Unraveling Binding Mechanism and Stability of Urease Inhibitors: A QM/MM MD Study.

Soil bacteria can produce urease, which catalyzes the hydrolysis of urea to ammonia (NH3) and carbamate. A variety of urease inhibitors have been proposed to reduce NH3 volatilization by interfering with the urease activity. We report a quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) study on the mechanism employed for the inhibition of urease by three representative competitive inhibitors; namely, acetohydroxamic acid (AHA), hydroxyurea (HU), and N-(n-butyl)phosphorictriamide (NBPTO). The possible connections between the structural and thermodynamical properties and the experimentally observed inhibition efficiency were evaluated and characterized. We demonstrate that the binding affinity decreases in the order NBPTO >> AHA > HU in terms of the computed activation and reaction free energies. This trend also indicates that NBPTO shows the highest inhibitory activity and the lowest IC50 value of 2.1 nM, followed by AHA (42 µM) and HU (100 µM). It was also found that the X=O moiety (X = carbon or phosphorous) plays a crucial role in the inhibitor binding process. These findings not only elucidate why the potent urease inhibitors are effective but also have implications for the design of new inhibitors.

Predictors of life-space mobility in patients with fracture 3 months after discharge from convalescent rehabilitation ward: a prospective longitudinal study.

[Purpose] To identify predictors of life-space mobility in patients with fracture three months after discharge from convalescent rehabilitation ward. [Participants and Methods] This is a prospective longitudinal study that included patients aged 65 or older with a fracture who were scheduled for discharge home from the convalescent rehabilitation ward. Baseline measurements included sociodemographic variables (age, gender, and disease), the Falls Efficacy Scale-International, maximum walking speed, the Timed Up & Go test, the Berg Balance Scale, the modified Elderly Mobility Scale, the Functional Independence Measure, the revised version of Hasegawa's Dementia Scale, and the Vitality Index up to two weeks before discharge. As a follow-up, the life-space assessment was measured three months after discharge. In the statistical analysis, multiple linear and logistic regression analyses were performed with the life-space assessment score and the life-space level of "places outside your town" as dependent variables. [Results] The Falls Efficacy Scale-International, the modified Elderly Mobility Scale, age, and gender were selected as predictors in the multiple linear regression analysis, whereas in the multiple logistic regression analysis, the Falls Efficacy Scale-International, age, and gender were selected as predictors. [Conclusion] Our study emphasized the importance of fall-related self-efficacy and motor function for life-space mobility. The findings of this study suggest that when considering post-discharge living, therapists should conduct an appropriate assessment and adequate planning.

Singlet O2 Reactions with Radical Cations of 8-Bromoguanine and 8-Bromoguanosine: Guided-Ion Beam Mass Spectrometric Measurements and Theoretical Treatments.

8-Bromoguanosine is generated in vivo as a biomarker for early inflammation. Its formation and secondary reactions lead to a variety of biological sequelae at inflammation sites, most of which are mutagenic and linked to cancer. Herein, we report the formation of radical cations of 8-bromoguanine (8BrG•+) and 8-bromoguanosine (8BrGuo•+) and their reactions toward the lowest excited singlet molecular oxygen (1O2)─a common reactive oxygen species generated in biological systems. This work aims to investigate synergistic, oxidatively generated damage of 8-brominated guanine and guanosine that may occur upon ionizing radiation, one-electron oxidation, and 1O2 oxidation. Capitalizing on measurements of reaction product ions and cross sections of 8BrG•+ and 8BrGuo•+ with 1O2 using guided-ion beam tandem mass spectrometry and augmented by computational modeling of the prototype reaction system, 8BrG•+ + 1O2, using the approximately spin-projected ωB97XD/6-31+G(d,p) density functional theory, the coupled cluster DLPNO-CCSD(T)/aug-cc-pVTZ and the multireference CASPT2(21,15)/6-31G**, probable reaction products, and potential energy surfaces (PESs) were mapped out. 8BrG•+ and 8BrGuo•+ present similar exothermic oxidation products, and their reaction efficiencies with 1O2 increase with decreasing collision energy. Both single- and multireference theories predicted that the two most energetically favorable reaction pathways correspond to 1O2-addition to the C8 and C5-positions of 8BrG•+, respectively. The CASPT2-calculated PES represents the best quantitative agreement with the experimental benchmark, in that the oxidation exothermicity is close to the water hydration energy of product ions and, thus, is able to eliminate a water ligand in the product ions.

