bluuebunny/crossref_metadata_2025 huggingface dataset:
The Interplay of Tissue Landscape and Extracellular Signaling during Drosophila Oogenesis
Authors: Starz‐Gaiano Michelle
Published: June 2021

Extracellular cues control developmental processes in a concentration‐dependent manner. Different amounts of morphogens elicit distinct cell fate decisions, and cells travel up chemoattractant gradients by assessing cue availability. Such signals must be tightly controlled to assure proper function. The concentration of signals in space and time depends on the parameters that govern the signal's release, diffusion, uptake, and decay rates. Chemical cues are often modeled as diffusing into open domains, but the structure of adjacent tissue could influence local signal availability, and thus cellular responses. The relatively simple tissue architecture of the Drosophila egg chamber, in which large germline cells are surrounded by a follicular epithelium, makes an ideal context to study how tissue contours influence extracellular signaling. In mid‐oogenesis, two anterior follicle cells secrete Unpaired (Upd) to activate the conserved factor Signal Transducer and Activator of Transcription (STAT) in nearby cells, and high STAT activity instructs a subset of those cells to become motile. Our genetic analysis identified specific vesicle trafficking components that modulate the secretion rate of Upd. While all cells near the source of Upd are responsive, we found that STAT activation and the resulting arrangements of motile cells often arose in unexpected, asymmetric patterns. We developed a mathematical model of the spatio‐temporal distribution of Upd in three‐dimensions to characterize signaling activity in the extracellular domain. Simulation results and genetic analysis of mutant phenotypes support the idea that irregular tissue domains result in asymmetric distributions of the Upd signal, consistent with outcomes in vivo. Additionally, modeling results suggest that the concentrations of chemoattractant signals that guide the motile cells can be spatially impacted by tissue structure. We conclude that the interstitial space generated by adjacent tissues creates complex distributions of signaling molecules, which impacts responding cells. This work illustrates novel aspects of how tissue topography alters signaling, and sheds light on cell motility during animal development.

Support or Funding Information

National Science Foundation IOS‐1656550

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Breaking new ground: the emergence of non‐canonical functions for telomerase subunits in plants
Authors: Shippen Dorothy
Published: June 2021

The telomere complex is highly conserved and essential for genome integrity. Recent studies reveal that core constituents of telomerase and the telomere cap can function off the telomere to promote DNA repair and regulate various metabolic processes in the cytoplasm. We are investigating how two telomere‐related factors in Arabidopsis contribute to genome stability through non‐canonical pathways. Protection of telomeres 1 (POT1) encodes a telomere binding protein implicated in chromosome end protection and telomerase regulation. Although most organisms harbor a single POT1 gene, in the few lineages where the POT1 family has expanded the duplicate genes evolved distinct functions. Arabidopsis thaliana provides an extreme example of divergence wherein the POT1a paralog retains the ancestral function of stimulating telomerase activity, while the POT1b paralog localizes to the cytoplasm and plays no direct role in telomere biology. Plants deficient in POT1b are hypersensitive to genotoxic stress as a result of environmental insult or intrinsic metabolic stress, and produce fewer, lower quality seeds. Notably, POT1b transcription is not regulated by genotoxic stress, but instead is developmentally regulated and concentrated in reproductive tissues. These findings indicate that POT1b evolved to serve a protective role for the genome, increasing plant fitness.

Arabidopsis also encodes a non‐canonical telomerase‐associated lncRNA called TER2. TER2 is a brand‐new gene unique to A. thaliana. TER2 is induced in response to DNA double‐strand breaks and its induction causes a rapid decrease in telomerase activity, likely to avert inappropriate telomerase action at sites of DNA damage. Unexpectedly, we found that plants doubly deficient in TER2 and POT1a exhibit a highly accelerated rate of telomere shortening that triggers massive genome instability and gross morphological defects earlier than in plants completely lacking telomerase. Thus, TER2 works in concert with the conventional telomerase enzyme to promote genome stability and telomere replication. Strikingly, like POT1b, TER2 can also be found in the cytoplasm and co‐expresses with POT1b in reproductive organs and seeds. As with pot1b mutants, plants lacking TER2 are hypersensitive to genotoxic stress and have reduced fitness. Finally, biochemical experiments indicate that POT1b physically interacts with TER2. Altogether, these data suggest that POT1b and TER2 are co‐evolving a novel protective mechanism for the plant genome.

