Selective hydroxylation of 1,8- and 1,4-cineole using bacterial P450 variants

This study has evaluated the use of the P450 metalloenzymes CYP176A1, CYP101A1 and CYP102A1, together with engineered protein variants of CYP101A1 and CYP102A1, to alter the regioselectivity of 1,8- and 1,4-cineole hydroxylation. CYP176A1 was less selective for 1,4-cineole oxidation when compared to its preferred substrate, 1,8-cineole. The CYP102A1 variants significantly improved the activity over the WT enzyme for oxidation of 1,4- and 1,8-cineole. The CYP102A1 R47L/Y51F/A74G/F87V/L188Q mutant generated predominantly (1S)-6α-hydroxy-1,8-cineole (78% e.e.) from 1,8-cineole. Oxidation of 1,4-cineole by the CYP102A1 R47L/Y51F/F87A/I401P variant generated the 3α product in >90% yield. WT CYP101A1 formed a mixture metabolites with 1,8-cineole and very little product was generated with 1,4-cineole. In contrast the F87W/Y96F/L244A/V247L and F87W/Y96F/L244A variants of CYP101A1 favoured formation of 5α-hydroxy-1,8-cineole (>88%, 1S 86% e.e.) while the F87V/Y96F/L244A variant generated (1S)-6α-hydroxy-1,8-cineole in excess (90% regioselective, >99% e.e.). The CYP101A1 F87W/Y96F/L244A/V247L and F87W/Y96F/L244A mutants improved the oxidation of 1,4-cineole generating an excess of the 3α metabolite (1S > 99% e.e. with the latter). The CYP101A1 F87L/Y96F variant also improved the oxidation of this substrate but shifted the site of oxidation to the isopropyl group, (8-hydroxy-1,4-cineole). When this 8-hydroxy metabolite was generated in significant quantities desaturation of C8C9 to the corresponding alkene was also detected

Contribution of siderophore systems to growth and urinary tract colonization of asymptomatic bacteriuria Escherichia coli

The molecular mechanisms that define asymptomatic bacteriuria (ABU) E. coli colonization of the human urinary tract remain to be properly elucidated. Here we utilize ABU E. coli strain 83972 as a model to dissect the contribution of siderophores to iron acquisition, growth, fitness and colonization of the urinary tract. We show that E. coli 83972 produces enterobactin, salmochelin, aerobactin and yersiniabactin, and examine the role of these systems using mutants defective in siderophore biosynthesis and uptake. Enterobactin and aerobactin contributed most to total siderophore activity and growth in defined iron-deficient media. No siderophores were detected in an 83972 quadruple mutant deficient in all four siderophore biosynthesis pathways; this mutant did not grow in defined iron-deficient media but grew in iron-limited pooled human urine due to iron uptake via the FecA ferric citrate receptor. In a mixed 1:1 growth assay with 83972 there was no fitness disadvantage of the 83972 quadruple biosynthetic mutant, demonstrating its capacity to act as a ‘cheater’ and utilize siderophores produced by the wild-type strain for iron uptake. An 83972 enterobactin/salmochelin double receptor mutant was outcompeted by 83972 in human urine and the mouse urinary tract, indicating a role for catecholate receptors in urinary tract colonization

Statistical Signatures of Structural Organization: The case of long memory in renewal processes

Identifying and quantifying memory are often critical steps in developing a mechanistic understanding of stochastic processes. These are particularly challenging and necessary when exploring processes that exhibit long-range correlations. The most common signatures employed rely on second-order temporal statistics and lead, for example, to identifying long memory in processes with power-law autocorrelation function and Hurst exponent greater than $1/2$. However, most stochastic processes hide their memory in higher-order temporal correlations. Information measures---specifically, divergences in the mutual information between a process' past and future (excess entropy) and minimal predictive memory stored in a process' causal states (statistical complexity)---provide a different way to identify long memory in processes with higher-order temporal correlations. However, there are no ergodic stationary processes with infinite excess entropy for which information measures have been compared to autocorrelation functions and Hurst exponents. Here, we show that fractal renewal processes---those with interevent distribution tails $\propto t^{-\alpha}$---exhibit long memory via a phase transition at $\alpha = 1$. Excess entropy diverges only there and statistical complexity diverges there and for all $\alpha < 1$. When these processes do have power-law autocorrelation function and Hurst exponent greater than $1/2$, they do not have divergent excess entropy. This analysis breaks the intuitive association between these different quantifications of memory. We hope that the methods used here, based on causal states, provide some guide as to how to construct and analyze other long memory processes.Comment: 13 pages, 2 figures, 3 appendixes; http://csc.ucdavis.edu/~cmg/compmech/pubs/lrmrp.ht

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an I

New stopping criteria for segmenting DNA sequences

We propose a solution on the stopping criterion in segmenting inhomogeneous DNA sequences with complex statistical patterns. This new stopping criterion is based on Bayesian Information Criterion (BIC) in the model selection framework. When this stopping criterion is applied to a left telomere sequence of yeast Saccharomyces cerevisiae and the complete genome sequence of bacterium Escherichia coli, borders of biologically meaningful units were identified (e.g. subtelomeric units, replication origin, and replication terminus), and a more reasonable number of domains was obtained. We also introduce a measure called segmentation strength which can be used to control the delineation of large domains. The relationship between the average domain size and the threshold of segmentation strength is determined for several genome sequences.Comment: 4 pages, 4 figures, Physical Review Letters, to appea

