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In nature and synthetic chemistry, stereoselective [2+1] cyclopropanation is the most prevalent strategy for the synthesis of chiral cyclopropanes, a class of key pharmacophores in pharmaceuticals and bioactive natural products. One of the most extensively studied reactions in the organic chemist’s arsenal, stereoselective [2+1] cyclopropanation, largely relies on the use of stereodefined olefins, which require elaborate laboratory synthesis or tedious separation to ensure high stereoselectivity. Here we report engineered hemoproteins derived from a bacterial cytochrome P450 that catalyze the synthesis of chiral 1,2,3-polysubstituted cyclopropanes, regardless of the stereopurity of the olefin substrates used. Cytochrome P450BM3 variant IC-G3 exclusively converts (Z)-enol acetates to enantio- and diastereoenriched cyclopropanes and in our model reaction delivers a leftover (E)-enol acetate with 98% stereopurity, using whole Escherichia coli cells. IC-G3 was further engineered with a single mutation to enable the biotransformation of (E)-enol acetates to α-branched ketones with high levels of enantioselectivity while simultaneously catalyzing the cyclopropanation of (Z)-enol acetates with excellent activities and selectivities. We conducted docking studies and molecular dynamics simulations to understand how active-site residues distinguish between the substrate isomers and enable the enzyme to perform these distinct transformations with such high selectivities. Computational studies suggest the observed enantio- and diastereoselectivities are achieved through a stepwise pathway. These biotransformations streamline the synthesis of chiral 1,2,3-polysubstituted cyclopropanes from readily available mixtures of (Z/E)-olefins, adding a new dimension to classical cyclopropanation methods.

An ambimodal transition state (TS) that leads to formation of four different pericyclic reaction products ([4 + 6]-, [2 + 8]-, [8 + 2]-, and [6 + 4]-cycloadducts) without any intervening minima has been designed and explored with DFT computations and quasiclassical molecular dynamics. Direct dynamics simulations propagated from the ambimodal TS show the evolution of trajectories to give the four cycloadducts. The topography of the PES is a key factor in product selectivity. A good correlation is observed between geometrical resemblance of the products to the ambimodal TS (measured by the RMSD) and the ratio of products formed in the dynamics simulations, We are grateful to the National Science Foundation (CHE1764328 to K.N.H.) for financial support of this research and for access to XSEDE and UCLA Hoffman 2 for computer time and for this study. A.M.S. thanks the Madrid Government (Comunidad de Madrid-Spain) under the Multiannual Agreement with Universidad Autónoma de Madrid in the line Support to Young Researchers, in the context of the V PRICIT (SI3-PJI-2021-00463) and “Ministerio de Educación Cultura y Deporte” for funding (CAS18/00458)

We have previously reported the identification of CghA, a proposed Diels–Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA–product complex at a resolution of 2.0 Å. Our result provides the second structural determination of eukaryotic Diels–Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exo adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels–Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications.

Sulfide oxidation is accomplished by a new class of dioxomolybdenum(VI) catalyst (1) that uses the tridentate 2,6-bis[hydroxyl(methyl)amino]-4-morpholino-1,3,5-triazine ligand to form a five-coordinate molybdenum(VI) center. Resonance Raman spectra show that the dioxo groups on the Mo(VI) oxygens readily exchange with water in an acetonitrile media that allows 18O labeling of catalyst 1. The model oxidation reaction was the conversion of thioanisole (2) to the corresponding sulfoxide with 4% of 1 using an equimolar amount of H2O2 in MeCN-d3. Oxygen-18 labeling experiments with either 18O-labeled 1 or 18O-labeled H2O2 are consistent with a sulfide oxygenation pathway that uses a η1-Mo(OOH) hydroxoperoxyl species (3). The hypothesized intermediate 3 is initially formed in a proton transfer reaction between 1 and H2O2. Oxidation is hypothesized via nucleophilic attack of the sulfide on 3 that is supported from a Hammett linear free-energy relationship for para-derivatives of 2. A Hammett reactivity constant (ρ) of -1.2 ± 0.2 was obtained, which is consistent with other ρ values found in prior sulfide oxidation reactions by group 6 complexes. An Eyring plot of the 2 oxidation by 1 gives an Ea of 63.0 ± 5.2 kJ/mol, which is slightly higher than that of a similar oxidation of 2 by the molybdenum(VI) complex, oxodiperoxo (pyridine-2-carboxylato)molybdate(VI) bis(pyridine-2-carboxylic acid) monohydrate (5). Computational modeling with density functional theory (DFT) of the complete reaction profile gave enthalpy and entropy of activations (64 kJ/mol and -120 J/mol·K, respectively) within 1 standard deviation of the experimental values, further supporting the hypothesized mechanism.

Computational studies with ωB97X-D density functional theory of the mechanisms of the steps in Trauner's biomimetic synthesis of preuisolactone A have elaborated and refined mechanisms of several unique processes. An ambimodal transition state has been identified for the cycloaddition between an o-quinone and a hydroxy-o-quinone; this leads to both (5 + 2) (with H shift) and (4 + 2) cycloaddition products, which can in principle interconvert via α-ketol rearrangements. The origins of periselectivity of this ambimodal cycloaddition have been investigated computationally with molecular dynamics simulations and tested further by an experimental study. In the presence of bicarbonate ions, the deprotonated hydroxy-o-quinone leads to only the (5 + 2) cycloaddition adduct. A new mechanism for a benzilic acid rearrangement resulting in ring contraction is proposed.

The cycloadditions of cyclopentadiene and cycloheptatriene with tropone are some of the earliest published examples of [6+4] cycloaddition reactions. We report quantum mechanical studies (ωB97X-D and DLPNO-CCSD(T)) of transition structures and products of these reactions, as well as quasi-classical molecular dynamics simulations of reaction trajectories. The study reveals that these cycloadditions involve ambimodal transition states resulting in a web of products by pericyclic interconversion pathways. Combined with these studies, calculations of simple parent systems and a thorough meta-analysis of literature examples reveal the general concept that all endo-[6+4] cycloadditions are ambimodal.

