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Open access publications by University of Delaware faculty, staff, postdocs, and graduate students at the Delaware Biotechnology Institute.
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Item A new approach for annotation of transposable elements using small RNA mapping(Oxford University Press, 2015-03-15) El Baidouri, Moaine; Do Kim, Kyung; Abernathy, Brian; Arikit, Siwaret; Maumus, Florian; Panaud, Olivier; Meyers, Blake C.; Jackson, Scott A.; Moaine El Baidouri, Kyung Do Kim, Brian Abernathy, Siwaret Arikit, Florian Maumus, Olivier Panaud, Blake C. Meyers and Scott A. Jackson; Arikit, Siwaret; Meyers, Blake C. (orcid.org/0000-0003-3436-6097)Transposable elements (TEs) are mobile genomic DNA sequences found in most organisms. They so densely populate the genomes of many eukaryotic species that they are often the major constituents. With the rapid generation of many plant genome sequencing projects over the past few decades, there is an urgent need for improved TE annotation as a prerequisite for genome-wide studies. Analogous to the use of RNA-seq for gene annotation, we propose a new method for de novo TE annotation that uses as a guide 24 nt-siRNAs that are a part of TE silencing pathways. We use this new approach, called TASR (for Transposon Annotation using Small RNAs), for de novo annotation of TEs in Arabidopsis, rice and soybean and demonstrate that this strategy can be successfully applied for de novo TE annotation in plants.Item Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana(Oxford University Press, 2015-04-06) Merret, R´emy; Nagarajan, Vinay K.; Carpentier, Marie-Christine; Park, Sunhee; Favory, Jean-Jacques; Descombin, Julie; Picart, Claire; Charng, Yee-yung; Green, Pamela J.; Deragon, Jean-Marc; Bousquet-Antonelli, C´ ecile; R´emy Merret, Vinay K. Nagarajan, Marie-Christine Carpentier, Sunhee Park, Jean-Jacques Favory, Julie Descombin, Claire Picart, Yee-yung Charng, Pamela J. Green, Jean-Marc Deragon and C´ ecile Bousquet-Antonelli; Nagarajan, Vinay K.; Park, Sunhee; Green, Pamela J.The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5′-ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5′-ribosome pausing leading to the XRN4-mediated 5′-directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20°C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘non-aberrant’ mRNAs.Item Heat-induced ribosome pausing triggers mRNA co-translational decay in Arabidopsis thaliana(Oxford University Press, 2015-04-06) Merret, R´emy; Nagarajan, Vinay K.; Carpentier, Marie-Christine; Park, Sunhee; Favory, Jean-Jacques; Descombin, Julie; Picart, Claire; Charng, Yee-yung; Green, Pamela J.; Deragon, Jean-Marc; Bousquet-Antonelli, C´ ecile; R´emy Merret, Vinay K. Nagarajan, Marie-Christine Carpentier, Sunhee Park, Jean-Jacques Favory, Julie Descombin, Claire Picart, Yee-yung Charng, Pamela J. Green, Jean-Marc Deragon and C´ ecile Bousquet-Antonelli; Nagarajan, Vinay K.; Park, Sunhee; Green, Pamela J.The reprogramming of gene expression in heat stress is a key determinant to organism survival. Gene expression is downregulated through translation initiation inhibition and release of free mRNPs that are rapidly degraded or stored. In mammals, heat also triggers 5 -ribosome pausing preferentially on transcripts coding for HSC/HSP70 chaperone targets, but the impact of such phenomenon on mRNA fate remains unknown. Here, we provide evidence that, in Arabidopsis thaliana, heat provokes 5 -ribosome pausing leading to the XRN4-mediated 5 -directed decay of translating mRNAs. We also show that hindering HSC/HSP70 activity at 20◦C recapitulates heat effects by inducing ribosome pausing and co-translational mRNA turnover. Strikingly, co-translational decay targets encode proteins with high HSC/HSP70 binding scores and hydrophobic N-termini, two characteristics that were previously observed for transcripts most prone to pausing in animals. This work suggests for the first time that stress-induced variation of translation elongation rate is an evolutionarily conserved process leading to the polysomal degradation of thousands of ‘nonaberrant’ mRNAs.Item Peptide hydrogels – versatile matrices for 3D cell culture in cancer medicine(Frontiers Media S.A., 2015-04-20) Worthington, Peter; Pochan, Darrin J.; Langhans, Sigrid A.; PeterWorthington, Darrin J. Pochan and Sigrid A. Langhans; Worthington, Peter; Pochan, Darrin J.Traditional two-dimensional (2D) cell culture systems have contributed tremendously to our understanding of cancer biology but have significant limitations in mimicking in vivo conditions such as the tumor microenvironment. In vitro, three-dimensional (3D) cell culture models represent a more accurate, intermediate platform between simplified 2D culture models and complex and expensive in vivo models. 