Publications by authors named "David Horn"

312 Publications

Oligo targeting for profiling drug resistance mutations in the parasitic trypanosomatids.

Nucleic Acids Res 2022 May 7. Epub 2022 May 7.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK.

Trypanosomatids cause the neglected tropical diseases, sleeping sickness, Chagas disease and the leishmaniases. Studies on these lethal parasites would be further facilitated by new and improved genetic technologies. Scalable precision editing methods, for example, could be used to improve our understanding of potential mutations associated with drug resistance, a current priority given that several new anti-trypanosomal drugs, with known targets, are currently in clinical development. We report the development of a simple oligo targeting method for rapid and precise editing of priority drug targets in otherwise wild type trypanosomatids. In Trypanosoma brucei, approx. 50-b single-stranded oligodeoxynucleotides were optimal, multiple base edits could be incorporated, and editing efficiency was substantially increased when mismatch repair was suppressed. Resistance-associated edits were introduced in T. brucei cyclin dependent kinase 12 (CRK12, L482F) or cleavage and polyadenylation specificity factor 3 (N232H), in the Trypanosoma cruzi proteasome β5 subunit (G208S), or in Leishmania donovani CRK12 (G572D). We further implemented oligo targeting for site saturation mutagenesis, targeting codon G492 in T. brucei CRK12. This approach, combined with amplicon sequencing for codon variant scoring, revealed fourteen resistance conferring G492 edits encoding six distinct amino acids. The outputs confirm on-target drug activity, reveal a variety of resistance-associated mutations, and facilitate rapid assessment of potential impacts on drug efficacy.
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http://dx.doi.org/10.1093/nar/gkac319DOI Listing
May 2022

CRISPR/Cas9-based precision tagging of essential genes in bloodstream form African trypanosomes.

Mol Biochem Parasitol 2022 Apr 1;249:111476. Epub 2022 Apr 1.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Electronic address:

Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent ≪0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; HAT2 or HDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.
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http://dx.doi.org/10.1016/j.molbiopara.2022.111476DOI Listing
April 2022

Control of Variant Surface Glycoprotein Expression by CFB2 in Trypanosoma brucei and Quantitative Proteomic Connections to Translation and Cytokinesis.

mSphere 2022 Apr 21;7(2):e0006922. Epub 2022 Mar 21.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundeegrid.8241.f, Dundee, United Kingdom.

Variant surface glycoproteins (VSGs) coat parasitic African trypanosomes and underpin antigenic variation and immune evasion. These VSGs are superabundant virulence factors that are subject to posttranscriptional gene expression controls mediated via the 3' untranslated region (UTR). To identify positive VSG regulators in bloodstream-form Trypanosoma brucei, we used genome-scale screening data to prioritize mRNA binding protein (mRBP) knockdowns that phenocopy VSG mRNA knockdown, displaying loss of fitness and precytokinesis accumulation. The top three candidates were CFB2 (cyclin F-box protein 2) (Tb927.1.4650), MKT1 (Tb927.6.4770), and PBP1 (polyadenylate binding protein 1) (Tb927.8.4540). Notably, CFB2 was recently found to regulate VSG transcript stability, and all three proteins were found to associate. We used data-independent acquisition for accurate label-free quantification and deep proteome coverage to quantify the expression profiles following the depletion of each mRBP. Only CFB2 knockdown significantly reduced VSG expression and the expression of a reporter under the control of the 3' UTR. CFB2 knockdown also triggered the depletion of cytoplasmic ribosomal proteins, consistent with translation arrest observed when VSG synthesis is blocked. In contrast, PBP1 knockdown triggered the depletion of CFB2, MKT1, and other components of the PBP1 complex. Finally, all three knockdowns triggered the depletion of cytokinesis initiation factors, consistent with a cytokinesis defect, which was confirmed here for all three knockdowns. Thus, genome-scale knockdown data sets facilitate the triage and prioritization of candidate regulators. Quantitative proteomic analysis confirms the 3'-UTR-dependent positive control of VSG expression by CFB2 and interactions with additional mRBPs. Our results also reveal new insights into the connections between VSG expression control by CFB2, ribosomal protein expression, and cytokinesis. VSG expression represents a key parasite virulence mechanism and an example of extreme biology. Posttranscriptional gene expression controls in trypanosomatids also continue to be the subject of substantial research interest. We have identified three candidate VSG regulators and used knockdown and quantitative proteomics, in combination with other approaches, to assess their function. CFB2 is found to control VSG expression via the 3' untranslated region, while other data support the view that MKT1 and PBP1 also form part of a CFB2 mRNA binding complex. Remarkably, we also find the depletion of cytoplasmic ribosomal proteins upon CFB2 knockdown, consistent with translation arrest observed when VSG synthesis is blocked. Proteomic profiles following knockdown further yield insights into cytokinesis defects. Taken together, our findings confirm and elaborate the role of CFB2 in controlling VSG expression and reveal new insights into connectivity with translation and cytokinesis controls.
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http://dx.doi.org/10.1128/msphere.00069-22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9044945PMC
April 2022

Repositioning of a Diaminothiazole Series Confirmed to Target the Cyclin-Dependent Kinase CRK12 for Use in the Treatment of African Animal Trypanosomiasis.

