bluuebunny/crossref_metadata_2025 huggingface dataset:
Allosteric Regulation of Coregulator Recruitment in FXR
Authors: Villalona Priscilla
Published: May 2022

The farnesoid x receptor (FXRα,NR1H4) is a ligand regulated transcription factor that plays a role in bile acid, carbohydrate, and lipid metabolism. The natural ligands of FXR are bile acids, a diverse class of cholesterol‐derived steroid acids. As a member of the nuclear receptor (NR) superfamily, FXR is comprised of modular domains, including a DNA binding domain (DBD) that is linked to a ligand binding domain (LBD) by a flexible hinge. The LBD contains the activation function 2 (AF‐2) surface that is the site of coregulator binding. Coregulators are an important class of proteins that are recruited to transcriptional complexes and promote up/down regulation of genes of interest by targeting chromatin. While it has been shown that coregulator recruitment to is modulated in a ligand‐specific manner, it is not understood how minor modifications on the bile acid scaffold drive differences in coregulator recruitment, and more broadly, diverse transcriptional outcomes. The goal of this project is to understand how minor differences in ligand structure gives rise to preferences in coregulator recruitment to FXR. To investigate this phenomenon, we are using dual luciferase assays and binding experiments to determine how various coregulators are utilized by FXR for transcription in the presence of multiple bile acids.
Nitric Oxide‐dependent Cutaneous Vasodilation is not Altered in Adults Following SARS‐CoV‐2 Infection
Authors: Dillon Gabrielle A. , Swanger Faith A. , Wolf S. T. , Alexander Lacy M.
Published: May 2022

Cardiovascular complications are part of the clinical sequelae following SARS‐CoV‐2 infection. Vascular dysfunction, one of the earliest indicators of cardiovascular disease (CVD), has been reported in adults who have recovered from COVID‐19. To date, no studies have investigated the underlying mechanisms of persistent COVID‐19‐associated vascular dysfunction. Additionally, physical activity (PA) status has not been accounted for when examining vascular function in COVID‐19 recovered (CR) adults.

PURPOSE

To directly quantify nitric oxide (NO)‐dependent vasodilation in the microcirculation in adults who have recovered from SARS‐CoV‐2 infection. Using the skin as a model circulation, we hypothesized that CR adults would have impaired NO‐mediated vasodilation compared to appropriate controls, and PA would be positively associated with NO‐dependent vasodilation in CR.

METHODS

We performed a cross‐sectional study including: 10 (5M/5W, 24 ± 4yrs) healthy control (HC) adults who were unvaccinated for COVID‐19, 11 (4M/7W, 25 ± 6yrs) healthy vaccinated (HV) adults, and 12 (5M/7W, 22 ± 3yrs) CR (19 ± 14 weeks post‐diagnosis) adults. COVID‐19 symptoms severity (survey) and PA (accelerometer) for 7 days prior to the experimental visit were assessed. A standardized 39°C local heating protocol was used to induce eNOS‐dependent vasodilation, quantified via perfusion (intradermal microdialysis) of 15 mM NG‐nitro‐l‐arginine methyl ester during the plateau of the heating response. Red blood cell flux was measured (laser‐Doppler flowmetry) and cutaneous vascular conductance (CVC = flux/mmHg) was expressed as a percentage of maximum (28mM sodium nitroprusside + 43°C). All data are means ± standard error.

RESULTS

The local heating plateau (HC: 61 ± 20 %CVCmax, HV: 60 ± 19 %CVCmax, CR: 67 ± 19 %CVCmax, p=0.80) and NO‐dependent vasodilation (HC: 77 ± 9%, HV: 71 ± 7%, CR: 70 ± 10%, p=0.36) were not different among groups. In the CR group neither peak symptom severity (25 ± 12 AU) nor time since diagnosis correlated with NO‐dependent vasodilation (R2=0.21, p=0.13 and R2=0.17, p=0.19, respectively). PA was not different among groups (see Table). PA did not correlate with the NO‐dependent vasodilation in CR (sedentary: R2<0.01, p=0.89; light: R2=0.04, p=0.63; moderate‐to‐very vigorous: R2=0.35, p=0.16).

CONCLUSION

Otherwise healthy young adults who have had COVID‐19 did not have impaired NO‐dependent cutaneous vasodilation. Additionally, in this cohort of CR adults, symptomology and physical activity was not related to microvascular function. As such, investigations in adults who have had more severe COVID‐19 cases are necessary. Discovering the underlying mechanisms linking SARS‐CoV‐2 infection and cardiovascular consequences are important for identifying additional treatment strategies for long haul COVID‐19.
Characterizing the Contribution of Adrenergic Signaling to Olaparib Resistance in Ovarian Cancer Cells
Authors: Acevedo‐Santiago Arelis , Cruz‐Robles Melanie , Monteiro Alvaro N. , Armaiz‐Pena Guillermo N.
Published: May 2022

