Intervention in gut microbiota increases intestinal γ-aminobutyric acid and alleviates anxiety behavior: a possible mechanism via the action on intestinal epithelial cells.

The role of the gut microbiota in the gut-brain axis has attracted attention in recent years. Some gut microbiota produces γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter in mammals, in vitro, but the correlation between gut microbiota composition and intestinal GABA concentration, as well as the action of intestinal GABA in vivo, are poorly understood. Herein, we found that the intestinal GABA concentration was increased in mice by the intervention of the gut microbiota with neomycin or Bifidobacterium bifidum TMC3115 (TMC3115). Administration of TMC3115 reduced anxiety without affecting serum levels of serotonin, corticosterone, or GABA. We further found that intestinal epithelial cells expressed GABA receptor subunits and mediated mitogen-activated protein kinase signaling upon GABA stimulation. In addition, administration of TMC3115 induced mitogen-activated protein kinase signaling in colonic epithelial cells but not in small intestinal epithelial cells in mice. These results indicate that GABA produced by the gut microbiota, mainly in the colon, may affect host behavioral characteristics via GABA receptors expressed in intestinal epithelial cells without being transferred to the blood. This study suggests a novel mechanism by which intestinal GABA exerts physiological effects, even in the presence of the blood-brain barrier.

Sugar and arginine facilitate oral tolerance by ensuring the functionality of tolerogenic immune cell subsets in the intestine.

Although oral tolerance is a critical system in regulating allergic disorders, the mechanisms by which dietary factors regulate the induction and maintenance of oral tolerance remain unclear. To address this, we explored the differentiation and function of various immune cells in the intestinal immune system under fasting and ad libitum-fed conditions before oral ovalbumin (OVA) administration. Fasting mitigated OVA-specific Treg expansion, which is essential for oral tolerance induction. This abnormality mainly resulted from functional defects in the CX3CR1+ cells responsible for the uptake of luminal OVA and reduction of tolerogenic CD103+ dendritic cells. Eventually, fasting impaired the preventive effect of oral OVA administration on asthma and allergic rhinitis development. Specific food ingredients, namely carbohydrates and arginine, were indispensable for oral tolerance induction by activating glycolysis and mTOR signaling. Overall, prior food intake and nutritional signals are critical for maintaining immune homeostasis by inducing tolerance to ingested food antigens.

A purified diet affects intestinal epithelial proliferation and barrier functions through gut microbial alterations.

The gut microbiota plays a crucial role in maintaining epithelial barrier function. Although multiple studies have demonstrated the significance of dietary factors on the gut microbiota and mucosal barrier function, the impact of a purified diet, which has long been used in various animal experiments, on intestinal homeostasis remains to be elucidated. Here, we compared the impact of two different types of diets, a crude diet and an AIN-93G-formula purified diet, on epithelial integrity and the gut microbiota. Purified diet-fed mice exhibited shorter villi and crypt lengths and slower epithelial turnover, particularly in the ileum. In addition, antimicrobial products, including REG3γ, were substantially decreased in purified diet-fed mice. Purified diet feeding also suppressed α1,2-fucosylation on the epithelial surface. Furthermore, the purified diet induced metabolic rewiring to fatty acid oxidation and ketogenesis. 16S ribosomal RNA gene sequencing of the ileal contents and mucus layer revealed distinct gut microbiota compositions between the purified and crude diet-fed mice. Purified diet feeding reduced the abundance of segmented filamentous bacteria (SFB), which potently upregulate REG3γ and fucosyltransferase 2 (Fut2) by stimulating group 3 innate lymphoid cells (ILC3s) to produce IL-22. These observations illustrate that the intake of a crude diet secures epithelial barrier function by facilitating SFB colonization, whereas a purified diet insufficiently establishes the epithelial barrier, at least partly owing to the loss of SFB. Our data suggest that the influence of purified diets on the epithelial barrier integrity should be considered in experiments using purified diets.

Gut microbiota-dependent adaptor molecule recruits DNA methyltransferase to the TLR4 gene in colonic epithelial cells to suppress inflammatory reactions.

