Crystal Structure of Pyrrolysyl-tRNA Synthetase from a Methanogenic Archaeon ISO4-G1 and Its Structure-Based Engineering for Highly-Productive Cell-Free Genetic Code Expansion with Non-Canonical Amino Acids.

Pairs of pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). Previously, we achieved full productivity of cell-free protein synthesis for bulky non-canonical amino acids, including Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-L-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS with structure-based mutations in and around the amino acid binding pocket (first-layer and second-layer mutations, respectively). Recently, the PylRS·tRNAPyl pair from a methanogenic archaeon ISO4-G1 was used for genetic code expansion. In the present study, we determined the crystal structure of the methanogenic archaeon ISO4-G1 PylRS (ISO4-G1 PylRS) and compared it with those of structure-known PylRSs. Based on the ISO4-G1 PylRS structure, we attempted the site-specific incorporation of Nε-(p-ethynylbenzyloxycarbonyl)-L-lysine (pEtZLys) into proteins, but it was much less efficient than that of TCO*Lys with M. alvus PylRS mutants. Thus, the first-layer mutations (Y125A and M128L) of ISO4-G1 PylRS, with no additional second-layer mutations, increased the protein productivity with pEtZLys up to 57 ± 8% of that with TCO*Lys at high enzyme concentrations in the cell-free protein synthesis.

Risk factors for myopia at 1-year corrected age following laser photocoagulation for retinopathy of prematurity.

BACKGROUND/OBJECTIVES: The prevalence of myopia is higher in preterm infants who underwent laser photocoagulation (LPC) for retinopathy of prematurity (ROP). The aim of this study was to investigate factors associated with myopia in preterm infants who undergo LPC for ROP. SUBJECTS/METHODS: We retrospectively analysed the medical records of preterm infants born at Kyushu University Hospital (October 2008-March 2018) at ≤32 weeks of gestational age or with birth weight ≤1500 g. We evaluated the associations between nine clinical factors and the spherical equivalent at 1-year corrected age by performing multivariable linear regression in LPC-treated ROP patients. RESULTS: Among the 485 infants enroled, 76 developed ROP requiring treatment. Of these, 71 underwent LPC, which was provided to 63 infants as the primary treatment (LPC alone or the combination therapy of LPC and intravitreal injection of bevacizumab [IVB]) and to eight infants as additional LPC after IVB monotherapy. The results of a refractive examination at 1-year corrected age were available for 110 eyes of 56 infants (78.9%). The mean ± standard deviation of the SE value was -0.5 ± 3.0 dioptres (D). Multivariable linear regression analysis revealed a significant association between laser spot count and SE value (ß = -0.081 ± 0.040 D per 100 spots [mean ± standard error], p = 0.045). CONCLUSIONS: Our results suggest that an increased laser spot count observed during ROP treatment associates with myopia.

Neurodevelopmental outcomes following intravitreal bevacizumab injection in Japanese preterm infants with type 1 retinopathy of prematurity.

PURPOSE: The purpose of this study was to evaluate neurodevelopmental outcomes in 18-month old (corrected age) preterm infants who received an intravitreal bevacizumab (IVB) injection for the treatment of type 1 retinopathy of prematurity (ROP). METHODS: In this ten-year retrospective study, we reviewed the medical records of patients who underwent ROP screening at Kyushu University Hospital. Among the patients who received IVB or laser photocoagulation (LPC) for the treatment of type 1 ROP, we included infants whose neurodevelopmental examination (the Kyoto Scale of Psychological Development [KSPD]) results at 18 months corrected age were available. Then, the effect of IVB on the developmental quotient (DQ) in each KSPD domain (Postural-Movement, Cognitive-Adaptive, or Language-Social domain) or the overall DQ was investigated by performing linear regression analysis. RESULTS: Out of the 513 patients reviewed, 53 were included in the study. IVB and LPC were performed for 14 and 39 patients, respectively. Administration of IVB was significantly associated with neurodevelopmental delay in the Language-Social domain (p = 0.01). The observed association remained even after adjusting for gestational age and birth weight (p = 0.03). CONCLUSIONS: Administration of IVB may introduce a risk of developmental impairment of interpersonal relationships, socializations, and/or verbal abilities of preterm children. We recommended that preterm infants who received IVB undergo a neurodevelopmental reassessment during their school years or in adulthood.

