Vesicle-like Nanocapsules Formed by Self-Assembly of Peptides with Oligoproline and -Leucine.

Since drug carriers are envisaged to be used in a wide variety of situations and environments, nanocarriers with diverse properties, such as biocompatibility, biodegradability, nonimmunogenicity, adequate particle size, robustness, and cell permeability, are required. Here, we report the construction of novel nanocapsules with the above-mentioned features by the self-assembly of peptides composed of oligoproline and oligoleucine (i.e., H-Pro10Leu4-NH2 and H-Pro10Leu6-NH2). The peptides self-organized via hydrogen bonds and hydrophobic interactions between oligoleucine moieties to form vesicle-like nanocapsules with cationic oligoproline exposed on the surface. The guest encapsulation experiments revealed that the nanocapsules were capable of uptake of both water-soluble and insoluble compounds. Furthermore, positively charged and/or oligoproline-based peptides are known to improve cell permeability and cellular uptake, suggesting that the peptide nanocapsules are good candidates for nanocarriers to complement liposomes and polymer micelles.

Kluyvera intermedia bacteremia with septic shock: A case report.

Background: Kluyvera intermedia is a bacterium indigenous to humans. But Kluyvera intermedia bacteremia has been not reported thus far. We report a case of Kluyvera intermedia bacteremia with septic shock due to left obstructive pyelonephritis as a result of urolithiasis. Case presentation: A 66-year-old woman with septic shock due to left obstructive pyelonephritis was transferred to our hospital. Tazobactam/Piperacillin 4.5 g was administered every 8 h for 5 days. The patient's condition improved, and she was transferred back to the previous hospital. Kluyvera intermedia was obtained by blood cultures. The patient was successfully treated with a two-week course of antibiotics. Conclusions: We describe the first case of bacteremia with septic shock caused by Kluyvera intermedia. Kluyvera intermedia can be a causative pathogen of septic shock. Since this bacterium has not been reported in the past, we expect further reports and the accumulation of cases in the future.

Catenane formation of a cyclic poly(alkyl sorbate) via chain-growth polymerization induced by an N-heterocyclic carbene and ring-closing without extreme dilution.

1,3-Di-tert-butylimidazol-2-ylidene (NHCtBu), a typical N-heterocyclic carbene (NHC), was previously found to induce the anionic chain-growth polymerization of ethyl sorbate (ES) in the presence of an aluminum Lewis acid, i.e., methylaluminum bis(2,6-di-tert-butyl-4-methylphenoxide) (MAD), in which the neighboring of α-terminal dienolate with a propagating anion induced cyclization without highly diluted conditions, after monomer depletion, to give the cyclic poly(ES). In this paper, we report that catenane formation occurs by two-step polymerization of ethyl sorbate (ES), in which, after complete monomer (ES) consumption ([ES]0/[NHCtBu]0 = 100/1) in toluene followed by purification by reprecipitation, a second addition of ES monomer ([ES]0/[ NHCtBu]0 = 20/1) in another pot (in toluene or tetrahydrofuran (THF)) resulted in catenane formation, namely a polycatenane. TEM images of a sample from the second step polymerization in THF revealed particles of polycatenane structure consisting of cyclic poly(ES) with sizes ranging from 200 to 1000 nm, showing that this NHCtBu triggered chain polymerization and successive cyclization without highly diluted conditions enabled us to fabricate the intended polycatenane in the successive two-step polymerization.

Usefulness of a medical interview support application for residents: A pilot study.

To conduct an appropriate medical interview, education and clinical experience are necessary. The usefulness of computer-based medical diagnostic support systems has been reported in medical interviewing. However, only a few reports have actually applied these systems and noted changes in the quality of the medical interview of residents. We aimed to examine how the use of a medical interview support application changes the medical interviews of residents. The study was conducted on 15 residents (with less than two years post-graduation) and ran from November 2020 to March 2021. Faculty members played the role of simulated patients in 20 cases, and the residents conducted the medical interviews. In 10 of the 20 cases, a medical interview support application was used. After the interview, the residents were asked to list up to 10 differential diseases; the interview was considered appropriate if it included the disease portrayed by the simulated patient. Furthermore, the duration of the medical interview, the number of questions asked, and changes in stress parameters were evaluated. The use of a medical interview support application increased the percentage of appropriate medical interviews. Considering the frequency, the use of a medical interview support application increased the rate of appropriate medical interviews in the rare disease group, as well as the number of questions and duration of the interviews. No stress reduction was observed. The medical interview support application may be a useful tool in identifying appropriate differential diseases during medical interviews by residents.

