Glucose stockpile in the intestinal apical brush border in C. elegans.

Revealing the mechanisms of glucose transport is crucial for studying pathological diseases caused by glucose toxicities. Numerous studies have revealed molecular functions involved in glucose transport in the nematode Caenorhabditis elegans, a commonly used model organism. However, the behavior of glucose in the intestinal lumen-to-cell remains elusive. To address that, we evaluated the diffusion coefficient of glucose in the intestinal apical brush border of C. elegans by using fluorescent glucose and fluorescence recovery after photobleaching. Fluorescent glucose taken in the intestine of worms accumulates in the apical brush border, and its diffusion coefficient of ∼10-8 cm2/s is two orders of magnitude slower than that in bulk. This result indicates that the intestinal brush border is a viscous layer. ERM-1 point mutations at the phosphorylation site, which shorten the microvilli length, did not significantly affect the diffusion coefficient of fluorescent glucose in the brush border. Our findings imply that glucose enrichment is dominantly maintained by the viscous layer composed of the glycocalyx and molecular complexes on the apical surface.

Out of the ancient lake: Multiple riverine colonizations and diversification of the freshwater snails in the genus Semisulcospira around Lake Biwa.

Ancient lakes are a hotspot of biodiversity. Freshwater species often experience spectacular species radiation after colonizing lakes from riverine habitats. Therefore, the relationship between the fauna of the ancient lakes and the surrounding riverine system has a special significance in understanding their origin and evolutionary history. The study of ancient lake species often focused on the lake colonization of riverine species. In contrast, far less attention has been placed on the reverse direction: the riverine colonization of the lake species, despite its importance in disentangling their complex evolutionary history. The freshwater snails in the genus Semisulcospira involve endemic groups that radiated in the ancient Lake Biwa. Using genetics and fossil records, we inferred that the ancestors of these lake-endemic Semisulcospira snails historically colonized the riverine habitats at least three times during the Middle Pleistocene. Each colonization resulted in the formation of a new lineage that was genetically and morphologically distinct from other lineages. Further, one of these colonizations was followed by hybridization with a cosmopolitan riverine species, which potentially facilitated the population persistence of the colonizers in the new environment. Despite their complex histories, all these colonizers were currently grouped within a single species, Semisulcospira kurodai, suggesting cryptic diversity in this species. This study highlights the significance of the riverine colonizations of the lake species to fully understand the diversification history of freshwater fauna in and around the ancient lakes.

The first intermediate host of the invasive frog trematode Glypthelmins quieta in Japan.

Glypthelmins quieta is a frog trematode native to North and Central America. This trematode was recently detected in Japan in the American bullfrog Lithobates catesbeianus, which was introduced from North America to Japan. As the first intermediate host of G. quieta, typically a snail, has not yet been identified in Japan, we conducted a snail survey in eastern Japan to screen for an intermediate host using DNA barcoding based on the nuclear 28S ribosomal RNA and mitochondrial cytochrome c oxidase subunit 1. We sampled 3 different snail species, Orientogalba ollula, Physella acuta, and Sinotaia quadrata histrica (157 individuals in total), and only the freshwater snail Physella acuta, which is also believed to have been introduced from North America to Japan, had sporocysts of G. quieta in its hepatopancreas. The introduction of the intermediate and definitive hosts from North America may have facilitated the invasion of G. quieta into Japan.

Changes in severity of pediatric suicide attempts before and after coronavirus disease 2019.

BACKGROUND: The recent coronavirus disease 2019 (COVID-19) pandemic and school closures were reported to have negatively impacted the mental health of children and adolescents. This study aimed to examine the change in the number and severity of pediatric suicide attempts before and after the COVID-19 pandemic. METHODS: This study enrolled 54 patients (26 vs. 28 patients before and after the COVID-19 pandemic, respectively) under 19 years of age who were transported to the emergency department as a result of suicide attempts between April 2017 and December 2021. The primary outcome includes the rate of serious suicide attempts (SSAs). RESULTS: The SSA rates were 19% (5/26) and 43% (12/28) before and after the COVID-19 pandemic, respectively (p = 0.62). The average number of transported suicide attempts per month almost doubled (0.72 vs. 1.33, respectively) and suicide attempts as a percentage of all ambulance transportations of individuals under 19 years old increased significantly from 0.95% (26/2729)to 1.98% (28/1414) (p = 0.006). CONCLUSIONS: The COVID-19 pandemic increased the severity of pediatric suicide attempts but not to a statistically significant degree. Social preventive support and early psychological intervention are therefore needed currently and in the future.

