bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of the O‐GlcNAc Transferase Interactome in X‐linked Intellectual Disability
Authors: Wells Lance
Published: April 2020

Both Ser/Thr phosphorylation and O‐linked beta‐N‐acetylglucosamine (O‐GlcNAc) modification occurs on thousands of nuclear and cytosolic proteins. The forces of evolution have resulted in gene duplication and divergence of substrate recognition for kinases to gain specificity. However there is only one mammalian gene encoding the O‐GlcNAc transferase (OGT). Missense mutations in this gene have been established as being causal for X‐linked intellectual disability. Working with collaborators, we have characterized several of the resulting OGT variants and found little to no defects in global O‐GlcNAc levels, enzyme kinetics, or stability. This led us to consider how is OGT regulated and targeted to substrates and whether the XLID associated variants had defects in protein‐protein interactions.

It has been suggested that the N‐terminal tetratricopeptide repeat (TPR) domain of OGT, where many XLID variants occur, rather than its C‐terminal catalytic domain, is responsible for OGT substrate selection by targeting to specific complexes and subcompartments of the cell. To assess the role of the TPR domain in OGT protein interaction, we generated a BioID proximity proteomics system. Utilizing a fusion protein, TPR‐BirA*, has allowed us to identify more than 100 OGT TPR domain‐interacting proteins. We also utilized BioID to identify proteins that fail to interact with the OGT XLID variants and are in the process of identifying cell type specific differences in protein:protein interactions of the variants focusing on neural lineages. This screen to date has already produced several candidates known to be involved in neurodevelopmental disorders that represent potential mechanisms for how OGT mutations could lead to XLID. These candidates set the stage for future work elucidating the function of specific OGT TPR interactions and how they contribute to OGT function and pathophysiology.

Support or Funding Information

This work is supported in part by NICHD/NIH (R21HD097652)
Genetic and acquired heterotopic ossification are driven by a self‐amplifying positive feedback loop of Hedgehog signaling
Authors: Yang Yingzi
Published: April 2020

Heterotopic ossification (HO), which refers to extra‐skeletal pathological bone formation, occurs as a common complication after injury or in genetic disorders. However, limited by poor understanding of the underlying cellular and molecular mechanisms, there is currently no effective treatment and surgical eviction often results in recurrence. We have found that in both genetic and injury‐induced HO, activated ectopic expression of a hedgehog ligand drives HO progression by forming a positive feedback loop that results in non‐cell‐autonomous and self‐propagation of osteoblast differentiation of wild type cells. In mouse models of progressive osseous heteroplasia (POH), a human disease caused by null mutations in GNAS that encodes Gαs, we found previously that Hedgehog signaling was activated, which is both necessary and sufficient to induce HO. We recently further found that progressively expanded ectopic bone was formed by progressively recruited wild type cells. Mechanistically, the Gnas−/− mesenchymal cells differentiate into osteoblasts and recruit wild type cells to form bone by activating expression of Hedgehog ligands. Secreted Hedgehog further induces Hedgehog expression and osteoblast differentiation in surrounding wild type cells. This self‐propagating positive feedback loop is both necessary and sufficient for ectopic bone formation and expansion and can act independently of Gαs. Importantly, Hedgehog activation was also found in fibrodysplasia ossificans progressive (FOP), Achilles tendon puncture (ATP)‐induced HO mouse models and in human HO samples. Genetic removal of Hedgehog abolished HO not only in POH, but also in FOP and aquired HO mouse models. We therefore identify a Hedgehog‐driven, self‐amplifying osteoblast differentiation as a common cellular and molecular mechanism underlying HO initiation and expansion. Our work highlights the importance of rare genetic disease studies in identifying a shared core pathological mechanism underlying a class of diseases. Our results further suggest that the Hedgehog pathway is a promising new therapeutic target for HO without affecting the normal bone.

Support or Funding Information

R01DE025866 from NIDCR, R01AR070877 from NIAMS
Crosstalk between mTOR and Estrogen Signaling
Authors: Holz Marina
Published: April 2020

Binding of estrogen to ERα triggers the regulation of the target genes (genomic pathway). Additionally, a cytoplasmic fraction of estrogen‐bound ERα activates oncogenic signaling pathways such as PI3K/AKT/mTOR (nongenomic pathway). The upregulation of the estrogenic and the PI3K/AKT/mTOR signaling pathways is frequently associated with a poor outcome in breast cancer. To better characterize the connection between these two pathways, we performed a phosphoproteome analysis of estrogen and mTORC1 signaling. We identified two inhibitory proteins, DEPTOR end eIF3f, as estrogen‐regulated mTORC1 targets. DEPTOR binds to mTOR and inhibits its kinase activity. Although estrogen enhances the phosphorylation of DEPTOR by mTORC1, DEPTOR levels increase in estrogen‐stimulated cells. We demonstrated that DEPTOR accumulation is the result of estrogen‐ERα‐mediated transcriptional upregulation of DEPTOR expression. Consequently, the elevated levels of DEPTOR partially counterbalance the estrogen‐induced activation of mTORC1/2. We also found that the expression of the translation pre‐initiation inhibitory protein eIF3f is tightly controlled by ERα at the transcriptional and translational levels. Specifically, estrogen‐bound ERα represses transcription of the EIF3F gene, while promoting eIF3f mRNA translation. To regulate translation, estrogen activates the mTORC1 pathway. These results underscore the critical role of estrogen‐ERα as a modulator of the PI3K/AKT/mTOR signaling pathway in ER‐positive breast cancer cells.

