Redirecting the specificity of tripartite motif containing-21 scaffolds using a novel discovery and design approach.

Hijacking the ubiquitin proteasome system to elicit targeted protein degradation (TPD) has emerged as a promising therapeutic strategy to target and destroy intracellular proteins at the post-translational level. Small molecule-based TPD approaches, such as proteolysis-targeting chimeras (PROTACs) and molecular glues, have shown potential, with several agents currently in clinical trials. Biological PROTACs (bioPROTACs), which are engineered fusion proteins comprised of a target-binding domain and an E3 ubiquitin ligase, have emerged as a complementary approach for TPD. Here, we describe a new method for the evolution and design of bioPROTACs. Specifically, engineered binding scaffolds based on the third fibronectin type III domain of human tenascin-C (Tn3) were installed into the E3 ligase tripartite motif containing-21 (TRIM21) to redirect its degradation specificity. This was achieved via selection of naïve yeast-displayed Tn3 libraries against two different oncogenic proteins associated with B-cell lymphomas, mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) and embryonic ectoderm development protein (EED), and replacing the native substrate-binding domain of TRIM21 with our evolved Tn3 domains. The resulting TRIM21-Tn3 fusion proteins retained the binding properties of the Tn3 as well as the E3 ligase activity of TRIM21. Moreover, we demonstrated that TRIM21-Tn3 fusion proteins efficiently degraded their respective target proteins through the ubiquitin proteasome system in cellular models. We explored the effects of binding domain avidity and E3 ligase utilization to gain insight into the requirements for effective bioPROTAC design. Overall, this study presents a versatile engineering approach that could be used to design and engineer TRIM21-based bioPROTACs against therapeutic targets.

Back to the future for drought tolerance.

Global agriculture faces increasing pressure to produce more food with fewer resources. Drought, exacerbated by climate change, is a major agricultural constraint costing the industry an estimated US$80 billion per year in lost production. Wild relatives of domesticated crops, including wheat (Triticum spp.) and barley (Hordeum vulgare L.), are an underutilized source of drought tolerance genes. However, managing their undesirable characteristics, assessing drought responses, and selecting lines with heritable traits remains a significant challenge. Here, we propose a novel strategy of using multi-trait selection criteria based on high-throughput spectral images to facilitate the assessment and selection challenge. The importance of measuring plant capacity for sustained carbon fixation under drought stress is explored, and an image-based transpiration efficiency (iTE) index obtained via a combination of hyperspectral and thermal imaging, is proposed. Incorporating iTE along with other drought-related variables in selection criteria will allow the identification of accessions with diverse tolerance mechanisms. A comprehensive approach that merges high-throughput phenotyping and de novo domestication is proposed for developing drought-tolerant prebreeding material and providing breeders with access to gene pools containing unexplored drought tolerance mechanisms.

Elevated CO2 improves phosphorus nutrition and growth of citrate-secreting wheat when grown under adequate phosphorus supply on an Al3+ -toxic soil.

BACKGROUND: Understanding how climate change affects the phosphorus (P) nutrition of crops grown on acid soils is important in optimizing the management of P, and to secure future food production on these soils. This study assessed the impact of elevated CO2 (eCO2 ) on the P nutrition of wheat (Triticum aestivum) grown on Al3+ -toxic and P-deficient soils or in hydroponics. The aluminium-resistant near-isogenic wheat lines EGA-Burke (malate efflux only) and EGA-Burke TaMATE1B (malate and citrate efflux) were grown under ambient (400 µmol mol-1 ) and elevated CO2 (800 µmol mol-1 ) in growth chambers for 4-6 weeks. RESULTS: Elevated CO2 enhanced shoot growth and total P uptake of both lines at P rates >250 mg kg-1 , which was associated with improved root biomass allocation and thus increased root growth, but these effects were not apparent at lower P rates. Elevated CO2 decreased specific P uptake (P uptake per unit root length) at P supply >250 mg kg-1 , but did not significantly affect external or internal P requirements. This effect on the specific P uptake was less for EGA-Burke TaMATE1B than for EGA-Burke, possibly due to the increased citrate efflux and decreased Al concentration in root tips of EGA-Burke TaMATE1B. Compared to EGA-Burke, citrate-exuding EGA-Burke TaMATE1B had greater shoot P concentration and greater specific P uptake. CONCLUSION: Elevated CO2 improved root growth, and thus total P uptake and plant production of both lines when high P alleviated Al3+ toxicity and improved P nutrition in acid soils. The decreased P uptake efficiency under eCO2 was less for EGA-Burke TaMATE1B than EGA-Burke. © 2022 Society of Chemical Industry.

