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Sialidases catalyze the release of sialic acid from the terminus of glycan chains. We previously characterized the sialidase from the opportunistic fungal pathogen, Aspergillus fumigatus, and showed that it is a Kdnase. That is, this enzyme prefers 3-deoxy-d-glycero-d-galacto-non-2-ulosonates (Kdn glycosides) as the substrate compared to N-acetylneuraminides (Neu5Ac). Here, we report characterization and crystal structures of putative sialidases from two other ascomycete fungal pathogens, Aspergillus terreus (AtS) and Trichophyton rubrum (TrS). Unlike A. fumigatus Kdnase (AfS), hydrolysis with the Neu5Ac substrates was negligible for TrS and AtS; thus, TrS and AtS are selective Kdnases. The second-order rate constant for hydrolysis of aryl Kdn glycosides by AtS is similar to that by AfS but 30-fold higher by TrS. The structures of these glycoside hydrolase family 33 (GH33) enzymes in complex with a range of ligands for both AtS and TrS show subtle changes in ring conformation that mimic the Michaelis complex, transition state, and covalent intermediate formed during catalysis. In addition, they can aid identification of important residues for distinguishing between Kdn and Neu5Ac substrates. When A. fumigatus, A. terreus, and T. rubrum were grown in chemically defined media, Kdn was detected in mycelial extracts, but Neu5Ac was only observed in A. terreus or T. rubrum extracts. The C8 monosaccharide 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) was also identified in A. fumigatus and T. rubrum samples. A fluorescent Kdn probe was synthesized and revealed the localization of AfS in vesicles at the cell surface.

Aim Traumatic Cardiac Arrest (TCA) is a potentially fatal consequence of penetrating or blunt trauma. Emergency-Department Resuscitative Thoracotomy (ED-RT) is the last resort procedure to restore cardiac output following TCA. This study reviews current UK guidelines of Major Trauma Centres (MTCs) to identify common indications for ED-RT and chest compressions following TCA. Method Departmental policies were requested from all UK MTCs. Data was extracted by three reviewers. A planned subgroup analysis was performed for blunt/penetrating trauma. Results 20 guidelines were identified covering 22 MTCs. All guidelines included recommendations regarding indications for ED-RT. Time thresholds from loss of output to undertaking RT in blunt trauma were given in 12 (60%) guidelines. Ranging from 5 (25%), 10 (50%) and 15 minutes (10%). Prehospital loss of output was a contraindication for blunt trauma ED-RT in 6 guidelines. ED-RT in penetrating trauma was indicated within 10 (50%) or 15 minutes (45%) from loss of output. Fewer than 50% of guidelines identified specific contra-indications including massive head injury and asystole. 3 guidelines stated chest compressions were not indicated in TCA. 8 stated that chest compressions may not be contra-indicated and 1 advised chest compressions to be restricted to blunt trauma. Conclusion In a survey of UK guidelines, we have found inconsistency in the indications for ED-RT, particularly associated with blunt trauma. A maximum time from loss of output to ED-RT for penetrating trauma of 15 minutes is compatible with most guidelines. This is less consistent with findings for blunt trauma. There is little consensus regarding use of chest compressions.

Hemin [Fe(III)-protoporphyrin IX] is known to bind tightly to single-stranded DNA and RNA molecules that fold into G-quadruplexes (GQ). Such complexes are strongly activated for oxidative catalysis. These heme•DNAzymes and ribozymes have found broad utility in bioanalytical and medicinal chemistry and have also been shown to occur within living cells. However, how a GQ is able to activate hemin is poorly understood. Herein, we report fast kinetic measurements (using stopped-flow UV–vis spectrophotometry) to identify the H2O2-generated activated heme species within a heme•DNAzyme that is active for the oxidation of a thioether substrate, dibenzothiophene (DBT). Singular value decomposition and global fitting analysis was used to analyze the kinetic data, with the results being consistent with the heme•DNAzyme's DBT oxidation being catalyzed by the initial Fe(III)heme–H2O2 complex. Such a complex has been predicted computationally to be a powerful oxidant for thioether substrates. In the heme•DNAzyme, the DNA GQ enhances both the kinetics of formation of the active intermediate as well as the oxidation step of DBT by the active intermediate. We show, using both stopped flow spectrophotometry and EPR measurements, that a classic Compound I is not observable during the catalytic cycle for thioether sulfoxidation.

