Time-lapse monitoring of fertilized human oocytes focused on the incidence of 0PN embryos in conventional in vitro fertilization cycles.

We aimed to investigate why the incidence of embryos derived from oocytes with no pronuclei (0PN) decreases using time-lapse monitoring (TLM) versus fixed-point assessment in conventional IVF cycles. We analyzed 514 embryos monitored with TLM 6-9 h after insemination and 144 embryos monitored using microscopic assessment 18-21 h after insemination. The primary endpoint of this study was the incidence of 0PN-derived embryos in short insemination followed by TLM. The secondary endpoint was the duration of insemination. As exploratory endpoints, we analyzed the blastulation rate and cryo-warmed blastocyst transfer outcome of embryos with early PN fading, whereby PN disappeared within < 20 h following the initiation of insemination. The incidence of 0PN-derived embryo reduced more significantly through TLM than through fixed-point observation. The microscopic assessment time was more significantly delayed in the 0PN-derived embryo than that in the 2PN-derived embryo. The embryo with early PN fading formed good-quality blastocysts, and their pregnancy outcomes were similar to those of other embryos. Most 0PN-derived embryos in the fixed-point assessment might have resulted from missed observation of PN appearance in the early-cleaved embryos. TLM or strict laboratory schedule management may reduce 0PN-derived embryos by reducing missed PN observations.

Menzerath's Law in the Syntax of Languages Compared with Random Sentences.

The Menzerath law is considered to show an aspect of the complexity underlying natural language. This law suggests that, for a linguistic unit, the size (y) of a linguistic construct decreases as the number (x) of constructs in the unit increases. This article investigates this property syntactically, with x as the number of constituents modifying the main predicate of a sentence and y as the size of those constituents in terms of the number of words. Following previous articles that demonstrated that the Menzerath property held for dependency corpora, such as in Czech and Ukrainian, this article first examines how well the property applies across languages by using the entire Universal Dependency dataset ver. 2.3, including 76 languages over 129 corpora and the Penn Treebank (PTB). The results show that the law holds reasonably well for x>2. Then, for comparison, the property is investigated with syntactically randomized sentences generated from the PTB. These results show that the property is almost reproducible even from simple random data. Further analysis of the property highlights more detailed characteristics of natural language.

Long-Range Correlation Underlying Childhood Language and Generative Models.

Long-range correlation, a property of time series exhibiting relevant statistical dependence between two distant subsequences, is mainly studied in the statistical physics domain and has been reported to exist in natural language. By using a state-of-the-art method for such analysis, long-range correlation is first shown to occur in long CHILDES data sets. To understand why, generative stochastic models of language, originally proposed in the cognitive scientific domain, are investigated. Among representative models, the Simon model is found to exhibit surprisingly good long-range correlation, but not the Pitman-Yor model. Because the Simon model is known not to correctly reflect the vocabulary growth of natural languages, a simple new model is devised as a conjunct of the Simon and Pitman-Yor models, such that long-range correlation holds with a correct vocabulary growth rate. The investigation overall suggests that uniform sampling is one cause of long-range correlation and could thus have some relation with actual linguistic processes.

Cross Entropy of Neural Language Models at Infinity-A New Bound of the Entropy Rate.

Neural language models have drawn a lot of attention for their strong ability to predict natural language text. In this paper, we estimate the entropy rate of natural language with state-of-the-art neural language models. To obtain the estimate, we consider the cross entropy, a measure of the prediction accuracy of neural language models, under the theoretically ideal conditions that they are trained with an infinitely large dataset and receive an infinitely long context for prediction. We empirically verify that the effects of the two parameters, the training data size and context length, on the cross entropy consistently obey a power-law decay with a positive constant for two different state-of-the-art neural language models with different language datasets. Based on the verification, we obtained 1.12 bits per character for English by extrapolating the two parameters to infinity. This result suggests that the upper bound of the entropy rate of natural language is potentially smaller than the previously reported values.

Do neural nets learn statistical laws behind natural language?

