Granulocyte colony-stimulating factor-induced aortitis with temporal arteritis and monoarthritis.

We present the case of a patient in his 80s receiving gemcitabine-cisplatin therapy for bladder cancer who developed neutropenia and was treated with filgrastim. In 10 days, the patient developed a mild fever with left jaw claudication and right knee arthritis. Contrast-enhanced CT findings indicated aortitis. Prednisolone was started for granulocyte colony-stimulating factor (G-CSF)-induced aortitis, and symptoms and elevated serum inflammatory markers resolved rapidly, allowing early discontinuation of prednisolone. Right knee arthritis relapsed at the initial follow-up. Contrast-enhanced CT revealed aortitis had disappeared. Therefore, recurrence of G-CSF-induced arthritis was suspected; prednisolone was resumed for 29 days without relapse. Most previous reports of G-CSF-induced aortitis have described inflammation of the aorta, carotid arteries and subclavian arteries; however, G-CSF-induced aortitis may present with more peripheral symptoms, such as temporal arteritis and knee arthritis. Furthermore, G-CSF-induced aortitis reportedly responds well and rapidly to prednisolone, although early discontinuation may lead to relapse.

Hydrophilic interaction chromatography-type sorbent prepared by the modification of methacrylate-base resin with polyethyleneimine for solid-phase extraction of polar compounds.

Hydrophilic interaction chromatography (HILIC)-type sorbents were newly developed for the solid-phase extraction (SPE) of polar compounds. Two methacrylate-base resins with different cross-linking monomers and pore properties were synthesized, and three polyethyleneimines (PEIs) with different molecular weights were modified onto each base resin. In both cases, PEIs with a molecular weight of 10,000 (PEI-10,000) exhibited the highest adsorption properties for polar compounds (uracil, uridine, adenosine, cytidine, and guanosine). To control the water-enriched layer at the surface of the PEI-10,000-modified sorbents, the additive amount of PEI-10,000 in the modified reaction was also optimized. When 10 times the amount of PEI-10,000 to each base resin was added, an improvement in adsorption property was observed. Moreover, the use of a nonaqueous sample solution (100% acetonitrile) during the sample loading process drastically improved adsorption, especially for uracil (about 80%) and adenosine (100%). These results indicate that the formation of a strong water-enriched layer at the surface of sorbents with an effective expression of hydrophilic interaction was an important factor in the adsorption properties of polar compounds in HILIC mode-SPE.

Development of a novel light-up probe for detection of G-quadruplexes in stress granules.

G-quadruplexes (G4s) regulate various biological processes in cells. However, cellular imaging of dynamically forming G4s in biomolecular condensates using small molecules has been poorly investigated. Herein, we present a fluorescent light-up probe with the ability to selectively stabilize G4s and enhance fluorescence upon G4 binding. The foci of the probe were mainly observed in the nucleoli. These were co-localized with anti-fibrillarin antibodies and anti-G4 antibodies (BG4). Moreover, we tested detection of G4 in stress granules using the developed probe. Stress granules were induced through treatment with not only thapsigargin, but also known G4 ligands (pyridostatin, RHPS4, and BRACO-19). In the stress granules, co-localization between the probe, BG4, and stress granule markers (TIA1 and G3BP1) was detected. We present a practical light-up probe for G4s in stress granules, providing potential targets for G4 ligands.

Preparation and evaluation of molding-type solid-phase extraction media binding with commercially available adhesives.

A fabrication method of molding-type solid-phase extraction media (M-SPEM) bound with commercially available adhesive is presented. Six pieces of M-SPEM were prepared by heating each kneaded product of a particulate sorbent and an adhesive inserted into a six-hole cylindrical mold for hardening under an open system and normal pressure. The particulate sorbent contained in M-SPEM was divinylbenzene-based reversed-phase mode solid-phase extractants that we have reported. An examination of several adhesives showed that the moldability of M-SPEM depended on the composition and properties of the adhesive. The optimized procedure can be used to prepare an M-SPEM containing an 85 wt% particulate sorbent (particulate sorbent/adhesive, 100 mg/17 mg; particle diameter, 90-150 µm), and the M-SPEM has a specific surface area of about 500 m2/g. The established procedure in this study can bind particulate sorbents together, which showed almost no reductions in the adsorption property and liquid permeability compared with those of the particulate sorbent.

