Volumetric imaging of the tumor microvasculature reflects outcomes and genomic states of clear cell renal cell carcinoma.

Tumor structure is heterogeneous and complex, and it is difficult to obtain complete characteristics by two-dimensional analysis. The aim of this study was to visualize and characterize volumetric vascular information of clear cell renal cell carcinoma (ccRCC) tumors using whole tissue phenotyping and three-dimensional light-sheet microscopy. Here, we used the diagnosing immunolabeled paraffin-embedded cleared organs pipeline for tissue clearing, immunolabeling, and three-dimensional imaging. The spatial distributions of CD34, which targets blood vessels, and LYVE-1, which targets lymphatic vessels, were examined by calculating three-dimensional density, vessel length, vessel radius, and density curves, such as skewness, kurtosis, and variance of the expression. We then examined those associations with ccRCC outcomes and genetic alteration state. Formalin-fixed paraffin-embedded tumor samples from 46 ccRCC patients were included in the study. Receiver operating characteristic curve analyses revealed the associations between blood vessel and lymphatic vessel distributions and pathological factors such as a high nuclear grade, large tumor size, and the presence of venous invasion. Furthermore, three-dimensional imaging parameters stratified ccRCC patients regarding survival outcomes. An analysis of genomic alterations based on volumetric vascular information parameters revealed that PI3K-mTOR pathway mutations related to the blood vessel radius were significantly different. Collectively, we have shown that the spatial elucidation of volumetric vasculature information could be prognostic and may serve as a new biomarker for genomic alterations. High-end tissue clearing techniques and volumetric immunohistochemistry enable three-dimensional analysis of tumors, leading to a better understanding of the microvascular structure in the tumor space.

Deep Learning-Driven Estimation of Centiloid Scales from Amyloid PET Images with 11C-PiB and 18F-Labeled Tracers in Alzheimer's Disease.

BACKGROUND: Standard methods for deriving Centiloid scales from amyloid PET images are time-consuming and require considerable expert knowledge. We aimed to develop a deep learning method of automating Centiloid scale calculations from amyloid PET images with 11C-Pittsburgh Compound-B (PiB) tracer and assess its applicability to 18F-labeled tracers without retraining. METHODS: We trained models on 231 11C-PiB amyloid PET images using a 50-layer 3D ResNet architecture. The models predicted the Centiloid scale, and accuracy was assessed using mean absolute error (MAE), linear regression analysis, and Bland-Altman plots. RESULTS: The MAEs for Alzheimer's disease (AD) and young controls (YC) were 8.54 and 2.61, respectively, using 11C-PiB, and 8.66 and 3.56, respectively, using 18F-NAV4694. The MAEs for AD and YC were higher with 18F-florbetaben (39.8 and 7.13, respectively) and 18F-florbetapir (40.5 and 12.4, respectively), and the error rate was moderate for 18F-flutemetamol (21.3 and 4.03, respectively). Linear regression yielded a slope of 1.00, intercept of 1.26, and R2 of 0.956, with a mean bias of -1.31 in the Centiloid scale prediction. CONCLUSIONS: We propose a deep learning means of directly predicting the Centiloid scale from amyloid PET images in a native space. Transferring the model trained on 11C-PiB directly to 18F-NAV4694 without retraining was feasible.

A general binary isotherm model for amines interacting with CO2 and H2O.

CO2 capture by primary or secondary amines has been a topic of great research interests for a century because of its industrial importance. Interest has grown even more, because of the need to eliminate CO2 emissions that lead to global warming. Experimental evidence shows that CO2 sorption by primary or secondary amines is accompanied by co-absorption of H2O. A quantitative analysis of such CO2-H2O co-absorption behavior is important for practical process design and theoretical understanding. Even though there is almost an experimental consensus that water enhances CO2 uptake capacity, an analytical model to explain this phenomenon is not well established. Instead, some empirical models such as the Toth model are used to describe the isotherm without accounting for the presence of water. Recently, we have demonstrated that the isotherm equation of CO2 sorption into strong-base anion exchange materials with quaternary ammonium can be derived from that of strong-base aqueous alkaline solutions by correcting for the drastic change in the water activity and by including an appropriate parameterization of the water activity terms. In this paper, we generalize this model from quaternary ammonium to primary, secondary and tertiary amines either in solutions or as functional groups in polymer resins. For primary, secondary and tertiary amines, the isotherm equation can be derived by extending that of a weak-base aqueous alkaline solution such as aqueous ammonia. The model has been validated using experimental data on CO2 sorption for aqueous ammonia from the literature. This general model even includes quaternary ammonium as a special limit. Hence, this general model offers a platform that can treat the isotherms of solid amines, aqueous amines and aqueous alkaline solutions in a unified manner.

