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The automated synthesis of small organic molecules from modular building blocks has the potential to transform our capacity to create medicines and materials 1-3 . Disruptive acceleration of this molecule-building strategy broadly unlocks its functional potential and requires the integration of many new assembly chemistries. Although recent advances in high-throughput chemistry 4-6 can speed up the development of appropriate synthetic methods, for example, in selecting appropriate chemical reaction conditions from the vast range of potential options, equivalent high-throughput analytical methods are needed. Here we report a streamlined approach for the rapid, quantitative analysis of chemical reactions by mass spectrometry. The intrinsic fragmentation features of chemical building blocks generalize the analyses of chemical reactions, allowing sub-second readouts of reaction outcomes. Central to this advance was identifying that starting material fragmentation patterns function as universal barcodes for downstream product analysis by mass spectrometry. Combining these features with acoustic droplet ejection mass spectrometry 7,8 we could eliminate slow chromatographic steps and continuously evaluate chemical reactions in multiplexed formats. This enabled the assignment of reaction conditions to molecules derived from ultrahigh-throughput chemical synthesis experiments. More generally, these results indicate that fragmentation features inherent to chemical synthesis can empower rapid data-rich experimentation., Competing Interests: Competing interests: D.J.B. is listed as an inventor on patents relating to TIDA boronates, and St Jude Children’s Research Hospital have filed patents relating to the new fragmentation patterns described in this study., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (-)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1's tert-butyl moiety as a PXR anchor and explains binding by (-)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (-)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

This study aimed to develop a streamlined method for evaluating the dilution ratio of drug dose-response plates created by automated liquid handlers in the early stages of drug discovery. The quantitative techniques commonly used for this purpose have restrictions due to their limited linear dynamic range and inaccuracies in assessing serial dilution performance. To address this challenge, we describe a method based on acoustic ejection mass spectrometry (AEMS). The method involves using standard compounds and an internal standard to evaluate each dilution point in quality control (QC) plates. The samples are transferred to a chromatography-free tandem mass spectrometry system through an acoustic source, enabling the analysis of one sample per three seconds from a microtiter plate. This approach provides precise, accurate, label-free, and rapid data acquisition to support high-throughput screening efforts., Competing Interests: Declaration of Competing Interest The authors state that they have no competing financial interests or personal relationships that may have influenced the work presented in this manuscript., (Copyright © 2023. Published by Elsevier Inc.)

Influenza is one of the leading causes of disease-related mortalities worldwide. Several strategies have been implemented during the past decades to hinder the replication cycle of influenza viruses, all of which have resulted in the emergence of resistant virus strains. The most recent example is baloxavir marboxil, where a single mutation in the active site of the target endonuclease domain of the RNA-dependent-RNA polymerase renders the recent FDA approved compound ∼1000-fold less effective. Raltegravir is a first-in-class HIV inhibitor that shows modest activity to the endonuclease. Here, we have used structure-guided approaches to create rationally designed derivative molecules that efficiently engage the endonuclease active site. The design strategy was driven by our previously published structures of endonuclease-substrate complexes, which allowed us to target functionally conserved residues and reduce the likelihood of resistance mutations. We succeeded in developing low nanomolar equipotent inhibitors of both wild-type and baloxavir-resistant endonuclease. We also developed macrocyclic versions of these inhibitors that engage the active site in the same manner as their 'open' counterparts but with reduced affinity. Structural analyses provide clear avenues for how to increase the affinity of these cyclic compounds., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Stephen White reports a relationship with Life Science Tennessee that includes: board membership. Zoran Rankovic reports a relationship with Revolution Medicines that includes: consulting or advisory. Zoran Rankovic reports a relationship with Orum Therapeutics that includes: consulting or advisory. Zoran Rankovic reports a relationship with Nyrada Inc that includes: consulting or advisory. Zoran Rankovic reports a relationship with Eli Lilly that includes: equity or stocks., (Copyright © 2022. Published by Elsevier Masson SAS.)

