Introduction Research

I. Water Biology

Understanding complex biological phenomena as complex systems is considered essential for the advancement of 21st-century life sciences. One of the characteristics of complex systems is the formation of dynamic order in open systems. Schrödinger (Nobel laureate in Physics, 1933), in his contemplation of this seemingly contradictory biological feature, likened it to "organisms feeding on negative entropy" ("What Is Life?"). We hypothesize that in living organisms, water may compensate for entropy, enabling the formation of dynamic order. In other words, we propose that the role of water in living organisms is entropy management, and we aim to elucidate the role of water molecules in biological phenomena.

Our bodies are composed of approximately 70% water molecules, making the dynamics and dynamic structure of water molecules in living organisms the best reflection of "maintenance and transformation of biological systems." Therefore, water can be considered the most excellent biomarker. In the medical field, for instance, MRI (magnetic resonance imaging) enables the diagnosis of conditions like cancer and inflammation by capturing changes in local dynamic water structures. We propose the concept of "Water Biology" and advance research based on the following three pillars to comprehensively understand complex biological phenomena from the perspective of water molecule dynamics:

"Three Pillars of Water Biology Research"

II. Visualize Water (Application of Nonlinear Optics)

~ Toward a New Frontier in Epithelial Transport Science ~

Living organisms interact with the external environment through epithelial tissues, facilitating the exchange of gases such as oxygen and carbon dioxide, water, ions, nutrients, and waste products. Epithelial tissues not only distinguish the individual organism from its surroundings but also play a crucial role in essential substance transport for maintaining the organism's life. Consequently, disruptions in these membrane transport functions are known to cause numerous significant diseases due to their vital importance.

To date, research on epithelial transport phenomena has been extensive and broad, driven by their medical and physiological significance. However, these studies have primarily focused on "solute" transporters such as ion channels and transporters, while the transport of "solvent," namely water, has remained largely unexplored due to the absence of direct observation methods for water movement.

Currently, our laboratory is addressing this challenge by applying microscopy techniques that enable the direct observation of water molecules without labeling. Furthermore, through the reconstruction (simulation) of temporal changes in observed images, we can quantitatively analyze the spatiotemporal aspects of water and lipid transport. This approach can be applied not only to isolated cells and epithelial tissues but also to tissue-engineered artificial tissues, offering the potential for gaining new insights into water transport and metabolism.

Additionally, in our department, we are conducting research on single-molecule-level structural-functional correlations, exploring unknown functions, and elucidating the roles of transporter molecule interactions in membrane transport. Ultimately, our goal is to achieve an integrated understanding of the water and solute transport system as a complex system, from the molecular level to tissue and organ levels, and to apply this knowledge to the medical field, including the elucidation of diseases, pathophysiology, and the development of therapeutic agents.

Research Highlights:

(1) Epithelial Tissues:

Measurement of Trans-Epithelial Water Diffusion Using CARS Microscopy: We used CARS microscopy to measure cell membrane and trans-epithelial water permeability in a 3D cyst formation model of MDCK cells. CARS microscopy is a nonlinear optical observation technique that amplifies signals by inducing resonant scattering of light with the inherent vibration frequency of OH bonds in water molecules, allowing direct imaging of water molecules. By observing MDCK cysts under conditions where they do not generate a CARS signal, unlike heavy water D2O, we successfully achieved the direct observation of water's trans-epithelial diffusion phenomenon. We also introduced computer modeling for data analysis, deepening our understanding of water diffusion within or between cells.

(2) Extracellular Space in Brain Cells:

Measurement of Substance Diffusion Inside and Outside Cells Using Two-Photon Microscopy with Brain Slice Samples: We used two-photon microscopy to analyze the distribution and diffusion speed of a dye injected from above acute brain slices. By overlaying this data with cell morphology (SR101: an astrocyte marker), we revealed that we could obtain maps of dye distribution (extracellular space distribution) and diffusion time constants (substance diffusion speed in the extracellular space) with high spatial resolution, less than 1 micron. Furthermore, by introducing non-fluorescent caged dyes into astrocytes and tracking and quantifying the diffusion of released fluorescent dyes from specific locations, we demonstrated that substance diffusion in astrocytic foot processes (B) is significantly slower than in cell bodies or processes (A), indicating the presence of independent compartments within cells (see figure).