Support or Funding Information

NIH R35GM128675
Anti‐glycation Effect and Advanced Glycation End‐products Cross‐link Breaking Ability of Sclerocarya birrea Stembark Extracts
Authors: Adeniran Oluwaseyefunmi Iyabo , Mogale Motetelo Alfred
Published: April 2020

Advanced glycation end‐products (AGEs) are implicated in the pathogenesis of late microvascular complications of diabetes mellitus. Currently, there is no clinically approved anti‐glycation agent with minimal side effects. The objective of the study was to investigate the anti‐glycation effect and AGE‐cross‐link breaking ability of Sclerocarya birrea (S. birrea) stembark extracts. Bovine serum albumin (BSA) was incubated with fructose in the presence of different stembark extracts of S. birrea at 37°C for 40 days. Fluorescent AGEs (FAGEs), total immunogenic AGEs (TIAGEs) and carboxymethyl lysine (CML) formed were measured using spectrofluorometry and enzyme‐linked immunosorbent assay (ELISA). The ability of S. birrea stembark extracts to break BSA‐AGE‐collagen cross‐links was also investigated by means of ELISA.

Ethyl acetate, methanol and water extracts significantly inhibited the formation of FAGEs than the standard inhibitor aminoguanidine. Hexane extract demonstrated significantly higher anti‐glycation activity than aminoguanidine against TIAGEs. The water extract exhibited higher inhibitory activity than aminoguanidine against the formation of CML. With regard to the ability to break down AGE‐cross‐links, S. birrea stembark methanol and water extracts demonstrated higher ability to break AGE‐cross‐links than the hexane and ethyl acetate extracts. In conclusion, crude S. birrea stembark extracts have the ability to inhibit the formation of AGEs and to breakdown AGEs‐protein cross‐links. The results of this study may lead to the isolation of bioactive phytochemicals from S. birrea stembark extracts that may be used for the prevention of vascular complications of diabetes.

Support or Funding Information

1. National Research Foundation (NRF), South Africa. 2. Sefako Makgatho Health Sciences University (SMU), South Africa
Developmental origins and evolution of bats
Authors: Sears Karen , Sadier Alexa , Urban Daniel
Published: April 2020

Through the evolution of novel wing structures, bats (Order Chiroptera) became the only mammalian group to achieve powered flight. This achievement proceeded the massive adaptive radiation of bats into diverse ecological niches. As a result, today’s bats exhibit diverse flight styles, the broadest range of dietary adaptations of any mammalian group, and comprise 20% of mammalian species. My lab is investigating the developmental mechanisms through which bats arose and then diversified. In this talk, I will discuss two of the systems we are studying in this regard: bat wings and teeth. In regard to the bat wing, we are investigating the developmental processes that underlie the evolutionary origination and diversification of two of its novel membranes: the plagiopatagium which connects the 5th digit to the body and hind limb in all bat species, and the uropatagium which connects the hind limbs in many species. Our results suggest that these structures arise through novel outgrowths of the body flank driven by increased rates of cellular proliferation. The diversification of membrane shape among bat species is driven by changes in the timing and levels of this cellular proliferation. In all bats, the cells of these novel outgrowths subsequently adhere and fuse to those of the limbs to generate the wing membranes. In regard to teeth, we are investigating the developmental processes behind the wide diversity of post‐canine dentitions among bat species. Our morphological, cellular, and computational analyses across diverse bats suggest that premolar and molar rows are established by two independent signaling mechanisms, and that the number of teeth and their size is controlled by local proliferative rates in jaw cells. Our findings support the hypothesis that an understanding of the evolution of cellular processes is a necessary prerequisite to an understanding of the developmental factors shaping evolution.
Investigating the impact of cell maturity on transplantation success for CNS injury
Authors: Payne Samantha L , Tuladhar Anup , Obermeyer Jaclyn , Varga Balazs V , Morshead Cindi M , Nagy Andras , Shoichet Molly S
Published: April 2020

Globally, a total of 82 million people suffer from injuries to the central nervous system (CNS) and to date there is no cure. However, while humans and other mammals have a limited capacity for functional tissue repair, various other species exhibit near‐perfect CNS regeneration following injury. Understanding the factors that regulate the reparative response can therefore provide important insight into strategies for promoting regeneration in mammals. An emerging approach involves the localized delivery of exogenous neural stem cells (NSCs). Although preclinical studies have shown that NSCs can survive once transplanted, whether they differentiate into neurons and functionally integrate into the host tissue remains unclear. In addition, there are questions as to what stage of NSC maturity will provide the most therapeutic benefit. To address these questions, we investigated cell survival, differentiation, and long‐term sensorimotor recovery in a rat model of stroke following the delivery of human NSCs at three different stages of maturity: 1) undifferentiated NSCs (early stage), 2) neuronal progenitors (mid stage), and 3) mature neurons (late stage). To facilitate cell survival and integration into host tissue, cultured cells were encapsulated into a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel prior to delivery. Our results demonstrated that transplantation of early stage cells results in the greatest tissue and functional repair, relative to transplantation of more mature neurons. Surprisingly, we found that the transplantation process itself resulted in acute cell death in the late stage cells. This negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Whereas each transplant cell group (early, mid and late) started the experiment with a distinctive maturity profile that was maintained after 7 days post‐transplantation, by 50 days the pattern of neural marker expression between groups was nearly identical, highlighting the influence of the microenvironment on the terminal cell phenotype. Overall, we demonstrate that the phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in CNS regenerative strategies.