Support or Funding Information

This research was supported by grants from NIH (R01‐GM065383) and NSF (MCB 1517817) to D.S.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Replication origin licensing deficiency and genome instability during cell cycle re‐entry from quiescence
Authors: Cook Jean , Matson Jacob , House Amy , Perez Joanna
Published: June 2021

Metazoan cells transition between active cell division cycles and periods of quiescence during development, regeneration, and homeostasis. Prior to cell division, DNA replication initiates during S phase at thousands of origins to ensure timely and complete genome duplication. Each origin that initiates must have been already licensed for replication in the preceding G1 phase by the DNA loading of Minichromosome Maintenance (MCM) complexes. Insufficient origin licensing in G1 causes incomplete replication and is associated with hypersensitivity to replication stress and DNA damage. We hypothesize that physiological differences in G1 length or G1 starting point impact origin licensing dynamics, and that such differences affect genome stability. We have explored origin licensing parameters in the first G1 phase during cell cycle re‐entry from quiescence relative to the G1 phases following mitosis in actively proliferating cells. We used single cell flow cytometry and live cell imaging to define the relationships among cell cycle re‐entry, G1 length, origin licensing dynamics and replication stress in untransformed human epithelial cells. Despite spending a long time in G1 phase, cells re‐entering the cell cycle are significantly underlicensed in the first cell cycle but not the second or subsequent cell cycles. Cells enter their first S phase before licensing is complete and are then hypersensitive to replication stress. Thus, G1 phase upon cell cycle re‐entry is characterized by slow (or delayed) origin licensing and an apparently weak licensing checkpoint. Strikingly, repeated transitions into and out of quiescence and active proliferation exacerbate replication stress and cause hypersensitivity to replication inhibitors Therefore, the transition from quiescence to active division is a particularly sensitive time for genome stability, and the number of transitions into and out of quiescence may play a role in long term cell viability and genome stability.

Support or Funding Information

The UNC Flow Cytometry Core Facility is supported in part by P30 CA016086. This work was supported by a fellowship from the NSF (DGE‐1144081) to J.P.M and by grants from the NIH to J.G.C. (GM083024 and GM102413).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crowdsourcing the Development of Assessments for Biomolecular Visual Literacy
Authors: Procko Kristen , Bednarski April , Beckham Josh T. , Dean Diane M. , Dries Daniel Robert , Franzen Margaret A. , Jakubowski Henry V. , Novak Walter R. P. , Roberts Rebecca A. , Roca Alberto I. , Terrell Cassidy R.
Published: June 2021

Biochemistry and molecular biology (BMB) education is rich in images of molecular structures. Yet, as often as we use these images for instruction, we seldom assess whether our students understand choices we make in the images we show (for example, ribbon versus ball‐and‐stick). To that end, the BioMolViz group (BioMolViz.org) is designing instruments and exercises to assess the molecular visual literacy of BMB students. With support from the National Science Foundation, the BioMolViz group partnered with institutions to host regional workshops in which participants wrote and refined more than 180 assessments to address specific learning objectives in the Molecular Visualization Framework. In pre‐ and post‐workshop surveys, participants reported shifts in their approach toward their instruction of molecular visualization literacy in the classroom. Through these workshops, a network of BMB educators collaborated to develop high‐quality assessment instruments, laying the foundation for crowdsourcing molecular visualization assessment development across the BMB education community.

Support or Funding Information

This work was supported by the National Science Foundation under award numbers DUE‐1022793, ‐1323414, ‐1503811 and IUSE‐1712268. Any opinions, findings, and conclusions or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the National Science Foundation.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of an Assessment Tool for Evaluating Undergraduate Student Understanding of Pedigree Analysis
Authors: Grimes Zachary , Boury Nancy , Reid Joshua , Wasendorf Chloe , Couch Brock , James Olena , Seipelt‐Thiemann Rebecca
Published: June 2021

Biology education is currently undergoing reform efforts to increase student retention and appreciation within the biological sciences. Both Vision and Change in Undergraduate Biology Education call for the increased use of evidence‐based pedagogical strategies to support learning in biology learning environments. However, integration of evidence‐based instructional strategies relies on biology instructors' knowledge of student pre‐conceptions and misconceptions. Genetics represents a critical area of biology education that presents many problems for student learning due to its abstract nature, the need to think through different spatial scales, and heavy reliance upon technical language. Pedigree analysis represents a convergence of topics in genetics and, therefore, has the potential to identify multiple student learning difficulties. Pedigree analysis requires an understanding of modes of inheritance, which requires a knowledge of the nature of both dominance and recessiveness of traits, as well as understanding of the connections between genotype and phenotype and use of the symbolic scale. This project sought to gain an understanding of students' misconceptions of pedigrees in order to promote the development of an assessment tool for students' misconceptions. The research team developed targeted questions and collected written responses to these open‐ended questions as well as conducting self‐selected student interviews. Student responses were coded to identify the most common misconceptions which were used as distractors a draft multiple choice concept inventory (CI). The draft CI was administered to freshman, sophomore, and upper division undergraduate students. These data were then used to further revise the finalized CI. Pre‐post data are currently being collected to ensure reliability and discriminatory power of this CI. Future studies will include analysis of the persistence of distinct misconceptions across the different student groups, as well as a more thorough analysis of student misconception types and their potential sources.

