bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulated Dephosphorylation of NCC Shapes the Renal Potassium Switch Pathway
Authors: Grimm P. Richard , Li Dimin , Delpire Eric , Welling Paul A.
Published: June 2021

The Renal Potassium Switch pathway stimulates the thiazide‐diuretic sensitive sodium chloride cotransporter (NCC) to limit urinary potassium loss at the expense of retaining sodium and elevating blood pressure. It has been established that Low [K+]o activates the WNK4‐SPAK kinases to drive NCC phosphorylation, but it remains mysterious how NCC is dephosphorylated in response to an acute rise in [K+]o. It has been generally assumed that high [K+]o turns off WNK4 kinase activation, allowing constitutive phosphatase activity to dephosphorylate WNK4 in states of dietary potassium loading. Here, we challenge the prevailing view.

Kinase‐activating mutations were introduced in SPAK, the terminal kinase in the WNK signaling pathway, and renal expression of the constitutively active (CA) SPAK mutant was specifically targeted in mice to the early DCT with a DCT‐driven Cre‐recombinase. NCC abundance (tNCC) and phosphorylation (pNCC) were evaluated in vivo and in isolated tubules, together with telemetric blood pressure (BP) measurements in response to changes in dietary potassium and [K+]o. CA‐SPAK were compared to Control mice.

CA‐SPAK mice display thiazide‐treatable hypertension, concurrent with NCC hyperphosphorylation. BP remained elevated in CA‐SPAK mice, compared to control, over a wide range of [K+]o, as predicted. However, BP dropped in CA‐mice when [K+]o exceeded 5 mM, and this was coincident with a reduction in pNCC/tNCC, revealing a [K+]‐regulated dephosphorylation mechanism. In isolated control DCT tubules, an acute increase in [K+]o rapidly reduced pNCC with IC50 of ~3.5 mM, indicating [K+]o directly modulates dephosphorylation of the switch pathway. [K+]o–regulated dephosphorylation of pNCC was also observed in DCT isolated from CA‐SPAK mice but the response displayed a rightward shift in the dependence on [K+]o.

Conclusion

Because CA‐SPAK drives NCC phosphorylation independent and downstream of WNK, these observations provide strong support for a potassium‐activated phosphatase that directly dephosphorylates NCC. Thus [K+]o–regulated phosphorylation as well as dephosphorylation of NCC shape the Renal Potassium Switch response. Regulated NCC dephosphorylation provides a new mechanism to explain why high dietary K+ consumption alleviates salt‐sensitive hypertension and associated cardiovascular complications.

Support or Funding Information

Funding for this project was provided National Institutes of Health Grants DK63049, DK54231, and DK093501

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inflammation Differentially Impacts Phrenic Long‐term Facilitation (pLTF) in Rats with Motor Neuron Death Induced by Intrapleural CTB‐saporin Injections
Authors: Nichols Nicole L. , Tanner Miles A.
Published: June 2021

Intrapleural injection of cholera toxin B fragment conjugated to saporin (CTB‐SAP) selectively kills respiratory motor neurons, and mimics aspects of neuromuscular disorders and neurodegenerative diseases including phrenic motor neuron death, decreased phrenic motor output, and increased microglial cell number. Our previous results demonstrated unexpectedly that respiratory plasticity is not only observed 7 days (d) after CTB‐SAP delivery, but that it is actually enhanced compared to control treatments. At 28d, however, respiratory plasticity is comparable to control levels, suggesting that the underlying mechanisms of plasticity at both time points may differ. Because inflammation is a hallmark of all neurodegenerative diseases, and it is known to prevent the development of acute intermittent hypoxia‐induced phrenic long‐term facilitation (pLTF), we predicted that inflammation would impair pLTF in CTB‐SAP‐treated rats and thus, anti‐inflammatory drugs would enhance pLTF at both time points. Using anesthetized, paralyzed and ventilated CTB‐SAP‐treated adult Sprague Dawley rats, we studied acute intermittent hypoxia (AIH; 3, 5 min bouts of 10.5% O2) induced pLTF following intrathecal C4 delivery of the nonsteroidal anti‐inflammatory drug ketoprofen to rats that received bilateral, intrapleural injections of: 1) CTB‐SAP (25μg), or 2) un‐conjugated CTB and SAP (control) in rats treated 7d or 28d prior. Surprisingly, preliminary results indicate that ketoprofen attenuates pLTF in 7d CTB‐SAP‐treated rats, but enhances it in the 28d CTB‐SAP‐treated rats. These observations suggest that inflammation potentially contributes to pLTF in 7d CTB‐SAP‐treated rats, whereas it may undermine pLTF in 28d CTB‐SAP‐treated rats. Studies are underway to assess cervical spinal levels of both pro‐inflammatory and anti‐inflammatory and neurotrophic molecules at both time points, and their cellular source. Future studies will investigate the potential contributions of microglia to the mechanisms whereby inflammation differentially impacts pLTF in CTB‐SAP‐treated rats. Results from this project will increase our understanding of how inflammation and microglial activities influence respiratory plasticity following motor neuron death, and will have broad implications for therapies restoring ventilatory function in many disorders in which motor neuron death is associated.

