Publications by authors named "Mark Field"

269 Publications

Exploring the potential of rapid evaporative ionization mass spectrometry (Intelligent Knife) for point-of-care testing in aortic surgery.

Eur J Cardiothorac Surg 2021 Apr 11. Epub 2021 Apr 11.

Liverpool Centre for Cardiovascular Science, Liverpool, UK.

Objectives: Many intraoperative decisions regarding the extent of thoracic aortic surgery are subjective and are based on the appearance of the aorta, perceived surgical risks and likelihood of early recurrent disease. Our objective in this work was to carry out a cross-sectional study to demonstrate that rapid evaporative ionization mass spectrometry (REIMS) of electrosurgical aerosol is able to empirically discriminate ex vivo aneurysmal human thoracic aorta from normal aorta, thus providing supportive evidence for the development of the technique as a point-of-care test guiding intraoperative surgical decision-making.

Methods: Human aortic tissue was obtained from patients undergoing surgery for thoracic aortic aneurysms (n = 44). Normal aorta was obtained from a mixture of post-mortem and punch biopsies from patients undergoing coronary surgery (n = 13). Monopolar electrocautery was applied to samples and surgical aerosol aspirated and analysed by REIMS to produce mass spectral data.

Results: Models generated from REIMS data can discriminate aneurysmal from normal aorta with accuracy and precision of 88.7% and 85.1%, respectively. In addition, further analysis investigating aneurysmal tissue from patients with bicuspid and tricuspid aortic valves was discriminated from normal tissue and each other with accuracies and precision of 93.5% and 91.4% for control, 83.8% and 76.7% for bicuspid aortic valve and 89.3% and 86.0% for tricuspid aortic valve, respectively.

Conclusions: Analysis of electrosurgical aerosol from ex vivo aortic tissue using REIMS allowed us to discriminate aneurysmal from normal aorta, supporting its development as a point-of-care test (Intelligent Knife) for guiding surgical intraoperative decision-making.
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http://dx.doi.org/10.1093/ejcts/ezab166DOI Listing
April 2021

Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase.

Protein Expr Purif 2021 Jul 25;183:105877. Epub 2021 Mar 25.

Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, Calle 25 #455 Entre I y J, Vedado, 10400, Havana, Cuba. Electronic address:

The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 °C with 400 μM IPTG and 250 μM l-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of ~10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appK = 30 μM, appk = 14.7 s). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co, Mg, Ca and Ba. However, rLAPLm was activated by Zn, Mn and Cd but is insensitive towards the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a K value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.
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http://dx.doi.org/10.1016/j.pep.2021.105877DOI Listing
July 2021

Evolution, function and roles in drug sensitivity of trypanosome aquaglyceroporins.

Parasitology 2021 Feb 19:1-6. Epub 2021 Feb 19.

School of Life Sciences, University of Dundee, DundeeDD1 5EH, UK.

Aquaglyceroporins (AQPs) are membrane proteins that function in osmoregulation and the uptake of low molecular weight solutes, in particular glycerol and urea. The AQP family is highly conserved, with two major subfamilies having arisen very early in prokaryote evolution and retained by eukaryotes. A complex evolutionary history indicates multiple lineage-specific expansions, losses and not uncommonly a complete loss. Consequently, the AQP family is highly evolvable and has been associated with significant events in life on Earth. In the African trypanosomes, a role for the AQP2 paralogue, in sensitivity to two chemotherapeutic agents, pentamidine and melarsoprol, is well established, albeit with the mechanisms for cell entry and resistance unclear until very recently. Here, we discuss AQP evolution, structure and mechanisms by which AQPs impact drug sensitivity, suggesting that AQP2 stability is highly sensitive to mutation while serving as the major uptake pathway for pentamidine.
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http://dx.doi.org/10.1017/S0031182021000354DOI Listing
February 2021

The life in their years versus the years in their life.

J Thorac Cardiovasc Surg 2021 Jan 12. Epub 2021 Jan 12.

Department of Cardiac Surgery, Liverpool Heart and Chest Hospital, Liverpool, United Kingdom; Liverpool Centre for Cardiovascular Science, University of Liverpool and Liverpool Heart and Chest Hospital, Liverpool, United Kingdom.

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http://dx.doi.org/10.1016/j.jtcvs.2020.11.113DOI Listing
January 2021

Evolution and diversification of the nuclear envelope.

Nucleus 2021 12;12(1):21-41

Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee , Dundee, UK.

