Kinetic characterization of the N-terminal domain of Malonyl-CoA reductase.

Climate change is driving a search for environmentally safe methods to produce chemicals used in ordinary life. One such molecule is 3-hydroxypropionic acid, which is a platform industrial chemical used as a precursor for a variety of other chemical end products. The biosynthesis of 3-hydroxypropionic acid can be achieved in recombinant microorganisms via malonyl-CoA reductase in two separate reactions. The reduction of malonyl-CoA by NADPH to form malonic semialdehyde is catalyzed in the C-terminal domain of malonyl-CoA reductase, while the subsequent reduction of malonic semialdehyde to 3-hydroxypropionic acid is accomplished in the N-terminal domain of the enzyme. A new assay for the reverse reaction of the N-terminal domain of malonyl-CoA reductase from Chloroflexus aurantiacus activity has been developed. This assay was used to determine the kinetic mechanism and for isotope effect studies. Kinetic characterization using initial velocity patterns revealed random binding of the substrates NADP+ and 3-hydroxypropionic acid. Isotope effects showed substrates react to give products faster than they dissociate and that the products of the reverse reaction, NADPH and malonic semialdehyde, have a low affinity for the enzyme. Multiple isotope effects suggest proton and hydride transfer occur in a concerted fashion. This detailed kinetic characterization of the reaction catalyzed by the N-terminal domain of malonyl-CoA reductase could aid in engineering of the enzyme to make the biosynthesis of 3-hydroxypropionic acid commercially competitive with its production from fossil fuels.

Antidepressants Trial in Parkinson's Disease (ADepT-PD): protocol for a randomised placebo-controlled trial on the effectiveness of escitalopram and nortriptyline on depressive symptoms in Parkinson's disease.

BACKGROUND: Depressive symptoms are common in patients with Parkinson's disease and depression is a significant predictor of functional impairment, reduced quality of life and general well-being in Parkinson's disease. Despite the high prevalence of depression, evidence on the effectiveness and tolerability of antidepressants in this population is limited. The primary aim of this trial is to establish the clinical and cost effectiveness of escitalopram and nortriptyline for the treatment of depression in Parkinson's disease. METHODS: This is a multi-centre, double-blind, randomised placebo-controlled trial in 408 people with Parkinson's disease with subsyndromal depression, major depressive disorder or persistent depressive disorder and a Beck Depression Inventory-II (BDI-II) score of 14 or above. Participants will be randomised into one of three groups, receiving either escitalopram, nortriptyline or placebo for 12 months. Trial participation is face-to-face, hybrid or remote. The primary outcome measure is the BDI-II score following 8 weeks of treatment. Secondary outcomes will be collected at baseline, 8, 26 and 52 weeks and following withdrawal, including severity of anxiety and depression scores as well as Parkinson's disease motor severity, and ratings of non-motor symptoms, cognitive function, health-related quality of life, levodopa-equivalence dose, changes in medication, overall clinical effectiveness, capability, health and social care resource use, carer health-related quality of life, adverse effects and number of dropouts. DISCUSSION: This trial aims to determine the effectiveness of escitalopram and nortriptyline for reducing depressive symptoms in Parkinson's disease over 8 weeks, to provide information on the effect of these medications on anxiety and other non-motor symptoms in PD and on impact on patients and caregivers, and to examine their effect on change in motor severity. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03652870 Date of registration - 29th August 2018.

Mechanism of biotin carboxylase inhibition by ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulphonylamino]benzoate.

The rise of antibacterial-resistant bacteria is a major problem in the United States of America and around the world. Millions of patients are infected with antimicrobial resistant bacteria each year. Novel antibacterial agents are needed to combat the growing and present crisis. Acetyl-CoA carboxylase (ACC), the multi-subunit complex which catalyses the first committed step in fatty acid synthesis, is a validated target for antibacterial agents. However, there are at present, no commercially available antibiotics that target ACC. Ethyl 4-[[2-chloro-5-(phenylcarbamoyl)phenyl]sulfonylamino]benzoate (SABA1) is a compound that has been shown to have antibacterial properties against Pseudomonas aeruginosa and Escherichia coli. SABA1 inhibits biotin carboxylase (BC), the enzyme that catalyses the first half reaction of ACC. SABA1 inhibits BC via an atypical mechanism. It binds in the biotin binding site in the presence of ADP. SABA1 represents a potentially new class of antibiotics that can be used to combat the antibacterial resistance crisis.

