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Serum proteins affect the in vivo fate and cellular uptake of arginine-rich cell-penetrating peptides (CPPs) and drugs delivered by CPPs. Although the binding of CPPs to serum proteins may possibly reduce their cellular uptake to some extent, it may also prolong their circulation half-life in vivo. We aimed to identify novel binding proteins of arginine-rich CPPs in serum to better understand their in vivo fate and develop more sophisticated drug delivery systems using CPPs. Isothermal titration calorimetry analysis suggests that albumin, the most abundant protein in serum, binds to d-forms of oligoarginine; however, the dissociation constants are several tens of a micromolar. Candidate proteins with the potential of binding to arginine-rich CPPs in serum were then explored using nondenaturing polyacrylamide gel electrophoresis followed by mass spectrometry analysis. Studies using fluorescence correlation spectroscopy determined hemopexin as a potential binding partner of d-forms of arginine-rich CPPs, including d-octaarginine (

ObjectiveTo establish a metagenomic profiling method using long-read sequencing for clinical diagnosis of ocular inflammation and detect the etiologic virus of herpetic uveitis.DesignA retrospective, cross-sectional study.ParticipantsThe participants were 44 uveitis patients with a suspected infectious etiology and 22 controls with cataract.MethodsThe anterior aqueous humor (10-20 µl) was subjected to DNA purification, followed by whole genome amplification. The Nanopore MinION™ using the Flongle Flow Cell was used to perform rapid long-read sequencing and the phylogenetic composition of the microorganisms in the specimen was evaluated.Main Outcomes and MeasuresThe detection of the DNA sequence reads of the etiologic virus of herpetic uveitis in the generated FASTQ files from nanopore sequencing and the evaluation of the limits of detection (LOD) of metagenomic analysis compared to multiplex polymerase chain reaction (mPCR) testing for etiologic virus detection of herpetic uveitis.ResultsThe detection rate of nanopore metagenomic analysis was approximately 59.0% as a result of validation against 22 mPCR-positive cases. The LOD was between 103.6and 106copies of virus DNA. The undetectable cases tended to have significantly lower copy numbers by mPCR, suggesting the lower metagenomic analysis sensitivity compared to mPCR. The nine pathogenic microorganisms evaluated by mPCR were also not detected by nanopore in all mPCR-negative cases and controls. The minimum time to obtain analysis results using this method was approximately 190 minutes.Conclusions and RelevanceOur established sequencing protocol from the anterior aqueous humor detected the DNA fragments of etiologic viruses in patients with herpes virus uveitis. Conversely, nanopore metagenomic results contained considerable noise and were found to be less sensitive compared to the conventional mPCR tests for ocular infections.

ImportanceTorque teno virus positivity in the aqueous humor of uveitis patients could be associated with systemic immunodeficiency.ObjectiveWe explored the correlation between the presence of torque teno virus in the aqueous humor of uveitis patients and clinical information, including immunodeficiency history.DesignThis was a retrospective, cross-sectional study.SettingAll participants were recruited at Nagoya University Hospital, Tokyo Medical University Hospital, or Yokkaichi Municipal Hospital between April 2017 and March 2022.ParticipantsThe participants were 58 uveitis patients with a suspected infectious etiology and 24 controls with cataract or age-related macular degeneration.Main Outcomes and MeasuresWe used quantitative polymerase chain reaction to test all subjects for torque teno virus and multiplex polymerase chain reaction to test uveitis subjects for common ocular pathogens. When possible, both serum and aqueous humor samples were tested. Ocular torque teno virus positivity was compared with age, sex, and a history of systemic immunodeficiency with logistic analysis.ResultsTorque teno virus positivity was found in 7 of 31 cases (23%) with herpetic uveitis, 3 of 27 cases (11%) with nonherpetic uveitis, and 0 of 24 controls (0%). Among patients with herpes infection, positivity for torque teno virus was found in 3 of 7 patients (43%) with cytomegalovirus retinitis, 1 of 12 (8%) patients with iridocyclitis, 1 of 7 patients (14%) with acute retinal necrosis, and 2 of 4 patients (50%) with Epstein–Barr virus–related uveitis. Cytomegalovirus retinitis patients showed a significantly higher rate of ocular torque teno virus infection than controls (P= .008). Serum analysis revealed torque teno virus positivity in 9 of 10 cases (90%) with uveitis and in all 8 controls (100%). Age- and gender-adjusted logistic analysis revealed a correlation between ocular torque teno virus positivity and systemic immunodeficiency (P= .01), but no correlations between ocular torque teno virus and age, gender, or viral pathogenic type.Conclusion and RelevanceThis study found that positivity for ocular torque teno virus was correlated with a clinical history of systemic immunodeficiency. This suggests that ocular torque teno virus is a biomarker of systemic immunity.Key pointsQuestionWhat is the correlation between torque teno virus (TTV) in the aqueous humor of patients with uveitis and the clinical characteristics of these patients?FindingsIn this retrospective, cross-sectional study that included 82 patients, ocular TTV was found to be present in 39% of uveitis patients with underlying immunodeficiency and 8% of patients without underlying immunodeficiency; these findings showed statistical significance.MeaningIn patients with uveitis, the presence of ocular TTV may represent systemic immunodeficiency.

