Leveraging a Phage-Encoded Noncanonical Amino Acid: A Novel Pathway to Potent and Selective Epigenetic Reader Protein Inhibitors.

Epigenetic reader proteins interpret histone epigenetic marks to regulate gene expression. Given their vital roles and the link between their dysfunction and various diseases, these proteins present compelling targets for therapeutic interventions. Nevertheless, designing selective inhibitors for these proteins poses significant challenges, primarily due to their unique properties such as shallow binding sites and similarities with homologous proteins. To overcome these challenges, we propose an innovative strategy that uses phage display with a genetically encoded noncanonical amino acid (ncAA) containing an epigenetic mark. This ncAA guides binding to the reader protein's active site, allowing the identification of peptide inhibitors with enhanced affinity and selectivity. In this study, we demonstrate this novel approach's effectiveness by identifying potent inhibitors for the ENL YEATS domain that plays a critical role in leukemogenesis. Our strategy involved genetically incorporating Nε-butyryl-l-lysine (BuK), known for its binding to ENL YEATS, into a phage display library for enriching the pool of potent inhibitors. One resultant hit was further optimized by substituting BuK with other pharmacophores to exploit a unique π-π-π stacking interaction with ENL YEATS. This led to the creation of selective ENL YEATS inhibitors with a KD value of 2.0 nM and a selectivity 28 times higher for ENL YEATS than its close homologue AF9 YEATS. One such inhibitor, tENL-S1f, demonstrated robust cellular target engagement and on-target effects to inhibit leukemia cell growth and suppress the expression of ENL target genes. As a pioneering study, this work opens up extensive avenues for the development of potent and selective peptidyl inhibitors for a broad spectrum of epigenetic reader proteins.

Diversification of Phage-Displayed Peptide Libraries with Noncanonical Amino Acid Mutagenesis and Chemical Modification.

Sitting on the interface between biologics and small molecules, peptides represent an emerging class of therapeutics. Numerous techniques have been developed in the past 30 years to take advantage of biological methods to generate and screen peptide libraries for the identification of therapeutic compounds, with phage display being one of the most accessible techniques. Although traditional phage display can generate billions of peptides simultaneously, it is limited to expression of canonical amino acids. Recently, several groups have successfully undergone efforts to apply genetic code expansion to introduce noncanonical amino acids (ncAAs) with novel reactivities and chemistries into phage-displayed peptide libraries. In addition to biological methods, several different chemical approaches have also been used to install noncanonical motifs into phage libraries. This review focuses on these recent advances that have taken advantage of both biological and chemical means for diversification of phage libraries with ncAAs.

Further investigation of lead exposure as a potential threatening process for a scavenging marsupial species.

There is a growing recognition of the harmful effects of lead exposure on avian and mammalian scavengers. This can lead to both lethal and non-lethal effects which may negatively impact wildlife populations. Our objective was to assess medium-term lead exposure in wild Tasmanian devils (Sarcophilus harrisii). Frozen liver samples (n = 41), opportunistically collected in 2017-2022, were analysed using inductively coupled plasma mass spectrometry (ICP-MS) to determine liver lead concentrations. These results were then used to calculate the proportion of animals with elevated lead levels (>5 mg/kg dry weight) and examine the role of explanatory variables that may have influenced the results. The majority of samples analysed were from the south-east corner of Tasmania, within 50 km of Hobart. No Tasmanian devil samples were found to have elevated lead levels. The median liver lead concentration was 0.17 mg/kg (range 0.05-1.32 mg/kg). Female devils were found to have significantly higher liver lead concentrations than males (P = 0.013), which was likely related to lactation, but other variables (age, location, body mass) were not significant. These results suggest that wild Tasmanian devil populations currently show minimal medium-term evidence of exposure to lead pollution, although samples were concentrated in peri-urban areas. The results provide a baseline level which can be used to assess the impact of any future changes in lead use in Tasmania. Furthermore, these data can be used as a comparison for lead exposure studies in other mammalian scavengers, including other carnivorous marsupial species.

Glycosaminoglycan-directed cobalt complexes.

