Synthesis of causal and surrogate models by non-equilibrium thermodynamics in biological systems.

We developed a model to represent the time evolution phenomena of life through physics constraints. To do this, we took into account that living organisms are open systems that exchange messages through intracellular communication, intercellular communication and sensory systems, and introduced the concept of a message force field. As a result, we showed that the maximum entropy generation principle is valid in time evolution. Then, in order to explain life phenomena based on this principle, we modelled the living system as a nonlinear oscillator coupled by a message and derived the governing equations. The governing equations consist of two laws: one states that the systems are synchronized when the variation of the natural frequencies between them is small or the coupling strength through the message is sufficiently large, and the other states that the synchronization is broken by the proliferation of biological systems. Next, to simulate the phenomena using data obtained from observations of the temporal evolution of life, we developed an inference model that combines physics constraints and a discrete surrogate model using category theory, and simulated the phenomenon of early embryogenesis using this inference model. The results show that symmetry creation and breaking based on message force fields can be widely used to model life phenomena.

Best practices for multimodal clinical data management and integration: An atopic dermatitis research case.

BACKGROUND: In clinical research on multifactorial diseases such as atopic dermatitis, data-driven medical research has become more widely used as means to clarify diverse pathological conditions and to realize precision medicine. However, modern clinical data, characterized as large-scale, multimodal, and multi-center, causes difficulties in data integration and management, which limits productivity in clinical data science. METHODS: We designed a generic data management flow to collect, cleanse, and integrate data to handle different types of data generated at multiple institutions by 10 types of clinical studies. We developed MeDIA (Medical Data Integration Assistant), a software to browse the data in an integrated manner and extract subsets for analysis. RESULTS: MeDIA integrates and visualizes data and information on research participants obtained from multiple studies. It then provides a sophisticated interface that supports data management and helps data scientists retrieve the data sets they need. Furthermore, the system promotes the use of unified terms such as identifiers or sampling dates to reduce the cost of pre-processing by data analysts. We also propose best practices in clinical data management flow, which we learned from the development and implementation of MeDIA. CONCLUSIONS: The MeDIA system solves the problem of multimodal clinical data integration, from complex text data such as medical records to big data such as omics data from a large number of patients. The system and the proposed best practices can be applied not only to allergic diseases but also to other diseases to promote data-driven medical research.

Multifaceted analysis of cross-tissue transcriptomes reveals phenotype-endotype associations in atopic dermatitis.

Atopic dermatitis (AD) is a skin disease that is heterogeneous both in terms of clinical manifestations and molecular profiles. It is increasingly recognized that AD is a systemic rather than a local disease and should be assessed in the context of whole-body pathophysiology. Here we show, via integrated RNA-sequencing of skin tissue and peripheral blood mononuclear cell (PBMC) samples along with clinical data from 115 AD patients and 14 matched healthy controls, that specific clinical presentations associate with matching differential molecular signatures. We establish a regression model based on transcriptome modules identified in weighted gene co-expression network analysis to extract molecular features associated with detailed clinical phenotypes of AD. The two main, qualitatively differential skin manifestations of AD, erythema and papulation are distinguished by differential immunological signatures. We further apply the regression model to a longitudinal dataset of 30 AD patients for personalized monitoring, highlighting patient heterogeneity in disease trajectories. The longitudinal features of blood tests and PBMC transcriptome modules identify three patient clusters which are aligned with clinical severity and reflect treatment history. Our approach thus serves as a framework for effective clinical investigation to gain a holistic view on the pathophysiology of complex human diseases.

Combined plasma metabolomic and transcriptomic analysis identify histidine as a biomarker and potential contributor in SLE pathogenesis.

OBJECTIVES: To investigate metabolite alterations in the plasma of SLE patients to identify novel biomarkers and provide insight into SLE pathogenesis. METHODS: Patients with SLE (n = 41, discovery cohort and n = 37, replication cohort), healthy controls (n = 30 and n = 29) and patients with RA (n = 19, disease control) were recruited. Metabolic profiles of the plasma samples were analysed using liquid chromatography-time-of-flight mass spectrometry and capillary electrophoresis-time-of-flight mass spectrometry. Transcriptome data was analysed using RNA-sequencing for 18 immune cell subsets. The importance of histidine (His) in plasmablast differentiation was investigated by using mouse splenic B cells. RESULTS: We demonstrate that a specific amino acid combination including His can effectively distinguish between SLE patients and healthy controls. Random forest and partial least squares-discriminant analysis identified His as an effective classifier for SLE patients. A decrease in His plasma levels correlated with damage accrual independent of prednisolone dosage and type I IFN signature. The oxidative phosphorylation signature in plasmablasts negatively correlated with His levels. We also showed that plasmablast differentiation induced by innate immune signals was dependent on His. CONCLUSIONS: Plasma His levels are a potential biomarker for SLE patients and are associated with damage accrual. Our data suggest the importance of His as a pathogenic metabolite in SLE pathogenesis.

