bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three‐dimensional visualization of cranial soft tissues in embryonic and early postnatal mice using phosphotungstic acid enhanced microCT
Authors: Lesciotto Kate M. , Motch Perrine Susan M. , Kawasaki Mizuho , Stecko Timothy , Ryan Timothy M. , Kawasaki Kazuhiko , Richstmeier Joan T. 
Published: June 2021

Acquiring three‐dimensional imaging of animal specimens utilized in genetic, developmental, and evolutionary studies has typically been based on microCT (bone and mineralized tissues) and magnetic resonance microscopy (soft and un‐mineralized tissues). However, recent advances in microCT imaging have allowed for 1–5 μm resolution for small biological specimens, while typical microMRI imaging can generally only produce data with a resolution of approximately 30–40 μm. In order to achieve high‐resolution 3D visualization of soft tissues, the use of various contrast agents have been proposed, including iodine, osmium, and phosphotungstic acid (PTA). We investigated protocols for using PTA to stain soft tissues of the head in embryonic and early postnatal mice to enhance visualization of soft tissues by microCT imaging. Protocols for mice between embryonic day 13.5 and postnatal day 7 have been developed that allow for the visualization, segmentation, quantification, and analysis of soft tissue structures. Stained specimens were mounted in a 50:50 mix of polyester and paraffin waxes within a small tube and scanned on the GE v|tome|x L300 scanner in the Center for Quantitative Imaging at Pennsylvania State University with voxel dimensions between 1 and 10 μm, depending on specimen size. Multiple soft tissue structures, including the eye, inner ear, cartilage, muscle, glandular tissue, and brain, are easily differentiated in the resultant scans. Images of PTA‐stained specimens can be superimposed with microCT images of the same specimen before PTA staining to enable the study of relationships between hard and soft tissues in the same individual. This approach to both 2D and 3D visualization of soft tissues in murine models has immediate implications for understanding developmental processes and integration between hard and soft tissues of the cranium.Support or Funding InformationNational Science Foundation Grant BCS‐1731909, and NIDCR R01DE027677 and R01 DE022988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The paediatric knee: A medical imaging assessment analysis
Authors: PATHER Nalini 
Published: June 2021

IntroductionIn recent years, there has been an increase in sport‐related injuries in the paediatric population. Improved imaging of musculoskeletal structures has also raised the need to better understand the changing morphology of the musculoskeletal structures during infant and child development. This study therefore aimed to investigate the morphology of bony elements of the paediatric knee at different ages from medical imaging data.MethodsErect knee CT (n=60) and MRI (n=72) DICOM images were obtained for 132 paediatric patients between 2–15 years (mean, 11.35 ±3.18) of age. The morphometry of nine parameters including notch width (NW), bicondylar width (BW), notch width index (NWI), tibial eminence height (TEH) and width (TEW), and femoral and tibial growth plate height (GPH) and width (GPW) were analysed and correlated with age and sex.ResultsIn summary, the mean±SD for each parameter was: NW, 9.55 ±2.6; BW, 69.63 ±8.56; NWI, 0.14 ±0.03; TEH, 8.53 ±1.73; TEW, 27.52 ±4.12; FGPH, 1.25 ±0.71; and TGPH, 1.21 ±0.72. NW, BW, NWI, TEH and TEW had a significant positive linear correlation with age. NW, NWI and TEH had strong positive linear correlations with all variables except tibial slope, and femoral and tibial GPH. Femoral and tibial GPH correlated well with each other.ConclusionsApplied anatomical knowledge of paediatric knee to clinical practice can possibly improve diagnosis and patient management, and assist in predicting injury patterns. Further longitudinal studies will assist in accurately mapping growth of each knee structure with age.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 Undergraduate Student Misconceptions Regarding Pedigree Analysis
Authors: James Olena , Boury Nancy , Grimes Zachary , Reid Joshua , Wasendorf Chloe , Couch Brock , Seipelt‐Thiemann Rebecca 
Published: June 2021