Spin-projected QM/MM Free Energy Simulations for Oxidation Reaction of Guanine in B-DNA by Singlet Oxygen.

Guanine is the most susceptible base to oxidation damage induced by reactive oxygen species including singlet oxygen (1 O2 , 1 Δg ). We clarify whether the first step of guanine oxidation in B-DNA proceeds via either a zwitterionic or a diradical intermediate. The free energy profiles are calculated by means of a combined quantum mechanical and molecular mechanical (QM/MM) method coupled with the adaptive biasing force (ABF) method. To describe the open-shell electronic structure of 1 O2 correctly, the broken-symmetry spin-unrestricted density functional theory (BS-UDFT) with an approximate spin projection (AP) correction is applied to the QM region. We find that the effect of spin contamination on the activation and reaction free energies is up to ∼8 kcal mol-1 , which is too large to be neglected. The QM(AP-ULC-BLYP)/MM-based free energy calculations also reveal that the reaction proceeds through a diradical transition state, followed by a conversion to a zwitterionic intermediate. Our computed activation energy of 5.2 kcal mol-1 matches experimentally observed range (0∼6 kcal mol-1 ).

Laparoscopic ventral rectopexy using the transanal vacuum test for complete rectal prolapse.

Laparoscopic ventral rectopexy was performed in 84 patients with complete rectal prolapse from January 2016 to December 2019. In the initial 27 cases, three cases had recurrence, especially in cases of a long rectal prolapse measuring over 10 cm. In order to avoid recurrence, the transanal vacuum test was performed following the dissection of the rectovaginal septum towards the pelvic floor. The disappearance of rectal prolapse is confirmed by the intraoperative transanal vacuum test. When the posterior wall of the rectum showed the presence of prolapse according to the transanal vacuum test, then laparoscopic ventral rectopexy was converted to laparoscopic posterior rectopexy. In 94 cases in which laparoscopic ventral rectopexy was attempted, laparoscopic ventral rectopexy was completed in 57 cases, while the procedure was converted to laparoscopic posterior rectopexy in 37 cases. The recurrence rate following laparoscopic ventral rectopexy decreased from 11.1% (3/27) to 1.7% (1/57) after beginning to use the transanal vacuum test. Laparoscopic ventral rectopexy using the transanal vacuum test is therefore considered to be a useful technique to reduce postoperative recurrence.

UNO DMRG CAS CI calculations of binuclear manganese complex Mn(IV)2 O2 (NHCHCO2 )4 : Scope and applicability of Heisenberg model.

Both direct exchange and super-exchange interactions cooperate to realize inter-spin magnetic interaction in binuclear manganese complex Mn(IV)2 O2 (NHCHCO2 )4 with a di-µ-oxo path. We revisited this spin system using DMRG CAS methods and CAS selection procedures. Our results indicate that our previous "dynamically extended spin polarization" (DE-SP) procedure for organic polyradicals and so forth does not work well. Thus, we have examined another selection procedure, the "dynamically extended super-exchange" (DE-SE) procedure. DMRG CASCI [18,18] by UB3LYP(HS)-UNO(DE-SE) can realize antiferromagnetic J values similar to experimental ones (-87 cm-1 ). In addition, all J values between all spin states (HS[septet],IS[quintet],IS[triplet],LS[singlet])were also shown to be correct under sufficiently large M values. © 2018 Wiley Periodicals, Inc.

Electrophysiological evaluation of a chronically implanted electrode for suprachoroidal transretinal stimulation in rabbit eyes.