Local injection of a hexametaphosphate formulation reduces heterotopic ossification in vivo.

Heterotopic ossification (HO), the pathological formation of ectopic bone, is a debilitating condition which can cause chronic pain, limit joint movement, and prevent prosthetic limb fitting. The prevalence of this condition has risen in the military population, due to increased survivorship following blast injuries. Current prophylaxes, which aim to target the complex upstream biological pathways, are inconsistently effective â€‹and have a range of side-effects that make them unsuitable for combat-injured personnel. As such, many patients must undergo further surgery to remove the formed ectopic bone. In this study, a non-toxic, U.S. Food and Drug Administration (FDA) -approved calcium chelator, hexametaphosphate (HMP), is explored as a novel treatment paradigm for this condition, which targets the chemical, rather that biological, â€‹bone formation pathways. This approach allows not only prevention of pathological bone formation â€‹but also uniquely facilitates reversal, which current drugs cannot achieve. Targeted, minimally invasive delivery is achieved by loading HMP into an injectable colloidal alginate. These formulations significantly reduce â€‹the length of the ectopic bone formed in a rodent model of HO, with no effect on the adjacent skeletal bone. This study demonstrates the potential of localized dissolution as a new treatment â€‹and an alternative to surgery â€‹for pathological ossification and calcification conditions.

Pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase.

Antimicrobial resistance is a growing global health and economic concern. Current antimicrobial agents are becoming less effective against common bacterial infections. We previously identified pyrrolocins A and C, which showed activity against a variety of Gram-positive bacteria. Structurally similar compounds, known as pyrrolidinediones (e.g., TA-289, equisetin), also display antibacterial activity. However, the mechanism of action of these compounds against bacteria was undetermined. Here, we show that pyrrolocin C and equisetin inhibit bacterial acetyl-CoA carboxylase (ACC), the first step in fatty acid synthesis. We used transcriptomic data, metabolomic analysis, fatty acid rescue and acetate incorporation experiments to show that a major mechanism of action of the pyrrolidinediones is inhibition of fatty acid biosynthesis, identifying ACC as the probable molecular target. This hypothesis was further supported using purified proteins, demonstrating that biotin carboxylase is the inhibited component of ACC. There are few known antibiotics that target this pathway and, therefore, we believe that these compounds may provide the basis for alternatives to current antimicrobial therapy.

Burns objective scar scale (BOSS): Validation of an objective measurement devices based burn scar scale panel.

AIMS: Hypertrophic scars in burn survivors are a major cause of morbidity but the development of evidence based treatments is hampered by the lack of objective measurements of these scars. The objective of our study is to investigate the most accurate parameters for objective scar assessment and to create a combination score to facilitate the use of a panel of objective scar measurement tools. METHODS: Three independent assessors evaluated fifty five scar sites on fifty five burn patients with both the subjective modified Vancouver Scar Scale (mVSS) and a panel of objective measurement tools including the DSM II Colormeter, Cutometer, Dermascan high frequency ultrasound. The sensitivity and specificity of the objective scar parameters in predicting a mVSS score of 6 or more using the Receiving Operator Characteristic Area under the curve (ROC AUC) was then calculated and the most accurate parameters were combined to create an objective global scar score. RESULTS: The ROC AUC values were found to be highest for the Dermascan scar thickness (0.897), dermal intensity and intensity ratio (0.914 and 0.919), Cutometer R0 value (0.942), and R0 ratio (0.944). For colour measurements, ratios of scar to normal skin performed better than the single parameters for both erythema and pigmentation measurements: DSM II Erythema ratio vs Erythema (0.885 vs 0.818), DSM II a* ratio vs a* (0.848 vs 0.741); DSM II Melanin ratio vs Melanin (0.854 vs 0.761), DSM II L* ratio vs L* (0.862 vs 0.767). Analysis of the ROC AUC with chi-square test values showed that the highest AUC (0.786) was obtained with the combination of the Cutometer R0, Dermascan scar thickness, intensity and their respective scar to normal skin ratios. A total score of 5 and above (out of 6 parameters) had the highest combined sensitivity (69.0%) and specificity (83.3%). CONCLUSION: The objective parameters for the DSM II Colormeter, Cutometer and Dermascan high frequency ultrasound were all found to have moderate to strong ROC AUC values and combination of the Cutometer R0 and Dermascan scar thickness and intensity values can be used to create an objective global scar scale that can accurately differentiate patients with hypertrophic burn scarring from non-hypertrophic scars or normal skin.

