Publications by authors named "Michelle Simon"

70 Publications

Generation and analysis of innovative genomically humanized knockin , (TDP-43), and mouse models.

iScience 2021 Dec 15;24(12):103463. Epub 2021 Nov 15.

UK MRC Harwell Institute, Harwell Campus, Oxfordshire OX11 0RD, UK.

Amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) is a fatal neurodegenerative disorder, and continued innovation is needed for improved understanding and for developing therapeutics. We have created next-generation knockin mouse models, by replacing the mouse genomic region of , (TDP-43), and , with their human orthologs, preserving human protein biochemistry and splicing with exons and introns intact. We establish a new standard of large knockin allele quality control, demonstrating the utility of indirect capture for enrichment of a genomic region of interest followed by Oxford Nanopore sequencing. Extensive analysis shows that homozygous humanized animals only express human protein at endogenous levels. Characterization of humanized FUS animals showed that they are phenotypically normal throughout their lifespan. These humanized strains are vital for preclinical assessment of interventions and serve as templates for the addition of coding or non-coding human ALS/FTD mutations to dissect disease pathomechanisms, in a physiological context.
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http://dx.doi.org/10.1016/j.isci.2021.103463DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8710557PMC
December 2021

Modifiable Risk Factors for SARS-CoV-2.

Integr Med (Encinitas) 2021 Oct;20(5):8-14

As the COVID-19 pandemic has raged on, considerable research has been performed around the world evaluating the environmental, genetic, lifestyle, and nutritional factors that significantly impact the COVID-19 pandemic. Many studies have now shown that key risk factors for SARS-CoV-2 infection, severity, and even death are modifiable. Patients, whether partially vaccinated, fully vaccinated, or not vaccinated, are expecting their clinicians to provide them with evidence-based guidance and to help them prioritize the factors most important for them. In this editorial we review the current state of the research on modifiable risk factors for SARS-CoV-2 infection, disease severity, and death.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8594967PMC
October 2021

Broad-spectrum XX and XY gonadal dysgenesis in patients with a homozygous L193S variant in PPP2R3C.

Eur J Endocrinol 2021 Dec 1;186(1):65-72. Epub 2021 Dec 1.

Department of Paediatric Endocrinology and Diabetes, Marmara University, School of Medicine, Istanbul, Turkey.

Context: Homozygous and heterozygous variants in PPP2R3C are associated with syndromic 46,XY complete gonadal dysgenesis (Myo-Ectodermo-Gonadal Dysgenesis (MEGD) syndrome), and impaired spermatogenesis, respectively. This study expands the role of PPP2R3C in the aetiology of gonadal dysgenesis (GD).

Method: We sequenced the PPP2R3C gene in four new patients from three unrelated families. The clinical, laboratory, and molecular characteristics were investigated. We have also determined the requirement for Ppp2r3c in mice (C57BL6/N) using CRISPR/Cas9 genome editing.

Results: A homozygous c.578T>C (p.L193S) PPP2R3C variant was identified in one 46,XX girl with primary gonadal insufficiency, two girls with 46,XY complete GD, and one undervirilised boy with 46,XY partial GD. The patients with complete GD had low gonadal and adrenal androgens, low anti-Müllerian hormone, and high follicle-stimulating hormone and luteinizing hormone concentrations. All patients manifested characteristic features of MEGD syndrome. Heterozygous Ppp2r3c knockout mice appeared overtly normal and fertile. Inspection of homozygous embryos at 14.5, 9.5, and 8.5 days post coitum(dpc) revealed evidence of dead embryos. We conclude that loss of function of Ppp2r3c is not compatible with viability in mice and results in embryonic death from 7.5 dpc or earlier.

Conclusion: Our data indicate the essential roles for PPP2R3C in mouse and human development. Germline homozygous variants in human PPP2R3C are associated with distinctive syndromic GD of varying severity in both 46,XY and 46,XX individuals.
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http://dx.doi.org/10.1530/EJE-21-0910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8679844PMC
December 2021

Comprehensive phenotypic analysis of the Dp1Tyb mouse strain reveals a broad range of Down syndrome-related phenotypes.

Dis Model Mech 2021 10 15;14(10). Epub 2021 Oct 15.

The Francis Crick Institute, London NW1 1AT, UK.