The performance of deep learning in natural language processing has been spectacular, but the reasons for this success remain unclear because of the inherent complexity of deep learning. This paper provides empirical evidence of its effectiveness and of a limitation of neural networks for language engineering. Precisely, we demonstrate that a neural language model based on long short-term memory (LSTM) effectively reproduces Zipf's law and Heaps' law, two representative statistical properties underlying natural language. We discuss the quality of reproducibility and the emergence of Zipf's law and Heaps' law as training progresses. We also point out that the neural language model has a limitation in reproducing long-range correlation, another statistical property of natural language. This understanding could provide a direction for improving the architectures of neural networks.

Long-Range Memory in Literary Texts: On the Universal Clustering of the Rare Words.

A fundamental problem in linguistics is how literary texts can be quantified mathematically. It is well known that the frequency of a (rare) word in a text is roughly inverse proportional to its rank (Zipf's law). Here we address the complementary question, if also the rhythm of the text, characterized by the arrangement of the rare words in the text, can be quantified mathematically in a similar basic way. To this end, we consider representative classic single-authored texts from England/Ireland, France, Germany, China, and Japan. In each text, we classify each word by its rank. We focus on the rare words with ranks above some threshold Q and study the lengths of the (return) intervals between them. We find that for all texts considered, the probability SQ(r) that the length of an interval exceeds r, follows a perfect Weibull-function, SQ(r) = exp(-b(ß)rß), with ß around 0.7. The return intervals themselves are arranged in a long-range correlated self-similar fashion, where the autocorrelation function CQ(s) of the intervals follows a power law, CQ(s) ∼ s-γ, with an exponent γ between 0.14 and 0.48. We show that these features lead to a pronounced clustering of the rare words in the text.

Fluorescence imaging of ATP in neutrophils from patients with sepsis using organelle-localizable fluorescent chemosensors.

BACKGROUND: The activation of polymorphonuclear neutrophils (PMNs) plays an important role in sepsis. Previously, we showed that ATP release and feedback via ATP receptors are essential for PMN activation; however, the dynamics remain poorly understood. Two new fluorescent chemosensors, PMAP-1 and MitoAP-1, were developed to detect ATP in the plasma membrane and mitochondria of living cells, respectively. In this study, we aimed to evaluate ATP localization using these chemosensors in PMNs of sepsis patients. METHODS: Live PMNs isolated from 16 sepsis patients and healthy controls (HCs) were stained with these chemosensors and observed by confocal microscopy, and their mean fluorescence intensities (MFIs) were evaluated using flow cytometry. CD11b expression in PMNs was also evaluated. RESULTS: The MFIs of PMAP-1 and MitoAP-1 and CD11b expression in PMNs from sepsis patients on days 0-1 were significantly higher than those of HCs. The MFI of PMAP-1 and CD11b expression on days 3-4 decreased significantly compared to those observed at days 0-1, whereas MitoAP-1 MFI was maintained at a high level. The PMAP-1 MFI was significantly positively correlated with CD11b expression, white blood cell counts, neutrophil counts, and C-reactive protein levels in patients. CONCLUSIONS: The higher MFIs of PMAP-1 and MitoAP-1 in sepsis patients suggest a pivotal role of ATP for PMN activation. The temporal difference in ATP levels suggests that ATP plays different roles in the mitochondria and on the cell surface. These data should contribute to the understanding of the dynamics of ATP in PMNs and help to develop a novel therapy for sepsis.

An association between dietary habits and traffic accidents in patients with chronic liver disease: A data-mining analysis.

