bluuebunny/crossref_metadata_2025_split huggingface dataset:
IMP1 loss in intestinal epithelial cells promotes altered Paneth cell morphology and autophagy defects (899.2)
Authors: Lundsmith Emma , Hamilton Kathryn , Noubissi Felicite , Hahn Christopher , Spiegelman Vladimir , Rustgi Anil 
Published: June 2021

Background Insulin‐like growth factor‐2 mRNA‐binding protein 1 (IMP1) is an oncofetal protein that exhibits high expression during embryonic development and lower expression in adult tissues. Mice with global Imp1‐/‐ gene deletion exhibit perinatal lethality due to altered gut morphology, suggesting a potential role in stem cell maintenance. We hypothesize that IMP1 contributes to intestinal homeostasis through modulation of the stem cell niche, specifically through a novel role in Paneth cells.Methods Using immunohistochemistry (IHC), immunofluorescense (IF), and immunoblotting, we evaluated mice with intestinal epithelial‐specific loss of Imp1 (VillinCre;Imp1floxed mice) for Paneth cell morphology (lysozyme), and alterations in markers of autophagy (LC3 and p62).Results VillinCre;Imp1floxed mice exhibit Paneth cell mislocalization and altered morphology. Furthermore, VillinCre;Imp1floxed mice exhibit increased LC3+ puncta and decreased p62 expression, representing up‐regulation in autophagic flux.Conclusions Our data suggest that IMP1 may mediate intestinal homeostasis through a novel role in Paneth cells. The intersection of Paneth cell function and autophagy pathway components is an emerging area of interest in patients with inflammatory bowel diseases. As such, Villin‐Cre;Imp1fl/fl mice provide a compelling model for elucidating this relationship.Grant Funding Source: Supported By HHMI Medical Fellowship (ETL), NIH grants (F32 DK093207‐01, RO1 DK056645‐12, UO1 DK0855
Did speech slow down the human face? Human facial muscles have a high proportion of slow myosin fibers compared to other primates (918.19)
Authors: Burrows Anne , Durham Emily , Smith Timothy , Parr Lisa 
Published: June 2021

Much is known about the comparative morphology of the human face relative to other primates but our understanding of comparative muscle physiology is poor. To further our insight, the present study used myofibrillar adenosine triphosphatase (mATPase) histochemical staining to investigate distribution of type I (slow) and type II (fast) myosin fibers in the zygomaticus major (ZM) and orbicularis oris muscles (OOM) of humans (n=3), chimpanzees (n=1), and rhesus macaques (n=3). Average percentages were created for fiber types from 5‐10 sections per specimen and average fiber cross‐sectional area and diameter were calculated for each muscle in each species. Non‐parametric tests were used to statistically compare averages since data were not normally distributed. Results of statistical testing showed that chimpanzees had a significantly higher percentage of fast fibers in the ZM relative to both macaques and humans. However, humans had a significantly higher percentage of slow fibers in both the ZM and OOM relative to chimpanzees and macaques, in excess of three times more than both macaques and chimpanzees. In the ZM, humans had the lowest cross‐sectional fiber area and diameter in both fast and slow fibers while chimpanzees had the greatest. Combined, these results may reflect adaptive divergence between chimpanzee and human facial muscles that may be influenced by the evolution of human speech where many facial muscles attaching to the lips act as speech articulators.Grant Funding Source: Supported by National Institutes of Health (contract grant number MH082282)
Mature dental enamel: a diverse repository of past and present information (918.2)
Authors: Gerlach Raquel , Line Sergio 
Published: June 2021

Dental enamel is the hardest, densest, and most mineralized tissue in vertebrates. It achieved its very high crystallinity with evolution of enamel specific proteins for more than 500 million years. During enamel formation (still inside the jaw bones), the proteins are degraded by the enamel‐resident proteinases and eventually “mature” enamel contains less than 0.1% of proteins 96% of minerals. This pre‐fossilized rocky tissue is essential for proper nutrition and survival, even today.The past century witnessed the characterization of 3 enamel structural proteins and 2 enamel proteinases, achieved by cumbersome work with samples of thousands of developing teeth to obtain micrograms of protein for mass spectrometry analyses and Edman degradation. Newer equipments enable today that hundreds of different peptides from all 3 enamel proteins can be obtained from enamel acid extracts, obtained with a much less invasive procedure compared to the ones used before. Mammal´s mature enamel harbors Y‐chromosome amelogenin specific peptides that allow the determination of sex, which we show at the EB2014 for the first time in humans samples. Enamel is also a good biomarker for toxic metals like lead and cadmium. Superficial enamel etch (for 2 minutes) was sufficient to obtain enamel specific peptides from shed primary teeth. Thus, dental enamel is an immense and diverse repository with relevant information for paleontology, archaeology, forensics, toxicology, medicine and developmental biology.Grant Funding Source: Supported by FAPESP (Brazil)
Jagged1 is essential and sufficient for osteoblast development during maxillary ossification (921.2)
Authors: Hill Cynthia , Yuasa Masato , Schoenecker Jon , Goudy Steven 
Published: June 2021