Support or Funding Information

NIH K99/R00 HL119606

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protection against hypoxia‐induced pulmonary hypertension in CX3CR1‐deficient mice correlates with decreased microglia activation
Authors: Oliveira Aline Cristina , Aquino Victor , Sharma Ravindra K. , Lobaton Gilberto , Harrison Jeffrey K. , Richards Elaine M. , Raizada Mohan K.
Published: June 2021

Background

Microglia are key modulators of the immune response in the brain and can be activated very early in pathology. Our previous studies have indicated that activation of microglial cells and neuro‐inflammation in autonomic brain regions plays a critical role in pulmonary hypertension (PH), possibly by increasing sympathetic activity. Microglial activation is regulated by the chemokine fractalkine, CX3CL1, and its receptor CX3CR1. Lack or deficiency of CX3CR1 was shown to significantly reduce inflammation, and suppress activation and neurotoxicity of microglia/macrophages in experimental ischemic stroke.

Aim

This study was designed to explore the role of CX3CR1 in microglial activation in PH induced by hypoxia.

Methods

CX3CR1‐deficient (CX3CR1GFP/GFP) mice and Wild‐type (WT) mice (C57BL/6J) were exposed to 3 weeks of hypoxia (H:10%O2) in a ventilated chamber (n=5–8/group). On day 21, animals were anesthetized and a Millar pressure catheter was inserted into the jugular vein and threaded down into the right ventricle to measure right ventricular systolic pressure (RVSP), dP/dt and collect data for spectral analysis, a measure of sympathetic activity. After measuring hemodynamic parameters, blood and tissues were collected and animals were perfused for histological analyses.

Results

Chronic hypoxia induced PH in WT mice, but not in CX3CR1GFP/GFP as demonstrated by hemodynamic analysis (shown in the table below) and sympathetic activity (LF/HF: WT‐N: 0.23 ± 0.8; WT‐H:1.19 ± 0.8; CX3CR1GFP/GFP‐N: 0.19 ± 0.1; CX3CR1GFP/GFP‐H: 0.29 ± 0.05; p<0.001). PH is associated with increase in activated microgial cells in the autonomic brain areas, predominantly in the paraventricular nucleus (PVN). However, exposure of the CX3CR1GFP/GFP mice to hypoxia did not change microglia activation in the PVN (CX3CR1GFP/GFP‐N: 1441 ± 51; CX3CR1GFP/GFP‐H: 1541 ± 68). Furthermore, a positive correlation between microglia and RVSP was also observed (R square: 0.4619 p=0.03).

Conclusion

These data demonstrate a relationship between RVSP and PVN microglia and suggest that microglia activation and neuroinflammation in the autonomic brain areas may be involved in development of PH.

Support or Funding Information

This research is supported by NIH grant HL102033.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Self‐Directed Learning Modules Enhance Mastery of Diagnostic Imaging in a Dissection‐Based Anatomy Course for Physician Assistant Students
Authors: Zhang Guiyun , Fenderson Bruce A. , Zuo Xuan
Published: June 2021

The virtues of using computer‐based learning modules as an alternative to traditional didactic lectures were tested in a human anatomy course for Physician Assistant (PA) students. Self‐directed learning modules on diagnostic imaging were created using Articulate™ software and provided online as homework assignments for PA students taking a human anatomy lecture and dissection laboratory course. These interactive self‐learning modules replaced the content of traditional didactic lectures on the same topics that had been given in previous years. For the PA class of 2016 (n=104), diagnostic imaging was taught via traditional lectures, whereas for the PA class of 2017 (n=100), students studied the same content online using self‐learning modules. An identical set of 21 questions on diagnostic imaging was used for both lecture (2016) and self‐learning (2017) groups of students, and student performance on these examinations was compared between the two groups. Our results indicate that PA students with access to online self‐learning modules performed as well or significantly better on the diagnostic imaging questions (p<0.05 for 5 of 21 questions). Moreover, the average on the diagnostic imaging questions improved from 86.29 (class of 2016) to 89.19 (class of 2017). The overall course grade for students using self‐learning modules for diagnostic imaging was also higher (class mean 87.76) than that of students receiving lectures for diagnostic imaging (class mean 86.08), suggesting that self‐learning modules may have a positive influence on students' overall understanding of the anatomical material. Our results indicate that online self‐study modules are highly effective teaching tools for pre‐clinical basic science medical education.

Support or Funding Information

None

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomy in a New Curriculum: Using Digital Media to Facilitate the Learning of Anatomy in the Medical Curriculum
Authors: Oberoi Vishesh , Hosseini Farshad , Doroudi Majid , Vo Lien
Published: June 2021

Introduction

The advances in technology have allowed teachers to develop new ways to teach their course materials to their students. This has ranged from using educational videos to developing online learning modules that students may complete at their pace and time. Although these advances in enhancing student knowledge have been made in many subjects, there are limited resources available to students for the purposes of learning anatomy. One area that needs to be expanded is one of educational anatomy video tutorials/lessons.

Objectives

This project was designed to create an educational video tutorial/lesson that provides a step by step tutorial that the students can follow to complete their anatomy lab. The video is designed to complement the lab component rather than replace it as we have realized that there is no substitute to viewing the material first hand. The video would allow students to prepare for the lab and upon its completion, they may refer back to the video and use it as a study tool.

