Publications by authors named "Robert Campbell"

676 Publications

Surgical Outcomes among Focused versus Diversified Cataract Surgeons.

Ophthalmology 2021 Feb 23. Epub 2021 Feb 23.

Department of Ophthalmology, Queen's University, Kingston, Canada; Department of Ophthalmology, Kingston Health Sciences Centre, Hotel Dieu Hospital site, Kingston, Canada.

Purpose: Narrowly focused surgical practice has become increasingly common in ophthalmology and may have an effect on surgical outcomes. Previous research evaluating the influence of surgical focus on cataract surgical outcomes has been lacking. This study aimed to evaluate whether surgeons' exclusive surgical focus on cataract surgery influences the risk of cataract surgical adverse events.

Design: Population-based cohort study.

Participants: All patients 66 years of age or older undergoing cataract surgery in Ontario, Canada, between January 1, 2002, and December 31, 2013.

Methods: Outcomes of isolated cataract surgery performed by exclusive cataract surgeons (no other types of surgery performed), moderately diversified cataract surgeons (1%-50% noncataract procedures), and highly diversified cataract surgeons (>50% noncataract procedures) were evaluated using linked healthcare databases and controlling for patient-, surgeon-, and institution-level covariates. Surgeon-level covariates included both surgeon experience and surgical volume.

Main Outcome Measures: Composite outcome incorporating 4 adverse events: posterior capsule rupture, dropped lens fragments, retinal detachment, and suspected endophthalmitis.

Results: The study included 1 101 864 cataract operations. Patients had a median age of 76 years, and 60.2% were female. Patients treated by the 3 groups of surgeons were similar at baseline. Adverse events occurred in 0.73%, 0.78%, and 2.31% of cases performed by exclusive cataract surgeons, moderately diversified surgeons, and highly diversified surgeons, respectively. The risk of cataract surgical adverse events for patients operated on by moderately diversified surgeons was not different than for patients operated on by exclusive cataract surgeons (odds ratio [OR], 1.08; 95% confidence interval [CI], 1.00-1.18). Patients operated on by highly diversified surgeons had a higher risk of adverse events than patients operated on by exclusive cataract surgeons (OR, 1.52; 95% CI, 1.09-2.14). This resulted in an absolute risk difference of 0.016 (95% CI, 0.012-0.020) and a number needed to harm of 64 (95% CI, 50-87).

Conclusions: Exclusive surgical focus did not affect the safety of cataract surgery when compared with moderate levels of surgical diversification. The risk of cataract surgical adverse events was higher among surgeons whose practice was dedicated mainly to noncataract surgery.
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http://dx.doi.org/10.1016/j.ophtha.2021.01.016DOI Listing
February 2021

Surgical Outcomes among Focused versus Diversified Cataract Surgeons.

Ophthalmology 2021 Feb 23. Epub 2021 Feb 23.

Department of Ophthalmology, Queen's University, Kingston, Canada; Department of Ophthalmology, Kingston Health Sciences Centre, Hotel Dieu Hospital site, Kingston, Canada.

Purpose: Narrowly focused surgical practice has become increasingly common in ophthalmology and may have an effect on surgical outcomes. Previous research evaluating the influence of surgical focus on cataract surgical outcomes has been lacking. This study aimed to evaluate whether surgeons' exclusive surgical focus on cataract surgery influences the risk of cataract surgical adverse events.

Design: Population-based cohort study.

Participants: All patients 66 years of age or older undergoing cataract surgery in Ontario, Canada, between January 1, 2002, and December 31, 2013.

Methods: Outcomes of isolated cataract surgery performed by exclusive cataract surgeons (no other types of surgery performed), moderately diversified cataract surgeons (1%-50% noncataract procedures), and highly diversified cataract surgeons (>50% noncataract procedures) were evaluated using linked healthcare databases and controlling for patient-, surgeon-, and institution-level covariates. Surgeon-level covariates included both surgeon experience and surgical volume.

Main Outcome Measures: Composite outcome incorporating 4 adverse events: posterior capsule rupture, dropped lens fragments, retinal detachment, and suspected endophthalmitis.

Results: The study included 1 101 864 cataract operations. Patients had a median age of 76 years, and 60.2% were female. Patients treated by the 3 groups of surgeons were similar at baseline. Adverse events occurred in 0.73%, 0.78%, and 2.31% of cases performed by exclusive cataract surgeons, moderately diversified surgeons, and highly diversified surgeons, respectively. The risk of cataract surgical adverse events for patients operated on by moderately diversified surgeons was not different than for patients operated on by exclusive cataract surgeons (odds ratio [OR], 1.08; 95% confidence interval [CI], 1.00-1.18). Patients operated on by highly diversified surgeons had a higher risk of adverse events than patients operated on by exclusive cataract surgeons (OR, 1.52; 95% CI, 1.09-2.14). This resulted in an absolute risk difference of 0.016 (95% CI, 0.012-0.020) and a number needed to harm of 64 (95% CI, 50-87).

Conclusions: Exclusive surgical focus did not affect the safety of cataract surgery when compared with moderate levels of surgical diversification. The risk of cataract surgical adverse events was higher among surgeons whose practice was dedicated mainly to noncataract surgery.
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http://dx.doi.org/10.1016/j.ophtha.2021.01.016DOI Listing
February 2021

A First-in-Class, Highly Selective and Cell-Active Allosteric Inhibitor of Protein Arginine Methyltransferase 6.

J Med Chem 2021 Feb 16. Epub 2021 Feb 16.

Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Oncological Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, New York 10029, United States.