Down syndrome (DS), trisomy 21, results in many complex phenotypes including cognitive deficits, heart defects and craniofacial alterations. Phenotypes arise from an extra copy of human chromosome 21 (Hsa21) genes. However, these dosage-sensitive causative genes remain unknown. Animal models enable identification of genes and pathological mechanisms. The Dp1Tyb mouse model of DS has an extra copy of 63% of Hsa21-orthologous mouse genes. In order to establish whether this model recapitulates DS phenotypes, we comprehensively phenotyped Dp1Tyb mice using 28 tests of different physiological systems and found that 468 out of 1800 parameters were significantly altered. We show that Dp1Tyb mice have wide-ranging DS-like phenotypes, including aberrant erythropoiesis and megakaryopoiesis, reduced bone density, craniofacial changes, altered cardiac function, a pre-diabetic state, and deficits in memory, locomotion, hearing and sleep. Thus, Dp1Tyb mice are an excellent model for investigating complex DS phenotype-genotype relationships for this common disorder.
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http://dx.doi.org/10.1242/dmm.049157DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8543064PMC
October 2021

Modulation of amyloid-β aggregation by metal complexes with a dual binding mode and their delivery across the blood-brain barrier using focused ultrasound.

Chem Sci 2021 Jul 15;12(27):9485-9493. Epub 2021 Jun 15.

Department of Chemistry, Imperial College London White City Campus, 82 Wood Lane London W12 0BZ UK

One of the key hallmarks of Alzheimer's disease is the aggregation of the amyloid-β peptide to form fibrils. Consequently, there has been great interest in studying molecules that can disrupt amyloid-β aggregation. While a handful of molecules have been shown to inhibit amyloid-β aggregation , there remains a lack of data reported due to their inability to cross the blood-brain barrier. Here, we investigate a series of new metal complexes for their ability to inhibit amyloid-β aggregation . We demonstrate that octahedral cobalt complexes with polyaromatic ligands have high inhibitory activity thanks to their dual binding mode involving π-π stacking and metal coordination to amyloid-β (confirmed a range of spectroscopic and biophysical techniques). In addition to their high activity, these complexes are not cytotoxic to human neuroblastoma cells. Finally, we report for the first time that these metal complexes can be safely delivered across the blood-brain barrier to specific locations in the brains of mice using focused ultrasound.
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http://dx.doi.org/10.1039/d1sc02273cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8278877PMC
July 2021

Linking the obesity rs1421085 variant circuitry to cellular, metabolic, and organismal phenotypes in vivo.

Sci Adv 2021 Jul 21;7(30). Epub 2021 Jul 21.

Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire OX11 0RD, UK.

Variants in FTO have the strongest association with obesity; however, it is still unclear how those noncoding variants mechanistically affect whole-body physiology. We engineered a deletion of the rs1421085 conserved cis-regulatory module (CRM) in mice and confirmed in vivo that the CRM modulates and gene expression and mitochondrial function in adipocytes. The CRM affects molecular and cellular phenotypes in an adipose depot-dependent manner and affects organismal phenotypes that are relevant for obesity, including decreased high-fat diet-induced weight gain, decreased whole-body fat mass, and decreased skin fat thickness. Last, we connected the CRM to a genetically determined effect on steroid patterns in males that was dependent on nutritional challenge and conserved across mice and humans. Together, our data establish cross-species conservation of the rs1421085 regulatory circuitry at the molecular, cellular, metabolic, and organismal level, revealing previously unknown contextual dependence of the variant's action.
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http://dx.doi.org/10.1126/sciadv.abg0108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8294759PMC
July 2021

It's not always postdural puncture headache: a case report and note to the astute anesthesiologist.

Braz J Anesthesiol 2021 Jun 24. Epub 2021 Jun 24.

University of Texas Medical Branch at Galveston, Department of Anesthesiology, Galveston, Texas, USA.

Dural puncture is either diagnosed by unexpectedly profound response to medication test dose or development of a postpartum postural headache. Epidural blood patch is the gold standard for treatment of PDPH when conservative management fails. However, postpartum headaches can be resistant to multiple epidural blood patches. In such cases, preexisting intracranial processes should be considered and ruled out. We report here the unique case of a pregnant patient who developed a resistant headache in the postpartum period related to an incidental intracranial aneurysm. Subsequent treatment with endovascular embolization adequately relieved her symptoms. Early surgical consultation and a multidisciplinary team approach involving neurology and neuroimaging is required for successful management of patients such as the one described here.
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http://dx.doi.org/10.1016/j.bjane.2021.06.003DOI Listing
June 2021

Unit and regression tests of scientific software: A study on SWMM.

J Comput Sci 2021 Jul;53

Department of Electrical Engineering and Computer Science, University of Cincinnati, Cincinnati, OH 45221, USA.

