Structural and Mechanical Characterization of DNA-Tethered Membranes on Graphene.

Lipid membranes that are separated from the surface of graphene by DNA tethers were prepared by surface functionalization with pyrene coupled to single-stranded DNA (ssDNA), followed by self-assembly of the mixture of ssDNA-functionalized phospholipid and the matrix phospholipids. The formation of uniform membranes was confirmed by fluorescence microscopy, and the structures of the systems before and after hybridization in the direction perpendicular to the global plane of the membranes were investigated using high-energy X-ray reflectivity. The thickness values of the DNA spacers (15 and 37 bp) calculated from the best-fit results were less than the expected thicknesses of the double-stranded DNA (dsDNA) chains taking the upright conformation, indicating that the DNA spacers are tilted with respect to the direction normal to the surface. The Young's moduli of the DNA-tethered membranes obtained by AFM nanoindentation showed higher values than the membranes with no DNA tethers, which suggests that the DNA layer resists against the compression, lifting up the membrane. Intriguingly, the presence of DNA tethers caused no increase in the yield depth. The smaller thickness values as well as the unchanged yield depth suggest that the dsDNA chains can tilt and rotate, which can be attributed to the flexible pyrene-DNA junction.

Hyperconfined bio-inspired Polymers in Integrative Flow-Through Systems for Highly Selective Removal of Heavy Metal Ions.

Access to clean water, hygiene, and sanitation is becoming an increasingly pressing global demand, particularly owing to rapid population growth and urbanization. Phytoremediation utilizes a highly conserved phytochelatin in plants, which captures hazardous heavy metal ions from aquatic environments and sequesters them in vacuoles. Herein, we report the design of phytochelatin-inspired copolymers containing carboxylate and thiolate moieties. Titration calorimetry results indicate that the coexistence of both moieties is essential for the excellent Cd2+ ion-capturing capacity of the copolymers. The obtained dissociation constant, KD ~ 1 nM for Cd2+ ion, is four-to-five orders of magnitude higher than that for peptides mimicking the sequence of endogenous phytochelatin. Furthermore, infrared and nuclear magnetic resonance spectroscopy results unravel the mechanism underlying complex formation at the molecular level. The grafting of 0.1 g bio-inspired copolymers onto silica microparticles and cellulose membranes helps concentrate the copolymer-coated microparticles in ≈3 mL volume to remove Cd2+ ions from 0.3 L of water within 1 h to the drinking water level (<0.03 µM). The obtained results suggest that hyperconfinement of bio-inspired polymers in flow-through systems can be applied for the highly selective removal of harmful contaminants from the environmental water.

Guttae Morphology After Cultured Corneal Endothelial Cell Transplant in Fuchs Endothelial Corneal Dystrophy.

Importance: Whether guttae in Fuchs endothelial corneal dystrophy (FECD) can be removed by polishing without Descemet stripping and whether postoperative maintenance of reduced guttae can be achieved through cultured corneal endothelial cell (CEC) transplant therapy are critical issues to be addressed. Objective: To investigate the decrease of guttae through polishing degenerated CECs and abnormal extracellular matrix (ECM) without Descemet stripping and to observe the behavior of guttae following cultured CEC transplant. Design, Setting, and Participants: This case series prospective observational study was conducted in a hospital outpatient clinic setting. Between December 2013 and January 2019, 22 eyes with corneal endothelial failure caused by FECD received cultured CEC transplant therapy at Kyoto Prefectural University Hospital. Of these, 15 eyes were consistently monitored at the same central corneal area during the preoperative phase, as well as in the early (within 1 year) and late (after 3 years) postoperative phases. The images from these phases were categorized into 3 groups: typical guttae, atypical guttae, and no guttae. Exposures: Cultured CEC transplant therapy. Main Outcomes: Proportion of guttae in the observable area was measured, comparing the early and late postoperative phases for each group. Results: The mean age of the patients at the time of surgery was 69 years (range, 49-79 years). All 15 eyes exhibited the presence of confluent guttae preoperatively (100%). Among these, 3 of 15 eyes belonged to male patients. The early postoperative phase of guttae morphologies was classified into 3 groups: 5 eyes with typical guttae, 7 with atypical guttae, and 3 with no guttae. The decrease in the number of these guttae was achieved by surgical procedures. The median percentage of guttae in the typical guttae, atypical guttae, and no guttae groups was 41.8%, 44.4%, and 16.2%, respectively, in the early phase, and 42.2%, 38.2%, and 18.8%, respectively, in the late phase. Conclusions and Relevance: The findings demonstrate that in some cases of FECD, guttae can be removed by scraping and polishing abnormal ECM and degenerated CECs, while preserving the Descemet membrane. Furthermore, cultured CEC transplant resulted in no increase in guttae for up to 3 years, providing insights into surgically eliminating guttae.

