Multistep Engineering of Pyrrolysyl-tRNA Synthetase to Genetically Encode Nɛ-(o-Azidobenzyloxycarbonyl) lysine for Site-Specific Protein Modification

SummaryPyrrolysyl-tRNA synthetase (PylRS) esterifies pyrrolysine to tRNAPyl. In this study, Nɛ-(tert-butyloxycarbonyl)-L-lysine (BocLys) and Nɛ-allyloxycarbonyl-L-lysine (AlocLys) were esterified to tRNAPyl by PylRS. Crystal structures of a PylRS catalytic fragment complexed with BocLys and an ATP analog and with AlocLys-AMP revealed that PylRS requires an Nɛ-carbonyl group bearing a substituent with a certain size. A PylRS(Y384F) mutant obtained by random screening exhibited higher in vitro aminoacylation and in vivo amber suppression activities with BocLys, AlocLys, and pyrrolysine than those of the wild-type PylRS. Furthermore, the structure-based Y306A mutation of PylRS drastically increased the in vitro aminoacylation activity for Nɛ-benzyloxycarbonyl-L-lysine (ZLys). A PylRS with both the Y306A and Y384F mutations enabled the large-scale preparation (>10 mg per liter medium) of proteins site-specifically containing Nɛ-(o-azidobenzyloxycarbonyl)-L-lysine (AzZLys). The AzZLys-containing protein was labeled with a fluorescent probe, by Staudinger ligation

The Kurashiki Prehospital Stroke Scale Is a Prehospital Scale That Can Predict Long-Term Outcome of Patients with Acute Cerebral Ischemia

Background and Purpose: Our aim was to confirm the clinical relationship between the Kurashiki Prehospital Stroke Scale (KPSS) scored by paramedics and favorable outcomes in patients with modified Rankin scale (mRS) scores of 0–1 assessed 3 months after symptom onset. Methods: We enrolled patients with acute stroke and transient ischemic attack showing symptoms on admission. Paramedics transferred patients to our hospital after estimating stroke severity using the KPSS. After categorizing patients into either the mRS 0–1 group (favorable outcome) or the mRS 2–6 group (no favorable outcome), we compared the background data between the two groups. We assessed KPSS scores predictive of a favorable outcome. Multivariate regression modeling was conducted to identify factors independently associated with a favorable outcome. Results: The study cohort comprised 147 patients with a premorbid status of mRS 0–1: 69 patients (47%) of them were in the mRS 0–1 group and 78 (53%) in the mRS 2–6 group at the follow-up 3 months after symptom onset. The median KPSS score was lower in the mRS 0–1 group than in the mRS 2–6 group (1 vs. 4, p Conclusion: KPSS score <3 apparently presents a reasonable cutoff for predicting a favorable outcome in patients with acute cerebral ischemia

Mechanisms of the inhibition of reverse transcription by unmodified and modified antisense oligonucleotides

AbstractWe demonstrated that unmodified and modified (phosphorothioate) oligonucleotides prevent cDNA synthesis by AMV or HIV reverse transcriptases. Antisense oligonucleotide/RNA hybrids specifically arrest primer extension. The blockage involves the degradation of the RNA fragment bound to the antisense oligonucleotide by the reverse transcriptase-associated RNase H activity. However, the phosphorothioate oligomer inhibited polymerization by binding to the AMV RT rather than to the template RNA, whereas there was no competitive binding of the phosphorothioate oligomer on the HIV RT during reverse transcription

Evaluation of Aortic Disease with Spiral CT Angiography and Multiplanar Reconstructions: Comparison with Catheter Angiography