Fully Productive Cell-Free Genetic Code Expansion by Structure-Based Engineering of Methanomethylophilus alvus Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS)/tRNAPyl pairs from Methanosarcina mazei and Methanosarcina barkeri are widely used for site-specific incorporations of non-canonical amino acids into proteins (genetic code expansion). In this study, we achieved the full productivity of cell-free protein synthesis for difficult, bulky non-canonical amino acids, such as Nε-((((E)-cyclooct-2-en-1-yl)oxy)carbonyl)-l-lysine (TCO*Lys), by using Methanomethylophilus alvus PylRS. First, based on the crystal structure of M. alvus PylRS, the productivities for various non-canonical amino acids were greatly increased by rational engineering of the amino acid-binding pocket. The productivities were further enhanced by using a much higher concentration of PylRS over that of M. mazei PylRS, or by mutating the outer layer of the amino acid-binding pocket. Thus, we achieved full productivity even for TCO*Lys. The quantity and quality of the cell-free-produced antibody fragment containing TCO*Lys were drastically improved. These results demonstrate the importance of full productivity for the expanded genetic code.

Structural Basis for Genetic-Code Expansion with Bulky Lysine Derivatives by an Engineered Pyrrolysyl-tRNA Synthetase.

Pyrrolysyl-tRNA synthetase (PylRS) and tRNAPyl have been extensively used for genetic-code expansion. A Methanosarcina mazei PylRS mutant bearing the Y306A and Y384F mutations (PylRS(Y306A/Y384F)) encodes various bulky non-natural lysine derivatives by UAG. In this study, we examined how PylRS(Y306A/Y384F) recognizes many amino acids. Among 17 non-natural lysine derivatives, Nɛ-(benzyloxycarbonyl)lysine (ZLys) and 10 ortho/meta/para-substituted ZLys derivatives were efficiently ligated to tRNAPyl and were incorporated into proteins by PylRS(Y306A/Y384F). We determined crystal structures of 14 non-natural lysine derivatives bound to the PylRS(Y306A/Y384F) catalytic fragment. The meta- and para-substituted ZLys derivatives are snugly accommodated in the productive mode. In contrast, ZLys and the unsubstituted or ortho-substituted ZLys derivatives exhibited an alternative binding mode in addition to the productive mode. PylRS(Y306A/Y384F) displayed a high aminoacylation rate for ZLys, indicating that the double-binding mode minimally affects aminoacylation. These precise substrate recognition mechanisms by PylRS(Y306A/Y384F) may facilitate the structure-based design of novel non-natural amino acids.

Cell-Free Protein Synthesis Using S30 Extracts from Escherichia coli RFzero Strains for Efficient Incorporation of Non-Natural Amino Acids into Proteins.

Cell-free protein synthesis is useful for synthesizing difficult targets. The site-specific incorporation of non-natural amino acids into proteins is a powerful protein engineering method. In this study, we optimized the protocol for cell extract preparation from the Escherichia coli strain RFzero-iy, which is engineered to lack release factor 1 (RF-1). The BL21(DE3)-based RFzero-iy strain exhibited quite high cell-free protein productivity, and thus we established the protocols for its cell culture and extract preparation. In the presence of 3-iodo-l-tyrosine (IY), cell-free protein synthesis using the RFzero-iy-based S30 extract translated the UAG codon to IY at various sites with a high translation efficiency of >90%. In the absence of IY, the RFzero-iy-based cell-free system did not translate UAG to any amino acid, leaving UAG unassigned. Actually, UAG was readily reassigned to various non-natural amino acids, by supplementing them with their specific aminoacyl-tRNA synthetase variants (and their specific tRNAs) into the system. The high incorporation rate of our RFzero-iy-based cell-free system enables the incorporation of a variety of non-natural amino acids into multiple sites of proteins. The present strategy to create the RFzero strain is rapid, and thus promising for RF-1 deletions of various E. coli strains genomically engineered for specific requirements.