Support or Funding Information

We are grateful for funding from: the Canada First Research Excellence Fund to Medicine by Design at the University of Toronto (to MSS, CMM, AN), the Canadian Institutes of Health Research (Foundation grant to MSS, Operating grant to CMM) and the Natural Sciences and Engineering Research Council (CGS‐D to SLP).
Current Use of Student Evaluations of Teaching in Anatomy Education
Authors: Hartley Rebecca S
Published: April 2020

Anatomy, histology, and embryology (or anatomical sciences) provide a framework for understanding normal function as well as disease and its underlying mechanisms. Thus, they are often taught (by basic scientists) in the context of their clinical relevance. Delivery formats and settings vary widely: anatomy, histology, and embryology can be combined in a single course or integrated into other courses at different points in a curriculum. Anatomy labs are taught with the expectation (by students) that faculty know every detail, be it taught in the form of dissection, prosection, or virtually. The uniqueness of teaching contexts in anatomical sciences education can make it challenging to document effective teaching and to obtain feedback from students that provide growth opportunities to develop as an educator. Educators in anatomical sciences are charged with facilitating learning of skills and knowledge that are foundational and can be applied to many health sciences fields. Because anatomical sciences can be taught virtually as well as practically, additional professional development needs arise. But carving out time for development activities is challenging. Some educators teach several different levels of learners, while managing donor programs, and coordinating laboratory use, all competing demands on their time. Simultaneously, curricular changes have reduced anatomical sciences curricular hours to an all‐time low, and education is shifting towards active learning modalities with increased digital learning and reduced classroom and laboratory teaching. These modalities, if not applied expertly, can increase student pushback. Topics explored in this presentation include if and how our methods of evaluating teaching effectiveness have evolved with the changed landscape in which anatomical sciences faculty teach, and how to support faculty in their development as educators by constructive use of student evaluations of teaching.
Multifaceted Roles of Primary Cilia in Polycystic Kidney Disease and Obesity
Authors: Tran Pamela V.
Published: April 2020

The primary cilium is a small, antenna‐like sensory organelle that protrudes from most mammalian cells and mediates signaling pathways. Cilia are built by intraflagellar transport (IFT) protein complexes B and A, which have separate roles. Dysfunction of IFT and cilia results in ciliopathies, disease syndromes which commonly cause renal cystic disease and can also lead to obesity.

Intriguingly, in mice, deletion of Ift‐B genes, which causes loss of primary cilia, can markedly attenuate Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most common renal cystic disease and a leading cause of renal failure. These data indicate that targeting primary cilia genes can have immense therapeutic potential in ADPKD through mechanisms that remain elusive. To help elucidate these mechanisms, we have ablated an Ift‐A gene in ADPKD mouse models. IFT‐A dysfunction partially rescues the ADPKD elongated renal ciliary phenotype and attenuates the disease in a renal tubular‐ and maturation‐dependent manner.

In adult mice, Ift gene deletion causes hyperphagia‐induced obesity. We have found that prior to an obese phenotype, IFT‐A dysfunction misregulates expression of appetite‐controlling neuropeptides in the hypothalamus. Additionally, in a pre‐obese state and through a non‐cell autonomous mechanism, hyperphagia induces hepatic insulin resistance, which drives systemic insulin resistance. These data uncouple obesity and insulin resistance, and indicate that hyperphagia is a major driver of both phenotypes. Thus, we are examining methods to counter hyperphagia. In collaboration with Zafgen Inc., we have found that pharmacological inhibition of MetAP2, which cleaves the N‐terminal methionine of nascent proteins, reduces food intake and body weight, and substantially improves metabolic parameters in an Ift‐A mutant mouse. To our knowledge, this is the first demonstration of rescue of an obese phenotype in a ciliopathy mouse model.

Support or Funding Information

This research is supported by NIH/NIDDK R01DK103033
Retinoid Exposure Decreases Foxp3 Expression in Cutaneous T‐cell Lymphoma
Authors: Stahly Samuel L. , Powers Andrea M. , Cline Reid , Griffin Lauren A. , Sarcar Bryce , Siddiqui Nabila A. , Johnson Lucas R. , Danekas Alexis N. , Martin Sykes , Geis Abby L. , Bridges Lance C.
Published: April 2020