Eukaryotic cells arose ~1.5 billion years ago, with the endomembrane system a central feature, facilitating evolution of intracellular compartments. Endomembranes include the nuclear envelope (NE) dividing the cytoplasm and nucleoplasm. The NE possesses universal features: a double lipid bilayer membrane, nuclear pore complexes (NPCs), and continuity with the endoplasmic reticulum, indicating common evolutionary origin. However, levels of specialization between lineages remains unclear, despite distinct mechanisms underpinning various nuclear activities. Several distinct modes of molecular evolution facilitate organellar diversification and  to understand which apply to the NE, we exploited proteomic datasets of purified nuclear envelopes from model systems for comparative analysis. We find enrichment of core nuclear functions amongst the widely conserved proteins to be less numerous than lineage-specific cohorts, but enriched in core nuclear functions. This, together with consideration of additional evidence, suggests that, despite a common origin, the NE has evolved as a highly diverse organelle with significant lineage-specific functionality.
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http://dx.doi.org/10.1080/19491034.2021.1874135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889174PMC
December 2021

Evolving Differentiation in African Trypanosomes.

Trends Parasitol 2021 Apr 11;37(4):296-303. Epub 2020 Dec 11.

School of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK; Institute of Parasitology, Biology Centre, Czech Academy of Sciences, 37005 Ceske Budejovice, Czech Republic. Electronic address:

Differentiation is a central aspect of the parasite life cycle and encompasses adaptation to both host and environment. If we accept that evolution cannot anticipate an organism's needs as it enters a new environment, how do parasite differentiation pathways arise? The transition between vertebrate and insect stage African trypanosomes is probably one of the better studied and involves a cell-cycle arrested or 'stumpy' form that activates metabolic pathways advantageous to the parasite in the insect host. However, a range of stimuli and stress conditions can trigger similar changes, leading to formation of stumpy-like cellular states. We propose that the origin and optimisation of this differentiation program represents repurposing of a generic stress response to gain considerable gain-of-fitness associated with parasite transmission.
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http://dx.doi.org/10.1016/j.pt.2020.11.003DOI Listing
April 2021

Systematic approach to diagnosis and management of infected prosthetic grafts in the proximal aorta.

J Card Surg 2021 Jan 10;36(1):145-152. Epub 2020 Nov 10.

Department of Cardiac Surgery, Liverpool Heart and Chest Hospital, Liverpool, UK.

Objectives: Management of infected prosthetic aortic grafts in the ascending and or root is complex and multifaceted. We report our diagnostic pathway, management and outcomes, identifying successful strategies.

Methods: This was a retrospective, single center, observational study. Consecutive patients who underwent management of infected aortic grafts in the ascending and/or root at our institution between October 1998 and December 2019 were included. The main outcome measures were: discharge from hospital alive with at least 1 year survival, operative mortality and success of primary treatment strategy.

Results: Twenty-six patients presented with infection of proximal aortic grafts and were managed through a number of strategies with an overall hospital-survival of 81% and 1 year survival of 69%. Twenty of them ultimately underwent redo surgery with 25% operative mortality (within 24 h of surgery). Five patients underwent washout and irrigation of which two were successfully treated and cured with adjunctive antibiotics and two went on to have staged explant and definitive surgery. Interval between surgery and infection was 42.5 ± 35.8 months. All patients had at least one major criterion and three minor criterions with no diagnostic uncertainty. The commonest primary strategy was 3a (definitive surgery), (13/26, 50%).

Conclusions: Adopting a systematic and flexible patient specific approach to the diagnosis and management of patients with proximal aortic graft infections results in reasonable overall 1 year survival. In the majority of patients surgery is ultimately required in an attempt to achieve a curative treatment; however this comes with high operative mortality risk.
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http://dx.doi.org/10.1111/jocs.15122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839687PMC
January 2021

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

PLoS Pathog 2020 11 3;16(11):e1008932. Epub 2020 Nov 3.

Wellcome Centre for Integrative Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.
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http://dx.doi.org/10.1371/journal.ppat.1008932DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7710103PMC
November 2020

Reductionist Pathways for Parasitism in Euglenozoans? Expanded Datasets Provide New Insights.

Trends Parasitol 2021 02 27;37(2):100-116. Epub 2020 Oct 27.

Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budějovice (Budweis), Czech Republic; Faculty of Sciences, University of South Bohemia, České Budějovice (Budweis), Czech Republic. Electronic address:

The unicellular trypanosomatids belong to the phylum Euglenozoa and all known species are obligate parasites. Distinct lineages infect plants, invertebrates, and vertebrates, including humans. Genome data for marine diplonemids, together with freshwater euglenids and free-living kinetoplastids, the closest known nonparasitic relatives to trypanosomatids, recently became available. Robust phylogenetic reconstructions across Euglenozoa are now possible and place the results of parasite-focused studies into an evolutionary context. Here we discuss recent advances in identifying the factors shaping the evolution of Euglenozoa, focusing on ancestral features generally considered parasite-specific. Remarkably, most of these predate the transition(s) to parasitism, suggesting that the presence of certain preconditions makes a significant lifestyle change more likely.
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http://dx.doi.org/10.1016/j.pt.2020.10.001DOI Listing
February 2021

Management of Lower Limb Ischemia During Operative Repair of Acute Type A Aortic Dissection by Distal Crossover Grafts: a Case Series.

