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Non-obstructive mesenteric ischemia (NOMI) is caused by reduced blood flow to the intestines without physical occlusion in the mesenteric artery. Previous reports show that drug overdose occasionally induces late-onset NOMI; however, in most cases, the reason for the delayed onset is unclear. Here, we present an autopsy case of late-onset NOMI that was induced by a drug overdose. An 80-year-old man was admitted to the intensive care unit because of severe hypotension after an overdose of antihypertensive drugs. He received vasopressor therapy and continuous hemodiafiltration dialysis; however, gastrointestinal decontamination was not performed. He began to recover but developed acute mesenteric ischemia on the evening of day 4 and died on day 5. Autopsy showed discontinuous submucosal bleeding from the duodenum to the colon; there was no thrombus in the mesenteric artery, which led to a diagnosis of NOMI. In the stomach, there was 250 mL of reddish-brown fluid with a muddy brown substance. Histologic examination revealed ischemic necrosis in the small intestine. Quantitative analysis of serum collected during hospitalization revealed that nifedipine and cilostazol levels had peaked on day 3 of hospitalization. The formulation of nifedipine was a controlled-release tablet, and the low water solubility of cilostazol may have caused the tablets to adhere and remain in the stomach. Therefore, the drugs had been released into the blood stream several days after hospitalization, leading to delayed-onset NOMI. In cases of overdose, it is crucial to consider the formulation and properties of the involved drugs when determining medical treatment.

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Analysis of volatile hydrocarbons in blood from fire-related deaths provides useful information such as whether the victim inhaled smoke from the fire before death or whether an accelerant was used in the fire. In this study, we used headspace gas chromatography-mass spectrometry to quantify volatile hydrocarbons in post-mortem heart blood from 121 fire victims. The cases were classified into the following four groups according to the detected volatile hydrocarbons: construction fires without accelerants, kerosene fires, gasoline fires, and a group with no fire-related hydrocarbons detected (other fires). We investigated the relationships between blood concentrations of carboxyhemoglobin (COHb) and volatile hydrocarbons, and between various volatile hydrocarbons. The mean COHb concentrations were higher in the construction fire group than in the kerosene and gasoline fire groups. In the construction fire group, there was a high correlation coefficient between the concentrations of benzene and COHb and relatively high coefficient correlations between the concentrations of benzene and toluene, benzene and xylene, toluene and styrene, and ethylbenzene and styrene. Our results indicate that the relationships between benzene, xylene, and toluene concentrations could be used to distinguish between deaths in construction fires, kerosene fires, and gasoline fires.

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Self-assembling peptides are a type of molecule with promise as scaffold materials for cancer cell engineering. We have reported a short self-assembling peptide, (FFiK)2, that had a symmetric structure connected via a urea bond. In this study, we functionalized (FFiK)2 by conjugation with various bioactive sequences for the 3D culture of cancer cells. Four sequences, RGDS and PHSRN derived from fibronectin and AG73 and C16 derived from laminin, were selected as bioactive sequences to promote cell adhesion, proliferation or migration. (FFiK)2, and its derivatives could co-assemble into supramolecular nanofibers displaying bioactive sequences and form hydrogels. MCF-7 cells were encapsulated in functionalized peptide hydrogels without significant cytotoxicity. Encapsulated MCF-7 cells proliferated under 3D culture conditions. MCF-7 cells proliferated with spheroid formation in hydrogels that displayed RGDS or PHSRN sequences, which will be able to be applied to drug screening targeting cancer stem cells. On the other hand, since MCF-7 cells migrated in a 3D hydrogel that displayed AG73, we could construct the metastatic model of breast cancer cells, which is helpful for the elucidation of breast cancer cells and drug screening against cancer cells under metastatic state. Therefore, functionalized (FFiK)2 hydrogels with various bioactive sequences can be used to regulate cancer cell function for tumor engineering and drug screening.