Selective CRAF Inhibition Elicits Transactivation.

Discovering molecules that regulate closely related protein isoforms is challenging, and in many cases the consequences of isoform-specific pharmacological regulation remains unknown. RAF isoforms are commonly mutated oncogenes that serve as effector kinases in MAP kinase signaling. BRAF/CRAF heterodimers are believed to be the primary RAF signaling species, and many RAF inhibitors lead to a "paradoxical activation" of RAF kinase activity through transactivation of the CRAF protomer; this leads to resistance mechanisms and secondary tumors. It has been hypothesized that CRAF-selective inhibition might bypass paradoxical activation, but no CRAF-selective inhibitor has been reported and the consequences of pharmacologically inhibiting CRAF have remained unknown. Here, we use bio-orthogonal ligand tethering (BOLT) to selectively target inhibitors to CRAF. Our results suggest that selective CRAF inhibition promotes paradoxical activation and exemplify how BOLT may be used to triage potential targets for drug discovery before any target-selective small molecules are known.

Exploiting genotype × management interactions to increase rainfed crop production: a case study from south-eastern Australia.

Crop yield must increase to keep pace with growing global demand. Past increases in crop production have rarely been attributable to an individual innovation but have occurred when technologies and practices combine to form improved farming systems. Inevitably this has involved synergy between genotypic and management improvements. We argue that research focused on developing synergistic systems that overcome clear production constraints will accelerate increases in yield. This offers the opportunity to better focus and multiply the impact of discipline-focused research. Here we use the rainfed grain production systems of south-eastern Australia as a case study of how transformational change in water productivity can be achieved with research focused on genotype × management synergies. In this region, rainfall is low and variable and has declined since 1990. Despite this, growers have maintained yields by implementing synergistic systems combining innovations in (i) soil water conservation, (ii) crop diversity, (iii) earlier sowing, and (iv) matching nitrogen fertilizer to water-limited demand. Further increases are emerging from synergies between genetic improvements to deliver flowering time stability, adjusted sowing times, and potential dual-purpose use. Collaboration between agronomists, physiologists, and crop breeders has led to development of commercial genotypes with stable flowering time that are in early phases of testing and adoption.

Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

Programmed cell death ligand-1 (PD-L1) expression levels in patient tumor samples have proven clinical utility across various cancer types. Several independently developed PD-L1 immunohistochemical (IHC) predictive assays are commercially available. Published studies using the VENTANA PD-L1 (SP263) Assay, VENTANA PD-L1 (SP142) Assay, Dako PD-L1 IHC 22C3 pharmDx assay, Dako PD-L1 IHC 28-8 pharmDx assay, and laboratory-developed tests utilizing the E1L3N antibody (Cell Signaling Technology), have demonstrated differing levels of PD-L1 staining between assays, resulting in conjecture as to whether antibody-binding epitopes could be responsible for discordance between assays. Therefore, to understand the performance of different PD-L1 predictive immunohistochemistry assays, we aimed to distinguish the epitopes within the PD-L1 protein responsible for antibody binding. The sites at which antibody clones SP263, SP142, 22C3, 28-8, and E1L3N bind to recombinant PD-L1 were assessed using several methods, including conformational peptide array, surface plasmon resonance, and/or hydrogen/deuterium exchange mass spectrometry. Putative binding sites were confirmed by site-directed mutagenesis of PD-L1, followed by western blotting and immunohistochemical analysis of cell lines expressing mutant constructs. Our results demonstrate that clones SP263 and SP142 bind to an identical epitope in the cytoplasmic domain at the extreme C-terminus of PD-L1, distinct from 22C3 and 28-8. Using mutated PD-L1 constructs, an additional clone, E1L3N, was also found to bind to the cytoplasmic domain of PD-L1. The E1L3N binding epitope overlaps considerably with the SP263/SP142 binding site but is not identical. Clones 22C3 and 28-8 have binding profiles in the extracellular domain of PD-L1, which differ from one another. Despite identifying epitope binding variance among antibodies, evidence indicates that only the SP142 assay generates significantly discordant immunohistochemical staining, which can be resolved by altering the assay protocol. Therefore, inter-assay discordances are more likely attributable to tumor heterogeneity, assay, or platform variables rather than antibody epitope.

Correction: Mapping the binding sites of antibodies utilized in programmed cell death ligand-1 predictive immunohistochemical assays for use with immuno-oncology therapies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Phenology and related traits for wheat adaptation.

Wheat is a major food crop, with around 765 million tonnes produced globally. The largest wheat producers include the European Union, China, India, Russia, United States, Canada, Pakistan, Australia, Ukraine and Argentina. Cultivation of wheat across such diverse global environments with variation in climate, biotic and abiotic stresses, requires cultivars adapted to a range of growing conditions. One intrinsic way that wheat achieves adaptation is through variation in phenology (seasonal timing of the lifecycle) and related traits (e.g., those affecting plant architecture). It is important to understand the genes that underlie this variation, and how they interact with each other, other traits and the growing environment. This review summarises the current understanding of phenology and developmental traits that adapt wheat to different environments. Examples are provided to illustrate how different combinations of alleles can facilitate breeding of wheat varieties with optimal crop performance for different growing regions or farming systems.

Duodenal eosinophils as predictors of symptoms in coeliac disease: a comparison of coeliac disease and non-coeliac dyspeptic patients with controls.

Introduction: Duodenal eosinophilia is a key feature of functional dyspepsia, particularly in those with early satiety. Duodenal eosinophilia is also recognised in coeliac disease, although its relevance to symptoms is not understood. We aimed to determine if duodenal eosinophilia is present in patients with coeliac disease presenting with dyspepsia, and whether other histological characteristics were associated with clinical features on presentation.Methods: The coeliac study population comprised 61 patients with a new presentation of coeliac disease to a single centre from 2003 to 2013. A standard symptom assessment was documented for all patients. The control population (55 adults) presenting for endoscopy without coeliac disease was drawn from the same centre with similar demographics for age and gender. Duodenal biopsies from both groups were assessed for eosinophil counts and histological features.Results: Dyspepsia was present in 18.0% of coeliac patients and early satiety in 24.6%. The eosinophil counts were significantly higher in the stomach (12.1/mm2 vs. 4.0/mm2, p < .001) and duodenum (60.4/mm2 vs. 18.0/mm2, p < .001) of coeliac patients compared with controls. There was no significant difference in the mean duodenal eosinophil count in coeliac disease with and without early satiety (55.4/mm2 vs. 66.9/mm2, p = .51). Duodenal eosinophilia was not associated with the severity of coeliac enteropathy. The degree of villous atrophy was associated with iron deficiency at presentation (p = .01), but not symptoms.Conclusions: Although duodenal eosinophil counts are higher in coeliac disease than controls, we were not able to demonstrate an association with presenting symptoms or markers of disease severity.

Hierarchical analysis of wild Atlantic salmon (Salmo salar) fecundity in relation to body size and developmental traits.