Laparoscopy-assisted nonexposed endoscopic full-thickness resection for local resection after endoscopic submucosal dissection.

Video 1Nonexposed endoscopic wall inversion surgery for local resection of microscopic residual tumor after endoscopic submucosal dissection.

Electrochemical Detection of Selective Anion Transport through Subnanopores in Liquid-Crystalline Water Treatment Membranes.

The anion-selective transport through subnanoporous liquid-crystalline (LC) water treatment membranes was quantitatively detected by the deposition and electrochemical analysis of the LC membrane on the GaN electrode. The time course of the capacitance and Warburg resistance of the LC membrane suggest that the interaction of the LC membrane with monovalent Cl- ions is distinctly different from that with SO42- ions. A continuous decay in capacitance suggests the condensation of Cl- ions in subnanopores, whereas the interaction between SO42- ions and the inner wall of subnanopores is much weaker. The chronoamperometry data further suggest that SO42- ions are transported through subnanoporous channels 10 times faster than Cl- ions. These results, together with the previous X-ray emission spectroscopy, suggest that SO42- ions, which possess similar hydrogen-bonded structures to the hydrogen-bonded networks inside the subnanopores, can exchange the associated water molecules and hop along the network of water molecules, but Cl- ions bind and accumulate inside subnanopores. The well-controlled supramolecular self-assembly of LC building blocks opens a large potential toward the fine adjustment of hydrogen-bonding networks in nanospace providing materials new functions, which cannot be realized by bulk water.

Structure-changeable luminescent Eu(III) complex as a human cancer grade probing system for brain tumor diagnosis.

Accurate determination of human tumor malignancy is important for choosing efficient and safe therapies. Bioimaging technologies based on luminescent molecules are widely used to localize and distinguish active tumor cells. Here, we report a human cancer grade probing system (GPS) using a water-soluble and structure-changeable Eu(III) complex for the continuous detection of early human brain tumors of different malignancy grades. Time-dependent emission spectra of the Eu(III) complexes in various types of tumor cells were recorded. The radiative rate constants (kr), which depend on the geometry of the Eu(III) complex, were calculated from the emission spectra. The tendency of the kr values to vary depended on the tumor cells at different malignancy grades. Between T = 0 and T = 3 h of invasion, the kr values exhibited an increase of 4% in NHA/TS (benign grade II gliomas), 7% in NHA/TSR (malignant grade III gliomas), and 27% in NHA/TSRA (malignant grade IV gliomas). Tumor cells with high-grade malignancy exhibited a rapid upward trend in kr values. The cancer GPS employs Eu(III) emissions to provide a new diagnostic method for determining human brain tumor malignancy.

Antibacterial Synthetic Nanocelluloses Synergizing with a Metal-Chelating Agent.

