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The ultrafast pump–probe measurement represents a key technique to study fs–ps dynamics. The asynchronous optical sampling (ASOPS) method realizes fast and long time-range measurement with high time resolution using different repetition frequency pump–probe light pulses. The frequency difference Δf is an important parameter, as it dictates the measurement time and time resolution. However, usual ASOPS measurements require a complex and precise stabilizer to control Δf or it is difficult to change Δf. In this study, we use two free-running titanium/sapphire pulse lasers to develop a variable repetition frequency ASOPS (VRF-ASOPS) method without a stabilizer or feedback loop, where we can easily alter Δf by changing the cavity length of the probe light laser. To detect the coincidences of the pump–probe light pulses, we cause the instantaneous reflectivity change in a 100 nm platinum film by irradiating the pump light and observe it by the probe light. We use this signal as the trigger signal to directly determine Δf, which enables us to average and convert the measured responses without a stabilizer or feedback loop. Using this VRF-ASOPS system, we obtain pulse echo signals and 100 GHz Brillouin oscillations, which are equivalent to those measured by the mechanical delay line method, confirming the validity of our developed method.

The FeSe compound is the simplest high-temperature superconductor (HTSc) possible, and relatively cheap, not containing any rare-earth material. Although the transition temperature, Tc, is just below 10 K, the upper critical fields are comparable with other HTSc. Preparing FeSe using solid-state sintering yields samples exhibiting strong ferromagnetic hysteresis loops (MHLs), and the superconducting contribution is only visible after subtracting MHLs from above Tc. Due to the complicated phase diagram, the samples are a mixture of several phases, the superconducting β-FeSe, and the non-superconducting δ-FeSe and γ-FeSe. Furthermore, antiferromagnetic Fe7Se8 and ferromagnetic α-Fe may be contained, depending directly on the Se loss during the sintering process. Here, we show MHLs measured up to ±7 T and determine the magnetic characteristics, together with the amount of superconductivity determined from M(T) measurements. We also performed a thorough analysis of the microstructures in order to establish a relation between microstructure and the resulting sample properties.

Small changes in the radiation budget at the earth's surface can lead to large climatological responses when persistent over time. With the increasing debate on anthropogenic influences on climatic processes during the 1980s the need for accurate radiometric measurements with higher temporal resolution was identified, and it was determined that the existing measurement networks did not have the resolution or accuracy required to meet this need. In 1988 the WMO therefore proposed the establishment of a new international Baseline Surface Radiation Network (BSRN), which should collect and centrally archive high-quality ground-based radiation measurements in 1 min resolution. BSRN began its work in 1992 with 9 stations; currently (status 2018-01-01), the network comprises 59 stations (delivering data to the archive) and 9 candidates (stations recently accepted into the network with data forthcoming to the archive) distributed over all continents and oceanic environments. The BSRN database is the World Radiation Monitoring Center (WRMC). It is hosted at the Alfred Wegener Institute (AWI) in Bremerhaven, Germany, and now offers more than 10 300 months of data from the years 1992 to 2017. All data are available at https://doi.org/10.1594/PANGAEA.880000 free of charge.

Structural and transport properties in the normal and superconducting states are investigated in a Ca0.8La0.2FeAs2 single crystal with Tc = 27 K, belonging to the newly discovered 112 family of iron based superconductors. The transport critical current density Jc for both field directions measured in a focused ion beam patterned microbridge reveals a weakly field dependent and low anisotropic behaviour with a low temperature value as high as Jc(B = 0) ∼ 105 A/cm2. This demonstrates not only bulk superconductivity but also the potential of 112 superconductors towards applications. Interestingly, this superconducting compound undergoes a structural transition below 100 K which is evidenced by temperature-dependent X-ray diffraction measurements. Data analysis of Hall resistance and magnetoresistivity indicate that magnetotransport properties are largely dominated by an electron band, with a change of regime observed in correspondence of the onset of a structural transition. In the low temperature regime, the contribution of a hole band to transport is suggested, possibly playing a role in determining the superconducting state.

Characterizing the mechanical properties of single colloids is a central problem in soft matter physics. It also plays a key role in cell biology through biopolymer condensates, which function as membraneless compartments. Such systems can also malfunction, leading to the onset of a number of diseases, including many neurodegenerative diseases; the functional and pathological condensates are commonly differentiated by their mechanical signature. Probing the mechanical properties of biopolymer condensates at the single particle level has, however, remained challenging. In this study, we demonstrate that acoustic trapping can be used to profile the mechanical properties of single condensates in a contactless manner. We find that acoustic fields exert the acoustic radiation force on condensates, leading to their migration to a trapping point where acoustic potential energy is minimized. Furthermore, our results show that the Brownian motion fluctuation of condensates in an acoustic potential well is an accurate probe for their bulk modulus. We demonstrate that this framework can detect the change in the bulk modulus of polyadenylic acid condensates in response to changes in environmental conditions. Our results show that acoustic trapping opens up a novel path to profile the mechanical properties of soft colloids at the single particle level in a non-invasive manner with applications in biology, materials science, and beyond.

Background: The relationship between the tumor-immune microenvironment and systemic inflammatory markers, including neutrophil-to-lymphocyte ratio (NLR), is unclear., Methods: We examined the characteristics of systemic inflammatory markers and tumor immune microenvironments in relation to treatment outcomes in 29 consecutive patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) who received pembrolizumab, using 14-marker multiplex immunohistochemistry and image cytometry., Results: NLR ≥4.5 (high NLR) at pretreatment status significantly correlated with short overall survival (OS) and progression-free survival-2 (PFS2) and malnutrition status. High NLR in peripheral blood was significantly correlated with low lymphoid cell and high tumor-associated macrophage counts in tissues, especially myeloid-to-lymphoid cell ratios, suggesting an association between circulating and intratumoral immune complexity profiles., Conclusions: This study suggests a link between NLR in circulating blood, systemic nutritional status, and immune composition within the tumor., (© 2024 Wiley Periodicals LLC.)

Background: In view of improving biomarkers predicting the efficacy of immunotherapy for head and neck squamous cell carcinoma (R/M HNSCC), this multicenter retrospective study aimed to identify clinical, tumor microenvironmental, and genomic factors that are related to therapeutic response to the anti- Programmed cell death protein 1 (PD-1) antibody, nivolumab, in patients with R/M HNSCC., Methods: The study compared 53 responders and 47 non-responders, analyzing formalin-fixed paraffin-embedded samples using 14-marker multiplex immunohistochemistry and targeted gene sequencing., Results: Of 100 patients included, responders had significantly lower smoking and alcohol index, higher incidence of immune related adverse events, and higher PD-1 ligand (PD-L1) expression in immune cells as well as PD-L1 combined positive score (CPS) than non-responders. The frequency of natural killer cells was associated with nivolumab response in patients with prior cetuximab use, but not in cetuximab-naïve status. Age-stratified analysis showed nivolumab response was linked to high CPS and lymphoid-inflamed profiles in patients aged ≥ 65. In contrast, lower NLR in peripheral blood counts was associated with response in patients aged < 65. Notably, TP53 mutation-positive group had lower CPS and T cell densities, suggesting an immune-excluded microenvironment. Patients with altered tumor suppressor gene pathways, including TP53 , CDKN2A , and SMAD4 mutations, had lower CPS, higher smoking index, and were associated with poor responses., Conclusion: Nivolumab treatment efficacy in HNSCC is influenced by a combination of clinical factors, age, prior treatment, immune environmental characteristics, and gene mutation profiles., Competing Interests: TT received lecture honoraria, consulting fees, and grants from Ono Pharmaceutical Co., Ltd. and lecture honoraria from Bristol Myers Squibb, and Merck Sharp & Dohme Corp. KO received grants from Ono Pharmaceutical Co., Ltd. HO and SS were employed by SCREEN Holdings Co., Ltd. YaS received lecture honoraria from Bristol Myers Squibb, Merck Sharp & Dohme Corp, and Ono Pharmaceutical Co., Ltd. RH received lecture honoraria from Rakuten Medical Inc. YuS received grants from Merck Sharp & Dohme Corp. KT received lecture honoraria from Bristol Myers Squibb, Merck Sharp & Dohme Corp, and Ono Pharmaceutical Co., Ltd. NO received lecture honoraria and grants from Ono Pharmaceutical Co., Ltd and lecture honoraria from Bristol-Myers Squibb. NH received lecture honoraria from Bristol Myers Squibb, and Ono Pharmaceutical Co., Ltd. KN received lecture honoraria from Merck Sharp & Dohme Corp, and Ono Pharmaceutical Co., Ltd. SI received lecture honoraria and grants from Ono Pharmaceutical Co., Ltd and lecture honoraria from Bristol Myers Squibb. KI received grants from SCREEN Holdings Co., Ltd. TAs received grants from Ono Pharmaceutical Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Tsujikawa, Ohno, Morita, Saburi, Mitsuda, Yoshimura, Kimura, Morimoto, Ogi, Shibata, Akashi, Kurata, Imoto, Shimizu, Kano, Watanabe, Yamazaki, Asada, Hayashi, Saito, Ozawa, Tsukahara, Oridate, Sano, Horii, Ueki, Maruo, Mukoyama, Hanai, Fukusumi, Iwai, Fujisawa, Fujii, Nibu, Iwae, Ueda, Chikuie, Yasumatsu, Matsuo, Umeno, Ono, Masuda, Toh, Itoh, Hirano and Asakage.)

