bluuebunny/crossref_metadata_2025 huggingface dataset:
Small Molecule Allosteric Modulation of Protein Tyrosine Kinases in Live Cells
Authors: Lasansky David , Bienick Matthew , Ghosh Indraneel
Published: June 2021

The protein kinase and phosphatase enzymes within a cell catalyze the addition or removal of a phosphate group onto a protein respectively, and previous research has shown their involvement in nearly all cell‐signaling pathways. A consequence of this is that the disruption of the function of a particular kinase or phosphatase may lead to the development of diseases such as cancer. A major goal of chemical biology is to develop methods that allow for a detailed understanding of cell‐signaling pathways. However, current methods for studying the roles of these enzymes in signaling such as CRISPR‐Cas9 and siRNA for gene knockout suffer from potential compensatory mechanisms in the cell. Our research has led to the development of a potentially general approach for controlling enzyme activity via insertion of small peptide chains (BH3 helices) which bind selectively to a protein partner (Bcl‐xL) (Figure 1) for allosteric inhibition of kinase activity. Kinase activity can then be modulated by the addition of a specific small molecule that disrupts BH3/Bcl‐xL interactions thus providing specific control over turning on a single type of protein kinase within a cell. This approach is distinct compared to an approach by Professor Shokat that allows turning off activity of individual protein kinases in cells. We have shown success with this model in live cells when applied to the tyrosine‐protein kinase c‐Src, and our current project focuses on applying the model to other homologous tyrosine‐protein kinases. If successful, this research will provide greater insight into the role of specific kinases in cell‐signaling pathways, and thus allow for the development of genetic and small molecule therapeutics targeted towards dysfunctional kinases.

Support or Funding Information

This work was supported by the National Institutes of Health [1R01GM115595‐01] and the National Science Foundation [CHE‐1506091].

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Engineering Starch Kinases For Increased Biofuel Production Efficiency
Authors: Rondon Alberto , Emanuelle Shane
Published: June 2021

The growth of the bioethanol industry over the last decade has been a result of the growing demand for gasoline fuels, which in the United States constitutes at least 10% by volume, and the implementation of higher Renewable Fuel Standards (RFS). Bioethanol is produced by the fermentation of simple sugars by yeasts and as a result starch feedstocks must undergo pretreatment steps. The various mechanical, thermal and chemical treatments used to degrade starch are both expensive and energy intensive. Alternatively, plants use a reversible phosphorylation mechanism to solubilize starch and access stored glucose for nocturnal metabolism. Starch is formed by linear α‐1,4 linked glucose units and branching glucose via α‐1,6 bonds which form insoluble helical structures. Starch kinases phosphorylate the crystalline layers, disrupting the helical structures, and exposing the polymer to hydrolysis by amylases. This mechanism could be adopted during biofuels production to decrease the need for solubilizing treatments. Starch kinases are characterized by two distinct domains: carbohydrate binding modules (CBMs) and a dikinase domain connected via a peptide linker. The CBM functions to locate the kinase on the starch substrate, which is subsequently phosphorylated by the kinase domain. In order to expand the substrate affinity of a kinase, we fused the CBM from a Neurospora crassa starch‐active polysaccharide monooxygenase to multiple dikinase domains. We defined the melting temperature of the chimeric proteins using differential scanning fluorimetry and found that the chimeric proteins had increased stability. Using [33P‐β‐]ATP, we radiolabeled starch with the chimeric proteins and found that they exhibited at least 7‐fold greater activity than wild type dikinase using maize, barley, and potato starch as the substrate.

Support or Funding Information

NSF IIA‐1355438 (R. Andrews), NSF MCB‐1252345 (M. Gentry)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Designed Metal‐Mediated Protein Dimerization
Authors: Maniaci Brian J. , Love John J.
Published: June 2021

The specificity and stability of protein/protein interactions is of vital interest to the pharmaceutical and biotechnological industries. Incorporation of metal‐binding sites at target protein interfaces may be one approach to improve the specificity and affinity of naturally occurring and designed protein‐protein interactions. The main goal for this research was to generate a metal‐mediated protein interface and study the effects of interfacial modification on protein‐protein affinity. Our approach recently resulted in the creation of a high‐affinity, zinc‐mediated protein homodimer.

Using a metal‐templated approach we engineered novel metal binding sites to generate a high affinity interaction using the β1 domain of Streptoccocal Protein G (Gβ1). Metal coordination enables focused surface redesign, allowing for the preservation of naturally occurring or designed interfaces. Incorporation of metal binding sites generates concerted interactions allowing for enhanced affinity and specificity for protein interactions. Our protein complex is driven by metal coordination through histidine side‐chains, allowing for a metal‐dependent interaction.

