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The development of safe crop protection products is a complex process that traditionally relies on intensive animal use for hazard identification. Methods that capture toxicity in early stages of agrochemical discovery programs enable a more efficient and sustainable product development pipeline. Here, we explored whether the zebrafish model can be leveraged to identify mammalian-relevant toxicity. We used transgenic zebrafish to assess developmental toxicity following exposures to known mammalian teratogens and captured larval morphological malformations, including bone and vascular perturbations. We further applied toxicogenomics to identify common biomarker signatures of teratogen exposure. The results show that the larval malformation assay predicted teratogenicity with 82.35% accuracy, 87.50% specificity, and 77.78% sensitivity. Similar and slightly lower accuracies were obtained with the vascular and bone assays, respectively. A set of 20 biomarkers were identified that efficiently segregated teratogenic chemicals from nonteratogens. In conclusion, zebrafish are valuable, robust, and cost-effective models for toxicity testing in the early stages of product development.

Utilization of in vitro (cellular) techniques, like Cell Painting and transcriptomics, could provide powerful tools for agrochemical candidate sorting and selection in the discovery process. However, using these models generates challenges translating in vitro concentrations to the corresponding in vivo exposures. Physiologically based pharmacokinetic (PBPK) modeling provides a framework for quantitative in vitro to in vivo extrapolation (IVIVE). We tested whether in vivo (rat liver) transcriptomic and apical points of departure (PODs) could be accurately predicted from in vitro (rat hepatocyte or human HepaRG) transcriptomic PODs or HepaRG Cell Painting PODs using PBPK modeling. We compared two PBPK models, the ADMET predictor and the httk R package, and found httk to predict the in vivo PODs more accurately. Our findings suggest that a rat liver apical and transcriptomic POD can be estimated utilizing a combination of in vitro transcriptome-based PODs coupled with PBPK modeling for IVIVE. Thus, high content in vitro data can be translated with modest accuracy to in vivo models of ultimate regulatory importance to help select agrochemical analogs in early stage discovery program.

High-quality reference genome assemblies, representative of global heterotic patterns, offer an ideal platform to accurately characterize and utilize genetic variation in the primary gene pool of hybrid crops. Here we report three platinum grade de-novo, near gap-free, chromosome-level reference genome assemblies from the active breeding germplasm in pearl millet with a high degree of contiguity, completeness, and accuracy. An improved Tift genome (Tift23D 2 B 1 -P1-P5) assembly has a contig N50 ~ 7,000-fold (126 Mb) compared to the previous version and better alignment in centromeric regions. Comparative genome analyses of these three lines clearly demonstrate a high level of collinearity and multiple structural variations, including inversions greater than 1 Mb. Differential genes in improved Tift genome are enriched for serine O-acetyltransferase and glycerol-3-phosphate metabolic process which play an important role in improving the nutritional quality of seed protein and disease resistance in plants, respectively. Multiple marker-trait associations are identified for a range of agronomic traits, including grain yield through genome-wide association study. Improved genome assemblies and marker resources developed in this study provide a comprehensive framework/platform for future applications such as marker-assisted selection of mono/oligogenic traits as well as whole-genome prediction and haplotype-based breeding of complex traits., (© 2023. Springer Nature Limited.)

Plate motion on shallow subduction megathrusts is accommodated by a spectrum of tectonic slip modes. However, the frictional properties and conditions that sustain these diverse slip behaviors remain enigmatic. Frictional healing is one such property, which describes the degree of fault restrengthening between earthquakes. We show that the frictional healing rate of materials entrained along the megathrust at the northern Hikurangi margin, which hosts well-characterized recurring shallow slow slip events (SSEs), is nearly zero (<0.0001 per decade). These low healing rates provide a mechanism for the low stress drops (<50 kilopascals) and short recurrence times (1 to 2 years) characteristic of shallow SSEs at Hikurangi and other subduction margins. We suggest that near-zero frictional healing rates, associated with weak phyllosilicates that are common in subduction zones, may promote frequent, small-stress-drop, slow ruptures near the trench.

Earthquakes occur in clusters or sequences that arise from complex triggering mechanisms, but direct measurement of the slow subsurface slip responsible for delayed triggering is rarely possible. We investigate the origins of complexity and its relationship to heterogeneity using an experimental fault with two dominant seismic asperities. The fault is composed of quartz powder, a material common to natural faults, sandwiched between 760 mm long polymer blocks that deform the way 10 meters of rock would behave. We observe periodic repeating earthquakes that transition into aperiodic and complex sequences of fast and slow events. Neighboring earthquakes communicate via migrating slow slip, which resembles creep fronts observed in numerical simulations and on tectonic faults. Utilizing both local stress measurements and numerical simulations, we observe that the speed and strength of creep fronts are highly sensitive to fault stress levels left behind by previous earthquakes, and may serve as on-fault stress meters., (© 2022. The Author(s).)

