Dynamic control over protein function is a central challenge in synthetic biology. To address this challenge, we describe the development of an integrated computational and experimental workflow to ...incorporate a metal-responsive chemical switch into proteins. Pairs of bipyridinylalanine (BpyAla) residues are genetically encoded into two structurally distinct enzymes, a serine protease and firefly luciferase, so that metal coordination biases the conformations of these enzymes, leading to reversible control of activity. Computational analysis and molecular dynamics simulations are used to rationally guide BpyAla placement, significantly reducing experimental workload, and cell-free protein synthesis coupled with high-throughput experimentation enable rapid prototyping of variants. Ultimately, this strategy yields enzymes with a robust 20-fold dynamic range in response to divalent metal salts over 24 on/off switches, demonstrating the potential of this approach. We envision that this strategy of genetically encoding chemical switches into enzymes will complement other protein engineering and synthetic biology efforts, enabling new opportunities for applications where precise regulation of protein function is critical.
Antibody discovery is bottlenecked by the individual expression and evaluation of antigen-specific hits. Here, we address this bottleneck by developing a workflow combining cell-free DNA template ...generation, cell-free protein synthesis, and binding measurements of antibody fragments in a process that takes hours rather than weeks. We apply this workflow to evaluate 135 previously published antibodies targeting the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including all 8 antibodies previously granted emergency use authorization for coronavirus disease 2019 (COVID-19), and demonstrate identification of the most potent antibodies. We also evaluate 119 anti-SARS-CoV-2 antibodies from a mouse immunized with the SARS-CoV-2 spike protein and identify neutralizing antibody candidates, including the antibody SC2-3, which binds the SARS-CoV-2 spike protein of all tested variants of concern. We expect that our cell-free workflow will accelerate the discovery and characterization of antibodies for future pandemics and for research, diagnostic, and therapeutic applications more broadly.
Plastic waste accumulating in the global ocean is an increasingly threatening environmental issue. To date, the floating and thus most visible fraction of ocean plastic pollution has been mapped at ...global scale. Yet, large knowledge gaps exist in our current understanding of the transport and transformation processes of positively buoyant plastic debris at the sea surface. Observations at sea typically report an apparent scarcity of microplastics (<5 mm) relative to the expected abundance-size distribution based on fragmentation of larger plastic objects. Here, we provide a comprehensive study on the relative abundance of microplastics (>500 µm) and mesoplastics (0.5-5 cm) in the surface waters of the eastern North Pacific Ocean using data from 1136 040 plastic fragments collected by 679 neuston trawl deployments between 2015 and 2019. Our results reveal that the apparent microplastic scarcity is not uniformly distributed across the region. Instead, we show that the relative abundance of floating microplastics increases from the outside to the inside of the North Pacific Garbage Patch. We hypothesize that this observation could be explained by (i) a spatially variable microplastic removal due to spatial differences in ocean productivity, (ii) a differential dispersal of micro- vs. mesoplastics with a preferential accumulation of microplastics in the subtropical gyre, and/or (iii) the timescales associated with transport and fragmentation of plastic objects at the ocean surface with older, more degraded, floating plastic accumulation in subtropical gyres. The results presented here highlight that global estimates of the accumulation and removal of positively buoyant microplastics need to consider spatial aspects such as variations in ocean productivity, the dominant physical transport processes in a given area, as well as the time needed for a plastic object to reach the specific offshore location.
•We report on the Consortium for Anthelmintic Resistance and Susceptibility 2013 meeting.•Recent advances in the identification of markers for anthelmintic resistance are described.•The use of ...markers for benzimidazole resistance in field studies with veterinary and human nematodes.•The application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.