J Med Chem 2022 04 18;65(7):5606-5624. Epub 2022 Mar 18.

Drug Discovery Unit, Wellcome Centre for Anti-Infectives Research, Division of Biological Chemistry and Drug Discovery, University of Dundee, Dow Street, Dundee DD1 5EH, United Kingdom.

African animal trypanosomiasis or nagana, caused principally by infection of the protozoan parasites and is a major problem in cattle and other livestocks in sub-Saharan Africa. Current treatments are threatened by the emergence of drug resistance and there is an urgent need for new, effective drugs. Here, we report the repositioning of a compound series initially developed for the treatment of human African trypanosomiasis. A medicinal chemistry program, focused on deriving more soluble analogues, led to development of a lead compound capable of curing cattle infected with both and via intravenous dosing. Further optimization has the potential to yield a single-dose intramuscular treatment for this disease. Comprehensive mode of action studies revealed that the molecular target of this promising compound and related analogues is the cyclin-dependent kinase CRK12.
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http://dx.doi.org/10.1021/acs.jmedchem.1c02104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9014415PMC
April 2022

Limits to the three domains of life: lessons from community assembly along an Antarctic salinity gradient.

Extremophiles 2022 Mar 16;26(1):15. Epub 2022 Mar 16.

Department of Biology, MSC03 2020 1UNM, University of New Mexico, Albuquerque, NM, 87131, USA.

Extremophiles exist among all three domains of life; however, physiological mechanisms for surviving harsh environmental conditions differ among Bacteria, Archaea and Eukarya. Consequently, we expect that domain-specific variation of diversity and community assembly patterns exist along environmental gradients in extreme environments. We investigated inter-domain community compositional differences along a high-elevation salinity gradient in the McMurdo Dry Valleys, Antarctica. Conductivity for 24 soil samples collected along the gradient ranged widely from 50 to 8355 µS cm. Taxonomic richness varied among domains, with a total of 359 bacterial, 2 archaeal, 56 fungal, and 69 non-fungal eukaryotic operational taxonomic units (OTUs). Richness for bacteria, archaea, fungi, and non-fungal eukaryotes declined with increasing conductivity (all P < 0.05). Principal coordinate ordination analysis (PCoA) revealed significant (ANOSIM R = 0.97) groupings of low/high salinity bacterial OTUs, while OTUs from other domains were not significantly clustered. Bacterial beta diversity was unimodally distributed along the gradient and had a nested structure driven by species losses, whereas in fungi and non-fungal eukaryotes beta diversity declined monotonically without strong evidence of nestedness. Thus, while increased salinity acts as a stressor in all domains, the mechanisms driving community assembly along the gradient differ substantially between the domains.
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http://dx.doi.org/10.1007/s00792-022-01262-3DOI Listing
March 2022

Spectral Resolution Development in Children With Normal Hearing and With Cochlear Implants: A Review of Behavioral Studies.

J Speech Lang Hear Res 2022 Apr 24;65(4):1646-1658. Epub 2022 Feb 24.

Virginia Merrill Bloedel Hearing Research Center, Department of Otolaryngology - Head and Neck Surgery, University of Washington, Seattle.

Purpose: This review article provides a theoretical overview of the development of spectral resolution in children with normal hearing (cNH) and in those who use cochlear implants (CIs), with an emphasis on methodological considerations. The aim was to identify key directions for future research on spectral resolution development in children with CIs.

Method: A comprehensive literature review was conducted to summarize and synthesize previously published behavioral research on spectral resolution development in normal and impaired auditory systems.

Conclusions: In cNH, performance on spectral resolution tasks continues to improve through the teenage years and is likely driven by gradual maturation of across-channel intensity resolution. A small but growing body of evidence from children with CIs suggests a more complex relationship between spectral resolution development, patient demographics, and the quality of the CI electrode-neuron interface. Future research should aim to distinguish between the effects of patient-specific variables and the underlying physiology on spectral resolution abilities in children of all ages who are hard of hearing and use auditory prostheses.
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http://dx.doi.org/10.1044/2021_JSLHR-21-00307DOI Listing
April 2022

A profile of research on the parasitic trypanosomatids and the diseases they cause.

Authors:
David Horn

PLoS Negl Trop Dis 2022 01 13;16(1):e0010040. Epub 2022 Jan 13.

The Wellcome Trust Centre for Anti-Infectives Research, Division of Biological Chemistry & Drug Discovery, School of Life Sciences, University of Dundee, Dundee, United Kingdom.

The parasitic trypanosomatids cause lethal and debilitating diseases, the leishmaniases, Chagas disease, and the African trypanosomiases, with major impacts on human and animal health. Sustained research has borne fruit by assisting efforts to reduce the burden of disease and by improving our understanding of fundamental molecular and cell biology. But where has the research primarily been conducted, and which research areas have received the most attention? These questions are addressed below using publication and citation data from the past few decades.
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http://dx.doi.org/10.1371/journal.pntd.0010040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8758061PMC
January 2022

Impact of Cost Information on Parental Decision Making: A Randomized Clinical Trial Evaluating Cast Versus Splint Selection for Pediatric Distal Radius Buckle Fractures.