Methods

To begin the cadaver was dissected and prepared so that the anatomical structures that needed to be identified were properly exposed for the filming. The filming was done in a stepwise manner, where the anatomical structures were identified and commented on throughout the video. During the editing, process animations were added to tag key anatomical structures and a quiz was added towards the end to allow students to test their knowledge of the materials in the video.

Results

To gather the efficacy of the videos and receive feedback we gave the students a survey to conduct prior to and after their lab. The results showed that more than 95% of students felt that the video helped them prepare for the upcoming lab dissection. While 98.9% of students felt that they would like to have a similar lab video for labs in the future. Also, 93% of the students stated that they would like to use the videos to study for their exams.

Conclusion

Due to the positive responses received it can be concluded that the use of video tutorials in teaching anatomy is an effective tool that can be implemented alongside the lecture material and cadaver dissections to enhance the learning of students.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of a Radiological Anatomy‐Based Intervention on Students in a Medical Gross Anatomy Course
Authors: Larsen Roxanne J. , Engle Deborah L.
Published: June 2021

Human gross anatomy is an integral aspect of medical education and serves as an important transition into the medical profession. Radiological imaging modalities serve as the link between gross anatomy content and clinical practice. At Duke University School of Medicine, anatomy faculty and radiologists collaborated to introduce radiological anatomy images into our gross anatomy course with the aim of reinforcing student learning of basic science content. We then investigated the effectiveness of this intervention from several perspectives.

Methods

In order to assess the impact of the radiological intervention, we administered: 1) a 12‐question pre‐test/post‐test, 2) an evaluation of the effectiveness of radiology‐based presentations, and 3) an evaluation of the usefulness of the radiology images used. Pre‐test/post‐test questions were selected from a previously validated question bank and were mapped to levels of cognitive processing as adapted from Bloom's Taxonomy. These questions spanned the content covered in our course (thorax, abdomen, pelvis, limbs, head/neck) and the radiological modalities learners would most likely be exposed to during their clinical training (X‐ray, MR, CT). At the conclusion of the course, Likert‐type questions were used to evaluate the learners' perceptions of the usefulness and effectiveness of the radiology presentations and images.

Results

Of 119 eligible learners, a total of 55 participants (46%) volunteered to take both the pre‐test and post‐test. Results of the paired t‐test showed there was a significant difference between the pre‐test (mean = 13.2%, SD = 8.9%) and post‐test (mean = 38.6%, SD = 17.7%) mean scores (t(54) = 9.9, p < 0.0001). Learners were most successful when questions included anatomy of the thorax and abdomen and MR or CT images, and on questions that were mapped to the analyze process. Additionally, survey feedback of all learners indicated all radiology presentations were rated as “adequately effective” (mean rating of 3 on a 5 point scale), and 65% of learners rated the radiological images as useful for studying in the course.

Conclusions

Results of this study suggest that the introduction of radiological images was an effective intervention and may have contributed to participants' increased knowledge of gross anatomy content. Given the success of this intervention as evidenced here, we continued to integrate radiological images into gross anatomy instruction for the 2017 cohort. More broadly, this work adds to the growing area of research that supports the integration of clinically relevant applications into basic science courses.

Support or Funding Information

Support and funding provided by the Office of Curricular Affairs and Duke Academy for Health Professions Education and Academic Development, Duke University School of Medicine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization and Quantification of Osteoarthritis in Cadaveric Elbow and Wrist Joints Using 3D Modeling
Authors: Anderson Jena Louise , Salmon Ciara , Lewis Christina
Published: June 2021

Osteoarthritis (OA) is the most common chronic disorder of the joints, affecting 10–13% of the U.S. population. OA can interfere with the quality of life by limiting the ability to participate in activities of daily living (ADLs) due to dysfunction and disability caused by reduced mobility and significant acute and chronic pain. While many OA studies have focused on the weight‐bearing joints of the lower limb, OA involving joints of the upper limb (UL), including those of the elbow and wrist, can just as significantly impact the quality of life, yet are underdeveloped. The purpose of the current study is to gain insight regarding the prevalence and patterns of OA in the distal joints of the UL, and to determine if there are relationships among frequency, location, and severity of occurrence, as well as an individual's sex, age, and prior occupations. For these purposes, the elbow and wrist joints were harvested bilaterally from 15 embalmed cadavers (7F, 8M; ages 63–100, with mean of 84 yo ±10.1), dissected, and the articulations examined. The articulating surfaces for the distal humerus, proximal and distal radius and ulna, and the scaphoid, lunate, and triquetrum carpal bones were each examined. Pitting and degeneration of both cartilage and bone of the articulating surfaces were digitized using the Microscribe 3D Digitizer, and3D models were created and subsequently analyzed via Rhinoceros 5 software. Quantitative analyses of the OA at each articulating surface was used to construct a descriptive comparison of the severity of degeneration on a 4‐point scale (i.e., 0 to 3) ranging from no arthritis to severe arthritis. Our preliminary data indicate that the presence and severity of OA typically affected multiple articulation sites, rather than single surfaces, suggesting that the pathogenesis of OA in any given individual may be more complex than is typically appreciated. Additionally, our assessments indicate that OA is present in significant degrees and on multiple surfaces of the elbow joints, and this region is not well explored in the current literature. Ongoing studies seek to include additional specimens and ad hoc analyses of OA patterns with sex, age, and prior occupations. In conclusion, the present study provides additional characterization and description of OA affecting the UL, and this information may guide therapeutic approaches to occupational therapy regimes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fine‐Tuning of an Evolutionarily Conserved Histone Chaperone, FACT, by Ubiquitin‐Proteasome System, and its Targeting to the Active Gene by mRNA Capping Machinery to Regulate Transcriptional Elongation
Authors: Kaja Amala , Sen Rwik , Ferdoush Jannatul , Lahudkar Shweta , Barman Priyanka , Bhaumik Sukesh R.
Published: June 2021