Braz J Cardiovasc Surg 2020 10 1;35(5):607-613. Epub 2020 Oct 1.

Liverpool Heart and Chest Hospital Department of Cardiothoracic Surgery Liverpool United Kingdom Department of Cardiothoracic Surgery, Liverpool Heart and Chest Hospital, Liverpool, United Kingdom.

Objective: To describe our experience of nine patients with extra-anatomical bypass for clinically ischemic distal limb during repair of acute Type A aortic dissection (ATAAD).

Methods: We retrospectively examined a series of nine patients who underwent surgery for ATAAD. We identified a subset of the patients who presented with concomitant radiographic and clinical signs of lower limb ischemia. All but one patient (axillobifemoral bypass) underwent femorofemoral crossover grafting by the cardiac surgeon during cooling.

Results: One hundred eighty-one cases of ATAAD underwent surgery during the study period with a mortality of 19.3%. Nine patients had persistent clinical evidence of lower limb ischemia (4.9%) and underwent extra-anatomical bypass during cooling. Two patients underwent additional fasciotomies. Mean delay from symptoms to surgery in these nine patients was 9.5 hours. Two patients had bilateral amputations despite revascularisation and, of note, had long delays in presentation for surgery (> 12 hours). There were no mortalities during these inpatient episodes. Outpatient radiographic follow-up at the first opportunity demonstrated 100% patency.

Conclusion: Our experience suggests that, during complicated aortic dissection, limb ischemia may have a devastating outcome including amputation when diagnosis and referral are delayed. Early diagnosis and surgery are crucial in preventing this potentially devastating complication.
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http://dx.doi.org/10.21470/1678-9741-2019-0259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7598955PMC
October 2020

Contemporary results of open thoracic and thoracoabdominal aortic surgery in a single United Kingdom center.

J Vasc Surg 2020 Oct 15. Epub 2020 Oct 15.

Liverpool Cardiovascular Surgery, Liverpool Heart and Chest Hospital, Liverpool, United Kingdom; Faculty of Health and Life Sciences, University of Liverpool, Liverpool, United Kingdom; Liverpool Centre for Cardiovascular Science, University of Liverpool, Liverpool, United Kingdom. Electronic address:

Objective: To report our outcomes and identify predictors of mortality after open descending thoracic aneurysm (DTA) and thoracoabdominal aortic aneurysm (TAAA) repair in a specialist aortic center.

Methods: This retrospective observational cohort study included consecutive patients who underwent surgery at our institution between October 1998 and December 2019. The main outcome measures were mortality and major morbidities. A multivariate analysis was used to identify predictors of mortality.

Results: There were 430 patients who underwent DTA (n = 157) and TAA (n = 273) repair; 151 underwent surgery nonelectively. Forty-eight patients (11%) died within 30 days of surgery. The 30-day mortality was lower after elective surgery (3.1% after DTA repair and 9.9% after TAAA repair), whereas nonelective surgery had a 30-day mortality of 17.9%. Fourteen additional patients died in hospital after 30 days, one after nonelective DTA repair and 13 after TAAA repair (10 elective), all but one extent II. In-hospital mortality for the whole cohort improved over time, as the activity volume increased, except for patients undergoing extent II TAAA repair. Predictors of in-hospital mortality were age ≥70 years (odds ratio [OR], 3.36; 95% confidence interval [CI], 1.79-6.32; P < .001), extent II repair (OR, 4.39; 95% CI, 2.34-8.21; P < .001), nonelective surgery (OR, 2.72; 95% CI, 1.44, 5.12; P = .002), out-of-hours surgery (OR, 8.17; 95% CI, 2.16-30.95; P = .002), a left ventricular ejection fraction of <30% (OR, 9.86; 95% CI, 1.91-50.86; P < .006), and surgery for a degenerative aneurysm (OR, 2.20; 95% CI, 1.12-4.31; P = .02). The incidence of stroke and paraplegia was 7.1% and 0% after DTA repair and 9.9% and 3.3% after TAAA repair. Hemodialysis was necessary in 5.1% of cases after DTA repair and 22.7% after TAAA repair.

Conclusions: Open thoracoabdominal aortic surgery carries significant risk to life, which is related to age, extent of aortic replacement, timing of surgery, and left ventricular function. Morbidity is considerable. Understanding these risks is fundamental for patient selection and the consent process of potential candidates for surgery, particularly in the elderly.
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http://dx.doi.org/10.1016/j.jvs.2020.09.027DOI Listing
October 2020

COVID-19 and cardiac surgery: A perspective from United Kingdom.

J Card Surg 2020 Sep 27. Epub 2020 Sep 27.