Antibacterial materials composed of biodegradable and biocompatible constituents that are produced via eco-friendly synthetic strategies will become an attractive alternative to antibiotics to combat antibiotic-resistant bacteria. In this study, we demonstrated the antibacterial properties of nanosheet-shaped crystalline assemblies of enzymatically synthesized aminated cellulose oligomers (namely, surface-aminated synthetic nanocelluloses) and their synergy with a metal-chelating antibacterial agent, ethylenediaminetetraacetic acid (EDTA). Growth curves and colony counting assays revealed that the surface-aminated cellulose assemblies had an antibacterial effect against Gram-negative Escherichia coli (E. coli). The cationic assemblies appeared to destabilize the cell wall of E. coli through electrostatic interactions with anionic lipopolysaccharide (LPS) molecules on the outer membrane. The antibacterial properties were significantly enhanced by the concurrent use of EDTA, which potentially removed metal ions from LPS molecules, resulting in synergistic bactericidal effects. No antibacterial activity of the surface-aminated cellulose assemblies was observed against Gram-positive Staphylococcus aureus even in the presence of EDTA, further supporting the contribution of electrostatic interactions between the cationic assemblies and anionic LPS to the activity against Gram-negative bacteria. Analysis using quartz crystal microbalance with dissipation monitoring revealed the attractive interaction of the surface-aminated cellulose assembly with LPS Ra monolayers artificially produced on the device substrate.

Reversible Host-Guest Crosslinks in Supramolecular Hydrogels for On-Demand Mechanical Stimulation of Human Mesenchymal Stem Cells.

Stem cells are regulated not only by biochemical signals but also by biophysical properties of extracellular matrix (ECM). The ECM is constantly monitored and remodeled because the fate of stem cells can be misdirected when the mechanical interaction between cells and ECM is imbalanced. A well-defined ECM model for bone marrow-derived human mesenchymal stem cells (hMSCs) based on supramolecular hydrogels containing reversible host-guest crosslinks is fabricated. The stiffness (Young's modulus E) of the hydrogels can be switched reversibly by altering the concentration of non-cytotoxic, free guest molecules dissolved in the culture medium. Fine-adjustment of substrate stiffness enables the authors to determine the critical stiffness level E* at which hMSCs turn the mechano-sensory machinery on or off. Next, the substrate stiffness across E* is switched and the dynamic adaptation characteristics such as morphology, traction force, and YAP/TAZ signaling of hMSCs are monitored. These data demonstrate the instantaneous switching of traction force, which is followed by YAP/TAZ signaling and morphological adaptation. Periodical switching of the substrate stiffness across E* proves that frequent applications of mechanical stimuli drastically suppress hMSC proliferation. Mechanical stimulation across E* level using dynamic hydrogels is a promising strategy for the on-demand control of hMSC transcription and proliferation.

Extracellular calcium functions as a molecular glue for transmembrane helices to activate the scramblase Xkr4.

The "eat me" signal, phosphatidylserine is exposed on the surface of dying cells by phospholipid scrambling. Previously, we showed that the Xkr family protein Xkr4 is activated by caspase-mediated cleavage and binding of the XRCC4 fragment. Here, we show that extracellular calcium is an additional factor needed to activate Xkr4. The constitutively active mutant of Xkr4 is found to induce phospholipid scrambling in an extracellular, but not intracellular, calcium-dependent manner. Importantly, other Xkr family members also require extracellular calcium for activation. Alanine scanning shows that D123 and D127 of TM1 and E310 of TM3 coordinate calcium binding. Moreover, lysine scanning demonstrates that the E310K mutation-mediated salt bridge between TM1 and TM3 bypasses the requirement of calcium. Cysteine scanning proves that disulfide bond formation between TM1 and TM3 also activates phospholipid scrambling without calcium. Collectively, this study shows that extracellular calcium functions as a molecular glue for TM1 and TM3 of Xkr proteins for activation, thus demonstrating a regulatory mechanism for multi-transmembrane region-containing proteins.

Thermodynamics and Viscoelastic Property of Interface Unravel Combined Functions of Cationic Surfactant and Aromatic Alcohol against Gram-Negative Bacteria.