Complex formation and assembly was evaluating using size exclusion chromatography and x‐ray crystallography. To accurately measure the molecular weight of the protein variants we have used size‐exclusion chromatography with multi‐angle light scattering (SEC‐MALS). This analytical technique first separates proteins based on size, and the molecular weight is determined from scattered laser light. The Gβ1 monomer and metal‐mediated dimers had molecular weight of 6.2 and 12.2–13.1 kDa, respectively. Crystal structure of the dimeric proteins proved the target design was achieved and that zinc was bound at the designed interface.

We have also engineered a library of rationally designed proteins that contain mutations in proximity to the metal binding sites. The goal is to exploit and explore key thermodynamic parameters that are essential for protein complex formation. We measured the binding affinity of the metal‐mediated protein‐protein interactions using a fluorescence polarization competition assay. The measured affinities were stronger than 100 nM, which is both physiologically relevant and provides additional support for the design of high‐affinity metal‐mediated protein‐protein interactions.

Support or Funding Information

This work was supported by the Department of Defense and by the California Metabolic Research Foundation.

Brian is a recipient of an Arne N. Wick Pre‐doctoral Research Fellowship from the California Metabolic Research Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting the Myristoylation of FRS2α Inhibits FGF/FGFRs‐mediated Oncogenic Signaling and Tumor Progression
Authors: Cai Houjian , Li Qianjin
Published: June 2021

Objective of the study

Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling facilitates tumor initiation and progression. Although there are currently approved inhibitors and agents in clinical trials targeting FGFR kinase activity that show therapeutic benefit, over‐expression and/or mutations of FGFRs eventually confer drug‐resistance and bypass the mechanisms of kinase inhibitors in a variety of cancer types. We hypothesize that a compound blocks the myristoylation of FRS2α, thus suppressing its interaction with FGFRs/FGFRsDRM and downstream proteins. As a result, it inhibits oncogenic signaling and tumor progression mediated by FGF/FGFRs or FGFRsDRM. The goal of this study is to investigate if the novel lead compound inhibits FGF/FGFRs oncogenic signaling and overcomes FGFRsDRM mediated drug resistance.

Statement of Methods

We used CRISPR/Cas9 mediated gene knockout to demonstrate that FRS2α is essential for FGF/FGFR signaling. The myristoylation of FRS2α was detected by Click chemistry. Additionally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were analyzed in numerous cancer cells. Prostate regeneration assay and xenograft tumor models were applied for in vivo studies.

Summary of the results

We demonstrate that loss of myristoylation of FRS2α, a scaffold protein key in FGFRs signaling, inhibits FGF/FGFRs‐mediated oncogenic signaling and FGF10‐induced tumorigenesis. Additionally, a previously synthesized myristoyl‐CoA analog, B13, which targets the activity of N‐myristoyltransferases, suppresses FRS2α myristoylation, and modulates the phosphorylation and localization of FRS2α at the cytoplasmic membrane. B13 exhibited an inhibitory effect on oncogenic signaling induced by wild type FGFRs and their drug resistant mutants (FGFRsDRM). The single agent or in collaboration with an FGFR inhibitor suppressed FGF induced wild type FGFR or FGFRDRM‐initiated PI3K and/or MAPK signaling, subsequently inhibiting cell proliferation and migration in a variety of cancer cell types by inducing cell cycle arrest. Finally, B13 significantly inhibited growth of xenograft tumors without pathological toxicity to the liver, kidney, or lung in vivo.

Conclusion

Our study demonstrates that B13, a myristoyl‐CoA analog, inhibits FGF/FGFR‐mediated oncogenic signaling and tumor progression. This study provides a novel therapeutic approach for inhibiting cancer progression mediated by FGF/FGFRs and their drug resistant mutants.

Support or Funding Information

NIH (R01CA172495) and DOD (W81XWH‐15‐1‐0507)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Seasonal Changes in the Central Nervous System of the Arctic Ground Squirrel
Authors: Frare Carla , Lokken Elisabeth , Drew Kelly
Published: June 2021

Hibernation is a seasonal adaptive strategy characterized by metabolic suppression and a decrease in body temperature (Tb). The Arctic Ground Squirrel (AGS) is an obligatory hibernator; the onset of hibernation is linked to the circannual rhythm even when AGS are housed at constant environmental conditions. This phenomenon suggests an endogenous circannual clock, which mechanism is unknown. We test the hypothesis that sleep‐awake pathways are differentially activated between summer and winter season. Summer season was defined by the lack of torpor bouts; winter season was defined by the presence of periodic torpor bouts. AGS, implanted with body temperature transmitter, were maintained at constant environmental conditions: 2°C and 4L:20D. Euthermic AGS, in winter and summer, were perfused with 4% paraformaldehyde and brains removed for immunohistochemical analysis. Free‐floating immunohistochemistry was used to localize active nuclei as indicated by cFos‐immunoreactivity (mouse anti‐cFos 1:20,000, Millipore). Data analysis was performed in R. Wake active neurons are localized in the Perifornical Nucleus and the Tuberomamillary Nucleus. Both nuclei show higher activation, measured as higher cFos+ count, in summer than winter. A lower wakefulness activity in winter suggests an increase in sleep propensity, supporting that hibernation is characterized by an increase in sleep. In addition the Raphe Pallidus, a region controlling thermoregulation, shows a lower activation in winter compared to summer. This result correlates with the lower euthermic body temperature of AGS in winter compared to summer. In conclusion, winter AGS are characterized by a seasonal decreased in wakefulness and thermogenic nuclei activity; these changes may underlie the mechanisms of the circannual endogenous clock.