Maxillary hypoplasia occurs due to insufficient maxillary intramembranous ossification, leading to poor dental occlusion, respiratory obstruction and cosmetic deformities. Conditional deletion of Jagged1 (Jag1) in cranial neural crest (CNC) cells using Wnt1‐cre (Jag1CKO) led to maxillary hypoplasia characterized by intrinsic differences in bone morphology and density using μCT evaluation. Jag1CKO maxillas had altered collagen deposition, delayed ossification, and reduced expression of early and late determinants of osteoblast development during maxillary ossification. In vitro bone cultures on Jag1CKO mouse embryonic maxillary mesenchymal (MEMM) cells demonstrated decreased mineralization that was also associated with diminished induction of osteoblast determinants. BMP receptor expression was reduced in the Jag1CKO MEMM cells suggesting that these cells were unable to respond to BMP‐induced differentiation. Jag1‐Fc rescued in vitro mineralization and osteoblast gene expression changes. These data suggest that Jag1 signaling in CNC‐derived MEMM cells is required for osteoblast development and differentiation during maxillary ossification.Grant Funding Source: 5K08DE17953‐5
Ephrin reverse signaling induces mouse palatal fusion without TGFβ3 signaling (921.4)
Authors: Serrano Maria , Hills Patricia , Pourmand Iksa , Varghese Allen , Svoboda Kathy , Benson M 
Published: June 2021

Orofacial clefts are the most prevalent craniofacial birth defects. The secondary palate forms from mesenchymal shelves covered with epithelium which adheres to form the midline epithelial seam (MES). MES cells then proceed through epithelial to mesenchymal transition (EMT) forming a fused palate. TGF‐β3 is essential for the disintegration of the palatal MES. Eph receptor tyrosine kinases and their ephrin ligands are responsible for many developmental processes. Binding of ephrins causes receptor activation in Eph‐bearing cells (forward signaling), and intracellular signaling inside ephrin‐bearing cells (reverse signaling). We asked if activation of ephrin reverse signaling is sufficient to fuse mouse palates if we blocked TGFβ receptors. We cultured E13.5 mouse palatal shelves in different conditions: Control IgG Fc; anti‐TGF‐β3 with or without clustered recombinant EphB2/Fc protein; TGF‐β RI kinase inhibitor (SB 431542) with or without EphB2/Fc. Palatal fusion was quantified and analyzed with a mean fusion score (MFS) in anterior, medial and posterior regions. Palates treated with anti‐TGF‐β3 or (SB 431542) did not fuse while those treated with SB 431542 and EphB2/Fc and those treated with anti‐TGF‐β3 and EphB2/Fc were partially fused. Palates from the control groups fused. These results demonstrate that ephrin reverse signaling induces fusion of the mammalian palate without TGF‐β3 signaling.Grant Funding Source: Biomedical Sciences Department, baylor College of Dentistry, Baylor Oral Health Foundation
A noncanonical nuclear role for fibroblast growth factor signaling in skeletal development (921.6)
Authors: Salva Joanna , Neben Cynthia , Merrill Amy 
Published: June 2021

Fibroblast Growth Factor (FGF) signaling plays crucial roles in osteochondral progenitor cell proliferation and differentiation as seen by the multiple skeletal dysplasias resulting from abnormal FGF signaling. We identified that the skeletal dysplasia Bent Bone Dysplasia Syndrome (BBDS) results from unique FGF Receptor 2 (FGFR2) mutations that reduce canonical membrane signaling, while also enhancing a noncanonical nuclear role for the receptor in ribosome biogenesis. To determine how canonical and noncanonical FGFR2 signaling individually contribute to skeletal development and the pathogenesis of BBDS, we blocked membrane FGFR2 signaling with a chemical inhibitor and enhanced nuclear FGFR2 signaling by employing the BBDS mutations. In a bone organ culture assay, avian mandibles grown with inhibitors of membrane FGF signaling displayed reduced bone formation and exhibited uncharacteristic hypertrophy of Meckel’s cartilage. Expression of the BBDS FGFR2 mutations in developing chicken embryos caused increased proliferation and abnormal patterning in bone and cartilage. These results suggest that membrane FGF signaling is important in osteochondral progenitor differentiation and maturation while nuclear FGF signaling regulates proliferation. Combined our research shows that canonical and noncanonical FGF signaling have distinct roles during skeletal development.Grant Funding Source: Supported by NIH#5T32HD060549‐02 to JES, MOD #5‐FY12‐166 to AEM, NIH #5P30DE020750‐02
Biophysical analysis of the human POT1‐TPP1 complex reveals functional protein‐protein and protein‐DNA interactions involved in telomere maintenance (926.1)
Authors: Taylor Derek , Rajavel Malligarjunan , Baus Diane , Orban Tivadar , Palczewski Krzysztof 
Published: June 2021

