Publications by authors named "Richard J Payne"

252 Publications

Glycosylation Regulates N-Terminal Proteolysis and Activity of the Chemokine CCL14.

ACS Chem Biol 2021 May 14. Epub 2021 May 14.

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.

Chemokines are secreted proteins that regulate leukocyte migration during inflammatory responses by signaling through chemokine receptors. Full length CC chemokine ligand 14, CCL14(1-74), is a weak agonist for the chemokine receptor CCR1, but its activity is substantially enhanced upon proteolytic cleavage to CCL14(9-74). CCL14 is O-glycosylated at Ser7, adjacent to the site of proteolytic activation. To determine whether glycosylation regulates the activity of CCL14, we used native chemical ligation to prepare four homogeneously glycosylated variants of CCL14(1-74). Each protein was assembled from three synthetic peptide fragments in "one-pot" using two sequential ligation reactions. We show that while glycosylation of CCL14(1-74) did not affect CCR1 binding affinity or potency of activation, sialylated variants of CCL14(1-74) exhibited reduced activity after treatment with plasmin compared to nonsialylated forms. These data indicate that glycosylation may influence the biological activity of CCL14 by regulating its conversion from the full-length to the truncated, activated form.
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http://dx.doi.org/10.1021/acschembio.1c00006DOI Listing
May 2021

Molecular testing for cytologically suspicious and malignant (Bethesda V and VI) thyroid nodules to optimize the extent of surgical intervention: a retrospective chart review.

J Otolaryngol Head Neck Surg 2021 Apr 28;50(1):29. Epub 2021 Apr 28.

Department of Otolaryngology Head and Neck Surgery, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada.

Background: Molecular testing has been used for cytologically indeterminate thyroid nodules (Bethesda III and IV), where the risk of malignancy is 10-40%. However, to date, the role of molecular testing in cytologically suspicious or positive for malignancy (Bethesda V and VI) thyroid nodules has been controversial. The aim of this study was to determine whether patients who had molecular testing in Bethesda V and VI thyroid nodules had the optimal extent of surgery performed more often than patients who did not have molecular testing performed.

Methods: A retrospective chart review of 122 cases was performed: 101 patients from the McGill University teaching hospitals and 21 patients from the Hillel Yaffe Medical center, Technion University. Patients included in the study were those with Bethesda V or VI thyroid nodules who underwent molecular testing (ThyGenext® or ThyroseqV3®) (McGill n = 72, Hillel Yaffe n = 14). Patients with Bethesda V or VI thyroid nodules who did not undergo molecular testing were used as controls (McGill n = 29, Hillel Yaffe n = 7). Each case was reviewed in order to determine whether the patient had optimal surgery. This was defined as total thyroidectomy in the presence of either a positive lymph node, extrathyroidal extension, or an aggressive pathological variant of papillary thyroid carcinoma (tall cell, hobnail, columnar cell, diffuse sclerosing, and solid/trabecular) documented on the final pathology report. In all other cases, a lobectomy/hemi/subtotal thyroidectomy was considered as optimal surgery. Chi-squared testing was performed to compare groups.

Results: When molecular testing was done, 91.86% (79/86) of surgeries in the molecular testing group were optimal, compared to 61.11% (22/36) in the control group. At McGill University teaching hospitals and at Hillel Yaffe, 91.67% (66/72) and 92.86% (13/14) of surgeries in the intervention group were considered as optimal, respectively. This compares to 58.62% (17/29) at McGill and 71.43% (5/7) at Hillel Yaffe when molecular testing was not performed (p = .001, p = .186).

Conclusions: In this study, molecular testing in Bethesda V and VI thyroid tumors significantly improved the likelihood of optimal surgery. Therefore, molecular testing may have an important role in optimizing surgical procedures performed in the setting of Bethesda V and VI thyroid nodules. Prospective studies with larger sample sizes are required to further investigate this finding.
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http://dx.doi.org/10.1186/s40463-021-00500-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082804PMC
April 2021

Key periods of peatland development and environmental changes in the middle taiga zone of Western Siberia during the Holocene.

Ambio 2021 Apr 6. Epub 2021 Apr 6.

Research Education Center of Environmental Dynamics and Climate Change (UNESCO Chair), Yugra State University, Chekhova str. 16, Khanty-Mansiysk, Russia, 628007.

The response of peatlands to climate change can be highly variable. Through understanding past changes we can better predict the response of peatlands to future climate change. We use a multi-proxy approach to reconstruct the surface wetness and carbon accumulation of the Mukhrino mire (Western Siberia), describing the development of the mire since peat formation in the early Holocene, around 9360 cal. year BP. The mire started as a rich fen which initiated after paludification of a spruce forest (probably in response to a wetter climate), while the Mukhrino mire progressed to ombrotrophic bog conditions (8760 cal. year BP). This transition coincided with the intensive development of mires in Western Siberia and was associated with active carbon accumulation (31 g m year). The ecosystem underwent a change to a tree-covered state around 5860 cal. year BP, likely in response to warming and possible droughts and this accompanied low carbon accumulation (12 g m year). If the future climate will be warmer and wetter, then regional mires are likely to remain a carbon sink, alternatively, a reversion to the wooded state with reduced carbon sink strength is possible.
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http://dx.doi.org/10.1007/s13280-021-01545-7DOI Listing
April 2021

Chemical Synthesis of Phosphorylated Insulin-like Growth Factor Binding Protein 2.