Previous studies showed that the FeII/α‐ketoglutarate dependent dioxygenase AsqJ induces a skeletal rearrangement in viridicatin biosynthesis in Aspergillus nidulans, generating a quinolone scaffold from benzo[1,4]diazepine‐2,5‐dione substrates. We report that AsqJ catalyzes an additional, entirely different reaction, simply by a change in substituent in the benzodiazepinedione substrate. This new mechanism is established by substrate screening, application of functional probes, and computational analysis. AsqJ excises H2CO from the heterocyclic ring structure of suitable benzo[1,4]diazepine‐2,5‐dione substrates to generate quinazolinones. This novel AsqJ catalysis pathway is governed by a single substituent within the complex substrate. This unique substrate‐directed reactivity of AsqJ enables the targeted biocatalytic generation of either quinolones or quinazolinones, two alkaloid frameworks of exceptional biomedical relevance., AsqJ catalyzes a complex rearrangement sequence in quinolone biosynthesis. We show the AsqJ biocatalytic potential to significantly exceed this natural function. An unprecedented reaction pathway leading to quinazolinones is uncovered, functionally and mechanistically characterized in detail, revealing a unique substrate‐dependent product selectivity in enzyme catalysis.

Psychoactive natural products play an integral role in the modern world. The tremendous structural complexity displayed by such molecules confers diverse biological activities of significant medicinal value and sociocultural impact. Accordingly, in the last two centuries, immense effort has been devoted towards establishing how plants, animals, and fungi synthesize complex natural products from simple metabolic precursors. The recent explosion of genomics data and molecular biology tools has enabled the identification of genes encoding proteins that catalyze individual biosynthetic steps. Once fully elucidated, the "biosynthetic pathways" are often comparable to organic syntheses in elegance and yield. Additionally, the discovery of biosynthetic enzymes provides powerful catalysts which may be repurposed for synthetic biology applications, or implemented with chemoenzymatic synthetic approaches. In this review, we discuss the progress that has been made toward biosynthetic pathway elucidation amongst four classes of psychoactive natural products: hallucinogens, stimulants, cannabinoids, and opioids. Compounds of diverse biosynthetic origin - terpene, amino acid, polyketide - are identified, and notable mechanisms of key scaffold transforming steps are highlighted. We also provide a description of subsequent applications of the biosynthetic machinery, with an emphasis placed on the synthetic biology and metabolic engineering strategies enabling heterologous production.

The Diels–Alder reactions of tropolone and its conjugate base with N-methylmaleimide have been explored computationally and experimentally. Previous studies of the [4+2] cycloaddition under basic conditions show that both endo- and exo-products are obtained in similar, but variable amounts. Density functional theory (ωB97X-D) explorations of potential energy surfaces, and molecular dynamics trajectories show that the reaction involves an ambimodal transition state for the reaction of the ammonium tropolonate with N-methylmaleimide, and that similar amounts of endo- and exo-products are obtained. The thermal reaction, studied experimentally in detail here for the first time, is predicted to form the endo-adduct through an ambimodal transition state. The exo-adduct can be formed from the same transition state, but requires a hydrogen shift, that hinders this reaction dynamically. Longer reaction times give a small excess of the exo-product, which is thermodynamically more stable.

CONSPECTUS: The concept of pericyclic reactions and the explanation of their specificity through orbital symmetries introduced a new way of understanding reactions and looking for new ones. One of the 1965 Woodward-Hoffmann communications described "the (as yet unobserved) symmetry-allowed 6 + 4 combination", the prediction of a new field of "higher-order" cycloadditions, involving more than six electrons. Later these authors predicted exo-stereoselectivity for the [6 + 4]-cycloaddition. Chemists rushed to test this prediction (for the most part successfully). For more than half a century, chemists have hunted for additional higher-order cycloadditions. The application of catalysis within organic chemistry allows the accomplishment of previously unattainable reactions, including higher-order cycloadditions.The many examples of [8 + 2], [6 + 4], and cycloadditions of even higher electron-counts discovered since the Woodward-Hoffmann rules were introduced illustrate the difficulty in predicting which of these transformations will occur when two highly unsaturated molecules react. Periselectivity has been a challenge, and the development of enantioselective variants has been elusive. While progress was made, the rise of organocatalysis in asymmetric synthesis has led to a surge of interest in stereoselective versions of higher-order cycloadditions. Through organocatalytic activation of conjugated cyclic polyenes and heteroaromatic compounds, asymmetric [8 + 2]-, [6 + 4]-, and [10 + 4]-cycloadditions have been realized by our groups. In this century, [6 + 4]-cycloadditions have been found also to occur in enzyme-catalyzed reactions for the biosynthesis of spinosyn A, heronamide, and streptoseomycin natural products. A whole new class of enzymes, the pericyclases that catalyze pericyclic reactions, has been discovered.A remarkable aspect of these recent developments is the cross-disciplinary research involved: from organic synthesis to computational studies integrated with experimental studies of reaction mechanisms, intermediates, and dynamics, to understanding mechanisms of enzyme catalysis and engineering of enzymes.This Account describes how our groups have been involved in the expansion of the higher-order cycloaddition frontiers. We describe both the history and recent progress in higher-order cycloadditions, and how these advances have been made by our collaborative experimental and computational studies. Progress in asymmetric organocatalysis, incorporating enantioselective higher-order cycloadditions in organic synthesis, and the stereoselective synthesis of important scaffolds will be highlighted. Experimental progress and computational modeling with density functional theory (DFT) has identified ambimodal cycloaddition pathways and led to the realization that multiple products of pericyclic reactions are linked by common transition states. Molecular dynamic simulations have provided fundamental understanding of factors controlling periselectivity and have led to discoveries of a group of enzymes, the pericyclases, which catalyze pericyclic reactions such as [6 + 4]-cycloadditions.

