bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of volume of interest placement and size in trabecular bone quantification
Authors: Palmer Jenna Elizabeth , Ragni Anna , Chirchir Habiba
Published: June 2021

Experimental and observational studies have shown that trabecular bone adapts to greater mechanical loading by increasing in volume and changing strut direction. These trabecular bone properties i.e., bone volume/total volume (BV/TV) and degree of anisotropy (DA) are typically quantified by selecting a volume of interest (VOI) in an anatomical element. Whether or not the VOIs should be cuboidal or spherical has been widely debated and multiple studies have argued for the benefit of using spherical VOIs. In this study we use spherical VOIs. Using human femoral heads as the elements of interest, our study compares BV/TV and DA in spherical VOIs placed in different locations, and of different sizes in the same epiphyseal element with the goal of understanding the effect of VOI size and placement on trabecular bone properties. Using the program Quant3D, we first quantified BV/TV in a single large VOI with a diameter of 200 mm centrally placed in the femoral head. Secondly, we placed three smaller VOIs with 80 mm diameters throughout the bone, sampling trabecular bone in somewhat similar locations as the single larger VOI. We used the inbuilt Full Width Height Maximum (FWHM) thresholding method in Quant3D to threshold images. We used the Wilcoxon rank sum test to identify differences in BV/TV and DA in the two sets of VOIs. Preliminary results show that mean BV/TV of the N=10 single large VOIs are significantly greater (p<0.05) than in the small VOIs N=10. In contrast, mean DA of the smaller three VOIs and the single large VOI are not significantly different (p>0.05) from one another. These initial findings of BV/TV have the potential to improve our understanding the effects of VOI size and placement on observed trabecular bone results. Testing the utility of VOI size and placement is critical in ensuring accuracy in data collection as well as quality of results obtained.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Three‐Dimensional Measurement of Lateral Ventricles of the Brain and Their Spatial Orientation
Authors: Yang Gongchao , Chen Jian , Palmer Sam , Cui Dongmei
Published: June 2021

Three‐dimensional measurement for the stereoscopic models has not been well established, especially for the anatomical education purpose. The ventricles of the human brain are complex in shape, including two lateral ventricles, a third ventricle and a fourth ventricle. Each ventricle has its unique size and serves as a landmark for the anatomy orientation. Measuring of the brain's compartments has many clinical implications for neurodegenerative disorders, particularly Alzheimer's disease. In this study, the curvature angle, length and volume of each lateral ventricle has been measured using 2D and 3D measurements, and their measurements are used to compare the length, volume and the curvature angle of the left and right ventricles. The 2D and 3D measurement and the spatial orientation of the ventricles were displayed in the virtual 3D stereoscopic presentation for visualization and comparison. The 3D measurement of virtual model can be helpful for understanding the shape and angle of the ventricles, and its stereoscopic visualization may help students to better understand the relationship among these ventricles and associated structures, ease of learning curve for understanding of radiographic images. The 3D measurement provides a new approach for stereo measurement and quantitative evaluation of the 3D virtual models.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Heat Transfer Modeling of the Artiodactyl Carotid Rete
Authors: Turner Skylar D , Jones Caleb D , Kay David I , Bourke Jason M , Alexander Aaron , O'Brien Haley D
Published: June 2021

Cerebral thermoregulation is vital to maintaining functionality of the central nervous system as well as maintaining homeostasis. Because of this, cerebral thermoregulatory structures have a high ecologic and conservation potential. Artiodactyls possess a series of anatomical structures that function to cool blood entering the cranial space. Before entering the brain, the major cerebral arteries split into numerous branches, forming the carotid rete which is surrounded by a venous sinus. The venous sinus contains blood that has been cooled by the nasal cavity. This coupling forms a countercurrent heat exchanger consisting of warm blood from the carotid rete encased by cooled blood from the venous sinus. Following heat transfer, cooler arterial blood flows to the hypothalamus, reducing the physiological need to evaporatively counter heat‐stress and significantly reducing water loss. The efficacy of heat‐exchange should depend on arterial surface area (SA) and volume (V); however, preliminary field‐based, in‐vivo studies do not identify a correlation between brain cooling magnitude and carotid rete height or volume. This contradicts the expected result based on countercurrent heat exchange models. In order to explore the relationship between artiodactyl carotid rete SA and V on heat exchange efficiency, we applied computational fluid dynamic analysis to digital vascular datasets. Anatomical data collection included radiopaque latex vascular injection and CT scanning of a domestic goat, followed by 3D modeling in Avizo (9.5, ThermoFisher) to build digital models of the carotid rete and its surrounding venous sinus. We then simulated countercurrent blood flow for 3 models: the original anatomical SA and V, as well as manipulated higher V / lower SA and lower V / higher SA. We identify a positive relationship between heat exchange and surface area. This suggests that non‐structural factors, such as adrenergic tone, may be responsible for the lack of correlation seen in in‐vivo animals.

