Publications by authors named "Robert Andrews"

301 Publications

Conventional and biological treatment for the removal of microplastics from drinking water.

Chemosphere 2021 Oct 15:132587. Epub 2021 Oct 15.

University of Toronto, Department of Civil and Mineral Engineering, 35 St George Street, Toronto, ON, M5S 1A4, Canada.

This study examines the removal of microplastics and other anthropogenic particles (>10 μm) from surface water by a full-scale conventional drinking water treatment plant. The treatment process is composed of coagulation with aluminum hydroxide, flocculation, anthracite-sand filtration, and chlorination. Samples were also collected from pilot-scale biological filters consisting of anthracite-sand or granular activated carbon (GAC) media operated with or without pre-ozonation and at a range of different empty-bed contact times (EBCTs). Particles in 10 L water samples collected in duplicate using a fully enclosed sampling apparatus were separated using sieves with 500 μm, 300 μm, 125 μm, and 45 μm openings followed by filtration through 10 μm polycarbonate filters. Particles were counted using stereomicroscopy and characterized using μ-Raman spectroscopy. Full-scale conventional treatment removed 52 % of anthropogenic particles when comparing raw (42 ± 18 particles/L) and finished water (20 ± 8 particles/L). Coagulation, flocculation, and sedimentation accounted for the highest removal (70 %) of any individual unit process. Overall removal was reduced to 52 %, the difference being attributed to airborne particle deposition that occurred while water was detained in a clearwell (exposed to atmosphere via ventilation) that was used to achieve the required contact time for disinfection. The majority of the particles (>80 %) were identified as fibers 10-45 μm; microplastics were predominantly composed of polyester while the non-plastic anthropogenic particles were primarily cellulose. None of the pilot filter configurations examined resulted in significantly fewer microplastics when compared to full-scale conventional filtration. This study illustrates that the removal efficiency of conventional treatment may be limited when considering microfibers <45 μm in size.
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http://dx.doi.org/10.1016/j.chemosphere.2021.132587DOI Listing
October 2021

Evaluation of enzyme activity for monitoring biofiltration performance in drinking water treatment.

Water Res 2021 Sep 9;205:117636. Epub 2021 Sep 9.

Department of Civil and Mineral Engineering, University of Toronto, 35 St. George St., Toronto, Ontario, M5S 1A4, Canada. Electronic address:

Many water providers monitor adenosine triphosphate (ATP) as an indicator of biological acclimation of their biofilters; however, strong correlations between ATP concentration and filter performance (e.g., organic matter or disinfection by-product precursor removal) are not typically observed. As an alternative, this study evaluated the use of enzyme activity for monitoring biological processes within filters. Recent studies have proposed that enzyme activity may be used as an indicator of biofilter function as it provides a means to quantify biodegradation which may allow for a more accurate measure of degradation potential and to gain a better understanding of biofilter performance. Sampling was completed from full- and pilot-scale biofilters to assess impacts associated with pre-treatments, varying sources waters, as well as pre-treatment and operating conditions. Enzyme activity (carboxylic esterase, phosphatase, ß-glucosidase, α-glucosidase, ß-xylosidase, chitinase, and cellulase) and ATP were measured from the top 5 cm of biofilter media representative of typical full-scale sampling; water quality parameters included dissolved organic carbon (DOC) and disinfection by-products (DBPs): trihalomethane (THM) formation potential (FP), and haloacetic acid FP (HAA FP). Results confirmed that ATP was not a reliable monitoring tool for DOC and DBP FP reduction in biofilters. A strong relationship was observed between esterase activity and DOC reduction; chitinase activity significantly correlated to THM FP reduction for filters treating three different source waters and HAA FP reduction achieved by filters treating the same source water with a range of pre-treatment and backwash conditions. This study showed that enzyme activity may be appropriate for monitoring biological processes within drinking water filters and may act as a surrogate for the removal of organic compounds.
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http://dx.doi.org/10.1016/j.watres.2021.117636DOI Listing
September 2021

Assessment of microplastic sampling and extraction methods for drinking waters.

Chemosphere 2021 Aug 12;286(Pt 3):131881. Epub 2021 Aug 12.

Department of Civil and Mineral Engineering, University of Toronto, 35 St. George St., Toronto, Ontario, M5S 1A4, Canada.

To date, no standardized methods have been proposed for conducting microplastic analyses in treated drinking waters, resulting in challenges associated with direct comparisons among studies. This study compares known methods for collecting and extracting microplastics from drinking waters: an in-laboratory (in-lab) filtration method and an in-line filtration method (i.e., water filtered on-site without an intermediate storage and/or transportation step). In-lab methods have been the predominant method for sample collection in drinking water matrices, and in-line methods are emerging due to the potential to sample large volumes of water on site and minimize contamination from airborne particles, but the two methods have yet to be directly compared using real samples. In response, this study evaluates both methods in terms of recovering spiked reference microplastics, collecting microplastics from tap water samples using the same water volume, and quantifying the removal of microplastics through a full-scale ultrafiltration system. In-line filtration was shown to have higher recoveries for all the reference microplastics examined (+37 % for PVC fragments, +23 % for PET fragments, +22 % for nylon fibers and +7 % for PET fibers) and a greater potential to reduce microplastic contamination. It also resulted in lower standard deviations for total microplastic counts in the tap water and UF influent and effluent samples. The filtration capacity of the proposed in-line filtration method could exceed 350 L of treated water, but this is highly dependent on the water quality. This study therefore supports the use of in-line filtration methods towards the standardization of microplastic collection procedures in drinking water.
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http://dx.doi.org/10.1016/j.chemosphere.2021.131881DOI Listing
August 2021

Measurement of Peak C-Peptide at Diagnosis Informs Glycemic Control but not Hypoglycemia in Adults With Type 1 Diabetes.

J Endocr Soc 2021 Oct 17;5(10):bvab127. Epub 2021 Jul 17.

Institute of Biomedical and Clinical Science, University of Exeter Medical School, Exeter, Devon EX2 5DW, UK.

Context: High-residual C-peptide in longer-duration type 1 diabetes (T1D) is associated with fewer hypoglycemic events and reduced glycemic variability. Little is known about the impact of C-peptide close to diagnosis.

Objective: Using continuous glucose monitoring (CGM) data from a study of newly diagnosed adults with T1D, we aimed to explore if variation in C-peptide close to diagnosis influenced glycemic variability and risk of hypoglycemia.

Methods: We studied newly diagnosed adults with T1D who wore a Dexcom G4 CGM for 7 days as part of the Exercise in Type 1 Diabetes (EXTOD) study. We examined the relationship between peak stimulated C-peptide and glycemic metrics of variability and hypoglycemia for 36 CGM traces from 23 participants.