An entropic intermediate is defined as a free energy minimum that is not a potential energy minimum; the favorable entropy in this region of the potential surface is responsible for the increased lifetime of this species. Enabled by molecular dynamics simulations, entropic intermediates have been identified for several types of reactions, particularly pericyclic reactions. This review highlights recent advances in the mechanistic and dynamic investigations of organic and biosynthetic pericyclic reactions that involve entropic intermediates. We have proposed timing criteria to differentiate dynamically concerted from dynamically stepwise mechanisms. These criteria complement the usual definitions of concertedness based on energetics and bonding changes along the potential surface.

The pericyclases are a growing superfamily of enzymes that catalyze pericyclic reactions. We report a pericyclase IccD catalyzing an inverse-electron demand Diels–Alder (IEDDA) reaction with a rate acceleration of 3 × 10(5) fold in the biosynthesis of fungal natural product ilicicolin H. We demonstrate IccD is highly periselective towards the IEDDA cycloaddition over a competing normal electron demand Diels-Alder (NEDDA) reaction from an ambimodal transition state. A predicted flavoenzyme IccE was identified to epimerize the IEDDA product 8-epi-ilicicolin H to ilicicolin H, a step that is critical for the observed antifungal activity of ilicicolin H. Our results reveal the ilicicolin H biosynthetic pathway and add to the collection of pericyclic reactions that are catalyzed by pericyclases.

We report an ambimodal trispericyclic transition state leading to [6+4]-, [4+6]-, and [8+2]-cycloadducts in the reactions of 8,8-disubstituted heptafulvenes with 6,6-dimethylfulvene. The potential energy surfaces for these reactions were explored with ωB97X-D density functional theory. Quasi-classical direct molecular dynamics simulations gave information on the ratios of products expected in these reactions.

Overcoming resistance to bioactive small molecules is a significant challenge for health care and agriculture. As a result, efforts to uncover the mechanisms of resistance are essential to the development of new antibiotics, anticancer drugs and pesticides. To study how nature evolves resistance to highly potent natural products, we examined the biosynthesis and mechanism of self-resistance of the fungal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inhibitor heptelidic acid (HA). HA is a nanomolar inhibitor of GADPH through the covalent modification of the active site cysteine thiol. The biosynthetic pathway of HA was elucidated, which uncovered the enzymatic basis of formation of the epoxide warhead. Structure–activity relationship study using biosynthetic intermediates established the importance of the fused lactone ring system in HA. The molecular basis of HA inhibiting human GAPDH was illustrated through the crystal structure of Hs-GAPDH covalently bound with HA. A GAPDH isozyme HepG encoded in the HA cluster was characterized to be less sensitive to HA, and therefore contribute to self-resistance for the producing host. Comparison of the crystal structures of human GAPDH and HepG showed mutations both within and remote to the active site can contribute to resistance of inactivation, which was confirmed through mutagenesis. Due to the critical role GAPDH plays in aerobic glycolysis and other cellular functions, knowledge of HA mode of action and self-resistance mechanism could accelerate the development of improved inhibitors., The structural basis and self-resistance mechanism of fungal glyceraldehyde-3-phosphate dehydrogenase inhibitor heptelidic acid are uncovered.

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A(2) (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane; while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.

The construction of libraries of stereoisomers of natural products serves as an important approach to investigating the correlation between the stereostructure and biological activity. However, the total synthesis and isomerzation of polycyclic scaffolds with multiple chrial centers are rare. Spirooliganin (1), a new skeleton natural product isolated from the plant Illicium oligandrum, was structurally characterized by comprehensive analysis of NMR spectroscopic data and ECD which revealed an unprecedented 5–6–6–6–7 polycyclic framework with six chiral centers. Here we report a 17-step total synthesis to prepare a library of stereochemically diverse isomers of spirooliganin, including 16 diastereoisomers and 16 regioisomers. In addition to a regioselective hetero-Diels–Alder cycloaddition, the synthetic strategy involves a photo-induced stereoselective Diels–Alder reaction, which gives only the abnormal trans-fused product as rationalized by density functional theory calculations. Preliminary biological evaluation showed that spirooliganin and regioisomers 39 exhibited potent inhibition of Coxsackievirus B3. It also revealed the pharmacophore effect of the D-ring (16R,18R,24R, and 26R) for their antiviral activities., Library construction of stereochemically diverse isomers to investigate the relationship between stereoconfiguration and anti-coxsackie virus B3 activity.

Hirsutellones are fungal natural products containing a macrocyclic para-cyclophane connected to a decahydrofluorene ring system. We have elucidated the biosynthetic pathway for pyrrocidine B (3) and GKK1032 A2 (4). Two small hypothetical proteins, an oxidoreductase and a lipocalin-like protein, function cooperatively in the oxidative cyclization of the cyclophane, while an additional hypothetical protein in the pyrrocidine pathway catalyzes the exo-specific cycloaddition to form the cis-fused decahydrofluorene.

The cycloadditions of cyclopentadiene and cycloheptatriene with tropone are some of the earliest published examples of [6+4] cycloaddition reactions. We report quantum mechanical studies (ωB97X-D and DLPNO-CCSD(T)) of transition structures and products of these reactions, as well as quasi-classical molecular dynamics simulations of reaction trajectories. The study reveals that these cycloadditions involve ambimodal transition states resulting in a web of products by pericyclic interconversion pathways. Combined with these studies, calculations of simple parent systems and a thorough meta-analysis of literature examples reveal the general concept that all

We have previously reported the identification of CghA, a proposed Diels–Alderase responsible for the formation of the bicyclic octalin core of the fungal secondary metabolite Sch210972. Here we show the crystal structure of the CghA–product complex at a resolution of 2.0 A. Our result provides the second structural determination of eukaryotic Diels–Alderases and adds yet another fold to the family of proteins reported to catalyse [4 + 2] cycloaddition reactions. Site-directed mutagenesis-coupled kinetic characterization and computational analyses allowed us to identify key catalytic residues and propose a possible catalytic mechanism. Most interestingly, we were able to rationally engineer CghA such that the mutant was able to catalyse preferentially the formation of the energetically disfavoured exo adduct. This work expands our knowledge and understanding of the emerging and potentially widespread class of natural enzymes capable of catalysing stereoselective Diels–Alder reactions and paves the way towards developing enzymes potentially useful in various bio/synthetic applications. Enzymatic Diels–Alder reactions are of high synthetic interest, but mechanistic insights remain scarce. Now, a structure of the Diels–Alderase CghA in complex with its product is reported, a catalytic mechanism proposed and the enzyme is engineered to form the energetically disfavoured exo adduct.

