Routine clozapine assay monitoring to improve the management of treatment-resistant schizophrenia.

AIMS AND METHOD: Routine therapeutic drug monitoring in clozapine therapy has previously not been considered justifiable. Using observational data, the clinical utility of annual clozapine assay monitoring is explored within a large mental health trust. RESULTS: After the introduction of routine monitoring, the rate of clozapine assays rose to 2.3 per patient per year, with a consistent reduction in high-risk clozapine assays (<0.1 mg/L or >1.0 mg/L or any result more than 24 months old). High-risk assays are associated with a mortality rate of 31.6 deaths per 1000 patients, more than twice that of those within the target range (0.35-0.60 mg/L and conducted within the past 12 months) (P = 0.048). CLINICAL IMPLICATIONS: Routine clozapine assay monitoring has significant clinical utility. Our simple but targeted approach can be readily implemented to reduce the number of patients with high-risk clozapine assay levels, potentially reduce all-cause mortality and provide optimal treatment for those with treatment-resistant schizophrenia.

Does Time to Theatre Affect the Ability to Achieve Fracture Reduction in Tibial Plateau Fractures?

Surgical management of displaced tibial plateau fracture (TPF) is often delayed due to accompanying soft tissue injuries sustained at the time of injury. The primary aim of this study was to assess the effect of time to surgery on fracture reduction in cases of TPF. The secondary aim was to assess the effect of preoperative demographics and residual articular step on Lysholm Scores and Knee Injury and Osteoarthritis Outcome Scores (KOOS) following fixation. Patients between 2006 and 2017, managed by a single surgeon, were prospectively enrolled in the study. Reduction of articular step, defined as <2 mm, was assessed by a single blinded examiner. A total of 117 patients were enrolled, 52 with Schatzker II, 4 with Schatzker IV, and 61 with Schatzker VI fractures. Patients were followed up to a mean of 3.9 years. Analysis showed that the ability to achieve fracture reduction was negatively influenced by time to theatre, with the odds of achieving reduction decreasing 17% with each subsequent day post injury (p = 0.002). Furthermore, an increased time to theatre was associated with a reduced Lysholm score at one year (p = 0.01). The ability to achieve fracture reduction did not influence PROMs within the study period. We conclude that delay in surgical fixation negatively affects fracture reduction in TPF and may delay recovery. However, residual articular step does not necessarily influence PROMs over the mid-term.

An uncharged amine in the transition state of the ribosomal peptidyl transfer reaction.

The ribosome has an active site comprised of RNA that catalyzes peptide bond formation. To understand how RNA promotes this reaction requires a detailed understanding of the chemical transition state. Here, we report the Brønsted coefficient of the alpha-amino nucleophile with a series of puromycin derivatives. Both 50S subunit- and 70S ribosome-catalyzed reactions displayed linear free-energy relationships with slopes close to zero under conditions where chemistry is rate limiting. These results indicate that, at the transition state, the nucleophile is neutral in the ribosome-catalyzed reaction, in contrast to the substantial positive charge reported for typical uncatalyzed aminolysis reactions. This suggests that the ribosomal transition state involves deprotonation to a degree commensurate with nitrogen-carbon bond formation. Such a transition state is significantly different from that of uncatalyzed aminolysis reactions in solution.

Fusion Assessment by MRI in Comparison With CT in Anterior Lumbar Interbody Fusion: A Prospective Study.