Support or Funding Information

Federal Work‐Study research assistantship to ST and CJ; Oklahoma State University and OSU Center for Health Sciences to DIK and HDO.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computationally Modeling Axonal Guidance Cue Expression in The Developing Olfactory Bulb
Authors: Desilets Bethany , Mobley Arie
Published: June 2021

Olfactory sensory neurons (OSNs) in most mammals have cell bodies in the olfactory epithelium (OE). OSNs project axons to glomeruli in the olfactory bulb (OB) of the brain. Developmentally, signal guidance cues direct axons to their target glomeruli. We created a program that displays a model of the OB using a series of sagittal section images based on in situ hybridization (ISH) images from the Allen Brain Atlas (ABA). The ABA's program, Brain Explorer 2, allows for the limited 3D visualization of gene expression data. Our program adds functionality by allowing users to clearly visualize and quantify the expression of several genes simultaneously via superimposition.

To optimize visualization of multiple ISH data sets simultaneously, we aimed to improve the signal to noise ration of those ISH data. We use color thresholding and density thresholding algorithms to computationally detect and filter guidance cue expression data. Color thresholds that establish gene expression from the background of the image were determined using ImageJ. The expression data were filtered using a density threshold in a 2D sliding window algorithm. The 2D sliding window algorithm is implemented for every pixel in the image. It distinguishes a window of the image around each pixel. The density of gene expression within that window is calculated. The expression for the current pixel is then decided using a density threshold. Methods for visualization of the superimposed genes were derived using the matplotlib and PIL libraries. Our program provides a tool for investigating olfactory axon targeting, glomeruli formation and glomeruli related pathologies in mammalian models. For example, users can examine the expression of different genes and investigate if guidance cue expression results in unique locations for mapping specific glomeruli. This program can also be adapted to examine guidance cue expression for other regions of nervous tissue.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of Field Intensity on Magnetic Resonance Imaging for Obtaining Three‐Dimensional Printed Models of Articular Patellar Cartilage
Authors: Vanni Fernando Pereira , Simão Marcelo Novelino , da Silva Airton Moreira , Nunes Amanda Amorin , Rezende Rodrigo Alvarenga , da Silva Jorge Vicente Lopes , Amaral André Capaldo
Published: June 2021

In recent research developed by our group, it was characterized and described the technical acquisition parameters of magnetic resonance imaging (MRI), with magnetic field intensity (MFi) of 1.5T, and its processing for printing three‐dimensional (3D) models of articular cartilage of patella. Such methodology should significantly contribute to the improvement of diagnosis, therapeutic planning and outcomes in the treatment of chondral patellar lesions. On the other hand, the influence of MFi in the processing and quality of 3D printed models was not established. The aim of this research was to characterize such influence, comparing qualitatively images and models generated from the intensities of 1.5 and 3T. For this, a healthy individual, female, 25 years old and with no orthopedic antecedents or complaints compatible with patellar chondropathy was evaluated. The patient was submitted to MRI of the knee in equipment of 1.5 T (HDxT ‐ General Electric®) and 3T (MD750 ‐ General Electric®). Volumetric axial sequences were established from 3D T2 Cube FS and 3D Fiesta HR sequences for both intensities. The 3D Merge FS sequence, available exclusively in the 3T equipment, was also considered in the analyzes. The images were processed in InVesalius® and Magics® software for the construction of 3D models in a virtual environment. The influence of MFi was established considering the image processing and the quality achieved in the virtual and printed models. The results showed that all sequences, in both intensities, allowed the generation of the models. In contrast, the quality achieved was higher in models obtained by the 3D Merge FS sequence. It was concluded that the MFi influences the process of obtaining 3D models of patellar articular cartilage. Therefore, the use of higher MFi is recommended due to the availability of more efficient acquisition sequences, such as Merge FS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A one semester biochemistry lab course with a partial research component
Authors: Park Chad K
Published: June 2021

We describe our continuing efforts to evolve a self‐directed, discovery based biochemical research laboratory curricula. As a required course for biochemistry undergraduate degrees, we emphasize science writing and literature review as well as molecular graphics and strucure‐function analysis. A challenge comes from training inexperienced students to become accomplished, troubleshooting scientists. The lab course provides instruction in scientific writing, as well as in the design, execution, and interpretation of biochemical research projects. The first goal is to train students in reading and understanding original peer‐reviewed scientific reports, as well as in writing in a scientific style (i.e. following Biochemistry format). The second goal is to bridge the gap between receiving detailed lab protocols and the ability to design, implement, troubleshoot and communicate protein based biochemical research investigations in small groups. Our efforts to develop and evolve this course in a land‐grant university are detailed here.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Visual Literacy: Using Art to Teach Biochemistry
Authors: Tansey John T
Published: June 2021

