Microvortex-induced turbulent mixing for reconstitution of high-density lipoprotein-mimicking nanoparticles with aggregation-prone phosphatidylcholine.

Lipid nanoparticles often contain a phosphatidylcholine with a long chain fatty acid, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). However, their preparation often encounters difficulties such as the inability to yield <20 nm nanoparticles due to the aggregation-prone behavior of DSPC. High-density lipoproteins (HDLs) are ∼10 nm protein-bound lipid nanoparticles in our body, and microfluidic preparations of HDL-mimicking nanoparticles (µHDL) have been reported. Herein, we report a new microfluidic mixing mode that enables preparation of µHDL with DSPC in high yield (≥90% on a protein basis). The critical mechanism of this mode is a spontaneous asymmetric distribution of the ethanol flow injected in a symmetric manner followed by turbulent mixing in a simple rectangular parallelepiped-shaped chip.

Carbon Nanotube-Based Printed All-Organic Microelectrode Arrays for Neural Stimulation and Recording.

In this paper, we report a low-cost printing process of carbon nanotube (CNT)-based, all-organic microelectrode arrays (MEAs) suitable for in vitro neural stimulation and recording. Conventional MEAs have been mainly composed of expensive metals and manufactured through high-cost and complex lithographic processes, which have limited their accessibility for neuroscience experiments and their application in various studies. Here, we demonstrate a printing-based fabrication method for microelectrodes using organic CNT/paraffin ink, coupled with the deposition of an insulating layer featuring single-cell-sized sensing apertures. The simple microfabrication processes utilizing the economic and readily available ink offer potential for cost reduction and improved accessibility of MEAs. Biocompatibility of the fabricated microelectrode was suggested through a live/dead assay of cultured neural cells, and its large electric double layer capacitance was revealed by cyclic voltammetry that was crucial for preventing cytotoxic electrolysis during electric neural stimulation. Furthermore, the electrode exhibited sufficiently low electric impedance of 2.49 Ω·cm2 for high signal-to-noise ratio neural recording, and successfully captured model electric waves in physiological saline solution. These results suggest the easily producible and low-cost printed all-organic microelectrodes are available for neural stimulation and recording, and we believe that they can expand the application of MEA in various neuroscience research.

Precise 3D Localization of Intracerebral Implants with a simple Brain Clearing Method.

Determining the localization of intracerebral implants in rodent brain stands as a critical final step in most physiological and behaviroral studies, especially when targeting deep brain nuclei. Conventional histological approaches, reliant on manual estimation through sectioning and slice examination, are error-prone, potentially complicating data interpretation. Leveraging recent advances in tissue-clearing techniques and light-sheet fluorescence microscopy, we introduce a method enabling virtual brain slicing in any orientation, offering precise implant localization without the limitations of traditional tissue sectioning. To illustrate the method's utility, we present findings from the implantation of linear silicon probes into the midbrain interpeduncular nucleus (IPN) of anesthetized transgenic mice expressing chanelrhodopsin-2 and enhanced yellow fluorescent protein under the choline acetyltransferase (ChAT) promoter/enhancer regions (ChAT-Chr2-EYFP mice). Utilizing a fluorescent dye applied to the electrode surface, we visualized both the targeted area and the precise localization, enabling enhanced inter-subject comparisons. Three dimensional (3D) brain renderings, presented effortlessly in video format across various orientations, showcase the versatility of this approach.

Comprehensive Analysis of Drug Loading into Engineered Lipoprotein Nanoparticles toward Their Eye Drop Application.

The drug loading capacity of an engineered lipoprotein (eLP1) and the colloidal stability of drug-loaded eLP1s were assessed with 12 drugs with different charges/hydrophobicities. The capacity was largely correlated with their log P values, and the binding to the protein moiety was suggested for two drugs. The size of drug-loaded eLP1 formulations after freeze-drying followed by resolubilization hardly changed. The eLP1 formulation of travoprost, a clinically used drug in eye drop formulations, maintained its small size (19 nm) for 1 h at 37 °C in an artificial tear solution, whereas the liposome counterpart of 112 nm in diameter aggregated.

