bluuebunny/crossref_metadata_2025 huggingface dataset:
Identifying the Location of the Foramen Ovale via the Articular Eminences, Molars, and Occipital Condyles
Authors: Daubenspeck Madison R. , Zdilla Matthew J.
Published: June 2021

The foramen ovale of the sphenoid bone is often cannulated for the treatment of trigeminal neuralgia. The success of foramen ovale cannulation procedures depends upon the properly locating the foramen, most often via fluoroscopy. Miscannulation may result in severe adverse events including intracranial hemorrhage and death. Unfortunately, despite fluoroscopic guidance, miscannulations continue to occur. Therefore, study of the location of the foramen ovale relative to anatomical landmarks that are easily identifiable through fluoroscopy is warranted. This study assessed the location of the foramen ovale in 228 crania. Three lines drawn upon digital photographs were assessed with regard to whether or not they traversed the foramen ovale. The lines included 1) a sagittal line drawn through the posterior‐most molar; 2) a line drawn through both the posterior‐most molar and the lateral‐most aspect of the ipsilateral occipital condyle; and 3) a transverse line drawn between the articular eminences. Of all the lines, the transverse line most consistently traversed the foramen ovale, bilaterally (left side: 159:228, 69.7%; right side: 157:228, 68.9%; bilateral: 316:456, 69.3%). Likewise, sagittal lines traversed the foramen more often than not. Because the articular eminence is easily identifiable via fluoroscopy from a lateral projection, the results of this study suggest that the articular eminence, in particular, may be a useful landmark for the cannulation of the foramen ovale. Moreover, using the articular eminence in conjunction with the molars and occipital condyles may improve localization of the foramen ovale from both lateral and AP views.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.17.06] and [HEPC.dsr.14.13]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Three‐Dimensional Infraorbital Foramen Location Relative to Facial Landmarks: Preliminary Results
Authors: Hartnell Lauren E. , Koons Aaron W. , Lynch Hannah L. , Ganoe Maria R. , Lambert H Wayne , Zdilla Matthew J.
Published: June 2021

Infraorbital nerve block injections are performed by either an intraoral approach per the oral vestibule or an extraoral approach through the skin near the infraorbital foramen. Many studies have assessed the location of the IOF relative to bony landmarks, teeth, as well as soft tissue landmarks with mixed results. Few studies have assessed the location of the IOF in three dimensions. This study analyzed the location of the IOF relative to a midpoint between the inferolateral nasal aperture and inferolateral orbital rim via three‐dimensional models rendered from 18 adult crania. The distance from the midpoint to the IOF averaged 4.16±1.43 mm (mean±SD) on the left and 3.93±1.43 mm on the right. Wilcoxon matched‐pairs signed rank tests revealed no significant difference among sides (p=0.325). This report also addresses differences among the x, y, and z axes. These preliminary data demonstrate the utility of three‐dimensional analysis for the assessment of IOF location. Understanding the location of the IOF in three dimensions may aid in determining the best patient‐specific approach for infraorbital nerve block.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.17.06] and [HEPC.dsr.14.13]

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 Relationship of the Articular Eminence with the Mandibular Fossa: Implications for Temporomandibular Joint Mechanics
Authors: Bodnar Sarah E. , Zdilla Matthew J.
Published: June 2021

The temporomandibular joint varies from most joints in that it is ginglymoarthrodial, both a hinge and a gliding joint. Early opening of the mouth produces a rotational movement at the joint; however, with wide mouth opening, the mandibular condyle translates anteriorly beyond the articular eminence. In some circumstances, the translation of the mandibular condyle over the articular eminence produces a dislocation of the mandible with concomitant spasm of local musculature, thereby holding the mandible in an open position. In certain circumstances, conservative treatments may prevent future dislocations. However, occasionally, surgeries such as eminectomy may be warranted. Because of the aforementioned relationship of the articular eminence with the movement of the mandible, the anatomy of the articular eminence warrants study in order to attain a better understanding of the temporomandibular joint mechanics. Therefore, this study assessed the angle formed by the anterior and posterior boundaries of the articular eminence and the angle formed between the posterior border of the mandibular fossa with the anterior border of the mandibular fossa (i.e., the posterior border of the articular eminence)—angles A and B, respectively. A total of 102 left sides and 99 right sides were analyzed. The average angle A was 112.5±14.49° (Mean±SD) and the average angle B was 112.4±13.28°. Angles A and B were correlated (r=0.41; p<0.0001). Paired t‐tests revealed no statistically significant difference between angle A, bilaterally (t=1.543(80); p=0.1268). However, there was a statistically significant difference between left‐ and right‐sided angle B (t=4.045(77); p=0.0001). The results of this study suggest that there is bilateral asymmetry among mandibular fossae. Similarly, the anterior and posterior boundaries of the articular eminence do not differ significantly from side‐to‐side. Further study should incorporate the size of the articular eminence and mandibular fossa in relation to the angular data presented here.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.17.06] and [HEPC.dsr.14.13]