Introduction Augmented urethroplasty with buccal mucosal graft remains one of the mainstays of urethral stricture management, and location of graft placement depends on surgeon preference as well as patient-specific factors. Historically, ventral graft placement has been preferred in the bulbar urethra due to a robust blood supply provided by a thicker corpus spongiosum at the bulb. While, in the penile urethra, dorsal placement is traditionally preferred due to limited spongiosum in this location and concern for poor graft take. Some have advocated for ventral onlay placement in the penile urethra, arguing this placement decreases dissection thereby preserving vascularization of the urethra. We present promising results and outcomes for ventral onlays in the penile urethra, which we cover with periurethral vascularized tissue as a “pseudospongioplasty” to provide a supportive, vascular bed for graft survival. Objective We aimed to assess the efficacy of ventral buccal mucosal graft positioning for treatment of penile urethral strictures using the pseudospongioplasty technique. Methods We retrospectively reviewed clinical and procedural characteristics for all patients that underwent urethroplasty at our institution from August 2016 through February 2021. Patients were divided into one of three groups based on their operative approach: (1) pseudospongioplasty in the penile urethra, (2) spongioplasty in the bulbar urethra, (3) and bulbopenile strictures requiring a combined approach. Key outcomes variables included recurrence rates and complication rates. Results 50 patients fit our inclusion criteria. We identified 18 patients that underwent isolated spongioplasty in the bulbar urethra, 19 patients that underwent isolated pseudospongioplasty in the penile urethra, and 13 patients with bulbopenile strictures that had simultaneous spongioplasty and pseudospongioplasty. All procedures used buccal mucosa as a ventral onlay. Median stricture length (cm) for spongioplasty, pseudospongioplasty and both were 4.0 (3-5) and 4.25 (3.5-6) and 8 (6-10), respectively. There were three 30-day complications in each group. Figure 1 shows the Kaplan Meier curves for recurrence in the three study groups, rates were not statistically different using spongioplasty as a reference group (p=0.83 and p=0.72 for pseudospongioplasty and combined, respectively). When compared to spongioplasty, the relative risk of recurrence in pseudospongioplasty and combined were 0.41 (0.05-2.44, p=0.344) and 1.56 (0.30-8.21, p=0.593), respectively. Meanwhile the relative risk for complications were 0.94 (0.15-5.77, p=0.942) and 1.5 (0.24-9.61, p=0.657), for pseudospongioplasty and combined respectively. Conclusions Supporting recent literature concerning ventral onlay graft positioning in the penile urethra, a pseudospongioplasty appears to provide enough of a supportive vascular bed for graft survival. There are few published series demonstrating its effectiveness; however, we show that pseudospongioplasty in the penile urethra has comparable outcomes to conventional spongioplasty in the bulbar urethra. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Coloplast, Boston Scientific

Medium-ring lactones are synthetically challenging due to unfavorable energetics involved in cyclization. We have discovered a thioesterase enzyme DcsB, from the decarestrictine C1 (1) biosynthetic pathway, that efficiently performs medium-ring lactonizations. DcsB shows broad substrate promiscuity towards linear substrates that vary in lengths and substituents, and is a potential biocatalyst for lactonization. X-ray crystal structure and computational analyses provide insights into the molecular basis of catalysis.

An ongoing challenge in chemical research is to design catalysts that select the outcomes of the reactions of complex molecules. Chemists rely on organocatalysts or transition metal catalysts to control stereoselectivity, regioselectivity and periselectivity (selectivity among possible pericyclic reactions). Nature achieves these types of selectivity with a variety of enzymes such as the recently discovered pericyclases-a family of enzymes that catalyse pericyclic reactions1. Most characterized enzymatic pericyclic reactions have been cycloadditions, and it has been difficult to rationalize how the observed selectivities are achieved2-13. Here we report the discovery of two homologous groups of pericyclases that catalyse distinct reactions: one group catalyses an Alder-ene reaction that was, to our knowledge, previously unknown in biology; the second catalyses a stereoselective hetero-Diels-Alder reaction. Guided by computational studies, we have rationalized the observed differences in reactivities and designed mutant enzymes that reverse periselectivities from Alder-ene to hetero-Diels-Alder and vice versa. A combination of in vitro biochemical characterizations, computational studies, enzyme co-crystal structures, and mutational studies illustrate how high regioselectivity and periselectivity are achieved in nearly identical active sites.

The combination of electrocyclizations and cycloadditions accounts for the formation of a range of fascinating natural products. Cascades consisting of 8π electrocyclizations followed by a 6π electrocyclization and a cycloaddition are relatively common. We now report the synthesis of the tetramic acid PF-1018 through an 8π electrocyclization, the product of which is immediately intercepted by a Diels-Alder cycloaddition. The success of this pericyclic cascade was critically dependent on the substitution pattern of the starting polyene and could be rationalized through DFT calculations. The completion of the synthesis required the instalment of a trisubstituted double bond by radical deoxygenation. An unexpected side product formed through 4-exo-trig radical cyclization could be recycled through an unprecedented triflation/fragmentation.

A method is presented for the determination of gamma-ray energies and transition rates in nuclei populated using the fusion-evaporation reaction mechanism and measured using the Doppler-shift attenuation method (DSAM). This method is applied to data collected for the stable benchmark nucleus 22Ne during a commissioning experiment at TRIUMF, Canada's National Laboratory for Particle and Nuclear Physics, employing a 12C(18O,2α)22Ne fusion-evaporation reaction. Gamma-ray energies were determined using offline reconstruction to correct for the Doppler shift. Mean lifetimes of the corresponding transitions were then measured via a comparison to Monte-Carlo lineshape simulations developed using the GEANT4 framework. Best fit lifetimes obtained using χ2 analysis were in general agreement with the existing literature, validating the analysis method used.