Effects of lipoprotein nanoparticles' composition and size on their internalization in plant and mammalian cells.

The internalization of engineered high-density lipoprotein nanoparticles (engineered lipoproteins [eLPs]) with different lipid and protein compositions, zeta potentials, and/or sizes were analyzed in representative plant and mammalian cells. The impact of the addition of a cell-penetrating peptide to eLPs on the internalization was very small in Bright Yellow (BY)-2 protoplasts compared with HeLa cells. When eLPs were prepared with one of the abundant lipids in BY-2 cells, digalactosyldiacylglycerol (DGDG) (eLP4), its internalization was dramatically increased only in HeLa cells. Such an increase in HeLa cells was also obtained for liposomes containing DGDG in a DGDG content-dependent manner. Increasing the size and zeta potential of eLPs improved their internalization in both HeLa cells and in BY-2 protoplasts but to quite varying degrees. Although eLPs tended to stay at the plasma membrane (PM) in BY-2 protoplasts with much less internalization, the PM-bound eLPs somehow promoted the internalization of coexisting nanobeads in cell culture media. These results provide fundamental insight into the future design of lipid nanoparticles for drug delivery in mammalian and plant cells.

Synthesis of Butterfly-Like Shaped Gold Nanomaterial: For the Regulation of Liquid-Liquid Phase-Separated Biomacromolecule Droplets.

Nanotechnology is a critical tool to manipulate the sophisticated behavior of biological structures and has provided new research fields. Liquid-liquid phase-separated (LLPS) droplets gather attention as basic reaction fields in a living cell. Droplets play critical roles in regulating protein behavior, including enzyme compartmentalization, stress response, and disease pathogenesis. The dynamic manipulation of LLPS droplet formation/deformation has become a crucial target in nanobiotechnology. However, the development of nanodevices specifically designed for this purpose remains a challenge. Therefore, this study presents butterfly-shaped gold nanobutterflies (GNBs) as novel nanodevices for manipulating LLPS droplet dynamics. The growth process of the GNBs is analyzed via time-lapse electroscopic imaging, time-lapse spectroscopy, and additives assays. Interestingly, GNBs demonstrate the ability to induce LLPS droplet formation in systems such as adenosine triphosphate/poly-l-lysine and human immunoglobulin G, whereas spherical and rod-shaped gold nanoparticles exhibit no such capability. This indicates that the GNB concave surface interacts with the droplet precursors facilitating the LLPS droplet formation. Near-infrared-laser irradiation applied to GNBs enables on-demand deformation of the droplets through localized heat effects. GNB regulates the enzymatic reaction of lysozymes. The innovative design of GNBs presents a promising strategy for manipulating LLPS dynamics and offers exciting prospects for future research.

Interpersonal predictors of loneliness in Japanese children: variable- and person-centered approaches.

BACKGROUND: Loneliness in children has been a major topic of interest in both clinical and developmental psychology. Further studies to investigate predictors of loneliness are needed for educational practices. METHODS: A total of 1088 late elementary school-aged children (48.81% boys, Mage = 10.35) participated in a 1-year longitudinal survey. We used hierarchical linear modeling and mover-stayer latent transition analysis. DISCUSSION: Findings from the variable- and person- centered approaches suggested that less positive peer relations, higher victimization, and higher relational aggression are predictors of higher future loneliness. Meanwhile, both approaches did not reach an agreement concerning predictors to reduce loneliness. This result highlighted a utility of a combined approach and sounded an alarm for overreliance on the variable-centered approach dominating child research. CONCLUSION: To protect young children from loneliness, it will be more beneficial to prevent the development of loneliness rather than to apply interventions to reduce loneliness once established. Preventive practices need to be implemented to protect children from loneliness.

Elucidation of the Mechanisms for the Underlying Depolarization and Reversibility by Photoactive Molecule.