J Am Chem Soc 2021 Apr 2;143(14):5336-5342. Epub 2021 Apr 2.

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.

Chemical protein synthesis is a powerful avenue for accessing homogeneously modified proteins. While a significant number of small modified proteins bearing native post-translational modifications and non-natural modifications have been generated to date, access to larger targets has proved challenging. Herein, we describe the use of two ligation manifolds, namely, diselenide-selenoester ligation and native chemical ligation, to assemble a 31.5 kDa phosphorylated insulin-like growth factor binding protein (IGFBP-2) that comprises 290 amino acid residues, a phosphoserine post-translational modification, and nine disulfide bonds.
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http://dx.doi.org/10.1021/jacs.1c02280DOI Listing
April 2021

BET-Family Bromodomains Can Recognize Diacetylated Sequences from Transcription Factors Using a Conserved Mechanism.

Biochemistry 2021 Mar 23;60(9):648-662. Epub 2021 Feb 23.

School of Life and Environmental Sciences, University of Sydney, Sydney, NSW 2006, Australia.

Almost all eukaryotic proteins receive diverse post-translational modifications (PTMs) that modulate protein activity. Many histone PTMs are well characterized, heavily influence gene regulation, and are often predictors of distinct transcriptional programs. Although our understanding of the histone PTM network has matured, much is yet to be understood about the roles of transcription factor (TF) PTMs, which might well represent a similarly complex and dynamic network of functional regulation. Members of the bromodomain and extra-terminal domain (BET) family of proteins recognize acetyllysine residues and relay the signals encoded by these modifications. Here, we have investigated the acetylation dependence of several functionally relevant BET-TF interactions using surface plasmon resonance, nuclear magnetic resonance, and X-ray crystallography. We show that motifs known to be acetylated in TFs E2F1 and MyoD1 can interact with all bromodomains of BRD2, BRD3, and BRD4. The interactions are dependent on diacetylation of the motifs and show a preference for the first BET bromodomain. Structural mapping of the interactions confirms a conserved mode of binding for the two TFs to the acetyllysine binding pocket of the BET bromodomains, mimicking that of other already established functionally important histone- and TF-BET interactions. We also examined a motif from the TF RelA that is known to be acetylated but were unable to observe any interaction, regardless of the acetylation state of the sequence. Our findings overall advance our understanding of BET-TF interactions and suggest a physical link between the important diacetylated motifs found in E2F1 and MyoD1 and the BET-family proteins.
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http://dx.doi.org/10.1021/acs.biochem.0c00816DOI Listing
March 2021

Synthetic protein conjugate vaccines provide protection against in mice.

Proc Natl Acad Sci U S A 2021 01;118(4)

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia;

The global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants PamCys-SK or PamCys-SK These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4 T cell responses against ESAT6 and provided significant protection in the lungs from virulent aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.
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http://dx.doi.org/10.1073/pnas.2013730118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7848748PMC
January 2021

Potent anti-SARS-CoV-2 activity by gallinamide A and analogues via inhibition of cathepsin L.

bioRxiv 2020 Dec 24. Epub 2020 Dec 24.

The emergence of SARS-CoV-2 in late 2019, and the subsequent COVID-19 pandemic, has led to substantial mortality, together with mass global disruption. There is an urgent need for novel antiviral drugs for therapeutic or prophylactic application. Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is recognized as a promising drug target. The marine natural product, gallinamide A and several synthetic analogues, were identified as potent inhibitors of cathepsin L activity with IC values in the picomolar range. Lead molecules possessed selectivity over cathepsin B and other related human cathepsin proteases and did not exhibit inhibitory activity against viral proteases Mpro and PLpro. We demonstrate that gallinamide A and two lead analogues potently inhibit SARS-CoV-2 infection , with EC values in the nanomolar range, thus further highlighting the potential of cathepsin L as a COVID-19 antiviral drug target.
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http://dx.doi.org/10.1101/2020.12.23.424111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781308PMC
December 2020

Potent Trivalent Inhibitors of Thrombin through Hybridization of Salivary Sulfopeptides from Hematophagous Arthropods.

Angew Chem Int Ed Engl 2021 03 28;60(10):5348-5356. Epub 2021 Jan 28.

School of Chemistry and ARC Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, Sydney, 2006, NSW, Australia.

Blood feeding arthropods, such as leeches, ticks, flies and mosquitoes, provide a privileged source of peptidic anticoagulant molecules. These primarily operate through inhibition of the central coagulation protease thrombin by binding to the active site and either exosite I or exosite II. Herein, we describe the rational design of a novel class of trivalent thrombin inhibitors that simultaneously block both exosites as well as the active site. These engineered hybrids were synthesized using tandem diselenide-selenoester ligation (DSL) and native chemical ligation (NCL) reactions in one-pot. The most potent trivalent inhibitors possessed femtomolar inhibition constants against α-thrombin and were selective over related coagulation proteases. A lead hybrid inhibitor possessed potent anticoagulant activity, blockade of both thrombin generation and platelet aggregation in vitro and efficacy in a murine thrombosis model at 1 mg kg . The rational engineering approach described here lays the foundation for the development of potent and selective inhibitors for a range of other enzymatic targets that possess multiple sites for the disruption of protein-protein interactions, in addition to an active site.
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http://dx.doi.org/10.1002/anie.202015127DOI Listing
March 2021

Active surveillance for low-risk small papillary thyroid cancer in North American countries: past, present and future (bridging the gap between North American and Asian practices).

