bluuebunny/crossref_metadata_2025 huggingface dataset:
Aging causes accumulation of non‐proliferative adipose derived stem cells that is attenuated by caloric restriction in mice
Authors: Schmuck Eric , Mulligan Jacob D , Saupe Kurt W
Published: June 2021

While white adipose tissue (WAT) is a promising source of mesenchymal stem cells, the effects of WAT remodeling factors such as aging and diet on these cells are unknown. To address this adipose‐derived stem cells (ADSC), defined as the non‐adipocyte cell fraction that is Lin‐/CD34+/CD31‐, were isolated from epididymal fat pads of adult (10–12 months, n=12) and aged (>24months, n=12) C57BL/6 mice fed ad lib or a calorie restricted (CR) diet known to retard aging and tumor development. ADSC abundance, single cell clonality, expression of stem cell associated genes and enzymatic activities were quantified. Aging increased ADSC abundance 3.2 fold and expression of the genes Cdkn2a 9.8 fold and Isl1 60.6 fold. However, clonality was decreased 33% suggesting that aging causes accumulation of non‐replicative ADSC. CR attenuated the age‐associated increase in ADCS abundance by greater than 50%, but decreased clonality to 10% and 7% of levels in adult and aged ad lib groups respectively. Therefore, CR appears to have anti‐proliferative effects on ADSC that may be advantageous from the perspective of cancer, but disadvantageous for regenerative medicine applications.

Grant Funding Source: 1R21HL092477‐01 Proposal Title: Impact of chronic caloric restriction on resident progenitor cells in the heart
Continuous Measurement of Negative Pressure Effects on Cell Migration in Wounded Monolayer Cell with Electrical Wound‐Healing Assay
Authors: Hsu Chih‐Chin , Tsai Wen‐Chung , Wang Jong‐Shyan
Published: June 2021

Background

Negative pressure wound therapy has recently gained popularity in chronic wound care. This study attempted to explore effects of different negative pressures on cell migration in wound healing process.

Methods

We used the electric cell‐substrate impedance sensing technique to create a 5×10−4 cm2 wound in the monolayer Madin‐Darby Canine Kidney cells and continuously monitor cell migration activities. The wounded cells were cultured in a self‐constructed negative pressure incubator at ambient pressure and negative pressures of 75 mmHg, 125 mmHg and 175 mmHg. The effective time, complete wound healing time, healing rate, cells radius and wound area over time were evaluated.

Findings

The effective time (1.25±0.27 hrs), complete healing time (1.76±0.32 hrs), healing rate (2.94±0.62 cm2/hr), cell radius (11.5±0.2 μm) at complete wound healing of cells treated at negative pressure of 125 mmHg were significantly different from those at other three pressure conditions.

Interpretation

The study has established a cellular model for observing cell migration at different negative pressures uninterruptedly. Results have shown that the negative pressure of 125 mmHg can enhance cell migration activites.
Effects of Negative Pressure on Cell‐Cell Junctions and Cell Migration
Authors: Lu Yun‐Mei , Hsu Chih‐Chin , Wang Jong‐Shyan
Published: June 2021

Background

Negative pressure wound therapy has been developed for treatment of chronic wounds. This study attempted to highlight effects of negative pressure of 125 mmHg on cell migration in wound healing process.

Methods

A monolayer of Madin‐Darby Canine Kidney cells were grown in the chambered coverglass system. We created a scratching wound on the well with a pipette tip. Then the prepared traditional wound‐healing assays were harvested in a self‐constructed negative pressure incubator at ambient pressure and negative pressure 125 mmHg for three hours separately. Immunocytochemical stain of cells vitality, tight junctions and actins were performed later.

Findings

No differences of cell viabilities between cells cultured at ambient pressure and negative pressure of 125 mmHg. While compared with cells at ambient pressure, nearly total loss of tight junction fluorescent intensities and lamellipodium together with filopodia could be identified in cells treated with negative pressure.

Interpretation

Loosening of cell‐cell junctions may induce actin assembly to enhance cell migration at negative pressure of 125 mmHg.
Upregulation of Sca‐1 in satellite cells during diaphragmatic skeletal muscle regeneration: Potential novel mechanism of skeletal muscle regeneration
Authors: Rezk Bashir Mahmoud , Yoshida Tadashi , Semprun‐Prieto Laura , Sukhanov Sergiy , Delafontane Patrice
Published: June 2021

Stem cell antigen‐1 (Sca‐1) is an 18kDa glycosyl phosphatidylinositol‐anchored cell surface protein of the Ly‐6 family that is involved in cell activation, proliferation and differentiation. Regeneration of skeletal muscle is a multifactorial process which includes proliferation and differentiation of satellite cells into new muscle fibers. Mechanisms of skeletal muscle regeneration, however, are poorly understood. We previously reported that angiotensin II (Ang II) is implicated in the induction of skeletal muscle atrophy that is characteristic of advanced heart failure. This study was designed to investigate the effects of Ang II on diaphragmatic skeletal muscle and determine mechanism(s) of skeletal muscle regeneration. FVB mice were infused with 1μg/Kg/min Ang II or sham‐infused for one week. Immunostaining for embryonic myosin heavy chain revealed multiple areas of regenerating muscle fibers and Sca‐1 expression was markedly upregulated in the satellite cells of these injured areas. Both proliferation markers PCNA and Ki67 were upregulated as detected by Western blot for PCNA and immunostaining for Ki67. The myogenic factor, M‐cadherin was also increased in the injured muscle. In conclusion, our results demonstrate that Ang II induced injury of diaphragmatic skeletal muscle is accompanied by increased proliferation and myogenesis that is potentially mediated, at least in part, by increased activity of Sca‐1 in muscle satellite cells.