The incidence of traffic accidents in patients with chronic liver disease (CLD) is high in the USA. However, the characteristics of patients, including dietary habits, differ between Japan and the USA. The present study investigated the incidence of traffic accidents in CLD patients and the clinical profiles associated with traffic accidents in Japan using a data-mining analysis. A cross-sectional study was performed and 256 subjects [148 CLD patients (CLD group) and 106 patients with other digestive diseases (disease control group)] were enrolled; 2 patients were excluded. The incidence of traffic accidents was compared between the two groups. Independent factors for traffic accidents were analyzed using logistic regression and decision-tree analyses. The incidence of traffic accidents did not differ between the CLD and disease control groups (8.8 vs. 11.3%). The results of the logistic regression analysis showed that yoghurt consumption was the only independent risk factor for traffic accidents (odds ratio, 0.37; 95% confidence interval, 0.16-0.85; P=0.0197). Similarly, the results of the decision-tree analysis showed that yoghurt consumption was the initial divergence variable. In patients who consumed yoghurt habitually, the incidence of traffic accidents was 6.6%, while that in patients who did not consume yoghurt was 16.0%. CLD was not identified as an independent factor in the logistic regression and decision-tree analyses. In conclusion, the difference in the incidence of traffic accidents in Japan between the CLD and disease control groups was insignificant. Furthermore, yoghurt consumption was an independent negative risk factor for traffic accidents in patients with digestive diseases, including CLD.

Targeting cholesterol in a liquid-disordered environment by theonellamides modulates cell membrane order and cell shape.

Roles of lipids in the cell membrane are poorly understood. This is partially due to the lack of methodologies, for example, tool chemicals that bind to specific membrane lipids and modulate membrane function. Theonellamides (TNMs), marine sponge-derived peptides, recognize 3ß-hydroxysterols in lipid membranes and induce major morphological changes in cultured mammalian cells through as yet unknown mechanisms. Here, we show that TNMs recognize cholesterol-containing liquid-disordered domains and induce phase separation in model lipid membranes. Modulation of membrane order was also observed in living cells following treatment with TNM-A, in which cells shrank considerably in a cholesterol-, cytoskeleton-, and energy-dependent manner. These findings present a previously unrecognized mode of action of membrane-targeting natural products. Meanwhile, we demonstrated the importance of membrane order, which is maintained by cholesterol, for proper cell morphogenesis.

Properties and functions of lactosylceramide from mouse neutrophils.

Lactosylceramide (LacCer), which is essential for many cellular processes, is highly expressed on the plasma membranes of human neutrophils and mediates innate immune functions. Less is known, however, about the properties and biological functions of LacCer in mouse neutrophils. This study therefore analyzed the properties of mouse neutrophil LacCer. LacCer was observed on the surface of these cells, with flow cytometry indicating that mouse neutrophil LacCer could be detected by the anti-LacCer mAb T5A7, but not by the anti-LacCer antibodies Huly-m13 and MEM-74. The molecular species of LacCer were nearly identical in mouse and human neutrophils, including C24:0 and C24:1 fatty acid chain-containing species, although the LacCer content in plasma membranes was ∼ 20-fold lower in mouse than in human neutrophils. Surface plasmon resonance analysis revealed that T5A7 bound to a lipid monolayer composed of LacCer, DOPC, cholesterol and sphingomyelin (molar ratio 0.1 : 10 : 10 : 1), whereas Huly-m13 did not. T5A7 induced neutrophil migration, which was abolished by inhibitors of Src-family kinases, PI-3 kinases, and trimeric G (o/i) proteins. T5A7 also inhibited phagocytosis of non-opsonized zymosans by neutrophils. Taken together, these findings suggest that in mouse neutrophils, (i) LacCer is expressed as LacCer-enriched microdomains in cell surface plasma membranes, (ii) these microdomains are recognized by T5A7 but not by other known anti-LacCer antibodies and (iii) LacCer is involved in cell migration and phagocytosis.

Basal autophagy is required for the efficient catabolism of sialyloligosaccharides.