Gland Surg 2020 Oct;9(5):1685-1697

Department of Otolaryngology-Head and Neck Surgery, Jewish General Hospital, McGill University, Montreal, Canada.

Papillary thyroid cancer (PTC) is increasingly being diagnosed worldwide; yet the mortality remains very low, suggesting widespread overdiagnosis. While traditional management of PTC includes thyroid surgery, sometimes followed by radioactive iodine treatment, there is a global trend towards more conservative approaches for patients who are considered as the lowest risk of recurrence or death from their disease. Active surveillance (AS), once called watchful waiting, involves close follow-up, with the intention to intervene if the cancer progresses, or on patient request. The Kuma Hospital in Japan was the first to introduce AS as an alternative to immediate thyroid surgery for low-risk papillary thyroid microcarcinomas (PTMC, <1 cm) in 1993. Accumulated evidence over the years has shown that AS is a safe and effective approach in select patients, with a low rate of cancer progression during AS. Consequently, the Japanese Clinical Guidelines for treatment of thyroid tumor approved AS as a first-line management for patients with asymptomatic PTMC in 2010. Subsequently, the latest 2015 American Thyroid Association guidelines endorsed AS as an alternative approach to immediate surgery for cytologically confirmed very low-risk PTC. However, the acceptance, feasibility and results of AS in patients with low-risk PTC outside of Japan are still largely unknown. Most guidelines recommend that thyroid nodules <1 cm should not be aspirated but instead monitored regardless of the ultrasonographic characteristics. In essence, these patients are also being subjected to AS. Specific recommendations and the role of molecular testing for the optimal selection of PTMC patients for an AS management approach are not well established. Furthermore, research is needed to assess the long-term clinical and psychosocial outcomes in patients with larger tumor sizes (>1 cm) who undergo screening and diagnosis according to the North American guidelines and practices. The first Canadian prospective observational study launched in 2016 is intended to complement the existing data for AS of small low-risk PTC (≤2 cm) and may provide insight into the different approaches in North American and Asian practices. This review intends to summarize the development and the rationale of AS for PTMC and highlights significant differences between North American and Japanese practices.
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http://dx.doi.org/10.21037/gs-20-389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667052PMC
October 2020

Sulfotyrosine-Mediated Recognition of Human Thrombin by a Tsetse Fly Anticoagulant Mimics Physiological Substrates.

Cell Chem Biol 2021 Jan 22;28(1):26-33.e8. Epub 2020 Oct 22.

IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal; Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal. Electronic address:

Despite possessing only 32 residues, the tsetse thrombin inhibitor (TTI) is among the most potent anticoagulants described, with sub-picomolar inhibitory activity against thrombin. Unexpectedly, TTI isolated from the fly is 2000-fold more active and 180 Da heavier than synthetic and recombinant variants. We predicted the presence of a tyrosine O-sulfate post-translational modification of TTI, prompting us to investigate the effect of the modification on anticoagulant activity. A combination of chemical synthesis and functional assays was used to reveal that sulfation significantly improved the inhibitory activity of TTI against thrombin. Using X-ray crystallography, we show that the N-terminal sulfated segment of TTI binds the basic exosite II of thrombin, establishing interactions similar to those of physiologic substrates, while the C-terminal segment abolishes the catalytic activity of thrombin. This non-canonical mode of inhibition, coupled with its potency and small size, makes TTI an attractive scaffold for the design of novel antithrombotics.
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http://dx.doi.org/10.1016/j.chembiol.2020.10.002DOI Listing
January 2021

Cyclic peptides can engage a single binding pocket through highly divergent modes.

Proc Natl Acad Sci U S A 2020 10 12;117(43):26728-26738. Epub 2020 Oct 12.

School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia;

Cyclic peptide library screening technologies show immense promise for identifying drug leads and chemical probes for challenging targets. However, the structural and functional diversity encoded within such libraries is largely undefined. We have systematically profiled the affinity, selectivity, and structural features of library-derived cyclic peptides selected to recognize three closely related targets: the acetyllysine-binding bromodomain proteins BRD2, -3, and -4. We report affinities as low as 100 pM and specificities of up to 10-fold. Crystal structures of 13 peptide-bromodomain complexes reveal remarkable diversity in both structure and binding mode, including both α-helical and β-sheet structures as well as bivalent binding modes. The peptides can also exhibit a high degree of structural preorganization. Our data demonstrate the enormous potential within these libraries to provide diverse binding modes against a single target, which underpins their capacity to yield highly potent and selective ligands.
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http://dx.doi.org/10.1073/pnas.2003086117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7604503PMC
October 2020

Are Bethesda III Thyroid Nodules More Aggressive than Bethesda IV Thyroid Nodules When Found to Be Malignant?

Cancers (Basel) 2020 Sep 9;12(9). Epub 2020 Sep 9.

Department of Otolaryngology Head and Neck Surgery, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada.