Sterically congested chiral alcohol and amines have gained tremendous attention in the design of asymmetric catalysts. Herein, the synthesis of a sterically congested bis-adamantane framework-based chiral alcohol, (1R,2S,3S,4R)-4-(2-adamantyl)adamantan-2-ol, and amine, (1R,2S,3S,4R)-4-(2-adamantyl)adamantan-2-amine, is described. Access to these sterically encumbered compounds is found via the preparation of an enantioenriched 4-adamantyladamantan-2-one intermediate, which was synthesized in 6 steps from adamantan-2-one. The key step involved enzyme-catalyzed ester hydrolysis in giving unsaturated alcohol with an enantiomeric excess of >95%. This adamantylidene adamantanol was subjected to an acid-catalyzed intramolecular [1,4] shift to give the key chiral intermediate without racemization. This ketone intermediate was transformed into the target compounds via reduction and reductive amination protocols.

The lungs are affected by illnesses including asthma, chronic obstructive pulmonary disease, and infections such as influenza and SARS-CoV-2. Physiologically relevant models for respiratory conditions will be essential for new drug development. The composition and structure of the lung extracellular matrix (ECM) plays a major role in the function of the lung tissue and cells. Lung-on-chip models have been developed to address some of the limitations of current two-dimensional in vitro models. In this review, we describe various ECM substitutes utilized for modeling the respiratory system. We explore the application of lung-on-chip models to the study of cigarette smoke and electronic cigarette vapor. We discuss the challenges and opportunities related to model characterization with an emphasis on in situ characterization methods, both established and emerging. We discuss how further advancements in the field, through the incorporation of interstitial cells and ECM, have the potential to provide an effective tool for interrogating lung biology and disease, especially the mechanisms that involve the interstitial elements.

In recent years, the need for sophisticated human in vitro models for integrative biology has motivated the development of organ-on-a-chip platforms. Organ-on-a-chip devices are engineered to mimic the mechanical, biochemical and physiological properties of human organs; however, there are many important considerations when selecting or designing an appropriate device for investigating a specific scientific question. Building microfluidic Brain-on-a-Chip (BoC) models from the ground-up will allow for research questions to be answered more thoroughly in the brain research field, but the design of these devices requires several choices to be made throughout the design development phase. These considerations include the cell types, extracellular matrix (ECM) material(s), and perfusion/flow considerations. Choices made early in the design cycle will dictate the limitations of the device and influence the end-point results such as the permeability of the endothelial cell monolayer, and the expression of cell type-specific markers. To better understand why the engineering aspects of a microfluidic BoC need to be influenced by the desired biological environment, recent progress in microfluidic BoC technology is compared. This review focuses on perfusable blood–brain barrier (BBB) and neurovascular unit (NVU) models with discussions about the chip architecture, the ECM used, and how they relate to the in vivo human brain. With increased knowledge on how to make informed choices when selecting or designing BoC models, the scientific community will benefit from shorter development phases and platforms curated for their application.