The intestine is inhabited by a large number of commensal bacteria that are immunologically non-self, potentially causing inflammation. However, in a healthy intestine, inflammation is strictly controlled at low levels to maintain homeostasis. We previously reported that the gut microbiota induce DNA methylation of the gene encoding Toll-like receptor (TLR) 4, a pattern recognition receptor that recognizes lipopolysaccharides of gram-negative bacteria, in colonic epithelial cells, suggesting its role in controlling intestinal inflammation. However, there remains a question of how gut microbiota cause methylation of only specific genes including TLR4, despite the fact that DNA methyltransferase (DNMT) is common to all genes targeted for methylation. Here, we identified RBM14 as an adaptor molecule that recruits DNMT to the TLR4 gene. RBM14 was shown to bind DNMT3 and be expressed at significantly higher levels in an intestinal epithelial cell (IEC) line with hypermethylated TLR4 gene than in an IEC line with hypomethylated TLR4 gene. In addition, RBM14 interacted with DNA regions of the TLR4 gene, and knockdown of RBM14 suppressed DNA methylation of the TLR4 gene in IECs. Furthermore, RBM14 expression was higher in colonic epithelial cells of conventional mice than in those of germ-free mice. Collectively, these results indicate that the gut microbiota induce methylation of the TLR4 gene in colonic epithelial cells by upregulating RBM14, which can recruit DNMT3 to the gene. The regulation of adaptor molecules such as RBM14, which bind to specific target genes and recruit DNMT, can explain, at least in part, how gut microbiota contribute to the maintenance of intestinal homeostasis through epigenetic control of specific gene expression in IECs.

Extremely large electro-optic effect of TPPS J-aggregates in the PVA or PVP polymer matrix and aqueous solution.

The molecules of tetra-phenyl porphyrin tetra-sulfonic acid (TPPS) form a J-aggregate via self-organization in aqueous solution. The J-aggregates formed in an aqueous solution with added hydrochloric acid were dispersed in PVP polymer and subjected to electric field modulation spectroscopy. The observed difference in the static polarizability Δαp between the excited and ground states in the TPPS J-aggregates reached a value 2.66 × 104 times and 1.22 × 105 times larger than in the monomer molecules in PVA and PVP polymer matrices, respectively. This large enhancement factor of the electro-optic effect (EOE) may be induced by the higher-order structure of the J-aggregate. The different enhancement factor for each polymer matrix may be introduced by the structural differences between the polymer matrices. Electric field modulation spectroscopy of the J-aggregate in aqueous solution showed an absorbance change of 25% of the magnitude of absorbance peak.

[A Case of GIST of Stomach with Peritoneal Dissemination-Long-Term Survival with Imatinib and Surgical Resection].

A 63-year-old female patient underwent resection of a gastrointestinal stromal tumor(GIST)at the age of 48 years. After surgery, she had adjuvant chemotherapy. She had been recurrence-free for 10 years. Two years after completion of medical therapy, local peritoneal recurrence of GIST was observed, and medical therapy with imatinib was restarted. The response was good, but 1 year after resumption of medical therapy, progression was observed, and imatinib resistance was suspected, and recurrent tumor resection was performed. After the reoperation, the patient continued medical treatment with imatinib. Two years after the reoperation, a tumor suspected to be recurrent was found in the abdominal cavity. Tumor resection was performed. Histopathological examination revealed c-kit and CD34 positivity, leading to a diagnosis of recurrence of GIST. Imatinib is the mainstay of treatment in patients with recurrent GISTs, and sunitinib may be considered if the patient becomes resistant to imatinib, or surgical treatment may be considered if the lesion can be resected. In this study, we report a case of GIST with peritoneal dissemination in which imatinib therapy was continued after surgery, but the disease recurred twice. We investigate the prognostic value of continued imatinib therapy after surgical resection of locally recurrent GIST.

Loss of GSTO2 contributes to cell growth and mitochondria function via the p38 signaling in lung squamous cell carcinoma.

