Accelerated drug-resistant variant discovery with an enhanced, scalable mutagenic base editor platform.

Personalized cancer therapeutics bring directed treatment options to patients based on their tumor's genetic signature. Unfortunately, tumor genomes are remarkably adaptable, and acquired resistance through gene mutation frequently occurs. Identifying mutations that promote resistance within drug-treated patient populations can be cost, resource, and time intensive. Accordingly, base editing, enabled by Cas9-deaminase domain fusions, has emerged as a promising approach for rapid, large-scale gene variant screening in situ. Here, we adapt and optimize a conditional activation-induced cytidine deaminase (AID)-dead Cas9 (dCas9) system, which demonstrates greater heterogeneity of edits with an expanded footprint compared to the most commonly utilized cytosine base editor, BE4. In combination with a custom single guide RNA (sgRNA) library, we identify individual and compound variants in epidermal growth factor receptor (EGFR) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) that confer resistance to established EGFR inhibitors. This system and analytical pipeline provide a simple, highly scalable platform for cis or trans drug-modifying variant discovery and for uncovering valuable insights into protein structure-function relationships.

The spatialization of decent work and the role of employability empowerment for minority ethnic young people in emerging economies.

Global rises in precarious labour conditions have prompted further empirical work in Decent Work, a special category of employment characterised by equitable pay, treatment, and healthy working conditions. Despite this, research has tended to be conducted in developed countries with privileged groups such as those with typical working arrangements and rely on psychologically framed individual characteristics to explain marginalising factors. We propose a more sociologically framed, spatialised perspective on Decent Work which posits that marginalising factors are spatially variable and determined but moderated by employability empowerment. We measure our propositions across three spatially different sites of Vietnam through (1) a survey of minority ethnic students and graduates (N = 1071) and (2) a survey of stakeholders involved in the recruitment and employment of this group (N = 204). We find support for most of our propositions and call for more spatialised empirical work in the field of Decent Work.

Allosteric Inhibitors of the SARS-COV-2 Papain-like Protease Domain Induce Proteasomal Degradation of Its Parent Protein NSP3.

The papain-like protease of SARS-COV-2 is essential for viral replication and pathogenesis. Its location within a much larger multifunctional protein, NSP3, makes it an ideal candidate for a targeted degradation approach capable of eliminating multiple functions with a single-molecule treatment. In this work, we have developed a HiBiT-based cellular model to study NSP3 degradation and used this platform for the discovery of monovalent NSP3 degraders. We present previously unreported degradation activity of published papain-like protease inhibitors. Follow-up exploration of structure-activity relationships and mechanism-of-action studies points to the recruitment of the ubiquitin-proteasome machinery that is solely driven by site occupancy, regardless of molecular features of the ligand. Supported by HDX data, we hypothesize that binding-induced structural changes in NSP3 trigger the recruitment of an E3 ligase and lead to proteasomal degradation.

Waveguide-integrated chip-scale optomechanical magnetometer.

Optomechanical magnetometers enable highly sensitive magnetic field sensing. However, all such magnetometers to date have been optically excited and read-out either via free space or a tapered optical fiber. This limits their scalability and integrability, and ultimately their range of applications. Here, we present an optomechanical magnetometer that is excited and read-out via a suspended optical waveguide fabricated on the same silicon chip as the magnetometer. Moreover, we demonstrate that thermomechanical noise limited sensitivity is possible using portable electronics and laser. The magnetometer employs a silica microdisk resonator selectively sputtered with a magnetostrictive film of galfenol (FeGa) which induces a resonant frequency shift in response to an external magnetic field. Experimental results reveal the retention of high quality-factor optical whispering gallery mode resonances whilst also demonstrating high sensitivity and dynamic range in ambient conditions. The use of off-the-shelf portable electronics without compromising sensor performance demonstrates promise for applications.

Computational remodeling of an enzyme conformational landscape for altered substrate selectivity.