Support or Funding Information

Funded by NSF Award #1710262

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improvements to Simplicity and Stability of the Cell‐Free Protein Synthesis Platform to Enable Applications in the Field and the Classroom
Authors: Gregorio Nicole E. , Kao Wesley Y. , Williams Layne C. , Oza Javin P.
Published: June 2021

E. coli‐based cell‐free protein synthesis (CFPS) is a flexible platform technology for on‐demand protein production that allows users to express traditionally intractable proteins, perform high‐throughput screening, and, with augmentation, supports applications such as metabolic engineering and genetic code expansion. The broad utility of the CFPS platform arises from the elimination of the cellular membrane and capture of transcription and translation machinery in vitro. This obviates the need for living cells and creates an open system for direct manipulation of the environment of protein production. However, broad dissemination to field and classroom applications remains limited, as CFPS is dependent on technical scientific expertise for proper reaction setup and laboratory infrastructure for proper storage of reagents. Here, we report our efforts to improve broad accessibility of CFPS by 1) simplifying reaction setup and 2) improving shelf stability of the cell‐extract at various storage conditions. In order to simplify reaction setup, which currently requires precise pipetting of 10 reagents, we have combined the CFPS reagents into two stable premixes. These premixes can be stored at −20 °C for at least 4 months and allow the user to pipette fewer times with larger volumes for more accurate reaction setup by non‐experts. To improve the shelf stability of cell extract, which contains the sensitive transcription and translation machinery and is traditionally stored at −80 °C, we have characterized the functional effects of 10 protein stabilizing additives such as sugars, osmolytes, surfactants, and molecular crowding agents. Tests of single additives and combinations of two or three have allowed us to identify the landscape of small molecule additives that can support a variety of storage conditions ranging from −80 °C to room temperature to suit a variety of applications. When combined, these advances to reaction setup and cell extract composition provide the foundation for broad implementation of CFPS. Improved ease of reaction setup enables the use of CFPS by non‐experts, such as early career students, and improves efficiency for researchers and field applications. Additionally, increased storage stability diminishes reliance on the cold chain, making CFPS reagents easier to disseminate, commercialize, and use in the field. Together, these modifications provide a foundation for democratization of the CFPS platform.

Support or Funding Information

Our research is funded by the Center for Applications in Biotechnology, Bill and Linda Frost Fund, and NSF‐1708919.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Study of Multidrug Efflux System Protein Degradation in E. coli Using Transposons Library
Authors: Rajapaksha Prasangi Irosha , Wei Yinan
Published: June 2021

AcrB is a trimeric multidrug efflux pump in E. coli belonging to the Resistance‐Nodulation‐Division (RND) super family. We have previously found that the introduction of a small peptide tag, the ssrA tag, to the C‐terminus of AcrB lead to efficient degradation of the protein. Soluble proteases ClpP and ClpX have been found to be involved in the degradation. However, a significant reduction in the amount of AcrB‐ssrA could still be detected in knockout strains lacking functional ClpXP. We speculate that the reduction of AcrB‐ssrA level is due to another protease/proteases. This study focuses on the identification of other proteins capable of degrading AcrB‐ssrA. Transposons libraries containing random gene disruption were generated and screened for clones displaying increased expression of AcrB‐ssrA. Our hypothesis is that the increased activity of AcrB‐ssrA protein is due to a mutation or inactivation of a gene that is involved in the degradation of AcrB‐ssrA. The elevated level of AcrB‐ssrA expression was then confirmed using anti‐AcrB antibody and minimum inhibitory concentration assays. Several candidate genes have been identified, which we are currently characterizing for their potential role in AcrB‐ssrA degradation.

Support or Funding Information

NSF grant CHE‐1709381

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hydrophobic Mutations Promote UBQLN2 Oligomerization And Phase Separation
Authors: Castaneda Carlos A , Dao Thuy P , Yang Yiran , Jones Holly , Lei Yongna , Martyniak Brian
Published: June 2021