Glutathione S-transferase omega 2 (GSTO2) lacks any appreciable GST activity, but it exhibits thioltransferase activity. The significance of GSTO2 in lung function has been reported; however, the precise expression and molecular function of GSTO2 in the lungs remain unclear. In the present study, we found that GSTO2 is expressed in airway basal cells, non-ciliated, columnar Clara cells, and type II alveolar cells, which have self-renewal capacity in the lungs. Contrastingly, no GSTO2 expression was observed in 94 lung squamous cell carcinoma (LSCC) samples. When human LSCC cell lines were treated with 5-aza-2'-deoxycytidine, a DNA-methyltransferase inhibitor, GSTO2 transcription was induced, suggesting that aberrant GSTO2 hypermethylation in LSCC is the cause of its downregulation. Forced GSTO2 expression in LSCC cell lines inhibited cell growth and colony formation in vitro. In a subcutaneous xenograft model, GSTO2-transfected cells formed smaller tumors in nude mice than mock-transfected cells. Upon intravenous injection into nude mice, the incidence of liver metastasis was lower in mice injected with GSTO2-transfected cells than in those injected with mock-transfected cells. In addition, GSTO2 induction suppressed the expression of ß-catenin and the oxygen consumption rate, but it did not affect the extracellular acidification rate. Furthermore, GSTO2-transfected cells displayed lower mitochondrial membrane potential than mock-transfected cells. When GSTO2-transfected cells were treated with a p38 inhibitor, ß-catenin expression and mitochondrial membrane potential were recovered. Our study indicated that the loss of GSTO2 via DNA hypermethylation contributes to the growth and progression of LSCC, probably by modulating cancer metabolism via the p38/ß-catenin signaling pathway.

Label-Free Imaging of Melanoma with Confocal Photothermal Microscopy: Differentiation between Malignant and Benign Tissue.

Label-free confocal photothermal (CPT) microscopy was utilized for the first time to investigate malignancy in mouse skin cells. Laser diodes (LDs) with 405 nm or 488 nm wavelengths were used as pumps, and a 638 nm LD was used as a probe for the CPT microscope. A Grey Level Cooccurrence Matrix (GLCM) for texture analysis was applied to the CPT images. Nine GLCM parameters were calculated with definite definitions for the intracellular super-resolved CPT images, and the parameters Entropy, Contrast, and Variance were found to be most suited among the nine parameters to discriminate clearly between healthy cells and malignant cells when a 405 nm pump was used. Prominence, Variance, and Shade were most suited when a pump wavelength of 488 nm was used.

Commensal microbiota-induced microRNA modulates intestinal epithelial permeability through the small GTPase ARF4.

The intestinal tract contains many commensal bacteria that modulate various physiological host functions. Dysbiosis of commensal bacteria triggers dysfunction of the intestinal epithelial barrier, leading to the induction or aggravation of intestinal inflammation. To elucidate whether microRNA plays a role in commensal microbiome-dependent intestinal epithelial barrier regulation, we compared transcripts in intestinal epithelial cells (IECs) from conventional and germ-free mice and found that commensal bacteria induced the expression of miR-21-5p in IECs. miR-21-5p increased intestinal epithelial permeability and up-regulated ADP ribosylation factor 4 (ARF4), a small GTPase, in the IEC line Caco-2. We also found that ARF4 expression was up-regulated upon suppression of phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4), which are known miR-21-5p targets, by RNAi. Furthermore, ARF4 expression in epithelial cells of the large intestine was higher in conventional mice than in germ-free mice. ARF4 suppression in the IEC line increased the expression of tight junction proteins and decreased intestinal epithelial permeability. These results indicate that commensal microbiome-dependent miR-21-5p expression in IECs regulates intestinal epithelial permeability via ARF4, which may therefore represent a target for preventing or managing dysfunction of the intestinal epithelial barrier.

Gray-level co-occurrence matrix analysis of several cell types in mouse brain using resolution-enhanced photothermal microscopy.

Qualifications of intracellular structure were performed for the first time using the gray-level co-occurrence matrix (GLCM) method for images of cells obtained by resolution-enhanced photothermal imaging. The GLCM method has been used to extract five parameters of texture features for five different types of cells in mouse brain; pyramidal neurons and glial cells in the basal nucleus (BGl), dentate gyrus granule cells, cerebellar Purkinje cells, and cerebellar granule cells. The parameters are correlation, contrast, angular second moment (ASM), inverse difference moment (IDM), and entropy for the images of cells of interest in a mouse brain. The parameters vary depending on the pixel distance taken in the analysis method. Based on the obtained results, we identified that the most suitable GLCM parameter is IDM for pyramidal neurons and BGI, granule cells in the dentate gyrus, Purkinje cells and granule cells in the cerebellum. It was also found that the ASM is the most appropriate for neurons in the basal nucleus.