Protein arginine methyltransferase 6 (PRMT6) catalyzes monomethylation and asymmetric dimethylation of arginine residues in various proteins, plays important roles in biological processes, and is associated with multiple cancers. To date, a highly selective PRMT6 inhibitor has not been reported. Here we report the discovery and characterization of a first-in-class, highly selective allosteric inhibitor of PRMT6, (SGC6870). is a potent PRMT6 inhibitor (IC = 77 ± 6 nM) with outstanding selectivity for PRMT6 over a broad panel of other methyltransferases and nonepigenetic targets. Notably, the crystal structure of the PRMT6- complex and kinetic studies revealed binds a unique, induced allosteric pocket. Additionally, engages PRMT6 and potently inhibits its methyltransferase activity in cells. Moreover, 's enantiomer, (SGC6870N), is inactive against PRMT6 and can be utilized as a negative control. Collectively, - is a well-characterized PRMT6 chemical probe and a valuable tool for further investigating PRMT6 functions in health and disease.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02160DOI Listing
February 2021

Heparanase expression and activity are increased in platelets during clinical sepsis.

J Thromb Haemost 2021 Feb 15. Epub 2021 Feb 15.

University of Utah, Department of Internal Medicine and Molecular Medicine Program, Salt Lake City, UT, United States.

Background: Heparanase (HPSE) is the only known mammalian enzyme that can degrade heparan sulfate. Heparan sulfate proteoglycans are essential components of the glycocalyx, and maintain physiological barriers between the blood and endothelial cells. HPSE increases during sepsis, which contributes to injurious glyocalyx degradation, loss of endothelial barrier function, and mortality.

Objectives: As platelets are one of the most abundant cellular sources of HPSE, we sought to determine whether HPSE expression and activity increases in human platelets during clinical sepsis. We also examined associations between platelet HPSE expression and clinical outcomes.

Patients/methods: Expression and activity of HPSE was determined in platelets isolated from septic patients (n=59) and, for comparison, sex matched healthy donors (n=46) using complementary transcriptomic, proteomic, and functional enzymatic assays. Septic patients were followed for the primary outcome of mortality, and clinical data was captured prospectively for Septic patients.

Results: The mRNA expression of HPSE was significantly increased in platelets isolated from septic patients. Ribosomal footprint profiling, followed by [S35] methionine labeling assays demonstrated that HPSE mRNA translation and HPSE protein synthesis were significantly upregulated in platelets during sepsis. While both the pro- and active forms of HPSE protein increased in platelets during sepsis, only the active form of HPSE protein significantly correlated with sepsis-associated mortality. Consistent with transcriptomic and proteomic upregulation, HPSE enzymatic activity was also increased in platelets during sepsis.

Conclusions: During clinical sepsis HPSE, translation, and enzymatic activity are increased in platelets. Increased expression of the active form of HPSE protein is associated with sepsis-associated mortality.
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http://dx.doi.org/10.1111/jth.15266DOI Listing
February 2021

Structure- and mechanism-guided design of single fluorescent protein-based biosensors.

Nat Chem Biol 2021 Feb 8. Epub 2021 Feb 8.

Department of Chemistry, Graduate School of Science, The University of Tokyo, Tokyo, Japan.

Intensiometric genetically encoded biosensors, based on allosteric modulation of the fluorescence of a single fluorescent protein, are powerful tools for enabling imaging of neural activities and other cellular biochemical events. The archetypical example of such biosensors is the GCaMP series of Ca biosensors, which have been steadily improved over the past two decades and are now indispensable tools for neuroscience. However, no other biosensors have reached levels of performance, or had revolutionary impacts within specific disciplines, comparable to that of the Ca biosensors. Of the many reasons why this has been the case, a critical one has been a general black-box view of biosensor structure and mechanism. With this Perspective, we aim to summarize what is known about biosensor structure and mechanisms and, based on this foundation, provide guidelines to accelerate the development of a broader range of biosensors with performance comparable to that of the GCaMP series.
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http://dx.doi.org/10.1038/s41589-020-00718-xDOI Listing
February 2021

Controlled Osteogenic Differentiation of Human Mesenchymal Stem Cells Using Dexamethasone-Loaded Light-Responsive Microgels.

ACS Appl Mater Interfaces 2021 Feb 2;13(6):7051-7059. Epub 2021 Feb 2.

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

Human mesenchymal stem cells (hMSCs), which have the ability to differentiate into osteoblasts, show promise for bone tissue engineering and bone defect treatment. While there are a number of approaches currently available to accomplish this, e.g., utilizing biodegradable materials loaded with the synthetic glucocorticoid osteogenic inducer dexamethasone (DEX), there are still many disadvantages with the current technologies. Here, we generated light-responsive microgels that we showed are capable of loading and releasing DEX in a light-triggered fashion, with the released DEX being able to induce hMSC differentiation into osteoblasts. Specifically, light-responsive poly(-isopropylacrylamide--nitrobenzyl methacrylate) (pNIPAm--NBMA) microgels were synthesized via free radical precipitation polymerization and their size, morphology, and chemical composition were characterized. We then went on to show that the microgels could be loaded with DEX (via what we think are hydrophobic interactions) and released upon exposure to UV light. We went on to show that the DEX released from the microgels was still capable of inducing osteogenic differentiation of hMSCs using an alamarBlue assay and normalized alkaline phosphatase (ALP) activity assay. We also investigated how hMSC differentiation was impacted by intermittent DEX released from UV-exposed microgels. Finally, we confirmed that the microgels themselves were not cytotoxic to hMSCs. Taken together, the DEX-loaded light-responsive microgels reported here may find a use for niche clinical applications, e.g., bone tissue repair.
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http://dx.doi.org/10.1021/acsami.0c17664DOI Listing
February 2021

Empowering local communities to make lifestyle changes: is the Health Mela a potential solution?

BMJ Nutr Prev Health 2020 Dec 21;3(2):143-150. Epub 2020 Jul 21.

Integrated Musculoskeletal Services, Lancashire and South Cumbria NHS Foundation Trust, Preston, UK.

Background: Health Melas are community-led public health events held in the North West of England that provide health information and free health checks. This descriptive observational study evaluates whether Health Melas are able to identify undiagnosed cardiovascular disease (CVD) risk factors in hard-to-reach communities and encourage individuals to make lifestyle changes.

Methods: Attendees ≥18 years at three separate Health Melas in 2016-2017 were invited to participate in screening and counselling for CVD risk factors as part of a Health MOT. Information was collected about demographics, CVD risk factors, blood pressure, total cholesterol, blood sugar and attendees' feedback. QRISK2 scoring system was used to estimate CVD risk.