Intratumoral immune profiles are related to prognosis and therapeutic efficacy, and could result in personalized treatments based on biomarkers. To develop a multiplex, quantitative, and rapid tissue evaluation method based on the clinically established standard immunohistochemistry (IHC), a 6-marker rapid multiplex IHC was developed based on our previously reported 14-marker multiplex IHC by reducing the number of labels and accelerating the staining procedure. First, fewer labels were required to identify the same immunological features linked to prognosis in 14-marker multiplex IHC analyses. The six selected markers showed a significant correlation with the 14 markers in the immune classification. Next, a rapid staining protocol was developed by optimizing the reaction temperature, chromogen, and washing time, allowing the completion of 6-marker analysis in 5 h and 49 min, as opposed to the several days required for conventional multiplex IHC. Validation of benign tonsil and head and neck cancer tissues revealed a significant correlation between rapid and conventional 6-makrer multiplex IHC in terms of staining intensities, densities of T cells, macrophages, lymphoid/myeloid immune cell ratios, and spatial profiles of intratumoral immune infiltrates. This method may enable quantitative assessment of the tumor-immune microenvironment on a clinically feasible time scale, which promotes the development of tissue biomarker-guided therapeutic strategies., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Takahiro Tsujikawa reports financial support was provided by Japanese 10.13039/100009950Ministry of Education, Culture, Sports, Science, and Technology. Takahiro Tsujikawa reports financial support was provided by 10.13039/100009619Japan Agency for Medical Research and Development. Takahiro Tsujikawa reports financial support was provided by Public Promoting Association Asano Foundation for Studies on Medicine. Takahiro Tsujikawa reports financial support was provided by Research Promotion Award from the Japanese Society of Otorhinolaryngology-Head and Neck Surgery, Inc. Takahiro Tsujikawa reports a relationship with 10.13039/100004334Merck Biopharma that includes: consulting or advisory and speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/100009947Merck Sharp & Dohme Corp that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/501100003769Eisai Co Ltd that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/501100013170Ono Pharmaceutical Co Ltd that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/100008021Bristol Myers Squibb Co that includes: speaking and lecture fees. Takahiro Tsujikawa reports a relationship with 10.13039/100030815Otsuka Pharmaceutical Factory Inc that includes: speaking and lecture fees. Saya Shibata reports a relationship with Screen Holdings Co Ltd that includes: employment. Hiroshi Ogi reports a relationship with Screen Holdings Co Ltd that includes: employment. Eiichi Konishi reports a relationship with Roche Diagnostics that includes: consulting or advisory. Eiichi Konishi reports a relationship with 10.13039/100010795Chugai Pharmaceutical Co Ltd that includes: speaking and lecture fees. Kyoko Itoh reports a relationship with Screen Holdings Co Ltd that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Rice production is seriously affected by saline-alkaline stress worldwide. To elucidate the saline-alkaline tolerance mechanisms in a novel tolerant rice variety, Shwe Nang Gyi (SNG), we investigated ion accumulation in SNG and Koshihikari (KSH), which is a saline-alkaline sensitive rice variety, and the candidates for saline-alkaline inducible genes in SNG using RNA-seq. SNG had superior ion accumulation capacity, such as K and Zn, compared to KSH. In contrast, SNG accumulated the same level of Na content in its leaf blades as KSH despite the higher dry weight of the SNG leaf blades. We further found that the expression of numerous genes, including several K + transporter/high-affinity K + transporter/K + uptake protein/K + transporter (HAK/KUP/KT) family members, were upregulated in SNG, and that OsHAK17 and OsHAK21 expression levels in the roots were significantly higher in SNG than in KSH. Moreover, yeast complementation analysis revealed that OsHAK17 was involved in K + uptake under high-Na conditions. These results suggested that SNG has an effective K + acquisition system supported by OsHAK17 functioning in saline-alkaline environments., (© 2024. The Author(s).)

The rapid amplification and sensitive detection of α-synuclein (αSyn) seeds is an efficient approach for the early diagnosis of Parkinson's disease. Ultrasonication stands out as a promising method for the rapid amplification of αSyn seeds because of its robust fibril fragmentation capability. However, ultrasonication also induces the primary nucleation of αSyn monomers, deteriorating the seed detection sensitivity by generating seed-independent fibrils. In this study, we show that an addition of surfactants to the αSyn monomer solution during αSyn seed detection under ultrasonication remarkably improves the detection sensitivity of the αSyn seeds by a factor of 100-1000. Chemical kinetic analysis reveals that these surfactants reduce the rate of primary nucleation while promoting the fragmentation of the αSyn fibrils under ultrasonication. These effects are attributed to the modification of the ultrasonic cavitation surface by the surfactants. Our study enhances the utility of ultrasonication in clinical assays targeting αSyn seeds as the Parkinson's disease biomarker.

Introduction: Biliary atresia (BA) is a cholestatic hepatopathy caused by fibrosing destruction of intrahepatic and extrahepatic bile ducts, and its etiology has not been clearly revealed. In BA, liver fibrosis progression is often observed even after Kasai portoenterostomy (KPE), and more than half of cases require liver transplantation in their lifetime in Japan. Macrophages play an important role in liver fibrosis progression and are classically divided into proinflammatory (M1) and fibrotic macrophages (M2), whose phenotypic transformation is called "macrophage polarity." The polarity has been reported to reflect the tissue microenvironment. In this study, we examined the relationship between macrophage polarity and the post-KPE clinical course., Materials and Methods: Thirty BA patients who underwent KPE in our institution from 2000 to 2020 were recruited. Multiple immunostainings for CD68, CD163, CK19, and α-SMA were carried out on liver biopsy specimens obtained at KPE. ROC curves were calculated based on each clinical event, and the correlation with the clinical data was analyzed., Results and Discussion: The M2 ratio, defined as the proportion of M2 macrophages (CD163-positive cells), was correlated inversely with the occurrence of postoperative cholangitis (AUC: 0.7602). The patients were classified into M2 high ( n  = 19) and non-high ( n  = 11) groups based on an M2 ratio value obtained from the Youden index ( = 0.918). As a result, pathological evaluations (Metavir score, αSMA area fraction, and CK19 area fraction) were not significantly different between these groups. In mild liver fibrosis cases (Metavir score = 0-2), the M2 non-high group had a significantly lower native liver survival rate than the high group ( p  = 0.02). Moreover, 4 out of 8 cases in the M2 non-high group underwent early liver transplantation within 2 years after KPE., Conclusions: Non-M2 macrophages, including M1 macrophages, may be correlated with postoperative cholangitis, and the M2 non-high group in mild liver fibrosis cases had a significantly lower native liver survival rate than the high group, requiring early liver transplantation in this study. Preventing advanced liver fibrosis is a key factor in improving native liver survival for BA patients, and liver macrophages may play important roles in liver homeostasis and the promotion of inflammation and fibrosis., Competing Interests: SS, MakH, and HO are employees of SCREEN Holdings Co., Ltd. KI received research funding from SCREEN Holdings Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Nagayabu, Fumino, Shimamura, Sengoku, Higashi, Iguchi, Aoi, Saya, Hirai, Ogi, Miyagawa-Hayashino, Konishi, Itoh, Tajiri and Ono.)