Modern students have grown up surrounded by stunning graphics and visual imagery. At the same time, advances in technology and how data are visualized has changed the practice of science. How do these two observations work for and against educators? A generation ago simple line drawings would have sufficed in a printed biochemistry text, but this is no longer the case. As digital media has progressed and ways that scientists represent data has changed, we as educators should ask if the two complement one another. In other words, can the images found in biochemistry help educators tell the story and enable student to better learn biochemistry? Are images a distraction, or, at worst, do figures in biochemistry teach misconceptions about biochemical principles? This talk will use examples of figures from a new work to show how art can help drive student understanding in biochemistry and help overall comprehension and higher‐order thinking.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Modeling of the PTF‐J and PTF‐L Heterotrimeric Complexes, 3‐D Printing, Overexpression, Purification and Structural Analysis of the Complexes using Cryo‐Electron Microscopy and Ptf1a gene Expression Patterns During Pancreas Development in Zebrafish
Authors: Coats Ward
Published: June 2021

Dallas Independent School District biomedical research students study the master regulatory transcription factor heterotrimeric complexes PTF‐J and PTF‐L that are required for the early and late developmental cascade of the pancreas. The PTF‐J and PTF‐L complexes are composed of a common E‐box protein E12/47, Ptf1a. and either RbpJ or RbpL. The PTF‐J complex is required for the early developmental stage of the pancreas and during the secondary transition of pancreas development, RbpJ is replaced by RbpL generating the PTF‐L complex, which is required for the final maturation steps for the development of the exocrine pancreas. In the current studies biomedical research student utilized the known structures for RbpJ, RpbL, the MyoD bHLH domain and E12/47 bHLH domain to generate molecular models bound to the enhancer sequence found in genes regulated by the heterotrimeric complexes during pancreas development. The PTF‐J and PTF‐L enhancer sequence is composed of an E‐box sequence separated by one to three helical turns from a TC‐box sequence. Biomedical research students performed energy minimization of the complexes and generate a model using 3‐D printing technology. Biomedical research students also study the temporal and spatial expression patterns of the PTF‐J and PTF‐L complexes during pancreas development in Zebrafish using immunofluorescence. The overall goals the program are threefold: first, to expose enthusiastic high school students to an authentic hands‐on biomedical research project, second, to solve the structure of the complexes using Cryo‐Electron Microscopy and third to identify the temporal and spatial expression pattern of the Ptf1a protein during pancreas development in Zebrafish.

Support or Funding Information

Junior League of Dallas

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unifying the Biochemistry Program of a Consolidated University through an ASBMB Student Chapter
Authors: Davis Nicole
Published: June 2021

In January 2017, the University System of Georgia Board of Regents announced and voted to consolidate two of its member institutions: Georgia Southern University (in Statesboro, GA) and Armstrong State University (in Savannah, GA). As of July 1, 2018, the two universities have fully merged. With the consolidation, the Biochemistry program on the Armstrong campus (formerly Armstrong State University) expanded to the Statesboro campus (Georgia Southern University). In an effort to unify the Biochemistry program and the two campuses, it was decided to create one student chapter of ASBMB. The campuses are 60 miles apart and there was little interaction between the student bodies. Here is presented how the chapter has organized and structured itself to be a single, unified chapter that bridges the physical gap and the cultural gap. We have used technology to allow students to participate in chapter meetings without needing to travel from their home campus and used both combined and campus‐separate social and outreach activities. We documented what we have done as a chapter, showing our successes, failures, student member feedback, and how we will move forward into the next academic year.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Statistical analysis of academic metrics as predictors of success on the MCAT for post‐baccalaureate pre‐medicine students
Authors: Dobrydneva Yuliya , Schwartz Lisa
Published: June 2021