Results: 375 attendees completed a questionnaire. The highest proportion (36.9%) of attendees were from areas of the lowest Index of Health Deprivation and Disability quintile; 38.8% were of South Asian ethnicity. Of the attendees who were eligible for a free National Health Service Health Check, 9.1% had received one. Overall, 57.5% of all attendees had a QRISK2 score ≥10% (of whom 56.9% were not on statins), 92.2% of attendees believed the Health Mela will help them to make lifestyle changes, 98.2% said they had improved their understanding of their health, and 99.6% thought the Health Mela was useful. 73.6% of those who had received a previous Health MOT reported making lifestyle changes. There was a positive correlation between South Asian ethnicity and QRISK2 score.

Conclusion: This study suggests the Health Melas successfully involve South Asian populations and people from a lower Index of Health Deprivation and Disability. Attendees felt the events were useful, improved understanding of their health needs and encouraged them to make lifestyle changes. High rates of modifiable CVD risk factors were newly identified and a high proportion of attendees were found to be at intermediate to high risk of CVD.
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http://dx.doi.org/10.1136/bmjnph-2020-000067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7841842PMC
December 2020

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

Sci Rep 2021 Jan 22;11(1):2121. Epub 2021 Jan 22.

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.
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http://dx.doi.org/10.1038/s41598-021-81486-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822874PMC
January 2021

The 2020 Top 10: Advancing the Science of Drug Discovery.

SLAS Discov 2021 Feb;26(2):163-164

Twentyeight-Seven Therapeutics, Watertown, MA, USA.

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http://dx.doi.org/10.1177/2472555220982581DOI Listing
February 2021

Switching between Ultrafast Pathways Enables a Green-Red Emission Ratiometric Fluorescent-Protein-Based Ca Biosensor.

Int J Mol Sci 2021 Jan 5;22(1). Epub 2021 Jan 5.

Department of Chemistry, Oregon State University, 153 Gilbert Hall, Corvallis, OR 97331-4003, USA.

Ratiometric indicators with long emission wavelengths are highly preferred in modern bioimaging and life sciences. Herein, we elucidated the working mechanism of a standalone red fluorescent protein (FP)-based Ca biosensor, REX-GECO1, using a series of spectroscopic and computational methods. Upon 480 nm photoexcitation, the Ca-free biosensor chromophore becomes trapped in an excited dark state. Binding with Ca switches the route to ultrafast excited-state proton transfer through a short hydrogen bond to an adjacent Glu80 residue, which is key for the biosensor's functionality. Inspired by the 2D-fluorescence map, REX-GECO1 for Ca imaging in the ionomycin-treated human HeLa cells was achieved for the first time with a red/green emission ratio change (ΔR/R) of ~300%, outperforming many FRET- and single FP-based indicators. These spectroscopy-driven discoveries enable targeted design for the next-generation biosensors with larger dynamic range and longer emission wavelengths.
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http://dx.doi.org/10.3390/ijms22010445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794744PMC
January 2021

Distribution and Predictors of Initial Glaucoma Care among Ophthalmologists and Optometrists: A Population-based Study.

J Glaucoma 2021 Jan 13. Epub 2021 Jan 13.

Department of Ophthalmology, Queen's University, Kingston, Canada Department of Ophthalmology, Kingston Health Sciences Centre, Kingston, Canada ICES, Ontario, Canada Division of Geriatric Medicine, Queen's University, Kingston, Canada Division of Geriatric Medicine, Kingston Health Sciences Centre, Kingston, Canada.

Purpose: To evaluate evolution in the distribution of new glaucoma patients between ophthalmologists and optometrists, and to examine factors predicting provider type, in the context of expansion in the scope of optometry practice.

Patients And Methods: A population-based study was undertaken using validated datasets in Ontario, Canada from 2007 to 2018, encompassing time before and after optometry practice scope expansion in 2011. All patients aged 66 and older receiving a glaucoma suspect diagnosis or first-line therapy for glaucoma from ophthalmologists or optometrists were enrolled. Predictors of provider type were evaluated using logistic regression.

Results: From 2007 to 2018, 401,560 patients received initial glaucoma care, including 303,440 by ophthalmologists and 98,120 by optometrists. Population rates of glaucoma suspect diagnosis increased for both providers over the study period. The rate of therapy initiation increased annually among optometrists after 2011, while the rate remained stable over that period among ophthalmologists. By 2018, 88% of patients initiating therapy and 59% of patients first diagnosed as a glaucoma suspect received that care from ophthalmologists. In the final study year, therapy initiations per provider were lower among optometrists (median: 2/provider; interquartile range [IQR]: 1-3) than among ophthalmologists (median: 26.5/provider, IQR: 10-53). Patients were more likely to receive care from an ophthalmologist than an optometrist if they were older, had higher ocular or systemic comorbidity, or lived in urban settings.

Conclusions: Optometrists have a large and growing role in diagnosing glaucoma suspects; however, despite scope expansion, optometrists play a much smaller role in initiating glaucoma therapy.
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http://dx.doi.org/10.1097/IJG.0000000000001792DOI Listing
January 2021

Vascular infusion with concurrent vascular rinsing on color, tenderness, and lipid oxidation of hog meat.

Meat Sci 2021 Apr 14;174:108409. Epub 2020 Dec 14.