The biological activity of the 6+ Co containing Werner's Complex has been described and mechanistic considerations suggest that the highly anionic glycosaminoglycans (heparan sulfate, HS, GAGs) are implicated in this activity [Paiva et al. 2021]. To examine in detail the molecular basis of Werner's Complex biological properties we have examined a selection of simple mononuclear Co3+ compounds for their interactions with HS and Fondaparinux (FPX). FPX is a highly sulfated synthetic pentasaccharide used as a model HS substrate [Mangrum et al. 2014, Peterson et al. 2017]. The Co complexes were chosen to be formally substitution-inert and/or have the potential for covalent binding to the biomolecule. Using both indirect competitive inhibition assays and direct mass spectrometric assays, formally substitution-inert complexes bound to FPX with protection from multiple sulfate loss in the gas phase through metalloshielding. Covalent binding of Co-Cl complexes as in [CoCl(NH3)5]2+ and cis-[CoCl2(en)2]+ was confirmed by mass spectrometry. Interestingly, the former complex was shown to be an effective inhibitor of bacterial heparinase enzyme activity and to inhibit heparanase-dependent cellular invasion through the extracellular matrix (ECM). Pursuing the theme of metalloglycomics, we have observed the hitherto unappreciated biological activity of the simple [CoCl(NH3)5]2+ compound, a staple of most inorganic chemistry lab curricula.

Seismic magnitude clustering is prevalent in field and laboratory catalogs.

Clustering of earthquake magnitudes is still actively debated, compared to well-established spatial and temporal clustering. Magnitude clustering is not currently implemented in earthquake forecasting but would be important if larger magnitude events are more likely to be followed by similar sized events. Here we show statistically significant magnitude clustering present in many different field and laboratory catalogs at a wide range of spatial scales (mm to 1000 km). It is universal in field catalogs across fault types and tectonic/induced settings, while laboratory results are unaffected by loading protocol or rock types and show temporal stability. The absence of clustering can be imposed by a global tensile stress, although clustering still occurs when isolating to triggered event pairs or spatial patches where shear stress dominates. Magnitude clustering is most prominent at short time and distance scales and modeling indicates >20% repeating magnitudes in some cases, implying it can help to narrow physical mechanisms for seismogenesis.

Novel Regioselective Approach to Cyclize Phage-Displayed Peptides in Combination with Epitope-Directed Selection to Identify a Potent Neutralizing Macrocyclic Peptide for SARS-CoV-2.

Using the regioselective cyanobenzothiazole condensation reaction with an N-terminal cysteine and the chloroacetamide reaction with an internal cysteine, a phage-displayed macrocyclic 12-mer peptide library was constructed and subsequently validated. Using this library in combination with iterative selections against two epitopes from the receptor binding domain (RBD) of the novel severe acute respiratory syndrome virus 2 (SARS-CoV-2) Spike protein, macrocyclic peptides that strongly inhibit the interaction between the Spike RBD and angiotensin-converting enzyme 2 (ACE2), the human host receptor of SARS-CoV-2, were identified. The two epitopes were used instead of the Spike RBD to avoid selection of nonproductive macrocyclic peptides that bind RBD but do not directly inhibit its interactions with ACE2. Antiviral tests against SARS-CoV-2 showed that one macrocyclic peptide is highly potent against viral reproduction in Vero E6 cells with an EC50 value of 3.1 µM. The AlphaLISA-detected IC50 value for this macrocyclic peptide was 0.3 µM. The current study demonstrates that two kinetically controlled reactions toward N-terminal and internal cysteines, respectively, are highly effective in the construction of phage-displayed macrocyclic peptides, and the selection based on the SARS-CoV-2 Spike epitopes is a promising methodology in the identification of peptidyl antivirals.

Decoupling anthropogenic vs. natural impacts at a wastewater treatment plant situated on acid sulfate soils.

Decoupling natural and anthropogenic impacts on the subsurface environment can be difficult, particularly when it has been subject to a wide range of influences over time and space. In this work we show how the use of hydrogeochemical plotting tools, time-series analysis of key contaminants of concern, and targeted isotopic analysis can be used to better understand the contamination sources/processes in a complex environment - a Wastewater Treatment Plant (WWTP) located on coastal acid sulfate soils (ASS). Analysis of soil profiles for potential oxidisable sulfur, acid neutralising capacity (ANC), and pHfox along with groundwater chemistry, revealed that oxidation of pyritic sediments, initially deposited during the mid-Holocene, have led to significant pH declines and the secondary mobilisation of metals into the groundwater environment. This is further complicated by historic anthropogenic inputs associated with the WWTP (e.g., effluent leakages) and the surrounding agricultural land uses. There is distinct separation between spatial and temporal trends in the nutrient and heavy metals data in groundwater, suggesting these reflect different contaminant sources and/or processes. Isotopic data indicate nutrients are largely derived from the WWTP, whereas time-series analysis of key contaminants of concern and hydrogeochemical plotting tools indicate metals are largely derived from the secondary mobilisation of ASS due to acidity generated during sulfide oxidation. This work highlights the importance of understanding the hydrogeological environment and need for careful planning and ongoing management of WWTP sites, particularly those constructed on potential acid sulfate soils (PASS), which, if disturbed or exposed, can lead to impacts beyond the area of ASS via groundwater discharge to nearby surface water bodies (in this case the site is adjacent to a Ramsar-listed wetland). The outcomes of this work have significant global application in the identification, assessment, and control of ASS, the practice of contaminant source attribution, and the siting and design of future WWTPs, which will continue to be sited in coastal areas to meet population needs.