Extracellular vesicles (EVs) have emerged as important targets in biological and medical studies because they are involved in diverse human diseases and bacterial pathogenesis. Although antibodies targeting the surface biomarkers are widely used to detect EVs, peptide-based curvature sensors are currently attracting an attention as a novel tool for marker-free EV detection techniques. We have previously created a curvature-sensing peptide, FAAV and applied it to develop a simple and rapid method for detection of bacterial EVs in cultured media. The method utilized the fluorescence/Förster resonance energy transfer (FRET) phenomenon to achieve the high sensitivity to changes in the EV amount. In the present study, to develop a practical and easy-to-use approach that can detect bacterial EVs by peptides alone, we designed novel curvature-sensing peptides, N-terminus-substituted FAAV (nFAAV) peptides. The nFAAV peptides exerted higher α-helix-stabilizing effects than FAAV upon binding to vesicles while maintaining a random coil structure in aqueous solution. One of the nFAAV peptides showed a superior binding affinity for bacterial EVs and detected changes in the EV amount with 5-fold higher sensitivity than FAAV even in the presence of the EV-secretory bacterial cells. We named nFAAV5, which exhibited the high ability to detect bacterial EVs, as an EV-sensing peptide. Our finding is that the coil-α-helix structural transition of the nFAAV peptides serve as a key structural factor for highly sensitive detection of bacterial EVs.

We demonstrate a fluid-fluid phase separation in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes using a metal complex lipid of type [Mn(L1)] (1; HL1 = 1-(2-hydroxybenzamide)-2-(2-hydroxy-3-formyl-5-hexadecyloxybenzylideneamino)ethane. Small amount of 1 produces two separated domains in DMPC, whose phase transition temperatures of lipids (Tc) are both lower than that of the pristine DMPC. Variable temperature fluorescent microscopy for giant-unilamellar vesicles of DMPC/1 hybrids demonstrates that visible phase separations remain in fluid phases up to 37 °C, which is clearly over the Tc of DMPC. This provides a new dimension for the application of metal complex lipids toward controlling lipid distributions in fluid membranes.

Endocytic pathways are practical routes for the intracellular delivery of biomacromolecules. Along with this, effective strategies for endosomal cargo release into the cytosol are desired to achieve successful delivery. Focusing on compositional differences between the cell and endosomal membranes and the pH decrease within endosomes, we designed the lipid-sensitive and pH-responsive endosome-lytic peptide HAad. This peptide contains aminoadipic acid (Aad) residues, which serve as a safety catch for preferential permeabilization of endosomal membranes over cell membranes, and His-to-Ala substitutions enhance the endosomolytic activity. The ability of HAad to destabilize endosomal membranes was supported by model studies using large unilamellar vesicles (LUVs) and by increased intracellular delivery of biomacromolecules (including antibodies) into live cells. Cerebral ventricle injection of Cre recombinase with HAad led to Cre/loxP recombination in a mouse model, thus demonstrating potential applicability of HAad in vivo.

Extracellular membrane vesicles (EMVs) are biogenic secretory lipidic vesicles that play significant roles in intercellular communication related to human diseases and bacterial pathogenesis. They are being investigated for their possible use in diagnosis, vaccines, and biotechnology. However, the existing methods suffer from a number of issues. High-speed centrifugation, a widely used method to collect EMVs, may cause structural artifacts. Immunostaining methods require several steps and thus the separation and detection of EMVs from the secretory cells is time-consuming. Furthermore, detection of EMVs using these methods requires specific and costly antibodies. To tackle these problems, development of a simple and rapid detection method for the EMVs in the cultured medium without separation from the secretory cells is a pressing task. In this study, we focused on the Gram-negative bacterium Shewanella vesiculosa HM13, which produces a large amount of EMVs including a cargo protein with high purity, as a model. Curvature-sensing peptides were used for EMV-detection tools. FAAV, a peptide derived from sorting nexin protein 1, selectively binds to the EMVs even in the presence of the secretory cells in the complex cultured medium. FAAV can fully detect the EMVs within a few minutes, and the resistance of FAAV to proteases enables it to withstand prolonged use in the cultured medium. Fluorescence/Förster resonance energy transfer was used to develop a method to detect changes in the amount of the EMVs with high sensitivity. Overall, our results indicate the potential applicability of FAAV for in situ EMV detection in cultured media.

Postoperative health care-associated infections are associated with a greater deterioration in patients' general health status and social and economic burden, with at least 1 occurring in approximately 4% of acute care hospital patients. Antimicrobial prophylaxis prevents surgical site infections in various orthopedic procedures; however, its relationship with health care-associated infections remains unknown.To examine whether a shorter antimicrobial prophylaxis duration of less than 24 hours after surgery is not inferior to a longer duration in preventing health care-associated infections after clean orthopedic surgery.This open-label, multicenter, cluster randomized, noninferiority clinical trial was conducted in 5 tertiary referral hospitals in greater Tokyo metropolitan area, Japan, from May to December 2018. Adult patients undergoing clean orthopedic surgery were recruited until the planned number of participants was achieved (500 participants per group). Statistical analysis was conducted from July to December 2019.Antimicrobial prophylaxis was discontinued within 24 hours after surgery in group 24 and 24 to 48 hours after surgery in group 48. Group allocation was switched every 2 or 4 months according to the facility-based cluster rule. Study-group assignments were masked from participants.The primary outcome was the incidence of health care-associated infections requiring antibiotic therapies within 30 days after surgery. The noninferiority margin was 4%.Of the 1211 participants who underwent cluster allocation, 633 participants were in group 24 (median [IQR] age, 73 [61-80] years; 250 men [39.5%] and 383 women [60.5%]), 578 participants were in group 48 (median [IQR] age, 74 [62-81] years; 204 men [35.3%] and 374 women [64.7%]), and all were eligible for the intention-to-treat analyses. Health care-associated infections occurred in 29 patients (4.6%) in group 24 and 38 patients (6.6%) in group 48. Intention-to-treat analyses showed a risk difference of -1.99 percentage points (95% CI, -5.05 to 1.06 percentage points; P .001 for noninferiority) between groups, indicating noninferiority. Results of adjusted intention-to-treat, per-protocol, and per designated procedure population analyses supported this result, without a risk of antibiotic resistance and prolonged hospitalization.This cluster randomized trial found noninferiority of a shorter antimicrobial prophylaxis duration in preventing health care-associated infections without an increase in antibiotic resistance risk. These findings lend support to the global movement against antimicrobial resistance and provide additional information on adequate antimicrobial prophylaxis for clean orthopedic surgery.Identifier: UMIN000030929.