Cell-Free Protein Synthesis for Multiple Site-Specific Incorporation of Noncanonical Amino Acids Using Cell Extracts from RF-1 Deletion E. coli Strains.

Cell-free protein synthesis (CFPS) is an effective method for the site-specific incorporations of noncanonical amino acids (ncAAs) into proteins. The nature of in vitro synthesis enables the use of experimental conditions that are toxic or reduce cellular uptake during in vivo site-specific incorporations of ncAAs. Using the Escherichia coli cell extract (S30) from the highly reproductive RF-1 deletion strains, B-60.∆A::Z and B-95.∆A, with orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pairs from Methanosarcina mazei, we have developed CFPS methods for the highly productive and efficient multiple incorporation of ncAAs. In this chapter, we describe our methods for the preparation of the S30 and the orthogonal tRNAPyl and PylRS pair, and two CFPS protocols for ncAA incorporation.

Radiographic changes and factors associated with subsequent progression of damage in weight-bearing joints of patients with rheumatoid arthritis under TNF-blocking therapies-three-year observational study.

OBJECTIVES: The long-term effects of tumor necrosis factor (TNF)-blocking therapies on weight-bearing joints in patients with rheumatoid arthritis (RA) have not been fully characterized. The purpose of this study was to assess the radiographic changes of weight-bearing joints in patients with RA during 3-year of TNF-blocking therapies and to identify factors related to the progression of joint damage. METHODS: Changes in clinical variables and radiological findings in 243 weight-bearing joints (63 hips, 54 knees, 71 ankles, and 55 subtalar joints) in 38 consecutive patients were investigated during three years of treatment with TNF-blocking agents. Multivariate logistic regression analysis was used to identify risk factors for the progression of weight-bearing joint damage. RESULTS: Seventeen (14.5%) of proximal weight-bearing joints (hips and knees) showed apparent radiographic progression during three years of treatment, whereas none of the proximal weight-bearing joints showed radiographic evidence of improvement or repair. In contrast, distal weight-bearing joints (ankle and subtalar joints) displayed radiographic progression and improvement in 20 (15.9%) and 8 (6.3%) joints, respectively. Multivariate logistic analysis for proximal weight-bearing joints identified the baseline Larsen grade (p < 0.001, OR:24.85, 95%CI: 5.07-121.79) and disease activity at one year after treatment (p = 0.003, OR:3.34, 95%CI:1.50-7.46) as independent factors associated with the progression of joint damage. On the other hand, multivariate analysis for distal weight-bearing joints identified disease activity at one year after treatment (p < 0.001, OR:2.13, 95%CI:1.43-3.18) as an independent factor related to the progression of damage. CONCLUSIONS: Baseline Larsen grade was strongly associated with the progression of damage in the proximal weight-bearing joints. Disease activity after treatment was an independent factor for progression of damage in proximal and distal weight-bearing joints. Early treatment with TNF-blocking agents and tight control of disease activity are necessary to prevent the progression of damage of the weight-bearing joints.

Investigating the Roles of the C-Terminal Domain of Plasmodium falciparum GyrA.

Malaria remains as one of the most deadly diseases in developing countries. The Plasmodium causative agents of human malaria such as Plasmodium falciparum possess an organelle, the apicoplast, which is the result of secondary endosymbiosis and retains its own circular DNA. A type II topoisomerase, DNA gyrase, is present in the apicoplast. In prokaryotes this enzyme is a proven, effective target for antibacterial agents, and its discovery in P. falciparum opens up the prospect of exploiting it as a drug target. Basic characterisation of P. falciparum gyrase is important because there are significant sequence differences between it and the prokaryotic enzyme. However, it has proved difficult to obtain soluble protein. Here we have predicted a new domain boundary in P. falciparum GyrA that corresponds to the C-terminal domain of prokaryotic GyrA and successfully purified it in a soluble form. Biochemical analyses revealed many similarities between the C-terminal domains of GyrA from E. coli and P. falciparum, suggesting that despite its considerably larger size, the malarial protein carries out a similar DNA wrapping function. Removal of a unique Asn-rich region in the P. falciparum protein did not result in a significant change, suggesting it is dispensable for DNA wrapping.