Students who aspire to enter medical school have to take the MCAT (Medical College Admission Test). In 2015, the MCAT was redesigned, covering a new set of foundational concepts. In 2015, a 12‐month post‐baccalaureate pre‐medicine certificate program at the GW School of Medicine and Health Sciences was launched and the curriculum designed to address the changes to the new MCAT. Furthermore, the program prepares students for medical school coursework, like biochemistry, histology, physiology and pharmacology. The GW Post‐bac Pre‐med program is intended for the career changers who typically majored as undergraduates in non‐STEM fields, and thus have little or no science background (“scientifically naïve”). Therefore, in our admissions process, the need to identify predictors of future success in heavily scientific courses for students who have minimal prior exposure to sciences was recognized. Data were analyzed of the first three cohorts of forty two students to determine what metrics appeared to be positively or negatively associated with MCAT performance. The metrics included undergraduate GPA, standardized testing scores in high school (SAT and ACT), undergraduate degree major and performance in science courses in the post‐baccalaureate program.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparison of DNA repair in human cell lines
Authors: Rivera Kristian , McCoy Daniel , Williams Robert , Podlutsky Andrej
Published: June 2021

The study of genome repair dynamics and their variance among cell lines refines our understanding of cancer and may lead to better gene‐targeting therapies. Our study compares the genotoxic repair rates of two cell types: the immortal cervical cancer line HeLa and human dermal fibroblasts, or HDFa. We are employing the comet assay, a single cell gel electrophoresis technique, to quantify genetic damage after subjecting cells to varying doses of UV and X‐ray radiation. To form a comparison of repair rates between the cell lines, we are tracking levels of damage at specific increments of recovery time.

HeLa exhibits greater genome instability than HDFa, presumably due to flawed DNA repair mechanisms. As such, the expectation is that the cell lines will show differing repair rates; however, prior observation has suggested the opposite. Our results demonstrate a similar repair dynamics between these two cell lines following X‐ray and UV‐C light exposures. An investigation into gene‐specific repair might shed light on specifics of nucleotide and base excision repair pathways in HeLa and HDFa cells.

Support or Funding Information

1) Research reported in this publication was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under Award Numbers UL1GM118991, TL4GM118992, or RL5GM118990. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. UA is an AA/EO employer and educational institution and prohibits illegal discrimination against any individual: www.alaska.edu/titleIXcompliance/nondiscrimination.

2) NASA EPSCoR, NNH15ZHA003C

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Effect of Chromatin Environment on Susceptibility to Double Strand Breaks and Choice of Repair Mechanism
Authors: Rodier Kendall Marie , Ani Danika Ngozika , Burgess Rebecca C
Published: June 2021

Double strand breaks are an extremely dangerous type of DNA damage. When a cell detects them they undergo apoptosis if it isn't repaired. If repaired improperly, mutations resulting in disease states can accumulate, so understanding the susceptibility and mechanism of DSB repair is important. While it is known how DSBs are repaired, it is unknown how the surrounding chromatin environment affects the DSB susceptibility and the choice of repair mechanism. The goal of this study is to compare DSB frequency and repair in regions with different chromatin environments around the genome. To induce DSBs, we first compared Galactose‐inducible and Tetracycline (tet)‐inducible CRISPR systems for cutting frequency, and found that the tet‐inducible system produces DSBs at a higher frequency in the time course examined. In our study, we will use this tet‐inducible system to measure DSB frequency at different genome sites as well as probe repair pathway choice, first comparing three cut sites near active genes and three near repressed genes.

We first measured DSB frequency using quantitative PCR across break sites. Preliminary data shows that the repressed genes have higher levels of DSBs after 3 h of DSB induction. This suggests that the specific chromatin environment that surrounds the cut site that may contribute to the increased susceptibility to DSBs, and correlation to histone modifications is underway. Alternatively, the increased amount of DSBs may be due to a reduced repair efficiency in the repressed gene regions compared to the active gene regions, thus more double strand breaks were present at each time point. This a topic of ongoing investigation.

The survivor analysis tests the ability of cells to become resistant to continuous DSB induction due to imprecise repair of the cut site and destruction of the Cas9‐guide RNA recognition site. Calculating percentage of survivor colonies can give an estimate of the mutation frequency of a genomic locus. Preliminary results suggest that active chromatin locations might generally have fewer survivors, but more data and genomic loci must be examined. Interestingly, a few active loci appear to be hypermutable. The TDH2gene, for example, showed a multitude of inaccuracies when repairing after cutting, one to two orders of magnitude higher than other active genes, including its paralog, TDH3. Sequencing of the imprecisely joined regions is underway to investigate this further. In addition, we will be determining if there is a correlation between specific histone modifications and mutation frequency.