V-shaped xanthene dyes capable of predicting absorption and emission wavelengths are described. These dyes were synthesized by bridging a xanthene ring and an aryl moiety of fluorescein through ether covalent bonds. These dyes showed longer absorption and emission wavelengths than those of the parent fluorescein. Furthermore, substituents introduced on the aryl moiety mainly affected the lowest unoccupied molecular orbital energy level of the molecule. Therefore, the Hammett substituent constants could be used to predict the absorption and emission wavelengths of the compound.

Background: The precise etiology of carpal tunnel syndrome (CTS) remains unclear. One of the accepted factors for CTS is the restriction of the median nerve. Previous reports using ultrasound had only observed and measured the movement of parts of the median nerve. In this study, we aimed to elucidate the difference in the movement of the entire median nerve in patients with CTS (before and after surgery) and healthy volunteers using a new measurement method. Methods: We expressed the amount of movement of the entire nerve by a new method creating the motion area of the median nerve (MAMn) from an ultrasonographic video image on the computer. We compared the MAMn, the real MAMn (RMMn) (the value obtained by subtracting the nerve cross-sectional area from the MAMn), and mobile ratio (MR) (the value obtained from dividing the MAMn by the nerve cross-sectional area) between six wrists of six cases of CTS (before and at an average of 3.5 months after surgery) and six wrists of six healthy volunteers. Results: During passive wrist flexion, the average MAMn, RMMn, and MR of healthy cases were 23.1 mm2, 16.4 mm2, and 3.52, respectively. The average MAMn, RMMn, and MR of cases of CTS were respectively 11.8 mm2, 5.4 mm2, and 1.86 preoperatively; and 16.2 mm2, 7.3 mm2, and 1.87, postoperatively. The MAMn, RMMn, and MR decreased more significantly in patients with CTS than in healthy volunteers (p < 0.01). The MAMn and RMMn increased postoperatively (p < 0.05), but the MR remained low. Conclusions: The new measurement method revealed that the mobility of the entire median nerve was significantly restricted in cases of CTS compared to healthy participants. However, after surgery, nerve restriction was not restored despite improvements in symptoms, suggesting that decreases in nerve mobility contribute to CTS but are not a direct cause of symptoms.

Membrane curvature formation is important for various biological processes such as cell motility, intracellular signal transmission, and cellular uptake of foreign substances. However, it remains still a challenging topic to visualize the membrane curvature formation on the cell membranes in real-time imaging. To develop and design membrane curvature-sensors, we focused on amphipathic helical peptides of proteins belonging to the Bin/Amphiphysin/Rvs (BAR) family as the starting point. BAR proteins individually have various characteristic structures that recognize different curvatures, and the derived peptides possess the potential to function as curvature sensors with a variety of recognition abilities. Peptide-based curvature sensors can have wide applications in biological research fields due to their small size, easy modification, and large production capability in comparison to protein-based sensors. In the present study, we found that an amphipathic peptide derived from sorting nexin1 (SNX1) has a curvature-recognition ability. The mutation studies of the initial peptide revealed a close correlation between the α-helicity and lipid binding ability of the peptides. In particular, the amino acids located on the hydrophobic face played a vital role in curvature recognition. The α-helix formation of the peptides was thought to serve to accommodate lipid-packing defects on the membrane surface and to maintain their binding to lipid vesicles. The structure-activity correlation found in this study have the potential to contribute to the design of peptide-based curvature sensors that will enable the capture of various life phenomena in cells.

Lipid packing has a strong influence on the formation and structural dynamics of cell membranes. Techniques to modulate lipid packing may thus enable modification of cellular functions and events. An 18-residue amphiphilic helical peptide derived from the N-terminal segment of epsin-1 (EpN18) is reported to induce positive membrane curvature and to loosen lipid packing in the cell membrane. In this study, it is shown that EpN18, crosslinked to a leucine-zipper peptide K4, is recruited to the cell surface by interacting with a cell-surface-expressed E3 leucine-zipper segment. Cell-surface tethering markedly enhanced loosening of lipid packing, which led to the promotion of membrane translocation of octaarginine. The loosening of lipid packing by EpN18 was also confirmed by analyzing the generalized polarization value with a membrane-environment-sensitive dye, 2-hydroxy-3-{2-[(2-hydroxyethyl)dimethylamino]ethyl}-4-{2-[6-(dibutylamino)-2-naphthyl]ethenyl}pyridiniumdibromide (di-4-ANEPPDHQ). This approach thus shows promise for the control of lipid packing and related cellular events.

