bluuebunny/crossref_metadata_2025_split huggingface dataset:
Clodronate Does Not Alter Bone Remodeling in Young Horses
Authors: Richbourg Heather A , Mitchell Colin F , McNulty Margaret A 
Published: November 2023

The bisphosphonate drug clodronate has been shown to treat diseases of abnormal bone remodeling (e.g., navicular disease) in horses, with the goal of systemically reducing osteoclast‐mediated bone loss. Recently FDA‐approved for use in horses, clinical trials have determined its effectiveness in reducing lameness in adult horses by 60 days post treatment. However, despite prolific clinical use, studies have not identified a systemic effect on normal bone remodeling in the horse, either in adults or young horses that are still undergoing tremendous bone growth and remodeling. Therefore, this study aimed to evaluate effects of clodronate on normal bone remodeling in young horses. In addition, the effect on bone healing was assessed using a bone defect model. Two‐ and three‐dimensional quantitative morphological evaluations of bone biopsies of the tuber coxae were utilized. We anticipated increases in bone volume, and decreases in bone remodeling and osteoclast number and function due to treatment compared to saline. Baseline biopsies were collected on Day 0 from nine horses aged 2–4 years old. Following the initial biopsy, horses were randomly assigned to a treatment group (control n=5, treated n=4) and treatment was administered intravenously. Oxytetracycline was administered at Day 47 and 57 as a fluorescent bone label. On Day 60, an additional bone biopsy was collected from the contralateral side, as well as a re‐biopsy of the initial side as a model of bone healing. Biopsies were evaluated via both micro‐computed tomography (microCT) and histomorphometry. Statistical analyses included paired and unpaired student's t‐tests. Horses tolerated surgery well with no post‐surgical complications. All biopsies were adequate for both microCT and histological evaluations. There was no significant effect of clodronate treatment on bone volume or trabecular structure parameters as evaluated by microCT. Clodronate did not significantly alter bone remodeling parameters after 60 days as evaluated by dynamic histomorphometry. Overall, clodronate does not appear to negatively affect normal bone structure and remodeling after 60 days in young horses. In addition, it appears that clodronate has little impact on bone remodeling short term, potentially due to its' relatively‐low potency compared to other bisphosphonates available. These data have significant clinical impact regarding the effectiveness of clodronate to treat diseases of bone remodeling in horses in general, and demonstrates that there is no negative effect of clodronate administration on normal remodeling processes in young horses.Support or Funding InformationLouisiana State University Charles V. Cusimano Equine Health Studies Program Grant
Palisade Endings in Cat Extraocular Muscles Develop Postnatally Following Different Time Courses
Authors: Streicher Johannes , Davis López de Carrizosa María A. , de la Cruz Rosa R. , Loro Ángel Manuel Pastor , Blumer Roland 
Published: November 2023

Proprioception from extraocular muscles (EOMs) provides the brain with eye position signals. Surprisingly, classical proprioceptors (muscle spindles and Golgi tendon organs) are absent in the EOMs of most mammals. Instead, palisade endings (PEs) are found as unique to EOMs of mammals. They are regularly present in frontal‐eyed but infrequent in lateral‐eyed species. PEs are formed by axons which extend onto the tendon where axons make a u‐turn and divide into axonal branches that establish nerve terminals around the tip of a single muscle fiber. For a century, PEs have been considered as sensory structures substituting classical proprioceptors in EOMs. The interest in PEs has newly aroused when molecular analysis and recent neuronal tracing experiments have shown that PEs are cholinergic and originate from the EOM motor nuclei.Since the development of PEs is unknown, we have analyzed their postnatal maturation in a frontal‐eyed species (cat). Cats of different ages (P0, P8, P22, and adult) were used and whole mount preparation of the rectus EOMs were triple‐immunolabelled and analyzed in the CLMS. Triple labelling included antibody against neurofilament (to label axons), anti‐synaptopysin (to label nerve terminals), anti‐growth associated protein43 (GAP43, to label developing axons), and phalloidin (to label muscle fibers).At P0, no PEs were found in the rectus muscles. However, in the medial rectus we found axons extending straight onto the tendon where they extensively sprouted to establish synaptophysin‐positive contacts. At P8, premature PEs were found in the medial rectus but not in the other rectus muscles. At P22, the complexity of PEs in the medial rectus had increased noticeably and formation of PEs had started in the inferior rectus. At this stage PEs were still absent in the lateral rectus and superior rectus muscle. In the adult animal, all rectus muscles contained PEs. GAP43, a marker for developing axons was highly expressed in premature PEs. GAP43 expression decreased with age and was nearly absent in PEs of adult animals.Here we show that the development of PEs in the rectus muscles occurs in a staggered fashion and is more accelerated in the medial rectus. Recently we have shown that in frontal‐eyed species many more PEs are present in the medial rectus compared with the other rectus muscles. The preponderance and the quicker maturation of PEs in the medial rectus support the notion that PEs could play a role in convergence.
Genomic cell lineage analysis uncovers novel endothelial gene expression programs for metabolic and immune regulation
Authors: Klomp Jeff , Malik Asrar 
Published: November 2023