Sen's Capability Approach is one of the most significant theoretical contributions to welfare analysis across a range of disciplines. A part of the literature argues that its conceptual linear flow-from resources to 'functionings', which result in well-being-potentially ignores more complex relations with the feedback loops where a single item could be viewed as having a different role by different people, in different contexts. We explore perceptions of existing feedback relationships in interviews with 74 women from nine seaweed farming villages in Indonesia, engaged in two distinct activities: seaweed farming and artisanal seaweed processing. We find that capability sets required for farming and for processing are distinct and in both cases we observe feedback loops. Several factors, notably social networks and transportation (motorbikes), were mentioned in almost all contexts indicating that not all resources are of equal 'value' and might yield different levels of well-being., (© 2021. Royal Swedish Academy of Sciences.)

We recently demonstrated that dietary supplementation with seaweed leads to dramatic improvements in immune responses in S. fuscescens , a candidate species for aquaculture development in Asia. Here, to assess whether the immunostimulatory effect was facilitated by changes to the gut microbiome, we investigated the effects of those same seaweed species and four commercial feed supplements currently used in aquaculture on the bacterial communities in the hindgut of the fish. Since we found no correlations between the relative abundance of any particular taxa and the fish enhanced innate immune responses, we hypothesised that S. fuscescens might have a core microbiome that is robust to dietary manipulation. Two recently published studies describing the bacteria within the hindgut of S. fuscescens provided an opportunity to test this hypothesis and to compare our samples to those from geographically distinct populations. We found that, although hindgut bacterial communities were clearly and significantly distinguishable between studies and populations, a substantial proportion (55 of 174 taxa) were consistently detected across all populations. Our data suggest that the importance of gut microbiota to animal health and the extent to which they can be influenced by dietary manipulations might be species-specific or related to an animals' trophic level.

Disease is one of the major bottlenecks for aquaculture development, costing the industry in excess of US $6 billion each year. The increase in pressure to phase out some traditional approaches to disease control (e.g. antibiotics) is pushing farmers to search for alternatives to treat and prevent disease outbreaks, which do not have detrimental consequences (e.g. antibiotic resistance). We tested the effects of eleven seaweed species and four established fish immunostimulants on the innate immune response (cellular and humoral immunity) of the rabbitfish Siganus fuscescens. All supplements including different seaweeds from the three groups (Chlorophyta, Phaeophyta and Rhodophyta) were included in the fish pellet at 3% (by weight) and had variably positive effects across the four innate immune parameters we measured compared to control fish. Diets supplemented with the red seaweed Asparagopsis taxiformis and the brown seaweed Dictyota intermedia led to the largest boosts in humoral and cellular innate immune defences, including particularly significant increases in haemolytic activity. Diets supplemented with Ulva fasciata also led to promising positive effects on the fish innate immune responses. We conclude that dietary seaweed supplements can boost the immune response of S. fuscescens and thus the top three species highlighted in this study should be further investigated for this emerging aquaculture species and other fish species., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Recent reports have shown that Gram-positive bacteria actively secrete spherical nanometer-sized proteoliposome membrane vesicles (MVs) into their surroundings. Though MVs are implicated in a broad range of biological functions, few studies have been conducted to examine their potential as delivery vehicles of antimicrobials. Here, we investigate the natural ability of Lactobacillus acidophilus MVs to carry and deliver bacteriocin peptides to the opportunistic pathogen, Lactobacillus delbrueckii . We demonstrate that upon treatment with lactacin B-inducing peptide, the proteome of the secreted MVs is enriched in putative bacteriocins encoded by the lab operon. Further, we show that purified MVs inhibit growth and compromise membrane integrity in L. delbrueckii , which is confirmed by confocal microscopy imaging and spectrophotometry. These results show that L. acidophilus MVs serve as conduits for antimicrobials to competing cells in the environment, suggesting a potential role for MVs in complex communities such as the gut microbiome. With the potential for controlling their payload through microbial engineering, MVs produced by L. acidophilus may be an interesting platform for effecting change in complex microbial communities or aiding in the development of new biomedical therapeutics., (Copyright © 2020 Dean, Rimmer, Turner, Phillips, Caruana, Hervey, Leary and Walper.)

In the tightly regulated glycogenolysis cascade, the breakdown of glycogen to glucose-1-phosphate, phosphorylase kinase (PhK) plays a key role in regulating the activity of glycogen phosphorylase. PhK is a 1.3 MDa hexadecamer, with four copies each of four different subunits (α, β, γ and δ), making the study of its structure challenging. Using hydrogen-deuterium exchange, we have analyzed the regulatory β subunit and the catalytic γ subunit in the context of the intact non-activated PhK complex to study the structure of these subunits and identify regions of surface exposure. Our data suggest that within the non-activated complex the γ subunit assumes an activated conformation and are consistent with a previous docking model of the β subunit within the cryoelectron microscopy envelope of PhK., (© 2017 The Protein Society.)

Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ) 4 , and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full-length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen-deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N-terminal glycoside hydrolase domain and a large C-terminal importin α/β-like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen-deuterium exchange results obtained for this subunit as part of the (αβγδ) 4 PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter-subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low-exchanging region in the C-terminus of α that is known to bind the regulatory domain of the catalytic γ subunit., (© 2017 The Protein Society.)

Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: (1) hydrogen/deuterium exchange (HDX), (2) limited proteolysis, and (3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography.

In the six decades since its discovery, phosphorylase kinase (PhK) from rabbit skeletal muscle has usually been studied at 30 °C; in fact, not a single study has examined functions of PhK at a rabbit's body temperature, which is nearly 10 °C greater. Thus, we have examined aspects of the activity, regulation, and structure of PhK at temperatures between 0 and 40 °C. Between 0 and 30 °C, the activity at pH 6.8 of nonphosphorylated PhK predictably increased; however, between 30 and 40 °C, there was a dramatic jump in its activity, resulting in the nonactivated enzyme having a far greater activity at body temperature than was previously realized. This anomalous change in properties between 30 and 40 °C was observed for multiple functions, and both stimulation (by ADP and phosphorylation) and inhibition (by orthophosphate) were considerably less pronounced at 40 °C than at 30 °C. In general, the allosteric control of PhK's activity is definitely more subtle at body temperature. Changes in behavior related to activity at 40 °C and its control can be explained by the near disappearance of hysteresis at physiological temperature. In important ways, the picture of PhK that has emerged from six decades of study at temperatures of ≤30 °C does not coincide with that of the enzyme studied at physiological temperature. The probable underlying mechanism for the dramatic increase in PhK's activity between 30 and 40 °C is an abrupt change in the conformations of the regulatory β and catalytic γ subunits between these two temperatures., Competing Interests: Notes The authors declare no competing financial interest.

Phosphorylase kinase (PhK) is a 1.3 MDa (αβγδ)4 enzyme complex, in which αβγδ protomers associate in D2 symmetry to form two large octameric lobes that are interconnected by four bridges. The approximate locations of the subunits have been mapped in low-resolution cryo-electron microscopy structures of the complex; however, the disposition of the subunits within the complex remains largely unknown. We have used partial proteolysis and chemical footprinting in combination with high-resolution mass spectrometry to identify surface-exposed regions of the intact nonactivated and phospho-activated conformers. In addition to the known interaction of the γ subunit's C-terminal regulatory domain with the δ subunit (calmodulin), our exposure results indicate that the catalytic core of γ may also anchor to the PhK complex at the bottom backside of its C-terminal lobe facing away from the active site cleft. Exposed loops on the α and β regulatory subunits within the complex occur at regions overlapping with tissue-specific alternative RNA splice sites and regulatory phosphorylatable domains. Their phosphorylation alters the surface exposure of α and β, corroborating previous biophysical and biochemical studies that detected phosphorylation-dependent conformational changes in these subunits; however, for the first time, specific affected regions have been identified., Competing Interests: Notes The authors declare no competing financial interest.

Hantaviruses, members of the Bunyaviridae family, are negative-stranded emerging RNA viruses and category A pathogens that cause serious illness when transmitted to humans through aerosolized excreta of infected rodent hosts. Hantaviruses have evolved a novel translation initiation mechanism, operated by nucleocapsid protein (N), which preferentially facilitates the translation of viral mRNAs. N binds to the ribosomal protein S19 (RPS19), a structural component of the 40 S ribosomal subunit. In addition, N also binds to both the viral mRNA 5' cap and a highly conserved triplet repeat sequence of the viral mRNA 5' UTR. The simultaneous binding of N at both the terminal cap and the 5' UTR favors ribosome loading on viral transcripts during translation initiation. We characterized the binding between N and RPS19 and demonstrate the role of the N-RPS19 interaction in N-mediated translation initiation mechanism. We show that N specifically binds to RPS19 with high affinity and a binding stoichiometry of 1:1. The N-RPS19 interaction is an enthalpy-driven process. RPS19 undergoes a conformational change after binding to N. Using T7 RNA polymerase, we synthesized the hantavirus S segment mRNA, which matches the transcript generated by the viral RNA-dependent RNA polymerase in cells. We show that the N-RPS19 interaction plays a critical role in the translation of this mRNA both in cells and rabbit reticulocyte lysates. Our results demonstrate that the N-mediated translation initiation mechanism, which lures the host translation machinery for the preferential translation of viral transcripts, primarily depends on the N-RPS19 interaction. We suggest that the N-RPS19 interaction is a novel target to shut down the N-mediated translation strategy and hence virus replication in cells.

