Distinct lactate metabolism between hepatocytes and myotubes revealed by live cell imaging with genetically encoded indicators.

The process of glycolysis breaks down glycogen stored in muscles, producing lactate through pyruvate to generate energy. Excess lactate is then released into the bloodstream. When lactate reaches the liver, it is converted to glucose, which muscles utilize as a substrate to generate ATP. Although the biochemical study of lactate metabolism in hepatocytes and skeletal muscle cells has been extensive, the spatial and temporal dynamics of this metabolism in live cells are still unknown. We observed the dynamics of metabolism-related molecules in primary cultured hepatocytes and a skeletal muscle cell line upon lactate overload. Our observations revealed an increase in cytoplasmic pyruvate concentration in hepatocytes, which led to glucose release. Skeletal muscle cells exhibited elevated levels of lactate and pyruvate levels in both the cytoplasm and mitochondrial matrix. However, mitochondrial ATP levels remained unaffected, indicating that the increased lactate can be converted to pyruvate but is unlikely to be utilized for ATP production. The findings suggest that excess lactate in skeletal muscle cells is taken up into mitochondria with little contribution to ATP production. Meanwhile, lactate released into the bloodstream can be converted to glucose in hepatocytes for subsequent utilization in skeletal muscle cells.

Lobular distribution of enhanced expression levels of heat shock proteins using in-situ hybridization in the mouse liver treated with a single administration of CCl4.

This study was conducted to visualize the lobular distribution of enhanced mRNA expression levels of heat shock proteins (HSPs) in liver samples from carbon tetra chloride (CCl4)-treated mice using in-situ hybridization (ISH). Male BALB/c mice given a single oral administration of CCl4 were euthanized 6 hours or 1 day after the administration (6 h or 1 day). Paraffin-embedded liver samples were obtained, ISH for HSPs was conducted, as well as hematoxylin-eosin staining and immunohistochemistry (IHC). At 6 h, centrilobular hepatocellular vacuolization was observed, and increased signals for Hspa1a, Hspa1b, and Grp78, which are HSPs, were noted in the centrilobular area using ISH. At 1 day, zonal hepatocellular necrosis was observed in the centrilobular area, but mRNA signal increases for HSPs were no longer observed there. Some discrepancies between ISH and IHC for HSPs were observed, and they might be partly caused by post-transcriptional gene regulation, including the ribosome quality control mechanisms. It is known that CCl4 damages centrilobular hepatocytes through metabolization by cytochrome P450, mainly located in the centrilobular region, and HSPs are induced under cellular stress. Therefore, our ISH results visualized increased mRNA expression levels of HSPs in the centrilobular hepatocytes of mice 6 hours after a single administration of CCl4 as a response to cellular stress, and it disappeared 1 day after the treatment when remarkable necrosis was observed there.

Noise Reduction Combining a General Microphone and a Throat Microphone.

In this study, we propose a method to reduce noise from speech obtained from a general microphone using the information of a throat microphone. A throat microphone records a sound by detecting the vibration of the skin surface near the throat directly. Therefore, throat microphones are less prone to noise than ordinary microphones. However, as the acoustic characteristics of the throat microphone differ from those of ordinary microphones, its sound quality degrades. To solve this problem, this study aims to improve the speech quality while suppressing the noise of a general microphone by using the information recorded by a throat microphone as reference information to extract the speech signal in general microphones. In this paper, the framework of the proposed method is formulated, and several experiments are conducted to evaluate the noise suppression and speech quality improvement effects of the proposed method.

Aurora Image Classification with Deep Metric Learning.

In recent years, neural networks have been increasingly used for classifying aurora images. In particular, convolutional neural networks have been actively studied. However, there are not many studies on the application of deep learning techniques that take into account the characteristics of aurora images. Therefore, in this study, we propose the use of deep metric learning as a suitable method for aurora image classification. Deep metric learning is one of the deep learning techniques. It was developed to distinguish human faces. Identifying human faces is a more difficult task than standard classification tasks because this task is characterized by a small number of sample images for each class and poor feature variation between classes. We thought that the face identification task is similar to aurora image classification in that the number of labeled images is relatively small and the feature differences between classes are small. Therefore, we studied the application of deep metric learning to aurora image classification. As a result, our experiments showed that deep metric learning improves the accuracy of aurora image classification by nearly 10% compared to previous studies.