Department of Cardiac Surgery, Liverpool Heart and Chest Hospital, Liverpool, UK.

The emergence of severe acute respiratory syndrome coronavirus 2 in December 2019, presumed from the city of Wuhan, Hubei province in China, and the subsequent declaration of the disease as a pandemic by the World Health Organization as coronavirus disease 2019 (COVID-19) in March 2020, had a significant impact on health care systems globally. Each country responded to this disease in different ways, however this was done broadly by fortifying and prioritizing health care provision as well as introducing social lockdown aiming to contain the infection and minimizing the risk of transmission. In the United Kingdom, a lockdown was introduced by the government on March 23, 2020 and all health care services were focussed to challenge the impact of COVID-19. To do so, the United Kingdom National Health Service had to undergo widespread service reconfigurations and the so-called "Nightingale Hospitals" were created de novo to bolster bed provision, and industries were asked to direct efforts to the production of ventilators. A government-led public health campaign was publicized under the slogan of: "Stay home, Protect the NHS (National Health Service), Save lives." The approach had a significant impact on the delivery of all surgical services but particularly cardiac surgery with its inherent critical care bed capacity. This paper describes the impact on provision for elective and emergency cardiac surgery in the United Kingdom, with a focus on aortovascular disease. We describe our aortovascular activity and outcomes during the period of UK lockdown and present a patient survey of attitudes to aortic surgery during COVID-19 pandemic.
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http://dx.doi.org/10.1111/jocs.15039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7537188PMC
September 2020

Management of a Compromised Frozen Elephant Trunk Due to Acute Type B Aortic Dissection.

Vasc Endovascular Surg 2020 Nov 13;54(8):756-759. Epub 2020 Aug 13.

Thoracic Aortic Aneurysm Service, Liverpool Centre for Cardiovascular Sciences, 156669Liverpool Heart and Chest Hospital, Liverpool, United Kingdom.

Purpose: To report a case who required a thoracic endovascular stenting (TEVAR) following the deployment of frozen elephant trunk due to false lumen expansion.

Case Report: A 47 years old male patient undergone emergency repair of acute type A aortic dissection in 2011 with bioprosthetic aortic root conduit. Seven years later he presented with moderate aortic valve disease and expanding chronic dissection of the aortic arch, therefore a redo operation with replacement of the prosthetic aortic valve, ascending aorta, total arch and deployment of frozen elephant trunk and he was discharged in good health. Several days post discharge he presented with new onset of chest pain and a new dissection involved the thoracoabdominal aorta was noted pressing on the true lumen and the frozen elephant trunk. Following a multi-disciplinary team meeting, TEVAR was deemed as a most appropriate approach and this was achieved successfully, and patient was discharged. At 1 year of follow up, he remains well and asymptomatic.

Conclusion: Close imaging follow-up following deployment of a FET is mandatory. A new acute Type B aortic dissection distal to the FET, that causes false lumen propagation parallel to the stented portion, is a surgical emergency and further intervention mandated.
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http://dx.doi.org/10.1177/1538574420949313DOI Listing
November 2020

Positively selected modifications in the pore of TbAQP2 allow pentamidine to enter .

Elife 2020 08 11;9. Epub 2020 Aug 11.

Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom.

Mutations in the aquaporin AQP2 are associated with resistance to pentamidine and melarsoprol. We show that TbAQP2 but not TbAQP3 was positively selected for increased pore size from a common ancestor aquaporin. We demonstrate that TbAQP2's unique architecture permits pentamidine permeation through its central pore and show how specific mutations in highly conserved motifs affect drug permeation. Introduction of key TbAQP2 amino acids into TbAQP3 renders the latter permeable to pentamidine. Molecular dynamics demonstrates that permeation by dicationic pentamidine is energetically favourable in TbAQP2, driven by the membrane potential, although aquaporins are normally strictly impermeable for ionic species. We also identify the structural determinants that make pentamidine a permeant although most other diamidine drugs are excluded. Our results have wide-ranging implications for optimising antitrypanosomal drugs and averting cross-resistance. Moreover, these new insights in aquaporin permeation may allow the pharmacological exploitation of other members of this ubiquitous gene family.
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http://dx.doi.org/10.7554/eLife.56416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473772PMC
August 2020

TEVAR in aortic dissection: A new standard for Marfan patients during COVID-19?

J Card Surg 2020 09 15;35(9):2443. Epub 2020 Jul 15.

Department of Cardiothoracic Surgery, Liverpool Heart and Chest Hospital, Liverpool, UK.

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http://dx.doi.org/10.1111/jocs.14863DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7405391PMC
September 2020

Instability of aquaglyceroporin (AQP) 2 contributes to drug resistance in Trypanosoma brucei.

PLoS Negl Trop Dis 2020 07 9;14(7):e0008458. Epub 2020 Jul 9.