J Pediatr Orthop 2022 Jan;42(1):e15-e20

Division of Orthopaedics, Children's Hospital of Philadelphia.

Background: Price transparency purports to help patients make high-value health care decisions, however, there is little data to support this. The pediatric distal radius buckle fracture (DRBF) has 2 equally efficacious but not equally priced treatment options (cast and splint), serving as an excellent potential model for studying price transparency. This study uses the DRBF model to assess the impact of up-front cost information on a family's treatment decisions when presented with clinically equivalent treatment options for a low-risk injury.

Methods: Participants age 4 to 14 presenting with an acute DRBF to a hospital-based pediatric orthopaedic clinic were recruited for this randomized controlled trial. Participants were randomized into cost-informed or cost-blind cohorts. All families received standardized information about the injury and treatment options. Cost-informed families received additional cost information. Both groups were allowed to freely choose a treatment. Families were surveyed regarding their decision factors. Cost-blinded families were subsequently presented with the cost information and could change their decision. Independent samples t tests and χ2 tests were utilized to evaluate differences.

Results: A total of 127 patients were enrolled (53% cost-informed, 47% cost-blind). The 2 groups did not significantly differ in demographics. Immobilization selection did not differ between groups, with 48% of the cost-informed families selecting the more expensive option (casting), compared with 47% of the cost-blind families. Cost was the least influential factor in the decision-making process according to participant survey, influencing only 9% of families. Only one family changed their decision after receiving cost information, from a splint to a cast.

Conclusion: Families appear to be cost-insensitive when making medical treatment decisions for low-risk injuries for their child. Price transparency alone may not help families arrive at a decision to pursue high-value treatment in low-risk orthopaedic injuries.

Level Of Evidence: Level I.
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http://dx.doi.org/10.1097/BPO.0000000000001980DOI Listing
January 2022

Post-Operative Splinting Versus Casting of Pediatric Supracondylar Humerus Fractures.

Cureus 2021 Sep 1;13(9):e17635. Epub 2021 Sep 1.

Orthopaedics, Children's Hospital of Philadelphia, Philadelphia, USA.

Background Supracondylar humerus fractures (SCH) are common upper extremity fractures in children and are usually treated by closed reduction and percutaneous pinning. Post-operative management may cause complications, but the difference between cast and splint has not been closely investigated. Purpose Our objective was to compare casting and splinting of SCH fractures with respect to post-operative complications. Patients and methods We reviewed 1,146 pediatric SCH fractures that were reduced, percutaneously pinned, and immobilized by cast or splint. Open fractures, openly reduced fractures, and pre-operative neurological injuries were excluded. Over the course of immobilization, we noted if the initial cast or splint was maintained and if the patient returned due to complications. Results Post-operative casting was performed on 1,091 (95.2%) fractures and 55 (4.8%) were splinted. Age was a significant factor, increasing the likelihood of splinting by 12% with each year of age (p = 0.023). A total of 28 patients (2.4%) returned for unscheduled visits due to immobilization complaints, infection, and pain, but the rate difference between cast and splint was negligible. Reoperation was required for five patients (0.4%), and more likely for splinted fractures (p = 0.021). After controlling for age, splinting was still associated with reoperation (OR: 15.1, p = 0.004). Conclusions Although complications inevitably exist, both casting and splinting are effective immobilization methods. Both resulted in few complications such as post-operative discomfort, pain, infection, loss of reduction, or damage. It was difficult to evaluate significance with few splinted cases, but considering no major differences between splinted and casted fractures, clinicians should consider splinting to reduce the cost associated with casting.
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http://dx.doi.org/10.7759/cureus.17635DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8486368PMC
September 2021

Identification of a Proteasome-Targeting Arylsulfonamide with Potential for the Treatment of Chagas' Disease.

Antimicrob Agents Chemother 2022 01 4;66(1):e0153521. Epub 2021 Oct 4.

Division of Biological Chemistry and Drug Discovery, Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundeegrid.8241.f, Dundee, United Kingdom.

Phenotypic screening identified an arylsulfonamide compound with activity against Trypanosoma cruzi, the causative agent of Chagas' disease. Comprehensive mode of action studies revealed that this compound primarily targets the T. cruzi proteasome, binding at the interface between β4 and β5 subunits that catalyze chymotrypsin-like activity. A mutation in the β5 subunit of the proteasome was associated with resistance to compound 1, while overexpression of this mutated subunit also reduced susceptibility to compound 1. Further genetically engineered and -selected clones resistant to proteasome inhibitors known to bind at the β4/β5 interface were cross-resistant to compound 1. Ubiquitinated proteins were additionally found to accumulate in compound 1-treated epimastigotes. Finally, thermal proteome profiling identified malic enzyme as a secondary target of compound 1, although malic enzyme inhibition was not found to drive potency. These studies identify a novel pharmacophore capable of inhibiting the T. cruzi proteasome that may be exploitable for anti-chagasic drug discovery.
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http://dx.doi.org/10.1128/AAC.01535-21DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8765320PMC
January 2022

Genome-scale RNAi screens in African trypanosomes.