BACKGROUND/AIMS: Light-induced control of the cell membrane potential has enabled important advances in the study of biological processes involving the nervous system and muscle activity. The use of these light-induced modifications is expected in various medical applications, including the control of physiological responses and the recovery of lost functions by regulating nerve activity. In particular, charge-separating linkage molecules (Charge-Separation (CS) molecules) can depolarize cells by photoexcitation without genetic processing. However, the molecular mechanisms underlying cell membrane depolarization are unknown and have hindered its application. Here, we show that CS molecules localized in the cell membrane of PC12 cells using a high-density lipoprotein (HDL)-based drug carrier can excite the cells through a novel membrane current regulation mechanism by light irradiation. METHODS: Membrane potential, channel activity, and membrane capacitance were measured by patch clamp method in rat adrenal gland pheochromocytoma (PC12) cells and KV-overexpressing PC12 cells. CS molecules localized in the cell membrane of PC12 cells using HDL-based drug carrier. The localization of CS molecule was measured by a confocal microscopy. The mRNA expression was tested by RT-PCR. RESULTS: Current clamp measurements revealed that the photo-activated CS molecule causes a sharp depolarization of about 15 mV. Furthermore, it was shown by voltage clamp measurement that this mechanism inactivates the voltage-dependent potassium current and simultaneously generates photo-activated CS molecule induced (PACS) current owing to the loss of the cell membrane capacitance. This activity continues the depolarization of the target cell, but is reversible via a regenerative mechanism such as endocytosis and exocytosis because the cell membrane is intact. CONCLUSION: Thus, the mechanism of photo-induced depolarization concludes that photo-activated TC1 causes depolarization by generating PACS current in parallel with the suppression of the K+ current. Moreover, the depolarization slowly restores by internalization of TC1 from the membrane and insertion of new lipids into the cell membrane, resulting in the restoration of KV to normal activity and eliminating PACS currents, without cell damage. These results suggest the possibility of medical application that can safely control membrane excitation.

Control of Lipid Bilayer Phases of Cell-Sized Liposomes by Surface-Engineered Plasmonic Nanoparticles.

Liquid-ordered (Lo)-phase domains, a cholesterol-rich area on lipid bilayers, have attracted significant attention recently because of their relevance to lipid rafts, the formation/collapse of which is associated with various kinds of information exchange through the plasma membrane. Here, we demonstrate that the formation/collapse of Lo-phase domains in cell-sized liposomes, that is, giant unilamellar vesicles (GUVs), can be controlled with bioactive plasmonic nanoparticles and light. The nanoparticles were prepared by surface modification of gold nanorods (AuNRs) using a cationized mutant of high-density lipoprotein (HDL), which is a natural cholesterol transporter. Upon the addition of surface-engineered AuNRs to GUVs with the mixed domains of Lo and liquid-disorder (Ld) phases, the Lo domains collapsed and solid-ordered (So)-phase domains were formed. The reverse phase transition was achieved photothermally, with the AuNRs loaded with cholesterol. During these transitions, the AuNRs appeared to be selectively localized on the less fluidic domain (Lo or So) in the phase-mixed GUVs. These results indicate that the phase transitions occur through the membrane binding of the AuNRs followed by spontaneous/photothermal transfer of cholesterol between the AuNRs and GUVs. Our strategy to develop bioactive AuNRs potentially enables spatiotemporal control of the formation/collapse of lipid rafts in living cells.

Formation of glyco-functionalized interfaces for protein binding using polyphenolic glycoside.