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fibrosis And Scarring of The Brachial And Sacral Plexus as Displayed by MRI/MRA/MRV
Authors: Collins James D. , Saxton Ernestina Howell , Gelabert Hugh Anthony , Carnes Alfred
Published: June 2021

Bilateral rounding of the shoulders (laxity) associated with kyphosis of the thoracic spine causes costoclavicular compression and brachial plexopathy. This form of thoracic outlet syndrome is usually not amenable to surgical treatment in older patients, particularly in severe kyphosis of the thoracic spine. Surgery alters fascial planes within the thorax and pelvis that causes fibrosis and scarring of the blood supply to the brachial and sacral plexus. The objective of this presentation is to display the sites of landmark anatomy compressing the brachial plexus that decreases venous return supply to the nerves causing a shortage of oxygen and glucose needed for cellular metabolism causing fibrosis and scarring of the soft tissues marginating the brachial and sacral plexus. Magnetic Resonance Imaging (MRI) is the only modality that displays fibrosis and scarring of fascial plane anatomy obstructing venous and lymphatic return not possible with ultrasound or Computerized Axial Tomography (CAT). The longer venous obstruction transient ischemia or permanent obstruction ischemia if unrelieved, progressively affect the nerve fibers increasing numbers to an increasing degree. Pathology develops with edematous swelling and vascular congestion. If the pressure is unrelieved and continues to increase, the nerve(s) suffer a first degree or a conduction block injury. Compression ischemia with degeneration and fibrosis develop. In absence of relief, the endoneurial tubes and funniculi atrophy increasing ischemia, fibrosis becomes marked. This presentation displays costoclavicular compression of the brachial and sacral plexus with fibrosis and scarring in two patients. One patient post multiple fractures of the thorax and pelvis complicated by thoracic outlet syndrome post pelvic hardware, and the second patient post cervical fusion and bilateral total shoulder and hip replacement with foot drop. Monitored bilateral MRI, MRA and MRV of the brachial plexus is the only modality of choice to display costoclavicular compression and fibrosis, and scarring of the brachial plexus.

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 MEK1/2 and MEK5 Pathway Disruption on Skeletal Phenotypes in Intact Female SCID Mice
Authors: Peacock Sarah J. , Middleton Kevin M. , Cavanaugh Jane , Wright Thomas , Burow Matthew , Barnes Van , Munmun Fahima , Witt‐Enderby Paula
Published: June 2021

Many receptor‐mediated cellular processes progress through mitogen‐activated protein kinase (MAPK) signaling pathways, which are initiated by activation of a surface receptor and ultimately lead to a change in cell physiology. Previous research has shown that the influence of melatonin on osteoblast activity is mediated in part through MAPK/ERK kinases (MEK) 1/2 and MEK5. Here we explore the possible effects of MEK 1/2 and MEK5 disruption on the skeletal structure in rodents with diurnal melatonin rhythms. To investigate the influence of MAPK signaling on bone mineral density and trabecular bone phenotypes, one group of SCID mice (SC; n = 4) was injected daily for a period of 30 days with a dual MEK1/2 and MEK5 inhibitor, SC‐1‐151. A second control group (n = 4) was injected daily with DMSO vehicle for the same period. At the end of the experimental period mice were euthanized, and tibiae were dissected free of soft tissues and fixed in formalin. Tibia were scanned at 9.61 μm3 voxel size for subsequent morphometric analyses. Regions of interest were identified on the scans and then analyzed for trabecular thickness (Tb.Th), trabecular spacing (Tb.Sp), bone volume fraction (BV/TV), and cortical bone mineral density (BMD) using ImageJ software. Resulting bone phenotypes were analyzed using Bayesian linear models. We found that the SC group showed credibly decreased BV/TV compared to the DMSO controls (Pr[SC < DMSO] = 0.83). However, comparisons of Tb.Th, Tb.Sp, and BMD indicated minimal credible differences between the SC and DMSO treatment groups. Overall, our results suggest that, while previous research has shown that disruption of the MAPK signaling pathway may have a negative effect on osteoblast activity, these effects may result in only subtle changes for trabecular bone phenotypes and no change in cortical bone mineralization. This project provides preliminary results for a larger‐scale project investigating the role of melatonin, MEK1/2, and MEK5 in bone formation.