Results: For every 100 pmol/L-increase in peak C-peptide, the percentage of time spent in the range 3.9 to 10 mmol/L increased by 2.4% (95% CI, 0.5-4.3),  = .01) with a reduction in time spent at level 1 hyperglycemia (> 10 mmol/L) and level 2 hyperglycemia (> 13.9 mmol/L) by 2.6% (95% CI, -4.9 to -0.4,  = .02) and 1.3% (95% CI, -2.7 to -0.006,  = .04), respectively. Glucose levels were on average lower by 0.19 mmol/L (95% CI, -0.4 to 0.02,  = .06) and SD reduced by 0.14 (95% CI, -0.3 to -0.02,  = .02). Hypoglycemia was not common in this group and no association was observed between time spent in hypoglycemia ( = .97) or hypoglycemic risk ( = .72). There was no association between peak C-peptide and insulin dose-adjusted glycated hemoglobin A ( = .45).

Conclusion: C-peptide is associated with time spent in the normal glucose range and with less hyperglycemia, but not risk of hypoglycemia in newly diagnosed people with T1D.
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http://dx.doi.org/10.1210/jendso/bvab127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8344843PMC
October 2021

Modest changes in Spi1 dosage reveal the potential for altered microglial function as seen in Alzheimer's disease.

Sci Rep 2021 07 22;11(1):14935. Epub 2021 Jul 22.

Division of Infection and Immunity, Cardiff University, Cardiff, UK.

Genetic association studies have identified multiple variants at the SPI1 locus that modify risk and age of onset for Alzheimer's Disease (AD). Reports linking risk variants to gene expression suggest that variants denoting higher SPI1 expression are likely to have an earlier AD onset, and several other AD risk genes contain PU.1 binding sites in the promoter region. Overall, this suggests the level of SPI1 may alter microglial phenotype potentially impacting AD. This study determined how the microglial transcriptome was altered following modest changes to Spi1 expression in primary mouse microglia. RNA-sequencing was performed on microglia with reduced or increased Spi1/PU.1 expression to provide an unbiased approach to determine transcriptomic changes affected by Spi1. In summary, a reduction in microglial Spi1 resulted in the dysregulation of transcripts encoding proteins involved in DNA replication pathways while an increased Spi1 results in an upregulation of genes associated with immune response pathways. Additionally, a subset of 194 Spi1 dose-sensitive genes was identified and pathway analysis suggests that several innate immune and interferon response pathways are impacted by the concentration of Spi1. Together these results suggest Spi1 levels can alter the microglial transcriptome and suggests interferon pathways may be altered in individuals with AD related Spi1 risk SNPs.
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http://dx.doi.org/10.1038/s41598-021-94324-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8298495PMC
July 2021

Lipidomic and transcriptional analysis of the linoleoyl-omega-hydroxyceramide biosynthetic pathway in human psoriatic lesions.

J Lipid Res 2021 Jun 22;62:100094. Epub 2021 Jun 22.

Institute of Infection and Immunity and Systems Immunity Research Institute, School of Medicine, Cardiff University, Nashville, TN, USA; School of Pharmacy and Pharmaceutical Sciences, Cardiff University, Aurora, CO, USA. Electronic address:

A complex assembly of lipids including fatty acids, cholesterol, and ceramides is vital to the integrity of the mammalian epidermal barrier. The formation of this barrier requires oxidation of the substrate fatty acid, linoleic acid (LA), which is initiated by the enzyme 12R-lipoxygenase (LOX). In the epidermis, unoxidized LA is primarily found in long-chain acylceramides termed esterified omega-hydroxy sphingosine (EOS)/phytosphingosine/hydroxysphingosine (collectively EOx). The precise structure and localization of LOX-oxidized EOx in the human epidermis is unknown, as is their regulation in diseases such as psoriasis, one of the most common inflammatory diseases affecting the skin. Here, using precursor LC/MS/MS, we characterized multiple intermediates of EOx, including 9-HODE, 9,10-epoxy-13-HOME, and 9,10,13-TriHOME, in healthy human epidermis likely to be formed via the epidermal LOX pathways. The top layers of the skin contained more LA, 9-HODE, and 9,10,13-TriHOME EOSs, whereas 9,10-epoxy-13-HOME EOS was more prevalent deeper in the stratum corneum. In psoriatic lesions, levels of native EOx and free HODEs and HOMEs were significantly elevated, whereas oxidized species were generally reduced. A transcriptional network analysis of human psoriatic lesions identified significantly elevated expression of the entire biosynthetic/metabolic pathway for oxygenated ceramides, suggesting a regulatory function for EOx lipids in reconstituting epidermal integrity. The role of these new lipids in progression or resolution of psoriasis is currently unknown. We also discovered the central coordinated role of the zinc finger protein transcription factor, ZIC1, in driving the phenotype of this disease. In summary, long-chain oxygenated ceramide metabolism is dysregulated at the lipidomic level in psoriasis, likely driven by the transcriptional differences also observed, and we identified ZIC1 as a potential regulatory target for future therapeutic interventions.
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http://dx.doi.org/10.1016/j.jlr.2021.100094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8326207PMC
June 2021

Single-Nucleus RNA Sequencing Identifies New Classes of Proximal Tubular Epithelial Cells in Kidney Fibrosis.

J Am Soc Nephrol 2021 Oct 21;32(10):2501-2516. Epub 2021 Jun 21.

Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, United Kingdom

Background: Proximal tubular cells (PTCs) are the most abundant cell type in the kidney. PTCs are central to normal kidney function and to regeneration versus organ fibrosis following injury. This study used single-nucleus RNA sequencing (snRNAseq) to describe the phenotype of PTCs in renal fibrosis.

Methods: Kidneys were harvested from naïve mice and from mice with renal fibrosis induced by chronic aristolochic acid administration. Nuclei were isolated using Nuclei EZ Lysis buffer. Libraries were prepared on the 10× platform, and snRNAseq was completed using the Illumina NextSeq 550 System. Genome mapping was carried out with high-performance computing.

Results: A total of 23,885 nuclei were analyzed. PTCs were found in five abundant clusters, mapping to S1, S1-S2, S2, S2-cortical S3, and medullary S3 segments. Additional cell clusters ("new PTC clusters") were at low abundance in normal kidney and in increased number in kidneys undergoing regeneration/fibrosis following injury. These clusters exhibited clear molecular phenotypes, permitting labeling as proliferating, New-PT1, New-PT2, and (present only following injury) New-PT3. Each cluster exhibited a unique gene expression signature, including multiple genes previously associated with renal injury response and fibrosis progression. Comprehensive pathway analyses revealed metabolic reprogramming, enrichment of cellular communication and cell motility, and various immune activations in new PTC clusters. In ligand-receptor analysis, new PTC clusters promoted fibrotic signaling to fibroblasts and inflammatory activation to macrophages.