Host-Shift Speciation Proceeded with Gene Flow in Algae Covering Shells.

AbstractHost shifts represent the advancement of a novel niche and often lead to speciation in symbionts. However, its mechanisms are not well understood. Here, we focused on the alga Pseudocladophora conchopheria growing on the shells of intertidal snails. Previous surveys have shown that the alga has host specificity-only attaching to the shell of Lunella correensis-but we discovered that the alga attaches to the shells of multiple sympatric snails. A genome-wide single-nucleotide polymorphism analysis (MIG-seq) was performed to determine whether host-associated speciation occurred in the algae. As a result, there was no gene flow or limited gene flow among the algae from different hosts, and some algae were genetically differentiated among hosts. In addition, the demographic estimate revealed that speciation with gene flow occurred between the algae from different hosts. Therefore, these results support the idea that host-shift speciation gradually proceeded with gene flow in the algae, providing insight into the early evolution of host shifts.

The emerging threat of methicillin-resistant Staphylococcus aureus (MRSA) clone ST22-PT, carrying both Panton-Valentine leucocidin and toxic shock syndrome toxin 1 genes.

OBJECTIVES: Some MRSA strains produce Panton-Valentine leucocidin (PVL) and/or toxic shock syndrome toxin 1 (TSST-1), which are associated with severe infectious diseases. Although PVL- or TSST-1-positive strains have been isolated worldwide, strains carrying both PVL and TSST-1 genes are rare and sporadic. The objective of this study was to characterize these strains from Japan. METHODS: A total of 6433 MRSA strains isolated in Japan between 2015 and 2021 were analysed. Molecular epidemiological and comparative genomic analyses were conducted on PVL- and TSST-1-positive MRSA strains. RESULTS: A total of 26 strains from 12 healthcare facilities were PVL positive and TSST-1 positive, and all were classified as clonal complex (CC) 22. These strains exhibited similar genetic features to each other and were named as ST22-PT according to a previous report. Twelve and one of the ST22-PT strains were identified in patients with deep-seated skin infections and toxic shock syndrome-like symptoms, which are typical clinical features of PVL-positive and TSST-1-positive Staphylococcus aureus, respectively. Whole-genome comparative analysis revealed that the ST22-PT strains were highly similar to PVL- and TSST-1-positive CC22 strains isolated in several countries. Evaluation of the genome structure showed that ST22-PT possessed ΦSa2 harbouring PVL genes and a unique S. aureus pathogenicity island harbouring the TSST-1 gene. CONCLUSIONS: ST22-PT strains have recently emerged from several healthcare facilities in Japan, and ST22-PT-like strains have been identified in several countries. Our report highlights that the risk of international spread of PVL- and TSST-1-positive MRSA clone ST22-PT needs to be further investigated.

Phylogenomic analyses reveal incongruences between divergence times and fossil records of freshwater snails in East Asia.

Fossils provide important insight into our understanding of phylogenetic history by serving as calibration points for divergence time estimation. However, uncertainties in the fossil record due to parallel evolution and convergent evolution can critically affect estimates of node ages. Here, we compare and contrast estimates of phylogenetic divergence with geologic and fossil history for two freshwater snail genera of the family Viviparidae in East Asia (Cipangopaludina and Margarya). Cipangopaludina species are commonly widely distributed species in East Asia, but extant Margarya species are endemic to the ancient lakes in Yunnan, China. According to some previous studies, parallel evolution or convergent evolution of shell morphology has occurred in the family several times which may affect divergence time estimation using fossil records. In this study, we used SNP data derived from ddRAD-seq loci to investigate population demographic history of both genera. Our results show a common pattern of lake endemic lineages diversifying from widely distributed lineages in the Miocene, and multiple colonization to a single ancient lake occurred in the Pleistocene. Our results indicate substantial incongruence among estimated phylogenomic divergence times, some fossil records, and formation ages of ancient lakes. These findings suggest some fossil records may be misidentified in these groups and highlight the need to carefully evaluate geological evidence and fossil records when using these for divergence time estimation.

Taxonomic insights and evolutionary history in East Asian terrestrial slugs of the genus Meghimatium.