In this study, we aimed to determine the electrophysiological efficacy, safety, and electrical stability of a chronically implanted electrode for suprachoroidal transretinal stimulation (STS) in rabbit eyes. A platinum microelectrode was implanted into the scleral pocket of rabbit eyes (n = 5) and followed-up for 6 months. To evaluate the electrophysiological efficacy, electrically evoked potentials (EEPs) were measured every month after implantation. To evaluate safety, fundus examinations, fluorescein angiograms, electroretinograms (ERGs), and visually evoked potentials (VEPs) were measured before and every month after the implantation. At the end of the experiment, histological examination of retinal tissue beneath the site of the electrode was performed. To evaluate electrical stability, the resistance of the circuit was measured every month after implantation. EEPs could be elicited from the STS electrodes at all testing times. The mean threshold current to evoke EEPs was 186.4 ± 47.0 µA at 6 months after implantation. There was no significant change in the threshold over the follow-up period. The resistance of the circuit was significantly increased at 1 months after implantation, with no further increase at 6 months. There was no statistically significant change in the relative amplitudes and implicit times of a- and b-waves of ERGs and VEPs. No intraocular infection, inflammation, or vitreoretinal proliferation was observed in any eye. Histological examination revealed no retinal damage beneath the electrode. We conclude that chronically implanted electrodes for STS appear to be effective, safe, and electrically stable.

Quantitative Assessment of rPM6 for Fluorine- and Chlorine-Containing Metal Complexes: Comparison with Experimental, First-Principles, and Other Semiempirical Results.

We report a reparameterization of PM6 parameters for fluorine and chlorine using our training set containing transition metal complexes. Spin unrestricted calculations with the resulting rPM6 (UrPM6) were examined quantitatively using two test sets: (i) the description of magnetic interactions in 25 dinuclear metal complexes and (ii) the prediction of barrier heights and reaction energies for epoxidation and fluorination reactions catalyzed by high-valent manganese-oxo species. The conventional UPM6 and UPM7 methods were also evaluated for comparison on the basis of either experimental or computational (the UB3LYP/SVP level) outcomes. The merits of UrPM6 are highlighted by both the test sets. As regards magnetic exchange coupling constants, the UrPM6 method had the smallest mean absolute errors from the experimental data (19 cm-1), followed by UPM7 (119 cm-1) and UPM6 (373 cm-1). For the epoxidation and fluorination reactions, all of the transition state searches were successful using UrPM6, while the success rates obtained by UPM6 and UPM7 were only 50%. The UrPM6-optimized stationary points also agreed well with the reference UB3LYP-optimized geometries. The accuracy for estimating reaction energies was also greatly remedied.

[A Case of Advanced Breast Cancer with Improvement in the Quality of Life by Local Treatment Using Mohs Paste and Systemic Pharmacotherapy].

Locally advanced breast cancer with skin invasion often causes malodor, bleeding, and massive exudates, which degrades patients' quality of life(QOL). A 61-year-old woman presented with locally advanced breast cancer with malodor and massive exudates, which had carcinomatous pleurisy causing dyspnea. We administered endocrine therapy and chemotherapy and used Mohs paste for local therapy. The exposed part of the tumor was fixed using Mohs paste. After continuing to apply Vaseline over the fixed part, the lesion spontaneously detached without surgical removal and completely epithelized, and malodor and exudates disappeared. Cancerous pleurisy also improved, and dyspnea disappeared. Local treatment using Mohs paste and systemic pharmacotherapy dramatically improved her QOL.

A Bis(µ-oxido)dinickel(III) Complex with a Triplet Ground State.

A bis(µ-oxido)dinickel(III) complex was synthesized and characterized by single crystal X-ray diffraction, resonance Raman, and ESI-mass measurements. Magnetic susceptibility measurements by SQUID and EPR spectroscopy reveal that the complex has a triplet ground state, which is unprecedented for high-valent metal (M) complexes with [M2 (µ-O)2 ] diamond core. DFT studies indicate ferromagnetic coupling of the nickel(III) centers. The complex exhibits hydrogen abstraction reactivity and oxygenation reactivity toward external substrates.

Reparameterization of PM6 Applied to Organic Diradical Molecules.

