bluuebunny/crossref_metadata_2025_split huggingface dataset:
Limb Strength and Efficacy of Strength Rehabilitation on an American Alligator
Authors: Wu Leslie Y. , Manfredi Kevin R. , Georgi Justin A. 
Published: June 2021

Several studies have looked at muscular strength in the jaws of crocodilians but there are little published data that examine the direct measurement of muscular strength in the limbs of crocodilians. When an Alligator mississippiensis was surrendered to the Phoenix Herpetological Society (PHS) with clear physical deficits, such as malnutrition and severe muscle atrophy, this subject was a suitable candidate for rehabilitation. We took this opportunity to measure the limb strength in healthy alligators over a range of sizes to assess the efficacy of the rehabilitation protocol we created for our afflicted alligator. Human physical therapy and companion animal rehabilitation are common and growing practices, but zoo animal rehabilitation is a relatively nascent field with very little published data. We designed rehabilitation exercises based on companion animal medicine.Three aspects of body strength and condition were compared: grip strength, limb strength, and body measurements. Grip strength was measured with an incline board by placing the alligators on an incline and measuring the angle before they slip; correlating angle with grip strength. Limb strength was measured by placed a cuff at the base of the tail and pulling the subject from behind and encouraging the subject to walk forward. Resistance was measured in Newtons. We tested alligators ranging from 74.1 cm to 114.4 cm allowing us to distinguish between growth related strength increases and our rehabilitation success.Data on normal alligators indicate an increase in limb strength with increasing size, however, with grip strength there is no indication that there is an increase with increasing size. Data from our rehabilitated subject indicate grip strength was far below standard levels initially, but is rapidly approaching normal levels. Limb strength was initially on the lower end of the normal levels for alligators smaller in size, and her growth in strength parallels that of the normal alligators. Rehabilitation is successful in companion animals and shows some degree of success in alligators. However, further investigation is required to explore why our limb strength results did not demonstrated a similar level of success to grip strength results. Rehabilitation is a rapidly growing field in both the human and veterinary profession; with our research we hope to expand current knowledge of alligator rehabilitation and improve biomechanical knowledge of alligator strength.Support or Funding InformationBiomedical Science Program at Midwestern University at Glendale, AZThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mylohyoid Variations and their Clinical Implications
Authors: Hartnell Layne S. , Bender‐Heine Adam N. , Lambert H Wayne , Zdilla Matthew J. 
Published: June 2021

The mylohyoid separates the sublingual and submandibular spaces and is, therefore, important with regard to the spread of infection and space occupying lesions. The mylohyoid may also be elevated in submental flap procedures. Therefore, mylohyoid variations are implicated in infection spread and are also important with regard to surgical planning. However, there have only been a few reported mylohyoid variations outside of mylohyoid boutonnieres. This report documents a case series of exotic mylohyoid variations including mylohyoid musculature that does not insert at the hyoid but rather inserts into the anterior digastric muscle belly, intermediate tendon of the digastric muscle, and geniohyoid. In each case, the mylohyoid is not a true mylohyoid because it does not insert into the hyoid. Understanding the potential for mylohyoid variation may improve clinical assessment, treatment, and prognosis in the suprahyoid region.Support or Funding InformationWV 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.
Wnt Pathway Involvement in Whole Body Regeneration in Botrylloides violaceus
Authors: Medina Elida , Blackmer Jacob , Roldan Isabella , Sumner Jack , Keeling Elena 
Published: June 2021

