 ± 2.6 for back pain, 6.8 ± 2.4 for leg pain, and 6.3 ± 2.8 for leg numbness. At the final follow-up, the mean postoperative VAS scores for leg pain, back pain, and leg numbness were 4.3 ± 2.5, 3.8 ± 2.6, and 4.6 ± 3.2, respectively. VAS scores for all three parameters were significantly improved (p < 0.05). There was no significant difference in operating time, intraoperative blood loss, or the complication rate between revision FES and primary FES. CONCLUSIONS: Clinical outcomes of revision TF-FES in patients with a history of posterior lumbar spine surgery were acceptable (excellent and good in 73.4% of cases). TF-FES can preserve the posterior structures and avoid scar tissue and adhesions. Therefore, TF-FES could be an effective procedure for patients who have previously undergone posterior lumbar spine surgery.

A Technical Pitfall of Decompression with Direct Repair of a Ragged Edge Using the Smiley-Face Rod Method†:†A Case Report.

The smiley-face rod method has been reported to be a successful technique for reducing slippage and repairing pars defects in lumbar spondylolisthesis. However, we encountered a patient who developed right L5 radiculopathy with muscle weakness after use of the smiley-face rod method. The patient was a 19-year-old female judo player who had undergone direct repair surgery using the smiley-face rod method for terminal-stage lumbar spondylolysis. Postoperatively, she developed paresthesia on the lateral side of the right thigh with weakness of the right tibialis anterior and extensor hallucis longus. Computed tomography showed right foraminal stenosis at L5 with the floating lamina shifted ventrally and apophyseal ring fracture. In this case, the spondylolysis fracture angle differed between the left and right sides, with the fracture line on the right side running more sagittally. As a result, the floating lamina was shifted ventrally on the right side by compression and the right L5 intervertebral foraminal space was narrowed due to the ventral shift in the floating lamina and the apophyseal ring bone fragment. The shape of the fracture line should be examined carefully before surgery to avoid this technical pitfall. J. Med. Invest. 69 : 308-311, August, 2022.

General Design Strategy to Precisely Control the Emission of Fluorophores via a Twisted Intramolecular Charge Transfer (TICT) Process.

Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.

Computational Analysis on the Allostery of Tryptophan Synthase: Relationship between α/ß-Ligand Binding and Distal Domain Closure.

Tryptophan synthase (TRPS) is a bifunctional enzyme consisting of α and ß-subunits and catalyzes the last two steps of l-tryptophan (L-Trp) biosynthesis, namely, cleavage of 3-indole-d-glycerol-3'-phosphate (IGP) into indole and glyceraldehyde-3-phosphate (G3P) in the α-subunit, and a pyridoxal phosphate (PLP)-dependent reaction of indole and l-serine (L-Ser) to produce L-Trp in the ß-subunit. Importantly, the IGP binding at the α-subunit affects the ß-subunit conformation and its ligand-binding affinity, which, in turn, enhances the enzymatic reaction at the α-subunit. The intersubunit communications in TRPS have been investigated extensively for decades because of the fundamental and pharmaceutical importance, while it is still difficult to answer how TRPS allostery is regulated at the atomic detail. Here, we investigate the allosteric regulation of TRPS by all-atom classical molecular dynamics (MD) simulations and analyze the potential of mean-force (PMF) along conformational changes of the α- and ß-subunits. The present simulation has revealed a widely opened conformation of the ß-subunit, which provides a pathway for L-Ser to enter into the ß-active site. The IGP binding closes the α-subunit and induces a wide opening of the ß-subunit, thereby enhancing the binding affinity of L-Ser to the ß-subunit. Structural analyses have identified critical hydrogen bonds (HBs) at the interface of the two subunits (αG181-ßS178, αP57-ßR175, etc.) and HBs between the ß-subunit (ßT110 - ßH115) and a complex of PLP and L-Ser (an α-aminoacrylate intermediate). The former HBs regulate the allosteric, ß-subunit opening, whereas the latter HBs are essential for closing the ß-subunit in a later step. The proposed mechanism for how the interdomain communication in TRPS is realized with ligand bindings is consistent with the previous experimental data, giving a general idea to interpret the allosteric regulations in multidomain proteins.

Segmental Arteries and Veins at Higher Lumbar Levels Can Intersect the Adjacent Caudal Intervertebral Disc in the Anterior Part of the Spinal Column: A Cadaveric Analysis.