School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.

Defining mode of action is vital for both developing new drugs and predicting potential resistance mechanisms. Sensitivity of African trypanosomes to pentamidine and melarsoprol is predominantly mediated by aquaglyceroporin 2 (TbAQP2), a channel associated with water/glycerol transport. TbAQP2 is expressed at the flagellar pocket membrane and chimerisation with TbAQP3 renders parasites resistant to both drugs. Two models for how TbAQP2 mediates pentamidine sensitivity have emerged; that TbAQP2 mediates pentamidine translocation across the plasma membrane or via binding to TbAQP2, with subsequent endocytosis and presumably transport across the endosomal/lysosomal membrane, but as trafficking and regulation of TbAQPs is uncharacterised this remains unresolved. We demonstrate that TbAQP2 is organised as a high order complex, is ubiquitylated and is transported to the lysosome. Unexpectedly, mutation of potential ubiquitin conjugation sites, i.e. cytoplasmic-oriented lysine residues, reduced folding and tetramerization efficiency and triggered ER retention. Moreover, TbAQP2/TbAQP3 chimerisation, as observed in pentamidine-resistant parasites, also leads to impaired oligomerisation, mislocalisation and increased turnover. These data suggest that TbAQP2 stability is highly sensitive to mutation and that instability contributes towards the emergence of drug resistance.
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http://dx.doi.org/10.1371/journal.pntd.0008458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7413563PMC
July 2020

EIF2α phosphorylation is regulated in intracellular amastigotes for the generation of infective Trypanosoma cruzi trypomastigote forms.

Cell Microbiol 2020 11 28;22(11):e13243. Epub 2020 Jul 28.

Departmento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.

Trypanosomatids regulate gene expression mainly at the post-transcriptional level through processing, exporting and stabilising mRNA and control of translation. In most eukaryotes, protein synthesis is regulated by phosphorylation of eukaryotic initiation factor 2 (eIF2) at serine 51. Phosphorylation halts overall translation by decreasing availability of initiator tRNA to form translating ribosomes. In trypanosomatids, the N-terminus of eIF2α is extended with threonine 169 the homologous phosphorylated residue. Here, we evaluated whether eIF2α phosphorylation varies during the Trypanosoma cruzi life cycle, the etiological agent of Chagas' disease. Total levels of eIF2α are diminished in infective and non-replicative trypomastigotes compared with proliferative forms from the intestine of the insect vector or amastigotes from mammalian cells, consistent with decreased protein synthesis reported in infective forms. eIF2α phosphorylation increases in proliferative intracellular forms prior to differentiation into trypomastigotes. Parasites overexpressing eIF2α or with an endogenous CRISPR/Cas9-generated eIF2α mutation were created and analysis revealed alterations to the proteome, largely unrelated to the presence of μORF in epimastigotes. eIF2α mutant parasites produced fewer trypomastigotes with lower infectivity than wild type, with increased levels of sialylated mucins and oligomannose glycoproteins, and decreased galactofuranose epitopes and the surface protease GP63 on the cell surface. We conclude that eIF2α expression and phosphorylation levels affect proteins relevant for intracellular progression of T. cruzi.
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http://dx.doi.org/10.1111/cmi.13243DOI Listing
November 2020

Frozen elephant trunk: reflections of the UK Aortic Group.

Ann Cardiothorac Surg 2020 May;9(3):228-229

Clinical Lead for Cardiac Surgery, Liverpool Heart and Chest Hospital, Liverpool, UK.

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http://dx.doi.org/10.21037/acs.2020.02.04DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7298240PMC
May 2020

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Using RapidFire Mass Spectrometry.

SLAS Discov 2020 10 13;25(9):1064-1071. Epub 2020 May 13.

Centre for Protein Studies, Faculty of Biology, University of Havana, La Habana, Cuba.

Leucyl aminopeptidases (LAPs) are involved in multiple cellular functions, which, in the case of infectious diseases, includes participation in the pathogen-host cell interface and pathogenesis. Thus, LAPs are considered good candidate drug targets, and the major M17-LAP from (LAPTc) in particular is a promising target for Chagas disease. To exploit LAPTc as a potential target, it is essential to develop potent and selective inhibitors. To achieve this, we report a high-throughput screening method for LAPTc. Two methods were developed and optimized: a Leu-7-amido-4-methylcoumarin-based fluorogenic assay and a RapidFire mass spectrometry (RapidFire MS)-based assay using the LSTVIVR peptide as substrate. Compared with a fluorescence assay, the major advantages of the RapidFire MS assay are a greater signal-to-noise ratio as well as decreased consumption of enzyme. RapidFire MS was validated with the broad-spectrum LAP inhibitors bestatin (IC = 0.35 μM) and arphamenine A (IC = 15.75 μM). We suggest that RapidFire MS is highly suitable for screening for specific LAPTc inhibitors.
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http://dx.doi.org/10.1177/2472555220923367DOI Listing
October 2020

Suramin exposure alters cellular metabolism and mitochondrial energy production in African trypanosomes.

