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Key Points O -GlcNAcylation is a nutrient- and stress-responsive post-translational modification (PTM) that involves the attachment of O -linked N -acetylglucosamine moieties to Ser and Thr residues of cytoplasmic, nuclear and mitochondrial proteins. A single pair of enzymes — O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA) — controls the dynamic cycling of this PTM. Potential mechanisms that enable a single OGT enzyme to recognize hundreds of protein substrates include substrate-specific interactions with the tetratricopeptide repeat (TPR) domain of OGT and context-dependent recruitment of OGT to its substrates by a hierarchy of conserved adaptor proteins. Furthermore, in response to cellular stress, O -GlcNAcylation may occur nonspecifically in unstructured regions of unfolded proteins in order to block their aggregation and degradation and facilitate their refolding. O -GlcNAcylation is involved in the spatiotemporal regulation of diverse cellular processes, which include transcription, epigenetic modifications and cell signalling dynamics. O -GlcNAcylation is highly dynamic and often transient, but the mechanisms underlying the temporal control of O -GlcNAc signalling are largely unknown. Nutrient availability regulates cellular O -GlcNAcylation levels not only by determining the abundance of the donor substrate uridine diphosphate GlcNAc (UDP-GlcNAc) but also by modulating the levels of OGT, OGA and their respective adaptor proteins and substrates. Hormones such as insulin, glucagon and ghrelin are secreted in response to systemic metabolic changes and modulate O -GlcNAc signalling in specific cell types and tissues to regulate key response pathways that help maintain metabolic homeostasis. Cellular O -GlcNAcylation levels may be maintained within an 'optimal zone' by a 'buffering system' that is generated by mutual regulation of OGT and OGA at the transcriptional and post-translational levels. Maintenance of O -GlcNAc homeostasis is essential for optimal cellular function, and disruption of the cellular O -GlcNAcylation 'buffer' may contribute to the pathogenesis of various human diseases. O -GlcNAcylation can be viewed as the essential 'grease and glue' of the cell: it acts as a 'grease' by coating target proteins (folded or unfolded, mature or nascent) and preventing unwanted protein aggregation or modification; it also acts as a 'glue' by modulating protein–protein interactions in time and space in response to internal and external cues, thereby affecting the functions of various proteins in the cell. Many cellular proteins are reversibly modified by O -linked N -acetylglucosamine ( O -GlcNAc) moieties on Ser and Thr residues. Studies on the mechanisms and functions of O -GlcNAcylation and its links to metabolism reveal the importance of this modification in the maintenance of cellular and organismal homeostasis. O -GlcNAcylation — the attachment of O -linked N -acetylglucosamine ( O -GlcNAc) moieties to cytoplasmic, nuclear and mitochondrial proteins — is a post-translational modification that regulates fundamental cellular processes in metazoans. A single pair of enzymes — O -GlcNAc transferase (OGT) and O -GlcNAcase (OGA) — controls the dynamic cycling of this protein modification in a nutrient- and stress-responsive manner. Recent years have seen remarkable advances in our understanding of O -GlcNAcylation at levels that range from structural and molecular biology to cell signalling and gene regulation to physiology and disease. New mechanisms and functions of O -GlcNAcylation that are emerging from these recent developments enable us to begin constructing a unified conceptual framework through which the significance of this modification in cellular and organismal physiology can be understood.