Meat Science and Muscle Biology Laboratory, Department of Animal Sciences, University of Wisconsin, Madison, WI 53706, USA. Electronic address:

Market hogs were conventionally chilled (CN, n = 12) or Rinse & Chill® processed (RC, n = 13, MPSC Inc.). Muscles (Longissimus lumborum, LL; picnic shoulder, PS) were processed (chops, ground), packaged, and displayed or stored in the dark. Color, pH, moisture fat free (MFF), expressible moisture (EM), oxygen consumption, Warner-Bratzler shear (WBS), total pigment, TBARS, and hexanal content were determined. RC generally resulted in a lower pH during the first 4 h compared to CN. RC compared to CN had lower fat, but were not different in moisture fat free, expressible moisture, and total pigments. RC did not affect cooler shrink, cook loss and WBS force. RC PS was redder than RC LM. RC had greater deoxymyoglobin than CN on 7 d display. RC chops (LL) were lighter and had less deoxymyoglobin compared to CN. RC ground pork had greater oxygen consumption, lower TBARS and hexanal values compared to CN. RC has the potential to improve color and reduce lipid oxidation.
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http://dx.doi.org/10.1016/j.meatsci.2020.108409DOI Listing
April 2021

Correlation of Pulmonary Function to Novel Radiographic Parameters of Collapsing Parasol Deformity in Spinal Muscular Atrophy.

Orthopedics 2020 Dec 29:1-7. Epub 2020 Dec 29.

Spinal muscular atrophy (SMA) is a neuromuscular disease with manifestations of scoliosis, pulmonary function decline, and, uniquely, collapse of the ribs. Methods to quantify rib deformity and its impact on pulmonary function are sparse. The authors propose new radiographic measurements to quantify the aspect of SMA known as collapsing parasol deformity and correlate these measurements with pulmonary function. Twenty-eight full-spine radiographs of pediatric SMA patients were measured twice by 3 independent investigators, with 2 weeks separating each measurement. Radiographic measurements, demographics, spirometry results, and assisted ventilation rating were obtained. Twenty-one patients with spirometry metrics were assessed to correlate pulmonary function and spinal measurements. The intrarater intraclass correlation coefficient (ICC) for the measurements ranged from 0.706 to 0.99, and the interrater ICC ranged from 0.64 to 0.97. Eighteen of 19 variables had ICC values greater than 0.75 for inter- and intrarater reliability. Twenty-one patients with forced expiratory volume in 1 second and forced vital capacity were assessed in terms of these measurements. Ratio of the concave hemithoracic width at T6/convex hemithoracic width at T6 (P=.004) and ratio of convex vertical rib displacement at the apical rib/concave vertical rib displacement (P=.021) were both significantly correlated with decreased pulmonary function. No significant correlation was found examining the average vertical rib displacement at the apical rib. High inter-and intrarater reliability can be obtained in a variety of spinal measurements of SMA patients. Various measurements are correlated to diminished pulmonary function, specifically variables showing asymmetric changes in the chest cavity. [Orthopedics. 2021;44(x):xx-xx.].
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http://dx.doi.org/10.3928/01477447-20201216-05DOI Listing
December 2020

Platelet electrical resistance for measuring platelet activation and adhesion in human health and disease.

Thromb Res 2021 Feb 18;198:204-209. Epub 2020 Dec 18.

University of Utah Molecular Medicine Program, Salt Lake City, UT 84112, United States of America; Department of Internal Medicine, University of Utah, Salt Lake City, UT 84132, United States of America. Electronic address:

Background: The ability to measure changes in platelet reactivity is important to identify novel aspects of platelet biology and develop targeted therapeutics to prevent bleeding or thrombosis. Current platelet function testing allows for single agonist analysis at a time. The ability to phenotype platelets in a single assay with multiple agonists and adhesion substrates could yield more insights into altered pathways than are feasible with current approaches. We hypothesized platelet electrical resistance (PER) could be used for more comprehensive phenotyping of platelets.

Methods: Platelets were isolated from male and female healthy donors (age 39.6 ± 6.9) and septic patients (age 44.0 ± 13.5). PER 96-well plates were coated with various substrates, including fibrinogen and collagen. Platelets were added to the coated plates in the presence or absence of thrombin or convulxin. Platelet activation and spreading was monitored by measuring changes in electrical impedance.

Results: Platelets adhesion to fibrinogen and collagen increased impedance. In addition, impedance increased in response to thrombin or convulxin. No changes in impedance were observed in the absence of platelets or when wells were uncoated, indicating changes in impedance were directly due to platelet adhesion and activation. Inhibiting integrin αIIbβ3 decreased impedance when fibrinogen was used as a substrate, consistent with platelet-dependent effects. Platelets from septic patients caused increased impedance compared to healthy donors, demonstrating this assay can be used to assess platelet hyperreactivity.

Conclusion: PER can be applied as a high throughput tool to measure platelet reactivity in health and disease, where platelet activation is increased.
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http://dx.doi.org/10.1016/j.thromres.2020.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7867577PMC
February 2021

Predictors of Initial Glaucoma Therapy with Laser Trabeculoplasty versus Medication: A Population-Based Study.

Ophthalmol Glaucoma 2020 Dec 21. Epub 2020 Dec 21.

Department of Ophthalmology, Queen's University, Kingston, Canada; Department of Ophthalmology, Kingston Health Sciences Centre, Kingston, Canada; ICES, Ontario, Canada. Electronic address:

Purpose: To investigate patient-level factors associated with first-line glaucoma therapy with laser trabeculoplasty (LT) versus topical medication.

Design: Population-based study.

Participants: All patients 66 years of age and older in Ontario, Canada, receiving first-ever therapy for glaucoma with either LT or topical medication between April 1, 2007, and March 31, 2019.

Methods: Linked health care databases were used to identify patients receiving first-line glaucoma therapy and to ascertain patient-level factors potentially associated with receipt of LT versus medication. Multivariate logistic regression analyses were undertaken.

Main Outcome Measures: Factors associated with receiving LT versus medications were evaluated using adjusted odds ratios (ORs) for age, gender, previous cataract surgery, previous corneal transplantation, previous retina surgery, level of systemic comorbidity, socioeconomic status (SES), and rural versus urban residence.