Conclusions: These data identify unrecognized PTC phenotype heterogeneity and reveal novel PTCs associated with kidney fibrosis.
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http://dx.doi.org/10.1681/ASN.2020081143DOI Listing
October 2021

Anti-inflammatory and immunoregulatory effects of pinolenic acid in rheumatoid arthritis.

Rheumatology (Oxford) 2021 Jun 3. Epub 2021 Jun 3.

Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK.

Objectives: In pre-clinical studies, pinolenic acid (PNLA), an omega-6-polyunsaturated fatty acid from pine nuts has shown anti-inflammatory effects. We aimed to investigate the effect of PNLA in human cell lines and peripheral blood mononuclear cells (PBMCs) from rheumatoid arthritis (RA) patients and healthy controls (HCs).

Methods: Modified Boyden chamber was used to assess chemokine-induced migration of THP-1 monocytes. Macropinocytosis was assessed using lucifer yellow and ox-LDL uptake using Dil-oxLDL in THP-1 macrophages and human monocyte-derived macrophages (HMDM). IL6, TNFα and PGE2 release by lipopolysaccharide (LPS) stimulated PBMCs from RA patients and HCs were measured by ELISA. The transcriptomic profile of PNLA treated, LPS activated PBMCs was investigated by RNA-sequencing.

Results: PNLA reduced THP-1 cell migration by 55% (p< 0.001). Macropinocytosis and Dil-oxLDL uptake were reduced by 50% (p< 0.001) and 40% (p< 0.01) in THP-1 macrophages and 40% (p< 0.01) and 25% (p< 0.05) in HMDM, respectively. PNLA reduced IL6 and TNFα release from LPS stimulated PBMCs from RA by 60% (p< 0.001) and by 50% and 35% respectively (p< 0.01) for HCs. PNLA also reduced PGE2 levels in such PBMCs from RA patients and HCs (p< 0.0001). Differentially expressed genes included upregulated expression of pyruvate dehydrogenase kinase-4, plasminogen activator inhibitor-1, fructose bisphosphatase 1 and N-Myc downstream-regulated gene, which have potential roles in regulating immune and metabolic pathways. Pathway analysis predicted upstream activation of nuclear receptors peroxisome proliferator-activated receptors involved in anti-inflammatory processes, and inhibition of NF-κB and STAT1.

Conclusions: PNLA has immune-metabolic effects on monocytes and PBMC which are pathogenic in RA and atherosclerosis. Dietary PNLA supplementation may be beneficial in RA.
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http://dx.doi.org/10.1093/rheumatology/keab467DOI Listing
June 2021

Trauma by-pass guideline: A data-driven conformance analysis for road trauma cases in Queensland.

Emerg Med Australas 2021 May 31. Epub 2021 May 31.

School of Information Systems, Queensland University of Technology, Brisbane, Queensland, Australia.

Objective: Study objectives were to (i) develop and test a whole-of-system method for identifying patients who meet a major trauma by-pass guideline definition; (ii) apply this method to assess conformance to the current 2006 guideline for a road trauma cohort; and (iii) leverage relevant findings to propose improvements to the guideline.

Methods: Retrospective analysis of existing, routinely collected data relating to Queensland road trauma patients July 2015 to June 2017. Data from ambulance, aero-medical retrievals, ED, hospital and death registers were linked and used for analysis. Processes of care measured included: frequency of pre-hospital triage criteria, distribution of destination (trauma service level), compliance with guideline (recommended vs actual destination), trauma service level by threat to life (injury severity) (all modes of transport and aero-medical in particular), proportion of patients requiring only ED, transport pathway (direct vs inter-hospital transfer).

Results: 3847 cases were identified from data as meeting criteria for major trauma by-pass. The top five most frequently used criteria for qualifying patients as meeting the major trauma by-pass guideline were pulse rate, vehicle rollover, possible spinal cord injury, respiration rate and entrapment. The study demonstrates a 65% conformance to the clinical guideline. Overtriaged patients (transported to higher trauma service than recommended) generally reveal International Classification of Disease Injury Severity Score representing a high threat to life.

Conclusion: Overall, the present study found good conformance, with overtriage rate as expected by clinicians. It is recommended to include data values to capture paramedics assessment of trauma level to enable more accurate assessment of conformance to guideline and future revision of the thresholds.
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http://dx.doi.org/10.1111/1742-6723.13807DOI Listing
May 2021

Determination of a microRNA signature of protective kidney ischemic preconditioning originating from proximal tubules.

Sci Rep 2021 05 10;11(1):9862. Epub 2021 May 10.

Wales Kidney Research Unit, School of Medicine, College of Biomedical & Life Sciences, Cardiff University, Heath Park Campus, Cardiff, CF14 4XN, UK.

Ischemic preconditioning (IPC) is effective in limiting subsequent ischemic acute kidney injury in experimental models. MicroRNAs are an important class of post-transcriptional regulator and show promise as biomarkers of kidney injury. We evaluated the time- and dose-dependence of benefit from IPC in a rat model of functional (bilateral) ischemia-reperfusion injury (IRI). We found optimal protection from subsequent injury following short, repetitive sequences of preconditioning insult. We subsequently used hybridization array and microRNA sequencing to characterize microRNA signatures of protective IPC and of IRI. These approaches identified a profile of microRNA changes consequent on IRI, that were limited by prior IPC. To localize these signals within the kidney, we used laser capture microdissection and RT-qPCR to measure microRNA abundance in nephron segments, pinpointing microRNA changes principally to glomeruli and proximal tubules. Our data describe a unique microRNA signature for IRI in the rat kidney. Pulsatile IPC reduces kidney damage following IRI and diminishes this microRNA signal. We have also identified candidate microRNAs that may act as biomarkers of injury and therapeutic targets in this context.
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http://dx.doi.org/10.1038/s41598-021-89195-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110756PMC
May 2021

Characterization of antimicrobial-resistant Gram-negative bacteria that cause neonatal sepsis in seven low- and middle-income countries.

Nat Microbiol 2021 04 29;6(4):512-523. Epub 2021 Mar 29.

The National Reference Laboratory, Rwanda Biomedical Centre, Kigali, Rwanda.