Authors:
David Horn

Trends Parasitol 2022 02 24;38(2):160-173. Epub 2021 Sep 24.

The Wellcome Trust Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Electronic address:

Genome-scale genetic screens allow researchers to rapidly identify the genes and proteins that impact a particular phenotype of interest. In African trypanosomes, RNA interference (RNAi) knockdown screens have revealed mechanisms underpinning drug resistance, drug transport, prodrug metabolism, quorum sensing, genome replication, and gene expression control. RNAi screening has also been remarkably effective at highlighting promising potential antitrypanosomal drug targets. The first ever RNAi library screen was implemented in African trypanosomes, and genome-scale RNAi screens and other related approaches continue to have a major impact on trypanosomatid research. Here, I review those impacts in terms of both discovery and translation.
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http://dx.doi.org/10.1016/j.pt.2021.09.002DOI Listing
February 2022

Inter-chromosomal k-mer distances.

BMC Genomics 2021 Sep 6;22(1):644. Epub 2021 Sep 6.

School of Physics and Astronomy, Tel Aviv University, 69978, Tel Aviv, Israel.

Background: Inversion Symmetry is a generalization of the second Chargaff rule, stating that the count of a string of k nucleotides on a single chromosomal strand equals the count of its inverse (reverse-complement) k-mer. It holds for many species, both eukaryotes and prokaryotes, for ranges of k which may vary from 7 to 10 as chromosomal lengths vary from 2Mbp to 200 Mbp. Building on this formalism we introduce the concept of k-mer distances between chromosomes. We formulate two k-mer distance measures, D and D, which depend on k. D takes into account all k-mers (for a single k) appearing on single strands of the two compared chromosomes, whereas D takes into account both strands of each chromosome. Both measures reflect dissimilarities in global chromosomal structures.

Results: After defining the various distance measures and summarizing their properties, we also define proximities that rely on the existence of synteny blocks between chromosomes of different bacterial strains. Comparing pairs of strains of bacteria, we find negative correlations between synteny proximities and k-mer distances, thus establishing the meaning of the latter as measures of evolutionary distances among bacterial strains. The synteny measures we use are appropriate for closely related bacterial strains, where considerable sections of chromosomes demonstrate high direct or reversed equality. These measures are not appropriate for comparing different bacteria or eukaryotes. K-mer structural distances can be defined for all species. Because of the arbitrariness of strand choices, we employ only the D measure when comparing chromosomes of different species. The results for comparisons of various eukaryotes display interesting behavior which is partially consistent with conventional understanding of evolutionary genomics. In particular, we define ratios of minimal k-mer distances (KDR) between unmasked and masked chromosomes of two species, which correlate with both short and long evolutionary scales.

Conclusions: k-mer distances reflect dissimilarities among global chromosomal structures. They carry information which aggregates all mutations. As such they can complement traditional evolution studies , which mainly concentrate on coding regions.
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http://dx.doi.org/10.1186/s12864-021-07952-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8422766PMC
September 2021

Validation of the Seattle Suprastomal Safety Score (5S): A Novel Measure in Pediatric Tracheostomy-Dependent Patients.

Otolaryngol Head Neck Surg 2022 05 7;166(5):970-975. Epub 2021 Sep 7.

Department of Otolaryngology-Head & Neck Surgery, University of Washington School of Medicine, Seattle, Washington, USA.

Objective: Suprastomal collapse and granulation are common sequelae of pediatric tracheostomy. We present the first measure of suprastomal obstructive pathology, the Seattle Suprastomal Safety Score (5S), an instrument with 2 domains: collapse and granulation.

Study Design: Cross-sectional repeated testing survey.

Setting: Electronic survey.

Methods: A library of images was assembled from still pictures of the suprastomal area in 50 patients who previously underwent trachea-bronchoscopy at a quaternary children's hospital. Five pediatric otolaryngologists and 2 pediatric pulmonologists reviewed the images in random, blinded fashion and provided 5S scores. Participants repeated this process 2 to 4 weeks later. Interrater agreement was calculated with an intraclass correlation coefficient (ICC) with a 2-way random-effects model and Fleiss's κ. Intrarater agreement was measured with an ICC using a 2-way mixed-effects model as well as with test-retest correlations using Spearman rank coefficient. All measures were performed separately on collapse and granulation domains.

Results: ICC for interrater agreement was 0.88 (95% CI, 0.82-0.93) for collapse and 0.97 (95% CI, 0.96-0.98) for granulation, indicating almost perfect agreement. Fleiss's κ demonstrated moderate agreement for collapse and almost perfect agreement for granulation. ICC for intrarater agreement was 0.95 (95% CI, 0.93-0.97) and 0.99 (95% CI, 0.98-0.99) for collapse and granulation, respectively, indicating almost perfect agreement. Spearman rank correlation for test-retest demonstrated substantial agreement for collapse and almost perfect agreement for granulation.

Conclusion: The 5S demonstrates excellent interrater and intrarater agreement, making it highly reliable as a novel measure of suprastomal collapse and granulation in tracheostomy-dependent pediatric patients.
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http://dx.doi.org/10.1177/01945998211037254DOI Listing
May 2022

INFERYS rescoring: Boosting peptide identifications and scoring confidence of database search results.