3D in vitro models can overcome 2D in vitro limitations caused by the oversupply of nutrients, and unphysiological cell–cell and cell–material interactions, and allow for dynamic interactions between cells, stroma, and extracellular matrix. In addition, 3D cultures allowfor the development of concentration gradients, including oxygen, metabolites, and growth factors, with chemical gradients playing an integral role in many cellular functions ranging from development to signaling in normal epithelia and cancer environments in vivo. Currently, the most common matrices used for 3D culture are biologically derived materials such as matrigel and collagen. However, in recent years, more defined, synthetic materials have become available as scaffolds for 3D culture with the advantage of forming well-defined, designed, tunable materials to control matrix charge, stiffness, porosity, nanostructure, degradability, and adhesion properties, in addition to other material and biological properties. One important area of synthetic materials currently available for 3D cell culture is short sequence, self-assembling peptide hydrogels. In addition to the review of recent work toward the control of material, structure, and mechanical properties, we will also discuss the biochemical functionalization of peptide hydrogels and how this functionalization, coupled with desired hydrogel material characteristics, affects tumor cell behavior in 3D culture.Item Complex and extensive post-transcriptional regulation revealed by integrative proteomic and transcriptomic analysis of metabolite stress response in Clostridium acetobutylicum(BioMed Central Ltd., 2015-06-10) Venkataramanan, Keerthi P.; Min, Lie; Hou, Shuyu; Jones, Shawn W.; Ralston, Matthew T.; Lee, Kelvin H.; Papoutsakis, E. Terry; Keerthi P. Venkataramanan, Lie Min, Shuyu Hou, Shawn W. Jones, Matthew T. Ralston, Kelvin H. Lee and E. Terry Papoutsakis; Venkataramanan, Keerthi P.; Min, Lie; Hou, Shuyu; Ralston, Matthew T.; Lee, Kelvin H.; Papoutsakis, E. TerryBACKGROUND: Clostridium acetobutylicum is a model organism for both clostridial biology and solvent production. The organism is exposed to its own toxic metabolites butyrate and butanol, which trigger an adaptive stress response. Integrative analysis of proteomic and RNAseq data may provide novel insights into post-transcriptional regulation. RESULTS: The identified iTRAQ-based quantitative stress proteome is made up of 616 proteins with a 15 % genome coverage. The differentially expressed proteome correlated poorly with the corresponding differential RNAseq transcriptome. Up to 31 % of the differentially expressed proteins under stress displayed patterns opposite to those of the transcriptome, thus suggesting significant post-transcriptional regulation. The differential proteome of the translation machinery suggests that cells employ a different subset of ribosomal proteins under stress. Several highly upregulated proteins but with low mRNA levels possessed mRNAs with long 5'UTRs and strong RBS scores, thus supporting the argument that regulatory elements on the long 5'UTRs control their translation. For example, the oxidative stress response rubrerythrin was upregulated only at the protein level up to 40-fold without significant mRNA changes. We also identified many leaderless transcripts, several displaying different transcriptional start sites, thus suggesting mRNA-trimming mechanisms under stress. Downregulation of Rho and partner proteins pointed to changes in transcriptional elongation and termination under stress. CONCLUSIONS: The integrative proteomic-transcriptomic analysis demonstrated complex expression patterns of a large fraction of the proteome. Such patterns could not have been detected with one or the other omic analyses. Our analysis proposes the involvement of specific molecular mechanisms of post-transcriptional regulation to explain the observed complex stress responseItem Quantification of Interfibrillar Shear Stress in Aligned Soft Collagenous Tissues via Notch Tension Testing(Nature Publishing Group, 2015-10-15) Szczesny, Spencer E.; Caplan, Jeffrey L.; Pedersen, Pal; Elliott, Dawn M.; Spencer E. Szczesny, Jeffrey L. Caplan, Pal Pedersen & Dawn M. Elliott; Caplan, Jeffrey L.; Elliott, Dawn M.The mechanical function of soft collagenous tissues is largely determined by their hierarchical organization of collagen molecules. While collagen fibrils are believed to be discontinuous and transfer load through shearing of the interfibrillar matrix, interfibrillar shear