Printing bone in a gel: using nanocomposite bioink to print functionalised bone scaffolds.

Free-form printing offers a novel biofabrication approach to generate complex shapes by depositing hydrogel materials within a temporary supportive environment. However, printed hydrogels typically lack the requisite mechanical properties and functionality of the desired tissue, limiting application and, more importantly, safety and efficacy of the implant. The study authors have developed an innovative nanoclay-based bioink to print high shape fidelity functional constructs for potential skeletal application. Laponite® (LAP) nanoclay was combined with gellan gum (GG) to generate a printable hydrogel that was highly stable in vitro, displayed limited swelling ability compared with the silicate-free control and remained stable over time. An agarose fluid gel was found to provide the requisite support for the deposition of the material ink and preservation of the printed structure before crosslinking. Printed C2C12 myoblasts remained viable and displayed extensive proliferation over 21 days in culture. Cell-laden scaffolds demonstrated functionality within 1 day of culture in vitro and that was preserved over 3 weeks. Analysis of absorption and release mechanisms from LAP-GG using model proteins (lysozyme and bovine serum albumin) demonstrated the retention capability of the clay-based materials for compound localisation and absence of burst release. Vascular endothelial growth factor â€‹was loaded within the agarose fluid gel and absorbed by the material ink via absorption during deposition. The 3D-printed constructs were implanted on the chorioallantoic membrane of a 10-day-old developing chick. Extensive and preferential vasculature infiltration was observed in LAP-GG-loaded vascular endothelial growth factor constructs compared with controls (p<0.01 and p<0.0001) after only 7 days of incubation. The current studies demonstrate, for the first time, the application of innovative LAP-GG 3D constructs in the generation of growth factor-loaded 3D constructs for potential application in skeletal tissue repair.

Investigating the intra- and inter-rater reliability of a panel of subjective and objective burn scar measurement tools.

BACKGROUND: Research into the treatment of hypertrophic burn scar is hampered by the variability and subjectivity of existing outcome measures. This study aims to measure the inter- and intra-rater reliability of a panel of subjective and objective burn scar measurement tools. METHODS: Three independent assessors evaluated 55 scar and normal skin sites using subjective (modified Vancouver Scar Scale [mVSS] & Patient and Observer Scar Assessment Scale [POSAS]) and objective tools. The intra-class correlation coefficient was utilised to measure reliability (acceptable when >0.70). Patient satisfaction with the different tools and scar parameter importance were assessed via questionnaires. RESULTS: The inter-rater reliabilities of the mVSS and POSAS were below the acceptable limit. For erythema and pigmentation, all of the Scanoskin and DSM II measures (except the b* value) had acceptable to excellent intra and inter-rater reliability. The Dermascan ultrasound (dermal thickness, intensity) had excellent intra- and inter-rater reliability (>0.90). The Cutometer R0 (firmness) had acceptable reliability but not R2 (gross elasticity). All objective measurement tools had good overall satisfaction scores. Patients rated scar related pain and itch as more important compared to appearance although this finding was not sustained when corrected for multiple comparisons. CONCLUSION: The objective scar measures demonstrated acceptable to excellent intra- and inter-rater reliability and performed better than the subjective scar scales.

Optimization and Mechanistic Characterization of Pyridopyrimidine Inhibitors of Bacterial Biotin Carboxylase.

A major challenge for new antibiotic discovery is predicting the physicochemical properties that enable small molecules to permeate Gram-negative bacterial membranes. We have applied physicochemical lessons from previous work to redesign and improve the antibacterial potency of pyridopyrimidine inhibitors of biotin carboxylase (BC) by up to 64-fold and 16-fold against Escherichia coli and Pseudomonas aeruginosa, respectively. Antibacterial and enzyme potency assessments in the presence of an outer membrane-permeabilizing agent or in efflux-compromised strains indicate that penetration and efflux properties of many redesigned BC inhibitors could be improved to various extents. Spontaneous resistance to the improved pyridopyrimidine inhibitors in P. aeruginosa occurs at very low frequencies between 10-8 and 10-9. However, resistant isolates had alarmingly high minimum inhibitory concentration shifts (16- to >128-fold) compared to the parent strain. Whole-genome sequencing of resistant isolates revealed that either BC target mutations or efflux pump overexpression can lead to the development of high-level resistance.