Rapid Commun Mass Spectrom 2021 May 20:e9128. Epub 2021 May 20.

MSAID GmbH, Garching b. München, Germany.

Database search engines for bottom-up proteomics largely ignore peptide fragment ion intensities during the automated scoring of tandem mass spectra against protein databases. Recent advances in deep learning allow the accurate prediction of peptide fragment ion intensities. Using these predictions to calculate additional intensity-based scores helps to overcome this drawback. Here, we describe a processing workflow termed INFERYS™ rescoring for the intensity-based rescoring of Sequest HT search engine results in Thermo Scientific™ Proteome Discoverer™ 2.5 software. The workflow is based on the deep learning platform INFERYS capable of predicting fragment ion intensities, which runs on personal computers without the need for graphics processing units. This workflow calculates intensity-based scores comparing peptide spectrum matches from Sequest HT and predicted spectra. Resulting scores are combined with classical search engine scores for input to the false discovery rate estimation tool Percolator. We demonstrate the merits of this approach by analyzing a classical HeLa standard sample and exemplify how this workflow leads to a better separation of target and decoy identifications, in turn resulting in increased peptide spectrum match, peptide and protein identification numbers. On an immunopeptidome dataset, this workflow leads to a 50% increase in identified peptides, emphasizing the advantage of intensity-based scores when analyzing low-intensity spectra or analytes with very similar physicochemical properties that require vast search spaces. Overall, the end-to-end integration of INFERYS rescoring enables simple and easy access to a powerful enhancement to classical database search engines, promising a deeper, more confident and more comprehensive analysis of proteomic data from any organism by unlocking the intensity dimension of tandem mass spectra for identification and more confident scoring.
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http://dx.doi.org/10.1002/rcm.9128DOI Listing
May 2021

Wildfires increasingly impact western US fluvial networks.

Nat Commun 2021 04 30;12(1):2484. Epub 2021 Apr 30.

Department of Biology, University of New Mexico, Albuquerque, NM, USA.

Wildfires are increasing globally in frequency, severity, and extent, but their impact on fluvial networks, and the resources they provide, remains unclear. We combine remote sensing of burn perimeter and severity, in-situ water quality monitoring, and longitudinal modeling to create the first large-scale, long-term estimates of stream+river length impacted by wildfire for the western US. We find that wildfires directly impact ~6% of the total stream+river length between 1984 and 2014, increasing at a rate of 342 km/year. When longitudinal propagation of water quality impacts is included, we estimate that wildfires affect ~11% of the total stream+river length. Our results indicate that wildfire activity is one of the largest drivers of aquatic impairment, though it is not routinely reported by regulatory agencies, as wildfire impacts on fluvial networks remain unconstrained. We identify key actions to address this knowledge gap and better understand the growing threat to fluvial networks, water security, and public health risks.
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http://dx.doi.org/10.1038/s41467-021-22747-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8087797PMC
April 2021

Natural History of Bilateral Congenital Absence of the L4 Pedicles.

JBJS Case Connect 2021 04 2;11(2). Epub 2021 Apr 2.

Children's Hospital of Philadelphia, Philadelphia Pennsylvania.

Case: A 5-month-old girl was diagnosed with congenital bilateral absence of the L4 pedicles and an absent right kidney. She developed a right thoracic scoliosis at age 3 that was treated with a brace. At her most recent follow-up at age 16, she was skeletally mature and had a residual 20° right thoracic scoliosis. She was pain-free, had a normal neurological examination, and was fully active.

Conclusion: This case presents a long-term follow-up of a patient with congenital bilateral absence of L4 pedicles. She developed scoliosis that was successfully treated with bracing. No other significant issues developed over 15 years of follow-up.
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http://dx.doi.org/10.2106/JBJS.CC.20.00663DOI Listing
April 2021

Spatial integration of transcription and splicing in a dedicated compartment sustains monogenic antigen expression in African trypanosomes.

Nat Microbiol 2021 03 11;6(3):289-300. Epub 2021 Jan 11.

Department of Veterinary Sciences, Experimental Parasitology, Ludwig-Maximilians-Universität München, Munich, Germany.

Highly selective gene expression is a key requirement for antigenic variation in several pathogens, allowing evasion of host immune responses and maintenance of persistent infections. African trypanosomes-parasites that cause lethal diseases in humans and livestock-employ an antigenic variation mechanism that involves monogenic antigen expression from a pool of >2,600 antigen-coding genes. In other eukaryotes, the expression of individual genes can be enhanced by mechanisms involving the juxtaposition of otherwise distal chromosomal loci in the three-dimensional nuclear space. However, trypanosomes lack classical enhancer sequences or regulated transcription initiation. In this context, it has remained unclear how genome architecture contributes to monogenic transcription elongation and transcript processing. Here, we show that the single expressed antigen-coding gene displays a specific inter-chromosomal interaction with a major messenger RNA splicing locus. Chromosome conformation capture (Hi-C) revealed a dynamic reconfiguration of this inter-chromosomal interaction upon activation of another antigen. Super-resolution microscopy showed the interaction to be heritable and splicing dependent. We found a specific association of the two genomic loci with the antigen exclusion complex, whereby VSG exclusion 1 (VEX1) occupied the splicing locus and VEX2 occupied the antigen-coding locus. Following VEX2 depletion, loss of monogenic antigen expression was accompanied by increased interactions between previously silent antigen genes and the splicing locus. Our results reveal a mechanism to ensure monogenic expression, where antigen transcription and messenger RNA splicing occur in a specific nuclear compartment. These findings suggest a new means of post-transcriptional gene regulation.
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http://dx.doi.org/10.1038/s41564-020-00833-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610597PMC
March 2021