Macroautophagy is an essential, homeostatic process involving degradation of a cell's own components; it plays a role in catabolizing cellular components, such as protein or lipids, and damaged or excess organelles. Here, we show that in Atg5(-/-) cells, sialyloligosaccharides specifically accumulated in the cytosol. Accumulation of these glycans was observed under non-starved conditions, suggesting that non-induced, basal autophagy is essential for their catabolism. Interestingly, once accumulated in the cytosol, sialylglycans cannot be efficiently catabolized by resumption of the autophagic process, suggesting that functional autophagy is important for preventing sialyloligosaccharides from accumulating in the cytosol. Moreover, knockdown of sialin, a lysosomal transporter of sialic acids, resulted in a significant reduction of sialyloligosaccharides, implying that autophagy affects the substrate specificity of this transporter. This study thus provides a surprising link between basal autophagy and catabolism of N-linked glycans.

Visualization of sterol-rich membrane domains with fluorescently-labeled theonellamides.

Cholesterol plays important roles in biological membranes. The cellular location where cholesterol molecules work is prerequisite information for understanding their dynamic action. Bioimaging probes for cholesterol molecules would be the most powerful means for unraveling the complex nature of lipid membranes. However, only a limited number of chemical or protein probes have been developed so far for cytological analysis. Here we show that fluorescently-labeled derivatives of theonellamides act as new sterol probes in mammalian cultured cells. The fluorescent probes recognized cholesterol molecules and bound to liposomes in a cholesterol-concentration dependent manner. The probes showed patchy distribution in the plasma membrane, while they stained specific organelle in the cytoplasm. These data suggest that fTNMs will be valuable sterol probes for studies on the role of sterols in the biological membrane under a variety of experimental conditions.

Nutritional assessments for ordinary medical care in patients with chronic liver disease.

AIM: In patients with chronic liver disease who are at risk of malnutrition, simple and useful assessments for nutritional status should be established for ordinary medical care. The prognostic nutritional index (PNI) and controlling nutritional status (CONUT) are simple assessments constructed of only two or three laboratory data. We aimed to describe the potential of PNI and CONUT as a nutritional assessment tool in patients with chronic liver disease. METHODS: We enrolled 165 patients, aged 18-85 years, with chronic liver disease. These patients were nutritionally assessed by PNI or CONUT, demonstrating the association with the severity of chronic liver disease or anthropometric values. RESULTS: The value of PNI or CONUT was significantly associated with the severity of chronic liver disease (P < 0.001, respectively). In addition, the value of CONUT was significantly associated with all the anthropometric values such as body mass index (BMI, P < 0.05), mid-arm circumference (AC, P < 0.001), mid-arm muscle circumference (AMC, P < 0.001), and triceps skinfold thickness (TSF, P < 0.001), whereas the value of PNI was significantly associated with the values of AC (P < 0.01), AMC (P < 0.05) and TSF (P < 0.05). Approximately 80% of cirrhotic patients were assessed by PNI or CONUT to have obvious malnutrition. CONCLUSION: PNI and CONUT are potential tools for nutritional assessment in patients with chronic liver disease, especially for ordinary medical care, because of their simplicity.

Identification of compounds that augment the lectin-sensitivity of Chinese Hamster Ovary cells.

Glycosylation is now recognized as one of the most important modifications of eukaryotic proteins. In cancer biology, alterations in cell surface glycosylation have been exploited as valuable biomarkers, and the relationship of this modification to the metastatic characteristics of cancer cells has also been well-documented. Chemicals that can alter cell surface glycosylation patterns will therefore become attractive lead compounds for controlling the metastatic characteristics of cancer cells, one of the critical factors in their malignancy and prognosis of the disease. In this study, we established a system for screening compounds that have the potential to alter cell surface glycosylation by taking advantage of the susceptibility of cells toward various lectins. Through our screening of a chemical library, we were able to identify two compounds that augment the sensitivity of Chinese Hamster Ovary (CHO-K1) cells against the L4-PHA lectin. Surprisingly, these compounds did not result in alterations in cell surface glycan structures. Instead, they appeared to render the cells to be more sensitive to various lectins with distinct carbohydrate specificities. These compounds promise to be valuable, not only as tools for providing insights into the intracellular signaling of lectin-mediated growth arrest, but also as potential lead compounds for use as therapeutic, anti-cancer drugs.

Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer.

Analyses of mice lacking glycosyltransferase have suggested that their pathological phenotypes are not attributable to the overall change of the sugar modification, but instead the result of changes of the glycan structures on a specific 'target' glycoprotein. Therefore, detecting or monitoring the glycosylation status of a specific protein in living cells is important, but no such methods are currently available. Here we demonstrate the detection of glycoforms of a specific glycoprotein using the fluorescence resonance energy transfer technique. Using model proteins, we detect characteristic fluorescence resonance energy transfer signals from the specific glycoform-bearing target glycoprotein. We also show that, upon insulin removal, sialylated glycoforms of green fluorescent protein-tagged GLUT4 seem to be internalized more slowly than non-sialylated GLUT4. This novel analytical imaging tool allows studying the roles of specific glycan modifications of a protein of interest.

Limonoid compounds inhibit sphingomyelin biosynthesis by preventing CERT protein-dependent extraction of ceramides from the endoplasmic reticulum.

To identify novel inhibitors of sphingomyelin (SM) metabolism, a new and selective high throughput microscopy-based screening based on the toxicity of the SM-specific toxin, lysenin, was developed. Out of a library of 2011 natural compounds, the limonoid, 3-chloro-8ß-hydroxycarapin-3,8-hemiacetal (CHC), rendered cells resistant to lysenin by decreasing cell surface SM. CHC treatment selectively inhibited the de novo biosynthesis of SM without affecting glycolipid and glycerophospholipid biosynthesis. Pretreatment with brefeldin A abolished the limonoid-induced inhibition of SM synthesis suggesting that the transport of ceramide (Cer) from the endoplasmic reticulum to the Golgi apparatus is affected. Unlike the Cer transporter (CERT) inhibitor HPA-12, CHC did not change the transport of a fluorescent short chain Cer analog to the Golgi apparatus or the formation of fluorescent and short chain SM from the corresponding Cer. Nevertheless, CHC inhibited the conversion of de novo synthesized Cer to SM. We show that CHC specifically inhibited the CERT-mediated extraction of Cer from the endoplasmic reticulum membranes in vitro. Subsequent biochemical screening of 21 limonoids revealed that some of them, such as 8ß-hydroxycarapin-3,8-hemiacetal and gedunin, which exhibits anti-cancer activity, inhibited SM biosynthesis and CERT-mediated extraction of Cer from membranes. Model membrane studies suggest that 8ß-hydroxycarapin-3,8-hemiacetal reduced the miscibility of Cer with membrane lipids and thus induced the formation of Cer-rich membrane domains. Our study shows that certain limonoids are novel inhibitors of SM biosynthesis and suggests that some biological activities of these limonoids are related to their effect on the ceramide metabolism.

N-glycosylation is critical for the stability and intracellular trafficking of glucose transporter GLUT4.

The facilitative glucose transporter GLUT4 plays a key role in regulating whole body glucose homeostasis. GLUT4 dramatically changes its distribution upon insulin stimulation, and insulin-resistant diabetes is often linked with compromised translocation of GLUT4 under insulin stimulation. To elucidate the functional significance of the sole N-glycan chain on GLUT4, wild-type GLUT4 and a GLUT4 glycosylation mutant conjugated with enhanced GFP were stably expressed in HeLa cells. The N-glycan contributed to the overall stability of newly synthesized GLUT4. Moreover, cell surface expression of wild-type GLUT4 in HeLa cells was elevated upon insulin treatment, whereas the glycosylation mutant lost the ability to respond to insulin. Subcellular distribution of the mutant was distinct from that of wild-type GLUT4, implying that the subcellular localization required for insulin-mediated translocation was impaired in the mutant protein. Interestingly, kifunensine-treated cells also lost sensitivity to insulin, suggesting the functional importance of the N-glycan structure for GLUT4 trafficking. The K(m) or turnover rates of wild-type and mutant GLUT4, however, were similar, suggesting that the N-glycan had little effect on transporter activity. These findings underscore the critical roles of the N-glycan chain in quality control as well as intracellular trafficking of GLUT4.