Using data from wild Atlantic salmon Salmo salar returning to spawn in seven Scottish rivers, we developed a model of fecundity based on individual body size and key developmental traits. We used a novel approach to model selection which maximises predictive accuracy for application to target river stocks to select the best from a suite of Bayesian hierarchical models. This approach aims to ensure the optimal model within the candidate set includes covariates that best predict out-of-sample data to estimate fecundity in areas where no direct observations are available. In addition to body size, the final model included the developmental characteristics of age at smolting and years spent at sea. Using two independent long-term monitoring datasets, the consequences of ignoring these characteristics was revealed by comparing predictions from the best model with models that omitted them.

Increase in coleoptile length and establishment by Lcol-A1, a genetic locus with major effect in wheat.

BACKGROUND: Good establishment is important for rapid leaf area development in wheat crops. Poor establishment results in fewer, later-emerging plants, reduced leaf area and tiller number. In addition, poorly established crops suffer from increased soil moisture loss through evaporation and greater competition from weeds while fewer spikes are produced which can reduce grain yield. By protecting the emerging first leaf, the coleoptile is critical for achieving good establishment, and its length and interaction with soil physical properties determine the ability of a cultivar to emerge from depth. RESULTS: Here we characterise a locus on chromosome 1AS, that increases coleoptile length in wheat, which we designate as Lcol-A1. We identified Lcol-A1 by bulked-segregant analysis and used a Halberd-derived population to fine map the gene to a 2 cM region, equivalent to 7 Mb on the IWGSC genome reference sequence of Chinese Spring (RefSeqv1.0). By sowing recently released cultivars and near-isogenic lines in the field at both conventional and deep sowing depths, we confirmed that Locl-A1 was associated with increased emergence from depth in the presence and absence of conventional dwarfing genes. Flanking markers IWB58229 and IWA710 were developed to assist breeders to select for long coleoptile wheats. CONCLUSIONS: Increased coleoptile length is sought in many global wheat production areas to improve crop emergence. The identification of the gene Lcol-A1, together with tools to allow wheat breeders to track the gene, will enable improvements to be made for this important trait.

Noninvasive 3D Field Mapping of Complex Static Electric Fields.

Many upcoming experiments in antimatter research require low-energy antiproton beams. With a kinetic energy in the order of 100 keV, the standard magnetic components to control and focus the beams become less effective. Therefore, electrostatic components are being developed and installed in transfer lines and storage rings. However, there is no equipment available to precisely map and check the electric field generated by these elements. Instead, one has to trust in simulations and, therefore, depend on tight fabrication tolerances. Here we present, for the first time, a noninvasive way to experimentally probe the electrostatic field in a 3D volume with a microsensor. Using the example of an electrostatic quadrupole focusing component, we find excellent agreement between a simulated and real field. Furthermore, it is shown that the spatial resolution of the probe is limited by the electric field curvature which is almost zero for the quadrupole. With a sensor resolution of 61 V/m/sqrt[Hz], the field deviation due to a noncompliance with the tolerances can be resolved. We anticipate that this compact and practical field strength probe will be relevant also for other scientific and technological disciplines such as atmospheric electricity or safeguarding near power infrastructure.

Elevated CO2 (free-air CO2 enrichment) increases grain yield of aluminium-resistant but not aluminium-sensitive wheat (Triticum aestivum) grown in an acid soil.