Development and validation of a deep-learning model for scoring of radiographic finger joint destruction in rheumatoid arthritis.

OBJECTIVE: The purpose of this research was to develop a deep-learning model to assess radiographic finger joint destruction in RA. METHODS: The model comprises two steps: a joint-detection step and a joint-evaluation step. Among 216 radiographs of 108 patients with RA, 186 radiographs were assigned to the training/validation dataset and 30 to the test dataset. In the training/validation dataset, images of PIP joints, the IP joint of the thumb or MCP joints were manually clipped and scored for joint space narrowing (JSN) and bone erosion by clinicians, and then these images were augmented. As a result, 11 160 images were used to train and validate a deep convolutional neural network for joint evaluation. Three thousand seven hundred and twenty selected images were used to train machine learning for joint detection. These steps were combined as the assessment model for radiographic finger joint destruction. Performance of the model was examined using the test dataset, which was not included in the training/validation process, by comparing the scores assigned by the model and clinicians. RESULTS: The model detected PIP joints, the IP joint of the thumb and MCP joints with a sensitivity of 95.3% and assigned scores for JSN and erosion. Accuracy (percentage of exact agreement) reached 49.3-65.4% for JSN and 70.6-74.1% for erosion. The correlation coefficient between scores by the model and clinicians per image was 0.72-0.88 for JSN and 0.54-0.75 for erosion. CONCLUSION: Image processing with the trained convolutional neural network model is promising to assess radiographs in RA.

Environmental epigenetic modifications and reprogramming-recalcitrant genes.

The term "environmental epigenetic modifications" refers to alterations in phenotype triggered by environmental stimuli via epigenetic mechanisms. Epidemiologic and animal model studies show that a subset of such environmental epigenetic marks may affect susceptibility to chronic diseases. A growing body of evidence regarding incompleteness of reprogramming indicates that the potential retention of pathogenic environmental epigenetics in human induced pluripotent stem cells (iPSCs) should be seriously considered. Given this possibility, the optimization of methods for the generation of human induc pluripotent stem cells may require the identification of epigenetically appropriate somatic cell sources. Similarly, techniques for controlling epigenetic modification by environmental factors may also play a critical role in the development of epigenetically stable sources of pluripotent stem cells.

Identification of stem cells during prepubertal spermatogenesis via monitoring of nucleostemin promoter activity.

The nucleostemin (NS) gene encodes a nucleolar protein found at high levels in several types of stem cells and tumor cell lines. The function of NS is unclear but it may play a critical role in S-phase entry by stem/progenitor cells. Here we characterize NS expression in murine male germ cells. Although NS protein was highly expressed in the nucleoli of all primordial germ cells, only a limited number of gonocytes showed NS expression in neonatal testes. In adult testes, NS protein was expressed at high levels in the nucleoli of spermatogonia and primary spermatocytes but at only low levels in round spermatids. To evaluate the properties of cells expressing high levels of NS, we generated transgenic reporter mice expressing green fluorescent protein (GFP) under the control of the NS promoter (NS-GFP Tg mice). In adult NS-GFP Tg testes, GFP and endogenous NS protein expression were correlated in spermatogonia and spermatocytes but GFP was also ectopically expressed in elongated spermatids and sperm. In testes of NS-GFP Tg embryos, neonates, and 10-day-old pups, however, GFP expression closely coincided with endogenous NS expression in developing germ cells. In contrast to a previous report, our results support the existence in neonatal testes of spermatogonial stem cells with long-term repopulating capacity. Furthermore, our data show that NS expression does not correlate with cell-cycle status during prepuberty, and that strong NS expression is essential for the maintenance of germline stem cell proliferation capacity. We conclude that NS is a marker of undifferentiated status in the germ cell lineage during prepubertal spermatogenesis.

Regenerative medicine and stem cell based drug discovery.

As William Shakespeare beautifully described, increasing age often causes loss of tissue and organ function. The increase in average life expectancy in many countries is generating an aging society and an increase in age-related health problems. Regenerative medicine is expected to be a powerful actor in this drama, and stem cell technology may hold the key to the development of innovative treatments for acute and chronic degenerative conditions. This Review surveys the present situation and some future prospects for regenerative medicine and stem cell based drug discovery.

