Streptococcus thermophilus iHA318 Improves Dry Eye Symptoms by Mitigating Ocular Surface Damage in a Mouse Model.

Dry eye is a complicated ocular surface disease that causes discomfort, visual disturbance, and frequently observed ocular surface damage. Emerging hypotheses suggest probiotics may help relieve dry eye symptoms by modulating inflammation and oxidative stress. This study aimed to investigate the therapeutic effects of Streptococcus thermophilus iHA318 probiotics on dry eye using in vitro assays and an in vivo murine model of ultraviolet B (UVB) radiation-induced dry eye. In vitro analyses revealed that S. thermophilus iHA318® exhibited antioxidant activity and anti-inflammatory effects by inhibiting reactive oxygen species production and suppressing inflammatory cytokines. For the in vivo study, female ICR mice were assigned to normal control, UVB-induced dry eye, and UVB+iHA318 treatment groups. UVB exposure significantly decreased tear volume and tear film breakup time (TBUT) compared to normal controls. Supplementation with S. thermophilus iHA318® via oral gavage markedly improved tear production and TBUT on day 7 post-UVB exposure. Ocular surface photography demonstrated improved gradings of corneal opacity, smoothness, and lissamine green staining in the iHA318 group versus the UVB group. Topographical analysis further revealed improvement in the UVB-induced corneal irregularities by iHA318 treatment. Collectively, these results indicate that S. thermophilus iHA318 exerts a protective effect against dry eye symptoms by mitigating oxidative stress and inflammation, thereby preserving tear film stability and ocular surface integrity. This probiotic strain represents a promising therapeutic approach for managing dry eye syndrome.

Encoder-Decoder Architecture for 3D Seismic Inversion.

Inverting seismic data to build 3D geological structures is a challenging task due to the overwhelming amount of acquired seismic data, and the very-high computational load due to iterative numerical solutions of the wave equation, as required by industry-standard tools such as Full Waveform Inversion (FWI). For example, in an area with surface dimensions of 4.5 km × 4.5 km, hundreds of seismic shot-gather cubes are required for 3D model reconstruction, leading to Terabytes of recorded data. This paper presents a deep learning solution for the reconstruction of realistic 3D models in the presence of field noise recorded in seismic surveys. We implement and analyze a convolutional encoder-decoder architecture that efficiently processes the entire collection of hundreds of seismic shot-gather cubes. The proposed solution demonstrates that realistic 3D models can be reconstructed with a structural similarity index measure (SSIM) of 0.9143 (out of 1.0) in the presence of field noise at 10 dB signal-to-noise ratio.

Mode of Action of Antimicrobial Peptides on E. coli Spheroplasts.

We investigated the phenomena of antimicrobial peptides (AMPs) directly attacking the cytoplasmic membranes of Escherichia coli spheroplasts. We developed a procedure for fluorescence recovery after photobleaching to examine dye leakage through bacterial membranes as AMPs in solution bound to the membranes. We found that the AMP binding did not increase the apparent membrane area of a spheroplast, contrary to the response of a lipid-bilayer vesicle, which always showed a membrane area expansion by AMP binding. The permeability through the bacterial membrane increased in a sigmoidal fashion as the AMP binding increased in time, exhibiting a cooperative behavior of AMPs. The analysis of fluorescence recovery after photobleaching showed that the fluxes of dye molecules into and out of the cell were consistent with diffusion of molecules through a number of pores that increased with binding of AMPs and then saturated to a steady level. We discovered a new, to our knowledge, experimental parameter called the flux rate that characterizes the AMP-induced permeability of dye molecules through bacterial membranes. The phenomena observed in bacterial membranes are consistent with the pore-forming activities of AMPs previously observed in lipid bilayers. The experimental value of the flux rate per pore is much smaller than a theoretical value that assumes no friction for the dye molecule's permeation through the pore. We believe that experimental studies of the flux rate will be useful for further analysis of AMPs' permeabilization mechanisms.

Application of the Blobo bluetooth ball in wrist rehabilitation training.

