SFTPB in serum extracellular vesicles as a biomarker of progressive pulmonary fibrosis.

Progressive pulmonary fibrosis (PPF), defined as the worsening of various interstitial lung diseases (ILDs), currently lacks useful biomarkers. To identify novel biomarkers for early detection of patients at risk of PPF, we performed a proteomic analysis of serum extracellular vesicles (EVs). Notably, the identified candidate biomarkers were enriched for lung-derived proteins participating in fibrosis-related pathways. Among them, pulmonary surfactant-associated protein B (SFTPB) in serum EVs could predict ILD progression better than the known biomarkers, serum KL-6 and SP-D, and it was identified as an independent prognostic factor from ILD-gender-age-physiology index. Subsequently, the utility of SFTPB for predicting ILD progression was evaluated further in 2 cohorts using serum EVs and serum, respectively, suggesting that SFTPB in serum EVs but not in serum was helpful. Among SFTPB forms, pro-SFTPB levels were increased in both serum EVs and lungs of patients with PPF compared with those of the control. Consistently, in a mouse model, the levels of pro-SFTPB, primarily originating from alveolar epithelial type 2 cells, were increased similarly in serum EVs and lungs, reflecting pro-fibrotic changes in the lungs, as supported by single-cell RNA sequencing. SFTPB, especially its pro-form, in serum EVs could serve as a biomarker for predicting ILD progression.

Galectin-10 in serum extracellular vesicles reflects asthma pathophysiology.

BACKGROUND: Novel biomarkers (BMs) are urgently needed for bronchial asthma (BA) with various phenotypes and endotypes. OBJECTIVE: We sought to identify novel BMs reflecting tissue pathology from serum extracellular vesicles (EVs). METHODS: We performed data-independent acquisition of serum EVs from 4 healthy controls, 4 noneosinophilic asthma (NEA) patients, and 4 eosinophilic asthma (EA) patients to identify novel BMs for BA. We confirmed EA-specific BMs via data-independent acquisition validation in 61 BA patients and 23 controls. To further validate these findings, we performed data-independent acquisition for 6 patients with chronic rhinosinusitis without nasal polyps and 7 patients with chronic rhinosinusitis with nasal polyps. RESULTS: We identified 3032 proteins, 23 of which exhibited differential expression in EA. Ingenuity pathway analysis revealed that protein signatures from each phenotype reflected disease characteristics. Validation revealed 5 EA-specific BMs, including galectin-10 (Gal10), eosinophil peroxidase, major basic protein, eosinophil-derived neurotoxin, and arachidonate 15-lipoxygenase. The potential of Gal10 in EVs was superior to that of eosinophils in terms of diagnostic capability and detection of airway obstruction. In rhinosinusitis patients, 1752 and 8413 proteins were identified from EVs and tissues, respectively. Among 11 BMs identified in EVs and tissues from patients with chronic rhinosinusitis with nasal polyps, 5 (including Gal10 and eosinophil peroxidase) showed significant correlations between EVs and tissues. Gal10 release from EVs was implicated in eosinophil extracellular trapped cell death in vitro and in vivo. CONCLUSION: Novel BMs such as Gal10 from serum EVs reflect disease pathophysiology in BA and may represent a new target for liquid biopsy approaches.

Idiopathic pulmonary fibrosis-specific Bayesian network integrating extracellular vesicle proteome and clinical information.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease characterized by severe lung fibrosis and a poor prognosis. Although the biomolecules related to IPF have been extensively studied, molecular mechanisms of the pathogenesis and their association with serum biomarkers and clinical findings have not been fully elucidated. We constructed a Bayesian network using multimodal data consisting of a proteome dataset from serum extracellular vesicles, laboratory examinations, and clinical findings from 206 patients with IPF and 36 controls. Differential protein expression analysis was also performed by edgeR and incorporated into the constructed network. We have successfully visualized the relationship between biomolecules and clinical findings with this approach. The IPF-specific network included modules associated with TGF-ß signaling (TGFB1 and LRC32), fibrosis-related (A2MG and PZP), myofibroblast and inflammation (LRP1 and ITIH4), complement-related (SAA1 and SAA2), as well as serum markers, and clinical symptoms (KL-6, SP-D and fine crackles). Notably, it identified SAA2 associated with lymphocyte counts and PSPB connected with the serum markers KL-6 and SP-D, along with fine crackles as clinical manifestations. These results contribute to the elucidation of the pathogenesis of IPF and potential therapeutic targets.