The Bethesda classification system for thyroid fine needle aspirate (FNA) is used to predict the risk of malignancy and to guide the management of thyroid nodules. We postulated that thyroid malignancies characterized as Bethesda III on FNA have more aggressive features than those classified as Bethesda IV. A retrospective chart review was performed to identify those who underwent thyroid surgery at a single tertiary hospital setting between 2015 and 2020. Associations between Bethesda category, molecular genetic test results, and histopathologic findings were examined. Out of 628 surgeries that were performed, 199 (54.2%) Bethesda III nodules and 216 (82.8%) Bethesda IV nodules were malignant. Of those that were malignant, 37 (18.6%) and 22 (10.2%) Bethesda III and Bethesda IV nodules showed aggressive features, respectively ( value = 0.014). There was a proportionally increased number of aggressive features in extra-thyroidal extension, lymph nodes metastasis, and all aggressive subtypes of papillary thyroid cancer in the Bethesda III category. Although Bethesda IV nodules are much more likely to be malignant ( value = 0.002), our study suggests that Bethesda III nodules that are resected are more likely to have aggressive features than Bethesda IV nodules, with a statistically significant increase in the solid variant of papillary thyroid cancer and lymph node metastasis.
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http://dx.doi.org/10.3390/cancers12092563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7564274PMC
September 2020

Total Synthesis of Glycosylated Human Interferon-γ.

Org Lett 2020 09 24;22(17):6863-6867. Epub 2020 Aug 24.

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.

Interferon-γ (IFN-γ) is a glycoprotein that is responsible for orchestrating numerous critical immune induction and modulation processes and is used clinically for the treatment of a number of diseases. Herein, we describe the total chemical synthesis of homogeneously glycosylated variants of human IFN-γ using a tandem diselenide-selenoester ligation-deselenization strategy in the C- to N-terminal direction. The synthetic glycoproteins were successfully folded, and the structures and antiviral functions were assessed.
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http://dx.doi.org/10.1021/acs.orglett.0c02401DOI Listing
September 2020

Revealing the functional roles of tyrosine sulfation using synthetic sulfopeptides and sulfoproteins.

Curr Opin Chem Biol 2020 10 7;58:72-85. Epub 2020 Aug 7.

School of Chemistry, The University of Sydney, Sydney, NSW, 2006, Australia; Australian Research Council Centre of Excellence for Innovations in Peptide and Protein Science, The University of Sydney, NSW, 2006, Australia. Electronic address:

The decoration of proteins with post-translational modifications (PTMs) serves as a mechanism to expand the functional repertoire of the proteome. Tyrosine sulfation is a PTM that has been shown to be a key regulator of extracellular protein-protein interactions in a select number of examples. However, the challenges associated with identifying and characterising the functional consequences of tyrosine sulfation have hindered our ability to understand the full scope of its role in the wider proteome when compared with that of other PTMs, for example, phosphorylation and glycosylation. In this account, we highlight recent advances in the prediction and detection of tyrosine sulfation and outline the need for continued innovation in this area. We also discuss the utility of emerging solid-phase synthesis and peptide ligation strategies for accessing libraries of homogeneously sulfated peptides and proteins to help reveal functional aspects of the sulfoproteome.
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http://dx.doi.org/10.1016/j.cbpa.2020.05.007DOI Listing
October 2020

Lactoferrin-Derived Peptide Lactofungin Is Potently Synergistic with Amphotericin B.

Antimicrob Agents Chemother 2020 09 21;64(10). Epub 2020 Sep 21.

School of Life and Environmental Sciences and the Marie Bashir Institute for Infectious Diseases and Biosecurity, University of Sydney, Sydney, NSW, Australia

Lactoferrin (LF) is an iron-binding glycoprotein with broad-spectrum antimicrobial activity. Previously, we discovered that LF synergistically enhanced the antifungal efficacy of amphotericin B (AMB) across a variety of yeast species and subsequently hypothesized that this synergy was enhanced by the presence of small peptides derived from the whole LF molecule. In this study, LF was digested with pepsin under a range of conditions. The resulting hydrolysates exhibited enhanced synergy with AMB compared to its synergy with undigested LF. Samples were analyzed using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and 14 peptides were identified. The sequences of these peptides were predicted by matching their molecular weights to those of a virtual digest with pepsin. The relative intensities of predicted peptides in each hydrolysate were compared with the activity of the hydrolysate, and the structural and physicochemical properties of the peptides were assessed. From this, a 30-residue peptide was selected for synthesis and dubbed lactofungin (LFG). Pure LFG was highly synergistic with AMB, outperforming native LF in all fungal species tested. With potential for further structural and chemical improvements, LFG is an excellent lead for development as an antifungal adjuvant.
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http://dx.doi.org/10.1128/AAC.00842-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7508585PMC
September 2020

Nutritional and metabolic regulation of the metabolite dimethylguanidino valeric acid: an early marker of cardiometabolic disease.

Am J Physiol Endocrinol Metab 2020 09 14;319(3):E509-E518. Epub 2020 Jul 14.

Charles Perkins Centre, The University of Sydney, Sydney, New South Wales, Australia.