Facilitates Chromatin Transcription (FACT), an evolutionarily conserved histone chaperone involved in multiple cellular processes, is upregulated in cancers, and its downregulation is associated with cellular death. However, it is not clearly understood how FACT is fine‐tuned for normal cellular functions. Here, we have used a variety of biochemical, genetic, and molecular biological methodologies to understand regulation of FACT and its abundance. We find that the FACT subunit, Spt16, is ubiquitylated by San1 (an E3 ubiquitin ligase) and degraded by the 26S proteasome. Enhanced abundance of Spt16 in the absence of San1 impairs transcriptional elongation. Likewise, decreased abundance of Spt16 also reduces transcription. Thus, an optimal level of Spt16 is required for efficient transcriptional elongation, which is maintained by San1 via ubiquitylation and proteasomal degradation. Consistently, San1 associates with the coding sequences of active genes to regulate Spt16's abundance. Further, our results reveal that enhanced abundance of Spt16 in the absence of San1 impairs chromatin reassembly at the coding sequence, similar to the results seen following inactivation of Spt16. Efficient chromatin reassembly enhances the fidelity of transcriptional elongation. These results demonstrate for the first time the fine‐tuning of FACT by ubiquitin proteasome system in promoting chromatin reassembly in the wake of elongating RNA polymerase II and transcriptional elongation. Further, it is not clear how FACT is targeted to the active gene. Our recent results reveal that FACT is targeted to the active gene by an mRNA capping enzyme, Cet1, independently of its mRNA capping activity. Subsequently, FACT enhances the release of paused RNA polymerase II from promoter proximal site via Paf1 complex (RNA polymerase II‐associated factor 1 complex) to promote transcriptional elongation. Collectively, these results shed much light on the regulation of FACT and its abundance in controlling transcription, thus providing novel regulatory mechanisms of gene expression.

Support or Funding Information

NIH 1R15GM088798‐01 and 2R15GM088798‐02

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Expression of mTOR Related Molecules in the Placenta of Gestational Diabetes Mellitus (GDM), Intrauterine Growth Restriction (IUGR) and Preeclampsia patients
Authors: Price Kate , Kimbler Brent , Knowlton Nekel , Franson Levi , Hirschi Kelsey M. , Reynolds Paul R. , Arroyo Juan A.
Published: June 2021

The mechanistic target of rapamycin (mTOR) pathway is involved in placental growth and function during pregnancy. The mTOR pathway responds to nutrient availability and growth factors that regulate protein expression and cell growth. mTOR disruptions are associated with the development of obstetric complications which often result in adverse health outcomes for the mother and/or fetus. The purpose of this study was to identify the differential placental expression of various mTOR‐associated proteins during normal gestation (Control), gestational diabetes mellitus (GDM), intrauterine growth restriction (IUGR) and preeclampsia (PE). Immunohistochemistry was used to stain human placenta for activated proteins (phospho; (p)AKT, (p)ERK, (p)mTOR, (p)p70 and (p)4EBP1. Real‐time PCR array was completed to show differing placental expression of additional mTOR‐associated genes during these conditions. We observed: 1) increased (p)AKT during GDM, 2) increased (p)ERK during IUGR, 3) increased (p)mTOR during GDM and decreased (p)mTOR during IUGR and PE, 4) increased (p)p70 during IUGR and decreased (p)p70 during GDM and PE, 5) increased (p)4EBP1 during GDM, IUGR, and PE, and 6) differential placental expression of mTOR pathway associated genes. We conclude that regulation of the mTOR pathway is uniquely involved in the development of these obstetric complications. These results may provide insight into the physiological relevance of these pathways, and if so, their modification during gestation may help alleviate these diseases.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining a Role for MUC2 Mucin‐Associated Proteins in Colitis and Restitution
Authors: Gorman Hayley , Moreau France , Chadee Kris
Published: June 2021

