Ratiometric sandwich-type assays for RNAs with a point mutation using benzo[a]pyrene-modified probes.

Benzo[a]pyrene-modified oligonucleotides were developed for the detection of RNAs with a point mutation. The probes produced two distinct fluorescence signals in response to single nucleotide differences in the RNA sequences, allowing for discrimination between the matched and single base mismatched RNA sequences in colorimetric and ratiometric manners.

A chemical platform for the efficient screening of arylazopyrazole-based photoswitchable CENP-E inhibitors using mild cyclization reactions.

A set of arylazopyrazole-based inhibitors targeting the mitotic motor protein CENP-E was discovered through the chemical platform using the quantitative cyclization of 1,3-diketone intermediate with various hydrazines under mild conditions. Through this efficient platform, the structure-activity relationship pertaining to the pyrazole photoswitch in photoswitchable CENP-E inhibitors not only in vitro but also in cells was successfully clarified.

Spatiotemporal regulation of CENP-E-guided chromosomes using a fast-relaxing arylazopyrazole photoswitch.

We developed a centromere-associated protein E (CENP-E) inhibitor employing trans to cis photoisomerization with 405 nm visible light illumination and fast thermal relaxation. This photoswitching characteristic of the inhibitor enabled selective blockage or release of the motion of particular chromosomes within a single mitotic cell. Using this technique, we successfully demonstrated targeted chromosome gain and loss in daughter cells by introducing asymmetric chromosome segregation.

In-cell chemical construction of a photoswitchable CENP-E using a photochromic covalent inhibitor.

An arylazopyrazole-based covalent inhibitor targeting the mitotic motor protein of centromere-associated protein E (CENP-E) was developed. Using this photoswitchable inhibitor, a photoswitchable CENP-E was chemically constructed in cells, which enabled to local control of mitotic cell division with light illumination.

Acetylmelodorinol isolated from Sphaerocoryne affinis seeds inhibits cell proliferation and activates apoptosis on HeLa cells.

BACKGROUND: Cervical cancer is a major global health concern with a high prevalence in low- and middle-income countries. Natural products, particularly plant-derived compounds, have shown immense potential for developing anticancer drugs. In this study, we aimed to investigate the anticancer properties of the pericarp and seeds of Sphaerocoryne affinis fruit on human cervical carcinoma cells (HeLa) and isolate the bioactive compound from the active fraction. METHODS: We prepared solvent fractions from the ethanol extracts of the pericarp and the seed portion by partitioning and assessing their cytotoxicity on HeLa cells. Subsequently, we collected acetylmelodorinol (AM), an anticancer compound, from the ethyl acetate fraction of seeds and determined its structure using nuclear magnetic resonance. We employed cytotoxicity assay, western blotting, Annexin V apoptosis assay, measurement of intracellular reactive oxygen species (ROS) levels, 4',6-diamidino-2-phenylindole (DAPI) staining, and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, to evaluate the anticancer properties of AM on HeLa. RESULTS: The solvent fractions from the seed displayed considerably higher cytotoxic activity against HeLa cells than those of the pericarp. We isolated and identified acetylmelodorinol as an anticancer compound from the ethyl acetate fraction from S. affinis seed extract. Treatment with acetylmelodorinol inhibited HeLa cell proliferation with an IC50 value of 2.62 ± 0.57 µg/mL. Furthermore, this study demonstrated that acetylmelodorinol treatment disrupted cell cycle progression by reducing the expression of cyclin E, CDK1/2, and AKT/mTOR pathways, increasing the intracellular ROS levels, reducing BCL-2/BCL-XL expression, causing DNA fragmentation and nuclear shrinkage, and triggering apoptosis through caspase 3 and 9 activation in a dose-and time-dependent manner. CONCLUSION: In contrast to previous reports, this study focuses on the inhibitory effects of AM on the AKT/mTOR pathway, leading to a reduction in cell proliferation in cervical cancer cells. Our findings highlight the promising potential of acetylmelodorinol as an effective treatment for cervical cancer. Additionally, this study establishes a foundation for investigating the molecular mechanisms underlying AM's properties, fostering further exploration into plant-based cancer therapies.

In Situ Raman Study of Neurodegenerated Human Neuroblastoma Cells Exposed to Outer-Membrane Vesicles Isolated from Porphyromonas gingivalis.