Polyamines polarized Th2/Th9 cell-fate decision by regulating GATA3 expression.

Polyamines produced by both prokaryotes and eukaryotes are bioactive substances with pleiotropic effects. Accumulating evidence has demonstrated that polyamines contribute to anti-inflammatory responses by suppressing the expression of proinflammatory cytokines in mononuclear cells and macrophages. However, the effects of polyamines on CD4+ T cell responses remain to be elucidated. Here, we investigated the effect of polyamines on cell fate decisions of naïve CD4+ T cells in vitro. We found that endogenously generated polyamines are essential for the development of T helper 2 (Th2) cells. Treatment with DL-2-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, diminished GATA3 expression in CD4+ T cells under Th2-skewed conditions. Supplementation of exogenous polyamines rescued GATA3 downregulation caused by DFMO treatment in CD4+ T cells. Transcriptome analysis revealed that deprivation of endogenous polyamines resulted in upregulated Th9-related genes, such as Il9, Irf4, and Batf3, even under the Th2-skewing conditions. Depletion of intracellular polyamines reduced GATA3 expression but increased IL-9-producing CD4+ T cells under both Th2 and Th9-skewing conditions. Furthermore, oral administration of DFMO increased IL-9-producing CD4+ T cells in small intestine in mice. Thus, our data indicate that polyamines play a critical role in the regulation of the Th2/Th9 balance.

Prevention of Atherosclerosis by the Induction of Microbial Polyamine Production in the Intestinal Lumen.

Low molecular weight metabolites produced by the intestinal microbiome that have been associated with health and disease as metabolites need to be constantly absorbed from the intestinal lumen and transported to intestinal epithelial cells and blood. Polyamines, especially spermidine and spermine, are bioactive chemicals which promote autophagy and suppress inflammation. The main source of exogenous polyamines is the intestinal lumen, where they are produced by intestinal microbiome. Considering the intestinal microbiome as a manufacturing plant for bioactive substances, we developed a novel hybrid putrescine biosynthesis system strategy, in which the simultaneous intake of Bifidobacterium animalis ssp. lactis LKM512 (Bifal) and arginine (Arg) upregulates the production of the putrescine, a precursor of spermidine and spermine, in the gut by controlling the bacterial metabolism beyond its vast diversity and inter-individual differences. In a clinical trial, healthy individuals with a body mass index near the maximum "healthy" range (25 kg/m3; n = 44) were randomized to consume either normal yogurt containing Bifal and Arg (Bifal + Arg YG) or placebo (normal yogurt) for 12 weeks. The change in reactive hyperemia index determined by EndoPAT from week 0 to 12 in the Bifal + Arg YG group was significantly higher than that in the placebo group, indicating that Bifal + Arg YG intake improved vascular endothelial function. In addition, the concentrations of fecal putrescine and serum spermidine in the Bifal+ Arg YG group were significantly higher than those in the placebo group. These findings suggest that consuming Bifal + Arg YG prevents or reduces atherosclerosis risk by upregulating blood spermidine levels, which subsequently induces autophagy.

Vision-referential speech enhancement of an audio signal using mask information captured as visual data.

This paper describes a vision-referential speech enhancement of an audio signal using mask information captured as visual data. Smartphones and tablet devices have become popular in recent years. Most of them not only have a microphone but also a camera. Although the frame rate of the camera in such devices is very low compared to the audio signal from the microphone, it will be useful to enhance the speech signal if both signals are used adequately. In the proposed method, the speaker broadcasts not only his/her speech signal through a loudspeaker but also its mask information through a display. The receiver can enhance the speech combining the speech signal captured by the microphone and the reference signal captured by the camera. Some experiments were conducted to evaluate the effectiveness of the proposed method compared to a typical sparse approach. It was confirmed that the speech could be enhanced even when there were different kinds of noise and a high level of real noise in the environments. Experiments were also conducted to check the sound quality of the proposed method. They were compared to clear audio data compressed with various bps mp3 format. The sound quality was sufficient for practical application.