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De novo designed self-assembling peptides (SAPs) are promising building blocks of supramolecular biomaterials, which can fulfill a wide range of applications, such as scaffolds for tissue culture, three-dimensional cell culture, and vaccine adjuvants. Nevertheless, the use of SAPs in intracellular spaces has mostly been unexplored. Here, we report a self-assembling peptide, Y15 (YEYKYEYKYEYKYEY), which readily forms ß-sheet structures to facilitate bottom-up synthesis of functional protein assemblies in living cells. Superfolder green fluorescent protein (sfGFP) fused to Y15 assembles into fibrils and is observed as fluorescent puncta in mammalian cells. Y15 self-assembly is validated by fluorescence anisotropy and pull-down assays. By using the Y15 platform, we demonstrate intracellular reconstitution of Nck assembly, a Src-homology 2 and 3 domain-containing adaptor protein. The artificial clusters of Nck induce N-WASP (neural Wiskott-Aldrich syndrome protein)-mediated actin polymerization, and the functional importance of Nck domain valency and density is evaluated.

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Fluorescent biosensors are indispensable tools for molecular imaging, detection, and drug screening. Conventionally, fluorescent biosensors were constructed by incorporating fluorophores into ligands. Here, to develop ligand-independent biosensors, we demonstrated biosensor selection from a fluorophore-modified peptide phage library. In this library, the peptides were designed to form α-helical structures, and one cysteine, the probe modification site, was located at the center of four randomized residues on the same face of the helix. By conjugation with 4-nitrobenzoxadiazole (NBD), we constructed an NBD-modified phage library. We conducted selection against galectin-3 (Gal-3), a galactose-specific lectin associated with various biological events such as tumor metastasis and insulin resistance. After biopanning, we obtained NBD-modified peptides that selectively bind to Gal-3 from the library. The fluorescence intensity of the hit biosensors increased with the concentration of Gal-3, and this fluorescent response was visually observed.

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We report a sudden death of an infant due to mirtazapine poisoning. A 15-day-old newborn boy was found dead when he was sleeping beside his mother who had suffered from panic disorder for approximately 1 year. After giving birth, she complained of palpitations and shaky hands, and was prescribed mirtazapine. The deceased newborn weighed 3,282 g and his height was 55 cm. There were no autopsy findings related to the death. The mirtazapine concentration as quantitated by liquid chromatography-tandem mass spectrometry analysis was 620 ng/mL in right heart blood, and was approximately 10 times higher than the therapeutic level in adults. Because transfer of mirtazapine into breast milk is low, mirtazapine was likely administered intentionally to the newborn. Based on the newborn's immature renal, liver, and blood-brain barrier function, the cause of death was attributed to mirtazapine poisoning. Poison-related homicide in the infant is rare. We report the first case of intentional mirtazapine poisoning case in a newborn.

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Chemically modified peptide ligands were identified from α-helix peptide phage libraries with different types of staple linkers. The hDM2-protein was used as a representative target of protein-protein interactions to screen ligands for p53 binding sites in hDM2. Two types of staple linkers were used for the chemical modification of the peptide phage display libraries before affinity selection. The identified stapled peptides could bind to hDM2 competitively with the p53 peptide. The stapled peptide phage libraries developed in this study will improve the discovery of protein-protein interaction inhibitors through the synergistic effect of peptide units and staple linkers.

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A stapled α-helix peptide library was designed and constructed using a chemically modified phage display system for screening stapled-peptide ligands against target proteins. The α-helix peptide library, with two cysteine residues on the opposite side of the randomized face, was modified with a rigid hydrocarbon staple linker on a phage. The stapled α-helix peptide phage library was screened against galectin-3 (Gal-3), a cancer-related galactose-binding protein. The obtained stapled peptides showed a high binding affinity (K d = 0.45 µM) despite being nonsugar ligands. The stapled modification played important roles in stabilizing the α-helical structure that contributed to the high binding affinity to Gal-3. In addition, the best stapled peptide ligands showed specific binding to Gal-3 among various carbohydrate-binding proteins. Thus, the designed α-helix peptide phage library with a constrained structure by the staple linker will advance the discovery of peptide ligands with improved specificity and affinity.