Lipopolysaccharides (LPSs), the major constituents of the outer membranes of Gram-negative bacteria, play a key role in protecting bacteria against antibiotics and antibacterial agents. In this study, we investigated how a mixture of cationic surfactants and aromatic alcohols, the base materials of widely used sanitizers, synergistically act on LPSs purified from Escherichia coli using isothermal titration calorimetry (ITC), surface tension measurements, and quartz crystal microbalance with dissipation (QCM-D). ITC data measured in the absence of Ca2+ ions showed the coexistence of exothermic and endothermic processes. The exotherm can be interpreted as the electrostatic binding of the cationic surfactant to the negatively charged LPS membrane surface, whereas the endotherm indicates the hydrophobic interaction between the hydrocarbon chains of the surfactants and LPSs. In the presence of Ca2+ ions, only an exothermic reaction was observed by ITC, and no entropically driven endotherm could be detected. Surface tension experiments further revealed that the co-adsorption of surfactants and LPS was synergistic, while that of surfactants and alcohol was negatively synergistic. Moreover, the QCM-D data indicated that the LPS membrane remained intact when the alcohol alone was added to the system. Intriguingly, the LPS membrane became highly susceptible to the combination of cationic surfactants and aromatic alcohols in the absence of Ca2+ ions. The obtained data provide thermodynamic and mechanical insights into the synergistic function of surfactants and alcohols in sanitation, which will enable the identification of the optimal combination of small molecules for a high hygiene level for the post-pandemic society.

Physical biomarkers for human hematopoietic stem and progenitor cells.

Adhesion of hematopoietic stem and progenitor cells (HSPCs) to the bone marrow niche plays critical roles in the maintenance of the most primitive HSPCs. The interactions of HSPC-niche interactions are clinically relevant in acute myeloid leukemia (AML), because (i) leukemia-initiating cells adhered to the marrow niche are protected from the cytotoxic effect by chemotherapy and (ii) mobilization of HSPCs from healthy donors' bone marrow is crucial for the effective stem cell transplantation. However, although many clinical agents have been developed for the HSPC mobilization, the effects caused by the extrinsic molecular cues were traditionally evaluated based on phenomenological observations. This review highlights the recent interdisciplinary challenges of hematologists, biophysicists and cell biologists towards the design of defined in vitro niche models and the development of physical biomarkers for quantitative indexing of differential effects of clinical agents on human HSPCs.

Wnt/ß-catenin signaling induces axial elasticity patterns of Hydra extracellular matrix.

The extracellular matrix (ECM) plays crucial roles in animal development and diseases. Here, we report that Wnt/ß-catenin signaling induces the ECM remodeling during Hydra axis formation. We determined the micro- and nanoscopic arrangement of fibrillar type I collagen along Hydra's body axis using high-resolution microscopy and X-ray scattering. Elasticity mapping of the ECM ex vivo revealed distinctive elasticity patterns along the body axis. A proteomic analysis of the ECM showed that these elasticity patterns correlate with a gradient-like distribution of metalloproteases along the body axis. Activation of the Wnt/ß-catenin pathway in wild-type and transgenic animals alters these patterns toward low ECM elasticity patterns. This suggests a mechanism whereby high protease activity under control of Wnt/ß-catenin signaling causes remodeling and softening of the ECM. This Wnt-dependent spatiotemporal coordination of biochemical and biomechanical cues in ECM formation was likely a central evolutionary innovation for animal tissue morphogenesis.

Modulation of wetting of stimulus responsive polymer brushes by lipid vesicles: experiments and simulations.

The interactions between vesicle and substrate have been studied by simulation and experiment. We grafted polyacrylic acid brushes containing cysteine side chains at a defined area density on planar lipid membranes. Specular X-ray reflectivity data indicated that the addition of Cd2+ ions induces the compaction of the polymer brush layer and modulates the adhesion of lipid vesicles. Using microinterferometry imaging, we determined the onset level, [CdCl2] = 0.25 mM, at which the wetting of the vesicle emerges. The characteristics of the interactions between vesicle and brush were quantitatively evaluated by the shape of the vesicle near the substrate and height fluctuations of the membrane in contact with brushes. To analyze these experiments, we have systematically studied the shape and adhesion of axially symmetric vesicles for finite-range membrane-substrate interaction, i.e., a relevant experimental characteristic, through simulations. The wetting of vesicles sensitively depends on the interaction range and the approximate estimates of the capillary length change significantly, depending on the adhesion strength. We found, however, that the local transversality condition that relates the maximal curvature at the edge of the adhesion zone to the adhesion strength remains rather accurate even for a finite interaction range as long as the vesicle is large compared to the interaction range.