Retinoids, natural and synthetic vitamin A derivatives, activate members of the RAR and RXR subfamilies of transcription factors to promote cellular differentiation. Exploiting retinoids to induce differentiation embodies a milestone in cancer therapeutics. The success is championed by remarkable outcomes achieved upon the advent of retinoid regimens with acute promyelocytic leukemia (APL). While retinoids have been used for over 3 decades to treat cutaneous T‐cell lymphoma (CTCL), it remains unknown if retinoid‐dependent changes in differentiation accounts for the clinical benefit in CTCL as occurs with APL. Delineating the mechanism of action of retinoid‐based treatment in CTCL merits addressing considering that a lack of specificity contributes to metabolic sequelae that require ceasing treatment. The heterogeneity of the CTCL etiology further frustrates efforts as the exact classification of the T‐cell nature of CTCL is varied and largely debated. We first identify that CTCL lines express the hallmark Treg marker Foxp3, whereas lymphoma lines not of CTCL origin lack Foxp3 expression. We found that various synthetic and natural retinoids, including Bexarotene, an RXR agonist and the only FDA‐approved retinoid for CTCL treatment, induce a dose‐dependent decrease in Foxp3 expression in six different human CTCL cell lines. Changes in Foxp3 expression occurred at lower retinoid doses and shorter exposure times than are required to induce apoptosis. In addition, RAR and RXR receptors function synergistically to reduce Foxp3 expression at very low doses and abbreviated exposure times. Indeed, the decrement in Foxp3 expression is abrogated upon retinoid removal, demonstrating that diminished Foxp3 protein expression is not an artifact of cell death. Prior work by our group has shown that the RAR‐alpha receptor subtype prompts gut‐tropic adhesion and chemotactic changes with CTCL. We now show that the RAR‐alpha receptor subtype, assessed using selective receptor agonists and antagonists, transduces the observed changes in Foxp3 expression. This work establishes that the differentiation marker of Tregs is routinely expressed by CTCL cell lines, and that therapeutic retinoids decrease the abundance of this protein marker. It remains to be determined if the retinoid‐induced differentiation of CTCL forces a phenotype that is incompatible with cutaneous localization and predisposes the cells to apoptosis. Alternatively, retinoid‐forced differentiation of cells with a terminally differentiated phenotype, like CTCL, may trigger apoptosis. Furthermore, future work will determine if this newly identified consequence of retinoid exposure to alter differentiation markers in CTCL has clinical ramifications reminiscent of APL.

Support or Funding Information

Funding provided by the BREA program at the Arkansas Colleges of Health Education
Deregulation of neuro‐developmental genes and primary cilium cytoskeleton anomalies in iPSc‐derived retinal sheets from human syndromic ciliopathies
Authors: Barabino Andrea , Flamier Anthony , Hanna Roy , Héon Elise , Freedman Benjamin , Bernier Gilbert
Published: April 2020

Modeling human developmental and degenerative diseases has been always logistically challenging in particular in the case of rare diseases where no exhaustive animal models are available. Generation of sustainable human disease models that allow in‐depth analysis of the molecular mechanism is one of the big challenges nowadays. Stem cell technology holds great potential in disease modeling and represents a new powerful tool for generating scalable and animal‐free models that can more accurately illustrate clinical phenotypes of complex human diseases. Ciliopathies are a group of heterogeneous genetic diseases affecting proteins involved in primary cilium structure and function. Syndromic ciliopathies have a broad spectrum of symptoms ranging from retinal degeneration to skeletal and neuro‐development anomalies, including polydactylism and mental retardation. Herein we describe the generation and molecular characterization of iPSC‐derived photoreceptor sheets from patients affected by ciliopathies. Photoreceptor sheets are characterized by a polarized, multi‐layered tissue expressing outer segment, connecting cilium, and nuclear photoreceptor markers. Ciliopathic photoreceptors displayed significant common alterations in the expression of hundreds of developmental genes. Moreover, they showed several anomalies in the formation and maintenance of cilia, the positioning of the mother centriole and the activation of a stress response to misfolded proteins. Furthermore, we observed genomic instabilities and accumulation of DNA damage in the photoreceptors progenitors of one of the patients. This study reveals how combining cell reprogramming and organogenesis technologies with nextgeneration sequencing enable the elucidation of molecular and cellular mechanisms involved in human ciliopathies. The same approach, combining photoreceptor sheet differentiation and wide genome expression profile could be applied to model many genetic, developmental and degenerative diseases affecting photoreceptors. These patient‐derived retinal sheets may be useful for elucidating the molecular mechanisms underlining these diseases, for drug screening of compounds with potential therapeutic effect and predicting drugs side effects.

Support or Funding Information

Stem Cell Network (SCN), Fondation de l’Hôpital Maisonneuve‐Rosemont (FHMR), Réseau de recherche en santé de la vision (RRSV), Université de Montréal (UdeM)
Developing Scientific Writing Skills in Upper Level Biochemistry Students through Extensive Practice and Feedback
Authors: Ponce Maria Laura Sosa , Moorhead Greg B.G.
Published: April 2020

Effective communication is one of the most marketable and transferable skills a graduate can have. Unfortunately, science programs rarely develop effective writing skills due to the time‐consuming nature of evaluating these skills. Here, we try to adapt tools from specifications grading to simplify marking and maximize student success in a third‐year biochemistry lab techniques course. We provided feedback to students on whether or not they were writing to the expected level on short lab reports so that they could implement it in a cumulative lab report. Students struggled to accept the all or none nature of specifications grading and did better with a writing workshop and one‐on‐one feedback. Overall, writing improved the most in sections where students received the most practice. We observed moderate success in improving writing skills in class size of 35, which is larger than most previous exercises of this nature.