stresses have never been quantified. Scaling traditional shear testing procedures down to the fibrillar length scale is impractical and would introduce substantial artifacts. Here, through the use of a novel microscopic variation of notch tension testing, we explicitly demonstrate the existence of interfibrillar shear stresses within tendon fascicles and provide the first measurement of their magnitude. Axial stress gradients along the sample length generated by notch tension testing were measured and used to calculate a value of 32 kPa for the interfibrillar shear stress. This estimate is comparable to the interfibrillar shear stress predicted by previous multiscale modeling of tendon fascicles, which supports the hypothesis that fibrils are discontinuous and transmit load through interfibrillar shear. This information regarding the structure-function relationships of tendon and other soft collagenous tissues is necessary to identify potential causes for tissue impairment with degeneration and provide the foundation for developing regenerative repair strategies or engineering biomaterials for tissue replacement.Item Mechanisms of extracellular S0 globule production and degradation in Chlorobaculum tepidum via dynamic cell–globule interactions(Microbiology Society, 2016-01-07) Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.; ; Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.The Chlorobiales are anoxygenic phototrophs that produce solid, extracellular elemental sulfur globules as an intermediate step in the oxidation of sulfide to sulfate. These organisms must export sulfur while preventing cell encrustation during S0 globule formation; during globule degradation they must find and mobilize the sulfur for intracellular oxidation to sulfate. To understand how the Chlorobiales address these challenges, we characterized the spatial relationships and physical dynamics of Chlorobaculum tepidum cells and S0 globules by light and electron microscopy. Cba. tepidum commonly formed globules at a distance from cells. Soluble polysulfides detected during globule production may allow for remote nucleation of globules. Polysulfides were also detected during globule degradation, probably produced as an intermediate of sulfur oxidation by attached cells. Polysulfides could feed unattached cells, which made up over 80% of the population and had comparable growth rates to attached cells. Given that S0 is formed remotely from cells, there is a question as to how cells are able to move toward S0 in order to attach. Time-lapse microscopy shows that Cba. tepidum is in fact capable of twitching motility, a finding supported by the presence of genes encoding type IV pili. Our results show how Cba. tepidum is able to avoid mineral encrustation and benefit from globule degradation even when not attached. In the environment, Cba. tepidum may also benefit from soluble sulfur species produced by other sulfur-oxidizing or sulfur-reducing bacteria as these organisms interact with its biogenic S0 globules.Item Mechanisms of extracellular S0 globule production and degradation in Chlorobaculum tepidum via dynamic cell–globule interactions(Microbiology Society, 2016-01-07) Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.; C. L. Marnocha, A. T. Levy, D. H. Powell, T. E. Hanson and C. S. Chan; Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.The Chlorobiales are anoxygenic phototrophs that produce solid, extracellular elemental sulfur globules as an intermediate step in the oxidation of sulfide to sulfate. These organisms must export sulfur while preventing cell encrustation during S0 globule formation; during globule degradation they must find and mobilize the sulfur for intracellular oxidation to sulfate. To understand how the Chlorobiales address these challenges, we characterized the spatial relationships and physical dynamics of Chlorobaculum tepidum cells and S0 globules by light and electron microscopy. Cba. tepidum commonly formed globules at a distance from cells. Soluble polysulfides detected during globule production may allow for remote nucleation of globules. Polysulfides were also detected during globule degradation, probably produced as an intermediate of sulfur oxidation by attached cells. Polysulfides could feed unattached cells, which made up over 80% of the population and had comparable growth rates to attached cells. Given that S0 is formed remotely from cells, there is a question as to how cells are able to move toward S0 in order to attach. Time-lapse microscopy shows that Cba. tepidum is in fact capable of twitching motility, a finding supported by the presence of genes encoding type IV pili. Our results show how