Destabilization of DNA and RNA G-quadruplex structures formed by GGA repeat due to N6-methyladenine modification.

N6-methyladenine (m6A) is the most abundant RNA modification in eukaryotic RNA. Further, m6A has been identified in the genomic DNA of both eukaryotes and prokaryotes. The G-quadruplex (G4) structure is a non-canonical nucleic acid structure formed by the stacking of G:G:G:G tetrads. In this study, we evaluated the effect of m6A modifications on G4 structures formed by GGA repeat oligonucleotides, d(GGA)8, d(GGA)4, and r(GGA)4. The d(GGA)8 forms an intramolecular tetrad:heptad:heptad:tetrad G4 structure, while d(GGA)4 forms a dimerized intermolecular tetrad:heptad:heptad:tetrad G4 structure. r(GGA)4 forms a dimerized intermolecular tetrad:hexad:hexad:tetrad G4 structure. Circular dichroism melting analysis demonstrated that (1) m6A modifications destabilized the G4 structure formed by d(GGA)8, (2) m6A modification at A3 disrupted the G4 structure formed by d(GGA)4, and (3) m6A modification at A3 destabilized the G4 structure formed by r(GGA)4. m6A modifications may be involved in controlling G4 structure formation to regulate biological functions.

The histamine H1 receptor antagonist hydroxyzine enhances sevoflurane and propofol anesthesia: A quantitative EEG study.

OBJECTIVE: The aim of this study was to determine the anesthesia-promoting effects of hydroxyzine on electroencephalograms during sevoflurane anesthesia and during propofol anesthesia. METHODS: We analyzed 40 patients scheduled for elective surgery under sevoflurane anesthesia (n = 20) or propofol anesthesia (n = 20). Anesthesia was adjusted at a bispectral index value of 50-60, and then 0.5 mg/kg of hydroxyzine was administered intravenously. We analyzed frontal electroencephalograms before and after hydroxyzine injection with power spectral and bicoherence analyses, which are suitable for assessing the anesthetic depth induced by γ-aminobutyric acid (GABA)ergic anesthetics. RESULTS: Hydroxyzine increased the α bicoherence peaks in both sevoflurane anesthesia (mean difference, 11.2%; 95% confidence interval (CI), 7.6 to 14.8; P < 0.001) and propofol anesthesia (mean difference, 5.6%; 95% CI, 1.7 to 9.4; P = 0.008). Hydroxyzine increased the averaged δ bicoherence values in both sevoflurane anesthesia (mean difference, 5.5%; 95% CI, 2.1 to 8.8; P = 0.003) and propofol anesthesia (mean difference, 3.9%; 95% CI, 1.0 to 6.8; P = 0.011). CONCLUSIONS: Hydroxyzine enhances both sevoflurane anesthesia and propofol anesthesia probably by facilitation of GABAergic neural circuit mechanisms. SIGNIFICANCE: The findings provide a new insight into the role of histaminergic neurons during general anesthesia in humans.

Successful Management of a Patient with Intraoperative Bleeding of More than 80,000 mL and Usefulness of QTc Monitoring for Calcium Correction.