Modified Intestinal Derotation Procedure with Reversed Kocherization to Facilitate Mesopancreas Excision During Pancreaticoduodenectomy.

BACKGROUND: Although intestinal derotation procedure has advantages of facilitating mesopancreas excision during pancreaticoduodenectomy, the wide mobilization takes time and risks injuring other organs. This article describes a modified intestinal derotation procedure in pancreaticoduodenectomy and its clinical impact on short-term outcomes. METHODS: The modified procedure comprised the pinpoint mobilization of the proximal jejunum following reversed Kocherization. Among 99 consecutive patients who underwent pancreaticoduodenectomy between 2016 and 2022, the short-term outcomes of pancreaticoduodenectomy with the modified procedure were compared with those of conventional pancreaticoduodenectomy. The feasibility of the modified procedure was investigated based on the vascular anatomy of the mesopancreas. RESULTS: Compared with conventional pancreaticoduodenectomy (n = 55), the modified procedure (n = 44) involved less blood loss and shorter operation time (p < 0.001 and 0.017, respectively). Severe morbidity, clinically relevant postoperative pancreatic fistula, and prolonged hospitalization occurred less often with the modified procedure compared with conventional pancreaticoduodenectomy (p = 0.003, 0.008, and < 0.001, respectively). According to preoperative image findings, most (72%) patients had a single inferior pancreaticoduodenal artery sharing a common trunk with the first jejunal artery. The inferior pancreaticoduodenal vein drained into the jejunal vein in 71% of the patients. The first jejunal vein ran behind the superior mesenteric artery in 77% of the patients. CONCLUSIONS: By combining our modified intestinal derotation procedure with preoperative recognition of the vascular anatomy of mesopancreas, mesopancreas excision during pancreaticoduodenectomy can be performed safely and accurately.

Lengthened Efferent Limb in Braun Enteroenterostomy Reduces Delayed Gastric Emptying After Pancreaticoduodenectomy.

BACKGROUND: Delayed gastric emptying (DGE) is a common complication after pancreaticoduodenectomy (PD), but a method to prevent DGE has not been established. This study aims to demonstrate a novel technique utilizing a lengthened efferent limb in Billroth-II (B-II) reconstruction during PD and to evaluate the impact of the longer efferent limb on DGE occurrence. METHODS: Patients who underwent PD with B-II reconstruction were divided into two groups: PDs with lengthened (50-60 cm) efferent limb (L group) and standard length (0-30 cm) efferent limb (S group). Postoperative outcomes were compared. DGE was defined and graded according to the International Study Group of Pancreatic Surgery criteria. RESULTS: Among 283 consecutive patients who underwent PD from 2002 to 2021, 206 patients were included in this study. Patients who underwent Roux-en-Y reconstruction (n = 77) were excluded. Compared with the S group, the L group included older patients and those who underwent PD after 2016 (p = 0.025, < 0.001, respectively). D2 lymphadenectomy, antecolic route reconstruction, and Braun enteroenterostomy were performed more frequently in the L group (p = 0.040, < 0.001, < 0.001, respectively). The rate of DGE was significantly decreased to 6% in the L group, compared with 16% in the S group (p = 0.027), which might lead to a shorter hospital stay in the L group (p < 0.001). Multivariable analysis identified two factors as independent predictors for DGE: intraabdominal abscess [odds ratio (OR) 5.530, p = 0.008] and standard efferent limb length (OR 2.969, p = 0.047). CONCLUSION: A lengthened efferent limb in Braun enteroenterostomy could reduce DGE after PD.

Ion-specific nanoscale compaction of cysteine-modified poly(acrylic acid) brushes revealed by 3D scanning force microscopy with frequency modulation detection.