Endothelial cells (ECs) take on a variety of cellular phenotypes that depend on their niches, making it difficult to discern lineage‐wide traits. Therefore, we sought to identify novel aspects of EC biology by defining a core EC transcriptional signature shared amongst ECs. We accomplished this by condensing multiple sources of EC transcriptomic and genomic data sets using multivariate statistics and phylogenetic analyses. Our comparative multi‐tissue analyses confirmed expected gene expression patterns such as Flt1, Pecam1, Cdh5, Tek, and Ptprb as core elements of ECs. We also observed strong statistical associations for a variety of genes without prominent roles in ECs, such as the oncostatin M receptor (Osmr), cysteine and tyrosine rich 1 (Cyyr1) and an uncharacterized cDNA sequence (BC028528) that were always positively associated with ECs compared to other cell types. This implied that associations with tightly regulated genes can be obscured by EC variability but may be important for fundamental EC biology. Strikingly, hallmark interferon alpha and gamma gene sets were associated with all EC subtypes and ECs showed highest enrichment in an interferon alpha activation gene signature (p<1e‐15). More broadly, glycolytic and mitochondrial metabolic gene sets were strongly down‐regulated in resting ECs while regulatory transcriptional and translational program gene sets were up‐regulated in these cells. Together, these findings support the existence of key metabolic and immune regulators that are necessary for constitutive EC function in all lineages that can be carefully scrutinized by gene modulation in experimental systems.Support or Funding InformationNIH T32
Deciphering the dynamics of alternative pre‐mRNA processing of glutaminase in ovarian cancer
Authors: LaFontaine Patrick R , Gibbs Bettine , Masamha Chioniso Patience 
Published: November 2023

Ovarian carcinoma is the fifth leading cause of cancer death among women in the United States and has the highest mortality rate of all gynecologic malignancies. Patients with ovarian cancer are often diagnosed with advanced stage, metastatic disease due to lack of specific symptoms and adequate reliable tumor specific biomarkers for early disease detection. An emerging hallmark of cancer is the metabolic reprogramming of transformed cells to support the energy and biosynthetic needs of the rapidly dividing and metastatic tumor phenotype. These metabolic changes facilitate increased glucose and glutamine uptake and metabolism. Recent studies suggest that highly invasive ovarian cancer cells show a remarkable dependence on glutamine, making glutamine metabolism a viable therapeutic target in ovarian cancer. There are several clinical trials focused on inhibiting glutaminase, the enzyme that catalyzes the first step of glutaminolysis and converts glutamine to glutamate. There are two genes that code for glutaminase in the human genome, glutaminase 1 (GLS1) located on chromosome 2 and glutaminase 2 (GLS2) encoded by chromosome 12. GLS1 is the enzyme most associated with tumor metabolism. There are two annotated alternative isoforms of GLS1, GAC and KGA, which result from alternative splicing. Different tumors express either or both isoforms. Under normal conditions the production of the KGA isoform is repressed by miR‐23 which binds to the long 3′UTR of KGA and silences its expression. We recently found that depletion of a 3′end processing factor, CFIm25, results in alternative polyadenylation of the KGA isoform resulting in a novel KGA transcript that has a truncated 3′UTR and is able to evade miR‐23 regulation. In addition, knockdown of CFIm25 also switched splicing to preferentially facilitate splicing to KGA instead of the GAC isoform. Our goal is to determine the different glutaminase isoforms in metastatic ovarian cancer cells and determine how they are regulated for therapeutic targeting using isoform specific siRNAs. We have developed isoform specific amplicons to measure levels of GAC, long KGA (with long 3′UTR) and short KGA (with short 3′UTR). Using qRT‐PCR we show that the highly metastatic ovarian cancer cell lines express high levels of the GAC isoform. The cisplatin resistant cell line SKOV3 also expresses KGA in addition to GAC and we detected this at both the mRNA and protein level. Inhibition of GLS1 using inhibitors resulted in decreased cell viability of highly metastatic ovarian cancer cell lines. Based on our findings, we hypothesize that expressing both the KGA and GAC isoforms facilitates the chemotherapeutic drug resistance of highly metastatic ovarian tumors.Support or Funding InformationFunding: Butler University Holcomb research award, Senior Research Grant from the Indiana Academy of Sciences
The Role of the C1q Domain of Otolin1 in Otolith Morphogenesis
Authors: Das Olipriya 
Published: November 2023