Intraoperative massive bleeding is associated with high rates of mortality and anesthetic management of massive bleeding is challenging because it is necessary to achieve volume resuscitation and electrolyte correction simultaneously during massive transfusion. We report a case of life-threatening bleeding of more than 80,000 mL during liver transplantation in which real-time QTc monitoring was useful for an extremely large amount of calcium administration for treatment of hypocalcemia. A 47-year-old female with a giant liver due to polycystic liver disease was scheduled to undergo liver transplantation. During surgery, life-threatening massive bleeding occurred. The maximum rate of blood loss was approximately 15,000 mL/hr and the total amount of estimated blood loss was 81,600 mL. It was extremely difficult to maintain blood pressure and a risk of cardiac arrest continued due to hypotension. In addition, even though administration of insulin and calcium was performed, electrolyte disturbances of hyperkalemia and hypocalcemia with prolongation of QTc interval occurred. At that time, we visually noticed that the QT interval was shortened in response to bolus calcium administration, and we used the change of real-time QTc interval as a supportive indicator for calcium correction. This monitoring allowed for us to administer calcium at an unusually high rate, by which progression of hypocalcemia was prevented. Levels of hemoglobin and coagulation factors were preserved both by restriction of crystalloid infusion and by a massive transfusion protocol. The patient was extubated without pulmonary edema or cardiac overload and was finally discharged without any sequelae. Intensive and cooperative management for massive transfusion and electrolyte correction using QTc monitoring was considered to be a key for successful management.

Dispersal history of Miniopterus fuliginosus bats and their associated viruses in east Asia.

In this study, we examined the role of the eastern bent-winged bat (Miniopterus fuliginosus) in the dispersion of bat adenovirus and bat alphacoronavirus in east Asia, considering their gene flows and divergence times (based on deep-sequencing data), using bat fecal guano samples. Bats in China moved to Jeju Island and/or Taiwan in the last 20,000 years via the Korean Peninsula and/or Japan. The phylogenies of host mitochondrial D-loop DNA was not significantly congruent with those of bat adenovirus (m2XY = 0.07, p = 0.08), and bat alphacoronavirus (m2XY = 0.48, p = 0.20). We estimate that the first divergence time of bats carrying bat adenovirus in five caves studied (designated as K1, K2, JJ, N2, and F3) occurred approximately 3.17 million years ago. In contrast, the first divergence time of bat adenovirus among bats in the 5 caves was estimated to be approximately 224.32 years ago. The first divergence time of bats in caves CH, JJ, WY, N2, F1, F2, and F3 harboring bat alphacoronavirus was estimated to be 1.59 million years ago. The first divergence time of bat alphacoronavirus among the 7 caves was estimated to be approximately 2,596.92 years ago. The origin of bat adenovirus remains unclear, whereas our findings suggest that bat alphacoronavirus originated in Japan. Surprisingly, bat adenovirus and bat alphacoronavirus appeared to diverge substantially over the last 100 years, even though our gene-flow data indicate that the eastern bent-winged bat serves as an important natural reservoir of both viruses.

Molding-type Solid-phase Extraction Media Glued with Commercially Available Adhesives.

The handling of a particulate sorbent for solid-phase extraction is often troublesome because it causes static clinging and scattering. To overcome this problem, a production method for a simple molding-type solid-phase extraction medium (M-SPEM) was developed in this study by using commercially available adhesives. The content of a particulate sorbent can increase to as much as 85 wt% in the M-SPEM. Because of the high content, the proposed M-SPEMs have a higher specific surface area than previous monolithic media.

(-)-Epigallocatechin gallate inhibits stemness and tumourigenicity stimulated by AXL receptor tyrosine kinase in human lung cancer cells.

Cancer stem cells (H1299-sdCSCs) were obtained from tumour spheres of H1299 human lung cancer cells. We studied low stiffness, a unique biophysical property of cancer cells, in H1299-sdCSCs and parental H1299. Atomic force microscopy revealed an average Young's modulus value of 1.52 kPa for H1299-sdCSCs, which showed low stiffness compared with that of H1299 cells, with a Young's modulus value of 2.24 kPa. (-)-Epigallocatechin gallate (EGCG) reversed the average Young's modulus value of H1299-sdCSCs to that of H1299 cells. EGCG treatment inhibited tumour sphere formation and ALDH1A1 and SNAI2 (Slug) expression. AXL receptor tyrosine kinase is highly expressed in H1299-sdCSCs and AXL knockdown with siAXLs significantly reduced tumour sphere formation and ALDH1A1 and SNAI2 (Slug) expression. An AXL-high population of H1299-sdCSCs was similarly reduced by treatment with EGCG and siAXLs. Transplantation of an AXL-high clone isolated from H1299 cells into SCID/Beige mice induced faster development of bigger tumour than bulk H1299 cells, whereas transplantation of the AXL-low clone yielded no tumours. Oral administration of EGCG and green tea extract (GTE) inhibited tumour growth in mice and reduced p-AXL, ALDH1A1, and SLUG in tumours. Thus, EGCG inhibits the stemness and tumourigenicity of human lung cancer cells by inhibiting AXL.