A Peptide Nanocage Constructed by Self-Assembly of Oligoproline Conjugates.

Cage-like supramolecular assemblies called molecular cages, which possess attractive functions, have been prepared. Although biomolecule-based nanocages are required for biological/medical applications such as drug delivery systems, the majority of nanocages are constructed using aromatic compounds with lower biocompatibility and biodegradability. In this study, the construction of a peptide nanocage consisting of an oligoproline conjugate is demonstrated. The conjugate was easy to prepare and had high biocompatibility. The oligoproline moiety of the conjugate had a rigid, rod-like structure suitable for the backbone of the supramolecular nanocage. The conjugates self-assembled to form peptide nanocages with a huge inner cavity.

Fabrication of CaCO3-Coated Vesicles by Biomineralization and Their Application as Carriers of Drug Delivery Systems.

We fabricated CaCO3-coated vesicles as drug carriers that release their cargo under a weakly acidic condition. We designed and synthesized a peptide lipid containing the Val-His-Val-Glu-Val-Ser sequence as the hydrophilic part, and with two palmitoyl groups at the N-terminal as the anchor groups of the lipid bilayer membrane. Vesicles embedded with the peptide lipids were prepared. The CaCO3 coating of the vesicle surface was performed by the mineralization induced by the embedded peptide lipid. The peptide lipid produced the mineral source, CO32-, for CaCO3 mineralization through the hydrolysis of urea. We investigated the structure of the obtained CaCO3-coated vesicles using transmission electron microscopy (TEM). The vesicles retained the spherical shapes, even in vacuo. Furthermore, the vesicles had inner spaces that acted as the drug cargo, as observed by the TEM tomographic analysis. The thickness of the CaCO3 shell was estimated as ca. 20 nm. CaCO3-coated vesicles containing hydrophobic or hydrophilic drugs were prepared, and the drug release properties were examined under various pH conditions. The mineralized CaCO3 shell of the vesicle surface was dissolved under a weakly acidic condition, pH 6.0, such as in the neighborhood of cancer tissues. The degradation of the CaCO3 shell induced an effective release of the drugs. Such behavior suggests potential of the CaCO3-coated vesicles as carriers for cancer therapies.

Development of a delirium predictive model for adult trauma patients in an emergency and critical care center: a retrospective study.

BACKGROUND: Delirium has been shown to prolong the length of intensive care unit stay, hospitalization, and duration of ventilatory control, in addition to increasing the use of sedatives and increasing the medical costs. Although there have been a number of reports referring to risk factors for the development of delirium, no model has been developed to predict delirium in trauma patients at the time of admission. This study aimed to create a scoring system that predicts delirium in trauma patients. METHODS: In this single-center, retrospective, observational study, trauma patients aged 18 years and older requiring hospitalization more than 48 hours were included and divided into the development and validation cohorts. Univariate analysis was performed in the development cohort to identify factors significantly associated with prediction of delirium. The final scoring system for predicting delirium was developed using multivariate analysis and internal validation was performed. RESULTS: Of the 308 patients in the development cohort, 91 developed delirium. Clinical Frailty Score, fibrin/fibrinogen degradation products, low body mass index, lactate level, and Glasgow Coma Scale score were independently associated with the development of delirium. We developed a scoring system using these factors and calculated the delirium predictive score, which had an area under the curve of 0.85. In the validation cohort, 46 of 206 patients developed delirium. The area under the curve for the validation cohort was 0.86, and the calibration plot analysis revealed the scoring system was well calibrated in the validation cohort. DISCUSSION: This scoring system for predicting delirium in trauma patients consists of only five risk factors. Delirium prediction at the time of admission may be useful in clinical practice. LEVEL OF EVIDENCE: Prognostic and epidemiological, level III.

Protein Sensing Device with Multi-Recognition Ability Composed of Self-Organized Glycopeptide Bundle.