Grant Funding Source: NIH
FACS Analysis and Immunohistochemical Analysis of Human Myogenic Stem Cell Number and Cell‐cycle Kinetics in Response to Acute Myotrauma
Authors: McKay Bryon , Toth Kyle , Tarnopolsky Mark , Parise Gianni
Published: June 2021

In humans, myogenic stem cell (SC) enumeration is an important measurement used to determine the SC response in vivo to various physiological stimuli. The current standard for enumeration is immunohistochemistry (IHC) with antibodies against common SC markers (i.e. Pax7, NCAM). Fluorescent activated cell sorting (FACS) analysis may provide a more accurate determination of changes in the SC pool and provide additional analysis unachievable with IHC. FACS analysis revealed Pax7+ cells/mg isolated from 50mg fresh tissue increased 36% 24h after injury. The number of Pax7+ cells/mg in G2/M phase of the cell cycle increased 202% after 24h and cells/mg in G1/G0 and S‐phase increased 32% and 59% respectively. IHC data illustrated, in relation to N‐CAM or C‐Met alone, Pax7 alone was expressed on a greater number of cells. Furthermore, all 3 markers appear to sufficiently and similarly report SC expansion after injury (26–36%). Here we illustrate the use of FACS as a precise method of enumerating SC number on a per milligram tissue basis, providing a more easily understandable relation to muscle mass as opposed to number of myonuclei or fiber number. Although IHC is a powerful tool for SC analysis, FACS is an objective, reliable and effective method for SC quantification and can provide additional information such as cell‐cycle kinetics more accurately than IHC alone.
Integrating Anatomical and Clinical Illustrations in a Teaching Format
Authors: Cui Dongmei , Lynch James C , Yang Gongchao , Haines Duane E
Published: June 2021

Integrating anatomical structures with appropriate clinical examples can enhance the understanding of basic medical sciences and speed up the learning process. We have integrated illustrations of normal tissues with illustrations of diseased to highlight the differences between normal and abnormal tissues, and to demonstrate the processes in which normal change into abnormal tissues. Examples include: (1) Connective tissue cells in normal tissue compared to the cells in the tissue during an allergic reaction. (2) Normal and abnormal endothelium in atherosclerosis in blood vessels. (3) Normal stratified columnar cells of the conjunctiva of the eye are compared to cells with inflammatory granulations of the conjunctiva. (4) A normal optic nerve papilla is compared with an optic nerve papilla that exhibits papilledema. (5) Comparison of normal dentinal tissue and abnormal dentin tissue in a case of dentinogenesis imperfecta. Clinical correlations with well‐designed illustrations benefit students in learning clinical concepts, enhance the student's understanding of basic medical sciences, and help the students acquire clinical skills. This learning process paves the road for medical students as well as dental students to develop critical thinking skills while they are still in their early years of medical education, thus guiding them in their future study.

Grant Funding Source: University of Mississippi Medical center
Peer teaching as an educational tool for learning basic concepts of the peripheral nervous system
Authors: Severson Arlen R
Published: June 2021

Although the lecture approach to educating students provides a means of disseminating subject material, it is a passive learning approach not particularly effective in engaging learners in critically comprehending and understanding the subject material. In our Applied Anatomy course, two student‐centered peer teaching sessions were conducted focusing on general concepts regarding the peripheral nervous system. Prior to each session, students were given a reading assignment, a handout emphasizing major points, learning objectives, and a narrated PowerPoint. At the beginning of the scheduled class period, the students took an ungraded Turning Point quiz to evaluate what they had learned. During the quiz the students discussed their answer choice with their peers in order to better understand the rationale for the correct answer. Following the student's response to each question, a short discussion by the instructor of the correct answer and the distractors followed. After the quiz, additional important points not covered by the quiz were reviewed in a short lecture. This was followed by a second quiz, the grades of which were recorded. Again, during the quiz the students discussed their answer choice with their peers. After the second session a survey was done to evaluate the effectiveness of the learning approach. Ninety‐five percent of the students agreed or strongly agreed that peer teaching is an effective learning approach. (Sponsored by a Herz Faculty Teaching Development Award, Minnesota Medical Foundation)
The BioNetLab Anatomy Collaboratory
Authors: Pescitelli Maurice J. , Lilagan C. , Huntley S. , Landers S. , Theiss E. , Doyle M.
Published: June 2021

The BioNetLab Anatomy Collaboratory enables an online community of anatomy annotators to expand upon the collection of anatomy annotations in the Eolas AnatLab Virtual Anatomy Laboratory, and other image datasets. The AnatLab system is a Web‐based multidimensional atlas, which allows the user to browse through the Visible Human male data in multiple planes. Users can slice through the data and interactively query the knowledge base. The system responds by displaying the name of the anatomical structure and highlighting the extent of the structure on the section image being viewed, as well as linking to related information from Wikipedia and other sources. Anatlab has over 2500 structures identified, involving a total of over 4600 sections and 700,000 annotations overall. The present project is directed at creating a way to leverage current Web collaboration, visualization and social network technologies to allow large loosely‐coordinated teams of anatomy experts to dramatically enhance and expand the existing knowledgebase concerning this important dataset, and to extend this approach to create similar knowledgebases from other equally‐important anatomical image resources.
Experience of true life: A qualitative inquiry of first‐year medical students’ experience of cadaver dissection.
Authors: Skinner Miki D , Morrow Sue , Morton David A
Published: June 2021