J Biol Chem 2020 06 30;295(24):8331-8347. Epub 2020 Apr 30.

School of Life Sciences, University of Dundee, Dundee, Scotland, United Kingdom

Introduced about a century ago, suramin remains a frontline drug for the management of early-stage East African trypanosomiasis (sleeping sickness). Cellular entry into the causative agent, the protozoan parasite , occurs through receptor-mediated endocytosis involving the parasite's invariant surface glycoprotein 75 (ISG75), followed by transport into the cytosol via a lysosomal transporter. The molecular basis of the trypanocidal activity of suramin remains unclear, but some evidence suggests broad, but specific, impacts on trypanosome metabolism ( polypharmacology). Here we observed that suramin is rapidly accumulated in trypanosome cells proportionally to ISG75 abundance. Although we found little evidence that suramin disrupts glycolytic or glycosomal pathways, we noted increased mitochondrial ATP production, but a net decrease in cellular ATP levels. Metabolomics highlighted additional impacts on mitochondrial metabolism, including partial Krebs' cycle activation and significant accumulation of pyruvate, corroborated by increased expression of mitochondrial enzymes and transporters. Significantly, the vast majority of suramin-induced proteins were normally more abundant in the insect forms compared with the blood stage of the parasite, including several proteins associated with differentiation. We conclude that suramin has multiple and complex effects on trypanosomes, but unexpectedly partially activates mitochondrial ATP-generating activity. We propose that despite apparent compensatory mechanisms in drug-challenged cells, the suramin-induced collapse of cellular ATP ultimately leads to trypanosome cell death.
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http://dx.doi.org/10.1074/jbc.RA120.012355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7294092PMC
June 2020

Validity of the cold pressor test and pain sensitivity questionnaire via online self-administration.

PLoS One 2020 16;15(4):e0231697. Epub 2020 Apr 16.

23andMe Inc., Mountain View, California, United States of America.

To determine the feasibility of complex home-based phenotyping, 1,876 research participants from the customer base of 23andMe completed an online version of a Pain Sensitivity Questionnaire (PSQ) as well as a cold pressor test (CPT) which is used in clinical assessments of pain. Overall our online version of the PSQ performed similarly to the original pen-and-paper version. Construct validity of the PSQ total was demonstrated by internal consistency and consistent discrimination between more and less painful items. Criterion validity was demonstrated by correlation with pain sensitivity as measured by the CPT. Within the same cohort we performed a cold pressor test using a layperson description and household equipment. Comparison with published reports from controlled studies revealed similar distributions of cold pain tolerance times (i.e., time elapsed before removing the hand from the water). Of those who elected to participate in the CPT, a large majority of participants did not report issues with the test procedure or noncompliance with the instructions (97%). We confirmed a large sex difference in CPT thresholds in line with published data, such that women removed their hands from the water at a median of 54.2 seconds, with men lasting for a median time of 82.7 seconds (Kruskal-Wallis statistic, p < 0.0001), but other factors like age or current pain treatment were at most weakly associated, and inconsistently between men and women. We introduce a new paradigm for performing pain testing, called testing@home, that, in the case of cold nociception, showed comparable results to studies conducted under controlled conditions and supervision of a health care professional.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231697PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7162430PMC
July 2020

Sorting the Muck from the Brass: Analysis of Protein Complexes and Cell Lysates.

Methods Mol Biol 2020 ;2116:645-653

School of Life Sciences, University of Dundee, Dundee, UK.

Reliable determination of protein complex composition or changes to protein levels in whole cells is challenging. Despite the multitude of methods now available for labeling, analysis, and the statistical processing of data, this large variety is of itself an issue: Which approach is most appropriate, where do you set cutoffs, and what is the most cost-effective strategy? One size does not fit all for such work, but some guidelines can help in terms of reducing cost, improving data quality, and ultimately advancing investigations. Here we describe two protocols and algorithms for facile sample preparation for mass spectrometric analysis, robust data processing, and considerations of how to interpret large proteomic datasets in a productive and robust manner.
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http://dx.doi.org/10.1007/978-1-0716-0294-2_38DOI Listing
February 2021

Postoperative Remote Automated Monitoring and Virtual Hospital-to-Home Care System Following Cardiac and Major Vascular Surgery: User Testing Study.

J Med Internet Res 2020 03 18;22(3):e15548. Epub 2020 Mar 18.

Population Health Research Institute, Hamilton, ON, Canada.

