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Covalent hit identification is a viable approach to identify chemical starting points against difficult-to-drug targets. While most researchers screen libraries of <2k electrophilic fragments, focusing on lead-like compounds can be advantageous in terms of finding hits with improved affinity and with a better chance of identifying cryptic pockets. However, due to the increased molecular complexity, larger numbers of compounds (>10k) are desirable to ensure adequate coverage of chemical space. Herein, the approach taken to build a library of 12k covalent lead-like compounds is reported, utilizing legacy compounds, robust library chemistry, and acquisitions. The lead-like covalent library was screened against the antiapoptotic protein Bfl-1, and six promising hits that displaced the BIM peptide from the PPI interface were identified. Intriguingly, X-ray crystallography of lead-like compound 8 showed that it binds to a previously unobserved conformation of the Bfl-1 protein and is an ideal starting point for the optimization of Bfl-1 inhibitors.

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INTRODUCTION: Assessment of the pupillary light reflex (PLR) is key in intensive care monitoring of neurosurgical patients, particularly for monitoring intracranial pressure (ICP). Quantitative pupillometry using a handheld pupillometer is a reliable method for PLR assessment. However, many variables are derived from such devices. We therefore aimed to assess the performance of these variables at monitoring ICP. METHODS: Sedated patients admitted to neurocritical care in a tertiary neurosurgical centre with invasive ICP monitoring were included. Hourly measurement of ICP, subjective pupillometry (SP) using a pen torch device, and quantitative pupillometry (QP) using a handheld pupillometer were performed. RESULTS: 561 paired ICP, SP and QP pupillary observations from nine patients were obtained (1122 total pupillary observations). SP and QP had a moderate concordance for pupillary size (κ=0.62). SP performed poorly at detecting pupillary size changes (sensitivity=24%). In 40 (3.6%) observations, SP failed to detect a pupillary response whereas QP did. Moderate correlations with ICP were detected for maximum constriction velocity (MCV), dilation velocity (DV), and percentage change in pupillary diameter (%C). Discriminatory ability at an ICP threshold of >22 mmHg was moderate for MCV (AUC=0.631), DV (AUC=0.616), %C (AUC=0.602), and pupillary maximum size (AUC=0.625). CONCLUSION: QP is superior to SP at monitoring pupillary reactivity and changes to pupillary size. Although effect sizes were moderate to weak across assessed variables, our data indicates MCV and %C as the most sensitive variables for monitoring ICP. Further study is required to validate these findings and to establish normal range cut-offs for clinical use.

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Penicillium roqueforti is used worldwide in the production of blue-veined cheese. The blue-green colour derives from pigmented spores formed by fungal growth. Using a combination of bioinformatics, targeted gene deletions, and heterologous gene expression we discovered that pigment formation was due to a DHN-melanin biosynthesis pathway. Systematic deletion of pathway genes altered the arising spore colour, yielding white to yellow-green to red-pink-brown phenotypes, demonstrating the potential to generate new coloured strains. There was no consistent impact on mycophenolic acid production as a result of pathway interruption although levels of roquefortine C were altered in some deletants. Importantly, levels of methyl-ketones associated with blue-cheese flavour were not impacted. UV-induced colour mutants, allowed in food production, were then generated. A range of colours were obtained and certain phenotypes were successfully mapped to pathway gene mutations. Selected colour mutants were subsequently used in cheese production and generated expected new colourations with no elevated mycotoxins, offering the exciting prospect of use in future cheese manufacture.

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Covalent hits for drug discovery campaigns are neither fantastic beasts nor mythical creatures, they can be routinely identified through electrophile-first screening campaigns using a suite of different techniques. These include biophysical and biochemical methods, cellular approaches, and DNA-encoded libraries. Employing best practice, however, is critical to success. The purpose of this review is to look at state of the art covalent hit identification, how to identify hits from a covalent library and how to select compounds for medicinal chemistry programmes.