Support or Funding Information

NIH R03 NS081637 NSF IOS‐1258179 Alaska INBRE P20GM103395 NIH TL4GM118992

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Correlating Enzymes to Antimicrobial Resistance in the Protein Data Bank
Authors: Salcedo Priscilla M. , Burley Stephen
Published: June 2021

Antibiotic resistant bacteria are predicted to be the leading cause of death worldwide, with an expected death rate of 10 million people, annually, by 2050. It is vital that antimicrobial resistance (AMR) be understood at the molecular level. The Protein Data Bank (PDB) archive holds over 130,000 3D structures of macromolecular machines that can be analyzed using the RCSB PDB website at RCSB.org. Studying these 3D structures enables scientific research and education in a variety of fields, including drug discovery, nanotechnology, green energy, and disease. In this study, PDB enzymes related to antibiotic inhibitors were identified and further analyzed using additional bioinformatics including The Comprehensive Antibiotic Resistance Database (CARD). CARD holds up to date clinically relevant antibiotic related information. CARD identified 4,688 Genbank IDs (protein accession codes) that are related to AMR. These codes were then put into the Uniprot website which holds protein sequences. As a result, 696 macromolecules related to AMR were found in the PDB. I specifically researched Metallo Beta Lactamases (MBL) because have a large correlation to AMR and have been studied in great detail, with many examples in the PDB. Prominent enzymes in this category include the New Delhi Beta Lactamase which can cleave most if not all antibiotics. This particular “superbug” was found to cleave penicillin, cephalosporin, and carbapenem antibiotics. Using 3D visualizations of beta lactamase enzymes a correlation between zinc ions and spectrum of antibiotic resistance was found. For example, if an enzyme had two zinc ions instead of one it could inhibit broad spectrum antibiotics. In conclusion, the findings of this research presented here will be used to develop novel AMR tab at RCSB.org that will provide antimicrobial details of enzymes that were unavailable before. In the near future a phylogenetic tree of AMR related enzymes and classification details will be implemented.

Support or Funding Information

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

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of gravidity on grasshopper oxygen delivery
Authors: Kirkton Scott D. , Altman Elizabeth K. , Zembrzuski Deanna
Published: June 2021

Although the insect tracheal system is extremely efficient, life history changes can reduce oxygen delivery. For example, intermolt growth results in compressed tracheae, lower tracheal volumes, and reduced femoral air sac ventilation rates. During gravidity, the egg mass of Schistocerca americana grasshoppers can reach up to 40% of their body mass. We investigated whether this increase in egg mass compressed tracheae, lowered tracheal volumes, and reduced ventilation rates in the abdomen and femur. Using micro‐dissections and live video analysis, we examined the tracheal system of thirty‐six female grasshoppers. We found that abdominal compression rates correlated with the inflation of proximal and distal femoral air sacs. In addition, abdominal compressibility decreased with gravidity, suggesting lower tidal volumes. However, there was a positive correlation between gravidity and tracheal compression rate. Furthermore, initial micro computed tomography findings also suggest that gravid females have reduced tracheal volumes. These findings suggest that gravid females compensate for reduced tidal and tracheal volumes by increasing tracheal ventilation rates to improve oxygen delivery.

Support or Funding Information

This research was funded in part by NSF award 1531850 to SDK.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acute Hypoxia Alters Ryanodine Receptor Activity in Pulmonary Arterial Myocytes of High Altitude Acclimatized Fetal and Adult Sheep
Authors: Thomas Jonathan , Yoo Timothy , Romero Monica , Puglisi Jose , Zhang Lubo , Wilson Sean M.
Published: June 2021