Goblet cells produce MUC2 mucin that forms the mucus bilayers in the colon that protect the single layer of epithelial cells lining the gut. The mucus layer serves not only as a viscous physical barrier to protect the epithelial cell surface, but also provides a substrate and nutrient source for colonic microbiota. MUC2 mucin together with microbiota plays a major role in maintaining colonic health and is severely altered in infectious colitis and inflammatory bowel diseases. MUC2 mucin also contains numerous mucus‐associated proteins (MAPs) that are uncharacterized and there is much interest in what role they play in mucosal health and disease. These proteins include calcium‐activated chloride channel regulator 1 (CLCA1), Fc fragment of IgG binding protein (FCGBP) and kallikrein 1 (KLK1). In this study we functionally characterized the expression of MAPs in colonic goblet cells and interrogated their role in colitis and restitution. To quantify MAPs expression we used human LS174T colonic goblet cells and generated CRISPR/Cas9 MUC2 KO cells to determine if MUC2 production regulated the expression of MAPs. Changes in mRNA and protein levels were determined by QPCR and Western blotting, respectively. To model epithelial injury in vitro, LS174T and CRISPR/Cas9 MUC2 KO cells were mechanically wounded. For in vivo studies we used Muc2+/+ and Muc2−/− littermates to basally characterize the expression of MAPs and to quantify what role they play in DSS‐induced colitis. In LS174T cells the temporal expression MUC2, FCGBP, CLCA1 and KLK1 peaked at day 5 and remained high up to day 9, whereas CLCA1 was drastically increased in CRISPR/Cas9 MUC2 KO cells. In LS174T wounded cells, all MAPs increased as early as 2 days post wound that was more pronounced in CRISPR/Cas9 MUC2 KO cells as compared to the LS174T controls. In Muc2+/+and Muc2−/− littermates and organoids derived from Muc2+/+and Muc2−/− colons, MAPs were expressed at similar levels regardless of if Muc2 was present or not. In DSS colitis, MAPs mRNA transcripts and protein levels steadily decreased up to day 15 in Muc2+/+ but not in Muc2−/− littermates. Clca1 expression disappeared during peak of colitis (days 8–12) and Fcgbp, Clca1 and Klk1 were not restored by day 15 during restitution. In active ulcerative colitis (UC), colonic biopsies showed a marked increase in MUC2, FCGBP and KLK1 and a decrease in CLCA1 mRNA expression that returned to basal levels as healthy controls and UC in remission. This study demonstrates that MUC2 mucin and MAPs are not mucus‐associated and are expressed and developmentally regulated independently. In DSS colitis and active UC, MAPs are dysregulated and their reappearance coincides with restitution.

Support or Funding Information

Supported by CIHR

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Development and Characterization of IFT20 knockout Mice
Authors: Krump Ryan , Anderson Aleesha , Hietpas Taylor , Pazour Gregory J , Fink Darci M
Published: June 2021

A primary cilium is a singular cilium on a cell that functions in cell signaling, development and homeostasis. Intraflagellar transport proteins are important for formation of primary cilia and trafficking of proteins to and from the distal tip of the cilium. In mice, tamoxifen‐inducible Cre recombinase‐mediated knockout (KO) of one of these intraflagellar transport proteins, IFT20, results in decreased formation of primary cilia and associated developmental defects. Phenotypic differences, including polydactyly, craniofacial defects, fluid accumulation, and lymphatic defects, were observed between the wildtype and the IFT20 KO, but the exact penetrance of tamoxifen on the inducible Cre‐mechanism that disrupts IFT20 was not known as the induction protocol was relatively conservative. A single dose of tamoxifen was delivered by gavage to pregnant dams at E7 and embryos were harvested and analyzed at E16.5. To infer penetrance of the IFT20 KO, we studied the penetrance of tamoxifen on dual‐reporter mTmG mouse embryos, which followed a similar mechanism as the IFT20 KO model. In this reporter system, cells constitutively expressed membrane‐bound tomato (mT) fluorescent protein; activation of the Cre enzyme in cells exposed to tamoxifen induced expression of green fluorescent protein (mG). Differential expression of mT and mG was assessed by two photon microscopy of whole mount E16.5 embryonic skin. This difference in fluorescence allowed the determination of the penetrance of recombination based on the volume of mG+ cells to the total volume of the tissue sample. The penetrance was quantified manually through ImageJ software. Quantification was difficult due to high background and bright red blood cell autofluorescence in whole mount skin samples. A series of MATLAB‐based clustering and other image analysis methods were used to improve detection of mG+ cells in these tissue samples. In related work, we have identified primary cilia on lymphatic endothelial cells for the first time both in vitro and in vivo and have developed a lymphatic‐specific inducible model of IFT20 disruption.

Support or Funding Information

The funding for this project was provided by the South Dakota State University Department of Chemistry and Biochemistry and BioSNTR of South Dakota.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An allosteric interaction network promotes conformation state‐dependent eviction of the Nas6 assembly chaperone from nascent 26S proteasomes
Authors: Nemec Antonia A. , Peterson Anna K. , Warnock Jennifer L. , Reed Randi G. , Tomko Robert J.
Published: June 2021

The 26S proteasome is the central ATP‐dependent protease in eukaryotes and is essential for organismal health. Proteasome assembly is mediated in part by several dedicated, evolutionarily conserved chaperone proteins. These chaperones associate transiently with assembly intermediates but are absent from mature proteasomes. Chaperone eviction upon completion of proteasome assembly is necessary for normal proteasome function, but how they are released remains unresolved. Here, we demonstrate that the Nas6 assembly chaperone, homolog of the human oncogene gankyrin, is evicted from nascent proteasomes during completion of assembly via a conformation‐specific allosteric interaction of the Rpn5 subunit with the proteasomal ATPase ring. Subsequent ATP‐binding by the ATPase subunit Rpt3 then promotes conformational remodeling of the ATPase ring that evicts Nas6 from the nascent proteasome. Our study demonstrates how assembly‐coupled allosteric signals promote chaperone eviction, and provide a framework for understanding the eviction of other chaperones from this biomedically important molecular machine.