Support or Funding Information

Research support: University of Missouri School of Medicine, National Institute of Arthritis and Musculoskeletal and Skin Diseases

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of an Evolutionarily Conserved RNA Polymerase II‐Associated Factor 1 (Paf1) Involved in Pancreatic Oncogenesis
Authors: Barman Priyanka , Ferdoush Jannatul , Kaja Amala , Karmakar Saswati , Uprety Bhawana , Batra Surinder K. , Bhaumik Sukesh R.
Published: June 2021

The multifunctional RNA polymerase II‐associated factor (Paf1) is highly conserved from yeast to humans and plays an important role in regulation of transcription and its associated processes. Hence, malfunctions and/or misregulations of Paf1 are associated with cellular pathologies. Recent studies found that Paf1 (also known as PD2 or pancreatic differentiation 2) is upregulated in poorly differentiated cancer cells, and such upregulation is involved in cellular transformation or oncogenesis. However, the basis for Paf1 upregulation in these cells remains largely unknown. In light of this, we have tested here the idea that the ubiquitin‐proteasome system (UPS) regulates the cellular abundance of Paf1. In this direction, we analyzed the role of UPS in regulation of Paf1's abundance in yeast. We find that Paf1 undergoes ubiquitylation and is degraded by the 26S proteasome in yeast, thus deciphering UPS regulation of an evolutionarily conserved factor, Paf1, involved in various cellular processes at the crossroads of the cancer networks. Likewise, Paf1 undergoes proteasomal degradation in well‐differentiated, but not poorly differentiated, pancreatic cancer cells, hence pointing to the UPS in upregulation of Paf1 in poorly differentiated cancers. Thus, our results reveal UPS regulation of Paf1 and suggest downregulation of UPS in elevating Paf1's abundance in poorly differentiated cancers. Further, we find that misregulation of Paf1's abundance alters transcription. Collectively, our results demonstrate that Paf1 is regulated by UPS to control transcription, thus providing a new regulatory mechanism of gene expression.

Support or Funding Information

NIH‐2R15GM088798‐02

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Construction of genome‐engineered mesenchymal stem cells secreting angiogenic or anti‐inflammatory factors for the treatment of acute kidney injury
Authors: Park Hye‐Jeong , Cho Jeong‐In , Jang Hyo‐Ju , Park Eui‐Jung , Choi Hyo‐Jung , Kwon Tae‐Hwan
Published: June 2021

Stem cell therapy has been proposed as a potential therapeutic strategy for acute kidney injury (AKI). By exploiting genome editing and cell sheet technology, we aimed to generate mesenchymal stem cells (MSCs) secreting angiogenic factors [vascular endothelial growth factor (VEGF) and angiopoietin‐1 (ANG1)] or anti‐inflammatory factors [erythropoietin (EPO) and a‐melanocyte stimulating hormone (a‐MSH)] for therapeutic application in AKI. To integrate each gene expression cassette into the safe harbor locus, AAVS1, of the human umbilical cord‐derived MSCs (hUC‐MSCs) chromosome, AAVS1‐targeting Zinc Finger Nuclease (ZFN) or AAVS1‐targeting CRISPR/Cas9 system was exploited. Junction PCR analysis demonstrated the ZFN‐ or CRISPR/Cas9‐aided gene integration in hUC‐MSCs. Flow cytometry and osteogenic and adipogenic differentiation assay revealed that stemness was maintained despite genome engineering. Protein measurement in conditioned media by ELISA and immunoblotting confirmed the production and secretion of each integrated gene product. In vitro assay demonstrated the angiogenic functions (increased HUVEC migration and increased mRNA expression of MMP‐9 and Tie‐2 in co‐cultured HUVEC) of genome‐engineered hUC‐MSCs secreting VEGF or ANG1. For the stem cell therapy in AKI, a scaffold‐free cell sheet system was established using a temperature‐responsive polymer (poly(N‐isopropylacrylamide). Taken together, cell sheet system of hUC‐MSCs secreting angiogenic or anti‐inflammatory factors is successfully established. This is to be examined in animal models of AKI to demonstrate the therapeutic effects of stem cell‐based regenerative strategy against AKI.

Support or Funding Information

This study was supported by the National Research Foundation of Korea Grants 2014R1A5A2009242 and 2016R1A2B4009365 funded by the Ministry of Science, ICT and Future Planning, Korea

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An mRNA‐rRNA Base Pairing Model for Efficient Protein Translation
Authors: Barr Will , Sheth Ruchi , Chatterji Om , Moon Hye Kyung , Weir Michael
Published: June 2021

The idea that base pairing between mRNAs and structural rRNAs of ribosomes might contribute to protein translation has long been an intriguing possibility. The 530 loop of 16S rRNA has been implicated in translation initiation, elongation and termination. This loop and the corresponding highly‐conserved loop in 18S rRNA are located in the mRNA entrance tunnel of ribosomes. As noted before high‐resolution ribosome structures were described, the 530 loop contains a 3‐nucleotide‐repeating pattern complementary to the 3‐nucleotide periodicity of protein open reading frames characterized by overrepresentation of (GCN)n. We find that the 3‐nucleotide periodicity is significantly enhanced downstream of translation start codons of highly expressed Saccharomyces cerevisiae genes with significant depression of G at nucleotides 2 and 3 of these codons. We propose that during translocation steps, exposed rRNA nucleotides of the 530 loop can transiently base pair with the second and third mRNA nucleotides of the incoming A‐site codon before engagement of the A‐site tRNA and that this promotes efficient launching of the ribosome through the start region codons permitting high protein expression. This cooperation between the A‐site and 530 loop requires a precise positioning of the mRNA reading frame, which if compromised in several adjacent codons leads to low translation levels.