STUDY DESIGN: A cadaveric study. PURPOSE: To investigate the anatomical features of segmental arteries and veins in the anterior part of the spinal column to prevent segmental vessel injury. OVERVIEW OF LITERATURE: The lateral transpsoas approach to the lumbar intervertebral discs (IVD) is associated with the risk of segmental vessel injury. Previous studies have described the vascular anatomy on the lateral part of the vertebral body. However, there are no studies that describe the segmental vessels on its anterior aspect. Here, we report the important anatomical features of the segmental arteries and veins that can intersect the anterior part of the IVD. These vessels are considered at risk of vascular injury when placing the anterior retractors during lateral lumbar interbody fusion or cutting the anterior longitudinal ligament during anterior column realignment. METHODS: Five formalin-embalmed human cadavers were used. We assessed the proportion of segmental arteries and veins that intersected the IVD in the L2-L5 range and their course on the anterior part of the spinal column. RESULTS: The segmental arteries and veins commonly intersect the anterior part of the IVD (artery, 28.1%; vein, 42.1%). Seven of 10 (70%) segmental arteries at L2 intersected the IVD, but only one artery intersected the IVD at L3 and L4. The proportions of segmental veins that intersected the IVD were 60%, 50%, and 16.7% at L2, L3, and L4, respectively. CONCLUSIONS: The segmental arteries and veins frequently intersect the IVD in the anterior part of the spinal column. Therefore, it is necessary to consider these individual anatomical features to prevent vascular damage during lateral lumbar interbody fusion surgery.

Weight average approaches for predicting dynamical properties of biomolecules.

Recent advances in atomistic molecular dynamics (MD) simulations of biomolecules allow us to explore their conformational spaces widely, observing large-scale conformational fluctuations or transitions between distinct structures. To reproduce or refine experimental data using MD simulations, structure ensembles, which are characterized by multiple structures and their statistical weights on the rugged free-energy landscapes, are often used. Here, we summarize weight average approaches for various experimental measurements. Weight average approaches are now applied to hybrid quantum mechanics/molecular mechanics MD simulations to predict fast vibrational motions in a protein with a high accuracy for better understanding of molecular functions from atomic structures.

Towards complete assignment of the infrared spectrum of the protonated water cluster H+(H2O)21.

The spectroscopic features of protonated water species in dilute acid solutions have been long sought after for understanding the microscopic behavior of the proton in water with gas-phase water clusters H+(H2O)n extensively studied as bottom-up model systems. We present a new protocol for the calculation of the infrared (IR) spectra of complex systems, which combines the fragment-based Coupled Cluster method and anharmonic vibrational quasi-degenerate perturbation theory, and demonstrate its accuracy towards the complete and accurate assignment of the IR spectrum of the H+(H2O)21 cluster. The site-specific IR spectral signatures reveal two distinct structures for the internal and surface four-coordinated water molecules, which are ice-like and liquid-like, respectively. The effect of inter-molecular interaction between water molecules is addressed, and the vibrational resonance is found between the O-H stretching fundamental and the bending overtone of the nearest neighboring water molecule. The revelation of the spectral signature of the excess proton offers deeper insight into the nature of charge accommodation in the extended hydrogen-bonding network underpinning this aqueous cluster.

Anharmonic Vibrational Calculations Based on Group-Localized Coordinates: Applications to Internal Water Molecules in Bacteriorhodopsin.

An efficient anharmonic vibrational method is developed exploiting the locality of molecular vibration. Vibrational coordinates localized to a group of atoms are employed to divide the potential energy surface (PES) of a system into intra- and inter-group contributions. Then, the vibrational Schrödinger equation is solved based on a PES, in which the inter-group coupling is truncated at the harmonic level while accounting for the intra-group anharmonicity. The method is applied to a pentagonal hydrogen bond network (HBN) composed of internal water molecules and charged residues in a membrane protein, bacteriorhodopsin. The PES is calculated by the quantum mechanics/molecular mechanics (QM/MM) calculation at the level of B3LYP-D3/aug-cc-pVDZ. The infrared (IR) spectrum is computed using a set of coordinates localized to each water molecule and amino acid residue by second-order vibrational quasi-degenerate perturbation theory (VQDPT2). Benchmark calculations show that the proposed method yields the N-D/O-D stretching frequencies with an error of 7 cm-1 at the cost reduced by more than five times. In contrast, the harmonic approximation results in a severe error of 150 cm-1. Furthermore, the size of QM regions is carefully assessed to find that the QM regions should include not only the pentagonal HBN itself but also its HB partners. VQDPT2 calculations starting from transient structures obtained by molecular dynamics simulations have shown that the structural sampling has a significant impact on the calculated IR spectrum. The incorporation of anharmonicity, sufficiently large QM regions, and structural samplings are of essential importance to reproduce the experimental IR spectrum. The computational spectrum paves the way for decoding the IR signal of strong HBNs and helps elucidate their functional roles in biomolecules.