The molecular mechanisms underlying insect gall formation remain unclear. A major reason for the inability to identify the responsible genes is that only a few systems can be experimentally validated in the laboratory. To overcome these problems, we established a new galling insect model, Smicronyx madaranus . Our manipulation experiments using nail polish sealing and insecticide treatment revealed an age-dependent change in gall formation by S. madaranus ; adult females and larvae are responsible for gall induction and enlargement, respectively. Furthermore, it has been suggested that substances released during oviposition and larval feeding are involved in each process. Phylogenetic analysis showed that gall-forming weevils, including S. madaranus , belong to two distinct lineages that utilize different host plants. This may indicate that gall-forming traits evolved independently in these Smicronyx lineages. The efficacy of RNA interference (RNAi) in S. madaranus was confirmed by targeting the multicopper oxidase 2 gene. It is expected that the mechanisms of gall formation will be elucidated by a comprehensive functional analysis of candidate genes using RNAi and the S. madaranus galling system in the near future.

We introduce a spectral analysis method in picosecond ultrasonics to derive strain pulse shapes in a opaque sample with known optical properties. The method makes use of both the amplitude and phase of optical transient relative reflectance changes obtained, for example, by interferometry. We demonstrate this method through numerical simulation and by analysis of experimental results for a chromium film., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author(s).)

Purpose: Though programmed cell death-1 (PD-1) inhibitors mainly target tumor-infiltrating lymphocytes (TILs) expressing PD-1, developing T cells in thymus also express PD-1 in their process of maturation. To predict the therapeutic effect of PD-1 inhibitors for thymoma, it is necessary to clarify the proportions of TILs and intratumoral developing T cells., Methods: The expressions of CD4, CD8, and PD-1 on T cells were analyzed by flow cytometry in 31 thymomas. The amount of T cell receptor excision circles (TRECs), which can be detected in newly formed naïve T cells in the thymus, was evaluated using sorted lymphocytes from thymomas by quantitative PCR. The expressions of granzyme B (GZMB) and lymphocyte activation gene-3 (LAG-3) in PD-1 + CD8 T cells were analyzed by image cytometry using multiplex immunohistochemistry., Results: The PD-1 + rate in both CD4 and CD8 T cells was significantly higher in type AB/B1/B2 than in type A/B3 thymomas. The amounts of TRECs in CD4 and CD8 T cells were significantly higher in type AB/B1/B2 than in type A/B3 thymomas and comparable to normal thymus. PD-1 expression at each stage of T cell development of type AB/B1/B2 thymomas was comparable to that of normal thymus. Both the percentages and cell densities of PD-1 + CD8 T cells expressing GZMB or LAG-3, which are known to contain tumor-reactive T cells, were significantly lower in type AB/B1/B2 thymomas., Conclusion: Most PD-1 + T cells in type AB/B1/B2 thymomas are intratumoral developing T cells and are not TILs., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We developed a multichannel wireless quartz-crystal-microbalance (QCM) biosensor for mechanically studying the on-surface aggregation reaction of α-synuclein (α-syn). We find a quite unusual change in the resonant frequency that eventually exceeds the baseline, which has never been observed during seeding aggregation reaction. By incorporating a growth-to-percolation theory for fibril elongation reaction, we have favorably reproduced this unusual response and found that it can be explained only with formation of an ultrastiff fibril network. We also find that the stiffness of the fibril network grown from artificially prepared twist-type seeds is significantly higher than that from rod-type seeds. Furthermore, the stiffnesses of fibril networks grown from seeds derived from brain tissues of Parkinson's disease (PD) and multiple system atrophy (MSA) patients show a very similar trend to those of rod and twist seeds, respectively, indicating that fibrils from MSA patients are stiffer than those from PD.

Quartz-crystal-microbalance (QCM) biosensor is a typical label-free biosensor, and its sensitivity can be greatly improved by removing electrodes and wires that would be otherwise attached to the surfaces of the quartz resonator. The wireless-electrodeless QCM biosensor was then developed using a microelectro-mechanical systems (MEMS) process, although challenges remain in the sensitivity, the coupling efficiency, and the miniaturization (or mass production). In this study, we establish a MEMS process to obtain a large number of identical ultrasensitive and highly efficient sensor chips with dimensions of 6 mm square. The fundamental shear resonance frequency of the thinned AT-cut quartz resonator packaged in the microchannel exceeds 160 MHz, which is excited by antennas deposited on inner walls of the microchannel, significantly improving the electro-mechanical coupling efficiency in the wireless operation. The high sensitivity of the developed MEMS QCM biosensors is confirmed by the immunoglobulin G (IgG) detection using protein A and ZZ-tag displaying a bionanocapsule (ZZ-BNC), where we find that the ZZ-BNC can provide more effective binding sites and higher affinity to the target molecules, indicating a further enhancement in the sensitivity of the MEMS QCM biosensor. We then perform the label-free C-reactive protein (CRP) detection using the ZZ-BNC-functionalized MEMS QCM biosensor, which achieves a detection limit of 1 ng mL -1 or less even with direct detection.

Dialysis-related amyloidosis (DRA), a serious complication among long-term hemodialysis patients, is caused by amyloid fibrils of β2-microglobulin (β2m). Although high serum β2m levels and a long dialysis vintage are the primary and secondary risk factors for the onset of DRA, respectively, patients with these do not always develop DRA, indicating that there are additional risk factors. To clarify these unknown factors, we investigate the effects of human sera on β2m amyloid fibril formation, revealing that sera markedly inhibit amyloid fibril formation. Results from over 100 sera indicate that, although the inhibitory effects of sera deteriorate in long-term dialysis patients, they are ameliorated by maintenance dialysis treatments in the short term. Serum albumin prevents amyloid fibril formation based on macromolecular crowding effects, and decreased serum albumin concentration in dialysis patients is a tertiary risk factor for the onset of DRA. We construct a theoretical model assuming cumulative effects of the three risk factors, suggesting the importance of monitoring temporary and accumulated risks to prevent the development of amyloidosis, which occurs based on supersaturation-limited amyloid fibril formation in a crowded milieu., (© 2022. The Author(s).)

The abnormal spindle-like, microcephaly-associated (ASPM) gene is a causative gene of autosomal recessive primary microcephaly (MCPH) 5 in humans, which is characterized by a reduction in brain volume. It was previously reported that truncated Aspm proteins in transgenic mice caused major defects in the germline, a severe reduction in ovary weight and the number of follicles accompanied by reduced fertility. However; it remains unknown whether a loss of Aspm induces abnormal ovarian function, resulting in female infertility. In order to assess the ovary function, we examined vaginal smear cytology from the age of 7 weeks to 100 weeks in CAG-mediated Cre-loxP conditional Aspm -/- knockout mice and control female mice. In addition, we evaluated the ovarian size, fibrosis ratio and the number of follicles (primordial, primary, secondary, antral and atretic follicles) in mice from 15 weeks to 100 weeks old by image analyses. Mann-Whitney U-test was used for statistical analysis. The size of the ovary was significantly reduced in Aspm knockout mice at 15-20 weeks, 40-50 weeks and 70-80 weeks old compared with the control mice. Furthermore, at all stages, we found a severe decrease in the number of developing follicles at 10-15 weeks, 40-50 weeks and 70-80 weeks old, accompanied by disrupted cyclic changes of vaginal cytology and an aberrant upregulation of Foxo3, Kitl, and Lhcgr in Aspm knockout female. These results suggested that Aspm might play an important role in the folliculogenesis and estrous cyclicity of the postnatal ovary during maturation and aging., (Copyright © 2022 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

