Interrogation of an Enzyme Library Reveals the Catalytic Plasticity of Naturally Evolved [4+2] Cyclases

\ua9 2023 The Authors. ChemBioChem published by Wiley-VCH GmbH.Stereoselective carbon-carbon bond forming reactions are quintessential transformations in organic synthesis. One example is the Diels-Alder reaction, a [4+2] cycloaddition between a conjugated diene and a dienophile to form cyclohexenes. The development of biocatalysts for this reaction is paramount for unlocking sustainable routes to a plethora of important molecules. To obtain a comprehensive understanding of naturally evolved [4+2] cyclases, and to identify hitherto uncharacterised biocatalysts for this reaction, we constructed a library comprising forty-five enzymes with reported or predicted [4+2] cycloaddition activity. Thirty-one library members were successfully produced in recombinant form. In vitro assays employing a synthetic substrate incorporating a diene and a dienophile revealed broad-ranging cycloaddition activity amongst these polypeptides. The hypothetical protein Cyc15 was found to catalyse an intramolecular cycloaddition to generate a novel spirotetronate. The crystal structure of this enzyme, along with docking studies, establishes the basis for stereoselectivity in Cyc15, as compared to other spirotetronate cyclases

Cognitive testing of physical activity and acculturation questions in recent and long-term Latino immigrants

<p>Abstract</p> <p>Background</p> <p>We ascertained the degree to which language (English versus Spanish), and residence time in the US influence responses to survey questions concerning two topics: self-reported acculturation status, and recent physical activity (PA). This topic is likely to be of general interest because of growing numbers of immigrants in countries worldwide.</p> <p>Methods</p> <p>We carried out qualitative (cognitive) interviews of survey items on acculturation and physical activity on 27 Latino subjects from three groups: (a) In Spanish, of those of low residence time (less than five years living in the U.S.) (n = 9); (b) In Spanish, of those of high residence time (15 or more years in the U.S) (n = 9); and (c) in English, of those of high residence time (n = 9).</p> <p>Results</p> <p>There were very few language translation problems; general question design defects and socio-cultural challenges to survey responses were more common. Problems were found for both acculturation and PA questions, with distinct problem types for the two question areas. Residence time/language group was weakly associated with overall frequency of problems observed: low residence time/Spanish (86%), high residence time/Spanish (67%), and English speaking groups (62%).</p> <p>Conclusions</p> <p>Standardized survey questions related to acculturation and physical activity present somewhat different cognitive challenges. For PA related questions, problems with such questions were similar regardless of subject residence time or language preference. For acculturation related questions, residence time/language or education level influenced responses to such questions. These observations should help in the interpretation of survey results for culturally diverse populations.</p

Mitochondrial and nuclear genes suggest that stony corals are monophyletic but most families of stony corals are not (Order Scleractinia, Class Anthozoa, Phylum Cnidaria)

Modern hard corals (Class Hexacorallia; Order Scleractinia) are widely studied because of their fundamental role in reef building and their superb fossil record extending back to the Triassic. Nevertheless, interpretations of their evolutionary relationships have been in flux for over a decade. Recent analyses undermine the legitimacy of traditional suborders, families and genera, and suggest that a non-skeletal sister clade (Order Corallimorpharia) might be imbedded within the stony corals. However, these studies either sampled a relatively limited array of taxa or assembled trees from heterogeneous data sets. Here we provide a more comprehensive analysis of Scleractinia (127 species, 75 genera, 17 families) and various outgroups, based on two mitochondrial genes (cytochrome oxidase I, cytochrome b), with analyses of nuclear genes (ßtubulin, ribosomal DNA) of a subset of taxa to test unexpected relationships. Eleven of 16 families were found to be polyphyletic. Strikingly, over one third of all families as conventionally defined contain representatives from the highly divergent "robust" and "complex" clades. However, the recent suggestion that corallimorpharians are true corals that have lost their skeletons was not upheld. Relationships were supported not only by mitochondrial and nuclear genes, but also often by morphological characters which had been ignored or never noted previously. The concordance of molecular characters and more carefully examined morphological characters suggests a future of greater taxonomic stability, as well as the potential to trace the evolutionary history of this ecologically important group using fossils

Screening of a Custom-Designed Acid Fragment Library Identifies 1-Phenylpyrroles and 1-Phenylpyrrolidines as Inhibitors of Notum Carboxylesterase Activity