Latent inter-organ mechanism of idiopathic pulmonary fibrosis unveiled by a generative computational approach.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease characterized by complex lung pathogenesis affecting approximately three million people worldwide. While the molecular and cellular details of the IPF mechanism is emerging, our current understanding is centered around the lung itself. On the other hand, many human diseases are the products of complex multi-organ interactions. Hence, we postulate that a dysfunctional crosstalk of the lung with other organs plays a causative role in the onset, progression and/or complications of IPF. In this study, we employed a generative computational approach to identify such inter-organ mechanism of IPF. This approach found unexpected molecular relatedness of IPF to neoplasm, diabetes, Alzheimer's disease, obesity, atherosclerosis, and arteriosclerosis. Furthermore, as a potential mechanism underlying this relatedness, we uncovered a putative molecular crosstalk system across the lung and the liver. In this inter-organ system, a secreted protein, kininogen 1, from hepatocytes in the liver interacts with its receptor, bradykinin receptor B1 in the lung. This ligand-receptor interaction across the liver and the lung leads to the activation of calmodulin pathways in the lung, leading to the activation of interleukin 6 and phosphoenolpyruvate carboxykinase 1 pathway across these organs. Importantly, we retrospectively identified several pre-clinical and clinical evidence supporting this inter-organ mechanism of IPF. In conclusion, such feedforward and feedback loop system across the lung and the liver provides a unique opportunity for the development of the treatment and/or diagnosis of IPF. Furthermore, the result illustrates a generative computational framework for machine-mediated synthesis of mechanisms that facilitates and complements the traditional experimental approaches in biomedical sciences.

A novel corpus of molecular to higher-order events that facilitates the understanding of the pathogenic mechanisms of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe and progressive chronic fibrosing interstitial lung disease with causes that have remained unclear to date. Development of effective treatments will require elucidation of the detailed pathogenetic mechanisms of IPF at both the molecular and cellular levels. With a biomedical corpus that includes IPF-related entities and events, text-mining systems can efficiently extract such mechanism-related information from huge amounts of literature on the disease. A novel corpus consisting of 150 abstracts with 9297 entities intended for training a text-mining system was constructed to clarify IPF-related pathogenetic mechanisms. For this corpus, entity information was annotated, as were relation and event information. To construct IPF-related networks, we also conducted entity normalization with IDs assigned to entities. Thereby, we extracted the same entities, which are expressed differently. Moreover, IPF-related events have been defined in this corpus, in contrast to existing corpora. This corpus will be useful to extract IPF-related information from scientific texts. Because many entities and events are related to lung diseases, this freely available corpus can also be used to extract information related to other lung diseases such as lung cancer and interstitial pneumonia caused by COVID-19.

Next-generation proteomics of serum extracellular vesicles combined with single-cell RNA sequencing identifies MACROH2A1 associated with refractory COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic is widespread; however, accurate predictors of refractory cases have not yet been established. Circulating extracellular vesicles, involved in many pathological processes, are ideal resources for biomarker exploration. METHODS: To identify potential serum biomarkers and examine the proteins associated with the pathogenesis of refractory COVID-19, we conducted high-coverage proteomics on serum extracellular vesicles collected from 12 patients with COVID-19 at different disease severity levels and 4 healthy controls. Furthermore, single-cell RNA sequencing of peripheral blood mononuclear cells collected from 10 patients with COVID-19 and 5 healthy controls was performed. RESULTS: Among the 3046 extracellular vesicle proteins that were identified, expression of MACROH2A1 was significantly elevated in refractory cases compared to non-refractory cases; moreover, its expression was increased according to disease severity. In single-cell RNA sequencing of peripheral blood mononuclear cells, the expression of MACROH2A1 was localized to monocytes and elevated in critical cases. Consistently, single-nucleus RNA sequencing of lung tissues revealed that MACROH2A1 was highly expressed in monocytes and macrophages and was significantly elevated in fatal COVID-19. Moreover, molecular network analysis showed that pathways such as "estrogen signaling pathway," "p160 steroid receptor coactivator (SRC) signaling pathway," and "transcriptional regulation by STAT" were enriched in the transcriptome of monocytes in the peripheral blood mononuclear cells and lungs, and they were also commonly enriched in extracellular vesicle proteomics. CONCLUSIONS: Our findings highlight that MACROH2A1 in extracellular vesicles is a potential biomarker of refractory COVID-19 and may reflect the pathogenesis of COVID-19 in monocytes.