According to the American Cancer Society, ovarian cancer is the deadliest cancer of the female reproductive system. Moreover, a significant number of ovarian cancer patients are clinically depressed or lack social support at the time of their diagnosis. Mounting data support an association between chronic stress and depression with sustained sympathetic nervous system activation (SNS). Furthermore, there is strong evidence supporting a relationship between prolonged SNS activation and cancer progression. In addition, work from our group and others has shown that psychological states such as chronic stress or depression accelerate the growth of existing tumors and promote chemoresistance. Also, recent data from our lab shows that exposure to norepinephrine (NE) leads to double‐stranded DNA breaks in epithelial ovarian cancer cells (EOC) in a β‐adrenergic receptor‐dependent manner. Interestingly, when EOC were co‐exposed to NE and cisplatin (a DNA damaging agent used in ovarian cancer therapy), we observed fewer double‐stranded DNA breaks than with either treatment alone. These data suggest that stress hormones could influence EOC responses to chemotherapeutic agents. Here, we sought to determine the contribution of adrenergic signaling to Olaparib (an FDA‐approved PARP inhibitor) resistance in EOC. We first determined the EOC viability after exposure to Olaparib. Here, we exposed ES‐2 ovarian cancer cells to serial dilutions of Olaparib and calculated their viability after 48 hrs, 72 hrs, 96 hrs, and 120 hrs. Following each experiment, we calculated the IC50 for each timepoint. Our preliminary data suggest an IC50 of 19µM at 48 hrs, 28.5 µM at 72 hrs, 14.5 µM at 96 hrs, and 8 µM at 120 hrs. Currently, we are performing additional experiments to expand these preliminary data to other EOC lines and further determine the contribution of NE to Olaparib resistance in EOC.
Proteome analysis of nuclear Lipid‐Associated Promyelocytic Leukemia (PML) Structures (LAPS) Using Biotin Proximity Labelling
Authors: Thompson Jordan , Salsman Jayme , Boisvert François‐Michel , Caradec Benjamin , Dellaire Graham , Ridgway Neale
Published: May 2022

Lipid droplets (LD) are organelles composed of a phospholipid monolayer surrounding a core of and triacylglycerol and cholesterol ester. LD have a primary role in energy homeostasis and lipotoxic stress through the regulated storage and release of fatty acids. While LD are cytoplasmic, lipoprotein secreting cells harbor nuclear LD (nLD) that associate with PML, the structural protein of PML nuclear bodies (PML‐NB) that regulate DNA repair and stress responses. Knockout studies in U2OS cells indicated that the 50% of nLDs contain PML isoform II (PML‐II) and have a unique lipid and protein composition marked by the lack of canonical PML‐NB‐associated proteins, such as DAXX, SP100 and SUMO. Owing to their unique composition, we term these subnuclear lipid:protein domains, lipid‐associated PML structures (LAPS). To gain insight into LAPS function, we conducted proximity labeling and mass spectrometry (MS) studies using PML‐I and PML‐II modified by fusion to the ascorbate peroxidase APEX2 that uses biotin‐phenol to biotinylate proteins. U2OS and Huh7 cells expressing PML‐APEX2 fusion proteins were treated with and without oleate, and biotinylated proteins were isolated and analyzed by LC‐MS/MS. Using standard filtering techniques (CRAPome, SAINT, Bayesian false discovery rate) we identified several high confidence interacting proteins associated with the COPII coatomer complex, including Sec24A, Sec23IP, Sec24B, and Sec23B. These Sec23/24 proteins were associated with PML‐I and, to a lesser extent, PML‐II under oleate‐treated conditions. Sec23B was observed to associate with both PML‐NBs as well as LAPS by immunofluorescence microscopy in U2OS cells expressing PML‐II‐APEX2. The COPII pathway has previously been implicated in protein delivery to cytoplasmic LD setting a precedence for the interaction we observed with LAPS. Thus, given our preliminary data showing interaction between Sec 23/24 heterodimer, PML‐NBs and LAPS we are now pursuing the hypothesis that lipid membrane deformation and/or cargo transport activity in the nucleus by COPII proteins could play a role in lipid homeostasis.
Sex differences in behavior and immune response revealed by a NSAID nanotherapeutic treatment during peripheral neuroinflammation
Authors: Deal Brooke S. , Reynolds Laura M. , Janjic Jelena M. , Pollock John A.
Published: May 2022

Until recently, the majority of preclinical neuroinflammatory studies were performed on males. Because of this, less is known about the female neuroinflammatory response to injury, formation of pain, or response to pain‐relieving therapies. A major focus has been on the macrophage and its contribution to the development of neuropathic pain. Under a typical response, the macrophage upregulates expression of cyclooxygenase‐2 (COX‐2), which in turn leads to the production of proinflammatory prostaglandin E2 (PGE2), which is linked to neuronal sensitization and pain. Non‐steroidal anti‐inflammatory drugs (NSAIDs) have been shown to attenuate PGE2 by the inhibition of COX‐2. However, a problem with NSAIDs is that due to the systemic dosage needed to achieve neuropathic pain relief there are risks of off‐target toxicity. This study evaluated a COX‐2 inhibiting nanotherapeutic in its ability to relieve neuropathic inflammation and pain‐like behavior of a chronic constriction injury (CCI) of the right sciatic nerve. We find that under the same injury, both males and females exhibit the same degree of hypersensitivity represented as pain‐like behavior. We also find that the nanotherapeutic was able to relieve pain‐like behavior in both sexes, but females experienced less relief. This behavioral response was reflected in the number of infiltrating macrophages at the site of injury; where again, both sexes had decreased inflammation but the females had significantly more macrophages present than the males.
Functional Characterization of a Human Choroid Plexus Cell Line and TRPV4 Stimulated Transepithelial Ion Flux
Authors: Hulme Louise , Hochstetler Alexandra , Schwerk Christian , Schroten Horst , Ishikawa Hiroshi , Blazer‐Yost Bonnie
Published: May 2022