Inhibitory effect of tacrolimus on progression of joint damage in patients with rheumatoid arthritis.

AIM: To examine the inhibitory effect of tacrolimus on radiographic joint damage in patients with rheumatoid arthritis (RA). METHODS: Thirty-eight patients with RA resistant or intolerant to conventional disease-modifying anti-rheumatic drugs were administered tacrolimus and analyzed retrospectively. Disease activity and clinical response were evaluated by Disease Activity Score in 28 joints and C-reactive protein (DAS28-CRP) and European League Against Rheumatism (EULAR) response criteria. The progression of joint destruction was evaluated by an estimated yearly change in modified Total Sharp Score (mTSS). RESULTS: Good or moderate response rate according to EULAR response criteria was seen in 63.2%, 63.2%, 73.7% and 65.8% of patients at 3, 6, 12, and 24 months, respectively. The rate of patients with low disease activity or remission reached 47.3% and 50.0% at 12 and 24 months, respectively. Progression of joint damage, evaluated as yearly change in mTSS (ΔmTSS), significantly decreased from 11.4 at baseline to 2.63 in the first year and 0.69 in the second year of tacrolimus treatment. CONCLUSION: These findings suggest tacrolimus has the potential to inhibit progression of joint damage in established RA.

Dislocation of the extensor carpi ulnaris tendon in rheumatoid wrists using three-dimensional computed tomographic imaging.

The purpose of this study was to evaluate dislocation of the extensor carpi ulnaris (ECU) tendon in patients with rheumatoid arthritis (RA) using three-dimensional computed tomography (3DCT). We then determined the value of 3DCT for predicting spontaneous extensor tendon rupture. 3DCT images of 102 wrists from 96 patients with RA were analyzed. Dislocation of the ECU tendon was evaluated from the 3DCT images with a soft tissue window. Dorsal subluxation of the ulnar head was evaluated as the dorsal subluxation ratio (DSR), and carpal supination was evaluated as the carpal supination angle (CSA) from the 3DCT images with a bone tissue window. Extensor tendon rupture was found in 43 of 102 wrists (42 %). Dislocation of the ECU tendon was found in 35 of 102 wrists (34 %). Dislocation of the ECU tendon was a strong risk factor for extensor tendon rupture in the multivariable logistic regression analysis, with an odds ratio of 26 (95 % confidence interval 7-99, p < 0.001). The DSR (p = 0.029) and CSA (p = 0.035) were significantly larger in wrists with a dislocated ECU tendon. Dislocation of the ECU tendon was well depicted using 3DCT images. It was strongly associated with extensor tendon rupture in the rheumatoid wrist.

Continuous peripheral nerve block in forearm for severe hand trauma.

We studied the use of a continuous peripheral nerve block (CPNB) in the distal forearm and wrist immediately after emergent surgery for severe hand trauma in 22 hands. After emergent surgery, a 2-3 cm longitudinal incision was made at the distal forearm and an 18-gauge catheter was inserted along the peripheral nerves. All patients received postoperative analgesia by continuous infusion of 0.2% ropivacaine at 2 ml/h for seven to 21 days. Pain score remained low during postoperative period and only a small number of analgesic rescues were needed. There were no major complications related to the CPNB and one patient showed mild superficial infection at the insertion site that immediately recovered after catheter removal. This method provides good postoperative analgesia without loss of motor function in extrinsic hand muscles and should be considered as a postoperative pain management for severe hand trauma.

Structural insight into the zinc finger CW domain as a histone modification reader.