Antimicrobial resistance in neonatal sepsis is rising, yet mechanisms of resistance that often spread between species via mobile genetic elements, ultimately limiting treatments in low- and middle-income countries (LMICs), are poorly characterized. The Burden of Antibiotic Resistance in Neonates from Developing Societies (BARNARDS) network was initiated to characterize the cause and burden of antimicrobial resistance in neonatal sepsis for seven LMICs in Africa and South Asia. A total of 36,285 neonates were enrolled in the BARNARDS study between November 2015 and December 2017, of whom 2,483 were diagnosed with culture-confirmed sepsis. Klebsiella pneumoniae (n = 258) was the main cause of neonatal sepsis, with Serratia marcescens (n = 151), Klebsiella michiganensis (n = 117), Escherichia coli (n = 75) and Enterobacter cloacae complex (n = 57) also detected. We present whole-genome sequencing, antimicrobial susceptibility and clinical data for 916 out of 1,038 neonatal sepsis isolates (97 isolates were not recovered from initial isolation at local sites). Enterobacterales (K. pneumoniae, E. coli and E. cloacae) harboured multiple cephalosporin and carbapenem resistance genes. All isolated pathogens were resistant to multiple antibiotic classes, including those used to treat neonatal sepsis. Intraspecies diversity of K. pneumoniae and E. coli indicated that multiple antibiotic-resistant lineages cause neonatal sepsis. Our results will underpin research towards better treatments for neonatal sepsis in LMICs.
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http://dx.doi.org/10.1038/s41564-021-00870-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007471PMC
April 2021

Examining the utility of extended laboratory panel testing in the emergency department for risk stratification of patients with COVID-19: a single-centre retrospective service evaluation.

J Clin Pathol 2021 Feb 19. Epub 2021 Feb 19.

Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK.

Background: The role of specific blood tests to predict poor prognosis in patients admitted with infection from SARS-CoV-2 remains uncertain. During the first wave of the global pandemic, an extended laboratory testing panel was integrated into the local pathway to guide triage and healthcare resource utilisation for emergency admissions. We conducted a retrospective service evaluation to determine the utility of extended tests (D-dimer, ferritin, high-sensitivity troponin I, lactate dehydrogenase and procalcitonin) compared with the core panel (full blood count, urea and electrolytes, liver function tests and C reactive protein).

Methods: Clinical outcomes for adult patients with laboratory-confirmed COVID-19 admitted between 17 March and 30 June 2020 were extracted, alongside costs estimates for individual tests. Prognostic performance was assessed using multivariable logistic regression analysis with 28-day mortality used as the primary endpoint and a composite of 28-day intensive care escalation or mortality for secondary analysis.

Results: From 13 500 emergency attendances, we identified 391 unique adults admitted with COVID-19. Of these, 113 died (29%) and 151 (39%) reached the composite endpoint. 'Core' test variables adjusted for age, gender and index of deprivation had a prognostic area under the curve of 0.79 (95% CI 0.67 to 0.91) for mortality and 0.70 (95% CI 0.56 to 0.84) for the composite endpoint. Addition of 'extended' test components did not improve on this.

Conclusion: Our findings suggest use of the extended laboratory testing panel to risk stratify community-acquired COVID-19 positive patients on admission adds limited prognostic value. We suggest laboratory requesting should be targeted to patients with specific clinical indications.
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http://dx.doi.org/10.1136/jclinpath-2020-207157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7898230PMC
February 2021

Prostaglandin E promotes intestinal inflammation via inhibiting microbiota-dependent regulatory T cells.

Sci Adv 2021 Feb 12;7(7). Epub 2021 Feb 12.

Centre for Inflammation Research, Queen's Medical Research Institute, The University of Edinburgh, Edinburgh EH16 4TJ, UK.

The gut microbiota fundamentally regulates intestinal homeostasis and disease partially through mechanisms that involve modulation of regulatory T cells (T), yet how the microbiota-T cross-talk is physiologically controlled is incompletely defined. Here, we report that prostaglandin E (PGE), a well-known mediator of inflammation, inhibits mucosal T in a manner depending on the gut microbiota. PGE through its receptor EP4 diminishes T-favorable commensal microbiota. Transfer of the gut microbiota that was modified by PGE-EP4 signaling modulates mucosal T responses and exacerbates intestinal inflammation. Mechanistically, PGE-modified microbiota regulates intestinal mononuclear phagocytes and type I interferon signaling. Depletion of mononuclear phagocytes or deficiency of type I interferon receptor diminishes PGE-dependent T inhibition. Together, our findings provide emergent evidence that PGE-mediated disruption of microbiota-T communication fosters intestinal inflammation.
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http://dx.doi.org/10.1126/sciadv.abd7954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880593PMC
February 2021

Loss of the exocyst complex component EXOC3 promotes hemostasis and accelerates arterial thrombosis.

Blood Adv 2021 02;5(3):674-686

School of Physiology, Pharmacology & Neuroscience, University of Bristol, Bristol, United Kingdom; and.

The exocyst is an octameric complex comprising 8 distinct protein subunits, exocyst complex components (EXOC) 1 to 8. It has an established role in tethering secretory vesicles to the plasma membrane, but its relevance to platelet granule secretion and function remains to be determined. Here, EXOC3 conditional knockout (KO) mice in the megakaryocyte/platelet lineage were generated to assess exocyst function in platelets. Significant defects in platelet aggregation, integrin activation, α-granule (P-selectin and platelet factor 4), dense granule, and lysosomal granule secretion were detected in EXOC3 KO platelets after treatment with a glycoprotein VI (GPVI)-selective agonist, collagen-related peptide (CRP). Except for P-selectin exposure, these defects were completely recovered by maximal CRP concentrations. GPVI surface levels were also significantly decreased by 14.5% in KO platelets, whereas defects in proximal GPVI signaling responses, Syk and LAT phosphorylation, and calcium mobilization were also detected, implying an indirect mechanism for these recoverable defects due to decreased surface GPVI. Paradoxically, dense granule secretion, integrin activation, and changes in surface expression of integrin αIIb (CD41) were significantly increased in KO platelets after protease-activated receptor 4 activation, but calcium responses were unaltered. Elevated integrin activation responses were completely suppressed with a P2Y12 receptor antagonist, suggesting enhanced dense granule secretion of adenosine 5'-diphosphate as a critical mediator of these responses. Finally, arterial thrombosis was significantly accelerated in KO mice, which also displayed improved hemostasis determined by reduced tail bleeding times. These findings reveal a regulatory role for the exocyst in controlling critical aspects of platelet function pertinent to thrombosis and hemostasis.
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http://dx.doi.org/10.1182/bloodadvances.2020002515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7876885PMC
February 2021

Immobilized collagen prevents shedding and induces sustained GPVI clustering and signaling in platelets.

Platelets 2021 Jan;32(1):59-73

Institute of Cardiovascular Sciences, College of Medical and Dental Sciences, University of Birmingham , Birmingham, UK.