Calystegine B3 as a specific inhibitor for cytoplasmic alpha-mannosidase, Man2C1.

Cytoplasmic α-mannosidase (Man2C1) has been implicated in non-lysosomal catabolism of free oligosaccharides derived from N-linked glycans accumulated in the cytosol. Suppression of Man2C1 expression reportedly induces apoptosis in various cell lines, but its molecular mechanism remains unclear. Development of a specific inhibitor for Man2C1 is critical to understanding its biological significance. In this study, we identified a plant-derived alkaloid, calystegine B(3), as a potent specific inhibitor for Man2C1 activity. Biochemical enzyme assay revealed that calystegine B(3) was a highly specific inhibitor for Man2C1 among various α-mannosidases prepared from rat liver. Consistent with this in vitro result, an in vivo experiment also showed that treatment of mammalian-derived cultured cells with this compound resulted in drastic change in both structure and quantity of free oligosaccharides in the cytosol, whereas no apparent change was seen in cell-surface oligosaccharides. Calystegine B(3) could thus serve as a potent tool for the development of a highly specific in vivo inhibitor for Man2C1.

Phosphatidylglucoside forms specific lipid domains on the outer leaflet of the plasma membrane.

Phosphatidylglucoside (PtdGlc) is a recently discovered unique glycophospholipid involved in granulocytic differentiation of human promyelocytic leukemia cell line HL60 and in astrocytic differentiation in developing rodent brains. Using a PtdGlc-specific monoclonal antibody in immunofluorescence and immunoelectron microscopy, we showed that PtdGlc forms distinct lipid domains on the outer leaflet of the plasma membrane of HL60 cells and the human alveolar epithelial cell line, A549. Similar to glycosphingolipid, glucosylceramide (GlcCer), the natural form of PtdGlc exhibited a high main phase transition temperature in differential scanning calorimetry (DSC). However, unlike GlcCer, PtdGlc did not exhibit a large difference in the main phase transition temperature between the heating and cooling scans. DSC further indicated that GlcCer, but not PtdGlc, was miscible with sphingomyelin. In addition, DSC and small-angle X-ray scattering (SAXS) experiments revealed that PtdGlc was poorly miscible with phosphatidylcholine. Our results suggest that the lack of tight intermolecular interaction excludes PtdGlc from other lipid domains on the plasma membrane.

Evidence for an essential deglycosylation-independent activity of PNGase in Drosophila melanogaster.

BACKGROUND: Peptide:N-glycanase (PNGase) is an enzyme which releases N-linked glycans from glycopeptides/glycoproteins. This enzyme plays a role in the ER-associated degradation (ERAD) pathway in yeast and mice, but the biological importance of this activity remains unknown. PRINCIPAL FINDINGS: In this study, we characterized the ortholog of cytoplasmic PNGases, PNGase-like (Pngl), in Drosophila melanogaster. Pngl was found to have a molecular weight of approximately 74K and was mainly localized in the cytosol. Pngl lacks a CXXC motif that is critical for enzymatic activity in other species and accordingly did not appear to possess PNGase activity, though it still retains carbohydrate-binding activity. We generated microdeletions in the Pngl locus in order to investigate the functional importance of this protein in vivo. Elimination of Pngl led to a serious developmental delay or arrest during the larval and pupal stages, and surviving mutant adult males and females were frequently sterile. Most importantly, these phenotypes were rescued by ubiquitous expression of Pngl, clearly indicating that those phenotypic consequences were indeed due to the lack of functional Pngl. Interestingly, a putative "catalytic-inactive" mutant could not rescue the growth-delay phenotype, indicating that a biochemical activity of this protein is important for its biological function. CONCLUSION: Pngl was shown to be inevitable for the proper developmental transition and the biochemical properties other than deglycosylation activity is important for its biological function.