bluuebunny/crossref_metadata_2025 huggingface dataset:
A Dynamic Spatio‐Temporal Expression Pattern of FGFR Isoforms During Scleral Ossicle Development
Authors: Kumar Shruti , Franz‐Odendaal Tamara A.
Published: June 2021

Fibroblast growth factors (FGFs) are an important family of proteins that play several roles during early vertebrate development. The FGF ligands bind to their membrane bound receptors (FGFRs) and activate downstream signalling pathways. Although the role of FGFRs has been widely studied in the development of endochondral bones, little is known about their role in the development of neural crest derived intramembranous bones. Scleral ossicles are a ring of 13–15 neural crest derived intramembranous bones in the eye of the chicken (Gallus gallus) and have been identified as a model for intramembranous bone formation. These bones are induced by a corresponding ring of epithelial thickenings, the conjunctival papillae. Using whole mount in situ hybridization, the spatio‐temporal expression patterns of ‘b’ and ‘c’ isoforms of FGFRs1–3 were determined during the stages of conjunctival papillae (HH30–33) as well as scleral ossicle development (HH34–37). The expression of both ‘b’ and ‘c’ isoforms of FGFRs1–3 were observed during conjunctival papillae development and their downregulation was observed during the stages of scleral ossicle induction. ImageJ was further used to study changes in the intensity of expression and to give a more precise indication of the stages of downregulation for each gene. Additionally, although all FGFRs were expressed in the epithelium, only FGFRs 1c, 2c, 3b and 3c were observed in the mesenchyme. The dynamic spatio‐temporal expression pattern of the FGFR isoforms during conjunctival papillae development implicates several FGF ligands in conjunctival papillae induction. This research will further our understanding of the signalling pathways potentially involved in the scleral ossicle system. FGF signaling is likely to interact with the Bone Morphogenetic Protein and Hedgehog pathways, already identified to play a role in the scleral ossicle system.

Support or Funding Information

This research was funded by the Natural Sciences and Engineering Research Council of Canada

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Untangling Sources of Phenotypic Variation Characterizing the Craniofacial Disease Holoprosencephaly
Authors: Lainoff Alexis , Young Nathan , Hallgrimsson Benedikt , Marcucio Ralph
Published: June 2021

Holoprosencaphly (HPE)—a craniofacial disorder in which the forebrain fails to develop into two hemispheres and the midface does not fully form—is characterized by a broad spectrum of severity. Phenotypes range from very mild midfacial narrowing to cyclopia. Mutations in Sonic hedgehog (SHH) and in SHH signaling pathway members are known to underlie HPE; however, the source of the highly variable craniofacial morphology manifesting in the HPE patient population is not understood. Previous research in a chick model suggests that reduced SHH‐signaling in the brain correlates with a continuous distribution of HPE‐like phenotypes, and that very small modifications in ligand concentration can result in highly heterogeneous morphologies. Here, we are probing the origins of morphological variation in a genetic mouse model. Although humans heterozygous for SHH mutations exhibit the full spectrum of HPE, mice heterozygous for Shh do not exhibit even mild HPE although homozygous mutants are cyclopic. Mice homozygous for mutations in the SHH‐signaling pathway member low‐density lipoprotein receptor‐related protein (Lrp) 2 however exhibit mild to severe forms of HPE, and are therefore a better model for examining sources of variation. LRP2 is an endocytic receptor required to induce proper SHH signaling in the forebrain. We used geometric morphometric analysis to quantify the shape of embryos with sequential, genetic loss of Shh and Lrp2 alleles, and qPCR was used to assess SHH signaling pathway activation. We found that there is no statistically significant difference in shape among wild‐type, Lrp2+/−, Shh+/−, and Lrp2+/−;Shh+/− embryos, while Lrp2−/− and Lrp2−/−;Shh+/− embryos diverged from the group, exhibiting a more narrow midface. Interestingly, there was increased phenotypic variance in Lrp2−/− and Lrp2−/−;Shh+/− embryos in comparison to the other genotypes, with a greater increase in variance seen in the Lrp2−/−;Shh+/− group. Our results support the hypothesis that very slight modifications to SHH pathway activation can yield large amounts of phenotypic variation.

Support or Funding Information

This work is supported by NIH/NIDCR‐R01DE018234, ‐R01DE019638, ‐T32DE730619, ‐F31DE02579001.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of a Novel Vomer Phenotype in the Fgfr2cC342Y/+ Mouse Model of Crouzon Syndrome
Authors: Lesciotto Kate M. , Motch Perrine Susan M. , Kawasaki Kazuhiko , Richtsmeier Joan T.
Published: June 2021