Intrauterine stress, such as long term high altitude hypoxia (LTH), can alter pulmonary function and reprogram fetal pulmonary vascular development, potentially causing respiratory distress and pulmonary hypertension after birth. Our recent data demonstrates that newborn LTH lambs exhibit exaggerated hypoxia‐induced pulmonary vasoconstriction (HPV). Previous evidence shows Ryanodine Receptor (RyR) activation is central to the HPV response. Furthermore, LTH disrupts RyR activated Ca2+ sparks in pulmonary arterial (PA) myocytes of sheep. Given the relationship between RyR and HPV responses, we tested the hypothesis that acute hypoxia (exposure to 4% O2) and LTH increase RyR mediated Ca2+ spark activity. This was tested by measuring the Ca2+ spark activity in PA myocytes of near‐term fetal lambs and adult sheep that resided at either low (335m, LA) or high (3801m, HA) altitude. Intracellular Ca2+ was recorded in myocytes of isolated PA tissues loaded with Fluo‐4 using line‐scan techniques on a confocal microscope. Line‐scan images were analyzed using custom software (SparkLab) to determine the number of cells displaying sparks, spark frequency, and spatial‐ temporal characteristics. Adult myocytes had greater prevalence and frequency of Ca2+ sparks relative to those of fetuses, confirming previous studies. Acute hypoxia significantly decreased spark prevalence and frequency in myocytes from both HA fetal and LA adult groups; however, acute hypoxia increased spark prevalence and frequency in HA adult sheep. Spark amplitude was decreased by acute hypoxia in the LA fetus but increased in the HA adult. Acute hypoxia also reduced spark duration in the fetus independent of altitude but increased the duration in HA adult. These results provide new insight into the influence of high altitude reprogramming of RyR function in PA of the fetus and adult and the roles their activity have during acute hypoxic exposures.

Support or Funding Information

This work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. JT was a Loma Linda University Summer Undergraduate Research Fellow while TY was a Walter E Macpherson Society Medical Student Summer Research Fellow.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cyclic Nucleotides Reduce Ryanodine Receptor Mediated Ca2+ Spark Activation Independent of Long Term Hypoxia in Ovine Fetal Pulmonary Arterial Myocytes
Authors: Guerrero Maria , Hough Steven , Romero Monica , Puglisi Jose , Zhang Lubo , Wilson Sean M.
Published: June 2021

Ryanodine receptors (RyR) release Ca2+ from smooth muscle sarcoplasmic reticulum in rapid events known as Ca2+ sparks. These Ca2+ sparks activate voltage and Ca2+ gated potassium channels (BK channels) that cause cell repolarization. Ca2+ spark‐BK channel mediated cell repolarization dilates many types of vessels and is essential to pulmonary arterial (PA) vasodilation during the fetal transition to breathing air. Infants who do not successfully negotiate this transition are at risk of developing pulmonary hypertension, which has significant morbidity and mortality. Previous work shows that long term hypoxia (LTH) decreases Ca2+ spark events in fetal pulmonary arterial smooth muscle cells (PASMCs) even though the Ca2+ stores have functional RyRs. Additional studies illustrate that cyclic nucleotides can increase RyR activity, which led us to test the hypothesis that cyclic nucleotides could rescue RyR activity in myocytes of LTH fetuses. This was examined by treating PAs isolated from low (700m; LA) and high altitude (3801m; HA) near‐term fetal sheep with exogenous cyclic AMP (cAMP) and cyclic GMP (cGMP) or the methylxanthine IBMX, a non‐specific cAMP and cGMP phosphodiesterase inhibitor. RyR activity was measured by confocal imaging of Ca2+ sparks in fluo‐4 labeled PASMCs. The spatial and temporal aspects to the Ca2+ sparks were evaluated using SparkLab, a custom software package. Contrary to our hypothesis, the prevalence of Ca2+ sparks decreased in the presence of cAMP, cGMP, and IBMX for PASMC from both LA and HA fetal sheep. These data suggest that cyclic nucleotides reduce instead of increase vasodilatory capacity via RyR‐BK channel coupling mechanisms. Furthermore, increased cyclic nucleotides had no effect on the spatial‐temporal aspects to the Ca2+ sparks. Even though the signaling pathway remains unclear, the reduction of Ca2+ sparks by cyclic nucleotides is potentially a novel mechanism of RyR modulation in sheep fetal PASMC. This data suggests that increasing cyclic nucleotides may be contra‐indicated in newborn infants who are at risk of developing pulmonary hypertension.

Support or Funding Information

This work is supported by The National Institutes of Health, Eunice Kennedy Shriver National Institute of Child Health and Human Development grant number HD083132, by the National Science Foundation under Grant No. MRI 0923559, and the Loma Linda University School of Medicine. SH was a Loma Linda University Summer Undergraduate Research Fellow.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects Of Ethynylestradiol‐3‐Sulfate (EE‐3‐S) On Blood Volume and Organ Function In Pigs With Severe Traumatic Hemorrhage
Authors: Martini Wenjun Z. , Xia Hui , Dubick Michael A.
Published: June 2021

Introduction

The beneficial effects of estrogens to improve survival from hemorrhage have been suggested in experimental studies of rodents and minipigs, as well as some clinical observations. However, the underlying possible protective mechanisms remain unclear. This study investigated the effects of ethynylestradiol‐3‐sulfate (EE‐3‐S) on blood volume and organ functions in pigs following severe traumatic hemorrhage and hypotensive resuscitation.

