ORCID Profile
0000-0003-1711-0815
Current Organisation
National Institutes of Health
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Publisher: Public Library of Science (PLoS)
Date: 29-07-2021
DOI: 10.1371/JOURNAL.PPAT.1009797
Abstract: Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii . Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that Pf PanK1 and Pf PanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf 14-3-3I. Similarly, we demonstrate that Tg PanK1 and Tg PanK2 form a single complex that possesses PanK activity. Both Tg PanK1 and Tg PanK2 are essential for T . gondii proliferation, specifically due to their PanK activity. Our study constitutes the first ex les of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.
Publisher: American Society for Microbiology
Date: 22-12-2020
Abstract: Malaria, caused by Plasmodium parasites, continues to be a devastating global health issue, causing 405,000 deaths and 228 million cases in 2018. Understanding key metabolic processes in malaria parasites is critical to the development of new drugs to combat this major infectious disease. The Plasmodium glycolytic pathway is essential to the malaria parasite, providing energy for growth and replication and supplying important biomolecules for other essential Plasmodium anabolic pathways. Despite this overreliance on glycolysis, no current drugs target glycolysis, and there is a paucity of information on critical glycolysis targets. Our work addresses this unmet need, providing new mechanistic insights into this key pathway.
Publisher: Royal Society of Chemistry (RSC)
Date: 2019
DOI: 10.1039/C9MD00312F
Abstract: Presenting an optimised synthesis of the fungus-derived antibiotic CJ-15,801 which shows selective activity against Staphylococcus aureus and Plasmodium falciparum .
Publisher: American Chemical Society (ACS)
Date: 04-2021
Publisher: American Society for Microbiology
Date: 06-2015
DOI: 10.1128/AAC.04970-14
Abstract: Pantothenamides inhibit blood-stage Plasmodium falciparum with potencies (50% inhibitory concentration [IC 50 ], ∼20 nM) similar to that of chloroquine. They target processes dependent on pantothenate, a precursor of the essential metabolic cofactor coenzyme A. However, their antiplasmodial activity is reduced due to degradation by serum pantetheinase. Minor modification of the pantothenamide structure led to the identification of α-methyl- N -phenethyl-pantothenamide, a pantothenamide resistant to degradation, with excellent antiplasmodial activity (IC 50 , 52 ± 6 nM), target specificity, and low toxicity.
Publisher: American Chemical Society (ACS)
Date: 06-05-2020
Publisher: Cold Spring Harbor Laboratory
Date: 16-05-2017
DOI: 10.1101/137182
Abstract: The malaria-causing blood stage of Plasmodium falciparum requires extracellular pantothenate for proliferation. The parasite converts pantothenate into coenzyme A (CoA) via five enzymes, the first being a pantothenate kinase ( Pf PanK). Multiple antiplasmodial pantothenate analogues, including pantothenol and CJ-15,801, kill the parasite by targeting CoA biosynthesis/utilisation. Their mechanism of action, however, remains unknown. Here, we show that parasites pressured with pantothenol or CJ-15,801 become resistant to these analogues. Whole-genome sequencing revealed mutations in one of two putative PanK genes ( Pfpank1 ) in each resistant line. These mutations significantly alter Pf PanK activity, with two conferring a fitness cost, consistent with Pfpank1 coding for a functional PanK that is essential for normal growth. The mutants exhibit a different sensitivity profile to recently-described, potent, antiplasmodial pantothenate analogues, with one line being hypersensitive . We provide evidence consistent with different pantothenate analogue classes having different mechanisms of action: some inhibit CoA biosynthesis while others inhibit CoA-utilising enzymes.
Publisher: Cold Spring Harbor Laboratory
Date: 16-03-2021
DOI: 10.1101/2021.03.16.435557
Abstract: Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites, Plasmodium falciparum and Toxoplasma gondii . Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate that Pf PanK1 and Pf PanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein, Pf 14-3-3I. Similarly, we demonstrate that Tg PanK1 and Tg PanK2 form a single complex that possesses PanK activity. Both Tg PanK1 and Tg PanK2 are essential for T. gondii proliferation, specifically due to their PanK activity. Our study constitutes the first ex les of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.
