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.

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.

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.

Genome-wide association study identifies CDH13 as a susceptibility gene for rhododendrol-induced leukoderma.

Racemic RS-4-(4-hydroxyphenyl)-2-butanol (rhododendrol; trade name: Rhododenol [RD]), which is used in topical skin-lightening cosmetics, was unexpectedly reported in Japan to induce leukoderma or vitiligo called RD-induced leukoderma (RIL) after repeated application. To our knowledge, no studies have investigated chemical-induced vitiligo pathogenesis on a genome-wide scale. Here, we conducted a genome-wide association study (GWAS) for 147 cases and 112 controls. CDH13, encoding a glycosylphosphatidylinositol-anchored protein called T-cadherin (T-cad), was identified as the strongest RIL susceptibility gene. RD sensitivity was remarkably increased by T-cad knockdown in cultured normal human melanocytes. Furthermore, we confirmed tyrosinase upregulation and downregulation of the anti-apoptotic molecules (BCL-2 and BCL-XL), suggesting that T-cad is associated with RD via tyrosinase or apoptotic pathway regulation. Finally, monobenzyl ether of hydroquinone sensitivity also tended to increase with T-cad knockdown, suggesting that the T-cad could be a candidate susceptibility gene for RIL and other chemical-induced vitiligo forms. This is the first GWAS for chemical-induced vitiligo, and it could be a useful model for studying the disease's genetic aspects.

[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 new in vivo analysis model to detect sexually dimorphic rat liver cytochrome P450 gene expression dependent on growth hormone secretory patterns.

Several drug-metabolizing cytochrome P450 (CYP) enzymes exhibit sexual dimorphism depending on the pituitary growth hormone (GH) secretory patterns. However, the mechanism underlying CYP sexual dimorphism remains unclear. We previously established a transgenic (Alb-DsRed2 Tg) rat that expressed red fluorescent DsRed2 protein, particularly in hepatocytes, to visualize cell differentiation and multiplication and found that hepatic DsRed2 expression exhibited sexual dimorphism that was limited to adult males. In this study, we compared the expression patterns between sexual dimorphic Cyps and DsRed2 in Tg rats after experimentally reversing the GH secretory patterns in males and females. Postnatal day 1 male and female Tg rats were gonadectomized and then testosterone propionate (0.25 mg/rat) was subcutaneously administered to ovariectomized females immediately after surgery. Cyp mRNA and DsRed2 expression levels were quantified using RT-PCR and an in vivo imaging system, respectively. GH-dependent Cyps and hepatic DsRed2 expression patterns were reversed in males and females at 9 weeks after birth and were significantly correlated (P<0.05). This suggested that DsRed2 expression in these Tg rats depended on GH secretory patterns. Based on DsRed2 fluorescence, this Tg rat model could become a tool to readily and effectively evaluate changes in GH-dependent Cyp expression.

[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.

Quantum mechanics/molecular mechanics study of oxygen binding in hemocyanin.

We report a combined quantum mechanics/molecular mechanics (QM/MM) study on the mechanism of reversible dioxygen binding in the active site of hemocyanin (Hc). The QM region is treated by broken-symmetry density functional theory (DFT) with spin projection corrections. The X-ray structures of deoxygenated (deoxyHc) and oxygenated (oxyHc) hemocyanin are well reproduced by QM/MM geometry optimizations. The computed relative energies strongly depend on the chosen density functional. They are consistent with the available thermodynamic data for oxygen binding in hemocyanin and in synthetic model complexes when the BH&HLYP hybrid functional with 50% Hartree-Fock exchange is used. According to the QM(BH&HLYP)/MM results, the reaction proceeds stepwise with two sequential electron transfer (ET) processes in the triplet state followed by an intersystem crossing to the singlet product. The first ET step leads to a nonbridged superoxo CuB(II)-O2(•-) intermediate via a low-barrier transition state. The second ET step is even more facile and yields a side-on oxyHc complex with the characteristic Cu2O2 butterfly core, accompanied by triplet-singlet intersystem crossing. The computed barriers are very small so that the two ET processes are expected to very rapid and nearly simultaneous.

Characterization of multiple first exons in murine prolactin receptor gene and the effect of prolactin on their expression in the choroid plexus.

Prolactin (Prl) receptor (Prlr) gene is expressed in various brain regions, with the highest level present in the choroid plexus, a site for receptor-mediated PRL transport from the blood to cerebrospinal fluid. We investigated the regulatory mechanism of Prlr gene expression by PRL in the murine choroid plexus. We first examined the organization of the alternative first exons in murine Prlr gene. In addition to the three known first exons, mE1(1), mE1(2), and mE1(3), two first exons, mE1(4) and mE1(5), were newly identified by cDNA cloning. Each first exon variant of Prlr mRNA exhibited tissue-specific or generic expression. In the choroid plexus of mice, the expression levels of mE1(3)-, mE1(4)-, and mE1(5)-Prlr mRNAs were increased in the lactating mice compared with those in the diestrus mice. Furthermore, the expression level of mE1(4)-Prlr mRNA was decreased in the PRL-deficient (Prl(-/-)) mice compared with the PRL-normal (Prl(+/+) and Prl(+/-)) mice. In the ovariectomized Prl(-/-) mice, the expression level of mE1(4)-Prlr mRNA was significantly increased by PRL administration but not by 17ß-estradiol administration. The expression levels of the two last exon variants of Prlr mRNAs, encoding the long and short cytoplasmic regions of PRLR, were also increased in the lactating mice and decreased in the Prl(-/-) mice. These findings suggest that PRL stimulates the Prlr gene expression through the transcriptional activation of mE1(4) first exon, leading to increases in the long- and short-form variants of Prlr mRNA in the murine choroid plexus.