Antisera to mammalian IGFs cross-react with several fish species, suggesting a long phylogenetic history of IGF-like peptides and their functional importance to all vertebrates. In this study, the tissue distribution of IGF-I-immunoreactivity (IGF-I-IR) was studied at various larval and juvenile stages of the life cycle of barramundi (Lates calcarifer). It was shown that the distribution of IGF-I-IR in this species was tissue-specific and age-dependent. In newly hatched larvae, presumptive musculature in the trunk and the pectoral fin rudiments reacted positively for IGF-I. As specimen age increased, however, IGF-I-IR in these tissues became less evident. In the retinas of barramundi 42 hr and older, a distinct band of IGF-I-IR was consistently detected between the presumptive outer nuclear and bacillary layers. Examination of sections from specimens 9 days and older revealed strong reactivity for IGF-I in a large proportion of renal tubule epithelial cells. In sections from larvae 13 to 28 days old, diffuse cytoplasmic IGF-I-IR was identified in cells lining the gill structures. At these same developmental stages, IGF-I reactive cells were also observed in the islets of Langerhans. In young juveniles (22 to 28 days posthatching), sparsely scattered clusters of neurons in the lower brain were observed which exhibited granular IGF-I-IR in perikarya. In all instances reported, IGF-I-IR in barramundi tissue was abolished by replacing antisera with normal rabbit serum or by preabsorption of antisera with purified IGF-I peptide, indicating the specificity of the reactions obtained. The distribution patterns of IGF-I-IR in barramundi tissues were broadly consistent with the reported distributions of IGF-I-like peptides and transcripts in other teleost species. The findings of this study are in general agreement with the hypothesis that IGF-I-like peptides may be involved in the regulation of tissue growth, differentiation, and function during early barramundi development.

The effect of chronic hyperglycemia and polyol pathway activation on the Schwann cell has not been resolved although injury to this cell has long been suspected in diabetic neuropathy. Hyperglycemia, resulting from galactose intoxication of four months duration, induces dose-dependent accumulations of endoneurial fluid sodium and chloride that are linked to polyol pathway activity and associated with dose-dependent increases in sciatic nerve water content, endoneurial fluid pressure and (Na+, K+)-ATPase activity. In order to understand the impact of these changes on the nerve microenvironment, cellular elements of the endoneurium were quantitatively and qualitatively assessed in rats receiving 0%, 10%, 20% or 40% galactose diets. After four months of galactose intoxication, dose-dependent changes in the size distribution of myelinated nerve fibers were apparent. A shift in size-frequency histograms of galactose-intoxicated animals towards smaller fibers was accompanied by a decrease in axon diameter and the volume fraction ratio of axon to myelinated nerve fibers. In the sciatic nerve of all 40% galactose-fed rats examined by electron microscopy, Schwann cells of myelinated fibers showed both reactive and degenerative changes. Demyelination was preceded by splitting at the intraperiod line. Remyelination was identified by axons with disproportionately thin myelin sheaths. Axonal dystrophy and degeneration were infrequently seen, but there was axonal regeneration. Dose-dependent increases in mast cell number were observed with degranulation apparent in rats receiving 20% and 40% galactose. Endothelial cell number and basal lamina thickness were increased in the endoneurial vessels of galactose-intoxicated rats. Increased cytoplasmic area and degenerative changes in pericytes were also noted. These observations indicate that significant morphologic changes accompany the hyperosmotic imbalance resulting from galactose intoxication of four months duration. Schwann cell injury and demyelination are present in a disorder linked to polyol metabolism since aldose reductase, the anabolic enzyme of the polyol pathway, is localized to this myelin-forming cell.