（２）脳細胞外空間：

２光子顕微鏡と脳スライス標本を用いた細胞内外における物質拡散の計測：

2光子顕微鏡を用いて、急性脳スライスの上から色素をパルス投与することで、その分布と拡散速度を各部位において解析している。これを細胞形態（SR101: アストロサイト・マーカー）と重ねる事により、アストロサイト周辺の各部位における細胞外環境を表す色素分布（細胞外空間の分布）と拡散時定数（細胞外空間の物質拡散速度）のマップが1ミクロンを切る高い空間分解能をもって得られることを明らかにした。一方、アストロサイトに無蛍光性ケージド色素を導入し、任意の部位において2光子アンケージングし、放出された蛍光色素のその部位からの拡散を追跡・定量化することで、アストロサイトの細胞体やプロセス（A）に比べて足突起（B）における物質拡散が非常に遅く、細胞内で独立したコンパートメントを作っていることが明らかにしてきた（図）。

Ⅲ. Understand Water (Near-Infrared Spectroscopy and Multivariate Analysis)

Analyzing water molecule dynamics within living organisms at nanoscale spatiotemporal resolutions presents an extreme challenge with current scientific technology. To deepen our understanding of water molecule dynamics in living organisms, we have introduced a technique called "Aqua-Photonics," which utilizes near-infrared water absorption spectra and multivariate analysis (developed by Professor Tsengova at Kobe University). We also aim to construct theoretical models of water molecule dynamics at the nanoscale through the application of molecular dynamics simulations and quantum chemistry calculations.

(1) Aqua-Photonics: Aqua-Photonics is a novel concept that comprehensively understands biological systems from the behavior of water molecules in aqueous solutions by leveraging the interaction between water and light, i.e., differences in water absorption spectrum patterns. Water molecules have three characteristic vibrations, which are known to be influenced by the hydrogen bonding state of water molecules. Therefore, understanding the distinctive water absorption bands (Water Matrix Coordinates, WAMACS) resulting from interactions with solute molecules (hydrogen bonds between solute molecules and water or hydrogen bonds between water molecules affected by solute) is the first step in Aqua-Photonics analysis. Previous research has revealed that specific physiological or pathological conditions are associated with unique combinations of these water absorption bands (Water Absorbance Patterns, WAPS). WAPS could potentially serve as biomarkers for health conditions or diseases. Aqua-Photonics is part of the "omics" research domain, which aims to comprehensively measure and analyze all molecular information in a biological system. By integrating this information with data from other "omics" research fields (genomics, proteomics, metabolomics, etc.), we aim to understand the entire biosystem. Using Aqua-Photonics, we have:

i) Analyzed dynamic changes in water structure in the presence of ions. ii) Discovered that the expression of aquaporins increases free water content both inside and outside cells. iii) Indicated the potential for predicting the ovulation day in healthy female volunteers using near-infrared spectra of urine, similar to female pandas (collaborative research with Professor Tsengova at Kobe University).

(2) Computer Simulations: Observing the dynamics of individual water molecules in practice is extremely challenging. However, the rise of supercomputers has made it possible to simulate water molecule dynamics at the nanoscale in both space and time. Therefore, in collaboration with Professor Yasuoka at Keio University, we aim to understand water molecule dynamics not only in aqueous solutions but also in the vicinity of cell membranes and even inside aquaporin pores using computational science. Specifically, we are advancing three projects:

Water Molecule Dynamics Inside Aquaporin Pores: With the proposed atomic models of aquaporins, we are analyzing in detail how water molecules pass through aquaporin pores.
Special Water Molecule Diffusion Near Cell Membranes: Using a system involving water molecules, ions, and cell membrane lipids, we are analyzing the dynamics of each molecule. Our results suggest that the interaction between cell membrane lipids and water molecules may create specific diffusion phenomena near cell membranes.
Interaction of Electrolytes and Biomolecules (e.g., Sugars, Amino Acids) with Water Molecules: Water-soluble small molecules exist surrounded by water molecules (hydration shell, bound water) in living organisms. Therefore, we are conducting detailed analysis of the interactions between important biomolecules like sugars and amino acids and water molecules in living systems.