Topologies of G-quadruplex: Biological functions and regulation by ligands.

G-Quadruplex (G4) is one of the higher-order structures occurring in guanine-rich sequences of nucleic acids, and plays critical roles in biological processes. The G4-forming sequences can generate three kinds of topologies, i.e., parallel, anti-parallel, and hybrid, and these polymorphic structures have an important influence on G4-related biological functions. In this review, we highlight variety of structures generated by G4s containing various sequences and under diverse conditions. We also discuss the G4 ligands which induce specific topologies and/or conversion between different topologies.

Rif1 promotes association of G-quadruplex (G4) by its specific G4 binding and oligomerization activities.

Rif1 is a conserved protein regulating replication timing and binds preferentially to the vicinity of late-firing/dormant origins in fission yeast. The Rif1 binding sites on the fission yeast genome have an intrinsic potential to generate G-quadruplex (G4) structures to which purified Rif1 preferentially binds. We previously proposed that Rif1 generates chromatin architecture that may determine replication timing by facilitating the chromatin loop formation. Here, we conducted detailed biochemical analyses on Rif1 and its G4 binding. Rif1 prefers sequences containing long stretches of guanines and binds preferentially to the multimeric G4 of parallel or hybrid/mix topology. Rif1 forms oligomers and binds simultaneously to multiple G4. We present a model on how Rif1 may facilitate the formation of chromatin architecture through its G4 binding and oligomerization properties.

Disulfide-Catalyzed Iodination of Electron-Rich Aromatic Compounds.

Herein, a disulfide-catalyzed electrophilic iodination of aromatic compounds using 1,3-diiodo-5,5-dimethylhydantoin (DIH) has been developed. The disulfide activates DIH as a Lewis base to promote the iodination reaction in acetonitrile under mild conditions. This system is applicable to a wide range of electron-rich aromatic compounds, including acetanilide, anisole, imidazole, and pyrazole derivatives.

Model studies for isolation of G-quadruplex-forming DNA sequences through a pull-down strategy with macrocyclic polyoxazole.

G-quadruplexes (G4s) are non-B DNA structures present in guanine-rich regions of gene regulatory areas, promoters and CpG islands, but their occurrence and functions remain incompletely understood. Thus, methodology to identify G4 sequences is needed. Here, we describe the synthesis of a novel cyclic hepta-oxazole compound, L1Bio-7OTD (1), bearing a biotin affinity-tag as a tool to pull down G4 structures from mixtures of G4-forming and non G4-forming DNA sequences. We confirmed that it could pull down G4s associated with telomeres, bcl-2 gene, and c-kit gene.

Synthesis and Telomeric G-Quadruplex-Stabilizing Ability of Macrocyclic Hexaoxazoles Bearing Three Side Chains.

G-quadruplexes (G4s), which are structures formed in guanine-rich regions of DNA, are involved in a variety of significant biological functions, and therefore "sequence-dependent" selective G4-stabilizing agents are required as tools to investigate and modulate these functions. Here, we describe the synthesis of a new series of macrocyclic hexaoxazole-type G4 ligand (6OTD) bearing three side chains. One of these ligands, 5b, stabilizes telomeric G4 preferentially over the G4-forming DNA sequences of c-kit and K-ras, due to the interaction of its piperazinylalkyl side chain with the groove of telomeric G4.

Binding of a Telomestatin Derivative Changes the Mechanical Anisotropy of a Human Telomeric G-Quadruplex.