The adsorbed structure of 1-pyrenebutanoic acid succinimidyl ester (PASE) on graphene was investigated based on density functional theory. We found two locally stable structures: a straight structure with the chainlike part of butanoic acid succinimidyl ester (BSE) lying down and a bent structure with the BSE part directed away from graphene, keeping the pyrene (Py) part adsorbed on graphene. Then, to elucidate the adsorption mechanism, we separately estimated the contributions of the Py and BSE parts to the entire PASE adsorption, and the adsorption effect of the BSE part was found to be secondary in comparison to the contribution of the Py. Next, the mobility of the BSE part at room temperature was confirmed by the activation energy barrier between straight and bent structures. To take account of the external environment, we considered the presence of amino acids and the hydration effect by a three-dimensional reference interaction site model. The contributions of glycine molecules and the solvent environment to stabilizing the bent PASE structure relative to the straight PASE structure were found. Therefore, the effect of the external environment around PASE is of importance when the standing-up process of the BSE part from graphene is considered., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background and Aim: Electrocardiography (ECG) is an adjunct for cardiac enlargement diagnosis. However, its efficacy in assessing left cardiac remodeling (left atrial and left ventricular enlargement) in dogs with myxomatous mitral valve disease (MMVD) remains unclear. This study aimed to evaluate the association between ECG parameters and left cardiac remodeling and to investigate whether the rate of change in ECG waveforms in the same individual reflected left cardiac remodeling in dogs with MMVD., Materials and Methods: This retrospective study included 20 healthy dogs and 140 dogs with MMVD. Data on clinical variables were obtained through physical examination, thoracic radiography, and echocardiography. The ECG parameters were the P-wave duration, PR interval, QRS complex duration, P-wave amplitude, R-wave amplitude, and mean electrical axis. Dogs with examination data that could be obtained multiple times during the study period were classified into the non-progressive and progressive groups., Results: Only the P-wave and QRS complex durations were selected as significant variables associated with imaging test parameters (p < 0.05); they had a relatively higher discriminatory ability for the left cardiac remodeling than other ECG parameters. The rates of change in the PR interval and R-wave amplitude were significantly higher in the progressive group than in the non-progressive group., Conclusion: In dogs with MMVD, the P-wave and QRS complex durations were significantly correlated with the left cardiac remodeling indicators. Furthermore, an increased rate of change in the PR interval and R-wave amplitude in the same individual may indicate left cardiac remodeling., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Ogawa, et al.)

Angiopoietin-like 4 (ANGPTL4) promotes cancer cell migration through vessels and has been implicated in cancer metastasis. Our previous study identified a robust increase in ANGPTL4 mRNA expression in lung-metastasized tongue cancer (TC) cells. Therefore, the present study investigated the association of ANGPTL4 with lung metastasis and outcomes of patient with TC. ANGPTL4 expression in TC cells was investigated by immunohistochemical staining. Patients were classified into 'low (0-30%)' and 'high (>30%)' ANGPTL4-expression groups based on the proportion of ANGPTL4-positive TC cells. The high ANGPTL4-expression group included 15 of 48 patients with TC. Notably, a significantly greater proportion of patients with lung metastasis exhibited a high rate of ANGPTL4-expressing cancer cells compared with patients without lung metastasis (P=0.029). The overall 5-year survival rate was lower in the high (27%) ANGPTL4-expression group compared with the low (68%) ANGPTL4-expression group. Univariate and multivariate analyses revealed that patients with high ANGPTL4 expression in TC cells exhibited significantly lower overall survival (OS) rates [hazard ratio (HR), 2.99; 95% confidence interval (95% CI), 1.34-6.69; P=0.008 and HR, 2.72; 95% CI, 1.14-6.51; P=0.024, respectively]. High plasma ANGPTL4 concentrations as measured by ELISA were associated with lung metastasis (P<0.001). The optimal cut-point for prediction of TC lung metastasis was 9.1 ng/ml (P<0.001; 95% CI, 7.2-10.9). The OS of patients with plasma ANPTL4 above the cut-point was significantly lower than that of patients with plasma ANGPTL4 ≤9.1 ng/ml (P<0.001). These results suggest that a high level of ANGPTL4 in cancer cells and plasma may predict lung metastasis and/or a poor prognosis of patients with TC., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Tanaka et al.)

Spatial profiles of the tumor-immune microenvironment are associated with disease progression and clinicopathological factors in various cancers. Follicular thyroid carcinoma (FTC) is the second most common thyroid cancer, where the presence of capsular invasion or angioinvasion determines the pathological diagnosis; however, little is known about the immune microenvironment profiles associated with the acquisition of invasive potential of FTC. In this study, we focused on FTC with minimal capsular invasion, and the spatially resolved immune microenvironment of FTC was studied in the discovery (n = 13) and validation cohorts (n = 40). CD8 + T cells, helper T cells, regulatory T cells, B cells, natural killer cells, tumor-associated macrophages, CD66 + granulocytes, mature dendritic cells, and mast cells were quantitatively evaluated in single tissue sections, via a 12-marker multiplex immunohistochemistry and image cytometry. Cell densities and compositions of immune cells were spatially stratified by six tissue regions including tumor center, subcapsular region, capsular invasion, adjacent stroma of capsular invasion, peritumoral stroma, and adjacent normal. Lymphoid cell lineages in the tumor center and subcapsular regions were significantly lower than those in adjacent normal and peritumoral stroma, potentially related to the lymphoid lineage exclusion from the intratumoral regions of FTC. Interestingly, immune cell composition profiles in the capsular invasive front were distinct from those of intratumoral region. The ratios of T cells to CD66b + granulocytes with capsular invasion were significantly higher than those without capsular invasion, suggesting the presence of a unique immune microenvironment at the invasive front between tumor foci and stroma. In addition, tumor cells at the capsular invasive front showed significantly higher expression of tumor programmed cell death ligand 1 (PD-L1) than those at the tumor center. This study revealed spatial immune profiles associated with capsular invasion of FTC, providing new insights into the mechanisms underlying its development and initial invasion., (© 2021. The Author(s), under exclusive licence to United States & Canadian Academy of Pathology.)

Amyloid fibrils involved in amyloidoses are crystal-like aggregates, which are formed by breaking supersaturation of denatured proteins. Ultrasonication is an efficient method of agitation for breaking supersaturation and thus inducing amyloid fibrils. By combining an ultrasonicator and a microplate reader, we developed the HANABI (HANdai Amyloid Burst Inducer) system that enables high-throughput analysis of amyloid fibril formation. Among high-throughput approaches of amyloid fibril assays, the HANABI system has advantages in accelerating and detecting spontaneous amyloid fibril formation. HANABI is also powerful for amplifying a tiny amount of preformed amyloid fibrils by seeding. Thus, HANABI will contribute to creating therapeutic strategies against amyloidoses by identifying their biomarkers., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recently, sleeping status has attracted attention for its relationship with oral health. In the present study, we have investigated the association between early childhood caries and sleeping status. A multicentre observational cross-sectional study was conducted among 332 preschoolers (aged 3-6 years) and their parents in Chitose, Japan. Dental caries and sleeping status were assessed in the children and the sleep quality and health literacy of the parents were also assessed. Univariate and multivariate regression analyses were used in order to investigate the effect of the sleeping status of the parents and their children on dental caries. Among the children, the prevalence of dental caries experience was 12.7%. The children without caries slept significantly longer and their parents had a better Pittsburgh Sleep Quality Index (PSQI) score than those with caries experience. The sleeping status and the numbers of caries in the children were significantly correlated. Health literacy was better in those without caries experience. Parents' PSQI was significantly positively correlated with the numbers of caries in the children (r = 0.19, p = 0.0004). The children's sleep durations, screen time, and parental smoking status were independently associated with early childhood caries. Poor sleeping status of children and their parents is related to dental caries among preschoolers.