（２）コンピューターシミュレーション
実際の水分子一つ一つの動態を観測することは、現状では極めて困難である。一方、スーパーコンピューターの台頭で、ナノスケールの時空間における水分子動態がシミュレーションできるようになった。そこで我々は、泰岡（慶応大理工学部）らと共同で、水溶液中のみならず、細胞膜近傍や更にはアクアポリンのポア内部における水分子動態を計算機科学で理解しようとしている。具体的には、以下３つのプロジェクトを推進している。
１．アクアポリンポア内の水分子動態：アクアポリンの原子モデルが提唱されたことで、水分子がいかにしてアクアポリンのポアを通過していくか、その様子を詳細に解析している。
２．細胞膜近傍の特殊な水分子拡散：我々は、水分子とイオンと細胞膜リン脂質を用いた系で、それぞれの分子動態を解析した。その結果、細胞膜リン脂質と水分子の相互作用が、細胞膜近傍における特殊な拡散現象を創出している可能性を示唆することが出来た。
３．電解質や生体低分子（糖質、アミノ酸など）と水分子の相互作用：水溶性小分子は、生体内において水分子に取り囲まれて存在している（水和殻、結合水）。そこで、生命現象に重要な糖質やアミノ酸と水分子の相互作用を詳細に解析していく。

Ⅳ. Manipulate Water (Aquaporin Research)

It is believed that life on Earth originated in the oceans and later evolved to colonize land. During this transition, the challenge of maintaining water and salt balance within the body became crucial. In the case of humans, the kidneys and skin play essential roles in water retention. With the discovery of water channels called "Aquaporins," our understanding of water regulation at the molecular level has deepened significantly.

Aquaporins were first discovered in 1992 by Professor Peter Agre at the Johns Hopkins University School of Medicine. They are selective channels that allow water to pass through membranes and were initially found in red blood cell membranes. Currently, 13 different types of Aquaporins have been identified in humans, and they are distributed throughout the body. Each Aquaporin exhibits unique tissue distribution and is associated with specific physiological significance. For example, AQP0 is associated with lens transparency, AQP2 with urine concentration, and AQP5 with saliva secretion, among others. Moreover, the connection between Aquaporins and human diseases is gradually becoming apparent. Diseases such as cataracts, diabetes insipidus, oral dryness syndrome, and dry skin have been linked to Aquaporin dysregulation. One of the most well-understood connections between Aquaporins and human diseases is in the case of AQP2, which plays a vital role in the regulation of the kidneys. Abnormalities in the AQP2 gene can lead to congenital diabetes insipidus. Recently, AQP4 has been identified as the antigen for autoantibodies in neuromyelitis optica (NMO), an autoimmune disease considered a subtype of multiple sclerosis. We are actively advancing Aquaporin research, with a focus on AQP4.

(1) AQP Structure-Function Correlations and Drug Discovery: The proposed atomic structure models of Aquaporins have enhanced our understanding of structure-function correlations. Recently, we discovered the involvement of zinc, copper, and the anesthetic propofol in the regulation of AQP4. We also utilize computer simulations in our research to facilitate efficient drug discovery efforts.

(2) AQP4 and Disease (NMO): To understand the pathophysiology of autoimmune diseases, particularly neuromyelitis optica (NMO), where AQP4 is the antigen, we conduct research on the development of disease models, diagnostic kits, and novel treatment strategies.

（２）AQP4と疾患（NMO）：AQP4を抗原とする自己免疫疾患、視神経脊髄炎（NMO）の病態生理を理解するため、疾患モデル動物の開発、診断キットの開発、および新規治療法の開発を目指した研究を行っている。

V. Research support

The biggest pleasure of research lies in the moments when, amidst the repeated process of hypothesizing and experimentation in the darkness, one suddenly has an epiphany, and the true meaning of the hypothesis becomes clear. Creating an intellectual space where such moments can be experienced is our goal, as it revitalizes research.

In our research laboratory, the members of the research support team engage with specialized researchers face to face, each contributing their unique qualities to advance cutting-edge research. We share research achievements and moments of inspiration. Additionally, we are exploring the use of web systems to facilitate smooth and efficient administrative processes.

We invite you to visit our research laboratory. Here, you can feel firsthand how our diverse team members embody an open, intellectual creative space, supporting advanced research.