Background Musculoskeletal (MSK) conditions are highly prevalent and represent a major burden in Australia and worldwide, yet undergraduate musculoskeletal education remains underrepresented in medical curricula. Near-peer teaching (NPT) has emerged as an effective adjunct to standard teaching, particularly in developing clinical examination skills. Objective To examine the effect of a near-peer-led, OSCE-style tutorial on medical student performance of the upper limb musculoskeletal examination, and to explore students’ perceptions of its clarity, relevance, and educational value. Methods A quasi-experimental pre–post study was conducted with 23 second year-year medical students in a problem-based learning program. Participants completed a pre-test OSCE, attended a near-peer-led tutorial, and undertook a post-test OSCE three days later. Assessments were scored using a 15-item rubric with percentage conversion. A post-hoc survey comprising Likert-scale and open-ended questions explored student perceptions. Quantitative data were analysed using paired statistical tests, while qualitative responses underwent content analysis. Results Improvements were observed in 14 of 15 OSCE items, with statistically significant gains in shoulder and elbow palpation, shoulder active and passive range of motion, patient comfort, and examination organisation ( p  < 0.05). The overall average performance improved from 63.8 ± 15.8% pre-test to 76.3 ± 10.5% post-test ( p  = 0.01). Survey responses ( n  = 17) indicated that all students perceived the tutorial as superior to lectures, textbooks, or videos, with 59% rating it extremely useful for clinical practice. Thematic analysis revealed strengths in logical organisation, integration of anatomy and pathology, demonstration, and opportunities for hands-on practice. Most students reported substantial gains in confidence and understanding. Findings support the utility of NPT in musculoskeletal education, with improvements potentially explained by cognitive and social congruence, experiential learning, and self-efficacy. While a single session significantly enhanced competence and confidence in many components of the physical examination, skills such as special testing requires repeated practice and longer-term follow up to observe similar gains. Conclusion A single near-peer-led, OSCE-style tutorial supports improvement in medical students’ objective performance and self-reported confidence in conducting the upper limb musculoskeletal examination. It is a beneficial adjunct to traditional musculoskeletal curricula for both learners and tutors. Broader, longitudinal research is needed to evaluate long-term knowledge retention and generalisability.

The conserved long noncoding RNA MeXis has anti-atherosclerotic effects in mice by acting with the nuclear hormone receptor LXR in macrophages to promote cholesterol efflux. Nuclear receptors regulate gene expression in response to environmental cues, but the molecular events governing the cell type specificity of nuclear receptors remain poorly understood. Here we outline a role for a long noncoding RNA (lncRNA) in modulating the cell type–specific actions of liver X receptors (LXRs), sterol-activated nuclear receptors that regulate the expression of genes involved in cholesterol homeostasis and that have been causally linked to the pathogenesis of atherosclerosis. We identify the lncRNA MeXis as an amplifier of LXR-dependent transcription of the gene Abca1 , which is critical for regulation of cholesterol efflux. Mice lacking the MeXis gene show reduced Abca1 expression in a tissue-selective manner. Furthermore, loss of MeXis in mouse bone marrow cells alters chromosome architecture at the Abca1 locus, impairs cellular responses to cholesterol overload, and accelerates the development of atherosclerosis. Mechanistic studies reveal that MeXis interacts with and guides promoter binding of the transcriptional coactivator DDX17. The identification of MeXis as a lncRNA modulator of LXR-dependent gene expression expands understanding of the mechanisms underlying cell type–selective actions of nuclear receptors in physiology and disease.

In cell models, changes in the ‘accessible’ pool of plasma membrane (PM) cholesterol are linked with the regulation of endoplasmic reticulum sterol synthesis and metabolism by the Aster family of nonvesicular transporters; however, the relevance of such nonvesicular transport mechanisms for lipid homeostasis in vivo has not been defined. Here we reveal two physiological contexts that generate accessible PM cholesterol and engage the Aster pathway in the liver: fasting and reverse cholesterol transport. During fasting, adipose-tissue-derived fatty acids activate hepatocyte sphingomyelinase to liberate sequestered PM cholesterol. Aster-dependent cholesterol transport during fasting facilitates cholesteryl ester formation, cholesterol movement into bile and very low-density lipoprotein production. During reverse cholesterol transport, high-density lipoprotein delivers excess cholesterol to the hepatocyte PM through scavenger receptor class B member 1. Loss of hepatic Asters impairs cholesterol movement into feces, raises plasma cholesterol levels and causes cholesterol accumulation in peripheral tissues. These results reveal fundamental mechanisms by which Aster cholesterol flux contributes to hepatic and systemic lipid homeostasis. Xiao and Kennelly et al. show that Aster-mediated nonvesicular cholesterol transport in the liver regulates hepatic and systemic lipid homeostasis during fasting, as well as reverse cholesterol transport.

