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Funding: This study was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/M010996/1) via EASTBIO Doctoral Training Partnership studentships to G.F. and B.J.R., and by the University of St Andrews via a StARIS summer research bursary to S.K.L. The bifunctional enzyme phosphoribosyl-ATP pyrophosphohydrolase/phosphoribosyl-AMP cyclohydrolase (HisIE) catalyzes the second and third steps of histidine biosynthesis: pyrophosphohydrolysis of N1-(5-phospho-β-D-ribosyl)-ATP (PRATP) to N1-(5-phospho-β-D-ribosyl)-AMP (PRAMP) and pyrophosphate in the C-terminal HisE-like domain, and cyclohydrolysis of PRAMP to N-(5′-phospho-D-ribosylformimino)-5-amino-1-(5″-phospho-D-ribosyl)-4-imidazolecarboxamide (ProFAR) in the N-terminal HisI-like domain. Here we use UV–VIS spectroscopy and LC–MS to show Acinetobacter baumannii putative HisIE produces ProFAR from PRATP. Employing an assay to detect pyrophosphate and another to detect ProFAR, we established the pyrophosphohydrolase reaction rate is higher than the overall reaction rate. We produced a truncated version of the enzyme-containing only the C-terminal (HisE) domain. This truncated HisIE was catalytically active, which allowed the synthesis of PRAMP, the substrate for the cyclohydrolysis reaction. PRAMP was kinetically competent for HisIE-catalyzed ProFAR production, demonstrating PRAMP can bind the HisI-like domain from bulk water, and suggesting that the cyclohydrolase reaction is rate-limiting for the overall bifunctional enzyme. The overall kcat increased with increasing pH, while the solvent deuterium kinetic isotope effect decreased at more basic pH but was still large at pH 7.5. The lack of solvent viscosity effects on kcat and kcat/KM ruled out diffusional steps limiting the rates of substrate binding and product release. Rapid kinetics with excess PRATP demonstrated a lag time followed by a burst in ProFAR formation. These observations are consistent with a rate-limiting unimolecular step involving a proton transfer following adenine ring opening. We synthesized N1-(5-phospho-β-D-ribosyl)-ADP (PRADP), which could not be processed by HisIE. PRADP inhibited HisIE-catalyzed ProFAR formation from PRATP but not from PRAMP, suggesting that it binds to the phosphohydrolase active site while still permitting unobstructed access of PRAMP to the cyclohydrolase active site. The kinetics data are incompatible with a build-up of PRAMP in bulk solvent, indicating HisIE catalysis involves preferential channeling of PRAMP, albeit not via a protein tunnel. Publisher PDF

IntroductionNemaline myopathies (NM) represent a group of clinically and genetically heterogeneous congenital muscle disorders with the common denominator of nemaline rods on muscle biopsy. NEB and ACTA1 are the most common causative genes. Currently, available treatments are supportive.Areas coveredWe explored experimental treatments for NM, identifying at least eleven mainly pre-clinical approaches utilizing murine and/or human muscle cells. These approaches target either i) the causative gene or associated genes implicated in the same pathway; ii) pathophysiologically relevant biochemical mechanisms such as calcium/myosin regulation of muscle contraction; iii) myogenesis; iv) other therapies that improve or optimize muscle function more generally; v) and/or combinations of the above. The scope and efficiency of these attempts is diverse, ranging from gene-specific effects to those widely applicable to all NM-associated genes.Expert OpinionThe wide range of experimental therapies currently under consideration for NM is promising. Potential translation into clinical use requires consideration of additional factors such as the potential muscle type specificity as well as the possibility of gene expression remodeling. Challenges in clinical translation include the rarity and heterogeneity of genotypes, phenotypes, and disease trajectories, as well as the lack of longitudinal natural history data and validated outcomes and biomarkers.