This work will help us better understand which locations in the genome are more susceptible to DSBs and to imprecise repair, and subsequent mutation, thus advancing our understanding of how DSB repair can drive genomic change.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modification of Ser7 of the RNA Polymerase II C‐terminal domain (CTD) regulates CTD phosphorylation patterns and transcription
Authors: Burkholder Nathaniel Tate , Sipe Sarah , Kumar Mukesh , Escobar Edwin , Irani Seema , Matthews Wendy , Sim Kelly , Brodbelt Jennifer , Zhang Yan Jessie
Published: June 2021

RNA Polymerase II subunit RPB1 in eukaryotes contains a conserved, repetitive C‐terminal Domain (CTD) consisting of predominantly Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 heptad repeats. While Ser2 and Ser5 are strongly conserved and serve as essential phosphorylation sites for recruiting specific transcription factors, the role of Ser7 is less understood as it is not well conserved nor seemingly required for general transcription. However, Ser7 is indeed phosphorylated across most genes in vivo suggesting that it is an important mark in transcription. We hypothesized that different mutations and modifications of the Ser7 site regulate the phosphorylation of other CTD residues which alter transcriptional outcomes. To test this hypothesis, we first generated GST‐CTD substrates with Ser7 mutated to residues with different charges like Glu and Lys to mimic specific chemical modifications or naturally occurring mutations. We then treated these substrates with different CTD kinases and used ultraviolet photodissociation mass spectrometry (UVPD‐MS) to analyze the resulting phosphorylation patterns. We found that the CTD kinase ABL1 preferentially targets Tyr1 adjacent to Glu7 while phosphorylating Tyr1 away from Lys7 or Arg7, showing that charge in the seventh CTD position can regulate CTD kinase activity. Furthermore, we observed increased ABL1 phosphorylation of Tyr1 adjacent to Gln7 which mimics Lys acetylation, suggesting that this CTD kinase regulation is reversible. To test how Ser7 modification can affect CTD phosphorylation and transcription in vivo, we used UVPD‐MS and ChIP‐Seq to analyze samples from cells expressing RPB1 with Ser7 mutated to Glu in every other repeat. Our analysis provides some of the first in vitro and in vivo evidence that Ser7 modification or mutation can control CTD phosphorylation patterns thereby regulating transcriptional outcome.

Support or Funding Information

NIH 1R01GM125882‐01A1 The Welch Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Cloning of a Fluorescence‐Based Reporter Plasmid for Cell‐Free Protein Synthesis
Authors: Dumeng Jeriel , Ferre Mariana , Rodriguez Jessica M , Rodriguez‐Martinez José A
Published: June 2021

Cell‐free protein expression, also known as in vitro protein synthesis is a convenient and fast method for protein production. It bypasses the need to culture living cells, which can be time and resource consuming. Among multiple cell‐free protein expression systems available, the wheat germ extract system has become a popular choice for producing eukaryotic proteins due to high yields and commercial availability. The goal of this work was to generate a fluorescence‐based reporter plasmid to be used in wheat germ based cell‐free protein expression system. Because of its unique optical properties, the Green Fluorescent Protein (deGFP) can be used as a positive control during high‐throughput synthesis of proteins by a cell‐free system. To construct the reporter plasmid, Gibson Assembly strategy was used to clone a deGFP gene in a wheat germ extract compatible plasmid. The deGFP gene was successfully cloned in two plasmids, bearing a hexahistidine tag on either the N‐, or C‐terminus of GFP. The clones were validated by Sanger DNA sequencing. After validation, protein expression was confirmed by western blotting using an anti‐His antibody. The reporter plasmid will be used as a positive control during high‐throughput production of eukaryotic transcription factors by wheat germ cell free protein synthesis.

Support or Funding Information

Research Initiative for Scientific Enhancement (RISE), GRANT 5R25GM061151‐17

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biophysical characterization of DNAJB1‐PRKACA in Fibrolamellar Hepatocellular Carcinoma
Authors: Le Quang Nguyen Trung
Published: June 2021

Fibrolamellar Hepatocellular Carcinoma (FL‐HCC) has historically been described as a rare and usually fatal pediatric liver cancer that almost exclusively affects adolescents and young adults without a history of underlying liver diseases. Occurring on a healthy background, FL‐HCC's lack of readily recognizable symptoms impedes early diagnosis and timely medical intervention that can prevent tumor's progression to more advanced and metastatic stages. Unfortunately, FL‐HCC is refractory to typically applicable curative regimens in traditional liver cancer such as radiation or chemotherapies, leaving surgical removal the only therapeutic mainstay. However, this approach is still unable to mitigate a 50% reoccurrence rate within 10–33 months.

A recent identification of a recurrent chimeric fusion between the catalytic subunit alpha (PRKACA) of protein kinase A, and the J‐domain of the heat shock protein 40 (DNAJB1) in virtually all patients marks an important milestone for the study of FL‐HCC's molecular pathogenesis. The DNAJB1‐PRKACA is central in several ongoing FL‐HCC studies as the primary tumorigenic driver.