A new method for the preparation of a purified glucosylceramide and ceramide from shiitake mushroom.

Ingestion of plant and fungal glucosylceramides is known to reduce colon carcinogenesis and skin barrier damage in mice and humans. However, such effects in animal experiments have not been revealed for plant and fungal ceramides because the content of ceramides contained in plants and fungi is so low that the large amount required for animal experiments is difficult to obtain. Noting that the fungus shiitake mushroom (Lentinula edodes) is rich in a glucosylceramide, (4E,8E)-N-d-2'-hydroxypalmitoyl-1-O-ß-d-glucopyranosyl-9-methyl-4,8-sphingadienine [Glc-d19:2(4E,8E,9Me)-h16:0], we developed a new method to purify this fungal glucosylceramide using ethanol precipitation and high-performance liquid chromatography. We also developed a new method to produce large amounts of a ceramide [d19:2(4E,8E,9Me)-h16:0] from this purified glucosylceramide using human glycoside hydrolase family 30 glucocerebrosidase (imiglucerase). These methods will be useful for elucidating the physiological function by ingestion of fungal ceramides in animal experiments.

Squamous cell carcinoma of the prostate with SMARCA4 alteration in a Japanese patient.

Introduction: Squamous cell carcinoma of the prostate is very rare. Due to difficulty in diagnosis, it is often detected in advanced stages. Effective treatment of squamous cell carcinoma and the genetic profiling has not yet been established. Case presentation: We experienced the case of a 79-year-old man who was diagnosed with primary squamous cell carcinoma of the prostate. He had multiple lymph node metastases, liver metastases, and lung metastases, and he was treated with gemcitabine and carboplatin. However, he had poor patient status: it became difficult to continue after one course, and best supportive care was provided. We also performed targeted next-generation sequencing of 160 cancer-related genes and detected SMARCA4 mutation. Conclusion: This is the first study reporting the genomic profiling of squamous cell carcinoma of prostate in Japan.

Kinetic model for moisture-controlled CO2 sorption.

The understanding of the sorption/desorption kinetics is essential for practical applications of moisture-controlled CO2 sorption. We introduce an analytic model of the kinetics of moisture-controlled CO2 sorption and its interpretation in two limiting cases. In one case, chemical reaction kinetics on pore surfaces dominates, in the other case, diffusive transport through the sorbent defines the kinetics. We show that reaction kinetics, which is dominant in the first case, can be expressed as a linear combination of 1st and 2nd order kinetics in agreement with the static isotherm equation derived and validated in a previous paper. The interior transport kinetics can be described by non-linear diffusion equations. By combining all carbon species into a single equation, we can eliminate - in certain limits - the source terms associated with chemical reactions. In this case, the governing equation is . For a sorbent in a form of a flat sheet or a membrane, one can maintain the same functional form of a diffusion equation by introducing a generalized effective diffusivity DM that combines contributions from both surface chemical reaction kinetics and interior diffusive transport kinetics. Experimental data of transient CO2 flux in a preconditioned commercial anion exchange membrane fit well to the 1st order model as long as very dry states are avoided, validating the theory. The observed DM for a preconditioned commercial anion exchange membrane ranges from 6.6 × 10-14 to 7.1 × 10-14 m2 s-1 at 35 °C. These small values compared to typical ionic diffusivities imply a very slow kinetics, which will be the largest issue that needs to be addressed for practical application. The collected transient CO2 flux data are used to predict the magnitude of a continuous CO2 pumping flux in an active membrane that transports CO2 against a CO2 concentration gradient. The pumped CO2 flux is supported by water flux due to a water concentration gradient.

Isotherm model for moisture-controlled CO2 sorption.

Moisture-controlled sorption of CO2, the basis for moisture-swing CO2 capture from air, is a novel phenomenon observed in strong-base anion exchange materials. Prior research has shown that Langmuir isotherms provide an approximate fit to moisture-controlled CO2 sorption isotherm data. However, this fit still lacks a governing equation derived from an analytic model. In this paper, we derive an analytic form for an isotherm equation from a bottom-up approach, starting with a fundamental theory for an alkali liquid. In the range of interest relevant to CO2 capture from air, an isotherm equation for an alkali liquid reduces to a simple analytic form with a single parameter, Keq. In the limit Keq ≫ 1, a 2nd order approximation simplifies to a Langmuir isotherm that, however, deviates from experimental data. The isotherm theory for an alkali liquid has been generalized to a strong-base anion exchange material. In a strong-base anion exchange material, water concentration inside a sorbent, [H2O], is not large enough to be regarded as constant, which allows us to extend Keq to Keq(AEM)eff = Keq(AEM) × [H2O]-n according to the law of mass action. The final isotherm formula has been validated by experimental data from the literature. For a moisture-controlled CO2 sorbent, Keq(AEM)eff varies significantly with moisture content of the sorbent. Depending on moisture level, the observed Keq(AEM)eff in a specific sorbent ranges from a few times to a few thousand times the value of Keq of a 2 mol L-1 alkali liquid.