Liquid‐liquid phase separation (LLPS) is hypothesized to be the dominant mechanism that underlies the formation of membraneless organelles, including stress granules formed under cellular stress containing sequestered RNA and proteins. Our lab has recently shown that Ubiquilin‐2 (UBQLN2), a protein involved in protein quality control through proteasomal degradation and autophagy pathways, is recruited to stress granules in vivo and undergoes LLPS in vitro under physiological conditions. Using NMR spectroscopy, we identified both intrinsically‐disordered and folded regions that are involved in UBQLN2 self‐association that also mediate UBQLN2 LLPS. One of these regions includes the proline‐rich (Pxx) segment, where known disease‐linked mutations have been shown to cause 1–2% of familial ALS or ALS/dementia cases. Using size‐exclusion chromatography, NMR spectroscopy, and microscopy, we demonstrated that Pxx mutations modulate UBQLN2 self‐association and phase separation in vitro. Pxx mutations at P497, P506 and P525 affected droplet morphology and dynamics to significantly different extents. Our data suggested that increased hydrophobicity of the amino acid promoted UBQLN2 LLPS and lowered the phase transition temperature at which UBQLN2 LLPS is first observed. To systematically investigate the molecular basis for how amino acid composition affects UBQLN2 phase separation, we generated all 19 possible amino acid replacements at residues P497, P506, P525, and V538. Our data indicate that the phase behavior of UBQLN2 is significantly modulated by mutations at positions 497 and 506, but weakly modulated by mutations at positions 525 and 538. These results suggest that positions 497 and 506 are ‘stickers’ whereas positions 525 and 538 are ‘spacers’ using the language of associative polymers. Consistent with our data above, hydrophobic amino acid substitutions promoted UBQLN2 LLPS. Our experiments suggest that UBQLN2 may be used as a model system to understand the sequence determinants of phase separation. Hydrophobic mutations modulate UBQLN2 oligomerization and LLPS, and potentially alter material properties of UBQLN2‐containing biomolecular condensates in the cell, promoting disease states.

Support or Funding Information

C.A.C. graciously acknowledges funding from the ALS Association via grants 17‐IIP‐369 and 18‐IIP‐400, as well as from the National Science Foundation (CAREER award 1750462).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Elucidation of Engineered Tissue Inhibitor of Metalloproteinases‐1 (TIMP‐1) Variants with Improved Binding Affinity toward Matrix Metalloproteinase‐3 (MMP‐3)
Authors: Raeeszadeh‐Sarmazdeh Maryam , Sankaran Banumathi , Radisky Derek , Radisky Evette
Published: June 2021

Tissue inhibitors of metalloproteinases (TIMPs) are natural inhibitors of matrix metalloproteinases (MMPs), enzymes known for contributions to diseases including cancer and fibrosis. The TIMP N‐terminal domain, regarded as the “inhibitory domain”, has been the center of attention both for understanding natural regulation of MMPs by TIMPs, and for developing protein‐based MMP inhibitors for therapeutic applications. The role of the TIMP C‐terminal domain in MMP inhibition has not been well studied. I have used directed evolution via yeast surface display for protein engineering of full‐length human TIMP‐1 toward development of MMP‐3‐targeted ultra‐binders. TIMP‐1 variants with up to 10‐fold improvement in MMP‐3 binding (compared to WT‐TIMP‐1) were isolated after six rounds of library screening using fluorescence‐activated cell sorting (FACS). Analysis of individual and paired mutations from the selected TIMP‐1 variants revealed a cooperative effect between distant mutations on the N‐ and C‐terminal TIMP domains, positioned on opposite sides of the interaction interface with MMP‐3. I have co‐crystallized two TIMP‐1 variants containing single and composite mutations in complex with the catalytic domain of MMP‐3 and solved the X‐ray crystal structures of these complexes. The protein structures reveal substantial conformational changes in the TIMP‐1 binding interfaces near the two cooperative mutations. Affinity appears to be strengthened by tighter cinching of a reciprocal tyrosine clasp formed between the N‐terminal loop of TIMP‐1 and the proximal MMP3 interface, and adoption of an alpha‐helical conformation by a segment of the TIMP‐1 C‐terminal domain, resulting in stabilized interactions between the two TIMP‐1 domains and improved hydrophobic interactions between TIMP‐1 and MMP‐3. Our protein engineering and structural studies provide novel insight into the cooperative function of N‐ and C‐terminal TIMP domains and the significance of peripheral TIMP epitopes in MMP recognition. Our results further point to new approaches for design of TIMP‐based protein therapeutics with improved affinity and selectivity.

Support or Funding Information

This work was supported by grants DOD W81XWH‐16‐2‐0030, and NIH R21 CA205471. Diffraction data were measured at the Berkeley National Synchrotron Light Source.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization of an in Vitro CYP450 Induction Assay Using Cryopreserved Cell Lines
Authors: Lim Tae Young , Rizkallah Amy , Capacio Benedict
Published: June 2021

Cytochrome P450 (CYP450) enzymes are responsible for the majority for Phase I metabolism. Induction occurs when a drug increases the rate of CYP450 enzyme production. As a result, the rates of metabolism of subsequent drugs increase, posing concerns for dosing and safety. To further expand the USAMRICD Absorption, Distribution, Metabolism, Excretion, and Toxicity Center of Excellence (ADMET CoE) profiling capabilities, a CYP450 induction assay is being developed using alternative cell lines to primary human hepatocytes to measure changes in gene expression of CYP1A2 and CYP3A4 isoenzymes.