Methods

Twenty anesthetized and instrumented pigs were randomized into saline (n=10) and EE‐3‐S(n=10) groups. Femur fracture was performed in each pig's left leg, followed byhemorrhage of 55% of estimated blood volume and 15 min shock period. Afterwards, pigs were hypotensively resuscitated with a small volume of saline(4 ml/kg) or EE‐3‐S (1 mg/kg at concentration of 1 mg/ml, plus 3 ml saline/kg). Indocyanine green dye (5 ml of 1 mg/ml) was injected before femur fracture and hemorrhage and 15 min after EE‐3‐S administration with serial blood samples taken accordingly to determine changes of blood volume. Pigs remained anesthetized and were monitored for 6 h or until death, and blood samples were taken every 2 h for measurements of blood gases and chemistry.

Results

All baseline measurements are similar between the two groups. Femur fracture and hemorrhage reduced mean arterial pressure to 54±6% of baseline values and reduced blood volume from 104±4 ml/kg to 67±4 ml/kg (both p<0.05) and reduced Hctfrom 31±1% to 28±1% in the saline resuscitated group. Similar reductions were observed with EE‐3‐S resuscitation in blood volume (from 109±4 ml/kg to 68±3 ml/kg, p<0.05) and Hct (30±1% to 27±1%, p<0.05). Compared to baseline value, creatinine, asparate aminotransferase (AST), and lactate dehydrogenase(LDH) were elevated at 6 h or death from 1.3±0.1 mg/dL to 3.1± 0.2 mg/dL, 33±3 U/L to 98±16 U/L, and 450± 55 U/L to 645±122 U/L, respectively (all p<0.05). Similar increases were observed in creatinine (1.4±0.2 mg/dL to 2.9±0.1 mg/dL), AST (32±3 U/L to 135±46 U/L) and LDH (450±35 U/L to 501±95 U/L) in the EE‐3‐S group (all p<0.05). Survival time was similar between the saline (212±43 min) and EE‐3‐S (212±39 min) groups.

Conclusions

Following severe traumatic hemorrhage and small volume resuscitation, administration of EE‐3‐S did not affect blood volume, organ function or short term survival in this model.

Support or Funding Information

US Army Medical Research and Materiel Command

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SYP73 Is a Novel Membrane‐Associated Actin‐Binding Protein that Anchors the ER to the Actin Cytoskeleton in Arabidopsis
Authors: Cao Pengfei , Renna Luciana , Stefano Giovanni , Brandizzi Federica
Published: June 2021

The endoplasmic reticulum (ER) is an essential organelle that spreads throughout the cytoplasm and undergoes extensive remodeling. In plants, the remodeling and motility of the ER rely mainly on actin and to a minor extent on microtubules. Although the actomyosin system has been suggested functional for ER streaming in plant cells, the mechanisms underlying stable interaction of the ER membrane with actin are unknown. We demonstrate that SYP73, a member of the plant Syp7 subgroup of SNARE proteins is a novel ER membrane‐associated actin‐binding protein. We show that overexpression of SYP73 causes a striking rearrangement of the ER over actin and that loss of SYP73 reduces ER streaming and affects overall ER network morphology and plant growth. A potential mechanism that regulates SYP73 functions has been investigated. Although conserved domain prediction tools did not detect any actin‐binding domain in SYP73, we identified a fragment that is mainly responsible for SYP73 actin binding. Furthermore, the role of SYP73 in regulating actin reorganization has also been examined. We propose a model for plant ER remodeling whereby the dynamic rearrangement and streaming of the ER network depend on the propelling action of myosin‐XI over actin coupled with a SYP73‐mediated bridging, which dynamically anchors the ER membrane with actin filaments.

Support or Funding Information

This work is support by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy, the National Science Foundation, and MSU AgBioResearch.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of Cellular Hyperosmotic Stress Response Pathways using CRISPR in Fishes
Authors: Hamar Jens Carlton , Kültz Dietmar
Published: June 2021

Osmotic stress is one of the primary challenges faced by aquatic animals and their capacity to osmoregulate in response will dictate its survivability within a given set of conditions. The life history of many fish species includes periods of hyperosmotic stress. Two primary physiological responses a fish can employ to these conditions are systemic, attempting to maintain internal osmotic homeostasis, or cellular by adjusting the intracellular environment to the new osmotic gradient. Cell cultures can serve as an efficient model to study these pathways as seen in a cell line derived from O. mossambicus brain tissue (OmB) that retains much of the osmotically induced proteome phenotypes observed in adult tissues of this animal. Furthermore, cell culture models can facilitate these studies by providing an enhanced means to isolate and examine individual components, such as proteins and the genes that encode them, of the mechanisms behind adaption to osmotic challenges. Valuable functional information can be obtained through observation of specific phenotypes caused by genetic manipulations of these components. CRISPR/Cas9 gene editing systems have proven to be a potent means to induce targeted genetic manipulations in cell culture from a variety of vertebrate taxa including fish. The objective of these experiments is to optimize strategies to implement CRISPR/Cas9 targeting of relevant previously identified genes in OmB cells to help decipher cellular hyperosmotic stress response pathways. In this work using genetically modified OmB cells, we have shown CRISPR/Cas9 gene targeting to be successful in this cell line through targeting and reduction of transgenic enhanced green fluorescent protein (EGFP) expression and established a puromycin selection system through stable integration of puromycin resistance. Additionally, this work has demonstrated multiple interspecies promoters, both fish and mammalian, to be suitable for ectopic gene expression in fish cells that can be used in future applications. The results indicate CRISPR/Cas9 has the potential to be a proficient means to manipulate osmotic stress response relevant genes in the OmB cell model.