Structural plasticity of enzymes dictates their function. Yet, our ability to rationally remodel enzyme conformational landscapes to tailor catalytic properties remains limited. Here, we report a computational procedure for tuning conformational landscapes that is based on multistate design of hinge-mediated domain motions. Using this method, we redesign the conformational landscape of a natural aminotransferase to preferentially stabilize a less populated but reactive conformation and thereby increase catalytic efficiency with a non-native substrate, resulting in altered substrate selectivity. Steady-state kinetics of designed variants reveals activity increases with the non-native substrate of approximately 100-fold and selectivity switches of up to 1900-fold. Structural analyses by room-temperature X-ray crystallography and multitemperature nuclear magnetic resonance spectroscopy confirm that conformational equilibria favor the target conformation. Our computational approach opens the door to targeted alterations of conformational states and equilibria, which should facilitate the design of biocatalysts with customized activity and selectivity.

The distribution of metal and petroleum-derived contaminants within sediments around oil and gas infrastructure in the Gippsland Basin, Australia.

As oil and gas infrastructure comes to the end of its working life, a decommissioning decision must be made: should the infrastructure be abandoned in situ, repurposed, partially removed, or fully removed? Environmental contaminants around oil and gas infrastructure could influence these decisions because contaminants in sediments could degrade the value of the infrastructure as habitat, enter the seafood supply if the area is re-opened for commercial and/or recreational fishing, or be made biologically available as sediment is resuspended when the structures are moved. An initial risk hypothesis, however, may postulate that these concerns are only relevant if contaminant concentrations are above screening values that predict the possibility of environmental harm or contaminant bioaccumulation. To determine whether a substantive contaminants-based risk assessment is needed for infrastructure in the Gippsland Basin (South-eastern Australia), we measured the concentration of metals and polycyclic aromatic hydrocarbons (PAHs) in benthic sediments collected around eight platforms earmarked for decommissioning. The measurements were compared to preset screening values and to background contaminant concentrations in reference sites. Lead (Pb), zinc (Zn), PAHs and other contaminants were occasionally measured at concentrations that exceeded reference values, most often within 150 m of the platforms. The exceedance of a few screening values by contaminants at some platforms indicates that these platforms require further analysis to determine the contaminant risks associated with any decommissioning option.

Ternary complex dissociation kinetics contribute to mutant-selective EGFR degradation.

Targeted degradation of proteins by chimeric heterobifunctional degraders has emerged as a major drug discovery paradigm. Despite the increased interest in this approach, the criteria dictating target protein degradation by a degrader remain poorly understood, and potent target engagement by a degrader does not strongly correlate with target degradation. In this study, we present the biochemical characterization of an epidermal growth factor receptor (EGFR) degrader that potently binds both wild-type and mutant EGFR, but only degrades EGFR mutant variants. Mechanistic studies reveal that ternary complex half-life strongly correlates with processive ubiquitination with purified components and mutant-selective degradation in cells. We present cryoelectron microscopy and hydrogen-deuterium exchange mass spectroscopy data on wild-type and mutant EGFR ternary complexes, which demonstrate that potent target degradation can be achieved in the absence of stable compound-induced protein-protein interactions. These results highlight the importance of considering target conformation during degrader development as well as leveraging heterobifunctional ligand binding kinetics to achieve robust target degradation.

Targeted degradation via direct 26S proteasome recruitment.

Engineered destruction of target proteins by recruitment to the cell's degradation machinery has emerged as a promising strategy in drug discovery. The majority of molecules that facilitate targeted degradation do so via a select number of ubiquitin ligases, restricting this therapeutic approach to tissue types that express the requisite ligase. Here, we describe a new strategy of targeted protein degradation through direct substrate recruitment to the 26S proteasome. The proteolytic complex is essential and abundantly expressed in all cells; however, proteasomal ligands remain scarce. We identify potent peptidic macrocycles that bind directly to the 26S proteasome subunit PSMD2, with a 2.5-Å-resolution cryo-electron microscopy complex structure revealing a binding site near the 26S pore. Conjugation of this macrocycle to a potent BRD4 ligand enabled generation of chimeric molecules that effectively degrade BRD4 in cells, thus demonstrating that degradation via direct proteasomal recruitment is a viable strategy for targeted protein degradation.