Traditional developmental toxicity testing practice examines fetal apical endpoints to identify a point of departure (POD) for risk assessment. A potential new testing paradigm involves deriving a POD from a comprehensive analysis of molecular-level change. Here, the rat ketoconazole endocrine-mediated developmental toxicity model was used to test the hypothesis that maternal epigenomic (miRNA) and transcriptomic (mRNA) PODs are similar to fetal apical endpoint PODs. Sprague-Dawley rats were exposed from gestation day (GD) 6-21 to 0, 0.063, 0.2, 0.63, 2, 6.3, 20, or 40 mg/kg/day ketoconazole. Dam systemic, liver, and placenta PODs, along with GD 21 fetal resorption, body weight, and skeletal apical PODs were derived using BMDS software. GD 21 dam liver and placenta miRNA and mRNA PODs were obtained using three methods: a novel individual molecule POD accumulation method, a first mode method, and a gene set method. Dam apical POD values ranged from 2.0 to 38.6 mg/kg/day; the lowest value was for placenta histopathology. Fetal apical POD values were 10.9-20.3 mg/kg/day; the lowest value was for fetal resorption. Dam liver miRNA and mRNA POD values were 0.34-0.69 mg/kg/day, and placenta miRNA and mRNA POD values were 2.53-6.83 mg/kg/day. Epigenomic and transcriptomic POD values were similar across liver and placenta. Deriving a molecular POD from dam liver or placenta was protective of a fetal apical POD. These data support the conclusion that a molecular POD can be used to estimate, or be protective of, a developmental toxicity apical POD., (© 2022 The Authors. Birth Defects Research published by Wiley Periodicals LLC.)

Tectonic faults fail through a spectrum of slip modes, ranging from slow aseismic creep to rapid slip during earthquakes. Understanding the seismic radiation emitted during these slip modes is key for advancing earthquake science and earthquake hazard assessment. In this work, we use laboratory friction experiments instrumented with ultrasonic sensors to document the seismic radiation properties of slow and fast laboratory earthquakes. Stick-slip experiments were conducted at a constant loading rate of 8 μm/s and the normal stress was systematically increased from 7 to 15 MPa. We produced a full spectrum of slip modes by modulating the loading stiffness in tandem with the fault zone normal stress. Acoustic emission data were recorded continuously at 5 MHz. We demonstrate that the full continuum of slip modes radiate measurable high-frequency energy between 100 and 500 kHz, including the slowest events that have peak fault slip rates <100 μm/s. The peak amplitude of the high-frequency time-domain signals scales systematically with fault slip velocity. Stable sliding experiments further support the connection between fault slip rate and high-frequency radiation. Experiments demonstrate that the origin of the high-frequency energy is fundamentally linked to changes in fault slip rate, shear strain, and breaking of contact junctions within the fault gouge. Our results suggest that having measurements close to the fault zone may be key for documenting seismic radiation properties and fully understanding the connection between different slip modes., (© 2022. The Authors.)

Understanding the temporal evolution of foreshocks and their relation to earthquake nucleation is important for earthquake early warning systems, earthquake hazard assessment, and earthquake physics. Laboratory experiments on intact rock and rough fractures have demonstrated that the number and size of acoustic emission (AE) events increase and that the Gutenberg-Richter b -value decreases prior to coseismic failure. However, for lab fault zones of finite width, where shear occurs within gouge, the physical processes that dictate temporal variations in frequency-magnitude ( F / M ) statistics of lab foreshocks are unclear. Here, we report on a series of laboratory experiments to illuminate the physical processes that govern temporal variations in b -value and AE size. We record AE data continuously for hundreds of lab seismic cycles and report F / M statistics. Our foreshock catalogs include cases where F / M data are not exponentially distributed, but we retain the concept of b -value for comparison with other works. We find that b -value decreases as the fault approaches failure, consistent with previous works. We also find that b -value scales inversely with shear velocity and fault slip rate, suggesting that fault slip acceleration during earthquake nucleation could impact foreshock F / M statistics. We propose that fault zone dilation and grain mobilization have a strong influence on foreshock magnitude. Fault dilation at higher shearing rates increases porosity and results in larger foreshocks and smaller b -values. Our observations suggest that lab earthquakes are preceded by a preparatory nucleation phase with systematic variations in AE and fault zone properties., (© 2021. The Authors.)