We are performing a biophysical characterization of the chimera versus the wild‐type protein kinase A. For example, Circular Dichroism is being used to monitor the secondary structure of the chimera in comparison to PRKACA. Differences in thermodynamics and kinase activity due to the J‐domain's fusion in DNAJB1‐PRKACA chimera may modify the protein's interaction with its downstream phosphorylation targets, therefore changing protein kinase A's signaling network implicated in FL‐HCC development. Analysis on changes in the chimera's phosphorylation network relative to the wild‐type protein will provide a crucial insight into FL‐HCC pathogenesis, promoting the development of highly efficacious chemotherapies in treating this disease.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for the Binding of Synthetic Cannabinoids to Human and Bovine Serum Albumin
Authors: Vanderpuye Oluseyi Adewale , Agu Chiagoziem Sylvester
Published: June 2021

Albumin is the most abundant protein in blood and binds to a large variety of small molecules such as drugs, fatty acids, and other organic compounds. The distribution and overall concentrations of some drugs in blood have been proposed to be affected by albumin because otherwise poorly soluble compounds may circulate in larger amounts by being bound to albumin. Synthetic cannabinoids comprise over 200 compounds with low aqueous solubility that bind and activate integral membrane cannabinoid receptors in the brain and other tissues. Although synthetic cannabinoid use is global and has been associated with serious side effects there is limited information on factors affecting their toxicity. It is hypothesized that despite their diversity of structures, synthetic cannabinoids bind to serum albumin and this renders them more soluble in plasma. The goal of the current research was to test for the formation of complexes between synthetic cannabinoids and albumin by using electrophoresis, uv‐vis and fluorescence spectroscopy. In the presence but not in the absence of BSA, fluorescent bands at the position of albumin were seen for mixtures of human serum and bovine serum albumin with Bay 59–3074 and JWH‐015, Nile Red, timolol, coumarin and eosin. In the absence of these compounds, the albumin band was not fluorescent or colored. The colors of eosin, bromophenol blue and Nile red changed in the presence of albumin indicating an influence of albumin on the spectral properties of small compounds. The binding of compounds to albumin was further examined and demonstrated by by uv‐vis and fluorescence spectroscopy. It was concluded that the binding of synthetic cannabinoids to serum albumin was observed for the first time. It was also shown that agarose gel electrophoresis can be used in a novel fashion to detect the interaction of certain colored or fluorescent compounds with albumin.

Support or Funding Information

Center for Undergraduate Research, Albany State University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Examination of HSF Activation in a Saccharomyces cerevisiae Model for TPI Deficiency
Authors: Skolnick Kathryn , Schlosser Lily Jean , Barnes Carol , Koziara Kortnee , Fisher Isaac , Hrizo Stacy
Published: June 2021

Triose Phosphate Isomerase (TPI) is a glycolytic enzyme that catalyzes the inter‐conversion of DHAP to G3P. Mutations in TPI have been identified that are associated with a neurodegenerative disease in humans known as TPI deficiency. To study the cause of TPI deficiency pathogenesis, we constructed Saccharomyces cerevisiae yeast strains expressing six different TPI alleles that are associated with the development of TPI deficiency in humans and Drosophila melanogaster. Interestingly, not all of the TPI alleles exhibit similar characteristics in terms of stability, temperature sensitivity and activity. Yeast with the M80T allele exhibits significant changes in growth and metabolic activity at all temperatures. Yeast with the E104D allele exhibit an acute temperature sensitive phenotype for growth and metabolic activity at 37C. All of the alleles except C41Y demonstrate protein instability at 37C. This study further examines the role of protein folding in TPI deficiency disease pathogenesis. Yeast expressing the mutant TPI alleles were examined for activation of HSF, a cytoplasmic protein folding stress response transcription factor. Understanding the activation of cell stress pathways in TPI deficient yeast may lead to understanding of disease pathogenesis in TPI deficiency patients and provide potential pharmacological targets.

Support or Funding Information

Research is supported by an SRU Student Faculty Research Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A possible connection between plant longevity and the absence of protein fibrillation: basis for identifying aggregation inhibitors in plants
Authors: Otzen Daniel E , Ward Jane , Mohammad‐Beigi Hossein
Published: June 2021