Collagen, the most thrombogenic constituent of blood vessel walls, activates platelets through glycoprotein VI (GPVI). In suspension, following platelet activation by collagen, GPVI is cleaved by A Disintegrin And Metalloproteinase (ADAM)10 and ADAM17. In this study, we use single-molecule localization microscopy and a 2-level DBSCAN-based clustering tool to show that GPVI remains clustered along immobilized collagen fibers for at least 3 hours in the absence of significant shedding. Tyrosine phosphorylation of spleen tyrosine kinase (Syk) and Linker of Activated T cells (LAT), and elevation of intracellular Ca, are sustained over this period. Syk, but not Src kinase-dependent signaling is required to maintain clustering of the collagen integrin α2β1, whilst neither is required for GPVI. We propose that clustering of GPVI on immobilized collagen protects GPVI from shedding in order to maintain sustained Src and Syk-kinases dependent signaling, activation of integrin α2β1, and continued adhesion.
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http://dx.doi.org/10.1080/09537104.2020.1849607DOI Listing
January 2021

Phosphoproteomic Analysis of Platelets in Severe Obesity Uncovers Platelet Reactivity and Signaling Pathways Alterations.

Arterioscler Thromb Vasc Biol 2021 01 5;41(1):478-490. Epub 2020 Nov 5.

Platelet Proteomics Group, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), Universidade Santiago de Compostela (M.N.B., L.H.-N., L.A.M., Á.G.).

Objective: Obesity is associated with a proinflammatory and prothrombotic state that supports atherosclerosis progression. The goal of this study was to gain insights into the phosphorylation events related to platelet reactivity in obesity and identify platelet biomarkers and altered activation pathways in this clinical condition. Approach and Results: We performed a comparative phosphoproteomic analysis of resting platelets from obese patients and their age- and gender-matched lean controls. The phosphoproteomic data were validated by mechanistic, functional, and biochemical assays. We identified 220 differentially regulated phosphopeptides, from at least 175 proteins; interestingly, all were up-regulated in obesity. Most of the altered phosphoproteins are involved in SFKs (Src-family kinases)-related signaling pathways, cytoskeleton reorganization, and vesicle transport, some of them validated by targeted mass spectrometry. To confirm platelet dysfunction, flow cytometry assays were performed in whole blood indicating higher surface levels of GP (glycoprotein) VI and CLEC (C-type lectin-like receptor) 2 in platelets from obese patients correlating positively with body mass index. Receiver operator characteristics curves analysis suggested a much higher sensitivity for GPVI to discriminate between obese and lean individuals. Indeed, we also found that obese platelets displayed more adhesion to collagen-coated plates. In line with the above data, soluble GPVI levels-indicative of higher GPVI signaling activation-were almost double in plasma from obese patients.

Conclusions: Our results provide novel information on platelet phosphorylation changes related to obesity, revealing the impact of this chronic pathology on platelet reactivity and pointing towards the main signaling pathways dysregulated.
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http://dx.doi.org/10.1161/ATVBAHA.120.314485DOI Listing
January 2021

Targeting shear gradient activated von Willebrand factor by the novel single-chain antibody A1 reduces occlusive thrombus formation in vitro.

Haematologica 2020 09 21;Online ahead of print. Epub 2020 Sep 21.

Baker Heart and Diabetes Institute, Melbourne; Australian Centre for Blood Diseases, Monash University, Melbourne; CEH and EW contributed equally to this work.

Intraluminal thrombus formation precipitates conditions such as acute myocardial infarction and disturbs local blood flow resulting in areas of rapidly changing blood flow velocities and steep gradients of blood shear rate. Shear rate gradients are known to be pro-thrombotic with an important role for the shear-sensitive plasma protein von Willebrand factor (VWF). Here, we developed a single-chain antibody (scFv) that targets a shear gradient specific conformation of VWF to specifically inhibit platelet adhesion at sites of SRGs but not in areas of constant shear. Microfluidic flow channels with stenotic segments were used to create shear rate gradients during blood perfusion. VWF-GPIbα interactions were increased at sites of shear rate gradients compared to constant shear rate of matched magnitude. The scFv-A1 specifically reduced VWF-GPIbα binding and thrombus formation at sites of SRGs but did not block platelet deposition and aggregation under constant shear rate in upstream sections of the channels. Significantly, the scFv A1 attenuated platelet aggregation only in the later stages of thrombus formation. In the absence of shear, direct binding of scFv-A1 to VWF could not be detected and scFV-A1 did not inhibit ristocetin induced platelet agglutination. We have exploited the pro-aggregatory effects of SRGs on VWF dependent platelet aggregation and developed the shear-gradient sensitive scFv-A1 antibody that inhibits platelet aggregation exclusively at sites of shear rate gradients. The lack of VWF inhibition in non-stenosed vessel segments places scFV-A1 in an entirely new class of anti-platelet therapy for selective blockade of pathological thrombus formation while maintaining normal haemostasis.
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http://dx.doi.org/10.3324/haematol.2020.250761DOI Listing
September 2020

Immune Remodeling of the Extracellular Matrix Drives Loss of Cancer Stem Cells and Tumor Rejection.

Cancer Immunol Res 2020 12 6;8(12):1520-1531. Epub 2020 Oct 6.

Infection and Immunity, School of Medicine, Cardiff University, Henry Wellcome Building, University Hospital of Wales, Cardiff, United Kingdom.

The nature of the tumor microenvironment (TME) influences the ability of tumor-specific T cells to control tumor growth. In this study, we performed an unbiased comparison of the TME of regulatory T-cell (Treg)-replete and Treg-depleted carcinogen-induced tumors, including Treg-depleted responding (regressing) and non-responding (growing) tumors. This analysis revealed an inverse relationship between extracellular matrix (ECM) and T-cell infiltrates where responding tumors were T-cell rich and ECM poor, whereas the converse was observed in non-responder tumors. For this reason, we hypothesized that the ECM acted as a barrier to successful T-cell infiltration and tumor rejection. However, further experiments revealed that this was not the case but instead showed that an effective T-cell response dramatically altered the density of ECM in the TME. Along with loss of ECM and high numbers of infiltrating T cells, responder tumors were distinguished by the development of lymphatic and blood vessel networks with specialized immune function. ECM-rich tumors exhibited a stem cell-like gene expression profile and superior tumor-initiating capacity, whereas such features were absent in responder tumors. Overall, these findings define an extended role for an effective immune response, not just in direct killing of tumor cells but in widescale remodeling of the TME to favor loss of ECM, elimination of cancer stem cells, and propagation of adaptive immunity.
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http://dx.doi.org/10.1158/2326-6066.CIR-20-0070DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7611107PMC
December 2020

Soluble glycoprotein VI is a predictor of major bleeding in patients with suspected heparin-induced thrombocytopenia.