Taxonomic classification for microbiome analysis, which correlates well with the metabolite milieu of the gut.

BACKGROUND: 16S rRNA gene amplicon sequencing analysis (16S amplicon sequencing) has provided considerable information regarding the ecology of the intestinal microbiome. Recently, metabolomics has been used for investigating the crosstalk between the intestinal microbiome and the host via metabolites. In the present study, we determined the accuracy with which 16S rRNA gene data at different classification levels correspond to the metabolome data for an in-depth understanding of the intestinal environment. RESULTS: Over 200 metabolites were identified using capillary electrophoresis and time-of-flight mass spectrometry (CE-TOFMS)-based metabolomics in the feces of antibiotic-treated and untreated mice. 16S amplicon sequencing, followed by principal component analysis (PCA) of the intestinal microbiome at each taxonomic rank, revealed differences between the antibiotic-treated and untreated groups in the first principal component in the family-, genus, and species-level analyses. These differences were similar to those observed in the PCA of the metabolome. Furthermore, a strong correlation between principal component (PC) scores of the metabolome and microbiome was observed in family-, genus-, and species-level analyses. CONCLUSIONS: Lower taxonomic ranks such as family, genus, or species are preferable for 16S amplicon sequencing to investigate the correlation between the microbiome and metabolome. The correlation of PC scores between the microbiome and metabolome at lower taxonomic levels yield a simple method of integrating different "-omics" data, which provides insights regarding crosstalk between the intestinal microbiome and the host.

A Novel Putrescine Exporter SapBCDF of Escherichia coli.

Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 µm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.

Noise reduction with complex bilateral filter.

This study introduces a noise reduction technique that uses a complex bilateral filter. A bilateral filter is a nonlinear filter originally developed for images that can reduce noise while preserving edge information. It is an attractive filter and has been used in many applications in image processing. When it is applied to an acoustical signal, small-amplitude noise is reduced while the speech signal is preserved. However, a bilateral filter cannot handle noise with relatively large amplitudes owing to its innate characteristics. In this study, the noisy signal is transformed into the time-frequency domain and the filter is improved to handle complex spectra. The high-amplitude noise is reduced in the time-frequency domain via the proposed filter. The features and the potential of the proposed filter are also confirmed through experiments.

Intestinal microbiota in neonates requiring urgent surgery: assessing the role of probiotics using fecal DNA sequencing.

PURPOSE: To assess the impact of urgent surgery on neonates and the value of an orally administered probiotic preparation of Bifidobacterium animalis subsp. lactis LKM512 (LKM) using fecal DNA sequencing to analyze intestinal microbiota. METHODS: Subjects for this study were 13 neonates born at our institution. Surgical cases required surgery within 3 days of birth. Groups studied were surgical cases administered LKM (n = 4; LKM+), surgical cases not administered have surgery and were not administered LKM (n = 2; CS), and normal healthy neonates (n = 3; CN). Stool specimens (20 mg) were collected five times (after birth, and on days 3, 7, 10, and 14 after surgery in surgical cases, and after birth, and on days 4, 8, 11, and 15 of life in controls). RESULTS: Clinical data were similar for LKM+ and LKM-. Enterobacteriaceae, Streptococcaceae, Staphylococcaceae and Bifidobacteriaceae were identified in the descending order of abundance in CS stool. Streptococcaceae, Staphylococcaceae, Enterococcaceae and Bifidobacteriaceae were identified in the descending order of abundance in LKM+ stool. Bifidobacteriaceae, Enterobacteriaceae, Staphylococcaceae and Streptococcaceae were identified in the descending order of abundance in LKM- stool. Unexpectedly, Bifidobacteriaceae was significantly more abundant in LKM- than LKM+ (p < 0.05). CONCLUSION: Surgical stress appears to affect intestinal microbiota considerably. Probiotic administration in neonates requires clarification.

Chemical structure of the cell wall-associated polysaccharide of Bifidobacterium animalis subsp. lactis LKM512.