On the Biology of Werner's Complex.

Werner's Complex, as a cationic coordination complex (CCC), has hitherto unappreciated biological properties derived from its binding affinity to highly anionic biomolecules such as glycosaminoglycans (GAGs) and nucleic acids. Competitive inhibitor and spectroscopic assays confirm the high affinity to GAGs heparin, heparan sulfate (HS), and its pentasaccharide mimetic Fondaparinux (FPX). Functional consequences of this affinity include inhibition of FPX cleavage by bacterial heparinase and mammalian heparanase enzymes with inhibition of cellular invasion and migration. Werner's Complex is a very efficient condensing agent for DNA and tRNA. In proof-of-principle for translational implications, it is demonstrated to display antiviral activity against human cytomegalovirus (HCMV) at micromolar concentrations with promising selectivity. Exploitation of non-covalent hydrogen-bonding and electrostatic interactions has motivated the unprecedented discovery of these properties, opening new avenues of research for this iconic compound.

An amber obligate active site-directed ligand evolution technique for phage display.

Although noncanonical amino acids (ncAAs) were first incorporated into phage libraries through amber suppression nearly two decades ago, their application for use in drug discovery has been limited due to inherent library bias towards sense-containing phages. Here, we report a technique based on superinfection immunity of phages to enrich amber-containing clones, thus avoiding the observed bias that has hindered incorporation of ncAAs into phage libraries. We then take advantage of this technique for development of active site-directed ligand evolution of peptides, where the ncAA serves as an anchor to direct the binding of its peptides to the target's active site. To demonstrate this, phage-displayed peptide libraries are developed that contain a genetically encoded butyryl lysine and are subsequently used to select for ligands that bind SIRT2. These ligands are then modified to develop low nanomolar inhibitors of SIRT2.

Identifying animal welfare impacts of livestock air transport.

Air transport of livestock occurs frequently from most Australian major cities. The total journey time starts with road transport from the farm or pre-export facility to the departing airport where livestock are crated, and ends with the unloading of animals at the premises or farm in the importing country. We reviewed the literature regarding airfreight and conclude that there was minimal information on current practices within this industry, particularly for procedures after arrival at the Australian airport, and during the on-board phase.

A Genetically Encoded, Phage-Displayed Cyclic-Peptide Library.

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands for therapeutic targets, phage-displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage-display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded Nϵ -acryloyl-lysine (AcrK). Using a randomized 6-mer library in which peptides were cyclized at two ends through a cysteine-AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4- to 6-fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.

Disciplinary boundaries and integrating care: using Q-methodology to understand trainee views on being a good doctor.

BACKGROUND: Rising numbers of patients with multiple-conditions and complex care needs mean that it is increasingly important for doctors from different specialty areas to work together, alongside other members of the multi-disciplinary team, to provide patient centred care. However, intra-professional boundaries and silos within the medical profession may challenge holistic approaches to patient care. METHODS: We used Q methodology to examine how postgraduate trainees (n = 38) on a range of different specialty programmes in England and Wales could be grouped based on their rankings of 40 statements about 'being a good doctor'. Themes covered in the Q-set include: generalism (breadth) and specialism (depth), interdisciplinarity and multidisciplinary team working, patient-centredness, and managing complex care needs. RESULTS: A by-person factor analysis enabled us to map distinct perspectives within our participant group (P-set). Despite high levels of overall commonality, three groups of trainees emerged, each with a clear perspective on being a good doctor. We describe the first group as 'generalists': team-players with a collegial and patient-centred approach to their role. The second group of 'general specialists' aspired to be specialists but with a generalist and patient-centred approach to care within their specialty area. Both these two groups can be contrasted to those in the third 'specialist' group, who had a more singular focus on how their specialty can help the patient. CONCLUSIONS: Whilst distinct, the priorities and values of trainees in this study share some important aspects. The results of our Q-sort analysis suggest that it may be helpful to understand the relationship between generalism and specialism as less of a dichotomy and more of a continuum that transcends primary and secondary care settings. A nuanced understanding of trainee views on being a good doctor, across different specialties, may help us to bridge gaps and foster interdisciplinary working.