Support or Funding Information

1R01GM118600 to R.J.T.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Ubiquitin‐dependent Switch during Assembly of the Proteasomal ATPases Mediated by Not4 Ubiquitin Ligase
Authors: Park Soyeon , Fu Xinyi , Sokolova Vladyslava
Published: June 2021

In the proteasome holoenzyme, the hexameric ATPases (Rpt1‐Rpt6) enable degradation of ubiquitinated proteins by unfolding and translocating them into the proteolytic core particle. During early‐stage proteasome assembly, individual Rpt proteins assemble into the hexameric ‘Rpt ring’ through binding to their cognate chaperones: Nas2, Hsm3, Nas6 and Rpn14. Here, we show that Rpt ring assembly employs a specific ubiquitination‐mediated control. An E3 ligase, Not4, selectively ubiquitinates Rpt5 during Rpt ring assembly. To access Rpt5, Not4 competes with Nas2 until the penultimate step, and then with Hsm3 at the final step of Rpt ring completion. Using the known Rpt‐chaperone co‐crystal structures, we show that Not4‐mediated ubiquitination sites in Rpt5 are obstructed by Nas2 and Hsm3. Thus, Not4 can distinguish a Rpt ring that matures without these chaperones, based on its accessibility to Rpt5. Rpt5 ubiquitination does not destabilize the ring, but hinders incorporation of incoming subunits—Rpn1 ubiquitin receptor and Ubp6 deubiquitinase, thereby blocking progression of proteasome assembly and ubiquitin regeneration from proteasome substrates. Our findings reveal an assembly checkpoint where Not4 monitors chaperone actions during hexameric ATPase ring assembly, thereby ensuring the accuracy of proteasome holoenzyme maturation.

Support or Funding Information

Boettcher Webb‐Waring Biomedical Research Award and 1R01GM127688‐01A1 from NIH (S.P.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanded Coverage of the 26S Proteasome Conformational Landscape Reveals Mechanisms of Peptidase Gating
Authors: Tomko Robert J. , Reed Randi G. , Eisele Markus R. , Rudack Till , Schweitzer Andreas , Beck Florian , Nagy Istvan , Pfeifer Guenter , Plitzko Juergen M. , Baumeister Wolfgang , Sakata Eri
Published: June 2021

The proteasome is the central protease for intracellular protein breakdown. Coordinated binding and hydrolysis of ATP by the six proteasomal ATPase subunits induces conformational changes that drive the unfolding and translocation of substrates into the proteolytic 20S core particle for degradation. Here, we combine genetic and biochemical approaches with cryo‐electron microscopy and integrative modeling to dissect the relationship between individual nucleotide binding events and proteasome conformational dynamics. We demonstrate unique impacts of ATP binding by individual ATPases on the proteasome conformational distribution, and report two as‐yet unidentified conformational states of the proteasome suggestive of a rotary ATP hydrolysis mechanism. These structures, coupled with functional analyses, reveal key roles for the ATPases Rpt1 and Rpt6 in gating substrate entry into the core particle. This deepened knowledge of proteasome conformational dynamics reveals key elements of intersubunit communication within the proteasome and clarifies the regulation of substrate entry into the proteolytic chamber.

Support or Funding Information

NIH 1R01GM118600 (RJT) German Science Foundation (Excellence Cluster CIPSM and SFB‐1035/ Project A01) Marie Curie Career Integration Grant (PCIG14‐GA‐2013‐631577) (ES)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recombinant production and structural studies of a serine peptidase from Scopulariopsis koningii
Authors: Pedezzi Rafael , Evangelista Danilo E. , Polikarpov Igor , Cabral Hamilton
Published: June 2021

Scopulariopsis koningii is an ascomycetes fungus with high biotechnological potential, due to its peptidase. Previous work showed high colagenolytic activity in S. koningii cultured in feather flour rich medium. A native serine peptidase was purified from this bioprocess and has been shown biotechnological potential. However, this fungus has a poor molecular data available in databanks. Thus, we proposed a transcriptomic study to better describing the S. koningii enzymatic arsenal and to find this serine peptidase gene. The gene was in silico selected and synthetized in pPICZα‐A vector to a Pichia pastoris recombinant production. The protein production and purification were carried out in methanol‐induced system followed by Ni‐affinity chromatography. A structural study was carried out by a 3D model construction, generated by homology, and SAXS data collection. The recombinant protein was produced with the pro‐region, which act as an activity inhibitor. The molecular weight of pro‐region plus catalytic region is 40 kDa. SAXS studies showed a globular monomeric protein with Rg ~ 22.5 Å and Dmax ~ 80.3 Å. N‐terminus region build a protuberance related to pro‐region. This region is flexible and, as has been shown elsewhere, acts as activity inhibitor in trans. This structural study has enlightened activation strategies that can bring more control of the peptide hydrolysis start.