Results: In total, 194 759 patients were included. Older patients were less likely to be treated with LT versus medication (≥81 years of age vs. 66-70 years of age: OR, 0.49; 95% confidence interval [CI], 0.48-0.50), whereas women were more likely than men to receive LT (OR, 1.42; 95% CI, 1.39-1.45). Previous ocular surgeries were associated with decreased probability of treatment with LT, including cataract surgery (OR, 0.31; 95% CI, 0.30-0.32), corneal transplantation (OR, 0.39; 95% CI, 0.31-0.49), and retina surgery (OR, 0.46; 95% CI, 0.41-0.51). Patients with high comorbidity were less likely to receive LT (highest vs. lowest level of comorbidity: OR, 0.94; 95% CI, 0.91-0.97). Laser trabeculoplasty use was less likely among patients at higher levels of SES (highest vs. lowest level: OR, 0.86; 95% CI, 0.84-0.89) and from a rural residence (versus urban: OR, 0.92; 95% CI, 0.90-0.95). Increasing utilization of LT over time was noted (for each additional calendar year: OR, 1.05 per year; 95% CI, 1.05-1.05 per year).

Conclusions: Our results identified patient characteristics associated with use of LT as primary therapy for glaucoma, including factors related to patient demographics, ocular history, and comorbidity. Many of these associations are unexpected based on efficacy data or evidence-based guidelines. These results are topical considering growing evidence supporting use of first-line LT.
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http://dx.doi.org/10.1016/j.ogla.2020.11.001DOI Listing
December 2020

FcγRIIA expression accelerates nephritis and increases platelet activation in systemic lupus erythematosus.

Blood 2020 Dec;136(25):2933-2945

Centre de Recherche du Centre Hospitalier Universitaire de Québec-Université Laval, Québec, QC, Canada.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by deposits of immune complexes (ICs) in organs and tissues. The expression of FcγRIIA by human platelets, which is their unique receptor for immunoglobulin G antibodies, positions them to ideally respond to circulating ICs. Whereas chronic platelet activation and thrombosis are well-recognized features of human SLE, the exact mechanisms underlying platelet activation in SLE remain unknown. Here, we evaluated the involvement of FcγRIIA in the course of SLE and platelet activation. In patients with SLE, levels of ICs are associated with platelet activation. Because FcγRIIA is absent in mice, and murine platelets do not respond to ICs in any existing mouse model of SLE, we introduced the FcγRIIA (FCGR2A) transgene into the NZB/NZWF1 mouse model of SLE. In mice, FcγRIIA expression by bone marrow cells severely aggravated lupus nephritis and accelerated death. Lupus onset initiated major changes to the platelet transcriptome, both in FcγRIIA-expressing and nonexpressing mice, but enrichment for type I interferon response gene changes was specifically observed in the FcγRIIA mice. Moreover, circulating platelets were degranulated and were found to interact with neutrophils in FcγRIIA-expressing lupus mice. FcγRIIA expression in lupus mice also led to thrombosis in lungs and kidneys. The model recapitulates hallmarks of human SLE and can be used to identify contributions of different cellular lineages in the manifestations of SLE. The study further reveals a role for FcγRIIA in nephritis and in platelet activation in SLE.
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http://dx.doi.org/10.1182/blood.2020004974DOI Listing
December 2020

FcγRIIA expression accelerates nephritis and increases platelet activation in systemic lupus erythematosus.

Blood 2020 Dec;136(25):2933-2945

Centre de Recherche du Centre Hospitalier Universitaire de Québec-Université Laval, Québec, QC, Canada.

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by deposits of immune complexes (ICs) in organs and tissues. The expression of FcγRIIA by human platelets, which is their unique receptor for immunoglobulin G antibodies, positions them to ideally respond to circulating ICs. Whereas chronic platelet activation and thrombosis are well-recognized features of human SLE, the exact mechanisms underlying platelet activation in SLE remain unknown. Here, we evaluated the involvement of FcγRIIA in the course of SLE and platelet activation. In patients with SLE, levels of ICs are associated with platelet activation. Because FcγRIIA is absent in mice, and murine platelets do not respond to ICs in any existing mouse model of SLE, we introduced the FcγRIIA (FCGR2A) transgene into the NZB/NZWF1 mouse model of SLE. In mice, FcγRIIA expression by bone marrow cells severely aggravated lupus nephritis and accelerated death. Lupus onset initiated major changes to the platelet transcriptome, both in FcγRIIA-expressing and nonexpressing mice, but enrichment for type I interferon response gene changes was specifically observed in the FcγRIIA mice. Moreover, circulating platelets were degranulated and were found to interact with neutrophils in FcγRIIA-expressing lupus mice. FcγRIIA expression in lupus mice also led to thrombosis in lungs and kidneys. The model recapitulates hallmarks of human SLE and can be used to identify contributions of different cellular lineages in the manifestations of SLE. The study further reveals a role for FcγRIIA in nephritis and in platelet activation in SLE.
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http://dx.doi.org/10.1182/blood.2020004974DOI Listing
December 2020

Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging.

PLoS Biol 2020 11 24;18(11):e3000965. Epub 2020 Nov 24.

Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada.

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.
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http://dx.doi.org/10.1371/journal.pbio.3000965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723245PMC
November 2020

COVID-19 and Sepsis Are Associated With Different Abnormalities in Plasma Procoagulant and Fibrinolytic Activity.

Arterioscler Thromb Vasc Biol 2021 01 16;41(1):401-414. Epub 2020 Nov 16.

University of Utah Molecular Medicine Program, Salt Lake City, UT (F.D., A.M.B., M.T.R., R.A.C.).

Objective: Coronavirus disease 2019 (COVID-19) is associated with derangement in biomarkers of coagulation and endothelial function and has been likened to the coagulopathy of sepsis. However, clinical laboratory metrics suggest key differences in these pathologies. We sought to determine whether plasma coagulation and fibrinolytic potential in patients with COVID-19 differ compared with healthy donors and critically ill patients with sepsis. Approach and Results: We performed comparative studies on plasmas from a single-center, cross-sectional observational study of 99 hospitalized patients (46 with COVID-19 and 53 with sepsis) and 18 healthy donors. We measured biomarkers of endogenous coagulation and fibrinolytic activity by immunoassays, thrombin, and plasmin generation potential by fluorescence and fibrin formation and lysis by turbidity. Compared with healthy donors, patients with COVID-19 or sepsis both had elevated fibrinogen, d-dimer, soluble TM (thrombomodulin), and plasmin-antiplasmin complexes. Patients with COVID-19 had increased thrombin generation potential despite prophylactic anticoagulation, whereas patients with sepsis did not. Plasma from patients with COVID-19 also had increased endogenous plasmin potential, whereas patients with sepsis showed delayed plasmin generation. The collective perturbations in plasma thrombin and plasmin generation permitted enhanced fibrin formation in both COVID-19 and sepsis. Unexpectedly, the lag times to thrombin, plasmin, and fibrin formation were prolonged with increased disease severity in COVID-19, suggesting a loss of coagulation-initiating mechanisms accompanies severe COVID-19.