Big Physics At Small Places: The Mongol Horde Model Of Undergraduate Research

A model for engaging undergraduates in cutting-edge experimental nuclear physics research at a national user facility is discussed.&nbsp; Methods to involve students and examples of their success are presented

Structural Basis for a Neutralizing Antibody Response Elicited by a Recombinant Hantaan Virus Gn Immunogen

Hantaviruses are a group of emerging pathogens capable of causing severe disease upon zoonotic transmission to humans. The mature hantavirus surface presents higher-order tetrameric assemblies of two glycoproteins, Gn and Gc, which are responsible for negotiating host cell entry and constitute key therapeutic targets. Here, we demonstrate that recombinantly derived Gn from Hantaan virus (HTNV) elicits a neutralizing antibody response (serum dilution that inhibits 50% infection [ID50], 1:200 to 1:850) in an animal model. Using antigen-specific B cell sorting, we isolated monoclonal antibodies (mAbs) exhibiting neutralizing and non-neutralizing activity, termed mAb HTN-Gn1 and mAb nn-ITN-Gn2, respectively. Crystallographic analysis reveals that these mAbs target spatially distinct epitopes at disparate sites of the N-terminal region of the HTNV Gn ectodomain. Epitope mapping onto a model of the higher order (Gn-Gc)(4) spike supports the immune accessibility of the mAb HTN-Gn1 epitope, a hypothesis confirmed by electron cryo-tomography of the antibody with virus-like particles. These data define natively exposed regions of the hantaviral Gn that can be targeted in immunogen design. IMPORTANCE The spillover of pathogenic hantaviruses from rodent reservoirs into the human population poses a continued threat to human health. Here, we show that a recombinant form of the Hantaan virus (HTNV) surface-displayed glycoprotein, Gn, elicits a neutralizing antibody response in rabbits. We isolated a neutralizing (HTN-Gn1) and a non-neutralizing (nn-ITN-Gn2) monoclonal antibody and provide the first molecular-level insights into how the Gn glycoprotein may be targeted by the antibody-mediated immune response. These findings may guide rational vaccine design approaches focused on targeting the hantavirus glycoprotein envelope.Peer reviewe

Identification of Mutant Genes and Introgressed Tiger Salamander DNA in the Laboratory Axolotl, \u3cem\u3eAmbystoma mexicanum\u3c/em\u3e

The molecular genetic toolkit of the Mexican axolotl, a classic model organism, has matured to the point where it is now possible to identify genes for mutant phenotypes. We used a positional cloning–candidate gene approach to identify molecular bases for two historic axolotl pigment phenotypes: white and albino. White (d/d) mutants have defects in pigment cell morphogenesis and differentiation, whereas albino (a/a) mutants lack melanin. We identified in white mutants a transcriptional defect in endothelin 3 (edn3), encoding a peptide factor that promotes pigment cell migration and differentiation in other vertebrates. Transgenic restoration of Edn3 expression rescued the homozygous white mutant phenotype. We mapped the albino locus to tyrosinase (tyr) and identified polymorphisms shared between the albino allele (tyra) and tyr alleles in a Minnesota population of tiger salamanders from which the albino trait was introgressed. tyra has a 142 bp deletion and similar engineered alleles recapitulated the albinophenotype. Finally, we show that historical introgression of tyrasignificantly altered genomic composition of the laboratory axolotl, yielding a distinct, hybrid strain of ambystomatid salamander. Our results demonstrate the feasibility of identifying genes for traits in the laboratory Mexican axolotl

Structure Determination, Functional Characterization, and Biosynthetic Implications of Nybomycin Metabolites from a Mining Reclamation Site-Associated \u3cem\u3eStreptomyces\u3c/em\u3e

We report the isolation and characterization of three new nybomycins (nybomycins B–D, 1–3) and six known compounds (nybomycin, 4; deoxynyboquinone, 5; α-rubromycin, 6; β-rubromycin, 7; γ-rubromycin, 8; and [2α(1E,3E),4β]-2-(1,3-pentadienyl)-4-piperidinol, 9) from the Rock Creek (McCreary County, KY) underground coal mine acid reclamation site isolate Streptomyces sp. AD-3-6. Nybomycin D (3) and deoxynyboquinone (5) displayed moderate (3) to potent (5) cancer cell line cytotoxicity and displayed weak to moderate anti-Gram-(+) bacterial activity, whereas rubromycins 6–8 displayed little to no cancer cell line cytotoxicity but moderate to potent anti-Gram-(+) bacterial and antifungal activity. Assessment of the impact of 3 or 5 cancer cell line treatment on 4E-BP1 phosphorylation, a predictive marker of ROS-mediated control of cap-dependent translation, also revealed deoxynyboquinone (5)-mediated downstream inhibition of 4E-BP1p. Evaluation of 1–9 in a recently established axolotl embryo tail regeneration assay also highlighted the prototypical telomerase inhibitor γ-rubromycin (8) as a new inhibitor of tail regeneration. Cumulatively, this work highlights an alternative nybomycin production strain, a small set of new nybomycin metabolites, and previously unknown functions of rubromycins (antifungal activity and inhibition of tail regeneration) and also provides a basis for revision of the previously proposed nybomycin biosynthetic pathway