The choroid plexus epithelium (CPe) establishes the blood‐cerebrospinal fluid barrier (BCSFB) and is the primary producer of cerebrospinal fluid (CSF). The CPe has a unique, polarized structure with distinct transporters and channels localized to the apical (CSF‐facing) or basolateral (blood‐facing) membrane. These proteins tightly regulate the composition of CSF by controlling transport of electrolytes and water from the blood into the intraventricular space. Significantly, CSF volume and composition varies on a diurnal basis, as well as in response to pathophysiological stresses. Developing an in vitromodel of the CPe that represents its unique epithelial structure and barrier function is thus key for studying the role of CSF in health and disease. The human choroid plexus papilloma (HIBCPP) cell line is a continuous culture model originally developed by Schwerk et al (2012). The current studies provide characterization of this line to determine its potential as a culture model for electrophysiological studies investigating the regulation of electrogenic ion transport. The HIBCPP cells expresses several CP markers via RT‐PCR, including E2f‐5, transthyretin, forkhead box protein J1 and orthodenticle homeobox 2, confirming lineage of the model. Characteristic of a barrier epithelium, the cells express appropriate junctional complex proteins, zonula occludens‐1, occludin, claudin 1 and claudin 2, via both RT‐PCR and immunofluorescence (IF). When cultured on 30mm Transwell filters, these cells predominately grow as a monolayer and have a transepithelial electrical resistance of ∼400 Ω*cm2. The cells are ciliated, determined by expression of ciliary markers Arl13B and acetylated tubulin. By RT‐PCR, the HIBCPP cells express numerous transporters previously implicated in regulating CSF production in the native CPe, including the Na‐K‐ATPase α1 and β2 subunits, transient receptor potential vanilloid 4 (TRPV4), Na‐K‐Cl cotransporter 1, Na2+ HCO3‐ cotransporter 2, Na2+ dependent Cl‐/ HCO3‐ exchanger, anion exchange protein 2 (AE2), K+ Cl‐ cotransporter 3, volume regulated ion channel, and aquaporin 1. Correct polarization of the N‐K‐ATPase pump, TRPV4 and AE2 suggests transporters in this in vitro model localize to the correct membrane. IF and 3D imaging was used to confirm apical localization of the pump and TRPV4, versus basolateral localization of AE2, consistent with expression patterns in the native tissue. Previous studies have implicated TRPV4 in the regulation of CSF production. In the HIBCPP model, addition of a TRPV4 agonist, GSK1016790A, stimulates a change in net transepithelial ion flux and increase in conductance that is inhibited by a TRPV4 specific antagonist, RN1734. These initial studies support the idea that TRPV4 contributes to CSF secretion by controlling solute movement across the CPe. Overall, this cell line holds great promise as an in vitro model for BCSFB‐based studies.
The Effect of Menstrual Cycle Phase on Neural Recruitment Patterns during Acute Chemoreflex Stress
Authors: Coovadia Yasmine , Shoemaker Kevin , Usselman Charlotte
Published: May 2022

We have shown previously that muscle sympathetic nerve activity (MSNA) responses to acute chemoreflex stress are augmented during the early follicular (EF) phase of the menstrual cycle relative to the midluteal (ML) phase in young healthy women. Interestingly, these differences were most pronounced in terms of MSNA burst amplitude, which may imply that neural recruitment strategies are affected by menstrual cycle phase. Therefore, the purpose of this retrospective analysis was to test the hypothesis that the recruitment of large action potential (AP) clusters occurs to a greater extent during the EF phase of the menstrual cycle compared with the ML phase. MSNA data (microneurography) from 7 young healthy women (aged 23±3 years; body mass index 23±3 kg/m2) were examined at rest and during a voluntary end‐inspiratory apnea which followed a rebreathing protocol to an end‐tidal PO2 of 65 Torr. These data were collected during the EF and ML phases. APs were identified from the filtered MSNA signal using a custom wavelet deconstruction approach which quantified AP firing frequencies (overall AP frequency and AP content per integrated burst). The identified APs were also binned in accordance to peak to peak amplitude to determine individual AP clusters (Scott’s rule). Resting AP frequency and AP content per MSNA burst were not different between menstrual cycle phases (APs/min: P=0.23; APs/burst: P=0.26). Similarly, the number of identified AP clusters at rest did not differ between the EF and ML phases (8±4 vs10±4 clusters; P=0.16). During apnea, changes in AP frequency and AP content per MSNA burst (i.e. expressed relative to baseline) were greater during the EF phase than the ML phase (ΔAPs/min: P=0.02; ΔAPs/burst: P=0.02). Similarly, apnea‐induced recruitment of new AP clusters (i.e. clusters which were not activated at baseline) was greater during the EF phase compared to the ML phase (ΔAP clusters: +6±8 vs+2±6 clusters; P=0.009). Taken together, these findings confirm that augmented integrated MSNA reactivity to chemoreflex stress during the EF phase of the menstrual cycle is mediated by a greater recruitment of latent subpopulations of larger sympathetic neurons relative to the ML phase. We speculate that the change in the gonadal hormone profile between EF (i.e. low estradiol, progesterone) and ML (i.e. elevated estradiol and progesterone) may be associated with centrally‐mediated alterations to neural outflow during chemoreflex stress, although further research is certainly required to address this hypothesis.
AP Profiling: Resolving Co‐Translational Protein Folding Pathways and Chaperone Interactions In Vivo
Authors: Chen Xiuqi , Kaiser Christian M.
Published: May 2022