Primary human hepatocytes are the gold standard for evaluating P450 induction in vitro; however, they have inherent limitations such as high costs, fastidious nature, and significant lot‐to‐lot variability. Induction assessments using cryopreserved hepatocytes and two immortalized cell lines, HepaRG and HepatoCells, are being conducted to determine a more robust, cost‐effective alternative model. Each cell line was exposed to the standard inducers rifampicin and omeprazole to assess CYP3A4 and CYP1A2 activity, respectively. Following a 72‐hour incubation period, RNA from the cell cultures was extracted and purified. Fold changes of gene expression were measured using real‐time quantitative PCR (RT‐qPCR). Hepatocytes displayed an average fold change of 92.7 for CYP3A4 and 49.6 for CYP1A2, whereas HepaRG displayed changes of 28.6 and 179.5, respectively. Average values for CYP3A4 and CYP1A2 for HepatoCells were 58.7 and 43.9. Based on the data, HepatoCells most closely mimicked cryopreserved hepatocytes in comparison to HepaRG cells. Additional data on assay parameters and technique optimization will be presented.

Support or Funding Information

Opinion disclaimer: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of the Army, Department of Defense, or the U.S. Government. This project was generously supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. Army Medical Research and Materiel Command. Support is also provided by the Defense Threat Reduction Agency – Joint Science and Technology Office, Medical S&T Division.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating Differential Effects of Cisplatin and Oxaliplatin on Nucleolar Stress using Platinum ‘Liaison’ Compounds
Authors: Prochneau Jack , McDevitt Christy , Sutton Emily , Cunningham Rachael , DeRose Victoria
Published: June 2021

Platinum(II) compounds are a versatile and effective class of chemotherapy drugs, employed extensively worldwide in single and combination therapy treatments. In one accepted mechanism of action these compounds bind to DNA, interfere with replication events and stimulate apoptosis. However, these small platinum compounds have multiple cellular targets and the three FDA‐approved platinum agents, cisplatin, carboplatin, and oxaliplatin have different activity, resistance and side effect profiles. Two of the FDA‐approved agents, cisplatin and oxaliplatin, exemplify this phenomenon with the former inducing more severe forms of ototoxicity and nephrotoxicity. One marked difference is that unlike cisplatin, oxaliplatin treatment induces markers of nucleolar stress response, including relocalization of nucleolar phosphoprotein NPM1 in mammalian cell culture. It is presently unknown which structural features of these square‐planar molecules, including aquation‐labile chloride/oxalate or non‐labile ammine/1,2‐diaminocyclohexane ligands, give rise to such different effects. To investigate how platinum ligands may influence the nucleolar stress response we have synthesized a series of ‘liaison’ Pt(II) compounds with different ligand combinations, and measured the effects of this series of compounds on NPM1 relocalization, DNA damage markers, and cell death. The results of this study will lead to an understanding of the structural features and molecular interactions underlying the ability of oxaliplatin to influence nucleolar processes.

Support or Funding Information

Support from the National Institutes of Health R25HD0708 (JP) and T32 GM007759 (ES), the National Science Foundation CHE 1710721 (VJD) and MRI 1531189 (for shared instrumentation), and the University of Oregon is gratefully acknowledged.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Genome Dynamics of Coevolving Podoviruses and Marine Cyanobacteria
Authors: Fenderson Brooke A , Marston Marcia F
Published: June 2021

Podoviruses are dsDNA bacteriophage with ~45 kb genomes that are abundant in coastal marine ecosystems. Many of these podoviruses infect cyanobacteria and are thought to undergo antagonistic coevolution with their host. During coevolution, the host evolves resistance to the virus and the viruses evolve to overcome this resistance. Coevolution can impact the fitness and genetic diversity of both host and virus. In this study, whole‐genome sequencing was used to examine the genome dynamics of two closely‐related podoviral strains coevolving with cyanobacteria Synechococcus WH7803 in continuous culture. For each podoviral strain, replicate chemostats containing Synechococcus WH7803 were inoculated with the virus and maintained for 24 weeks. Two additional chemostats of Synechococcus without virus served as the controls. Monthly samples were taken from all chemostats and used to isolate individual viral plaques and Synechococcus colonies. The genomes of viral and host isolates from various time points were then fully sequenced and compared to the genomes of the ancestral strains. Viruses in all four chemostats accumulated mutations as time progressed. These mutations consisted mostly of single nucleotide polymorphisms leading to amino acid changes in the tail fiber region of the viral genome. In addition, large deletions, ranging from 500 to 1600 nt occurred in isolates from later time points in three of the four chemostats. Interestingly, these deletions occurred in regions of the genome that are not shared by the two podoviral strains. In contrast to the viruses, host cells accumulated far fewer mutations and no gene deletions were observed. Only a few mutations were needed for the host to become resistant to the podoviral strains. One of these mutations was located in an intergenic region likely involved in gene regulation and provided broad‐spectrum resistance to both podoviruses and myoviruses. Studying the genome dynamics that occur during coevolution will lead to a better understanding of how viruses and their hosts interact and coexist in natural ecosystems.

