Fluoromethylketone-Fragment Conjugates Designed as Covalent Modifiers of EcDsbA are Atypical Substrates.

Disulfide bond protein A (DsbA) is an oxidoreductase enzyme that catalyzes the formation of disulfide bonds in Gram-negative bacteria. In Escherichia coli, DsbA (EcDsbA) is essential for bacterial virulence, thus inhibitors have the potential to act as antivirulence agents. A fragment-based screen was conducted against EcDsbA and herein we describe the development of a series of compounds based on a phenylthiophene hit identified from the screen. A novel thiol reactive and "clickable" ethynylfluoromethylketone was designed for reaction with azide-functionalized fragments to enable rapid and versatile attachment to a range of fragments. The resulting fluoromethylketone conjugates showed selectivity for reaction with the active site thiol of EcDsbA, however unexpectedly, turnover of the covalent adduct was observed. A mechanism for this turnover was investigated and proposed which may have wider ramifications for covalent reactions with dithiol-disulfide oxidoreducatases.

Conserving a threatened North American walnut: a chromosome-scale reference genome for butternut (Juglans cinerea).

With the advent of affordable and more accurate third-generation sequencing technologies, and the associated bioinformatic tools, it is now possible to sequence, assemble, and annotate more species of conservation concern than ever before. Juglans cinerea, commonly known as butternut or white walnut, is a member of the walnut family, native to the Eastern United States and Southeastern Canada. The species is currently listed as Endangered on the IUCN Red List due to decline from an invasive fungus known as Ophiognomonia clavigignenti-juglandacearum (Oc-j) that causes butternut canker. Oc-j creates visible sores on the trunks of the tree which essentially starves and slowly kills the tree. Natural resistance to this pathogen is rare. Conserving butternut is of utmost priority due to its critical ecosystem role and cultural significance. As part of an integrated undergraduate and graduate student training program in biodiversity and conservation genomics, the first reference genome for Juglans cinerea is described here. This chromosome-scale 539 Mb assembly was generated from over 100 × coverage of Oxford Nanopore long reads and scaffolded with the Juglans mandshurica genome. Scaffolding with a closely related species oriented and ordered the sequences in a manner more representative of the structure of the genome without altering the sequence. Comparisons with sequenced Juglandaceae revealed high levels of synteny and further supported J. cinerea's recent phylogenetic placement. Comparative assessment of gene family evolution revealed a significant number of contracting families, including several associated with biotic stress response.

First report of severe tolpyralate sensitivity in corn (Zea mays) discovers a novel genetic factor conferring crop response to a herbicide.

BACKGROUND: Tolpyralate, a relatively new inhibitor of 4-hydroxyphenylpyruvate dioxygenase (HPPD), is registered for postemergence use in all types of corn (Zea mays L.) and has a record of excellent crop tolerance. A report of severe crop injury to sweet corn inbred (XSEN187) led to the following objectives: (i) determine whether sensitivity to tolpyralate in XSEN187 exists, and if confirmed, (ii) determine the genetic basis of tolpyralate sensitivity, and (iii) screen other corn germplasm for sensitivity to tolpyralate. RESULTS: Inbred XSEN187 was confirmed sensitive to tolpyralate. Inclusion of methylated seed oil or nonionic surfactant in the spray volume was necessary for severe crop injury. Tolpyralate sensitivity in XSEN187 is not conferred by alleles at Nsf1, a cytochrome P450-encoding gene (CYP81A9) conferring tolerance to many corn herbicides. Evidence suggests that tolpyralate sensitivity in XSEN187 is conferred by a single gene mapped to the Chr05: 283 240-1 222 909 bp interval. Moreover, tolpyralate sensitivity was observed in 48 other sweet corn and field corn inbreds. CONCLUSIONS: Severe sensitivity to tolpyralate exists in sweet corn and field corn germplasm when the herbicide is applied according to label directions. Whereas the corn response to several other herbicides, including HPPD-inhibitors, is conferred by the Nsf1 locus, corn sensitivity to tolpyralate is the result of a different locus. The use of tolpyralate should consider herbicide tolerance in inbred lines from which corn hybrids were derived, whereas alleles that render corn germplasm sensitive to tolpyralate should be eliminated from breeding populations, inbreds, and commercial cultivars. © 2023 Illinois Foundation Seeds, Inc and The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

The silver bullet that wasn't: Rapid agronomic weed adaptations to glyphosate in North America.