Aspergillus fumigatus is one of the main causative agents of invasive aspergillosis, an often-lethal fungal disease that affects immunocompromised individuals. A. fumigatus produces a sialidase that cleaves the nine-carbon carbohydrate Kdn from glycoconjugates. This enzyme plays a critical role in A. fumigatus pathogenicity, and is thus a target for the development of new therapeutics. In order to understand the reactivity of this Kdnase, and to develop a sensitive and selective assay for its catalytic activity we determined whether, like its close structural homolog the excreted sialidase produced by Micromonospora viridifaciens, this enzyme can efficiently hydrolyze thioglycoside substrates. We synthesized a panel of seven aryl 2-thio-d-glycero-α-d-galacto-non-2-ulopyranosonides and measured the activity of the A. fumigatus Kdnase towards these substrates. Four of these substrates were hydrolyzed by the A. fumigatus enzyme, although M. viridifaciens sialidase-catalyzed the hydrolysis of these Kdn thioglycosides with higher catalytic efficiencies (kcat/Km). We also tested an enzyme that was evolved from MvNA to improve its activity against Kdn glycosides (Glycobiology 2020, 30, 325). All three enzymes catalyzed the hydrolysis of the four most reactive Kdn thioglycosides and their second-order rate constants (kcat/Km) display a concave downwards Bronsted plot. The kinetic data, for each enzyme, is consistent with a change in rate-limiting step from CS bond cleavage for thioglycosides in which the pKa of the corresponding aryl thiol is >3.6, to a non-chemical step, which is likely a conformational change, that occurs prior to CS bond cleavage for the 2,3,4,5,6-pentafluorothiophenyl glycoside.

Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreographs a series of conformational changes during the catalytic cycle. As a result, some GH families, including α-amylases (GH13), have their chemical steps concealed kinetically. To address this issue for a GH13 enzyme, we prepared seven cyclohexenyl-based carbasugars of α-d-glucopyranoside that we show are good covalent inhibitors of a GH13 yeast α-glucosidase. The linear free energy relationships between rate constants and p

We report a microfluidic cell culture model that incorporates a novel 3-dimensional extracellular matrix composed of a biodegradable membrane and lumen-patterned, cell-embedded hydrogel. Additionally, we developed a novel approach to produce a hydrogel mixture containing gelatin methacrylate (GelMA) and ultrasonicated Matrigel microparticles. The novel fabrication approach creates a hybrid hydrogel where the Matrigel is well distributed within the GelMA, it promotes fibroblast cell elongation, and it does not affect the photopolymerization of GelMA.

Carbasugars are structural mimics of naturally occurring carbohydrates that can interact with and inhibit enzymes involved in carbohydrate processing. In particular, carbasugars have attracted attention as inhibitors of glycoside hydrolases (GHs) and as therapeutic leads in several disease areas. However, it is unclear how the carbasugars are recognized and processed by GHs. Here, we report the synthesis of three carbasugar isotopologues and provide a detailed transition state (TS) analysis for the formation of the initial GH-carbasugar covalent intermediate, as well as for hydrolysis of this intermediate, using a combination of experimentally measured kinetic isotope effects and hybrid QM/MM calculations. We find that the α-galactosidase from Thermotoga maritima effectively stabilizes TS charge development on a remote C5-allylic center acting in concert with the reacting carbasugar, and catalysis proceeds via an exploded, or loose, SN2 transition state with no discrete enzyme-bound cationic intermediate. We conclude that, in complement to what we know about the TS structures of enzyme-natural substrate complexes, knowledge of the TS structures of enzymes reacting with non-natural carbasugar substrates shows that GHs can stabilize a wider range of positively charged TS structures than previously thought. Furthermore, this enhanced understanding will enable the design of new carbasugar GH transition state analogues to be used as, for example, chemical biology tools and pharmaceutical lead compounds., Positive charge stabilized on remote C5-allylic center with catalysis occurring via a loose SN2 transition state.

Sialic acid (N-acetylneuraminic acid) is a carbohydrate that possess a nine carbon backbone, and it is often found at the termini of glycoconjugates in biological systems. Because of this prominence many syntheses have reported routes to sialic acid and many of its derivatives. Most of these compounds retain the endocyclic oxygen atom that becomes part of the ketal glycosidic linkage that joins sialic acid to the penultimate residue in the glycoconjugate. With respect to carba-sialic acid (replacement of the ring oxygen atom with a methylene group) a single synthesis has been reported (Ogawa et al. (Carbohydr. Res., 1995, 269, 53–78) in 30 steps and 0.5% yield. The current report details a robust synthesis of 6a-carba-α- d -sialic acid that involves 18 steps and give a 5% yield using d -quinic acid as the starting material.