BACKGROUND AND AIMS: Soil acidity currently limits root growth and crop production in many regions, and climate change is leading to uncertainties regarding future food supply. However, it is unknown how elevated CO2 (eCO2) affects the performance of wheat crops in acid soils under field conditions. We investigated the effects of eCO2 on plant growth and yield of three pairs of near-isogenic hexaploid wheat lines differing in alleles of aluminium-resistant genes TaALMT1 (conferring root malate efflux) and TaMATE1B (conferring citrate efflux). METHODS: Plants were grown until maturity in an acid soil under ambient CO2 (aCO2; 400 µmol mol-1) and eCO2 (550 µmol mol-1) in a soil free-air CO2 enrichment facility (SoilFACE). Growth parameters and grain yields were measured. KEY RESULTS: Elevated CO2 increased grain yield of lines carrying TaMATE1B by 22 % and lines carrying only TaALMT1 by 31 %, but did not increase the grain yield of Al3+-sensitive lines. Although eCO2 promoted tiller formation, coarse root length and root biomass of lines carrying TaMATE1B, it did not affect ear number, and it therefore limited yield potential. By contrast, eCO2 decreased or did not change these parameters for lines carrying only TaALMT1, and enhanced biomass allocation to grains thereby resulting in increased grain yield. Despite TaMATE1B being less effective than TaALMT1 at conferring Al3+ resistance based on root growth, the gene promoted grain yield to a similar level to TaALMT1 when the plants were grown in acid soil. Furthermore, TaALMT1 and TaMATE1B were not additive in their effects. CONCLUSIONS: As atmospheric CO2 increases, it is critical that both Al3+-resistance genes (particularly TaALMT1) should be maintained in hexaploid wheat germplasm in order for yield increases from CO2 fertilization to be realized in acid soils.

Direct Monitoring Reveals Initiation of Turbidity Currents From Extremely Dilute River Plumes.

Rivers (on land) and turbidity currents (in the ocean) are the most important sediment transport processes on Earth. Yet how rivers generate turbidity currents as they enter the coastal ocean remains poorly understood. The current paradigm, based on laboratory experiments, is that turbidity currents are triggered when river plumes exceed a threshold sediment concentration of ~1 kg/m3. Here we present direct observations of an exceptionally dilute river plume, with sediment concentrations 1 order of magnitude below this threshold (0.07 kg/m3), which generated a fast (1.5 m/s), erosive, short-lived (6 min) turbidity current. However, no turbidity current occurred during subsequent river plumes. We infer that turbidity currents are generated when fine sediment, accumulating in a tidal turbidity maximum, is released during spring tide. This means that very dilute river plumes can generate turbidity currents more frequently and in a wider range of locations than previously thought.

Clinical measurements performed during alfaxalone total intravenous anaesthesia for radiography and neurophysiological investigations in dogs.

OBJECTIVE: To describe clinically relevant, physiological measurements collected during a 3 hour duration of alfaxalone total intravenous anaesthesia. STUDY DESIGN: Case series. ANIMALS: A total of 112 client-owned middle-aged or older dogs. METHODS: Dogs were premedicated with intramuscular acepromazine (0.03 mg kg-1). Anaesthesia was induced and subsequently maintained for up to 3 hours with alfaxalone administered intravenously. Dogs breathed 100% oxygen via an endotracheal tube. Heart rate, respiratory rate and blood pressure were evaluated 30 minutes after administration of acepromazine and used as baseline values for comparisons of intra-anaesthetic data. Blood glucose was measured 1 week prior to anaesthesia and every hour during alfaxalone anaesthesia. Quality and duration of recovery were recorded. Mean data for physiological variables were compared over three time points-before induction of anaesthesia, for the first hour of anaesthesia and from 60 minutes to discontinuation of anaesthesia. RESULTS: Mean induction dose of alfaxalone was 1.4 mg kg-1 [95% confidence interval (CI) 1.3-1.5). Post induction apnoea for >60 seconds occurred in 13 (11.6%) dogs. Mean alfaxalone infusion rate during the first 60 minutes of anaesthesia was 0.099 mg kg-1 minute-1; mean infusion rate was 0.092 mg kg-1 minute-1 from 60 minutes until discontinuation of anaesthesia. Heart rate was well maintained; hypotension (mean arterial blood pressure < 60 mmHg) was encountered in 23 (21%) dogs. Blood glucose levels did not alter during anaesthesia. Median time between discontinuation of alfaxalone infusion and extubation was 17 (7-35 minutes), time to assuming sternal recumbency was 75 (58-110 minutes), and time to standing was 109 (88-140 minutes). CONCLUSIONS AND CLINICAL RELEVANCE: Alfaxalone infusion provided effective anaesthesia in this population. In a minority of cases, respiratory and haemodynamic support of the patient was required.