Mouse tissue glycome atlas 2022 highlights inter-organ variation in major N-glycan profiles.

This study presents "mouse tissue glycome atlas" representing the profiles of major N-glycans of mouse glycoproteins that may define their essential functions in the surface glycocalyx of mouse organs/tissues and serum-derived extracellular vesicles (exosomes). Cell surface glycocalyx composed of a variety of N-glycans attached covalently to the membrane proteins, notably characteristic "N-glycosylation patterns" of the glycocalyx, plays a critical role for the regulation of cell differentiation, cell adhesion, homeostatic immune response, and biodistribution of secreted exosomes. Given that the integrity of cell surface glycocalyx correlates significantly with maintenance of the cellular morphology and homeostatic immune functions, dynamic alterations of N-glycosylation patterns in the normal glycocalyx caused by cellular abnormalities may serve as highly sensitive and promising biomarkers. Although it is believed that inter-organs variations in N-glycosylation patterns exist, information of the glycan diversity in mouse organs/tissues remains to be elusive. Here we communicate for the first-time N-glycosylation patterns of 16 mouse organs/tissues, serum, and serum-derived exosomes of Slc:ddY mice using an established solid-phase glycoblotting platform for the rapid, easy, and high throughput MALDI-TOFMS-based quantitative glycomics. The present results elicited occurrence of the organ/tissue-characteristic N-glycosylation patterns that can be discriminated to each other. Basic machine learning analysis using this N-glycome dataset enabled classification between 16 mouse organs/tissues with the highest F1 score (69.7-100%) when neural network algorithm was used. A preliminary examination demonstrated that machine learning analysis of mouse lung N-glycome dataset by random forest algorithm allows for the discrimination of lungs among the different mouse strains such as the outbred mouse Slc:ddY, inbred mouse DBA/2Crslc, and systemic lupus erythematosus model mouse MRL-lpr/lpr with the highest F1 score (74.5-83.8%). Our results strongly implicate importance of "human organ/tissue glycome atlas" for understanding the crucial and diversified roles of glycocalyx determined by the organ/tissue-characteristic N-glycosylation patterns and the discovery research for N-glycome-based disease-specific biomarkers and therapeutic targets.

Integrative Network Modeling Highlights the Crucial Roles of Rho-GDI Signaling Pathway in the Progression of non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most prevalent form of lung cancer and a leading cause of cancer-related deaths worldwide. Using an integrative approach, we analyzed a publicly available merged NSCLC transcriptome dataset using machine learning, protein-protein interaction (PPI) networks and bayesian modeling to pinpoint key cellular factors and pathways likely to be involved with the onset and progression of NSCLC. First, we generated multiple prediction models using various machine learning classifiers to classify NSCLC and healthy cohorts. Our models achieved prediction accuracies ranging from 0.83 to 1.0, with XGBoost emerging as the best performer. Next, using functional enrichment analysis (and gene co-expression network analysis with WGCNA) of the machine learning feature-selected genes, we determined that genes involved in Rho GTPase signaling that modulate actin stability and cytoskeleton were likely to be crucial in NSCLC. We further assembled a PPI network for the feature-selected genes that was partitioned using Markov clustering to detect protein complexes functionally relevant to NSCLC. Finally, we modeled the perturbations in RhoGDI signaling using a bayesian network; our simulations suggest that aberrations in ARHGEF19 and/or RAC2 gene activities contributed to impaired MAPK signaling and disrupted actin and cytoskeleton organization and were arguably key contributors to the onset of tumorigenesis in NSCLC. We hypothesize that targeted measures to restore aberrant ARHGEF19 and/or RAC2 functions could conceivably rescue the cancerous phenotype in NSCLC. Our findings offer promising avenues for early predictive biomarker discovery, targeted therapeutic intervention and improved clinical outcomes in NSCLC.

Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the ß-Subunit of Acid Ceramidase.

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the ß-subunit of aCDase, which consists of α- and ß-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the ß-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the ß-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading ß-subunit that evokes the ceramide deficiency in AD skin.