Crouzon syndrome has been linked with several FGFR2 mutations, including the Cys342Tyr mutation. The Fgfr2c C342Y/+ mouse model displays many of the phenotypic traits associated with Crouzon syndrome, including premature coronal suture fusion, a dome shaped neurocranium, and midfacial retrusion. This study was designed to perform a systematic evaluation of the morphological variation of the vomer in 3D micro‐computed tomography (3DμCT) scans of Fgfr2c C342Y/+ mice and their unaffected littermates. Vomer morphology was evaluated in mice at three ages: E17.5, P0, and P2. First, 3D reconstructions and slice images of 3DμCT scans were evaluated for evidence of bridging or fusion between the presphenoid bone and either the left or right vomer alae (N=81 Fgfr2cC342Y/+ mice, 92 unaffected littermates). None of the unaffected mice at any age exhibited any bridging or fusion between the vomer alae and presphenoid bone, while affected mice were increasingly likely to show bridging or fusion as age progressed. At P2, 60% of affected mice exhibited at least unilateral bridging between one of the vomer ala and the presphenoid. Second, geometric morphometric analysis was conducted on a set of 6 3D landmarks describing vomer shape, including landmarks on the vomer body and tips of the left and right alae (N=45 Fgfr2cC342Y/+ mice, 55 unaffected littermates). Mann‐Whitney U testing revealed that at each of the three ages tested, the anteroposterior lengths of the vomer bodies and vomer alae were significantly longer (p < 0.05) and the mediolateral distance between the tips of the vomer alae was also significantly increased (p < 0.001) in Fgfr2cC342Y/+ mice as compared to their unaffected littermates. The vomer is an element of the dermatocranium that interfaces with elements of the cranial base and the facial skeleton, which are components of the endocranium. This novel phenotype provides insight into the morphogenesis of the complex craniofacial traits typically associated with Crouzon syndrome and suggests the importance of evolutionary considerations in the interpretation of dysmorphogenesis associated with this FGFR2 mutation.

Support or Funding Information

This research was supported in part by NIDCR/NIH R01DE018500 R01DE022988 NICHD/NIH P01HD078233 and NSF BCS 1731909.

This 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 the Bent Bone Dysplasia Mouse: conditional knock‐in of FGFR2M391R
Authors: Rigueur Diana , Merrill Amy
Published: June 2021

Fibroblast Growth Factor Receptor 2 (FGFR2) regulates osteoprogenitor proliferation, self‐renewal and terminal differentiation during skeletal development, yet it remains unclear how the receptor distinctly elicits these cellular processes. We discovered a nuclear feature of FGF signaling that will elucidate with more specificity how FGFR2 regulates skeletal development. This mechanism emerged from our analysis of Bent Bone Dysplasia Syndrome (BBDS), an FGFR2‐disorder where the cell‐fate decisions of osteoprogenitor cells are biased towards self‐renewal over differentiation. We developed a mouse with a conditional knock‐in of the FGFR2 M391R mutation that causes BBDS in humans. DNA sequencing of cultured primary chondrocytes treated with Adeno‐GFP or Adeno‐Cre, confirmed efficient Cre‐mediated recombination of the knock‐in allele. Cell culture experiments and immunofluorescence further showed that the knock‐in FGFR2M391R allele functions like that of BBDS, aberrantly translocating FGFR2 to the nucleus. Moreover, generation of a Wnt1‐Cre; FGFR2M391R/+ mouse renders a phenotype, craniosynostosis, along with other defects in neural crest derived tissues, which strongly suggests that the mutation is dominant in mouse as it is in humans. Overall our preliminary data shows that the conditional knock‐in FGFR2M391R mouse can be used to model human BBDS and can also be used to parse out the unknown endogenous and pathogenic functions of nuclear FGFR2.

Support or Funding Information

NIH RO1, NIH Diversity Supplement Award, and March of Dimes

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cholesterol regulates craniofacial development by modulating the WNT signaling pathway.
Authors: Gonzalez Cesar
Published: June 2021

The cholesterol synthesis pathway produces two independent lipid molecules, cholesterol and isoprenoids, both of which have diverse cellular functions. Mutations that inhibit the cholesterol synthesis pathway result in at least 8 different human syndromes, some of which are characterized by defects in facial development. The presence of facial dysmorphia in these disorders strongly suggests that the cholesterol synthesis pathway facilitates appropriate facial development. Recently, it was demonstrated that mutation or inhibition of this pathway in zebrafish results in craniofacial abnormalities by interfering with appropriate neural crest cell differentiation. However, whether these deficits in differentiation are cholesterol independent, cholesterol dependent, or a combination of both has not been fully elucidated. In order to better understand the independent functions of cholesterol and isoprenoids during neural crest cell differentiation, we performed a combination of loss of function and pharmacological inhibition studies. Mutation of the zebrafish ortholog of hmgcs1, which encodes the first enzyme in the cholesterol synthesis pathway, disrupts the differentiation of cranial neural crest cells, but does not affect the specification and migration of these cells. Furthermore, pharmacological inhibition of each branch of the cholesterol synthesis pathway revealed a novel function for isoprenoids in neural crest differentiation. Interestingly, the cholesterol dependent mechanisms modulating cranial facial development were sonic hedgehog independent. Moreover, our preliminary analysis suggests that the inhibition of cholesterol synthesis reduces activation of the WNT signaling pathway, an essential mediator of craniofacial development. Taken together these data demonstrate a novel mechanisms by which the cholesterol synthesis pathway regulates craniofacial development.