Telomeres are nucleoprotein complexes that reside at the ends of linear chromosomes. Six specialized proteins interact specifically with telomere DNA and one another to form a protective moiety called shelterin. Telomere repeat‐binding factors (TRF) 1 and 2 bind to the double‐stranded telomere DNA sequence and the Protection of telomeres 1 (POT1) protein binds to the single‐stranded DNA overhang. RAP1, TIN2, and TPP1 interact with TRF1, TRF2, and POT1 to form shelterin. TPP1, specifically, interacts with POT1 to increase its affinity and selectivity for telomere DNA. In addition, TPP1 recruits telomerase to the telomere, where it can synthesize telomere DNA. X‐ray crystallography has revealed the atomic structures of individual domains of POT1 and TPP1, but the molecular interactions between the two proteins with and without DNA remains obscure. Using amide hydrogen‐deuterium exchange and biochemical analysis, we have determined the regions of TPP1 that are responsible for POT1‐interactions and those that are altered upon DNA binding. Combining these experimental data with homology modeling allowed us to construct a model of the POT1‐TPP1‐DNA complex and interpret its biological function in DNA recognition and in regulating telomerase activity.Grant Funding Source: Supported by American Heart Association 11SDB5580010 and American Cancer Society RSG‐13‐211‐01‐DM
Biological activity of a set of new copper(II) complexes: toward understanding structure‐reactivity correlations (928.2)
Authors: Aitken Chelsea , Tanski Joseph , Fox Kristin , Tyler Laurie 
Published: June 2021

Cisplatin, carboplatin and oxaliplatin are all platinum‐containing complexes that have been approved for use as chemotherapy drugs. The success of these complexes as well as their downfalls have sparked interest into alternative transition metal based compounds as potential chemotherapeutic agents. The use of platinum based complexes in cancer treatment has faced complications including cytotoxicity and drug resistance. The use of copper(II) as an alternative metal center is of specific interest because of copper’s bioavailability and elevated concentrations within tumor cells. We have synthesized and characterized several copper complexes that utilize a nitrogen based ligand system including some that contain a thiazole moiety. The biological activity and potential cytotoxicity of these complexes was measured on the basis of nuclease activity, DNA binding, and interaction with BSA. These characteristics were determined by quantitative analysis of studies using ethidium bromide, gel electrophoresis, and UV‐Vis spectroscopy. We present comparisons between the activity of these newly synthesized compounds and those previously studied to aid in developing a correlation between ligand structure and complex activity.Grant Funding Source: Supported by Union College
Proteasomal inhibition effects Notch signaling (937.2)
Authors: Chambers Elaina , Hsu Aaron , White Brandon 
Published: June 2021

T‐cell acute lymphoblastic leukemia (T‐ALL) is an aggressive cancer with poor clinical outcome and aberrant Notch signaling has been shown to play a central role in disease pathogenesis. Therefore, it is crucial to elucidate the mechanism underlying Notch deregulation. Upon release from the membrane, the Notch Intracellular Domain (NICD) translocates to the nucleus where is associates with CSL, displacing co‐repressors and recruiting co‐activators (such as Mastermind and CBP/p300), thus resulting in the transcription of target genes (Hes1 and Myc). Our earlier work demonstrated that the NICD could be turned over through a phosphorylation, and subsequent ubiquitination mechanism of degradation. However, it remains unclear if turnover occurs on chromatin or in the nucleoplasm. It is also known that Notch translocation can be pharmacologically inhibited by the drug DAPT. The current work examines the interplay at hand between the inhibition of Notch translocation and the ubiquitin‐mediated proteasomal degradation of Notch. Current data suggest that proteasomal inhibition enhances or “rescues” the transcription of Notch target genes which have been inhibited by DAPT, thus implying that Notch has the ability to “recycle” itself, despite the ubiquitin signal, and that Notch continues participating in gene transcription. These data clearly indicate that Notch signaling is more complex than previously believed, and elucidating the mechanisms underlying its deregulation will greatly benefit the biomedical community as it will effectively enable us to target the Notch pathway in cancer treatment.Grant Funding Source: Support of research was through NSF‐RUI Grant#1052039 to JBW.
The hunt for a PAX3‐FOXO1 motif (939.4)
Authors: Johanson Kelly , Collins Chantelle , Stewart Desmond , Cherlin Tess 
Published: June 2021