The zinc finger CW (zf-CW) domain is a motif of about 60 residues that is frequently found in proteins involved in epigenetic regulation. Here, we determined the NMR solution structure of the zf-CW domain of the human zf-CW and PWWP domain containing protein 1 (ZCWPW1). The zf-CW domain adopts a new fold in which a zinc ion is coordinated tetrahedrally by four conserved Cys ligand residues. The tertiary structure of the zf-CW domain partially resembles that adopted by the plant homeo domain (PHD) finger bound to the histone tail, suggesting that the zf-CW domain and the PHD finger have similar functions. The solution structure of the complex of the zf-CW domain with the histone H3 tail peptide (1-10) with trimethylated K4 clarified its binding mode. Our structural and biochemical studies have identified the zf-CW domain as a member of the histone modification reader modules for epigenetic regulation.

Rheumatoid wrist deformity and risk of extensor tendon rupture evaluated by 3DCT imaging.

OBJECTIVE: Extensor tendon rupture on the dorsum of the wrist is commonly seen in patients with rheumatoid arthritis (RA). It causes immediate dysfunction of the hand and surgical reconstruction is usually required. The purpose of this study was to clarify the risk of extensor tendon rupture by quantifying wrist deformity on three-dimensional computed tomography (3DCT) images. MATERIALS AND METHODS: Three-dimensional CT images of 108 wrists in 102 patients with RA and 38 wrists in 38 healthy volunteers were analyzed retrospectively. All of the rheumatoid wrists had caused persistent pain for more than 6 months despite ongoing medical treatment. Extensor tendon rupture was noted in 49 wrists in 47 patients, and no rupture was noted in 59 wrists in 56 patients. The dorsal subluxation ratio (DSR) of the ulnar head and the carpal supination angle (CSA) were measured utilizing a new technique. RESULTS: The average DSR and CSA in the rupture group (n = 49), the non-rupture group (n = 59), and the normal wrist group (n = 38) were 37%, 19%, and 26%, and 15 degrees , 11 degrees , and 6 degrees respectively. The cut-off values for extensor tendon rupture in the wrists of patients with RA were 32% (sensitivity; 70%, specificity; 75%) in the DSR, and 14 degrees (71%, 68%) in the CSA. CONCLUSION: By utilizing 3DCT imaging of the rheumatoid wrist, these parameters can help improve our ability to predict extensor tendon rupture.

Multiple inhibitory factor removal from an Escherichia coli cell extract improves cell-free protein synthesis.

Cell-free protein synthesis systems are generally influenced by the nature of the cell extract, which contains various factors on the chromosomal DNA. Some of the Escherichia coli cell extract factors are essential, despite their negative effects on protein synthesis, because they are required during the cell growth and/or extract preparation stage. In this study, modified E. coli strains were generated by inserting a streptavidin binding peptide (SBP) tag sequence at the 3' termini of the genes encoding polynucleotide phosphorylase (PNPase) and/or Exodeoxyribonuclease V alpha chain (RecD) on the chromosomal DNA. The SBP-tagged target gene products were specifically removed from the cell extract prepared from modified E. coli cells using SBP affinity resin. The linear DNA-directed cell-free protein synthesis using the treated extract achieved higher productivity, especially when removing both the PNPase and RecD factors. Using this strategy to remove multiple inhibitory factors in a cell extract will be widely applicable to improve cell-free protein synthesis.

Solution structure of the RNA binding domain in the human muscleblind-like protein 2.