α-Defensin 5 gene expression is regulated by gut microbial metabolites.

α-Defensin 5 is important to both maintenance of a gut microbiota and host immunity. While previous reports have shown that gut bacteria are able to upregulate α-defensin 5 through Toll-like receptor signaling, we demonstrate here that α-defensin 5 expression can also be regulated by microbial metabolites. Among these, lactate appeared to significantly suppress α-defensin 5 gene transcription. Actually, fractions of <3 kD compounds obtained from the ceca of SPF mice were suppressed α-defensin 5 gene transcription at specific concentrations. Our results also suggest that cecal content may include as yet unidentified factors that can enhance α-defensin 5 expression. Our data point to a novel function for the gut microbial metabolites in controlling the expression of antimicrobial peptides in the intestine.

Post-Transcriptional Regulation of Toll-Interacting Protein in the Intestinal Epithelium.

Immune responses against gut microbiota should be minimized to avoid unnecessary inflammation at mucosal surface. In this study, we analyzed the expression patterns of Toll-interacting protein (Tollip), an inhibitor of TLRs and IL-1 family cytokine-related intracellular signaling, in intestinal epithelial cells (IECs). Comparable mRNA expression was observed in murine small and large IECs (S-IECs and L-IECs). However, Tollip protein was only detected in L-IECs, but not in S-IECs. Similar results were obtained in germ-free mice, indicating that L-IEC-specific TOLLIP expression does not depend on bacterial colonization. Next, to understand the mechanisms underlying the post-transcriptional repression of Tollip, 3´-UTR-mediated translational regulation was evaluated. The region +1876/+2398 was responsible for the repression of Tollip expression. This region included the target sequence of miR-31. The inhibition of miR-31 restored the 3´-UTR-meditaed translational repression. In addition, miR-31 expression was significantly higher in S-IECs than in L-IECs, suggesting that miR-31 represses the translation of Tollip mRNA in S-IECs. Collectively, we conclude that the translation of Tollip is inhibited in S-IECs, at least in part, by miR-31 to yield L-IEC-specific high-level expression of the Tollip protein, which may contribute to the maintenance of intestinal homeostasis.

Scan-Free Absorbance Spectral Imaging A(x, y, λ) of Single Live Algal Cells for Quantifying Absorbance of Cell Suspensions.

Label-free, non-invasive, rapid absorbance spectral imaging A(x,y,λ) microscopy of single live cells at 1.2 µm × 1.2 µm resolution with an NA = 0.85 objective was developed and applied to unicellular green algae Chlamydomonas reinhardtii. By introducing the fiber assembly to rearrange a two-dimensional image to the one-dimensional array to fit the slit of an imaging spectrograph equipped with a CCD detector, scan-free acquisition of three-dimensional information of A(x,y,λ) was realized. The space-resolved absorbance spectra of the eyespot, an orange organelle about 1 µm, were extracted from the green-color background in a chlorophyll-rich single live cell absorbance image. Characteristic absorbance change in the cell suspension after hydrogen photoproduction in C. reinhardtii was investigated to find a single 715-nm absorption peak was locally distributed within single cells. The formula to calculate the absorbance of cell suspensions from that of single cells was presented to obtain a quantitative, parameter-free agreement with the experiment. It is quantitatively shown that the average number of chlorophylls per cell is significantly underestimated when it is evaluated from the absorbance of the cell suspensions due to the package effect.

Vibrational energy flow between modes by dynamic mode coupling in THIATS J-aggregates.