The rapid adoption of glyphosate-resistant crops at the end of the 20th century caused a simplification of weed management that relied heavily on glyphosate for weed control. However, the effectiveness of glyphosate has diminished. A greater understanding of trends related to glyphosate use will shed new light on weed adaptation to a product that transformed global agriculture. Objectives were to (1) quantify the change in weed control efficacy from postemergence (POST) glyphosate use on troublesome weeds in corn and soybean and (2) determine the extent to which glyphosate preceded by a preemergence (PRE) improved the efficacy and consistency of weed control compared to glyphosate alone. Herbicide evaluation trials from 24 institutions across the United States of America and Canada from 1996 to 2021 were compiled into a single database. Two subsets were created; one with glyphosate applied POST, and the other with a PRE herbicide followed by glyphosate applied POST. Within each subset, mean and variance of control ratings for seven problem weed species were regressed over time for nine US states and one Canadian province. Mean control with POST glyphosate alone decreased over time while variability in control increased. Glyphosate preceded by a labeled PRE herbicide showed little change in mean control or variability in control over time. These results illustrate the rapid adaptation of agronomically important weed species to the paradigm-shifting product glyphosate. Including more diversity in weed management systems is essential to slowing weed adaptation and prolonging the usefulness of existing and future technologies.

Long-acting refillable nanofluidic implant confers protection against SHIV infection in nonhuman primates.

The impact of pre-exposure prophylaxis (PrEP) on slowing the global HIV epidemic hinges on effective drugs and delivery platforms. Oral drug regimens are the pillar of HIV PrEP, but variable adherence has spurred development of long-acting delivery systems with the aim of increasing PrEP access, uptake, and persistence. We have developed a long-acting subcutaneous nanofluidic implant that can be refilled transcutaneously for sustained release of the HIV drug islatravir, a nucleoside reverse transcriptase translocation inhibitor that is used for HIV PrEP. In rhesus macaques, the islatravir-eluting implants achieved constant concentrations of islatravir in plasma (median 3.14 nM) and islatravir triphosphate in peripheral blood mononuclear cells (median 0.16 picomole per 106 cells) for more than 20 months. These drug concentrations were above the established PrEP protection threshold. In two unblinded, placebo-controlled studies, islatravir-eluting implants conferred 100% protection against infection with SHIVSF162P3 after repeated low-dose rectal or vaginal challenge in male or female rhesus macaques, respectively, compared to placebo control groups. The islatravir-eluting implants were well tolerated with mild local tissue inflammation and no signs of systemic toxicity over the 20-month study period. This refillable islatravir-eluting implant has potential as a long-acting drug delivery system for HIV PrEP.

Biotinylation of reducing and non-reducing termini to create plug-and-play polysaccharides.

Single-molecule studies continue to grow in popularity. In cases where biopolymer samples of interest exhibit variations in fine-structure between individual chains such single-molecule studies uniquely offer the promise of revealing deep structure-function relationships. Polysaccharides are typically studied in bulk and, as such, their study could greatly benefit from the application of single-molecule techniques. However, while for example single-molecule optical tweezers (OT) studies have become commonplace for DNA, studies of polysaccharides have lagged behind somewhat, complicated by the difficulty of studying molecules that amongst other things have more complex end-group chemistry. Recently, divalent streptavidin linkers have been shown to be capable of concatenating two pieces of biotin-terminated DNA to produce robust composite strings that run intact through conventional gels, and can be used in single-molecule OT experiments (Mohandas, Kent, Raudsepp, Jameson, & Williams, 2022). By using two such streptavidin linkers, biotin-terminated polymers could be inserted between two sections of DNA in order to facilitate single-molecule experiments on biopolymers that are currently difficult to address by other means. Here, we describe a generic approach for placing the required biotin moieties at both ends of polysaccharide chains, producing plug-and-play polysaccharide inserts that can be incorporated into composite polymer strings using streptavidin linking hubs.

Cryogenic storage increases the longevity of butternut (Juglans cinerea, L.) seed embryogenic axes.