Blood Adv 2020 09;4(18):4327-4332

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

We have shown that patients with suspected heparin-induced thrombocytopenia (HIT) have a high incidence of major bleeding. Recent studies have implicated elevated soluble glycoprotein VI (sGPVI) levels as a potential risk factor for bleeding. We sought to determine if elevated sGPVI plasma levels are associated with major bleeding events in patients with suspected HIT. We used a cohort of 310 hospitalized adult patients with suspected HIT who had a blood sample collected at the time HIT was suspected. Plasma sGPVI levels were measured by using enzyme-linked immunosorbent assay. Patients were excluded who had received a platelet transfusion within 1 day of sample collection because of the high levels of sGPVI in platelet concentrates. We assessed the association of sGPVI (high vs low) with International Society on Thrombosis and Haemostasis major bleeding events by multivariable logistic regression, adjusting for other known risk factors for bleeding. Fifty-four patients were excluded due to recent platelet transfusion, leaving 256 patients for analysis. Eighty-nine (34.8%) patients had a major bleeding event. Median sGPVI levels were significantly elevated in patients with major bleeding events compared with those without major bleeding events (49.09 vs 31.93 ng/mL; P < .001). An sGPVI level >43 ng/mL was independently associated with major bleeding after adjustment for critical illness, sepsis, cardiopulmonary bypass surgery, and degree of thrombocytopenia (adjusted odds ratio, 2.81; 95% confidence interval, 1.51-5.23). Our findings suggest that sGPVI is associated with major bleeding in hospitalized patients with suspected HIT. sGPVI may be a novel biomarker to predict bleeding risk in patients with suspected HIT.
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http://dx.doi.org/10.1182/bloodadvances.2020002861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509866PMC
September 2020

Pharmacological Blockade of Glycoprotein VI Promotes Thrombus Disaggregation in the Absence of Thrombin.

Arterioscler Thromb Vasc Biol 2020 09 23;40(9):2127-2142. Epub 2020 Jul 23.

From the Université de Strasbourg, INSERM, EFS Grand-Est, BPPS UMR-S1255, FMTS, F-67065 Strasbourg, France (M.U.A., N.R., M.L.B., E.J.-B., F.L., C.G., P.H.M.).

Objective: Atherothrombosis occurs upon rupture of an atherosclerotic plaque and leads to the formation of a mural thrombus. Computational fluid dynamics and numerical models indicated that the mechanical stress applied to a thrombus increases dramatically as a thrombus grows, and that strong inter-platelet interactions are essential to maintain its stability. We investigated whether GPVI (glycoprotein VI)-mediated platelet activation helps to maintain thrombus stability by using real-time video-microscopy. Approach and Results: We showed that GPVI blockade with 2 distinct Fab fragments promoted efficient disaggregation of human thrombi preformed on collagen or on human atherosclerotic plaque material in the absence of thrombin. ACT017-induced disaggregation was achieved under arterial blood flow conditions, and its effect increased with wall shear rate. GPVI regulated platelet activation within a growing thrombus as evidenced by the loss in thrombus contraction when GPVI was blocked, and the absence of the disaggregating effect of an anti-GPVI agent when the thrombi were fully activated with soluble agonists. The GPVI-dependent thrombus stabilizing effect was further supported by the fact that inhibition of any of the 4 key immunoreceptor tyrosine-based motif signalling molecules, src-kinases, Syk, PI3Kβ, or phospholipase C, resulted in kinetics of thrombus disaggregation similar to ACT017. The absence of ACT017-induced disaggregation of thrombi from 2 afibrinogenemic patients suggests that the role of GPVI requires interaction with fibrinogen. Finally, platelet disaggregation of fibrin-rich thrombi was also promoted by ACT017 in combination with r-tPA (recombinant tissue plasminogen activator).

Conclusions: This work identifies an unrecognized role for GPVI in maintaining thrombus stability and suggests that targeting GPVI could dissolve platelet aggregates with a poor fibrin content.
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http://dx.doi.org/10.1161/ATVBAHA.120.314301DOI Listing
September 2020

Effective enzyme activity: A proposed monitoring methodology for biofiltration systems with or without ozone.

Water Res 2020 Sep 1;183:116069. Epub 2020 Jul 1.

University of Toronto, Department of Civil and Mineral Engineering, Canada.

"Effective Enzyme Activity", or simply "Effective Activity", is proposed as a biofiltration monitoring tool which combines enzyme activity with empty bed contact time (EBCT) to quantify biodegradation potential. The primary objective of this study was to evaluate the applicability of the Effective Activity concept for predicting water quality in biofiltration systems. This pilot-scale study evaluated eight different biofilter configurations in order to quantify impacts associated with filter media (anthracite/sand or granular activated carbon), pre-treatment (settled water with or without ozonation) and operating conditions (15- and 30-min EBCT, and backwash with or without chlorine). Microbial characterization included biomass concentration, as measured by adenosine triphosphate (ATP), in addition to esterase and phosphatase activity. Water quality parameters included dissolved organic carbon (DOC), trihalomethane (THM) formation potential (FP), haloacetic acid (HAA) FP, haloacetonitrile (HAN) FP, iodinated DBP FP (THMs and HAAs) and inorganic nutrients (phosphorus and nitrogen). Results confirmed the benefits to treated water quality associated with the application of an ozone residual of 0.5 mg/L, utilization of GAC filter media, eliminating chlorinated backwash, and extending EBCT. This study demonstrated a good relationship between effective esterase activity and reductions in DOC and THM FP, including those systems which incorporate pre-ozonation. As such, this study showed that Effective Activity may be appropriate for relating biomass characterization to treated water quality and highlights the importance of quantifying biomass activity in addition to quantity.
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http://dx.doi.org/10.1016/j.watres.2020.116069DOI Listing
September 2020

CD4 T Cells Recognize Conserved Influenza A Epitopes through Shared Patterns of V-Gene Usage and Complementary Biochemical Features.

Cell Rep 2020 07;32(2):107885

Cardiff University, School of Medicine, Heath Park, Cardiff, UK. Electronic address:

T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8 T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4 T cells. Here, we investigate CD4 T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4 T cells in five HLA-DR1 subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies.
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http://dx.doi.org/10.1016/j.celrep.2020.107885DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7370177PMC
July 2020

Sampling and Quality Assurance and Quality Control: A Guide for Scientists Investigating the Occurrence of Microplastics Across Matrices.

Appl Spectrosc 2020 Sep;74(9):1099-1125

Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, Canada.

Plastic pollution is a defining environmental contaminant and is considered to be one of the greatest environmental threats of the Anthropocene, with its presence documented across aquatic and terrestrial ecosystems. The majority of this plastic debris falls into the micro (1 μm-5 mm) or nano (1-1000 nm) size range and comes from primary and secondary sources. Its small size makes it cumbersome to isolate and analyze reproducibly, and its ubiquitous distribution creates numerous challenges when controlling for background contamination across matrices (e.g., sediment, tissue, water, air). Although research on microplastics represents a relatively nascent subfield, burgeoning interest in questions surrounding the fate and effects of these debris items creates a pressing need for harmonized sampling protocols and quality control approaches. For results across laboratories to be reproducible and comparable, it is imperative that guidelines based on vetted protocols be readily available to research groups, many of which are either new to plastics research or, as with any new subfield, have arrived at current approaches through a process of trial-and-error rather than in consultation with the greater scientific community. The goals of this manuscript are to (i) outline the steps necessary to conduct general as well as matrix-specific quality assurance and quality control based on sample type and associated constraints, (ii) briefly review current findings across matrices, and (iii) provide guidance for the design of sampling regimes. Specific attention is paid to the source of microplastic pollution as well as the pathway by which contamination occurs, with details provided regarding each step in the process from generating appropriate questions to sampling design and collection.
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http://dx.doi.org/10.1177/0003702820945713DOI Listing
September 2020

Loss of GPVI and GPIbα contributes to trauma-induced platelet dysfunction in severely injured patients.