[Purpose] The introduction of emerging technologies such as the wireless Blobo bluetooth ball with multimedia features can enhance wrist physical therapy training, making it more fun and enhancing its effects. [Methods] Wrist injuries caused by fatigue at work, improper exercise, and other conditions are very common. Therefore, the reconstruction of wrist joint function is an important issue. The efficacy of a newly developed integrated wrist joint rehabilitation game using a Blobo bluetooth ball with C# software installed was tested in wrist rehabilitation (Flexion, Extension, Ulnar Deviation, Radial Deviation). [Results] Eight subjects with normal wrist function participated in a test of the system's stability and repeatability. After performing the Blobo bluetooth ball wrist physical therapy training, eight patients with wrist dysfunction experienced approximately 10° improvements in range of motion (ROM) of flexion extension, and ulnar deviation and about 6° ROM improvement in radial deviation. The subjects showed progress in important indicators of wrist function. [Conclusion] This study used the Blobo bluetooth ball in wrist physical therapy training and the preliminary results were encouraging. In the future, more diverse wrist or limb rehabilitation games should be developed to meet the needs of physical therapy training.

Membrane-mediated amyloid formation of PrP 106-126: A kinetic study.

PrP 106-126 conserves the pathogenic and physicochemical properties of the Scrapie isoform of the prion protein. PrP 106-126 and other amyloidal proteins are capable of inducing ion permeability through cell membranes, and this property may represent the common primary mechanism of pathogenesis in the amyloid-related degenerative diseases. However, for many amyloidal proteins, despite numerous phenomenological observations of their interactions with membranes, it has been difficult to determine the molecular mechanisms by which the proteins cause ion permeability. One approach that has not been undertaken is the kinetic study of protein-membrane interactions. We found that the reaction time constant of the interaction between PrP 106-126 and membranes is suitable for such studies. The kinetic experiment with giant lipid vesicles showed that the membrane area first increased by peptide binding but then decreased. The membrane area decrease was coincidental with appearance of extramembranous aggregates including lipid molecules. Sometimes, the membrane area would increase again followed by another decrease. The kinetic experiment with small vesicles was monitored by circular dichroism for peptide conformation changes. The results are consistent with a molecular simulation following a simple set of well-defined rules. We deduced that at the molecular level the formation of peptide amyloids incorporated lipid molecules as part of the aggregates. Most importantly the amyloid aggregates desorbed from the lipid bilayer, consistent with the macroscopic phenomena observed with giant vesicles. Thus we conclude that the main effect of membrane-mediated amyloid formation is extraction of lipid molecules from the membrane. We discuss the likelihood of this effect on membrane ion permeability.

Physical properties of Escherichia coli spheroplast membranes.

We investigated the physical properties of bacterial cytoplasmic membranes by applying the method of micropipette aspiration to Escherichia coli spheroplasts. We found that the properties of spheroplast membranes are significantly different from that of laboratory-prepared lipid vesicles or that of previously investigated animal cells. The spheroplasts can adjust their internal osmolality by increasing their volumes more than three times upon osmotic downshift. Until the spheroplasts are swollen to their volume limit, their membranes are tensionless. At constant external osmolality, aspiration increases the surface area of the membrane and creates tension. What distinguishes spheroplast membranes from lipid bilayers is that the area change of a spheroplast membrane by tension is a relaxation process. No such time dependence is observed in lipid bilayers. The equilibrium tension-area relation is reversible. The apparent area stretching moduli are several times smaller than that of stretching a lipid bilayer. We conclude that spheroplasts maintain a minimum surface area without tension by a membrane reservoir that removes the excessive membranes from the minimum surface area. Volume expansion eventually exhausts the membrane reservoir; then the membrane behaves like a lipid bilayer with a comparable stretching modulus. Interestingly, the membranes cease to refold when spheroplasts lost viability, implying that the membrane reservoir is metabolically maintained.

Interaction of daptomycin with lipid bilayers: a lipid extracting effect.

Daptomycin is the first approved member of a new structural class of antibiotics, the cyclic lipopeptides. The peptide interacts with the lipid matrix of cell membranes, inducing permeability of the membrane to ions, but its molecular mechanism has been a puzzle. Unlike the ubiquitous membrane-acting host-defense antimicrobial peptides, daptomycin does not induce pores in the cell membranes. Thus, how it affects the permeability of a membrane to ions is not clear. We studied its interaction with giant unilamellar vesicles (GUVs) and discovered a lipid-extracting phenomenon that correlates with the direct action of daptomycin on bacterial membranes observed in a recent fluorescence microscopy study. Lipid extraction occurred only when the GUV lipid composition included phosphatidylglycerol and in the presence of Ca(2+) ions, the same condition found to be necessary for daptomycin to be effective against bacteria. Furthermore, it occurred only when the peptide/lipid ratio exceeded a threshold value, which could be the basis of the minimal inhibitory concentration of daptomycin. In this first publication on the lipid extracting effect, we characterize its dependence on ions and lipid compositions. We also discuss possibilities for connecting the lipid extracting effect to the antibacterial activity of daptomycin.