The aim of this study was to elucidate the chemistry of cellular degeneration in human neuroblastoma cells upon exposure to outer-membrane vesicles (OMVs) produced by Porphyromonas gingivalis (Pg) oral bacteria by monitoring their metabolomic evolution using in situ Raman spectroscopy. Pg-OMVs are a key factor in Alzheimer's disease (AD) pathogenesis, as they act as efficient vectors for the delivery of toxins promoting neuronal damage. However, the chemical mechanisms underlying the direct impact of Pg-OMVs on cell metabolites at the molecular scale still remain conspicuously unclear. A widely used in vitro model employing neuroblastoma SH-SY5Y cells (a sub-line of the SK-N-SH cell line) was spectroscopically analyzed in situ before and 6 h after Pg-OMV contamination. Concurrently, Raman characterizations were also performed on isolated Pg-OMVs, which included phosphorylated dihydroceramide (PDHC) lipids and lipopolysaccharide (LPS), the latter in turn being contaminated with a highly pathogenic class of cysteine proteases, a key factor in neuronal cell degradation. Raman characterizations located lipopolysaccharide fingerprints in the vesicle structure and unveiled so far unproved aspects of the chemistry behind protein degradation induced by Pg-OMV contamination of SH-SY5Y cells. The observed alterations of cells' Raman profiles were then discussed in view of key factors including the formation of amyloid ß (Aß) plaques and hyperphosphorylated Tau neurofibrillary tangles, and the formation of cholesterol agglomerates that exacerbate AD pathologies.

Retardation of freezing of precooled, impinged water droplets on glass surfaces with microgrooves and silane coating.

Freezing impinged water droplets on glass surfaces cause serious problems such as reduced visibility of traffic lights and surveillance cameras. Droplets in the air associated with these issues are often at subzero temperatures. However, experimental results on the freezing of precooled impinged droplets are limited. In this study, we measured the freezing of precooled and impinged water droplets on cold glass surfaces. Two types of lattice-patterned microscale grooves were formed on glass surfaces to reduce the contact area of droplets and growth of frosts, which contributed to droplet freezing. In addition, the surfaces were coated with a silane coupling agent to further reduce the contact area. We analyzed the images of droplets captured using a high-speed video camera. The results of the linear relationships between the frozen droplet height, freezing front velocity, and freezing time (for the impinged droplets) indicated that the grooves and coating were effective in retarding the freezing of impinged droplets. This retardation was more evident for frost-free glass surfaces, and it was less evident for precooled droplets. Moreover, a simple heat transfer analysis was conducted to effectively estimate the overall heat flux and freezing front velocity. The sublimation of frost (adjacent to the impinged droplets) and supercool elimination of the precooled droplets significantly contributed to the heat flux and caused an increase in the freezing front velocity.

Therapeutic effect of a novel curcumin derivative GT863 on a mouse model of amyotrophic lateral sclerosis.

The present study investigated the therapeutic effects of the curcumin derivative 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy)phenyl]ethenyl]-1H-pyrazole (GT863) in amyotrophic lateral sclerosis (ALS). The inhibitory effect of GT863 on superoxide dismutase 1 (SOD1) aggregation was evaluated in cell-free assays. GT863 interfered with the conformational changes of the SOD1 protein and later, oligomeric aggregation. Furthermore, its antioxidant, anti-inflammatory, and neuroprotective effects were evaluated in cell-free and cultured cell assays. GT863 inhibited H2O2- and glutamate-induced cytotoxicity and activated an antioxidant responsive element pathway. Additionally, in vivo effects of GT863 in the ALS mice model were evaluated by its oral administration to H46R mutant SOD1 transgenic mice. Rotarod test showed that GT863 administration significantly slowed the progression of motor dysfunction in the mice. In addition, GT863 substantially reduced highly-aggregated SOD1, further preserving large neurons in the spinal cord of GT863-treated mice. Collectively, these results indicated that GT863 could be a viable therapeutic agent with multiple vital actions for the treatment of ALS.

Distribution of class IId bacteriocin-producing Virgibacillus salexigens in various environments.

We performed several experiments using three strains of Virgibacillus salexigens, namely, P2, NT N53, and C-20MoT (DSM 11483T), which were isolated from completely different sources, in relation to bacteriocin production ability. Results of whole-genome sequencing analysis revealed that all strains have very similar sequences encoding class IId bacteriocin. Although a partial amino acid sequence of the purified bacteriocin produced by strain P2 isolated from fermented food was previously reported, whole-genome sequencing and the N-terminal sequencing results in this study showed that its complete amino acid sequence consisted of 48 residues, which corresponded to that of the hypothetical bacteriocin encoded by the gene in Virgibacillus massiliensis strain Vm-5T (DSM 28587T) isolated from the human gut. From the results of 16S rRNA gene sequencing and whole-genome sequencing analyses, we taxonomically confirmed Vm-5T to be a strain of V. salexigens, and its broth culture showed antibacterial activity. Strain NT N53 isolated from the deep-sea floor produced two bacteriocins, namely, NTN-A and NTN-B. The results of N-terminal sequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and whole-genome sequencing analyses showed that their amino acid sequences differed in only one residue, and NTN-A showed the same sequence as the bacteriocin produced by strain P2. Although strain C-20MoT isolated from a solar saltern had the coding sequence very similar to that of NTN-A, its broth culture showed no antibacterial activity. This finding suggests that class IId bacteriocin-producing or bacteriocin-gene-encoding V. salexigens strains are widely distributed in distinct environment sources with different geographical and material properties.