Background: Functional interactions between immune cells and neoplastic cells in the tumor immune microenvironment have been actively pursued for both biomarker discovery for patient stratification, as well as therapeutic anti-cancer targets to improve clinical outcomes. Although accumulating evidence indicates that intratumoral infiltration of immune cells has prognostic significance, limited information is available on the spatial infiltration patterns of immune cells within intratumoral regions. This study aimed to understand the intratumoral heterogeneity and spatial distribution of immune cell infiltrates associated with cell phenotypes and prognosis in head and neck squamous cell carcinoma (HNSCC)., Methods: A total of 88 specimens of oropharyngeal squamous cell carcinoma, categorized into discovery (n = 38) and validation cohorts (n = 51), were analyzed for immune contexture by multiplexed immunohistochemistry (IHC) and image cytometry-based quantification. Tissue segmentation was performed according to a mathematical morphological approach using neoplastic cell IHC images to dissect intratumoral regions into tumor cell nests versus intratumoral stroma., Results: Tissue segmentation revealed heterogeneity in intratumoral T cells, varying from tumor cell nest-polarized to intratumoral stroma-polarized distributions. Leukocyte composition analysis revealed higher ratios of T H 1/T H 2 in tumor cell nests with higher percentages of helper T cells, B cells, and CD66b + granulocytes within intratumoral stroma. A discovery and validation approach revealed a high density of programmed death receptor-1 (PD-1) + helper T cells in tumor cell nests as a negative prognostic factor for short overall survival. CD163 + tumor-associated macrophages (TAM) provided the strongest correlation with PD-1 + helper T cells, and cases with a high density of PD-1 + helper T cells and CD163 + TAM had a significantly shorter overall survival than other cases., Conclusion: This study reveals the significance of analyzing intratumoral cell nests and reports that an immune microenvironment with a high density of PD-1 + helper T cells in tumoral cell nests is a poor prognostic factor for HNSCC., Competing Interests: TT is a paid consultant for Ono Pharmaceutical and receives speaker fees from Merck Sharp & Dohme Corp, Ono Pharmaceutical, and Bristol-Myers Squibb. HO is an employee of SCREEN Holdings Co., Ltd. SSh is an employee of SCREEN Holdings Co., Ltd. EK is a paid consultant for Roche Diagnostics and receives speaker fees from Chugai Pharmaceutical. KI received research funding from the SCREEN Holdings Co., Ltd. LC is a paid consultant for Cell Signaling Technologies, AbbVie Inc., and Shasqi Inc., received reagent and/or research support from Plexxikon Inc., Pharmacyclics, Inc., Acerta Pharma, LLC, Deciphera Pharmaceuticals, LLC, Genentech, Inc., Roche Glycart AG, Syndax Pharmaceuticals Inc., Innate Pharma, NanoString Technologies, and Cell Signaling Technologies, is a member of the Scientific Advisory Boards of Syndax Pharmaceuticals, Carisma Therapeutics, Zymeworks, Inc, Verseau Therapeutics, Cytomix Therapeutics, Inc., Hibercell, Inc., Alkermes, Inc., Genenta Sciences, and Kineta Inc, and is a member of the Lustgarten Therapeutics Advisory working group, and the AstraZeneca Partner of Choice Network. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yoshimura, Tsujikawa, Mitsuda, Ogi, Saburi, Ohmura, Arai, Shibata, Thibault, Chang, Clayburgh, Yasukawa, Miyagawa-Hayashino, Konishi, Itoh, Coussens and Hirano.)

Objective: According to a World Health Organization report, in 2016, 41 million young children globally were overweight or obese. The connection between parents' social capital and their children's health has been studied, but associations between parental social capital and children's weight are largely unexamined. Hence, we assessed the relationship between preschool children's weight and parents' social capital., Methods: We used BMI assessment data for 357 children (46.5% girls; mean age 5.3 years) in Japan. We examined parents' structural and cognitive social capital using a self-report questionnaire., Results: Multiple logistic regression analysis revealed associations between parents' social capital and children's BMI. Seventy-two (20.2%) of the children had poor BMI (body mass index; overweight or thin). Interpersonal trust was significantly associated with normal BMI in children after adjustment for all confounding factors (OR 2.68; 95% CI, 1.33-5.44; P = .006) and was independently associated with other social capital factors, including norm of reciprocity (OR 3.38; 95% CI, 1.68-6.79; P < .001) and trust in organization (OR 1.24; 95% CI, 1.09-1.42; P = .001)., Conclusion: Social capital factors were independently associated with each other. Japanese parents' high social capital was an independent predictor of normal BMI among preschool children., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Ultrasonication has been recently adopted in amyloid-fibril assays because of its ability to accelerate fibril formation, being promising in the early stage diagnosis of amyloidoses in clinical applications. Although applications of this technique are expanding in the field of protein science, its effects on the aggregation reactions of amyloidogenic proteins are poorly understood. In this study, we comprehensively investigated the morphology and structure of resultant aggregates, kinetics of fibril formation, and seed-detection sensitivity under ultrasonication using β 2 -microglobulin and compared these characteristics under shaking, which has been traditionally adopted in amyloid-fibril assays. To discuss the ultrasonic effects on the amyloid-fibril formation, we propose the half-time heat map, which describes the phase diagram of the aggregation reaction of amyloidogenic proteins. The experimental results show that ultrasonication greatly promotes fibril formation, especially in dilute monomer solutions, induces short-dispersed fibrils, and is capable of detecting ultra-trace-concentration seeds with a detection limit of 10 fM. Furthermore, we indicate that ultrasonication highly alters the energy landscape of an aggregation reaction due to the effect of ultrasonic cavitation. These insights contribute not only to our understanding of the effects of agitation on amyloidogenic aggregation reactions but also to their effective application in the clinical diagnosis of amyloidoses.

Recent advances made in treatment for head and neck squamous cell carcinoma (HNSCC) highlight the need for new prediction tools to guide therapeutic strategies. In this study, we aimed to develop a HNSCC-targeting multiplex immunohistochemical (IHC) panel that can evaluate prognostic factors and the intratumor heterogeneity of HNSCC. To identify IHC-based tissue biomarkers that constitute new multiplex IHC panel, a systematic review and meta-analysis were performed to analyze reported IHC biomarkers in laryngeal and pharyngeal SCC in the period of 2008-2018. The Cancer Genome Atlas (TCGA) and Reactome pathway databases were used to validate the prognostic and functional significance of the identified biomarkers. A 14-marker chromogenic multiplex IHC panel including identified biomarkers was used to analyze untreated HNSCC tissue. Forty-five high-quality studies and thirty-one candidate tissue biomarkers were identified (N = 7062). Prognostic validation in TCGA laryngeal and pharyngeal SCC cohort (N = 205) showed that β-catenin, DKK1, PINCH1, ADAM10, and TIMP1 were significantly associated with poor prognosis, which were related to functional categories such as immune system, cellular response, cell cycle, and developmental systems. Selected biomarkers were assembled to build a 14-marker panel, evaluating heterogeneity and polarized expression of tumor biomarkers in the tissue structures, which was particularly related to activation of Wnt/β-catenin pathway. Integrated IHC analysis based on a systemic review and meta-analysis provides an in situ proteomics tool to assess the aggressiveness and intratumor heterogeneity of HNSCC., Competing Interests: HO is an employee for SCREEN Holdings Co., Ltd. KI has received research funding from SCREEN Holdings Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mitsuda, Tsujikawa, Yoshimura, Saburi, Suetsugu, Kitamoto, Takenaka, Ohmura, Arai, Ogi, Itoh and Hirano.)

Amyloid fibrils are formed from various proteins, some of which cause the corresponding neurodegenerative disorders, such as Alzheimer's and Parkinson's diseases. It has been reported that many compounds inhibit the formation of amyloid fibrils. Anthocyanins are flavonoid pigments present in fruits and vegetables, which are known to suppress symptoms related with Alzheimer's disease. However, the influence of anthocyanins on the amyloid fibril remains unclear. Here, we succeeded in the direct monitoring of the disaggregation reaction of single amyloid β (Aβ) fibrils by anthocyanins using total-internal-reflection-fluorescence microscopy with a quartz-crystal microbalance (TIRFM-QCM). It is found that the disassembly activity to the Aβ fibrils depends on the number of hydroxyl groups in six-membered ring B of anthocyanin, and only delphinidin-3-galactoside, possessing three hydroxyl groups there, shows high disassembly activity. Our results show the importance of the number of hydroxyl groups and demonstrate the usefulness of TIRFM-QCM as a powerful tool in studying interactions between amyloid fibrils and compounds.