STUDY DESIGN: Prospective cohort study. OBJECTIVES: To evaluate the role of magnetic resonance imaging (MRI) in evaluation of fusion status following anterior lumbar interbody fusion (ALIF) and compare agreement and confidence in assessing fusion or its absence on MRI to the current standard computed tomography (CT). METHODS: A prospective follow up of patients undergoing surgery by 2 spine surgeons between 2012 and 2015 at a single institution. Fusion was assessed at different time points in these patients by 2 independent musculoskeletal radiologists. Fusion was analyzed in coronal and sagittal planes using both imaging modalities, with confidence being attributed on a scale of 0 to 3. Assessors were blinded to patient data. RESULTS: Fourteen patients (25 levels) with mean follow-up of 10.2 months (range 2.4-20.3 years) and age of 41 years (range 20.7-61.5 years) were assessed. MRI within the interbody cage in coronal (κ = .58) and sagittal (κ = .50) planes had the highest interobserver agreement. CT anterior to the cage in coronal (κ = .48) and sagittal (κ = .44) planes, as well as within the cage in coronal (κ = .50) and sagittal planes (κ = .44) showed moderate agreement. Confidence anterior to the interbody cage using MRI scan was reduced when compared with remaining angles and imaging modalities. CONCLUSIONS: The study demonstrates that MRI may be a useful tool in the assessment of fusion following ALIF with results comparable to CT, and that it may have a useful role in select patients especially considering marked radiation exposure reduction.

Time dependent loss of trabecular bone in human tibial plateau fractures.

We investigated if time between injury and surgery affects cancellous bone properties in patients suffering tibial plateau fractures (TPF), in terms of structural integrity and gene expression controlling bone loss. A cohort of 29 TPF, operated 1-17 days post-injury, had biopsies from the fracture and an equivalent contralateral limb site, at surgery. Samples were assessed using micro-computed tomography and real-time RT-PCR analysis for the expression of genes known to be involved in bone remodeling and fracture healing. Significant decreases in the injured vs control side were observed for bone volume fraction (BV/TV, -13.5 ± 6.0%, p = 0.011), trabecular number (Tb.N, -10.5 ± 5.9%, p = 0.041) and trabecular thickness (Tb.Th, -4.6 ± 2.5%, p = 0.033). Changes in these parameters were more evident in patients operated 5-17 days post-injury, compared to those operated in the first 4 days post-injury. A significant negative association was found between Tb.Th (r = -0.54, p < 0.01) and BV/TV (r = -0.39, p < 0.05) in relation to time post-injury in the injured limb. Both BV/TV and Tb.Th were negatively associated with expression of key molecular markers of bone resorption, CTSK, ACP5, and the ratio of RANKL:OPG mRNA. These structure/gene expression relationships did not exist in the contralateral tibial plateau of these patients. This study demonstrated that there is a significant early time-dependent bone loss in the proximal tibia after TPF. This bone loss was significantly associated with altered expression of genes typically involved in the process of osteoclastic bone resorption but possibly also bone resorption by osteocytes. The mechanism of early bone loss in such fractures should be a subject of further investigation. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2865-2875, 2018.

Solid phase synthesis and binding affinity of peptidyl transferase transition state mimics containing 2'-OH at P-site position A76.

All living cells are dependent on ribosomes to catalyze the peptidyl transfer reaction, by which amino acids are assembled into proteins. The previously studied peptidyl transferase transition state analog CC-dA-phosphate-puromycin (CCdApPmn) has important differences from the transition state, yet current models of the ribosomal active site have been heavily influenced by the properties of this molecule. One significant difference is the substitution of deoxyadenosine for riboadenosine at A76, which mimics the 3' end of a P-site tRNA. We have developed a solid phase synthetic approach to produce inhibitors that more closely match the transition state, including the critical P-site 2'-OH. Inclusion of the 2'-OH or an even bulkier OCH3 group causes significant changes in binding affinity. We also investigated the effects of changing the A-site amino acid side chain from phenylalanine to alanine. These results indicate that the absence of the 2'-OH is likely to play a significant role in the binding and conformation of CCdApPmn in the ribosomal active site by eliminating steric clash between the 2'-OH and the tetrahedral phosphate oxygen. The conformation of the actual transition state must allow for the presence of the 2'-OH, and transition state mimics that include this critical hydroxyl group must bind in a different conformation from that seen in prior analog structures. These new inhibitors will provide valuable insights into the geometry and mechanism of the ribosomal active site.

RNA oligonucleotide synthesis via 5'-silyl-2'-orthoester chemistry.