Co‐translational folding and molecular chaperone action are crucial for productive protein folding in the cell, but how these processes shape folding pathways remains largely unknown. We have utilized translation arrest peptides (APs) to monitor co‐translational folding in live bacterial cells, combined with single‐molecule optical tweezers experiments, to define the co‐translational folding pathway of the GTPase domain (G‐domain) from E. coli elongation factor G (EF‐G). Surprisingly, the 293 amino acid long domain remains unfolded, without forming stable intermediate structures, until it is fully extruded from the ribosome. The full‐length G‐domain transitions to its stable native structure via obligate folding intermediates both in isolation and while bound to the ribosome. Folding therefore follows a strictly sequential pathway that initiates at the very C‐terminus, which is likely imposed by the structure and topology of the G‐domain from EF‐G. Consequently, folding and synthesis proceed in opposite directions. G‐domains represent a common element in a number of multi‐domain proteins. To determine whether their folding pathways are conserved, we have combined our AP approach with cell sorting and deep sequencing into a method that we term “AP profiling”. Homologous G‐domains exhibited distinct folding patterns that are conserved across distant bacterial species. Individual deletion of the primary nascent chain‐binding chaperones, trigger factor and DnaK (Hsp70), resulted in numerous localized changes to co‐translational folding while preserving overall G‐domain folding, highlighting the functional redundancy of cellular chaperone systems. In summary, we have developed AP profiling as a technique for monitoring co‐translational folding in the native cellular environment with high throughput. Combined with single‐molecule force spectroscopy, AP profiling yields a unique view of co‐translational folding and chaperone function.
Fentanyl‐Induced Sleep Disruption in C57BL/6J (B6) Mice is not Secondary to Increased Motor Activity or Hypothermia
Authors: Zebadúa Unzaga Diana , Zhu Xiaojuan , Price Josh , Lydic Ralph , Baghdoyan Helen A.
Published: May 2022

Studies of mice have enhanced the understanding of opioid‐induced respiratory depression (10.1152/jn.00017.2021) and opioid abuse (10.1016/j.neuropharm.2013.08.028). The potential that mechanistic insights gained from studies of mice may be relevant for humans is consistent with the close genetic homology between these two species. Features of mouse biology, however, may limit the extent to which studies of opioid effects can be generalized. Fentanyl administered to mice can increase motor activity (10.1152/jn.00711.2020), cause hypothermia (10.1124/jpet.102.037655), alter cortical electroencephalogram (EEG) power, and disrupt the temporal organization of sleep (10.1152/jn.00266.2021). Our recent finding that fentanyl causes dose‐dependent sleep disruption in B6 mice (10.1096/fasebj.2021.35.S1.01706) led us to test the hypothesis that fentanyl‐induced increases in wakefulness are not caused by changes in motor activity or body temperature. The FASEB Statement of Principles for the use of Animals in Research and Education was followed. Adult males (n=8) were housed in a temperature‐ and humidity‐controlled room with a 12:12h light:dark cycle. Mice were implanted with electrodes and subcutaneous telemeters (DSI HD‐X02) to record EEG, electromyogram, motor activity, and body temperature. Seven to 10 days after recovery from surgery each mouse received a series of subcutaneous injections (0.3 mL) of saline (vehicle) and increasing half‐log doses of fentanyl (0.001 to 3 mg/kg). All injections were made within 60 min of light onset and mice were recorded for 4 h. Injections in the same mouse were separated by 3 or 4 days. Repeated measures mixed model ANCOVAs were performed to evaluate percent time awake between fentanyl dose while accounting for the effects of motor activity and body temperature. Each ANCOVA model was evaluated to determine if the motor activity and body temperature slopes at each level of fentanyl were equal to zero by including the fentanyl main effect and the fentanyl by motor activity (p = 0.11) or fentanyl by body temperature (p = 0.19) interaction. This established the lack of dependence between fentanyl dose and motor activity or body temperature when explaining differences in percent of time in wakefulness. Subsequent common slope models were performed including the main effects for fentanyl with body temperature and fentanyl with motor activity to confirm no effects of either covariate on percent time awake. Motor activity (p = 0.611) and body temperature (p = 0.614) were not found to be significant predictors of increased wakefulness. This finding supports the interpretation that fentanyl causes sleep disruption that is not secondary to enhanced motor activity or hypothermia.
Anthraquinone derivatives inhibit telomerase activity by interaction with G quadruplex DNA and acts as a promising anticancerous agent
Authors: Dey Arpita , Prasad Ramasaree , Barthwal Ritu , Pandav Kumud
Published: May 2022

G‐quadruplex defines as an evolutionarily conserved sequence, non‐canonical secondary structures found at the telomeric region and in promoter regions of various oncogenes like cMYC, CKIT, and BCL2, etc. Telomerase enzyme activity is inhibited by a small molecule that selectively interacts with G‐quadruplex. Understanding the binding mechanism of the ligand‐G quadruplex complex is crucial for assessing anti‐tumor therapeutic efficacy. We have synthesized different anthraquinone derivative‐based G4 ligands which stabilize quadruplex sequence and influence various cellular processes. We have explored the interaction of synthesized anthraquinone derivatives with G4 DNA sequences comprising human telomeric DNA sequence by using absorption, fluorescence, circular dichroism (CD) spectroscopy, surface plasmon resonance (SPR) and thermal melting. Each ligand binds to the G4 DNA in monomeric form. The absorption intensity decreases with a shift of 6‐8 nm. Fluorescence quenching studies and sensorgram indicated a significant binding affinity (Kb) in the order of 102 to 108 M−1. A shift of ~6‐10 nm indicates partial stacking of aromatic chromophores with nucleic acid bases. Similar studies with CD spectroscopy yielded Kb in the range of 103 to 107 M−1. Thermal melting profiling of DNA saturated at Drug/DNA = 5 indicated a stabilization (ΔTm) of 21 to 34 °C in presence of different ions indicating more thermal stabilization in the presence of Na+ ions as compared to K+. Molecular docking analysis concluded that external binding of the ligands to G4 DNAs. The formation of a complex G4 structure was further established by polymerase stop assay. The compounds showed in vitro cytotoxicity in cancer cells (MCF) based on a cell viability assessment which undergoes oxidative lesions at a greater extent. Apoptosis is the mechanical pathway behind the cytotoxicity of cancer cells from the live‐dead staining trials. Modifications of cellular morphs, nuclear condensation, and fragmentation were observed in fluorescence microscopy upon treatment of these compounds. qRT‐PCR highlights gene transcriptional regulation genes containing G4 sequence after treatment of ligands. Apart from damaging G4 DNA, thermal stabilization caused by selective interactions is thought to block telomerase enzyme associated with telomere end and contribute to drug‐induced death in cancerous cell types. The insights expedite therapeutic development due to the extensive possibilities of changing ensuing groups on anthraquinones to improve efficacy, reduce cell toxicity, and increase specificity for G‐quadruplex DNA.
Hyperactive Striatal Dopamine Signaling Observed in Huntington’s Disease Mouse Models Prior to Motor Symptom Onset Can be Attenuated via Activation of M4 Receptors
Authors: Rodriguez Roland M. , Foster Daniel J. , Lavery Madigan L. , Johnston Curran M. , Moehle Mark S. , Dickerson Jonathon W. , Rook Jerri M. , Conn P. J.
Published: May 2022