East Asia, specifically the Japanese Archipelago, is a biodiversity hotspot of both vertebrates and invertebrates. Mollusks represent a burst of species diversity in this region due to the effects of biotic and abiotic factors on their morphological traits, such as shell shape and size. However, the evolutionary history of terrestrial slugs in East Asia remains unknown. In the present study, we investigated the molecular phylogeny of terrestrial slugs of the genus Meghimatium. This genus includes three described and eight undescribed species, and our study used all except for two. Based on phylogeny and the species delimitation tests, the genus Meghimatium was split into many putative species, suggesting higher species diversity than previously thought based on morphological and anatomical studies and that almost undescribed species may be inappropriate. Therefore, morphological traits, such as body size and colour, conventionally considered for classification may easily vary or be similar across geographic region. Moreover, the divergence time of this genus is almost concordant with the geographical time scale of the formation of the Japanese mainland. Our findings suggest that molecular phylogenetics helps classify Japanese Meghimatium slugs, but comprehensive taxonomic revisions using multi-locus analyses are needed.

Heterogeneous Aggregation of Humic Acids Studied by Small-Angle Neutron and X-ray Scattering.

Aggregation of humic acids (HAs) was studied by small-angle neutron and X-ray scattering techniques. The combination of these techniques enables us to examine the aggregation structures of HA particles. Two HAs with distinctive compositions were examined: a commercial HA (PAHA) and a HA extracted from deep sedimentary groundwater (HHA). While macroscopic coagulation tests showed that these HAs were stable in solutions except for HHA at pH < 6, small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) revealed that they formed aggregates with sizes exceeding the sub-micrometer length scale. The SAXS curves of PAHA remarkably varied with pD = log aD+, where aD+ stands for the activity of deuterium ions, whereas the SANS curves did not. With the help of theoretical fittings, it was revealed that PAHA aggregates consisted of two domains: poorly hydrated cores and well-hydrated proton-rich shells. The cores were (dis)aggregated with pD inside the aggregates of the shells. The SANS and SAXS curves of HHA resembled each other, and their intensities at low q, where q stands for the scattering vector, increased with a decrease of pD, indicating the formation of homogeneous aggregates within the spatial resolutions of SANS and SAXS. This study revealed that distinctive aggregation behaviors exist in humic substances with nm-scale heterogeneous structures like PAHA, which is important for their roles in the fate of contaminants or nutrients in aqueous environments.

Adsorption of cesium and strontium on mesoporous silicas.

Studies are underway on the adsorption reactions of metal ions in confined spaces at the solid-water interface, but it is unclear how the effects of confinement differ for different types of ions. We investigated the effect of the pore size on the adsorption of two cations with different valence, Cs+ and Sr2+, on mesoporous silicas with different pore size distributions. The amount of Sr2+ adsorbed per unit surface area did not differ significantly among the silicas, whereas that of Cs+ was particularly high for silicas with a larger fraction of micropores. The results of X-ray absorption fine structure analysis showed that both ions form outer-sphere complexes with the mesoporous silicas. The results of adsorption experiments were analyzed by fitting using a surface complexation model with the cylindrical Poisson-Boltzmann equation and optimized capacitance of the Stern layer for different pore sizes, and we found that the intrinsic equilibrium constant for the adsorption of Sr2+ is constant regardless of the pore size, whereas that of Cs+ increases as the pore size decreases. The decrease in the relative permittivity of water inside pores with a decrease of the pore size can be interpreted to cause a change in the hydration energy of Cs+ in the second coordination sphere upon adsorption. The reasons for the different confinement effects on the adsorption reactions of Cs+ and Sr2+ were discussed based on the distance of the adsorbed ions from the surface and the chaotropic and kosmotropic nature of Cs+ and Sr2+, respectively.

Responses to oxidative stress and antioxidant capacity in rats at different growth stages.

[Purpose] Oxidative stress is regulated by antioxidant capacity in vivo. However, its impact on aging characteristics remains debatable. This study is first to report oxidative stress, antioxidant capacity, and their ratio in five age groups of rats, and aimed to provide basic data useful for disease prevention. [Materials and Methods] Sixty male Wistar rats of different ages were used as experimental animals, grouped as follows: weaned (three weeks), growth (eight weeks), adulthood (six months), middle-age (12 months), and old-age (24 months). To assess oxidative stress and antioxidant capacity, derivatives of reactive oxygen metabolites and biological antioxidant potential were measured. [Results] The lowest level of oxidative stress and the highest level of antioxidant capacity were observed during the weaning stage, and remarkable dynamic changes were observed until adulthood. The highest oxidative stress and lowest antioxidant capacity were observed in the old-age group. [Conclusion] In vivo oxidative stress and antioxidant capacity are largely reflected in the characteristics of aging, and this ratio is greatly influenced by the dynamics of oxidative stress and antioxidant capacity with age.