The glycoside hydrolase family 4 (GH4) α-galactosidase from Citrobacter freundii (MelA) catalyzes the hydrolysis of fluoro-substituted phenyl α-d-galactopyranosides by utilizing two cofactors, NAD+ and a metal cation, under reducing conditions. In order to refine the mechanistic understanding of this GH4 enzyme, leaving group effects were measured with various metal cations. The derived βlg value on V/K for strontium activation is indistinguishable from zero (0.05 ± 0.12). Deuterium kinetic isotope effects (KIEs) were measured for the activated substrates 2-fluorophenyl and 4-fluorophenyl α-d-galactopyranosides in the presence of Sr2+, Y3+, and Mn2+, where the isotopic substitution was on the carbohydrate at C-2 and/or C-3. To determine the contributing factors to the virtual transition state (TS) on which the KIEs report, kinetic isotope effects on isotope effects were measured on these KIEs using doubly deuterated substrates. The measured DV/K KIEs for MelA-catalyzed hydrolysis of 2-fluorophenyl α-d-gal...

Here we describe the synthesis of two specifically labelled 13C isotopologues of cis-2-(4-nitrophenoxy)bicyclo[4.1.0]heptane and their solvolysis reactions in trifluoroethanol. By using one- and two-dimensional 1H- and 13C-NMR spectroscopy, we characterized the pathways for the rearrangement of these isotopologues to give 13C-labelled 4-(2,2,2-trifluoroethoxy)cycloheptene. We show that the initially formed cationic intermediate undergoes a degenerate rearrangement, which does not reach equilibrium before nucleophilic capture of the cation. Moreover, we show that the nonclassical carbocation, cyclohept-3-ene(3,1,4-deloc)ylium, gives an approximate 6:1 ratio of the cis- to trans-diastereomeric 2-(2,2,2-trifluoroethoxy)bicyclo[4.1.0]heptane as reaction intermediates that subsequently solvolyze to the 4-(2,2,2-trifluoroethoxy)cycloheptene product.

In the late 1950s and early 1960s, the number of universities across all countries of the developed world saw a dramatic expansion. This was in response to the anticipated role that post-secondary education was to play in the rapidly developing knowledge-based economy. Within Canada, several new universities were established: Victoria, Calgary, Regina, Guelph, Waterloo, Trent, York, and in 1963, the Province of British Columbia passed legislation creating Simon Fraser University (SFU). In contrast with many of the other new universities across the country, which grew out of existing colleges associated with the major universities, SFU was to be a completely new institution with a campus and faculty built from scratch. SFU’s role was to provide for the rapid increase anticipated in university-bound students in the Lower Mainland, relieving some of the pressure on the University of British Columbia’s (UBC) growth.

We describe the formation of a bis-cyclopropane product, a tricyclic[4.1.0.02,4]heptane, that is formed during a Johnson–Corey–Chaykovsky reaction on a cyclopentenone. Two (of four possible) bicyclic products are selectively formed by addition of a COOEt-stabilized sulfur ylide onto the Michael acceptor. The tricyclic product is formed subsequently via a retro Michael elimination of a hindered ether followed by addition of a further cyclopropyl moiety, affecting only one of the two bicyclic products initially formed. The experimental reaction outcome was rationalized using density functional theory (DFT), investigating the different Michael-addition approaches of the sulfur ylide, the transition state (TS) energies for the formation of possible zwitterionic intermediates and subsequent reactions that give rise to cyclopropanation. Selective formation of only two of the four possible products occurs due to the epimerization of unreactive intermediates from the other two pathways, as revealed by energy barr...