Publisher: Springer Science and Business Media LLC
Date: 15-01-2014
DOI: 10.1038/NATURE12914
Abstract: Forests are major components of the global carbon cycle, providing substantial feedback to atmospheric greenhouse gas concentrations. Our ability to understand and predict changes in the forest carbon cycle--particularly net primary productivity and carbon storage--increasingly relies on models that represent biological processes across several scales of biological organization, from tree leaves to forest stands. Yet, despite advances in our understanding of productivity at the scales of leaves and stands, no consensus exists about the nature of productivity at the scale of the in idual tree, in part because we lack a broad empirical assessment of whether rates of absolute tree mass growth (and thus carbon accumulation) decrease, remain constant, or increase as trees increase in size and age. Here we present a global analysis of 403 tropical and temperate tree species, showing that for most species mass growth rate increases continuously with tree size. Thus, large, old trees do not act simply as senescent carbon reservoirs but actively fix large amounts of carbon compared to smaller trees at the extreme, a single big tree can add the same amount of carbon to the forest within a year as is contained in an entire mid-sized tree. The apparent paradoxes of in idual tree growth increasing with tree size despite declining leaf-level and stand-level productivity can be explained, respectively, by increases in a tree's total leaf area that outpace declines in productivity per unit of leaf area and, among other factors, age-related reductions in population density. Our results resolve conflicting assumptions about the nature of tree growth, inform efforts to undertand and model forest carbon dynamics, and have additional implications for theories of resource allocation and plant senescence.
Publisher: Wiley
Date: 04-06-2014
DOI: 10.1111/GCB.12622
Abstract: Established forests currently function as a major carbon sink, sequestering as woody biomass about 26% of global fossil fuel emissions. Whether forests continue to act as a global sink will depend on many factors, including the response of aboveground wood production (AWP MgC ha(-1 ) yr(-1) ) to climate change. Here, we explore how AWP in New Zealand's natural forests is likely to change. We start by statistically modelling the present-day growth of 97 199 in idual trees within 1070 permanently marked inventory plots as a function of tree size, competitive neighbourhood and climate. We then use these growth models to identify the factors that most influence present-day AWP and to predict responses to medium-term climate change under different assumptions. We find that if the composition and structure of New Zealand's forests were to remain unchanged over the next 30 years, then AWP would increase by 6-23%, primarily as a result of physiological responses to warmer temperatures (with no appreciable effect of changing rainfall). However, if warmth-requiring trees were able to migrate into currently cooler areas and if denser canopies were able to form, then a different AWP response is likely: forests growing in the cool mountain environments would show a 30% increase in AWP, while those in the lowland would hardly respond (on average, -3% when mean annual temperature exceeds 8.0 °C). We conclude that response of wood production to anthropogenic climate change is not only dependent on the physiological responses of in idual trees, but is highly contingent on whether forests adjust in composition and structure.
Publisher: Public Library of Science (PLoS)
Date: 03-04-2018
Publisher: Cold Spring Harbor Laboratory
Date: 09-10-2019
DOI: 10.1101/798967
Abstract: Intracellular parasites, such as the apicomplexan Toxoplasma gondii , are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. Tg ApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T. gondii . Here, we show that the abundance of Tg ApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite’s external environment, increasing in response to decreased [Arg]. Using a luciferase-based ‘biosensor’ strain of T. gondii , we demonstrate that parasites vary the expression of Tg ApiAT1 in different organs within their host, indicating that parasites are able to modulate Tg ApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo . Finally, we show that Arg-dependent regulation of Tg ApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the Tg ApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.
Publisher: Wiley
Date: 07-2014
DOI: 10.1002/ECE3.1087
Publisher: Wiley
Date: 29-11-2018
Abstract: Pantothenamides are potent growth inhibitors of the malaria parasite Plasmodium falciparum. Their clinical use is, however, hindered due to the ubiquitous presence of pantetheinases in human serum, which rapidly degrade pantothenamides into pantothenate and the corresponding amine. We previously reported that replacement of the labile amide bond with a triazole ring not only imparts stability toward pantetheinases, but also improves activity against P. falciparum. A small library of new triazole derivatives was synthesized, and their use in establishing structure-activity relationships relevant to antiplasmodial activity of this family of compounds is discussed herein. Overall it was observed that 1,4-substitution on the triazole ring and use of an unbranched, one-carbon linker between the pantoyl group and the triazole are optimal for inhibition of intraerythrocytic P. falciparum growth. Our results imply that the triazole ring may mimic the amide bond with an orientation different from what was previously suggested for this amide bioisostere.
Publisher: Cold Spring Harbor Laboratory
Date: 05-10-2020
DOI: 10.1101/2020.10.02.324079
Abstract: The Plasmodium parasites that cause malaria are adept at developing resistance to antimalarial drugs, necessitating the search for new antiplasmodials. Although several amide analogs of pantothenate (pantothenamides) show potent antiplasmodial activity, hydrolysis by pantetheinases (or vanins) present in blood rapidly inactivates them. We report herein the facile synthesis and biological activity of a small library of pantothenamide analogs in which the labile amide group is replaced with a variety of heteroaromatic rings. Several of the new analogs display antiplasmodial activity in the nanomolar range against P. falciparum and/or P. knowlesi in the presence of pantetheinase. A previously reported triazole and an isoxazole derivative presented here were further characterized and found to possess high selectivity indices, medium or high Caco-2 permeability, and medium or low microsomal clearance in vitro . Although we show here that the two compounds fail to suppress proliferation of P. berghei in vivo , pharmacokinetic and contact time data presented provide a benchmark for the compound profile required to achieve antiplasmodial activity in mice and should facilitate lead optimization.