Identifying transcriptional programs underlying cancer drug response with TraCe-seq.

Genetic and non-genetic heterogeneity within cancer cell populations represent major challenges to anticancer therapies. We currently lack robust methods to determine how preexisting and adaptive features affect cellular responses to therapies. Here, by conducting clonal fitness mapping and transcriptional characterization using expressed barcodes and single-cell RNA sequencing (scRNA-seq), we have developed tracking differential clonal response by scRNA-seq (TraCe-seq). TraCe-seq is a method that captures at clonal resolution the origin, fate and differential early adaptive transcriptional programs of cells in a complex population in response to distinct treatments. We used TraCe-seq to benchmark how next-generation dual epidermal growth factor receptor (EGFR) inhibitor-degraders compare to standard EGFR kinase inhibitors in EGFR-mutant lung cancer cells. We identified a loss of antigrowth activity associated with targeted degradation of EGFR protein and an essential role of the endoplasmic reticulum (ER) protein processing pathway in anti-EGFR therapeutic efficacy. Our results suggest that targeted degradation is not always superior to enzymatic inhibition and establish TraCe-seq as an approach to study how preexisting transcriptional programs affect treatment responses.

Sample size requirements for genetic studies on yellowfin tuna.

In population genetics, the amount of information for an analytical task is governed by the number of individuals sampled and the amount of genetic information measured on each of those individuals. In this work, we assessed the numbers of individual yellowfin tuna (Thunnus albacares) and genetic markers required for ocean-basin scale inferences. We assessed this for three distinct data analysis tasks that are often employed: testing for differences between genetic profiles; stock delineation, and; assignment of individuals to stocks. For all analytical tasks, we used real (not simulated) data from four sampling locations that span the tropical Pacific Ocean. Whilst spatially separated, the genetic differences between the sampling sites were not substantial, a maximum of approximately Fst = 0.02, which is quite typical of large pelagic fish. We repeatedly sub-sampled the data, mimicking a new survey, and performed the analyses. False positive rates were also assessed by re-sampling and randomly assigning fish to groups. Varying the sample sizes indicated that some analytical tasks, namely profile testing, required relatively few individuals per sampling location (n ≳ 10) and single nucleotide polymorphisms (SNPs, m ≳ 256). Stock delineation required more individuals per sampling location (n ≳ 25). Assignment of fish to sampling locations required substantially more individuals, more in fact than we had available (n > 50), although this sample size could be reduced to n ≳ 30 when individual fish were assumed to belong to one of the groups sampled. With these results, designers of molecular ecological surveys for yellowfin tuna, and users of information from them, can assess whether the information content is adequate for the required inferential task.

Predicting the spread and persistence of genetically modified dominant sterile male mosquitoes.

BACKGROUND: Reproductive containment provides an opportunity to implement a staged-release strategy for genetic control of malaria vectors, in particular allowing predictions about the spread and persistence of (self-limiting) sterile and male-biased strains to be compared to outcomes before moving to (self-sustaining) gene-drive strains. METHODS: In this study, we: (i) describe a diffusion-advection-reaction model of the spread and persistence of a single cohort of male mosquitoes; (ii) elicit informative prior distributions for model parameters, for wild-type (WT) and genetically modified dominant sterile strains (DSM); (iii) estimate posterior distributions for WT strains using data from published mark-recapture-release (MRR) experiments, with inference performed through the Delayed-Rejection Adaptive Metropolis algorithm; and (iv) weight prior distributions, in order to make predictions about genetically modified strains using Bayes factors calculated for the WT strains. RESULTS: If a single cohort of 5000 genetically modified dominant sterile male mosquitoes are released at the same location as previous MRR experiments with their WT counterparts, there is a 90% probability that the expected number of released mosquitoes will fall to < 1 in 10 days, and that by 12 days there will be a 99% probability that no mosquitoes will be found more than 150 m from the release location. CONCLUSIONS: Spread and persistence models should form a key component of risk assessments of novel genetic control strategies for malaria vectors. Our predictions, used in an independent risk assessment, suggest that genetically modified sterile male mosquitoes will remain within the locality of the release site, and that they will persist for a very limited amount of time. Data gathered following the release of these mosquitoes will enable us to test the accuracy of these predictions and also provide a means to update parameter distributions for genetic strains in a coherent (Bayesian) framework. We anticipate this will provide additional insights about how to conduct probabilistic risk assessments of stage-released genetically modified mosquitoes.