Metabolomic analysis of fibrotic mice combined with public RNA-Seq human lung data reveal potential diagnostic biomarker candidates for lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a severe lung disease with poor survival that warrants early and precise diagnosis for timely therapeutic intervention. Despite accumulating genomic, transcriptomic, proteomic, and lipidomic data on IPF, evidence from water-soluble metabolomics is limited. To identify biomarkers for IPF from water-soluble metabolomic data, we measured the levels of various metabolites in bronchoalveolar lavage fluid (BALF) and serum samples from a bleomycin-induced murine pulmonary fibrotic model using gas chromatography/mass spectrometry. Thirty-two of 73 BALF metabolites and 29 of 74 serum metabolites were annotated. We observed that the levels of proline and methionine were higher in BALF but lower in serum than those in the control. Furthermore, analysis of public RNA-Seq data from the lungs of patients with IPF revealed that proline- and methionine-related genes were significantly upregulated compared to those in the lungs of healthy controls. These results suggest that proline and methionine may be potential biomarkers for IPF and may help to deepen our understanding of the pathophysiology of the disease. Based on our results, we propose a model capable of recapitulating the proline and methionine metabolism of fibrotic lungs, thereby providing better means for studying the disease and developing novel therapeutic strategies for IPF.

Analysis of steryl glucosides in rice bran-based fermented food by LC/ESI-MS/MS.

Steryl glucosides (SGs) and acylated steryl glucosides (ASGs) are phytochemicals found in plant-based foods and are known as bioactive compounds with potential health benefits. These include anti-inflammatory properties, anti-diabetic effects, and modulation of immunoregulatory functions as well as having cholesterol lowering effects. In this study, three major SGs, i.e., glucosides of ß-sitosterol, stigmasterol, and campesterol, were synthesized and used as standards for measurement of their contents in rice bran (RB)-based fermented food (FBRA) utilizing Aspergillus oryzae and raw material (RM). The compounds were quantified using liquid chromatography/electrospray ionization-tandem mass spectrometry. It was found that ß-sitosteryl glucoside was most abundant among the analyzed glucosides in both samples, and the contents of each SG in FBRA decreased about 35% from those of RM. In contrast to SGs, the contents of ASGs in FBRA increased 1.5-fold during the fermentation process as evidenced by an alkaline hydrolysis. The present results suggest that the FBRA might have greater beneficial effects than the RM, since ASGs have shown to have more potent cholesterol lowering effects and stronger anti-diabetic properties than SGs.

Integrative Analysis Reveals Common and Unique Roles of Tetraspanins in Fibrosis and Emphysema.

While both chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are multifactorial disorders characterized by distinct clinical and pathological features, their commonalities and differences have not been fully elucidated. We sought to investigate the preventive roles of tetraspanins Cd151 and Cd9 -that are involved in diverse cellular processes in lung pathophysiology- in pulmonary fibrosis and emphysema, respectively, and to obtain a deeper understanding of their underlying molecular mechanisms toward facilitating improved therapeutic outcomes. Using an integrative approach, we examined the transcriptomic changes in the lungs of Cd151- and Cd9-deficient mice using functional-enrichment-analysis, pathway-perturbation-analysis and protein-protein-interaction (PPI) network analysis. Circadian-rhythm, extracellular-matrix (ECM), cell-adhesion and inflammatory responses and associated factors were prominently influenced by Cd151-deletion. Conversely, cellular-junctions, focal-adhesion, vascular-remodeling, and TNF-signaling were deeply impacted by Cd9-deletion. We also highlighted a "common core" of factors and signaling cascades that underlie the functions of both Cd151 and Cd9 in lung pathology. Circadian dysregulation following Cd151-deletion seemingly facilitated progressive fibrotic lung phenotype. Conversely, TGF-ß signaling attenuation and TNF-signaling activation emerged as potentially novel functionaries of Cd9-deletion-induced emphysema. Our findings offer promising avenues for developing novel therapeutic treatments for pulmonary fibrosis and emphysema.

The TargetMine Data Warehouse: Enhancement and Updates.