The ability of proteins to aggregate to form well‐organized b‐sheet rich amyloid fibrils is increasingly viewed as a general if regrettable property of the polypeptide chain. Aggregation leads to diseases such as amyloidosis and neurodegeneration in humans and various mammalian species but is also found in a functional variety in both animals and microbes. However, there are to our knowledge no reports of amyloid formation in plants. Plants are also the source of a large number of aggregation‐inhibiting compounds. We reasoned that the two phenomena are connected and that the longer‐lived the plant, the more efficiently it can suppress unwanted protein aggregation. In support of this, we show that while protein extracts from the sugar maple tree A. saccharum fibrillate readily on their own, this process is efficiently abolished by addition of small molecule extracts from the same plant. Further analysis of 44 plants showed a correlation between plant longevity and ability to inhibit protein aggregation. Extracts from the best performing plant, the sugar maple, were subjected to chromatographic fractionation, leading to the identification of a large number of compounds, many of which were shown to inhibit aggregation in vitro. We suggest that plants have been under evolutionary pressure to maintain an efficient collection of aggregation‐inhibiting metabolites; consequently, long‐lived plants may be particularly appropriate sources of new anti‐aggregation compounds with therapeutic potential.

Support or Funding Information

D.E.O. was supported by a grant to D.E.O. from The Danish Council for Independent Research | Technology and Production Sciences (grant number: 12–126186), and Parkinson Foreningen (the Danish Parkinson Society). Rothamsted Research receives grant‐aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recruitment and Regulation of RPN13 in the 26S Proteasome
Authors: Hemmis Casey W , VanderLinden Ryan T , Yao Tingting , Robinson Howard , Hill Christopher P
Published: June 2021

The 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anti‐cancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain that is recognized by proteasome‐associated ubiquitin receptors. One of these receptors, RPN13, was identified as the most commonly overexpressed gene in solid tumors and has been directly implicated in cancers such as hepatocellular carcinoma, ovarian cancer, and leukemia.

RPN13 is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2. To better understand the interactions between RPN13, RPN2, and its substrates, we mapped the RPN13‐binding epitope in RPN2, and using structural and biochemical methods, validated the RPN2‐binding epitope, quantified binding interactions, and revealed details of RPN13 substrate binding. Examination of our structure revealed that the primary binding determinants in RPN2 bind into a small, surface exposed groove of RPN13. These features suggest that the RPN13‐RPN2 interaction could be modulated by small molecule compounds, thus making it an attractive target for developing orthogonal approaches to proteasome inhibition.

Our findings provide a detailed characterization of interactions that are important for proteasome function, and have major implications for the development of novel anti‐cancer therapeutics.

Support or Funding Information

This work was supported in part by National Institutes of Health Grants R01 GM059135 and R21 CA191929 (to C. P. H.) and American Cancer Society Postdoctoral Fellowship PF‐16‐100‐01 (to C.W.H.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenetic regulation of osteoblastogenesis by blackcurrant fruit extracts in vitro and in vivo
Authors: Mahady Gail B , Raut Nishikant , Lawal Temitope O , Patel Shital
Published: June 2021

Osteoporosis is a major public health concern and is characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to bone fragility and an increased susceptibility to fractures. Osteoporosis and osteopenia affect over 45 million Americans annually with an estimated cost of > $20 billion USD. Over 50% of women and ~13% of men over the age of 50 will suffer an osteoporosis‐related fracture in their lifetime resulting in loss of independence, health and productivity, thus the research and discovery of compounds that can enhance bone formation would be clinically significant. Blackcurrants (Ribes nigrum) are native to Central and Eastern Europe and Northern Asia and are used in traditional medicine for the treatment of cardiovascular disease, bone and muscle disorders, diabetes, viral infections and inflammation. The berries contain four major anthocyanins: delphinidin‐3‐O‐glucoside, delphinidin‐3‐O‐rutinoside, cyanidin‐3‐O‐glucoside, cyanidin‐3‐O‐rutinoside. We have previously shown that blackcurrant extracts (BCE) increase myoblastogenesis, and in this work, we investigate its effects in cultured hFOB human osteoblasts and osterix/Sp7:mCherry transgenic medaka. hFOB human osteoblasts were obtained from ATCC and cultured in 1:1 mixture of Ham's F12 Medium Dulbecco's Modified Eagle's medium, with 2.5 mM L‐glutamine (without phenol red). Double transgenic medaka (dT; osterix/Sp7:mCherry were maintained at UIC under protocol #17‐166. Larvae at 9–12 dpf were staged and then treated for 5 days with the extracts and compounds (1–10 mg/ml). Osteoblast formation and distribution were analyzed by fluorescence microscopy, bone mineralization was visualized by calcein. The results of this investigation demonstrate that BCE and delphinidin‐3‐glucose increased osteoblast proliferation and reduced apoptosis in cultured human osteoblasts (hFOB) by reducing Bax and p53 expression and altering HDAC1, HDAC3, SIRT 3 and PGC1amRNA expression. In osterix/Sp7:mCherry medaka, BCE treatment (10 mg/ml) increased osteoblast proliferation by increasing osterix/Sp7 expression. These data suggest that BCE and D3G increase osteoblastogenesis through epigenetic regulation of HDACs and mitochondrial biogenesis.