Unitary operator discrimination is a fundamental problem in quantum information theory. The basic version of this problem can be described as follows: given a black box implementing a quantum operator U, and the promise that the black box implements either the unitary operator \(U_1\) or the unitary operator \(U_2\), the goal is to decide whether \(U=U_1\) or \(U=U_2\). In this paper, we consider the query complexity of this problem. We show that there exists a quantum algorithm that solves this problem with bounded-error probability using \(\left\lceil \frac{\pi }{3\theta _\mathrm{cover}} \right\rceil \) queries to the black-box, where \(\theta _\mathrm{cover}\) represents the “closeness” between \(U_1\) and \(U_2\) (this parameter is determined by the eigenvalues of the matrix \(U_1^\dag U_2\)). We also show that this upper bound is essentially tight: we prove that there exist operators \(U_1\) and \(U_2\) such that any quantum algorithm solving this problem with bounded-error probability requires at least \(\left\lceil \frac{2}{3\theta _\mathrm{cover}} \right\rceil \) queries.

EpN18 is a curvature-inducing peptide, which loosens lipid packing upon interaction with the cell membrane, and facilitates cell-membrane penetration by arginine-rich cell-penetrating peptides, including octaarginine (R8). In the present study, we conjugated the N-terminal of EpN18 with a pyrenebutyryl (pBu) moiety, which acts as an anchoring unit that increases membrane interactions. Enhanced lipid-packing loosening and cytosolic translocation of R8 were observed by the pBu anchoring of EpN18.

Cell-penetrating peptides (CPPs) are promising delivery vehicles. These short peptides can transport wide range of cargos into cells, although their usage has often limitations. One of them is the endosomatic internalisation and thus the vesicular entrapment. Modifications which increases the direct delivery into the cytosol is highly researched area. Among the oligoarginines the longer ones (n > 6) show efficient internalisation and they are well-known members of CPPs. Herein, we describe the modification of tetra- and hexaarginine with (4–((4–(dimethylamino)phenyl)azo)benzoyl) (Dabcyl) group. This chromophore, which is often used in FRET system increased the internalisation of both peptides, and its effect was more outstanding in case of hexaarginine. The modified hexaarginine may enter into cells more effectively than octaarginine, and showed diffuse distribution besides vesicular transport already at low concentration. The attachment of Dabcyl group not only increases the cellular uptake of the cell-penetrating peptides but it may affect the mechanism of their internalisation. Their conjugates with antitumor drugs were studied on different cells and showed antitumor activity. Supplementary Information The online version contains supplementary material available at 10.1007/s00726-021-03003-w.

Case Axillary nerve rupture without shoulder joint fracture or dislocation in contact sports is very rare. To date, there has been no detailed report on such cases. We present 2 rare cases of axillary nerve rupture in contact sports who were successfully treated with free nerve grafting. Conclusion In contact sports, the deltoid muscle is sometimes paralyzed temporarily after a collision. However, similar to our cases, the axillary nerve can be lacerated without fracture or dislocation. It is necessary to watch the course of paralysis carefully and consider nerve reconstruction if it does not recover.

Cell membranes contain lateral systems that consist of various lipid compositions and actin cytoskeleton, providing two-dimensional (2D) platforms for chemical reactions. However, such complex 2D environments have not yet been used as a synthetic platform for artificial 2D nanomaterials. Herein, we demonstrate the direct synthesis of 2D coordination polymers (CPs) at the liquid-cell interface of the plasma membrane of living cells. The coordination-driven self-assembly of networking metal complex lipids produces cyanide-bridged CP layers with metal ions, enabling "pseudo-membrane jackets" that produce long-lived micro-domains with a size of 1-5 μm. The resultant artificial and visible phase separation systems remain stable even in the absence of actin skeletons in cells. Moreover, we show the cell application of the jackets by demonstrating the enhancement of cellular calcium response to ATP.

Marked promotion of membrane permeation of a cell-penetrating peptide, octaarginine (R8), was attained by attachment to a single 2,2'-dipicolylamine moiety (DPA-R8) that forms 1:1 complexes with metal ions. Studies using giant unilamellar vesicles demonstrated that DPA targets phospholipids and enhances R8 binding to the membranes in the presence of metal ions. While DPA/Zn(II) complex has been most frequently employed for chelate formation with phosphates, Ni(II) had the most prominent effect on the membrane binding and penetration of DPA-R8. Facile cytosolic distribution of DPA-R8 was also attained in a few minutes in the presence of Ni(II). Analysis of the cellular uptake methods of DPA-R8/Ni(II) suggested the involvement of direct permeation through cell membrane without the use of endocytosis. The applicability of this system to the intracellular delivery of bioactive compounds was exemplified using a peptidomimetic farnesyltransferase inhibitor, FTI277.

Suffusion or internal instability is a major form of internal erosion in cohesionless soils. Hypotheses based on experimental work suggest complex micro-scale influences of stress and material fabric on suffusion. This study presents the results of coupled discrete element method – computational fluid dynamics (DEM-CFD) simulations of permeameter tests. These simulations were carried out to study the influence of micro-scale variables on the initiation of particle transport in gap-graded cohesionless soils with varying fines contents and relative densities. The results highlight the importance of both particle stress and connectivity on the initiation of suffusion. Particles which do not transfer externally applied stress and those with low initial connectivity are shown to be particularly susceptible to suffusion.

Fluorescent dyes possessing a variety of arylacetylenes at the 9-position of a xanthene skeleton were synthesized and their optical properties were investigated. The π system effectively expanded over the xanthene skeleton and the aryl group through the triple bond. Starting from the emission wavelength (λem) of 9-methyl xanthene 20 in basic DMSO solution at 536 nm, the emission wavelengths gradually shifted to the red region for methylacetylene 17 (λem = 600 nm), phenylacetylene 5 (λem = 636 nm), and p-CF3-phenylacetylene 11 (λem = 660 nm). On the basis of these data, we estimated the substituent effects for the red shift on the emission wavelength and rationally explained the results by DFT calculations. Furthermore, potential applicability of these fluorescent dyes to cell staining was exemplified.