Support or Funding Information

Research reported in this poster was supported by the National Institute Of General Medical Sciences of the National Institutes of Health under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. Funding for this project was obtained from NIMH Grant no. 2G12MD007592 to the University of Texas El Paso, University of Texas El Paso Office of Research and Sponsored Projects New Investigator start‐up funding, NIGMS linked awards RL5GM118969, TL4GM118971, UL1GM118970 and the NIMH Grant no. T32MH015442 to the University of Colorado School of Medicine, Department of Psychology

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development, Innervation, and Involution of a Transient Cement Gland in a Giant Danio (D. cf. Malabaricus): the Role of Retinoic Acid
Authors: Nelson Hannah M. , Coffing Gabrielle , Lafontant Pascal J.
Published: June 2021

Cement glands are transient evolutionarily adaptive adhesive organs. They mediate the attachment of a number aquatic larvae to substrates in their environments. There is significant interest in understanding the mechanisms that orchestrate their development and involution, as well as the chemical constituent of the biological adhesive they produce. We have identified and studied a cephalic and ectodermally derived cement gland in a giant danio (GD), by light microscopy, immune and lectin histochemistry. We hypothesized that early inductive embryonic events regulates the development of the gland, and that exogenous retinoic acid may disrupt its proper development. Our studies revealed that the cement gland is readily identified at hatching time, and mediates attachment of larvae until they are able to swim. At the swimming stage, the gland begins its involution and disappears by day 14. Using lectin histochemistry we demonstrate that the first differentiated goblet‐like cells that constitute the gland appear as early as 12 hours post fertilization. Using acetylated tubulin staining we also show that the gland is innervated by branches of the emerging trigeminal nerve. Lectin histochemistry also indicates that the adhesive substance within the gland is composed in part of various glycoconjugated products. Finally, treatment of GD embryos during gastrulation with exogenous retinoic acid (RA) leads to posteriorization and progressive disappearance of the gland in a dose‐dependent manner. This is the first detailed description of the development, maturation, its innervation and involution of the cement gland in a GD. Our results suggest that the cement gland is one of the earliest formed organs in the GD and that its proper development depends on maintenance of a stable RA gradient. Further studies are required for greater insights onto the function the gland.

Support or Funding Information

DePauw FDC, SRF.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Impact of Selection for Facial Reduction in Dogs
Authors: Selba Molly Chisholm , Oechtering Gerhard , Heng Hock Gan , DeLeon Valerie Burke
Published: June 2021

Through artificial selection, humans have altered the morphology of domestic dogs and produced the broad range of morphological variation seen in modern dog breeds. The purpose of this study was to analyze the effects of artificial selection for short faces in domestic dogs (Canis lupus familiaris). The morphological characteristics associated with years of selection in favor of smushed, paedomorphic faces often result in Brachycephalic Airway Syndrome (BAS), which is characterized by problems with olfaction, respiration, and thermoregulation. We used clinically obtained CT scans of dogs representing brachycephalic breeds (pugs and bulldogs), normocephalic breeds (beagles), and dogs of unknown breed. We established two groups of dogs (brachycephalic and normocephalic) based on cephalic index. In both groups, we recorded a total of 62 cranial and mandibular landmarks. Through geometric morphometric analysis, we determined that the greatest shape variation in this sample was correlated with cephalic index. The shape changes in the crania and mandibles co‐vary, and are both associated with cephalic index. Within the hard palate, the majority of the shape change associated with brachycephaly is centered on the maxilla, specifically at the carnassials. The shape change seen on the hard palate is a unique lateral displacement and rotation of the carnassials, associated with shortening of the hard palate. In contrast, the shape change seen in the mandible is mainly a uniform rostral‐caudal shortening and a medial‐lateral widening. In addition, the soft palate does not appear to be reduced in these breeds. From a functional perspective, the maxillary and mandibular carnassials become malaligned in brachycephalic dogs. The shape change seen in the carnassials demonstrates that morphological integration of the carnassial complex is disrupted in brachycephalic dogs. Despite the facial reduction observed in these breeds, the soft palate does not appear to be reduced, and these soft tissues may crowd the reduced pharyngeal space. Selection for the aesthetic traits associated with brachycephaly in domestic dog breeds results in changes to the carnassial complex and pharynx that are functionally suboptimal and might limit survival outside of a domesticated environment.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Evaluating the Effects of the Obturator Externus Tendon on the Posterior Femoral Neck”
Authors: Campeau Lisa M. , Schachner Emma R. , Sedlmayr Jayc C.
Published: June 2021