Conclusions: Both COVID-19 and sepsis are associated with endogenous activation of coagulation and fibrinolysis, but these diseases differently impact plasma procoagulant and fibrinolytic potential. Dysregulation of procoagulant and fibrinolytic pathways may uniquely contribute to the pathophysiology of COVID-19 and sepsis.
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http://dx.doi.org/10.1161/ATVBAHA.120.315338DOI Listing
January 2021

Mucosal-associated invariant T (MAIT) cells mediate protective host responses in sepsis.

Elife 2020 11 9;9. Epub 2020 Nov 9.

Division of Infectious Diseases, University of Utah, Salt Lake City, United States.

Sepsis is a systemic inflammatory response to infection and a leading cause of death. Mucosal-associated invariant T (MAIT) cells are innate-like T cells enriched in mucosal tissues that recognize bacterial ligands. We investigated MAIT cells during clinical and experimental sepsis, and their contribution to host responses. In experimental sepsis, MAIT-deficient mice had significantly increased mortality and bacterial load, and reduced tissue-specific cytokine responses. MAIT cells of WT mice expressed lower levels of IFN-γ and IL-17a during sepsis compared to sham surgery, changes not seen in non-MAIT T cells. MAIT cells of patients at sepsis presentation were significantly reduced in frequency compared to healthy donors, and were more activated, with decreased IFN-γ production, compared to both healthy donors and paired 90-day samples. Our data suggest that MAIT cells are highly activated and become dysfunctional during clinical sepsis, and contribute to tissue-specific cytokine responses that are protective against mortality during experimental sepsis.
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http://dx.doi.org/10.7554/eLife.55615DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7679140PMC
November 2020

Is there a role for the ACE2 receptor in SARS-CoV-2 interactions with platelets?

J Thromb Haemost 2021 01 18;19(1):46-50. Epub 2020 Nov 18.

University of Utah Molecular Medicine Program, Salt Lake City, UT, USA.

There is an urgent need to understand the underlying mechanisms contributing to thrombotic and inflammatory complications during COVID-19. Data from independent groups have identified that platelets are hyperreactive during COVID-19. Platelet hyperreactivity is accompanied by changes in platelet gene expression, and enhanced interactions between platelets and leukocytes. In some patients, SARS-CoV-2 mRNA has been detected in platelets. Together, this suggests that SARS-CoV-2 may interact with platelets. However, controversy remains on which receptors mediate SARS-CoV-2 platelet interactions. Most, but not all, transcriptomic and proteomic analyses fail to observe the putative SARS-CoV-2 receptor, angiotensin converting enzyme-2, or the cellular serine protease necessary for viral entry, TMPRSS2, on platelets and megakaryocytes. Interestingly, platelets express other known SARS-CoV-2 receptors, which induce similar patterns of activation to those observed when platelets are incubated with SARS-CoV-2. This article explores these findings and discusses ongoing areas of controversy and uncertainty with regard to SARS-CoV-2 platelet interactions.
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http://dx.doi.org/10.1111/jth.15156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7899240PMC
January 2021

Hyperglycemia exacerbates ischemic stroke outcome independent of platelet glucose uptake.

J Thromb Haemost 2021 Feb 27;19(2):536-546. Epub 2020 Nov 27.

University of Utah Molecular Medicine Program, Salt Lake City, UT, USA.

Objective: Hyperglycemia is a common comorbidity for ischemic stroke and is associated with worsened neurological outcomes. Platelets are central mediators of ischemic stroke and hyperglycemia mediates platelet hyperactivity. In this study, we investigated the contribution of platelet glucose metabolism to ischemic stroke.

Methods: Mice lacking both Glut1 and Glut3 specifically in platelets (DKO) and their littermate controls (WT) were subjected to 1-hour transient middle cerebral artery occlusion under normoglycemic and streptozotocin-induced hyperglycemic conditions after which stroke outcomes, platelet activation, and platelet-neutrophil aggregate (PNA) formation were examined.

Results: Under normoglycemic conditions, DKO mice were protected from ischemic stroke with smaller brain infarct volumes and improved cerebral blood flow. In addition, DKO mice had reduced platelet activation, PNA, and cerebral neutrophil recruitment after stroke. Hyperglycemia significantly increased infarct size and cerebral Evans blue extravasation and worsened neurological outcomes and cerebral blood flow in both WT and DKO mice, abolishing the protective effect witnessed under normoglycemic conditions. Flow cytometric analysis after stroke demonstrated increased platelet activation and neutrophil trafficking to the brain, independent of platelet glucose metabolism. Finally, platelets from healthy DKO mice were unable to become procoagulant upon dual agonist stimulation. Conversely, hyperglycemia increased platelet mitochondrial reactive oxygen species production which potentiated procoagulant platelet formation in WT mice and restored procoagulant platelet formation in DKO mice.

Conclusion: Hyperglycemia aggravates ischemic stroke outcome independent of platelet glucose uptake. Furthermore, we demonstrated that hyperglycemia primes procoagulant platelet formation. This underlines the therapeutic potential for strategies targeting procoagulant platelet formation for the treatment of acute ischemic stroke.
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http://dx.doi.org/10.1111/jth.15154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902465PMC
February 2021

Role of Platelets in Detection and Regulation of Infection.

Arterioscler Thromb Vasc Biol 2021 01 29;41(1):70-78. Epub 2020 Oct 29.

University of Utah Molecular Medicine Program, Salt Lake City (I.P., R.A.C.).