Alveolar Rhabdomyosarcoma (ARMS) patients carrying a chromosomal translocation of PAX3 and FOXO1 have the highest mortality rates, indicating the importance of understanding how this transcription factor influences gene expression. Many studies have been performed to identify PAX3‐FOXO1 target genes and while some overlapping gene sets have been identified, there exists great variation in the genes identified. The search for these target genes is hampered by a lack of understanding of the full DNA‐binding capabilities of this fusion protein. We have examined the truncated FOXO1 DNA‐binding domain in PAX3‐FOXO1 and found that it retains activity, but does not have the same sequence specifications for binding as full length FOXO1. A search of 1463 PAX3‐FOXO1 putative binding sites revealed that 37% contained both a PAX3 paired domain and FOXO1 recognition sequence within close proximity. The sequences containing the dual motif mapped to 160 genes with the most interesting of these known to be regulated by PAX3‐FOXO1 such as MET or upregulated in response to PAX3‐FOXO1 expression such as ABAT and PBK. Here we present additional data exploring the sequence variations on the PAX3 and FOXO1 recognition sequences found within Pax3‐FOXO1 putative binding sites as well as in vitro confirmation of the interaction.Grant Funding Source: Supported by NIH SC2GM095430
Vitamin D hormone regulates the expression of hedgehog coreceptor genes Boc and Cdon in osteocytes (946.4)
Authors: Osinski Victoria , St. John Hillary , Pike J 
Published: June 2021

Osteocytes are terminally differentiated cells descended from mesenchymal stem cells that are embedded in bone. Osteocytes are known to play an essential role in maintaining bone health, acting as mechanical sensors in bone: translating mechanical load into biochemical signals. Osteocytes have diverse regulatory functions that extend beyond bone, such as lipid regulation and immune system development. These cells are regulated by a number of hormones and cofactors, including the active form of vitamin D, 1,25 dihydroxyvitamin D3 (1,25D3). 1,25D3 binds and activates the vitamin D receptor (VDR) and this complex serves as a transcriptional regulator of various genes. Previously, chromatin immuno‐precipitation coupled to high throughput sequencing (ChIP‐seq) revealed that VDR binds to regions upstream and downstream of the genes coding for Cell adhesion molecule‐related/down‐regulate by oncogenes protein (Cdon) and Brother of Cdon (Boc). These two genes encode cell surface receptor proteins in the Hedgehog signaling pathway, that play an important role in animal tissue development. To better understand 1,25D3’s role in regulating Boc and Cdon, MC3T3‐E1 cells were transfected with luciferase reporter plasmids containing regions of VDRE occupancy identified by ChIP‐seq. These results revealed that these regions of Boc and Cdon mediate up‐regulation by 1,25D3. Specific vitamin D response elements (VDREs) within the enhancer regions were then identified by in silico analyses and selectively mutated. Analysis of these VDRE mutated luciferase reporters revealed an elimination of 1,25D3 activity. In summary, we have shown 1,25D3’s novel role in the up‐regulation of Boc and Cdon and identified the specific VDREs that are responsible for the 1,25D3 response of these genes in osteocytes. Further studies are underway to define the impact of 1,25D3 on Hedgehog signaling in osteocytes. These results contribute to an understanding of 1,25D3’s role in osteocyte and bone health and development.Grant Funding Source: Supported by NIH DK072281
Experimental and computational analysis of peroxisome protein targeting (948.10)
Authors: Berg Jeremy 
Published: June 2021