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AIM: To explain the process by which nurses' roles are negotiated in general practice. BACKGROUND: Primary care nurses do important work within a social model of health to meet the needs of the populations they serve. Latterly, in the face of increased demand and workforce shortages, they are also taking on more medical responsibilities through task-shifting. Despite the increased complexity of their professional role, little is known about the processes by which it is negotiated. DESIGN: Constructivist grounded theory. METHODS: Semi-structured interviews were conducted with 22 participants from 17 New Zealand general practices between December 2020 and January 2022. Due to COVID-19, 11 interviews were via Zoom™. Concurrent data generation and analysis, using the constant comparative method and common grounded theory methods, identified the participants' main concern and led to the construction of a substantive explanatory theory around a core category. RESULTS: The substantive explanatory theory of creating place proposes that the negotiation of nurse roles within New Zealand general practice is a three-stage process involving occupying space, positioning to do differently and leveraging opportunity. Nurses and others act and interact in these stages, in accordance with their conceptualizations of need-responsive nursing practice, towards the outcome defining place. Defining place conceptualizes an accommodation between the values beliefs and expectations of individuals and pre-existing organizational norms, in which individual and group-normative concepts of need-responsive nursing practice are themselves developed. CONCLUSION: The theory of creating place provides new insights into the process of nurses' role negotiation in general practice. Findings support strategies to enable nurses, employers and health system managers to better negotiate professional roles to meet the needs of the populations they serve, while making optimum use of nursing skills and competencies. IMPLICATIONS FOR THE PROFESSION AND/OR PATIENT CARE: Findings can inform nurses to better negotiate the complexities of the primary care environment, balancing systemic exigencies with the health needs of populations. IMPACT: What Problem Did the Study Address? In the face of health inequity, general practice nurses in New Zealand, as elsewhere, are key to meeting complex primary health needs. There is an evidence gap regarding the processes by which nurses' roles are negotiated within provider organizations. A deeper understanding of such processes may enable better use of nursing skills to address unmet health need. What Were the Main Findings? Nurses' roles in New Zealand general practice are determined through goal-driven negotiation in accordance with individual concepts of need-responsive nursing practice. Individuals progress from occupying workspaces defined by the care-philosophies of others to defining workplaces that incorporate their own professional beliefs, values and expectations. Negotiation is conditional upon access to role models, scheduled dialogue with mentors and decision-makers, and support for safe practice. Strong clinical and organizational governance and individuals' own positive personal self-efficacy are enablers of effective negotiation. Where and on Whom Will the Research Have Impact? The theory of Creating Space can inform organizational and individual efforts to advance the roles of general practice nurses to meet the health needs of their communities. General practice organizations can provide safe, supported environments for effective negotiation; primary care leaders can promote strong governance and develop individuals' sense of self-efficacy by involving them in key decisions. Nurses themselves can use the theory as a framework to support critical reflection on how to engage in active negotiation of their professional roles. REPORTING METHOD: The authors adhered to relevant EQUATOR guidelines using the COREQ reporting method. PATIENT OR PUBLIC CONTRIBUTION: Researchers and participants currently working in general practice were involved in the development of this study. By the process of theoretical sampling and constant comparison, participants' comments helped to shape the study design. WHAT DOES THIS PAPER CONTRIBUTE TO THE WIDER GLOBAL CLINICAL COMMUNITY?: An understanding of the processes by which health professionals negotiate their roles is important to support them to meet the challenges of increased complexity across all health sectors globally.

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BACKGROUND: Prior research findings are mixed regarding whether prosocial behavior is positively or negatively related to socioeconomic status and its correlates, such as economic pressure. This may be due to the lack of considerations for the type of prosocial behavior. AIMS: In this study, we aimed to examine how six types of prosocial behavior (i.e., public, anonymous, compliant, emotional, dire, and altruistic) are related to economic pressure among early adolescents. We hypothesized that family economic pressure would be associated with each type of prosocial behavior in differing ways. MATERIALS & METHODS: Participants were 11-14 years old (N = 143, Mage = 12.2 years, SDage = 0.87, 63 boys, 1 trans-identified boy, 55 girls), early adolescents and their parents. Among them, 54.6% were non-Hispanic/Latinx (NH/L) White, 23.8% were NH/L Black, 11.2% were NH/L Asian, 2.1% were NH/L Multiracial, and 8.4% were Hispanic/Latinx. Parents reported family economic pressure and adolescents' six types of prosocial behavior. RESULTS: Path analysis revealed that economic pressure was negatively associated with emotional and dire prosocial behavior over and above age, gender, and race/ethnicity. Family economic pressure was unrelated to public, anonymous, compliant, and altruistic prosocial behavior. DISCUSSION: These findings show some support for the Family Stress Model, such that economic stress might hinder youth's prosocial development. At the same time, youth may have similar levels of certain types of prosocial behavior regardless of their family's economic pressure. CONCLUSION: This research provided insight into the complex relation between economic pressure and youth's prosocial behavior which varies depending on the type of behavior.