Platelets are classically known as essential mediators of hemostasis and thrombosis. However, in recent years, platelets have gained recognition for their inflammatory functions, which modulate the immune response during infectious diseases. Platelets contain various immunoreceptors that enable them to act as sentinels to recognize intravascular pathogens. Upon activation, platelets directly limit pathogen growth through the release of AMPs (antimicrobial proteins) and ensure pathogen clearance through activation of immune cells. However, aberrant platelet activation can lead to inflammation and thrombotic events.
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http://dx.doi.org/10.1161/ATVBAHA.120.314645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770024PMC
January 2021

Different glycoforms of alpha-1-acid glycoprotein contribute to its functional alterations in platelets and neutrophils.

J Leukoc Biol 2020 Oct 18. Epub 2020 Oct 18.

Department of Studies in Biochemistry, University of Mysore, Manasagangothri, Mysuru, Karnataka, India.

Alpha-1-acid glycoprotein (AGP-1) is a positive acute phase glycoprotein with uncertain functions. Serum AGP-1 (sAGP-1) is primarily derived from hepatocytes and circulates as 12-20 different glycoforms. We isolated a glycoform secreted from platelet-activating factor (PAF)-stimulated human neutrophils (nAGP-1). Its peptide sequence was identical to hepatocyte-derived sAGP-1, but nAGP-1 differed from sAGP-1 in its chromatographic behavior, electrophoretic mobility, and pattern of glycosylation. The function of these 2 glycoforms also differed. sAGP-1 activated neutrophil adhesion, migration, and neutrophil extracellular traps (NETosis) involving myeloperoxidase, peptidylarginine deiminase 4, and phosphorylation of ERK in a dose-dependent fashion, whereas nAGP-1 was ineffective as an agonist for these events. Furthermore, sAGP-1, but not nAGP-1, inhibited LPS-stimulated NETosis. Interestingly, nAGP-1 inhibited sAGP-1-stimulated neutrophil NETosis. The discordant effect of the differentially glycosylated AGP-1 glycoforms was also observed in platelets where neither of the AGP-1 glycoforms alone stimulated aggregation of washed human platelets, but sAGP-1, and not nAGP-1, inhibited aggregation induced by PAF or ADP, but not by thrombin. These functional effects of sAGP-1 correlated with intracellular cAMP accumulation and phosphorylation of the protein kinase A substrate vasodilator-stimulated phosphoprotein and reduction of Akt, ERK, and p38 phosphorylation. Thus, the sAGP-1 glycoform limits platelet reactivity, whereas nAGP-1 glycoform also limits proinflammatory actions of sAGP-1. These studies identify new functions for this acute phase glycoprotein and demonstrate that the glycosylation of AGP-1 controls its effects on 2 critical cells of acute inflammation.
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http://dx.doi.org/10.1002/JLB.3A0720-422RDOI Listing
October 2020

Culture, teamwork and engagement: 40 years of finding the right ingredients.

Curr Opin Cardiol 2021 Jan;36(1):105-109

Emory University School of Medicine, Sibley Heart Center Cardiology, Atlanta, Georgia, USA.

Purpose Of Review: Changing demands in healthcare, new leadership structures and physician preferences on work-life balance have made culture, teamwork and engagement vitally important for the long-term success of medical practices. With recent emphasis placed on culture, teamwork and engagement, leaders have had to acquire management skills that extend beyond the scope of being a successful clinician, educator and researcher.

Recent Findings: Over the last two decades, experts throughout the business and medical fields have worked to define culture. Furthermore, these authors have shown that success in businesses is often rooted in a strong organizational culture. Large surveys have confirmed that physicians value culture. They may join or leave a practice based on the culture. Furthermore, creating, defining and preserving culture requires leaders to be exemplary citizens and to inspire colleagues to be engaged.

Summary: Practices and businesses that have been shown to have a strong culture with engaged employees form stronger teams which ultimately confers a competitive advantage. In the current era, culture fit should be considered during any hire. An organization's unique culture actively needs to be taught and cultivated by leaders. Employees will become more engaged and adopt a practice's culture with education and by following the example of others.
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http://dx.doi.org/10.1097/HCO.0000000000000805DOI Listing
January 2021

COVID-19 patients exhibit reduced procoagulant platelet responses.

J Thromb Haemost 2020 11 6;18(11):3067-3073. Epub 2020 Oct 6.

University of Utah Molecular Medicine Program, Salt Lake City, UT, USA.

Background: Emerging evidence implicates dysfunctional platelet responses in thrombotic complications in COVID-19 patients. Platelets are important players in inflammation-induced thrombosis. In particular, procoagulant platelets support thrombin generation and mediate thromboinflammation.

Objectives: To examine if procoagulant platelet formation is altered in COVID-19 patients and if procoagulant platelets contribute to pulmonary thrombosis.

Patients/methods: Healthy donors and COVID-19 patients were recruited from the University of Utah Hospital System. Platelets were isolated and procoagulant platelet formation measured by annexin V binding as well as mitochondrial function were examined. We utilized mice lacking the ability to form procoagulant platelets (CypD ) to examine the role of procoagulant platelets in pulmonary thrombosis.

Results And Conclusions: We observed that platelets isolated from COVID-19 patients had a reduced ability to become procoagulant compared to those from matched healthy donors, as evidenced by reduced mitochondrial depolarization and phosphatidylserine exposure following dual stimulation with thrombin and convulxin. To understand what impact reduced procoagulant platelet responses might have in vivo, we subjected mice with a platelet-specific deletion of cyclophilin D, which are deficient in procoagulant platelet formation, to a model of pulmonary microvascular thrombosis. Mice with platelets lacking cyclophilin D died significantly faster from pulmonary microvascular thrombosis compared to littermate wild-type controls. These results suggest dysregulated procoagulant platelet responses may contribute to thrombotic complications during SARS-CoV-2 infection.
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http://dx.doi.org/10.1111/jth.15107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646270PMC
November 2020

Cytokine release syndrome in COVID-19: Innate immune, vascular, and platelet pathogenic factors differ in severity of disease and sex.