This research project explored the experience of first‐year medical students studying anatomy through full cadaver dissection. Literature suggests that cadaver dissection is a learning experience comprised of multiple variables that do not lend themselves to quantitative analysis. The goal of this research was to gain a greater understanding of the meaning that medical students take from cadaver dissection. The study was qualitative in nature. Perspectives of 16 first‐year medical students were drawn together through individual interviews and one focus group to produce a grounded theory hierarchy of the meaning‐making process associated with cadaver dissection. Themes emerging from the data were congruent with those identified in the literature. However the predominant theme of Balancing Respect is not reflected in the current scholarly literature regarding the use of cadaver dissection as an educational tool. These results suggest that the cadaver lab is the initial training ground for doctors to learn how to balance personal values connected with the human body with the invasive procedures they are required to do in order to be competent physicians.

Grant Funding Source: AAA Student Travel Award
Testing knowledge of clinically‐relevant surface anatomy
Authors: Hankin Mark , Baptista Carlos A.C. , Lane Richard D. , Morse Dennis E. , Bennett‐Clarke Carol A.
Published: June 2021

INTRODUCTION

A thorough knowledge of surface anatomy is essential for diagnosis and management of patients. For many students, “instruction” in this subject is limited to an introductory lecture. While surface anatomy is available for study on cadavers, dissection unavoidably removes skin making advanced study and learning difficult. Therefore, students’ knowledge of surface anatomy is rarely assessed on practical exams but instead measured in a limited way on written exams.

METHODS

This report describes the use of undissected cadavers in practical exams given to physician assistant students (N=34) to assess their knowledge of surface anatomy on written and practical exams. Four pairs of subject‐matched, multiple‐choice surface anatomy questions on three exams were used to assess the association between performance on the subject‐matched questions and exam scores.

RESULTS

Spearman non‐parametric analysis showed strong correlations (0.79≤ r≤ 0.88, p<0.001) between overall performance on written and practical exams. While there was no correlation between performance on subject‐matched questions for Exam 1, significant correlations were observed for Exam 2 (r=0.37 p=0.3) and Exam 3 (r=0.39, p=0.02). Performance on subject‐matched questions also correlated with overall performance for Exams 2 and 3, but only for practical surface anatomy questions in Exam 1.

CONCLUSION

This data suggests that it is possible to accurately assess clinically‐relevant surface anatomy on a practical exam. Since physical examination is an important clinical application of surface anatomy, it is essential that we have an accurate way to assess our students' ability to recognize essential surface landmarks.
CORRELATING NUMERICAL COURSE GRADES BETWEEN GROSS ANATOMY AND CLINICAL COURSES WITH STRONG ORTHOPEDIC CONTENT IN THE PHYSICAL THERAPY CURRICULUM
Authors: Bukoskey Thomas Patrick , Barredo Ronald
Published: June 2021

Objective

The purpose of this study is to determine the presence, strength, and significance of correlations between the Gross Anatomy course and the Orthopedics course in the Doctor of Physical Therapy (DPT) curriculum.

Subjects

Numerical course grades of students in the first DPT class were used to determine the correlations.

Methods

Correlations were conducted using Pearson r. Numerical course grades in the Gross Anatomy course (Year 1, Semester 1) were correlated with those from the Orthopedics course (Year 2, Semester 2). In addition, correlations were determined between the Gross Anatomy course and courses with strong orthopedic content which were taken prior to the actual Orthopedics course, including: Tests and Measurements (Year 1, Semester 2), Biomechanics and Movement Science I (Year 1, Semester 2), and Biomechanics and Movement Science II (Year 1, Semester 3).

Results

Correlation significant at the 0.05 level was identified between Gross Anatomy and Orthopedics. Strong correlations significant at the 0.01 level were identified between the Gross Anatomy course and the following courses: Tests & Measurements, Biomechanics and Movement Science I, and Biomechanics and Movement Science II. Regression analysis between and among these courses revealed significance at the 0.00 level.

Conclusions

Strong correlations existed between and among the courses foundational to the Orthopedics course.

Relevance

This study underscored the foundational role of the Gross Anatomy course in courses with strong orthopedic content. A strong background in Gross Anatomy was important when taking courses with strong orthopedic content.
Sem1 is required for TREX2 stability
Authors: Oliver Encarna García , García Pau Pascual , Navarro Susana Rodríguez
Published: June 2021

Sem1 is a conserved component of the lid subcomplex of the 19S regulatory particle of the 26S proteasome. Several studies have established links between components of the mRNA export machinery and Sem1 in a proteasome‐independent manner. Here we provide additional data that support this functional and physical association. We show that Sem1 is genetically linked to Sus1, a bona fide component of TREX2 complex. Biochemical analyses revealed that, Sem1 and Sus1 interact physically, which depends on the presence of Sac3. Reciprocally Sus1‐Sac3 interaction depends on Sem1. Moreover, mRNA export is drastically inhibit in the double mutant sus1Δsem1Δ. Here we will present new data concerning Sem1 proteasome‐independent role in mRNA export as a functional component of the TREX2 complex.
Regulation of Alpha‐fetoprotein expression in the mouse liver by HNF1 and NF1
Authors: Ren Hui , Spear Brett
Published: June 2021