Analysis of chemomechanical behavior of stress fibers by continuum mechanics-based FRAP.

Fluorescence recovery after photobleaching (FRAP) is a common technique to analyze the turnover of molecules in living cells. Numerous physicochemical models have been developed to quantitatively evaluate the rate of turnover driven by chemical reaction and diffusion that occurs in a few seconds to minutes. On the other hand, they have limitations in interpreting long-term FRAP responses where intracellular active movement inevitably provides target molecular architectures with additional effects other than chemical reaction and diffusion, namely directed transport and structural deformation. To overcome the limitations, we develop a continuum mechanics-based model that allows for decoupling FRAP response into the intrinsic turnover rate and subcellular mechanical characteristics such as displacement vector and strain tensor. Our approach was validated using fluorescently labeled ß-actin in an actomyosin-mediated contractile apparatus called stress fibers, revealing spatially distinct patterns of the multi-physicochemical events, in which the turnover rate, which represents effective off-rate of ß-actin, was significantly higher at the center of the cell. We also found that the turnover rate is negatively correlated with the rate of displacement or velocity along stress fibers but, interestingly, not with the absolute magnitude of strain. Moreover, stress fibers are subjected to centripetal flow that is facilitated by the circulation of actin molecules. Taken together, this novel framework for long-term FRAP analysis allows for unveiling the contribution of overlooked microscopic mechanics to molecular turnover in living cells.

Patterns of diversification of the operculate land snail genus Cyclophorus (Caenogastropoda: Cyclophoridae) on the Ryukyu Islands, Japan.

The Ryukyu Islands, an island chain in southwestern Japan, originated from land masses that separated from the Eurasian continent due to the formation of sea barriers about 1.55 million years ago. In this study, we investigated the phylogenetic relationships of the operculate land snail genus Cyclophorus (Caenogastropoda: Cyclophoridae) in the Ryukyu Archipelago and surrounding regions based on DNA sequence data. According to our results, all studied Cyclophorus specimens from Japan form a monophyletic group containing eight subclades. Six of these subclades were found only on the Ryukyu Islands. On most islands of the Ryukyu Archipelago, no more than one Cyclophorus subclade was recorded, which may be due to limited ecological niche space and competition. No subclade was found to occur on both sides of the Watase Line, a regional zoogeographical boundary. Divergence times were estimated based on a time-calibrated phylogeny. We found that multiple splits among the Japanese Cyclophorus subclades predate the emergence of major sea barriers in the Ryukyu Archipelago. Vicariance due to sea barrier formation, as assumed for many other taxa from the region, was thus likely not the main driver for subclade divergence in these snails. Instead, certain geographical features might have shaped the diversification of subclades prior to sea barrier formation. Given that Cyclophorus populations were also present on islands that have never been connected to other land masses, the snails must have colonized them via oversea dispersal. As not all nominal taxa corresponded to monophyletic groups, our molecular phylogenetic approach revealed that a taxonomic revision of the Japanese Cyclophorus fauna is necessary. The eight subclades may be regarded as potential species-level groups based on COI p-distances. A canonical discriminant analysis using shell morphological data revealed slight differences among the subclades.

Resolving species-level diversity of Beringiana and Sinanodonta mussels (Bivalvia: Unionidae) in the Japanese archipelago using genome-wide data.