Testing helps assure software quality by executing a program and uncovering bugs. Scientific software developers often find it challenging to carry out systematic and automated testing due to reasons like inherent model uncertainties and complex floating-point computations. Extending the recent work on analyzing the unit tests written by the developers of the Storm Water Management Model (SWMM) [32], we report in this paper the investigation of both unit and regression tests of SWMM. The results show that the 2953 unit tests of SWMM have a 39.7% statement-level code coverage and a 82.4% user manual coverage. Meanwhile, an examination of 58 regression tests of SWMM shows a 44.9% statement-level code coverage and a near 100% user manual coverage. We also observe a "getter-setter-getter" testing pattern from the SWMM unit tests, and suggest a diversified way of executing regression tests.
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http://dx.doi.org/10.1016/j.jocs.2021.101347DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8128694PMC
July 2021

Scientific Software Testing Goes Serverless: Creating and Invoking Metamorphic Functions.

IEEE Softw 2020 Oct;38(1):61-67

University of Cincinnati.

Our function-as-a-service (FaaS) framework transformed end users' questions into automated tests for scientific software. Our case study illustrates the FaaSification of scientific software testing and the importance of value-driven evaluations by focusing on real-world defect detection.
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http://dx.doi.org/10.1109/ms.2020.3029468DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129919PMC
October 2020

Mouse mutant phenotyping at scale reveals novel genes controlling bone mineral density.

PLoS Genet 2020 12 28;16(12):e1009190. Epub 2020 Dec 28.

Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.
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http://dx.doi.org/10.1371/journal.pgen.1009190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822523PMC
December 2020

Improving agreement of ASA physical status class between pre-anesthesia screening and day of surgery by adding institutional-specific and ASA-approved examples: a quality improvement project.

Perioper Med (Lond) 2020 Nov 19;9(1):34. Epub 2020 Nov 19.

Department of Anesthesiology, University of Texas Medical Branch, Medical Branch, 301 University Blvd. Rt 0877, Galveston, TX, 77555, USA.

Background: A successful anesthesia pre-assessment clinic needs to identify patients who need further testing, evaluation, and optimization prior to the day of surgery to avoid delays and cancelations. Although the ASA Physical Status Classification system (ASA PS) has been used widely for over 50 years, it has poor interrater agreement when only using the definitions. In 2014, ASA-approved examples for each ASA physical status class (ASA PS). In this quality improvement study, we developed and evaluated the effectiveness of institutional-specific examples on interrater reliability between anesthesia pre-anesthesia clinic (APAC) and the day of surgery evaluation (DOS).

Methods: A multi-step, multi-year quality improvement project was performed. Step 1, pre-intervention, was a retrospective review to determine the percentage agreement of ASA PS assignment between APAC and DOS for adult and pediatric patients. Step 2 was a retrospective review of the step 1 cases where the ASA PS assignment differed to determine which medical conditions were valued differently and then develop institutional-specific examples for medical conditions not addressed by ASA-approved examples. Step 3 was to educate clinicians about the newly implemented examples and how they should be used as a guide. Step 4, post-intervention, was a retrospective review to determine if the examples improved agreement between APAC and DOS ASA PS assignments. Weighted Kappa coefficient was used to measure of interrater agreement excluding chance agreement.

Results: Having only ASA PS definitions available, APAC and DOS agreement was only 74% for adults (n = 737) and 63% for pediatric patients (n = 216). For adults, 20 medical co-morbidity categories and, for pediatric patients, 9 medical co-morbidity categories accounted for > 90% the differences in ASA PS. After development and implementation of institutional-specific examples with ASA-approved examples, the percentage agreement increased for adult patients (n = 795) to 91% and for pediatric patients (n = 239) to 84%. Weighted Kappa coefficients increased significantly for all patients (from 0.62 to 0.85, p < .0001), adult patients (from 0.62 to 0.86, p < .0001), and pediatric patients (from 0.48 to 0.78, p < .0001).

Conclusions: ASA-approved examples do not address all medical conditions that account for differences in the assignment of ASA PS between pre-anesthesia screening and day of anesthesia evaluation at our institution. The process of developing institutional-specific examples addressed the medical conditions that caused differences in assignment at one institution. The implementation of ASA PS examples improved consistency of assignment, and therefore communication of medical conditions of patients presenting for anesthesia care.
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http://dx.doi.org/10.1186/s13741-020-00162-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677831PMC
November 2020

A holistic view of mouse enhancer architectures reveals analogous pleiotropic effects and correlation with human disease.

BMC Genomics 2020 Nov 2;21(1):754. Epub 2020 Nov 2.

Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire, OX11 0RD, UK.

Background: Efforts to elucidate the function of enhancers in vivo are underway but their vast numbers alongside differing enhancer architectures make it difficult to determine their impact on gene activity. By systematically annotating multiple mouse tissues with super- and typical-enhancers, we have explored their relationship with gene function and phenotype.