Support or Funding Information

FAPESP Proc. n° 2016/20385‐2 and 2015/16084‐4 and CAPES.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying the BMP Pathway Type II Receptors in Decidualization
Authors: Hamor Clark , Monsivais Diana , Matzuk Marty
Published: June 2021

Decidualization is a process driven by hormones and growth factors that results in the differentiation of endometrial stromal cells into decidual cells that are capable of supporting a developing embryo. The overall effect of decidualization is the differentiation of the endometrial stromal cells into decidual cells, which facilitate early blastocyst development, placental function and parturition. Despite the importance of decidualization in reproduction, the molecular mechanisms controlling decidualization are not fully understood. Identifying the signaling pathways responsible for decidualization will provide opportunity to enhance the process of decidualization, which can benefit women experiencing infertility, early pregnancy loss or other pregnancy‐related complications. Past research has shown that Bone Morphogenetic Protein (BMP) signaling is one of the mechanisms controlling decidualization, but the roles of each member of this pathway in decidualization are unknown. Identifying the precise genes in the BMP pathway responsible for decidualization will enable us to develop drugs with clinical applications. The BMP signaling pathway is initiated when a BMP dimer associates with a tetrameric complex of two type one (ALK2, ALK3, ALK6) and two type two receptor proteins (BMPR2, ACVR2A, ACVR2B), which then phosphorylate the SMAD1 and SMAD5 transcription factors. Human endometrial stromal cell (HESC) decidualization can be recapitulated in vitro by exposure to progesterone, estrogen and 8‐bromo‐cyclic‐AMP. Using this technique, we performed siRNA experiments in primary cultures of HESC's to determine the roles of various BMP signaling pathway members in decidualization. We found that SMAD1 and SMAD5 transcription factors and the BMPR2 receptor are essential for in vitro HESC cell decidualization. We also identified that ACVR2A and ACVR2B receptors may be playing a redundant role in decidualization as well. These results indicate that the BMP signaling receptor complex responsible for HESC decidualization is controlled by the concerted action of cell surface receptors that act redundantly but require the activity of both SMAD1 and SMAD5 transcription factors.

Support or Funding Information

NCI P30 CURE Grant (CA125123)IRACDA Training GrantPDEP Burroughs Wellcome Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Substrate Binding, Kinetics and Regulation of Glucan Phosphatase Starch EXcess4
Authors: Raththagala Madushi Upendrika , Henao Tiffany
Published: June 2021

Starch is the main energy reserve in plants, and the activity of dual specificity phosphatase Starch EXcess4 (SEX4) is essential for the degradation of starch via its involvement in the reversible phosphorylation mechanism. In plants, SEX4 acts as the main glucan phosphatase, facilitating the continuous breakdown of the starch granule by amylases. High‐resolution structural studies of SEX4 with the model substrate maltohexaose have provided important insights into the mechanisms of glucan dephosphorylation in plant systems. However, the details of how SEX4 recognizes substrate and binds to the regions of phosphorylated amylopectin within the heterogenous starch granule is not very well understood. Our newly developed Concavalin based assay for determining protein‐carbohydrate affinity increases our understanding of how glucan phosphatases recognize different substrate domains within its microenvironment. Further, enzyme kinetics and the regulatory mechanisms of the SEX4 enzyme are currently not well understood. Here we show the kinetics for SEX4 enzyme using the physiological relevant substrates, amylopectin, and starch. Using these kinetic data, we aim to provide experimental evidence for a possible allosteric regulatory mechanism of SEX4, which may be important for sensing energy status in plants.

Support or Funding Information

Skidmore College Startup fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of an agonist binding site motif that regulates biased signaling of GPCRs through altered conformations of intracellular loop 2
Authors: Soto Marta Sanchez , Verma Ravi Kumar , Willette Blair K.A. , Gonye Elizabeth C , Moore Annah M , Chun Lani S , Moritz Amy E , Yano Hideaki , Free R Benjamin , Shi Lei , Sibley David R
Published: June 2021

We recently identified a signaling‐biased agonist of the D2 dopamine receptor (D2R), MLS1547, that stimulates G protein‐mediated signaling, but is ineffective in recruiting β‐arrestin. MLS1547 was found to uniquely interact with a hydrophobic binding pocket formed by residues I184, F189, and V190 at the interface between the fifth transmembrane segment (TM5) and the second extracellular loop (EL2) of the D2R. Detailed investigations on the role of these specific residues lead to the identification of F189 (residue 5.38 using Ballesteros‐Weinstein numbering) as a micro‐switch for regulating D2R interactions with β‐arrestin. As position 5.38 is relatively conserved (frequently Phe or Tyr) in class A GPCRs, we constructed alanine mutations (Phe/Tyr to Ala) at this position within all D2‐like receptors (D2R, D3R, and D4R), the β2‐adrenergic receptor (β2R) and the V2 vasopressin receptor (V2R). Strikingly, we found that the alanine 5.38 mutations negated the receptors' ability to recruit β‐arrestin in response to agonists, while G protein‐signaling efficacy was maintained. These data suggest that the presence of a Phe or Tyr residue at position 5.38 in these GPCRs is critical for stabilizing an activate state for recruiting β‐arrestin. To investigate how alterations at this position produce conformational rearrangements resulting in signaling bias, we used the β2R, for which active state crystal structures are available, to build both β2R‐WT and β2R‐Y199A models in complex with the full agonist BI‐167107, and performed extensive molecular dynamics simulations. Using this approach, we identified residues that differentially interact with BI‐167107 in the β2R‐WT vs. β2R‐Y199A leading to conformational rearrangements that propagate through the TM3‐TM4‐TM5 interface to the intracellular side of the receptor. These coordinated changes result in a different tilt of TM4, face shift of TM4 and TM5 on their extracellular sides, and an altered orientation of IL2 in the β2R‐Y199A compared to the β2R‐WT. Strikingly, such coordinated changes and altered IL2 conformations are reminiscent of the differences between the recently solved cryo‐EM structure of the rhodopsin‐Gi complex and the crystal structure of the rhodopsin‐β‐arrestin complex. These results describe a structural basis for how ligand binding site alterations can modulate GPCR coupling to different transducers resulting in biased signaling.