Support or Funding Information

This research was supported by the National Science Foundation (OCE‐0314523, OCE‐1029684 and OIA‐1736030) to M. Marston.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Illuminating DYRK1A Substrates Through a Multi‐omic Drug Based Approach
Authors: Guard Steven , Poss Zachary , Ebmeier Chris , Webb Kristofor , Old William
Published: June 2021

Protein kinases comprise only ~2.5% of human protein coding genes yet are responsible for the regulation of nearly every cellular process through their catalytic activity. The kinome thus represents one of the most druggable classes of regulatory proteins for human disease. However, despite major advances in protein identification through mass‐spectrometry based techniques, it remains difficult to uncover substrate profiles for individual kinases using pharmacological manipulation. Undesirable off‐target inhibition and diverse secondary events muddle the phosphorylation landscape that can be detected using phosphoproteomic methods. Further, phosphorylation events and target engagement are often cell type specific confounding our ability to discern contributions of off‐target kinases. Using the protein kinase DYRK1A and inhibitor harmine as a model, we utilize orthogonal mass spectrometry‐based approaches to distinguish kinase‐substrates. Thermal proteome profiling revealed that harmine differentially stabilizes proteins in a cell type and dose dependent manner. Drug target stabilization in combination with substrate motif analysis were used to elucidate high‐confidence DYRK1A substrates from a SILAC‐based harmine phosphoproteome. The substrates revealed in this study will be crucial to understanding DYRK1A function and contribution to human health.

Support or Funding Information

DARPA cooperative agreement 13‐34‐RTA‐FP‐007 to W.M.O

Linda Crnic Institute for Down syndrome‐ Grand Challenge Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Analysis of the C‐terminal domain of MtlA and its role in cell wall metabolism in the fungus Aspergillus nidulans
Authors: Nichols Stewart , Cannavo Matthew M. , Jackson‐Hayes Loretta M. , Loprete Darlene M.
Published: June 2021

The Mid2‐like protein MtlA is a putative sensor that affects cell wall integrity in the filamentous fungus Aspergillus nidulans. Plasmid‐based, overexpression of MtlA can suppress the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin‐binding agent Calcofluor White. In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity. MtlA is predicted to have a single transmembrane domain, an extracellular domain rich in serine and threonine amino acids residues, and a C‐terminal domain of 59 amino acids. A MtlA ‐GFP hybrid localizes to the plasma membrane of vegetative hyphae. In the cell wall integrity pathway in yeast, the C‐terminal domain of the stress receptors, Wsc1 and Mid2, have been shown to play an important role in the signal transduction pathway. We have shown that the C‐terminal domain of MtlA plays an important role in cell wall integrity. We have created and analyzed C‐terminal truncation mutants of MtlA and have shown that complete removal of the C‐terminal domain obliterates its function. We have also shown that amino acids 241–251 are critical for the function of MtlA. Site directed mutagenesis of key amino acids will be elucidated.

Support or Funding Information

Rhodes College

National Science Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Analyses of Myc and β‐catenin during Echinoderm Intestinal Regeneration using RNAi
Authors: Alicea‐Delgado Miosotis , Salem‐Hernández Joseph , Garcia‐Arraras Jose E
Published: June 2021

Molecular approaches have been widely used to understand and characterize regenerative processes in different animal systems. Nevertheless, characterizing the genetic component of echinoderms using RNAi techniques represent a tremendous challenge. We have now designed and implemented the use of Dicer‐substrate RNAi technology (DsiRNA) to knockdown the expression of Myc and β‐catenin transcripts on tissue explants of the sea cucumber Holothuria glaberrima. Quantification of Myc and β‐catenin transcripts showed a decrease following electroporation of specific DsiRNAs when compared to explants electroporated with control DsiRNA. Explants were then assayed for the effects on cell proliferation, cell dedifferentiation and apoptosis. No effect on cellular dedifferentiation was observed. In contrast, the number of apoptotic cells was significantly different in explants electroporated with DsiRNAs‐targeting Myc and β‐catenin when compared to control DsiRNA. Finally, a decrease in cell proliferation was observed following the reduction of Myc transcript, but not following the reduction of β‐catenin. These results suggest that Myc has a significant role on cell proliferation and apoptosis, but not on cell dedifferentiation during the regeneration of the intestine. Similar to Myc, β‐catenin has a significant role on apoptosis, but not on cell proliferation and cell dedifferentiation. Moreover, our results show that DsiRNAs and intestinal tissue explants are a valuable tool to manipulate and explore the role of genes of interest in echinoderms.