Accurate species identification is of primary importance in ecology and evolutionary biology. For a long time, the unionid mussels Beringiana and Sinanodonta have puzzled researchers trying to unravel their diversity because of their poorly discernible morphologies. A recent study conducted species delineation of unionid mussels based on mitochondrial DNA variation, opening up a new avenue to grasp species diversity of the mussels. However, mtDNA-based classification may not align with species boundaries because mtDNA is prone to introgression and incomplete lineage sorting that cause discordance between species affiliation and gene phylogeny. In this study, we evaluated the validity of the mtDNA-based classification of unionid mussels Beringiana and Sinanodonta in Japan using mitochondrial sequence data, double digest restriction site-associated DNA library (ddRAD) sequencing, and morphological data. We found significant inconsistencies in the mitochondrial and nuclear DNA phylogenies, casting doubt on the reliability of the mtDNA-based classification in this group. In addition, nuclear DNA phylogeny revealed that there are at least two unionid lineages hidden in the mtDNA phylogeny. Although molecular dating technique indicates that Beringiana and Sinanodonta diverged >35 million years ago, their shell morphologies are often indistinguishable. Specifically, morphological analyses exhibited the parallel appearance of nearly identical ball-like shell forms in the two genera in Lake Biwa, which further complicates species identification and the morphological evolution of unionid mussels. Our study adds to a growing body of literature that accurate species identification of unionid mussels is difficult when using morphological characters alone. Although mtDNA-based classification is a simple and convenient way to classify unionid mussels, considerable caution is warranted for its application in ecological and evolutionary studies.

Uncovering overlooked diversity using molecular phylogenetic approach: A case of Japanese sphaeriid clams (Bivalvia: Sphaeriidae).

Revealing the species and lineage diversity of a taxon is important for many biological studies of wildlife. In recent decades, DNA-based approaches have been widely utilised to elucidate the diversity of taxa, especially those that are difficult to distinguish based on morphological traits. This study focused on freshwater clams (Sphaeriidae) in Japan, a biodiversity hotspot of freshwater molluscs. Molecular phylogenetic approaches, including divergence time estimation, species delimitation, rarefaction, and biogeographic area estimation, were used to reveal the nature of the species diversity and its formation process, which are largely unknown. Our delimitation and rarefaction analyses suggest that Japanese sphaeriid clams consist of at least 18 delimitated lineages. This lineage diversity is relatively high compared to other Japanese freshwater molluscs, and in addition, the majority of the Japanese lineage appears to have high endemicity despite the possibility of long-distance dispersal in sphaeriid clams. Our biogeographical analyses suggest that this diversity may be due to the combination of colonisation, during the period when Japan was connected to the continent, and the relatively recent dispersal. Our results highlight the overlooked biodiversity of Japan and provide a basis for further Japanese sphaeriid research, including conservation perspectives.

Pore size effects on surface charges and interfacial electrostatics of mesoporous silicas.

Water in confinement becomes more structured than bulk water, and its properties, such as the dielectric constant, change. It remains unclear, however, how the interfacial reactions in confinement, such as the adsorption of ions on the surfaces of small pores, differ from those in larger spaces. We focused on the deprotonation reaction of hydroxyl groups, a fundamental surface reaction, and investigated the dependence of the surface charge density on pore size by determining the surface charge densities of six types of mesoporous silicas with micropores and mesopores at different ionic strengths and pH levels from batch titration tests. The surface complexation model assuming a potential distribution based on the Poisson-Boltzmann equation in cylindrical coordinates was fitted to the obtained surface charge densities to relate the electrostatics near the surface to the surface reaction. The results showed that the absolute values of the surface charge densities decreased with decreasing pore diameter due to the overlap of the electrical double layers. Furthermore, the capacitance of the Stern layer optimized by fitting decreased with decreasing pore diameter, especially in pores smaller than 4 nm in diameter, which suggested that the dielectric constants of water decreased near the surfaces of small pores.

Proton binding to humic nano particles: electrostatic interaction and the condensation approximation.

Proton binding to "carboxylic" and "phenolic" sites of humic nano particles (HNPs) is determined by the total proton affinity that is due to a specific and an electrostatic affinity. Both affinities are accounted for in the bi-modal Langmuir-Freundlich (bi-LF)-equation extended with a Boltzmann factor that includes the electrostatic site potential(s), y. For y → 0 the equation reduces to the bi-LF Master Curve (MC). Commonly, an electrical double layer model is used to obtain y, e.g., the bi-LF-Donnan-Vapp (monocomponent NICA-Donnan) model and bi-LF-soft-particle-Poison-Boltzmann-Theory (SPBT). A new method is presented that combines the "condensation approximation" (CA) with the MC concept (CA-MC). With the CA, the proton binding curve and MC can be transformed in, respectively, the total and specific affinity distribution. The difference at a given charge density provides the electrostatic affinity and CA-potentials vs. charge density. The MC can be obtained theoretically or by using the convention that the electrostatic interaction is negligible at 1 M salt concentration. For five HNPs CA-potentials corresponding with the bi-LF-SPBT are compared with results of the bi-LF-Donnan-Vapp model using the MC(SPBT). The bi-LF-Donnan-Vapp model fails when the Debye length > hydrated particle radius. The CA-MC(1M) method does not require characteristics of the HNPs. Combination of the bi-LF-eq. with the CA-MC(1 M) method gives the bi-LF-CA-MC(1 M) model. The CA-MC(1 M) differs from the MC(SPBT); therefore, resulting parameters can only be compared when the same method is used.