The anatomical literature describes the insertion tendon of obturator externus (OE) as leaving a distinct groove on the posterior aspect of the femoral neck as it travels distally to insert in the intertrochanteric fossa, a trait proposed to be directly associated with the hyper‐extended position of the human hip. The absence of this osteological correlate from the femoral neck of chimpanzees, a sister taxon to humans and a brachiating quadruped, has resulted in the use of this trait as an indicator for the evolution of bipedalism in extinct hominins. The aim of this study was to dissect and record the path of the OE tendon and adjacent ischiofemoral ligament as well as reassess the reliability of the osteological correlate in order to determine if the groove created by the OE tendon is an acceptable indicator of the origin of bipedalism in extinct hominins. The posterior hip region of embalmed (n=27) and fresh (n=3) cadavers were dissected, and the posterior aspect of the femoral neck was cleared of periosteum to evaluate the presence or absence of the OE groove. We found that 2 cadavers had a groove consistent with the OE, and 24 did not have a groove consistent with the OE. The osteological findings varied, ranging from no bony defects, a small, medially‐placed divot near the lesser trochanter, to a variety of both horizontally and vertically positioned groves on the posterior aspect of the femoral neck. The morphology of the OE as it traveled across the posterior femoral neck also varied, ranged from predominantly muscular, a thin, mostly inferiorly‐placed tendon, to a very thick, rigid, tendinous insertion. In the specimens that demonstrated a clear indication or groove on the posterior surface of the femoral neck that could be aligned with the OE, there was no consistency in the overall morphology of that groove, the OE muscle, or its insertion tendon. Additionally, a groove parallel to the OE tendon often appeared to correlate with a thickened area in the ischiofemoral ligament. As a result, we conclude that this anatomical characteristic of the posterior femoral neck is highly variable and should not be used as an osteological correlate to indicate bipedalism in extinct hominins.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Saccadic Eye Movement and Cognition
Authors: Manuel Morganne , Tzeng Tony , Aubin Ashleigh , Devier Deidre , Canavier Carmen , Lovera Jesus
Published: June 2021

Background

Horizontal saccadic eye movements (saccades) have been shown to improve performance on memory and attentional control tasks. These cognitive improvements are a result of enhanced intra‐hemispheric processing capabilities following saccades. Our goal was to determine if adding horizontal saccadic eye movements during a cognitive task would improve performance on a working memory and an attentional control, when compared to adding a control task (central eye fixation).

Methods

Fifty healthy, right handed students were recruited on LSUHSC–NO and UNO campuses. They attended two sessions and were randomly assigned to perform a memory or attentional control task during the first visit. The other task was then completed during the second session. In addition, they were randomized 1:1 to either saccades or central eye fixation for each session. Participants were asked to either follow a dot that changed from left and right position at 1Hz or a dot that blinked in the center of a computer screen. The memory task involved recalling a short story. The attention task involved participants responding to an arrow that appeared on the computer screen flanked by distractors. Interspersed throughout each task, participants were asked to either perform saccades or central fixation.

Results

On the memory task, there was no group difference based on eye movements) on immediate recall (15.4±3.7), delayed recall (14.0±2.9), and 24 hour delayed recall (14.4±3.0); control group immediate recall (15.1±3.3), delayed recall (14.2±3.2), and 24 hour delayed recall (14.1±3.4), p>0.05. On the attention task, the saccades improved accuracy on incongruent flankers (97.6%±2.5) when compared to the control group (95.5%±4.0), p=0.03; but eye movements did not have an effect on accuracy for congruent flankers (99.3%±2.0) when compared to the control group (99.3%±0.8), p=0.856. Although participants performed horizontal saccadic eye movements had a faster reaction time than controls, this difference was not significance.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative analysis of V1a and V1b receptor distribution in the mammalian brain
Authors: Knappe Carolin , Giesecke Torsten , Koshimizu Taka‐aki , Gimber Niclas , Schmoranzer Jan , Storm Robert , Bachmann Sebastian , Mutig Kerim
Published: June 2021

Vasopressin (AVP) induces antidiuresis via its renal V2 receptors but also acts as a neuropeptide through activation of central V1a and V1b receptors (V1aR, V1bR). Despite a large body of functional evidence for roles of these receptors in motivated behavior, little information is available on their distribution in the brain tissue. This study was designed to provide a comparative analysis of the V1aR and V1bR distribution patterns in rodent and human brain tissues using new antisera.

Antibodies to individual AVP receptors were generated and validated using respective mouse knockout tissues or overexpression of the human V1aR and V1bR in HEK293 cells. Distribution of the receptors was studied by immunofluorescence in vivo in perfused rat or mouse brains and human brain samples obtained from clinical section. Confocal microscopy or super resolution imagingwere applied for signal detection. AVP receptors expressing cell types were identified by double‐labeling for markers of neurons (CTIP2 or SATB2) or glial cells (GFAP).

Application of the anti‐V1aR antibody on rat or mouse brain sections produced signal in pyramidal cells of the isocortical layers II and V, astrocytes, neurons of the medial habenula, the suprachiasmatic and supraoptic hypothalamic nuclei, as well as in the CA2 and CA3 fields of hippocampus, whereas no significant signal was detected in the choroid plexus. V1bR signal was present in pyramidal cells of the isocortical layers II and V, neurons of the medial and lateral habenula, the suprachiasmatic and supraopticus nuclei of hypothalamus and in basolateral cellular aspects of the choroid epithelia. Evaluation of human brain samples revealed similar V1aR and V1bR distribution patterns, as compared to rodents.