Genomic and Metabolomic Analyses of a Piezosensitive Mutant of Saccharomyces cerevisiae and Application for Generation of Piezosensitive Niigata-Sake Yeast Strains.

A sparkling-type draft cloudy sake (Japanese rice wine), AWANAMA, was recently developed using high hydrostatic pressure (HHP) treatment as a non-thermal pasteurization method. This prototype sake has a high potential market value, since it retains the fresh taste and flavor similar to draft sake while avoiding over-fermentation. From an economic point of view, a lower pressure level for HHP pasteurization is still required. In this study, we carried out a genome analysis of a pressure-sensitive (piezosensitive) mutant strain, a924E1, which was generated by UV mutagenesis from a laboratory haploid Saccharomyces cerevisiae strain, KA31a. This mutant strain had a deletion of the COX1 gene region in the mitochondrial DNA and had deficient aerobic respiration and mitochondrial functions. A metabolomic analysis revealed restricted flux in the TCA cycle of the strain. The results enabled us to use aerobic respiration deficiency as an indicator for screening a piezosensitive mutant. Thus, we generated piezosensitive mutants from a Niigata-sake yeast strain, S9arg, which produces high levels of ethyl caproate but does not produce urea and is consequently suitable for brewing a high-quality sake. The resultant piezosensitive mutants showed brewing characteristics similar to the S9arg strain. This study provides a screening method for generating a piezosensitive yeast mutant as well as insight on a new way of applying HHP pasteurization.

Field emission characteristics of metal nanoparticle-coated carbon nanowalls.

Metal nanoparticles are deposited on nitrogen-incorporated carbon nanowalls (CNWs) using Ag, Au, In, and Mg as metal species for enhancing field emission. Morphology, coverage, chemical composition, and crystallinity of the metal coatings on CNW surfaces are examined by varying nominal thickness of metals within 10 nm. The emission characteristics reveal that coating CNWs with any metal species lowers emission turn-on fields and thus increases emission efficiency. The inverse dependence of field enhancement factor and turn-on field upon nominal thickness of metals confirms that additional field amplification at metal nanoparticles governs emission efficiency regardless of work functions of the metals. The Ag-coated CNWs retain the highest current density for long-time emission at a constant applied field, while the non-coated CNWs have higher emission stability and a larger time constant of current degradation than the metal-coated ones.

Asymmetric Arginine Dimethylation Modulates Mitochondrial Energy Metabolism and Homeostasis in Caenorhabditis elegans.

Protein arginine methyltransferase 1 (PRMT-1) catalyzes asymmetric arginine dimethylation on cellular proteins and modulates various aspects of biological processes, such as signal transduction, DNA repair, and transcriptional regulation. We have previously reported that the null mutant of prmt-1 in Caenorhabditis elegans exhibits a slightly shortened life span, but the physiological significance of PRMT-1 remains largely unclear. Here we explored the role of PRMT-1 in mitochondrial function as hinted by a two-dimensional Western blot-based proteomic study. Subcellular fractionation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that PRMT-1 is almost entirely responsible for asymmetric arginine dimethylation on mitochondrial proteins. Importantly, isolated mitochondria from prmt-1 mutants represent compromised ATP synthesis in vitro, and whole-worm respiration in prmt-1 mutants is decreased in vivo Transgenic rescue experiments demonstrate that PRMT-1-dependent asymmetric arginine dimethylation is required to prevent mitochondrial reactive oxygen species (ROS) production, which consequently causes the activation of the mitochondrial unfolded-protein response. Furthermore, the loss of enzymatic activity of prmt-1 induces food avoidance behavior due to mitochondrial dysfunction, but treatment with the antioxidant N-acetylcysteine significantly ameliorates this phenotype. These findings add a new layer of complexity to the posttranslational regulation of mitochondrial function and provide clues for understanding the physiological roles of PRMT-1 in multicellular organisms.

Non-transcriptional Function of FOXO1/DAF-16 Contributes to Translesion DNA Synthesis.

