Translational genomics using Arabidopsis as a model enables the characterization of pennycress genes through forward and reverse genetics.

Thlaspi arvense (pennycress) has the potential for domestication as a new oilseed crop. Information from an extensive body of research on the related plant species Arabidopsis can be used to greatly speed this process. Genome-scale comparisons in this paper documented that pennycress and Arabidopsis share similar gene duplication. This finding led to the hypothesis that it should be possible to isolate Arabidopsis-like mutants in pennycress. This proved to be true, as forward genetic screens identified floral and vegetative pennycress mutants that were similar to mutants found in Arabidopsis. Extending this approach, it was shown that most of the pennycress genes responsible for the formation of oxidized tannins could be rapidly identified. The causative mutations in the pennycress mutants could be identified either by PCR amplification of candidate genes or through whole-genome sequencing (WGS) analysis. In all, WGS was used to characterize 95 ethyl methane sulfonate mutants, which revealed a mutation rate of 4.09 mutations per megabase. A sufficient number of non-synonymous mutations were identified to create a mutant gene index that could be used for reverse genetic approaches to identify pennycress mutants of interest. As proof of concept, a Ta-max3-like dwarf mutant and Ta-kcs5/cer60-like wax mutants deficient in the biosynthesis of long chain fatty acids were identified. Overall, these studies demonstrate that translational genomics can be used to promote the domestication of pennycress. Furthermore, the ease with which important findings could be made in pennycress makes this species a new potential model plant.

Fast and Efficient Root Phenotyping via Pose Estimation.

Image segmentation is commonly used to estimate the location and shape of plants and their external structures. Segmentation masks are then used to localize landmarks of interest and compute other geometric features that correspond to the plant's phenotype. Despite its prevalence, segmentation-based approaches are laborious (requiring extensive annotation to train) and error-prone (derived geometric features are sensitive to instance mask integrity). Here, we present a segmentation-free approach that leverages deep learning-based landmark detection and grouping, also known as pose estimation. We use a tool originally developed for animal motion capture called SLEAP (Social LEAP Estimates Animal Poses) to automate the detection of distinct morphological landmarks on plant roots. Using a gel cylinder imaging system across multiple species, we show that our approach can reliably and efficiently recover root system topology at high accuracy, few annotated samples, and faster speed than segmentation-based approaches. In order to make use of this landmark-based representation for root phenotyping, we developed a Python library (sleap-roots) for trait extraction directly comparable to existing segmentation-based analysis software. We show that pose-derived root traits are highly accurate and can be used for common downstream tasks including genotype classification and unsupervised trait mapping. Altogether, this work establishes the validity and advantages of pose estimation-based plant phenotyping. To facilitate adoption of this easy-to-use tool and to encourage further development, we make sleap-roots, all training data, models, and trait extraction code available at: https://github.com/talmolab/sleap-roots and https://osf.io/k7j9g/.

In vivo effects of anacetrapib on preß HDL: improvement in HDL remodeling without effects on cholesterol absorption.

Cholesteryl ester transfer protein (CETP) transfers cholesteryl ester and triglyceride between HDL and apoB-containing lipoproteins. Anacetrapib (ANA), a reversible inhibitor of CETP, raises HDL cholesterol and lowers LDL cholesterol in dyslipidemic patients. We previously demonstrated that ANA increases macrophage-to-feces reverse cholesterol transport and fecal cholesterol excretion in hamsters, and increased preß HDL-dependent cholesterol efflux via ABCA1 in vitro. However, the effects of ANA on in vivo preß HDL have not been characterized. In vitro, ANA inhibited the formation of preß, however in ANA-treated dyslipidemic hamsters, preß HDL levels (measured by two-dimensional gel electrophoresis) were increased, in contrast to in vitro findings. Because changes in plasma preß HDL have been proposed to potentially affect markers of cholesterol absorption with other CETP inhibitors, a dual stable isotope method was used to directly measure cholesterol absorption in hamsters. ANA treatment of hamsters (on either dyslipidemic or normal diet) had no effect on cholesterol absorption, while dalcetrapib-treated hamsters displayed an increase in cholesterol absorption. Taken together, these data support the notion that ANA promotes preß HDL functionality in vivo, with no effects on cholesterol absorption.

NADPH oxidase-dependent and -independent mechanisms of reported inhibitors of reactive oxygen generation.

NADPH oxidase isoform-2 (NOX2) generates reactive oxygen species (ROS) that contribute to neurodegenerative and cardiovascular pathologies. However, validation of NOX2 as a pharmacotherapeutic target has been hampered by a lack of mechanistically-defined inhibitors. Using cellular and biochemical assays, we explored previously reported inhibitors of ROS production (perhexiline, suramin, VAS2870 and two Shionogi patent compounds) as direct NOX2 inhibitors. All but suramin, which presumably lacks cell penetrance, inhibit cellular ROS production. However, only perhexiline and suramin inhibit biochemical NOX2 activity. Indeed, our data suggest that NOX2 inhibition by perhexiline may contribute significantly to its demonstrated cardioprotective effects. Inhibition of protein kinase CßII explains the cellular activity of the Shionogi compounds, whereas VAS2870 inhibits by an as-yet unidentified mechanism unrelated to direct NOX2 function or subunit assembly. These data delineate the mechanisms of action of these compounds and highlight their strengths and limitations for use in future target validation studies.

Fast and efficient root phenotyping via pose estimation.