Preferential coupling of diamond NV centres in step-index fibres.

Diamonds containing the negatively charged nitrogen-vacancy centre are a promising system for room-temperature magnetometry. The combination of nano- and micro-diamond particles with optical fibres provides an option for deploying nitrogen-vacancy magnetometers in harsh and challenging environments. Here we numerically explore the coupling efficiency from nitrogen-vacancy centres within a diamond doped at the core/clad interface across a range of commercially available fibre types so as to inform the design process for a diamond in fibre magnetometers. We determine coupling efficiencies from nitrogen-vacancy centres to the guided modes of a step-index fibre and predict the optically detected magnetic resonance (ODMR) generated by a ensemble of four nitrogen-vacancy centres in this hybrid fibre system. Our results show that the coupling efficiency is enhanced with a high refractive index difference between the fibre core and cladding and depends on the radial position of the nitrogen-vacancy centres in the fibre core. Our ODMR simulations show that due to the preferential coupling of the nitrogen-vacancy emission to the fibre guided modes, certain magnetometry features such as ODMR contrast can be enhanced and lead to improved sensitivity in such diamond-fibre systems, relative to conventional diamond only ensemble geometries.

Effects of ignoring survey design information for data reuse.

Data are currently being used, and reused, in ecological research at an unprecedented rate. To ensure appropriate reuse however, we need to ask the question: "Are aggregated databases currently providing the right information to enable effective and unbiased reuse?" We investigate this question, with a focus on designs that purposefully favor the selection of sampling locations (upweighting the probability of selection of some locations). These designs are common and examples are those designs that have uneven inclusion probabilities or are stratified. We perform a simulation experiment by creating data sets with progressively more uneven inclusion probabilities and examine the resulting estimates of the average number of individuals per unit area (density). The effect of ignoring the survey design can be profound, with biases of up to 250% in density estimates when naive analytical methods are used. This density estimation bias is not reduced by adding more data. Fortunately, the estimation bias can be mitigated by using an appropriate estimator or an appropriate model that incorporates the design information. These are only available however, when essential information about the survey design is available: the sample location selection process (e.g., inclusion probabilities), and/or covariates used in their specification. The results suggest that such information must be stored and served with the data to support meaningful inference and data reuse.

Monitoring the resilience of a no-take marine reserve to a range extending species using benthic imagery.

Global climate change is driving the redistribution of marine species and thereby potentially restructuring endemic communities. Understanding how localised conservation measures such as protection from additional human pressures can confer resilience to ecosystems is therefore an important area of research. Here, we examine the resilience of a no-take marine reserve (NTR) to the establishment of urchin barrens habitat. The barrens habitat is created through overgrazing of kelp by an invading urchin species that is expanding its range within a hotspot of rapid climate change. In our study region, a multi-year monitoring program provides a unique time-series of benthic imagery collected by an Autonomous Underwater Vehicle (AUV) within an NTR and nearby reference areas. We use a Bayesian hierarchical spatio-temporal modelling approach to estimate whether the NTR is associated with reduced formation of urchin barrens, and thereby enhances local resilience. Our approach controls for the important environmental covariates of depth and habitat complexity (quantified as rugosity derived from multibeam sonar mapping), as well as spatial and temporal dependence. We find evidence for the NTR conferring resilience with a strong reserve effect that suggests improved resistance to the establishment of barrens. However, we find a concerning and consistent trajectory of increasing barrens cover in both the reference areas and the NTR, with the odds of barrens increasing by approximately 32% per year. Thus, whereas the reserve is demonstrating resilience to the initial establishment of barrens, there is currently no evidence of recovery once barrens are established. We also find that depth and rugosity covariates derived from multibeam mapping provide useful predictors for barrens occurrence. These results have important management implications as they demonstrate: (i) the importance of monitoring programs to inform adaptive management; (ii) that NTRs provide a potential local conservation management tool under climate change impacts, and (iii) that technologies such as AUVs and multibeam mapping can be harnessed to inform regional decision-making. Continuation of the current monitoring program is required to assess whether the NTR can provide long term protection from a phase shift that replaces kelp with urchin barrens.