Results: Though super-enhancers drive high total- and tissue-specific expression of their associated genes, we find that typical-enhancers also contribute heavily to the tissue-specific expression landscape on account of their large numbers in the genome. Unexpectedly, we demonstrate that both enhancer types are preferentially associated with relevant 'tissue-type' phenotypes and exhibit no difference in phenotype effect size or pleiotropy. Modelling regulatory data alongside molecular data, we built a predictive model to infer gene-phenotype associations and use this model to predict potentially novel disease-associated genes.

Conclusion: Overall our findings reveal that differing enhancer architectures have a similar impact on mammalian phenotypes whilst harbouring differing cellular and expression effects. Together, our results systematically characterise enhancers with predicted phenotypic traits endorsing the role for both types of enhancers in human disease and disorders.
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http://dx.doi.org/10.1186/s12864-020-07109-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7607678PMC
November 2020

Sizing, stabilising, and cloning repeat-expansions for gene targeting constructs.

Methods 2021 07 25;191:15-22. Epub 2020 Jul 25.

Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire OX11 0RD, UK. Electronic address:

Aberrant microsatellite repeat-expansions at specific loci within the human genome cause several distinct, heritable, and predominantly neurological, disorders. Creating models for these diseases poses a challenge, due to the instability of such repeats in bacterial vectors, especially with large repeat expansions. Designing constructs for more precise genome engineering projects, such as engineering knock-in mice, proves a greater challenge still, since these unstable repeats require numerous cloning steps in order to introduce homology arms or selection cassettes. Here, we report our efforts to clone a large hexanucleotide repeat in the C9orf72 gene, originating from within a BAC construct, derived from a C9orf72-ALS patient. We provide detailed methods for efficient repeat sizing and growth conditions in bacteria to facilitate repeat retention during growth and sub-culturing. We report that sub-cloning into a linear vector dramatically improves stability, but is dependent on the relative orientation of DNA replication through the repeat, consistent with previous studies. We envisage the findings presented here provide a relatively straightforward route to maintaining large-range microsatellite repeat-expansions, for efficient cloning into vectors.
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http://dx.doi.org/10.1016/j.ymeth.2020.07.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8215685PMC
July 2021

Testing of the Storm Water Management Model Low Impact Development Modules.

J Am Water Resour Assoc 2020 Apr;56(2):283-296

United States Environmental Protection Agency, National Risk Management Research Laboratory, Cincinnati, Ohio 45268.

Stormwater infrastructure designers and operators rely heavily on the United States Environmental Protection Agency's Storm Water Management Model (SWMM) to simulate stormwater and wastewater infrastructure performance. Since its inception in the late 1970s, improvements and extensions have been tested and evaluated rigorously to verify the accuracy of the model. As a continuation of this progress, the main objective of this study was to quantify how accurately SWMM simulates the hydrologic activity of low impact development (LID) storm control measures. Model performance was evaluated by quantitatively comparing empirical data to model results using a multievent, multiobjective calibration method. The calibration methodology utilized the PEST software, a Parameter ESTimation tool, to determine unmeasured hydrologic parameters for SWMM's LID modules. The calibrated LID modules' Nash-Sutcliffe efficiencies averaged 0.81; average percent bias (PBIAS) -9%; average ratio of root mean square error to standard deviation of measured values 0.485; average index of agreement 0.94; and the average volume error, simulated vs. observed, was +9%. SWMM accurately predicted the timing of peak flows, but usually underestimated their magnitudes by 10%. The average volume reduction, measured outflow volume divided by inflow volume, was 48%. We had more difficulty in calibrating one study, an infiltration trench, which identified a significant limitation of the current version of the SWMM LID module; it cannot simulate lateral exfiltration of water out of the storage layers of a LID storm control measure. This limitation is especially severe for a deep LIDs, such as infiltration trenches. Nevertheless, SWMM satisfactorily simulated the hydrologic performance of eight of the nine LID practices.
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http://dx.doi.org/10.1111/1752-1688.12832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7321851PMC
April 2020

Comparison of the Effectiveness of Different Barrier Enclosure Techniques in Protection of Healthcare Workers During Tracheal Intubation and Extubation.

A A Pract 2020 Jun;14(8):e01252

From the Department of Anesthesiology, The University of Texas Medical Branch at Galveston, Galveston, Texas.