Colonial ascidians, or sea squirts, are invertebrate urochordates noted for their remarkable regenerative potential. One such organism, Botrylloides violaceus, is capable of undergoing whole body regeneration (WBR) from its peripheral vasculature. Studying the cellular mechanisms of regeneration in B. violaceus could provide insight into regenerative mechanisms in vertebrates. Specifically, since the Wnt signaling pathway has been consistently linked with regeneration in other organisms, we investigated its possible involvement in WBR for B. violaceus. Our results demonstrate that B. violaceus completed WBR an average of 4.5 days faster than control regenerates when treated with a Wnt agonist. This suggests that the Wnt pathway is involved in activating cellular signals that facilitate WBR in B. violaceus. We continue to investigate effects of other pharmacological manipulations of the Wnt pathway. We are also attempting to amplify possible Wnt homologs using PCR with degenerate primers derived from conserved Wnt sequences. Obtaining DNA sequence for B. violaceus Wnts will allow future experiments on Wnt gene expression during regeneration.Support or Funding InformationCalifornia Polytechnic State University, Biological Sciences Department; Frost Summer 2017 Undergraduate Research ProgramThis 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 female sexual development‐1 (fsd‐1) Transcript Structure, Expression, and Localization in the Fungus Neurospora crassa
Authors: Hurysz Thomas , Gebhardt Bryce , Pyatt Mary , Emmens Kathryn , Toufexis Christine , Hutchison Elizabeth 
Published: June 2021

Neurospora crassa is a filamentous ascomycete fungus that can reproduce sexually and asexually. The signaling pathways that control sexual development in N. crassa are not well characterized. FSD‐1 is an N. crassa transcription factor that controls both early and late stages of sexual development. Although we know that fsd‐1 regulates female sexual development (including fertility and spore maturation), little is known about (1) its transcriptional regulation, (2) RNA isoforms, (3) protein expression, and (4) localization. Intron and exon boundaries of fsd‐1 were mapped using reverse transcription (RT)‐PCR and sequencing and we determined that fsd‐1 can be expressed as three different transcripts, which differ in length and intron/exon structure. Quantitative RT‐PCR during a time course of sexual development showed that expression of fsd‐1 transcripts is not detectable until N. crassa initiates sexual development. In addition, one transcript variant is predominantly expressed during sexual development, and expression of this transcript peaks approximately five days after crossing (mating). It is unknown whether all three fsd‐1 transcripts are translated into protein, and thus we will monitor FSD‐1 protein expression using immunoprecipitation and western blotting. The three fsd‐1 transcript variants encode for predicted proteins of slightly different sizes, which can be visualized via western blot. To investigate FSD‐1 localization, we constructed a GFP‐tagged version of FSD‐1 using overlap PCR and targeted this construct to the native locus. FSD‐1‐GFP localization was monitored via fluorescence microscopy throughout a time course of development. We found that FSD‐1‐GFP localized to the nuclei of female reproductive tissues, such as paraphyses, but not to tissues where meiosis occurs. In addition, we confirmed that the FSD‐1‐GFP strain complements the sexual development‐related phenotypes that occur in an fsd‐1 knockout strain, such as sterility and lack of ascospore melanization. Finally, we constructed strains where fsd‐1 has an overexpression promoter, and we will determine whether overexpression of fsd‐1 has a dominant negative effect on wild‐type strains during mating. Little is known about the regulatory pathways that function in non‐meiotic filamentous fungal female sexual tissues, and our work on fsd‐1 provides an important first step in characterizing this aspect of fungal development.Support or Funding InformationGeneseo FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Systematic Screening for Transcriptional Regulators of Adult Myogenesis in Drosophila by RNAi
Authors: Soudachanh Tommy , Oas Sandy , Mendes Tyler , Byrantsev Anton , Cripps Richard 
Published: June 2021