Support or Funding Information

Thank you to Kyle McDade and Ryan Toth for their help in grading.
Signaling Diversity Enabled by Rap1 and cAMP/PKA‐Regulated Plasma Membrane ERK with Distinct Temporal Dynamics
Authors: Keyes Jeremiah , Ganesan Ambhighainath , Molinar-Inglis Olivia , Hamidzadeh Archer , Ling Megan , Trejo JoAnn , Levchenko Andre , Zhang Jin
Published: April 2020

A variety of different signals induce specific responses through a common, ERK‐dependent kinase cascade. It has been suggested that signaling specificity can be achieved through precise temporal regulation of ERK activity. Given the wide distrubution of ERK susbtrates across different subcellular compartments, it is important to understand how ERK activity is temporally regulated at specific subcellular locations. To address this question, we expanded the toolbox of FRET‐based ERK biosensors by creating a series of improved biosensors targeted to various subcellular regions via sequence specific motifs to measure spatiotemporal changes in ERK enzymatic activity. Using these sensors, we showed that EGF induces sustained ERK activity near the plasma membrane in sharp contrast to the transient activity observed in the cytopolasm and nucleus. Furthermore, EGF‐induced plasma membrane ERK activity is regulated by Rap1, a noncanonical activator, and the cAMP/PKA signaling axis. We report that plasma membrane ERK controls cell morphology and EGF‐induced membrane protrusion dynamics. Our work strongly supports that spatial and temporal regulation of ERK activity is integrated to control signaling specificity from a single extracellular signal to multiple cellular processes.

Support or Funding Information

This work is supported by NIH R35 CA197622, R01 DK073368 (to J.Z). J.K. and O.M.I are supported by a post‐doctoral fellowship from NIH/NIGMS (K12GM068524). The development of Rap1 sensors at CISMM was supported by 5P41‐EB002025.
Chemical Biology Tools to Elucidate the Solution Conformation and Cellular Interactions of Cyclic Adenosine 5′‐Diphosphate Ribose
Authors: Uwakwe Chiemeka David , Grega Sarah , Serrano Inis , Graham Steven
Published: April 2020

The movement of calcium ions (Ca2+) is intimately involved in regulating many physiological processes, and so there is great interest in the structure of molecules, especially second messengers, that regulate this flow. One such messenger is cyclic adenosine 5′‐diphosphate ribose (cADPR), which is suspected to play a role in Ca2+ release in cardiomyocytes. The speculated mechanism of action involves the binding of cADPR to FKBP12.6, releasing FKBP12.6 from RyR2 channels, through which Ca2+ enters the sarcoplasm from the sarcoplasmic reticulum during ventricular systole. Guided by the principle that structure determines function, we aim to advance the investigation of regulatory cADPR interactions by synthesizing and studying cADPR and its analogs using the combined tools of liquid chromatography‐mass spectrometry (LCMS) and nuclear magnetic resonance (NMR) spectroscopy. The endogenous cyclization of nicotinamide adenine dinucleotide (NAD+) to cADPR by CD38 is mimicked in vitro using ADP‐ribosylcyclase (ADP‐RC, sourced from Aplysia californica). The low substrate specificity of ADP‐RC conveniently permits the cyclization of various cADPR analogs. Kinetic assays were prompted by an observed feedback inhibition of ADP‐RC by nicotinamide (IC50 ~ 1mM), and performed using LCMS. Following synthesis and purification, NMR experimental data provided insight into favored structural conformations of cADPR (Saatori et al., 2018) Photoaffinity labeling (PAL) was aimed at identifying the cognate binding partners of cADPR, some of which have been previously described by Walseth et al. (1993). Conformational analyses of cADPR analogs revealed structural trends with correlations to the biological activity of the analogs.

Support or Funding Information

Department of Chemistry, Saint John’s University
Expression and Purification of Human Circadian Protein hRORγ for Structural and Functional Studies
Authors: Van Sanchez Sebastian , Madej Alexander , Moreno Brenda , Xian Yuejiao , Yoo Seung-Hee , Chen Zheng , Xiao Chuan
Published: April 2020

The circadian rhythm is an internal biological clock that runs in many living organisms. It regulates the sleep wake cycle among other physiological functions. Long term disruption of this internal clock can cause sleep disorders, metabolic diseases, and high risks of cancer. The circadian rhythm is controlled at molecular level by the transcriptional‐translational feedback loop (TTFL) that involve positive transcriptional activators CLOCK/BMAL1 and negative feedback suppressors PERs/CRYs. The Retinoic Acid Receptor‐Related Orphan Receptors (RORs) function as ligand‐dependent transcription factors which regulate BMAL1 production to improve circadian rhythm stability and robustness. It was shown that the naturally occurring flavonoid Nobiletin can bind to human ROR gamma (hRORγ) and increase its function through a yet unknown mechanism. Understanding the interaction between hRORγ and Nobiletin would provide insights in enhancing the circadian rhythm and ameliorating its related diseases. This project focuses on solving the three‐dimensional structure of hRORγ/Nobiletin complex to establish the mechanism of their interaction. The hRORγ gene was cloned and expressed using E. coli BL21 cells. Expression protocols were optimized to produce large amounts of hRORγ recombinant proteins. The soluble portion of expressed protein was purified using affinity chromatography and size exclusion chromatography, and the purified recombinant hRORγ is properly folded indicating from circular dichroism data. Once the required concentration is reached, the purified hRORγ will be co‐crystalized with Nobiletin. The crystals would be used for structural determination using X‐ray crystallography. The structural information will shed light on Nobiletin’s enhancement mechanism on circadian rhythm and provide guidance in related drug development.