Machine learning (ML) techniques have become increasingly important in seismology and earthquake science. Lab-based studies have used acoustic emission data to predict time-to-failure and stress state, and in a few cases, the same approach has been used for field data. However, the underlying physical mechanisms that allow lab earthquake prediction and seismic forecasting remain poorly resolved. Here, we address this knowledge gap by coupling active-source seismic data, which probe asperity-scale processes, with ML methods. We show that elastic waves passing through the lab fault zone contain information that can predict the full spectrum of labquakes from slow slip instabilities to highly aperiodic events. The ML methods utilize systematic changes in P-wave amplitude and velocity to accurately predict the timing and shear stress during labquakes. The ML predictions improve in accuracy closer to fault failure, demonstrating that the predictive power of the ultrasonic signals improves as the fault approaches failure. Our results demonstrate that the relationship between the ultrasonic parameters and fault slip rate, and in turn, the systematically evolving real area of contact and asperity stiffness allow the gradient boosting algorithm to "learn" about the state of the fault and its proximity to failure. Broadly, our results demonstrate the utility of physics-informed ML in forecasting the imminence of fault slip at the laboratory scale, which may have important implications for earthquake mechanics in nature., (© 2021. The Authors.)

Machine learning can predict the timing and magnitude of laboratory earthquakes using statistics of acoustic emissions. The evolution of acoustic energy is critical for lab earthquake prediction; however, the connections between acoustic energy and fault zone processes leading to failure are poorly understood. Here, we document in detail the temporal evolution of acoustic energy during the laboratory seismic cycle. We report on friction experiments for a range of shearing velocities, normal stresses, and granular particle sizes. Acoustic emission data are recorded continuously throughout shear using broadband piezo-ceramic sensors. The coseismic acoustic energy release scales directly with stress drop and is consistent with concepts of frictional contact mechanics and time-dependent fault healing. Experiments conducted with larger grains (10.5 μm) show that the temporal evolution of acoustic energy scales directly with fault slip rate. In particular, the acoustic energy is low when the fault is locked and increases to a maximum during coseismic failure. Data from traditional slide-hold-slide friction tests confirm that acoustic energy release is closely linked to fault slip rate. Furthermore, variations in the true contact area of fault zone particles play a key role in the generation of acoustic energy. Our data show that acoustic radiation is related primarily to breaking/sliding of frictional contact junctions, which suggests that machine learning-based laboratory earthquake prediction derives from frictional weakening processes that begin very early in the seismic cycle and well before macroscopic failure., (©2020. The Authors.)

