In-Situ Observation of Membrane Protein Folding during Cell-Free Expression

Proper insertion, folding and assembly of functional proteins in biological membranes are key processes to warrant activity of a living cell. Here, we present a novel approach to trace folding and insertion of a nascent membrane protein leaving the ribosome and penetrating the bilayer. Surface Enhanced IR Absorption Spectroscopy selectively monitored insertion and folding of membrane proteins during cell-free expression in a label-free and non-invasive manner. Protein synthesis was performed in an optical cell containing a prism covered with a thin gold film with nanodiscs on top, providing an artificial lipid bilayer for folding. In a pilot experiment, the folding pathway of bacteriorhodopsin via various secondary and tertiary structures was visualized. Thus, a methodology is established with which the folding reaction of other more complex membrane proteins can be observed during protein biosynthesis (in situ and in operando) at molecular resolution.     

Mtu1-Mediated Thiouridine Formation of Mitochondrial tRNAs Is Required for Mitochondrial Translation and Is Involved in Reversible Infantile Liver Injury

Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in MTU1 have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm⁵s²U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1^LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1^LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.     

Month of birth is associated with mortality among older people in Japan: Findings from the JAGES cohort

Month of birth (MOB) has been linked to a variety of health conditions in adulthood. This study examined the association between MOB and mortality among the healthy elderly in Japan, where a practice of traditional age reckoning was employed up until the late 1940s. The results showed male participants born in December were more likely to die earlier while those born in January had lower mortality. It is possible that social factors in early life, such as the time period when a birth is officially registered, may have implications for health that stretch across the life course.     

Evaluation of a non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold as a SARS 3CL protease inhibitor

A non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold was evaluated as a novel inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CL^pro). The decahydroisoquinolin scaffold has been demonstrated to be an effective hydrophobic center to interact with S2 site of SARS 3CL^pro, but the lack of interactions at S3 to S4 site is thought to be a major reason for the moderate inhibitory activity. In this study, the effects of an additional non-prime site substituent on the scaffold as well as effects of several warheads are evaluated. For the introduction of a desired non-prime site substituent, amino functionality was introduced on the decahydroisoquinolin scaffold, and the scaffold was constructed by Pd(II) catalyzed diastereoselective ring formation. The synthesized decahydroisoquinolin inhibitors showed about 2.4 times potent inhibitory activities for SARS 3CL^pro when combined with a non-prime site substituent. The present results indicated not only the expected additional interactions with the SARS 3CL^pro but also the possibility of new inhibitors containing a fused-ring system as a hydrophobic scaffold and a new warhead such as thioacetal.     

EcoBalance 2018—Nexus of ideas: innovation by linking through life cycle thinking (9–12 October 2018, Tokyo,Japan)

The International Journal of Life Cycle Assessment -     

Spatial heterogeneity in induced defense of Brachionus calyciflorus within a single lake caused by a bed of floating-leaved macrophyte Trapa species

Limnology - While induced defense of aquatic organisms against predators has been considerably studied by both laboratory and field research, our understanding is still limited about, for example,...     

Antifibrotic effect of methylated quercetin derivatives on TGFβ-induced hepatic stellate cells

Quercetin (QCT) and isorhamnetin (ISO), natural flavonoids, were both shown to possess antifibrotic activity in in vivo and in vitro models of hepatic fibrosis. Although ISO is a direct metabolite of QCT differing by a methyl group, it has been reported to be absorbed more adequately and eliminated slower than QCT after oral administration. Our aim of the study was to investigate biological effect of mono-methylated QCT derivatives against fibrosis using rat hepatic stellate cells (HSC-T6). All test derivatives were synthesized from QCT. HSC-T6 cells were induced by TGFβ and treated with derivatives followed by cell proliferation assay, immunofluorescence staining of αSMA, and gene expression analysis of fibrosis markers. All compounds showed a dose- and time-dependent antiproliferation effect. ISO, 3-O-methylquercetin (3MQ), and rhamnetin (RHA) reduced αSMA mRNA; 3MQ prevented the augmentation of collagen I mRNA; and compounds, except azaleatin and 3MQ, reduced Timp1 mRNA expression in TGFβ-induced HSCs. In conclusion, each compound had singular effect against different features of fibrosis depending on the position of methyl group although the further mechanism of action of compounds during fibrosis development remains to be investigated. These findings suggest that antifibrotic effect of quercetin can be enhanced by adding methyl group on functionally important position.     

Cyclin E2 is required for embryogenesis in Xenopus laevis

In mammalian cells, E-type cyclins (E1 and E2) are generally believed to be required for entry into S phase. However, in mice, cyclin E is largely dispensable for normal embryogenesis. Moreover, Drosophila cyclin E plays a critical role in cell fate determination in neural lineages independently of proliferation. Thus, the functions of cyclin E, particularly during early development, remain elusive. Here, we investigated the requirement for E-type cyclins during Xenopus embryogenesis. Although cyclin E1 has been reported as a maternal cyclin, inhibition of its translation in the embryo caused no serious defects. We isolated a Xenopus homologue of human cyclin E2, which was zygotically expressed. Sufficient inhibition of its expression led to death at late gastrula, while partial inhibition allowed survival. These observations indicate distinct roles for Xenopus cyclins E1 and E2, and an absolute requirement of cyclin E2 for Xenopus embryogenesis.     

Lower extremity muscle activity during different types and speeds of underwater movement

To compare the activity of lower extremity muscles during land walking (LW), water walking (WW), and deep-water running (DWR), 9 healthy young subjects were tested at self-selected low, moderate, and high intensities for 8 sec with two repetitions. Surface EMG electrodes were placed on the tibialis anterior (TA), soleus (SOL), medial gastrocnemius (GAS), rectus femoris (RF), and biceps femoris (BF). During DWR, the SOL and GAS activities were lower than LW and WW. The BF activities were higher during DWR than LW and WW. It was considered that the lower activity of SOL and GAS depended on water depth, and higher activity of BF occurred by greater flexion of the knee joint or extension of the hip joint during exercise.