Support or Funding Information

Funded by NIH R15GM124595, NSF IOS‐1252679 and the University of Puerto Rico.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of TRPV4‐TAZ signaling axis in epithelial‐mesenchymal transition
Authors: Rahaman Shaik , Sharma Shweta , Goswami Rishov
Published: June 2021

Epithelial to mesenchymal transition (EMT) is a critical event in development, fibrogenesis, and oncogenesis. Emerging data support a role for both a mechanical signal, and a biochemical signal, e.g., transforming growth factor β1 (TGFβ1) in EMT. Here, we report evidence that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel sensitive to both matrix stiffness and TGFβ1 stimuli, is a likely mediator of EMT. We found that antagonism of TRPV4 blocked both matrix stiffness‐ and TGFβ1‐induced EMT in normal human primary epidermal keratinocytes (NHEKs), as determined by changes in morphology and alterations in expression of EMT markers including E‐cadherin, N‐cadherin, and α‐smooth muscle actin (α‐SMA). In a murine skin fibrosis model, TRPV4 deletion resulted in decreased expression of the mesenchymal marker, α‐SMA, and increased expression of epithelial marker, E‐cadherin. Mechanistically, we found that: i) TRPV4 activity was essential for the nuclear translocation of TAZ (transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1; ii) Antagonism of TRPV4 activity inhibited both matrix stiffness‐induced and TGFβ1‐induced expression of TAZ proteins; and iii) TRPV4 antagonism suppressed both matrix stiffness‐induced and TGFβ1‐induced activation of Smad2/3, but not of AKT. Altogether, these data identify a novel role for TRPV4 in regulating EMT in response to both matrix stiffness and TGFβ1.

Support or Funding Information

AHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik Rahaman

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hof1 of Aspergillus nidulans is Essential for Cytokinesis and Septum Formation
Authors: Rowland Lauren , Brookover Zoe , Ward Rachel , Hill Terry , Jackson‐Hayes Loretta
Published: June 2021

Cytokinesis is an essential component of cellular development in filamentous fungi. The process parallels that of cell division in animal cells in requiring assembly and constriction of a contractile actomyosin ring (CAR) in the cell periphery. However, the process differs from that in animals by involving the simultaneous construction of a chitin‐rich cross wall termed a septum, which separates semi‐independent compartments of the fungal hypha. Through randomized mutations, we have generated a temperature‐sensitive mutant defective in septum formation, designated strain RCH59. Mendelian crossing confirmed that the temperature sensitive phenotype results from a single locus mutation. Next Generation Whole Genome Sequencing identified a mutation that is located in gene AN4566 encoding the homolog of Saccharomyces cerevisiae's Hof1. The mutation in Aspergillus nidulans’ version of Hof1 (AnHof1) is at base 2436 out of 5536 and is a transition from cytosine to thymine. The mutation is predicted to remove an Arginine and introduce a premature stop codon after residue 738. The missing 342 C‐terminal residues includes the entire SH3 domain of AnHof1. We cloned the wildtype version of AnHof1 into plasmid pRG3 and complemented the phenotype in RCH59, confirming that the mutation occurs in AnHof1. We also deleted AnHof1 to demonstrate that a AnHof1 minus strain has an aseptate phenotype. In the yeasts (S. cerevisiae and S. pombe), Hof1 forms a ring structure that co‐localizes with the CAR and is involved with mediating the cytoskeletal rearrangements necessary for cytokinesis. We GFP tagged AnHof1 to demonstrate that the protein product localizes to sites of septum formation and co‐localizes with actin during at least part of CAR assembly and constriction. In further work, we have generated double‐mutant strains in which a GFP‐tagged septation‐related protein is expressed in the RCH59 strain to demonstrate a network of genetics interactions between AnHof1 and other proteins involved in septation.

Support or Funding Information

This research was supported by NSF grant RUI‐0742907 to TWH and LJH and NSF grant RUI‐1615192 to LJH and TWH.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inositol Pyrophosphates and Phosphate Sensing in Plants
Authors: Gillaspy Glenda , Adepoju Olusegun , Land Eric , Williams Sarah Phoebe , Donahue Janet , Cridland Caitlin , Freed Catherine , Perera Imara
Published: June 2021

Phosphate (Pi) is an essential nutrient for plants, required for plant growth and seed viability. Under Pi stress, plants undergo dynamic changes to leverage available Pi. One class of signaling molecules implicated in the Pi sensing pathway is the inositol pyrophosphates (PPx‐InsPs). PPx‐InsPs have high energy bonds, and have been linked to maintaining Pi and energy homeostasis in plants, yeast, and humans. In plants, PPx‐InsPs are thought to function in sensing Pi changes by binding to proteins containing an SPX domain. One particular SPX protein binds to PPx‐InsPs which prevents a transcription factor from activating transcription of the so‐called phosphate starvation response (PSR) genes. Plants are known to accumulate two common PPx‐InsPs, PP‐InsP5 (called InsP7) and PP2‐InsP4 (called InsP8), but the synthesis pathway has not yet been elucidated. We have identified and characterized the plant VIP kinases, enzymes that likely function as PP‐InsP5 kinases. The identity of a plant inositol kinase that can phosphorylate InsP6, resulting in PP‐InsP5, however, has remained elusive. We will describe our recent results in characterizing this pathway, and genetic mutants key for understanding inositol kinase gene function in plants. Our results support a model in which InsP7 and InsP8 function to turn off the PSR in plants.