Purpose: To determine whether the macula is displaced after vitrectomy with internal limiting membrane (ILM) peeling in eyes with an idiopathic macular hole. Design: A retrospective, observational case series. Methods: Optical coherence tomography angiography was used to obtain 3 mm × 3 mm en face images before and 2, 4, and 8 weeks after the vitrectomy with ILM peeling for 20 eyes of 20 patients with an idiopathic macular hole. The displacements of easily identifiable retinal vessel bifurcations were measured relative to the fovea and the optic disc. Results: The distance between the optic disc and vessel bifurcations was significantly shorter in all 4 quadrants throughout the postoperative period (P < .001). This distance was significantly greater in the temporal quadrant (P < .001). The distance of the bifurcations was significantly correlated with the preoperative distance from the optic disc (r = −0.579, P < .001). A significantly greater downward displacement was observed in the superior quadrant (P < .001). The change in the angle of bifurcations was significantly correlated with the preoperative angle to the optic disc (r = −0.632, P < .001). The change in the distance in the inner region was significantly greater than that in the outer region in all quadrants. A significant reduction was observed in the mean foveal avascular zone area during the follow-up period. Conclusions: The retina in the macular region was displaced nasally, probably owing to movement of the retina toward the optic disc because of a contraction after the ILM removal. There is also a possibility of a rotation downward by buoyancy from gas tamponade, and centripetal contraction during the process of hole closure., ファイル公開：2019-05-01

New V-shaped xanthene dyes 3–6 containing piperidino groups at the resonance sites on their skeletons were constructed as second-generation V-shaped dyes and their functions were investigated. The non-symmetric compound 5 had higher solubility and gave a higher fluorescence quantum yield and longer emission wavelength compared with those of previously reported V-shaped xanthene dyes with hydroxyl groups 1 and 2. Furthermore, compound 5 stained living cells, enabling bright fluorescence imaging.

G-protein-coupled receptors (GPCRs) form the largest family of transmembrane receptors, and their oligomerization has been suggested to be related to their functions. Despite extensive studies, their oligomeric states are highly controversial. One of the reasons is the overestimation of oligomerization by conventional methods. We recently established a stoichiometric analysis method for precisely determining the oligomeric state of membrane proteins on living cells with the combined use of the coiled-coil labeling method and a spectral imaging technique and showed that the prototypical class-A GPCR β2-adrenergic receptor (β2AR) did not form functional oligomers. In this study, we expanded our study to three well-studied class-A GPCRs: C-X-C chemokine receptor of stromal cell-derived factor-1α (CXCR4), dopamine receptor D2 short isotype (D2R), and prostaglandin E receptor subtype 1 (EP1R). We found that these receptors did not form constitutive homooligomers. The receptors exhibited calcium signaling upon agonist stimulation as monomers, although CXCR4 and EP1R gradually clustered after fast signaling. We conclude that homooligomerization is not necessary for the signal transductions of these four class-A GPCRs. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Purpose To investigate the regeneration of the cone outer segments in eyes after surgery for fovea-off rhegmatogenous retinal detachment with an adaptive optics (AO) fundus camera and to correlate these findings with the findings of optical coherence tomography (OCT). Design Retrospective, observational case series. Methods Medical charts of 21 eyes of 21 patients who had undergone surgery for fovea-off rhegmatogenous retinal detachment were retrospectively studied. Cone mosaic images were obtained with an AO fundus camera. Cone packing density at 2 degrees from the fovea within the previously detached area was measured 6 and 12 months after surgery. Retinal thicknesses between the interdigitation zone and the retinal pigment epithelium (IZ-RPE) and between the ellipsoid zone and the retinal pigment epithelium (EZ-RPE) were measured in OCT images. Results Cone density 12 months after surgery was significantly increased from that at 6 months ( P = .001), but was still significantly lower than that of normal fellow eyes ( P P = .045, P = .033, respectively), and these values were not significantly different from those of normal fellow eyes. Multivariate analysis showed that cone density at 12 months was significantly associated with IZ-RPE thickness ( P = .002), and increases in cone packing density were significantly associated with increases in IZ-RPE thickness ( P = .001). Conclusions Recovery of cone packing density measured by AO was associated with structural recovery of the outer retina observed in OCT, suggesting regeneration of the photoreceptor outer segment after surgery.

To evaluate the relationship between morphological changes and functional improvements assessed using focal macular electroretinograms after intravitreal aflibercept (IVA) injections in eyes with wet age-related macular degeneration. The clinical records of 42 eyes of 42 consecutive patients with naive, wet age-related macular degeneration received 3 monthly IVA were reviewed. The best-corrected visual acuity, central foveal thickness, outer retinal thickness, inner retinal thickness at baseline and 1 month after each IVA, and focal macular electroretinograms at baseline and 1 month after the first and third IVA were compared. Best-corrected visual acuity was improved after the third IVA (P = 0.0091). Central foveal thickness and outer retinal thickness showed decreases after every IVA (P < 0.001, respectively). Inner retinal thickness showed a decrease after the second IVA (P = 0.002), after and third IVA (P = 0.001). On focal macular electroretinograms, a- and b-wave amplitudes showed increases after the third IVA (P = 0.0028, P = 0.0012, respectively). Significant correlations were observed between best-corrected visual acuity and central foveal thickness, a-wave amplitude and outer retinal thickness, and b-wave amplitude and inner retinal thickness changes after the third IVA. All parameters significantly recovered after three monthly IVA, with a correlation between functional improvements and morphological changes.