Cba. tepidum is able to avoid mineral encrustation and benefit from globule degradation even when not attached. In the environment, Cba. tepidum may also benefit from soluble sulfur species produced by other sulfur-oxidizing or sulfur-reducing bacteria as these organisms interact with its biogenic S0 globules.Item Genome assembly with in vitro proximity ligation data and whole-genome triplication in lettuce(Nature Publishing Group, 2017-04-12) Reyes-Chin-Wo, Sebastian; Wang, Zhiwen; Yang, Xinhua; Kozik, Alexander; Arikit, Siwaret; Song, Chi; Xia, Liangfeng; Froenicke, Lutz; Lavelle, Dean O.; Truco, Marı´a-Jose´; Xia, Rui; Zhu, Shilin; Xu, Chunyan; Xu, Huaqin; Xu, Xun; Cox, Kyle; Korf, Ian; Meyers, Blake C.; Michelmore, Richard W.; Sebastian Reyes-Chin-Wo, Zhiwen Wang, Xinhua Yang, Alexander Kozik, Siwaret Arikit, Chi Song, Liangfeng Xia, Lutz Froenicke, Dean O. Lavelle, Marı´a-Jose´ Truco, Rui Xia, Shilin Zhu, Chunyan Xu, Huaqin Xu, Xun Xu, Kyle Cox, Ian Korf, Blake C. Meyers & Richard W. Michelmore; Arikit, Siwaret; Meyers, Blake CLettuce (Lactuca sativa) is a major crop and a member of the large, highly successful Compositae family of flowering plants. Here we present a reference assembly for the species and family. This was generated using whole-genome shotgun Illumina reads plus in vitro proximity ligation data to create large superscaffolds; it was validated genetically and superscaffolds were oriented in genetic bins ordered along nine chromosomal pseudomolecules. We identify several genomic features that may have contributed to the success of the family, including genes encoding Cycloidea-like transcription factors, kinases, enzymes involved in rubber biosynthesis and disease resistance proteins that are expanded in the genome. We characterize 21 novel microRNAs, one of which may trigger phasiRNAs from numerous kinase transcripts. We provide evidence for a whole-genome triplication event specific but basal to the Compositae. We detect 26% of the genome in triplicated regions containing 30% of all genes that are enriched for regulatory sequences and depleted for genes involved in defence.Item Nanocrystalline protein domains via salting-out(Acta Crystallographica Section F: Structural Biology Communications, 2021-11-02) Greene, D. G.; Modla, S.; Sandler, S. I.; Wagner, N. J.; Lenhoff, A. M.Protein salting-out is a well established phenomenon that in many cases leads to amorphous structures and protein gels, which are usually not considered to be useful for protein structure determination. Here, microstructural measurements of several different salted-out protein dense phases are reported, including of lysozyme, ribonuclease A and an IgG1, showing that salted-out protein gels unexpectedly contain highly ordered protein nanostructures that assemble hierarchically to create the gel. The nanocrystalline domains are approximately 10–100 nm in size, are shown to have structures commensurate with those of bulk crystals and grow on time scales in the order of an hour to a day. Beyond revealing the rich, hierarchical nanoscale to mesoscale structure of protein gels, the nanocrystals that these phases contain are candidates for structural biology on next-generation X-ray free-electron lasers, which may enable the study of biological macromolecules that are difficult or impossible to crystallize in bulk.Item Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton(FASEB Journal, 2022-02-23) Ghosh, Arit; Coffin, Megan; West, Richard; Fowler, Velia M.Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3’s function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,β1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.Item Maize brace root mechanics vary by whorl, genotype, and reproductive stage(Annals of Botany, 2022-03-03) Hostetler, Ashley N.; Erndwein, Lindsay; Ganji, Elahe; Reneau, Jonathan W.; Killian, Megan L.; Sparks, Erin E.Background and Aims: Root lodging is responsible for significant crop losses world-wide. During root lodging, roots fail by breaking, buckling, or pulling out of the ground. In maize, above-ground roots, called brace roots, have been shown to reduce root lodging susceptibility. However, the underlying structural-functional properties of brace roots that prevent root lodging are poorly defined. In this study, we quantified structural mechanical properties, geometry, and bending moduli for brace roots from different whorls, genotypes, and reproductive stages. Methods: Using 3-point bend tests, we show that brace root mechanics are variable by whorl, genotype, and reproductive stage. Key Results: Generally, we find that within each genotype and reproductive stage, the brace roots from the first