Biological data analysis is the key to new discoveries in disease biology and drug discovery. The rapid proliferation of high-throughput 'omics' data has necessitated a need for tools and platforms that allow the researchers to combine and analyse different types of biological data and obtain biologically relevant knowledge. We had previously developed TargetMine, an integrative data analysis platform for target prioritisation and broad-based biological knowledge discovery. Here, we describe the newly modelled biological data types and the enhanced visual and analytical features of TargetMine. These enhancements have included: an enhanced coverage of gene-gene relations, small molecule metabolite to pathway mappings, an improved literature survey feature, and in silico prediction of gene functional associations such as protein-protein interactions and global gene co-expression. We have also described two usage examples on trans-omics data analysis and extraction of gene-disease associations using MeSH term descriptors. These examples have demonstrated how the newer enhancements in TargetMine have contributed to a more expansive coverage of the biological data space and can help interpret genotype-phenotype relations. TargetMine with its auxiliary toolkit is available at https://targetmine.mizuguchilab.org. The TargetMine source code is available at https://github.com/chenyian-nibio/targetmine-gradle.

Changes in Polyamine Content in Rice Bran due to Fermentation with Aspergillus oryzae Analyzed by LC/ESI-MS/MS Combined with Derivatization.

Many studies have demonstrated that the dietary supplementation of polyamines, especially spermidine (SPD), prevents age-related diseases. Rice bran is rich in polyamines and their amounts could be increased by fermentation with Aspergillus oryzae (A. oryzae). In this study, we developed a method for the determination of putrescine (PUT), SPD and spermine (SPM) in rice bran samples by liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) after derivatization with 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F). The derivatization improved the LC retention and ESI-MS/MS detectability of the polyamines, and consequently enabled precise and accurate quantification. Using this method, we found that the SPD content increased to 158% due to fermentation with A. oryzae, while the content of PUT and SPM decreased. SPD is known as the polyamine playing a central role in cell proliferation and growth, and therefore has health benefits. The fermented rice bran might be a good material for functional foods aimed at SPD supplementation.

Identification of a neuronal population in the telencephalon essential for fear conditioning in zebrafish.

BACKGROUND: Fear conditioning is a form of learning essential for animal survival and used as a behavioral paradigm to study the mechanisms of learning and memory. In mammals, the amygdala plays a crucial role in fear conditioning. In teleost, the medial zone of the dorsal telencephalon (Dm) has been postulated to be a homolog of the mammalian amygdala by anatomical and ablation studies, showing a role in conditioned avoidance response. However, the neuronal populations required for a conditioned avoidance response via the Dm have not been functionally or genetically defined. RESULTS: We aimed to identify the neuronal population essential for fear conditioning through a genetic approach in zebrafish. First, we performed large-scale gene trap and enhancer trap screens, and created transgenic fish lines that expressed Gal4FF, an engineered version of the Gal4 transcription activator, in specific regions in the brain. We then crossed these Gal4FF-expressing fish with the effector line carrying the botulinum neurotoxin gene downstream of the Gal4 binding sequence UAS, and analyzed the double transgenic fish for active avoidance fear conditioning. We identified 16 transgenic lines with Gal4FF expression in various brain areas showing reduced performance in avoidance responses. Two of them had Gal4 expression in populations of neurons located in subregions of the Dm, which we named 120A-Dm neurons. Inhibition of the 120A-Dm neurons also caused reduced performance in Pavlovian fear conditioning. The 120A-Dm neurons were mostly glutamatergic and had projections to other brain regions, including the hypothalamus and ventral telencephalon. CONCLUSIONS: Herein, we identified a subpopulation of neurons in the zebrafish Dm essential for fear conditioning. We propose that these are functional equivalents of neurons in the mammalian pallial amygdala, mediating the conditioned stimulus-unconditioned stimulus association. Thus, the study establishes a basis for understanding the evolutionary conservation and diversification of functional neural circuits mediating fear conditioning in vertebrates.

Activation of the hypothalamic feeding centre upon visual prey detection.

The visual system plays a major role in food/prey recognition in diurnal animals, and food intake is regulated by the hypothalamus. However, whether and how visual information about prey is conveyed to the hypothalamic feeding centre is largely unknown. Here we perform real-time imaging of neuronal activity in freely behaving or constrained zebrafish larvae and demonstrate that prey or prey-like visual stimuli activate the hypothalamic feeding centre. Furthermore, we identify prey detector neurons in the pretectal area that project to the hypothalamic feeding centre. Ablation of the pretectum completely abolishes prey capture behaviour and neurotoxin expression in the hypothalamic area also reduces feeding. Taken together, these results suggest that the pretecto-hypothalamic pathway plays a crucial role in conveying visual information to the feeding centre. Thus, this pathway possibly converts visual food detection into feeding motivation in zebrafish.

Objective Predictive Score as a Feasible Biomarker for Short-term Survival in TerminalIy Ill Patients with Cancer.