Dimethylguanidino valeric acid (DMGV) is a marker of fatty liver disease, incident coronary artery disease, cardiovascular mortality, and incident diabetes. Recently, it was reported that circulating DMGV levels correlated positively with consumption of sugary beverages and negatively with intake of fruits and vegetables in three Swedish community-based cohorts. Here, we validate these results in the Framingham Heart Study Third Generation Cohort. Furthermore, in mice, diets rich in sucrose or fat significantly increased plasma DMGV concentrations. DMGV is the product of metabolism of asymmetric dimethylarginine (ADMA) by the hepatic enzyme AGXT2. ADMA can also be metabolized to citrulline by the cytoplasmic enzyme DDAH1. We report that a high-sucrose diet induced conversion of ADMA exclusively into DMGV (supporting the relationship with sugary beverage intake in humans), while a high-fat diet promoted conversion of ADMA to both DMGV and citrulline. On the contrary, replacing dietary native starch with high-fiber-resistant starch increased ADMA concentrations and induced its conversion to citrulline, without altering DMGV concentrations. In a cohort of obese nondiabetic adults, circulating DMGV concentrations increased and ADMA levels decreased in those with either liver or muscle insulin resistance. This was similar to changes in DMGV and ADMA concentrations found in mice fed a high-sucrose diet. Sucrose is a disaccharide of glucose and fructose. Compared with glucose, incubation of hepatocytes with fructose significantly increased DMGV production. Overall, we provide a comprehensive picture of the dietary determinants of DMGV levels and association with insulin resistance.
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http://dx.doi.org/10.1152/ajpendo.00207.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509244PMC
September 2020

Total Synthesis and Antimycobacterial Activity of Ohmyungsamycin A, Deoxyecumicin, and Ecumicin.

Chemistry 2020 Nov 22;26(66):15200-15205. Epub 2020 Oct 22.

School of Chemistry, The University of Sydney, Sydney, NSW, 2006, Australia.

The ohmyungsamycin and ecumicin natural product families are structurally related cyclic depsipeptides that display potent antimycobacterial activity. Herein the total syntheses of ohmyungsamycin A, deoxyecumicin, and ecumicin are reported, together with the direct biological comparison of members of these natural product families against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). The synthesis of each of the natural products employed a solid-phase strategy to assemble the linear peptide precursor, involving a key on-resin esterification and an optional on-resin dimethylation step, before a final solution-phase macrolactamization between the non-proteinogenic N-methyl-4-methoxy-l-tryptophan amino acid and a bulky N-methyl-l-valine residue. The synthetic natural products possessed potent antimycobacterial activity against Mtb with MIC 's ranging from 110-360 nm and retained activity against Mtb in Mtb-infected macrophages. Deoxyecumicin also exhibited rapid bactericidal killing against Mtb, sterilizing cultures after 21 days.
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http://dx.doi.org/10.1002/chem.202002408DOI Listing
November 2020

Semisynthesis of an evasin from tick saliva reveals a critical role of tyrosine sulfation for chemokine binding and inhibition.

Proc Natl Acad Sci U S A 2020 06 27;117(23):12657-12664. Epub 2020 May 27.

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia;

Blood-feeding arthropods produce antiinflammatory salivary proteins called evasins that function through inhibition of chemokine-receptor signaling in the host. Herein, we show that the evasin ACA-01 from the tick can be posttranslationally sulfated at two tyrosine residues, albeit as a mixture of sulfated variants. Homogenously sulfated variants of the proteins were efficiently assembled via a semisynthetic native chemical ligation strategy. Sulfation significantly improved the binding affinity of ACA-01 for a range of proinflammatory chemokines and enhanced the ability of ACA-01 to inhibit chemokine signaling through cognate receptors. Comparisons of evasin sequences and structural data suggest that tyrosine sulfation serves as a receptor mimetic strategy for recognizing and suppressing the proinflammatory activity of a wide variety of mammalian chemokines. As such, the incorporation of this posttranslational modification (PTM) or mimics thereof into evasins may provide a strategy to optimize tick salivary proteins for antiinflammatory applications.
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http://dx.doi.org/10.1073/pnas.2000605117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293604PMC
June 2020

Discovery of Potent Cyclic Sulfopeptide Chemokine Inhibitors via Reprogrammed Genetic Code mRNA Display.

J Am Chem Soc 2020 05 7;142(20):9141-9146. Epub 2020 May 7.

School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia.

Targeting chemokine signaling is an attractive avenue for the treatment of inflammatory disorders. Tyrosine sulfation is an important post-translational modification (PTM) that enhances chemokine-receptor binding and is also utilized by a number of pathogenic organisms to improve the binding affinity of immune-suppressive chemokine binding proteins (CKBPs). Here we report the display selection of tyrosine-sulfated cyclic peptides using a reprogrammed genetic code to discover high-affinity ligands for the chemokine CCL11 (eotaxin-1). The selected cyclic sulfopeptides possess high affinity for the target chemokine (as well as one or more of the related family members CCL2, CCL7 and CCL24) and inhibit CCL11 activation of CC chemokine receptor 3 (CCR3). This work demonstrates the utility of exploiting native PTMs as binding motifs for the generation of new leads for medicinal chemistry.
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http://dx.doi.org/10.1021/jacs.0c03152DOI Listing
May 2020

Total Synthesis and Antitrypanosomal Activity of Janadolide and Simplified Analogues.

Org Lett 2020 04 6;22(8):3089-3093. Epub 2020 Apr 6.