Comparative transcriptomics of convergent evolution: different genes but conserved pathways underlie caste phenotypes across lineages of eusocial insects.

An area of great interest in evolutionary genomics is whether convergently evolved traits are the result of convergent molecular mechanisms. The presence of queen and worker castes in insect societies is a spectacular example of convergent evolution and phenotypic plasticity. Multiple insect lineages have evolved environmentally induced alternative castes. Given multiple origins of eusociality in Hymenoptera (bees, ants, and wasps), it has been proposed that insect castes evolved from common genetic "toolkits" consisting of deeply conserved genes. Here, we combine data from previously published studies on fire ants and honey bees with new data for Polistes metricus paper wasps to assess the toolkit idea by presenting the first comparative transcriptome-wide analysis of caste determination among three major hymenopteran social lineages. Overall, we found few shared caste differentially expressed transcripts across the three social lineages. However, there is substantially more overlap at the levels of pathways and biological functions. Thus, there are shared elements but not on the level of specific genes. Instead, the toolkit appears to be relatively "loose," that is, different lineages show convergent molecular evolution involving similar metabolic pathways and molecular functions but not the exact same genes. Additionally, our paper wasp data do not support a complementary hypothesis that "novel" taxonomically restricted genes are related to caste differences.

Nourishment level affects caste-related gene expression in Polistes wasps.

BACKGROUND: Social insects exhibit striking phenotypic plasticity in the form of distinct reproductive (queen) and non-reproductive (worker) castes, which are typically driven by differences in the environment during early development. Nutritional environment and nourishment during development has been shown to be broadly associated with caste determination across social insect taxa such as bees, wasps, and termites. In primitively social insects such as Polistes paper wasps, caste remains flexible throughout adulthood, but there is evidence that nourishment inequalities can bias caste development with workers receiving limited nourishment compared to queens. Dominance and vibrational signaling are behaviors that have also been linked to caste differences in paper wasps, suggesting that a combination of nourishment and social factors may drive caste determination. To better understand the molecular basis of nutritional effects on caste determination, we used RNA-sequencing to investigate the gene expression changes in response to proteinaceous nourishment deprivation in Polistes metricus larvae. RESULTS: We identified 285 nourishment-responsive transcripts, many of which are related to lipid metabolism and oxidation-reduction activity. Via comparisons to previously identified caste-related genes, we found that nourishment restriction only partially biased wasp gene expression patterns toward worker caste-like traits, which supports the notion that nourishment, in conjunction with social environment, is a determinant of developmental caste bias. In addition, we conducted cross-species comparisons of nourishment-responsive genes, and uncovered largely lineage-specific gene expression changes, suggesting few shared nourishment-responsive genes across taxa. CONCLUSION: Overall, the results from this study highlight the complex and multifactorial nature of environmental effects on the gene expression patterns underlying plastic phenotypes.

Site-specific dual labeling of proteins by using small orthogonal tags at neutral pH.

To expand the utility of proteinaceous FRET biosensors, we have developed a dual-labeling approach based on two small bio-orthogonal tags: pyrroline-carboxy-lysine (Pcl) and the S6 peptide. The lack of cross-reactivity between those tags enables site-specific two-color protein conjugation in a one-pot reaction. Moreover, Pcl/S6 dual-tagged proteins can be produced in both bacterial and mammalian expression systems, as demonstrated for Z domain and IgE-Fc, respectively. Both proteins could be efficiently dual-labeled with FRET-compatible fluorescent dyes at neutral pH. In the case of IgE-Fc, the resulting conjugate enabled the monitoring of IgE binding to its high-affinity receptor FcεRI, which is a key event in allergic disease.