Drug-induced sleep endoscopy directed surgery improves polysomnography measures in overweight and obese children with obstructive sleep apnea.

Acta Otolaryngol 2021 Apr 29;141(4):397-402. Epub 2020 Dec 29.

Department of Otolaryngology-Head and Neck Surgery, University of Washington, Seattle, WA, USA.

Background: Obstructive sleep apnea affects approximately 1-4% of all children, with increased prevalence amongst overweight and obese children.

Objective: To assess the effects of drug-induced sleep endoscopy (DISE)-directed surgery on polysomnography parameters in obese and overweight children.

Material/methods: A retrospective case-series was performed on obese and overweight pediatric patients who underwent clinically indicated DISE-directed surgery. Forty children met the inclusion criteria, including: body mass index ≥85%, DISE-study, and pre- and post-DISE polysomnography. Patients were divided into surgically naïve ( = 23) and prior adenotonsillectomy ( = 17) groups. Demographic and clinical characteristics were examined with chi-square and Wilcoxon rank-sum test. Polysomnography parameters were compared with Wilcoxon signed rank test.

Results: Of 40 children with mean BMI 94% and mean age 8 ± 6 years old, 17 (43%) underwent a previous adenotonsillectomy. Overall, significant improvements were observed in the apnea-hypopnea index (AHI; 25.0 to 9.9 events/hour,  < .01) and oxygen nadir (82.7% to 88.5%,  < .01). A similar pattern was observed among the surgically naïve (AHI: 35.9 to 12.7 events/hour,  = .04; oxygen nadir: 79.7% to 86.4%,  = .2) and post-adenotonsillectomy groups (AHI: 10.4 to 6.2 events/hour,  = .02; oxygen nadir: 86.7% to 91.2%,  < .01).

Conclusions/significance: Polysomnography parameters significantly improved following DISE-directed interventions in obese and overweight children with obstructive sleep apnea.
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http://dx.doi.org/10.1080/00016489.2020.1863465DOI Listing
April 2021

Experimental nitrogen and phosphorus enrichment stimulates multiple trophic levels of algal and detrital-based food webs: a global meta-analysis from streams and rivers.

Biol Rev Camb Philos Soc 2020 Dec 17. Epub 2020 Dec 17.

Department of Biological Sciences, University of Alabama, Tuscaloosa, AL, 35487, U.S.A.

Anthropogenic increases in nitrogen (N) and phosphorus (P) concentrations can strongly influence the structure and function of ecosystems. Even though lotic ecosystems receive cumulative inputs of nutrients applied to and deposited on land, no comprehensive assessment has quantified nutrient-enrichment effects within streams and rivers. We conducted a meta-analysis of published studies that experimentally increased concentrations of N and/or P in streams and rivers to examine how enrichment alters ecosystem structure (state: primary producer and consumer biomass and abundance) and function (rate: primary production, leaf breakdown rates, metabolism) at multiple trophic levels (primary producer, microbial heterotroph, primary and secondary consumers, and integrated ecosystem). Our synthesis included 184 studies, 885 experiments, and 3497 biotic responses to nutrient enrichment. We documented widespread increases in organismal biomass and abundance (mean response = +48%) and rates of ecosystem processes (+54%) to enrichment across multiple trophic levels, with no large differences in responses among trophic levels or between autotrophic or heterotrophic food-web pathways. Responses to nutrient enrichment varied with the nutrient added (N, P, or both) depending on rate versus state variable and experiment type, and were greater in flume and whole-stream experiments than in experiments using nutrient-diffusing substrata. Generally, nutrient-enrichment effects also increased with water temperature and light, and decreased under elevated ambient concentrations of inorganic N and/or P. Overall, increased concentrations of N and/or P altered multiple food-web pathways and trophic levels in lotic ecosystems. Our results indicate that preservation or restoration of biodiversity and ecosystem functions of streams and rivers requires management of nutrient inputs and consideration of multiple trophic pathways.
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http://dx.doi.org/10.1111/brv.12673DOI Listing
December 2020

Publisher Correction: Mutation-selection balance and compensatory mechanisms in tumour evolution.

Nat Rev Genet 2021 Apr;22(4):263

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.

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http://dx.doi.org/10.1038/s41576-020-00313-9DOI Listing
April 2021

Mutation-selection balance and compensatory mechanisms in tumour evolution.

Nat Rev Genet 2021 04 30;22(4):251-262. Epub 2020 Nov 30.

National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA.