ObjectiveLarge studies of knee osteoarthritis (KOA) require well-characterized efficient methods to assess progression. We previously developed the local-area cartilage segmentation (LACS) software method, to measure cartilage volume on magnetic resonance imaging (MRI) scans. The present study further validates this method in a larger patient cohort and assesses predictive validity in a case-control study.MethodThe OA Biomarkers Consortium FNIH Project, a case-control study of KOA progression nested within the Osteoarthritis Initiative (OAI), includes 600 subjects in four subgroups based on radiographic and pain progression. Our software tool measured change in medial femoral cartilage volume in a central weight-bearing region. Different sized regions of cartilage were assessed to explore their sensitivity to change. The readings were performed on MRI scans at the baseline and 24-month visits. We used standardized response means (SRMs) for responsiveness and logistic regression for predictive validity.ResultsCartilage volume change was associated strongly with radiographic progression (odds ratios (OR)=4.66; 95% confidence intervals (CI)=2.85-7.62). OR were significant but of lesser magnitude for the combined radiographic and pain progression outcome (OR=1.70; 95% CI=1.40-2.07). For the full 600 subjects, theSRM was -0.51 for the largest segmented area. Smaller areas of cartilage segmentation were also able to predict the case-control status. The average reader time for the largest area was less than 20min per scan. Smaller areas could be assessed with less reader time.ConclusionWe demonstrated that the LACS method is fast, responsive, and associated with radiographic and pain progression, and is appropriate for existing and future large studies of KOA.

Aim To evaluate the clinical impact of combined 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) positron-emission tomography/computed tomography (PET/CT) brain imaging performed in selected patients with cognitive impairment at a tertiary referral centre in the UK, and to assess the accuracy of FDG PET/CT to correctly establish the diagnosis of Alzheimer's dementia (AD) in “real-world” clinical practice. Methods and materials Using an institutional radiology database, 136 patients were identified for inclusion in the study. FDG PET/CT was performed using a standard technique and interpreted by dual-trained radiologists and nuclear medicine physicians. Standardised questionnaires were sent to the referring clinicians to establish the final clinical diagnosis and to obtain information about the clinical impact of FDG PET/CT. Results There was a 72% questionnaire return (98/136), with mean patient follow-up of 471 (standard deviation 205) days. FDG PET/CT had an impact on patient management in 81%, adding confidence to the pre-test diagnosis in 43%, changing the pre-test diagnosis in 35%, reducing the need for further investigations in 42%, and resulting in a change in therapy in 32%. There was substantial correlation between the PET/CT diagnosis and final clinical diagnosis with a correlation (k) coefficient of 0.78 (p

Background Various designs of total knee arthroplasty (TKA) have provided satisfactory outcomes for the treatment of knee osteoarthritis for many years. The aim of the study is to evaluate the success and failure rate of the medial stabilized (MS) TKA design through national joint registries and the current literature. Materials and Methods A comprehensive literature review using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses was carried out through PubMed and MEDLINE database. The international registries analyzed included the National Joint Registry, the Australian Orthopedic Association National Joint Replacement Registry, the Dutch Arthroplasty Register, the New Zealand Orthopedic Association Joint Registry, and the Michigan Arthroplasty Registry Collaborative Quality Initiative. We are reporting descriptive data which include means and percentages of survival rates and revision rates and number of years after the primary procedure. The revision rates and the cumulative revision rates are reported separately for each MS implant. Results Our detailed review identified 5 of 12 registries and 25 of 550 studies reporting on the survivorship of an MS TKA design. There were a total of 3684 procedures between the 25 studies, with an average survivorship free of aseptic loosening of 99% at 6.9 years. There are 26,693 (2.5%) MS TKAs in the current National Joint Registry with a mean cumulative revision rate of 2.63% at 5 years, 3.35% at 10 years, and 4.6% at 15 years. The Australian Orthopedic Association National Joint Replacement Registry provides survivorship data on 19,249 (2.9%) MS TKAs, with a mean cumulative revision rate of 3.34% at 5 years, 7.4% at 10 years, and 8.1% at 15 years. The Dutch Arthroplasty Register contains survivorship data on 1490 MS designs and a mean revision rate of these implants is 0.8% at 1 year, 5.95% at 5 years, and 9.8% at 10 years. The Evolution MP is the only implant reported in the Michigan Arthroplasty Registry Collaborative Quality Initiative and has a revision rate of 2.28% at 3 years. Conclusion The MS TKA design has comparable results to traditional TKA designs across several joint registries and 25 studies in the literature.

The combination of electrocyclizations and cycloadditions accounts for the formation of a range of fascinating natural products. Cascades consisting of 8π electrocyclizations followed by a 6π electrocyclization and a cycloaddition are relatively common. We now report the synthesis of the tetramic acid PF-1018 through an 8π electrocyclization, the product of which is immediately intercepted by a Diels-Alder cycloaddition. The success of this pericyclic cascade was critically dependent on the substitution pattern of the starting polyene and could be rationalized through DFT calculations. The completion of the synthesis required the instalment of a trisubstituted double bond by radical deoxygenation. An unexpected side product formed through 4-exo-trig radical cyclization could be recycled through an unprecedented triflation/fragmentation.

Diels-Alder reactions are among the most powerful synthetic transformations to construct complex natural products. Despite that increasing of enzymatic intramolecular Diels-Alder reactions have been discovered, natural intermolecular Diels-Alderases are rarely described. Here, we report an intermolecular hetero-Diels-Alder reaction in the biosynthesis of tropolonic sesquiterpenes and functionally characterize EupfF as the first fungal intermolecular hetero-Diels-Alderase. We demonstrate that EupfF catalyzed the dehydration of a hydroxymethyl-containing tropolone (5) to generate a reactive tropolone o-quinone methide (6) and might further stereoselectively control the subsequent intermolecular hetero-Diels-Alder reaction with (1E,4E,8Z)-humulenol (8) to produce enantiomerically pure neosetophomone B (1). Our results reveal the biosynthetic pathway of 1 and expand the repertoire of activities of Diels-Alder cyclases.