Support or Funding Information

National Institutes of Health 1R15GM096228

Beckman Scholars Program, Arnold & Mabel Beckman 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.
Atypical Binding Behavior of Epsin‐like Clathrin Adaptor 1 to Diacylglycerol Pyrophosphate
Authors: Putta Priya , Kooijman Edgar E.
Published: June 2021

Epsin N‐terminal homology (ENTH) and AP‐180 N‐terminal homology (ANTH) domain proteins are membrane binding proteins known to interact with polyphosphoinositides (PIPs). They share function, structure and sequence similarity, and are grouped together as A/ENTH domain proteins. The ENTH domain contains a distinctive N‐terminal helix (Ho) region that plays a crucial role in membrane bending. This Ho helix is not usually found in ANTH domains. Epsin‐like Clathrin Adaptor 1 (ECA1) is one such A/ENTH protein that acts as an adaptor protein in clathrin mediated endocytosis. It interacts with clathrin via its C‐terminus, and PIPs via its N‐terminus. In addition to having ANTH domain sequence similarity, ECA1 also contains the distinctive Ho helix region that is generally found in the ENTH domain. ECA1 is therefore best characterized as an N‐ANTH domain protein with a unique combination of both ANTH and ENTH regions. ECA1 is known to play a role in stress signaling in plants and also binds to phosphatidic acid (PA) upon salt stress. Like proteins, membrane lipids undergo drastic changes during stress stimuli. PA is a lipid second messenger that rapidly and transiently increases under stress stimuli. Upon rise of PA levels another lipid, diacylglycerol pyrophosphate (DGPP) starts to accumulate. DGPP is phosphorylated PA. It is suggested to be synthesized at higher levels as a signal for PA attenuation during stress. We recently showed in vitro that ECA1‐PA ‐affinity is modulated as a function of membrane curvature stress. In this research, we investigate ECA1 affinity to DGPP. We hypothesize that the PA target ECA1 has affinity for its metabolite DGPP which is highly negatively charged and this affinity is variable at varying pH. We tested ECA1 affinity to DGPP using liposome binding assay along with other anionic lipids; i.e., phosphatidylserine, phosphatidylinositol (4)‐phosphate and phosphatidylinositol (4,5)‐bisphosphate in the presence and absence of phosphatidylethanolamine. Our results show striking evidence that protein‐lipid interactions are not simply based on electrostatics. We also show that higher charge density on membranes does not always correspond to higher protein affinity. In fact, DGPP showed lesser protein binding to ECA1 compared to PA although DGPP has a higher negative charge. This study brings us a step closer towards understanding the complexity of protein interactions with anionic lipids and opens the discussion to lipid homeostasis upon salt stress.

Support or Funding Information

Department of Biological Science, Kent State University and NSF CHE 1412920

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interactome mapping of the pathogen Helicobacter pylori using All‐vs‐All sequencing (AVA‐seq)
Authors: Ramadan Stephanie , Andrews Simeon , Al‐Thani Nayra , Ahmed Ikhlak , Malek Joel
Published: June 2021

A first step in discovering new drug targets is to identify protein‐protein interactions specific to pathogenic bacteria that could be disrupted by small molecules (Valkov et al., 2011; Kuzmanov and Emili 2013). We recently developed a novel method for studying protein‐protein interactions, termed All‐vs‐All sequencing (AVA‐seq), which is rapid and sensitive. This new technique dramatically reduces the effort needed to map all protein interactions in an organism and, for the first time in protein interaction mapping, utilizes the full power of next‐generation sequencing.

While this method is new, the bacterial two‐hybrid system it is based on is well validated (Dove et al., 2004). Our innovations on this original system are multiple. They include the fusion of two vectors to allow the genes encoding interacting proteins to remain fused to each other. This critical change is what allows the system to be compatible with next‐generation sequencing. Further changes include a liquid based selection rather than colony based. This allows us to detect protein interactions by changes in a population of potential interactors much the same way that RNA‐seq detect differentially expressed genes. The method returns very rich interaction data including the ability to localize the interacting domains between two proteins, quantitate their interaction strength and observe the interaction in multiple fusion forms for validation. AVA‐seq will revolutionize the way genome‐wide protein interaction maps (Interactomes) are produced.

Here we show method validation using Helicobacter pylori, both for its clinical importance and, more importantly, because its interactome has been mapped with the old, labor‐intensive yeast two‐hybrid approach so it offers an excellent validation set. This older method took 14 years to complete for H. pylori and we are close to completing a similar map in just a matter of months. Additionally, our preliminary results with known interactors in H. pylori attest to the sensitivity of the method.

Support or Funding Information

Funding for this research was provided by the Biomedical Research Program Grant from Qatar Foundation Research to Weill Cornell Medicine.