whorl (closest to the ground) had higher structural mechanical properties and a lower bending modulus than brace roots from the second whorl. There was additional variation between genotypes and reproductive stages. Specifically, genotypes with higher structural mechanical properties also had a higher bending modulus, and senesced brace roots had lower structural mechanical properties than hydrated brace roots. Conclusions: Collectively these results highlight the importance of considering whorl-of-origin, genotype, and reproductive stage for quantification of brace root mechanics, which is important for mitigating crop loss due to root mechanical failure.Item Maize plants and the brace roots that support them(New Phytologist, 2022-09-14) Sparks, Erin E.Brace roots are a unique but poorly understood set of organs found in some large cereal crops such as maize. These roots develop from aerial stem nodes and can remain aerial or grow into the ground. Despite their name, the function of these roots to brace the plant was only recently shown. In this article, I discuss the current understanding of brace root function and development, as well as the multitude of open questions that remain about these fascinating organs.Item Matrix Adhesiveness Regulates Myofibroblast Differentiation from Vocal Fold Fibroblasts in a Bio-orthogonally Cross-linked Hydrogel(ACS Applied Materials and Interfaces, 2022-11-23) Song, Jiyeon; Gao, Hanyuan; Zhang, He; George, Olivia J.; Hillman, Ashlyn S.; Fox, Joseph M.; Jia, XinqiaoRepeated mechanical and chemical insults cause an irreversible alteration of extracellular matrix (ECM) composition and properties, giving rise to vocal fold scarring that is refractory to treatment. Although it is well known that fibroblast activation to myofibroblast is the key to the development of the pathology, the lack of a physiologically relevant in vitro model of vocal folds impedes mechanistic investigations on how ECM cues promote myofibroblast differentiation. Herein, we describe a bio-orthogonally cross-linked hydrogel platform that recapitulates the alteration of matrix adhesiveness due to enhanced fibronectin deposition when vocal fold wound healing is initiated. The synthetic ECM (sECM) was established via the cycloaddition reaction of tetrazine (Tz) with slow (norbornene, Nb)- and fast (trans-cyclooctene, TCO)-reacting dienophiles. The relatively slow Tz–Nb ligation allowed the establishment of the covalent hydrogel network for 3D cell encapsulation, while the rapid and efficient Tz–TCO reaction enabled precise conjugation of the cell-adhesive RGDSP peptide in the hydrogel network. To mimic the dynamic changes of ECM composition during wound healing, RGDSP was conjugated to cell-laden hydrogel constructs via a diffusion-controlled bioorthognal ligation method 3 days post encapsulation. At a low RGDSP concentration (0.2 mM), fibroblasts residing in the hydrogel remained quiescent when maintained in transforming growth factor beta 1 (TGF-β1)-conditioned media. However, at a high concentration (2 mM), RGDSP potentiated TGF-β1-induced myofibroblast differentiation, as evidenced by the formation of an actin cytoskeleton network, including F-actin and alpha-smooth muscle actin. The RGDSP-driven fibroblast activation to myofibroblast was accompanied with an increase in the expression of wound healing-related genes, the secretion of profibrotic cytokines, and matrix contraction required for tissue remodeling. This work represents the first step toward the establishment of a 3D hydrogel-based cellular model for studying myofibroblast differentiation in a defined niche associated with vocal fold scarring.Item Transcriptomic Signature of the Simulated Microgravity Response in Caenorhabditis elegans and Comparison to Spaceflight Experiments(Cells, 2023-01-10) Çelen, İrem; Jayasinghe, Aroshan; Doh, Jung H.; Sabanayagam, Chandran R.Given the growing interest in human exploration of space, it is crucial to identify the effects of space conditions on biological processes. Here, we analyze the transcriptomic response of Caenorhabditis elegans to simulated microgravity and observe the maintained transcriptomic response after returning to ground conditions for four, eight, and twelve days. We show that 75% of the simulated microgravity-induced changes on gene expression persist after returning to ground conditions for four days while most of these changes are reverted after twelve days. Our results from integrative RNA-seq and mass spectrometry analyses suggest that simulated microgravity affects longevity-regulating insulin/IGF-1 and sphingolipid signaling pathways. Finally, we identified 118 genes that are commonly