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Sulfotransferases constitute a ubiquitous class of enzymes which are poorly understood due to the lack of a convenient tool for screening their activity. These enzymes use the anion PAPS (adenosine-3'-phosphate-5'-phosphosulfate) as a donor for a broad range of acceptor substrates, including carbohydrates, producing sulfated compounds and PAP (adenosine-3',5'-diphosphate) as a side product. We present a europium(III)-based probe that binds reversibly to both PAPS and PAP, producing a larger luminescence enhancement with the latter anion. We exploit this greater emission enhancement with PAP to demonstrate the first direct real-time assay of a heparan sulfate sulfotransferase using a multi-well plate format. The selective response of our probe towards PAP over structurally similar nucleoside phosphate anions, and over other anions, is investigated and discussed. This work opens the possibility of investigating more fully the roles played by this enzyme class in health and disease, including operationally simple inhibitor screening.

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A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral LnIII tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with TbIII or TmIII ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron-electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the TbIII complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.

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Anion receptors can be used to transport ions across lipid bilayers, which has potential for therapeutic applications. Synthetic bicarbonate transporters are of particular interest, as defects in transmembrane transport of bicarbonate are associated with various diseases. However, no convenient method exists to directly observe bicarbonate transport and study the mechanisms involved. Here, an assay is presented that allows the kinetics of bicarbonate transport into liposomes to be monitored directly and with great sensitivity. The assay utilises an encapsulated europium(III) complex, which exhibits a large increase in emission intensity upon binding bicarbonate. Mechanisms involving CO2 diffusion and the dissipation of a pH gradient are shown to be able to lead to an increase in bicarbonate concentration within liposomes, without transport of the anion occurring at all. By distinguishing these alternative mechanisms from actual bicarbonate transport, this assay will inform the future development of bicarbonate transporters.

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Invited for the cover of this issue are Dr. Stephen Butler, Dr. Hennie Valkenier and co-workers at Université Libre de Bruxelles, Loughborough University, Masaryk University, and the University of Bristol. The image depicts the transport of bicarbonate anions versus the spontaneous diffusion of CO2 across the lipid bilayer of a liposome. Read the full text of the article at 10.1002/chem.202100491.

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Rapid ascent to high altitude imposes an acute hypoxic and acid-base challenge, with ventilatory and renal acclimatization countering these perturbations. Specifically, ventilatory acclimatization improves oxygenation, but with concomitant hypocapnia and respiratory alkalosis. A compensatory, renally mediated relative metabolic acidosis follows via bicarbonate elimination, normalizing arterial pH(a). The time course and magnitude of these integrated acclimatization processes are highly variable between individuals. Using a previously developed metric of renal reactivity (RR), indexing the change in arterial bicarbonate concentration (Δ[HCO3-]a; renal response) over the change in arterial pressure of CO2 (Δ[Formula: see text]; renal stimulus), we aimed to characterize changes in RR magnitude following rapid ascent and residence at altitude. Resident lowlanders (n = 16) were tested at 1,045 m (day [D]0) prior to ascent, on D2 within 24 h of arrival, and D9 during residence at 3,800 m. Radial artery blood draws were obtained to measure acid-base variables: [Formula: see text], [HCO3-]a, and pHa. Compared with D0, [Formula: see text] and [HCO3-]a were lower on D2 (P < 0.01) and D9 (P < 0.01), whereas significant changes in pHa (P = 0.072) and RR (P = 0.056) were not detected. As pHa appeared fully compensated on D2 and RR did not increase significantly from D2 to D9, these data demonstrate renal acid-base compensation within 24 h at moderate steady-state altitude. Moreover, RR was strongly and inversely correlated with ΔpHa on D2 and D9 (r≤ -0.95; P < 0.0001), suggesting that a high-gain renal response better protects pHa. Our study highlights the differential time course, magnitude, and variability of integrated ventilatory and renal acid-base acclimatization following rapid ascent and residence at high altitude.NEW & NOTEWORTHY We assessed the time course, magnitude, and variability of integrated ventilatory and renal acid-base acclimatization with rapid ascent and residence at 3,800 m. Despite reductions in [Formula: see text] upon ascent, pHa was normalized within 24 h of arrival at 3,800 m through renal compensation (i.e., bicarbonate elimination). Renal reactivity (RR) was unchanged between days 2 and 9, suggesting a lack of plasticity at moderate steady-state altitude. RR was strongly correlated with ΔpHa, suggesting that a high-gain renal response better protects pHa.