Almost all Huntington’s Disease (HD) patients die within 10‐30 years after symptom onset and currently there are no available treatments to slow, stop, or reverse its progression. HD is a neurodegenerative disease that causes a biphasic signaling pattern in the striatum in which there is hyperactive signaling during the early stages, followed by hypoactive signaling later on. Among the many neurotransmitters shown to follow the biphasic signaling pattern found in HD, dopamine (DA) and its inputs from the mid‐brain to the striatum are thought to be critical for motor and action planning. However, there is currently a lack of studies that directly examine DA signaling in animal models before symptom onset, leaving room for investigation in a critical area of the disease. Here we use a combination of techniques to assess dopamine signaling and its regulation during pre‐symptomatic disease stages. We hypothesized that we would observe increased dopamine transients in YAC128 positive mice when compared to littermate controls at this time point, and that activation of the M4 receptor with the M4 selective positive allosteric modulator (PAM) VU’154 would normalize transient amplitude in YAC128 mice. Using fast scanning cyclic voltammetry (FSCV), we recorded dopamine signaling in pre‐symptomatic YAC128 mice via ex vivo preparations. We also used, fiber photometry to record dopamine signaling in vivo, in the same mouse strain. We found that indeed, DA transient amplitudes are increased in pre‐symptomatic YAC128 mice when compared to littermate controls in both ex vivo and in vivo settings. Additionally, we report that activation of the M4 receptor is able to normalize the enhanced DA signaling in HD mice. These findings are some of the first to directly show increased DA signaling in the basal ganglia in early stage HD rodent models and support previous evidence that M4 ­­­­­­­is potentially an exciting target for normalizing aberrant neurotransmission in HD.
Interaction Sites of the Nem1‐Spo7/Pah1 Phosphatase Cascade in Yeast Lipid Synthesis
Authors: Jog Ruta , Mirheydari Mona , Han Gil‐Soo , Carman George
Published: May 2022

In the yeast Saccharomyces cerevisiae, Pah1 phosphatidate (PA) phosphatase catalyzes the dephosphorylation of PA to produce the diacylglycerol used to synthesize triacylglycerol that is stored in lipid droplets. Pah1 is inactive as a phosphorylated form in the cytosol and becomes active as a dephosphorylated form on the nuclear/ER membrane following its recruitment and dephosphorylation by the Nem1‐Spo7 protein phosphatase complex. Spo7, the regulatory subunit in this protein phosphatase complex, is required for stability and function of the catalytic subunit Nem1. In this work, we examined regions of Spo7 that are involved with the interaction with Nem1 for Nem1‐Spo7 complex formation and interaction with Pah1. By deletion analyses and site‐directed mutagenesis, we found that the Spo7 C‐terminal residues 240‐259 are important for Nem1‐Spo7/Pah1 phosphatase function as indicated by a dramatic reduction in triacylglycerol synthesis and lipid droplet formation, along with a temperature‐sensitive phenotype. We are currently investigating whether this sequence is required for physical interaction with Nem1 or Pah1.
Alternative Chloride Channels are Increased in the CFTR‐/‐ Rat Airway
Authors: Birket Susan , Green Morgan , Doty Ian , Oden Ashley , Keith Johnanthan
Published: May 2022

Cystic fibrosis (CF) is a genetic disease caused by absence of the cystic fibrosis transmembrane conductance regulator (CFTR) affecting multiple organs. CFTR is an anion transporter that moves chloride and bicarbonate ions to the apical side of the airway epithelial surface. CFTR deficiency results in decreased airway surface liquid depth and abnormally acidic airway pH, leading to decreased mucociliary transport and subsequent airway infections with pathogens such as Pseudomonas aeruginosa. While a major anion transporter in the epithelium, CFTR is not the only channel responsibly for efflux of chloride to the apical surface of the airway. Additional chloride transporters such as TMEM16A, SLC26A9, and CLC2 are present on the airway and have been detected to be potential disease‐modifying genes. Recently, these transporters have been postulated to be therapeutic targets for supportive therapies in CF and other airway diseases. Here, we sought to determine the role of these chloride transporters in the development of lung disease of the CFTR‐/‐ rat model. Short‐circuit current data suggest that the largest portion of chloride transport function in the rat lung is caused by CFTR; however there remains some residual non CFTR‐dependent current that may be due to additional chloride transporters. To determine which were important in the airway, CFTR‐/‐ rats at 1, 3, and 6 months of age were assayed for mRNA and protein concentrations of TMEM16A, SCL26A9, and CLC2 compared to their wild‐type (WT) littermates. Tracheae from these rats were excised and mucus particles tracked to determine mucociliary transport. We also assayed rats before and after lung infection with P. aeruginosa. Initial results indicated that TMEM16A and SLC26A9 mRNA and protein expression are increased in CFTR‐/‐ compared to their WT littermates at each age tested and remain elevated compared to control following infection with P. aeruginosa. CLC2 has increased expression in CFTR‐/‐ rats only following infection with P. aeruginosa. We found no correlation between expression of these transporters and mucociliary transport rates. However, we believe that results obtained suggest that these transporters may be exploited via agonists to improve airway surface liquid depth and pH. Future studies will examine the role of these transporters in correction of CF airway pathophysiology.
RAGE Signaling and Models of COPD Pathogenesis
Authors: Reynolds Paul R.
Published: May 2022