Remote chemical immobilisation method for free-ranging Australian cattle.

BACKGROUND: Many situations are encountered in Australia where the capture and restraint of free-ranging cattle (Bos taurus/Bos indicus) is required. Chemical immobilisation via darting is a potentially useful tool for managing and researching large wild herbivores; however, there is no reliable method for its application to Australian cattle. The aim of this study was to develop an efficacious, humane, cost-effective ground darting method for free-ranging cattle. METHODS: The 30 female cattle were darted and captured on a pastoral station in north-west Australia from a vehicle. Xylazine (0.59 mg/kg) and ketamine (3.59 mg/kg) were used to capture animals and yohimbine (0.10 mg/kg) was used as an antagonist to xylazine to reduce recumbent time. RESULTS: Cattle became recumbent at a mean time of 8 min and a mean distance of 260 m from darting. The mortality rate was zero on the day of capture and 7% at 14 days post-capture. CONCLUSIONS: The majority of darted cattle were successfully immobilised with one dart and recovered within 30 min, with consumables costing approximately A$30 per captured animal. The technique developed represents a rapid and humane method for capturing free-ranging cattle and, with consideration for legislation surrounding use of veterinary chemicals, could be applied in many contexts across Australia.

Management of Acute Lower Gastrointestinal Bleeding: Principles and Current Practice in the United Kingdom.

Acute lower gastrointestinal bleeding (ALGIB) is a common cause for hospital admission that results in significant morbidity and mortality. The major objectives of all involved in the management of ALGBI patients are to reduce mortality and the need for major surgery. A secondary objective is to prevent unnecessary hospital admission for patients presenting with bleeding that is not life-threatening. The management of ALGBI has evolved over last decade with the changing modalities of diagnostic facilities. On review of the published literature, there is paucity of randomised control trials in relation to the diagnostic tools and management of ALGBI. The aim of this review is to summarise the principles and current methods available for the diagnosis and treatment of ALGIB and based on the available evidence and the current practice in the United Kingdom, outline an algorithm for the management of ALGIB.

Effects of elevated CO2 and temperature on seed quality.

Successful crop production depends initially on the availability of high-quality seed. By 2050 global climate change will have influenced crop yields, but will these changes affect seed quality? The present review examines the effects of elevated carbon dioxide (CO2) and temperature during seed production on three seed quality components: seed mass, germination and seed vigour. In response to elevated CO2, seed mass has been reported to both increase and decrease in C3 plants, but not change in C4 plants. Increases are greater in legumes than non-legumes, and there is considerable variation among species. Seed mass increases may result in a decrease of seed nitrogen (N) concentration in non-legumes. Increasing temperature may decrease seed mass because of an accelerated growth rate and reduced seed filling duration, but lower seed mass does not necessarily reduce seed germination or vigour. Like seed mass, reported seed germination responses to elevated CO2 have been variable. The reported changes in seed C/N ratio can decrease seed protein content which may eventually lead to reduced viability. Conversely, increased ethylene production may stimulate germination in some species. High-temperature stress before developing seeds reach physiological maturity (PM) can reduce germination by inhibiting the ability of the plant to supply the assimilates necessary to synthesize the storage compounds required for germination. Nothing is known concerning the effects of elevated CO2 on seed vigour. However, seed vigour can be reduced by high-temperature stress both before and after PM. High temperatures induce or increase the physiological deterioration of seeds. Limited evidence suggests that only short periods of high-temperature stress at critical seed development stages are required to reduce seed vigour, but further research is required. The predicted environmental changes will lead to losses of seed quality, particularly for seed vigour and possibly germination. The seed industry will need to consider management changes to minimize the risk of this occurring.

Modeling paternal attentiveness: distressed pups evoke differential neurobiological and behavioral responses in paternal and nonpaternal mice.