Mechanical anisotropy is an essential property for biomolecules to assume structural and functional roles in mechanobiology. However, there is insufficient information on the mechanical anisotropy of ligand-biomolecule complexes. Herein, we investigated the mechanical property of individual human telomeric G-quadruplexes bound to telomestatin, using optical tweezers. Stacking of the ligand to the G-tetrad planes changes the conformation of the G-quadruplex, which resembles a balloon squeezed in certain directions. Such a squeezed balloon effect strengthens the G-tetrad planes, but dislocates and weakens the loops in the G-quadruplex upon ligand binding. These dynamic interactions indicate that the binding between the ligand and G-quadruplex follows the induced-fit model. We anticipate that the altered mechanical anisotropy of the ligand-G-quadruplex complex can add additional level of regulations on the motor enzymes that process DNA or RNA molecules.

Encephalomyocarditis virus is potentially derived from eastern bent-wing bats living in East Asian countries.

Bats are reservoir hosts of many zoonotic viruses and identification of viruses that they carry is important. This study aimed to use high throughput screening to identify the viruses in fecal guano of Taiwanese insectivorous bats caves in order to obtain more information on bat-derived pathogenic viruses in East Asia. Guano samples were collected from two caves in Taiwan, pooled, and then subjected to Multiplex PCR-based next generation sequencing for viral identification. Subsequently, encephalomyocarditis virus (EMCV) sequence was detected and confirmed by reverse transcription PCR. EMCV is considered as rodent virus and thus, animal species identification through cytochrome oxidase I (COI) barcoding was further done to identify the viral source. Finally, determination of distribution and verification of the presence of EMCV in guano obtained from Japanese and South Korean caves was also done. We concluded that the guano collected was not contaminated with the excrement of rodents which were reported and presumed to live in Taiwan. Also, EMCV genome fragments were found in guanos of Japanese and South Korean caves. It is possible that the eastern bent-wing bat (Miniopterus fuliginosus) is one of the natural hosts of EMCV in East Asia.

Development of G-quadruplex ligands for selective induction of a parallel-type topology.

G-Quadruplex structures (G4s) in guanine-rich regions of DNA play critical roles in various biological phenomena, including replication, translation, and gene expression. The G4-forming DNAs can form three kinds of topologies, i.e., parallel, anti-parallel, and hybrid. In this paper, we present G4 ligands L2H2-2M2EA-6OTD (3) and L2G2-2M2EG-6OTD (4) bearing tetra-aminoalkyl and -guanidinylalkyl side chains, respectively, in a macrocyclic hexaoxazole structure. These ligands efficiently induce the parallel-type topology of telomeric G4 regardless of the effects of cations. Titration with 4 results in a drastic topology switch to the parallel topology from the anti-parallel structure induced by the structurally related ligand L2H2-6OTD (1).

Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences.

G-quadruplexes (G4s) are four-stranded DNA structures comprising stacks of four guanines, are prevalent in genomes, and have diverse biological functions in various chromosomal structures. A conserved protein, Rap1-interacting factor 1 (Rif1) from fission yeast (Schizosaccharomyces pombe), binds to Rif1-binding sequence (Rif1BS) and regulates DNA replication timing. Rif1BS is characterized by the presence of multiple G-tracts, often on both strands, and their unusual spacing. Although previous studies have suggested generation of G4-like structures on duplex Rif1BS, its precise molecular architecture remains unknown. Using gel-shift DNA binding assays and DNA footprinting with various nuclease probes, we show here that both of the Rif1BS strands adopt specific higher-order structures upon heat denaturation. We observed that the structure generated on the G-strand is consistent with a G4 having unusually long loop segments and that the structure on the complementary C-strand does not have an intercalated motif (i-motif). Instead, we found that the formation of the C-strand structure depends on the G4 formation on the G-strand. Thus, the higher-order structure generated at Rif1BS involved both DNA strands, and in some cases, G4s may form on both of these strands. The presence of multiple G-tracts permitted the formation of alternative structures when some G-tracts were mutated or disrupted by deazaguanine replacement, indicating the robust nature of DNA higher-order structures generated at Rif1BS. Our results provide general insights into DNA structures generated at G4-forming sequences on duplex DNA.