Purpose: To assess the usefulness of spiral CT angiography (CTA) in the evaluation of aortic aneurysm (AA) or dissection (AD). Methods: Ninety-eight patients with AA (n = 78) or AD (n = 20) were examined with CTA. Imaging results were correlated with angiographic (n = 98) findings in all cases and surgical findings in AA cases (n = 64). The spiral CT angiography were analyzed by an experienced radiologist without knowledge of the result of the catheter angiography, to evaluate the same features. The catheter angiograms were individually interpreted by two experienced radiologists. Results: In AA, all of major aortic branches were depicted on CTA except two of seven accessory renal arteries and six of 26 inferior mesenteric arteries. CTA correctly assessed aneurysm involvement of left subclavian (LSA), renal (RA), and iliac arteries (IA) in all patients. In AD, CTA correctly assessed Stanford classification in all patients, and the relationship between 70 major aortic branches and true/false lumen in all but two branches. CTA showed 23 of 30 intimal tears in double barreled AD. Conclusion: CTA might replace catheter angiography in evaluation of AA and AD except in cases of type A dissection. Index Terms: computed tomography - CT angiography - aorta, aneurysm - aorta, dissection - Catheter angiograph

Genetic Encoding of 3-Iodo-l-Tyrosine in Escherichia coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography

SummaryWe developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-l-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-l-tyrosine. The 1.7 Å crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-l-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-l-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation

Functional replacement of the endogenous tyrosyl-tRNA synthetase–tRNATyr pair by the archaeal tyrosine pair in Escherichia coli for genetic code expansion

Non-natural amino acids have been genetically encoded in living cells, using aminoacyl-tRNA synthetase–tRNA pairs orthogonal to the host translation system. In the present study, we engineered Escherichia coli cells with a translation system orthogonal to the E. coli tyrosyl-tRNA synthetase (TyrRS)–tRNATyr pair, to use E. coli TyrRS variants for non-natural amino acids in the cells without interfering with tyrosine incorporation. We showed that the E. coli TyrRS–tRNATyr pair can be functionally replaced by the Methanocaldococcus jannaschii and Saccharomyces cerevisiae tyrosine pairs, which do not cross-react with E. coli TyrRS or tRNATyr. The endogenous TyrRS and tRNATyr genes were then removed from the chromosome of the E. coli cells expressing the archaeal TyrRS–tRNATyr pair. In this engineered strain, 3-iodo-l-tyrosine and 3-azido-l-tyrosine were each successfully encoded with the amber codon, using the E. coli amber suppressor tRNATyr and a TyrRS variant, which was previously developed for 3-iodo-l-tyrosine and was also found to recognize 3-azido-l-tyrosine. The structural basis for the 3-azido-l-tyrosine recognition was revealed by X-ray crystallography. The present engineering allows E. coli TyrRS variants for non-natural amino acids to be developed in E. coli, for use in both eukaryotic and bacterial cells for genetic code expansion

Background Factors Affecting Visual Acuity at Initial Visit in Eyes with Central Retinal Vein Occlusion : Multicenter Study in Japan

Purpose: To determine the baseline characteristics of patients with central retinal vein occlusion (CRVO) that were significantly associated with the best-corrected visual acuity (BCVA) at the initial examination. Methods: This was a retrospective multicenter study using the medical records registered in 17 ophthalmological institutions in Japan. Patients with untreated CRVO (≥20-years-of-age) who were initially examined between January 2013 and December 2017 were studied. The patients’ baseline factors that were significantly associated with the BCVA at the initial examination were determined by univariate and multivariate linear regression analyses. Results: Data from 517 eyes of 517 patients were analyzed. Univariate analyses showed that an older age (r = 0.194, p < 0.001) and the right eye (r = −0.103, p < 0.019) were significantly associated with poorer BCVA at the initial visit. Multivariate analyses also showed that an older age (β = 0.191, p < 0.001) and the right eye (β = −0.089, p = 0.041) were significantly associated with poorer BCVA at the initial visit. Conclusions: The results indicate that an older age, a known strong factor, and the right eye were significantly associated with poorer BCVA at the initial visit to the hospital. These results suggest that functional and/or anatomical differences between the right and left eyes may be involved in these results

Genetic Code Expansion: Another Solution to Codon Assignments

This Special Issue is intended to highlight recent advances in genetic code expansion, particularly the site-specific incorporation of noncanonical amino acids (ncAAs) into proteins [...