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: The effects of rapid hemorrhage on coagulopathy have been reported. However, the effects of different hemorrhage speeds on the blood coagulation/fibrinolysis system have not been investigated. This study aimed to compare different hemorrhage speeds for clarifying their effects on the coagulation/fibrinolysis system and circulation disorders in rats. Male Sprague-Dawley rats (301-396 g) were randomly assigned to five groups depending on hemorrhage speed and length of procedure: first, rapid (1.4 ml/min, 30-min bleeding); second, rapid-L (1.4 ml/min, 30-min bleeding and observation until 6 h); third, slow (0.1 ml/min, intermittently, 6-h bleeding); fourth, control (30-min observation); and fifth, control-L (6-h observation). Hemorrhage was induced by withdrawing blood until 40% of the estimated blood volume from the femoral artery. We measured vital signs, hematology, general chemistry, blood gas status, coagulation parameters, fibrinolytic markers [tissue-type plasminogen activator and plasminogen activator inhibitor one (PAI-1)], vascular endothelial damage (syndecan-1), and liver PAI-1 mRNA expression. Rapid hemorrhage induced elevation of lactate and syndecan-1 levels and prolonged prothrombin time and activated partial thromboplastin time in the rapid group. In contrast, slow hemorrhage did not induce these changes. Hemorrhage speed had no effect on plasma tissue-type plasminogen activator and hematology. Plasma PAI-1 levels were significantly increased in the rapid-L group, while liver PAI-1 mRNA levels were increased in the slow group. This study shows changes in the circulatory and fibrinolysis systems, depending on the hemorrhage speed. Hemorrhage might promote production of PAI-1, while tissue hypoxia due to rapid hemorrhage might promote release of PAI-1.

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A neuropsychiatric drug overdose impairs physiological function via central nervous system (CNS) depression. In drug-related deaths, only the drug concentration can currently provide information regarding CNS depression in victims. In this study, using a drug overdose model, we investigated the ability of neurotransmitters in the cerebrospinal fluid (CSF) to serve as biomarkers for CNS depression. Four groups of rats were orally administered diazepam (200 mg/kg) and/or phenobarbital (100 mg/kg) or vehicle. In a hot plate test performed to assess physiological impairment, drug-administered animals showed prolongation of the response latency. Serum drug concentrations were also sufficient to observe the effect of drug overdose. The levels of benzoyl-derivatized neurotransmitters were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Noradrenaline, adrenaline, serotonin, melatonin, phosphoethanolamine, and histamine levels in the CSF decreased as the response latencies in the hot plate test increased. These reduced CSF neurotransmitter levels may represent physiological dysfunction through CNS depression.

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The misuse of psychotropic drugs intended for medical treatment represents a recent worldwide public health concern. Quenchbody (Q-body) is a novel fluoroimmunosensor that can detect an antigen immediately without additional reagents or washing steps. Here, we describe creating Q-bodies for the detection of the antidepressant fluvoxamine (FLV) and determining optimal conditions to achieve the highest fluorescence intensity (FI). We prepared five Q-bodies with the fluorophore labeled at either the N- or C- terminus and with different linker lengths. Fluorescence was measurable within minutes, indicating the interaction of Q-bodies with FLV. The normalized FI (FI ratio) of the N-terminus labeled Q-body increased approximately 1.5-fold upon FLV addition; Q-bodies labeled at the C-terminus did not significantly increase FI. Among the fluorescence dyes used in this study, Rhodamine 6G labeled Q-body showed the best FI ratio. EC50 values of the N-terminus labeled Q-bodies were similar (23.2-224nM) regardless of linker length or labeling dye. We examined whether the Q-body could be applicable to serum matrix instead of phosphate-buffered saline. The intact serum interfered strongly with the Q-body fluorescence. However, the FI ratios of the Q-body for FLV-spiked serum filtrate, for which proteins were removed by filtration, showed a dose-dependency for detecting FLV levels. Deproteinization, which does not interfere with Q-body fluorescence measurements, is likely necessary to detect serum FLV with high sensitivity. This study demonstrates the potential of Q-body probes as a tool towards developing creative immunoassay applications.