The coronavirus disease 2019 (COVID-19; SARS-CoV-2) pandemic has created serious challenges to anesthesiologists. As hospitalized patients' respiratory function deteriorates, many will require endotracheal intubation. Airway management of infected patients risks aerosolization of viral-loaded droplets that pose serious hazards to the anesthesiologist and all health care personnel present. The addition of an enclosure barrier during airway management minimizes the hazard by entrapping the droplets and possibly the aerosols within an enclosed space adding additional protection for health care workers. The aim of this study was to compare the effectiveness of different barrier enclosure techniques during tracheal intubation and extubation.
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http://dx.doi.org/10.1213/XAA.0000000000001252DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302074PMC
June 2020

Genetic background influences tumour development in heterozygous Men1 knockout mice.

Endocr Connect 2020 May;9(5):426-437

Academic Endocrine Unit, Radcliffe Department of Medicine, University of Oxford, Oxford Centre for Diabetes, Endocrinology and Metabolism (OCDEM), Churchill Hospital, Headington, Oxford, UK.

Multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant disorder caused by MEN1 germline mutations, is characterised by parathyroid, pancreatic and pituitary tumours. MEN1 mutations also cause familial isolated primary hyperparathyroidism (FIHP), a milder condition causing hyperparathyroidism only. Identical mutations can cause either MEN1 or FIHP in different families, thereby implicating a role for genetic modifiers in altering phenotypic expression of tumours. We therefore investigated the effects of genetic background and potential for genetic modifiers on tumour development in adult Men1+/- mice, which develop tumours of the parathyroids, pancreatic islets, anterior pituitary, adrenal cortex and gonads, that had been backcrossed to generate C57BL/6 and 129S6/SvEv congenic strains. A total of 275 Men1+/- mice, aged 5-26 months were macroscopically studied, and this revealed that genetic background significantly influenced the development of pituitary, adrenal and ovarian tumours, which occurred in mice over 12 months of age and more frequently in C57BL/6 females, 129S6/SvEv males and 129S6/SvEv females, respectively. Moreover, pituitary and adrenal tumours developed earlier, in C57BL/6 males and 129S6/SvEv females, respectively, and pancreatic and testicular tumours developed earlier in 129S6/SvEv males. Furthermore, glucagon-positive staining pancreatic tumours occurred more frequently in 129S6/SvEv Men1+/- mice. Whole genome sequence analysis of 129S6/SvEv and C57BL/6 Men1+/- mice revealed >54,000 different variants in >300 genes. These included, Coq7, Dmpk, Ccne2, Kras, Wnt2b, Il3ra and Tnfrsf10a, and qRT-PCR analysis revealed that Kras was significantly higher in pituitaries of male 129S6/SvEv mice. Thus, our results demonstrate that Kras and other genes could represent possible genetic modifiers of Men1.
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http://dx.doi.org/10.1530/EC-20-0103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7274560PMC
May 2020

Additional Barrier to Protect Health Care Workers During Intubation.

Anesth Analg 2020 07;131(1):e47-e48

Department of Anesthesiology, The University of Texas Medical Branch at Galveston, Galveston, Texas, Department of Outcomes Research, Outcomes Research Consortium, Cleveland Clinic, Cleveland, Ohio, Department of Anesthesiology, The University of Texas Medical Branch at Galveston, Galveston, Texas.

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http://dx.doi.org/10.1213/ANE.0000000000004904DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7188029PMC
July 2020

Discovering Metamorphic Relations for Scientific Software From User Forums.

Comput Sci Eng 2020 ;23(2):65-72

University of Cincinnati, Cincinnati, OH, 45221, USA.

Scientific software can be used for decades and is constantly evolving. Recently, metamorphic testing, a property-based testing technique, has shown to be effective in testing scientific software, and the necessary properties are expressed as metamorphic relations. However, the development of metamorphic relations is difficult: it requires considerable practical expertise for the software tester. In this article, we report our experience of uncovering metamorphic relations from a user forum's questions of the United States Environmental Protection Agency's Storm Water Management Model (SWMM). Our study not only illustrates a wealth of end users' expertise in interpreting software results, but also demonstrates the usefulness of classifying the user-oriented metamorphic relations into a nominal, ordinal, and functional hierarchy mainly from the software output perspective.
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http://dx.doi.org/10.1109/mcse.2020.3046973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129917PMC
January 2020

Protection Against XY Gonadal Sex Reversal by a Variant Region on Mouse Chromosome 13.

Genetics 2020 02 13;214(2):467-477. Epub 2019 Dec 13.

Mammalian Genetics Unit, MRC Harwell Institute, Oxfordshire, OX11 0RD, UK.

