The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.

Cleft palate and minor metabolic disturbances in a mouse global Arl15 gene knockout.

ARL15, a small GTPase protein, was linked to metabolic traits in association studies. We aimed to test the Arl15 gene as a functional candidate for metabolic traits in the mouse. CRISPR/Cas9 germline knockout (KO) of Arl15 showed that homozygotes were postnatal lethal and exhibited a complete cleft palate (CP). Also, decreased cell migration was observed from Arl15 KO mouse embryonic fibroblasts (MEFs). Metabolic phenotyping of heterozygotes showed that females had reduced fat mass on a chow diet from 14 weeks of age. Mild body composition phenotypes were also observed in heterozygous mice on a high-fat diet (HFD)/low-fat diet (LFD). Females on a HFD showed reduced body weight, gonadal fat depot weight and brown adipose tissue (BAT) weight. In contrast, in the LFD group, females showed increased bone mineral density (BMD), while males showed a trend toward reduced BMD. Clinical biochemistry analysis of plasma on HFD showed transient lower adiponectin at 20 weeks of age in females. Urinary and plasma Mg2+ concentrations were not significantly different. Our phenotyping data showed that Arl15 is essential for craniofacial development. Adult metabolic phenotyping revealed potential roles in brown adipose tissue and bone development.

Genic constraint against nonsynonymous variation across the mouse genome.

BACKGROUND: Selective constraint, the depletion of variation due to negative selection, provides insights into the functional impact of variants and disease mechanisms. However, its characterization in mice, the most commonly used mammalian model, remains limited. This study aims to quantify mouse gene constraint using a new metric called the nonsynonymous observed expected ratio (NOER) and investigate its relationship with gene function. RESULTS: NOER was calculated using whole-genome sequencing data from wild mouse populations (Mus musculus sp and Mus spretus). Positive correlations were observed between mouse gene constraint and the number of associated knockout phenotypes, indicating stronger constraint on pleiotropic genes. Furthermore, mouse gene constraint showed a positive correlation with the number of pathogenic variant sites in their human orthologues, supporting the relevance of mouse models in studying human disease variants. CONCLUSIONS: NOER provides a resource for assessing the fitness consequences of genetic variants in mouse genes and understanding the relationship between gene constraint and function. The study's findings highlight the importance of pleiotropy in selective constraint and support the utility of mouse models in investigating human disease variants. Further research with larger sample sizes can refine constraint estimates in mice and enable more comprehensive comparisons of constraint between mouse and human orthologues.

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

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.

Mouse genomic and cellular annotations.

Mice have emerged as one of the most popular and valuable model organisms in the research of human biology. This is due to their genetic and physiological similarity to humans, short generation times, availability of genetically homologous inbred strains, and relatively easy laboratory maintenance. Therefore, following the release of the initial human reference genome, the generation of the mouse reference genome was prioritised and represented an important scientific resource for the mouse genetics community. In 2002, the Mouse Genome Sequencing Consortium published an initial draft of the mouse reference genome which contained ~ 96% of the euchromatic genome of female C57BL/6 J mice. Almost two decades on from the publication of the initial draft, sequencing efforts have continued to increase the completeness and accuracy of the C57BL/6 J reference genome alongside advances in genome annotation. Additionally new sequencing technologies have provided a wealth of data that has added to the repertoire of annotations associated with traditional genomic annotations. Including but not limited to advances in regulatory elements, the 3D genome and individual cellular states. In this review we focus on the reference genome C57BL/6 J and summarise the different aspects of genomic and cellular annotations, as well as their relevance to mouse genetic research. We denote a genomic annotation as a functional unit of the genome. Cellular annotations are annotations of cell type or state, defined by the transcriptomic expression profile of a cell. Due to the wide-ranging number and diversity of annotations describing the mouse genome, we focus on gene, repeat and regulatory element annotation as well as two relatively new technologies; 3D genome architecture and single-cell sequencing outlining their utility in genetic research and their current challenges.

Modelling the genetic aetiology of complex disease: human-mouse conservation of noncoding features and disease-associated loci.

Understanding the genetic aetiology of loci associated with a disease is crucial for developing preventative measures and effective treatments. Mouse models are used extensively to understand human pathobiology and mechanistic functions of disease-associated loci. However, the utility of mouse models is limited in part by evolutionary divergence in transcription regulation for pathways of interest. Here, we summarize the alignment of genomic (exonic and multi-cell regulatory) annotations alongside Mendelian and complex disease-associated variant sites between humans and mice. Our results highlight the importance of understanding evolutionary divergence in transcription regulation when interpreting functional studies using mice as models for human disease variants.

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

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.

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

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.

A genetic modifier suggests that endurance exercise exacerbates Huntington's disease.