Stimuli-responsive polyelectrolyte brushes adapt their physico-chemical properties according to pH and ion concentrations of the solution in contact. We synthesized a poly(acrylic acid) bearing cysteine residues at side chains and a lipid head group at the terminal, and incorporated them into a phospholipid monolayer deposited on a hydrophobic silane monolayer. The ion-specific, nanoscale response of polyelectrolyte brushes was detected by using three-dimensional scanning force microscopy (3D-SFM) combined with frequency modulation detection. The obtained topographic and mechanical landscapes indicated that the brushes were uniformly stretched, undergoing a gradual transition from the brush to the bulk electrolyte in the absence of divalent cations. When 1 mM calcium ions were added, the brushes were uniformly compacted, exhibiting a sharper brush-to-bulk transition. Remarkably, the addition of 1 mM cadmium ions made the brush surface significantly rough and the mechanical landscape highly heterogeneous. Currently, cadmium-specific nanoscale compaction of the brushes is attributed to the coordination of thiol and carboxyl side chains with cadmium ions, as suggested for naturally occurring, heavy metal binding proteins.

Stimulus-Responsive, Gelatin-Containing Supramolecular Nanofibers as Switchable 3D Microenvironments for Cells.

Polymer- and/or protein-based nanofibers that promote stable cell adhesion have drawn increasing attention as well-defined models of the extracellular matrix. In this study, we fabricated two classes of stimulus-responsive fibers containing gelatin and supramolecular crosslinks to emulate the dynamic cellular microenvironment in vivo. Gelatin enabled cells to adhere without additional surface functionalization, while supramolecular crosslinks allowed for the reversible switching of the Young's modulus through changes in the concentration of guest molecules in culture media. The first class of nanofibers was prepared by coupling the host-guest inclusion complex to gelatin before electrospinning (pre-conjugation), while the second class of nanofibers was fabricated by coupling gelatin to polyacrylamide functionalized with host or guest moieties, followed by conjugation in the electrospinning solution (post-conjugation). In situ AFM nano-indentation demonstrated the reversible switching of the Young's modulus between 2-3 kPa and 0.2-0.3 kPa under physiological conditions by adding/removing soluble guest molecules. As the concentration of additives does not affect cell viability, the supramolecular fibers established in this study are a promising candidate for various biomedical applications, such as standardized three-dimensional culture matrices for somatic cells and the regulation of stem cell differentiation.

Receptor-Functionalized Lipid Membranes as Biomimetic Surfaces for Adhesion of Plasmodium falciparum-Infected Erythrocytes.

Here, we describe a detailed protocol of how to manufacture biomimetic, host receptor-functionalized membranes and how to use them in adhesion assays. Receptor-functionalized membranes have the advantage that the receptor identity and the receptor density can be controlled, which, in turn, enables studies on the kinetics, dynamics and biomechanics of receptor/ligand interactions. Such information is difficult to obtain from currently used in vitro systems, including cultured primary human microvascular endothelial cells or receptor-coated surfaces, which often display either multiple receptors or receptors with uncertain density and arrangement.

Time-strain inseparability in multiaxial stress relaxation of supramolecular gels formed via host-guest interactions.

Supramolecular hydrogels utilizing host-guest interactions (HG gels) exhibit large deformability and pronounced viscoelasticity. The inclusion complexes between ß-cyclodextrin (host) and adamantane (guest) units on the water-soluble polymers form transient bonds. The HG gels show significant stress relaxation with finite equilibrium stress following the step strain. The stress relaxation process reflects the detachment dynamics of the transient bonds which sustain the initial stress, while the finite equilibrium stress is preserved by the permanent topological cross-links with a rotaxane structure. Nonlinear stress relaxation experiments in biaxial stretching with various combinations of two orthogonal strains unambiguously reveal that time and strain effects on stress are not separable. The relaxation is accelerated for a short time frame (<102 s) with an increase in the magnitude of strain, whereas it is retarded for a longer time window with an increase in the anisotropy of the imposed biaxial strain. The time-strain inseparability in the HG gels is in contrast to the simple nonlinear viscoelasticity of a dual cross-link gel with covalent and transient cross-links in which the separability was previously validated by the same assessment. We currently interpret that the significant susceptibility of the detachment dynamics to the deformation type results from the structural characteristics of the HG gels, i.e., the host and guest moieties covalently connected to the network chains, the considerably low concentrations (<0.1 M) of these moieties, and the slidability of the permanent rotaxane cross-links.