Sandwich-type detection of nucleic acids by bioorthogonal SERS probes.

Nucleic acids in body fluids, such as circulating cell-free nucleic acids, viral DNA, and RNA have received much attention for their great potential as biomarkers in liquid biopsies of serious diseases. Although quantitative polymerase chain reaction (qPCR) has been traditionally used as a laboratory-based assay for measuring nucleic acids, there is a strong demand for techniques to qualitatively, rapidly, and simply measure the extremely low-abundance nucleic acids in order to realize the nucleic acid-based liquid biopsies. With this aim in mind, we developed a simple and highly sensitive sandwich-type assay for nucleic acids using a combination of surface-enhanced Raman scattering (SERS), which enhances Raman scattering by 108- to 1010-fold, and bioorthogonal Raman tags, which generate signals in the biologically silent region (1800-2800 cm-1). Using gold nanorods having approximately 240 strands of oligonucleotides and 4-cyano-N-(2-mercaptoethyl)benzamide (4CMB) as the bioorthogonal Raman tag, we successfully detected target nucleic acids in a sequence-selective manner.

Investigation on the Interactions between Self-Assembled ß-Sheet Peptide Nanofibers and Model Cell Membranes.

Self-assembled peptide nanofibers (NFs) obtained from ß-sheet peptides conjugated with drugs, including antigenic peptides, have recently attracted significant attention. However, extensive studies on the interactions of ß-sheet peptide NFs with model cell membranes have not been reported. In this study, we investigated the interactions between three types of NFs, composed of PEG-peptide conjugates with different ethylene glycol (EG) lengths (6-, 12- and 24-mer), and dipalmitoylphosphatidylcholine (DPPC) Langmuir membranes. When increasing the EG chain length, those interactions significantly decreased considering measurements in the presence of the NFs of: (i) changes in surface pressure of the DPPC Langmuir monolayers and (ii) surface pressure-area (π-A) compression isotherms of DPPC. Because the observed trend was similar to the EG length dependency with regard to cellular association and cytotoxicity of the NFs that was reported previously, the interaction of NFs with phospholipid membranes represented a crucial factor to determine the cellular association and toxicity of the NFs. In contrast to NFs, no changes were observed with varying EG chain length on the interaction of the building block peptide with the DPPC membrane. The results obtained herein can provide a design guideline on the formulation of ß-sheet peptide NFs, which may broaden its potential.

Development of a brain-permeable peptide nanofiber that prevents aggregation of Alzheimer pathogenic proteins.

Alzheimer's disease (AD) is proposed to be induced by abnormal aggregation of amyloidß in the brain. Here, we designed a brain-permeable peptide nanofiber drug from a fragment of heat shock protein to suppress aggregation of the pathogenic proteins. To facilitate delivery of the nanofiber into the brain, a protein transduction domain from Drosophila Antennapedia was incorporated into the peptide sequence. The resulting nanofiber efficiently suppressed the cytotoxicity of amyloid ßby trapping amyloid ß onto its hydrophobic nanofiber surface. Moreover, the intravenously or intranasally injected nanofiber was delivered into the mouse brain, and improved the cognitive function of an Alzheimer transgenic mouse model. These results demonstrate the potential therapeutic utility of nanofibers for the treatment of AD.

The Inhibition of Icing and Frosting on Glass Surfaces by the Coating of Polyethylene Glycol and Polypeptide Mimicking Antifreeze Protein.