Genetics is a difficult topic for undergraduate biology students to comprehend because the topics are abstract, complex, use specific terminology, and require thinking across multiple scales, including the symbolic scale. Education regarding the most basic genetics concepts begins in mid‐elementary grades with distinct concepts repeated and built upon through high school. However, students also transfer information from popular culture and through making their own meaning of observed phenomena. Therefore, when students arrive to a genetics course, they have pre‐instructional knowledge about genetics topics some of which are misconceptions and/or naïve conceptions. The aim of this project was to investigate common misconceptions and naïve conceptions in undergraduate courses for pedigree analysis. Pedigree analysis is a common tool that involves thinking across the symbolic scale, distinct terminology, and the application of Mendelian and non‐Mendelian genetic principles. Open‐ended questions were used to gather student answers for three distinct learning objectives. Student answers were analyzed and grouped according to each student's conceptual errors. Terminology, the ability to gather the appropriate information from questions, and the symbolic nature of pedigree analysis were major problems for students and represent major, cross‐cutting problems with many genetics concepts. In addition, students' answers also revealed problems with understanding autosomal versus sex‐linked inheritance as well as confusion about the nature and relationship of genes and alleles. Furthermore, some students used reasoning associated with population level genetics, which shows a lack of understanding about mathematical probability and its relationship to inheritance for an individual. Future studies aim to utilize these data to investigate a schema theory approach to formation and organization of conceptual understanding for pedigree analysis.Support or Funding InformationNSF Award : 1710262This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Integrating civic scientific literacy skills in a Biochemistry course
Authors: Taylor Ann TS 
Published: June 2021

Individuals and organizations have to make regular decisions about biochemical data in their everyday lives. At many higher education institutions, all students are required to take science courses with the goal of developing scientific literacy. Scientific literacy has several distinct meanings, but this project emphasizes civic scientific literacy, the understanding of contemporary science‐related issues necessary for informed community engagement. The hypothesis is that consumer and citizenship skills for evaluating data sources can be integrated into Biochemistry courses, specifically a one semester survey course of biochemistry for non‐science (liberal arts) majors. Parallel strategies were integrated to teach students to access biochemical and health related scientific information, analyze arguments, and decide on personal or community action. Students were taught how research studies are conducted and the limits of each model through a website called Study Design 101 (https://himmelfarb.gwu.edu/tutorials/studydesign101/) and to evaluate arguments with the Claim‐Evidence‐Reasoning model. These approaches were integrated into classroom activities and homework assignments. Personal and community action opportunities were provided through outside of class activities that students chose for themselves. The impact of these activities was assessed through performance on the activities, open ended exam questions, and an end of course survey. Students gained both biochemical content and skills in evaluating claims with evidence, and reported the course helped them better understand what scientists do and how to make decisions based upon scientific evidence.Support or Funding InformationThanks to the Haines Fund for the Study of Biochemistry at Wabash College.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of DNA damage suppression from the tardigrade (Ramazzottius varieornatus) gene Dsup in E. coli through ultraviolet radiation and hydrogen peroxide exposure
Authors: Pough Jason D , Wolyniak Michael J 
Published: June 2021