The derivation of an apical endpoint point of departure (POD) from animal-intensive testing programs has been the traditional cornerstone of human health risk assessment. Replacement of in vivo chronic studies with novel approaches, such as toxicogenomics, holds promise for future alternative testing paradigms that significantly reduce animal testing. We hypothesized that a toxicogenomic POD following a 14 day exposure in the rat would approximate the most sensitive apical endpoint POD derived from a battery of chronic, carcinogenicity, reproduction and endocrine guideline toxicity studies. To test this hypothesis, we utilized myclobutanil, a triazole fungicide, as a model compound. In the 14 day study, male rats were administered 0 (vehicle), 30, 150, or 400 mg/kg/day myclobutanil via oral gavage. Endpoints evaluated included traditional apical, hormone, and liver and testis transcriptomic (whole genome RNA sequencing) data. From the transcriptomic data, liver and testis biological effect POD (BEPOD) values were derived. Myclobutanil exposure for 14 days resulted in increased liver weight, altered serum hormones, liver histopathology, and differential gene expression in liver and testis. The liver and testis BEPODs from the short-term study were 22.2 and 25.4 mg/kg/day, respectively. These BEPODs were approximately an order of magnitude higher than the most sensitive apical POD identified from the two year cancer bioassay based on testis atrophy (1.4 mg/kg/day). This study demonstrates the promise of using a short-term study BEPOD to derive a POD for human health risk assessment while substantially reducing animal testing., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Recent advances in genome engineering technologies based on designed endonucleases (DE) allow specific and predictable alterations in plant genomes to generate value-added traits in crops of choice. The EXZACT Precision technology, based on zinc finger nucleases (ZFN), has been successfully used in the past for introduction of precise mutations and transgenes to generate novel and desired phenotypes in several crop species. Current methods for delivering ZFNs into plant cells are based on traditional genetic transformation methods that result in stable integration of the nuclease in the genome. Here, we describe for the first time, an alternative ZFN delivery method where plant cells are transfected with ZFN protein that eliminates the need for stable nuclease genomic integration and allows generation of edited, but not transgenic cells or tissues. For this study, we designed ZFNs targeting the wheat IPK1 locus, purified active ZFN protein from bacterial cultures, complexed with cell-penetrating peptides (CPP) and directly transfected the complex into either wheat microspores or embryos. NGS analysis of ZFN-treated material showed targeted edits at the IPK1 locus in independent experiments. This is the first description of plant microspore genome editing by a ZFN when delivered as a protein complexed with CPP., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Fetal rat exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) reduces epididymal sperm number involving altered pituitary-testicular hormonal signaling as the proposed mode-of-action (MOA). To evaluate this MOA and compare TCDD to 2,3,7,8-tetrachlorodibenzofuran (TCDF), an in utero rat exposure and study was conducted. Endpoints included congener tissue levels and transcriptomes of maternal liver and fetal liver, testis, and pituitary. Decreased gonadotropin subunit mRNAs levels (Lhb and Fshb) and enriched signaling pathways including GNRH Signaling and Calcium Signaling were observed in fetal pituitary after TCDD (but not TCDF) exposure. TCDD (but not TCDF) decreased fetal testis cholesterologenic and steroidogenic pathway genes. TCDD tissue concentrations in dam liver, dam adipose, and whole fetus were approximately 3- to 6-fold higher than TCDF. These results support a MOA for dioxin-induced rat male reproductive toxicity involving key events in both the fetal pituitary (e.g., reduced gonadotropin production) and fetal testis (e.g., reduced Leydig cell cholesterologenesis and steroidogenesis)., Competing Interests: Declaration of Competing Interest The authors acknowledge prior or current employment by The Dow Chemical Company and that The Dow Chemical Company is currently engaged in dioxin remediation activities., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Slow slip events (SSEs) accommodate a significant proportion of tectonic plate motion at subduction zones, yet little is known about the faults that actually host them. The shallow depth (<2 km) of well-documented SSEs at the Hikurangi subduction zone offshore New Zealand offers a unique opportunity to link geophysical imaging of the subduction zone with direct access to incoming material that represents the megathrust fault rocks hosting slow slip. Two recent International Ocean Discovery Program Expeditions sampled this incoming material before it is entrained immediately down-dip along the shallow plate interface. Drilling results, tied to regional seismic reflection images, reveal heterogeneous lithologies with highly variable physical properties entering the SSE source region. These observations suggest that SSEs and associated slow earthquake phenomena are promoted by lithological, mechanical, and frictional heterogeneity within the fault zone, enhanced by geometric complexity associated with subduction of rough crust., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Oilseed crops, especially soybean (Glycine max) and canola/rapeseed (Brassica napus), produce seeds that are rich in both proteins and oils and that are major sources of energy and nutrition worldwide. Most of the nutritional content in the seed is accumulated in the embryo during the seed filling stages of seed development. Understanding the metabolic pathways that are active during seed filling and how they are regulated are essential prerequisites to crop improvement. In this review, we summarize various omics studies of soybean and canola/rapeseed during seed filling, with emphasis on oil and protein traits, to gain a systems-level understanding of seed development. Currently, most (80-85%) of the soybean and rapeseed reference genomes have been sequenced (950 and 850 megabases, respectively). Parallel to these efforts, extensive omics datasets from different seed filling stages have become available. Transcriptome and proteome studies have detected preponderance of starch metabolism and glycolysis enzymes to be the possible cause of higher oil in B. napus compared to other crops. Small RNAome studies performed during the seed filling stages have revealed miRNA-mediated regulation of transcription factors, with the suggestion that this interaction could be responsible for transitioning the seeds from embryogenesis to maturation. In addition, progress made in dissecting the regulation of de novo fatty acid synthesis and protein storage pathways is described. Advances in high-throughput omics and comprehensive tissue-specific analyses make this an exciting time to attempt knowledge-driven investigation of complex regulatory pathways.

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

We developed PolyA-seq, a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts, and used it to globally map polyadenylation (polyA) sites in 24 matched tissues in human, rhesus, dog, mouse, and rat. We show that PolyA-seq is as accurate as existing RNA sequencing (RNA-seq) approaches for digital gene expression (DGE), enabling simultaneous mapping of polyA sites and quantitative measurement of their usage. In human, we confirmed 158,533 known sites and discovered 280,857 novel sites (FDR < 2.5%). On average 10% of novel human sites were also detected in matched tissues in other species. Most novel sites represent uncharacterized alternative polyA events and extensions of known transcripts in human and mouse, but primarily delineate novel transcripts in the other three species. A total of 69.1% of known human genes that we detected have multiple polyA sites in their 3'UTRs, with 49.3% having three or more. We also detected polyadenylation of noncoding and antisense transcripts, including constitutive and tissue-specific primary microRNAs. The canonical polyA signal was strongly enriched and positionally conserved in all species. In general, usage of polyA sites is more similar within the same tissues across different species than within a species. These quantitative maps of polyA usage in evolutionarily and functionally related samples constitute a resource for understanding the regulatory mechanisms underlying alternative polyadenylation.

The efficacy of supravital staining in the detection of malignancies in oro and oropharyngeal lesions and its role in the detection of malignant changes in premalignant lesions were studied. This prospective study comprises 90 cases of clinically suspicious lesions and it was done over a period of 3 years. Most of the patients had multiple risk factors for the development of malignancy. All underwent staining with a modified solution of 1% toluidine blue (TB). In our study the overall sensitivity was 97.29% and the specificity was 62.5%.