The development of anti-icing, anti-frosting transparent plates is important for many reasons, such as poor visibility through the ice-covered windshields of vehicles. We have fabricated new glass surfaces coated with polypeptides which mimic a part of winter flounder antifreeze protein. We adopted glutaraldehyde and polyethylene glycol as linkers between these polypeptides and silane coupling agents applied to the glass surfaces. We have measured the contact angle, the temperature of water droplets on the cooling surfaces, and the frost weight. In addition, we have conducted surface roughness observation and surface elemental analysis. It was found that peaks in the height profile, obtained with the atomic force microscope for the polypeptide-coated surface with polyethylene glycol, were much higher than those for the surface without the polypeptide. This shows the adhesion of many polypeptide aggregates to the polyethylene glycol locally. The average supercooling temperature of the droplet for the polypeptide-coated surface with the polyethylene glycol was lower than for the polypeptide-coated surface with glutaraldehyde and the polyethylene-glycol-coated surface without the polypeptide. In addition, the average weight of frost cover on the specimen was lowest for the polypeptide-coated surface with the polyethylene glycol. These results argue for the effects of combined polyethylene glycol and polypeptide aggregates on the locations of ice nuclei and condensation droplets. Thus, this polypeptide-coating with the polyethylene glycol is a potential contender to improve the anti-icing and anti-frosting of glasses.

Effect of the Hydrophilic-Hydrophobic Balance of Antigen-Loaded Peptide Nanofibers on Their Cellular Uptake, Cellular Toxicity, and Immune Stimulatory Properties.

Recently, nanofibers (NFs) formed from antigenic peptides conjugated to ß-sheet-forming peptides have attracted much attention as a new generation of vaccines. However, studies describing how the hydrophilic-hydrophobic balance of NF components affects cellular interactions of NFs are limited. In this report, three different NFs were prepared by self-assembly of ß-sheet-forming peptides conjugated with model antigenic peptides (SIINFEKL) from ovalbumin and hydrophilic oligo-ethylene glycol (EG) of differing chain lengths (6-, 12- and 24-mer) to investigate the effect of EG length of antigen-loaded NFs on their cellular uptake, cytotoxicity, and dendritic cell (DC)-stimulation ability. We used an immortal DC line, termed JAWS II, derived from bone marrow-derived DCs of a C57BL/6 p53-knockout mouse. The uptake of NFs, consisting of the EG 12-mer by DCs, was the most effective and activated DC without exhibiting significant cytotoxicity. Increasing the EG chain length significantly reduced cellular entry and DC activation by NFs. Conversely, shortening the EG chain enhanced DC activation but increased toxicity and impaired water-dispersibility, resulting in low cellular uptake. These results show that the interaction of antigen-loaded NFs with cells can be tuned by the EG length, which provides useful design guidelines for the development of effective NF-based vaccines.

Bioluminescence Microplate Assay of Cyanide with Escherichia coli Harboring a Plasmid Responsible for Cyanide-dependent Light Emission in Alginate Microenvironment.

We describe the bioluminescence of a genetically engineered Escherichia coli harboring a recombined plasmid with a catalase gene promoter fused lux gene cluster, responsible for the generation of photons closely associated with respiratory inhibition, with the aim of applying it for cyanide sensing. This E. coli construct was favorably utilized for the microplate assay of cyanide by leveraging the microenvironment of the biocompatible alginate. The brightness of the bioluminescence, induced by cyanide stimulation of the respiration causative of the production of hydrogen peroxide, positively correlates with its concentration. Moreover, visualization of cyanide with a consumer digital camera, ranging in concentration from about 0.01 mg CN·L-1 in the alginate sol to around 100 mg CN·L-1 in its gel, was attained.

Effects of Winter Flounder Antifreeze Protein on the Growth of Ice Particles in an Ice Slurry Flow in Mini-Channels.

The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of the promising methods for the control is to use a substance such as antifreeze protein. We have observed and report here growth states of ice particles in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in mini-channels with a microscope. We also measured ice growth rates. Our aim was to improve the levels of ice growth inhibition by subjecting the antifreeze protein solution both to preheating and to concentrating by ultrafiltration. We have found that the ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. In addition, unlike unidirectional freezing experiments, the preheating of the antifreeze protein solution reduced the ice growth inhibition properties. This is because the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, and thus the probability of interaction between HPLC6 and ice surfaces does not increase. In contrast to this, ultrafiltration after preheating the solution improved the ice growth inhibition. This may be due to the interaction between ice surfaces and many aggregates in the concentrates.

Modulation of Cell Adhesion and Differentiation on Collagen Gels by the Addition of the Ovalbumin Secretory Signal Peptide.