To report the clinical course and high resolution images of autosomal recessive retinitis pigmentosa (RP) associated with a variant of the RP1 gene (c.4052_4053ins328/p.Tyr1352Alafs*9; m1), a high frequency founder variant in Japanese RP patients.; Retrospective case series.; Nine patients from 5 unrelated Japanese families were studied. Five patients had the m1 variant homozygously, and 4 patients had the m1 variant compound heterozygously with another frameshift variant (c.4196delG/p.Cys1399Leufs*5). Ophthalmic examinations including adaptive optics (AO) fundus imaging were performed periodically.; The fundus photographs, fundus autofluorescence (FAF) images, and optical coherence tomographic (OCT) images indicated severe retinal degeneration in all the patients involving the macula even at a young age (20 s). The areas of surviving photoreceptors in the central macula were seen as hyper-autofluorescent regions in the FAF images and preserved outer retinal structure in the OCT images; they were identifiable in the AO fundus images in 8 eyes. The borders of the surviving photoreceptor areas were surrounded by hyporeflective clumps, presumably containing melanin, and the size of these areas decreased progressively during the 4-year follow-up period. The disappearance of the surviving photoreceptor areas was associated with complete blindness.; Patients with RP associated with the m1 variant have a progressive and severe retinal degeneration that begins at an early age. Monitoring the surviving photoreceptor areas by AO fundus imaging can provide a more precise pathological record of retinal degeneration.

Purpose To report a novel finding in a case of idiopathic recurrent branch retinal arterial occlusions. Observations We report our findings in a 31-year-old woman with an idiopathic recurrent branch retinal arterial occlusions (BRAOs) who was followed in our clinic for 16 years. Repeated fluorescein angiographies were performed to evaluate the idiopathic recurrent BRAOs. The findings showed multiple segmental arteriolar staining in the fundus. These staining were found at locations with and without branch retinal arterial occlusions. Repeated fluorescein angiography showed that these staining migrated toward the periphery. The speed of the migration of the staining was about 0.64 ± 0.22 mm/day with a range of 0.43–0.85 mm/day in the posterior pole of the eye. In each of the BRAO, the staining did not cause branch retinal arterial occlusions in the peripheral retina again. Conclusions and importance Segmental arteriolar staining that migrates towards the periphery may be a clue to the etiology of this disease process.

Modulating the structural dynamics of biomembranes by inducing bilayer curvature and lipid packing defects has been highlighted as a practical tool to modify membrane-dependent cellular processes. Previously, we have reported on an amphipathic helical peptide derived from the N-terminal segment (residues 1-18, EpN18) of epsin-1, which can promote membrane remodeling including lipid packing defects in cell membranes. However, a high concentration is required to exhibit a pronounced effect. In this study, we demonstrate a significant increase in the membrane-remodeling effect of EpN18 by constructing a branched EpN18 homotrimer. Both monomer and trimer could enhance cell internalization of octaarginine (R8), a cell-penetrating peptide. The EpN18 trimer, however, promoted the uptake of R8 at an 80-fold lower concentration than the monomer. Analysis of the generalized polarization of a polarity-sensitive dye (di-4-ANEPPDHQ) revealed a higher efficacy of trimeric EpN18 in loosening the lipid packing in the cell membrane. Circular dichroism measurements in the presence of lipid vesicles showed that the EpN18 trimer has a higher α-helix content compared with the monomer. The stronger ability of the EpN18 trimer to impede negative bilayer curvature is also corroborated by solid-state 31P NMR spectroscopy. Hence, trimerizing peptides can be considered a promising approach for an exponential enhancement of their membrane-remodeling performance.

The epidermal growth factor receptor (EGFR) is a well-studied receptor tyrosine kinase and an important anticancer therapeutic target. The activity of EGFR autophosphorylation and transphosphorylation, which induces several cell signaling pathways, has been suggested to be related to its oligomeric state. However, the oligomeric states of EGFRs induced by EGF binding and the receptor–ligand stoichiometry required for its activation are still controversial. In the present study, we performed Förster resonance energy transfer (FRET) measurements by combining the coiled-coil tag–probe labeling method and spectral imaging to quantitatively analyze EGFR oligomerization on living CHO-K1 cell membranes at physiological expression levels. In the absence of its ligands, EGFRs mainly existed as monomers with a small fraction of predimers (~10%), whereas ~70% of the EGFRs formed dimers after being stimulated with the ligand EGF. Ligand-induced dimerization was not significantly affected by the perturbation of membrane components (cholesterol or monosialoganglioside GM3). We also investigated both dose and time dependences of EGF-dependent EGFR dimerization and autophosphorylation. The formation of dimers occurred within 20s of the ligand stimulation and preceded its autophosphorylation, which reached a plateau 90s after the stimulation. The EGF concentration needed to evoke half-maximum dimerization (~1nM) was lower than that for half-maximum autophosphorylation (~8nM), which suggested the presence of an inactive dimer binding a single EGF molecule.