School of Chemistry, The University of Sydney, Sydney, New South Wales 2006, Australia.

Janadolide is a cyclic depsipeptide natural product isolated from the marine cyanobacterium . Herein, we describe the total synthesis of janadolide, along with eight simplified analogues, via an efficient solid-phase strategy. Crucial to the synthesis of the natural product was the construction of a key polyketide fragment via an enantioselective (-)--chlorodiisopinocampheylborane-mediated reduction and a alkyl Suzuki reaction. Janadolide and the simplified analogues exhibited antitrypanosomal activity against pathogenic and parasites.
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http://dx.doi.org/10.1021/acs.orglett.0c00840DOI Listing
April 2020

CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation.

Nat Commun 2020 03 23;11(1):1519. Epub 2020 Mar 23.

School of Life and Environmental Sciences, University of Sydney, Sydney, NSW, 2006, Australia.

Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and it is unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family. We demonstrate that the binding energy for CHD4-nucleosome complex formation-even in the absence of nucleotide-triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4-6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. Based on our work and previous studies, we propose a mechanism for nucleosome sliding.
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http://dx.doi.org/10.1038/s41467-020-15183-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7090039PMC
March 2020

The Role of the ThyroSeq v3 Molecular Test in the Surgical Management of Thyroid Nodules in the Canadian Public Health Care Setting.

Thyroid 2020 09 5;30(9):1280-1287. Epub 2020 May 5.

Department of Otolaryngology-Head and Neck Surgery, Royal Victoria Hospital, McGill University, Montreal, Canada.

Although the current gold standard for diagnosing thyroid nodule malignancy is ultrasound-guided fine-needle aspiration (FNA) cytology, about 20-25% of cytological evaluations are considered indeterminate for malignancy. This limitation has led to the emergence of next-generation sequencing panels, for example, ThyroSeq v3 (TSv3), which recognize highly diagnostic genetic mutations of common thyroid carcinomas in FNA samples and classify them as test-negative or test-positive, helping optimize treatment for indeterminate thyroid nodules (ITNs). Our goals were to evaluate the benign call rate (BCR) of TSv3 and assess its diagnostic performance and clinical utility while highlighting the points of consideration for a public Canadian institution. This is a single-center study conducted at the Royal Victoria Hospital (McGill University Health Centre) in Montreal, Canada, between January and February 2019. Patients were offered TSv3 following the McGill algorithm for ITN workup, a novel protocol developed at our institution to select only diagnostic surgery candidates to minimize waste of public resources, considering the single-payer health care system. Patient demographics, cytopathology results, TSv3 data, treatment plan, and final histopathology result were reviewed. A total of 50 ITNs underwent TSv3 testing; molecular analysis yielded 20 (40%) "positive" results and 24 (48%) "negative" results. Six (12%) results were classified as "currently negative" or "negative but limited." "Currently negative" results indicate a low-risk mutation that alone is insufficient for development of a malignant lesion. "Negative but limited" results indicate a sample that is nondiagnostic for malignancy due to low cell count. BCR was calculated as ("negative" and "currently negative")/total, resulting in a BCR of 58%. Twenty-three (46%) patients were scheduled for surgery and 27 (54%) patients continued with surveillance. Ninety-one percent (20 of 22) of the resected target nodules were malignant on final pathology. TSv3 proved beneficial in classifying ITNs as positive or negative, avoiding surgery in the latter cases. We found a lower reduction rate in surgery and BCR than the previously published studies, which is attributable to the criteria of the McGill algorithm. In the Canadian public health care system, preventing unnecessary surgery represents significant cost savings for the provincial government while also improving patient quality of life.
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http://dx.doi.org/10.1089/thy.2019.0539DOI Listing
September 2020

Annual gaseous carbon budgets of forest-to-bog restoration sites are strongly determined by vegetation composition.

Sci Total Environ 2020 Feb 2;705:135863. Epub 2019 Dec 2.

Edge Hill University, Geography Department, St Helens Road, Ormskirk, Lancashire L39 4QP, UK.

Large areas of naturally open peatland in western Europe were drained and planted with non-native conifers in the twentieth century. Efforts are currently underway to restore many of these sites. Ultimately, forest-to-bog restoration aims to bring back functional peatlands that can sequester carbon but there is a lack of empirical evidence for whether current approaches are effective. Using a chronosequence design, we compared the annual gaseous carbon balance of two forest-to-bog restoration areas with an open area not subject to afforestation. A closed chamber method was used to determine gas fluxes (Net Ecosystem Respiration, Gross Primary Productivity, Net Ecosystem Exchange (NEE) and methane (CH)) over a twelve-month period for locations spanning the range of peatland microtopography and vegetation communities. Relationships between gas fluxes, vegetation/cover and environmental factors were analysed and regression models used to estimate annual CO and CH budgets. During the study period, NEE estimates (total gaseous C expressed as CO-eq) showed a net sink for the unafforested (-102 g C m yr) and oldest (-131 g C m yr) restoration area (17 years post-restoration 'RES 17 YRS'), whilst the youngest restoration area (6 years post-restoration 'RES 6YRS'), was a net source (35 g C m yr). We observed significantly higher CH emissions from restoration areas dominated by Eriophorum angustifolium compared with other peatland vegetation types. Sampling points with higher cover of Sphagnum were found to be most effective for C sequestration. Overall, vegetation composition/cover was observed to be an important factor determining C emissions from forest-to-bog restoration areas. These results suggest that restoration is effective in returning the carbon sink function of peatlands damaged by commercial forestry and - depending on restoration techniques - timescales of >10 years may be required.
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http://dx.doi.org/10.1016/j.scitotenv.2019.135863DOI Listing
February 2020

Synthesis and Utility of β-Selenophenylalanine and β-Selenoleucine in Diselenide-Selenoester Ligation.