Detailed spectroscopic measurements of excited states in $^{50}\mathrm{Cr}$ and $^{62}\mathrm{Zn}$ were performed using 24-MeV $(p,t)$ transfer reactions on $^{52}\mathrm{Cr}$ and $^{64}\mathrm{Zn}$, respectively. In total, 45 states in $^{50}\mathrm{Cr}$ and 67 states in $^{62}\mathrm{Zn}$ were observed up to excitation energies of 5.5 MeV, including several previously unobserved states. These experimental results are compared to ab-initio shell-model calculations using chiral effective field theory with the valence-space in-medium similarity renormalization group method. This comparison demonstrates good agreement in the level orderings with these new theoretical methods, albeit with a slight overbinding in the calculations. This work is part of a continued push to benchmark ab-initio theoretical techniques to nuclear-structure data in ${0}^{+}\ensuremath{\rightarrow}{0}^{+}$ superallowed Fermi $\ensuremath{\beta}$-decay systems.

Objectives:This article aimed to address the feasibility of mentalization-based treatment (MBT) for patients with personality disorder in a non-specialist setting. The development and implementation of an MBT Programme is described.Methods:A multidisciplinary Consult Group met to plan the implementation of the programme. Participants attended a psychoeducation group (MBT Introductory Group), then weekly individual and group therapy. Fourteen participants started the full programme with eight completing at least 9 months, complete data are available for five participants who completed 27 months (first cohort) and 21 months (second cohort). Data include quantitative measures and qualitative questionnaires/interviews. All had a diagnosis of personality dysfunction with co-morbid disorder including anxiety/depressive disorder, post-traumatic stress disorder and eating disorder.Results:Data on five participants revealed reductions in global level of distress, improvements in psychological well-being, less interpersonal difficulties and better work and social functioning. Qualitative data from feedback questionnaires (n = 18) and in-depth interview (n = 2) are discussed under the themes of mentalizing, treatment feedback/outcomes and group factors. Therapist reflections on the process identify the challenges involved in implementing a specialist psychotherapy programme within a general service and learning points from this are discussed.Conclusions:MBT is an acceptable treatment for patients with personality dysfunction. Prior to the implementation of a programme, factors at the therapist, team and organizational level, as well as the wider context, need to be examined. This is to ensure that conditions are in place for proper adherence to the model to achieve the positive outcomes demonstrated in the RCT studies.

Covering: 2000 to 2018 Pericyclic reactions are a distinct class of reactions that have wide synthetic utility. Before the recent discoveries described in this review, enzyme-catalyzed pericyclic reactions were not widely known to be involved in biosynthesis. This situation is changing rapidly. We define the scope of pericyclic reactions, give a historical account of their discoveries as biosynthetic reactions, and provide evidence that there are many enzymes in nature that catalyze pericyclic reactions. These enzymes, the “pericyclases,” are the subject of this review.

Pericyclases are an emerging family of enzymes catalyzing pericyclic reactions. A class of lipocalin-like enzymes recently characterized as Diels-Alderases (DAses) catalyze decalin formation through intramolecular Diels-Alder (IMDA) reactions between electron-rich dienes and electron-deficient dienophiles. Using this class of enzyme as a beacon for genome mining, we discovered a biosynthetic gene cluster from Penicillium variabile and identified that it encodes for the biosynthesis of varicidin A (1), a new antifungal natural product containing a cis-octahydrodecalin core. Biochemical analysis reveals a carboxylative deactivation strategy used in varicidin biosynthesis to suppress the nonenzymatic IMDA reaction of an early acyclic intermediate that favors trans-decalin formation. A P450 oxidizes the reactive intermediate to yield a relatively unreactive combination of an electron-deficient diene and an electron-deficient dienophile. The DAse PvhB catalyzes the final stage IMDA on the carboxylated intermediate to form the cis-decalin that is important for the antifungal activity.

Background CT perfusion (CTP) and diffusion or perfusion MRI might assist patient selection for endovascular thrombectomy. We aimed to establish whether imaging assessments of irreversibly injured ischaemic core and potentially salvageable penumbra volumes were associated with functional outcome and whether they interacted with the treatment effect of endovascular thrombectomy on functional outcome.Methods In this systematic review and meta-analysis, the HERMES collaboration pooled patient-level data from all randomised controlled trials that compared endovascular thrombectomy (predominantly using stent retrievers) with standard medical therapy in patients with anterior circulation ischaemic stroke, published in PubMed from Jan 1,2010, to May 31, 2017. The primary endpoint was functional outcome, assessed by the modified Rankin Scale (mRS) at 90 days after stroke. Ischaemic core was estimated, before treatment with either endovascular thrombectomy or standard medical therapy, by CTP as relative cerebral blood flow less than 30% of normal brain blood flow or by MRI as an apparent diffusion coefficient less than 620 mu m(2)/s. Critically hypoperfused tissue was estimated as the volume of tissue with a CTP time to maximum longer than 6 s. Mismatch volume (ie, the estimated penumbral volume) was calculated as critically hypoperfused tissue volume minus ischaemic core volume. The association of ischaemic core and penumbral volumes with 90-day mRS score was analysed with multivariable logistic regression (functional independence, defined as mRS score 0-2) and ordinal logistic regression (functional improvement by at least one mRS category) in all patients and in a subset of those with more than 50% endovascular reperfusion, adjusted for baseline prognostic variables. The meta-analysis was prospectively designed by the HERMES executive committee, but not registered.Findings We identified seven studies with 1764 patients, all of which were included in the meta-analysis. CTP was available and assessable for 591 (34%) patients and diffusion MRI for 309 (18%) patients. Functional independence was worse in patients who had CTP versus those who had diffusion MRI, after adjustment for ischaemic core volume (odds ratio [OR] 0.47 [95% CI 0.30-0.72], p=0.0007), so the imaging modalities were not pooled. Increasing ischaemic core volume was associated with reduced likelihood of functional independence (CTP OR 0.77 [0.69-0.86] per 10 mL, p(interaction)=0.29; diffusion MRI OR 0.87 [0.81-0.94] per 10 mL, p(interaction)=0.94). Mismatch volume, examined only in the CTP group because of the small numbers of patients who had perfusion MRI, was not associated with either functional independence or functional improvement. In patients with CTP with more than 50% endovascular reperfusion (n=186), age, ischaemic core volume, and imaging-to-reperfusion time were independently associated with functional improvement. Risk of bias between studies was generally low.Interpretation Estimated ischaemic core volume was independently associated with functional independence and functional improvement but did not modify the treatment benefit of endovascular thrombectomy over standard medical therapy for improved functional outcome. Combining ischaemic core volume with age and expected imagingto-reperfusion time will improve assessment of prognosis and might inform endovascular thrombectomy treatment decisions. Copyright (C) 2018 Elsevier Ltd. All rights reserved.