We designed and synthesized amphiphilic glycopeptides with glucose or galactose at the C-terminals. We observed the protein-induced structural changes of the amphiphilic glycopeptide assembly in the lipid bilayer membrane using transmission electron microscopy (TEM) and Fourier transform infrared reflection-absorption spectra (FTIR-RAS) measurements. The glycopeptides re-arranged to form a bundle that acted as an ion channel due to the interaction among the target protein and the terminal sugar groups of the glycopeptides. The bundle in the lipid bilayer membrane was fixed on a gold-deposited quartz crystal microbalance (QCM) electrode by the membrane fusion method. The protein-induced re-arrangement of the terminal sugar groups formed a binding site that acted as a receptor, and the re-binding of the target protein to the binding site induced the closing of the channel. We monitored the detection of target proteins by the changes of the electrochemical properties of the membrane. The response current of the membrane induced by the target protein recognition was expressed by an equivalent circuit consisting of resistors and capacitors when a triangular voltage was applied. We used peanut lectin (PNA) and concanavalin A (ConA) as target proteins. The sensing membrane induced by PNA shows the specific response to PNA, and the ConA-induced membrane responded selectively to ConA. Furthermore, PNA-induced sensing membranes showed relatively low recognition ability for lectin from Ricinus Agglutinin (RCA120) and mushroom lectin (ABA), which have galactose binding sites. The protein-induced self-organization formed the spatial arrangement of the sugar chains specific to the binding site of the target protein. These findings demonstrate the possibility of fabricating a sensing device with multi-recognition ability that can recognize proteins even if the structure is unknown, by the protein-induced self-organization process.

Intracellular Oxygen Concentration Determined By Mitochondrial Respiration Regulates Production of Reactive Oxygen Species.

Oxidative phosphorylation not only generates cellular energy via ATP synthesis, but also controls the intracellular oxygen level to minimize oxygen toxicity resulting from reactive oxygen species (ROS). These species include superoxide (O2 -), hydrogen peroxide (H2O2), and hydroxyl radical (•OH). While the rate of mitochondrial respiration determines the intracellular oxygen concentration, the relationship between oxygen concentration and ROS generation is not fully understood. We hypothesized that mitochondrial respiration controls intracellular oxygen concentration which in turn regulates ROS generation. To test this hypothesis, we used two prostate cancer cell lines; PC-3 cells, which have low mitochondrial genome (mtDNA) content and low mitochondrial respiratory activity, and LNCaP cells, which have high mtDNA content and high mitochondrial respiratory activity. PC-3 cells exhibited high mitochondrial oxygen concentration and generated more O2 - as well as •OH when compared to LNCaP cells which showed low mitochondrial oxygen concentration and reduced levels of O2 - and •OH. Exogenous hypoxic conditions (0.2% O2) reduced mitochondrial oxygen concentration and the levels of ROS, whereas exogenous hyperoxic conditions (40% O2) increased mitochondrial oxygen concentration and increased the levels of ROS. These results support the hypothesis that mitochondrial respiration regulates the intracellular oxygen concentration and in turn the generation of ROS.

Mineralization of Calcium Carbonate on Multifunctional Peptide Assembly Acting as Mineral Source Supplier and Template.

Crystal phase and morphology of biominerals may be precisely regulated by controlled nucleation and selective crystal growth through biomineralization on organic templates such as a protein. We herein propose new control factors of selective crystal growth by the biomineralization process. In this study, a designed ß-sheet Ac-VHVEVS-CONH2 peptide was used as a multifunctional template that acted as mineral source supplier and having crystal phase control ability of calcium carbonate (CaCO3) during a self-supplied mineralization. The peptides formed three-dimensional nanofiber networks composed of assembled bilayer ß-sheets. The assembly hydrolyzed urea molecules to one carbonate anion and two ammonium cations owing to a charge relay effect between His and Ser residues under mild conditions. CaCO3 was selectively mineralized on the peptide assembly using the generated carbonate anions on the template. Morphology of the obtained CaCO3 was fiber-like structure, similar to that of the peptide template. The mineralized CaCO3 on the peptide template had aragonite phase. This implies that CaCO3 nuclei, generated using the carbonate anions produced by the hydrolysis of urea on the surface of the peptide assembly, preferentially grew into aragonite phase, the growth axis of which aligned parallel to the direction of the ß-sheet fiber axis.