A modified flavonoid accelerates oligodendrocyte maturation and functional remyelination.

Myelination delay and remyelination failure following insults to the central nervous system (CNS) impede axonal conduction and lead to motor, sensory and cognitive impairments. Both myelination and remyelination are often inhibited or delayed due to the failure of oligodendrocyte progenitor cells (OPCs) to mature into myelinating oligodendrocytes (OLs). Digestion products of the glycosaminoglycan hyaluronan (HA) have been implicated in blocking OPC maturation, but how these digestion products are generated is unclear. We tested the possibility that hyaluronidase activity is directly linked to the inhibition of OPC maturation by developing a novel modified flavonoid that functions as a hyaluronidase inhibitor. This compound, called S3, blocks some but not all hyaluronidases and only inhibits matrix metalloproteinase activity at high concentrations. We find that S3 reverses HA-mediated inhibition of OPC maturation in vitro, an effect that can be overcome by excess recombinant hyaluronidase. Furthermore, we find that hyaluronidase inhibition by S3 accelerates OPC maturation in an in vitro model of perinatal white matter injury. Finally, blocking hyaluronidase activity with S3 promotes functional remyelination in mice with lysolecithin-induced demyelinating corpus callosum lesions. All together, these findings support the notion that hyaluronidase activity originating from OPCs in CNS lesions is sufficient to prevent OPC maturation, which delays myelination or blocks remyelination. These data also indicate that modified flavonoids can act as selective inhibitors of hyaluronidase activity and can promote OPC maturation, making them excellent candidates to accelerate myelination or promote remyelination following perinatal and adult CNS insults.

Diastereoselective Synthesis of Arabino- and Ribo-like Nucleoside Analogues Bearing a Stereogenic C3' All-Carbon Quaternary Center.

The synthesis of novel nucleoside analogues bearing a C3' all-carbon quaternary center and a C2'-hydroxy substituent is described. The all-carbon stereogenic center was generated through an intramolecular 7-endo attack of a silyl-tethered allyl moiety on a tertiary radical using photoredox catalysis. Subsequent allylic oxidation and diastereoselective hydride reductions provided the hydroxy substituent at C2', which then controls the stereoselective introduction of pyrimidine nucleobases on the corresponding furanose scaffold. Density functional theory (DFT) calculations provided insights into the origin of the high syn diastereoselectivity resulting from the radical cyclization. This original methodology grants access to a wide range of 1',2'-cis and 1',2'-trans arabino- and ribo-like analogues bearing an all-carbon quaternary center at C3'. These molecules are currently being tested for their antiviral and anticancer properties.

The role of halogens in the catalyst transfer polycondensation for π-conjugated polymers.

Catalyst transfer polycondensation is the only method to prepare π-conjugated polymers in a chain-growth manner, yet several aspects that underlie this polymerization are not fully understood. Here, we investigate the nickel-catalyzed polymerization mechanisms of a series of thiophene monomers bearing different halogen functionalities (Cl, Br, I). We have discovered the significant role that halogens and magnesium salts play in this polymerization. More specifically, the catalyst resting state changes depending on the type of halogenated monomer. For chlorinated monomers a mixture of Ni(ii)-dithienyl and dissociated Ni(phosphine) complexes are the resting states, which results in uncontrolled polymerization. For brominated monomers, a Ni(ii)-dithienyl complex is the resting state, which leads to controlled polymerization. For iodinated monomers, a Ni(ii)-thienyl iodide complex is the resting state, and notable inhibition by magnesium salt by-products is observed. The catalyst resting state changes to a Ni(ii)-dithienyl complex when a turbo Grignard reagent (i-PrMgCl·LiCl) is used. These findings are used to guide the design of a new monomer, 2-bromo-3-(2-ethylhexyl)-5-iodotellurophene, which enables the first controlled polymerization of a tellurophene monomer containing a sterically encumbered 2-ethylhexyl side chain. These insights are crucial for deepening the mechanistic understanding of Kumada cross coupling reactions and the controlled synthesis of π-conjugated polymers.