J Org Chem 2020 02 31;85(3):1567-1578. Epub 2019 Dec 31.

School of Chemistry , The University of Sydney , Sydney , New South Wales 2006 , Australia.

The synthesis of suitably protected β-selenophenylalanine and β-selenoleucine amino acids was accomplished from Garner's aldehyde as a common starting point. These selenoamino acids were incorporated into model peptides and shown to facilitate rapid diselenide-selenoester ligation (DSL) with peptide selenoesters which, when coupled with deselenization, afforded native peptide products. The utility of one-pot DSL-deselenization chemistry at phenylalanine and leucine was demonstrated through the rapid synthesis of a glycosylated interferon-γ fragment and the chemokine-binding protein UL22A, respectively.
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http://dx.doi.org/10.1021/acs.joc.9b02665DOI Listing
February 2020

Peptide Ligation at High Dilution via Reductive Diselenide-Selenoester Ligation.

J Am Chem Soc 2020 01 31;142(2):1090-1100. Epub 2019 Dec 31.

School of Chemistry , The University of Sydney , Sydney , NSW 2006 , Australia.

Peptide ligation chemistry has revolutionized protein science by providing access to homogeneously modified peptides and proteins. However, lipidated polypeptides and integral membrane proteins-an important class of biomolecules-remain enormously challenging to access synthetically owing to poor aqueous solubility of one or more of the fragments under typical ligation conditions. Herein we describe the advent of a reductive diselenide-selenoester ligation (rDSL) method that enables efficient ligation of peptide fragments down to low nanomolar concentrations, without resorting to solubility tags or hybridizing templates. The power of rDSL is highlighted in the efficient synthesis of the FDA-approved therapeutic lipopeptide tesamorelin and palmitylated variants of the transmembrane lipoprotein phospholemman (FXYD1). Lipidation of FXYD1 was shown to critically modulate inhibitory activity against the Na/K pump.
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http://dx.doi.org/10.1021/jacs.9b12558DOI Listing
January 2020

Synthesis and structure-activity relationships of teixobactin.

Ann N Y Acad Sci 2020 01 2;1459(1):86-105. Epub 2019 Dec 2.

Department of Pharmacology & Therapeutics, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, the University of Melbourne, Parkville, Victoria, Australia.

The discovery of antibiotics has led to the effective treatment of bacterial infections that were otherwise fatal and has had a transformative effect on modern medicine. Teixobactin is an unusual depsipeptide natural product that was recently discovered from a previously unculturable soil bacterium and found to possess potent antibacterial activity against several Gram positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. One of the key features of teixobactin as an antibiotic lead is that resistance could not be generated in a laboratory setting. This is proposed to be a result of a mechanism of action that involves binding to essential cell wall synthesis building blocks, lipid II and lipid III. Since the initial isolation report in 2015, significant efforts have been made to understand its unique mechanism of action, develop efficient synthetic routes for its production, and thus enable the generation of analogues for structure-activity relationship studies and optimization of its pharmacological properties. Our review provides a comprehensive treatise on the progress in understanding teixobactin chemistry, structure-activity relationships, and mechanisms of antibacterial activity. Teixobactin represents an exciting starting point for the development of new antibiotics that can be used to combat multidrug-resistant bacterial ("superbug") infections.
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http://dx.doi.org/10.1111/nyas.14282DOI Listing
January 2020

Molecular mutations as a possible factor for determining extent of thyroid surgery.

J Otolaryngol Head Neck Surg 2019 Oct 17;48(1):51. Epub 2019 Oct 17.

Department of Otolaryngology Head and Neck Surgery, Sir Mortimer B. Davis-Jewish General Hospital, McGill University, Montreal, QC, Canada.

Background: Molecular testing of thyroid nodules is a diagnostic tool used to better understand the nature of thyroid nodules. The aim of this study is to better comprehend the relationship between specific mutations and aggressive behavior of the tumour as demonstrated on postoperative pathological analysis.

Methods: A retrospective chart review of 103 cases was performed. Included were patients who had undergone molecular testing using a panel that tests for 9 mutations (ThyGenX®) and were found to have malignant tumours. The following gene alterations were found pre-operatively in the nodules: BRAF V600E (n = 32), BRAF K601E (n = 4), NRAS (n = 11), HRAS (n = 4), KRAS (n = 3), RET/PTC1 rearrangement (n = 1), TERT promoter (n = 2), PAX8-PPARγ rearrangement (n = 1), and 45 cases where no mutation was detected. Aggressive behavior was defined by extra-thyroidal extension (ETE), lymph node metastasis (LN+), and the following variants of papillary thyroid carcinoma: tall cell, solid, diffuse sclerosing, columnar cell and hobnail. Chi-squared testing was performed to compare groups.