We hypothesized that endothelial progenitor cells derived from individuals with diabetes would exhibit functional defects including inability to respond to hypoxia and altered paracrine/autocrine function that would impair the angiogenic potential of these cells. Circulating mononuclear cells isolated from diabetic (n = 69) and nondiabetic (n = 46) individuals were used to grow endothelial colony forming cells (ECFC), early endothelial progenitor cells (eEPCs) and isolate CD34+ cells. ECFCs and eEPCs were established from only 15% of the diabetic individuals tested thus directing our main effort toward examination of CD34+ cells. CD34+ cells were plated in basal medium to obtain cell-free conditioned medium (CM). In CM derived from CD34+ cells of diabetic individuals (diabetic-CM), the levels of stem cell factor, hepatocyte growth factor, and thrombopoietin were lower, and IL-1β and tumor necrosis factor (TNFα) levels were higher than CM derived from nondiabetic individuals (nondiabetic-CM). Hypoxia did not upregulate HIF1α in CD34+ cells of diabetic origin. Migration and proliferation of nondiabetic CD34+ cells toward diabetic-CM were lower compared to nondiabetic-CM. Attenuation of pressure-induced constriction, potentiation of bradykinin relaxation, and generation of cGMP and cAMP in arterioles were observed with nondiabetic-CM, but not with diabetic-CM. Diabetic-CM failed to induce endothelial tube formation from vascular tissue. These results suggest that diabetic subjects with microvascular complications exhibit severely limited capacity to generate ex-vivo expanded endothelial progenitor populations and that the vasoreparative dysfunction observed in diabetic CD34+ cells is due to impaired autocrine/paracrine function and reduced sensitivity to hypoxia.

Pentametaphosphate is the little studied cyclic pentamer of the metaphosphate ion, [PO3]5 5–. We show that the doubly protonated form of this pentamer can be selectively dehydrated to provide the anhydride [P5O14]3– (1). This trianion is the well-defined condensed phosphate component of a novel reagent for attachment of a pentaphosphate chain to biomolecules all in one go. Here, we demonstrate by extending adenosine monophosphate (AMP) and uridine monophosphate (UMP) to their corresponding nucleoside hexaphosphates, while adenosine diphosphate (ADP) and uridine diphosphate (UDP) are phosphate chain-extended to the corresponding nucleoside heptaphosphates. Such constructs are of interest for their potential biological function with respect to RNA-processing enzymes. Thus, we go on to investigate in detail the interaction of the polyanionic constructs with ribonuclease A, a model protein containing a polycationic active site and for which X-ray crystal structures are relatively straightforward to obtain. This work presents a combined experimental and quantum chemical approach to understanding the interactions of RNase A with the new nucleoside hexa- and heptaphosphate constructs.

At the outset, Chapter 1 begins by examining the central importance of phosphorus in both nature and industry, situating the chemistry of polyphosphates within a broader historical and conceptual context. A brief overview of the history of polyphosphate research in the life sciences is given, as well as a discussion on the persistent ambiguities in condensed phosphate nomenclature. To frame the work in the following chapters, the idea of the \"hydrocarbon analogy\" is introduced: a conceptual strategy that draws parallels between the structure and reactivity of organic molecules and that of inorganic phosphate constructs, thus offering a new way of thinking about molecular complexity in this underexplored chemical space.Building from this foundation, Chapter 2 details the discovery of a diphosphorylation reagent, identified to be a mixture of neutral zwitterionic adducts of P4O10 and pyridine. This reagent emerged serendipitously from our efforts to activate trimetaphosphate and has proved to be a powerful tool for synthesizing functionalized cyclic metaphosphates. Chapter 3 is the extrapolation of our strategy to activate otherwise inert metaphosphates by forming ring-strained bicyclic ultraphosphates. We discuss the reactivity of [P5O14]3–, the oligophosphate analog of the bicyclic hydrocarbon housane. Attempts to push this strategy further toward the synthesis of a hexaphosphorylation reagent were ultimately unsuccessful but provided valuable insight into the limitations of this ring-strain activation paradigm. The methods developed in earlier chapters set the stage for our collaboration with the Fielder group, described in Chapter 4. We designed new reagents to chemoselectively conjugate polyphosphates to densely functionalized peptides and proteins. These synthetic strategies pave the way for the study of recently discovered, but poorly characterized post-translational modifications featuring novel phosphorylation modes. Finally, Chapter 5 presents a series of unpublished studies that expand on the themes of the previous chapter. Together, these investigations contribute to a growing body of knowledge aimed at broadening the chemical space of condensed inorganic phosphates.