Background: Cardiac and major vascular surgeries are common surgical procedures associated with high rates of postsurgical complications and related hospital readmission. In-hospital remote automated monitoring (RAM) and virtual hospital-to-home patient care systems have major potential to improve patient outcomes following cardiac and major vascular surgery. However, the science of deploying and evaluating these systems is complex and subject to risk of implementation failure.

Objective: As a precursor to a randomized controlled trial (RCT), this user testing study aimed to examine user performance and acceptance of a RAM and virtual hospital-to-home care intervention, using Philip's Guardian and Electronic Transition to Ambulatory Care (eTrAC) technologies, respectively.

Methods: Nurses and patients participated in systems training and individual case-based user testing at two participating sites in Canada and the United Kingdom. Participants were video recorded and asked to think aloud while completing required user tasks and while being rated on user performance. Feedback was also solicited about the user experience, including user satisfaction and acceptance, through use of the Net Promoter Scale (NPS) survey and debrief interviews.

Results: A total of 37 participants (26 nurses and 11 patients) completed user testing. The majority of nurse and patient participants were able to complete most required tasks independently, demonstrating comprehension and retention of required Guardian and eTrAC system workflows. Tasks which required additional prompting by the facilitator, for some, were related to the use of system features that enable continuous transmission of patient vital signs (eg, pairing wireless sensors to the patient) and assigning remote patient monitoring protocols. NPS scores by user group (nurses using Guardian: mean 8.8, SD 0.89; nurses using eTrAC: mean 7.7, SD 1.4; patients using eTrAC: mean 9.2, SD 0.75), overall NPS scores, and participant debrief interviews indicated nurse and patient satisfaction and acceptance of the Guardian and eTrAC systems. Both user groups stressed the need for additional opportunities to practice in order to become comfortable and proficient in the use of these systems.

Conclusions: User testing indicated a high degree of user acceptance of Philips' Guardian and eTrAC systems among nurses and patients. Key insights were provided that informed refinement of clinical workflow training and systems implementation. These results were used to optimize workflows before the launch of an international RCT of in-hospital RAM and virtual hospital-to-home care for patients undergoing cardiac and major vascular surgery.
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http://dx.doi.org/10.2196/15548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7113803PMC
March 2020

A Uniquely Complex Mitochondrial Proteome from Euglena gracilis.

Mol Biol Evol 2020 08;37(8):2173-2191

Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budějovice, Budweis, Czech Republic.

Euglena gracilis is a metabolically flexible, photosynthetic, and adaptable free-living protist of considerable environmental importance and biotechnological value. By label-free liquid chromatography tandem mass spectrometry, a total of 1,786 proteins were identified from the E. gracilis purified mitochondria, representing one of the largest mitochondrial proteomes so far described. Despite this apparent complexity, protein machinery responsible for the extensive RNA editing, splicing, and processing in the sister clades diplonemids and kinetoplastids is absent. This strongly suggests that the complex mechanisms of mitochondrial gene expression in diplonemids and kinetoplastids occurred late in euglenozoan evolution, arising independently. By contrast, the alternative oxidase pathway and numerous ribosomal subunits presumed to be specific for parasitic trypanosomes are present in E. gracilis. We investigated the evolution of unexplored protein families, including import complexes, cristae formation proteins, and translation termination factors, as well as canonical and unique metabolic pathways. We additionally compare this mitoproteome with the transcriptome of Eutreptiella gymnastica, illuminating conserved features of Euglenida mitochondria as well as those exclusive to E. gracilis. This is the first mitochondrial proteome of a free-living protist from the Excavata and one of few available for protists as a whole. This study alters our views of the evolution of the mitochondrion and indicates early emergence of complexity within euglenozoan mitochondria, independent of parasitism.
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http://dx.doi.org/10.1093/molbev/msaa061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403612PMC
August 2020

Expression of a specific variant surface glycoprotein has a major impact on suramin sensitivity and endocytosis in .

FASEB Bioadv 2019 Oct 30;1(10):595-608. Epub 2019 Sep 30.

Swiss Tropical and Public Health Institute Basel Switzerland.

Suramin was introduced into the clinic a century ago and is still used to treat the first stage of acute human sleeping sickness. Due to its size and sixfold negative charge, uptake is mediated through endocytosis and the suramin receptor in trypanosomes is thought to be the invariant surface glycoprotein 75 (ISG75). Nevertheless, we recently identified a variant surface glycoprotein (VSG) that confers strong in vitro resistance to suramin in a line. In this study, we introduced into the active bloodstream expression site of a line. This caused suramin resistance and cross resistance to trypan blue. We quantified the endocytosis of different substrates by flow cytometry and showed that the expression of VSG strongly impairs the uptake of low-density lipoprotein (LDL) and transferrin, both imported by receptor-mediated endocytosis. However, bulk endocytosis and endocytosis of the trypanolytic factor were not affected, and the -expressors did not exhibit a growth phenotype in the absence of suramin. Knockdown of ISG75 was synergistic with expression, indicating that these two proteins are mediating distinct suramin resistance pathways. In conclusion, VSG causes suramin resistance in bloodstream forms by decreasing specific, receptor-mediated endocytosis pathways.
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http://dx.doi.org/10.1096/fba.2019-00033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996322PMC
October 2019

The Artemisinin Susceptibility-Associated AP-2 Adaptin μ Subunit is Clathrin Independent and Essential for Schizont Maturation.

mBio 2020 02 25;11(1). Epub 2020 Feb 25.