The muscles of Drosophila show similar structure and pathway of development to the muscles of humans, and allows Drosophila to model human muscle disorders. However, little is known about many of the genes and their potential role in muscle development. Thus, the focus of this project is to efficiently screen for potential transcriptional regulators of muscle development and identify associated disorder phenotypes. Genes were selected based on having a potential role in transcription, which were silenced using RNA interference (RNAi). The 1151‐gal4 driver was utilized to induce RNAi in developing adult muscles. Combined with the driver, the flies harbored enhancers of either Flightin‐LacZ or TpnC41C‐LacZ. The resulting transgenic flies were collected and assayed to measure the effects of this genetic loss. All 101 genes in the latest set have been assayed for a change of enhancer activity of less than 70% or greater than 130%, in Flightin and TpnC41C; the genes selected for immunostaining exhibited semi‐lethality or lethality, and indicated a loss or gain of LacZ. The immunostaining would reveal several factors: muscle morphology, and expansion or loss of enhancer activity associated with specific flight and jump muscle genes. The resulting data was classified into three categories, severe loss, moderate loss, or no change in muscle development. Genes with a severe loss indicates a potential issue with myoblasts fusion, and genes with a moderate loss indicates issues with adult muscle formation and maturation. Therefore, the results demonstrate that the screen is efficient in identifying potential transcriptional regulators and muscle disorder phenotypes.Support or Funding InformationNational Institute of Health ROI GM061738This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Interaction of Positive Coactivator 4 with Histone 3.3 Protein is Essential for Transcriptional Activation of the Luteinizing Hormone Receptor Gene
Authors: Kavarthapu Raghuveer , Zhao Peng , Liao Mingjuan , Dufau Maria 
Published: June 2021

The luteinizing hormone receptor (LHR) is a member of the G protein‐coupled receptor family which is essential for sexual development and reproduction in mammals. Previous studies from our laboratory established that Sp1 is important for basal LHR promoter activity and has central role in derepression of LHR gene transcription induced by the histone deacetylase inhibitor Trichostatin A (TSA) in MCF‐7 cancer cells. Moreover, our studies revealed that the co‐activator PC4 which associates directly with Sp1 at the LHR promoter is essential for TSA‐mediated LHR transcription. The present study explores interactions of PC4 with histone proteins which presumably triggers chromatin modifications during LHR transcriptional activation induced by TSA. TSA treatment of MCF‐7 cells transiently expressing PC4‐Flag protein induces acetylation of H3 and immunoprecipitation (IP) studies revealed its interaction with PC4‐Flag protein. Mass spectrometry analysis from the protein complex obtained after IP with PC4 flag in TSA treated MCF‐7 samples detected H3.3 acetylated at lysine residues (K9, K14, K18, K23, K27 and K36) as a PC4 interacting protein. This association of PC4 with H3.3 was corroborated by re‐ChIP. In subsequent studies by IP and reChip analysis using specific antibodies against acetylated H3 protein for the lysine residues, also confirmed PC4 interaction with acetylated H3 protein. Knockdown of PC4 in MCF‐7 cells significantly reduced H3 protein acetylation at these sites and LHR promoter activity in TSA treated cells despite an increase in H3 and H3.3, linking PC4 expression to H3.3 recruitment and its acetylation. Depletion of H3.3 A/B in MCF7 cells significantly impair recruitment of Pol II and TFIIB to the LHR promoter and its activation induced by TSA. In this study, we have shown interaction between PC4 and acetylated H3.3 in LHR induced derepression by TSA. Together these findings point to the critical role of PC4 and it is associated acetylated H3.3 protein in TSA‐induced LHR gene transcription.Support or Funding InformationIntramural Research Program, National Institute of Child Health and Human Development, NIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the Regulatory Mechanism of the Transcription Factor Krüppel homolog 1 in Mosquito Reproduction
Authors: Ahmed Tahmina Hossain , Zhu Jinsong 
Published: June 2021