During the deposition of a metallic material on a substrate, a nanocluster-array structure with an extremely narrow gap is formed transiently at the transition between isolated clusters and the continuous film. It is known that the nanocluster array shows a unique electrical property different from that of isolated clusters and the continuous film. The electrical property of the nanocluster array changes significantly depending on its morphology, and precise control of the deposition time is indispensable to obtain a desired electrical property. However, the detection of the transition is not straightforward. To overcome this problem, we develop the multi-mode resistive spectroscopy. It evaluates the morphological change during deposition using resonant vibrations of a piezoelectric material and enables the fabrication of nanocluster arrays with a slightly different morphology. Palladium nanocluster arrays with different morphologies are fabricated using this method, and the availability of the multi-mode resistive spectroscopy is demonstrated by evaluating their electrical response to hydrogen gas.

Ultrasonication to supersaturated protein solutions forcibly forms amyloid fibrils, thereby allowing the early-stage diagnosis for amyloidoses. Previously, we constructed a high-throughput sonoreactor to investigate features of the amyloid-fibril nucleation. Although the instrument substantiated the ultrasonication efficacy, several challenges remain; the key is the precise control of the acoustic field in the reactor, which directly affects the fibril-formation reaction. In the present study, we develop the optimized sonoreactor for the amyloid-fibril assay, which improves the reproducibility and controllability of the fibril formation. Using β 2 -microglobulin, we experimentally demonstrate that achieving identical acoustic conditions by controlling oscillation amplitude and frequency of each transducer results in identical fibril-formation behavior across 36 solutions. Moreover, we succeed in detecting the 100-fM seeds using the developed sonoreactor at an accelerated rate. Finally, we reveal that the acceleration of the fibril-formation reaction with the seeds is achieved by enhancing the primary nucleation and the fibril fragmentation through the analysis of the fibril-formation kinetics. These results demonstrate the efficacy of the developed sonoreactor for the diagnosis of amyloidoses owing to the accelerative seed detection and the possibility for further early-stage diagnosis even without seeds through the accelerated primary nucleation., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nucleation by sedimentation of colloidal particles on a flat surface is experimentally observed, and effect of attractive depletion force generated by polymers on nucleation is investigated. Sedimentation forms polycrystalline colloidal crystal on a flat surface, and above the threshold polymer concentration, ratio of the spontaneous nucleation increases, resulting in a decrease in the grain size, whereas dependence of the contact angle on the polymer concentration was not observed. We show that the interaction between particles and the flat surface mainly affects the spontaneous nucleation, not the interaction between the particles, and it is demonstrated that the nucleation process can be numerically reproduced using the rate equations.

Background: Sleep problems in preschool children can stunt their health and growth. However, the factors that cause sleep problems in children are not well understood. The aim of this study was to determine the relationship between parents' health literacy (HL) and children's sleep problems. The study was conducted at two kindergartens, two nursery schools, and a center for early childhood education in Chitose-city, Hokkaido, Japan., Method: This study used a multicenter cross-sectional design. The sample comprised 354 preschoolers (aged 3-6 years) and their parents. In families with two or more children attending the same facility, only the oldest child was asked to participate in the study. Exclusion criteria included participants whose completed questionnaires had missing values. Children's sleep problems were assessed using the Japanese version of the Children's Sleep Habits Questionnaire (CSHQ-J). Parents' HL was assessed using the 14-item Health Literacy Scale (HLS-14). The parents were classified into two groups (high HL group and low HL group). Multiple regression modelling was used to determine the association between HLS-14 and CSHQ-J scores., Results: Of the 354 parents, 255 (72%) were in the high HL group and 99 (28%) in the low HL group. The mean CSHQ-J score was significantly lower in the high HL group than in the low HL group (45.3 ± 6.0 points vs. 46.8 ± 5.9 points, p = 0.043). In multiple regression analyses, parents' HL was independently associated with their CSHQ-J score after adjusting for all confounding factors (adjusted R 2  = 0.22, β = - 0.11; p = 0.043)., Conclusions: Parents' HL appears to affect their children's sleep problems. This finding suggests that parents' HL may be a target for intervention to improve children's sleep problems.

Differential expressions of estrogen/progesterone receptors (ER/PR) and individual component of extracellular matrices derived from fibroid are reported. Information on the pattern of change in ER/PR expression and amount of tissue fibrosis after hormonal treatment is unclear. We investigated pattern of change in ER/PR expression and percentage of tissue fibrosis in different uterine leiomyomas after gonadotropin-releasing hormone agonist (GnRHa) treatment. Biopsy specimens from fibroids and adjacent myometria were collected after surgery from women with submucosal myoma (SMM, n = 18), intramural myoma (IMM, n = 16) and subserosal myoma (SSM, n = 17). A proportion of women in each group of fibroid underwent treatment with GnRHa for a variable period of 3-6 months. Tissue expression of ER and PR was analyzed by immunohistochemistry. In vitro cell proliferation effect of GnRHa on human umbilical vein endothelial cells (HUVECs) was examined. Distribution of tissue fibrosis was examined by Masson's trichrome staining with computer-captured image analysis of fibrosis derived from different types of fibroid. PR content was significantly higher than ER in tissues derived from GnRHa-untreated women with SMM and SSM (p = 0.04 for both). Comparing to untreated group, GnRHa-treatment significantly decreased either ER or PR expression in different fibroids. Exogenous treatment with GnRHa dose-dependently decreased proliferation of HUVECs. No significant difference was observed in the percentage of fibrosis in tissues collected from GnRHa-treated and -untreated women with fibroids. The distribution of fibrosis in myoma/myometria and occurrence of fibrosis in perivascular area showed an increasing trend with higher age of the women and with larger size of fibroids. Our findings suggest that despite estrogen dependency, higher PR content in GnRHa-untreated group may indicate a potential role of progesterone in leiomyoma growth. Although GnRHa therapy may shrink fibroids and reduce risk of bleeding during surgery, the occurrence of diffuse tissue fibrosis may impair effective reduction of fibroid size after hormonal treatment., Competing Interests: The authors have declared that no competing interests exist.

Immune-based tumor characteristics in the context of tumor heterogeneity are associated with suppression as well as promotion of cancer progression in various tumor types. As immunity typically functions based on intercellular contacts and short-distance cytokine communications, the location and spatial relationships of the tumor immune microenvironment can provide a framework to understand the biology and potential predictive biomarkers related to disease outcomes. Immune spatial analysis is a newly emerging form of cancer research based on recent methodological advances in in situ single-cell analysis, where cell-cell interaction and the tissue architecture can be analyzed in relation to phenotyping the tumor immune heterogeneity. Spatial characteristics of tumors can be stratified into the tissue architecture level and the single-cell level. At the tissue architecture level, the prognostic significance of the density of immune cell lineages, particularly T cells, is leveraged by understanding longitudinal changes in cell distribution in the tissue architecture such as intra-tumoral and peri-tumoral regions, and invasive margins. At the single-cell level, the proximity of the tumor to the immune cells correlates with disease aggressiveness and therapeutic resistance, providing evidence to understand biological interactions and characteristics of the tumor immune microenvironment. In this review, we summarize recent findings regarding spatial information of the tumor immune microenvironment and review advances and challenges in spatial single-cell analysis toward developing tissue-based biomarkers rooted in the immune spatial landscape., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The aggregation behavior of amyloid-β (Aβ) peptides remains unclarified despite the fact that it is closely related to the pathogenic mechanism of Alzheimer's disease. Aβ peptides form diverse oligomers with various diameters before nucleation, making clarification of the mechanism involved a complex problem with conventional macroscopic analysis methods. Time-resolved single-molecule level analysis in bulk solution is thus required to fully understand their early stage aggregation behavior. Here, we perform time-resolved observation of the aggregation dynamics of Aβ oligomers in bulk solution using liquid-state transmission electron microscopy. Our observations reveal previously unknown behaviors. The most important discovery is that a salt crystal can precipitate even with a concentration much lower than its solubility, and it then dissolves in a short time, during which the aggregation reaction of Aβ peptides is significantly accelerated. These findings will provide new insights in the evolution of the Aβ oligomer.