Membrane permeability of hydrocarbon-cross-linked peptides.

Schafmeister, Po, and Verdine (another study) introduced a method using a hydrocarbon linker (staple) to stabilize a peptide in a helical configuration. One intended goal of this scheme is to facilitate the delivery of peptide drugs into target cells. Here, we investigate whether stapled peptides are intrinsically membrane permeable, by performing a case study on a stapled 12-mer peptide named NYAD-1. We found that the native peptide CAI (an HIV-1 inhibitor) does not bind to lipid bilayers, however NYAD-1 indeed permeates through lipid bilayers even at low solution concentrations. To understand the reason for the membrane permeability, we investigated the physical properties of NYAD-1 as a function of bound peptide/lipid molar ratio P/L. We found that NYAD-1 spontaneously binds to a lipid bilayer. At low P/L, the peptide primarily binds on the polar-apolar interface with its helical axis parallel to the bilayer, which has the effect of stretching the membrane area and thinning the membrane. The membrane thinning reaches its maximum at P/L ∼1/15-1/12 in DOPC bilayers. Additional bound peptides have little thinning effect and their helical axes are normal to the plane of bilayers. Thus, the stapled peptide has a membrane interaction behavior similar to helical antimicrobial peptides, such as magainin and melittin. We emphasize that not all peptides that bind to lipid bilayers in the α-helical form behave this way.

How type II diabetes-related islet amyloid polypeptide damages lipid bilayers.

A leading hypothesis for the decimation of insulin-producing ß-cells in type 2 diabetes attributes the cause to islet amyloid polypeptide (IAPP) for its deleterious effects on the cell membranes. This idea has produced extensive investigations on human IAPP (hIAPP) and its interactions with lipid bilayers. However, it is still difficult to correlate the peptide-lipid interactions with its effects on islet cells in culture. The hIAPP fibrils have been shown to interact with lipids and damage lipid bilayers, but appear to have no effect on islet cells in culture. Thus, a modified amyloid hypothesis assumes that the toxicity is caused by hIAPP oligomers, which are not preamyloid fibrils or protofibrils. However, so far such oligomers have not been isolated or identified. The hIAPP monomers also bind to lipid bilayers, but the mode of interaction is not clear. Here, we performed two types of experiments that, to our knowledge, have not been done before. We used x-ray diffraction, in conjunction with circular dichroism measurement, to reveal the location of the peptide bound to a lipid bilayer. We also investigated the effects of hIAPP on giant unilamellar vesicles at various peptide concentrations. We obtained the following qualitative results. Monomeric hIAPP binds within the headgroup region and expands the membrane area of a lipid bilayer. At low concentrations, such binding causes no leakage or damage to the lipid bilayer. At high concentrations, the bound peptides transform to ß-aggregates. The aggregates exit the headgroup region and bind to the surface of lipid bilayers. The damage by the surface bound ß-aggregates depends on the aggregation size. The initial aggregation extracts lipid molecules, which probably causes ion permeation, but no molecular leakage. However, the initial ß-aggregates serve as the seed for larger fibrils, in the manner of the Jarrett-Lansbury seeded-polymerization model, that eventually disintegrate lipid bilayers by electrostatic and hydrophobic interactions.

Transmembrane pores formed by human antimicrobial peptide LL-37.

Human LL-37 is a multifunctional cathelicidin peptide that has shown a wide spectrum of antimicrobial activity by permeabilizing microbial membranes similar to other antimicrobial peptides; however, its molecular mechanism has not been clarified. Two independent experiments revealed LL-37 bound to membranes in the α-helical form with the axis lying in the plane of membrane. This led to the conclusion that membrane permeabilization by LL-37 is a nonpore carpet-like mechanism of action. Here we report the detection of transmembrane pores induced by LL-37. The pore formation coincided with LL-37 helices aligning approximately normal to the plane of the membrane. We observed an unusual phenomenon of LL-37 embedded in stacked membranes, which are commonly used in peptide orientation studies. The membrane-bound LL-37 was found in the normal orientation only when the membrane spacing in the multilayers exceeded its fully hydrated value. This was achieved by swelling the stacked membranes with excessive water to a swollen state. The transmembrane pores were detected and investigated in swollen states by means of oriented circular dichroism, neutron in-plane scattering, and x-ray lamellar diffraction. The results are consistent with the effect of LL-37 on giant unilamellar vesicles. The detected pores had a water channel of radius 23-33 Å. The molecular mechanism of pore formation by LL-37 is consistent with the two-state model exhibited by magainin and other small pore-forming peptides. The discovery that peptide-membrane interactions in swollen states are different from those in less hydrated states may have implications for other large membrane-active peptides and proteins studied in stacked membranes.