Tardigrades (genus Ramazzottius), also known as water bears, are microscopic aquatic multicellular organisms that can be found in just about any environment. The creatures are known to be extremotolerant, which is an oddity since most extremotolerant organisms are single‐celled bacteria or archaea. With well over 1000 different species, these stoic creatures can withstand dehydration, freezing, high pressures, and even the vacuum of outer space (Hashimoto 2017). Interestingly, while most mammals would find roughly 5–10 grays of ionizing radiation fatal, and some prokaryotes such as E. coli could survive roughly 50 grays, tardigrades can survive up to 5,000 grays of radiation. What allows tardigrades to survive radiation that would be fatal to most other organisms?In this research, we have chosen to focus on the Ramazzottius varieornatus Damage suppressor gene (Dsup). The gene produces proteins that contribute to an organism's tolerance to harsh environmental stresses by suppressing both single strand and double strand breaks in DNA and therefore improving overall radiotolerance (Hashimoto 2017). What makes the gene even more intriguing, however, is that it has been cultured in human and animal cells and found to improve radiotolerance and viability of irradiated cells. With the information known, there holds some potential for having this gene produce a practical method for fixing DNA breaks and preventing mutation from ionizing radiation (i.e. UV rays from the sun).To confirm the original findings as seem in Hashimoto et al. (2017) as well as refine them for the context of our laboratory, we transformed E. coli with a plasmid containing Dsup and subjected the resulting transformants to either ultraviolet light or hydrogen peroxide with the goal of challenging the DNA repair machinery as well as the ability of Dsup to prevent DNA breaks. DNA damage was assessed through the comet assay, a single‐cell gel electrophoresis technique for the detection of damage through silver nitrate staining. Overall, E. coli cells with the Dsup plasmid showed significantly lower levels of DNA damage when insulted with ultraviolet radiation. Ongoing research is assessing the effectiveness of Dsup in response to hydrogen peroxide damage. Going forward, we are working towards site‐directed mutagenesis experiments with the goal of better characterizing Dsup's role in suppressing DNA damage.Support or Funding InformationHampden‐Sydney College Office of Undergraduate ResearchThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New approach for the DNA damage/repair analysis: vertical comet assay
Authors: Williams Robert , McCoy Daniel , Chen Chenf‐fu , Podlutsky Andrej 
Published: June 2021

Ina traditional Comet Assay, the fluorescence of the “head” and “tail” regions of the comet is used to assess the level of DNA damage. The traditional Comet Assay technique issensitive and wildly used, however, also time‐consuming and results are inherently variable between different Las. The Vertical Comet Assay (V‐Comet, patent pending) aims to quantify the extent of DNA damage by collecting and analyzing the tail‐portions of comets. This is achieved through the collection of damaged DNA pieces into abuffer solution via electrophoresis, which can then be used to quantify the concentration of DNA or be used as a template for the qPCR. The collection of damaged DNA in this manner would allow for the improvement of bioanalytical and clinical assessments of genotoxic agents. Due to the capability to analyze the DNA fragments with qPCR, a gene‐specific damage profile can be established for a wide variety of genes, not just a whole‐genome assessment like in the traditional Comet Assay. Presently we use our technique for the comparison of isolated DNA from HeLa and HDFa cell lines following the X‐ray exposure conducted with/without the repair time. For the first time we could answer the question: do cells discriminate between genome regions to perform DNA repair? Our results are based on a comparison between DNA repair in the following regions: “junk” DNA, housekeeping genes and cancer‐related genes.Support or Funding Information1) NASA EPSCoR, NNH15ZHA003C 2) 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.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hookah and Vaping: Safe Nicotine Delivery Systems?
Authors: Dunham Rolaun , Bethishou Britiel , Skochko Alexander , Stone Koni K 
Published: June 2021