Synthesis of Glycopolymer Containing Cell-Penetrating Peptides as Inducers of Recombinant Protein Expression under the Control of Lactose Operator/Repressor Systems.

We recently reported on newly synthesized S-galactosyl oligo(Arg) conjugates to overcome the serious problem of the passage through the E. coli cell membrane. Following in vivo expression of green fluorescent protein (GFP) induced by each of the S-galactosyl (Arg)n constructs (n = 5, 6, 8) at the T5 promoter in E. coli for 18 h, we visually observed that the cultures fluoresced green light when excited with UV light. The fluorescence intensities for these cultures were greater than that found for a control culture, indicating that the peptides had induced GFP expression. In order to accomplish higher expression efficiency, we investigated the cluster effect and structural fine-tuning of new poly(2-oxazoline) containing CysArgArg as the cell-penetrating peptide (CPP) and S-galactosides when acting as inducers of recombinant protein expression under the control of lac operator/repressor systems in this article. Quantitative fluorescence intensities (calculated per molecule) also supported the observations that the cell-penetrating glyco poly(2-oxazoline)s were better inducers of GFP expression than glyco poly(2-oxazoline) containing no CPP or isopropyl ß-d-thiogalactoside. Because the level of GFP expression was directly related to the number of sugar residues in each glyco poly(2-oxazoline), we propose that a cluster effect of the S-galactosides attached to the cell-penetrating poly(2-oxazoline) is responsible for how well the galactosides inhibited the lac repressor to activate the protein expression under the control of the lac operator/repressor system. A similar tendency was observed when the T7 promoter was placed upstream of the gene for an artificial extracellular matrix protein and glyco poly(2-oxazoline)s-CPP conjugates were used as inducers. To assess how the glyco poly(2-oxazoline) penetrate the cell membrane, we labeled the glyco poly(2-oxazoline) using 1-amino pyrene and directly observed the penetration process. Furthermore, we could visualize protein expression under the control of a lac promoter/operator/repressor system using transmission electron microscopy combined with energy dispersive X-ray analysis mapping.

Mitochondrial respiratory function induces endogenous hypoxia.

Hypoxia influences many key biological functions. In cancer, it is generally believed that hypoxic condition is generated deep inside the tumor because of the lack of oxygen supply. However, consumption of oxygen by cancer should be one of the key means of regulating oxygen concentration to induce hypoxia but has not been well studied. Here, we provide direct evidence of the mitochondrial role in the induction of intracellular hypoxia. We used Acetylacetonatobis [2-(2'-benzothienyl) pyridinato-kN, kC3'] iridium (III) (BTP), a novel oxygen sensor, to detect intracellular hypoxia in living cells via microscopy. The well-differentiated cancer cell lines, LNCaP and MCF-7, showed intracellular hypoxia without exogenous hypoxia in an open environment. This may be caused by high oxygen consumption, low oxygen diffusion in water, and low oxygen incorporation to the cells. In contrast, the poorly-differentiated cancer cell lines: PC-3 and MDAMB231 exhibited intracellular normoxia by low oxygen consumption. The specific complex I inhibitor, rotenone, and the reduction of mitochondrial DNA (mtDNA) content reduced intracellular hypoxia, indicating that intracellular oxygen concentration is regulated by the consumption of oxygen by mitochondria. HIF-1α was activated in endogenously hypoxic LNCaP and the activation was dependent on mitochondrial respiratory function. Intracellular hypoxic status is regulated by glucose by parabolic dose response. The low concentration of glucose (0.045 mg/ml) induced strongest intracellular hypoxia possibly because of the Crabtree effect. Addition of FCS to the media induced intracellular hypoxia in LNCaP, and this effect was partially mimicked by an androgen analog, R1881, and inhibited by the anti-androgen, flutamide. These results indicate that mitochondrial respiratory function determines intracellular hypoxic status and may regulate oxygen-dependent biological functions.

Silica Mineralization by a Peptide Template Having a High Charge Relay Effect.

The influence of the charge relay effects of peptide organic templates on silica mineralization has been investigated. ß-Sheet-type peptide showed higher catalytic activity towards the condensation of trimethylsilanol than random-coil peptides. The higher activity of the ß-sheet peptide was attributed to an effective charge relay effect between the His and Glu residues of the ß-sheet assembly. Furthermore, the silica mineralized on the VHVEVS peptide template formed a flat surface, which was affected by the peptide morphology. This result implied that the growth of the silica occurred at the surface of the peptide template in a manner very similar to that of the enzyme reaction. Thus, by using a specific structural design strategy, this process could be used for the preparation of specific silica particles.