This work was supported by a Wellcome Trust Seed Award in Science [208980/Z/17/Z] to RGdS; a University of St Andrews/Scottish Funding Council St Andrews Restarting Research Fund to RGdS; and a Wellcome Trust Institutional Strategic Support Fund [204821/Z/16/Z] to the University of St Andrews. ES is the recipient of a Cunningham Trust PhD studentship (PhD-CT-18-41). The enzyme m1A22-tRNA methyltransferase (TrmK) catalyzes the transfer of a methyl group to the N1 of adenine 22 in bacterial tRNAs. TrmK is essential for Staphylococcus aureus survival during infection but has no homolog in mammals, making it a promising target for antibiotic development. Here, we characterize the structure and function of S. aureus TrmK (SaTrmK) using X-ray crystallography, binding assays, and molecular dynamics simulations. We report crystal structures for the SaTrmK apoenzyme as well as in complexes with methyl donor SAM and co-product product SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favorable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated for by a large favorable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. In addition, activity assays for SaTrmK-catalyzed methylation of A22 mutants of tRNALeu demonstrate that the adenine at position 22 is absolutely essential. In silico screening of compounds suggested the multifunctional organic toxin plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS data indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92 in the vicinity of the SAM-binding site as the sole residue modified. These results identify a cryptic binding pocket of SaTrmK, laying a foundation for future structure-based drug discovery. Publisher PDF

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (Grant BB/M010996/1) via EASTBIO Doctoral Training Partnership studentships to B.J.R. and G.F., and by the Engineering and Physical Sciences Research Council (EPSRC) [grant number EP/L016419/1] via a CRITICAT Centre for Doctoral Training studentship to T.F.G.M. ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate. Catalytic (HisGS) and regulatory (HisZ) subunits assemble in a hetero-octamer where HisZ activates HisGS and mediates allosteric inhibition by histidine. In Acinetobacter baumannnii, HisGS is necessary for the bacterium to persist in the lung during pneumonia. Inhibition of ATPPRT is thus a promising strategy for specific antibiotic development. Here, A. baumannii ATPPRT is shown to follow a rapid equilibrium random kinetic mechanism, unlike any other ATPPRT. Histidine noncompetitively inhibits ATPPRT. Binding kinetics indicates histidine binds to free ATPPRT and to ATPPRT:PRPP and ATPPRT:ATP binary complexes with similar affinity following a two-step binding mechanism, but with distinct kinetic partition of the initial enzyme:inhibitor complex. The dipeptide histidine-proline inhibits ATPPRT competitively and likely uncompetitively, respectively, against PRPP and ATP. Rapid kinetics analysis shows His-Pro binds to the ATPPRT:ATP complex via a two-step binding mechanism. A related HisZ that shares 43% sequence identity with A. baumannii HisZ is a tight-binding allosteric inhibitor of A. baumannii HisGS. These findings lay the foundation for inhibitor design against A. baumannii ATPPRT. Postprint

Spinal muscular atrophy (SMA) is a rare autosomal recessive neuromuscular disease which is characterised by muscle atrophy and early death in most patients. Risdiplam is the third overall and first oral drug approved for SMA with disease-modifying potential. Risdiplam acts as a survival motor neuron 2 (SMN2) pre-mRNA splicing modifier with satisfactory safety and efficacy profile. This review aims to critically appraise the place of risdiplam in the map of SMA therapeutics. This review gives an overview of the current market for SMA and presents the mechanism of action and the pharmacological properties of risdiplam. It also outlines the development of risdiplam from early preclinical stages through to the most recently published results from phase 2/3 clinical trials. Risdiplam has proved its efficacy in pivotal trials for SMA Types 1, 2, and 3 with a satisfactory safety profile. In the absence of comparative data with the other two approved drugs, the role of risdiplam in the treatment algorithm of affected individuals is examined in three different patient populations based on the age and diagnosis method (newborn screening or clinical, symptom-driven diagnosis). Long-term data and real-world data will play a fundamental role in its future.

The enzyme m

The enzyme m1A22-tRNA methyltransferase (TrmK) catalyses the transfer of a methyl group from SAM to the N1 of adenine 22 in tRNAs. TrmK is essential for Staphylococcus aureus survival during infection, but has no homologue in mammals, making it a promising target for antibiotic development. Here we describe the structural and functional characterisation of S. aureus TrmK. Crystal structures are reported for S. aureus TrmK apoenzyme and in complexes with SAM and SAH. Isothermal titration calorimetry showed that SAM binds to the enzyme with favourable but modest enthalpic and entropic contributions, whereas SAH binding leads to an entropic penalty compensated by a large favourable enthalpic contribution. Molecular dynamics simulations point to specific motions of the C-terminal domain being altered by SAM binding, which might have implications for tRNA recruitment. Activity assays for S. aureus TrmK-catalysed methylation of WT and position 22 mutants of tRNALeu demonstrate that the enzyme requires an adenine at position 22 of the tRNA. Intriguingly, a small RNA hairpin of 18 nucleotides is methylated by TrmK depending on the position of the adenine. In-silico screening of compounds suggested plumbagin as a potential inhibitor of TrmK, which was confirmed by activity measurements. Furthermore, LC-MS indicated the protein was covalently modified by one equivalent of the inhibitor, and proteolytic digestion coupled with LC-MS identified Cys92, in the vicinity of the SAM-binding site, as the sole residue modified. These results these results identify a cryptic binding pocket of S. aureus TrmK and lay the foundation for future structure-based drug discovery.