Hookah and vaping have recently been popularized as alternative to smoking tobacco. Hookah heats tobacco with a very hot piece of charcoal and then the air is pulled through the hot tobacco, this smoke is then pulled through water before it reaches the user's mouth and lungs. Vaping uses electricity to vaporize a propylene glycol/glycerine matrix that contains nicotine and flavoring agents. This vapor is then pulled directly into the mouth and lungs. The mainstream vapors from both hookah and vaping were collected on Cambridge filters, the filters were soaked in water and then the aqueous solutions were dried via vacuum centrifugation. After weighing the dried mass, the samples were reconstituted with water and then analyzed by UV spectroscopy; the Hookah samples had absorbance at 275 nm. Our original hypothesis was that the water soluble tar components would be trapped in the water that the hookah smoke is pulled through. Thus, there should have been no absorbance at 275nm. The vaping samples did not absorb at 275; since vaping does not burn or heat tobacco, we did not expect to find the same absorbance as the cigarette tar radical. Gel electrophoresis was used to analyze the nicking of plasmid (pUC18) DNA. Aqueous solutions of Hookah and vaping solutions were tested in our DNA nicking assay. Briefly, we use supercoiled pUC18 DNA, this DNA unwinds after a single nick. The supercoiled and nicked (open circular) DNA migrate as two distinct bands in the agarose gel. The density of each band is then quantitated to determine the amount of DNA nicking. The amount of nicking by both vaping and hookah solutions is orders of magnitude less that the nicking we have detected with cigarette smoke. However, we have detected aldehydes in both vaping and hookah solutions. Aldehydes are known to be toxic, primarily by their facile reaction with primary amines and thus their ability to wreck havoc with protein structure. We have implemented an assay to detect aldehydes in both hookah and vaping solutions. The reagent, Purpald, 4‐Amino‐3‐hydrazino‐5‐mercapto‐1,2,4‐triazole readily reacts with aldehydes and then the oxidized product of the reaction produces purple solutions. We then analyzed the purple solutions by UV/VIS. Based on the UV spectra, we have detected acetaldehydes in both vaping and hookah solutions.Support or Funding InformationR. Dunham is supported by the CSU, Stanislaus McNair Scholars 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.
Roles of High Mobility Group Box Proteins in Nucleotide Excision Repair‐associated Processing of DNA Interstrand Crosslinks
Authors: Gerberich Jillian , Mukherjee Anirban , Vasquez Karen 
Published: June 2021

Many traditional anti‐cancer chemotherapeutic agents induce DNA interstrand crosslinks (ICLs), lesions that form covalent bonds between the DNA strands. ICLs are highly cytotoxic if not properly repaired because they can block basic cellular processes such as transcription and replication. Nucleotide excision repair (NER) is a critical repair mechanism in processing ICLs in human cells. In addition to NER, ICLs are also processed through DNA double‐strand break repair pathways (e.g. homologous recombination) in S‐phase. We have previously demonstrated that the high mobility group box protein (HMGB1), a non‐histone architectural protein, can bind to site‐directed ICLs with high affinity and modulate error‐free repair processing of such lesions. In addition, we have found that HMGB1 associates with the NER damage recognition protein XPA in human cells and facilitates the recruitment of XPA to the damaged site. Further, we have demonstrated that HMGB1 can architecturally modify the damaged substrate by inducing negative supercoils, which may facilitate DNA damage processing by NER. We hypothesize that HMGB1 can modulate NER processing of ICLs by aiding NER damage‐recognition factors in locating damaged DNA. To test this hypothesis we are using a mutation‐reporter system to measure mutation frequencies and spectra generated by site‐directed ICLs in human cells. There are two NER subpathways that recognize DNA damage via different mechanisms. I am investigating in which pathway(s) HMGB1 is involved by utilizing various cell lines that are proficient or deficient for an essential recognition protein in each pathway. Additionally, we are investigating if HMGB2 and HMGB3, protein family members that are highly similar in structure to HMGB1, are also co‐factors in NER. Preliminary evidence suggests that HMGB1 may be involved in transcription coupled‐NER of site‐directed ICLs. If future evidence supports these data, this would indicate that HMGB1 could associate with transcription machinery and aid in recruiting other NER co‐factors, such as XPA. Future genetic, molecular biological, and biochemical assays will provide additional insight as to the roles of the HMGB proteins in ICL repair. The processing and repair of ICLs in human cells is not completely understood, and therefore a detailed study to define the molecular mechanisms of ICL processing is warranted. Identifying new proteins involved in ICL processing may lead to new pharmacological targets, which may help us to improve the outcome of cancer treatment.Support or Funding InformationNIH/NCI to K.V. (CA093729) and NSF GRFP to J.G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cruciform DNA structure formed at short inverted repeats: A source of genetic instability in vivo
Authors: Mandke Pooja P , Kompella Pallavi , Lu Steve , Wang Guliang , Vasquez Karen 
Published: June 2021