Blood Adv 2020 06;4(12):2623-2630

Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom.

Trauma-induced coagulopathy (TIC) is a complex, multifactorial failure of hemostasis that occurs in 25% of severely injured patients and results in a fourfold higher mortality. However, the role of platelets in this state remains poorly understood. We set out to identify molecular changes that may underpin platelet dysfunction after major injury and to determine how they relate to coagulopathy and outcome. We performed a range of hemostatic and platelet-specific studies in blood samples obtained from critically injured patients within 2 hours of injury and collected prospective data on patient characteristics and clinical outcomes. We observed that, although platelet counts were preserved above critical levels, circulating platelets sampled from trauma patients exhibited a profoundly reduced response to both collagen and the selective glycoprotein VI (GPVI) agonist collagen-related peptide, compared with those from healthy volunteers. These responses correlated closely with overall clot strength and mortality. Surface expression of the collagen receptors GPIbα and GPVI was reduced on circulating platelets in trauma patients, with increased levels of the shed ectodomain fragment of GPVI detectable in plasma. Levels of shed GPVI were highest in patients with more severe injuries and TIC. Collectively, these observations demonstrate that platelets experience a loss of GPVI and GPIbα after severe injury and translate into a reduction in the responsiveness of platelets during active hemorrhage. In turn, they are associated with reduced hemostatic competence and increased mortality. Targeting proteolytic shedding of platelet receptors is a potential therapeutic strategy for maintaining hemostatic competence in bleeding and improving the efficacy of platelet transfusions.
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http://dx.doi.org/10.1182/bloodadvances.2020001776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7322946PMC
June 2020

A Comparative Process Mining Analysis of Road Trauma Patient Pathways.

Int J Environ Res Public Health 2020 05 14;17(10). Epub 2020 May 14.

Queensland Ambulance Service (QAS), Brisbane 4034, Australia.

In this paper we report on key findings and lessons from a process mining case study conducted to analyse transport pathways discovered across the time-critical phase of pre-hospital care for persons involved in road traffic crashes in Queensland (Australia). In this study, a case is defined as being an individual patient's journey from roadside to definitive care. We describe challenges in constructing an event log from source data provided by emergency services and hospitals, including record linkage (no standard patient identifier), and constructing a unified view of response, retrieval, transport and pre-hospital care from interleaving processes of the individual service providers. We analyse three separate cohorts of patients according to their degree of interaction with Queensland Health's hospital system (C1:no transport required, C2:transported but no Queensland Health hospital, C3:transported and hospitalisation). Variant analysis and subsequent process modelling show high levels of variance in each cohort resulting from a combination of data collection, data linkage and actual differences in process execution. For Cohort 3, automated process modelling generated 'spaghetti' models. Expert-guided editing resulted in readable models with acceptable fitness, which were used for process analysis. We also conduct a comparative performance analysis of transport segment based on hospital `remoteness'. With regard to the field of process mining, we reach various conclusions including (i) in a complex domain, the current crop of automated process algorithms do not generate readable models, however, (ii) such models provide a starting point for expert-guided editing of models (where the tool allows) which can yield models that have acceptable quality and are readable by domain experts, (iii) process improvement opportunities were largely suggested by domain experts (after reviewing analysis results) rather than being directly derived by process mining tools, meaning that the field needs to become more prescriptive (automated derivation of improvement opportunities).
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http://dx.doi.org/10.3390/ijerph17103426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7277496PMC
May 2020

Metabolic Dysregulation of the Lysophospholipid/Autotaxin Axis in the Chromosome 9p21 Gene SNP rs10757274.

Circ Genom Precis Med 2020 06 12;13(3):e002806. Epub 2020 May 12.

Division of Infection and Immunity, Systems Immunity Research Institute (S.W.M., J.I.H., D.W., R.A., P.R., A.O., J.A.-J., V.J.T., C.H., Y.Z., M.A., W.J.W., D.A.S., V.B.O.), Cardiff University, United Kingdom.

Background: Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA.

Methods: Untargeted and targeted lipidomics was applied to plasma from NPHSII (Northwick Park Heart Study II) homozygotes for AA or GG in rs10757274, followed by correlation and network analysis. To identify candidate genes, transcriptomic data from shRNA downregulation of ANRIL in HEK-293 cells was mined. Transcriptional data from vascular smooth muscle cells differentiated from induced pluripotent stem cells of individuals with/without Chr9p21 risk, nonrisk alleles, and corresponding knockout isogenic lines were next examined. Last, an in-silico analysis of miRNAs was conducted to identify how ANRIL might control lysoPL (lysophosphospholipid)/lysoPA (lysophosphatidic acid) genes.

Results: Elevated risk GG correlated with reduced lysoPLs, lysoPA, and ATX (autotaxin). Five other risk SNPs did not show this phenotype. LysoPL-lysoPA interconversion was uncoupled from ATX in GG plasma, suggesting metabolic dysregulation. Significantly altered expression of several lysoPL/lysoPA metabolizing enzymes was found in HEK cells lacking ANRIL. In the vascular smooth muscle cells data set, the presence of risk alleles associated with altered expression of several lysoPL/lysoPA enzymes. Deletion of the risk locus reversed the expression of several lysoPL/lysoPA genes to nonrisk haplotype levels. Genes that were altered across both cell data sets were , and The in-silico analysis identified 4 ANRIL-regulated miRNAs that control lysoPL genes as miR-186-3p, miR-34a-3p, miR-122-5p, and miR-34a-5p.

Conclusions: A Chr9p21 risk SNP associates with complex alterations in immune-bioactive phospholipids and their metabolism. Lipid metabolites and genomic pathways associated with coronary heart disease pathogenesis in Chr9p21 and ANRIL-associated disease are demonstrated.
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http://dx.doi.org/10.1161/CIRCGEN.119.002806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7299226PMC
June 2020

Shedding of soluble glycoprotein VI is neither affected by animal-derived antibeta-2-glycoprotein 1 antibodies nor IgG fractions from patients with systemic lupus erythematosus.

Blood Coagul Fibrinolysis 2020 Jun;31(4):258-263

College of Science, Health, Engineering and Education, Murdoch University, Murdoch, Western Australia, Australia.