Anthelmintic resistance has a great impact on livestock production systems worldwide, is an emerging concern in companion animal medicine, and represents a threat to our ongoing ability to control human soil-transmitted helminths. The Consortium for Anthelmintic Resistance and Susceptibility (CARS) provides a forum for scientists to meet and discuss the latest developments in the search for molecular markers of anthelmintic resistance. Such markers are important for detecting drug resistant worm populations, and indicating the likely impact of the resistance on drug efficacy. The molecular basis of resistance is also important for understanding how anthelmintics work, and how drug resistant populations arise. Changes to target receptors, drug efflux and other biological processes can be involved. This paper reports on the CARS group meeting held in August 2013 in Perth, Australia. The latest knowledge on the development of molecular markers for resistance to each of the principal classes of anthelmintics is reviewed. The molecular basis of resistance is best understood for the benzimidazole group of compounds, and we examine recent work to translate this knowledge into useful diagnostics for field use. We examine recent candidate-gene and whole-genome approaches to understanding anthelmintic resistance and identify markers. We also look at drug transporters in terms of providing both useful markers for resistance, as well as opportunities to overcome resistance through the targeting of the transporters themselves with inhibitors. Finally, we describe the tools available for the application of the newest high-throughput sequencing technologies to the study of anthelmintic resistance.
New platforms for the rapid and sensitive detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern are urgently needed. Here we report the development of a ...nanomechanical sensor based on the deflection of a microcantilever capable of detecting the SARS-CoV-2 spike (S) glycoprotein antigen using computationally designed multivalent minibinders immobilized on a microcantilever surface. The sensor exhibits rapid (<5 min) detection of the target antigens down to concentrations of 0.05 ng/mL (362 fM) and is more than an order of magnitude more sensitive than an antibody-based cantilever sensor. Validation of the sensor with clinical samples from 33 patients, including 9 patients infected with the Omicron (BA.1) variant observed detection of antigen from nasopharyngeal swabs with cycle threshold (Ct) values as high as 39, suggesting a limit of detection similar to that of the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate the use of minibinders and nanomechanical sensors for the rapid and sensitive detection of SARS-CoV-2 and potentially other disease markers.
New variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue to arise and prolong the coronavirus disease 2019 (COVID-19) pandemic. Here, we used a cell-free expression ...workflow to rapidly screen and optimize constructs containing multiple computationally designed miniprotein inhibitors of SARS-CoV-2. We found the broadest efficacy was achieved with a homotrimeric version of the 75-residue angiotensin-converting enzyme 2 (ACE2) mimic AHB2 (TRI2-2) designed to geometrically match the trimeric spike architecture. Consistent with the design model, in the cryo-electron microscopy structure TRI2-2 forms a tripod at the apex of the spike protein that engaged all three receptor binding domains simultaneously. TRI2-2 neutralized Omicron (B.1.1.529), Delta (B.1.617.2), and all other variants tested with greater potency than the monoclonal antibodies used clinically for the treatment of COVID-19. TRI2-2 also conferred prophylactic and therapeutic protection against SARS-CoV-2 challenge when administered intranasally in mice. Designed miniprotein receptor mimics geometrically arrayed to match pathogen receptor binding sites could be a widely applicable antiviral therapeutic strategy with advantages over antibodies in greater resistance to viral escape and antigenic drift, and advantages over native receptor traps in lower chances of autoimmune responses.
Breast implant anaplastic large cell lymphoma (ALCL) is a T-cell neoplasm arising around textured breast implants that was recognized recently as a distinct entity by the World Health Organization. ...Rarely, other types of lymphoma have been reported in patients with breast implants, raising the possibility of a pathogenetic relationship between breast implants and other types of lymphoma. We report eight cases of Epstein-Barr virus (EBV)-positive large B-cell lymphoma associated with breast implants. One of these cases was invasive, and the other seven neoplasms were noninvasive and showed morphologic overlap with breast implant ALCL. All eight cases expressed B-cell markers, had a non-germinal center B-cell immunophenotype, and were EBV+ with a latency type III pattern of infection. We compared the noninvasive EBV+ large B-cell lymphoma cases with a cohort of breast implant ALCL cases matched for clinical and pathologic stage. The EBV+ large B-cell lymphoma cases more frequently showed a thicker capsule, and more often were associated with calcification and prominent lymphoid aggregates outside of the capsule. The EBV+ B-cell lymphoma cells were more often arranged within necrotic fibrinoid material in a layered pattern. We believe that this case series highlights many morphologic similarities between EBV+ large B-cell lymphoma and breast implant ALCL. The data presented suggest a pathogenetic role for breast implants (as well as EBV) in the pathogenesis of EBV+ large B-cell lymphoma. We also provide some histologic findings useful for distinguishing EBV+ large B-cell lymphoma from breast implant ALCL in this clinical setting.