Calcium carbonate biomineralization utilizing a multifunctional ß-sheet peptide template.

We designed a novel multifunctional ß-sheet peptide template for calcium carbonate mineralization. The template self-supplies the mineral source, a carbonate ion, by hydrolysis of urea, and regulates the crystal phase and morphology of the obtained calcium carbonate.

Design of a nanocarrier with regulated drug release ability utilizing a reversible conformational transition of a peptide, responsive to slight changes in pH.

We investigated the drug releasing behavior of a novel nanocarrier system, utilizing a peptide to act as a nanogate to the mesopore, on a mesoporous silica nanoparticle. The surface peptide on mesoporous silica displayed pH-dependant mesopore cap-uncap switching behavior, enabled by the reversible ß-sheet-to-random coil conformational transition resulting from slight pH changes between 8.0 and 6.0. The peptide adopted a ß-sheet structure under weakly basic conditions (pH 8.0) and a random coil conformation under weakly acidic conditions (pH 6.0). We demonstrated the pH-dependant regulation of the material's drug release property by the reversible conformational transition of the surface peptide. Under basic pH conditions, the drug release from the nanocarrier was significantly inhibited. However, under acidic pH conditions, the drug in the mesopore was gradually released.

Electric-field-enhanced oriented cobalt coordinated peptide monolayer and its electrochemical properties.

The monolayer composed of cobalt coordinated peptides having lipoic acid at the amino terminals was fabricated on gold substrate by a self-assembly method under the electric field. For comparison, the self-assembled peptide monolayer was also prepared without applying a voltage. A leucine-rich hexadecapeptide, Leu(2)HisLeu(6)HisLeu(6), was chosen as the cobalt coordinated peptide. Histidines, His, were introduced as metal ligands for cobalt to the sequential peptide. The complexation between the cobalt and imidazole groups of His residues formed a stable α-helical peptide bundle, which oriented perpendicularly to the substrate surface. In the case of the self-assembled peptide monolayer (SAM), which was fabricated under the electric field, the peptide macro-dipole moments aligned unidirectionally along to the direction of the electric field, and the cobalt complexes were fixed in the monolayer to form the ordered arrangement. On the other hand, the SAM prepared without applying the voltage formed the mixture of parallel and antiparallel packing owing to the dipole-dipole interaction. As the result, the efficient non-linear electron flow through the SAM, which was fabricated under the electric field, was achieved by the regular alignment of the peptide macro-dipole moment and the cobalt complexes. This result implied that the self-assembly under the electric field is an efficient method to obtain stable oriented α-helical peptide monolayers. This method may be useful for the fabrication of the nano-devices capable of transferring information.

Synthesis of Cell-Penetrating S-Galactosyl-Oligoarginine Peptides as Inducers of Recombinant Protein Expression under the Control of lac Operator/Repressor Systems.

The synthesis of glycodendrimers and glycopoly(oxazoline)s as inducers of recombinant protein expression has recently been reported; however, these compounds induced the expression of only small amounts of the green fluorescence protein (GFP), which was used as the model recombinant protein, because of their poor ability to penetrate the Escherichia coli cell membrane. Therefore, S-galactosyl-oligo(Arg) conjugates have now been synthesized to overcome this problem. Following in vivo expression of GFP induced by each of the S-galactosyl (Arg)n constructs (n=5, 6, 8) at the T5 promoter in E. coli for 18 hours, we visually observed that the cultures fluoresced green light when excited with UV light. The fluorescent intensities for these cultures were greater than that found for a control culture, which indicates that the peptides had induced GFP expression. Quantitative fluorescent measurements also supported the observations that the peptides were better inducers of GFP expression than the galactosyl dendrimers and the poly(oxazoline)s and the natural inducer lactose. Because the level of GFP expression was directly related to the number of arginine moieties in each peptide, we propose that the number of arginine moieties is responsible for how well each peptide passes through the E. coli membrane, which affects the expression level. A similar tendency was observed when the T7 promoter was placed upstream from the gene for an artificial extracellular matrix protein and the S-Gal-oligo(Arg) peptides were used as inducers. To assess how the distance between two galactosyl moieties as well as how the multivalent effect (cluster effect) in an oligo(Arg) inducer affects the expression level of GFP, we synthesized a conjugate of Lys(Arg)8 (Lys=lysine) and two S-galactosyls, which enhanced the expression of GFP in comparison with that obtained for S-Gal(Arg)8 .

