Branchpoints as potential targets of exon-skipping therapies for genetic disorders.

Fukutin (FKTN) c.647+2084G>T creates a pseudo-exon with a premature stop codon, which causes Fukuyama congenital muscular dystrophy (FCMD). We aimed to ameliorate aberrant splicing of FKTN caused by this variant. We screened compounds focusing on splicing regulation using the c.647+2084G>T splicing reporter and discovered that the branchpoint, which is essential for splicing reactions, could be a potential therapeutic target. To confirm the effectiveness of branchpoints as targets for exon skipping, we designed branchpoint-targeted antisense oligonucleotides (BP-AONs). This restored normal FKTN mRNA and protein production in FCMD patient myotubes. We identified a functional BP by detecting splicing intermediates and creating BP mutations in the FKTN reporter gene; this BP was non-redundant and sufficiently blocked by BP-AONs. Next, a BP-AON was designed for a different FCMD-causing variant, which induces pathogenic exon trapping by a common SINE-VNTR-Alu-type retrotransposon. Notably, this BP-AON also restored normal FKTN mRNA and protein production in FCMD patient myotubes. Our findings suggest that BPs could be potential targets in exon-skipping therapeutic strategies for genetic disorders.

Antagonist of sphingosine 1-phosphate receptor 3 reduces cold injury of rat donor hearts for transplantation.

Cold storage is widely used to preserve an organ for transplantation; however, a long duration of cold storage negatively impacts graft function. Unfortunately, the mechanisms underlying cold exposure remain unclear. Based on the sphingosine-1-phosphate (S1P) signal involved in cold tolerance in hibernating mammals, we hypothesized that S1P signal blockage reduces damage from cold storage. We used an in vitro cold storage and rewarming model to evaluate cold injury and investigated the relationship between cold injury and S1P signal. Compounds affecting S1P receptors (S1PR) were screened for their protective effect in this model and its inhibitory effect on S1PRs was measured using the NanoLuc Binary Technology (NanoBiT)-ß-arrestin recruitment assays. The effects of a potent antagonist were examined via heterotopic abdominal rat heart transplantation. The heart grafts were transplanted after 24-hour preservation and evaluated on day 7 after transplantation. Cold injury increased depending on the cold storage time and was induced by S1P. The most potent antagonist strongly suppressed cold injury consistent with the effect of S1P deprivation in vitro. In vivo, this antagonist enabled 24-hour preservation, and drastically improved the beating score, cardiac size, and serological markers. Pathological analysis revealed that it suppressed the interstitial edema, inflammatory cell infiltration, myocyte lesion, TUNEL-positive cell death, and fibrosis. In conclusion, S1PR3 antagonist reduced cold injury, extended the cold preservation time, and improved graft viability. Cold preservation strategies via S1P signaling may have clinical applications in organ preservation for transplantation and contribute to an increase in the donor pool.

Layer-by-Layer Growth Control of Metal-Organic Framework Thin Films Assembled on Polymer Films.

We demonstrate growth control of Cu-based metal-organic framework (MOF) (HKUST-1) thin films assembled by the layer-by-layer technique on polymer films. The crystallinity and crystal face of MOF thin films were found to be controlled by reaction sites in polymer films such as hydroxy groups (the (100) crystal face), carbonyl groups (the (111) crystal face), and amide groups (the (100) crystal face). The HKUST-1 film growth amount is highly correlated with the polar component of the surface free energy, indicating that polymer sites, which afford hydrogen and coordination bonding, are important for the initial adsorption of Cu complexes. We also demonstrated a resistive switching device application using an HKUST-1 thin film on the poly(vinyl alcohol) dip-coated film at 40 deposition cycles, which suggests that the HKUST-1 thin film serves as a resistive switching layer with good film formation capability.

Intracardiac Thrombus Following Rivaroxaban Treatment in a Patient with Atrial Fibrillation Associated with Rheumatic Heart Disease.

We report a case of atrial fibrillation with rheumatic heart disease (RHD) who had intracardiac thrombus and cardiogenic cerebral embolism with rivaroxaban therapy. Intracardiac thrombus disappeared after switching from rivaroxaban to warfarin. Patients of RHD have the possibility of gradual progression of valvular disease even if they are old, so we need to distinguish nonvalvular atrial fibrillation from RHD before starting direct oral anticoagulants.

Cellulose Nanofiber Nanosheet Multilayers by the Langmuir-Blodgett Technique.

We describe a systematic approach for producing cellulose nanofiber (CNF) nanosheets using the Langmuir-Blodgett (LB) technique. The CNFs were obtained from sulfuric acid hydrolysis of commercially available microfibrillated cellulose. Needle-like CNFs, negatively charged by grafted sulfate groups, were maintained at the air-water interface, assisted by amphiphilic polymer, poly( N-dodecyl acrylamide) (pDDA). The CNFs produced a stable monolayer. The surface pressure increased steadily with a high collapse pressure of 50 mN m-1 when spread with formic acid and pDDA. The composite monolayers were transferred onto solid substrates as Y-type LB films using a vertical dipping method. Upstroke and downstroke transfer ratios of the films were, respectively, unity and 0.88, indicating that full coverage was achieved by the monolayer even for more than 200 layers. Results obtained using atomic force microscopy, Fourier transform infrared, and X-ray photoelectron spectroscopy showed that CNF nanosheets possess well-defined layer structures with average monolayer thickness of 5.3 nm. The relative amount of CNFs in the nanosheets was calculated as 62.6 wt % using the quartz crystal microbalance technique. The as-prepared nanosheets are optically transparent to visible light and have high hydrophobicity. In fact, the nanosheet transparency was higher than 88% at 600 nm wavelength for 24 layers. A miniscule amount of pDDA enables demonstration of free-standing CNF nanosheets with 1 cm width and 45.6 nm thickness (23 layers).