Image segmentation is commonly used to estimate the location and shape of plants and their external structures. Segmentation masks are then used to localize landmarks of interest and compute other geometric features that correspond to the plant's phenotype. Despite its prevalence, segmentation-based approaches are laborious (requiring extensive annotation to train), and error-prone (derived geometric features are sensitive to instance mask integrity). Here we present a segmentation-free approach which leverages deep learning-based landmark detection and grouping, also known as pose estimation. We use a tool originally developed for animal motion capture called SLEAP (Social LEAP Estimates Animal Poses) to automate the detection of distinct morphological landmarks on plant roots. Using a gel cylinder imaging system across multiple species, we show that our approach can reliably and efficiently recover root system topology at high accuracy, few annotated samples, and faster speed than segmentation-based approaches. In order to make use of this landmark-based representation for root phenotyping, we developed a Python library (sleap-roots) for trait extraction directly comparable to existing segmentation-based analysis software. We show that landmark-derived root traits are highly accurate and can be used for common downstream tasks including genotype classification and unsupervised trait mapping. Altogether, this work establishes the validity and advantages of pose estimation-based plant phenotyping. To facilitate adoption of this easy-to-use tool and to encourage further development, we make sleap-roots, all training data, models, and trait extraction code available at: https://github.com/talmolab/sleap-roots and https://osf.io/k7j9g/.

Mortality rate and prognostic factors for dogs with severe anaphylaxis: 67 cases (2016-2018).

OBJECTIVE: To determine mortality rates for dogs with severe anaphylaxis and identify potential prognostic factors. ANIMALS: 67 dogs with suspected anaphylaxis graded as severe. PROCEDURES: Dogs were classified on the basis of outcome as survivors and nonsurvivors. Medical records were reviewed, and data were extracted including signalment, examination findings, time to hospital admission from onset of clinical signs, CBC results, serum biochemical analysis results, coagulation testing results, and findings on abdominal ultrasonography. Initial treatment within the first 6 hours after hospital admission was recorded for analysis, specifically including the use of epinephrine, diphenhydramine, corticosteroids, antimicrobials, fresh-frozen plasma, and supplemental dextrose. RESULTS: The overall mortality rate was 14.9% (10/67) for dogs with anaphylaxis graded as severe. Serum phosphorus concentration and prothrombin time (PT) were significantly higher in nonsurvivors, compared with survivors. Nonsurvivors had lower presenting body temperatures than survivors. Serum phosphorus concentration ≥ 12.0 mmol/L, hypoglycemia within 6 hours after hospital admission, high PT value, concurrently high PT and partial thromboplastin time (PTT) values > 50% above the reference range limit, and the need for supplemental dextrose were associated with death. The incidences of coagulopathy and peritoneal effusion were unexpectedly high (85.2% and 65.5% of dogs, respectively) but were not indicative of survival. CONCLUSIONS AND CLINICAL RELEVANCE: Despite the poor presenting clinical condition seen in dogs with severe anaphylaxis, the rate of survival with treatment was fairly high. Coagulopathy and the presence of peritoneal effusion were common findings in dogs with severe anaphylaxis. Serum phosphorus concentration ≥ 12.0 mmol/L, high PT value, concurrent increases of PT and PTT values > 50% above reference range limits, hypoglycemia within 6 hours after hospital admission, and the need for supplemental dextrose were associated with death.

Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans.

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult "hidden" in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

Spring flowering habit in field pennycress (Thlaspi arvense) has arisen multiple independent times.

Field pennycress (Thlaspi arvense L.) is currently being developed as a new cold-tolerant oilseed crop. In natural populations, pennycress, like many Brassicaceae relatives, can exhibit either a winter or spring annual phenotype. Pennycress is a diploid relative of Arabidopsis thaliana, a model species that has been used to study many adaptive phenotypes, including flowering time and developmental timing. In Arabidopsis and other Brassicaceae species, mutations in negative regulators of flowering, including FLOWERING LOCUS C and FRIGIDA can cause the transition to a spring annual habit. The genetics underlying the difference between spring and winter annual pennycress lines are currently unknown. Here, we report the identification of four natural alleles of FLC in pennycress that confer a spring annual growth habit identified through whole genome sequencing, cosegregation analyses, and comparative genomics. The global distribution of these spring annual alleles of FLC suggests that the spring annual growth habit has arisen on several independent occasions. The two spring annual FLC alleles present in European accessions were only identified in North American accessions collected in southern Montana, which indicates accessions harboring these two alleles were introduced to North America, likely after pennycress became a widespread species on the continent. These findings provide new information on the natural history of the introduction and spread of spring annual pennycress accessions from Europe into North America. At the molecular level, these findings are important for the ongoing development of pennycress as a winter annual crop. An enhanced understanding of the regulation of flowering in this species should allow for the fine-tuning of flowering in commercial varieties.

Hypercalcemia: Pathophysiology, Clinical Signs, and Emergent Treatment.

Hypercalcemia is uncommonly encountered in veterinary patients. When it does occur, the effects can be severe, resulting in significant morbidity and mortality if not recognized and addressed in a timely manner. Causes of hypercalcemia are varied and include pituitary-dependent and pituitary-independent causes. A diagnosis of hypercalcemia should be made based on documentation of ionized hypercalcemia. The mainstay of emergency treatment usually involves aggressive IV fluid diuresis, the use of diuretics, and, often, glucocorticoids. The use of bisphosphonates has become increasingly more common in veterinary medicine.