Butternut (Juglans cinerea L.), a species listed as endangered, is currently undergoing rapid decline due to habitat loss and the introduction of Ophiognomonia clavigignenti-juglandacearum, a non-native pathogen causing butternut canker. The decline of butternut has led to the development of cryobiological methods for the ex-situ conservation of the species since viability is not maintained using conventional methods. In this study, we assess the survivability and growth of butternut embryogenic axes into plantlets after 7 years of cryopreservation. Results show that cryopreservation is a highly effective method for the long-term storage of embryogenic axes since both survival and subsequent acclimatization into plantlets was successful with an average of 76% survival and 71% acclimatization, respectively. These results surpass the actual duration viability for non-cryogenic storage of butternut seed and we hope that future testing will result in longer term success of this cryobiological method and provide much needed germplasm for future restoration.

A comparison of 2 cementing techniques in hip resurfacing using human femoral heads.

INTRODUCTION: Hip resurfacing remains a valid option in young male patients. The creation of the optimum cement mantle aids fixation of the femoral component. If the cement mantle is too thick the prosthesis can remain proud leading to early failure or if it penetrates too far into the femoral head, it may cause osteonecrosis. METHOD: 18 of 96 femoral heads collected from patients undergoing total hip arthroplasty were matched for their surface porosity. They were randomly allocated into 2 different cementing groups. Group 1 had the traditional bolus of cement technique, while group 2 had a modified cementing technique (swirl) where the inside of the femoral component was lined with an even layer of low viscosity cement. RESULTS: The traditional bolus technique had significantly greater cement mantle thickness in 3 of 4 zones of penetration (p = 0.002), greater and larger air bubble formation (6 of 9 in bolus technique vs. 1 in 9 in swirl technique, p = 0.05) and more incomplete cement mantles compared with the swirl technique. There was no relationship to femoral head porosity. CONCLUSION: The swirl technique should be used to cement the femoral component in hip resurfacing. Long-term clinical studies would conform if this translates into increased survivorship of the femoral component.

Modeling multiple duplex DNA attachments in a force-extension experiment.

Optical tweezers-based DNA stretching often relies on tethering a single end-activated DNA molecule between optically manipulated end-binding beads. Measurement success can depend on DNA concentration. At lower DNA concentrations tethering is less common, and many trials may be required to observe a single-molecule stretch. At higher DNA concentrations tethering is more common; however, the resulting force-extensions observed are more complex and may vary from measurement to measurement. Typically these more complex results are attributed to the formation of multiple tethers between the beads; however, to date there does not appear to have been a critical examination of this hypothesis or the potential usefulness of such data. Here we examine stretches at a higher DNA concentration and use analysis and simulation to show how the more complex force-extensions observed can be understood in terms of multiple DNA attachments.

The greatest air quality experiment ever: Policy suggestions from the COVID-19 lockdown in twelve European cities.

COVID-19 (Coronavirus disease 2019) hit Europe in January 2020. By March, Europe was the active centre of the pandemic. As a result, widespread "lockdown" measures were enforced across the various European countries, even if to a different extent. Such actions caused a dramatic reduction, especially in road traffic. This event can be considered the most significant experiment ever conducted in Europe to assess the impact of a massive switch-off of atmospheric pollutant sources. In this study, we focus on in situ concentration data of the main atmospheric pollutants measured in twelve European cities, characterized by different climatology, emission sources, and strengths. We propose a methodology for the fair comparison of the impact of lockdown measures considering the non-stationarity of meteorological conditions and emissions, which are progressively declining due to the adoption of stricter air quality measures. The analysis of these unmatched circumstances allowed us to estimate the impact of a nearly zero-emission urban transport scenario on air quality in 12 European cities. The clearest result, common to all the cities, is that a dramatic traffic reduction effectively reduces NO2 concentrations. In contrast, each city's PM and ozone concentrations can respond differently to the same type of emission reduction measure. From the policy point of view, these findings suggest that measures targeting urban traffic alone may not be the only effective option for improving air quality in cities.

Evidence of sweet corn yield losses from rising temperatures.

Crop production is sensitive to anomalous weather conditions, but vegetable crops can be highly sensitive to environmental changes. Using sweet corn data collected on 16,040 fields over a 27-year period, we: (a) estimate yield sensitivities to changes in growing season temperature and total precipitation, (b) estimate critical thresholds in non-linear temperature effects on sweet corn yield across diverse environments, and (c) quantify yield losses from surpassing the upper temperature threshold during anthesis in sweet corn. Our results show growing-season temperatures exceeding 30 [Formula: see text] were detrimental to crop yield. Each additional degree day spent above 30 [Formula: see text] during anthesis reduced crop yields by 0.5% and 2% in irrigated and rainfed fields, respectively. This study shows evidence for sweet corn yield losses across broad spatial domains in the wake of climate change and underscores the urgency to accelerate crop adaptation strategies to sustain production of this highly popular crop.