Capillary electrophoretic assay of human acetyl-coenzyme A carboxylase 2.

Human acetyl-coenzyme A carboxylase 2 catalyzes the carboxylation of acetyl coenzyme A to form malonyl coenzyme A, along with the conversion of magnesium-adenosine triphosphate complex to magnesium-adenosine diphosphate complex. A simple off-column capillary electrophoresis assay for human acetyl-coenzyme A carboxylase 2 was developed based on the separation of magnesium-adenosine triphosphate complex, magnesium-adenosine diphosphate complex, acetyl coenzyme A and malonyl coenzyme A with detection by ultraviolet absorption at 256 nm. When Mg2+ was absent from the separation buffer, the zones due to magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex both split and migrated as two separate peaks. With Mg2+ added to the separation buffer, magnesium-adenosine triphosphate complex and magnesium-adenosine diphosphate complex produced single peaks, and the reproducibility of peak shape and area improved for human acetyl-coenzyme A carboxylase 2 assay components. The final separation buffer used was 30.0 mM HEPES, 3.0 mM MgCl2 , 2.5 mM KHCO3 , and 2.5 mM potassium citrate at pH 7.50. The same buffer was used for the enzyme-catalyzed reaction (off-column). Inhibition of human acetyl-coenzyme A carboxylase 2 by CP-640186, a known inhibitor, was detected using the capillary electrophoresis assay.

A gellan-based fluid gel carrier to enhance topical spray delivery.

Autologous cell transplantation was introduced to clinical practice nearly four decades ago to enhance burn wound re-epithelialisation. Autologous cultured or uncultured cells are often delivered to the surface in saline-like suspensions. This delivery method is limited because droplets of the sprayed suspension form upon deposition and run across the wound bed, leading to uneven coverage and cell loss. One way to circumvent this problem would be to use a gel-based material to enhance surface retention. Fibrin systems have been explored as co-delivery system with keratinocytes or as adjunct to 'seal' the cells following spray delivery, but the high costs and need for autologous blood has impeded its widespread use. Aside from fibrin gel, which can exhibit variable properties, it has not been possible to develop a gel-based carrier that solidifies on the skin surface. This is because it is challenging to develop a material that is sprayable but gels on contact with the skin surface. The manuscript reports the use of an engineered carrier device to deliver cells via spraying, to enhance retention upon a wound. The device involves shear-structuring of a gelling biopolymer, gellan, during the gelation process; forming a yield-stress fluid with shear-sensitive behaviours, known as a fluid gel. In this study, a formulation of gellan gum fluid gels are reported, formed with from 0.75 or 0.9% (w/v) polymer and varying the salt concentrations. The rheological properties and the propensity of the material to wet a surface were determined for polymer modified and non-polymer modified cell suspensions. The gellan fluid gels had a significantly higher viscosity and contact angle when compared to the non-polymer carrier. Viability of cells was not impeded by encapsulation in the gellan fluid gel or spraying. The shear thinning property of the material enabled it to be applied using an airbrush and spray angle, distance and air pressure were optimised for coverage and viability. STATEMENT OF SIGNIFICANCE: Spray delivery of skin cells has successfully translated to clinical practice. However, it has not yet been widely accepted due to limited retention and disputable cell viability in the wound. Here, we report a method for delivering cells onto wound surfaces using a gellan-based shear-thinning gel system. The viscoelastic properties allow the material to liquefy upon spraying and restructure rapidly on the surface. Our results demonstrate reduced run-off from the surface compared to currently used low-viscosity cell carriers. Moreover, encapsulated cells remain viable throughout the process. Although this paper studies the encapsulation of one cell type, a similar approach could potentially be adopted for other cell types. Our data supports further studies to confirm these results in in vivo models.

The many types of carbonic anhydrases in photosynthetic organisms.