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Limited knowledge about the nursing workforce in New Zealand general practice inhibits the optimal use of nurses in this increasingly complex setting. Using workforce survey data published biennially by the Nursing Council of New Zealand, this study describes the characteristics of nurses in general practice and contrasts them with the greater nursing workforce, including consideration of changes in the profiles between 2015 and 2019. The findings suggest the general practice nursing workforce is older, less diverse, more predominately New Zealand trained and very much more likely to work part-time than other nurses. There is evidence that nurses in general practice are increasingly primary health care focused, as they take on expanded roles and responsibilities. However, ambiguity about terminology and the inability to track individuals in the data are limitations of this study. Therefore, it was not possible to identify and describe cohorts of nurses in general practice by important characteristics, such as prescribing authority, regionality and rurality. A greater national focus on defining and tracking this pivotal workforce is called for to overcome role confusion and better facilitate the use of nursing scopes of practice.

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Mitochondrial DNA (mtDNA) in yeast is biparentally inherited, but colonies rapidly lose one type of parental mtDNA, thus becoming homoplasmic. Therefore, hybrids between the yeast species possess two homologous nuclear genomes, but only one type of mitochondrial DNA. We hypothesise that the choice of mtDNA retention is influenced by its contribution to hybrid fitness in different environments, and the allelic expression of the two nuclear sub-genomes is affected by the presence of different mtDNAs in hybrids. Saccharomyces cerevisiae/S. uvarum hybrids preferentially retained S. uvarum mtDNA when formed on rich media at colder temperatures, while S. cerevisiae mtDNA was primarily retained on non-fermentable carbon source, at any temperature. Transcriptome data for hybrids harbouring different mtDNA showed a strong environmentally dependent allele preference, which was more important in respiratory conditions. Co-expression analysis for specific biological functions revealed a clear pattern of concerted allelic transcription within the same allele type, which supports the notion that the hybrid cell works preferentially with one set of parental alleles (or the other) for different cellular functions. Given that the type of mtDNA retained in hybrids affects both nuclear expression and fitness, it might play a role in driving hybrid genome evolution in terms of gene retention and loss.

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Recent findings suggest that dopamine oxidation contributes to the development of Parkinson's disease (PD); however, the mechanistic details remain elusive. Here, we compare 6-hydroxydopamine (6-OHDA), a product of dopamine oxidation that commonly induces dopaminergic neurodegeneration in laboratory animals, with a synthetic alkyne-functionalized 6-OHDA variant. This synthetic molecule provides insights into the reactivity of quinone and neuromelanin formation. Employing Huisgen cycloaddition chemistry (or "click chemistry") and fluorescence imaging, we found that reactive 6-OHDA p-quinones cause widespread protein modification in isolated proteins, lysates and cells. We identified cysteine thiols as the target site and investigated the impact of proteome modification by quinones on cell viability. Mass spectrometry following cycloaddition chemistry produced a large number of 6-OHDA modified targets including proteins involved in redox regulation. Functional in vitro assays demonstrated that 6-OHDA inactivates protein disulfide isomerase (PDI), which is a central player in protein folding and redox homeostasis. Our study links dopamine oxidation to protein modification and protein folding in dopaminergic neurons and the PD model.

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The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.