Alpha‐fetoprotein (AFP) is highly expressed in the fetal liver but silenced at birth. A G‐A substitution at −119 of the human AFP promoter was associated with persistent AFP expression in the adult liver. The −119 region harbors overlapping sites for Hepatocyte Nuclear Factor 1 (HNF1) and Nuclear Factor I (NFI); the G‐A substitution increases HNF1 binding. This overlapping HNF1/NFI site is conserved in other mammals, including the mouse. In this study, we have used a combination of biochemical, tissue culture, and transgenic mouse studies to explore further the role of this HNF1/NFI site in AFP regulation. Transient co‐transfections in Hep3B hepatoma cells indicate that HNF1 activates while NFI represses the AFP promoter. EMSAs indicate that HNF1 and NF1 compete for binding to this site. Transgenes regulated by the wild‐type AFP promoter are expressed at low levels in the adult liver. Transgenes with a GG‐AA mutation (similar to the G‐A human mutation) are more active in the adult liver. In EMSAs, the GG‐AA mutation enhances HNF1 binding and reduces NF1 binding. Our data indicate that HNF1 and NFI compete for binding to the AFP promoter and this competition is involved in postnatal AFP repression..
An Androgen Response‐Like Element Half‐Site Is Essential For Androgen Regulation Of Gonadotropin Regulated Testicular RNA Helicase (GRTH/DDX25) Transcription
Authors: Villar Joaquin , Tsai‐Morris Chon‐Hwa , Dufau Maria L
Published: June 2021

GRTH is a testis‐specific member of the DEAD‐box family of RNA helicases which is present in Leydig and germ cells and is essential for spermatogenesis. It is the only known RNA helicase to be hormonally regulated. GRTH is transcriptionally up‐regulated by hCG via cAMP‐induced androgen (A) in Leydig cells. To gain insights into the mechanism of androgen regulation, immortalized rat hypothalamic GnRH neurons that express GRTH mRNA were used to generate a stable cell line expressing the A receptor (GT1‐7AR). The significantly A‐induced GRTH expression (mRNA/protein) in GT1‐7AR was prevented by the AR antagonist Nilutamide. Two putative ARE atypical half sites were present at −800 (ARE2) and −200 bp (ARE1) of GRTH gene. Functional analysis of sequential deleted GRTH 5' flanking sequence in GT1‐7AR treated with dihydrotestosterone (DHT) and/or Nilutamide revealed an active AR response region at −1085/−500 bp. Point mutation of ARE2 prevented A‐induced up‐regulation of GRTH transcription. ChIP using AR antibody in GT1‐7AR cells treated with DHT revealed AR recruitment to GRTH (−980/−702 bp) containing ARE2 compared to control. No binding activity was associated with ARE1. We conclude that ARE2 plays a key role in the androgen regulation of the GRTH gene transcription. Our findings provide valuable insights for future elucidation of androgen regulated GRTH function during germ cell development. Supported‐IRP, NICHD
Identification of a receptor‐induced binding site (RIBS) in plasminogen induced by its interaction with cells
Authors: Han Jaena , Parmer Robert J , Miles Lindsey A
Published: June 2021

When plasminogen is bound to cells its activation is markedly enhanced compared to the reaction in solution, a key step in regulation of thrombolysis and cell migration. To study the mechanism of cell‐surface plasminogen activation we developed a monoclonal antibody (mAb 51) that reacts with cell‐associated plasminogen in blood in the presence of a high molar excess of soluble plasminogen. Thus, mAb 51 detects receptor‐induced binding sites (RIBS) induced in plasminogen upon its interaction with cells. The objective of our study was to define the RIBS epitope in plasminogen recognized by mAb 51. Plasminogen was denatured, reduced and alkylated and then digested with trypsin. Using western blotting and a specific Mab 51‐based ELISA for plasminogen, we established the concentration of trypsin that resulted in maximal digestion of plasminogen, while still retaining reactivity with mAb 51. The plasminogen digest was applied to a mAb 51 immunoaffinity column. MALDI analysis of the eluate yielded a single peak with a mass of 3,076 m/z. In LC‐MS‐MS, a peak with the same mass was obtained and assigned to a peptide corresponding to K177‐Y154, spanning a domain linking plasminogen kringles 1 and 2. Our data suggest that this domain becomes accessible when plasminogen interacts with cells. Thus, this conformational change detected by mAb 51 is likely to contribute to the ability of plasminogen to be activated on the cell surface.
Interrupted catalysis: the EF4 (LepA) effect on back translocation
Authors: Liu Hanqing , Pan Dongli , Pech Markus , Cooperman Barry S.
Published: June 2021

EF4 (LepA) promotes back translocation of tRNAs on the ribosome, and is important for bacterial growth under certain conditions. Here, using a coordinated set of in vitro kinetic measures, including changes in the puromycin reactivity of peptidyl tRNA and in the fluorescence of labeled tRNAs and mRNA, we elucidate the kinetic mechanism of EF4‐catalyzed back translocation. EF4‐dependent back translocation proceeds from post‐translocation complex (POST) to pre‐translocation complex (PRE) via a four‐step kinetic scheme, i.e., POST → I1 → I2 → I3 → PRE, that is not the simple reverse of translocation, and during which movement of the 3’‐end of peptidyl–tRNA is partially decoupled from movements of the tRNA core regions and of mRNA. EF4 may be thought of as performing an interrupted catalysis of this process, stopping at the formation of I3 rather than catalyzing the complete process of back translocation culminating in PRE complex formation. The delay in polypeptide elongation resulting from transient accumulation of I3 is likely to be important for optimizing functional protein biosynthesis. Supported by NIH grant GM071014 to B. S. C.
Nontraditional Careers for Scientists
Authors: Balbes Lisa Marie
Published: June 2021