Insect juvenile hormone (JH) plays a crucial role during reproduction in adult mosquitoes. Krüppel homolog 1 (Kr‐h1) is a transcription factor that is upregulated by JH. However, the molecular action of AaKr‐h1 in the JH‐regulated gene expression has not yet been well studied in adult mosquitoes. Our recent ChIP‐seq study has indicated that Kr‐h1 in Aedes aegypti binds to the promoter regions of genes that are under the control of JH or another crucial insect hormone, 20‐hydroxyecdysone (20E). Therefore, we hypothesize that AaKr‐h1 modulates the JH and 20E responses in adult mosquitoes. We have performed RNAi mediated gene silencing to examine the regulatory function of AaKr‐h1 on the selected JH (Met, Early Trypsin) and 20E (EcRA, HR3, E75A, βFTZ‐F1, and E93) responsive genes. Successful knockdown at the mRNA and protein levels was confirmed by Quantitative PCR (qPCR) and Western blot analyses respectively. In the AaKr‐h1 depleted mosquitoes, most of the selected JH and 20E responsive genes showed considerable alteration in their expression. Knockdown of AaKr‐h1 led to an increase in the expression of Early Trypsin and a significant reduction in the expression of Met, EcRA, HR3, and E93. In conclusion, our study showed that AaKr‐h1 regulates the expression of diverse genes that are crucial for egg maturation in adult female mosquitoes. Ae. aegypti is a vector of several tropical diseases including yellow fever, dengue, chikungunya and zika fever. The growing resistance to many of the currently used insecticides in mosquitoes creates an urgent need to develop novel insecticides with unique modes of action. This study will advance our understanding of the regulatory mechanism of AaKr‐h1 during reproduction and might provide a new target for novel insecticides to suppress mosquito population.Support or Funding InformationNIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Rapid, Simple, High‐throughput Compatible Approach to Generating CRISPR/Cas9 Knock‐out Cell Lines
Authors: Wu Meiye , Okino Steven , Uy Gerald , Woo Deanna , Shulewitz Mark , Wang Yan 
Published: June 2021

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and its associated endonuclease Cas9 have emerged as a revolutionary genome engineering tool. Using CRISPR/Cas9 to induce mutations in protein encoding genomic DNA regions is a powerful method for studying protein function in the native cellular context. However, the existing workflows for generating CRISPR/Cas9 knock‐out cell lines are cumbersome and labor intensive, comprising of arduous rounds of cloning, limiting dilutions, and surveyor assays performed on dozens if not hundreds of clones. In order for large scale CRISPR/Cas9 studies to become practical and accurate for the genomics community, a streamlined process must be developed and validated at both genomic and protein levels. Here, we report a novel workflow that leverages the power of droplet digital PCR (ddPCR) and high‐resolution melt analysis (HRM) to produce DNA sequence and western blot validated CRISPR/Cas9 knock‐out cell lines in under 5 weeks.Support or Funding InformationBio‐Rad, Inc Internal R&D support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluorescence Kinetic Studies of DNA Unwrapping in Transcription Initiation with NTP addition and in Open Complex Dissociation by high salt
Authors: Callies Katelyn , McNerney Christina , Cimperman Clare Kai , Xue Andrew , Kan Hertina , Sreenivasan Raashi , Chhabra Munish , Record Tom 
Published: June 2021

λPR promoter DNA is bent and wrapped around E. coli RNA polymerase (RNAP) in the stable open complex (RPo), as demonstrated by Cy3‐Cy5 fluorescent energy transfer (FRET) between dyes attached 115 bp apart on the far‐upstream (−100) and downstream (+14) position of DNA. Here we report the use of FRET and PIFE (single‐dye fluorescence enhancements that indicate close contacts between RNAP and the far‐upstream or downstream DNA) to investigate unwrapping of upstream DNA after NTP addition to initiate transcription, form a short RNA‐DNA hybrid and escape from the promoter. We observe two kinetic phases of roughly equal amplitude in the decrease in FRET in transcription initiation. The first kinetic phase (0.1 s to 10 s) of DNA unbending/unwrapping appears slower than the rate of NTP binding and dinucleotide synthesis, indicating that this first phase of unbending/unwrapping occurs in the processes of translocation and hybrid extension. The second kinetic phase (at times > 10 s) corresponds to that of production of full‐length (14 bp) RNA transcripts, as determined in parallel single‐round transcription assays. These results indicate that the second phase of DNA unbending and unwrapping occurs in promoter escape. In addition, we report the kinetics of unwrapping in dissociation by salt‐upshifts. These experiments reveal that salt‐induced unwrapping occurs during the conversion of the stable open complex to the unstable open intermediate I2, prior to DNA closing.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
USP24 Induces IL‐6 in Tumor‐associated Microenvironment through stabilizing p300 and β‐TrCP to Promote the Malignancy of Lung Cancer
Authors: HUNG Jan‐Jong 
Published: June 2021