This work was supported by the Natural Sciences and Engineering Research Council of Canada award to MM (grant number 611181). C. Carroll thanks Simon Fraser University for a travel and research award. Iron is essential for growth and in low iron environments such as serum many bacteria and fungi secrete ferric iron-chelating molecules called siderophores. All fungi produce hydroxamate siderophores with the exception of Mucorales fungi, which secrete rhizoferrin, a polycarboxylate siderophore. Here we investigated the biosynthesis of rhizoferrin by the opportunistic human pathogen, Rhizopus delemar. We searched the genome of R. delemar 99–880 for a homologue of the bacterial NRPS-independent siderophore (NIS) protein, SfnaD that is involved in biosynthesis of staphyloferrin A in Staphylococcus aureus. A protein was identified in R. delemar with 22% identity and 37% similarity with SfnaD, containing an N-terminal IucA/IucC family domain, and a C-terminal conserved ferric iron reductase FhuF-like transporter domain. Expression of the putative fungal rhizoferrin synthetase (rfs) gene was repressed by iron. The rfs gene was cloned and expressed in E.coli and siderophore biosynthesis from citrate and diaminobutane was confirmed using high resolution LC–MS. Substrate specificity was investigated showing that Rfs produced AMP when oxaloacetic acid, tricarballylic acid, ornithine, hydroxylamine, diaminopentane and diaminopropane were employed as substrates. Based on the production of AMP and the presence of a mono-substituted rhizoferrin, we suggest that Rfs is a member of the superfamily of adenylating enzymes. We used site-directed mutagenesis to mutate selected conserved residues predicted to be in the Rfs active site. These studies revealed that H484 is essential for Rfs activity and L544 may play a role in amine recognition by the enzyme. This study on Rfs is the first characterization of a fungal NIS enzyme. Future work will determine if rhizoferrin biosynthesis is required for virulence in Mucorales fungi. Postprint

Introduction The aim was to determine whether there is a significant difference in clinical outcomes when using short vs. long nails for pertrochanteric femur fractures. Methods A systematic literature search was undertaken of Pubmed and Embase in April 2016. All papers published in English reporting studies comparing long vs. short nails for pertrochanteric femur fractures were studied. Data were pooled for blood loss and transfusion requirement, operative time, length of stay, incidence of delayed or non-union, incidence of avascular necrosis or infection, fracture or metalware failure and was analysed to examine the differences between long and short nails. Results From 90 potential studies, nine were considered to be eligible for inclusion. The short nail resulted in a statistically significant benefit when looking at transfusion requirement (p = 0.02); however, blood loss was not statistically different (p = 0.33) Operative time was also quicker with the short nail (p = 0.004), but the length of stay was no different (p = 0.41). When examining complications and fixation outcomes, there was no difference in delayed union rates (p = 0.29) and non-union rates (p = 0.19) nor when looking at metalware failure (p = 0.41) and fracture (p = 0.14). Discussion The use of short intramedullary nails for pertrochanteric femur fractures appeared to be beneficial when looking at transfusion requirements and operative time. When looking at complications such as fracture and metalware failure, despite there being a slightly higher incidence of these in the short nail group, it is not statistically significant. This study advocates that both devices are safe to use, however, the short nail may have some perceived benefits in reducing transfusion requirements and have cost benefits with a shorter operative time.

N-acetylneuraminic acid (5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid), which is the principal sialic acid family member of the non-2-ulosonic acids and their various derivatives, is often found at the terminal position on the glycan chains that adorn all vertebrate cells. This terminal position combined with subtle variations in structure and linkage to the underlying glycan chains between humans and other mammals points to the importance of this diverse group of nine-carbon sugars as indicators of the unique aspects of human evolution and is relevant to understanding an array of human conditions. Enzymes that catalyze the removal N-acetylneuraminic acid from glycoconjugates are called neuraminidases. However, despite their documented role in numerous diseases, due to the promiscuous activity of many neuraminidases, our knowledge of the functions and metabolism of many sialic acids and the effect of the attachment to cellular glycans is limited. To this end, through a concerted effort of generation of random and site-directed mutagenesis libraries, subsequent screens and positive and negative evolutionary selection protocols, we succeeded in identifying three enzyme variants of the neuraminidase from the soil bacterium Micromonospora viridifaciens with markedly altered specificity for the hydrolysis of natural Kdn (3-deoxy-d-glycero-d-galacto-non-2-ulosonic acid) glycosidic linkages compared to those of N-acetylneuraminic acid. These variants catalyze the hydrolysis of Kdn-containing disaccharides with catalytic efficiencies (second-order rate constants: kcat/Km) of greater than 105 M−1 s−1; the best variant displayed an efficiency of >106 M−1 s−1 at its optimal pH.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/KM) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism., Mannosidases of glycoside hydrolase family 99 use a neighboring-group participation mechanism involving the substrate 2-hydroxyl.