XY C57BL/6J (B6) mice harboring a -type Y chromosome (Y ), known as B6.Y mice, commonly undergo gonadal sex reversal and develop as phenotypic females. In a minority of cases, B6.Y males are identified and a proportion of these are fertile. This phenotypic variability on a congenic B6 background has puzzled geneticists for decades. Recently, a B6.Y colony was shown to carry a non-B6-derived region of chromosome 11 that protected against B6.Y sex reversal. Here. we show that a B6.Y colony bred and archived at the MRC Harwell Institute lacks the chromosome 11 modifier but instead harbors an ∼37 Mb region containing non-B6-derived segments on chromosome 13. This region, which we call , protects against B6.Y sex reversal in a proportion of heterozygous animals through its positive and negative effects on gene expression during primary sex determination. We discuss 's influence on the testis determination process and its possible origin in light of sequence similarities to that region in other mouse genomes. Our data reveal that the B6.Y sex reversal phenomenon is genetically complex and the explanation of observed phenotypic variability is likely dependent on the breeding history of any local colony.
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http://dx.doi.org/10.1534/genetics.119.302786DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017026PMC
February 2020

Genomic Mutation Identification in Mice Using Illumina Sequencing and Linux-Based Computational Methods.

Curr Protoc Mouse Biol 2019 Sep;9(3):e64

MRC Harwell Institute, Mammalian Genetics Unit, Harwell Campus, Oxfordshire, United Kingdom.

Genetically modified mice are an essential tool for modeling disease-causing mechanisms and discovering gene function. SNP genotyping was traditionally used to associate candidate regions with traits in the mouse, but failed to reveal novel variants without further targeted sequencing. Using a robust set of computational protocols, we present a platform to enable scientists to detect variants arising from whole-genome and exome sequencing experiments. This article guides researchers on aligning reads to the mouse genome, quality-assurance strategies, mutation discovery, comparing mutations to previously discovered mouse SNPs, and the annotation of novel variants, in order to predict mutation consequences on the protein level. Challenges unique to the mouse are discussed, and two protocols use self-contained containers to maintain version control and allow users to adapt our approach to new techniques by upgrading container versions. Our protocols are suited for servers or office workstations and are usable by non-bioinformatics specialists. © 2019 by John Wiley & Sons, Inc.
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http://dx.doi.org/10.1002/cpmo.64DOI Listing
September 2019

Clarin-2 is essential for hearing by maintaining stereocilia integrity and function.

EMBO Mol Med 2019 09 26;11(9):e10288. Epub 2019 Aug 26.

Department of Biomedical Science, University of Sheffield, Sheffield, UK.

Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2 mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance.
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http://dx.doi.org/10.15252/emmm.201910288DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6728604PMC
September 2019

Peripartum cardiomyopathy: a single institution 5-yr experience.

Br J Anaesth 2019 10 20;123(4):e491-e493. Epub 2019 Aug 20.

Galveston, TX, USA.

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http://dx.doi.org/10.1016/j.bja.2019.07.006DOI Listing
October 2019

Mice with a Brd4 Mutation Represent a New Model of Nephrocalcinosis.

J Bone Miner Res 2019 07 4;34(7):1324-1335. Epub 2019 Mar 4.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology, and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Nephrolithiasis (NL) and nephrocalcinosis (NC), which comprise renal calcification of the collecting system and parenchyma, respectively, have a multifactorial etiology with environmental and genetic determinants and affect ∼10% of adults by age 70 years. Studies of families with hereditary NL and NC have identified >30 causative genes that have increased our understanding of extracellular calcium homeostasis and renal tubular transport of calcium. However, these account for <20% of the likely genes that are involved, and to identify novel genes for renal calcification disorders, we investigated 1745 12-month-old progeny from a male mouse that had been treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for radiological renal opacities. This identified a male mouse with renal calcification that was inherited as an autosomal dominant trait with >80% penetrance in 152 progeny. The calcification consisted of calcium phosphate deposits in the renal papillae and was associated with the presence of the urinary macromolecules osteopontin and Tamm-Horsfall protein, which are features found in Randall's plaques of patients with NC. Genome-wide mapping located the disease locus to a ∼30 Mbp region on chromosome 17A3.3-B3 and whole-exome sequence analysis identified a heterozygous mutation, resulting in a missense substitution (Met149Thr, M149T), in the bromodomain-containing protein 4 (BRD4). The mutant heterozygous (Brd4 ) mice, when compared with wild-type (Brd4 ) mice, were normocalcemic and normophosphatemic, with normal urinary excretions of calcium and phosphate, and had normal bone turnover markers. BRD4 plays a critical role in histone modification and gene transcription, and cDNA expression profiling, using kidneys from Brd4 and Brd4 mice, revealed differential expression of genes involved in vitamin D metabolism, cell differentiation, and apoptosis. Kidneys from Brd4 mice also had increased apoptosis at sites of calcification within the renal papillae. Thus, our studies have established a mouse model, due to a Brd4 Met149Thr mutation, for inherited NC. © 2019 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6658219PMC
July 2019

Multimodal Pain Management for Cesarean Delivery: A Double-Blinded, Placebo-Controlled, Randomized Clinical Trial.