Otolin1 is an extracellular matrix protein of otoliths (“ear stones” of fishes), and otoconia (“ear dust” of higher vertebrates). These acellular biominerals are essential for the sense of balance; dislodging results in the most common human balance disorder, benign paroxysmal positional vertigo. Otolin1 comprises of a collagen and a C1q domain, similar to atypical collagens VIII and X. We propose that Otolin1 forms a scaffold to which other otolith proteins, Ca2+ and CO2− ions bind during otolith morphogenesis, with C1q trimers as hubs and collagen triple‐helices as spokes. To test this model, we investigated whether the Otolin1 C1q domain is necessary and sufficient to form trimers and higher complexes in vitro. The zebrafish Otolin1a C1q domain was expressed in bacteria either alone or fused to thioredoxin. Multimerization of affinity purified recombinant proteins was assessed by size‐exclusion chromatography and gel electrophoresis. Under denaturing conditions, recombinant C1q proteins formed monomers and trimers, but not dimers or higher‐order complexes. Under native conditions, they formed only higher‐order complexes. In contrast, thioredoxin alone only appeared as a monomer. Our results are consistent with the proposed ability of the Otolin C1q domain to form trimers that, in turn, assemble into higher‐order complexes. Experiments are underway to characterize the various complexes in more detail and to test whether the C1q domain is necessary to trimerize of full‐length Otolin1a.Support or Funding InformationSupported by NIH R01DC006962
Transport of the Alzheimer's Disease Associated Amyloid‐Beta Peptide by P‐Glycoprotein
Authors: McCormick James Warren , Nanayakkara Amila K , Vogel Pia D , Wise John G 
Published: November 2023

The broad range of substrates that can be transported by the transmembrane protein, P‐glycoprotein (P‐gp), has been well established. While best known for the ability to confer general multidrug resistance to a population of cancer cells and for its pharmacokinetic implications in being a critical transporter in the blood brain barrier (BBB), P‐gp action has also been implicated in Alzheimer's disease (AD). One clearance pathway across the BBB for amyloid‐β, which is thought to be a causative agent in neurodegeneration and symptom onset in AD, was hypothesized to be catalyzed by P‐gp. A dynamic P‐gp model similar to that used in McCormick et al. (Biochemistry 2015) was used here to allow docking of amyloid‐β and an unrelated peptide of the same size to P‐gp. Simulations of the catalytic cycles through four different crystallographic conformations of P‐gp‐related proteins using targeted molecular dynamics (TMD) were performed. Even though movement of amyloid‐β was not targeted by the method, transport of amyloid‐β through the membrane was observed during the putative catalytic cycles. These simulations started with amyloid‐β bound to P‐gp in conformations that were open to the cytoplasm and ended with amyloid‐β bound to P‐gp in conformations that would allow amyloid‐β access to the extracellular space. In simulations using a control peptide of the same length, no transport was observed. In experiments to physically evaluate the computational results, synthetic amyloid‐β 42 was fluorescently labeled with carboxyfluorescein and incubated with cells having P‐gp overexpressed. The experiments showed that cell lines with high levels of P‐gp had measurably less accumulation of the fluorescent amyloid‐β 42 than cells with low expression levels of P‐gp. Amyloid‐β accumulation was inhibited in the presence of P‐gp specific inhibitors, supporting the hypothesis that P‐gp plays a role in the clearance of amyloid‐β 42 from the brain.Support or Funding InformationThis work is supported by NIH NIGMS [R15GM09477102] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.
Genetic Requirements for the Degradation of Translocon‐Associated Proteins
Authors: Indovina Christopher Joseph , Engle Sarah M , Watts Sheldon G , Rubenstein Eric M 
Published: November 2023