We outline a quantum-enabled blockchain architecture based on a consortium of quantum servers. The network is hybridised, utilising digital systems for sharing and processing classical information combined with a fibre--optic infrastructure and quantum devices for transmitting and processing quantum information. We deliver an energy efficient interactive mining protocol enacted between clients and servers which uses quantum information encoded in light and removes the need for trust in network infrastructure. Instead, clients on the network need only trust the transparent network code, and that their devices adhere to the rules of quantum physics. To demonstrate the energy efficiency of the mining protocol, we elaborate upon the results of two previous experiments (one performed over 1km of optical fibre) as applied to this work. Finally, we address some key vulnerabilities, explore open questions, and observe forward--compatibility with the quantum internet and quantum computing technologies., 25 pages, 5 figures

Our understanding of the mechanisms of glycopyranosyl transfer that occur in solution, both for the chemical synthesis of complex structures and that for the cleavage of glycosidic bonds has allowed us to design biologically active molecules. Recent efforts on the reactivity of glycopyranosides, which are critical entities in all biological systems, coupled with the advent of modern spectroscopic instrumentation have allowed physical organic chemists to broaden our knowledge of glycosyl transfer reaction transition states, both in solution and for enzyme-catalyzed processes, and of critical high energy intermediates. This review details recent physical organic, kinetic and structural studies that have led to elucidation of several different mechanism for the transfer of glycopyranosyl moieties from various substrates to acceptors, such as water or a sugar hydroxyl group.

UV1C, a 42-nt DNA oligonucleotide, is a deoxyribozyme (DNAzyme) that optimally uses 305 nm wavelength light to catalyze photoreactivation of a cyclobutane thymine dimer placed within a gapped, unnatural DNA substrate, TDP. Herein we show that UV1C is also capable of photoreactivating thymine dimers within an authentic single-stranded DNA substrate, LDP. This bona fide UV1C substrate enables, for the first time, investigation of whether UV1C catalyzes only photoreactivation or also the de novo formation of thymine dimers. Single-turnover experiments carried out with LDP and UV1C, relative to control experiments with LDP alone in single-stranded and double-stranded contexts, show that while UV1C does modestly promote thymine dimer formation, its major activity is indeed photoreactivation. Distinct photostationary states are reached for LDP in its three contexts: as a single strand, as a constituent of a double-helix, and as a 1:1 complex with UV1C. The above results on the cofactor-independent photoreactivation capabilities of a catalytic DNA reinforce a series of recent, unexpected reports that purely nucleotide-based photoreactivation is also operational within conventional double-helical DNA.

The hydroxide-catalyzed hydrolysis of aryl 1,2-trans-glycosides proceeds through a mechanism involving neighboring group participation by a C2-oxyanion and rate-limiting formation of a 1,2-anhydro sugar (oxirane) intermediate. The transition state for the hydroxide-catalyzed hydrolysis of 4-nitrophenyl α-d-mannopyranoside in aqueous media has been studied by the use of multiple kinetic isotope effect (KIE) measurements in conjunction with ab initio theoretical methods. The experimental KIEs are C1-2H (1.112 ± 0.004), C2-2H (1.045 ± 0.005), anomeric 1-13C (1.026 ± 0.006), C2-13C (0.999 ± 0.005), leaving group oxygen 2-18O (1.040 ± 0.012), and C2-18O (1.044 ± 0.006). The transition state for the hydrolysis reaction was modeled computationally using the experimental KIE values as constraints. Taken together, the reported kinetic isotope effects and computational modeling are consistent with the reaction mechanism involving rate-limiting formation of a transient oxirane intermediate that opens in water to giv...