Intratumour heterogeneity and phenotypic plasticity, sustained by a range of somatic aberrations, as well as epigenetic and metabolic adaptations, are the principal mechanisms that enable cancers to resist treatment and survive under environmental stress. A comprehensive picture of the interplay between different somatic aberrations, from point mutations to whole-genome duplications, in tumour initiation and progression is lacking. We posit that different genomic aberrations generally exhibit a temporal order, shaped by a balance between the levels of mutations and selective pressures. Repeat instability emerges first, followed by larger aberrations, with compensatory effects leading to robust tumour fitness maintained throughout the tumour progression. A better understanding of the interplay between genetic aberrations, the microenvironment, and epigenetic and metabolic cellular states is essential for early detection and prevention of cancer as well as development of efficient therapeutic strategies.
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http://dx.doi.org/10.1038/s41576-020-00299-4DOI Listing
April 2021

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

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

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

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

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

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

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

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

Displaced Supracondylar Humerus Fractures in Toddlers.

Orthopedics 2020 Sep 7;43(5):e421-e424. Epub 2020 Jul 7.

Gartland type III fracture is the most troublesome type of supracondylar humerus fracture. These injuries most often occur in school age children, but they are seen in pediatric patients of all ages. The goal of this study was to analyze toddlers with Gartland type III fractures to identify clinically significant differences compared with older children. A retrospective cohort study was conducted with 94 toddlers (<3 years) and 378 older children (3 to 12 years). Factors including demographics, mechanism of injury, additional injuries, location of trauma, pin configuration, postoperative complications, follow-up time, and compliance with the treatment plan were collected and compared. The study included 94 toddlers (59% girls, 2.11±0.64 years) and 378 older children (48% girls, 6.32±1.89 years), chosen at random, who were treated between 2000 and 2015. Among toddlers, fractures were more likely to occur at home (P<.001) and to be the result of suspected nonaccidental trauma (P<.001). Older children had more additional injuries (P<.001), but were no more likely to have an open fracture (P=.59) or a flexion-type fracture (P=.42). Older children were more likely to undergo open reduction (P=.03), whereas toddlers were more likely to be treated with a medial pin (P<.001). Toddlers experienced more cubitus varus (P<.001) and loss of reduction (P=.02). No difference was found in length of follow-up (P=.83) or compliance with the treatment plan (P=.11). This study provides novel insights into clinical differences between toddlers and older children with Gartland type III fractures. Knowledge of these differences can facilitate the delivery of targeted, age-specific care for patients with type III supracondylar humerus fractures. [Orthopedics. 2020;43(5);e421-e424.].
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http://dx.doi.org/10.3928/01477447-20200619-13DOI Listing
September 2020

Epidemiology and management of appendicular fractures occurring in neonatal intensive care patients.

Acta Paediatr 2021 02 1;110(2):489-494. Epub 2020 Jul 1.

Division of Orthopaedics, The Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Aim: To describe the epidemiology and management of appendicular fractures occurring in the neonatal ICU in a large series of patients treated a single, quaternary care neonatal intensive care unit.

Methods: Patients <1 years old with appendicular fractures treated from 2012 to 2016 at a quaternary-level NICU were identified. Bivariate testing compared fractures, work-up and management based on designated mechanism (presumed birth-related vs unknown). In patients with unknown mechanism, factors with potential fracture association were analysed in a descriptive fashion.

Results: Eighty-five fractures (54 patients) were included. Mechanistic cohorts differed by birthweight (P < .001) and gestational age at birth (P < .001). Presumed birth-related fractures were more commonly upper extremity (P < .001), solitary (P = .001) and radiographically diagnosed in the acute state (<.001). The biochemical profile of the cohorts differed significantly. The prevalence of factors with potential fracture association was high in patients with unknown mechanism. Only one patient required surgery, while all others resolved with minimal orthopaedic intervention.

Conclusion: Findings indicate these injuries rarely require operative intervention and that two distinct injury profiles appear to exist based on fracture mechanism. Steroid use, ventilation use, diuretic use, nutritional supplementation and recent bedside procedures were common among patients without known fracture mechanism.

Level Of Evidence: Level III-Retrospective Cohort Study.
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http://dx.doi.org/10.1111/apa.15430DOI Listing
February 2021

Water quality impacts of urban and non-urban arid-land runoff on the Rio Grande.

Sci Total Environ 2020 Aug 24;729:138443. Epub 2020 Apr 24.

Department of Civil, Construction & Environmental Engineering, University of New Mexico, Albuquerque, NM 87131, USA.