TiO2 synthesis inspired by biomineralization: control of morphology, crystal phase, and light-use efficiency in a single process.

Hydroxyapatite is mineralized along the long axis of collagen fiber during osteogenesis. Mimicking such biomineralization has great potential to control inorganic structures and is fast becoming an important next-generation inorganic synthesis method. Inorganic matter synthesized by biomineralization can have beautiful and functional structures that cannot be created artificially. In this study, we applied biomineralization to the synthesis of the only photocatalyst in practical use today, titanium dioxide (TiO(2)). The photocatalytic activity of TiO(2) mainly relates to three properties: morphology, crystal phase, and light-use efficiency. To optimize TiO(2) morphology, we used a simple sequential peptide as an organic template. TiO(2) mineralized by a ß-sheet peptide nanofiber template forms fiber-like shapes that are not observed for mineralization by peptides in the shape of random coils. To optimize TiO(2) crystal phase, we mineralized TiO(2) with the template at 400 °C to transform it into the rutile phase and at 700 °C to transform it into a mixed phase of anatase and rutile. To optimize light-use efficiency, we introduced nitrogen atoms of the peptide into the TiO(2) structure as doped elemental material during sintering. Thus, this biomineralization method enables control of inorganic morphology, crystal phase, and light-use efficiency in a single process.

Mitochondrial dysfunction induced by sertraline, an antidepressant agent.

Sertraline, a selective serotonin reuptake inhibitor, has been used for the treatment of depression. Although it is generally considered safe, cases of sertraline-associated liver injury have been documented; however, the possible mechanism of sertraline-associated hepatotoxicity is entirely unknown. Here, we report that mitochondrial impairment may play an important role in liver injury induced by sertraline. In mitochondria isolated from rat liver, sertraline uncoupled mitochondrial oxidative phosphorylation and inhibited the activities of oxidative phosphorylation complexes I and V. Additionally, sertraline induced Ca(2+)-mediated mitochondrial permeability transition (MPT), and the induction was prevented by bongkrekic acid (BA), a specific MPT inhibitor targeting adenine nucleotide translocator (ANT), implying that the MPT induction is mediated by ANT. In freshly isolated rat primary hepatocytes, sertraline rapidly depleted cellular adenosine triphosphate (ATP) and subsequently induced lactate dehydrogenase leakage; both were attenuated by BA. Our results, including ATP depletion, induction of MPT, inhibition of mitochondrial respiration complexes, and uncoupling oxidative phosphorylation, indicate that sertraline-associated liver toxicity is possibly via mitochondrial dysfunction.

The awakening of an advanced malignant cancer: an insult to the mitochondrial genome.

BACKGROUND: In only months-to-years a primary cancer can progress to an advanced phenotype that is metastatic and resistant to clinical treatments. As early as the 1900s, it was discovered that the progression of a cancer to the advanced phenotype is often associated with a shift in the metabolic profile of the disease from a state of respiration to anaerobic fermentation - a phenomenon denoted as the Warburg Effect. SCOPE OF REVIEW: Reports in the literature strongly suggest that the Warburg Effect is generated as a response to a loss in the integrity of the sequence and/or copy number of the mitochondrial genome content within a cancer. MAJOR CONCLUSIONS: Multiple studies regarding the progression of cancer indicate that mutation, and/or, a flux in the copy number, of the mitochondrial genome content can support the early development of a cancer, until; the mutational load and/or the reduction-to-depletion of the copy number of the mitochondrial genome content induces the progression of the disease to an advanced phenotype. GENERAL SIGNIFICANCE: Collectively, evidence has revealed that the human cell has incorporated the mitochondrial genome content into a cellular mechanism that, when pathologically actuated, can de(un)differentiate a cancer from the parental tissue of origin into an autonomous disease that disrupts the hierarchical structure-and-function of the human body. This article is part of a Special Issue entitled: Biochemistry of Mitochondria.