ATP phosphoribosyltransferase (ATPPRT) catalyzes the first step of histidine biosynthesis in bacteria, namely, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate

Structural Maintenance of Chromosomes (SMC) complexes contribute ubiquitously to chromosome organization-segregation. SMC proteins have a conserved architecture, with a dimerization hinge and an ATPase head domain separated by a long antiparallel intramolecular coiled-coil. Dimeric SMC proteins interact with essential accessory proteins, kleisins that bridge the two subunits of an SMC dimer, and HAWK/KITE accessory proteins that interact with kleisins. The ATPase activity of the Escherichia coli SMC protein, MukB, is essential for in vivo function and is regulated by interactions with its dimeric kleisin, MukF, and KITE, MukE. Here we demonstrate that, in addition, MukB interacts with Acyl Carrier Protein (AcpP) that has essential functions in fatty acid synthesis. We characterize the AcpP interaction site at the joint of the MukB coiled-coil and show that the interaction is essential for MukB ATPase and for MukBEF function in vivo. Therefore, AcpP is an essential co-factor for MukBEF action in chromosome organization-segregation.

Biallelic mutations in SNORD118, encoding the small nucleolar RNA U8, cause leukoencephalopathy with calcifications and cysts (LCC). Given the difficulty in interpreting the functional consequences of variants in nonprotein encoding genes, and the high allelic polymorphism across SNORD118 in controls, we set out to provide a description of the molecular pathology and clinical spectrum observed in a cohort of patients with LCC. We identified 64 affected individuals from 56 families. Age at presentation varied from 3 weeks to 67 years, with disease onset after age 40 years in eight patients. Ten patients had died. We recorded 44 distinct, likely pathogenic, variants in SNORD118. Fifty two of 56 probands were compound heterozygotes, with parental consanguinity reported in only three families. Forty nine of 56 probands were either heterozygous (46) or homozygous (three) for a mutation involving one of seven nucleotides that facilitate a novel intramolecular interaction between the 5' end and 3' extension of precursor-U8. There was no obvious genotype-phenotype correlation to explain the marked variability in age at onset. Complementing recently published functional analyses in a zebrafish model, these data suggest that LCC most often occurs due to combinatorial severe and milder mutations, with the latter mostly affecting 3' end processing of precursor-U8.

This study examined developmental differences in the acoustics of pharyngeal swallowing. Thirty-one young children (M = 4.5 years) and 29 adults (M = 22.5 years) were recorded swallowing thin liquid and puree boluses. In comparison with adults, children showed longer total swallow sound duration and duration to peak intensity, as well as greater variability in the duration to peak intensity and mean of the averaged spectrum in Hz. Thin and puree boluses differed in measures of duration, intensity and frequency of the averaged sound spectrum, although these effects did not interact with age. The increased variability in swallowing observed in children paralleled that found in acoustic measures of vowel formants, although speech and swallowing acoustic measures were uncorrelated. Using Formant 2 frequency as a proxy measure of vocal tract length, the age differences in swallowing acoustics appear to be independent of physical size, although associations between duration to peak intensity and pharyngeal size warrant further investigation. These findings suggest acoustic measures of swallowing are sensitive to developmental status, possibly reflecting ongoing refinement of the pharyngeal swallow across childhood, and support continued research into the use of digital cervical auscultation as a tool to assess the efficiency and stability of the swallowing neuromuscular control system in children and adults.