Department of Infection Biology, Faculty of Infectious Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom

The efficacy of current antimalarial drugs is threatened by reduced susceptibility of to artemisinin, associated with mutations in Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in , we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent. We examine in detail the AP-2 adaptin complex from the malaria parasite In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.
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http://dx.doi.org/10.1128/mBio.02918-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042695PMC
February 2020

Diversification of CORVET tethers facilitates transport complexity in .

J Cell Sci 2020 02 12;133(3). Epub 2020 Feb 12.

Department of Molecular Genetics and Cell Biology, 920 E 58th Street, The University of Chicago, Chicago, IL, 60637, USA

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/jcs.238659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7033735PMC
February 2020

Evolution of late steps in exocytosis: conservation and specialization of the exocyst complex.

Wellcome Open Res 2019 29;4:112. Epub 2019 Nov 29.

School of Life Sciences, University of Dundee, Dow Street, Dundee, DD1 5EH, UK.

The eukaryotic endomembrane system most likely arose paralogous expansions of genes encoding proteins that specify organelle identity, coat complexes and govern fusion specificity. While the majority of these gene families were established by the time of the last eukaryotic common ancestor (LECA), subsequent evolutionary events has moulded these systems, likely reflecting adaptations retained for increased fitness. As well as sequence evolution, these adaptations include loss of otherwise canonical components, the emergence of lineage-specific proteins and paralog expansion. The exocyst complex is involved in late exocytosis and additional trafficking pathways and a member of the complexes associated with tethering containing helical rods (CATCHR) tethering complex family. CATCHR includes the conserved oligomeric Golgi (COG) complex, homotypic fusion and vacuole protein sorting (HOPS)/class C core vacuole/endosome tethering (CORVET) complexes and several others. The exocyst is integrated into a complex GTPase signalling network in animals, fungi and other lineages. Prompted by discovery of Exo99, a non-canonical subunit in the excavate protist and availability of significantly increased genome sequence data, we re-examined evolution of the exocyst. We examined the evolution of exocyst components by comparative genomics, phylogenetics and structure prediction. The exocyst composition is highly conserved, but with substantial losses of subunits in the Apicomplexa and expansions in Streptophyta plants, Metazoa and land plants, where for the latter, massive paralog expansion of Exo70 represents an extreme and unique example. Significantly, few taxa retain a partial complex, suggesting that, in general, all subunits are probably required for functionality. Further, the ninth exocyst subunit, Exo99, is specific to the Euglenozoa with a distinct architecture compared to the other subunits and which possibly represents a coat system. These data reveal a remarkable degree of evolutionary flexibility within the exocyst complex, suggesting significant diversity in exocytosis mechanisms.
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http://dx.doi.org/10.12688/wellcomeopenres.15142.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784791PMC
November 2019

SUMOylated SNF2PH promotes variant surface glycoprotein expression in bloodstream trypanosomes.

EMBO Rep 2019 12 6;20(12):e48029. Epub 2019 Nov 6.

Instituto de Parasitología y Biomedicina "López-Neyra", CSIC (IPBLN-CSIC), Granada, Spain.

SUMOylation is a post-translational modification that positively regulates monoallelic expression of the trypanosome variant surface glycoprotein (VSG). The presence of a highly SUMOylated focus associated with the nuclear body, where the VSG gene is transcribed, further suggests an important role of SUMOylation in regulating VSG expression. Here, we show that SNF2PH, a SUMOylated plant homeodomain (PH)-transcription factor, is upregulated in the bloodstream form of the parasite and enriched at the active VSG telomere. SUMOylation promotes the recruitment of SNF2PH to the VSG promoter, where it is required to maintain RNA polymerase I and thus to regulate VSG transcript levels. Further, ectopic overexpression of SNF2PH in insect forms, but not of a mutant lacking the PH domain, induces the expression of bloodstream stage-specific surface proteins. These data suggest that SNF2PH SUMOylation positively regulates VSG monoallelic transcription, while the PH domain is required for the expression of bloodstream-specific surface proteins. Thus, SNF2PH functions as a positive activator, linking expression of infective form surface proteins and VSG regulation, thereby acting as a major regulator of pathogenicity.
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http://dx.doi.org/10.15252/embr.201948029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893287PMC
December 2019