We have performed a reparameterization of PM6 (called rPM6) to compute open-shell species, specifically organic diradical molecules, within a framework of the spin-unrestricted semiempirical molecular orbital (SE-UMO) method. The parameters for the basic elements (hydrogen, carbon, nitrogen, and oxygen) have been optimized simultaneously using the training set consisting of 740 reference data. On the basis of the GMTKN30 database, the mean absolute error of rPM6 is decreased from 16.1 to 14.1 kcal/mol, which reassures its accuracy for ground-state properties. Applications of the spin-unrestricted rPM6 (UrPM6) method to small diradicals and relatively large polycyclic aromatic hydrocarbons have provided substantial improvement over the standard SE-UMO methods like UAM1, UPM3, and the original UPM6. The UrPM6 calculation is much less susceptible to spin contamination and, therefore, reproduces geometric parameters and adiabatic singlet-triplet energy gaps obtained by UDFT (UB3LYP and/or UBHandHLYP) at much lower computational cost.

Effects of Hachimijiogan, a Kampo powder, on epididymidis sperm characteristics in healthy male rats.

Purpose: Hachimijiogan (HJG, Rehmannia Eight Formula), a kidney-replenishing Kampo formula, is clinically known to be effective in the treatment of male infertility with oligozoospermia. The purpose of this study was to evaluate the effect of HJG on the epididymal sperm characteristics and related serum hormone changes in rats in an attempt to determine its mechanism. Methods: Male Wistar-Imamichi rats (233.4 ± 5.2 g, nine weeks old) were assigned randomly to four groups (n = 6 for each group). Apart from one control group treated with distilled water, the other groups were administered HJG consecutively for 9-11 days with doses of 250, 500 and 1,000 mg/kg. After the last administration the caude epididymides were quickly removed under anesthesia for assessing sperm characteristics. Additionally, the testes, seminal vesicles, prostate and adrenal glands were removed surgically and their wet weights measured. Results: Results showed that HJG increased sperm numbers and motility as well as the weights of seminal vesicles and adrenal glands at lower doses. Moreover, HJG decreased serum levels of testosterone and luteinizing hormone while increasing follicle-stimulating hormone levels. Conclusions: Our findings may support the conclusion that a lower dosage of HJG has an effect on improving local spermatogenous environments by activating adrenal functions and/or promoting local androgen activity.

[Neuroendocrine tumor of the terminal ileum observed by magnifying endoscopy with narrow-band imaging: a case report].

We report the case of an 88-year-old woman with localized intestinal obstruction caused by a midgut neuroendocrine tumor (NET) without endocrine symptoms. She was referred to our hospital for lower abdominal pain. Abdominal enhanced computed tomography revealed a thickened wall in the terminal ileum with dilated small bowel and multiple hepatic metastases upstream. Although the presenting symptoms resolved with short-term fasting and defecation, we performed further investigation. Colonoscopy confirmed the presence of submucosal tumors in the terminal ileum with a yellow-discolored surface but without ulceration or erosion. Magnifying endoscopy with narrow-band imaging clearly showed extended and dilated vessels, with the existing vessels maintained under the epithelium. Biopsies from these lesions were immunohistochemically positive for all neuroendocrine markers, and the Ki-67 index was 10%. Therefore, the patient was diagnosed with NET, and she underwent laparoscopic surgery to relieve the intestinal obstruction. Pathological examination of the resected specimen confirmed grade 2 NET with intramural metastasis and dissemination. After follow-up for a month, octreotide long-acting repeatable therapy was initiated and the patient was free of symptoms at the 6-month follow-up. This is the first report of midgut NET observed by magnifying endoscopy with narrow-band imaging.

Time-dependent changes in cardiovascular function during copulatory behavior induced by the hand method in the male dog.

PURPOSE: Ejaculation in the male dog consists of three fractions. Observation of behavior and measurement of heart rate (HR), and plasma noradrenaline (NA) and adrenaline (Ad) concentrations were researched sequentially, and a fundamental examination of the features of sympathetic nerve activity during copulatory behavior induced by the hand method in the male dog was undertaken. METHODS: We investigated the breeding capability of male dogs. HR, plasma NA level and plasma Ad levels were measured during ejaculation induced by the hand method. RESULTS: HR was 125.8 ± 6.0 beats/min at rest, and peaked during mounting at 195.2 ± 8.2 beats/min. Moreover, HR at 3 min after the first fraction decreased to values similar to those at rest. Plasma NA and Ad concentrations during copulatory behavior induced by the hand method did not differ significantly from those at rest. However, although there was no significant difference, plasma NA concentration during ejaculation of the third fraction peaked at about 1.8 times the baseline value. CONCLUSIONS: In the male dog, excitation of sympathetic nerves of long duration during erection of the penis and ejaculation is questionable. However, inhibition of sympathetic nerves and activation of parasympathetic nerves is thought to occur during erection of the penis and ejaculation.