Quantitative estimates of the abundance of surface materials and of water ice particle grain sizes at five widely separated locations on the surface of Europa have been obtained by two independent methods in order to search for possible discrepancies that may be attributed to differences in the methods employed. Results of radiative transfer (RT) compositional modeling (also known as intimate mixture modeling) from two prior studies are here employed without modification. Areal (or “checkerboard”) mixture modeling, also known as linear mixture (LM) modeling, was performed to allow direct comparisons. The failure to model scattering processes (whose effects may be strongly nonlinear) in the LM approach is recognized as a potential source of errors. RT modeling accounts for nonlinear spectral responses due to scattering, but is subject to other uncertainties. By comparing abundance estimates for H2SO4•nH2O and water ice, obtained through both methods as applied to identical spectra, we may gain some insight into the importance of ‘volume scattering’ effects for investigations of Europa's surface composition. We find that both methods return similar abundances for each location analyzed; linear correlation coefficients of ≥ 0.98 are found between the derived H2SO4•nH2O and water ice abundances returned by both methods. We thus find no evidence of a significant influence of volume scattering on the compositional solutions obtained by LM modeling for these locations. Some differences in the results obtained for water ice grain sizes are attributed to the limited selection of candidate materials allowed in the RT investigations. This article is protected by copyright. All rights reserved.

Predictive central cues generate location-based expectancies, voluntary shifts of attention, and facilitate target processing. Often, location-based expectancies and voluntary attention are confounded in cueing tasks. Here we vary the predictability of central cues to determine whether they can evoke the inhibition of target processing in three go/no-go experiments. In the first experiment, the central cue was uninformative and did not predict the target's location. Importantly, these cues did not seem to affect target processing. In the second experiment, the central cue indicated the most or the least likely location of the target. Surprisingly, both types of cues facilitated target processing at the cued location. In the third experiment, the central cue predicted the most likely location of a no-go target, but it did not provide relevant information pertaining to the location of the go target. Again, the central cue facilitated processing of the go target. These results suggest that efforts to strategically allocate inhibition may be thwarted by the paradoxical monitoring of the cued location. The current findings highlight the need to further explore the relationship between location-based expectancies and spatial attention in cueing tasks.

LepI is an S-adenosylmethionine (SAM)-dependent pericyclase that catalyzes the formation of 2-pyridone natural product leporin C. Biochemical characterization showed LepI can catalyze the stereoselective dehydration to yield a reactive (E)-quinone methide that can undergo bifurcating intramolecular Diels-Alder (IMDA) and hetero-Diels-Alder (HDA) cyclizations from an ambimodal transition state, as well as a [3,3]-retro-Claisen rearrangement to recycle the IMDA product into leporin C. Here we solved the X-ray crystal structures of SAM-bound LepI and in complex with a substrate analog, the product leporin C, and a retro-Claisen reaction transition-state analog to understand the structural basis for the multitude of reactions. Structural and mutational analysis revealed how Nature evolves a classic methyltransferase active site into one that can serve as a dehydratase and a multifunctional pericyclase. Catalysis of both sets of reactions employs H133 and R295, two active site residues that are not found in canonical methyltransferases. An alternative role of SAM, which is not found to be in direct contact with the substrate, is also proposed., Graphical Abstract

The nuclear structure of $^{112}\mathrm{Cd}$ has been investigated with the $^{111}\mathrm{Cd}(\stackrel{P\vec}{d},p)^{112}\mathrm{Cd}$ reaction. Isotopically enriched targets of $^{111}\mathrm{Cd}$ were bombarded with 22 MeV polarized deuterons, and reaction products were analyzed with a magnetic spectrograph. Angular distributions and analyzing powers were determined for 129 states, 49 of which are newly observed, up to approximately 4.2 MeV in excitation energy. The observed angular distributions were compared with distorted wave Born approximation (DWBA) and adiabatic distorted wave approximation (ADWA) calculations to extract the spectroscopic factors. Two-quasineutron configurations involving coupling to the ${s}_{\frac{1}{2}}$ orbital are suggested. The sum of spectroscopic strengths extracted by using the ADWA for the individual $l$ transfers are combined with previous results from the $^{111}\mathrm{Cd}(\stackrel{P\vec}{d},t)$ reaction and show good agreement with the $2j+1$ sum rule, whereas those extracted with the DWBA calculations are significantly less.

Purpose Efforts to promote the health and well-being of military veterans have been criticised for being inadequately informed of veterans’ most pressing needs as they separate from military service, as well as the programmes that are most likely to meet these needs. The current article summarises limitations of the current literature and introduces The Veterans Metrics Initiative (TVMI) study, a longitudinal assessment of US veterans’ well-being and programme use in the first three years after they separate from military service. Veterans were assessed within 3 months of military separation and will complete five additional assessments at 6-month intervals during the subsequent period. Participants The TVMI study cohort consists of a national sample of 9566 newly separated US veterans that were recruited in the fall of 2016. Findings to date The TVMI sample includes representation from all branches of service, men and women, and officers and enlisted personnel. Although representative of the larger population on many characteristics, differential response rates were observed for some subgroups, necessitating the development of non-response bias weights. Comparisons between unweighed and weighted results suggest that the weighting procedure adequately adjusts for observed differences. Future plans Analyses are under way to examine veterans’ well-being and programme use in the period following separation after military service, as well as factors associated with poor outcomes. We have also begun to decompose programmes into their core components to facilitate examination of how these components relate to well-being. Once our third data collection is complete, we will examine factors related to different patterns of readjustment over time.