A scientific background prepares you for much more than just a laboratory career. The broad science education, analytical thinking, research methods, and other skills learned are of value to a wide variety of types of employers, and essential for a plethora of types of positions. By understanding your own personal values and interests, you can make informed decisions about what career paths to explore, and identify positions that match your needs. This talk provide an introduction to the wide variety of nontraditional career possibilities for scientists, including chemical information, patent work, technical writing, education, human resources, sales and marketing, and much more. We will discuss typical tasks, education and/or training requirements, and personal characteristics that make for a successful career in each field, illustrated with specific examples. Valuable tips and advice about planning career transitions will also be provided.
SUMO modification of the Polycomb group protein Sex comb on midleg (Scm) counteracts its function as a repressor of homeotic genes
Authors: Courey Albert J , Smith Matthew , Simon Jeffrey A. , Mallin Daniel R.
Published: June 2021

The Drosophila protein Sex Comb on Midleg (Scm) is a member of the Polycomb group (PcG), a set of transcriptional repressors that maintain silencing of homeotic genes during development. We have found that the SUMO conjugating enzyme Ubc9 interacts with the Scm C‐terminal Sterile alpha motif (SAM) domain and this interaction is crucial for the efficient sumoylation of Scm. Scm is associated with the major Polycomb response element (PRE) of the homeotic gene Ultrabithorax (Ubx) and efficient PRE recruitment requires an intact Scm SAM domain. Scm is found in discrete intranuclear foci known as PcG bodies and global changes in sumoylation disrupt formation of these structures. Sumoylation‐defective Scm mutants are recruited to the PRE at higher than normal levels and globally perturbing sumoylation augments the Scm‐PRE association. In the metathorax, Ubx expression promotes haltere formation and suppresses wing development. When SUMO levels are reduced we observe decreased expression of Ubx and partial haltere‐to‐wing transformation phenotypes. These observations suggest that SUMO negatively regulates Scm function by impeding its recruitment to the Ubx major PRE.
Molecular Mechanisms of TRAF6 Ubiquitination and Activation
Authors: Wang Kent Z.Q. , Su An‐Jey , Adamik Juraj , Galson Deborah L. , Auron Philip E.
Published: June 2021

Tumor Necrosis Factor Receptor Associated Factor 6 (TRAF6) is an E3 ubiquitin ligase catalyzing K63‐linked polyubiquitination of itself and other molecules upon association with upstream effectors possessing a short TRAF Interaction Motif (TIM) peptide. TRAF6 regulates biological processes such as inflammation, innate and adaptive immunity, osteoclastogenesis, and nerve growth. Ectopic expression of TRAF6 is sufficient as a dominant‐positive activator. However, the mechanism by which a TIM activates TRAF6 has been unclear. We now demonstrate that two critical domains of TRAF6, the MATH domain required for TIM binding and the RING‐Zinc for downstream signaling, mutually interact and render the molecule structurally closed and inactive. We propose that TIM engagement disrupts this interaction and that autoubiquitination provides a means of sustaining the open conformation necessary for downstream signaling. However, excessive ubiquitination results in inactivation of TRAF6 by trafficking into subcellular sequestosomes. Furthermore, the inferred cis nature of TRAF autoubiquitination is now demonstrated to act in trans and is regulated via its RING‐Zinc and coiled‐coil domains, providing a new mechanism for TRAF regulation and its ubiquitination target diversity.
Isotope‐Reinforced Polyunsaturated Fatty Acids Protect Yeast Cells from Oxidative Stress
Authors: Clarke Catherine Freitag , Shmanai Vadim V , Hirano Kathleen , Hill Shauna , Kay Bradley , Tse Vincent , Marbois Beth N. , Molinari Robert J. , Shchepinov Mikhail
Published: June 2021

Polyunsaturated fatty acids (PUFAs) are exquisitely sensitive to autoxidation damage. The enhanced vulnerability of PUFAs to such autoxidation stems from the labile nature of the bis‐allylic hydrogen atoms. PUFAs synthesized to contain Deuterium atoms uniquely at the bis‐allylic sites (termed isotope‐reinforced PUFAs) would be expected to be more resistant to autoxidation reactions due to the isotope effect. This hypothesis was tested by making use of yeast coq mutants with defects in biosynthesis of coenzyme Q (CoQ, or ubiquinone). CoQ plays a well‐known role in respiratory energy metabolism and also functions as a lipid soluble chain terminating antioxidant. Although yeast cannot synthesize PUFAs, they are able to incorporate exogenously supplied PUFAs. Yeast coq mutants incubated in the presence of linolenic acid (C18:3) exhibit profound loss of viability as ascertained by greater than 99% loss of colony formation at 4 hours. In contrast, the coq mutants treated with the isotope‐reinforced linolenic acid (bis‐allylic D4‐C18:3) retain 80–90% viability, a value similar to wild‐type or CoQ‐replete yeast. These results indicate that isotope reinforced PUFAs are stabilized as compared to standard PUFAs, and the coq mutant yeast cells containing the D4‐linolenic acid are protected against the toxic effects of lipid autoxidation products. This research was supported by NIH GM45952.
Modeling changes in energy substrate usage in response to acute stress in birds.
Authors: Fokidis Bobby H , Sparr Richard , Deviche Pierre , Sweazea Karen
Published: June 2021