The muscleblind-like (MBNL) proteins 1, 2, and 3, which contain four CCCH zinc finger motifs (ZF1-4), are involved in the differentiation of muscle inclusion by controlling the splicing patterns of several pre-mRNAs. Especially, MBNL1 plays a crucial role in myotonic dystrophy. The CCCH zinc finger is a sequence motif found in many RNA binding proteins and is suggested to play an important role in the recognition of RNA molecules. Here, we solved the solution structures of both tandem zinc finger (TZF) motifs, TZF12 (comprising ZF1 and ZF2) and TZF34 (ZF3 and ZF4), in MBNL2 from Homo sapiens. In TZF12 of MBNL2, ZF1 and ZF2 adopt a similar fold, as reported previously for the CCCH-type zinc fingers in the TIS11d protein. The linker between ZF1 and ZF2 in MBNL2 forms an antiparallel beta-sheet with the N-terminal extension of ZF1. Furthermore, ZF1 and ZF2 in MBNL2 interact with each other through hydrophobic interactions. Consequently, TZF12 forms a single, compact global fold, where ZF1 and ZF2 are approximately symmetrical about the C2 axis. The structure of the second tandem zinc finger (TZF34) in MBNL2 is similar to that of TZF12. This novel three-dimensional structure of the TZF domains in MBNL2 provides a basis for functional studies of the CCCH-type zinc finger motifs in the MBNL protein family.

Radiographic progression in weight-bearing joints of patients with rheumatoid arthritis after TNF-blocking therapies.

The aim of the present study was to assess the influence of tumor necrosis factor (TNF)-blocking therapies on weight-bearing joints in patients with rheumatoid arthritis. Changes in clinical variables and radiological findings in 213 weight-bearing joints (69 hip joints, 63 knee joints, and 81 ankle joints) of 42 consecutive patients were investigated at baseline and at 1 year of TNF-blocking therapies. Structural damage to the weight-bearing joints was assessed using the Larsen scoring method. Detailed comparisons of the sizes and locations of erosions were performed for each set of radiographs of the respective joints. Assessment of radiographs of the 213 weight-bearing joints indicated progression of the Larsen grade in eight joints. Another five joints without Larsen grade progression showed apparent radiographic progression of joint damage based on increases in bony erosions. Overall, 13 joints (6%) of eight patients (19%) showed progression of joint damage after 1 year of TNF-blocking therapies. Analysis of each baseline grade indicated that radiographic progression of joint damage was inhibited in most grade 0-II joints. On the other hand, all hip and knee joints with pre-existing damage of grade III/IV showed apparent progression even in patients with good response. The results further suggested that radiographic progression may occur in less damaged joints when the patients were non-responders to the therapy. Among the weight-bearing joints, ankle joints showed different radiographic behavior and four ankle joints displayed improvement of radiographic damage. Early initiation of anti-TNF therapy should be necessary especially when the patients are starting to show early structural damage in weight-bearing joints.

Structural basis for controlling the dimerization and stability of the WW domains of an atypical subfamily.

The second WW domain in mammalian Salvador protein (SAV1 WW2) is quite atypical, as it forms a beta-clam-like homodimer. The second WW domain in human MAGI1 (membrane associated guanylate kinase, WW and PDZ domain containing 1) (MAGI1 WW2) shares high sequence similarity with SAV1 WW2, suggesting comparable dimerization. However, an analytical ultracentrifugation study revealed that MAGI1 WW2 (Leu355-Pro390) chiefly exists as a monomer at low protein concentrations, with an association constant of 1.3 x 10(2) M(-1). We determined its solution structure, and a structural comparison with the dimeric SAV1 WW2 suggested that an Asp residue is crucial for the inhibition of the dimerization. The substitution of this acidic residue with Ser resulted in the dimerization of MAGI1 WW2. The spin-relaxation data suggested that the MAGI1 WW2 undergoes a dynamic process of transient dimerization that is limited by the charge repulsion. Additionally, we characterized a longer construct of this WW domain with a C-terminal extension (Leu355-Glu401), as the formation of an extra alpha-helix was predicted. An NMR structural determination confirmed the formation of an alpha-helix in the extended C-terminal region, which appears to be independent from the dimerization regulation. A thermal denaturation study revealed that the dimerized MAGI1 WW2 with the Asp-to-Ser mutation gained apparent stability in a protein concentration-dependent manner. A structural comparison between the two constructs with different lengths suggested that the formation of the C-terminal alpha-helix stabilized the global fold by facilitating contacts between the N-terminal linker region and the main body of the WW domain.