We have demonstrated that Bifidobacterium animalis subsp. lactis LKM512 had some probiotic properties in vivo and in vitro. To further understand their mechanisms, the chemical structure of the extracellular polysaccharide that constructs the cell envelope was determined. The strain was anaerobically cultured in MRS broth at 37 °C for 20 h, then the bacterial cells were harvested by centrifugation and washed. The cell wall-associated polysaccharide (CPS) was prepared from the cell wall component digested by lysozyme. The results of anion exchange and gel filtration chromatography showed that the polysaccharide was negatively charged and had a high molecular mass. The CPS was found to compose of galactopyranosyl, galactofuranosyl, glucopyranosyl and rhamnopyranosyl residues in the molar ratio of 1:1:1:3 by using methylation analysis with GC-MS and HPLC profiling. From the results of the structural characterization by 1 dimensional and 2 dimensional NMR spectroscopy, the polysaccharide was established to be a hexasaccharide repeating unit with the following structure: LKM512.

Antipruritic effects of the probiotic strain LKM512 in adults with atopic dermatitis.

BACKGROUND: Evidence suggests that intestinal microbiota play an important role in the pathogenesis of atopic dermatitis (AD) through induction of immunosuppression and immune tolerance; however, the exact underlying mechanism is unclear. Few studies to date have examined the effects of probiotics on adult-type AD. OBJECTIVE: To examine the effects of the probiotic Bifidobacterium animalis subsp lactis LKM512 on adult-type AD and the expression of metabolites that are known to be influenced by gut microbiota in fecal samples. METHODS: Forty-four patients were randomly assigned to receive LKM512 or a placebo and underwent medical examinations. Fecal microbiota were analyzed with real-time polymerase chain reaction. Metabolomic analysis was conducted to search for antipruritic metabolites produced by intestinal bacteria using feces derived from 3 patients whose itch scores had improved using capillary electrophoresis with time-of-flight mass spectrometry. Antipruritic effects of kynurenic acid were observed using AD-induced NC/Nga mice. RESULTS: LKM512 administration alleviated itch in AD patients compared with controls and improved the dermatology-specific quality-of-life scores when compared with the controls. Administration of LKM512 also increased the expression of the antipruritic and antinociceptive metabolite kynurenic acid (KYNA) in patients whose itch score had improved after LKM512 treatment. In mouse experiments, scratching behavior counts tended to be decreased by KYNA injection when compared with no treatment. CONCLUSION: LKM512 administration may exert antipruritic effects by increasing KYNA production. LKM512 could therefore be a potentially effective therapeutic candidate for the reduction of pruritus. TRIAL REGISTRATION: umin.ac.jp/ctr Identifier: UMIN000005695.

A Sheet-Shaped Transforming Robot That Can Be Thrown from the Air.

In this paper, we describe a sheet-shaped throwable transforming robot. Sheet-type robots can change their shape to perform tasks according to the situation. Therefore, they are expected to be useful in places with many restrictions, such as disaster sites. However, most of them can only move slowly on the ground. Therefore, in order to actually deliver the robot to the disaster site, it must be carried manually. To solve this problem, we are developing a sheet-shaped robot that can be thrown from the sky. Previously developed prototypes could only move in the forward direction, and the transition from falling to walking was complicated and uncertain. In this paper, we report on a new prototype that improves on these shortcomings.

Mitochondrial ATP concentration decreases immediately after glucose administration to glucose-deprived hepatocytes.

Hepatocytes can switch their metabolic processes in response to nutrient availability. However, the dynamics of metabolites (such as lactate, pyruvate, and ATP) in hepatocytes during the metabolic switch remain unknown. In this study, we visualized metabolite dynamics in primary cultured hepatocytes during recovery from glucose-deprivation. We observed a decrease in the mitochondrial ATP concentration when glucose was administered to hepatocytes under glucose-deprivation conditions. In contrast, there was slight change in the cytoplasmic ATP concentration. A decrease in mitochondrial ATP concentration was associated with increased protein synthesis rather than glycogen synthesis, activation of urea cycle, and production of reactive oxygen species. These results suggest that mitochondrial ATP is important in switching metabolic processes in the hepatocytes.

Bifidobacterium animalis subsp. lactis and arginine mixture intake improves cognitive flexibility in mice.