Forkhead box O (FOXO; DAF-16 in nematode) transcription factors activate a program of genes that control stress resistance, metabolism, and lifespan. Given the adverse impact of the stochastic DNA damage on organismal development and ageing, we examined the role of FOXO/DAF-16 in UV-induced DNA-damage response. Knockdown of FOXO1, but not FOXO3a, increases sensitivity to UV irradiation when exposed during S phase, suggesting a contribution of FOXO1 to translesion DNA synthesis (TLS), a replicative bypass of UV-induced DNA lesions. Actually, FOXO1 depletion results in a sustained activation of the ATR-Chk1 signaling and a reduction of PCNA monoubiquitination following UV irradiation. FOXO1 does not alter the expression of TLS-related genes but binds to the protein replication protein A (RPA1) that coats single-stranded DNA and acts as a scaffold for TLS. In Caenorhabditis elegans, daf-16 null mutants show UV-induced retardation in larval development and are rescued by overexpressing DAF-16 mutant lacking transactivation domain, but not substitution mutant unable to interact with RPA-1. Thus, our findings demonstrate that FOXO1/DAF-16 is a functional component in TLS independently of its transactivation activity.

CTF18 interacts with replication protein A in response to replication stress.

Replication stress response is a protective mechanism against defects in chromosome replication for maintaining genome integrity in eukaryotic cells. An alternative clamp loader complex termed chromosome transmission fidelity protein 18 and replication factor C (CTF18­RFC) has been shown to act as a positive regulator of two types of replication stress response: S­phase checkpoint signaling and translesion DNA synthesis. However, it remains largely unknown how CTF18­RFC responds to replication stress and is recruited to stalled replication forks. The present study demonstrated that endogenous CTF18 forms a physical complex with a single­stranded DNA­binding protein replication protein A (RPA) in mammalian cells. Using an in situ proximity ligation assay (PLA), it was demonstrated that the interaction between CTF18 and RPA occurs in chromatin when replication stress is elicited by treatment with hydroxyurea during S phase. Similar results were obtained after exposure to ultraviolet irradiation, which triggers translesion DNA synthesis. Furthermore, the PLA demonstrated that the kinetics of the interaction between CTF18 and RPA was positively correlated with that of checkpoint kinase 1 phosphorylation, which is an indicator of activation of the ATM and Rad3­related pathway. These findings provide novel insights into the molecular mechanism underlying the participation of CTF18­RFC in the regulation of replication stress response.

Conserved SAMS function in regulating egg-laying in C. elegans.

S-adenosyl-L-methionine (SAM) is an intermediate metabolite of methionine and serves as the methyl donor for many biological methylation reactions. The synthesis of SAM is catalyzed by SAM synthetase (SAMS), which transfers the adenosyl moiety of adenosine-5'-triphosphate to methionine. In the nematode Caenorhabditis elegans, four sams family genes, sams-1, -3, -4 and -5, are predicted to encode SAMS proteins. However, their physiological roles remain unclear. Here we show that the four predicted SAMS proteins in fact have the ability to catalyze the formation of SAM in vitro, and revealed that only sams-1 mutant animals among the family genes exhibited a significant reduction in egg-laying. Using transgenic animals carrying a transcriptional reporter for each sams gene promoter, we observed that each sams promoter confers a distinct expression pattern with respect to tissue, time of expression and expression level (i.e. promoter specificity). Promoter-swap experiments revealed that the ectopic expression of SAMS-3, -4 or -5 driven by the sams-1 promoter completely rescued egg-laying in sams-1 mutants. These data indicate that SAMS protein function is conserved throughout the entire family.

GSK3ß regulates gluconeogenic gene expression through HNF4α and FOXO1.

Hepatic gluconeogenesis is important for the maintenance of blood glucose homeostasis under fasting condition. Hepatocyte nuclear factor 4α (HNF4α) and FOXO1 transcription factors have implicated in this process through transcriptional regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), which are rate-limiting enzymes in gluconeogenesis. In this study, we demonstrate that glycogen synthase kinase 3ß (GSK3ß) regulates the expression of gluconeogenic genes through HNF4α and FOXO1. Silencing of GSK3ß leads to reduction in the expression of gluconeogenic genes, including G6Pase, PEPCK, and peroxisome proliferator-activated receptor γ coactivator-1α. We show that GSK3ß directly binds to both HNF4α and FOXO1. Inhibition of GSK3 by SB-216763 abolishes HNF4α-mediated activation of G6Pase promoter. We also found that overexpression of GSK3ß potentiates G6Pase promoter activation by FOXO1 in a manner dependent on its kinase activity. Treatment of SB-216763 diminishes FOXO1-mediated activation of G6Pase promoter. Taken together, these results reveal a previously unrecognized mechanism for the regulation of gluconeogenic gene expression.