A new surgical strategy for the intractable chronic low back pain due to type 1 Modic change using transforaminal full-endoscopic disc cleaning (FEDC) surgery under the local anesthesia†:†A case report and literature review.

It has been reported that Modic change of the lumbar spine endplate includes three types: i.e. . edema or inflammation for type 1, fatty marrow change for type 2 and sclerotic change for type 3. Basically, type 1 Modic change may be related to the chronic low back pain. There are two kinds of the treatment for the type 1 Modic change to heal the pain : the anti-inflammatory drugs, and intra-discal injection of steroid. When the inflammatory change would be intractable, surgical intervention is needed. The gold standard for the surgical intervention is the segmental fusion of the affected level. The fusion surgery may cause the adjacent degeneration ; thus, motion preservation surgery is better, if possible. Our department started the motion preservation full-endoscopic intradiscal debridement surgery for this pathology, since some of the type 1 Modic change may be chronic discitis by P. Acnes. In this paper, we describe the first patient of type 1 Modic change who was successfully treated by the full-endoscopic intra-discal debridement and drainage under the local anesthesia. We named this procedure as transforaminal full-endoscopic disc cleaning surgery (FEDC). Finally, pathology, conservative and surgical intervention of Modic change was discussed. J. Med. Invest. 68 : 1-5, February, 2021.

Exploring the Minimum-Energy Pathways and Free-Energy Profiles of Enzymatic Reactions with QM/MM Calculations.

Understanding molecular mechanisms of enzymatic reactions is of vital importance in biochemistry and biophysics. Here, we introduce new functions of hybrid quantum mechanical/molecular mechanical (QM/MM) calculations in the GENESIS program to compute the minimum-energy pathways (MEPs) and free-energy profiles of enzymatic reactions. For this purpose, an interface in GENESIS is developed to utilize a highly parallel electronic structure program, QSimulate-QM (https://qsimulate.com), calling it as a shared library from GENESIS. Second, algorithms to search the MEP are implemented, combining the string method (E et al. J. Chem. Phys. 2007, 126, 164103) with the energy minimization of the buffer MM region. The method implemented in GENESIS is applied to an enzyme, triosephosphate isomerase, which converts dihyroxyacetone phosphate to glyceraldehyde 3-phosphate in four proton-transfer processes. QM/MM-molecular dynamics simulations show performances of greater than 1 ns/day with the density functional tight binding (DFTB), and 10-30 ps/day with the hybrid density functional theory, B3LYP-D3. These performances allow us to compute not only MEP but also the potential of mean force (PMF) of the enzymatic reactions using the QM/MM calculations. The barrier height obtained as 13 kcal mol-1 with B3LYP-D3 in the QM/MM calculation is in agreement with the experimental results. The impact of conformational sampling in PMF calculations and the level of electronic structure calculations (DFTB vs B3LYP-D3) suggests reliable computational protocols for enzymatic reactions without high computational costs.

p38 Mitogen-Activated Protein Kinase Is Involved in Interleukin-6 Secretion from Human Ligamentum Flavum-Derived Cells Stimulated by Tumor Necrosis Factor-α.

STUDY DESIGN: Human ligamentum flavum-derived cells (HFCs) were obtained from surgical samples for a basic experimental study. PURPOSE: We sought to evaluate the inflammatory response of human ligamentum flavum cells to investigate hypertrophic changes occurring in the ligamentum flavum. OVERVIEW OF LITERATURE: Lumbar spinal stenosis (LSS) is a disease commonly observed in the elderly. The number of patients with LSS has increased over time, yet the pathomechanisms of LSS still have not been fully elucidated. One of the clinical features of LSS is hypertrophy of the ligamentum flavum, which results in narrowing of the lumbar spinal canal. Some reports have suggested that ligamentum flavum hypertrophy is associated with inflammation and fibrosis; meanwhile, the p38 mitogen-activated protein (MAP) kinase is involved in the hypertrophy of human ligamentum flavum cells. METHODS: HFCs were obtained from patients with LSS who underwent surgery. HFCs were stimulated by tumor necrosis factor-α (TNF-α) and a p38 MAP kinase inhibitor, SB203580. Phosphorylation of the p38 MAP kinase was analyzed by western blotting. The concentration of interleukin-6 (IL-6) in the conditioned medium was measured by enzyme-linked immunoassay and IL-6 messenger RNA expression levels were determined by real-time polymerase chain reaction. RESULTS: TNF-α induced the phosphorylation of p38 MAP kinase in a time-dependent manner, which was suppressed by the p38 MAP kinase inhibitor, SB203580. TNF-α also stimulated IL-6 release in both a time- and dose-dependent manner. On its own, SB203580 did not stimulate IL-6 secretion from HFCs; however, it dramatically suppressed the degree of IL-6 release stimulated by TNF-α from HFCs. CONCLUSIONS: This is the first report suggesting that TNF-α stimulates the gene expression and protein secretion of IL-6 via p38 MAP kinase in HFCs. A noted association between tissue hypertrophy and inflammation suggests that the p38 MAP kinase inflammatory pathway may be a therapeutic molecular target for LSS.