Adhesion and merging of lipid bilayers: a method for measuring the free energy of adhesion and hemifusion.

Lipid bilayers can be induced to adhere to each other by molecular mediators, and, depending on the lipid composition, such adhesion can lead to merging of the contacting monolayers in a process known as hemifusion. Such bilayer-bilayer reactions have never been systematically studied. In the course of our studies of membrane-active molecules, we encountered such reactions. We believe that they need to be understood whenever bilayer-bilayer interactions take place, such as during membrane fusion. For illustration, we discuss three examples: spontaneous adhesion between phospholipid bilayers induced by low pH, polymer-induced osmotic depletion attraction between lipid bilayers, and anionic lipid bilayers cross-bridged by multicationic peptides. Our purpose here is to describe a general method for studying such interactions. We used giant unilamellar vesicles, each of which was aspirated in a micropipette so that we could monitor the tension of the membrane and the membrane area changes during the bilayer-bilayer interaction. We devised a general method for measuring the free energy of adhesion or hemifusion. The results show that the energies of adhesion or hemifusion of lipid bilayers could vary over 2 orders of magnitude from -1 to -50 × 10(-5) J/m(2) in these examples alone. Our method can be used to measure the energy of transition in each step of lipid transformation during membrane fusion. This is relevant for current research on membrane fusion, which focuses on how fusion proteins induce lipid transformations.

Kinetic process of beta-amyloid formation via membrane binding.

Recently we have studied thermodynamics of membrane-mediated beta-amyloid formation in equilibrium experiments using penetratin-lipid mixtures. The results showed that penetratin bound to the membrane interface in the alpha-helical conformation when the peptide/lipid (P/L) ratios were below a lipid-dependent critical value P/L*. When P/L reached P/L*, small beta-aggregates emerged, which served as the nuclei for large beta-aggregates. Here we studied the corresponding kinetic process to understand the potential barriers for the membrane-mediated beta-amyloid formation. We performed kinetic experiments using giant unilamellar vesicles made of 7:3 DOPC/DOPG. The observed time behavior of individual giant unilamellar vesicles, although complex, exhibited the physical effects seen in equilibrium experiments. Most interestingly, a potential barrier appeared to block penetratin from translocating across the bilayer. As a result, the kinetic value for the critical threshold P/L* is roughly one-half of the value measured in equilibrium where peptides bind symmetrically on both sides of lipid bilayers. We also investigated the similarity and differences between the charged and neutral lipids in their interactions with penetratin. We reached an important conclusion that the bound states of peptides in lipid bilayers are largely independent of the charge on the lipid headgroups.

Membrane-mediated peptide conformation change from alpha-monomers to beta-aggregates.

Jarrett and Lansbury's nucleation-dependent polymerization model describes the generic process of beta-amyloid formation for a large number of diverse proteins and peptides. Here, we discuss a case of membrane-mediated nucleation that leads to beta-aggregation. We studied the membrane-mediated conformation changes of the peptide penetratin, and the results of our study led us to a free-energy description for a membrane-mediated version of the Jarrett-Lansbury model. Like the prototype beta-amyloid peptide Alzheimer's Abeta 1-40, penetratin is a random-coil monomer in solution but changes to alpha-helical or beta-like conformations in the presence of anionic lipid membranes. We measured the correlations between the membrane-bound conformation of penetratin and its effect on the bilayer thickness in four different lipids with various degrees of chain unsaturation. We found a new lipid chain effect on peptide conformation. Our results showed that the interface of a lipid bilayer provided energetically favorable binding sites for penetratin in the alpha-helical form. However, increasing the bound molecules/lipid ratio elevated the energy level of the bound states toward a higher level that favored creation of small beta-aggregates. The binding to the beta-aggregate became more energetically favorable as the aggregate grew larger. The peptide aggregates were visible on the surface of giant unilamellar vesicles. Thus, membrane binding facilitates nucleation-dependent beta-aggregation, which could be the prototype for the general membrane-mediated pathway to beta-amyloid formation.

Interaction of tea catechin (-)-epigallocatechin gallate with lipid bilayers.