Physiological responses to stress, imposed by intrinsic or environmental challenges, result in a rapid mobilization of energy. However, how an organism's current energetic state affects the stress response and the energy substrates used is poorly understood. We measured plasma metabolites (glucose, free glycerol, triglycerides, and the ketone β‐OH‐butyrate) associated with energy storage and mobilization in response to a 30 min long mild stress in five species of free‐ranging birds. We predicted that individuals with greater lipid stores rely more on intrinsic energy stores during stress than individuals with low lipid stores and this difference would be reflected in differential energy substrate utilization. In all species, acute stress was associated with increased total plasma corticosterone and decreased plasma triglycerides. Changes in plasma concentrations of the other metabolites in response to stress reflected differences in phylogeny and/or diet type. Granivorous species (House Sparrows, Abert's and Canyon Towhees) showed a decrease in free glycerol and no change in glucose. By contrast, primarily insectivorous species (Curve‐billed Thrashers and Northern Mockingbirds) showed increased glucose and β‐OH‐butyrate. Metabolic pathways for energy mobilization and usage during stress may, therefore, be related to specific life‐history traits.
Cotranslational regulation of calcium sensing receptor biosynthesis by an allosteric agonist
Authors: Cavanaugh Alice , Breitwieser Gerda E
Published: June 2021

Calcium sensing receptor (CaSR) biosynthesis incorporates quality control checkpoints common to all G protein‐coupled receptors as well as a unique conformational checkpoint. The membrane‐permeant allosteric agonist NPS R‐568 can increase net and surface abundance of wt CaSR and a class of loss‐of‐function mutants. Here we use [35S]cysteine labeling to characterize the mechanism of NPS R‐568 action. [35S]CaSR is cotranslationally glycosylated with slow and incomplete maturation to the final glycosylated form. [35S]CaSR is relatively stable (half‐life > 8–12 hrs). Incorporation of [35S]cysteine into CaSR is linear over 60 min and the rate of [35S]CaSR biosynthesis varies as a function of conformation, which can be biased toward the ‘active’ state by cotranslational exposure to NPS R‐568 in cells with little or no plasma membrane CaSR. Similar effects on net [35S]CaSR biosynthesis are observed with loss‐ or gain‐of‐function mutations. Thus NPS R‐568 can cotranslationally regulate [35S]CaSR stability. [35S]cysteine pulse‐chase analysis suggests that prolonged exposure to NPS R‐568 not only increases cotranslational stability but also facilitates maturation of [35S]CaSR through the secretory pathway. Overall, these results suggest that CaSR is subject to cotranslational quality control which includes a pharmacochaperone‐sensitive conformational checkpoint. NIH GM077563 and Geisinger Clinic.
Protocadherin of the liver, kidney and colon associates with detergent‐resistant membranes during cellular differentiation
Authors: Rizk Sandra , Krahn Michael P. , Alfalah Marwan , Behrendt Marc , Naim Hassan Y.
Published: June 2021

Protocadherin LKC (PLKC) is a member of the heterogeneous subgroup of protocadherins, which was identified and described as a potential tumor‐suppressor gene involved in contact inhibition. Several aspects of the structure, posttranslational processing, targeting and function of this new protocadherin are still not known. Here, we generated MDCK cell lines that stably express PLKC and investigated its cellular distribution, biosynthesis, glycosylation and association with detergent‐resistant membranes, particularly during cellular differentiation. We demonstrate that PLKC is localized at the apical membrane and adherens junctions, but it does not colocalize with the tight junctions proteins ZO‐1 and occludin. Unlike E‐cadherin, PLKC is not redistributed from the cell membrane to submembranous and vesicular structures upon Ca2+ removal. Interestingly, PLKC associates during differentiation with detergent‐resistant membranes that trigger its redistribution from intracellular membranes to the cell surface. This association occurs concomitant with alterations in the O‐glycosylation pattern that is increased in postconfluent, i.e. well differentiated MDCK cells. Finally, we demonstrate that PLKC is directly involved in the establishment of a proper epithelial cell polarity.

This work has been supported by grants from the German Research Council, DFG (SFB 621) to HYN.
Role of SNARE proteins in the pathogenesis of HIV
Authors: Achuthan Adrian , Zhou Jing Ling , Crowe Suzanne , Jaworowski Anthony
Published: June 2021

Tuberculosis (TB) is the leading cause of death among HIV‐positive individuals. Indeed, HIV and M. tuberculosis (Mtb) co‐infected individuals have a 10% annual risk of reactivating latent Mtb infection. Macrophages are both the host cell for Mtb infection and also the cell directly responsible for Mtb killing. Hence, evasion of macrophage intracellular destruction mechanisms is pivotal to Mtb virulence. IFNγ promotes macrophage immune functions (e.g. cytokine secretion and phagocytosis) relevant to Mtb killing and immunity, in part by modulating expression and/or function of effector proteins (e.g. SNARE, autophagy and Rab proteins), controlling membrane vesicle fusion events. We report here that IFNγ‐induced STAT1 phosphorylation is impaired in HIV‐1‐infected primary human monocyte‐derived macrophages. We found IFNγ up‐regulates the expression of SNARE proteins Stx11 and VAMP8 in macrophages. Significantly, CD14+ monocytes from HIV‐1‐infected individuals displayed decreased protein levels of VAMP8 and Stx11 as compared to controls. The effects of IFNγ‐inducible Stx11 and VAMP8 on secreting cytokines and killing Mtb in macrophages will be presented and discussed.
Downregulation of EGFR Signaling Requires Gαs
Authors: Beas Anthony , Osipov Arsen , Farquhar Marilyn G.
Published: June 2021