: Antibeta-2-glycoprotein 1 (antiβ2GP1) antibodies are associated with increased risk of thrombosis in patients with systemic lupus erythematosus (SLE). The specific effect(s) of antiβ2GP1 antibodies on platelets are unclear. Platelet activation in response to antiplatelet antibodies has been shown to induce shedding of the ectodomain of the platelet collagen receptor, glycoprotein VI (GPVI), releasing soluble GPVI (sGPVI). The aim of this study was to therefore determine whether antiβ2GP1 antibodies, and/or purified IgG fractions, from patients with SLE shed sGPVI from platelets. We determined sGPVI levels in platelet poor plasma from SLE patients with/without antiβ2GP1 antibodies (n = 37), as well as in platelet-rich plasma from healthy donors treated with either SLE-derived IgG fractions containing antiβ2GP1, animal-derived antiβ2GP1, or isotype control antibodies. Levels of sGPVI were higher in three SLE-derived platelet poor plasma with antiβ2GP1 antibodies (103.52 ± 12.32 ng/ml) compared with those without (28.11 ± 12.73 ng/ml). Neither SLE-derived IgG fractions containing antiβ2GP1 antibodies, nor animal-derived antiβ2GP1 antibodies induced significant shedding of sGPVI from healthy donor platelets compared with isotype controls. These results suggest that antiβ2GP1 antibodies do not affect shedding of sGPVI, and therefore collagen-mediated platelet signalling pathways. The shedding activity in SLE patients may be due to factors other than antiβ2GP1 antibodies, for example, metalloproteinases.
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http://dx.doi.org/10.1097/MBC.0000000000000909DOI Listing
June 2020

Fibrin exposure triggers αIIbβ3-independent platelet aggregate formation, ADAM10 activity and glycoprotein VI shedding in a charge-dependent manner.

J Thromb Haemost 2020 06 16;18(6):1447-1458. Epub 2020 Apr 16.

ACRF Department of Cancer Biology and Therapeutics, The John Curtin School of Medical Research, The Australian National University, Canberra, ACT, Australia.

Background: Collagen and fibrin engagement and activation of glycoprotein (GP) VI induces proteolytic cleavage of the GPVI ectodomain generating shed soluble GPVI (sGPVI). Collagen-mediated GPVI shedding requires intracellular signalling to release the sGPVI, mediated by A Disintegrin And Metalloproteinase 10 (ADAM10); however, the precise mechanism by which fibrin induces GPVI shedding remains elusive. Plasma sGPVI levels are elevated in patients with coagulopathies, sepsis, or inflammation and can predict onset of sepsis and sepsis-related mortality; therefore, it is clinically important to understand the mechanisms of GPVI shedding under conditions of minimal collagen exposure.

Objectives: Our aim was to characterize mechanisms by which fibrin-GPVI interactions trigger GPVI shedding.

Methods: Platelet aggregometry, sGPVI ELISA, and an ADAM10 fluorescence resonance energy transfer assay were used to measure fibrin-mediated platelet responses.

Results: Fibrin induced αIIbβ3-independent washed platelet aggregate formation, GPVI shedding, and increased ADAM10 activity, all of which were insensitive to pre-treatment with inhibitors of Src family kinases but were divalent cation- and metalloproteinase-dependent. In contrast, treatment of washed platelets with other GPVI ligands, collagen, and collagen-related peptide caused αIIbβ3-dependent platelet aggregation and GPVI release but did not increase constitutive ADAM10 activity.

Conclusions: Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.Fibrin engages GPVI in a manner that differs from other GPVI ligands. Inclusion of polyanionic molecules disrupted fibrin-induced platelet aggregate formation and sGPVI release, suggesting that electrostatic charge may play a role in fibrin/GPVI engagement. It may be feasible to exploit this property and specifically disrupt GPVI/fibrin interactions whilst sparing GPVI/collagen engagement.
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http://dx.doi.org/10.1111/jth.14797DOI Listing
June 2020

Cancer Antigen Discovery Is Enabled by RNA Sequencing of Highly Purified Malignant and Nonmalignant Cells.

Clin Cancer Res 2020 07 2;26(13):3360-3370. Epub 2020 Mar 2.

Division of Infection and Immunity, Henry Wellcome Building, Cardiff University, Cardiff, United Kingdom.

Purpose: Broadly expressed, highly differentiated tumor-associated antigens (TAA) can elicit antitumor immunity. However, vaccines targeting TAAs have demonstrated disappointing clinical results, reflecting poor antigen selection and/or immunosuppressive mechanisms.

Experimental Design: Here, a panel of widely expressed, novel colorectal TAAs were identified by performing RNA sequencing of highly purified colorectal tumor cells in comparison with patient-matched colonic epithelial cells; tumor cell purification was essential to reveal these genes. Candidate TAA protein expression was confirmed by IHC, and preexisting T-cell immunogenicity toward these antigens tested.

Results: The most promising candidate for further development is DNAJB7 [DnaJ heat shock protein family (Hsp40) member B7], identified here as a novel cancer-testis antigen. It is expressed in many tumors and is strongly immunogenic in patients with cancers originating from a variety of sites. DNAJB7-specific T cells were capable of killing colorectal tumor lines , and the IFNγ response was markedly magnified by control of immunosuppression with cyclophosphamide in patients with cancer.

Conclusions: This study highlights how prior methods that sequence whole tumor fractions (i.e., inclusive of alive/dead stromal cells) for antigen identification may have limitations. Through tumor cell purification and sequencing, novel candidate TAAs have been identified for future immunotherapeutic targeting.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3087DOI Listing
July 2020

Impact of backwash on biofiltration-related nitrogenous disinfection by-product formation.

Water Res 2020 May 25;174:115641. Epub 2020 Feb 25.

Department of Civil and Mineral Engineering, University of Toronto, 35 St. George St., Toronto, Ontario, M5S 1A4, Canada. Electronic address:

Previous studies have reported that biofilm extracted from full-scale biofilters can serve as nitrogenous disinfection by-product (N-DBP) precursors. Detached biofilm materials could escape during filter ripening and form N-DBP upon chloramination. This study examined the potential breakthrough of biofilm and N-DBP precursors during filter ripening at two water treatment plants (WTPs). The presence of biofilm material in aqueous samples was estimated by total adenosine triphosphate (tATP) levels; N-DBP formation potential (FP) tests were conducted under uniform formation conditions to quantify N-nitrosodimethylamine (NDMA) and haloacetonitrile (HAN) precursors. While tATP peaks in filter effluent were observed post backwash at both WTPs, temporary increases of effluent NDMA FP were only observed during filter ripening where particle-associated NDMA precursors served as the dominant contributor. Overall, biofilters examined in this study demonstrated a consistent removal of NDMA FP regardless of the filter ripening process.
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http://dx.doi.org/10.1016/j.watres.2020.115641DOI Listing
May 2020
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