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and it is characterized by inflammation and loss of respiratory tissue. Tobacco smoke is the major cause of COPD. Studies identified receptors for advanced glycation end‐products (RAGE) as a pattern recognition receptor with potent pro‐inflammatory characteristics that is increased in the lung parenchyma following first or secondhand smoke (SHS) exposure. The recent generation of a conditional RAGE transgenic mouse that increases RAGE expression manifests characteristics of COPD and may provide a smokeless model of a smoker’s lung. We evaluated inflammatory effects of SHS with and without semi‐synthetic glycosaminoglycan ethers (SAGEs), a family of anionic, partially lipophilic sulfated polysaccharide derivatives that inhibit RAGE signaling. The current research evaluated the in vivo effects of smoke exposure in RAGE null, conditional RAGE over‐expressing, and control mice via a nose‐only exposure platform for 1, 3, and 6 months (Sireq Scientific) and compared them to animals exposed to room air. Groups of mice were also co‐treated with SAGEs via weekly ip injections (a 30mg/kg body weight). Molecular characterization of smoke exposure revealed significant pulmonary inflammation and apoptosis mediated in part by RAGE. Inflammatory cell behaviors were assessed by determining the activation of Ras, intracellular signaling kinases, and cellularity/cytokine secretion in bronchoalveolar lavage fluid (BALF). Inflammatory signaling intermediates and pulmonary remodeling were induced by SHS and influenced by the availability of RAGE, as evidenced by RAGE targeted mice and SAGE treatment. We also observed compromised lung mechanics (FEV1, FVC, elastance, compliance) following exposure, but improved physiology when RAGE was concomitantly targeted. These data reveal captivating information suggesting a role for RAGE signaling in lungs exposed to tobacco smoke and implicates plausible therapeutic modalities that may attenuate lung pathology.
Pro‐Inflammatory Profiling Of RAGE Targeted Mice Following Short‐Term Tobacco Exposure
Authors: Curtis Katrina , Clarke Derek , Grajeda Samuel , Hanegan Makayla , Neciosup Katie , Black Joe , Theurer Ben , Arroyo Juan A. , Reynolds Paul
Published: May 2022

Inflammation underpins pulmonary disease progression during tobacco smoke exposure that may culminate in irreversdible pulmonary remodeling. While primary smoke poses notable risk, nearly half of the US population is also at risk due to exposure to secondhand smoke (SHS). In the present study, we assessed a potential role for RAGE, a cell‐surface pattern recognition receptor implicated in pro‐inflammatory signaling, following exposure to SHS. Specifically, we used wild type, RAGE null, and lung‐specific RAGE overexpressing transgenic (TG) mice and evaluated the elaboration of inflammatory mediators in bronchoalveolar lavage fluid (BALF). Select mice were administered semi‐synthetic glycosaminoglycan ethers (SAGEs), a family of anionic, partially lipophilic sulfated polysaccharide derivatives known to inhibit RAGE signaling. Mice were exposed to room air (RA) or SHS from three Kentucky 3R4F research cigarettes via a nose‐only delivery system (Sireq Scientific, Montreal, Canada) five days a week and ip injections of PBS or SAGE (a 30mg/kg body weight) occurred three times per week from PN40 until sacrifice date on PN70. RAGE mRNA and protein expression was elevated in wild type and TG mice following SHS exposure and no expression was detected in RAGE nulls. BALF analyses revealed RAGE‐mediated control of leukocyte extravasation and a multiplex cytokine array confirmed a role for RAGE in the coordination of pro‐inflammatory chemokine/cytokine secretion. Among other mediators, TNF‐a, MIP‐2, and IL‐1b were each differentially secreted by lung tissue following SHS exposure and concentrations were significantly decreased in BALF from exposed RAGE null or wild type mice concomitantly administered SAGEs. In summary, inflammatory responses induced by SHS exposure were influenced by the availability of RAGE, as evidenced by RAGE nulls and SAGE treatment. These data reveal fascinating data suggesting the utility of RAGE abrogation in lessening smoke‐induced pulmonary exacerbations.
The Effects of Prune Extract on Cellular Models of Bone Cancer
Authors: Miller Chelsie , Adam Mowaffaq , Chao Grace , Hooshmand Shirin , Sohl Christal
Published: May 2022

There is strong evidence for prunes as a health food for improvement in digestion due to their high fiber content. More recent work has shown that prunes also have a positive impact on bone health, attributed in part to the antioxidant compounds and polyphenols they possess. Despite their important antioxidant role, the anticancer properties of prunes has yet to be determined. We hypothesize that treatment of osteosarcoma cell lines with prune extracts will lead to a decrease in cell proliferation and migration, driven at least in part due to the antioxidant compounds. We will treat C28, Saos‐2, and hFOB 1.19 bone cell lines with varying concentrations of prune extract, and use the CellTiterGlo assay and scratch assays to measure the effects on cell proliferation and migration, respectively. A key component of our work is establishing the optimum cell culturing conditions and developing robust protocols for important cancer phenotype assays. Upon completion of these experiments, we hope to better understand the effects of prunes on bone health and disease.
Probing the potential interactions between two protein vaccine candidates from Nontypeable Haemophilus influenzae
Authors: McGinnity Grace , Perdue Janai , Labbe Natalie , Kaur Ravinder , Holmquist Melody , Michel Lea
Published: May 2022