Carbonic anhydrases (CAs) are enzymes that catalyze the interconversion of CO2 and HCO3-. In nature, there are multiple families of CA, designated with the Greek letters α through θ. CAs are ubiquitous in plants, algae and photosynthetic bacteria, often playing essential roles in the CO2 concentrating mechanisms (CCMs) which enhance the delivery of CO2 to Rubisco. As algal CCMs become better characterized, it is clear that different types of CAs are playing the same role in different algae. For example, an α-CA catalyzes the conversion of accumulated HCO3- to CO2 in the green alga Chlamydomonas reinhardtii, while a θ-CA performs the same function in the diatom Phaeodactylum tricornutum. In this review we argue that, in addition to its role of delivering CO2 for photosynthesis, other metabolic roles of CA have likely changed as the Earth's atmospheric CO2 level decreased. Since the algal and plant lineages diverged well before the decrease in atmospheric CO2, it is likely that plant, algae and photosynthetic bacteria all adapted independently to the drop in atmospheric CO2. In light of this, we will discuss how the roles of CAs may have changed over time, focusing on the role of CA in pH regulation, how CAs affect CO2 supply for photosynthesis and how CAs may help in the delivery of HCO3- for other metabolic reactions.

Acetyl-CoA carboxylase from Escherichia coli exhibits a pronounced hysteresis when inhibited by palmitoyl-acyl carrier protein.

Acetyl-CoA carboxylase (ACC) in bacteria is composed of three components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. ACC catalyzes the first committed step in fatty acid synthesis: the carboxylation of acetyl-CoA to form malonyl-CoA via a two-step reaction. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of the vitamin biotin covalently linked to biotin carboxyl carrier protein. In the second half-reaction, the carboxyl group is transferred from biotin to acetyl-CoA by the enzyme carboxyltransferase, to form malonyl-CoA. In most Gram-negative and Gram-positive bacteria, the three components of ACC form a complex that requires communication for catalysis, and is subject to feedback inhibition by acylated-acyl carrier proteins. This study investigated the mechanism of inhibition of palmitoyl-acyl carrier protein (PACP) on ACC. Unexpectedly, ACC was found to exhibit a significant hysteresis, meaning ACC was subject to inhibition by PACP in a time dependent manner. Pull-down assays demonstrated PACP does not prevent formation of the multiprotein complex, while steady-state kinetic analyses showed PACP inhibited ACC activity allosterically. Structure-activity analyses revealed that the pantothenic acid moiety of PACP is responsible for the inhibition of ACC. This study provides the first evidence of the hysteretic nature of ACC.

Annexin-enriched osteoblast-derived vesicles act as an extracellular site of mineral nucleation within developing stem cell cultures.

The application of extracellular vesicles (EVs) as natural delivery vehicles capable of enhancing tissue regeneration could represent an exciting new phase in medicine. We sought to define the capacity of EVs derived from mineralising osteoblasts (MO-EVs) to induce mineralisation in mesenchymal stem cell (MSC) cultures and delineate the underlying biochemical mechanisms involved. Strikingly, we show that the addition of MO-EVs to MSC cultures significantly (P < 0.05) enhanced the expression of alkaline phosphatase, as well as the rate and volume of mineralisation beyond the current gold-standard, BMP-2. Intriguingly, these effects were only observed in the presence of an exogenous phosphate source. EVs derived from non-mineralising osteoblasts (NMO-EVs) were not found to enhance mineralisation beyond the control. Comparative label-free LC-MS/MS profiling of EVs indicated that enhanced mineralisation could be attributed to the delivery of bridging collagens, primarily associated with osteoblast communication, and other non-collagenous proteins to the developing extracellular matrix. In particular, EV-associated annexin calcium channelling proteins, which form a nucleational core with the phospholipid-rich membrane and support the formation of a pre-apatitic mineral phase, which was identified using infrared spectroscopy. These findings support the role of EVs as early sites of mineral nucleation and demonstrate their value for promoting hard tissue regeneration.

Visualising phase change in a brushite-based calcium phosphate ceramic.

The resorption of brushite-based bone cements has been shown to be highly unpredictable, with strong dependence on a number of conditions. One of the major factors is phase transformation, with change to more stable phases such as hydroxyapatite affecting the rate of resorption. Despite its importance, the analysis of phase transformation has been largely undertaken using methods that only detect crystalline composition and give no information on the spatial distribution of the phases. In this study confocal Raman microscopy was used to map cross-sections of brushite cylinders aged in Phosphate Buffered Saline, Foetal Bovine Serum, Dulbecco's - Minimum Essential Medium (with and without serum). Image maps showed the importance of ageing medium on the phase composition throughout the ceramic structure. When aged without serum, there was dissolution of the brushite phase concomitant to the deposition of octacalcium phosphate (OCP) around the periphery of the sample. The deposition of OCP was detectable within five days and reduced the rate of brushite dissolution from the material. The use of serum, even at a concentration of 10vol% prevented phase transformation. This paper demonstrates the value of confocal Raman microscopy in monitoring phase change in biocements; it also demonstrates the problems with assessing material degradation in non-serum containing media.

