To analyze the mechanism of formation of tubular myelin (TM), we reconstituted TM from synthetic lipids and two surfactant-associated proteins (SP15 and SP35). SP15 was extracted from lyophilized pig pulmonary surfactant with 5% Triton X-100 and purified by DEAE-cellulose, CM-cellulose, and affinity chromatography with a specific antibody. SP35 was extracted from the precipitate of the 5% Triton X-100 extraction with pH 10 borate buffer and purified by DEAE-cellulose column chromatography. Lipid-SP15 complex was formed by a detergent dialysis method using octylglucopyranoside, and to this complex were added various concentrations of SP35 at 37 degrees C. Structures similar to TM were formed when lipid-SP15 complex containing dipalmitoylphosphatidylcholine:phosphatidylglycerol from egg lecithin (2:1) and SP15 (lipid/protein, 5:1) was incubated with SP35 at concentrations of 0.15 to 0.22 mg/ml in CaCl2-containing buffer. At higher concentrations of SP35, many six-sided lattices were formed; the addition of EDTA abolished the formation of these lattice structures. The results suggest that SP15 and SP35 have an important function in the structural organization of lipid membranes to form lattices.

Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hardto-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

The 2-5A system is one of the major pathways for antiviral and antitumor functions that can be induced by interferons (IFNs). The 2-5A system is modulated by 5-triphosphorylated, 2,5-phosphodiester-linked oligoadenylates (2-5A), which are synthesized by 2,5-oligoadenylate synthetases (2,5-OASs), inactivated by 5-phosphatase and completely degraded by 2-phosphodiesterase (2-PDE). Generated 2-5A activates 2-5A-dependent endoribonuclease, RNase L, which induces RNA degradation in cells and finally apoptosis. Although 2,5-OASs and RNase L have been molecularly cloned and studied well, the identification of 2-PDE has remained elusive. Here, we describe the first identification of 2-PDE, the third key enzyme of the 2-5A system. We found a putative 2-PDE band on SDS-PAGE by successive six-step chromatographies from ammonium sulfate precipitates of bovine liver and identified a partial amino acid sequence of the human 2-PDE by mass spectrometry. Based on the full-length sequence of the human 2-PDE obtained by in silico expressed sequence tag assembly, the gene was cloned by reverse transcription-PCR. The recombinant human 2-PDE expressed in mammalian cells certainly cleaved the 2,5-phosphodiester bond of 2-5A trimer and 2-5A analogs. Because no sequences with high homology to this human 2-PDE were found, the human 2-PDE was considered to be a unique enzyme without isoform. Suppression of 2-PDE by a small interfering RNA and a 2-PDE inhibitor resulted in significant reduction of viral replication, whereas overexpression of 2-PDE protected cells from IFN-induced antiproliferative activity. These observations identify 2-PDE as a key regulator of the 2-5A system and as a potential novel target for antiviral and antitumor treatments.

Sperm motility patterns are continuously changed after ejaculation to fertilization in the female tract. Hyperactivated motility is induced with high glucose medium in vitro or the oviduct fluids in vivo, whereas sperm maintain linear motility in the seminal plasma or the uterine fluids containing low glucose. Therefore, it is estimated that sperm motility patterns are dependent on the energy sources, and the mitochondrial oxidative phosphorylation is activated to produce ATP in low glucose condition. To elucidate these hypotheses, boar sperm was incubated in different energy conditions with the transcription and translation inhibitors in vitro. Sperm motility parameters, mitochondrial activity, ATP level, gene expression and protein synthesis were analyzed. Sperm progressive motility and straight-line velocity were significantly increased with decreasing glucose level in the incubation medium. Moreover, the mitochondrial protein turnover meaning transcription and translation from mitochondrial genome in sperm is activated during incubation. Incubation of sperm with mitochondrial translation inhibitor (D-chloramphenicol) suppressed mitochondrial protein synthesis, mitochondrial activity and ATP level in sperm and consequently reduced the linear motility speed, but not the motility. Thus, it is revealed that the mitochondrial central dogma is active in sperm, and the high-speed linear motility is induced in low glucose condition via activating the mitochondrial activity for ATP generation.

A-503083 B, a capuramycin-type antibiotic, contains an L-aminocaprolactam and an unsaturated hexuronic acid that are linked via an amide bond. A putative class C beta-lactamase (CapW) was identified within the biosynthetic gene cluster that-in contrast to the expected beta-lactamase activity-catalyzed an amide-ester exchange reaction to eliminate the L-aminocaprolactam with concomitant generation of a small but significant amount of the glyceryl ester derivative of A-503083 B, suggesting a potential role for an ester intermediate in the biosynthesis of capuramycins. A carboxyl methyltransferase, CapS, was subsequently demonstrated to function as an S-adenosylmethionine-dependent carboxyl methyltransferase to form the methyl ester derivative of A-503083 B. In the presence of free L-aminocaprolactam, CapW efficiently converts the methyl ester to A-503083 B, thereby generating a new amide bond. This ATP-independent amide bond formation using methyl esterification followed by an ester-amide exchange reaction represents an alternative to known strategies of amide bond formation.

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Lip reading: The biosynthetic gene cluster for A‐90289, a fatty acid nucleoside antibiotic, was cloned and sequenced. The sulfotransferase LipB is demonstrated to be essential for A‐90289 biosynthesis, and in vitro characterization revealed LipB utilizes p‐nitrophenylsulfate as an aryl sulfate donor and a variety of nonaryl acceptors including caprazamycin A to give the 2′‐O‐sulfated product, A‐90289A.

Photoluminescence ͑PL͒ behavior of peapod-derived double-walled carbon nanotubes ͑DWNTs͒-the simplest form among multiwalled carbon nanotubes-is investigated. Even though the optical absorption and the resonant Raman spectra show the characteristic features of DWNTs, the PL signals originated from DWNTs are severely suppressed. This suppression is a consequence of an interlayer interaction between the inner and the outer tubes that efficiently quenches the PL signals of the DWNTs.