Methyl probes in proteins for determining ligand binding mode in weak protein-ligand complexes.

Structures of protein-ligand complexes provide critical information for drug design. Most protein-ligand complex structures are determined using X-ray crystallography, but where crystallography is not able to generate a structure for a complex, NMR is often the best alternative. However, the available tools to enable rapid and robust structure determination of protein-ligand complexes by NMR are currently limited. This leads to situations where projects are either discontinued or pursued without structural data, rendering the task more difficult. We previously reported the NMR Molecular Replacement (NMR2) approach that allows the structure of a protein-ligand complex to be determined without requiring the cumbersome task of protein resonance assignment. Herein, we describe the NMR2 approach to determine the binding pose of a small molecule in a weak protein-ligand complex by collecting sparse protein methyl-to-ligand NOEs from a selectively labeled protein sample and an unlabeled ligand. In the selective labeling scheme all methyl containing residues of the protein are protonated in an otherwise deuterated background. This allows measurement of intermolecular NOEs with greater sensitivity using standard NOESY pulse sequences instead of isotope-filtered NMR experiments. This labelling approach is well suited to the NMR2 approach and extends its utility to include larger protein-ligand complexes.

Interfacial colloidal assembly guided by optical tweezers and tuned via surface charge.

HYPOTHESIS: The size, shape and dynamics of assemblies of colloidal particles optically-trapped at an air-water interface can be tuned by controlling the optical potential, particle concentration, surface charge density and wettability of the particles and the surface tension of the solution. EXPERIMENTS: The assembly dynamics of different colloidal particle types (silica, polystyrene and carboxyl coated polystyrene particles) at an air-water interface in an optical potential were systematically explored allowing the effect of surface charge on assembly dynamics to be investigated. Additionally, the pH of the solutions were varied in order to modulate surface charge in a controllable fashion. The effect of surface tension on these assemblies was also explored by reducing the surface tension of the supporting solution by mixing ethanol with water. FINDINGS: Silica, polystyrene and carboxyl coated polystyrene particles showed distinct assembly behaviours at the air-water interface that could be rationalised taking into account changes in surface charge (which in addition to being different between the particles could be modified systematically by changing the solution pH). Additionally, this is the first report showing that wettability of the colloidal particles and the surface tension of the solution are critical in determining the resulting assembly at the solution surface.

Deteriorating weed control and variable weather portends greater soybean yield losses in the future.

Since the 1950's much of the US soybean growing region has experienced rising temperatures, more variable rainfall, and increased carbon emissions. These trends are predicted to continue throughout the 21st century. Variable weather and weed interference influence crop performance; however, their combined effects on soybean yield are poorly understood. Using machine learning techniques on a database of herbicide trials spanning 28 years and 106 weather environments we modeled the most important relationships among weed control, weather variability, and crop management on soybean yield loss. When late-season weeds were poorly controlled, average soybean yield losses of 48% were observed. Additionally, when weeds were not completely controlled, low rainfall and high temperatures during seed fill exacerbated soybean yield loss due to weeds. Since much of the US soybean growing region is heading towards drier, warmer conditions, coupled with growing herbicide resistance, future soybean yield loss will increase without significant improvements in weed management systems.

Estimating orientation of optically trapped, near vertical, microsphere dimers using central moments and off-focus imaging.

Near vertical optically trapped dimers, composed of pairs of microspheres, and constructed in situ, were imaged in bright-field in flow and at rest, and with displacement Δz from the transverse xy imaging plane of an inverted microscope. Image first central moments µ01 were measured, and their dependence on the imposed flow velocity of the surrounding fluid was calculated. This dependence was related to the at-rest restricted diffusion statistics. It was assumed that, for small perturbations, the torque T on the dimer was proportional to the velocity of flow v and resulting angular deflection Δθ so that T∝v∝Δθ. Displacements Δz at which vâˆΔµ01∝Δθ, which are typically off focus, were examined in more detail; in this range, Δθ=hΔµ01. The hydrodynamics of the dimer were modeled as that of a prolate ellipsoid, and the constant of proportionality h was determined by comparing the short-time mean-squared variation measured during diffusion to that predicted by the model calculation: h2⟨Δµ012(t)⟩=⟨Δθ2(t)⟩. With h determined, the optical trap stiffness kθ was determined from the long-time restricted diffusion of the dimer. The measured kθ and Δθ can then be used compute torque: T=kθΔθ, potentially enabling the near vertical optically trapped dimer to be used as a torque probe.