Activated Akt expression is associated with the recurrence of primary melanomas and further refines the prognostic and predictive values for relapse in acral melanomas.

A PTEN deficiency leads to the activation of phospho-Akt at serine 473 (p-Akt) and promotes the tumorigenesis of melanomas by coupling with NUAK2 amplification. We tested the prognostic impact of p-Akt and/or NUAK2 expression on the relapse-free survival (RFS) and overall survival (OS) of melanoma patients. Primary tumors from patients with acral melanomas (112), Low-cumulative sun damage (CSD) melanomas (38), and High-CSD melanomas (18) were examined using immunohistochemistry and their prognostic significance was analyzed statistically. The expression of p-Akt was found in 32.1%, 68.4%, and 55.6% of acral, Low-CSD, and High-CSD melanomas, while NUAK2 expression was found in 46.4%, 76.3%, and 50.0%, respectively. Either p-Akt or NUAK2 expression was inversely correlated with the RFS of primary melanoma patients and acral melanoma patients (p-Akt: p < .0001, p < .0001; NUAK2; p = .0005, p < .0001, respectively). Strikingly, multivariate analyses revealed that p-Akt had a significant impact on RFS (Hazard ratio = 4.454; p < .0001), while NUAK2 did not. Further subset analyses revealed that p-Akt expression had an inferior RFS of patients with acral melanomas (Hazard ratio = 4.036; p = .0005). We conclude that the expression of p-Akt has a significant impact on RFS of patients with primary melanomas and can predict the relapse of patients with acral melanomas.

Analytical gradients for density functional calculations with approximate spin projection.

We have derived and implemented analytical gradients for broken-symmetry unrestricted density functional calculations (BS-UDFT) with removal of spin contamination by Yamaguchi's approximate spin projection method. Geometry optimizations with these analytical gradients (AGAP-opt) yield results consistent with those obtained with the previously available numerical gradients (NAP-opt). The AGAP-opt approach is found to be more precise, efficient, and robust than NAP-opt. It allows full geometry optimizations for large open-shell systems. We report results for three types of organic diradicals and for a binuclear vanadium(II) complex to demonstrate the merits of removing the spin contamination effects during geometry optimization (AGAP-opt vs BS-UDFT) and to illustrate the superior performance of the analytical gradients (AGAP-opt vs NAP-opt). The results for the vanadium(II) complex indicate that the AGAP-opt method is capable of handling pronounced spin contamination effects in large binuclear transition metal complexes with two magnetic centers.

QM/MM Molecular Dynamics Simulations Revealed Catalytic Mechanism of Urease.

Urease catalyzes the hydrolysis of urea to form ammonia and carbamate, inducing an overall pH increase that affects both human health and agriculture. Inhibition, mutagenesis, and kinetic studies have provided insights into its enzymatic role, but there have been debates on the substrate binding mode as well as the reaction mechanism. In the present study, we report quatum mechanics-only (QM-only) and quantum mechanics/molecular mechanics molecular dynamics (QM/MM MD) calculations on urease that mainly investigate the binding mode of urea and the mechanism of the urease-catalyzed hydrolysis reaction. Comparison between the experimental data and our QM(GFN2-xTB)/MM metadynamics results demonstrates that urea hydrolysis via a complex with bidentate-bound urea is much more favorable than via that with monodentate-bound urea for both nucleophilic attack and the subsequent proton transfer steps. We also indicate that the bidentate coordination of urea fits the active site with a closed conformation of the mobile flap and can facilitate the stabilization of transition states and intermediates by forming multiple hydrogen bonds with certain active site residues.