In summary, our results document the distribution patterns of V1aR and V1bR in the rodent brain, thus corroborating previous functional data on the role of AVP in motivated behavior.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Maternal immune activation affects precursor cell populations and developmental processes in the rodent spinal cord in a temporally and spatially dependent manner
Authors: Mcdermott Kieran W. , Anderson Rebecca C. , Foley Tara , Radford Jay , Allardyce Joanna M. , O'Hallaron Sophie , Uzun Burak , O'Keeffe Gerard W.
Published: June 2021

Studies suggest that maternal immune activation (MIA) during pregnancy may be implicated in development of neurological disorders with timing of immune challenge and spatial differences playing an important role. However, further research is necessary in order to elucidate fully the CNS regions, cell populations and timing of gestational vulnerability. As the development of the spinal cord (SC) is well characterised, here we investigate how MIA affects cell populations and developmental processes in this CNS region.

Time‐mated dams received an intraperitoneal injection of 100μg/kg lipopolysaccharide (LPS) or saline on embryonic day (E)12 or 16. Embryos were harvested 5hr post‐injection, or delivered and sacrificed at postnatal day (P)14. Offspring were fixed, frozen whole or following SC dissection and cryosectioned. In order to determine how MIA affects oligodendrocyte precursors, microglia, and reelin expression during development, sections were immunostained with Olig2, Iba‐1 and reelin. In addition total RNA was extracted from fresh frozen SCs and prepared for microarray, RNAseq and small RNAseq.

At E12, numbers of Olig2+ and Iba‐1+ cells were unchanged 5hrs post‐LPS injection but Olig2+ cells were decreased in the ventral grey matter of the rostral SC at P14. The number of Iba‐1+ cells were unchanged at P14. At E16, Olig2+ and Iba‐1+ cells were decreased in grey and white matter 5 hrs post‐LPS, however by P14 there was no difference between the LPS and saline treated groups. Reelin expression following MIA at E12 appeared unchanged at 5hrs and P14 however, at E16, reelin expression was decreased 5hrs after MIA. Total cell numbers at E16, at both the 5hr and P14 survival times, were unchanged following LPS injection.

These findings suggest MIA affects development in the SC in a temporally and spatially precise manner. At E12 MIA does not appear to affect olig2+ oligodendrocyte precursors acutely but may affect their survival to P14. In contrast, at E16, MIA affects Olig2+, Iba‐1+ and reelin expression acutely. Ongoing work is characterising postnatal reelin expression following LPS at E16, and the phenotype of Iba1+ cells to understand more fully the detailed cellular perturbations resulting from MIA.

Support or Funding Information

Anatomical Society, Graduate Entry Medical School, Health Research Institute, University of Limerick

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A New Pathway of Transcription‐Coupled Repair
Authors: Myka Kamila , Washburn Robert , Kusters Kira , Gottesman Max
Published: June 2021

Transcription‐coupled repair (TCR) of damaged DNA in E. coli can involve several different pathways. We propose that one pathway entails release of RNAP at the site of DNA damage without dissociation of the RNA:DNA hybrid. The RNA can serve as a primer for PolI, which initiates the repair process. We are investigating how TCR repairs nalidixic acid‐induced DNA damage. Nalidixic acid, like Etoposide generates a DNA double‐strand break bearing a TopA adduct at each 5′ DNA end. We show that efficient repair requires two transcription factors. The first DksA, is a coiled‐coil protein that inserts into the RNAP secondary channel and, in concert with ppGpp, regulates the activity of certain promoters. The role of DksA in transcription elongation is undefined, although it is known to suppresses replisome – RNAP clashes. DksA plays a direct role in repair, rather than blocking access of GreA to RNAP. Thus, a ΔdksA mutation is not suppressed by a greA deletion. Recent work from the Murakami laboratory reveals that DksA binding distorts the structure of RNAP, opening the pincers, thus potentially reducing the processivity of transcription elongation. DksA in association with ppGpp, however, does not affect RNAP structure. We find that repair is more efficient in strains carrying an RNAP mutation that reduces ppGpp binding to site 2, the location of DksA insertion. We have isolated a suppressor of ΔdksA based on resistance to nalidixic acid. The mutation, rpoBQ148H, phenocopies RNAP bound to DksA in the absence of ppGpp, i.e. it opens the pincers and may favor release of elongating RNAP. These findings suggest that DksA may act as a transcription termination factor that can release RNAP without removing the RNA:DNA hybrid.

The second, transcription termination protein Rho, is an RNA‐dependent DNA:RNA helicase. The defects in TCR of rho and dksA mutations are additive, suggesting that they act on different substrates. Whereas Rho is thought to release RNAP by unwinding the RNA:DNA helix, rho mutants that terminate transcription without releasing RNA have been isolated.

Another reaction that entails elongation of an RNA:DNA hybrid by PolI is constitutive stable DNA replication (cSDR). In cSDR, elongation of an R‐loop by PolI creates a new origin of replication. We find that cSDR is dependent on DksA. Presumably the mechanisms of nalidixic acid DNA damage and cSDR both involve allowing access of PolI to the 3′ end of an RNA:DNA hybrid.