Urban surface runoff from storms impacts the water quality dynamics of downstream ecosystems. While these effects are well-documented in mesic regions, they are not well constrained for arid watersheds, which sustain longer dry periods, receive intense but short-lived storms, and where stormwater drainage networks are generally isolated from sewage systems. We used a network of high-frequency in situ water quality sensors located along the Middle Rio Grande to determine surface runoff origins during storms and track rapid changes in physical, chemical, and biological components of water quality. Specific conductivity (SpCond) patterns were a reliable indicator of source, distinguishing between runoff events originating primarily in urban (SpCond sags) or non-urban (SpCond spikes) catchments. Urban events were characterized by high fluorescent dissolved organic matter (fDOM), low dissolved oxygen (including short-lived hypoxia <2 mg/L), smaller increases in turbidity and varied pH response. In contrast, non-urban events showed large turbidity spikes, smaller dissolved oxygen sags, and consistent pH sags. Principal component analysis distinguished urban and non-urban events by dividing physical and biogeochemical water quality parameters, and modeling of DO along the same reach demonstrated consistently higher oxygen demand for an urban event compared to a non-urban event. Based on our analysis, urban runoff poses more potential ecological harm, while non-urban runoff poses a larger problem for drinking water treatment. The comparison of our results to other reports of urban stormwater quality suggest that water quality responses to storm events in urban landscapes are consistent across a range of regional climates.
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http://dx.doi.org/10.1016/j.scitotenv.2020.138443DOI Listing
August 2020

Reliability of Measuring Insertion Depth in Cochlear Implanted Infants and Children Using Cochlear View Radiography.

Otolaryngol Head Neck Surg 2020 Oct 26;163(4):822-828. Epub 2020 May 26.

Department of Otolaryngology-Head and Neck Surgery, University of Washington, Seattle, Washington, USA.

Objectives: Cochlear implant depth of insertion affects audiologic outcomes and can be measured in adults using plain films obtained in the "cochlear view." The objective of this study was to assess interrater and intrarater reliability of measuring depth of insertion using cochlear view radiography.

Study Design: Prospective, observational.

Setting: Tertiary referral pediatric hospital.

Subjects And Methods: Patients aged 11 months to 20 years (median, 4 years; interquartile range [IQR], 1-8 years) undergoing cochlear implantation at our institution were studied over 1 year. Children underwent cochlear view imaging on postoperative day 1. Films were deidentified and 1 image per ear was selected. Two cochlear implant surgeons and 2 radiologists evaluated each image and determined angular depth of insertion. Images were re-reviewed 6 weeks later by all raters. Inter- and intrarater reliability were calculated with intraclass correlation coefficients (ICCs).

Results: Fifty-seven ears were imaged from 42 children. Forty-nine ears (86%) had successful cochlear view x-rays. Median angular depth of insertion was 381° (minimum, 272°; maximum, 450°; IQR, 360°-395°) during the first round of measurement. Measurements of the same images reviewed 6 weeks later showed median depth of insertion of 382° (minimum, 272°; maximum, 449°; IQR, 360°-397°). Interrater and intrarater reliability ICCs ranged between 0.81 and 0.96, indicating excellent reliability.

Conclusions: Postoperative cochlear view radiography is a reliable tool for measurement of cochlear implant depth of insertion in infants and children. Further studies are needed to determine reliability of intraoperatively obtained cochlear view radiographs in this population.
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http://dx.doi.org/10.1177/0194599820921857DOI Listing
October 2020

Defining Essential Services for Deaf and Hard of Hearing Children during the COVID-19 Pandemic.

Otolaryngol Head Neck Surg 2020 07 5;163(1):91-93. Epub 2020 May 5.

Department of Otolaryngology-Head and Neck Surgery, School of Medicine, University of Washington, Seattle, Washington, USA.

COVID-19 is a rapidly growing global pandemic caused by a novel coronavirus. With no vaccine or definitive treatment, public health authorities have recommended a strategy of "social distancing," reducing individual interaction, canceling elective procedures, and limiting nonessential services. Health care providers must determine what procedures are considered "elective," balancing risk of treatment delays with that of coronavirus exposure to patient, family, and providers. Given critical periods for language development and the long-term impact of auditory deprivation, some audiologic and otologic services should be considered essential. In this article, we describe the experience of a quaternary referral pediatric hospital in Seattle, the epicenter of COVID-19 in the United States, and share strategies for risk minimization employed by Seattle Children's Hospital. We hope that this work can be a reference for other centers continuing care for children who are deaf and hard of hearing during the COVID-19 and future resource-limiting crises.
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http://dx.doi.org/10.1177/0194599820925058DOI Listing
July 2020

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

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

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

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

Burnout and Self Care for Palliative Care Practitioners.

Med Clin North Am 2020 May 2;104(3):561-572. Epub 2020 Mar 2.

Division of Geriatrics, General Internal Medicine and Palliative Medicine, University of Arizona College of Medicine, PO Box 245036, 1501 North Campbell Avenue, Tucson, AZ 85724-5036, USA; Department of Medicine, University of Arizona, Tucson, AZ, USA. Electronic address:

Burnout is common in physicians who care for patients with serious illness, with rates greater than 60% in some studies. Risk factors for burnout include working on small teams and/or in small organizations, working longer hours and weekends, being younger than 50 years, burdensome documentation requirements, and regulatory issues. Personal factors that can protect against burnout include mindfulness, exercise, healthy sleep patterns, avoiding substance abuse, and having adequate leisure time. Institutional and work factors that can buffer against burnout include working on adequately staffed teams, having a manageable workload, and minimally burdensome electronic health record documentation.
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http://dx.doi.org/10.1016/j.mcna.2019.12.007DOI Listing
May 2020
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