BACKGROUND: In palliative care, prediction of life expectancy is one of the most crucial issues for patients, family and medical staff, in order to provide appropriate end-of-life care. The aim of this study was to formulate a new objective score to predict life expectancy within 1 week for terminally ill patients with cancer. PATIENTS AND METHODS: Medical records were obtained from 187 terminally-ill patients with cancer who were admitted for palliative care. The biomarkers for a potential 'Objective Predictive Score' were assessed. RESULTS: Profiling of blood parameters demonstrated that elevated levels of alanine aminotransferase (ALT), total bilirubin (T-bil), blood urea nitrogen (BUN), creatinine (Cr) and a decreased platelet count were significantly correlated with death within 1 week in a training cohort. Our formulated Objective Predictive Score was able to predict death within 1 week with high accuracy in a training and a validation cohort. CONCLUSION: Our scoring system might enable the assessment of prognostication with higher accuracy in a terminal care setting.

Fermented Brown Rice Extract Stimulates BDNF Gene Transcription in C6 Glioma Cells: Possible Connection with HO-1 Expression.

Fermented brown rice with Aspergillus oryzae, designated as FBRA, is known to be commercially available dietary fiber-rich food, which is appreciated as prebiotics to improve intestinal microflora, and also shown to contain various biologically active substances including polyphenolic compounds. On the other hand, polyphenolic compounds have been suggested to stimulate the expression of brain-derived neurotrophic factor (BDNF) gene in connection with the expression of heme oxidase-1 (HO-1) gene in glial cells, thus resulting in the augmentation of BDNF production in the brain, thereby being anticipated to have a putative effect on the brain function. Then, the effect of FBRA extract on HO-1 and BDNF messenger ribonucleic acid (mRNA) levels in C6 glioma cells was examined, and the extract was shown to stimulate both HO-1 and BDNF gene transcription in the glioma cells. Further studies showed that the stimulatory effect of FBRA extract on BDNF gene transcription was almost completely suppressed by silencing HO-1 gene expression with an HO-1 antisense oligodeoxynucleotide and also inhibiting HO-1 activity with an inhibitor zinc protoporphyrin, thus suggesting that FBRA might have a potential ability to induce BDNF gene expression through HO-1 activity in glial cells.

Fluorescence-Activated Cell Sorting and Gene Expression Profiling of GFP-Positive Cells from Transgenic Zebrafish Lines.

Gene expression profiling is a useful approach for deeper understanding of the specificity of cells, tissues, and organs in the transcriptional level. Recent development of high-throughput next-generation sequence (NGS) allows the RNA-seq method for this profiling. This method provides precise information of transcripts about the quantitation and the structure such as the splicing variants. In this chapter, we describe a method for gene expression profiling of GFP-positive cells from transgenic zebrafish by RNA-seq. We labeled specific cells in the brain with GFP by crossing a Gal4 driver line with the UAS:GFP line, isolated those cells by fluorescence-activated cell sorting (FACS), and analyzed by RNA-seq.

Fermented Brown Rice Extract Causes Apoptotic Death of Human Acute Lymphoblastic Leukemia Cells via Death Receptor Pathway.

Mixture of brown rice and rice bran fermented with Aspergillus oryzae, designated as FBRA, has been reported to reveal anti-carcinogenic and anti-inflammatory effects in rodents. Then, to test its potential anti-cancer activity, the aqueous extract was prepared from FBRA powder, and the effect of this extract on human acute lymphoblastic leukemia Jurkat cells was directly examined. The exposure to FBRA extract reduced the cell viability in a concentration- and time-dependent manner. The reduction of the cell viability was accompanied by the DNA fragmentation, and partially restored by treatment with pan-caspase inhibitor. Further studies showed that FBRA extract induced the cleavage of caspase-8, -9, and -3, and decreased Bcl-2 protein expression. Moreover, the expression of tBid, DR5, and Fas proteins was enhanced by FBRA extract, and the pretreatment with caspase-8 inhibitor, but not caspase-9 inhibitor, restored the reduction of the cell viability induced by FBRA extract. These findings suggested that FBRA extract could induce the apoptotic death of human acute lymphoblastic leukemia cells probably through mainly the death receptor-mediated pathway and supplementarily through the tBid-mediated mitochondrial pathway, proposing the possibility that FBRA was a potential functional food beneficial to patients with hematological cancer.