Polyglutamine expansions in the huntingtin gene cause Huntington's disease (HD). Huntingtin is ubiquitously expressed, leading to pathological alterations also in peripheral organs. Variations in the length of the polyglutamine tract explain up to 70% of the age-at-onset variance, with the rest of the variance attributed to genetic and environmental modifiers. To identify novel disease modifiers, we performed an unbiased mutagenesis screen on an HD mouse model, identifying a mutation in the skeletal muscle voltage-gated sodium channel (Scn4a, termed 'draggen' mutation) as a novel disease enhancer. Double mutant mice (HD; Scn4aDgn/+) had decreased survival, weight loss and muscle atrophy. Expression patterns show that the main tissue affected is skeletal muscle. Intriguingly, muscles from HD; Scn4aDgn/+ mice showed adaptive changes similar to those found in endurance exercise, including AMPK activation, fibre type switching and upregulation of mitochondrial biogenesis. Therefore, we evaluated the effects of endurance training on HD mice. Crucially, this training regime also led to detrimental effects on HD mice. Overall, these results reveal a novel role for skeletal muscle in modulating systemic HD pathogenesis, suggesting that some forms of physical exercise could be deleterious in neurodegeneration.

A mutation in Nischarin causes otitis media via LIMK1 and NF-κB pathways.

Otitis media (OM), inflammation of the middle ear (ME), is a common cause of conductive hearing impairment. Despite the importance of the disease, the aetiology of chronic and recurrent forms of middle ear inflammatory disease remains poorly understood. Studies of the human population suggest that there is a significant genetic component predisposing to the development of chronic OM, although the underlying genes are largely unknown. Using N-ethyl-N-nitrosourea mutagenesis we identified a recessive mouse mutant, edison, that spontaneously develops a conductive hearing loss due to chronic OM. The causal mutation was identified as a missense change, L972P, in the Nischarin (NISCH) gene. edison mice develop a serous or granulocytic effusion, increasingly macrophage and neutrophil rich with age, along with a thickened, inflamed mucoperiosteum. We also identified a second hypomorphic allele, V33A, with only modest increases in auditory thresholds and reduced incidence of OM. NISCH interacts with several proteins, including ITGA5 that is thought to have a role in modulating VEGF-induced angiogenesis and vascularization. We identified a significant genetic interaction between Nisch and Itga5; mice heterozygous for Itga5-null and homozygous for edison mutations display a significantly increased penetrance and severity of chronic OM. In order to understand the pathological mechanisms underlying the OM phenotype, we studied interacting partners to NISCH along with downstream signalling molecules in the middle ear epithelia of edison mouse. Our analysis implicates PAK1 and RAC1, and downstream signalling in LIMK1 and NF-κB pathways in the development of chronic OM.

Discovering Metamorphic Relations for Scientific Software From User Forums.

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.

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

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.

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

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.

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

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.

Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants.

BACKGROUND: Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing have led to the use of long single-stranded DNA (lssDNA) molecules for generating conditional mutations. However, there is still limited available data on the efficiency and reliability of this method. RESULTS: We generated conditional mouse alleles using lssDNA donor templates and performed extensive characterization of the resulting mutations. We observed that the use of lssDNA molecules as donors efficiently yielded founders bearing the conditional allele, with seven out of nine projects giving rise to modified alleles. However, rearranged alleles including nucleotide changes, indels, local rearrangements and additional integrations were also frequently generated by this method. Specifically, we found that alleles containing unexpected point mutations were found in three of the nine projects analyzed. Alleles originating from illegitimate repairs or partial integration of the donor were detected in eight projects. Furthermore, additional integrations of donor molecules were identified in four out of the seven projects analyzed by copy counting. This highlighted the requirement for a thorough allele validation by polymerase chain reaction, sequencing and copy counting of the mice generated through this method. We also demonstrated the feasibility of using lssDNA donors to generate thus far problematic point mutations distant from active CRISPR cutting sites by targeting two distinct genes (Gckr and Rims1). We propose a strategy to perform extensive quality control and validation of both types of mouse models generated using lssDNA donors. CONCLUSION: lssDNA donors reproducibly generate conditional alleles and can be used to introduce point mutations away from CRISPR/Cas9 cutting sites in mice. However, our work demonstrates that thorough quality control of new models is essential prior to reliably experimenting with mice generated by this method. These advances in genome editing techniques shift the challenge of mutagenesis from generation to the validation of new mutant models.

The digital revolution in phenotyping.

Phenotypes have gained increased notoriety in the clinical and biological domain owing to their application in numerous areas such as the discovery of disease genes and drug targets, phylogenetics and pharmacogenomics. Phenotypes, defined as observable characteristics of organisms, can be seen as one of the bridges that lead to a translation of experimental findings into clinical applications and thereby support 'bench to bedside' efforts. However, to build this translational bridge, a common and universal understanding of phenotypes is required that goes beyond domain-specific definitions. To achieve this ambitious goal, a digital revolution is ongoing that enables the encoding of data in computer-readable formats and the data storage in specialized repositories, ready for integration, enabling translational research. While phenome research is an ongoing endeavor, the true potential hidden in the currently available data still needs to be unlocked, offering exciting opportunities for the forthcoming years. Here, we provide insights into the state-of-the-art in digital phenotyping, by means of representing, acquiring and analyzing phenotype data. In addition, we provide visions of this field for future research work that could enable better applications of phenotype data.

The International Mouse Phenotyping Consortium (IMPC): a functional catalogue of the mammalian genome that informs conservation.

The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied.

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

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.

Exploratory Metamorphic Testing for Scientific Software.

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 exploratory metamorphic testing, 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).

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity.

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.