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 Related Roles of a Ubiquitin Processing Protease, Nutrient Sensor, and Cytidine Deaminase in the Growth‐to‐Development Transition of Dictyostelium Development
Authors: Nelson Brianna , Pandoy Ronald , Cook Kelia , Lindsey David
Published: June 2021

The ubiquitin processing protease, UbpA (a homolog of yeast Ubp14 and human IsoT/USP5), is required for the growth‐to‐development transition of Dictyostelium. To understand the role of UbpA in regulating this transition, ubpA− cells were used to elucidate UbpA‐dependent pathways. ubpA− cells were hypersensitive to oxidative and nitrosative stresses, and miss‐regulated genes at the growth‐to‐development transition, including lmcB. ubpA is required for growth‐stage expression of lmcB which is involved in sensing nutrient levels and signaling cells to develop upon starvation. Exogenous expression of ubpA in ubpA− cells restored lmcB transcript levels, while exogenous expression of lmcB partially restored the developmental defect in ubpA− cells. Interestingly, lmcB is downregulated in wild‐type cells once the growth‐to‐development transition has occurred. Pull down experiments utilizing His‐tagged LmcB and TAP‐tagged LmcB expressed in Dictyostelium identified putative interacting proteins that included LmcA, PsmC5, CdaA, and a cAMP‐inducible protein. Direct binding experiments confirmed the interaction between LmcB and LmcA, and between LmcB and CdaA, a cytidine deaminase. To investigate cytidine deaminase (CDA) activity levels at the growth‐to‐development transition, a CDA assay was adapted for Dictyostelium. Wild‐type cells showed relatively low levels of CDA activity when growing and substantially increased levels upon starvation. Both growing and starving ubpA− cells have high CDA levels similar to the starving wild type cells. It is possible that LmcB binds CdaA to repress CDA activity during growth; then, upon starvation, LmcB is downregulated releasing CdaA. These results will lead to a better understanding of the mechanisms cells use to sense starvation stress and respond by making the transition from growth to development.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biochemical Characterization of DszD, the Flavin Reductase Involved in Bacterial Biodesulfurization
Authors: Mendez Gilberto
Published: June 2021

Dibenzothiophene (DBT) is a compound found in crude oil that upon combustion releases sulfur dioxide. The current chemical process used to remove sulfur from crude oil, hydrodesulfurization (HDS), is inefficient because it fails to work effectively on heterocyclic organosulfur compounds, such as DBT. Biodesulfurization (BDS) is a promising alternative to HDS for industrial sulfur removal. Microorganisms, like the bacterium Rhodococcus erythropolis, utilize BDS enzymes to remove sulfur from these organosulfur compounds while retaining the hydrocarbon structure of the fossil fuel components. In our work, we focus on the flavin reductase from the 4S BDS pathway, dibenzothiophene flavin reductase (DszD) from R. erythropolis, which catalyzes the reduction of flavin for subsequent use of dibenzothiophene monooxygenases DszC and DszA. This project entails the use of biochemical and structural experimentation to fully comprehend catalysis of DszD. DszD has been successfully overexpressed and purified to homogeneity from E. coli. Kinetic parameters have been determined using steady‐state and transient‐state kinetics. Weare currently looking at binding interactions between DszD and its substrate, DBT. Our preliminary experiments suggest that DBT may be acting as an effector; which means that DBT is selectively binding to DszD and regulating its biological activity. We plan to follow similar experiments with other sulfur‐containing compounds found in crude oil to see there is a direct interaction between substrate and DszD. Our current work will ultimately propel our research towards the long‐term goal of improving the BDS pathway for widespread industrial use.

Support or Funding Information

This work was funded by the Research Corporation through CCSA 22672 and the NIH through Grant 5SC2AI109500

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Mutant Sunflower Acetoacetyl CoA Thiolase
Authors: Dyer James
Published: June 2021

Sunflower (Helianthus annus L) cotyledons have two thiolase activities that have been identified previously. Thiolase II (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase I (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism and other residues have been implicated in the stabilization of the enolate intermediate in the synthetic reaction. Site‐directed mutagenesis of the various amino acid residues was done and the effect of these mutations on the catalytic activity was measured.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Kinetic characterization of Staphylococcus aureus SpeG polyamine N‐acetyltransferase
Authors: Boeck Paloma , Renolo Rossellini , Forwood Jade , Kuhn Misty L.
Published: June 2021