Spinel type nano-sized ferrite compounds AFe2O4 (A = Co, Mg and Mn) have been successfully prepared by self-propagating combustion method using glycine as fuel at 400 °C under air atmosphere for 4 h. The crystal structure, chemical composition, morphology and magnetic properties of the synthesized samples were characterized by X−ray diffraction, Fourier transform infrared spectroscopy, X−ray photoelectron spectroscopy, Energy dispersive X−ray, Scanning and Transmission electron microscopy and vibrating sample magnetometer. The chemical reaction and role of fuel on the nanoparticles formation were discussed. The XRD pattern of the synthesized samples shows the formation of pure phase with average crystallite size of 97, 57 and 98 nm from Scherrer formula and 86, 54 and 87 nm from Williamson and Hall (W–H) formula respectively. FTIR absorption spectra revealed that the presence of strong absorption peaks near 400–600 cm−1 corresponds to tetrahedral and octahedral complex of spinel ferrites. The relative concentrations of electronic states of elements such as cobalt (Co2+ and Co3+), iron (Fe2+ and Fe3+) and manganese (Mn2+ and Mn3+) oxidation states were studied from XPS and it is found that 55% of Fe ions are in Fe2+ state and the remaining is in Fe3+ state and thus the cationic distribution of Fe ions occurred in both tetrahedral and octahedral sites. SEM analysis indicates the presence of pore like morphology which is nearly homogenous because of combustion process. EDS analysis confirms the presence of elements in the ferrite samples. By replacing the active ‘A’ site cations in AFe2O4 (A = Co, Mg and Mn) samples show the different magnetic properties. The parameters like saturation magnetization, coercivity and remnant magnetization obtained from M−H loops are studied in room temperature.

This manuscript describes a novel class of derivatives based on a bicyclo[3.1.0]hexane scaffold, proposed as mimics of sialic acid in a distorted boat conformation that is on the catalytic pathway of neuraminidases (sialidases). A general synthetic route for these constrained-ring molecules was developed using a photochemical reaction followed by a Johnson-Corey-Chaykovsky cyclopropanation. Functionalization with the goal of occupying the 150-cavity was also exploited. Inhibition assays demonstrated low micromolar inhibition against both group-1 (H5N1) and group-2 (H9N2) influenza neuraminidase subtypes, indicating good affinity for the alpha and beta sialic acid mimics and 150-cavity-targeted derivatives. These results provide a validation of a bicyclo[3.1.0]hexane scaffold as a mimic of a distorted sialic acid bound in the neuraminidase active site during catalysis.

Summary Bone morphogenetic proteins (BMPs) are a group of signalling molecules that belong to the transforming growth factor-β superfamily of proteins. Initially identified for their ability to induce bone formation, recent advances in the understanding of cellular and molecular mechanisms regarding BMPs have led to the use of the growth factor to accelerate bone healing. Recent clinical trials have demonstrated that BMPs, BMP-7 in particular, may present an alternative line of treatment other than the gold standard, autogenous bone grafting, in the treatment of fracture nonunion. We performed a literature search in September 2014 of PubMed and Embase using search terms, including “bone morphogenetic proteins”, “BMP-7”, “non-union”, “fracture healing” and “cost-effectiveness”, reviewing the efficacy, safety, and cost of treatment of nonunions with BMP-7. The authors further canvassed the reference lists of selected articles and used online search tools, such as Google Scholar. BMP-7 uses both the canonical and noncanonical signalling pathways. The treatment of fracture nonunion with recombinant human BMP-7 (rhBMP-7) has a comparable efficacy with that of autogenous bone grafting with an average union rate of 87% compared with 93% for bone grafting. Furthermore, fewer complications have been described with the use of rhBMP-7 compared with traditional bone grafting. We describe the signalling pathways that BMP-7 uses to exert its effect on bone. In nonunions, rhBMP-7 has been shown to have a similar efficacy to bone grafting with fewer complications.