In this study, a polyphenolic glycoside (α-glucosyl rutin) was used to form glyco-functionalized interfaces for protein binding. α-Glucosyl rutin was coated onto precious metals, metal oxides, and synthetic polymers, including polyethylene and polytetrafluoroethylene with poor surface modifiability. The glyco-functionalized interfaces bound strongly and specifically to concanavalin A and Bauhinia purpurea lectin, which have different carbohydrate specificities. Competitive adsorption tests demonstrated that the binding sites for the abovementioned lectins were glucosyl and rhamnosyl residues, respectively. The glyco-functionalized interfaces maintained the protein binding ability after being stored in aqueous solution for 1 day and in air for 160 days. Once the glyco-functionalized interfaces were formed on gold, silicon dioxide, polystyrene, and polytetrafluoroethylene using α-glucosyl rutin, all the glyco-functionalized interfaces bound to concanavalin A rather than peanut agglutinin.

Potential of Lipoprotein-Based Nanoparticulate Formulations for the Treatment of Eye Diseases.

Lipoproteins are naturally occurring nanoparticles and their main physiological function is the promotion of lipid metabolism. They can be prepared in vitro for use as drug carriers, and these reconstituted lipoproteins show similar biological activity to their natural counterparts. Some lipoproteins can cross the blood-retinal barrier and are involved in intraocular lipid metabolism. Drug-loaded lipoproteins can be delivered to the retina for the treatment of posterior eye diseases. In this review, we have discussed the therapeutic applications of lipoproteins for eye diseases and introduced the emerging animal models used for the evaluation of their therapeutic effects.

Urea-Assisted Reconstitution of Discoidal High-Density Lipoprotein.

High-density lipoprotein (HDL) is a naturally occurring composite of lipids and lipid-binding proteins. The cholate dialysis method, first reported by Jonas in 1969, is the most widely used approach for reconstituting discoidal HDL (dHDL) in test tubes with phospholipids and the most dominant protein, apolipoprotein A-1 (apoA-I). Here, we show that a dHDL-relevant complex can also be prepared by gently mixing 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and apoA-I or its mutants in ethanol/H2O solutions containing urea at a concentration of a few molar and then incubating the mixture at the gel-liquid crystalline phase transition temperature in test tubes. Subsequent purification steps involve quick dialysis following size exclusion chromatography. The yields (73 ± 3% and 70 ± 1% protein and DMPC, respectively) of the resulting HDL-like nanoparticles, designated as uHDL, were comparable to the values of 68 ± 9% and 71 ± 12% obtained in the cholate dialysis method. Using apoA-I and two mutants, the key factor in this method was found to be urea at the folded and unfolded transition midpoint concentration. By using this urea-assisted method in the presence of a hydrophobic drug, all-trans-retinoic acid (ATRA), one-step preparation of ATRA-loaded uHDL was also possible. The loading efficiency was comparable to that in the mixing of ATRA and uHDL or dHDL reconstituted by the cholate dialysis method. Atomic force microscopy analysis revealed that uHDL and ATRA-loaded uHDL were discoidal. Our urea-assisted method is an easy and efficient method for reconstituting dHDL and can be utilized to prepare various drug-dHDL complexes.

Advanced CUBIC tissue clearing for whole-organ cell profiling.

Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (>0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g., >100 mouse brains per study), providing a platform for next-generation biomedical research.

[Coronary Artery Bypass Grafting in an Elderly Patient with Anomalous Origin of the Left Coronary Artery from the Pulmonary Artery].

We report a case of a 73-year-old woman who was diagnosed with anomalous origin of the left coronary artery(LCA) from the pulmonary artery(ALCAPA) by coronary angiography. Drug stress myocardial perfusion scintigraphy demonstrated myocardial ischemia in the left anterior descending coronary artery (LAD) region. She underwent single coronary artery bypass grafting to LAD using left internal thoracic artery (LITA) and direct closure of the origin of the anomalous LCA. Postoperative coronary catheterization revealed a patent graft showing no residual shunt from the pulmonary artery into the left coronary artery. Myocardial scintigraphy proved improvement of the ischemia. In general, once ALCAPA is diagnosed, early surgical intervention is recommended. However, since there are few reports regarding surgical treatment for ALCAPA in elderly patients, the optimal treatment strategy is not completely established. Therefore careful long-term follow-up is mandatory.

[Mycobacterium Mageritense Bacteremia after Implantation of Pacemaker;Report of a Case].