differentially expressed in simulated microgravity- and space-exposed worms. Overall, this work provides insight into the effect of microgravity on biological systems during and after exposure.Item Costs of parthenogenesis on growth and longevity in ex situ zebra sharks Stegostoma tigrinum(Endangered Species Research, 2023-02-16) Adams, Lance; Lyons, Kady; Monday, Janet; Larkin, Elizabeth; Wyffels, JenniferThe zebra shark Stegostoma tigrinum, a popular aquarium fish, is an endangered species that is known to readily reproduce both sexually and through facultative parthenogenesis while in human care. Artificial insemination trials that took place between 2011 and 2013 resulted in the hatching of 2 sexually produced (herein heterozygotes) and 10 parthenogenetic sharks that allowed for a retrospective comparison of growth, feeding and longevity between offspring produced from 2 distinct reproductive modes. Parthenogenetic offspring were generally smaller at hatch than their heterozygous counterparts and, after the first several months post-hatch, failed to increase in mass and length at the same rate as heterozygotes. Parthenogenetic offspring exhibited non-normal swimming behaviors such as spiraling, spy hopping and head standing, which may have been correlated with a gradual decline in the ability of some sharks to properly suction feed. Median lifespan for the parthenotes was 1.05 yr (range: 0.27-6.64 yr); one of the heterozygotes lived to 2.37 yr of age, and the other was alive at the time of this writing in August 2022 and had reached reproductive maturity. By contrast, the 2 longest surviving parthenotes perished just prior to reaching sexual maturity (~5.5 and ~6.5 yr). Parthenogenesis has been documented among ex situ S. tigrinum maintained in aquariums across the globe, and this study demonstrates substantial negative costs to fitness in parthenogenetic offspring compared with their heterozygous siblings. The reduced fitness of parthenotes has implications for managing populations in human care as well as for in situ conservation efforts.Item Limiting Mrs2-dependent mitochondrial Mg2+ uptake induces metabolic programming in prolonged dietary stress(Cell Reports, 2023-03-28) Madaris, Travis R.; Venkatesan, Manigandan; Maity, Soumya; Stein, Miriam C.; Vishnu, Neelanjan; Venkateswaran, Mridula K.; Davis, James G.; Ramachandran, Karthik; Uthayabalan, Sukanthathulse; Allen, Cristel; Osidele, Ayodeji; Stanley, Kristen; Bigham, Nicholas P.; Bakewell, Terry M.; Narkunan, Melanie; Le, Amy; Karanam, Varsha; Li, Kang; Mhapankar, Aum; Norton, Luke; Ross, Jean; Aslam, M. Imran; Reeves, W. Brian; Singh, Brij B.; Caplan, Jeffrey; Wilson, Justin J.; Stathopulos, Peter B.; Baur, Joseph A.; Madesh, MuniswamyHighlights: • Mitochondrial Mg2+ channel Mrs2 rheostats MCU Ca2+ signals to maintain bioenergetic circuit • DNL precursor and cellular Mg2+ chelator citrate curbs HIF1α signal and oxidative metabolism • Lowering mMg2+ mitigates prolonged dietary-stress-induced obesity and metabolic syndrome • Mrs2 channel blocker CPACC reduces lipid accumulation and promotes browning and weight loss Summary The most abundant cellular divalent cations, Mg2+ (mM) and Ca2+ (nM-μM), antagonistically regulate divergent metabolic pathways with several orders of magnitude affinity preference, but the physiological significance of this competition remains elusive. In mice consuming a Western diet, genetic ablation of the mitochondrial Mg2+ channel Mrs2 prevents weight gain, enhances mitochondrial activity, decreases fat accumulation in the liver, and causes prominent browning of white adipose. Mrs2 deficiency restrains citrate efflux from the mitochondria, making it unavailable to support de novo lipogenesis. As citrate is an endogenous Mg2+ chelator, this may represent an adaptive response to a perceived deficit of the cation. Transcriptional profiling of liver and white adipose reveals higher expression of genes involved in glycolysis, β-oxidation, thermogenesis, and HIF-1α-targets, in Mrs2−/− mice that are further enhanced under Western-diet-associated metabolic stress. Thus, lowering mMg2+ promotes metabolism and dampens diet-induced obesity and metabolic syndrome. Graphical abstract Available at: https://doi.org/10.1016/j.celrep.2023.112155Item RNA degradome analysis reveals DNE1 endoribonuclease is required for the turnover of diverse mRNA substrates in Arabidopsis(The Plant Cell, 2023-04-20) Nagarajan, Vinay K.; Stuart, Catherine J.; DiBattista, Anna T.; Accerbi, Monica; Caplan, Jeffrey L.; Green, Pamela J.In plants, cytoplasmic mRNA decay is critical for posttranscriptionally controlling gene expression and for maintaining cellular RNA homeostasis. Arabidopsis