The endoplasmic reticulum (ER) translocon is a molecular channel responsible for moving secreted and endomembrane system proteins across the ER membrane. Proteins that stall within or otherwise aberrantly engage the translocon channel may impair transport of other proteins across the membrane. Proteins that persistently or aberrantly engage the translocon are targeted for degradation via the conserved ER‐resident ubiquitin ligase Hrd1. A better understanding of this system may prove useful in treating elevated cholesterol in humans as, under certain circumstances, the major protein component of low‐density lipoproteins (LDL; bad cholesterol) stalls within the translocon prior to LDL synthesis. Protein co‐factors that assist Hrd1 in targeting other substrates for degradation are dispensable for Hrd1‐dependent degradation of translocon‐obstructing proteins. We hypothesize that Hrd1 requires the function of a distinct cohort of protein co‐factors to recognize and ubiquitylate proteins that aberrantly engage the translocon. We have developed a growth‐based screen to identify genes in Saccharomyces cerevisiae that mediate the degradation of proteins that aberrantly engage the translocon. We fused the His3 enzyme, which is required for histidine biosynthesis, to a model protein that aberrantly engages with the translocon. In this way, histidine prototrophy is an indicator of impaired protein degradation. We introduced this fusion protein into the non‐essential yeast gene knockout collection using Synthetic Genetic Array (SGA) technology. Genes found to be required for degradation of the model translocon‐associated protein may represent therapeutic targets for patients with cholesterol‐related pathology.Support or Funding InformationNational Institutes of Health Grant R15 GM111713 (to EMR). Funds from the Ball State University Provost's Office and Department of Biology (to EMR). Ball State University Chapter of Sigma Xi Research Award (to CJI).
Deciphering the Selectivity and Reactivity of Ebselen Derivatives toward Mycobacterium tuberculosis Antigen 85C
Authors: Goins Christopher M , Dajnowicz Steven , Thanna Sandeep , Sucheck Steven J , Parks Jerry M , Ronning Donald R 
Published: November 2023

Ebselen was previously found to potently inhibit the essential Mycobacterium tuberculosis (Mtb) Antigen 85s through the allosteric, covalent oxidation of a solvent exposed, conserved cysteine residue. In this study, we sought to better understand the in vitro inhibitory properties of a recently synthesized 1st generation library of ebselen derivatives with respect to Ag85C. To better understand protein‐inhibitor interactions, Ag85C was successfully co‐crystalized with two bulky, chemically dissimilar derivatives, resulting in two X‐ray crystal structures of 2.01 Å and 1.30 Å. Both inhibitors displayed an aromatic stacking interaction with Phe253 and the aromatic ring of the ebselen scaffold in addition to a cationic interaction between the side chain of Arg239 and the respective chemical derivative. Aside from these interactions with Ag85C, the ebselen derivative was oriented towards solvent. To better understand how these two interactions potentially facilitate initial drug binding, resulting in the unique in vitro properties of the various derivatives, interaction energetics were computed for both interactions with respect to the two derivatives using Density Functional Theory. When comparing the energetic values to inhibitory activities, we found derivatives capable of forming favorable interactions with Arg239 have higher levels of inhibition. Furthermore, we looked into the electronic effects the derivatives have on the reactivity of selenium towards the oxidation of Cys209 using Density Functional Theory. The various derivatives were found to have little too no effect on reactivity, strengthening the importance of interactions with Arg239. Both novel structures displayed a greater shift in movement to helix α9 than what was observed in the previous Ag85C Ebselen structure. This movement caused the elongation and restructuring of a loop connecting helix α9 to the preceding β‐strand. We questioned whether this movement affected protein stability and found using differential scanning fluorimetry that Ag85C modified by ebselen or its derivatives decreased the melting temperature by 14 °C. This shift results in the unfolding of protein at physiological temperatures. Upon closer inspection of both structures, a solvent channel leading from the active site to the modified Cys209 is observable. This channel is of great intrigue to us as a future path for drug development towards Mtb Ag85s.Support or Funding InformationNIH grant AI105084
AF4‐AF9 Protein‐Protein Interaction Inhibitor: Synthesis and Biological Evaluation
Authors: Roseman Shelby , Bao Wei , Koh John 
Published: November 2023