Rapid, reliable, and cost-efficient methods of ribonucleic acid (RNA) oligonucleotide synthesis are in demand owing to an increasing awareness of critical structural, functional, and regulatory roles of RNA throughout biology. The most promising area of growth and development is in RNA interference as an emerging technology for facilitating research in drug discovery and therapeutic intervention. Traditional methods of RNA synthesis, which are based on 2'-silyl protection strategies derived from deoxyribonucleic acid (DNA) synthesis strategies, are limited in their ability to produce oligos of sufficient purity and length for high-throughput applications. The more recently developed 5'-silyl-2'-acetoxy ethyl orthoester chemistry (2'-ACE trade mark ), circumvents several limitations of the 2'-silyl approaches. A clear improvement in RNA synthesis technology, 2'-ACE results in faster coupling rates, higher yields, greater purity, and superior ease of handling. Another advantage of the 2'-ACE protecting group strategy is that the molecules can be produced in an intermediately protected form that is soluble in aqueous solutions but resistant to nuclease attack. The chemistry can be scaled up or down and is flexible enough to allow for the incorporation of modifying groups if desired. A detailed description of the 2'-ACE protocol and procedures for end product analysis are presented.

Chromophoric 5'-O-Silyl protection of N-protected 2'-ACE ribonucleosides for solid-phase RNA synthesis.

Recent advances in the understanding of the pivotal roles played by endogenous small RNAs in gene regulation have resulted in a substantial and rapidly growing market for synthetic RNA. 5'-Silyl-2'-ACE chemistry has proven to be a robust and reliable technology for the synthesis of oligoribonucleotides. This unit describes an important improvement to this chemistry, by adding a cycle-to-cycle traceability analogous to that inherent in 5'-dimethoxytrityl-based approaches. This is achieved by first regioselectively introducing a 5'-alkynylsilyl protecting group onto the 2'-ACE-protected nucleosides. The 5'-alkynylsilyl group is then reacted with an azide derivative of the chromophore Disperse Red 1, which enables spectrophotometric interrogation of each coupling step following 5'-deprotection. Finally, the protected nucleosides are elaborated into their 3'-phosphoramidite derivatives for use in solid-phase RNA synthesis.

Structural insights into the roles of water and the 2' hydroxyl of the P site tRNA in the peptidyl transferase reaction.

Peptide bond formation is catalyzed at the peptidyl transferase center (PTC) of the large ribosomal subunit. Crystal structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with several analogs that represent either the substrates or the transition state intermediate of the peptidyl transferase reaction show that this reaction proceeds through a tetrahedral intermediate with S chirality. The oxyanion of the tetrahedral intermediate interacts with a water molecule that is positioned by nucleotides A2637 (E. coli numbering, 2602) and (methyl)U2619(2584). There are no Mg2+ ions or monovalent metal ions observed in the PTC that could directly promote catalysis. The A76 2' hydroxyl of the peptidyl-tRNA is hydrogen bonded to the alpha-amino group and could facilitate peptide bond formation by substrate positioning and by acting as a proton shuttle between the alpha-amino group and the A76 3' hydroxyl of the peptidyl-tRNA.

Preparation of 5'-silyl-2'-orthoester ribonucleosides for use in oligoribonucleotide synthesis.

The recent discovery that small interfering RNAs (siRNAs) induce gene suppression in mammalian cells has sparked tremendous interest in using siRNA-based assays and high-throughput screens to study gene function. As a result, research programs at leading academic and commercial institutions have become a substantial and rapidly growing market for synthetic RNA. Important considerations in synthesizing RNA for biological gene function studies are sequence integrity, purity, scalability, and resistance to nucleases; ease of chemical modification, deprotection, and handling; and cost. Of the well-established RNA synthesis methods, 2'-ACE chemistry is the only one that meets all of these criteria. 2'-ACE technology employs a unique class of silyl ethers to protect the 5'-hydroxyl, in combination with an acid-labile orthoester protecting group on the 2'-hydroxyl (2'-ACE). 2'-ACE-protected phosphoramidite monomers are joined using standard solid-phase technology to achieve RNA synthesis at efficiencies rivaling those for DNA. This unit describes the synthesis of standard 5'-silyl-2'-ACE-protected phosphoramidites.