Results: The group with BRAF V600E, RET/PTC1 rearrangement, and TERT promoter mutations was associated with ETE 37.1%, and LN+ 45.7% of the time compared to 4.3 and 13.0% in the group with other mutations, and 4.4 and 4.4% in the group with no mutations (p-value 0.02, p-value < 0.001, p-value 0.006). In addition, the BRAF V600E, RET/PTC1 rearrangement, and TERT mutations group demonstrated tall cell variants (17.1%), columnar cell variants (5.7%), and hobnail variants (3%). The other mutations group demonstrated columnar cell variants (4.3%), and the no mutations group demonstrated solid variants (2.2%).

Conclusions: In this study, BRAF V600E, RET/PTC1 rearrangement, and TERT mutations were associated with aggressive behaving thyroid malignancies as defined above. Molecular testing may be a useful method to anticipate aggressive tumour types and therefore assist in planning the extent and timing of surgery.
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http://dx.doi.org/10.1186/s40463-019-0372-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796357PMC
October 2019

Dissecting the Binding Interactions of Teixobactin with the Bacterial Cell-Wall Precursor Lipid II.

Chembiochem 2020 03 12;21(6):789-792. Epub 2019 Nov 12.

Department of Chemistry, University of Alberta, 11227 Saskatchewan Drive, Edmonton, Alberta, T6G 2G2, Canada.

The prevalence of life-threatening, drug-resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered "resistance-proof" antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram-positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram-positive and Gram-negative bacteria. Furthermore, we show that when combined with known outer membrane-disrupting peptides, teixobactin is active against Gram-negative organisms.
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http://dx.doi.org/10.1002/cbic.201900504DOI Listing
March 2020

Preoperative Risk Index Among Patients Undergoing Thyroid or Parathyroid Surgery.

JAMA Otolaryngol Head Neck Surg 2020 01;146(1):7-12

Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal, Quebec, Canada.

Importance: Frailty represents a multidimensional syndrome that is increasingly being used to stratify risk in surgical patients. Current frailty risk models are limited among those undergoing thyroid or parathyroid surgery.

Objective: To develop and compare preoperative risk indices to determine factors associated with short-term major postoperative adverse events in patients undergoing thyroid or parathyroid surgery.

Design, Setting, And Participants: This cohort study evaluated 154 895 patients in the American College of Surgeons National Surgical Quality Improvement Program who underwent thyroid or parathyroid surgery from January 1, 2007, to December 31, 2016.

Exposures: Preoperative frailty-related and surgical factors from a derivation cohort were evaluated using simple and multiple logistic regression. Variables potentially associated with postoperative adverse events were subsequently combined into a personalized preoperative Cervical Endocrine Surgery Risk Index (CESRI) and compared with existing risk models using the validation cohort.

Main Outcomes And Measures: Composite variable of any major postoperative adverse event, including death, within 30 days of surgery.

Results: Of the 154 895 operations reviewed, 3318 patients (2.1%; 2296 women and 1022 men; mean [SD] age, 56.1 [15.6] years) experienced a major postoperative adverse event, with 163 deaths (0.1%). Older age (age, ≥80 years: odds ratio [OR], 2.35; 95% CI, 1.74-3.13), inpatient status (OR, 3.55; 95% CI, 3.08-4.11), male sex (OR, 1.49; 95% CI, 1.29-1.71), current tobacco smoking (OR, 1.25; 95% CI, 1.05-1.48), dyspnea (OR, 1.58; 95% CI, 1.29-1.91), recent weight loss (OR, 1.88; 95% CI, 1.23-2.78), functional dependence (OR, 2.77; 95% CI, 2.05-3.69), obesity (OR, 1.33; 95% CI, 1.10-1.60), anemia (OR, 2.14; 95% CI, 1.82-2.52), leukocytosis (OR, 1.73; 95% CI, 1.38-2.14), hypoalbuminemia (OR, 1.87; 95% CI, 1.56-2.23), use of anticoagulation (OR, 2.16; 95% CI, 1.64-2.81), and length of surgery (>4 hours: OR, 2.92; 95% CI, 2.37-3.59) were independently associated with major adverse events or death on multiple regression analysis (C statistic, 0.77; 95% CI, 0.76-0.78). The area under the curve of the CESRI to determine major adverse events, including death, using the validation cohort was 0.63 (95% CI, 0.61-0.64), with a sensitivity of 0.66 (95% CI, 0.64-0.68) and specificity of 0.66 (95% CI, 0.65-0.66). The CESRI outperformed other risk models for determining adverse events (CESRI vs 5-Factor Modified Frailty Index: delta C index, 0.11; 95% CI, 0.09-0.13; CESRI vs American Society of Anesthesiologists Physical Status Classification System: delta C index, 0.05; 95% CI, 0.03-0.07; CESRI vs American College of Surgeons Risk Calculator: delta C index, 0.02; 95% CI, 0.01-0.03; and CESRI vs Head and Neck Surgery Risk Index: delta C index, 0.04; 95% CI, 0.03-0.06).

Conclusions And Relevance: This study suggests that the CESRI is able to determine major postoperative adverse events in patients undergoing thyroid or parathyroid surgery.
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http://dx.doi.org/10.1001/jamaoto.2019.2413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735422PMC
January 2020