Support or Funding Information

NINDS Intramural Research Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Contribution of Mg2+ and Solute Carrier Family 41 to Pulmonary Hypertension in Rat Models
Authors: Lin Mo‐Jun , Wang Dan , Lin Da‐Cen , Jiao Hai‐Xia , Sham James SK
Published: June 2021

Pulmonary hypertension (PH) is a debilitating disease and characterized by profound vascular remodeling. Many studies indicate that it is related in part to the alterations of Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Mg2+ ion is closely related to Ca2+ ion and is the second‐most abundant divalent cation in animal cells. It is required for the regulation of diverse cellular functions, including cell growth, proliferation, and migration. The Solute Carrier 41 family (SLC41) members including A1, A2 and A3 are Mg2+ transporters, and they have been identified in lung vascular tissue. However, the changes in their expression and functions of SLC41 transporters in PH have not been examined. The present study was designed to investigate whether Mg2+ and its transporters are involved in PH development. PH rats were established by a single injection of MCT or exposed to normobaric hypoxia (CH, 10% O2) for 21 days. Another set of animals received the same treatment were fed with Mg2+ diet supplement (10 mL/kg/d) for 21 days. The expression of SLC41A1/2/3 mRNA and protein in PAs of control and PH rats with or without Mg2+ supplement were determined by Q‐PCR and Western blot. SLC41A1‐2 expressions were significantly increased in PAs of MCT or CH‐treated rats, while mRNA and protein of SLC41A3 were decreased in PH rats. Moreover, PH was attenuated and the changes in SLC41A1‐3 expression were reversed in rats treated with Mg2+ diet supplement. The roles of Mg2+ and SLC41 proteins in PASMCs proliferation and migration were further examined in PASMCs isolated from control rat. They were treated with a control sequence or a SLC41A1/2 targeting siRNA and cultured under low (0.15 mM), normal (1.2 mM) and high (4.8 mM) level of Mg2+. PASMC proliferation monitored using MTS reagent or EdU staining and, migration assessed with Transwell or wound healing assay were markedly inhibited after incubation with high Mg2+ for 48h. Furthermore, siRNA targeting SLC41A1/2 attenuated PASMC proliferation. Our results, hence, clearly show that the SLC41 transporters contribute to PH, and Mg2+ diet supplement could effectively alleviate PH; In addition, Mg2+ and SLC41A1/2 actively participate in PASMC proliferation and migration. These results provide the basis for future study on the roles of Mg2+ and its transporters in PH development.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China NSFC31571179 and NSFC31371165

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
G‐Quadruplex Loop Length Regulates PARP‐1 Enzymatic Activation
Authors: Edwards Andrea , Marecki John , Byrd Alicia , Raney Kevin
Published: June 2021

Guanine‐rich sequences can interact by Hoogsteen‐hydrogen bonding to form planar quartets that stack into four‐stranded structures known as G‐Quadruplexes (G4DNA). G4DNA is non‐random, overrepresented at key regulatory regions of the cell such as promoters of proto‐oncogenes and telomeres. The overrepresentation in such sites suggests G4DNA serves a significant role in the cell. Poly(ADP‐ribose) polymerase 1 (PARP‐1) is an enzyme that mediates production of polymers of ADP‐ribose units (PAR) that serve as a signal for important cellular processes. PARP‐1 plays a role in the regulation of gene expression, transcription, chromatin remodeling, and DNA repair. Interestingly, PARP‐1 interacts with G4DNA. We show that PARP‐1 binds several G4DNA structures with tight affinity. We also confirm previous evidence revealing that PARP‐1 is enzymatically activated by c‐KIT G4DNA in a DNA structure‐specific manner. In addition, our activity assays reveal that while PARP‐1 binds various G4DNA structures only a subset promote PARP‐1‐mediated PARylation. We show that PARP‐1 enzymatic activation is regulated by G4DNA loop length where longer loops, at least >1 nucleotide, confer higher PARP‐1 activation while shorter loops reduce PARP‐1 activation. Our approach supports a PARP‐1 and G4DNA interaction but emphasizes PARP‐1's catalytic activity is G4DNA loop length‐dependent.

Support or Funding Information

National Institutes of Health Grant R35 GM122601 (to K.D.R.) UAMS Initiative for Maximizing Student Development Program (IMSD) Grant R25 GM083247

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.