Support or Funding Information

Supported by NSF award 1656371 and BARD award IS‐4800‐15 R.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Trabecular bone structural variation in the human postcranial skeleton
Authors: Ryan Timothy M , Stephens Nicholas , Doershuk Lily J. , Saers Jaap P. P. , Jashashvili Tea , Carlson Kristian J. , Stock Jay T. , Gordon Adam D.
Published: June 2021

Modern humans have relatively lightly built skeletons compared to other primates and earlier hominins which may predispose recent humans to bone‐related health risks such as osteopenia and osteoporosis. Although recent work suggests a relationship between higher activity/mobility and increased postcranial trabecular bone structure, the amount of variation in skeletal phenotype across modern humans and the role of declining activity/mobility and other factors in driving this variation remain unclear. The goal of this study is to characterize variation in trabecular bone structure in the human postcranial skeleton in relation to variation in subsistence strategy, mobility, and physical activity. The seventh cervical vertebral body, proximal humerus, proximal femur, and distal tibia from 60 young adult individuals from six populations were microCT scanned. These groups represented a behavioral/mobility gradient spanning from forager to post‐industrial populations. Bone volume fraction (BV/TV) and degree of anisotropy (DA) were quantified in the entire trabecular volume for each bone using Medtool 4.2. Results for each bone were mapped onto a 3D tetrahedral mesh and these meshes were registered using common morphological characters for visualization and analysis. For all groups BV/TV was highest in the cervical vertebra, followed by the proximal femur, distal tibia, and proximal humerus. DA was highest in the distal tibia, followed by the femur, humerus, and vertebra. The hunter‐gatherers had the highest BV/TV and lowest DA. Statistical comparisons of BV/TV and DA found significant differences among groups for the cervical vertebra (BV/TV) and distal tibia (BV/TV, DA). Comparisons of trabecular structure within each bone indicate broad similarities between groups with some unique patterns potentially reflective of behavioral and activity differences. The results demonstrate the potential for identifying functionally‐relevant trabecular bone patterns among skeletal elements, but indicate significant within and between individual variation that may obscure behavioral signals. The data may be useful in medical contexts looking to identify which postcranial elements are more diagnostic of or susceptible to osteopenia/osteoporosis, allowing for more targeted intervention.

Support or Funding Information

Funding: NSF BCS‐1719187, NSF BCS‐1719140 RCUK/BBSRC grant BB/R01292X/1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The plant social network: Volatiles and their release
Authors: Doudareva Natalia
Published: June 2021

Plants have exploited the language of small chemicals for interacting with their environment more extensively than any other types of living organisms. An amazing diversity of volatile organic compounds (VOCs) released by plants play essential roles in their reproduction, defense, and communication, and influence atmospheric chemistry and climate. Plant VOCs are also used by humans as flavors, fragrances, biofuels, insecticides and pharmaceuticals. To date, the chemistry of plant volatiles is well understood, however, little is known about the biosynthesis of this diverse group of compounds and their release from the cell to the atmosphere. Presented results will show how the integration of metabolic profiling, functional genomic approach, targeted metabolic engineering with metabolic flux analysis and modeling can provide a comprehensive understanding of the regulation of flux through VOC metabolic networks. This presentation will also cover different aspects of volatile emission: from function in protecting plant reproductive organs via natural fumigation to promoting stigma development and seed yield. This presentation will also discuss molecular mechanisms responsible for deploying VOC from plant cells into the atmosphere.

Support or Funding Information

This work was supported by Grant IOS‐16555438 from the National Science Foundation and by the USDA National Institute of Food and Agriculture Hatch project 177845.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution and the single cell: Metabolic diversity in tomato
Authors: Last Robert
Published: June 2021

Research in the Last group focuses on specialized metabolism in glandular trichomes of tomato and other plants in the Solanaceae. Acylsugars – relatively simple metabolites typically composed of sucrose or glucose and C2 to C12 acylesters – are produced in the tip cell of the long hairs of domesticated and wild tomato. Despite being derived from simple chemical building blocks, tremendous variation in acylsugar structures is observed within and across species. Some of the changes in products and enzymatic activities have occurred over remarkably short evolutionary timeframes. The talk will describe our results documenting ‘recruitment’ of enzymes of central metabolism to acylsugar biosynthesis, as well as studies of BAHD acyltransferase evolution. I will emphasize how comparative biochemistry led to insights into how gene duplication, neofunctionalization and gene loss contributed to the impressive acylsugar diversity observed in domesticated and wild tomato species. Work towards understanding the evolutionary history of this metabolic network over tens of millions of years of evolution of the Solanaceae will be described.

