α‐Synuclein (α‐syn) has been known to be a key player of the pathogenesis of Parkinson’s disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of α‐syn into cells has also been observed. In this study, we observed that dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric α‐syn into BV‐2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of α‐syn internalization in dl‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol‐treated BV‐2 cells. Furthermore, inhibition of clathrin‐, caveolae‐, and dynamin‐dependent endocytosis did not prevent the internalization of α‐syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of α‐syn. In addition, we further confirmed these observations by co‐culture system of BV‐2 cells and α‐syn‐over‐expressing SH‐SY5Y cells. These findings suggest that extracellular α‐syn is internalized into microglia via GM1 as well as hitherto‐unknown protein receptors in clathrin‐, caveolae‐, and dynamin‐independent, but lipid raft‐dependent manner. Elucidation of the mechanism involved in internalization of α‐syn should be greatly helpful in the development of new treatments of α‐syn‐related neurodegenerative diseases.

The ring-shaped structural-maintenance-of-chromosomes (SMC) complexes condensin and cohesin extrude loops of DNA as a key motif in chromosome organization. It remains, however, unclear how these SMC motor proteins can extrude DNA loops in chromatin that is bound with proteins. Here, using in vitro single-molecule visualization, we show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to DNA loop extrusion by yeast condensin. Strikingly, we find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, can be translocated into DNA loops during condensin-driven extrusion. Similarly, human cohesin can pass 200 nm particles during loop extrusion, which even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open up to entrap DNA. These findings disqualify all common loop-extrusion models where DNA passes through the SMC rings (pseudo)topologically, and instead point to a nontopological mechanism for DNA loop extrusion.

Despite the recent development in the design of DNA origami, its folding yet relies on thermal or chemical annealing methods. We here demonstrate mechanical folding of the DNA origami structure via a pathway that has not been accessible to thermal annealing. Using magnetic tweezers, we stretch a single scaffold DNA with mechanical tension to remove its secondary structures, followed by base pairing of the stretched DNA with staple strands. When the force is subsequently quenched, folding of the DNA nanostructure is completed through displacement between the bound staple strands. Each process in the mechanical folding is well defined and free from kinetic traps, enabling us to complete folding within 10 min. We also demonstrate parallel folding of DNA nanostructures through multiplexed manipulation of the scaffold DNAs. Our results suggest a path towards programmability of the folding pathway of DNA nanostructures.

BackgroundSerine/arginine-rich splicing factors (SRSFs) and HNRNPA1 have oncogenic properties. However, their proteomic expressions and practical priority in gastric cancer (GC) and colorectal cancer (CRC) are mostly unknown. To apply SFs in clinics, effective marker selection and characterization of properties in the target organ are essential.MethodsWe concurrently analyzed SRSF1, 3, and 5–7, and HNRNPA1, together with the conventional tumor marker carcinoembryonic antigen (CEA), in stomach and colorectal tissue samples (n = 420) using semiquantitative immunoblot, subcellular fractionation, and quantitative real-time polymerase chain reaction methods.ResultsIn the semiquantitative immunoblot analysis, HNRNPA1 and SRSF7 levels were significantly higher in GC than in gastric normal mucosa, and SRSF7 levels were higher in intestinal-type compared with diffuse-type of gastric adenocarcinoma. Of the SFs, only HNRNPA1 presented greater than 50 % upregulation (cancer/normal mucosa > 2-fold) incidences and CEA-comparable, acceptable (>70 %) detection accuracy (74 %) for GC. All SF protein levels were significantly higher in CRC than in colorectal normal mucosa, and HNRNPA1 levels were higher in low-stage CRC compared with high-stage CRC. Among the SFs, HNRNPA1 and SRSF3 presented the two highest upregulation incidences (88 % and 74 %, respectively) and detection accuracy (90 % and 84 %, respectively) for CRC. The detection accuracy of HNRNPA1 was comparable to that of CEA in low (≤ II)-stage CRC but was inferior to that of CEA in high (>II)-stage CRC. Extranuclear distributions of HNRNPA1 and SRSF6 (cytosol/microsome) differed from those of other SRSFs (membrane/organelle) in both cancers. In an analysis of the six SF mRNAs, all mRNAs presented unacceptable detection accuracies (≤70 %) in both cancers, and all mRNAs except SRSF6 were disproportionate to the corresponding protein levels in GC.ConclusionOur results provide a comprehensive insight into the six SF expression profiles in GC and indicate that, among the SFs, HNRNPA1, but not HNRNPA1 mRNA, is the most effective, novel GC marker. Regardless of the good to excellent detection accuracy of SRSF3 and HNRNPA1 in CRC, the SFs have lower practical priority than CEA, especially for high-stage CRC detection.

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The pharmacokinetics and metabolism of 2'-benzoyloxycinnamaldehyde (BCA) was characterized in male Sprague-Dawley rats as part of the preclinical evaluations for developing this compound as an antitumour agent. BCA was not detected in the plasma following either intravenous or oral dose, whereas its putative metabolites 2'-hydroxycinnamaldehyde (HCA) and o-coumaric acid were present at considerable levels. In separate pharmacokinetics studies, HCA exhibited a high systemic clearance and a large volume of distribution, whereas both pharmacokinetic parameters were much lower for o-coumaric acid. The terminal half-life of both metabolites was approximately 2 h. BCA was converted rapidly to HCA in rat serum, liver microsomes and cytosol in vitro; HCA was subsequently converted to o-coumaric acid in a quantitative manner only in the liver cytosol. In addition, the formation of o-coumaric acid was inhibited significantly by menadione, a specific inhibitor for aldehyde oxidase. Taken collectively, the results suggest that the rapid systemic clearance of HCA is likely due mainly to hepatic clearance occurring from aldehyde oxidase-catalysed biotransformation to o- coumaric acid. In conclusion, the present work demonstrates that the anticancer drug candidate BCA is highly likely to work as its active metabolite HCA in the body.

The results of our study show that prostate biopsy leads to objective voiding impairment. Therefore, the use of alpha-1 blocker tamsulosin before biopsy in patients without prior BPH medication may decrease this morbidity.