Progress toward Plug-and-Play Polymer Strings for Optical Tweezers Experiments: Concatenation of DNA Using Streptavidin Linkers.

Streptavidin is a tetrameric protein that is renowned for its strong binding to biotin. The robustness and strength of this noncovalent coupling has led to multitudinous applications of the pairing. Within the streptavidin tetramer, each protein monomer has the potential to specifically bind one biotin-bearing moiety. Herein, by separating various streptavidin species that have had differing numbers of their four potential binding sites blocked, several different types of "linking hub" were obtained, each with a different valency. The identification of these species and the study of the plugging process used to block sites during their preparation were carried out using capillary electrophoresis. Subsequently, a specific species, namely, a trans-divalent linker, in which the two open biotin-binding pockets are approximately opposite one another, was used to concatenate two ∼5 kb pieces of biotin-terminated double-stranded DNA. Following the incubation of this DNA with the prepared linker, a fraction of ∼10 kb strings was identified using gel electrophoresis. Finally, these concatenated DNA strings were stretched in an optical tweezer experiment, demonstrating the potential of the methodology for coupling and extending molecules for use in single-molecule biophysical experiments.

Selective Binding of Small Molecules to Vibrio cholerae DsbA Offers a Starting Point for the Design of Novel Antibacterials.

DsbA enzymes catalyze oxidative folding of proteins that are secreted into the periplasm of Gram-negative bacteria, and they are indispensable for the virulence of human pathogens such as Vibrio cholerae and Escherichia coli. Therefore, targeting DsbA represents an attractive approach to control bacterial virulence. X-ray crystal structures reveal that DsbA enzymes share a similar fold, however, the hydrophobic groove adjacent to the active site, which is implicated in substrate binding, is shorter and flatter in the structure of V. cholerae DsbA (VcDsbA) compared to E. coli DsbA (EcDsbA). The flat and largely featureless nature of this hydrophobic groove is challenging for the development of small molecule inhibitors. Using fragment-based screening approaches, we have identified a novel small molecule, based on the benzimidazole scaffold, that binds to the hydrophobic groove of oxidized VcDsbA with a KD of 446±10 µM. The same benzimidazole compound has ∼8-fold selectivity for VcDsbA over EcDsbA and binds to oxidized EcDsbA, with KD >3.5 mM. We generated a model of the benzimidazole complex with VcDsbA using NMR data but were unable to determine the structure of the benzimidazole bound EcDsbA using either NMR or X-ray crystallography. Therefore, a structural basis for the observed selectivity is unclear. To better understand ligand binding to these two enzymes we crystallized each of them in complex with a known ligand, the bile salt sodium taurocholate. The crystal structures show that taurocholate adopts different binding poses in complex with VcDsbA and EcDsbA, and reveal the protein-ligand interactions that stabilize the different modes of binding. This work highlights the capacity of fragment-based drug discovery to identify inhibitors of challenging protein targets. In addition, it provides a starting point for development of more potent and specific VcDsbA inhibitors that act through a novel anti-virulence mechanism.

Changes in the properties of membrane tethers in response to HP1α depletion in MCF7 cells.

Plasma membrane tension is known to regulate many cell functions, such as motility and membrane trafficking. Membrane tether pulling is an effective method for measuring the apparent membrane tension of cells and exploring membrane-cytoskeleton interactions. In this article, the mechanical properties of HP1α-depleted MCF7 breast cancer cells are explored in comparison to controls, by pulling membrane tethers using optical tweezers. These studies were inspired by previous findings that a loss of HP1α correlates with an increase in the invasive potential of malignant cancer cells. Specifically, the membrane tension and force relaxation curves for tethers pulled from MCF7 breast cancer cells with HP1α knockdown and their matched controls were measured, and shown to be significantly different.