All-hydrocarbon stapled peptides make up a promising class of protein-protein interaction regulators; their potential therapeutic benefit arises because they have a high binding affinity and specificity for intracellular molecules. The cell permeation efficacy of these peptides is a critical determinant of their bioactivity. However, the factors that determine their cellular uptake remain an active area of research. In this study, we evaluated the effect of stapled (or cross-linked) formation on the cellular uptake of six known all-hydrocarbon stapled peptides. We found that the rate of cellular uptake of unstapled peptides (i.e., those bearing olefinic non-natural amino acids that are not subjected to olefin metathesis) was higher than that for the corresponding stapled peptides. Additionally, the insertion of these olefinic non-natural amino acids into peptide sequences significantly increased their rate of cellular uptake. According to the high-performance liquid chromatography retention times, the overall hydrophobicity of unstapled peptides was greater than that of stapled peptides, followed by that of the original peptides without olefinic non-natural amino acids. There was not a close correlation between helical content and the rate of cellular uptake of these peptides. Therefore, the increase in overall hydrophobicity resulting from the introduction of non-natural amino acids, rather than the structural stabilization resulting from staple formation, is the key driver promoting cellular uptake. Macropinocytosis, a form of fluid-phase endocytosis, was involved in the cellular uptake of all six peptides.

Many membrane proteins are responsible for signaling and ionic transport necessary to maintain biological func- tions in vivo. Recently, not only conformational changes but also oligomerization have been proposed to regulate pro- tein activation. Thus, the study of membrane protein oligomerization is crucial for new drug development. The existing destructive methodologies such as immunoprecipitation, however, are not suitable to determine oligomeric states pre- cisely because of the articial aggregation of proteins after detergent solubilization. In the present study, the coiled-coil tag-probe labeling method and spectral imaging wererst combined to establish a new methodology based on ‰uores- cence resonance energy transfer (FRET) for stoichiometric analysis of the oligomeric states of membrane proteins on living cells. After validating the method for mono-, di-, and tetrameric standard membrane proteins, the oligomeric state of b2-adrenergic receptors (b2ARs) was examined to clarify its functional signicance. It was found that b2ARs could transduce cyclic adenosine 5' -monophosphate (cAMP) signals and internalize them upon treatment with ligands without showing any FRET signals. Thus, b2ARs do not form constitutive homooligomers, and homooligomerization is not necessary for the receptor function of b2ARs. Finally, the oligomeric state of full-length M2 proton-selective chan- nels of in‰uenza A virus was investigated. Although the results of X-ray crystallography and NMR studies using frag- ment peptides suggested that M2 stably forms a tetrameric channel, the full-length M2 proteins formed proton-conduct- ing dimers at neutral pH and these dimers were converted to tetramers at acidic pH, indicating that the minimal func- tional unit of the M2 channel is a dimer.

Genetic fusion of fluorescent/luminescent proteins to a target protein for specific labeling in living cells has been widely used to investigate the intracellular trafficking and oligomerization of the proteins. However, several limitations of fluorescent/luminescent proteins, such as considerable size, difficulty in controlling labeling ratio in multicolor labeling, can obscure true behaviors of the target proteins. To overcome these difficulties, post-translational labeling methods using pairs of small genetically-encodable 'tags' and synthetic 'probes' targeting the tags have been widely studied in recent years. We have developed a quick tag-probe labeling method using a high-affinity heterodimeric coiled-coil formation between the E3 tag (EIAALEK)3 attached to the target protein and the K4 probe (KIAALKE)4 labeled with a fluorophore. The labeling is cell-surface-specific and completed within 1 min, therefore suitable for monitoring oligomerization/internalization of membrane proteins on living cell surface. Taking advantage of easiness in multicolor labeling, we show that the oligomeric state of membrane proteins can be precisely analyzed based on fluorescence resonance energy transfer. By using this method, we found that β2 adrenergic receptors do not form constitutive homooligomers, and homooligomerization is not necessary for the receptor function. Furthermore, the degree of internalization of the β2 receptors following agonist stimulation was evaluated by ratiometric detection of pH decrease in endosomes.

To determine whether there is a displacement of the fovea toward the optic disc after successful macular hole (MH) surgery with internal limiting membrane (ILM) peeling. The medical records of 54 eyes of 53 patients that had undergone pars plana vitrectomy with ILM peeling and gas or air tamponade for an idiopathic MH were evaluated. Spectral-domain optical coherence tomography (OCT) had been performed before and >6 months after the surgery. The preoperative distances between the center of the MH and the optic disc (MH-OD), center of the MH and the bifurcation or crossing of retinal vessels (MH-RV) were measured in the OCT images. In addition, the postoperative distance between the center of the fovea and optic disc (F-OD) and the center of the fovea and the same bifurcation or crossing of retinal vessels (F-RV) were measured in the OCT images. The F-OD was 2.67±0.33 disc diameters (DD), which was significantly shorter than that of the MH-OD of 2.77±0.33 DD (P

Many membrane proteins are proposed to work as oligomers; however, the conclusion is sometimes controversial, as for β2-adorenergic receptor (β2AR), which is one of the best-studied family A G-protein-coupled receptors. This is due to the lack of methods for easy and precise detection of the oligomeric state of membrane proteins on living cells. Here, we show that a combination of the coiled-coil tag-probe labeling method and spectral imaging enable a stoichiometric analysis of the oligomeric state of membrane proteins on living cells using monomeric, dimeric, and tetrameric standard membrane proteins. Using this method, we found that β2ARs do not form constitutive homooligomers, while they exhibit their functions such as the cyclic adenosine 5'-monophosphate (cAMP) signaling and internalization upon agonist stimulation.