High performance computing libraries provide efficient implementations of common computational kernels. Traditionally, such libraries are written in C or assembly. User-schedulable languages provide performance engineers a productive way to optimize these kernels with welldesigned interfaces which provide users control over performance-relevant decisions and automate unnecessary concerns. Often, this is a tradeoff: too much control with too little automation is tedious to program, and too much automation with too little control will hinder obtaining peak performance. The principle of exocompilation advocates for one end of the extreme: to give performance engineers maximal control over code execution so they can maximize performance, its current implementation in existing systems is impractical to use. This thesis broadly explores ways to make exocompilation a practical solution for performance engineers. We show that providing more control does not necessitate sacrificing automation, as long as the language is designed so that users can build their own automation. We explore the necessary design features to enable such a system, demonstrate the types of automation users can build in the system, and brainstorm ways to further push the amount of control user-schedule languages expose to the user.

Multilocular adipocytes are a hallmark of thermogenic adipose tissue 1 , 2 , but the factors that enforce this cellular phenotype are largely unknown. Here, we show that an adipocyte-selective product of the Clstn3 locus (CLSTN3β) present in only placental mammals facilitates the efficient use of stored triglyceride by limiting lipid droplet (LD) expansion. CLSTN3β is an integral endoplasmic reticulum (ER) membrane protein that localizes to ER–LD contact sites through a conserved hairpin-like domain. Mice lacking CLSTN3β have abnormal LD morphology and altered substrate use in brown adipose tissue, and are more susceptible to cold-induced hypothermia despite having no defect in adrenergic signalling. Conversely, forced expression of CLSTN3β is sufficient to enforce a multilocular LD phenotype in cultured cells and adipose tissue. CLSTN3β associates with cell death-inducing DFFA-like effector proteins and impairs their ability to transfer lipid between LDs, thereby restricting LD fusion and expansion. Functionally, increased LD surface area in CLSTN3β-expressing adipocytes promotes engagement of the lipolytic machinery and facilitates fatty acid oxidation. In human fat, CLSTN3B is a selective marker of multilocular adipocytes. These findings define a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes. An adipocyte-selective product of the Clstn3 locus (CLSTN3β) facilitates the use of stored triglyceride by limiting lipid droplet (LD) expansion, defining a molecular mechanism that regulates LD form and function to facilitate lipid utilization in thermogenic adipocytes.

Cancer cells are known to adopt aerobic glycolysis in order to fuel tumor growth, but the molecular basis of this metabolic shift remains largely undefined. O-GlcNAcase (OGA) is an enzyme harboring O-linked β-N-acetylglucosamine (O-GlcNAc) hydrolase and cryptic lysine acetyltransferase activities. Here, we report that OGA is upregulated in a wide range of human cancers and drives aerobic glycolysis and tumor growth by inhibiting pyruvate kinase M2 (PKM2). PKM2 is dynamically O-GlcNAcylated in response to changes in glucose availability. Under high glucose conditions, PKM2 is a target of OGA-associated acetyltransferase activity, which facilitates O-GlcNAcylation of PKM2 by O-GlcNAc transferase (OGT). O-GlcNAcylation inhibits PKM2 catalytic activity and thereby promotes aerobic glycolysis and tumor growth. These studies define a causative role for OGA in tumor progression and reveal PKM2 O-GlcNAcylation as a metabolic rheostat that mediates exquisite control of aerobic glycolysis.