In normal human cells, the Mixed Lineage Leukemia (MLL) gene is believed to encode proteins involved in the transcriptional regulation of developmental processes. However, MLL can undergo chromosomal translocations to join with over sixty partner genes and produce fusion proteins that play key roles in the transformation of hematopoietic cells into leukemia cells. These genetic fusions are observed in eighty percent of infant Acute Lymphoblastic Leukemia patients and sixty percent of infant Acute Myeloid Leukemia patients, and the most common fusion partner is AF4. MLL‐AF4 interacts with transcription factor AF9, and it has been shown that inhibiting this interaction leads to leukemia cell death. As such, the AF4‐AF9 interaction is a target for disrupting the MLL fusion pathway and a potential point of intervention for pediatric leukemia.This study aims to evaluate and improve upon a small molecule inhibitor of the AF4‐AF9 interaction identified from high‐throughput screening. Though the compound disrupts AF4‐AF9 association, its mechanism of action is unclear and difficult to study. AF4 and AF9 are intrinsically unstable proteins and the initial hit compound is prone to oxidation. In this study, more stable analogs of the hit compound were synthesized. In addition, AF9 was expressed in high yield as a fusion with AF4 containing a proteolytic site in the linker. The fusion protein can be cleaved, but separation of AF4 and AF9 has proved challenging due to strong intermolecular association. Interactions of the complex with the target molecule were evaluated by circular dichroism and fluorescence anisotropy. Surface plasmon resonance will also be employed to study inhibitor binding to AF4 and AF9.Support or Funding InformationSupport for this project is provided by Alex's Lemonade Stand Foundation. Additionally, summer research funding was provided by the Plastino Scholars program and University of Delaware Summer Scholars.
Purification and Quantification of the Chemical Markers of Melanin to Enhance Early Diagnosis of Melanoma
Authors: Weddle Carly , Sienerth Karl , Glass Keely 
Published: November 2023

Melanoma is characterized by overproduction of the pigment melanin, which is present in two forms in human skin – eumelanin and pheomelanin. High discordance rates among experienced pathologists in the diagnosis of questionable malignant epidermal samples demonstrate a need for more robust and objective diagnostic tools. Although optical imaging qualitatively suggests that a high ratio of eumelanin to pheomelanin corresponds to the incidence of malignant melanoma, no chemical analysis to date has successfully quantified this ratio. Reproducible techniques for quantifying the natural forms of melanin may provide insight into the molecular mechanisms underlying melanoma and may be used to determine whether a high eumelanin: pheomelanin ratio is indicative of malignant melanoma. Current protocols for analyzing melanin entail chemical degradation of the pigment into markers specific to each of the two natural forms. These chemical markers are separated and quantified by high performance liquid chromatography with ultraviolet detection (HPLC‐UV). While this characterization has proved useful, identification of the chemical markers in complex biological matrices is limited by interfering signals. This study introduces two new methods for identifying and quantifying the chemical markers of melanin: (1) sample purification with solid‐phase extraction (SPE) and (2) selective identification of degradation markers with mass spectrometry (HPLC‐MS). Application of these techniques to melanoma cells derived from a metastatic lung site and normal skin fibroblast cells demonstrate enhanced selectivity and reproducibility in the quantification of chemical markers of melanin in complex biological matrices. Sample preparation with SPE improves clarity in HPLC chromatograms and MS detection allows unambiguous assignment of individual peaks to specific degradation markers of melanin. Enhanced selectivity and accuracy in the quantification of melanin may ultimately contribute to standardization of techniques in pigment cell research and be used as a robust diagnostic tool for detecting early stage, thin lesion melanomas.Support or Funding InformationElon University Honors ProgramElon University Undergraduate Research ProgramElon University Lumen Prize