J Leukoc Biol 2021 01 15;109(1):55-66. Epub 2020 Sep 15.

Division of Microbiology and Immunology, Department of Pathology, University of Utah, Salt Lake City, Utah, USA.

COVID-19 rapidly emerged as a crippling public health crisis in the last few months, which has presented a series health risk. Understanding of the immune response and biomarker analysis is needed to progress toward understanding disease pathology and developing improved treatment options. The goal of this study is to identify pathogenic factors that are linked to disease severity and patient characteristics. Patients with COVID-19 who were hospitalized from March 17 to June 5, 2020 were analyzed for clinical features of disease and soluble plasma cytokines in association with disease severity and sex. Data from COVID-19 patients with acute illness were examined along with an age- and gender-matched control cohort. We identified a group of 16 soluble factors that were found to be increased in COVID-19 patients compared to controls, whereas 2 factors were decreased. In addition to inflammatory cytokines, we found significant increases in factors known to mediate vasculitis and vascular remodeling (PDGF-AA, PDGF-AB-BB, soluble CD40L (sCD40L), FGF, and IP10). Four factors such as platelet-derived growth factors, fibroblast growth factor-2, and IFN-γ-inducible protein 10 were strongly associated with severe disease and ICU admission. Th2-related factors (IL-4 and IL-13) were increased with IL-4 and sCD40L present at increased levels in males compared with females. Our analysis revealed networking clusters of cytokines and growth factors, including previously unknown roles of vascular and stromal remodeling, activation of the innate immunity, as well activation of type 2 immune responses in the immunopathogenesis of COVID-19. These data highlight biomarker associations with disease severity and sex in COVID-19 patients.
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http://dx.doi.org/10.1002/JLB.3COVA0820-410RRRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7902354PMC
January 2021

Organochlorine pesticide bioaccumulation in wild Nile crocodile (Crocodylus niloticus) fat tissues: Environmental influences on changing residue levels and contaminant profiles.

Sci Total Environ 2021 Jan 28;753:142068. Epub 2020 Aug 28.

Nature Conservation Department, Tshwane University of Technology, South Africa.

Biologically significant concentrations of organochlorine pesticides (OCPs) continue to be reported in wildlife populations and are of particular concern in species that occupy the highest trophic levels. Nile crocodiles (Crocodylus niloticus) are important apex predators occurring throughout much of tropical and subtropical sub-Saharan Africa, where they inhabit estuarine and freshwater habitats often impacted by contamination. In this study we examined pesticide residue accumulation in fat tissue from Nile crocodiles at Lake St Lucia, South Africa, where historically large quantities of OCPs have been used for agriculture and disease control. During 2019, we collected tail fat samples from wild (n = 21) and captive (n = 3) individuals to examine the influence of habitat, body size and sex on variations in bioaccumulation. The principal contaminant found was p,p'-DDE, a major persistent metabolite of DDT, which continues to be used in the region for combating malaria. Tissue p,p'-DDE concentrations in wild crocodiles (95-1200 ng g ww) were significantly (p < 0.05) higher compared to captive individuals (23-68 ng g ww) and strongly correlated (R > 0.70) to body length. Male (n = 14) and female (n = 7) wild crocodiles exhibited similar contaminant body burdens, however, total concentrations were substantially lower than those measured in the same population during 2016/2017. Marked differences in residue levels and profiles appear to reflect changes in food availability and dietary exposure associated with a shift in environmental conditions. These findings suggest that periods of environmental stress may be associated with enhanced toxicological risk in crocodiles. Additional work is needed to better understand contaminant accumulation and elimination mechanisms in crocodiles, and their potential effects on reproductive health.
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http://dx.doi.org/10.1016/j.scitotenv.2020.142068DOI Listing
January 2021

Engineering Photosensory Modules of Non-Opsin-Based Optogenetic Actuators.

Int J Mol Sci 2020 Sep 7;21(18). Epub 2020 Sep 7.

Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada.

Optogenetic (photo-responsive) actuators engineered from photoreceptors are widely used in various applications to study cell biology and tissue physiology. In the toolkit of optogenetic actuators, the key building blocks are genetically encodable light-sensitive proteins. Currently, most optogenetic photosensory modules are engineered from naturally-occurring photoreceptor proteins from bacteria, fungi, and plants. There is a growing demand for novel photosensory domains with improved optical properties and light-induced responses to satisfy the needs of a wider variety of studies in biological sciences. In this review, we focus on progress towards engineering of non-opsin-based photosensory domains, and their representative applications in cell biology and physiology. We summarize current knowledge of engineering of light-sensitive proteins including light-oxygen-voltage-sensing domain (LOV), cryptochrome (CRY2), phytochrome (PhyB and BphP), and fluorescent protein (FP)-based photosensitive domains (Dronpa and PhoCl).
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http://dx.doi.org/10.3390/ijms21186522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7555876PMC
September 2020

High-Performance Intensiometric Direct- and Inverse-Response Genetically Encoded Biosensors for Citrate.

ACS Cent Sci 2020 Aug 9;6(8):1441-1450. Epub 2020 Jul 9.

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

Motivated by the growing recognition of citrate as a central metabolite in a variety of biological processes associated with healthy and diseased cellular states, we have developed a series of high-performance genetically encoded citrate biosensors suitable for imaging of citrate concentrations in mammalian cells. The design of these biosensors was guided by structural studies of the citrate-responsive sensor histidine kinase and took advantage of the same conformational changes proposed to propagate from the binding domain to the catalytic domain. Following extensive engineering based on a combination of structure guided mutagenesis and directed evolution, we produced an inverse-response biosensor (Δ/ ≈ 18) designated Citroff1 and a direct-response biosensor (Δ/ ≈ 9) designated Citron1. We report the X-ray crystal structure of Citron1 and demonstrate the utility of both biosensors for qualitative and quantitative imaging of steady-state and pharmacologically perturbed citrate concentrations in live cells.
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http://dx.doi.org/10.1021/acscentsci.0c00518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7453566PMC
August 2020