A major component of green tea extracts, catechin (-)-Epigallocatechin gallate (EGCg), has been reported to be biologically active and interacting with membranes. A recent study reported drastic effects of EGCg on giant unilamellar vesicles (GUVs). In particular, EGCg above 30 microM caused GUVs to burst. Here we investigated the effect of EGCg on single GUVs at lower concentrations, believing that its molecular mechanism would be more clearly revealed. We used the micropipette aspiration method, by which the changes of surface area and volume of a GUV could be measured as a result of interaction with EGCg. We also used x-ray diffraction to measure the membrane thinning effect by EGCg. To understand the property of EGCg, we compared its effect with other membrane-active molecules, including pore-forming peptide magainin, the turmeric (curry) extract curcumin, and detergent Triton X100. We found the effect of EGCg somewhat unique. Although EGCg readily binds to lipid bilayers, its membrane area expansion effect is one order of magnitude smaller than curcumin. EGCg also solubilizes lipid molecules from lipid bilayers without forming pores, but its effect is different from that of Triton X100.

The bound states of amphipathic drugs in lipid bilayers: study of curcumin.

Drug-membrane interactions are well known but poorly understood. Here we describe dual measurements of membrane thickness change and membrane area change due to the binding of the amphipathic drug curcumin. The combined results allowed us to analyze the binding states of a drug to lipid bilayers, one on the water-membrane interface and another in the hydrocarbon region of the bilayer. The transition between the two states is strongly affected by the elastic energy of membrane thinning (or, equivalently, area stretching) caused by interfacial binding. The data are well described by a two-state model including this elastic energy. The binding of curcumin follows a common pattern of amphipathic peptides binding to membranes, suggesting that the binding states of curcumin are typical for amphipathic drugs.

Membrane-thinning effect of curcumin.

Interaction of curcumin with lipid bilayers is not well understood. A recent experiment showed that curcumin significantly affected the single-channel lifetime of gramicidin in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer without affecting its single-channel conductance. We performed two experiments to understand this result. By isothermal titration calorimetry, we measured the partition coefficient of curcumin binding to DOPC bilayers. By x-ray lamellar diffraction, we measured the thickness change of DOPC bilayers as a function of the curcumin/lipid ratio. A nonlinear membrane-thinning effect by curcumin was discovered. The gramicidin data were qualitatively interpreted by the combination of isothermal titration calorimetry and x-ray results. We show that not only does curcumin thin the lipid bilayer, it might also weaken its elasticity moduli. The result implies that curcumin may affect the function of membrane proteins by modifying the properties of the host membrane.

Simple HPLC-UV determination of nucleosides and its application to the authentication of Cordyceps and its allies.

A simple HPLC-UV method combined with a simple extraction procedure of nucleosides (adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine) was developed and applied to the authentication of Cordyceps and its allies. The separation was performed on a C(18) column by isocratic elution with acetonitrile-water, and UV detection at 260 nm. The amounts of adenosine, cordycepin, 2'-deoxyadenosine, guanosine and uridine in Cordyceps were 0.28-14.15, 0.006-6.36, 0.01-0.14, 0.68-14.79 and 0.19-20.29 mg/g, respectively. Among the nucleosides studied, cordycepin was characteristically included in Cordyceps militaris (L.) Link. (CM), which is one of key Cordyceps allies, and might be a good marker for authenticating CM. The ratio of nucleosides to adenosine contents in Cordyceps seemed to be a useful marker for authentication and quality control of Cordyceps.

Combination of multiphoton and reflective confocal imaging of cornea.

We combine reflective confocal microscopy with multiphoton microscopy to form a minimally invasive technique to observe the cornea. The two imaging modalities allow detection of complementary information from the cornea. The autofluorescence signal shows the cytoplasm of epithelial cells, and the second harmonic generation signal is used to detect collagen, found mostly in the stroma of the cornea. The reflective confocal imaging allows detection of epithelial cells and keratocytes in the stroma. The system is first tested on bovine cornea. Assessment of the result on the bovine eye will be used to evaluate the potential of the system as a technique for in vivo clinical application.

Imaging tissue engineering scaffolds using multiphoton microscopy.

In this study, we combined two-photon autofluorescence and second harmonic generation imaging to investigate the three-dimensional microstructure and nonlinear optical properties of tissue engineering scaffolds. We focused on five different types of scaffold materials commonly used in tissue engineering, including: open-cell polylactic acid, polyglycolic acid, collagen composite scaffold, collagraft bone graft matrix strip, and nylon. By the use of multiphoton microscopy and a motorized stage, we obtained high resolution, spectrally resolved structural information of the scaffolds over large areas or in three-dimensions. Our results show that the nonlinear optical properties of the scaffolds will enable us to spectrally and morphologically distinguish the different types of scaffold materials investigated. We envision multiphoton microscopy to be a useful technique in tissue engineering applications in understanding the interplay between cultured cells and the scaffold materials.