A Randomized, Double-Blind, Placebo-Controlled Phase 3 Trial of Oral Brincidofovir for Cytomegalovirus Prophylaxis in Allogeneic Hematopoietic Cell Transplantation.

Cytomegalovirus (CMV) infection is a common complication of allogeneic hematopoietic cell transplantation (HCT). In this trial, we randomized adult CMV-seropositive HCT recipients without CMV viremia at screening 2:1 to receive brincidofovir or placebo until week 14 post-HCT. Randomization was stratified by center and risk of CMV infection. Patients were assessed weekly through week 15 and every third week thereafter through week 24 post-HCT. Patients who developed clinically significant CMV infection (CS-CMVi; CMV viremia requiring preemptive therapy or CMV disease) discontinued the study drug and began anti-CMV treatment. The primary endpoint was the proportion of patients with CS-CMVi through week 24 post-HCT; patients who discontinued the trial or with missing data were imputed as primary endpoint events. Between August 2013 and June 2015, 452 patients were randomized at a median of 15 days after HCT and received study drug. The proportion of patients who developed CS-CMVi or were imputed as having a primary endpoint event through week 24 was similar between brincidofovir-treated patients and placebo recipients (155 of 303 [51.2%] versus 78 of 149 [52.3%]; odds ratio, .95 [95% confidence interval, .64 to 1.41]; P = .805); fewer brincidofovir recipients developed CMV viremia through week 14 compared with placebo recipients (41.6%; P < .001). Serious adverse events were more frequent among brincidofovir recipients (57.1% versus 37.6%), driven by acute graft-versus-host disease (32.3% versus 6.0%) and diarrhea (6.9% versus 2.7%). Week 24 all-cause mortality was 15.5% among brincidofovir recipients and 10.1% among placebo recipients. Brincidofovir did not reduce CS-CMVi by week 24 post-HCT and was associated with gastrointestinal toxicity.

Antiviral activity of brincidofovir on parvovirus B19.

Parvovirus B19 (B19V), a single-stranded DNA virus in the family Parvoviridae, is a human pathogenic virus responsible for a wide range of clinical manifestations. Currently there is no approved antiviral therapy for parvovirus infection. The acyclic nucleoside phosphonate cidofovir (CDV) has been demonstrated to inhibit replication of B19V in vitro. The aim of the present study was to evaluate whether brincidofovir (BCV), a novel lipid conjugate of CDV, could also inhibit B19V replication. Experiments were carried out in erythroid progenitor cells (EPCs) and UT7/EpoS1 cells, infected with B19V and cultured in the presence of different concentrations of BCV and CDV for comparison. The dynamics of viral replication was evaluated by a qPCR-based assay and the extent of inhibition of viral replication exerted by the compounds determined, along with the effect of the compounds on cell viability and cell proliferation rates. Results confirmed that BCV showed significantly higher antiviral activity against B19V compared to CDV in both cell-based systems. For BCV, the calculated EC50 values were in the range 6.6-14.3 µM in EPCs and 0.22-0.63 µM in UT7/EpoS1 cells. In comparison, the EC50 values for CDV were >300 µM in EPCs and 16.1 µM in UT7/EpoS1 cells. Concurrently, the effects on cell viability were observed at a much higher concentration of BCV, with calculated CC50 values in the range 93.4-102.9 µM in EPCs and 59.9-66.8 µM in UT7/Epos1. The antiviral activity was observed specifically with the metabolically active stereoisomer of BCV suggesting that CDV-diphosphate, the metabolite of both BCV and CDV, was the active antiviral. Our results support a selective role for BCV in the inhibition of B19 viral replication.