Multireference character of 1,3-dipolar cycloaddition of ozone with ethylene and acrylonitrile.

In the present study, the concerted and stepwise reaction mechanisms for 1,3-dipole cycloaddition of ozone with ethylene (1) and acrylonitrile (2) are investigated. The stationary points are optimized by using four hybrid R(U)DFT methods. A geometry optimization method based on an approximate spin projection (AP-opt method) is applied to eliminate a spin contamination from the broken-symmetry (BS) solution. The AP-opt method reveals that a diradical intermediate for the stepwise pathway is spurious due to the spin contamination. The revised reaction profile with no diradical intermediate supports the stereospecificity. On the basis of the experimental data, the RCCSD(T) method outperforms AP-UCCSD(T), AP-UBD(T), and MkMRCCSD(4e,4o) for the systems, indicating that the RCCSD(T) method can describe the diradical character of ozone within a framework of single reference wave function. The subsequent single point energy calculations show that the highly synchronous transition state is much more favorable than the asynchronous one for 1. In the case of 2, there is not much difference between two transition states because of its asymmetric structure and charge separations in the transition states.

Reinvestigation of the reaction of ethylene and singlet oxygen by the approximate spin projection method. Comparison with multireference coupled-cluster calculations.

We quantify a spin contamination error caused by a broken-symmetry (BS) method on the geometry at the stationary points and barrier heights of the [2 + 2] reaction between singlet oxygen and ethylene, which goes through a diradical intermediate. Several hybrid GGA, hybrid meta-GGA, and long-range corrected hybrid functionals, O3LYP, B3LYP, PBE0, MPW1B95, BHandHLYP, and omegaB97X, are examined to elucidate their original nature without the spin contamination error. For that purpose, the geometry of each reaction step for the BS state as well as its total energy is corrected by using an approximate spin projection method. The CCSD and CCSD(T) single-point calculations are also carried out at optimized geometries at the DFT level to confirm the results of the DFT methods. The single-point calculations by means of Mukherjee's multireference coupled cluster with single and double excitations at CASSCF(10e,8o)-optimized geometries are also presented to assess the DFT methods. After the energy and geometry corrections, the barrier height of each functional is consistent with conventional closed-shell-type reactions even in the reaction involving singlet diradical species. We also find that the spin contamination error on the geometric change is not negligible especially at the early stage of the reaction ( approximately 3 kcal/mol), where the triplet state is the ground state.

Morphological and histochemical study of the nasal cavity and fused olfactory bulb of the brown-eared bulbul, Hysipetes amaurotis.

The brown-eared bulbul (Hysipetes amaurotis) is commonly found in Japan where it is regarded as a harmful bird that causes damage to agricultural products. Few studies have investigated the sensory apparatus of this bird, and consequently little is known of the sensory modalities it uses. Here we analyzed the anatomical and histological properties of the nasal cavity and olfactory bulb (OB) of the bulbul in order to investigate the functional level of olfaction in this species. Although both anterior and maxillary conchae were observed in the bulbul nasal cavity, there was no structure equivalent to the posterior concha. The OB located on the ventral side of the anterior extremity of the cerebrum and the ratio of olfactory bulb size to that of the cerebral hemisphere were very small. Interestingly, the left and right OBs were completely fused at the midline of the cerebrum. Furthermore, certain types of lectins that bind to the olfactory nerve of vertebrates with a well-developed sense of smell also bound positively to the olfactory nerve and glomerular layers of the bulbul OB. These findings suggest that the brown-eared bulbul has an anatomically and functionally less well developed sense of smell compared to other avian species. Although the molecular and developmental mechanisms underlying the fusion of the OB remain unknown, we suggest that the fused OB may offer a unique model for studying the evolution and development of the central olfactory nervous system in vertebrates.

Investigation of the chemical-induced selective type II (T(H)2) allergic response in mice: effect of the length of the sensitizing phase.

Allergies are immune system disorders characterized by abnormal, acquired sensitivity to various environmental chemicals. We investigated the mechanism of chemical-induced selective type II (T(H)2) allergy by using three different sensitization protocols and the well-known respiratory sensitizer trimellitic anhydride (TMA). Mice were sensitized for either 1, 2, or 3 weeks. For each sensitization schedule, the mice were allocated into 3 or 4 groups: -/- group, both sensitized and challenged with vehicle; -/+ group, sensitized with vehicle and challenged with 0.1% TMA; +/- group, sensitized with 1% TMA and challenged with vehicle; and +/+ group, both sensitized and challenged with 0.1% TMA. After challenge, we assayed the auricular lymph nodes of all mice for number of lymphocytes, surface antigen expression of B-cells, and local cytokine production, and we measured TMA-specific serum IgE levels. Some parameters in mice sensitized for 1 or 2 wk showed, at most, mild changes. In contrast, all parameters in animals receiving 3-wk sensitization showed marked increases, as well as marked increases in the IgE/major histocompatibility complex (MHC) Class II-positive B-cell population and T(H)2 cell production of IL-10 and IL-13. These results indicate that 3 wk of sensitization according to our protocol led to overt respiratory allergic reactions. While these studies showed that using the approach here, positive reactions were elicited using a typical allergen; whether the same events occur after sensitization by other chemicals that are found in the environment remains uncertain. These findings here should be regarded moreover as preliminary in scope and that additional studies with irritants, dermal sensitizers and other respiratory sensitizers are needed to further evaluate the overall sensitivity and selectivity of this novel protocol.