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We recently developed an amphiphilic peptide, E1Y9 (Ac-E-YEYKYEYKY-NH2), that self-assembles into nanofibers and forms a hydrogel in the presence of Ca2+ ion. Four E1Y9-derivatives (E1Y9-ALK, E1Y9-DGR, E1Y9-PRG and E1Y9-RGD) were designed as conjugates of E1Y9 with bioactive peptide sequences named as ALK (ALKRQGRTLYGF), DGR (DGRDSVAYG), PRG (PRGDSGYRGDS) and RGD (RGDS), respectively, and stimulated osteoblast cells growth as well as differentiation. In this study, E1Y9/E1Y9-derivative mixed hydrogels were constructed to serve as scaffolds for osteoblastic differentiation of MC3T3-E1 cells. E1Y9 and E1Y9-derivatives co-assembled into networked nanofibers and formed hydrogels in response to Ca2+ ion. The pre-osteoblast cell line MC3T3-E1 was cultured and differentiated on mixed hydrogels. An E1Y9/E1Y9-ALK mixed hydrogel exhibited the highest cell proliferation and differentiation activity among the peptide hydrogels. The peptide sequence ALK promoted expression of RUNX2 and osteopontin, a key transcription factor and bone tissue matrix protein, respectively, during the differentiation stage. During the later stage, localization of RUNX2 and osteopontin was regulated in the cytosol and extracellularly, respectively, indicating that the E1Y9/E1Y9-ALK mixed hydrogel controlled the differentiation of MC3T3-E1 cells. Thus, the E1Y9/E1Y9-ALK mixed hydrogel developed in this study showed potential for the culture and regulation of differentiation of osteoblast cells for bone regeneration.

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We report a forensic autopsy case of an elderly man who ingested unknown amount of germicidal disinfectant containing 50% benzalkonium chloride (BZK). He survived for 18 days after BZK ingestion and then died because of pneumonia. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used to detect three BZK compounds (C12-BZK, C14-BZK and C16-BZK) in the blood. Extraction of BZK was carried out according to a modified QuEChERS method. Chromatographic separation was achieved on an ODS column and detection was performed in selected reaction monitoring mode. The accuracy and the precision were acceptable for quantitative analysis in the concentration range of 10-200 ng/mL for the three BZK compounds. BZK was detected in heart and femoral vein blood samples even 18 days after BZK ingestion. Taking into consideration clinical information during 18 days hospitalization and the autopsy findings, the cause of death was attributed to BZK poisoning. Several toxico-kinetic factors regarding absorption and excretion of BZK in the body were also discussed to elucidate the detection of BZK such a long time after ingestion.