Full-endoscopic Spine Surgery for Discogenic Low Back Pain with High-intensity Zones and Modic Type 1 Change in a Professional Baseball Player.

Non-specific low back pain in athletes can be caused by discogenic back pain, Modic type 1 change, and facet joint arthritis. In this report, we describe a full-endoscopic surgical strategy that we have used to treat a patient with both discogenic pain and Modic type 1 change. The patient was a 32-year-old professional baseball player who played an infield position and had a 2-year history of low back pain. Three years earlier, he had undergone micro-endoscopic discectomy for left herniated nucleus pulposus at L5/S1. His leg symptoms resolved postoperatively, and he returned to playing baseball the following season. However, his low back pain gradually increased. Two years after the initial surgery, he was experiencing low back pain in daily life and found it very difficult to play baseball. Short T1 inversion recovery (STIR) magnetic resonance imaging (MRI) revealed Modic type 1 change and high-signal intensity zones in degenerated discs at L4/5 and L5/S1. Injection of xylocaine 1% reduced the pain temporarily, confirming that the pain generator was at L4/5 and L5/S1. The pathological diagnosis was discogenic pain with Modic type 1 change. We performed full-endoscopic disc cleaning (FEDC) surgery for the Modic type 1 change and thermal annuloplasty (TA) for the discogenic pain at these levels. The patient's low back pain decreased steadily thereafter. Six months after surgery, he returned to baseball, playing for a full season without pain. We have successfully treated a professional baseball player with discogenic pain and Modic type 1 change by full-endoscopic surgery.

Retinal Vibrations in Bacteriorhodopsin are Mechanically Harmonic but Electrically Anharmonic: Evidence From Overtone and Combination Bands.

Fundamental vibrations of the chromophore in the membrane protein bacteriorhodopsin (BR), a protonated Schiff base retinal, have been studied for decades, both by resonance Raman and by infrared (IR) difference spectroscopy. Such studies started comparing vibrational changes between the initial BR state (all-trans retinal) and the K intermediate (13-cis retinal), being later extended to the rest of intermediates. They contributed to our understanding of the proton-pumping mechanism of BR by exploiting the sensitivity of fundamental vibrational transitions of the retinal to its conformation. Here, we report on new bands in the 2,500 to 1,800 cm-1 region of the K-BR difference FT-IR spectrum. We show that the bands between 2,500 and 2,300 cm-1 originate from overtone and combination transitions from C-C stretches of the retinal. We assigned bands below 2,300 cm-1 to the combination of retinal C-C stretches with methyl rocks and with hydrogen-out-of-plane vibrations. Remarkably, experimental C-C overtone bands appeared at roughly twice the wavenumber of their fundamentals, with anharmonic mechanical constants ≤3.5 cm-1, and in some cases of ∼1 cm-1. Comparison of combination and fundamental bands indicates that most of the mechanical coupling constants are also very small. Despite the mechanical quasi-harmonicity of the C-C stretches, the area of their overtone bands was only ∼50 to ∼100 times smaller than of their fundamental bands. We concluded that electrical anharmonicity, the second mechanism giving intensity to overtone bands, must be particularly high for the retinal C-C stretches. We corroborated the assignments of negative bands in the K-BR difference FT-IR spectrum by ab initio anharmonic vibrational calculations of all-trans retinal in BR using a quantum-mechanics/molecular mechanics approach, reproducing reasonably well the small experimental anharmonic and coupling mechanical constants. Yet, and in spite accounting for both mechanical and electrical anharmonicities, the intensity of overtone C-C transitions was underestimated by a factor of 4-20, indicating room for improvement in state-of-the-art anharmonic vibrational calculations. The relatively intense overtone and combination bands of the retinal might open the possibility to detect retinal conformational changes too subtle to significantly affect fundamental transitions but leaving a footprint in overtone and combination transitions.