Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme

Mapping protein dynamics at high spatial resolution with temperature-jump X-ray crystallography

温度による酵素の構造変化を分子動画撮影 様々な生体高分子のダイナミクスを決定する新たな方法論. 京都大学プレスリリース. 2023-09-19.Understanding and controlling protein motion at atomic resolution is a hallmark challenge for structural biologists and protein engineers because conformational dynamics are essential for complex functions such as enzyme catalysis and allosteric regulation. Time-resolved crystallography offers a window into protein motions, yet without a universal perturbation to initiate conformational changes the method has been limited in scope. Here we couple a solvent-based temperature jump with time-resolved crystallography to visualize structural motions in lysozyme, a dynamic enzyme. We observed widespread atomic vibrations on the nanosecond timescale, which evolve on the submillisecond timescale into localized structural fluctuations that are coupled to the active site. An orthogonal perturbation to the enzyme, inhibitor binding, altered these dynamics by blocking key motions that allow energy to dissipate from vibrations into functional movements linked to the catalytic cycle. Because temperature jump is a universal method for perturbing molecular motion, the method demonstrated here is broadly applicable for studying protein dynamics

MicroRNA-338-3p and microRNA-451 contribute to the formation of basolateral polarity in epithelial cells

MicroRNAs are small noncoding RNA species, some of which are playing important roles in cell differentiation. However, the level of participations of microRNAs in epithelial cell differentiation is largely unknown. Here, utilizing an epithelial differentiation model with T84 cells, we demonstrate that miR-338-3p and miR-451 contribute to the formation of epithelial basolateral polarity by facilitating translocalization of β1 integrin to the basolateral membrane. Among 250 microRNAs screened in this study, the expression levels of four microRNAs (miR-33a, 210, 338-3p and 451) were significantly elevated in the differentiated stage of T84 cells, when epithelial cell polarity was established. To investigate the involvement of these microRNAs in terms of epithelial cell polarity, we executed loss-of- and gain-of-function analyses of these microRNAs. The blockade of endogenous miR-338-3p or miR-451 via each microRNA-specific antisense oligonucleotides inhibited the translocalization of β1 integrin to the basolateral membrane, whereas inhibition of miR-210 or miR-33a had no effect on it. On the other hand, simultaneous transfection of synthetic miR-338-3p and miR-451 accelerated the translocalization of β1 integrin to the basolateral membrane, although the introduction of individual synthetic microRNAs exhibited no effect. Therefore, we concluded that both miR-338-3p and miR-451 are necessary for the development of epithelial cell polarity

胆石患者における血清スクアレン

Serum squalene concentration, which represents the degree of cholesterol synthesis,　and other serum and biliary lipids were determined and characteristic abnormalities of　lipid metabolism were discussed in patients with gallstones having high serum squalene. The serum squalene concentration was significantly high in patients with pure　cholesterol and mixed stones. It remained in normal range in patients with combination　and bilirubinate stones. The high serum squalene concentration was accompanied by　a high lithogenic index and serum triglyceride concentration. Patients with high serum　squalene were also accompanied by increased percent cholic and decreased percent　chenodeoxycholic acid. These results may indicate that patients with high serum squalene in our present　study have similar lipid abnormalities as type IV hyperlipoproteinemia which has　increased cholesterol synthesis, hypertriglyceridemia and increased cholic acid pool and　normal chenodeoxycholic acid pool size

肝静脈－股動脈間の血流中に起る高比重リポ蛋白及びその亜分画構成成分の変化

The difference in HDL-lipid constituents was examined in sera from the hepatic vein and femoral artery. In HDL, HDL2 and HDL3 the most pronounced difference existed in the triglyceride concentration which was significantly higher in the femoral artery as compared to the hepatic vein. The cholesterol and phospholipid concentrations of HDL2 alone but not of HDL3 were also significantly higher in the femoral artery than hepatic vein. The concentrations of apo A-I and A-II, on the other hand, remained unaltered in the blood circulation. These results, in combination with the finding of a significant decrease in triglycerides of VLDL and LDL fraction (d. = 1.006－1.063) in the femoral artery as compared to the hepatic vein suggest that the accumulation of triglycerides in HDL is due to their transfer from other triglyceride-rich lipoprotein and the accumulation of HDL2b occurs in the blood stream from the hepatic vein to the femoral artery

USV-Observed Turbulent Heat Flux Induced by Late Spring Cold Dry Air Incursion over Sub-Mesoscale Warm Regions off Sanriku, Japan

We performed oceanic and atmospheric observations in the region off the Sanriku coast, Japan, from May 11 to 5 July 2022, using a wave-propelled unmanned surface vehicle, a Wave Glider (WG). Despite the severe weather conditions of atmospheric low-pressure system crossings, we successfully measured wind, air temperature, humidity, and sea surface temperature over the course of 55 days to calculate the turbulent heat flux. The WG observed that the atmosphere became more humid due to the southerly wind along the northwestern rim of the North Pacific subtropical high. The warm Kuroshio water expanded to the southeast of Hokkaido as a result of the northward shedding of an anticyclonic mesoscale (~100 km) eddy, called a warm-core ring, from the Kuroshio Extension. The WG traversed smaller (sub-mesoscale) water regions that were warmer and saltier than the surrounding Kuroshio water. The observations indicate that cold, dry air masses advected by northerly winds following the passage of atmospheric low-pressure systems generate a substantial upward turbulent heat flux over sub-mesoscale warm water regions, contrasting to no heat flux in the surrounding Kuroshio water region