The Wnt family of proteins are secreted signaling proteins that play key roles in regulating cellular functions. Recently, carboxylesterase Notum was shown to act as a negative regulator of Wnt signaling by mediating the removal of an essential palmitoleate. Here we disclose two new chemical scaffolds that inhibit Notum enzymatic activity. Our approach was to create a fragment library of 250 acids for screening against Notum in a biochemical assay followed by structure determination by X-ray crystallography. Twenty fragments were identified as hits for Notum inhibition, and 14 of these fragments were shown to bind in the palmitoleate pocket of Notum. Optimization of 1-phenylpyrrole 20, guided by structure-based drug design, identified 20z as the most potent compound from this series. Similarly, the optimization of 1-phenylpyrrolidine 8 gave acid 26. This work demonstrates that inhibition of Notum activity can be achieved by small, drug-like molecules possessing favorable in vitro ADME profiles

Capsular profiling of the Cronobacter genus and the association of specific Cronobacter sakazakii and C. malonaticus capsule types with neonatal meningitis and necrotizing enterocolitis

Background: Cronobacter sakazakii and C. malonaticus can cause serious diseases especially in infants where they are associated with rare but fatal neonatal infections such as meningitis and necrotising enterocolitis. Methods: This study used 104 whole genome sequenced strains, covering all seven species in the genus, to analyse capsule associated clusters of genes involved in the biosynthesis of the O-antigen, colanic acid, bacterial cellulose, enterobacterial common antigen (ECA), and a previously uncharacterised K-antigen. Results: Phylogeny of the gnd and galF genes flanking the O-antigen region enabled the defining of 38 subgroups which are potential serotypes. Two variants of the colanic acid synthesis gene cluster (CA1 and CA2) were found which differed with the absence of galE in CA2. Cellulose (bcs genes) were present in all species, but were absent in C. sakazakii sequence type (ST) 13 and clonal complex (CC) 100 strains. The ECA locus was found in all strains. The K-antigen capsular polysaccharide Region 1 (kpsEDCS) and Region 3 (kpsMT) genes were found in all Cronobacter strains. The highly variable Region 2 genes were assigned to 2 homology groups (K1 and K2). C. sakazakii and C. malonaticus isolates with capsular type [K2:CA2:Cell+] were associated with neonatal meningitis and necrotizing enterocolitis. Other capsular types were less associated with clinical infections. Conclusion: This study proposes a new capsular typing scheme which identifies a possible important virulence trait associated with severe neonatal infections. The various capsular polysaccharide structures warrant further investigation as they could be relevant to macrophage survival, desiccation resistance, environmental survival, and biofilm formation in the hospital environment, including neonatal enteral feeding tubes

Bilateral femoral neck fractures due to transient osteoporosis of pregnancy: a case report

We describe a case of bilateral femoral neck fractures secondary to transient osteoporosis of pregnancy, which were diagnosed after delivery due to the desire to avoid ionising radiation. These fractures were presumed to be secondary to transient osteoporosis of pregnancy and were treated successfully with internal fixation despite delayed presentation. We discuss the role of MRI in the evaluation of hip pain in pregnancy

Prevalence of Disorders Recorded in Dogs Attending Primary-Care Veterinary Practices in England

Purebred dog health is thought to be compromised by an increasing occurence of inherited diseases but inadequate prevalence data on common disorders have hampered efforts to prioritise health reforms. Analysis of primary veterinary practice clinical data has been proposed for reliable estimation of disorder prevalence in dogs. Electronic patient record (EPR) data were collected on 148,741 dogs attending 93 clinics across central and south-eastern England. Analysis in detail of a random sample of EPRs relating to 3,884 dogs from 89 clinics identified the most frequently recorded disorders as otitis externa (prevalence 10.2%, 95% CI: 9.1-11.3), periodontal disease (9.3%, 95% CI: 8.3-10.3) and anal sac impaction (7.1%, 95% CI: 6.1-8.1). Using syndromic classification, the most prevalent body location affected was the head-and-neck (32.8%, 95% CI: 30.7-34.9), the most prevalent organ system affected was the integument (36.3%, 95% CI: 33.9-38.6) and the most prevalent pathophysiologic process diagnosed was inflammation (32.1%, 95% CI: 29.8-34.3). Among the twenty most-frequently recorded disorders, purebred dogs had a significantly higher prevalence compared with crossbreds for three: otitis externa (P = 0.001), obesity (P = 0.006) and skin mass lesion (P = 0.033), and popular breeds differed significantly from each other in their prevalence for five: periodontal disease (P = 0.002), overgrown nails (P = 0.004), degenerative joint disease (P = 0.005), obesity (P = 0.001) and lipoma (P = 0.003). These results fill a crucial data gap in disorder prevalence information and assist with disorder prioritisation. The results suggest that, for maximal impact, breeding reforms should target commonly-diagnosed complex disorders that are amenable to genetic improvement and should place special focus on at-risk breeds. Future studies evaluating disorder severity and duration will augment the usefulness of the disorder prevalence information reported herein

