Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.     

Self‐Degradable Lipid‐Like Materials Based on “Hydrolysis accelerated by the intra‐Particle Enrichment of Reactant (HyPER)” for Messenger RNA Delivery

RNA‐based therapeutics is a promising approach for curing intractable diseases by manipulating various cellular functions. For eliciting RNA (i.e., mRNA and siRNA) functions successfully, the RNA in the extracellular space must be protected and it must be delivered to the cytoplasm. In this study, the development of a self‐degradable lipid‐like material that functions to accelerate the collapse of lipid nanoparticles (LNPs) and the release of RNA into cytoplasm is reported. The self‐degradability is based on a unique reaction “Hydrolysis accelerated by intra‐Particle Enrichment of Reactant (HyPER).” In this reaction, a disulfide bond and a phenyl ester are essential structural components: concentrated hydrophobic thiols that are produced by the cleavage of the disulfide bonds in the LNPs drive an intraparticle nucleophilic attack to the phenyl ester linker, which results in further degradation. An oleic acid‐scaffold lipid‐like material that mounts all of these units (ssPalmO‐Phe) shows superior transfection efficiency to nondegradable or conventional materials. The insertion of the aromatic ring is unexpectedly revealed to contribute to the enhancement of endosomal escape. Since the intracellular trafficking is a sequential process that includes cellular uptake, endosomal escape, the release of mRNA, and translation, the improvement in each process synergistically enhances the gene expression.     

Strong and Specific Recognition of CAG/CTG Repeat DNA (5’-dWGCWGCW-3’) by a Cyclic Pyrrole-Imidazole Polyamide

Abnormally expanded CAG/CTG repeat DNA sequences lead to a variety of neurological diseases, such as Huntington's disease. Here, we synthesized a cyclic pyrrole-imidazole polyamide (cPIP), which can bind to the minor groove of the CAG/CTG DNA sequence. The double-stranded DNA melting temperature (T_m) and surface plasmon resonance assays revealed the high binding affinity of the cPIP. In addition, next-generation sequencing showed that the cPIP had high specificity for its target DNA sequence.     

PEG Modification Effect of Silica on the Suzuki–Miyaura Coupling Reaction Using Silica-immobilized Palladium Catalysts

Abstract  

The Pd phosphine complex catalysts immobilized onto polyethylene glycol (PEG)-modified silica were prepared in order to clarify the effect of the PEG modification on the Suzuki–Miyaura coupling reaction in organic solvents. For the reaction of ethyl p-bromobenzoate and phenylboronic acid in the presence of potassium carbonate in toluene, the PEG-modified silica-immobilized Pd catalysts exhibited much higher activities than the catalysts without PEG modification.     

Biological Evaluation of Double Point Modified Analogues of 1,25-Dihydroxyvitamin D2 as Potential Anti-Leukemic Agents

Structurally similar double-point modified analogues of 1,25-dihydroxyvitamin D₂ (1,25D₂) were screened in vitro for their pro-differentiating activity against the promyeloid cell line HL60. Their affinities towards human full length vitamin D receptor (VDR) and metabolic stability against human vitamin D 24-hydroxylase (CYP24A1) were also tested. The analogues (PRI-1730, PRI-1731, PRI-1732, PRI-1733 and PRI-1734) contained 5,6-trans modification of the A-ring and of the triene system, additional hydroxyl or unsaturation at C-22 in the side chain and reversed absolute configuration (24-epi) at C-24 of 1,25D₂. As presented in this paper, introduction of selected structural modifications simultaneously in two distinct parts of the vitamin D molecule resulted in a divergent group of analogues. Analogues showed lower VDR affinity in comparison to that of the parent hormones, 1,25D₂ and 1,25D₃, and they caused effective HL60 cell differentiation only at high concentrations of 100 nM and above. Unexpectedly, introducing of a 5,6-trans modification combined with C-22 hydroxyl and 24-epi configuration switched off entirely the cell differentiation activity of the analogue (PRI-1734). However, this analogue remained a moderate substrate for CYP24A1, as it was metabolized at 22%, compared to 35% for 1,25D₂. Other analogues from this series were either less (12% for PRI-1731 and PRI-1733) or more (52% for PRI-1732) resistant to the enzymatic deactivation. Although the inactive analogue PRI-1734 failed to show VDR antagonism, when tested in HL60 cells, its structure might be a good starting point for our design of a vitamin D antagonist.     

Comparative Analysis of DNA‐Binding Selectivity of Hairpin and Cyclic Pyrrole‐Imidazole Polyamides Based on Next‐Generation Sequencing

Many long pyrrole‐imidazole polyamides (PIPs) have been synthesized in the search for higher specificity, with the aim of realizing the great potential of such compounds in biological and clinical areas. Among several types of PIPs, we designed and synthesized hairpin and cyclic PIPs targeting identical sequences. Bind‐n‐Seq analysis revealed that both bound to the intended sequences. However, adenines in the data analyzed by the previously reported Bind‐n‐Seq method appeared to be significantly higher in the motif ratio than thymines, even though the PIPs were not expected to distinguish A from T. We therefore examined the experimental protocol and analysis pipeline in detail and developed a new method based on Bind‐n‐Seq motif identification with a reference sequence (Bind‐n‐Seq‐MR). High‐throughput sequence analysis of the PIP‐enriched DNA data by Bind‐n‐Seq‐MR presented A and T comparably. Surface plasmon resonance assays were performed to validate the new method.     