Neurofibromatosis type 2 (NF2) is a rare autosomal dominant disorder (incidence 1:33 000-40 000) characterized by formation of central nervous system tumors, due to mutation in the NF2 gene on chromosome 22q12. Vestibular schwannomas are the hallmark lesion, affecting 95% of individuals and typically occur bilaterally. Schwannomas commonly occur on other nerves intracranially and in the spinal compartment, along with meningiomas, ependymomas, and gliomas. Although histologically benign, tumors are associated with significant morbidity due to multiple problems including hearing and vision loss, gait abnormalities, paralysis, pain, and seizures. Risk of early mortality from brainstem compression and other complications is significant. Severity of disease is higher when NF2 presents during childhood. Children have a more variable presentation, which can be associated with significant delays in recognition of the condition. Careful examination of the skin and eyes can identify important clinical signs of NF2 during childhood, allowing timely initiation of disease-specific surveillance and treatment. Monitoring for complications comprises clinical evaluation, along with functional testing including audiology and serial neuroimaging, which together inform decisions regarding treatment. Evidence for disease-specific medical treatment options is increasing, nevertheless most patients will benefit from multimodal treatment including surgery during their lifetime. Patient enrolment in international natural history and treatment trials offers the best opportunity to accelerate our understanding of the complications and optimal treatment of NF2, with a view to improving outcomes for all affected individuals.

This literature review aimed to determine the risk factors being used to identify children and young people who are at increased risk of engaging in self-harm and suicidal behaviour, so that optimal care can be provided for this patient group in children's medical ward settings. The two main themes that emerged were mental and neurodevelopmental disorders, and external factors. Management strategies to aid healthcare professionals in caring for this patient group were also identified. The review concludes by highlighting the need to provide healthcare professionals with continuing education about the mental health problems of children and young people, including risk factors and management strategies.

This article reports on the process and findings from a small-scale qualitative research study. The study intended to develop an evidence-based care plan/pathway for children and young people in paediatric inpatient settings presenting with self-harm/suicidal behaviour. The article includes a critical review of unanticipated challenges of recruiting to the study, and the learning from this process. Data are reported from three data collection strategies. A critical review of literature and policy was undertaken. The Delphi Technique was used to generate clinician-reported evidence from healthcare professionals within a paediatric general ward caring for children and young people presenting with self-harm/suicidal behaviour. Face-to-face consultations were undertaken in order to gain greater understanding of challenges to recruitment that were encountered. Despite evidence of participants possessing sufficient knowledge to care for young people engaging in self-harm/suicidal behaviour, participants...

Short-form ATP phosphoribosyltransferase (ATPPRT) is a hetero-octameric allosteric enzyme comprising four catalytic subunits (HisGS) and four regulatory subunits (HisZ). ATPPRT catalyzes the Mg2+-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, the first reaction of histidine biosynthesis. While HisGS is catalytically active on its own, its activity is allosterically enhanced by HisZ in the absence of histidine. In the presence of histidine, HisZ mediates allosteric inhibition of ATPPRT. Here, initial velocity patterns, isothermal titration calorimetry, and differential scanning fluorimetry establish a distinct kinetic mechanism for ATPPRT where PRPP is the first substrate to bind. AMP is an inhibitor of HisGS, but steady-state kinetics and 31P NMR spectroscopy demonstrate that ADP is an alternative substrate. Replacement of Mg2+ by Mn2+ enhances catalysis by HisGS but not by the holoenzyme, suggesting different rate-limiting steps for nonactivated and activated enzyme forms. Density functional theory calculations posit an SN2-like transition state stabilized by two equivalents of the metal ion. Natural bond orbital charge analysis points to Mn2+ increasing HisGS reaction rate via more efficient charge stabilization at the transition state. High solvent viscosity increases HisGS’s catalytic rate, but decreases the hetero-octamer’s, indicating that chemistry and product release are rate-limiting for HisGS and ATPPRT, respectively. This is confirmed by pre-steady-state kinetics, with a burst in product formation observed with the hetero-octamer but not with HisGS. These results are consistent with an activation mechanism whereby HisZ binding leads to a more active conformation of HisGS, accelerating chemistry beyond the product release rate.