We previously found that Gαs promotes EGF‐induced EGFR degradation (Zheng, et al. MBC 2004, 15:5538). Since degradation is key step in the downregulation of EGFR signaling, we predict that Gαs facilitates signal downregulation. To determine if this is the case, control, Gαs, or Gαi3 depleted HeLa cells stimulated with EGF were monitored for EGFR autophosphosphorylation by immunoblotting using tyrosine site‐specific antibodies (pY1045, Y1068). We found that Gαs but not Gαi3 depletion increases EGFR autophosphorylation at 5 and 15 min after EGF stimulation, and the effects of Gαs depletion were partially rescued by transfection with siRNA resistant Gαs. These data indicate that Gαs is specifically required to downregulate EGFR autophosphorylation. Delayed downregulation and degradation could result from disrupted EGFR trafficking. To determine the effect(s) of Gαs on EGFR trafficking, we monitored the distribution of 647‐EGF and EGFR (pY1068) in control, Gαs, or Gαi3 depleted HeLa cells by immunofluoresence. At 5 min after stimulation, we found that EGFR and 647‐EGF localize to EEA1 and Hrs labeled endosomes in control cells. In contrast, more EGFR and 647‐EGF are found near the PM and show less colocalization in Gαs depleted cells. At 15 min after stimulation, pY1068 and 647‐EGF colocalize with EEA1 and Hrs in control and Gαs depleted cells, indicating that trafficking of EGFR from the PM to endosomes is delayed in the absence of Gαs, which may result in delayed receptor degradation. Overall, our novel data suggest that Gαs affects the downregulation of EGFR signaling by modulating an early (e.g., internalization and/or recycling) step in receptor trafficking.
Clathrin binding to the DLLRKN region of Huntingtin‐interacting protein 1 (HIP1) requires coiled coil instability
Authors: Ybe Joel Alcasid , Ignatius Arun Alphonse , Giedroc David , Illingworth Melissa , Poorfarahani Sara
Published: June 2021

Huntingtin‐interacting protein 1 (HIP1) is a coiled coil protein involved in clathrin‐mediated endocytosis and plays a role in Huntington's disease. The binding determinants for proteins that associate with HIP1, such as huntingtin, HIP1‐protein interactor (HIPPI), and clathrin are all located in the middle of the HIP1 coiled coil that we discovered was naturally flexible. The objective of the study is to identify those amino acids that destabilize HIP1 to find out if molecular flexibility is a requirement for clathrin binding. Circular dichroism (CD) studies on wild type and mutant HIP1 482–586 constructs showed K510, T528 and Q531 to be major determinants of HIP1 stability. GST pull down experiments demonstrated the binding of clathrin was significantly reduced when HIP1 was engineered to be more stable. 2D‐HSQC NMR experiments on wild type HIP1 482–586 and HIP1 2M (T528L and Q531L mutations in HIP1 482–586) showed HIP1 482–586 and HIP1 2M behaved differently in solution, suggesting the mutations impacted the topology of HIP1. HIP1 2M was in a higher ordered oligomeric state compared to wild type HIP1 482–586. The 2D‐HSQC spectra of HIP1 2M also displayed strong peaks from a dynamic region in the protein. Together our studies argue the intrinsic instability of HIP1 is necessary for HIP1 to interact productively with clathrin.
Two members of the unusually large family of protein phosphatase 2B (PP2B, calcineurin) in Paramecium have distinct roles in calcium dependent processes.
Authors: Fraga Dean , Sehring Ivonne , Kissmehl Roland , Reiss Martina , Gaines Raymond , Hinrichsen Robert
Published: June 2021

We have characterized the calcineurin (CaN) gene family including subunits CaN‐A and CaN‐B based on sequence information obtained from the Paramecium genome project. Paramecium tetraurelia has 7 subfamilies of the catalytic CaN‐A subunit and one subfamily of the regulatory CaN‐B subunit, each with two members of considerable identity on the amino acid level (≥55% between subfamilies, and ≥94% within CaN‐A subfamilies, and full identity in the CaN‐B subfamily). At 14 members, the size of the CaN‐A family is unprecedented. We hypothesized that the different CaN‐A subfamilies were not strictly redundant and fulfill different roles in the cell. This was tested by selecting two phylogenetically distinct members of this large family for posttranscriptional silencing by RNAi. The two targets resulted in differing effects in exocytosis, calcium dynamics, and backward swimming behavior that supported our hypothesis that the large, highly‐conserved CaN‐A family members are not strictly redundant and at least two members have evolved diverse but overlapping functions. The fact that CaN regulates similar processes in more complex organisms suggests that these functional roles were acquired early in the evolution of this protein family.
Regulator of G‐protein signalling 4 (RGS4) KO mice show increased susceptibility to atrial fibrillation
Authors: Cifelli Carlo , Dissanayake Kaveesh , Zhang Hangjun , Liu Jie , Nattel Stanley , Backx Peter H , Heximer Scott
Published: June 2021

Susceptibility to atrial fibrillation (AF) depends strongly on parasympathetic activity. Since RGS4 inhibits parasympathetic signaling in the sinoatrial node, we explore whether changes in RGS4 levels altered the susceptibility of atrial fibrillation. Consistent with this possibility, RGS4 levels were decreased in atria of tachypaced dogs prior to their development of chronic AF. Moreover, in vivo ECG recordings of anaesthetized mice showed greater susceptibility to AF while optical mapping of isolated atrial preparations using a voltage‐sensitive dye revealed greatly increased susceptibility to rotor formation when rgs4 was ablated. Consistent with altered parasympathetic signaling in the myocardium of rgs4−/− mice, acetylcholine‐sensitive potassium currents (IKACh) evoked by carbachol application were greater in isolated atrial myocytes from rgs4−/− mice. These IKACh changes were, as expected, associated with marked enhancements of action potential duration shortening in response to parasympathetic activation, but not to slower conduction velocities. Together, our findings establish that RGS4 protects atrial tissues from excess parasympathetic signaling that predispose to atrial fibrillation.