An octogenarian female patient underwent implantation of pacemaker for sick sinus syndrome. Seventeen days after implantation, she came back to our hospital with pain and redness of the skin around the pacemaker generator. It was difficult to differentiate the infection from a subcutaneous hemorrhage. She did not have high fever. The blood test showed only a slight elevation of C-reactive protein(CRP). Cefazolin was administered after readmission. Five days after readmission, the report of the blood culture showed nontuberculous Mycobacterium positive. We immediately removed the generator and the leads completely. Antibiotics were changed to clarithromycin and ciprofloxacin. The culture reports of blood, generator, leads, and subcutaneous tissue were first Mycobacterium peregrinum( M. peregrinum) but was later corrected to be Mycobacterium mageritense (M. mageritense). Twenty-seven days after the removal of the device, she was transferred to another hospital near her family and antibiotics were ceased due to allergic reaction the day after transfer. Ten days after transfer, a new device was implanted, and she does not have any signs of infection now. The possibility of mycobacterial infection, including nontuberculous mycobacterial infection, should be considered for device contamination, especially for the aging population, and quick and proper treatments are required.

Membrane fusogenic high-density lipoprotein nanoparticles.

Membrane fusion under mildly acidic pH occurs naturally during viral infection in cells and has been exploited in the field of nanoparticle-mediated drug delivery to circumvent endosomal entrapment of the cargo. Herein, we aimed to confer virus-like fusogenic activity to HDL in the form of a ca. 10-nm disc comprising a discoidal lipid bilayer and two copies of a lipid-binding protein at the edge. A series of HDL mutants were prepared with a mixture of three lipids and a cell-penetrating peptide (TAT, penetratin, or Arg8) fused to the protein. In a lipid-mixing assay with anionic liposomes at pH 5.5, one HDL mutant showed the fusogenic activity higher than known fusogenic liposomes. In live mammalian cells, this HDL mutant showed high plasma membrane-binding activity in the presence of serum independent of pH. In the absence of serum, a mildly acidic pH dependency for binding to the plasma membrane and the subsequent lipid mixing between them was observed for this mutant. We propose a novel strategy to develop HDL-based drug carriers by taking advantage of the HDL lipid/protein composite structure.

Novel approach to enhance sensitivity in surface-assisted laser desorption/ionization mass spectrometry imaging using deposited organic-inorganic hybrid matrices.

Sample pretreatment is key to obtaining good data in matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). Although sublimation is one of the best methods for obtaining homogenously fine organic matrix crystals, its sensitivity can be low due to the lack of a solvent extraction effect. We investigated the effect of incorporating a thin film of metal formed by zirconium (Zr) sputtering into the sublimation process for MALDI matrix deposition for improving the detection sensitivity in mouse liver tissue sections treated with olanzapine. The matrix-enhanced surface-assisted laser desorption/ionization (ME-SALDI) method, where a matrix was formed by sputtering Zr to form a thin nanoparticle layer before depositing MALDI organic matrix comprising α-cyano-4-hydroxycinnamic acid (CHCA) by sublimation, resulted in a significant improvement in sensitivity, with the ion intensity of olanzapine being about 1800 times that observed using the MALDI method, comprising CHCA sublimation alone. When Zr sputtering was performed after CHCA deposition, however, no such enhancement in sensitivity was observed. The enhanced sensitivity due to Zr sputtering was also observed when the CHCA solution was applied by spraying, being about twice as high as that observed by CHCA spraying alone. In addition, the detection sensitivity of these various pretreatment methods was similar for endogenous glutathione. Given that sample preparation using the ME-SALDI-MSI method, which combines Zr sputtering with the sublimation method for depositing an organic matrix, does not involve a solvent, delocalization problems such as migration of analytes observed after matrix spraying and washing with aqueous solutions as sample pretreatment are not expected. Therefore, ME-Zr-SALDI-MSI is a novel sample pretreatment method that can improve the sensitivity of analytes while maintaining high spatial resolution in MALDI-MSI.