This work was supported by the University of St Andrews, a Leverhulme Trust grant (RL - 2012 - 025) to G.J.F, the Engineering and Physical Sciences Research Council (EPSRC) [grant number EP/L016419/1] via a CRITICAT Centre for Doctoral Training studentship to TFGM, and the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/M010996/1] via an EASTBIO Doctoral Training Partnership studentship to GF. Allosteric modulation of catalysis is a common regulatory strategy of flux-controlling biosynthetic enzymes. The enzyme ATP phosphoribosyltransferase (ATPPRT) catalyzes the first reaction in histidine biosynthesis, the magnesium-dependent condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate (PRPP) to generate N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate (PPi). ATPPRT is allosterically inhibited by the final product of the pathway, histidine. Hetero-octameric ATPPRT consists of four catalytic subunits (HisGS) and four regulatory subunits (HisZ) engaged in intricate catalytic regulation. HisZ enhances HisGS catalysis in the absence of histidine while mediating allosteric inhibition in its presence. Here we report HisGS structures for the apoenzyme and complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP), product (PRATP), and inhibitor (AMP), along with ATPPRT holoenzyme structures in complexes with substrates (PRPP, PRPP-ATP, PRPP-ADP) and product (PRATP). These 10 crystal structures provide an atomic view of the catalytic cycle and allosteric activation of Psychrobacter arcticus ATPPRT. In both ternary complexes with PRPP-ATP, the adenine ring is found in an anticatalytic orientation, rotated 180° from the catalytic rotamer. Arg32 interacts with phosphate groups of ATP and PRPP, bringing the substrates in proximity for catalysis. The negative charge repulsion is further attenuated by a magnesium ion sandwiched between the α- and β-phosphate groups of both substrates. HisZ binding to form the hetero-octamer brings HisGS subunits closer together in a tighter dimer in the Michaelis complex, which poises Arg56 from the adjacent HisGS molecule for cross-subunit stabilization of the PPi leaving group at the transition state. The more electrostatically preorganized active site of the holoenzyme likely minimizes the reorganization energy required to accommodate the transition state. This provides a structural basis for allosteric activation in which chemistry is accelerated by facilitating leaving group departure. Postprint

Background: Qualitative research has suggested that spousal carers of someone with dementia differ in terms of whether they perceive their relationship with that person as continuous with the premorbid relationship or as radically different, and that a perception of continuity may be associated with more person-centered care and the experience of fewer of the negative emotions associated with caring. The aim of the study was to develop and evaluate a quantitative measure of the extent to which spousal carers perceive the relationship to be continuous.Methods: An initial pool of 42 questionnaire items was generated on the basis of the qualitative research about relationship continuity. These were completed by 51 spousal carers and item analysis was used to reduce the pool to 23 items. The retained items, comprising five subscales, were then administered to a second sample of 84 spousal carers, and the questionnaire's reliability, discriminative power, and validity were evaluated.Results: The questionnaire showed good reliability: Cronbach's α for the full scale was 0.947, and test–retest reliability was 0.932. Ferguson's δ was 0.987, indicating good discriminative power. Evidence of construct validity was provided by predicted patterns of subscale correlations with the Closeness and Conflict Scale and the Marwit–Meuser Caregiver Grief Inventory.Conclusion: Initial psychometric evaluation of the measure was encouraging. The measure provides a quantitative means of investigating ideas from qualitative research about the role of relationship continuity in influencing how spousal carers provide care and how they react emotionally to their caring role.

Background The temporomandibular joint (TMJ) is arguably one of the most complex joints in the human body but its intricate structure and mobile nature make dysfunction in this region extremely common. With a reported prevalence of up to 85% of the general population experiencing at least one sign of temporomandibular dysfunction (TMD) at some point in their lives, a wide range of treatment options must be available to manage the array of disorders that may occur at this joint, in order to provide a tailored approach to suit the individuality of patients. The TMJ has a close topographical and functional relationship with the upper cervical spine (Csp) and dysfunction in one area is often concomitant with reduced function in the other. There is significant research supporting the use of physical therapy for Csp dysfunction but more definitive research needs to objectively assess the effectiveness of cervical treatment for TMD and the reciprocal approach. Aim To investigate whether it is possible to affect active depression of the TMJ via a High-Velocity Low-Amplitude Thrust (HVLAT) technique of the C1-2 spinal segment within a symptomatic population, in order to contribute to the current knowledge base. Methods A pilot study for a single-blind, randomised controlled trial (RCT), was carried out using 30 volunteers with current TMD symptoms, as defined by the National Health Service. The volunteers were randomly assigned to either the intervention or control group and their TMJ and upper Csp range of motion (ROM) were assessed three times pre- and post-intervention. The Csp was assessed via expert palpation and passive range of motion (PROM) testing and the TMJ was assessed using Silicon Coach video analysis software. The treatment consisted of an HVLAT technique of the C1/2 spinal segment and the control group did not receive any treatment. The data was analysed using Statistical Package for Social Sciences (SPSS) software via parametric t -tests. Results The results of the study demonstrated a statistically significant increase in TMJ AROM from baseline to post intervention ( p Conclusion This study demonstrated that an HVLAT of the C1/2 spinal segment significantly increases active depression of the TMJ in a symptomatic population. Larger RCTs of this nature are recommended, with the use of a TMD assessment tool in order to investigate the effect of this technique on particular sub-groups of TMD.