bluuebunny/crossref_metadata_2025 huggingface dataset:
The Fickle Cheerleader Hysteresis: Explaining Haemoglobin‐Oxygen Equilibrium through Non‐traditional Case‐Based Instruction in Physiology.
Authors: Harris Michael B.
Published: June 2021

Modern teaching in physiology is greatly enhanced by case‐based instruction, and the explanation of complex processes may be enhanced by metaphor or analogy. It is challenging to select cases that engage a wide audience, and analogies that resonate. “The Fickle Cheerleader Hysteresis” is an analogy exemplifying cooperativity in hemoglobin‐oxygen binding. It is designed to illustrate basic core principles relevant to cardiovascular and respiratory physiology. The analogy considers how hemoglobin monomers interact in a tetramer, implications of transitions between tense and relaxed state configurations, O2 binding site availability, binding site limitation, and the overall the pattern of hemoglobin‐oxygen saturation and desaturation underlying the sigmoidal O2‐equilibrium curve. The context is particularly relevant to undergraduate students and may allow better student engagement and concept retention.

Support or Funding Information

Work reported in this publication was supported by the National Heart Lung and Blood Institute and National Institute of General Medical Sciences of the National Institutes of Health under awards 1R15HL126105 and 1SC2GM112570. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health or any other funding body.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Introducing Physiology of Diabetes to Middle School and High School Students
Authors: Zuercher Jennifer L. , Gopalan Chaya
Published: June 2021

Diabetes, a chronic condition that impacts millions, is a complex disease. Understanding the disease can contribute to both increasing awareness about this debilitating condition as well as preventing occurrences. In pursuit of both of these goals, we set out to educate middle school and high school students about diabetes during PhUn Week. PhUn Week is an annual outreach program sponsored by the American Physiological Society (APS), partnering physiologists and K‐12 students, introducing students to physiological concepts. Thirty (16 high school; 14 middle school) students from a Sunday school program at an urban religious center completed a 10‐question pretest, measuring initial knowledge about diabetes, followed by a one‐hour education session presented by a local physician. A post‐test was administered following this presentation. The post‐test consisted of ten questions where all but one question were the same as the pre‐test, measuring improvement of prior knowledge and engagement in the presentation. The overall post‐test average score increased by approximately two correct responses, which was a significant improvement (p <0.0001), suggesting that the students were motivated to and did learn diabetes concepts. This study also suggests that exposing students to educational activities related to physiology is beneficial and may lead to an increase in interest in such topics.

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.
The Effect of a Limbic System and Emotions Educational Program on Elementary and Middle School Learning
Authors: Bhatnagar Samarth , Phan Sydney , Puder Barb
Published: June 2021

The effectiveness of scientific educational outreach among children has been difficult to measure because of many confounding variables such as teaching style, material taught and many other factors. To find clarity with this, an experiment was designed involving two 5th grade classes from Martinez, CA, John Swett Elementary and Las Juntas Elementary, and one 7th grade class from Pleasant Hill, CA, Sequoia Middle School. The experiment entailed using an educational poster with characters from a well‐known Disney movie, Inside Out, to teach students about emotional regulation and control guided by the Limbic system and its main components (hypothalamus, amygdala, hippocampus and thalamus). All three schools were approached using the same protocol: a pre‐test, an educational portion with the poster, a hands‐on activity, and then a post‐test. The hands‐on activity, creating emotion stress balls, was specifically meant to reinforce the material to the children and give them a broader understanding of how to positively deal with their own emotions. In addition to the quantitative pre‐and‐post‐test, a qualitative survey was also administered using the Likert scale to assess how the poster was perceived, the ease of understanding and other factors. The 5th grade students showed a statistically significant quantitative improvement between their pre‐and‐post‐test average results and the 7th graders showed a higher qualitative mean score, indicating they had a better appreciation of the outreach.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“An Ethical Dilemma?: How Anatomy Education Impacts Healthcare Students' Ethics”
Authors: Stephens Georgina Caitlin , Paynter Sophie , Rees Charlotte , Lazarus Michelle
Published: June 2021

The objective of this study was to explore how anatomy education impacts healthcare students' ethics. Donor dissection remains debated anatomy pedagogy. While short‐term anatomy knowledge gains may not be impacted by dissection, studies also suggest that students' anxiety and stress levels are. However, compared to clinical settings, donor dissection may provide a lower stress environment for introductions to death and dying. To improve the knowledge base related to this anatomy education discourse, we evaluated the longitudinal impacts of donor dissection on healthcare students' professional identity (PI).