We present the fiducial main-sequence stellar locus traced by 10 photometric colours observed by Sloan Digital Sky Survey (SDSS), Two Micron All Sky Survey (2MASS), and Wide-field Infrared Survey ...Explorer (WISE). Median colours are determined using 1052 793 stars with r-band extinction less than 0.125. We use this locus to measure the dust extinction curve relative to the r band, which is consistent with previous measurements in the SDSS and 2MASS bands. The WISE band extinction coefficients are larger than predicted by standard extinction models. Using 13 lines of sight, we find variations in the extinction curve in H, K
s
, and WISE bandpasses. Relative extinction decreases towards Galactic anticentre, in agreement with prior studies. Relative extinction increases with Galactic latitude, in contrast to previous observations. This indicates a universal mid-IR extinction law does not exist due to variations in dust grain size and chemistry with Galactocentric position. A preliminary search for outliers due to warm circumstellar dust is also presented, using stars with high signal-to-noise ratio in the W3 band. We find 199 such outliers, identified by excess emission in K
s
− W3. Inspection of SDSS images for these outliers reveals a large number of contaminants due to nearby galaxies. Six sources appear to be genuine dust candidates, yielding a fraction of systems with infrared excess of 0.12 ± 0.05 per cent.
•Larval development assay able to define sensitivity to monepantel in Haemonchus contortus.•Resistant isolate readily distinguishable from susceptible isolate.•Resistant isolate showed presence of ...two subpopulations.•Sub-population 2 with IC50 elevated over 1000-fold.•Suggests presence of at least two resistance mechanisms.
Resistance to the amino-acetonitrile derivative monepantel has been reported in several species of gastrointestinal nematodes over recent years. We were interested in the use of in vitro assays with free-living worm life-stages to detect resistance to this drug. We therefore used larval development and larval migration assays to examine dose response relationships for the drug against two susceptible and one resistant isolate of Haemonchus contortus. The resistant isolate was established by laboratory propagation of the survivors of a field treatment with Zolvix® that had originally resulted in a drug efficacy of over 99%. Drug efficacy against this field—derived laboratory-propagated resistant isolate in vivo was approximately 15%. The larval development assay proved able to discriminate between the susceptible and resistant isolates, with larvae of the resistant isolate showing an ability to develop at higher drug concentrations than the two susceptible isolates. The resistant isolate showed the presence of two distinct subpopulations, separated by a plateau in the dose-response curve. Sub-population 1 (approximately 40% of the total population) showed a low level of resistance with an IC50 increased approximately 7-fold compared to the baseline susceptible isolate, while sub-population 2 (the remaining 60% of the total population) showed an IC50 increased over 1000-fold compared to the baseline susceptible isolate. This level of resistance is unusually high for any gastrointestinal nematode species in drug dose-response in vitro assays. In contrast, the migration assay could not discriminate between the three isolates, with migration not reduced to zero at any of the drug concentrations tested. This study demonstrates that a larval development assay is able to detect resistance to monepantel in H. contortus, and that resistance can exist in two distinct forms. This suggests that at least two separate monepantel resistance mechanisms are acting within the worm isolate studied here, with one or more mechanisms conferring a much higher level of resistance than the other(s).
PDF