Support or Funding Information

GM037219 GM037219 NIH: GM037219 NIH: GM037219 GM037219

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Escherichia coli RarA commits cells to post‐replication repair pathways by facilitating replisome skipping
Authors: Gopal Kamya , Stanage Tyler , Cox Michael M.
Published: June 2021

Members of the highly conserved RarA/Mgs1/WRNIP1 protein family have been implicated in the maintenance of genomic stability, but their exact functions remain unknown. Loss of one of these proteins rarely results in an altered phenotype, but a knockout mutation in genes encoding proteins of this family in tandem with the loss of other DNA metabolism functions can adversely affect cell survival. We hypothesize that the RarA protein of E. coli commits the cell to post‐replication repair pathways, namely translesion DNA synthesis (TLS) and daughter strand gap synthesis. RarA overexpression is toxic to the cell and knocking out DNA Polymerase IV and DNA Polymerase V, TLS‐competent polymerases, alleviates this toxicity. Furthermore, the presence of RarA induces sensitivity in cells lacking TLS polymerases to the DNA‐damaging agents nitrofurazone (NFZ) and 4‐nitroquinoline‐1‐oxide (4‐NQO). Deletion of RarA suppresses DNA Polymerase IV mediated sensitivity to NFZ and 4‐NQO. These data suggest that RarA can promote rapid replisome progress by committing cells to using post‐replication repair pathways and blocking access to other repair pathways when the replisome encounters certain types of lesions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the role of viral RNA sequence in host shut‐off by SARS‐CoV
Authors: Nag Anita , Chockalingam Prem K. , Adams Jessica L. , Gonzalez Fabiola G. , Abrar Fareeha
Published: June 2021

Nonstructural Protein 1 (nsp1), generated by Severe Acute Respiratory Syndrome Coronavirus (SARS‐CoV), is responsible for host cell mRNA degradation via endonucleolytic cleavage. While the cellular mRNA is targeted for cleavage, few viral mRNAs are resistant. These mRNAs include mRNA from SARS‐CoV, hepatitis C virus and cricket paralysis virus. Other studies concluded that a stem‐loop forming sequence in the 5′‐UTR (untranslated region) may be responsible for this cleavage resistance. We designed multiple mutations in the UTR to partially or fully disrupt the stem‐loop, or reinstate a stem‐loop with mutated sequence. These RNAs were subjected to nsp1 mediated cleavage and RNA stability was quantified using reverse transcription followed by quantitative polymerase chain reaction. We used 18S ribosomal RNA as a control since it does not undergo nsp1 mediated cleavage. Surprisingly, our results show that a partially disrupted stem‐loop leads to enhanced stability of the RNA. In order to understand any sequence driven stability that arises due to stronger binding by nsp1, we are currently investigating RNA binding properties of nsp1.

Support or Funding Information

SC INBRE faculty fellows award

SCICU summer fellows award

Furman fellows award

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An in vivo method to study post‐transcriptional regulation in germ stem cells
Authors: Doenier Jon , Aoki Scott Takeo , Kimble Judith
Published: June 2021

Stem cells in regenerative tissues must balance factors promoting self‐renewal with those driving differentiation. Faulty regulation of this balance can lead to tumor growth or loss of regenerative ability. The tractable germline of the nematode C. elegans has allowed the identification of a wealth of key stem cell regulatory factors and remarkably, virtually all are RNA binding proteins (RBP). Despite this insight, a mechanistic understanding of how these RBPs regulate their target mRNAs is lacking. Here, we develop an in vivo method to investigate their mechanisms. Our assay probes how a given RBP affects expression of a reporter mRNA when recruited to its 3′ untranslated region (3′UTR). To perform the assay, an RBP of interest is genetically engineered to contain a small λN peptide. The λN peptide binds a specific RNA hairpin, called boxB, with high affinity. Thus, a reporter mRNA engineered to contain boxB hairpins in its 3′UTR will bind the λN tagged RBP and either increase or decrease expression, depending on the RBP regulatory function. By imaging reporter expression in germline stem cells, we can gain insight into how the RBP regulates target transcripts in its normal biological context. The simple and rapid readout of the assay allows it to be used to probe how an RBP accomplishes its RNA regulation or screen for other factors necessary for RBP function. We have applied this assay to several of the key stem cell RBP regulators to test their functions in vivo. Our results demonstrate the tractability and ease of this method to study RBPs in the C. elegans germline as well as providing mechanistic insights into how the RBPs regulate stem cell fate decisions.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mass Spectrometry Analysis of RBM20 Phosphorylation and Its Role in Titin Splicing
Authors: Sun Mingming , Jin Yutong , Zhu Chaoqun , Rexiati Maimaiti , Cai Hanfang , Chen Zhilong , Ge Ying , Guo Wei
Published: June 2021

Objective

The SR protein family has a characteristic domain which is rich in arginine and serine residues. They play significant roles in constitutive pre‐mRNA splicing and the phosphorylation status of them is important in the regulation of alternative splicing. RNA binding motif 20 (RBM20), a newly identified SR protein, mainly regulates splicing of a giant muscle gene titin, a major heart disease‐causing gene. However, the mechanism of RBM20 modulation on titin splicing is not well defined. We hypothesize that RBM20 phosphorylation plays an important role in titin splicing. In this study, we employed middle‐down mass spectrometry and in vitro splicing assay to explore the phosphorylation status of RBM20 and its role in titin splicing.