Our previous study indicated that increase in USP24 level in lung cancer facilitates lung cancer metastasis. Our recent study found that higher USP24 level was found in tumor‐associated microenvironment (TAM) than surrounding lung cancer cells in lung cancer patient specimens. USP24 level was significantly increased in M2‐macrophages. Knockdown of USP24 in M2‐macrophages decreased the migratory and chemotactic activity of lung cancer cells and angiogenetic ability of human umbilical vein endothelial cells (HUVEC) induced by M2‐derived conditioned medium. In vivo experiments also showed USP24 knockdown decreased M2‐macrophages induced lung cancer cells metastasis and angiogenesis. IL‐6 mRNA and protein levels were significantly decreased in USP24 knockdown M2‐macrophages and lung cancer cells. IL‐6 replenishment can rescue migratory and chemotactic ability of conditioned medium derived from USP24 knockdown M2‐macrophages, suggesting that increase in USP24 in M2‐macrophages induce the metastasis activity of lung through promoting IL‐6 expression. To address the molecular mechanism of how USP24 regulated IL‐6 in M2‐macrophage and lung cancer cells, we found that USP24 stabilized p300, thereby increased the transcriptional activity of IL‐6. USP24 could also decreased DNM1 and IkB level through stabilizing β‐TrCP and resulted in upregulated IL‐6. Metastatic cytokines in tumor‐associated microenvironment are important for cancer progression, in this study, we provide the direct evidence to support that USP24 increased in M2‐macrophages and lung cancer cells positive regulates the metastasis of lung cancer through enhancing the IL‐6 expression, which might be beneficial for the drug development for cancer therapy 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.
Characterizing the Spatial Variance of Autophagic Cargo Receptor, OPTN
Authors: Najera Susana Sarai , Heo Jin‐Mi , Harper Wade 
Published: June 2021

The mitochondria are required for various cellular processes spanning from cellular energy generation to signal transduction, growth and survival. Not surprisingly, cumulative mitochondrial damage is tightly linked to the development of multiple human diseases including various neurodegenerative diseases such as Parkinson's and Huntington's disease. In order to maintain functional mitochondria, cells have coped with multiple mitochondrial quality control pathways such as mitochondrial Unfolded Protein Response (mt‐UPR), the ubiquitin proteasome system, and mitochondrial autophagy (mitophagy). The most well‐characterized mitophagy is the PINK1‐PARKIN pathway, where damaged mitochondria are selectively marked with ubiquitin chains by the PINK1 kinase and PARKIN ubiquitin E3 ligase, engulfed by autophagosome, and cleared by the lysosome. While we now have a better understanding on this pathway, it is still unclear how autophagic cargo adaptor proteins, such as optineurin (OPTN), get recruited to damaged mitochondria, synthesized or recycled throughout the process. We hypothesized that the availability of OPTN would be the rate‐limiting factor to achieve complete removal of damaged mitochondria and this would require synthesis of new OPTN molecule. To test this hypothesis, we generated a cell line containing a genomic Halo tag at OPTN N‐terminus by utilizing CRISPR/cas9, where differentially fluorescent labeled Halo tag can distinguish older versus newer OPTN populations over time. After successfully generating a CRISPR/Cas9 knock‐in cell line for OPTN, single cell imaging was performed to determine the spatial variance and emergence of OPTN recruited to mitochondria. We were able to conclude that OPTN localizes to a fraction of damaged mitochondria despite the fact that the entire mitochondria are coated with ubiquitin chains, suggesting specificity of this recruitment. Additionally, our preliminary results indicated that newly synthesized OPTN is recruited to mitochondria for the continuation of mitophagy. Together, these observations suggest that OPTN is the rate‐limiting factor and that in order to completely remove all dysfunctional mitochondria, cells need to synthesize new OPTN. In the future, we would like to further explore the underlying mechanism of OPTN's binding specificity and determine what drives this selective recruitment of OPTN to mitochondria.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.