Activation of adenosine A1 receptor-induced neural stem cell proliferation via MEK/ERK and Akt signaling pathways.

Adenosine, a modulator of neuronal function in the mammalian central nervous system, exerts a neuroprotective effect via the adenosine A(1) receptor; however, its effect on neural stem cells (NSCs) remains unclear. Because adenosine is released in response to pathological conditions and NSCs play a key role in neuroregeneration, we tested the hypothesis that adenosine is capable of stimulating NSC proliferation. We demonstrated that NSCs dominantly express adenosine A(1) and A(2B) receptors. Adenosine and the adenosine A(1) receptor agonist cyclopentyladenosine (CPA) increased proliferation of NSCs, and this CPA-induced cell proliferation was attenuated by the A(1) antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPA). CPA also induced phosphorylation of extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase/ERK kinase (MEK), and Akt, and their phosphorylation was inhibited by DPCPA. In addition, CPA-induced cell proliferation was inhibited by MEK and Akt inhibitors. These results suggest that activation of adenosine A(1) receptor-stimulated proliferation of NSCs occurs via MEK/ERK and Akt signaling pathways.

Identification of NR5A1 (SF-1/AD4BP) gene expression modulators by large-scale gain and loss of function studies.

Nuclear receptor subfamily 5, group A, member 1 (NR5A1 previously known as SF-1/AD4BP) is a transcription factor involved in the development of adrenal/gonadal tissues and steroidogenic lineage cell differentiation in adult somatic stem cells. To understand the cellular signaling network that regulates NR5A1 gene expression, loss of function screening with an siRNA kinome library, and gain of function screening with an addressable full-length cDNA library representing one quarter of the human genome was carried out. The NR5A1 gene expression was activated in mesenchymal stem cells by siRNA directed against protein kinase C (PKC)-delta, erb-B3, RhoGAP (ARHGAP26), and hexokinase 2, none of which were previously known to be involved in the NR5A1 gene expression. Among these, we identified crosstalk between erb-B3 and PKC-delta signaling cascades. In addition, the gain of function studies indicated that sex-determining region Y (SRY)-box 15 (SOX15), TEA domain family member 4, KIAA1257 (a gene of unknown function), ADAM metallopeptidase with thrombospondin type 1 motif 6, Josephin domain containing 1, centromere protein, TATA box-binding protein-associated factor 5-like RNA polymerase, and inducible T-cell co-stimulator activate NR5A1 gene expression. These results provide new insights into the molecular mechanisms of NR5A1 gene expression.

Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells.

Bone marrow-derived stromal cells can give rise to cardiomyocytes as well as adipocytes, osteocytes, and chondrocytes in vitro. The existence of mesenchymal stem cells has been proposed, but it remains unclear if a single-cell-derived stem cell stochastically commits toward a cardiac lineage. By single-cell marking, we performed a follow-up study of individual cells during the differentiation of 9-15c mesenchymal stromal cells derived from bone marrow cells. Three types of cells, i.e., cardiac myoblasts, cardiac progenitors and multipotent stem cells were differentiated from a single cell, implying that cardiomyocytes are generated stochastically from a single-cell-derived stem cell. We also demonstrated that overexpression of Csx/Nkx2.5 and GATA4, precardiac mesodermal transcription factors, enhanced cardiomyogenic differentiation of 9-15c cells, and the frequency of cardiomyogenic differentiation was increased by co-culturing with fetal cardiomyocytes. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 behaved like cardiac transient amplifying cells, and still retained their plasticity in vivo.

Heterogeneity of pluripotent marker gene expression in colonies generated in human iPS cell induction culture.

Induction of pluripotent stem cells from human fibroblasts has been achieved by the ectopic expression of two different sets of four genes. However, the mechanism of the pluripotent stem cell induction has not been elucidated. Here we identified a marked heterogeneity in colonies generated by the four-gene (Oct3/4, Sox2, c-Myc, and Klf4) transduction method in human neonatal skin-derived cells. The four-gene transduction gave a higher probability of induction for archetypal pluripotent stem cell marker genes (Nanog, TDGF, and Dnmt3b) than for marker genes that are less specific for pluripotent stem cells (CYP26A1 and TERT) in primary induction culture. This tendency may reflect the molecular mechanism underlying the induction of human skin-derived cells into pluripotent stem cells. Among the colonies induced by the four-gene transduction, small cells with a high nucleus-to-cytoplasm ratio could be established by repeated cloning. Subsequently established cell lines were similar to human embryonic stem cells as well as human induced pluripotent stem (iPS) cells derived from adult tissue in morphology, gene expression, long-term self-renewal ability, and teratoma formation. Genome-wide single-nucleotide polymorphism array analysis of the human iPS cell line indicates that the induction process did not induce DNA mutation.