The relationship between intestinal microbiota and cognitive function has been investigated as one of the major topics within the intestinal microbiota-gut-brain axis. Although an increasing number of studies have demonstrated an improvement in learning and memory when using probiotics or prebiotics, to date, there are no studies that target the cognitive flexibility observed in the early stages of several neuropsychiatric diseases, including dementia. We have recently developed a novel behavioral task using the touchscreen operant system to assess cognitive flexibility. We found that the disruption of the intestinal microbiota in mice induced a decline in cognitive flexibility. In the present study, we investigated the effects of treatments consisting of Bifidobacterium animalis subsp. lactis and arginine (Bifal + Arg), which promote the production of intestinal bacterial polyamine, on cognitive flexibility in the mouse model. Male C57BL6 mice orally treated with Bifal + Arg three times a week gradually decreased the 1st-choice incorrect diagonal rate with repeated reversals compared with the control group. Furthermore, in serial reversal phases, Bifal + Arg-treated mice shifted to the behavior of choosing a new correct spot more quickly after the reversal, and this was faster with repeated reversals. These results indicate that this treatment adapts to change and improves cognitive flexibility. This is the first report to show that intestinal environmental control, including probiotics and prebiotics, improves cognitive flexibility in mice.

Probiotic treatment with Bifidobacterium animalis subsp. lactis LKM512 + arginine improves cognitive flexibility in middle-aged mice.

Cognitive flexibility, the ability of adapting to an ever-changing environment, declines with aging and impaired in early stages of dementia. Although recent studies have indicated there is a relationship between the intestinal microbiota and cognitive function, few studies have shown relationships between intestinal microbiota and cognitive flexibility because of limited behavioural tasks in mice. We recently established a novel cognitive flexibility task for mice using a touchscreen operant apparatus and found that probiotic treatment with a mixture of Bifidobacterium animalis subsp. lactis LKM512 and arginine improved cognitive flexibility in young adult mice. To confirm the effects of the probiotic treatment on cognitive flexibility and to determine whether it is effective even in older age, we here examined the effects of long-term treatment with Bifidobacterium animalis subsp. lactis LKM512 and arginine on cognitive flexibility in middle-aged mice. From 8 to 15 months of age, mice received LKM + Arg or vehicle (controls) orally three times per week and were subjected to the cognitive flexibility task at 13-15 months old. In one of indices of cognitive flexibility, both Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice and vehicle-treated mice showed progressively improved performance by repeating reversal tasks, with a small trend that Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice showed better learning performance through reversal phases. With respect to the other index of cognitive flexibility, Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice showed significantly fewer error choices than control mice at the reversal phase, i.e. Bifidobacterium animalis subsp. lactis LKM512 and arginine improved the performance of behavioural sequencing acquired in the previous phase, which allowed Bifidobacterium animalis subsp. lactis LKM512 and arginine-treated mice to show an early onset of shift to reversal contingency. Taken together, long-term treatment with Bifidobacterium animalis subsp. lactis LKM512 and arginine was found to improve cognitive flexibility in middle-aged mice, indicating that probiotic treatment might contribute to prevention of age-related cognitive decline.

Strain-level detection of Fusobacterium nucleatum in colorectal cancer specimens by targeting the CRISPR-Cas region.

IMPORTANCE: Fusobacterium nucleatum is one of the predominant oral bacteria in humans. However, this bacterium is enriched in colorectal cancer (CRC) tissues and may be involved in CRC development. Our previous research suggested that F. nucleatum is present in CRC tissues originating from the oral cavity using a traditional strain-typing method [arbitrarily primed polymerase chain reaction (AP-PCR)]. First, using whole-genome sequencing, this study confirmed an exemplary similarity between the oral and tumoral strains derived from each patient with CRC. Second, we successfully developed a method to genotype this bacterium at the strain level, targeting the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated system, which is hypervariable (defined as F. nucleatum-strain genotyping PCR). This method can identify F. nucleatum strains in cryopreserved samples and is significantly superior to traditional AP-PCR, which can only be performed on isolates. The new methods have great potential for application in etiological studies of F. nucleatum in CRC.

A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: design and synthesis of benzylpyrazole acylsulfonamides.

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.