Am J Perinatol 2019 09 1;36(11):1097-1105. Epub 2019 Mar 1.

Department of Obstetrics and Gynecology, The University of Texas Medical Branch, Galveston, Texas.

Objective: Our objective was to evaluate the efficacy of perioperative multimodal pain management in reducing opioid use after elective cesarean delivery (CD).

Study Design: A single-center, double-blinded, placebo-controlled randomized trial of women undergoing elective CD. Participants were allocated 1:1 to receive the multimodal protocol or matching placebos. The multimodal protocol consisted of a preoperative dose of intravenous acetaminophen, preincision injection of subcutaneous bupivacaine, and intraoperative injection of intramuscular ketorolac. Primary outcome was total opioid intake at 48 hours postoperatively. Secondary outcomes were pain scores, time to first opioid intake, neonatal outcomes, and total outpatient opioid intake on postoperative day (POD) 7. Data were analyzed using parametric and nonparametric tests and quantile regression as appropriate.

Results: A total of 242 women were screened with 120 randomized, 60 to the multimodal group and 60 to control group. There was no significant difference in the primary outcome of opioid use nor in the secondary outcomes. Smokers and patients with a history of drug use had higher median postoperative opiate use and earlier administration. On POD 7, only 40% of prescribed opioids had been used, and there was no difference between the groups.

Conclusion: This perioperative multimodal pain regimen did not reduce opioid use in 48 hours after CD. Patients who smoke or with a history of drug use required more opioids in the postoperative period. Providers significantly overprescribed opioids after CD.
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http://dx.doi.org/10.1055/s-0039-1681096DOI Listing
September 2019

A Wars2 Mutant Mouse Model Displays OXPHOS Deficiencies and Activation of Tissue-Specific Stress Response Pathways.

Cell Rep 2018 12;25(12):3315-3328.e6

MRC Harwell Institute, Mammalian Genetics Unit and Mary Lyon Centre, Harwell Campus, Oxfordshire OX11 0RD, UK. Electronic address:

Mutations in genes essential for mitochondrial function have pleiotropic effects. The mechanisms underlying these traits yield insights into metabolic homeostasis and potential therapies. Here we report the characterization of a mouse model harboring a mutation in the tryptophanyl-tRNA synthetase 2 (Wars2) gene, encoding the mitochondrial-localized WARS2 protein. This hypomorphic allele causes progressive tissue-specific pathologies, including hearing loss, reduced adiposity, adipose tissue dysfunction, and hypertrophic cardiomyopathy. We demonstrate the tissue heterogeneity arises as a result of variable activation of the integrated stress response (ISR) pathway and the ability of certain tissues to respond to impaired mitochondrial translation. Many of the systemic metabolic effects are likely mediated through elevated fibroblast growth factor 21 (FGF21) following activation of the ISR in certain tissues. These findings demonstrate the potential pleiotropy associated with Wars2 mutations in patients.
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http://dx.doi.org/10.1016/j.celrep.2018.11.080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315286PMC
December 2018

Embedding co-production and addressing uncertainty in watershed modeling decision-support tools: successes and challenges.

Environ Model Softw 2018 Nov;109:368-379

United States Environmental Protection Agency, National Risk Management Research Laboratory, Water Supply and Water Resources Division, Cincinnati, Ohio, 45268.

Decision-support tools (DSTs) are often produced from collaborations between technical experts and stakeholders to address environmental problems and inform decision making. Studies in the past two decades have provided key insights on the use of DSTs and the importance of bidirectional information flows among technical experts and stakeholders - a process that is variously referred to as co-production, participatory modeling, structured decision making, or simply stakeholder participation. Many of these studies have elicited foundational insights for the broad field of water resources management; however, questions remain on approaches for balancing co-production with uncertainty specifically for watershed modeling decision support tools. In this paper, we outline a simple conceptual model that focuses on the DST development process. Then, using watershed modeling case studies found in the literature, we discuss successful outcomes and challenges associated with embedding various forms of co-production into each stage of the conceptual model. We also emphasize the "3 Cs" (i.e., characterization, calculation, communication) of uncertainty and provide evidence-based suggestions for their incorporation in the watershed modeling DST development process. We conclude by presenting a list of best practices derived from current literature for achieving effective and robust watershed modeling decision-support tools.
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http://dx.doi.org/10.1016/j.envsoft.2018.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260939PMC
November 2018

Mouse mutations cause retinal degeneration and reduced mitochondrial function.