Publisher: Wiley
Date: 10-2022
DOI: 10.1002/CPZ1.575
Abstract: The Sleeping Beauty (SB) transposon system is an efficient non‐viral tool for gene transfer into a variety of cells, including human cells. Through a cut‐and‐paste mechanism, your favorite gene (YFG) is integrated into AT‐rich regions within the genome, providing stable long‐term expression of the transfected gene. The SB system is evolving and has become a powerful tool for gene therapy. There are no safety concerns using this system, the handling is easy, and the time required to obtain a stable cell line is significantly reduced compared to other systems currently available. Here, we present a novel application of this system to generate, within 8 days, a stable producer HEK293T cell line capable of constitutively delivering enveloped virus‐like particles (eVLPs) for vaccination. We provide step‐by‐step protocols for generation of the SB transposon constructs, transfection procedures, and validation of the produced eVLPs. We next describe a method to pseudotype the constitutively produced eVLPs using the Spike protein derived from the SARS‐CoV‐2 virus (by coating the eVLP capsid with the heterologous antigen). We also describe optimization methods to scale up the production of pseudotyped eVLPs in a laboratory setting (from 100 µg to 5 mg). © Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1 : Generation of the SB plasmids Basic Protocol 2 : Generation of a stable HEK293T cell line constitutively secreting MLV‐based eVLPs Basic Protocol 3 : Evaluation of the SB constructs by immunofluorescence assay Basic Protocol 4 : Validation of eVLPs by denaturing PAGE and western blot Alternate Protocol 1 : Analysis of SARS‐CoV‐2 Spike protein oligomerization using blue native gel electrophoresis and western blot Alternate Protocol 2 : Evaluation of eVLP quality by electron microscopy (negative staining) Basic Protocol 5 : Small‐scale production of eVLPs Alternate Protocol 3 : Large‐scale production of eVLPs (up to about 1 to 3 mg VLPs) Alternate Protocol 4 : Large‐scale production of eVLPs (up to about 3 to 5 mg VLPs) Support Protocol : Quantification of total protein concentration by Bradford assay
Publisher: Wiley
Date: 13-08-2013
DOI: 10.1016/J.FEBSLET.2013.07.052
Abstract: To characterise plasmodial glycolysis, we generated two transgenic Plasmodium falciparum lines, one expressing P. falciparum hexokinase (PfHK) tagged with GFP (3D7-PfHK(GFP)) and another overexpressing native PfHK (3D7-PfHK(+)). Contrary to previous reports, we propose that PfHK is cytosolic. The glucose analogue, 2-deoxy-d-glucose (2-DG) was nearly 2-fold less toxic to 3D7-PfHK(+) compared with control parasites, supporting PfHK as a potential drug target. Although PfHK activity was higher in 3D7-PfHK(+), they accumulated phospho-[(14)C]2-DG at the same rate as control parasites. Transgenic parasites overexpressing the parasite's glucose transporter (PfHT) accumulated phospho-[(14)C]2-DG at a higher rate, consistent with glucose transport limiting glucose entry into glycolysis.
Publisher: Public Library of Science (PLoS)
Date: 05-08-2021
DOI: 10.1371/JOURNAL.PPAT.1009816
Abstract: Intracellular parasites, such as the apicomplexan Toxoplasma gondii , are adept at scavenging nutrients from their host. However, there is little understanding of how parasites sense and respond to the changing nutrient environments they encounter during an infection. Tg ApiAT1, a member of the apicomplexan ApiAT family of amino acid transporters, is the major uptake route for the essential amino acid L-arginine (Arg) in T . gondii . Here, we show that the abundance of Tg ApiAT1, and hence the rate of uptake of Arg, is regulated by the availability of Arg in the parasite’s external environment, increasing in response to decreased [Arg]. Using a luciferase-based ‘biosensor’ strain of T . gondii , we demonstrate that the expression of Tg ApiAT1 varies between different organs within the host, indicating that parasites are able to modulate Tg ApiAT1-dependent uptake of Arg as they encounter different nutrient environments in vivo . Finally, we show that Arg-dependent regulation of Tg ApiAT1 expression is post-transcriptional, mediated by an upstream open reading frame (uORF) in the Tg ApiAT1 transcript, and we provide evidence that the peptide encoded by this uORF is critical for mediating regulation. Together, our data reveal the mechanism by which an apicomplexan parasite responds to changes in the availability of a key nutrient.
No related grants have been discovered for Erick Tjhin.