Nontypeable Haemophilus influenzae (NTHi) is the leading cause of AOM (acute otitis media) among children in the US, and NTHi infections contribute to the $6 billion in AOM‐related medical costs each year. NTHi is also a leading cause of COPD (Chronic Obstructive Pulmonary Disease), the third leading cause of death in adults in the US, underlining the need for a vaccine to protect us against NTHi. Two well studied protein vaccine candidates from NTHi are OMP26 and Protein D. In preliminary studies, mice produced a robust antibody response to OMP26 and Protein D when injected individually. However, when both proteins were mixed in a single solution, Protein D antibody production was significantly suppressed. We hypothesized that an interaction between the proteins prevented Protein D’s interaction with immune cells thus suppressing antibody production. Here, we discuss our biochemical approaches to study the potential OMP26‐Protein D interaction, including native mass spectrometry. Preliminary results from those studies suggest OMP26 and Protein D do not form a complex in vitro.
D1 Dopamine/Mu Opioid Receptor Interactions in Operant Conditioning Assays of Pain‐Depressed Responding and Drug‐Induced Rate Suppression: Assessment of Therapeutic Index in Rats
Authors: Asmus Francesca , LaCourse Hannah , Smith Meghan , Davis Ravin , Harrington Kylee , Bergman Jack , Stevenson Glenn
Published: May 2022

In vivo receptor interactions vary as a function of behavioral endpoint. Our laboratory has begun investigating the effects of D1 dopamine receptor agonists and D1 dopamine/mu opioid receptor interactions. This decision is based on studies that have shown (1) dopamine D1 receptors produce pain relief in rodent models of acute pain, (2) a mixed literature indicating D1 agonists either do or do not possess abuse liability, perhaps due in part to behavioral endpoint selection, and (3) co‐localization of D1 and mu receptors in brain. This is the first report of D1/mu receptor interactions using an assay of pain depressed behavior. A multiple‐cycle FR10 operant schedule and a simple FR10 schedule were utilized in the presence of (antinociception) and in the absence of (rate suppression / sedation) a lactic acid inflammatory pain‐like manipulation, with antinociception defined as the therapeutic effect, and sedation defined as a side effect. SKF82958 and methadone were used as selective/high efficacy D1 dopamine and mu opioid agonists, respectively. Both SKF82958 and methadone alone produced dose‐dependent restoration of pain‐depressed responding and response rate suppression in the mult‐cycle schedule, but SKF82958 was ineffective in restoring pain‐depressed responding in the simple FR10 schedule. Equal potency 1:1 SKF82958/methadone mixtures and 1:3 and 3:1 mixtures are currently being tested in both operant schedules to determine the nature of the interaction (additive, superadditive, subadditive). Results will indicate if therapeutic index varies as a function of operant schedule, and the degree to which the addition of SKF82958 produces opioid‐sparing effects.
An Evaluation of the Stability of Splicing Protein Dib1 Through Circular Dichroism Spectroscopy
Authors: Dunn Richard , Conde Anabelle , Lee Grace , Meeks Garrison , Pittner Nick , Maeder Corina
Published: May 2022

Pre‐messenger RNA (pre‐mRNA) splicing is the process in which introns, noncoding regions of DNA, are removed and exons, coding regions of DNA, are ligated to produce a mature mRNA transcript. This process is mediated by the spliceosome, a dynamic macromolecular complex composed of five small nuclear ribonucleoproteins and other protein factors. At the catalytic center of the splicing machinery is the evolutionarily conserved yeast protein, Dib1, composed of 143 amino acids essential for splicing and cell viability. Dib1 and its human ortholog, hDim1, have demonstrated peptidase activity, specifically autocleavage of the last thirteen and fourteen amino acids, respectively, of their carboxy‐terminal tails. The mechanism and function of this autocleavage activity of the carboxy‐terminal tail is uncharacterized.

Previous yeast growth assays comparing mutants to wildtype Dib1 have established that deletion of fifteen and sixteen amino acids off the carboxy‐terminal tail results in temperature‐sensitivity of yeast cell growth at 37ºC. Since Dib1 and splicing are essential for cell viability, the temperature‐sensitive mutants suggest that truncations to the carboxy‐terminal tail of Dib1 may result in defective splicing. Thus, the carboxy‐terminal tail may play a direct role in splicing or destabilize splicing complexes. To address these two possible hypotheses, we are investigating the effects of the carboxy‐terminal truncations on Dib1 stability. Protein purification of wild type and a series of truncated Dib1 mutants were performed. Circular dichroism spectroscopy employing increasing concentrations of guanidinium hydrochloride, a protein denaturant, was used to determine the stability of Dib1 and its truncated mutants. Overall, our findings show that Dib1 stability is directly linked to the length of the tail region and lead us to speculate that the tail is critical for stabilization of the spliceosomal complex.
Using Mouse Metatarsal and Pisiform Ossification to Identify Genes Underlying Growth Plate Formation
Authors: Reno Philip L. , Kjosness Kelsey M. , Park Sungdae , Fared Daved , Stubblebine Mallory B. , Fitzwater Meghan T. , Menke Douglas B.
Published: May 2022