We performed ultrafast pump-probe spectroscopy of J-aggregates of 3,3'-disulfopropyl-5,5'-dichloro-9-ethyl thiacarbocyanine triethylammonium (THIATS), one of the most typical cyanine dyes, and detected excited molecular vibrations, using a sub-10 fs pulse laser. The time-resolved two-dimensional difference absorption (ΔA) spectra are observed between -314 and 1267 fs. By performing the Fourier transform and spectrogram analysis, vibrational modes in THIATS are observed at 285, 485, 555, 824, and 1633 cm(-1) and there was a modulation of the vibrational frequencies around 1633 cm(-1) which depend on the delay time, respectively. By the analysis of the modulation, energy flow is found to take place from other modes to the 1633 cm(-1) mode through the low frequency mode with ∼50 cm(-1). Also, by fitting the real-time traces of ΔA with the sum of two exponential functions and a constant term, the average lifetimes of three electronically excited states were found to be τ1 = 52 ± 5 fs and τ2 = 540 ± 78 fs. By performing single-exponential fitting around the stationary absorption peak at 1.990 eV, in the negative time range, the electronic dephasing time, T2(ele), is determined to be 18.30 fs.

Spectral modulation observed in Chl-a by ultrafast laser spectroscopy.

Broadband real-time dynamic vibronic coupling in Chl-a were experimentally studied using few cycle laser pulses of 6.8fs duration and a 128-channnel lock-in amplifier. Thanks to the extreme temporal resolution benefitting from the ultrashort laser pulse, the real-time modulation of the electronic transition energy induced by the molecular vibrations were calculated by the time dependent first moments of the bleaching band. The transition energy was found to be modulated periodically with the same frequencies of molecular vibration found in the Fourier amplitude spectrum of the difference absorbance real-time traces. This was interpreted to be due to the difference in the effective transition energy associated with the wavepacket motion induced by the equilibrium positions of potential curves between the ground state and the excited state. Using the values, Huang-Rhys factors for several vibrational modes involved in the spectral modulation at the room-temperature have been determined.

Electric field-controlled dissociation and association of porphyrin J-aggregates in aqueous solution.

The electrooptic effects of porphyrin J-aggregates of tetraphenyl porphyrin tetrasulfonic acid (TPPS) in aqueous solution were studied using electroabsorption (EA) spectroscopy. When the J-aggregates were three-dimensionally distributed, the EA spectra exhibited broadening in the exciton band. When a DC or AC electric field was applied for a long time, the J-aggregates with KCl were dissociated into monomers via N-mers (N = 2-4) as intermediate states, while those without KCl had an increase in aggregation. The EA spectra showed a red shift in the exciton band for N-mers, which indicates that N-mers are isolated microaggregates with a coherent aggregation number N, and isolated microaggregates have not been microscopically or spectrally observed until now. The estimated third-order nonlinear optical susceptibility χ((3)) for EA spectra in aqueous solution was 10(4) times larger than that in a polymer film. The molecular rearrangement model was applied to a variety of orientational distributions and the results were explained fairly well. The contribution of the electric double layer is the most probable reason for the large enhancement of χ((3)) for the solution sample. The dynamic equilibrium between two types of monomers, J-aggregates of various aggregation numbers and cations such as K(+) and H(+) was investigated to reveal that K(+) is more loosely bound to the constituent monomers in J-aggregates than H(+). Equilibrium equations also show that well-grown aggregates with N > 15 tend to dominate in a solution of J-aggregates, which explains why only well-developed aggregates can be observed spectroscopically.

Real-time vibrational dynamics in chlorophyll a studied with a few-cycle pulse laser.

We use a 6.8-fs laser as the light source for broad-band femtosecond pump-probe real-time vibrational spectroscopy to investigate both electronic relaxation and vibrational dynamics of the Q(y)-band of Chl-a at 293 K. More than 25 vibrational modes coupled to the Q(y) transition are observed. Eleven of them have been clarified predominantly due to the excited state, and six of them are concluded to be nearly exclusively resulting from the ground-state wave-packet motion. Moreover, thanks to the broad-band detection over 5000 cm⁻¹, the modulated signals due to the excited state vibrational coherence are observed on both sides of the 0-0 transition with equal separation. The corresponding nonlinear process has been studied using a three-level model, from which the probe wavelength dependence of the phase of the periodic modulation can be calculated. The probe wavelength dependence of the vibrational amplitude is interpreted in terms of the interaction between the "pump" or "laser," Stokes, and anti-Stokes field intermediated by the molecular vibrations. In addition, an excited state absorption peak at ~709 nm has been observed. To the best of our knowledge, this is the first study of broad-band real-time vibrational spectroscopy in Chl-a.