Introduction/objectives: The American College of Veterinary Internal Medicine (ACVIM) guidelines suggest that pimobendan should be initiated in dogs which meet all criteria of stage B2 myxomatous mitral valve disease (MMVD): murmur intensity ≥ 3/6, left atrial-to-aortic ratio ≥ 1.6, normalized left ventricular internal diameter in diastole ≥ 1.7, and vertebral heart size > 10.5. Recently, a new radiographic index for left atrial enlargement, vertebral left atrial size (VLAS), was proposed. The objective of the present study was to evaluate whether VLAS is useful in staging MMVD and if it can distinguish between ACVIM stages B1 and B2., Animals: Ninety-seven client-owned dogs with MMVD were evaluated and classified as ACVIM stage B1, B2, or C-D., Materials and Methods: The echocardiographs and radiographs of all the dogs were retrospectively evaluated to obtain left atrial-to-aortic ratio, normalized left ventricular internal diameter in diastole, and VLAS values. The data were analyzed to assess the correlation between these measurements and VLAS, and the optimal cutoff value of VLAS was determined., Results: A VLAS cutoff value of 2.6 provided the greatest diagnostic accuracy for identification of dogs with ACVIM stage B2 MMVD (area under the curve, 0.96; sensitivity, 95%; specificity, 84%). A VLAS ≥2.5 exhibited the highest sensitivity (sensitivity, 100%; specificity, 78%), and a VLAS ≥ 3.1 exhibited the highest specificity (sensitivity, 47%; specificity, 100%)., Conclusions: VLAS is a helpful index for monitoring MMVD using radiography. A VLAS cutoff value of 2.5 could be used to identify dogs that may benefit from echocardiography to determine if they have reached ACVIM stage B2., Competing Interests: Conflict of Interest Statement The authors do not have any conflicts of interest to disclose., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objectives: Whether the extent of expression of programmed death-ligand 1 (PD-L1) is clinically significant remains uncertain, although immuno-oncological features have been studied in thymic epithelial cell tumors (TETs). We investigated the histological features of PD-L1 expression in TETs, and assessed PD-L1 expression using digital image analysis., Materials and Methods: Participants comprised 66 patients with TET who underwent surgical resection between 2001 and 2016. We calculated tumor cell-positive ratio as total proportion score (TPS) with immunohistochemistry using SP263 anti-PD-L1 monoclonal antibody. PD-L1 expression was also quantified using digital image analysis of whole-slide images. We evaluated the relationship between conventional visual TPS using optical microscopy (TPS-V) and TPS from digital image analysis (TPS-IA). We further classified all TETs into high or low PD-L1 expression groups and assessed the clinical significance of PD-L1 expression level using TPS-V and TPS-IA., Results: WHO histological types were Type A (n = 8), AB (n = 18), B1 (n = 5), B2 (n = 16), B3 (n = 6), metaplastic thymoma (n = 2), and thymic carcinoma (TC) (n = 11). Median TPS-Vs were 2%, 2%, 10 %, 65 %, 90 %, 1%, and 20 %, respectively. TPS-IAs correlated with TPS-Vs in TETs overall and in thymomas, but not in TCs. PD-L1 expression levels in TETs differed significantly among histological types. Whether TPS-V or TPS-IA were used, the PD-L1 high group included more cases of the more aggressive histological types. Recurrence-free survival (RFS) was shorter in the PD-L1 high group than in the PD-L1 low group in thymoma using TPS-IA, whereas RFS of the PD-L1 high group was shorter in all TETs using TPS-V., Conclusion: PD-L1 expression levels depended on the histological type of TET. Extensive PD-L1 expression in TETs was associated with poor prognosis. Digital image analysis is feasible for evaluating PD-L1 expression in TETs and might offer clinically relevant features of thymomas., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Pulmonary hypoplasia is an important cause of morbidity and mortality in infants with congenital diaphragmatic hernia (CDH). This study aimed to verify our hypothesis that the abnormal development of bronchial cartilage as well as alveolar immaturity, might play a central role in hypoplasia of the lung in human CDH., Method: We retrospectively analyzed autopsied lungs from 10 CDH cases and compared with nine age-matched controls to assess the bronchial cartilage and alveolar maturity using morphological techniques., Result: Ki-67 and thyroid transcription factor-1 (TTF-1) expression in the alveoli significantly increased in bilateral lungs with CDH. The shortest distance from the bronchial cartilage to the pleura was significantly shorter in ipsilateral (left) lungs with CDH, showing a positive correlation with the radial alveolar count (RAC). Regarding the small bronchial cartilages less than 20 000 μm 2 , the average cartilage area significantly decreased in left lungs with CDH, and tended to decrease in right lungs with CDH. In addition, cartilage around the bronchi less than 200 μm in diameter tended to be smaller in left lungs with CDH. In contrast, regarding the cartilage around the bronchi 200 to 400 μm in diameter, the ratio of the total cartilage area relative to the bronchial diameter tended to be higher in left lungs with CDH, although there was a large variation., Conclusions: These opposite directional cartilage abnormalities around the distal and more proximal bronchi support our hypothesis that abnormal development of bronchial cartilage might play an important role in the hypoplastic lung in CDH., (© 2020 Wiley Periodicals, Inc.)

Crystallization from amorphous solids is generally caused by activating phonons in a wide frequency range during heat treatment. In contrast, the activation of phonons in a narrow frequency range using ultrasonic treatment also causes crystallization below the glass transition temperature. These behaviors indicate that crystallization is related to the atomic motion in the glass state, and it is suggested that the activation of specific atomic motion can cause crystallization without increasing temperature. In this study, we observe nucleation and nuclei growth caused by mechanical oscillation in a hard-sphere colloidal glass and evaluate the effect of mechanical oscillation on the structural evolution in the early stage of the crystallization. Oscillation between 5 and 100 Hz is applied to the colloidal glass, and it is observed that the nucleation rate increases under the 70 Hz oscillation, resulting in formation of stable nuclei in a short amount of time. The nuclei growth is also accelerated by the 70 Hz oscillation, whereas increases in the nucleation rate and nuclei growth were not observed at other frequencies. Finally, activation of the diffusion-based rattling of particles by caging is considered as a possible mechanism of the observations.

Label-free biosensors, including conventional quartz-crystal-microbalance (QCM) biosensors, are seriously affected by nonspecific adsorption of contaminants involved in analyte solution, and it is exceptionally difficult to extract the sensor responses caused only by the targets. In this study, we reveal that this difficulty can be overcome with an ultrahigh-frequency, wireless QCM biosensor. The sensitivity of a QCM biosensor dramatically improves when the quartz resonator is thinned, which also makes the resonance frequency higher, causing high-speed surface movement. Contaminants weakly (nonspecifically) interact with the quartz surface, but they fail to follow the fast surface movement and cannot be detected as the loaded mass. The targets are, however, tightly captured by the receptor proteins immobilized on the surface, and they can move with the surface, contributing to the loaded mass and decreasing the resonant frequency. We have developed a MEMS QCM biosensor in which an AT-cut quartz resonator, 26 μm thick, is packaged without fixing, and we demonstrate this phenomenon by comparing the frequency changes of the fundamental (∼64 MHz) and ninth (∼576 MHz) modes. At ultrahigh-frequency operation with the ninth mode, the sensor response is independent of the amount of impurity proteins, and the binding affinity is unchanged. We then applied this method to the label-free and sandwich-free, direct detection of C-reactive protein (CRP) in serum and confirmed its applicability.