Does lumbar spinal degeneration begin with the anterior structures? A study of the observed epidemiology in a community-based population

<p>Abstract</p> <p>Background-</p> <p>Prior studies that have concluded that disk degeneration uniformly precedes facet degeneration have been based on convenience samples of individuals with low back pain. We conducted a study to examine whether the view that spinal degeneration begins with the anterior spinal structures is supported by epidemiologic observations of degeneration in a community-based population.</p> <p>Methods-</p> <p>361 participants from the Framingham Heart Study were included in this study. The prevalences of anterior vertebral structure degeneration (disk height loss) and posterior vertebral structure degeneration (facet joint osteoarthritis) were characterized by CT imaging. The cohort was divided into the structural subgroups of participants with 1) no degeneration, 2) isolated anterior degeneration (without posterior degeneration), 3) combined anterior and posterior degeneration, and 4) isolated posterior degeneration (without anterior structure degeneration). We determined the prevalence of each degeneration pattern by age group < 45, 45-54, 55-64, ≥65. In multivariate analyses we examined the association between disk height loss and the response variable of facet joint osteoarthritis, while adjusting for age, sex, BMI, and smoking.</p> <p>Results-</p> <p>As the prevalence of the no degeneration and isolated anterior degeneration patterns decreased with increasing age group, the prevalence of the combined anterior/posterior degeneration pattern increased. 22% of individuals demonstrated isolated posterior degeneration, without an increase in prevalence by age group. Isolated posterior degeneration was most common at the L5-S1 and L4-L5 spinal levels. In multivariate analyses, disk height loss was independently associated with facet joint osteoarthritis, as were increased age (years), female sex, and increased BMI (kg/m²), but not smoking.</p> <p>Conclusions-</p> <p>The observed epidemiology of lumbar spinal degeneration in the community-based population is consistent with an ordered progression beginning in the anterior structures, for the majority of individuals. However, some individuals demonstrate atypical patterns of degeneration, beginning in the posterior joints. Increased age and BMI, and female sex may be related to the occurrence of isolated posterior degeneration in these individuals.</p

Consistent phenological shifts in the making of a biodiversity hotspot: the Cape flora

Background The best documented survival responses of organisms to past climate change on short (glacial-interglacial) timescales are distributional shifts. Despite ample evidence on such timescales for local adaptations of populations at specific sites, the long-term impacts of such changes on evolutionary significant units in response to past climatic change have been little documented. Here we use phylogenies to reconstruct changes in distribution and flowering ecology of the Cape flora - South Africa's biodiversity hotspot - through a period of past (Neogene and Quaternary) changes in the seasonality of rainfall over a timescale of several million years. Results Forty-three distributional and phenological shifts consistent with past climatic change occur across the flora, and a comparable number of clades underwent adaptive changes in their flowering phenology (9 clades; half of the clades investigated) as underwent distributional shifts (12 clades; two thirds of the clades investigated). Of extant Cape angiosperm species, 14-41% have been contributed by lineages that show distributional shifts consistent with past climate change, yet a similar proportion (14-55%) arose from lineages that shifted flowering phenology. Conclusions Adaptive changes in ecology at the scale we uncover in the Cape and consistent with past climatic change have not been documented for other floras. Shifts in climate tolerance appear to have been more important in this flora than is currently appreciated, and lineages that underwent such shifts went on to contribute a high proportion of the flora's extant species diversity. That shifts in phenology, on an evolutionary timescale and on such a scale, have not yet been detected for other floras is likely a result of the method used; shifts in flowering phenology cannot be detected in the fossil record

Optimal timing for managed relocation of species faced with climate change

Managed relocation is a controversial climate-adaptation strategy to combat negative climate change impacts on biodiversity. While the scientific community debates the merits of managed relocation(1-12), species are already being moved to new areas predicted to be more suitable under climate change(13,14). To inform these moves, we construct a quantitative decision framework to evaluate the timing of relocation in the face of climate change. We find that the optimal timing depends on many factors, including the size of the population, the demographic costs of translocation and the expected carrying capacities over time in the source and destination habitats. In some settings, such as when a small population would benefit from time to grow before risking translocation losses, haste is ill advised. We also find that active adaptive management(15,16) is valuable when the effect of climate change on source habitat is uncertain, and leads to delayed movement