Dis Model Mech 2018 12 18;11(12). Epub 2018 Dec 18.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK

Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, one β and one γ subunit. Loss-of-function and missense mutations in both and have previously been implicated in families exhibiting retinal degeneration. Using mouse models, we investigated the role of IDH3 in retinal disease and mitochondrial function. We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying an ENU-induced mutation, E229K, in Mice homozygous for this mutation exhibit signs of retinal stress, indicated by GFAP staining, as early as 3 months, but no other tissues appear to be affected. We produced a knockout of and found that homozygous mice do not survive past early embryogenesis. compound heterozygous mutants exhibit a more severe retinal degeneration compared with homozygous mutants. Analysis of mitochondrial function in mutant cell lines highlighted a reduction in mitochondrial maximal respiration and reserve capacity levels in both and cells. Loss-of-function mutants do not exhibit the same retinal degeneration phenotype, with no signs of retinal stress or reduction in mitochondrial respiration. It has previously been reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed in mutant mice.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.036426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6307916PMC
December 2018

Exploratory Metamorphic Testing for Scientific Software.

Comput Sci Eng 2018 Nov;22(2):78-87

University of Cincinnati, USA.

Scientific model developers are able to verify and validate their software via metamorphic testing, even when the expected output of a given test case is not readily available. The tenet is to check whether certain relations hold among the expected outputs of multiple related inputs. Contemporary approaches require the relations to be defined before tests. Our experience shows that it is often straightforward to first define the multiple iterations of tests for performing continuous simulations, and then keep multiple and even competing metamorphic relations open for investigating the testing-result patterns. We call this new approach , and report our experience of applying it to detect bugs, mismatches, and constraints in automatically calibrating parameters for the United States Environmental Protection Agency's Storm Water Management Model (SWMM).
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http://dx.doi.org/10.1109/MCSE.2018.2880577DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252536PMC
November 2018

An N-Ethyl-N-Nitrosourea (ENU)-Induced Tyr265Stop Mutation of the DNA Polymerase Accessory Subunit Gamma 2 (Polg2) Is Associated With Renal Calcification in Mice.

J Bone Miner Res 2019 03 14;34(3):497-507. Epub 2018 Dec 14.

Academic Endocrine Unit, Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, UK.

Renal calcification (RCALC) resulting in nephrolithiasis and nephrocalcinosis, which affects ∼10% of adults by 70 years of age, involves environmental and genetic etiologies. Thus, nephrolithiasis and nephrocalcinosis occurs as an inherited disorder in ∼65% of patients, and may be associated with endocrine and metabolic disorders including: primary hyperparathyroidism, hypercalciuria, renal tubular acidosis, cystinuria, and hyperoxaluria. Investigations of families with nephrolithiasis and nephrocalcinosis have identified some causative genes, but further progress is limited as large families are unavailable for genetic studies. We therefore embarked on establishing mouse models for hereditary nephrolithiasis and nephrocalcinosis by performing abdominal X-rays to identify renal opacities in N-ethyl-N-nitrosourea (ENU)-mutagenized mice. This identified a mouse with RCALC inherited as an autosomal dominant trait, designated RCALC type 2 (RCALC2). Genomewide mapping located the Rcalc2 locus to a ∼16-Mbp region on chromosome 11D-E2 and whole-exome sequence analysis identified a heterozygous mutation in the DNA polymerase gamma-2, accessory subunit (Polg2) resulting in a nonsense mutation, Tyr265Stop (Y265X), which co-segregated with RCALC2. Kidneys of mutant mice (Polg2 ) had lower POLG2 mRNA and protein expression, compared to wild-type littermates (Polg2 ). The Polg2 and Polg2 mice had similar plasma concentrations of sodium, potassium, calcium, phosphate, chloride, urea, creatinine, glucose, and alkaline phosphatase activity; and similar urinary fractional excretion of calcium, phosphate, oxalate, and protein. Polg2 encodes the minor subunit of the mitochondrial DNA (mtDNA) polymerase and the mtDNA content in Polg2 kidneys was reduced compared to Polg2 mice, and cDNA expression profiling revealed differential expression of 26 genes involved in several biological processes including mitochondrial DNA function, apoptosis, and ubiquitination, the complement pathway, and inflammatory pathways. In addition, plasma of Polg2 mice, compared to Polg2 littermates had higher levels of reactive oxygen species. Thus, our studies have identified a mutant mouse model for inherited renal calcification associated with a Polg2 nonsense mutation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.
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http://dx.doi.org/10.1002/jbmr.3624DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6446808PMC
March 2019
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