Support or Funding Information

NSF IOS‐1546617 NIH 1T32GM110523 NSF CBET‐1565232

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mutation of a predicted DNA binding site on bacteriophage T4 Rad50 creates a constitutively active ATPase
Authors: Teklemariam Tibebe Antonios , Finnerty Ryan Michael , Nelson Scott William
Published: June 2021

The Mre11/Rad50 (MR) complex uses ATP to repair DNA double strand breaks. The mechanistic details of how ATP hydrolysis relates to DNA binding and processing, as well as the associated structural changes that occur within the MR complex heterotetramer are not fully understood. An amino acid sequence‐based analysis predicted a patch of positively charged residues on bacteriophage T4 Rad50 as a potential DNA binding site. Specific mutations were generated to neutralize the charge at this site and the mutant proteins were biochemically characterized. Consistent with the bioinformatic predictions, the mutant proteins bound double‐stranded DNA with lesser affinity than the wild‐type enzyme. Surprisingly, ATP hydrolysis assays indicated that the mutations created an enzyme that was fully activated, even in the absence of bound activator DNA. DNA exonuclease assays suggest that the ATP hydrolyzed by the mutant proteins is somehow uncoupled from normal repetitive cleavage of nucleotides by the MR complex. All together, these findings suggest that the point mutations have stabilized a conformational state of Rad50 that is normally induced by DNA binding and clearly demonstrate the allosteric relationship between the substrate DNA and the activator ATP. A complete investigation of these residues and their effect on allostery will lead to a more complete picture of the steps involved in the repair of harmful DNA breaks.

Support or Funding Information

NSF. Award number 1716269. The molecular mechanisms of the Mre11/Rad50 DNA repair complex

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Rfa2 N‐Terminal Phosphorylation on Adaptation Deficient Yeast
Authors: Hernandez Cristian Antonio , Baumgartner Trevor , Haring Stuart
Published: June 2021

Determining the role of the Replication Factor A2 (Rfa2) N‐terminus on cell cycle regulation and DNA damage checkpoint progression is crucial for studying how cells prevent genetic defects. Cells must be able to efficiently and accurately repair DNA damage in a temporally‐controlled manner. If cells are unable to do so, they are at a very high risk of developing permanent DNA mutations, which are directly correlated with cancer and other cellular diseases. To prevent mutations, it is especially important for cells to be able to address DNA damage before they replicate their DNA or segregate their chromosomes by mitosis. Before the phases of the cell cycle that involve DNA metabolism (i.e., DNA synthesis and mitosis), checkpoints are elicited to provide the necessary time to do one last check to ensure that the genome is intact. The N‐terminus of the single‐stranded DNA‐binding protein Rfa2 plays a crucial role in the response to DNA damage, especially in regulating checkpoint function. Checkpoints are utilized by all eukaryotic cells and require precise coordination of many proteins. Using yeast as a model eukaryotic organism, gene deletions of checkpoint machinery required for checkpoint exit (adaptation) were generated to assess the ability of cells to progress through cell cycle checkpoints. We show here how Rfa2 modification affects adaptation when these genes are deleted, to determine the pathway through which Rfa2 mediates checkpoint exit and the return to cell growth.

Support or Funding Information

National Science Foundation (NSF‐CAREER‐1253723) to Stuart Haring.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proteomic Dissection of the Spindle Assembly Checkpoint
Authors: Garcia Yenni Alejandra , Velasquez Erick , Gao Lucy , Whitelegge Julian , Torres Jorge
Published: June 2021

Human cell division is a critical process that ensures the accurate transmission of the genetic material from one mother cell to two cells. The mitotic Spindle Assembly Checkpoint (SAC) ensures the fidelity of this process by responding to errors in microtubule‐kinetochore attachment and tension, and arresting the cells in prometaphase to allow time to repair the errors before progressing into anaphase. The SAC core proteins include MAD2, MAD1, BUB1, BUBR1, and BUB3. To gain insight into the protein associations that are critical for regulating the SAC in a spatial‐temporal manner, we have employed a label‐free proximity‐based association mapping approach. We have generated BIOID2 stable cell lines for all of the core SAC proteins and the BioID2 tag alone as our control. We have verified the inducible expression of these BioID2 tagged SAC proteins and their localization to the kinetochore region at the time when the SAC is active. We have performed purifications from mitotic arrested cells and have generated proximity association networks for these SAC proteins. Although, proteomic approaches, like BioID2, have been widely used to identify novel cellular pathway components and to interrogate their functions, a common problem that we and other researchers face is what to call a “Hit” and what to call a “contaminant” in mass proteomic data, therefore we have developed a statistical proteomic pipeline in R that 1) is user friendly and 2) can be used to analyze mass proteomic data with any lab specific control and lab specific conditions to generate statistically significant results to inform on hits vs. contaminants. We have analyzed our proximity‐based purifications with this new computational approach and have identified known and novel associations. Our current and future work is focused on better understanding these identified associations by validating them, and assessing their roles in the activation and maintenance of the SAC pathway.