The purpose of this study was to determine the subfoveal scleral thickness in highly myopic eyes by enhanced depth imaging spectral-domain optical coherence tomography (EDI-OCT) and to identify the ocular parameters significantly associated with the scleral thickness.The subfoveal scleral thickness of myopic eyes (≥-8 diopters (D) or axial length ≥26.5 mm) was examined by EDI-OCT. The correlations between the thickness and the best-corrected visual acuity (BCVA), refractive error, axial length (AL), the subfoveal retinal thickness, choroidal thickness, and posterior staphyloma height 2 mm from the fovea were investigated.A total of 75 eyes of 54 patients (21 men, 33 women; mean age, 62.3±11.3 years; mean AL, 30.2±1.68 mm) were studied. Eighteen eyes had no pathological retinochoroidal lesions, and 57 eyes had retinochoroidal lesion, that is, myopic schisis, choroidal neovascularization, and other retinochoroidal pathologies. The mean subfoveal scleral thickness was 284.0±70.4 μm, and the thickness was significantly correlated negatively with the absolute value of the nasal and overall average posterior staphyloma height (P0.05 and P0.01, respectively). The subfoveal scleral thickness was also significantly correlated negatively with the relative value of the superior, nasal, and overall average posterior staphyloma height (P0.05, P0.01, and P0.001, respectively). Stepwise analyses showed that the factor most significantly associated with the scleral thickness was the average relative posterior staphyloma height (F=16.0, P0.001). The scleral thickness was not significantly different between eyes with and without myopic retinochoroidal pathologies (P0.05).Posterior staphyloma formation was a key factor associated with a posterior scleral thinning in highly myopic eyes.

Underfilled and gap-graded soils are known to be susceptible to suffusion; a form of internal instability in which the finer fraction of a soil is washed out from the coarser matrix under the action of seepage. This phenomenon poses a risk to embankment dams and flood embankments. The processes and mechanisms operate at the particle scale, and insight can be gained via the particulate discrete element method (DEM). Vir-tual samples can be created using DEM and simulation results can provide information on particle stresses, as well as quantitative information on the fabric of the particulate material. This is important as the amount of stress carried by the finer particles is thought to govern the susceptibility of a given material to suffusion. DEM modelling can also provide information on variation in properties within samples as well as the detailed data needed to quantify the material fabric. DEM models are, however, an idealization of reality and con-strained in particular by the number of particles used and sample preparation method. This study examines key issues relating to the development of virtual samples for use in DEM analysis and also examines the proportion of the applied stress that is carried by the finer particles.

Purpose: We compared 1-year outcomes of 1 + pro re nata (PRN) versus 3 + PRN of intravitreal aflibercept injection (IAI) for age-related macular degeneration (AMD). Methods: Forty-two eyes with naïve AMD received 3 + PRN IAI treatment and 47 eyes with naïve AMD received 1 + PRN IAI treatment. Visual acuity (VA), central retinal thickness (CRT), and central choroidal thickness (CCT) and number of administered IAIs during 12 months were compared. Results: VAs improved, and CRTs reduced significantly at any given month from baseline in both groups (p < 0.01, respectively). CCT reduced significantly at 3 months in the 3 + PRN group (p = 0.024) but not in the 1 + PRN group. The 1 + PRN group received fewer injections than the 3 + PRN group (p < 0.01). Conclusions: Aflibercept leads to equivalent VA and morphologic retinal improvement without administering 3 injections.

Naturally occurring cationic antimicrobial peptides exhibit not only antimicrobial activity, but also anticancer activity and are expected to be new weapons in cancer treatment. The selectivity for cancer cells over normal cells is at least partly due to the more negative surface of cancer cells. A lower pH in tumor tissue (pH 6.2-6.9) than that in normal tissues (pH 7.3-7.4) has also been utilized to develop anticancer agents. However, cytotoxicity against normal cells at physiological pH is often an issue. Furthermore, acidic regions can be found in some normal tissues such as the kidneys. Therefore, existing approaches to cancer targeting are not fully satisfactory. In this study, we designed a peptide, HE (GIHHWLHSAHEFGEHFVHHIMNS-amide), with a charge that reverses from -1.5 at pH 7.4 to +6 at pH 5.5 for cancer targeting at low pH based on the antimicrobial peptide magainin 2 by introducing 6 His, an additional Glu, and an amidated terminal. HE interacted with cancer-mimicking negatively charged liposomes in a pH-dependent fashion with a midpoint with a pH of 6.5 just above the membrane surface. The peptide killed human renal adenocarcinoma ACHN cells at pH 6.0, but not at pH 7.4, and was nontoxic against human normal glomerular mesangial cells even at this low pH. Thus, the novel peptide may be a promising lead peptide for cancer therapy, although this derivatization resulted in weakened cytotoxicity.

To investigate the relationship between the size of the peripapillary crescent and different ocular parameters in highly myopic healthy eyes; in addition, to determine whether the area of the peripapillary crescent enlarged significantly during one year of observation.The medical records of 49 highly myopic healthy eyes whose fellow eyes had myopic complications were reviewed. The area of the peripapillary crescent and other ocular parameters were measured initially and after one year. The changes in the area of the peripapillary crescent and their association with other ocular parameters during the natural course of the pathological myopia were determined.The area of the peripapillary crescent was significantly associated with the choroidal thickness (P 0.001), axial length (P 0.001), and foveal thickness (P 0.01). Stepwise regression analyses found that the factors most associated with the area of the peripapillary crescent were the choroidal thickness (P 0.01) and the absolute nasal staphyloma height (P 0.05). The factor most associated with the increase in the area of the peripapillary crescent was the increase in the axial length (P 0.01).The size of the peripapillary crescent may be affected by changes in the axial length, the height of the posterior staphyloma, and the choroidal thickness.