Variation in growth rate underlies, in large part, the great diversity in mammalian skeletal shape. Longitudinal growth occurs at growth plates, which amplify and maintain the process of chondrocyte differentiation throughout ontogeny. Experiments using genetic inactivation in the mouse model have expanded knowledge of growth plate physiology; yet, little is known about how growth plate location and behavior are patterned to produce the normal diversity in skeletal shapes. Fortunately, substantial anatomical variation exists within the mammalian skeleton that can be used to identify the mechanisms underlying growth plate formation. Third metatarsals (MT3) form only a single growth plate located at the distal end, and the pisiform (in non‐human mammals) forms an active growth plate unlike the other carpals. In both cases, growth plate containing tissues can be paired with those undergoing generalized endochondral ossification which can control for the effects of age, systemic growth factors, and biomechanical environment. We performed global transcriptome sequencing (RNA‐seq) on 4‐ and 9‐day old mouse samples comparing either the distal and proximal MT3s or the pisiform and other carpals to identify growth plate‐specific differentially expressed genes (DEGs). Gene ontology (GO) analysis revealed DEGs from the MT3 are disproportionately associated with anatomical structure morphogenesis, developmental growth, and limb development, in addition to the Fzd8‐Ror2‐Wnt5a pathway. Significant GO terms for the pisiform‐carpal comparison include skeletal system development, cartilage development, anatomical structure morphogenesis, and abnormalities of the carpal bones. We identified a limited set of DEGs shared by both the MT3 and Pisiform‐Carpal datasets, including Wnt5a. Using immunohistochemistry, we observed Wnt5a expression in both the distal and proximal MT3 but identified that intense expression in the bone collar is unique to the distal end adjacent to the growth plate. This conforms with previously identified roles for Wnt5a in regulating osteogenesis and cell polarity within the growth plate and validates this approach for identifying growth plate specific chondrogenic and osteogenic factors.
Interspecific variation in avian postcranial pneumaticity: A pilot study
Authors: Martinez Aracely , Lawson Adam B. , Echols M. S. , Hedrick Brandon P. , Schachner Emma R.
Published: May 2022

The avian respiratory system is composed of a unidirectionally ventilated, volume‐constant gas‐exchanging lung, and a series of compliant, flexible air sacs. Diverticula from the lungs and air sacs invade adjacent bone and create air‐filled cavities within the skeleton through the process of pneumatization. While previous studies have addressed the presence or absence of pneumatic bones in multiple species, the pattern of pneumatization has been vaguely generalized with respect to which lung elements are responsible for pneumatizing different skeleton regions. Here, we aim to address which components of the avian respiratory system are pneumatizing each component of the postcranial skeleton and how the patterns vary between different taxa. Computed tomography (CT) and microCT data are used to visualize the pneumatized bones and to segment 3D digital surface models of the skeletal and respiratory systems. Specimens used in this study include the African grey parrot (Psittacus erithacus), ostrich (Struthio camelus), red‐tailed hawk. (Buteo jamaicensis), and tundra swan (Cygnus columbianus). We found substantial differences in the pneumatization patterns between the birds. In all taxa examined, there is an extensive supramedullary diverticulum that travels through the vertebral canal which directly pneumatizes each vertebra in the parrots but does not contribute to any pneumatization in the swan or hawk. The sacral, pelvic, and femoral elements are pneumatized solely by pelvic diverticula in the ostrich, but predominantly by the abdominal sacs in parrots. These data indicate that the pneumatization patterns in birds are highly variable and warrant further study.
Developing a Mouse Model to Test the Impact of Metabolic Syndrome on Systemic Lupus Erythematosus‐Induced Organ Damage
Authors: Kakalij Rahul M. , Boesen Erika I.
Published: May 2022

Background/hypothesis of the study

Metabolic syndrome (MetS) is characterized by a group of conditions that include obesity, impaired glucose tolerance, dyslipidemia and hypertension. MetS is common in systemic erythematosus (SLE) patients and associated with increased cardio‐renal risk. The present study was carried out to develop a model to use in future investigations of whether MetS accelerates SLE‐induced organ damage.

Methods and Results

Female C57BL/6 mice were untreated or treated epicutaneously with the TLR7 agonist imiquimod (IMQ) and fed a high‐fat (HF) “Western” diet (fat 42% kcal, sucrose 34% kcal) or control diet (fat 12.6% kcal, sucrose 34% kcal) for 6 weeks. Data at 6 weeks were compared by two‐way ANOVA, with n=8 mice per group. Supporting early‐stage induction of autoimmunity, spleen weights were significantly increased in IMQ‐treated mice (PTreatment<0.001), and renal cortex IgG deposition was confirmed by western blot. Increased body weight, gonadal fat pad mass, and plasma leptin levels were observed between HF and control animals (PDiet<0.001) for both IMQ and untreated mice. At the 6th week, HF animals showed an inrease in 24‐hour calorie intake (PDiet<0.05) in contrast to control animals. At the same time, HF diet increased total cholesterol (PDiet<0.05) when compared with control diet‐fed animals. However, the increase in these parameters with HF diet was significantly diminished in IMQ‐treated mice (PDiet*Treatment<0.05). Both the HF diet (PDiet<0.001) and IMQ treatment (PTreatment<0.05) significantly increased fasting blood glucose. IMQ treatment affected fasting insulin concentrations in a diet‐dependent manner (PDiet*Treatment<0.01), with hyperinsulinemia observed in IMQ‐HF treated mice (178±8 μIU/ml) compared to IMQ‐treated control diet mice (49±8 μIU/ml; P<0.05 by Bonferroni post‐hoc test), with plasma insulin similar between untreated mice on HF (112±19 μIU/ml) and control diets (98±20 μIU/ml; P>0.05).

Conclusion

Together, these data indicate that the IMQ model of SLE is associated with metabolic alterations, impaired glycemic control, hypercholesterolemia, and hyperinsulinemia under HF intake conditions. In the future, this model may be helpful to understand the complex relationship between MetS and organ damage in SLE.