Determining the domain-level reaction-diffusion properties of an actin-binding protein transgelin-2 within cells.

Proteins in cells undergo repeated binding to other molecules, thereby reducing the apparent extent of their intracellular diffusion. While much effort has been made to analytically decouple these combined effects of pure diffusion and chemical binding, it is difficult with conventional approaches to attribute the measured quantities to the nature of specific domains of the proteins. Motivated by the common goal in cell signaling research aimed at identifying the domains responsible for particular intermolecular interactions, here we describe a framework for determining the local physicochemical properties of cellular proteins associated with immobile scaffolds. To validate this new approach, we apply it to transgelin-2, an actin-binding protein whose intracellular dynamics remains elusive. We develop a fluorescence recovery after photobleaching (FRAP)-based framework, in which comprehensive combinations of domain-deletion mutants are created, and the difference among them in FRAP response is analyzed. We demonstrate that transgelin-2 in actin stress fibers (SFs) interacts with F-actin via two separate domains, and the chemical properties are determined for the individual domains. Its pure diffusion properties independent of the association to F-actin is also obtained. Our approach will thus be useful, as presented here for transgelin-2, in addressing the signaling mechanism of cellular proteins associated with SFs.

Mechanosensitive myosin II but not cofilin primarily contributes to cyclic cell stretch-induced selective disassembly of actin stress fibers.

Cells adapt to applied cyclic stretch (CS) to circumvent chronic activation of proinflammatory signaling. Currently, the molecular mechanism of the selective disassembly of actin stress fibers (SFs) in the stretch direction, which occurs at the early stage of the cellular response to CS, remains controversial. Here, we suggest that the mechanosensitive behavior of myosin II, a major cross-linker of SFs, primarily contributes to the directional disassembly of the actomyosin complex SFs in bovine vascular smooth muscle cells and human U2OS osteosarcoma cells. First, we identified that CS with a shortening phase that exceeds in speed the inherent contractile rate of individual SFs leads to the disassembly. To understand the biological basis, we investigated the effect of expressing myosin regulatory light-chain mutants and found that SFs with less actomyosin activities disassemble more promptly upon CS. We consequently created a minimal mathematical model that recapitulates the salient features of the direction-selective and threshold-triggered disassembly of SFs to show that disassembly or, more specifically, unbundling of the actomyosin bundle SFs is enhanced with sufficiently fast cell shortening. We further demonstrated that similar disassembly of SFs is inducible in the presence of an active LIM-kinase-1 mutant that deactivates cofilin, suggesting that cofilin is dispensable as opposed to a previously proposed mechanism.

Stoichiometry between Humate Unit Molecules and Metal Ions in Supramolecular Assembly Induced by Cu2+ and Tb3+ Measured by Gel Electrophoresis Techniques.

Humic acid (HA), a fraction of humic substances, can strongly complex with metal ions to form a supramolecular assembly via coordination binding and other intermolecular forces. However, determining the supramolecular size distribution and stoichiometry between small HA unit molecules constituting HA supramolecules and metal ions has proven to be challenging. Here, we investigated the changes in the size distributions of HAs induced by Cu2+ and Tb3+ ions using unique PAGE for the separation and quantification of HA complexes and metal ions bound, followed by UV-vis spectroscopy and excitation-emission matrix-parallel factor analysis. By determining the concentrations of HA and metal ions, it was possible to estimate the stoichiometry of the HA unit molecule to metal ions in supramolecular complexes. It was found that the supramolecular behaviors of Cu2+ and Tb3+ complexes with HA collected from peat (PAHA) and deep groundwater (HHA) differed. For example, two HHA unit molecules form a supramolecule via cross-linking by a Cu2+ ion in the case of Cu2+-HHA. Our results suggest that this supramolecular stoichiometry is related to the abundance of sulfur atoms in the elemental composition of HHA. Our experimental results and analysis provide new insights into HA supramolecules formed via metal complexation.