The targeting of proteins from the cytoplasm to the peroxisomal matrix is determined largely by targeting signals with the sequences of the proteins to be targeted. The Peroxisomal Targeting Signal‐1 (PTS1) is particularly important and comprises sequences at the carboxyl terminus of the protein that approximate X‐X‐X‐Ser‐Lys‐Leu‐COO‐. PTS1s are recognized by the protein Pex5p which binds them and carries the targeted protein to the peroxisome membrane at which point the Pex5p‐protein complexes translocate to the peroxisomal matrix. The structures of Pex5p‐PTS1 peptide and protein complexes as well as apo‐Pex5p have been determined. An experimental human proteome‐wide survey of Pex5p‐PTS1 peptides using a fluorescence anisotropy‐based assay revealed dissociation constants for bona fide peroxisomal matrix proteins ranging from 1.6 nm to > 1 micromolar. Further analysis revealed correlation between protein tissue‐specific expression levels and these dissociation constants, suggesting co‐evolution of PTS1‐Pex5p affinities and expression levels. These observations also suggest testable differences in tissue‐specific peroxisome composition and function. The Pex5p‐PTS1 system also presents a well‐characterized system for the exploration of free energy perturbation methods for calculating binding free energies from structural data. It has been possible to calculate relative free energies with reasonable agreement with experimental results for a series of peptides with variations within the PTS1 sequence as well as some mutated forms of Pex5p.Grant Funding Source: Supported in part by NIGMS and NIDDK
Alternative autophagy alleviates A1AT‐Z accrual (949.2)
Authors: Wen John , Dankle Geoff , Glenn Kevin 
Published: June 2021

Alpha‐1 antitrypsin deficiency is the leading cause of childhood liver failure, one of the most common lethal genetic diseases, and is present in 1 in 2,500 people of European descent. The disease‐causing mutant A1AT‐Z fails to fold correctly and accumulates in the endoplasmic reticulum of the liver, resulting in hepatic fibrosis and hepatocellular carcinoma. A1AT‐Z sequestration in hepatocytes leads to reduced secretion, causing centrolobular emphysema in adults. The purpose of this work was to study the degradation of A1AT‐Z in hepatic tissue and cultured cell lines. We identified the ubiquitin ligase FBG1, which has been previously shown to degrade proteins by both the ubiquitin proteasome pathway and autophagy, as being key to A1AT‐Z degradation. Using chemical and genetic approaches we show that FBG1 degrades A1AT‐Z via both the ubiquitin proteasome system and autophagy. Overexpression of FBG1 decreases the half‐life of A1AT‐Z. Knocking down FBG1 in a hepatic cell line results in an increase in ATAT. Heterozygous A1AT‐Z mutant mice have more Periodic Acid Schiff Base positive inclusions in an FBG1 heterozygous knockdown background compared to mouse lines containing the full amount of FBG1. Finally, we show that FBG1 degrades A1AT‐Z through an ATG5‐independent arm of autophagy, suggesting that alternative autophagy may be important in treating A1AT‐Z deficiency.Grant Funding Source: VA‐Career Development Award
Structural and biophysical analysis of UCH37: ubiquitin recognition and binding to ADRM1 (952.3)
Authors: Morrow Marie , Kim Myung‐Il , Paul Lake , Ronau Judith , Das Chittaranjan 
Published: June 2021

Ubiquitin C‐terminal hydrolase 37 (UCH37) is a deubiquitinating enzyme capable of dismantling Lys48‐linked polyubiquitin chains when associated with the 26S proteasome. Upon binding to the proteasomal subunit ADRM1, UCH37 is activated for cleavage of the distal monomer in a chain, an exo‐specific activity. Prior to binding with ADRM1, it is thought that UCH37 is inhibited by its own C‐terminal UCHL5‐like domain (ULD). Our interests lie in UCH37’s mechanism of activation by ADRM1 as well as its exo‐specificity. Previously we have reported the crystal structure of T. spiralis UCH37 bound to ubiquitin vinyl methyl ester, which revealed that the ULD domain interacts with ubiquitin’s Lys48, an event we believe accounts for its exo‐specificity. In order to further examine specificity and activation, we have generated a series of ULD mutants of UCH37. To probe exo‐specificity, we are purifying the 19S regulatory particle of the proteasome from rabbit skeletal muscle, will reconstitute each ULD mutant within the 19S, and then test their cleavage with an asymmetric triubiquitin substrate. When mutated, ULD residues involved in ubiquitin recognition may lose exo‐specificity against the asymmetric substrate. Additionally, we have analyzed UCH37 and ADRM1’s biophysical interactions through isothermal titration calorimetry, finding that the two bind with 1:1 stoichiometry and a dissociation constant of 22 nM. Due to this tight binding and our structural data, we hypothesize that ADRM1’s activation of UCH37 may not be due to an enhancement of UCH37’s turnover rate, but rather due to an increase in ubiquitin’s koff rate; that is, the ULD merely slows product dissociation. We hope to characterize this phenomenon through surface plasmon resonance studies of ubiquitin binding to ULD mutants in the presence of ADRM1. These findings should not only provide a better understanding of UCH37’s activation and exo‐specificity, but will further probe the role of deubiquitination at the proteasome.Grant Funding Source: Supported by National Institutes of Health Grant 1R01RR026273 (C.D.)