Crystal Structure of Carboxyltransferase from Staphylococcus aureus Bound to the Antibacterial Agent Moiramide B.

The dramatic increase in the prevalence of antibiotic-resistant bacteria has necessitated a search for new antibacterial agents against novel targets. Moiramide B is a natural product, broad-spectrum antibiotic that inhibits the carboxyltransferase component of acetyl-CoA carboxylase, which catalyzes the first committed step in fatty acid synthesis. Herein, we report the 2.6 Å resolution crystal structure of moiramide B bound to carboxyltransferase. An unanticipated but significant finding was that moiramide B bound as the enol/enolate. Crystallographic studies demonstrate that the (4S)-methyl succinimide moiety interacts with the oxyanion holes of the enzyme, supporting the notion that an anionic enolate is the active form of the antibacterial agent. Structure-activity studies demonstrate that the unsaturated fatty acid tail of moiramide B is needed only for entry into the bacterial cell. These results will allow the design of new antibacterial agents against the bacterial form of carboxyltransferase.

Carbon Fixation Driven by Molecular Hydrogen Results in Chemolithoautotrophically Enhanced Growth of Helicobacter pylori.

UNLABELLED: A molecular hydrogen (H2)-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H2-supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [(14)C]bicarbonate (on a per cell basis) in a 10-h period than cells without H2 Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H2-supplied cells contained 3-fold more ACC activity than cells without H2 Other possible carbon dioxide (CO2) fixation enzymes were not up-expressed under the H2-containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCE: Many pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen.

Identifying the Cellular Mechanisms Leading to Heterotopic Ossification.

Heterotopic ossification (HO) is a debilitating condition defined by the de novo development of bone within non-osseous soft tissues, and can be either hereditary or acquired. The hereditary condition, fibrodysplasia ossificans progressiva is rare but life threatening. Acquired HO is more common and results from a severe trauma that produces an environment conducive for the formation of ectopic endochondral bone. Despite continued efforts to identify the cellular and molecular events that lead to HO, the mechanisms of pathogenesis remain elusive. It has been proposed that the formation of ectopic bone requires an osteochondrogenic cell type, the presence of inductive agent(s) and a permissive local environment. To date several lineage-tracing studies have identified potential contributory populations. However, difficulties identifying cells in vivo based on the limitations of phenotypic markers, along with the absence of established in vitro HO models have made the results difficult to interpret. The purpose of this review is to critically evaluate current literature within the field in an attempt identify the cellular mechanisms required for ectopic bone formation. The major aim is to collate all current data on cell populations that have been shown to possess an osteochondrogenic potential and identify environmental conditions that may contribute to a permissive local environment. This review outlines the pathology of endochondral ossification, which is important for the development of potential HO therapies and to further our understanding of the mechanisms governing bone formation.

Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase.

Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. This finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO2 from the carboxyphosphate intermediate to biotin.

Development of UK guidance on the management of erectile dysfunction resulting from radical radiotherapy and androgen deprivation therapy for prostate cancer.

AIM: To develop a management strategy (rehabilitation programme) for erectile dysfunction (ED) after radiotherapy (RT) or androgen deprivation therapy (ADT) for prostate cancer that is suitable for use in a UK NHS healthcare context. METHODS: PubMed literature searches of ED management in this patient group together with a survey of 28 experts in the management of treatment-induced ED from across the UK were conducted. RESULTS: Data from 19 articles and completed questionnaires were collated. The findings discussed in this article confirm that RT/ADT for prostate cancer can significantly impair erectile function. While many men achieve erections through PDE5-I use, others need combined management incorporating exercise and lifestyle modifications, psychosexual counselling and other erectile aids. This article offers a comprehensive treatment algorithm to manage patients with ED associated with RT/ADT. CONCLUSION: Based on published research literature and survey analysis, recommendations are proposed for the standardisation of management strategies employed for ED after RT/ADT. In addition to implementing the algorithm, understanding the rationale for the type and timing of ED management strategies is crucial for clinicians, men and their partners.