Risk factors for residual mitral regurgitation after aortic valve replacement in patients with severe aortic valve stenosis and moderate mitral regurgitation.

OBJECTIVES: While it was reported that patients with residual moderate mitral regurgitation (MR) after surgical aortic valve replacement (SAVR) had a poorer prognosis than those without it, the risk factors for residual MR have not been fully elucidated. The aim of the study was to evaluate risk factors for residual MR after SAVR. METHODS: Of the 222 patients who underwent isolated SAVR from 2001 to 2018, 33 (11 men; age: 74 ± 7 years) had functional moderate MR before surgery. The risk factors for residual MR were evaluated by comparing patients with residual moderate MR (n = 11, 33%) with those who exhibited improved post-surgery MR (n = 22, 67%). RESULTS: The left atrial diameter was significantly larger in the residual MR group (51 ± 7 mm) than in the improved MR group (46 ± 5 mm; P = 0.049). The mean pressure gradient at the aortic valve was significantly smaller in the residual MR group (52 ± 18 mmHg) than in the improved MR group (69 ± 22 mmHg; P = 0.043). A ratio of left atrial diameter (mm) and mean aortic valve pressure gradient (mmHg) greater than 0.9 predicted residual MR with a sensitivity of 70% and a specificity of 74% (area under the ROC curve: 0.779; P = 0.015). CONCLUSIONS: In patients with severe aortic valve stenosis and moderate MR, a high ratio of preoperative left atrial diameter and mean aortic valve pressure gradient would be a parameter predicting residual moderate MR post-SAVR.

The Japanese Version of the Adolescent Time Inventory-Time Attitudes (ATI-TA) Scale: Internal Consistency, Structural Validity, and Convergent Validity.

In this study, we translated the Adolescent Time Inventory-Time Attitudes (ATI-TA) scale into Japanese and examined the internal consistency and structural and convergent validity of ATI-TA scores in a sample of 383 Japanese high school students aged 15 to 18 years. The ATI-TA consists of six subscales assessing two valences (positive and negative) for each of three time periods (past, present, and future). Internal consistency estimates for scores on the ATI-TA were over .79. Confirmatory factor analysis indicated that a six-factor model yielded the best fit to scores. Correlations among ATI-TA scores, time attitudes assessed by the semantic differential method, educational career planning, and self-esteem were consistent with our hypotheses. Our results provide evidence that scores on the Japanese version of the ATI-TA are reliable and valid, and thus potentially useful for research in this cultural context.

Effects of the Peripheral Substituents, Central Metal, and Solvent on the Photochemical and Photophysical Properties of 5,15-Diazaporphyrins.

Metal complexes of 3,7,13,17-tetrakis(di(4-carboxyphenyl)amino)-5,15-diazaporphyrin (MDAP-COOH; M=Pd, Cu) and their ethyl ester precursors (MDAP-COOEt; M=Pd, Cu) have been synthesized for use as near-infrared (NIR)-light-responsive photosensitizers. Under irradiation with visible or NIR light, PdDAP-COOEt in toluene generated singlet oxygen (1 O2 ) with an excellent quantum yield (ΦΔ =0.99), whereas CuDAP-COOEt exhibited a lower efficiency (ΦΔ =0.21). The water-soluble PdII complex PdDAP-COOH also behaved as a photosensitizer (ΦΔ =0.20) in a micellar solution. The photophysical properties of these dyes were measured by transient absorption techniques. It was found that the efficiency of the energy transfer from the triplet state of MDAP-COOR (R=Et, H) to the ground state of dioxygen was highly dependent on the peripheral substituents, the central metal, and the solvent. Furthermore, the phototoxicity of PdDAP-COOH toward HeLa cells under irradiation of NIR light (720 nm) was evaluated. As expected, PdDAP-COOH exhibited good photodynamic activity, and control experiments confirmed that 1 O2 was generated as the active oxygen species.