With the exception of parturition and lactation, male California deer mice (Peromyscus californicus) exhibit the same parental responses toward offspring as conspecific females. A closely related species, Peromyscus maniculatus, however, rarely exhibits paternal responses. In the current study, a comparative species approach was used to assess paternal responses in both Peromyscus species with varying levels of paternal experience (biological fathers, pup-exposed virgins, and pup-naïve virgins). Of special interest was the persistence of the males to direct their attention toward a distressed pup housed in a small enclosure (i.e., a barrier existed between males and pups). In addition to pup-directed responses, non-pup-directed responses such as grooming, resting and jumping were recorded. Subsequently, all animals' brains were assessed for fos-immunoreactivity (ir) in several areas previously associated with the paternal brain circuit. Overall, P. californicus exhibited more pup-directed responses as well as less fos-ir in brain areas involved in emotional integration and processing such as the insula and anterior cingulate. In addition to increased activation of emotional regulatory areas, P. maniculatus males, observed to direct their behavior away from the pup, exhibited higher fos-ir in the nucleus accumbens (involved in goal acquisition), perhaps due to a heightened motivation to avoid the pups. Interestingly, experience with pups altered the lateral septum and amygdala activation of P. maniculatus to levels similar to P. californicus biological fathers. Finally, fos-ir was increased in the medial preoptic area, involved in the maintenance of maternal behavior, in the biological fathers of both species. Thus, although biological predispositions toward pup-directed behaviors were observed in P. californicus males, evidence of a few shifts toward the paternal neural activation profile was apparent in P. maniculatus males. Specifically, modifications in fear responses and social processing may represent the cornerstones of the gradual shift from social tentativeness to social attentiveness in the presence of pups.

The apelin receptor inhibits the angiotensin II type 1 receptor via allosteric trans-inhibition.

BACKGROUND AND PURPOSE: The apelin receptor (APJ) is often co-expressed with the angiotensin II type-1 receptor (AT1) and acts as an endogenous counter-regulator. Apelin antagonizes Ang II signalling, but the precise molecular mechanism has not been elucidated. Understanding this interaction may lead to new therapies for the treatment of cardiovascular disease. EXPERIMENTAL APPROACH: The physical interaction of APJ and AT1 receptors was detected by co-immunoprecipitation and bioluminescence resonance energy transfer (BRET). Functional and pharmacological interactions were measured by G-protein-dependent signalling and recruitment of ß-arrestin. Allosterism and cooperativity between APJ and AT1 were measured by radioligand binding assays. KEY RESULTS: Apelin, but not Ang II, induced APJ : AT1 heterodimerization forced AT1 into a low-affinity state, reducing Ang II binding. Likewise, apelin mediated a concentration-dependent depression in the maximal production of inositol phosphate (IP(1) ) and ß-arrestin recruitment to AT1 in response to Ang II. The signal depression approached a limit, the magnitude of which was governed by the cooperativity indicative of a negative allosteric interaction. Fitting the data to an operational model of allosterism revealed that apelin-mediated heterodimerization significantly reduces Ang II signalling efficacy. These effects were not observed in the absence of apelin. CONCLUSIONS AND IMPLICATIONS: Apelin-dependent heterodimerization between APJ and AT1 causes negative allosteric regulation of AT1 function. As AT1 is significant in the pathogenesis of cardiovascular disease, these findings suggest that impaired apelin and APJ function may be a common underlying aetiology. LINKED ARTICLE: This article is commented on by Goupil et al., pp. 1101-1103 of this issue. To view this commentary visit http://dx.doi.org/10.1111/bph.12040.

Reproductive experience facilitates recovery from kainic acid-induced neural insult in female Long-Evans rats.

The hormones of pregnancy and lactation (e.g., estrogen, progesterone, and oxytocin) have been shown to modulate learning, memory, and the restructuring of brain areas not traditionally associated with maternal behavior. Given the impact of reproductive experience on plasticity of brain areas such as the hippocampus, kainic acid (KA) was used in the current study to induce hippocampal-specific neurotoxic insult in adult multiparous and virgin Long-Evans rats. In Experiment I, Fluoro-Jade B, an indicant of degenerating cells, revealed significant neuronal damage in KA-treated hippocampi at 16 h post-injection in both maternal and virgin rats. In Experiment II, maternal and virgin rats were assessed in spatial and novel object preference tasks to determine the effects of KA on subsequent behavioral and cognitive responses. Twenty-four hours post injection, saline maternal animals exhibited superior memory in a spatial task. Further, maternal saline-injected rats were more similar to maternal KA-injected rats than both the virgin groups. Forty-eight hours following the KA or saline injection, compared to virgins, maternal animals demonstrated enhanced memory in the novel object memory test, regardless of type of injection. Further, neurobiological assessments in Experiment II indicated that virgin KA exposed rats had significantly more glial fibrillary acidic protein (GFAP)-immunoreactivity in the hippocampus, suggesting that they were in an earlier stage of neural recovery compared to maternal animals or, alternatively, may have exhibited more trauma than maternal animals. Together, these data suggest that the previously reported plasticity of the maternal brain may facilitate neural and behavioral recovery from neural insults.