Simultaneous display of bacterial and fungal lipases on the cell surface of Bacillus subtilis

A small cell wall-binding domain (CWB(c)) of the Bacillus subtilis peptidoglycan hydrolase CwlC fused to B. subtilis lipase B was able to be localized on the cell wall of B. subtilis. With the aim of developing an efficient lipid-hydrolyzing bacterium with two different lipases on the cell surface, plasmids possessing genes for LipB-CWB(c) and CWB(b)-CutL (the cell wall-binding domain of CwlB fused to the Aspergillus oryzae lipolytic enzyme CutL) were constructed. B. subtilis harboring these plasmids accumulated considerable amounts of both lipases on the cell surface.     

Ozonation of cholesterol in the presence of ethanol: Identification of a cytotoxic ethoxyhydroperoxide molecule

Cholesterol ozonation was carried out in ethanol-containing aqueous or nonaqueous solvent, and the ozonized products were analyzed by chemiluminescence detection-HPLC with on-line electrospray MS (HPLC-CL-MS) and characterized on the basis of NMR and FABMS. After the ozonolysis of cholesterol in water/ethanol (aqueous system) as well as in chloroform/ethanol (nonaqueous system), a unique ethoxyhydroperoxide molecule (7α-ethoxy-3β-hydroxy-5α-B-homo-6-oxacholestane-5-hydroperoxide, termed “7α-ethoxy-5-OOH”) appeared as main ozonation product. In addition to structural analysis, we confirmed the remarkable cytotoxicity of 7α-ethoxy-5-OOH toward human lung adenocarcinoma A549 cells and found that its cytotoxicity is superior to that of the commonly known autoxidized cholesterol (3β-hydroxycholest-5-ene-7-one). Hence, 7α-ethoxy-5-OOH is a toxic molecule of primary importance, arising during cholesterol ozonation in the presence of ethanol.     

An absorption-based surface plasmon resonance sensor applied to sodium ion sensing based on an ion-selective optode membrane

A surface plasmon resonance (SPR) sodium ion sensor using an ion optode membrane film was experimentally and theoretically described based on an absorption-based SPR principle proposed in our previous article (Kurihara, K; Suzuki, K. AnaL Chem. 2002, 74, 696-701). The sodium ion concentrations from 10(-6) to 10(-1) have been successfully determined not only by the resonance angle diagnosis of the SPR curve but also by the minimum reflectance one. The ion optode film was plasticized poly(vinyl chloride) including a neutral sodium ionophore, a pH-sensitive cationic dye, and an anionic additive. Its optical absorption intensity changed with the sodium ion concentrations. The SPR ion sensor physically measured the complex refractive index caused by the absorption in the ion optode film. We have exhaustively investigated the experimental response behavior of the SPR curve relative to the sodium ion concentrations by comparison with numerically simulated SPR curves using a three-layer Fresnel equation including experimental values for the sodium ion optode membrane film. As predicted by the absorption-based SPR principle, the SPR curve behavior of the SPR ion sensors depended on two factors: one was the relation between the excitation frequency of the light source and the absorption maximum frequency in the ion optode film while the other was the gold metallic thickness in the Kretchmann configuration. The concept and practical theory of an absorption-based SPR sensor not only have been proved by the experimental results of the SPR sodium ion sensor but also have successfully allowed flexible ion sensing in an SPR sensor, which would be very difficult without the absorption mechanism in the ion optode film.     

Acute kidney injury in a postpartum woman with paroxysmal nocturnal hemoglobinuria: A case report and literature review

Paroxysmal nocturnal hemoglobinuria is a rare clonal hematopoietic stem cell disorder characterized by intravascular hemolysis, hemoglobinuria, and inflammatory thrombotic state. Intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) can lead to acute and chronic renal injury through hemoglobin‐mediated toxicity. A 32‐year‐old pregnant woman with myelodysplastic syndrome was admitted to our hospital with severe preeclampsia. Shortly after an urgent caesarean section, she became obtunded and showed signs of acute kidney injury (AKI) with anuria, severe intravascular hemolysis, and hypermagnesemia. She was diagnosed with PNH with a positive Ham test and flow cytometry analysis. Renal magnetic resonance imaging revealed decreased signal intensity in the renal cortex due to hemosiderin deposition. Hemodialysis, plasma exchange, and administration of corticosteroids ameliorated her clinical condition and renal function. This case illustrates that careful management is required to prevent postpartum AKI in pregnant women with PNH.