Support or Funding Information

This work was supported by an award from the NSF to GG and IP:MCB 1615953

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transgenic analysis of maize endosperm metabolism
Authors: Juhl Emily , Myers Alan M. , Hennen‐Bierwagen Tracie A.
Published: June 2021

Maize seeds provide a major component of the calories in the human diet, are critical as a feed source in animal agriculture, and are an important source of biorenewable raw materials. Endosperm tissue accounts for approximately 80% of seed weight, and is the site where carbohydrate and protein storage end products are produced in this net anabolic system. Sucrose supplied from photosynthetic tissues of the maternal plant is transported to growing seeds where it is divided between catabolic pathways that generate ATP and anabolic biosynthetic processes that utilize ATP to generate starch and storage proteins. Considering the high degree of ATP requirement, the fact that maize endosperm is an extremely hypoxic environment raises questions about how energy is generated, and the net efficiency of the system for product formation. This project tested the hypothesis that fully oxidative metabolism is not required for normal kernel development, and accordingly that ATP is generated exclusively or substantially through fermentative processes. To do so, RNAi technology was utilized to inactivate the 2‐oxoglutarate dehydrogenase (ODH) step of the citric acid cycle. T‐DNA vectors were constructed that expressed a double stranded RNA structure containing sequences homologous to both of the maize mRNAs encoding the E2 subunit of this enzyme. Highly active, tissue specific promoters were used to restrict the effects of the transgene exclusively to endosperm tissue. Plants were generated containing the anti‐ODH transgene, and subsequent crosses have established segregating populations of sibling kernels that either contain or lack that element. ODH activity and protein abundance in endosperm tissue will be measured to verify that the transgene is effective in inactivating the targeted enzyme activity. Subsequently, segregating kernel populations will be compared regarding growth rate over the course of development, mature kernel weight and composition, and global metabolite profiles. These studies will reveal the extent to which ODH is required for starch and storage protein synthesis to proceed in developing maize endosperm. This will reveal whether normal ATP generation in this tissue requires oxidative processes or, alternatively, whether fermentative processes can be sufficient to provide the energy required for the massive amount of starch and protein synthesis that makes maize such an important agronomic resource. Answering this question will enable future efforts to improve maize yields and grain quality.

Support or Funding Information

This research was supported by NSF award 1517256 to A.M.M. and T.A.H.‐B.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Data Mining Scientific Literature Demonstrates Use of Biological and Medical Data Across Scientific Disciplines
Authors: Verdiguel Natalie , Feng Zukang , Westbrook John , Zardecki Christine
Published: June 2021

The Protein Data Bank (PDB) was established as the 1st open access digital data resource in biology and medicine. Today, the PDB archive contains over 140,000 atomic‐level biomolecular structures determined by crystallography, NMR spectroscopy, and 3D electron microscopy. About 20 new structures are deposited daily. PDB data is used in basic and applied research, patent applications, discovery of lifesaving drugs, product innovation, and education. About 80% of all PDB structures deposited have a “primary citation,” which is the first paper to describe the molecule and its structure and function. In turn, these primary citations have contributed to more than 1 million publications. An initial study by Clarivate Analytics on the Nucleic Acids Research publication demonstrated that PDB data drive high‐impact research in diverse scientific fields. To build on this study, we used Python programming to perform several data mining studies to understand the impact of these structures in fundamental biology, biomedicine, and energy in the scientific literature. The scientific literature database, Web of Science, tracks scientific article metrics such as the number of times cited, subject category, year published, funding source, and keywords. Web of Science data for PDB primary citations was compiled as used as the data set. Two main investigations were performed. In the first, we examined the number of citations per article (data overall and by individual subject categories) to develop “top 10 cited structures” lists and analyzed for trends. In the second investigation, we extended our data mining analysis to consider for each main subject category, the papers with the most citations and the structures they cite, and the most frequently occuring keywords. We have observed different aspects of biological data utility in various subject areas, at times in ways unanticipated from the structures' primary citation. This research demonstrates the value of biological data from the PDB by showing the diverse ways in which the data are being used.

Support or Funding Information

RCSB PDB is funded by a grant (DBI‐1338415) from the National Science Foundation, the National Institutes of Health, and the US Department of Energy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.