Research Question: Is there a biological difference between intrinsic and extrinsic adenomyosis with coexisting deep infiltrating endometriosis (DIE)?, Design: In this prospective controlled study, biopsy specimens were collected after surgery from 23 women with intrinsic adenomyosis and 10 women with extrinsic adenomyosis with coexisting DIE lesions. Histological evaluation was carried out by immunoreaction to Ber-EP4 (epithelial cell marker) and CD10 (stromal cell marker). Tissue expression of oestrogen and progesterone receptors was analysed by immunohistochemistry. Tissue fibrosis was examined by Masson's trichrome staining with computer-based image analysis of fibrosis in respective samples., Results: The detection rate of coexistent DIE was significantly higher in women with extrinsic adenomyosis (9/10 [90.0%]) than in women with intrinsic adenomyosis (3/23 [13.0%]; P < 0.001). The pattern of Ber-EP4-stained glands and CD10-stained stromal cells of extrinsic adenomyosis was similar to that of coexistent DIE lesions. In contrast, the pattern of gland and stromal cells was similar to the endometrium in the cases with intrinsic adenomyosis. Unlike extrinsic adenomyosis, progesterone receptor expression was significantly decreased in both gland cells (P < 0.05) and stromal cells (P < 0.05) of intrinsic adenomyosis. Although relatively more fibrosis was seen in biopsy samples of extrinsic adenomyosis and coexistent DIE than in intrinsic adenomyosis and their coexistent DIE, no significant difference was found., Conclusions: Extrinsic adenomyosis may be considered as adenomyosis externa based on a close histological and biological relationship between extrinsic adenomyosis and coexistent DIE. Our findings may contribute to the understanding of a possible biological origin of two newly classified intrinsic and extrinsic adenomyosis., (Copyright © 2019 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

α-Synuclein aggregates, a key hallmark of the pathogenesis of Parkinson's disease, can be amplified by using their seeding activity, and the evaluation of the seeding activity of cerebrospinal fluid (CSF) is reportedly useful for diagnosis. However, conventional shaking-based assays are time-consuming procedures, and the clinical significance of the diversity of seeding activity among patients remains to be clarified. Previously, we reported a high-throughput ultrasonication-induced amyloid fibrillation assay. Here, we adapted this assay to amplify and detect α-synuclein aggregates from CSF, and investigated the correlation between seeding activity and clinical indicators. We confirmed that this assay could detect α-synuclein aggregates prepared in vitro and also aggregates released from cultured cells. The seeding activity of CSF correlated with the levels of α-synuclein oligomers measured by an enzyme-linked immunosorbent assay. Moreover, the seeding activity of CSF from patients with Parkinson's disease was higher than that of control patients. Notably, the lag time of patients with Parkinson's disease was significantly correlated with the MIBG heart-to-mediastinum ratio. These findings showed that our ultrasonication-based assay can rapidly amplify misfolded α-synuclein and can evaluate the seeding activity of CSF.

Research Question: Is there any difference in ovarian steroid receptor expression and pattern of fibrosis in focal and diffuse adenomyosis and response to hormonal treatment?, Design: Prospective controlled study where biopsy samples were prospectively collected after surgery from 30 women with focal adenomyosis, 21 women with diffuse adenomyosis and 20 women with uterine myoma. Some of these women underwent 3-6 months of treatment with gonadotrophin-releasing hormone agonist (GnRHa) before surgery. Tissue expression of oestrogen receptor (ER) and progesterone receptor (PR) was analysed by immunohistochemistry. Distribution of tissue fibrosis was examined by Masson's trichrome staining with computer-based image analysis of fibrosis in tissues derived from women with and without adenomyosis., Results: There was no difference in ER/PR expression in gland cells/stromal cells of adenomyotic lesions on the ipsilateral side of focal adenomyosis and the anterior/posterior walls of diffuse adenomyosis. Compared to myoma tissues, a relatively decreased expression of ovarian steroid receptors was observed in both focal and diffuse adenomyosis. Image analysis of tissue fibrosis indicated more fibrosis in both focal and diffuse adenomyosis compared to fibrosis in the myometrium derived from women with uterine myoma. The pattern of fibrosis was no different in tissues derived from GnRHa-treated and -untreated women with focal and diffuse adenomyosis., Conclusions: No difference was found in the expression of ER/PR and entity of fibrosis between women with focal and diffuse adenomyosis regardless of GnRHa treatment. A lower expression of ER/PR compared to myoma tissue potentially clarifies the biological rationale of non-response to hormonal therapies for adenomyosis., (Copyright © 2018 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Due to a growing demand for a viable label-free observation method in the biomedical field, many techniques, such as quantitative phase imaging and Raman spectroscopy, have been studied, and a complementary approach, hyperspectral imaging, has also been introduced. We developed a high-speed hyperspectral imaging microscopy imaging method with commercially available apparatus, employing a liquid crystal tunable bandpass filter combined with a pixel-wise machine learning classification. Next, we evaluated the feasibility of the application of this method for stem cell research utilizing neural stem cells. Employing this microscopy method, with a 562 × 562 μm 2 field of view, 2048 × 2048 pixel resolution images containing 63 wavelength pixel-wise spectra could be obtained in 30 seconds. The neural stem cells were differentiated into neurons and astroglia (glia), and a four-class cell classification evaluation (including neuronal cell body, glial cell body, process and extracellular region) was conducted under co-cultured conditions. As a result, an average of 88% of the objects of interest were correctly classified, with an average precision of 94%, and more than 99% of the extracellular pixels were correctly segregated. These results indicated that the proposed hyperspectral imaging microscopy is feasible as a label-free observation method for stem cell research.

Autoimmune diseases are caused by immune complex-induced activation of the complement system and subsequent inflammation. Recent studies have revealed an association between autoimmune diseases and worse survival in patients with cancer; however, the underlying mechanism is still unknown. The C5a-C5a receptor (C5aR) system has been shown to enhance cancer activity and recruit myeloid-derived suppressor cells (MDSCs) that suppress the anti-tumor immune response. The Arthus reaction is inflammation caused by complement system activation by the immune complex and thus is a model of autoimmune diseases. To explore the effect of the Arthus reaction on cancer progression, mouse cancer cells were inoculated in syngeneic mouse skin, where the Arthus reaction was induced simultaneously. The Arthus reaction enhanced invasion and tumor growth of C5aR-positive cancer cells, but not control cells, and induced MDSC recruitment. Intravenous injection of C5a-stimulated C5aR-positive cancer cells into nude mice resulted in more lung nodules than injection of nontreated C5aR-positive cells and C5a-stimulated C5aR-negative cells, supporting C5a-C5aR-mediated enhancement of cancer growth. C5aR expression in uterine cervical carcinoma stage I cells, which invade into the deeper tissues, was significantly higher than that in CIN3 cells, which remain in the epithelium. These results indicate that cancer promotion by the C5a-C5aR system may underlie poor prognosis in cancer patients with autoimmune diseases, particularly in patients with C5aR-positive cancer, and may be associated with cervical cancer invasion. The enhancement of cancer cell invasion and growth by the C5a-C5aR system suggests that this system is a possible target of cancer therapy.

Background: Diabetes decreases bone strength, possibly because of cortical bone changes. Sweep imaging with Fourier transform (SWIFT) has been reported to be useful for cortical bone evaluation., Purpose: To evaluate cortical bone changes in diabetic rats using SWIFT, assess the usefulness of this technique through comparisons with microcomputed tomography (μCT) and conventional MRI, and clarify the mechanism underlying cortical bone changes using histomorphometry STUDY TYPE: Animal cohort., Animal Model: 8-week-old male Wistar/ST rats (N = 36) were divided into diabetes (induced by streptozotocin injection) and control groups., Field Strength/sequence: 7.04T MRI, SWIFT., Assessment: Six animals from each group were sacrificed at 2, 4, and 8 weeks after injection. Tibial bones were extracted and evaluated using μCT and MRI. The cortical bone mineral density (BMD) was measured using μCT. Proton density-weighted imaging (PDWI) and SWIFT were also performed. The signal-to-noise ratio (SNR) was calculated for each acquisition. The bone formation rate was evaluated using histomorphometry., Statistical Tests: Findings at each timepoint were compared using Mann-Whitney U-tests., Results: Cortical BMD was significantly lower in the diabetes group than in the control group only at 8 weeks (P < 0.05). At all timepoints, PDWI-SNR showed no significant differences between groups (P = 0.59, 0.70, and 0.82 at 2, 4, and 8 weeks, respectively). SWIFT-SNR was significantly lower in the diabetes group than in the control group (P < 0.05 at 2 and 4 weeks and P < 0.01 at 8 weeks), and the bone formation rate was significantly lower in the diabetes group than in the control group (P < 0.01 for all)., Data Conclusion: SWIFT can detect cortical bone changes even before a decline in the cortical BMD in a diabetic model., Level of Evidence: 1 Technical Efficacy: Stage 3 J. MAGN. RESON. IMAGING 2018;48:389-397., (© 2018 International Society for Magnetic Resonance in Medicine.)