Ambimodal reactions involve a single transition state leading to multiple products. In such reactions, transition state theory gives no information about the ratio of products that are formed, and molecular dynamics must be performed to predict this ratio. Understanding the relationship between the transition structure and the product ratio is a long-standing problem in molecular dynamics. We have studied 15 ambimodal pericyclic reactions and investigated the relationship between the TS bond lengths in the saddle points and the product ratios from trajectory simulations. A linear correlation, ln(B:A) = −9.4(Bond 3 – Bond 2), is found with R2 = 0.92, where A and B refer to the products formed upon formation of bonds 2 and 3, respectively. The correlation shows that the ratio of products formed after the bifurcation is related to the partial bond lengths, and corresponding bond orders, in the transition state.

(10,8)CASPT2/6-31G**//(10,8)CASSCF/6-31G** and CCSD(T)/cc-pVDZ//(10,8)-CASSCF/6-31G** calculations have been performed on the potential surface for the [3,3] sigmatropic allyl ester rearrangements of cis-3-penten-2-yl acetate (16) to trans-3-penten-2-yl acetate (17) and 3-buten-2-yl acetate (21) to trans-2-buten-1-yl acetate (22). The results are compared to DFT (B3LYP/6-31G**) calculations on the known 16 → 17 rearrangement that reported it to be concerted and pseudopericyclic through a boat-shaped transition structure ( Birney, D. M. et al. J. Am. Chem. Soc. 2009 , 131 , 528 - 537 ). The CASSCF calculations, on the other hand, uncovered competitive concerted pathways for both the 16 → 17 and 21 → 22 rearrangements, though it was necessary to apply certain approximations in the former case. While one CASSCF pathway in each case involves a boat-shaped transition structure, similar to the one located through DFT calculations, the other pathway involves a chair-shaped transition structure. Preference for chair or boat is shown to be method dependent. Moreover, examination of the CASSCF active-space orbitals clearly confirms that the boat-shaped transition structures are pseudopericyclic but significantly also established that the chair-shaped transition structures are clearly pericyclic. Conclusions based on these results, and regarding our understanding of pericyclic vs pseudopericyclic reactions, are proffered.

Many efforts have been made in nuclear structure physics to interpret the nature of low-lying excited 0+ states in well-deformed rare-earth nuclei. However, one of the difficulties in resolving the nature of these states is that there is a paucity of data. In this work, excited 0+ states in the N = 92 nucleus 160Er were studied via the 162Er(p, t)160Er two-neutron transfer reaction, which is ideal for probing 0+ → 0+ transitions, at the Maier-Leibnitz-Laboratorium in Garching, Germany. Reaction products were momentum-analyzed with a Quadrupole-3-Dipole magnetic spectrograph. The 0+2 state was observed to be strongly populated with 18% of the ground state strength.

BACKGROUND: General anaesthesia (GA) during endovascular thrombectomy has been associated with worse patient outcomes in observational studies compared with patients treated without GA. We assessed functional outcome in ischaemic stroke patients with large vessel anterior circulation occlusion undergoing endovascular thrombectomy under GA, versus thrombectomy not under GA (with or without sedation) versus standard care (ie, no thrombectomy), stratified by the use of GA versus standard care.METHODS: For this meta-analysis, patient-level data were pooled from all patients included in randomised trials in PuMed published between Jan 1, 2010, and May 31, 2017, that compared endovascular thrombectomy predominantly done with stent retrievers with standard care in anterior circulation ischaemic stroke patients (HERMES Collaboration). The primary outcome was functional outcome assessed by ordinal analysis of the modified Rankin scale (mRS) at 90 days in the GA and non-GA subgroups of patients treated with endovascular therapy versus those patients treated with standard care, adjusted for baseline prognostic variables. To account for between-trial variance we used mixed-effects modelling with a random effect for trials incorporated in all models. Bias was assessed using the Cochrane method. The meta-analysis was prospectively designed, but not registered.FINDINGS: Seven trials were identified by our search; of 1764 patients included in these trials, 871 were allocated to endovascular thrombectomy and 893 were assigned standard care. After exclusion of 74 patients (72 did not undergo the procedure and two had missing data on anaesthetic strategy), 236 (30%) of 797 patients who had endovascular procedures were treated under GA. At baseline, patients receiving GA were younger and had a shorter delay between stroke onset and randomisation but they had similar pre-treatment clinical severity compared with patients who did not have GA. Endovascular thrombectomy improved functional outcome at 3 months both in patients who had GA (adjusted common odds ratio (cOR) 1·52, 95% CI 1·09-2·11, p=0·014) and in those who did not have GA (adjusted cOR 2·33, 95% CI 1·75-3·10, pINTERPRETATION: Worse outcomes after endovascular thrombectomy were associated with GA, after adjustment for baseline prognostic variables. These data support avoidance of GA whenever possible. The procedure did, however, remain effective versus standard care in patients treated under GA, indicating that treatment should not be withheld in those who require anaesthesia for medical reasons.FUNDING: Medtronic.

Uncontrolled bleeding and infection are the major causes of death and morbidity from traumatic wounds during military conflicts, disasters, and accidents. Because immediate treatment is critical to survival, it is desirable to have a lightweight and rapidly applicable bandage—one capable of delivering a hemostat that can quickly resolve bleeding while addressing infection over short and longer time frames. It is challenging to design thin film coatings capable of multidrug release, particularly when the drugs are quite different in nature (biologic versus small molecule, charged versus neutral) and the desired release profiles are different for each drug. Herein we have adopted a layer-by-layer film assembly technique to create a linear combination of two independently functional films capable of rapidly releasing thrombin within minutes while sustaining vancomycin elution for more than 24 h. By conjugating vancomycin to a hydrolytically degradable polyacid, poly(β-L-malic acid), we were able to create a robust thin film with loading and release kinetics that remain unaffected by the additional deposition of a thrombin-based film, demonstrating the possibility for future multitherapeutic films with independently tunable release kinetics.