SpeG is a bacterial polyamine N‐acetyltransferase that belongs to the Gcn5‐related N‐acetyltransferase (GNAT) superfamily. Members of this family have a diverse set of functions and are present in all domains of life. Polyamines are cationic molecules present in all organisms, with roles in nucleic acid and protein synthesis, the cell cycle, stress responses, and biofilm regulation. While SpeGs from Gram‐negative bacteria have been studied previously, SpeGs from Gram‐positive bacteria have not been kinetically characterized. Here, we present the kinetic characterization of SpeG from Staphylococcus aureus and compare our results with those of other bacterial polyamine acetyltransferases. Additionally, we present a structural comparison of all SpeG proteins that have been deposited into the Protein Data Bank from both Gram‐positive and Gram‐negative bacteria. An understanding of the structure/function relationships of these enzymes is critical to advancing our understanding of how polyamine concentrations are regulated across both Gram‐positive and Gram‐negative bacteria. This may lead to a deeper understanding of the evolution of polyamine acetyltransferases across domains of life and offer opportunities to apply this knowledge toward production of effective pharmaceuticals for infectious bacteria in the future.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Resolving the mechanism of glycine oxidation by GoxA using X‐ray crystallography
Authors: Avalos Dante Merced , Mamounis Kyle , Davidson Victor L. , Yukl Erik T.
Published: June 2021

GoxA, responsible for the oxidation of glycine, is the second oxidase observed to utilize a cysteine tryptophylquinone (CTQ) cofactor after the lysine oxidase LodA. All other known enzymes containing a CTQ cofactor are involved in dehydrogenation reactions. The CTQ cofactor is produced in a post‐translation modification on Trp697 of Pseudoalteromonas luteoviolacea GoxA by the flavoenzyme GoxB. GoxA catalyzes an oxidative reaction, resulting in a deamination of glycine, producing glyoxalate, H2O2, and ammonia. The mechanism of action is unknown for this process, and revealing it could provide insight for amino acids involved in an oxidative active site containing a CTQ cofactor. A proposed mechanism is supported by using high resolution X‐ray crystal structures of GoxA in its native and substrate bound forms. Overall, the crystal structure of GoxA is observed to be a dimer of dimers, with two chains working cooperatively in the active site of each other. Native GoxA crystals are yellow in color due to the CTQ cofactor. Upon anaerobic soaking in glycine, these crystals turned a blue color, suggesting a change in the electronic structure of CTQ. Indeed, the crystal structure of this intermediate reveals the substrate glycine covalently bound to the CTQ cofactor. The bond angles of the covalently bound intermediate as well as the color change upon its formation allow us to suggest the nature of this species along the GoxA catalytic pathway. The active site environments of these two crystal structures reveals residues that may provide key interactions with the glycine for the completion of the reaction. The importance of each residue will be confirmed in future work using site‐directed mutagenesis. Other intermediates in the reaction will be investigated using methods such as resonance Raman spectroscopy. These results will shed light on the vital amino acids necessary for oxidation by a CTQ cofactor. Comparing these results with those of LodA could determine how specificity arises in these active sites.

Support or Funding Information

This work was supported by National Institutes of Health Grant 1SC2GM111170‐01 from NIGMS. Dante Avalos Supported by New Mexico State University's Howard Hughes Medical Institute Research Scholars Program.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Safety and Efficacy of a Novel Curculigo orchioides Extract in Boosting Testosterone Levels in Male Rats
Authors: Swaroop Anand , Preuss Harry G. , Bagchi Manashi , Bagchi Debasis
Published: June 2021

Curculigo orchioides Gaertn (family Hypoxidaceae, also known as black or kali musli), is an endangered medicinal plant used for diverse medicinal purposes including impotency, aphrodisiac, diuretic, tonic, jaundice, and skin ailments. Phytochemical investigations of rhizomes revealed the presence of a novel phenolic glycoside, curculigoside, triterpenoid, saponins, flavones, cellulose, hemicellulose, and calcium oxalate. We developed a novel extract of Curculigo orchioides (Blamus™, standardized to 30% curculigosides) and assessed its dose‐and time‐dependent efficacy (0, 10, 25 and 50 mg/kg body weight p.o.) on body weight, serum free and total testosterone levels in male rats (200–230 grams; n = 6) over a period of 28 days. Blamus™ didn't cause any marked elevation in serum free testosterone levels at either10 or 25 mg/kg body weight doses, however, a 50 mg/kg body weight dose of Blamus™ induced a significant increase in serum free testosterone level (*p < 0.0001). However, no significant increases were observed in serum total testosterone levels at 0, 10, 25 or 50 mg/kg body weight doses of Blamus™. Extensive testicular histopathological analyses including investigations on the seminiferous tubules, spermatogenesis, sperm cell morphology, Leydig cells and Sertoli cells following treatment with either 0, 10, 25 or 50 mg/kg body weight doses of Blamus™ demonstrated dose‐dependent improvement in structural integrity. Furthermore, no significant changes were observed in serum SGOT, SGPT, BUN and creatinine levels following treatment with any of the given doses, which demonstrated its safety profiles. These data demonstrated that Blamus™ may serve as a safe and novel, natural testosterone booster and provide broad spectrum application is sports nutrition, muscle building and exercise pathophysiology.