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BACKGROUND: Lectins are carbohydrate binding proteins and related to various biological events and diseases including virus infection and cancer metastasis. In particular, galactose-binding lectins have attracted attention as targets for drug delivery and cancer markers. We, previously, demonstrated that sugar-modified peptides (glycopeptides) were useful ligands for the detection and characterization of lectins compared to the sugar unit alone. Gold nanoparticles (GNPs) conjugated with mannose-modified glycopeptides were useful in detection of concanavalin A, a mannose binding lectin. OBJECTIVES: The main objective of this study was to expand our glycopeptide-GNP conjugates for detection and imaging of galactose-binding lectins. METHODS: Four galactose-modified peptides (glycopeptides) were synthesized by Fmoc-based solid peptide synthesis method. Synthesized glycopeptides were conjugated with PEG-coated GNPs using thiol-maleimide chemistry. The interaction between glycopeptide-GNPs (GP/GNPs) (0.5 nM) and RCA120, a galactose binding lectin, (0.5-1000 nM) was evaluated by mesuring absorption spectra of GNPs. The inhibition experiment in the interaction between GP/GNPs (0.5 nM) and RCA120 (100 nM) was performed in the presence of 60 mM α- methyl mannose or 60 mM lactose. HepG2 and MCF7 cells were placed on 22×22 mm cover slip in 6 well cell culture plates (2×105 cells / well) and cultured overnight at 37°C under 5% CO2 condition. 1 mL of GP/GNPs (0.2 nM) were added in each well and incubated for 18 h at 37°C under 5% CO2 condition. After incubation, cells were washed twice with PBS and fixed with 4% paraformaldehyde solution. The cover slips were coated with 90% glycerol and sealed to slide glass. Dark-field images based on elastic light scattering were taken using a Nikon microscope (TieU) with an immersion dark field condenser. RESULTS: In the titration experiment of RCA120, GP/GNPs showed a decrease of absorbance according to the addition of RCA120, suggesting that the aggregation of GP/GNPs is induced through the binding to RCA120. The EC50 values of AA(Gal)/GNP, WF(Gal)/GNP, TS(Gal)/GNP and ED(Gal)/ GNP were estimated as 66.2 nM, 43.2 nM, 38.6 nM and 104.4 nM, respectively. TS(Gal)/GNP showed the lowest EC50 value among GP/GNPs. RCA120 has several binding sites for the galactose, and there are hydrophilic amino acids (Thr24, Glu26, Gln35, Asn42 and Asp44) around one of galactose binding sites. This result indicates that the hydrogen bonds between these amino acids and Thr/Ser residues of TS(Gal) contribute to the efficient aggregation of TS(Gal)/GNP. Next, inhibition experiments in the aggregation of WF(Gal)/GNP with RCA120 revealed that lactose inhibits the WF(Gal)/GNP binding with RCA120, but α-methyl mannose does not, and that WF(Gal)/GNP selectively interacts with RCA120 and forms the aggregate. Finally, a galactose binding protein on the surface of HepG2 cells was successfully visualized by using GP/GNPs as optical probes. CONCLUSION: Our results demonstrated that GP/GNPs could detect RCA120 by the selective binding and the aggregation formation. Furthermore, a galactose binding protein on the surface of HepG2 cells is successfully visualized using WF(Gal)/GNP as an optical probe. Thus, GNPs conjugated with glycopeptides will be useful probes for the selective detection and imaging of lectins.

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Mannose-modified lectin-binding peptides were obtained from an α-helical-designed peptide phage library. Concanavalin A (ConA) was used as a representative target protein for the lectin family. The identified glycopeptides could selectively bind to ConA with micromolar affinity. With these results, the methodologies described in this study will enhance the selection of saccharide-modified ligands through the synergistic effects of sugar and peptide units, with better specificity and affinity towards lectin proteins.

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We recently developed a self-assembling peptide, E1Y9, that self-assembles into nanofibers and forms a hydrogel in the presence of Ca(2+) . E1Y9 derivatives conjugated with functional peptide sequences derived from extracellular matrices (ECMs) reportedly self-assemble into peptide nanofibers that enhance cell adhesion and differentiation. In this study, E1Y9/E1Y9-IKVAV-mixed hydrogels were constructed to serve as artificial ECMs that promote cell differentiation. E1Y9 and E1Y9-IKVAV co-assembled into networked nanofibers, and hydrogels with disk and string shapes were formed in response to Ca(2+) treatment. The neuronal differentiation of PC12 cells was facilitated on hydrogels of both shapes that contained the IKVAV motifs. Moreover, long neurites extended along the long axis of the string-shaped gel, suggesting that the structure of hydrogels of this shape can affect cellular orientation. Thus, E1Y9 hydrogels can potentially be used as artificial ECMs with desirable bioactivities and shapes that could be useful in tissue engineering applications. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 476-483, 2016.