Singlet O2 Oxidation of the Radical Cation versus the Dehydrogenated Neutral Radical of 9-Methylguanine in a Watson-Crick Base Pair. Consequences of Structural Context.

In DNA, guanine is the most susceptible to oxidative damage by exogenously and endogenously produced electronically excited singlet oxygen (1O2). The reaction mechanism and the product outcome strongly depend on the nucleobase ionization state and structural context. Previously, exposure of a monomeric 9-methylguanine radical cation (9MG•+, a model guanosine compound) to 1O2 was found to result in the formation of an 8-peroxide as the initial product. The present work explores the 1O2 oxidation of 9MG•+ and its dehydrogenated neutral form [9MG - H]• within a Watson-Crick base pair consisting of one-electron-oxidized 9-methylguanine-1-methylcytosine [9MG·1MC]•+. Emphasis is placed on entangling the base pair structural context and intra-base pair proton transfer with and consequences thereof on the singlet oxygenation of guanine radical species. Electrospray ionization coupled with guided-ion beam tandem mass spectrometry was used to study the formation and reaction of guanine radical species in the gas phase. The 1O2 oxidation of both 9MG•+ and [9MG - H]• is exothermic and proceeds barrierlessly either in an isolated monomer or within a base pair. Single- and multi-referential theories were tested for treating spin contaminations and multi-configurations occurring in radical-1O2 interactions, and reaction potential energy surfaces were mapped out to support experimental findings. The work provides a comprehensive profile for the singlet oxygenation of guanine radicals in different charge states and in the absence and the presence of base pairing. All results point to an 8-peroxide as the major oxidation product in the experiment, and the oxidation becomes slightly more favorable in a neutral radical form. On the basis of a variety of reaction pathways and product profiles observed in the present and previous studies, the interplay between guanine structure, base pairing, and singlet oxygenation and its biological implications are discussed.

Impact of a SLC24A5 variant on the retinal pigment epithelium of a Japanese patient with oculocutaneous albinism type 6.

Oculocutaneous albinism (OCA) 6 is a non-syndromic type of OCA that has distinct ocular symptoms and variable cutaneous hypopigmentation. The causative gene of OCA6 is SLC24A5, which encodes NCKX5, a K+ -dependent Na+ /Ca2+ exchanger 5. NCKX5 is involved in the maturation of melanosomes, but its function is still unclear. In this study, we characterized a Japanese patient with OCA6. Genetic analysis revealed compound heterozygous variants in SLC24A5, c.590 + 1dupG, and c.598G>A (p.G200R). To clarify the functional significance of the missense variant, we generated a knock-in (KI) mouse model carrying the mouse homolog of the G200R variant using the CRISPR/Cas9 system. Chemical analysis showed decreased amounts of eumelanin in the hair and skin of KI mice, while levels of benzothiazine units in pheomelanin were significantly increased in their hair. Retinal pigment was also decreased in KI mice. Notably, a histopathologic study revealed a significant pigment loss in the retinal pigment epithelium (RPE) but not in the choroid. Immunohistochemically, the expression of NCKX5 in the RPE was decreased but was maintained in the choroid of KI mice. These findings could explain the difference in phenotypic severity between eye symptoms and hypopigmentation in the skin/hair.

Potential energy curve for ring-opening reactions: comparison between broken-symmetry and multireference coupled cluster methods.

The spin-unrestricted Hartree-Fock (UHF)-based coupled cluster singles and doubles (UHF-CCSD) and Mukherjee's state-specific multireference CCSD (MkCCSD) methods are applied to four ring-opening reactions. The spin-restricted Hartree-Fock (RHF)-based CCSD (RHF-CCSD) calculations are also performed for comparison. In the case of the UHF-CCSD method, an approximate spin-projection (AP) method is applied to the broken-symmetry (BS) singlet solution to remove the spin contamination effect. For potential energy curves (PECs) of all reactions presented in this study, the results of RHF-CCSD and UHF-CCSD are substantially different from those of MkCCSD, while the results after the AP method (AP-UCCSD) reproduce the MkCCSD results well. It strongly suggests that the spin contamination effect should be removed by the AP correction even at the UHF-CCSD level to predict reliable energetics of these reactions.