Support or Funding Information

This Project was Funded by Cepham Inc., Piscataway, NJ

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Modified DNA Aptamers Targeting ENOX2
Authors: Fetter Lisa , Bonham Andrew J.
Published: June 2021

Human Ecto‐NOX Disulfide‐Thiol Exchanger 2 (ENOX2) is a cell‐surface metalocatalyst which is expressed solely on malignancies of all major human cancer types. ENOX2, along with its ubiquitous parent protein ENOX1, serve as terminal oxidases in plasma membrane electron transport chains, suggesting that such proteins are energetically essential for normal and malignant cell growth. Since ENOX2 is shed from malignancies into the sera and demonstrates site‐specific transcript variants, it presents an attractive target for both diagnostic blood testing and anti‐cancer therapeutic treatments. To better facilitate detection and targeting of ENOX2 in patient sera, a handful of next‐generation aptamers were identified and characterized to determine their specificity and affinity for human ENOX2. These so‐called X‐Aptamers include DNA oligonucleotides that have been chemically modified to further enhance target affinity while greatly diversifying oligonucleotide functionality. Ultimately, these novel ENOX2‐specific X‐Aptamers will be incorporated into an electrode‐based electrochemical biosensor platform to create a rapid, dose‐responsive readout for the presence of ENOX2.

Support or Funding Information

This work is supported by internal grant funding from the Metropolitan State University of Denver.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolite Concentrations Are Unstable during Cell Sorting
Authors: Llufrio Elizabeth M. , Wang Lingjue , Naser Fuad , Patti Gary J.
Published: June 2021

There is increasing interest in the application of metabolomics to animal models. When performing metabolomics with animal models, however, often researchers are focused on only one specific cell lineage. A common approach for purifying one cell type from a multicellular sample is fluorescent‐activated cell sorting (FACS). Cells can be sorted according to morphology or fluorescent markers, such as GFP. Although necessary to purify cell populations, FACS is a timely process that could potentially influence metabolism. In this study, we examined whether the process of cell sorting causes changes in metabolism, which may complicate interpretation in metabolic studies employing FACS. We used a simplified model in which astrocytes grown in a mono‐culture were either metabolically quenched with methanol immediately after harvesting or metabolically quenched after cell sorting. Samples were analyzed using tandem liquid chromatography/mass spectrometry (LC/MS) with both targeted and untargeted methods. We found that many metabolite concentrations, including central carbon metabolites, amino acids, and nucleotides, were altered after cell sorting. Some structural lipid pool sizes were unchanged. We conclude that cell sorting is not compatible with measuring metabolite concentrations by conventional metabolomics, and we explore several alternative metabolomics strategies that may provide more robust results.

Support or Funding Information

GJP received financial support for this work from the National Institutes of Health Grants R21 CA191097 and R35 ES028365, the Alfred P. Sloan Foundation, the Pew Scholars Program in the Biomedical Sciences, the Camille Dreyfus Foundation, and the Edward Mallinckrodt, Jr 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.
Increases in transmembrane glycoprotein NMB (GPNMB), phospho‐ERK1/2, and matrix metallopeptidase (MMP)‐9 follow decline in arylsulfatase B in cystic fibrosis
Authors: Tobacman Joanne Kramer , Bhattacharyya Sumit , Feferman Leo , Sharma Girish
Published: June 2021

In previous reports, decline in the enzyme arylsulfatase B (ARSB; N‐acetylgalactosamine‐ 4‐sulfatase) was identified in cystic fibrosis (CF) cells in culture and in circulating leukocytes from CF patients. Recently, the transmembrane glycoprotein NMB (GPNMB) was shown to be increased in a CF gene array and in Arylsulfatase B (ARSB; N‐acetylgalactosamine‐4‐sulfatase)‐null mice. These findings are consistent with previous reports that ARSB is reduced in cystic fibrosis (CF). A mechanism by which decline in ARSB, and the resultant increase in chondroitin 4‐sulfate, increased GPNMB expression has been reported. Subsequently, experiments were designed to assess the potential role of GPNMB in CF and the mechanisms by which GPNMB might contribute to CF pathophysiology. In serum and circulating leukocytes from CF and control patients, GPNMB levels were determined by ELISA. GPNMB was also measured in cells and spent media of CFTR‐corrected, CFTR‐uncorrected, and normal human bronchial epithelial cells (BEC) following ARSB siRNA or control siRNA. The GPNMB which co‐immunoprecipitated with β‐1 integrin was determined by ELISA. Matrix metalloproteinase (MMP)‐9 was measured, and the correlations between GPNMB and MMP‐9 in serum and spent media were determined. Phospho‐ERK1/2 in the circulating leukocytes and in the cultured cells was measured. The impact of excess exogenous RGD peptide and GPNMB siRNA on phospho‐ERK1/2 content was determined, and the impact of the RGD peptide, GPNMB siRNA, and ERK phosphorylation inhibitor on MMP‐9 mRNA expression was determined. GPNMB was markedly increased in cells and serum of CF patients compared to controls (p<0.0001, unpaired t‐test, two‐tailed). In uncorrected CF cells and in BEC following ARSB silencing, the GPNMB was significantly increased (p<0.0001; p<0.001). Silencing GPNMB or treatment with excess RGD peptide significantly inhibited the ARSB‐silencing induced increase in phospho‐ERK1/2 in the BEC. Silencing GPNMB, treatment with excess RGD peptide, or treatment with ERK phosphorylation inhibitor blocked the ARSB silencing‐induced increases in MMP‐9 expression in the normal BEC. Findings suggest that decline in ARSB activity caused by decline in CFTR function leads to increased GPNMB, phospho‐ERK1/2, and MMP‐9 expression, which may contribute to disruption of cell signaling and to organ dysfunction in CF.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Gαq/11 localization and trafficking in uveal melanoma
Authors: Randolph Clinita , Wedegaertner Philip
Published: June 2021