Support or Funding Information

1. NSF MCB1243645(JZT), NIH R01GM117475‐01 (JZT),

2. USHHS Ruth L. Kirschtein Institutional National Research Service Award #T32 CA009056 (YAG)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thinking Outside the Informatics Box: Computed Chemical Properties for Protein Function Annotation
Authors: Ondrechen Mary Jo , Mills Caitlyn L. , Ruffner Lydia A. , Beuning Penny J.
Published: June 2021

There are now over 14,000 Structural Genomics (SG) protein structures deposited in the Protein Data Bank (PDB) and most of these are of unknown or uncertain biochemical function. Reliable computational methods for the prediction of the function of protein structures is an important current need. Typically, functions are assigned using informatics‐based approaches. The annotation of protein function by automated means has led to high rates of misannotations in some databases. Here we present a complementary and powerful approach based on computed chemical properties of the individual residues in a protein structure. Partial Order Optimum Likelihood (POOL) is used to predict the residues in the query protein structure that are important for catalysis. Typically these include the residues in the first layer that make direct contact with the substrate molecule(s) and also some residues in the second and third layers that play supporting roles in the catalytic process. Then, for proteins of known biochemical function, Graph Representation of Active Sites for Prediction of Function (GRASP‐Func) establishes local arrays of POOL‐predicted residues that are common to proteins of the same function. Then local arrays of POOL‐predicted residues of the query (SG) protein are aligned with those of the proteins of known function for the different functional types. These alignments, each SG protein against each functional family, are scored in order to predict the most likely function of the SG proteins. Results are reported for the SG members of the Ribulose Phosphate Binding Barrel (RPBB), Clp‐Crotonase, and Haloacid Dehalogenase superfamilies. While we find the SG proteins in the RPBB superfamily to be well annotated, we predict very high annotation error rates (about 70%) in the Clp‐Crotonase superfamily. Of particular interest are cases of predicted misannotation, where our prediction differs from that of the assigned function. Experimental testing of our predictions is performed by direct biochemical assays to verify our predictions.

Support or Funding Information

National Science Foundation under grant number CHE‐1305655, MathWorks, Inc. and a PhRMA Foundation Fellowship awarded to CLM.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel metabolome and breath platforms to trace microbiome contribution to host metabolism
Authors: Assadi‐Porter Fariba M , Tonelli Marco , Regan Matthew D , Carey Hannah V
Published: June 2021

Host diet influences the structure and function of the microbiota in species that regularly consume food. To measure the functional contribution of microbiotas, we developed a metabolome‐microbiome platform (MMP) and novel stable isotopic labeling to trace microbiotas' metabolism in breath, cecum, and host tissues. We combined cavity ringdown spectroscopy to measure real‐time breath biomarkers and NMR‐based metabolomics to analyze metabolome profiles in tissues. We applied MMP to hibernating ground squirrels to assess the role of bacterial metabolism contributing to seasonal changes in diet. Previously, we showed that the long‐term fast of hibernation decreases relative abundance of bacterial taxa that are adept at degrading abundant plant glycans in summer versus their reduction in winter. Here, we determined the functional significance of seasonally changing microbiotas by gavaging Spring and Summer squirrels and aroused hibernators in Winter with 13C‐labeled complex and simple carbohydrates, and comparing against their respective intraperitoneal and antibiotics applications. Measurement of 13CO2/12CO2(δ13C) in breath was used as an index of bacterial degradation of 13C‐substrates and linked to observed changes in 1H‐[13C]‐metabolome profiles. Compared with robust responses in Summer changes in δ13C after 13C‐inulin gavage in hibernators were nearly abolished, whereas squirrels in Early Winter and Spring showed variable responses consistent with their transitional microbiotas. Metabolome profiles mirrored seasonal changes in 13C‐labeled short‐chain fatty acids. These suggest that during hibernation, the gut microbiota gradually loses the capacity to degrade a plant‐derived glycan, and a shift away from taxa that prefer dietary substrates. The results suggest MMP is a powerful tool in analysis of host‐microbe relationships.

Support or Funding Information

Supported by funds from NSF (IOS1558044), UW‐Madison Graduate and Veterinary Medicine.

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