Methods

Both bacteria and insect cell expressed RBM20 were used for ProQ diamond staining and middle down mass spectrometry analysis. In vitro approaches with co‐transfection of mutated RBM20 and titin minigene, and a dual luciferase splice reporter assay were performed to explore the role of RBM20 phosphorylation in titin splicing.

Results

Insect cell expressed RBM20 was stained by ProQ diamond, a dye which is specifically used for phosphorylated protein staining in SDS‐PAGE gel, whereas ProQ diamond stained bands were not observed with bacteria expressed RBM20. Mass spectrometry analysis on the insect cell expressed RBM20 identified 16 phosphorylation sites with four of them located in RS domain. 16 mutated RBM20 plasmids on individual phosphorylation sites co‐transfected with titin minigene in HeLA cells, and the results indicated that only phosphorylation in RS domain affects titin splicing, and splice report assay further confirmed the results.

Conclusion

RBM20 is massively phosphorylated with 16 phosphorylation sites, and only RS domain phosphorylation plays a role in titin splicing. These results reveal that the phosphorylation sites on RS domain residues could be potential therapeutic targets for titin splicing‐induced heart disease.

Support or Funding Information

NIFA‐USDA 1009266, NIH NIGMSP20GM103432, AHA BGIA and Faculty‐Grant‐in‐Aid from University of Wyoming

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Monitoring Innate Immune Receptor Stability via Post‐Translational Modification by OGT
Authors: Drake Walter R. , Hou Ching‐Wen , Zachara Natasha E. , Grimes Catherine Leimkuhler
Published: June 2021

The human body's first line of defense against infectious disease is the innate immune system, which is tasked with distinguishing harmful pathogens from commensal and beneficial microbes. On a molecular level, this system contains different pattern recognition receptors (PRRs) that detect microbe associated molecular patterns (MAMPs) specific to particular microorganisms, and the interaction between a PRR and its respective MAMP triggers an immune response. The Nod‐like receptors (NLRs) are one major family of innate immune receptors that contain the proteins Nod1 and Nod2, which use different bacterial cell wall peptidoglycan (PGN) fragments as their designated MAMPs. Binding between the NLR and PGN fragment is mediated by the leucine rich repeat domain (LRR), which exists at the C‐termini of Nod1 and Nod2. Upon LRR‐MAMP stimulation, NF‐kB and other signaling cascades are initiated, ending with the production of proinflammatory cytokines and chemokines. Misregulation of signaling by NLRs can lead to disease, and mutations in Nod1 and Nod2 are associated with gastric cancer and Crohn's disease, respectively. These NLRs are critically important for maintaining the relationship between the human body and the trillions of microorganisms that it hosts, yet so much about their functions and regulation in the body remain unclear. Recently, Nod2 was identified as being O‐‐GlcNAcylated, which is a post‐translational modification mediated by two enzymes that affect thousands of proteins in diverse ways. An emerging cellular thermal shift assay (CETSA) that is used for identifying protein‐ligand binding was repurposed to measure the stabilizing effect of O‐GlcNAc modification on Nod1 and Nod2. When protein O‐GlcNAc levels are increased, melting temperatures of both NLRs increase as well. These data are supported by cycloheximide half‐life experiments and NF‐kB luciferase assays, which demonstrated that the modification also led to increased NLR half‐lives and NF‐kB activity. Additionally, two Nod2 Crohn's associated variants were studied, and both G908R‐Nod2 and 1007fs‐Nod2 are O‐GlcNAcylated in the cell. While the modification led to similar increases in melting temperature and NF‐kB activity for 1007fs‐Nod2, its effects on G908R‐Nod2 are less clear. These results show that CETSA is a useful tool for measuring stability changes caused by O‐GlcnAc modification and that NLRs may be commonly post‐translationally modified to regulate stability.

Support or Funding Information

MITZUTANI GLYCOSCIENCE FOUNDATION, NATIONAL SCIENCE FOUNDATION (CAREER CHE 1554967), NIH NATIONAL HEART, LUNG, AND BLOOD INSTITUTE (P01HL107153)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Palmitoylation Regulates the Functions of NHE1
Authors: Pitsch Emily , Wallert Mark , Provost Joseph
Published: June 2021

The sodium hydrogen exchanger isoform 1 (NHE1) is a ubiquitously expressed transmembrane protein that regulates intercellular pH (pHi) and interacts with proteins involved in cell migration and adhesion. Within the extended cytosolic tail of NHE1, there are multiple sites of post‐translational modification including phosphorylation and lipidation. Our understanding of impact of reversible palmitoylation on membrane transporters is growing. 2‐bromopalmitate (2BP) irreversibly inhibits the enzyme, palmitoyl acyltransferase, that transfer palmitate to cysteines. We found NHE1 is reversibly palmitoylated and here, demonstrate the impact of the lipidation on NHE1 function. Lung fibroblasts expressing NHE1 (PSN) showed minimal decrease (8.5 ± 2.5% SEM) in cell viability after treatment with 50 μM 2BP after 72 h. Palmitoylation plays a critical role in cell adhesion and motility. Cells that were incubated in 15 μM 2BP for 18 hrs displayed a significant decrease cell adhesion in low serum conditions and cells treated with 2BP did not fully migrate into vacated space after 24 hrs in a wounding assay. Impact on cytoskeletal structure by 2BP treatment was also investigated. Lysophosphatidic acid (LPA) increased actin stress fiber formation 40% over control cells. This LPA‐induced stress fiber formation was inhibited more than two‐fold in the presence of 2BP. This is important as LPA‐induced stress fiber formation requires NHE1; therefore, palmitoylation is required for NHE1‐dependent actin stress fiber formation. To determine the effect of palmitoylation on NHE1 transport activity, PSN cells were treated with 2BP and the pHi was measured. Addition of increasing concentrations of 2BP inhibited NHE1 transport in cells with and without serum, but the effect was enhanced in low serum conditions. Interestingly, LPA stimulation of NHE1 activity was inhibited by 60%, but not to baseline. This indicates that in addition to cell adherence, motility, and cytoskeletal formation, palmitoylation is critical for NHE1 transport.