Constitutively activating mutations in Gαq and Gα11 (Gαq/11) have been reported in up to 93% of uveal melanomas. Although constitutively active Gαq/11 promote uveal melanoma tumorigenesis through activation of multiple downstream pathways, no therapies inhibit constitutively active Gαq/11. Studies suggest that upon activation of some Gα subunits increased palmitate turnover and increased cytoplasmic localization occur. The purpose of our studies is to understand the role of palmitoylation in trafficking and signaling of constitutively active Gαq/11 in uveal melanoma. Using live cell imaging and cellular fractionation of HEK 293 cells and uveal melanoma cells, constitutively active GαqQ209L and GαqQ209P show decreased localization at membranes compared to wild type Gαq, suggesting increased turnover of attached palmitate in constitutively active mutants. Moreover, immunofluorescence microscopy revealed that a palmitoylation‐deficient constitutively active mutant displays a complete loss of plasma membrane localization and an inability to signal as measured by Rho‐ and Rac‐dependent YAP translocation into the nucleus, TEAD‐dependent luciferase activity, and ERK phosphorylation. These studies demonstrate that palmitoylation of mutationally activated Gαq/11 is required for its signaling functions. Although live cell imaging and cellular fractionation experiments reveal decreased membrane localization of constitutively active Gαq compared to wild‐type Gαq, our studies suggest that complete loss of membrane localization abolishes constitutively active Gαqdependent signaling. Disruption of constitutively active Gαq/11 palmitoylation and localization may be an effective strategy for inhibiting constitutively active Gαq/11 oncogenic signaling in uveal melanoma.

Support or Funding Information

This research was funded by NIH grants R01GM056444‐16, R01GM056444‐16S1 (Research Supplements to Promote Diversity in Health‐Related Research) and T32 GM100836.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Trans‐phosphorylation Activates The Yeast Protein Kinase Kin2, An Ortholog Of Human Microtubule Affinity Regulating Kinase
Authors: Ghosh Chandrima , Sathe Leena , Dey Madhusudan
Published: June 2021

Yeast protein kinases Kin1 and its isoform Kin2 are orthologs of the human microtubule affinity‐regulating kinase (MARK), which are known to regulate multiple cellular processes, including cellular exocytosis and polarity. Recently, we show that Kin kinases regulate cellular protein homeostasis or proteostasis by an unknown mechanism. To decipher the role of Kin kinases in proteostasis network, we focused our studies on the Kin2 kinase. We observed that the N‐terminal half of Kin2 protein (residues 94–510, referred to here as the Kin2mini) fully complemented the Kin2 protein null strain. These data suggest that Kin2mini plays a primary role in cellular processes. The subsequent mass spectrometric analysis of the Kin2mini protein identified three phosphorylated peptides with five phospho‐acceptor residues. Mutations of only two phospho‐acceptor residues Y275 and T281 by non‐phosphorylatable alanines in a single protein (i.e., Kin2mini‐Y275A, T281A) completely abolished the kinase activity both in vivo and in vitro. These results suggest that phosphorylation of the residues Y275 and T281 are important for its kinase function. We also observed that substitution of the residue T281 by a phosphomimetic glutamate (i.e., Kin2mini‐Y275A, T281E) restored the Kin2 kinase function. These data suggest that T281 is the key phosphorylation residue for the Kin2 kinase function. Furthermore, we found that the kinase‐dead Kin2mini‐D248A mutant (D of the conserved catalytic HRD motif) was phosphorylated on the residue T281 in vivo and the recombinant glutathione S‐transferase (GST)‐fused Kin2‐T281E mutant protein, but not the GST‐Kin2 wild type protein, was an active kinase in vitro. Collectively, our data demonstrate that Kin2 kinase is activated by transphosphorylation on the residue T281 by an upstream kinase. Currently we are studying how an active Kin2 kinase initiates a signaling cascade to fine‐tune the proteostasis network. Characterization of the Kin‐kinase signaling pathway in yeast, we believe, will illuminate the inner workings of the MARK signaling pathway in human cells.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.