Support or Funding Information

NCCIH at the National Institutes of Health

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Evaluation of a Photoswitchable ABL Kinase Inhibitor as a Biochemical Tool
Authors: Swartz Brock , Streu Craig
Published: June 2021

With so much to learn about the various biological pathways that allow complex organisms to function, researchers are constantly working to expand their biochemical toolkit. A common way to study the role of an enzyme is to inhibit the protein with a drug and monitor effects such as metabolite buildup or increased activity in other enzymes. To assist these processes, the use of photoswitch molecules is beneficial. The electronics of these molecules allow them to change conformation in response to specific wavelengths of light. With the shape of a drug being so crucial to its binding affinity, molecules capable of changing their shape may be active in one conformation but inactive in the other. This allows these drugs to be activated and inactivated at will, allowing for more in‐depth studies of the spatial and temporal role of proteins. Azo‐stilbenes are among the most well established photoswitches. The azo‐stilbene structure, a chromophore commonly present in industrial dyes due to its vibrant colors, consists of two aromatic rings connected by an azo (N=N) bond. When irradiated with blue light (wavelength of 450–490 nm) these molecules undergo a trans‐to‐cis isomerization about the azo bond. Exposure to heat or red light (wavelength 620–750 nm) incites these molecules to relax back into their more thermodynamically favorable trans state. This poster will outline the synthesis and photophysical characterization of a photoswitchable inhibitor of ABL, a tyrosine kinase. A key component of the synthesis is the use of the Mills reaction, a condensation process that forms an azo bond between an amine and a nitroso compound. UV/Vis absorption spectroscopy was used to characterize properties such as the lambda max values for the cis and trans conformations as well as the thermal relaxation time. This molecule may also have potential for more targeted cancer treatments since the BCR‐ABL fusion gene causes chronic myeloid leukemia. By administering the drug in its inactive conformation and then irradiating sites to induce isomerization where cancer cells are present, unnecessary cell death may be avoided. Treatment of this sort could reduce the side effects of current CML treatments, namely low blood cell counts, which may result in infection and/or anemia.

Support or Funding Information

Albion College Foundation for Undergraduate Research, Scholarship, and Creative Activities

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of Antigen Binding Protein Monobodies as a Tool for Antigen Specific Treatment for Autoimmune Kidney Disease
Authors: Stocks Omar , Goldman Liam , Stoddard Shana Victoria
Published: June 2021

Autoimmune disease (AD) is a disease where antibodies incorrectly attack healthy self cells by binding to antigens. As a result of this self attacking patients experience long term sickness or even death. Current treatment methods include the use of non‐specific immunosuppressive medicines, which are somewhat effective at diminishing the effects of the disease. While effective, these therapies leave the patient with a significantly weakened immune system and decreased ability to fight off simple pathogens. This emphasizes the need for a more targeted treatment for AD. The work here focuses on the disease primary membranous nephropathy (PMN), which targets three domains of the phospholipase A2 receptor (PLA2R): the ricin domain and C‐type lectin domains numbers 1 and 7 (CTLD1 and CTLD7). The goal of this project is design of antigen specific treatment for PMN, which involves preventing autoantibodies from binding to the PLA2R antigen, through design of protein monobodies. In this work, the epitope regions on CTLD7 were predicted using the programs Epitopia and EPCES to define target sites for the epitope caps. The data suggest that there are three distinct epitope locations on CTLD7 that could be targeted by PMN autoantibodies. Currently, 26 epitope caps have been designed using the templates 3UYO and 5A40. The results show that in silico mutagenesis of protein monobodies enhanced binding of epitope caps to epitope sites on the CTLD7 domain from −4.94 Rosetta Energy Units (REU) on the 3UYO template to −5.094 REU, and −5.081 in 3UYO‐004‐OS and 3UYO‐022‐OS, respectively. In silico mutagenesis to the 5A40 template, producing the 5A40‐001‐OS cap, improved binding by approximately 2 REUs: improving the REU score from −5.618 to −7.596 to the CTLD7 domain. Results indicate that in the 5A40‐001‐OS cap, introduction of a negatively charged aspartate created an electrostatic interaction with CTLD7, which improved the potency of binding. The 5A40‐001‐OS cap along with 3UYO‐004‐OS and 3UYO‐022‐OS caps are being expressed in E. coli for further evaluation through ELISA assays. The results of this work could lead to a much safer and more antigen specific treatment for autoimmune disease.

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