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.

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.

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.

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.

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.

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.

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.

Testing of Newly Developed Wide-Field Dual-Array Suprachoroidal-Transretinal Stimulation Prosthesis in Dogs.

Purpose: This study was conducted to investigate the feasibility of a newly developed wide-field dual-array suprachoroidal-transretinal stimulation (STS) prosthesis in dogs and to examine its biocompatibility and stability over a 4-month period. Methods: Three types of STS dual arrays were designed and tested. The STS dual-array was implanted into a scleral pocket of the left eye of six healthy beagle dogs. Ophthalmic examinations, fundus photography, fluorescein angiography (FA), electroretinography (ERG), and functional testing of this system were conducted postoperatively. The dogs were euthanatized at the end of the experiment, and their eyes were enucleated and histologically examined. Results: All prostheses were successfully implanted without complications, and no serious adverse event occurred during the postoperative period. Fundus photographs and FA showed no serious damage in the retina surrounding the arrays. The ERGs recorded from the implanted eyes showed no significant differences from those from control eyes. Histological evaluations demonstrated good preservation of the retina over the array. However, system failure occurred in 50% of the dogs owing to dog-specific habits. Conclusions: Implantation of this prosthesis system in dogs is feasible and can be performed without significant damage to the eye. The biocompatibility and stability of the array were good during the observation period, but the low durability of the system against dogs (not humans) is an issue to be resolved in the future. Translational Relevance: This study suggests that this wide-field dual-array prosthesis might widen the visual field and might be useful for patients with retinitis pigmentosa.

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 ).

Five novel mutations in SASH1 contribute to lentiginous phenotypes in Japanese families.

SASH1 has been reported as a causal gene of lentiginous phenotypes with and without heredity, including an autosomal dominant type characterized by lentigines predominantly on sun-exposed areas such as the face and limbs. Recently, cases of dyschromatosis with SASH1 mutations have been reported worldwide; however, only one case has been reported from Japan. Here, we analyzed six Japanese patients who characteristically showed many lentigines on sun-exposed areas, using next-generation sequencing. We identified five novel heterozygous mutations in SASH1 (p.I586M, p.S531Y, p.R644W, p.T525R, and p.S516I) in our patients and their families. The p.R644W substitution identified in two unrelated families was the first mutation located in the sterile alpha motif 1 (SAM1) domain. The degree and location of the lentigines were variable across individuals, even if they shared the same SASH1 mutation. All mutations were predicted to be deleterious by six different algorithms used to evaluate the functional impact of a variation. In addition, immunohistopathological findings and RNA sequencing results suggested that SASH1 mutations were associated with an increase in the number of melanocytes, acceleration of melanogenesis, and upregulated hair keratin expression.

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.