Genomic instability is a major driving force for many human diseases including cancer. Certain mutation “hotspot” regions of the human genome are susceptible to cancer‐associated instability. These regions often co‐localize with repetitive DNA sequences that can adopt alternative structures (i.e. non‐B DNA). One such structure is cruciform DNA which forms at inverted repeat sequences. Short inverted repeats (<100bp) are abundant in the human genome and our laboratory has shown that they are substantially enriched at translocation breakpoints in human cancer genomes. Also, they are mutagenic and stimulate the formation of DNA double strand breaks (DSBs) leading to deletions in mammalian and yeast cells. The DSBs can be caused by fork stalling (i.e. a replication‐dependent mechanism) or by cleavage of cruciform structures by the repair enzyme XPF (i.e. a replication‐independent mechanism). Together, these studies have provided a mechanistic insight into the role of short inverted repeats in disease etiology. To further delineate the biological significance of these sequences, we have developed a transgenic mouse model containing human inverted repeats. We hypothesize that short inverted repeats capable of forming cruciform structures increase genetic instability in vivo. To test this, we have introduced a recoverable mutation‐reporter shuttle vector, carrying either a GC‐rich short inverted repeat (GC mice) or B‐DNA‐forming sequence (control mice) into the mouse genome. Both male and female GC‐containing mice (n=7) and control mice (n=9) were aged for 2 years and the shuttle‐vector was subsequently recovered from the genomic DNA of spleen tissue. Transformation into competent cells was performed to obtain blue (wild‐type) and white (mutant) colonies. Mutation frequencies were calculated and mutation spectra were obtained by direct DNA sequencing. Preliminary data reveals that GC rich cruciform‐forming sequences induce a significantly higher mutation frequency than the control B‐DNA‐forming sequences. These preliminary results suggest that cruciform‐forming sequences contribute to genetic instability in vivo. The contribution of this study to cancer etiology is significant, as the results provide insights into the molecular changes induced by cruciform structures.Support or Funding InformationNIH/NCI support to KMV (CA193124 and CA093729)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Post‐transcriptional processing at the promoter proximal RNA polymerase II pausing. A possible mechanism for premature termination
Authors: Koney Nii Koney‐Kwaku , Perley Danielle , Guo Kai , Hur Junguk , Nechaev Sergei 
Published: June 2021

Promoter‐proximal RNA Polymerase II pausing, which occurs within the first 100 nucleotides of a gene, refers to a property of the Polymerase II to halt transcription temporarily but remain transcriptionally competent. Knowledge of proximally paused Polymerase II dynamics is important for understanding the regulation of mRNA production.At the paused site, Polymerase II can either elongate to resume mRNA synthesis or terminate to release a short RNA. We hypothesize that both events occur on genes and, furthermore, suggest that the equilibrium between elongation rate and termination rate at the pause site reflects regulatory inputs at the promoter.We adopted the small RNA sequencing approach to detect short RNA generated by paused Pol II genome‐wide on chromatin. Analysis of short RNA in subcellular fractions revealed genes that display RNA species with a 5′ monophosphate ends downstream of transcription start sites. The 3′ ends of these monophosphate RNAs reside within the location of the 3′ ends of RNAs from paused Polymerase II demonstrating they may be products of paused Polymerase II. Short RNAs generated by Paused Pol II may represent a novel class of small RNAs derived from promoter‐proximal transcription termination. Future experiments will focus on identity and functional roles of these transcripts.Support or Funding InformationNIH COBRE P20GM104360NIH R21CA217751This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.