Regulation of oxidative stress by ATM is required for self-renewal of haematopoietic stem cells.

The 'ataxia telangiectasia mutated' (Atm) gene maintains genomic stability by activating a key cell-cycle checkpoint in response to DNA damage, telomeric instability or oxidative stress. Mutational inactivation of the gene causes an autosomal recessive disorder, ataxia-telangiectasia, characterized by immunodeficiency, progressive cerebellar ataxia, oculocutaneous telangiectasia, defective spermatogenesis, premature ageing and a high incidence of lymphoma. Here we show that ATM has an essential function in the reconstitutive capacity of haematopoietic stem cells (HSCs) but is not as important for the proliferation or differentiation of progenitors, in a telomere-independent manner. Atm-/- mice older than 24 weeks showed progressive bone marrow failure resulting from a defect in HSC function that was associated with elevated reactive oxygen species. Treatment with anti-oxidative agents restored the reconstitutive capacity of Atm-/- HSCs, resulting in the prevention of bone marrow failure. Activation of the p16(INK4a)-retinoblastoma (Rb) gene product pathway in response to elevated reactive oxygen species led to the failure of Atm-/- HSCs. These results show that the self-renewal capacity of HSCs depends on ATM-mediated inhibition of oxidative stress.

Differential effects of DNA tumor virus genes on the expression profiles, differentiation, and morphogenetic reprogramming potential of epithelial cells.

The availability of cell lines that retain their differentiation programs is important for the study of differentiated cell types and the development of cell therapies. DNA tumor virus genes are often used to establish cell lines from primary culture for the analysis of cell-specific functions. To ascertain whether viral immortalizing or transforming genes differed in their effects on cellular differentiation programs, the E1A 12S (WT12S) gene of adenovirus and the large T antigen (LT) gene of SV40 were used to derive stable cell lines from primary kidney. The resultant cell types exhibited very different morphologies, growth and behavior patterns, differentiation states, and plasticities. Renal cells immortalized by LT exhibited branching tubulogenesis in response to Matrigel. This was in contrast to their behavior under normal culture conditions, wherein they were less differentiated, very nonadhesive, very rapidly growing, and transformed. These cells coexpressed adult epithelial (keratin) and embryonic mesenchymal (vimentin, osteopontin, FSP1, PAX-2, and WT1) genes. WT12S-immortalized cells grown on or in Matrigel formed cysts or tubules, consistent with their expression profiles, which consisted of both epithelial and adult kidney markers (E-cadherin, alpha-catenin, circumferential actin filaments (CAF), alkaline phosphatase, aminopeptidase M, BMP7, or podocalyxin), but not embryonic/mesenchymal markers (PAX-2 or WT1). The WT12S-expressing cells were well differentiated, adhesive, slow growing, and nontransformed. Thus, cells expressing WT12S maintained their original differentiation status and were less sensitive to reprogramming, while cells expressing LT were dedifferentiated, but had the potential for reprogramming by exogenous factors.

Large scale isolation of non-uniform shear stress-responsive genes from cultured human endothelial cells through the preparation of a subtracted cDNA library.

To investigate the molecular mechanisms responsible for the regional selectivity of early atherogenesis, we have applied a non-uniform shear stress to cultured human umbilical vein endothelial cells (HUVEC). We used a microcarrier culture system and a combination of subtraction and reverse-subtraction methods to isolate a number of genes upregulated by shear stress. The resultant subtracted library includes several known genes (e.g. MCP-1, TM) whose responsiveness to shear stress has been previously reported, indicating that the library is enriched for genes upregulated by shear stress. Also included are atherosclerosis-related genes (e.g. CTGF, IL-8) whose responsiveness to shear stress had not been demonstrated, other known genes whose relationship to atherosclerosis had not been reported, and novel genes. Some responsive to centrifugal force and shear stress (RECS) genes are also upregulated following stimulation by steady laminar shear stress in a parallel plate chamber. Interestingly, the library includes ET-1 and PAI, which are well known atherogenic factors that are downregulated by laminar shear stress. This implies that turbulent shear stress has effects on HUVEC that are different from those elicited by laminar shear stress. Importantly, analysis of specimens taken from human aorta showed that several RECS genes are transcriptionally upregulated in atherosclerotic lesions, suggesting that the subtracted library includes novel therapeutic targets for the treatment of atherosclerosis.