Support or Funding Information

This research was partially funded by the Beckman Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural comparison of the Arabidopsis thaliana family of β‐amylases
Authors: Chandrasekharan Nithesh , Monroe Jonathan , Berndsen Christopher
Published: June 2021

The β‐amylase (BAM) family in Arabidopsis thaliana has nine members, some of which are known to hydrolyze starch to maltose. The members of this family show unique functional, regulatory, and catalytic behaviors due to changes in sequence or domain composition. However, the molecular basis for functional specificity and regulation is not always clear. Some of the inactive members of the lack the catalytic amino acids required for hydrolysis. However, this is not the case for all the enzymes such as BAM2, which appears to be allosterically regulated and requires KCl for activity. Given the lack of structural information on any member of this protein family, we homology modeled all 9 BAM proteins from Arabidopsis and performed molecular dynamics simulations to propose how these proteins may differ at a functional level. All simulations were equilibrated for at least 50 ns in explicit solvent, the equilibrated models were aligned structurally, and then analyzed to compare the structure, dynamics, and chemical properties. All 9 proteins modeled well as TIM barrel proteins with most of the structural deviations occurring in predicted loops. Comparison of the simulated dynamics shows that overall the BAM proteins show similar areas of high and low motion. Interestingly, BAM4 and BAM9 showed fluctuation profiles that were distinct from the other BAM proteins, which matches their predicted lack of catalytic activity and distinct active site sequences. BAM7 and BAM8 which also have distinct active sites however these enzymes show dynamics similar to those of the catalytically active BAM1 and BAM3 suggesting that BAM7 and BAM8 may also have activity, however the proper conditions are not known. We further modeled BAM2 as a tetramer based on recent biochemical information to identify how allostery and solution conditions may influence the activity of this protein. We identified predicted changes in the dynamics upon binding to a ligand in the starch‐binding site as well as changes in hydrogen bonding within the protein in potassium. Both findings provide testable proposals for the regulation of BAM2 activity.

Support or Funding Information

This work was supported by a National Science Foundation Research at Undergraduate Institutions Grant MCB‐1616467 and National Science Foundation Research Experience for Undergraduates Grant CHE‐1461175

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Distinct functions for the flexible loops of zinc specific solute binding proteins from Paracoccus denitrificans
Authors: NEUPANE DURGA P. , YUKL ERIK T.
Published: June 2021

In bacteria, ATP binding cassette (ABC) transporters of cluster A‐I exist for the acquisition of the essential trace element zinc from limiting environments. These transporters are crucial for bacterial survival, and the gene knockout strains often demonstrate a pronounced growth defect in minimal media and attenuated virulence in the case of pathogens. Thus such systems are attractive targets for the development of novel antibiotics. Zinc binding with high affinity and specificity is mediated by the solute binding protein (SBP) component of ABC transporters. Zinc‐specific SBPs are characterized by the presence of a flexible loop near zinc binding pocket of the protein. P. denitrificans encodes two zinc ABC transporter operons, znuABC and aztABCD. The SBPs for these systems (AztC and ZnuA) are distinct in the length and sequence of flexible loop regions. The function of the flexible loop in each protein was investigated using fluorescence binding assays and structural methods for WT and loop mutant proteins. The flexible loop of AztC has no impact on zinc binding affinity, stoichiometry or protein structure, yet it is essential for zinc transfer from the metallochaperone AztD. Site specific mutagenesis was employed to evaluate the roles of specific residues in this process and the results showed that three His residues in the loop temporarily coordinate zinc and then convey it to the high‐affinity binding site. Consistent with this, mutation of any of these residues to Ala abrogated zinc transfer from AztD. AztD does not transfer zinc to ZnuA, despite the fact that ZnuA also contains a flexible loop rich in His residues. The ZnuA flexible loop contains 15 His, 11 Glu and 3 Asp residues, providing multiple additional zinc binding sites. In fact, P. denitrificans ZnuA binds up to 6 zinc ions, the most for any zinc SBP studied to date. The results demonstrate distinct roles for zinc SBP flexible loops depending on loop structure and context, potentially allowing for acquisition of zinc from different environments.

Support or Funding Information

National Institute of General Medical Science of the National Institutes of Health under award number 1SC2GM111170‐01.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.