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Enzyme inhibitory peptides with a loop structure stabilized using an antiparallel ß-sheet scaffold (ß-loop peptide) were obtained from a designed peptide phage library. Human dihydrofolate reductase (hDHFR) was used as the target enzyme. The obtained ß-loop peptides were competitive inhibitors of hDHFR with micromolar inhibition constants and dissociation constants.

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Cell-penetrating peptides (CPPs) are able to be taken up by cells and can deliver macromolecular cargos. However, the mechanism of this internalization is not yet fully understood. Recent theories suggest that the binding of cationic CPPs to negatively charged extracellular glycosaminoglycans, such as heparan sulfate (HS), is a possible mechanism of cellular uptake (CU). Our group has screened the CU activities of 54 systematically designed amphiphilic α-helical peptides in HeLa cells. Notably, a mutation in even a single residue significantly alters the CU ability of a peptide. To determine the structure-CU activity relationship of CPPs, four peptides, which contain a difference in one or two amino acids (i.e., Arg/Glu and Ala/Phe), were chosen from our CPP library to examine their interactions with HS. Fluorescence spectroscopy, isothermal titration calorimetry (ITC) and dynamic light scattering analysis indicated that the HS-binding affinities and HS-clustering abilities of the four CPPs correlated well with their CU activities in HeLa and A549 cells. The heat capacities of the CPPs, determined using ITC and binding free energy decomposition analyses in molecular dynamics simulations, revealed that electrostatic interactions were more dominant in the HS-binding processes of Arg-containing peptides in comparison to Glu-containing peptides, whereas hydrophobic contributions were the primary mode of interaction of Phe-containing peptides in comparison to Ala-containing peptides. Furthermore, it was implied that hydrophobic interactions may be more favourable than electrostatic interactions during the CU process.

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Cell penetrating peptides (CPPs), which can enter a cell through the cell membrane, have potential research applications in the fields of drug delivery, gene therapy, and cancer therapy. However, CPPs are associated with problems such as low cell selectivity, low cell penetrating activity, and cell toxicity. To overcome the disadvantages of CPPs, we constructed a drug delivery system by developing 25 nm gold nanospheres (GNSs) conjugated to four α-helical CPPs from our peptide library. We examined the applicability of this cell-selective drug delivery system by evaluating its cell-penetrating and cell death activities and comparing them with those activities of the TAT peptide. Using the 25 nm GNS, we obtained higher cell death induction activity by the anti-cancer drug doxorubicin compared with our previous study using a 41 nm GNS. After entering the cell, the peptide-conjugated 25 nm GNS accumulated around the cell nucleus. High cell selectivity by α-helical CPP sequences was also demonstrated. Our results indicate that these α-helical peptide and 25 nm GNS conjugates are useful elements in an efficient cell-selective drug delivery system.

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The human immunodeficiency virus type 1 (HIV-1) uses CD4 and the co-receptor CCR5 or CXCR4 in the process of cell entry. The negatively charged extracellular domains of CXCR4 (CXCR4-ED) interact with positive charges on the V3 loop of gp120, facilitating binding via electrostatic interactions. The presence of highly conserved positively charged residues in the V3 loop suggests that CXCR4-ED-derived inhibitors might be broadly effective inhibitors. Synthetic peptide derivatives were evaluated for anti-HIV-1 activity. The 39-mer extracellular N-terminal region (NT) was divided into three fragments with 10-mer overlapping sites (N1-N3), and these linear peptides were synthesized. Peptide N1 contains Met 1-Asp 20 and shows significant anti-HIV-1 activity. Extracellular loops 1 and 2 (ECL1 and 2) were mimicked by cyclic peptides C1 and C2, which were synthesized by chemoselective cyclization. Cyclic peptides C1 and C2 show higher anti-HIV-1 activity than their linear peptide counterparts, L1 and L2. The cytotoxicities of C1 and C2 are lower than those of L1 and L2. These results indicate that Met 1-Asp 20 segments of the NT and cyclic peptides of ECL1 and ECL2 are potent anti-HIV-1 drug candidates.

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