Technical and financial assistance for this work was provided by the Heart and Stroke Foundation of Ontario Grant‐in‐Aid Program grants T6799, and Canadian Institutes of Health Research (CIHR) Canada Graduate Scholarship Program.
Structural analysis of the Gβ1‐CCT complex provides insight into the mechanism of Gβ1 folding
Authors: Plimpton Rebecca , Lai Chun Wan J. , Haines Kara , Stowell Caleb , Cuellar Jorge , Martin‐Benito Jaime , Valpuesta Jose M. , Willardson Barry M.
Published: June 2021

The cytosolic chaperonin containing TCP‐1 (CCT) carries out the final folding steps for the G protein ß1 (Gß1) subunit, enabling Gß1 to assemble into the G protein heterotrimer and act in downstream signaling. We employed cryo‐electron (cryo‐EM) microscopy to elucidate the CCT‐Gß1 interaction, using CCT‐Gß1 complexes purified from insect cells. Reconstructions of cryo‐EM images reveal Gß1 bound to the apical domains of two adjacent CCT subunits, CCTß and CCT?. Gß1 forms a globular structure and sits perpendicular to the axis of the CCT toroid. Thus, Gß1 appears to be mostly folded and bound to CCT through one or two of its propeller blades. We identified the Gß1 binding sites on CCTß and CCT? through mutagenesis and co‐immunoprecipitation techniques. Two regions of these CCT subunits contribute strongly to Gß1 binding: a hydrophobic loop and a hydrophobic groove between two helices above the loop. CCT binds many of its substrates through hydrophobic interactions, yet molecular‐level understanding of specific binding contacts has remained elusive. We identify specific sequences in this hydrophobic chaperone‐substrate interaction that serve to stabilize one or two Gß1 propeller blades, thereby allowing the remaining blades to reach their native conformation.
Is b‐arrestin‐2 a determining factor in PAR‐2 stimulated allergic asthma?
Authors: Richards Heddie L , Wilson Emma , DeFea Kathryn
Published: June 2021

PAR‐2 exhibits pro‐ and anti‐inflammatory effects in the airway and lungs. From a molecular standpoint, the anti‐inflammatory effects signal via PAR‐2/Gαq resulting in mobilization of intracellular calcium and prostaglandin production. Pro‐inflammatory effects include leukocyte migration and cytokine production. Our lab has demonstrated that PAR‐2 promotes leukocyte migration in a β‐arrestin‐dependent pathway as β‐arrestins scaffold the actin filament severing protein, cofilin, with its upstream activator, CIN at the leading edge. This brings CIN and cofilin into proximity leading to cofilin dephosphorylation and activation and is independent of classic Gαq signaling. We believe the PAR‐2 pro‐inflammatory effects are mediated by β‐arrestin‐dependent actin reorganization and chemotaxis, and will be abolished in mice lacking β‐arrestins.

These studies illustrate β‐arrestin‐2 mediation of an allergic asthma inflammatory response to intranasal administration of allergen and PAR‐2 activating peptides. Wild type mice exposed to allergen and treated with PAR‐2 activating peptide have increased leukocyte recruitment to BAL and lung epithelium; while β‐arrestin‐2−/− mice do not. These data suggest that β‐arrestins facilitate PAR‐2 stimulated cell migration by recruitment of actin machinery to the leading edge. This pathway is important for PAR‐2 stimulated inflammatory responses in vivo.
Post‐endocytotic trafficking of ETB receptors in cardiomyocytes: effects of pressure‐overload hypertrophy on their subcellular distribution.
Authors: MERLEN Clemence , FARHAT Nada , Villeneuve Louis R. , Gillis Marc‐Antoine , Thorin Eric , Allen Bruce G.
Published: June 2021

Endothelins are implicated in both cardiac function and pathophysiology via their actions on ETA (selective for ET‐1>>ET‐3) and ETB (non selective) receptors. To date, the cellular signaling evoked by ET‐1 has been studied by activating receptors at the cell surface. However, ETB are primarily located on the nuclear membrane in adult cardiac ventricular myocytes (ACVMs). The present study was to determine the origin of these receptors. In ACVMs, which express ETA and ETB, fluorescently‐labeled ET‐1 was internalized and localized to lysosomes plus perinuclear structures. ET‐3 was transported soley to lysosomes. In rat aortic endothelial cells, which express ETB, endocytosed ET‐1 and ET‐3 were transported to lysosomes. To determine if the subcellular distribution of ETRs was altered during hypertrophy, mice were subjected to transverse aortic constriction (TAC) for 7 days and the distribution of ETRs examined by immunocytofluorescence. Whereas ETB immunofluorescence was associated with the nuclear membrane in sham animals, additional perinuclear staining was observed in TAC hearts. The distribution of ETA immunofluorescence was unaffected. Hence, localization of ETB to the nuclear membrane is not a result of post‐endocytotic ligand/receptor complex trafficking and the subcellular distribution, and possibly trafficking, of ETB is altered during hypertrophy.

(Supported by the CIHR, FICM and HSFC)