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The development of synthetic receptors for the selective binding and discrimination of anions in water requires an understanding of how anions interact with these synthetic receptors. Molecules designed to differentiate nucleoside phosphate anions (e.g. ATP, ADP, GTP, GDP, UDP) under physiological conditions could underpin exciting new sensing tools for biomedical research and drug discovery, but it is very challenging due to the similarities in anion structure, size and charge. We present a series of lanthanide-based anion receptors and establish key structural elements that impact on nucleoside phosphate anion binding and sensing. Structural evidence of anion binding using X-ray crystallographic and NMR data, supported by DFT calculations indicate the binding modes between the lanthanide complexes and certain phosphoanions, revealing a bidentate (α-, γ-) binding mode to ATP. We further use four of the receptors to allow discrimination of eight nucleoside phosphate anions in the first array-based assay using lanthanide complexes, taking advantage of the multiple emission bands and long emission lifetimes associated with luminescent lanthanide complexes.

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Enzymes that consume and produce nucleoside polyphosphate (NPP) anions represent major targets in drug discovery. For example, protein kinases are one of the largest classes of drug targets in the fight against cancer. The accurate determination of enzyme kinetics and mechanisms is a critical aspect of drug discovery research. To increase confidence in the selection of lead drug compounds it is crucial that pharmaceutical researchers have robust, affordable assays to measure enzyme activity accurately. We present a simple, sensitive microplate assay for real-time monitoring of a range of pharmaceutically important enzyme reactions that generate NPP anions, including kinases and glycosyltransferases. Our assay utilises a single, stable europium(iii) complex that binds reversibly to NPP anions, signalling the dynamic changes in NPP product/substrate ratio during an enzyme reaction using time-resolved luminescence. This supramolecular approach to enzyme monitoring overcomes significant limitations in existing assays, obviating the need for expensive antibodies or equipment, chemically labelled substrates or products and isolation or purification steps. Our label and antibody-free method enables rapid and quantitative analysis of enzyme activities and inhibition, offering a potentially powerful tool for use in drug discovery, suitable for high-throughput screening of inhibitors and accurate measurements of enzyme kinetic parameters.

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Molecular diagnostic investigations, following the identification of foetal abnormalities, are routinely performed using array comparative genomic hybridisation (aCGH). Despite the utility of this technique, contemporary approaches for the detection of copy number variation are typically based on next-generation sequencing (NGS). We sought to compare an in-house NGS-based workflow (CNVseq) with aCGH, for invasively obtained foetal samples from pregnancies complicated by foetal structural abnormality. DNA from 40 foetuses was screened using both 8 × 60 K aCGH oligoarrays and low-coverage whole genome sequencing. Sequencer-compatible libraries were combined in a ten-sample multiplex and sequenced using an Illumina HiSeq2500. The mean resolution of CNVseq was 29 kb, compared to 60 kb for aCGH analyses. Four clinically significant, concordant, copy number imbalances were detected using both techniques, however, genomic breakpoints were more precisely defined by CNVseq. This data indicates CNVseq is a robust and sensitive alternative to aCGH, for the prenatal investigation of foetuses with structural abnormalities. Impact statement What is already known about this subject? Copy number variant analysis using next-generation sequencing has been successfully applied to investigations of tumour specimens and patients with developmental delays. The application of our approach, to a prospective prenatal diagnosis cohort, has not hitherto been assessed. What do the results of this study add? Next-generation sequencing has a comparable turnaround time and assay sensitivity to copy number variant analysis performed using array CGH. We demonstrate that having established a next-generation sequencing facility, high-throughput CNVseq sample processing and analysis can be undertaken within the framework of a regional diagnostic service. What are the implications of these findings for clinical practice and/or further research? Array CGH is a legacy technology which is likely to be superseded by low-coverage whole genome sequencing, for the detection of copy number variants, in the prenatal diagnosis of structural abnormalities.

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Dynamic combinatorial chemistry (DCC) represents an approach, whereby traditional supramolecular scaffolds used for protein surface recognition might be exploited to achieve selective high affinity target recognition. Synthesis, in situ screening and amplification under selection pressure allows the generation of ligands, which bear different moieties capable of making multivalent non-covalent interactions with target proteins. Generic tetracarboxyphenyl porphyrin scaffolds bearing four hydrazide moieties have been used to form dynamic combinatorial libraries (DCLs) using aniline-catalyzed reversible hydrazone exchange reactions, in 10 % DMSO, 5 mm NH4OAc, at pH 6.75. High resolution mass spectrometry (HRMS) was used to monitor library composition and establish conditions under which equilibria were established.