Support or Funding Information

Research reported is supported by: National Research of General Medical Sciences of NIH under award number SC3GM109870. Researcher has used instruments sponsored by the BUILDing SCHOLARS program funded by the National Institute of General Medical Sciences of NIH under linked Award Numbers RL5GM118969, TL4GM118971, and UL1GM118970. Researcher is supported by Maximizing Access to Research Careers Grant # T34GM008048
Urolithin A, a Product of the Microbiome, Attenuates the Palmitate‐TLR4 Inflammatory Pathway in Renal Tubule Cells
Authors: Chappell Mark , Zhu Xuewei , Pirro Nancy , Tallant Ann , Gallagher Patricia
Published: April 2020

Obesity is a major risk factor for cardiovascular disease and chronic renal injury. Tubulointerstial inflammation may reflect increased exposure of saturated fatty acids (sFA) to the renal epithelium and activation of the toll‐like receptor 4 (TLR4). Obesity‐induced inflammation may also facilitate gut microbiome dysbiosis and potentially alter the processing of exogenous polyphenolic compounds to metabolites with enhanced protective effects. Indeed, Urolithin A, the primary product of the microbiome metabolism of ellagitanins, promotes gut barrier function and attenuates colonic inflammation. However, the role of Urolithin A to attenuate tubular injury and inflammation has not been assessed. We propose that Urolithin A has anti‐inflammatory actions in rat proximal tubule cells (PTCs) chronically exposed to the sFA palmitate.

PTCs (NRK‐52e cells) were exposed to palmitate [200 μM] for a 24 hours in the presence or absence of Urolithin A [50 μM], the TLR4 inhibitor TAK242 [1 μM], the EGFR inhibitor AG1478 [1 μM] and cSRC kinase inhibitor Dasatinib [10 μM] as both EGFR and cSRC may be integral to TLR4 activation. Cytokine expression in the PTCs was quantified by separate ELISAs.

Palmitate exposure stimulated the robust release of PAI‐1 6‐fold [4.3 ± 0.3 vs. 24.2 ± 0.4 ng/ml; P<0.001, n=3], CCL2 150‐fold [0.04 ± 0.01 vs. 6.2 ± 0.4 ng/ml; P<0.001, n=3] and CCL5 10‐fold [23.4 ± 0.1 vs. 255 ± 11 pg/ml; P<0.001, n=3]. The TLR4 inhibitor abolished the increase in CCL2 and CCL5, and markedly attenuated PAI‐1 release [70%, P<0.01]. The EGFR inhibitor partially inhibited all three cytokines [40–50%, P<0.01] while the cSRC inhibitor abolished PAI‐1 and partially inhibited CCL5, but failed to inhibit CCL2 release. Urolithin A essentially abolished the increase in PAI‐1 and CCL5, and markedly inhibited CCL2 release to palmitate [70%, P<0.01].

We conclude that Urolithin A may be renoprotective in obesity‐induced injury by targeting activation of TLR4 within the renal epithelium to attenuate the cytokine release. Moreover, a deficit in the microbiome‐dependent generation of Urolithin A may potentially promote renal inflammation.

Support or Funding Information

Chronic Disease Research Fund, AHA Transformative Grant 18TPA34170522; NIH R01HL146818, the Farley Hudson Foundation, the Hypertension & Vascular Research Center
Impact of Maternal Obesogenic Diet on Offspring Bile Acid Homeostasis and Disease Progression in Non‐Alcoholic Fatty Liver Disease is Transmitted to Next Generation by Male Offspring
Authors: Thompson Michael D. , Cowen Jamie , Hinrichs Holly , Derse Alaina , Ferey Jeremie , Moley Kelle , Davidson Nicholas
Published: April 2020

Background

Non‐alcoholic fatty liver disease (NAFLD) affects approximately one‐quarter of the global population, with up to 20–25% developing progressive liver disease. Recent evidence in both humans and animal models show that maternal obesity is a risk factor for the development of NAFLD and progression to NASH. We have shown in a murine model of maternal obesity the offspring develop altered bile acid homeostasis and a varied response to a western diet which is passed by female offspring across generations. In this study we tested the hypothesis that male offspring exposed to maternal obesogenic diet pass an altered bile acid homeostasis phenotype and susceptibility to western diet induced liver disease to their offspring.

Methods

Female C57Bl6 mice were fed chow (CON) or HF/HS diet for 6 weeks to induce obesity and bred with lean males. Male F1 offspring were then bred with lean females to create a paternal second generation (PF2C from maternal CON lineage, PF2H from maternal HF/HS lineage). Tissues were collected from PF2 offspring to measure bile acid pool size and hepatic bile acid species profile. Additional PF2 offspring were fed high trans‐fat, cholesterol, fructose (HTFC) diet for 16 weeks to induce progressive NAFLD. Metabolic and histopathologic analyses were performed.

Results

In chow fed PF2 offspring from the maternal HF/HS (mHF/HS) lineage, total BA pool size was increased. No change in intrahepatic BA composition was observed. After HTFC diet, PF2H offspring showed decreased liver weight and liver weight to body weight ratio compared to PF2C offspring. PF2H offspring exhibited lower levels of intrahepatic triglyceride and free fatty acids. Conversely, PF2H offspring liver showed increased Sirius red staining.