Ovalbumin (OVA) is the most abundant protein in egg whites that is unnecessary in the egg yolk-based food industry. The development of OVA-based functional materials is of great interest. Collagen is a major component of the extracellular matrix. In this study, an OVA fragment, the OVA secretory signal peptide (OVA SP), was loaded in collagen gels, which were used as a cell scaffold for various types of cells. We examined the effect of OVA SP loaded in collagen gels to cell properties. The peptide was initially bound to the collagen fibers and then released from the gel. Our results indicate that the released OVA SP suppressed the integrin-mediated cell adhesion and focal adhesion formation. However, the adhesion of NIH3T3 cells was not suppressed by treatment with a chelating agent and an anti-ß1 antibody. Our results suggest that OVA SP nonspecifically interacts with cell surface proteins. The adhesion of various cell types on collagen gels were changed by the addition of OVA SP, depending on their integrin expression pattern. Additionally, the differentiation of MC3T3-E1 osteoblastic cells was promoted on the OVA SP-loaded collagen gels. This suggests that OVA SP may modulate both the differentiation and the adhesion of cells cultured on the collagen gels.

Asiatic Acid, Corosolic Acid, and Maslinic Acid Interfere with Intracellular Trafficking and N-Linked Glycosylation of Intercellular Adhesion Molecule-1.

The pentacyclic triterpenoid ursolic acid was previously shown to inhibit the intracellular trafficking of intercellular adhesion molecule-1 (ICAM-1) from the endoplasmic reticulum (ER) to the Golgi apparatus. In the present study, we further investigated the biological activities of three pentacyclic triterpenoids closely related to ursolic acid on the interleukin 1α-induced expression and intracellular trafficking of ICAM-1. In human lung adenocarcinoma A549 cells, asiatic acid, corosolic acid, and maslinic acid interfered with the intracellular transport of ICAM-1 to the cell surface. Endoglycosidase H-sensitive glycans were linked to ICAM-1 in asiatic acid-, corosolic acid-, and maslinic acid-treated cells. Unlike corosolic acid, asiatic acid and maslinic acid increased the amount of the ICAM-1 protein. Moreover, asiatic acid increased the co-localization of ICAM-1 with calnexin (an ER marker), but not GM130 (a cis-Golgi marker). Asiatic acid, corosolic acid, and maslinic acid inhibited yeast α-glucosidase activity, but not Jack bean α-mannosidase activity. These results indicate that asiatic acid, corosolic acid, and maslinic acid interfere with the intracellular transport of ICAM-1 to the cell surface and cause the accumulation of ICAM-1 linked to endoglycosidase H-sensitive glycans.

Suppression of droplets freezing on glass surfaces on which antifreeze polypeptides are adhered by a silane coupling agent.

The development of ice-phobic, glass-substrate surfaces is important for many reasons such as poor visibility through the ice-covered windshields of vehicles. The present authors have developed new glass surfaces coated with a silane coupling agent and polypeptides whose amino-acid sequence is identical to a partial sequence of winter flounder antifreeze protein. We have conducted experiments on the freezing of sessile water droplets on the glass surfaces, and measured the droplet temperature, contact angle, contact area and surface roughness. The results show that the supercooling temperature decreased noticeably in the case where a higher concentration solution of polypeptide was used for the coating. The adhesion strength of frozen droplets was lowest in the same case. In addition, we observed many nanoscale humps on the coated surface, which were formed by polypeptide aggregates in the solution. We argue that the combination of the hydrophilic humps and the hydrophobic base surfaces causes water molecules adjacent to the surfaces to have a variety of orientations in that plane, even after the ice layer started to grow. This then induces a misfit of water-molecule spacing in the ice layers and consequent formation of fragile polycrystalline structure. This explains the lower values of ice adhesion strength and supercooling enhancement in the cases of the polypeptide-coated glass plates.

Morphological Transformation of Peptide Nanoassemblies through Conformational Transition of Core-forming Peptides.

Morphological control of nanostructures that are composed of amphiphilic di- or tri-block molecules by external stimuli broadens their applications for molecular containers, nanoreactors, and controlled release materials. In this study, triblock amphiphiles comprising oligo(ethylene glycol), oligo(l-lysine), and tetra(l-phenylalanine) were prepared for the construction of nanostructures that can transform accompanying α-to-ß transition of core-forming peptides. Circular dichroic (CD) measurements showed that the triblock amphiphiles adopted different secondary structures depending on the solvent environment: they adopt ß-sheet structures in aqueous solution, while α-helix structures in 25% 2,2,2-trifluoroethanol (TFE) solution under basic pH conditions. Transmission electron microscopic (TEM) observation revealed that the triblock amphiphiles formed vesicle structures in 25% TFE aq. Solvent exchange from 25% TFE to water induced morphological transformation from vesicles to arc-shaped nanostructures accompanying α-ß conformational transition. The transformable nanostructures may be useful as novel smart nanomaterials for molecular containers and micro reactors.