DCP1-ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1) is a cytoplasmic mRNA decay factor that interacts with proteins involved in mRNA decapping and nonsense-mediated mRNA decay (NMD). There is limited information on the functional role of DNE1 in RNA turnover, and the identities of its endogenous targets are unknown. In this study, we utilized RNA degradome approaches to globally investigate DNE1 substrates. Monophosphorylated 5′ ends, produced by DNE1, should accumulate in mutants lacking the cytoplasmic exoribonuclease XRN4, but be absent from DNE1 and XRN4 double mutants. In seedlings, we identified over 200 such transcripts, most of which reflect cleavage within coding regions. While most DNE1 targets were NMD-insensitive, some were upstream ORF (uORF)-containing and NMD-sensitive transcripts, indicating that this endoribonuclease is required for turnover of a diverse set of mRNAs. Transgenic plants expressing DNE1 cDNA with an active-site mutation in the endoribonuclease domain abolished the in planta cleavage of transcripts, demonstrating that DNE1 endoribonuclease activity is required for cleavage. Our work provides key insights into the identity of DNE1 substrates and enhances our understanding of DNE1-mediated mRNA decay.Item Matrix Degradability Contributes to the Development of Salivary Gland Progenitor Cells with Secretory Functions(ACS Applied Materials and Interfaces, 2023-07-12) Metkari, Apoorva S.; Fowler, Eric W.; Witt, Robert L.; Jia, XinqiaoSynthetic matrices that are cytocompatible, cell adhesive, and cell responsive are needed for the engineering of implantable, secretory salivary gland constructs to treat radiation induced xerostomia or dry mouth. Here, taking advantage of the bioorthogonality of the Michael-type addition reaction, hydrogels with comparable stiffness but varying degrees of degradability (100% degradable, 100DEG; 50% degradable, 50DEG; and nondegradable, 0DEG) by cell-secreted matrix metalloproteases (MMPs) were synthesized using thiolated HA (HA-SH), maleimide (MI)-conjugated integrin-binding peptide (RGD-MI), and MI-functionalized peptide cross-linkers that are protease degradable (GIW-bisMI) or nondegradable (GIQ-bisMI). Organized multicellular structures developed readily in all hydrogels from dispersed primary human salivary gland stem cells (hS/PCs). As the matrix became progressively degradable, cells proliferated more readily, and the multicellular structures became larger, less spherical, and more lobular. Immunocytochemical analysis showed positive staining for stem/progenitor cell markers CD44 and keratin 5 (K5) in all three types of cultures and positive staining for the acinar marker α-amylase under 50DEG and 100DEG conditions. Quantitatively at the mRNA level, the expression levels of key stem/progenitor markers KIT, KRT5, and ETV4/5 were significantly increased in the degradable gels as compared to the nondegradable counterparts. Western blot analyses revealed that imparting matrix degradation led to >3.8-fold increase in KIT expression by day 15. The MMP-degradable hydrogels also promoted the development of a secretary phenotype, as evidenced by the upregulation of acinar markers α-amylase (AMY), aquaporin-5 (AQP5), and sodium-potassium chloride cotransporter 1 (SLC12A2). Collectively, we show that cell-mediated matrix remodeling is necessary for the development of regenerative pro-acinar progenitor cells from hS/PCs.Item Phylogenetic Analysis to Detect COVID Superspreaders(Microbiology Research Journal International, 2023-10-12) Jungck, John R.; Ko, HajaeAims: Detection of superspreading events by phylogenetic analysis of nucleotide sequences from a population of individuals collected from a narrow time interval. Study Design: Retrieve nucleic acid sequences, construct multiple sequence alignments, and build phylogenetic networks to determine sources of infection. Place and Duration of Study: This study was performed at the Delaware Biotechnology Institute of the University of Delaware over the period: June-August, 2022. The data used were from the GIS AID database. Methodology: Sequences for analysis were sampled from the GISAID initiative’s open-access SARS-CoV-2 genome database. We selected high-quality nucleotide sequences submitted by Delaware labs between March 18 and April 14, 2021, an important period of 4 weeks which saw the Alpha variant spread rapidly in the Delaware population. Results: Four sources accounted for 215 of the 401 sequences. In other words, 54% of all cases were rooted in just five sources. Conclusion: Thus, superspreading seems to have a major impact on the proportion of individuals in a population affected with COVID.