Conclusions

Male offspring exposed to maternal obesogenic diet transmit a phenotype of altered BA homeostasis to their offspring. After exposure to a western diet, PF2H offspring exhibited worse fibrosis despite having less steatosis. Passage of these phenotypes through male offspring would suggest an epigenetic phenomenon as a mechanistic etiology. Future studies will evaluate the mechanism for transmission of maternal obesity driven changes in the liver across generations in the male lineage.
IPMK is Indispensable for Activation of AKT: Redefining PI3kinase pathway
Authors: Guha Prasun
Published: April 2020

Inositol polyphosphate multikinase (IPMK) can catalyze production of IP5 and PIP3. Deletion of IPMK abolish AKT phosphorylation at both T 308 and Ser 473 residues even when PI3kinase activity of classical PI3kinase is intact. Akt activation requires its membrane localization followed by phosphorylation by PDK1 and mTORC2. We found that classical PI3kinase exclusively mediates AKT localization to the membrane but dispensable for PDK1 membrane localization and activation of mTORC2. IPMK interacts with both PDK1 and mTORC2 complex. Generation of PIP3 by IPMK promotes membrane localization of PDK1 and also activation of mTORC2, however, shows no effect in AKT localization to the membrane. In summary, PIP3 generated by IPMK exclusively controls PDK1 and mTORC2 functioning, however, classical PI3Kinase only controls AKT localization to the membrane.

Support or Funding Information

NIH grant 5R01MH018501‐48
Tau Protein in Human Brain Vascular Smooth Muscle
Authors: Seeherman Sarah , Sariipek Nurefsan , Rybka Vladyslava , Shults Nataliia V. , Gychka Sergiy G. , Suzuki Yuichiro
Published: April 2020

Background

Ischemic stroke is a leading cause of long‐term disability and death worldwide. Thickening of the vascular wall and the reduction of lumen increase the incidence of ischemic stroke. Thus, therapeutic strategies targeting brain vascular narrowing should reduce the mortality and morbidity associated with ischemic stroke. Vascular smooth muscle cell (SMC) proliferation contributes to the development of vascular narrowing, however, cell signaling mechanisms of brain vascular SMC proliferation has not been defined. Tau is a microtubule‐associated protein indicated in various neurological pathologies, including Alzheimer’s disease. Although Tau protein is known to be expressed in neuronal cells, the role of Tau in brain vasculature is unknown. The present study tested the hypothesis that Tau is expressed in brain vascular SMCs.

Methods & Results

Immunohistochemistry of postmortem brain tissues from human patients died of ischemic stroke demonstrated the expression of Tau protein in the vascular smooth muscle media layer. Western blotting of cell lysates from cultured human brain vascular SMCs revealed the occurrence of a Tau isoform with a molecular weight of ~45 kD that was knocked down by Tau siRNA. Treatment of cells with a potent mitogen of SMCs, platelet‐derived growth factor (PDGF), promoted the phosphorylation of Tau at Thr181, but not, Ser198, Ser199, Ser202, Thr205, Thr212, Ser214, Ser356, Ser404, or Ser422. A Tau protein aggregation inhibitor, methylene blue, inhibited human brain vascular SMC growth.

Conclusions

This is the first demonstration that Tau protein is expressed in any kind of vascular SMCs. Moreover, results showing that PDGF causes the phosphorylation of Tau at a specific amino acid and a Tau aggregation inhibitor suppresses cell growth suggest that Tau may play a role in cell growth signaling in brain vascular SMCs.

Support or Funding Information

Supported by NIH
Chronic Intermittent Hypoxia Blunts Phrenic Motor Plasticity: Role of Inflammation
Authors: El-Chami Mohamad , Mitchell Gordon , Gonzalez-Rothi Elisa
Published: April 2020

Acute intermittent hypoxia (AIH) elicits a form of spinal, respiratory motor plasticity known as phrenic long‐term facilitation (pLTF). Preconditioning with a single day of chronic intermittent hypoxia (8 hours, 2 min hypoxic episodes with 2 min intervals) abolishes AIH‐induced pLTF through a mechanism that requires systemic inflammation and spinal p38 MAP kinase activation (Huxtable et al., 2015). pLTF is restored by high‐dose, systemic administration of the non‐steroidal anti‐inflammatory drug, ketoprofen, or spinal p38 MAP kinase inhibition. Similarly, preconditioning with 7 days of CIH (8 hours per day, 2 min episodes, 2 min intervals) abolishes pLTF, but we observed that phospho‐p38 within phrenic motor neurons is unchanged from control rats (Gonzalez‐Rothi and colleagues, unpublished). Thus, mechanisms whereby 7 days of CIH undermines AIH‐induced pLTF are unclear. Since CIH induces time‐dependent spinal inflammatory gene expression profiles over time (Smith et al., 2013), we hypothesize that prolonged CIH (versus a single day) blunts LTF through an inflammation‐dependent mechanism independent of p‐38MAP kinase. In ongoing experiments, we are evaluating whether anti‐inflammatory drugs restore AIH‐induced pLTF in rats preconditioned with prolonged CIH (7–28 days). Preliminary analyses in rats preconditioned with CIH for 7 days indicate that pLTF is expressed when ketoprofen is given on each day of the CIH exposure (67% baseline), but not when a single ketoprofen dose is given 3 hours prior to assessment of pLTF (−6% baseline). These experiments are the first to explore shifting mechanisms whereby prolonged CIH and associated inflammation undermine phrenic motor plasticity. Such experiments are essential since CIH is a hallmark of sleep apnea, a condition highly prevalent in individuals living with neurological disorders (spinal cord injury, ALS, etc.). It is therefore important to identify and mitigate factors which may undermine the potential for therapeutic interventions (i.e. repetitive AIH) to harness endogenous plasticity and ultimately improve breathing.