Synthetic Tyrosine tRNA Molecules with Noncanonical Secondary Structures

The L-shape form of tRNA is maintained by tertiary interactions occurring in the core. Base changes in this domain can cause structural defects and impair tRNA activity. Here, we report on a method to safely engineer structural variations in this domain utilizing the noncanonical scaffold of tRNAPyl. First, we constructed a naïve hybrid between archaeal tRNAPyl and tRNATyr, which consisted of the acceptor and T stems of tRNATyr and the other parts of tRNAPyl. This hybrid tRNA efficiently translated the UAG codon to 3-iodotyrosine in Escherichia coli cells, when paired with a variant of the archaeal tyrosyl-tRNA synthetase. The amber suppression efficiency was slightly lower than that of the “bench-mark” archaeal tRNATyr suppressor assuming the canonical structure. After a series of modifications to this hybrid tRNA, we obtained two artificial types of tRNATyr: ZtRNA had an augmented D (auD) helix in a noncanonical form and the D and T loops bound by the standard tertiary base pairs, and YtRNA had a canonical auD helix and non-standard interloop interactions. It was then suggested that the ZtRNA scaffold could also support the glycylation and glutaminylation of tRNA. The synthetic diversity of tRNA would help create new tRNA–aminoacyl-tRNA synthetase pairs for reprogramming the genetic code

Negative Fluid-Attenuated Inversion Recovery- Based Intravenous Thrombolysis Using Recombinant Tissue Plasminogen Activator in Acute Stroke Patients with Unknown Onset Time

Background: Approximately 25% of acute stroke patients were excluded from intravenous thrombolysis using recombinant tissue plasminogen activator (IV-tPA) because of unknown onset time. Recent studies have shown that patients with unknown onset time would be able to receive IV-tPA when showing no ischemia on fluid-attenuated inversion recovery (negative FLAIR). The present study evaluated the safety and feasibility of IV-tPA in patients with unknown onset time and negative FLAIR compared to those with standard IV-tPA. Methods: Stroke patients with unknown onset time were prospectively enrolled. Only patients with an occlusion of the internal carotid artery (ICA) and/or middle cerebral artery (M1 and M2) with a Diffusion-Weighted Imaging-Alberta Stroke Program Early CT Score (DWI-ASPECTS) ≥5 were analyzed. IV-tPA was performed within 3 h from the ‘first found abnormal time' if the patient showed negative FLAIR. Standard IV-tPA patients were extracted from our registry as controls after having been matched by age and occluded artery to the negative FLAIR (N-F) group. Results: Twenty patients in the N-F group and 60 in the control group were included. National Institutes of Health Stroke Scale (NIHSS) scores [median 18 (interquartile range 13-20) vs. 17 (12-20), p = 0.609] and DWI-ASPECTS [9 (7-9) vs. 8 (5-9), p = 0.213] were similar between the 2 groups. ICA occlusion was seen in 35%, M1 in 50%, and M2 in 15% in both groups. None of the N-F group and 1 (2%) of the control group experienced symptomatic intracerebral hemorrhage (p = 1.000). Recanalization within 1 h after IV-tPA was achieved in 6 (30%) patients in the N-F group and 24 (40%) in the control group (p = 0.595). Recanalization at 24 h after IV-tPA was seen in 13 (65%) patients in the N-F group and 43 (72%) in the control group (p = 0.584). At 7 days, 8 (40%) in the N-F group and 28 (47%) in the control group had a dramatic recovery (defined as a ≥10-point reduction in the total NIHSS score or a score of 0 or 1) (p = 0.796). At 3 months, a favorable outcome (modified Rankin scale score, 0-2) was seen in 47% in the N-F group and 33% in the control group (p = 0.365). Conclusion: IV-tPA in negative FLAIR patients with unknown onset time appears safe and feasible