Mechanism-based glycoside hydrolase inhibitors are carbohydrate analogs that mimic the natural substrate’s structure. Their covalent bond formation with the glycoside hydrolase makes these compounds excellent tools for chemical biology and potential drug candidates. Here we report the synthesis of cyclohexene-based α-galactopyranoside mimics and the kinetic and structural characterization of their inhibitory activity toward an α-galactosidase from Thermotoga maritima (TmGalA). By solving the structures of several enzyme-bound species during mechanism-based covalent inhibition of TmGalA, we show that the Michaelis complexes for intact inhibitor and product have half-chair (2H3) conformations for the cyclohexene fragment, while the covalently linked intermediate adopts a flattened half-chair (2H3) conformation. Hybrid QM/MM calculations confirm the structural and electronic properties of the enzyme-bound species and provide insight into key interactions in the enzyme-active site. These insights should stimulate the design of mechanism-based glycoside hydrolase inhibitors with tailored chemical properties., Mechanism-based inhibitors of glycoside hydrolases are useful probes for basic research and represent potential drug candidates. Here, the authors present a series of mechanism-based covalent α-galactosidase inhibitors and elucidate the kinetic and structural basis of their inhibitory activity.

Aspergillus fumigatus is a filamentous fungus that can cause a life-threatening invasive pulmonary aspergillosis (IPA) in immunocompromised individuals. We previously characterized an exo-sialidase from A. fumigatus that prefers the sialic acid substrate, 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (Kdn); hence it is a Kdnase. Sialidases are known virulence factors in other pathogens; therefore, the goal of our study was to evaluate the importance of Kdnase in A. fumigatus. A kdnase knockout strain (Δkdnase) was unable to grow on medium containing Kdn and displayed reduced growth and abnormal morphology. Δkdnase was more sensitive than wild type to hyperosmotic conditions and the antifungal agent, amphotericin B. In contrast, Δkdnase had increased resistance to nikkomycin, Congo Red and Calcofluor White indicating activation of compensatory cell wall chitin deposition. Increased cell wall thickness and chitin content in Δkdnase were confirmed by electron and immunofluorescence microscopy. In a neutropenic mouse model of invasive aspergillosis, the Δkdnase strain had attenuated virulence and a significantly lower lung fungal burden but only in animals that received liposomal amphotericin B after spore exposure. Macrophage numbers were almost twofold higher in lung sections from mice that received the Δkdnase strain, possibly related to higher survival of macrophages that internalized the Δkdnase conidia. Thus, A. fumigatus Kdnase is important for fungal cell wall integrity and virulence, and because Kdnase is not present in the host, it may represent a potential target for the development of novel antifungal agents.

Here we describe the measurement of the inactivation rate constants for the mechanism-based inactivator 2,3-difluorosialic acid acting upon the sialidase from Micromonospora viridifaciens. Using double mixing stopped-flow experiments conducted in a 3-(N-morpholino)propanesulfonic acid buffer (100 mmol/L, pH 7.00) at 25 °C, the derived kinetic parameters are kinact/Ki= (3.9 ± 0.8) × 106(mol/L)–1s–1and Ki= 1.7 ± 0.4 μmol/L. We demonstrate that the inhibitory efficiency of the inactivation event is similar to the catalytic efficiency for this sialidase acting upon a typical substrate, 4-methylumbelliferone α-d-sialoside, kcat/Km= (7.2 ± 2.8) × 106(mol/L)–1s–1. Furthermore, we show that the catalytic efficiencies for inactivation and hydrolysis by the Kdnase from Aspergillus fumigatus are similar for the corresponding Kdn-analogues. We conclude that the deactivating effect of incorporating an axial 3-fluoro substituent onto the sialic acid scaffold is comparable to the enhanced activation that occurs when the 4-methylumbelliferone leaving group is changed to the more nucleofugal fluoride ion.