Support or Funding Information

Supported by: Department of Defense: SCI160123 and NIH R01 HL147554
Exposure to Cigarette Smoke is Linked to Platelet‐Activating Factor Accumulation in Myocardial Tissue
Authors: Kispert Shannon E. , Adney Jacob , Hui Dawn S. , Kennedy Kelsey R. , McHowat Jane
Published: April 2020

Smoking is a lifestyle risk factor for heart disease and is responsible for one‐third of all cardiovascular deaths. Although many studies have investigated the correlation between smoking and coronary artery disease, there are few studies examining the direct effect of smoking on the myocardium. In this study, we examined the effect of smoking on myocardial accumulation of platelet‐activating factor (PAF), a phospholipid mediator that causes a negative inotropic effect, arrhythmias, apoptosis and is involved in inflammation and atherosclerosis. Ventricular samples from mice exposed up to six months of cigarette smoke (48 min/day, 5 days/week), and human right atrial samples from smokers and nonsmokers scheduled for coronary artery bypass graft surgery were analyzed for biochemical and histochemical changes using activity assays, ELISA, immunoblot, and immunohistochemistry. Mice exposed to cigarette smoke showed a decrease in PAF‐acetylhydrolase (PAF‐AH, the enzyme responsible for PAF metabolism) activity that was significant after 1 month and decreased progressively over the 6 month period (5.8 ± 0.3 to 3.2 ± 0.5 nmol/mg protein/min, p<0.01, n=6). This inhibition in PAF‐AH activity was accompanied by increased myocardial PAF content (0.23 ± 0.04 to 0.35 ± 0.03 at 6 months smoking, p<0.05, n=6). In humans, smoking was associated with an increase in PAF content (3.19 ± 0.28 vs 4.29 ± 0.37 nmol/mg protein, p<0.05, n=18) and inhibition of PAF‐AH activity (30.7 ± 2.0 vs 23.2 ± 1.8 nmol/mg protein/min, p<0.01, n=18) when comparing atrial tissue from never smokers to current smokers. We did not detect a change in phospholipase A2 activity (the enzyme responsible for PAF production) in human or mouse myocardium exposed to cigarette smoke or room air. Thus, we hypothesize that the increase in PAF content in the myocardium is likely a result of smoking‐induced inhibition of PAF‐AH activity. The inhibition of PAF‐AH and subsequent accumulation of PAF in the myocardium of cigarette smokers may play a role in the pathogenesis of heart disease in this population.

Support or Funding Information

Lottie Caroline Hardy Foundation (to J.McH.)
Eating a high fat diet enhances sensitivity of rats to serotonin syndrome
Authors: Beltran Nina M. , Landavazo Antonio , Blough Bruce E. , Serafine Katherine M.
Published: April 2020

Eating a high fat diet can lead to obesity, type 2 diabetes, and dysfunction to neurotransmitter systems. For example, eating high fat chow enhances sensitivity of rats to unconditioned behavioral effects (e.g., lower lip retraction) induced by 8‐OH‐DPAT, a serotonin (5‐HT)1A receptor agonist. It is not known if eating high fat chow also impacts sensitivity of rats to other types of 5‐HT receptor agonists, or other behaviors induced by these drugs, collectively referred to as animal models of 5‐HT syndrome (e.g., flat body posture and forepaw treading). Recently, the FDA approved lorcaserin, a 5‐HT2C receptor agonist for the treatment of obesity. Lorcaserin induces some 5‐HT syndrome behaviors in rats including forepaw treading; however, it is not known if diet impacts sensitivity of rats to these lorcaserin‐induced behaviors. Further, previous research suggests that females might be more sensitive to the effects of 5‐HT receptor agonists, and to the effects of eating a high fat diet. To test the hypothesis that eating high fat chow will enhance the sensitivity of rats to the behavioral effects of 5‐HT2C and 5‐HT1A receptor agonists, 16 male and 16 female rats eating high fat chow (60% kcal from fat) or standard chow (17% kcal from fat) were tested once weekly with cumulative doses of 8‐OH‐DPAT (0.01–1.0 mg/kg, s.c.), WAY 163909 (1.0–32.0 mg/kg, i.p.), or lorcaserin (1.0–32.0 mg/kg, i.p.). 8‐OH‐DPAT induced lower lip retraction, flat body posture, and forepaw treading, while WAY 163909 and lorcaserin only induced forepaw treading. Rats eating high fat chow were more sensitive to lower lip retraction (induced by 8‐OH‐DPAT) and forepaw treading (induced by all three serotonergic drugs), as compared to male rats eating standard chow. These experiments are still ongoing; however, preliminary data and previous reports suggest that female rats will be more sensitive than males to diet‐induced enhancements in 5‐HT syndrome behaviors. Future directions will use selective 5‐HT receptor antagonists to examine the specific receptor subtypes mediating these behavioral effects in male and female rats eating different diets.

Support or Funding Information

The authors have no conflicts to disclose. N.M.B. was awarded summer funding through the American Society for Pharmacology and Experimental Therapeutics (ASPET) Summer Undergraduate Research Fellowship (SURF). N.M.B is also supported by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under R25GM069621.