A longitudinal qualitative study with Monash University student participants from physiotherapy and medicine involved online discussion forums during semester and inter‐professional focus groups at the end of semesters. Preliminary themes were developed using framework analysis.

Six themes related to ethics in anatomical education were identified: 1. Informed consent, 2. Non‐maleficence, 3. Beneficence, 4. Dichotomy of personification versus objectification, 5. Duty of care, and 6. Culture. Interestingly, the dominant themes of students' ethical perceptions changed with time and varied between student groups utilising whole body donors and those using prosected specimens.

The impact of donor dissection on students' perception of ethics is seen to be complicated. Prior to this study, most investigations focused only on pre‐ and post‐ scenarios. The longitudinal approach presented here suggests a strong impact of donor dissection on priming students' focus on medical ethics, but also suggests that over time, this effect fades. This fading effect may be mitigated with intervention, which will be a focus of future studies. This research does suggest that donor dissection may have broader impacts on students than simply anatomical knowledge acquisition and that anatomy can play a vital role in early professional identity developmental of healthcare students.

Support or Funding Information

Monash Education Academy

Monash University Faculty of Medicine, Nursing and Health Sciences Learning and Teaching Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Developing a Dissection‐based Human Anatomy Laboratory Manual to Target Course and Laboratory Learning Objectives at the University of Guelph
Authors: Marrelli Kristina M. , Forster Victoria Nicole , Valencia Jason , Danielli Ethan , Robson Naomi , Jadeski Lorraine C.
Published: June 2021

Dissection‐based human anatomy is an inherently time‐consuming process. Accordingly, students must use their time in the laboratory as efficiently as possible. There are many commercial resources that are currently available to students for pre‐laboratory preparation at home; however, preliminary data gathered from student questionnaires (n = 217) indicate that a significant proportion of third‐year undergraduate students enrolled in human anatomy at the University of Guelph choose not to use these resources to prepare for their laboratory sessions (p<0.01). Anecdotal student feedback revealed that their avoidance of the resources was because the students typically perceived the commercial resources to be overly text‐based and lacking alignment with the course curriculum. Therefore, we created a preparatory dissection‐based laboratory manual as a part of an ongoing project in which we are developing, evaluating, and refining educational tools to enhance our human anatomy program. To maximize the potential impact of all such laboratory resources, we aim to develop the dissection‐based laboratory manual to: 1) specifically target the University of Guelph's human anatomy course and laboratory learning objectives, 2) bridge lecture and laboratory content, and 3) enable students to address basic practical and clinical applications of anatomy. To achieve these objectives, the manual incorporates various educational components, including dissection‐based cadaveric images, digital illustrations and schematics, dissection instructions, and active‐learning exercises. These components will be integrated to help students visualize and understand concepts such as: 1) the origins and pathways of nerves and arteries that are initially discovered and examined in distal sites, 2) compartmentalization of the limbs, and 3) relative levels of depth between structures and their organization within the human body. In addition, the manual targets course concepts that are not easily visualized or understood with a regional anatomy approach, given the time constraints and the schedules of our dissection‐based courses. By making this resource available to students outside of the laboratory, our goal is to promote student preparedness for each session and enhance the quality of learning within the human anatomy laboratory itself. Student feedback sessions will be used to inform further development of the laboratory companion and improve future editions of the resource.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Implementation of a Claymation Video to Explain Heart Septation
Authors: Van Winkle Katherine , Krimsier Jonathan , Elzie Carrie
Published: June 2021

Embryology is critical for understanding many developmental diseases and their processes, and provides a solid foundation for comprehending adult anatomical structure and its abnormalities. Despite our technological advancements, there is still a lack of adequate visual representation of the dynamic, three dimensional (3D) events that occur during development. In an effort to close this critical learning gap a claymation video of an embryological event (heart septation) was created. Heart septation is an extremely important event to conceptualize in the medical field as it lays the groundwork for many adult abnormalities. It has also proven to be one of the most difficult topics for students to comprehend. Therefore, a video was generated to provide students with a readily accessible and effective learning platform to better represent embryological events not easily depicted through flat, two dimensional (2D) images. Using stop‐motion animations of clay models and drawings to represent the different aspects of heart septation, a video was developed and voiced over to provide context. Data on its efficacy will be collected within the next few months through the distribution of post‐viewing surveys. It is hypothesized that this video and its future counterparts will dramatically increase the level of embryologic comprehension and thus improve upon the student's education. This video will be the beginning of a series of videos both representing normal and abnormal developmental events. The end goal being the creation of a visual library to aid in student understanding of embryology and its clinical applications.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Incorporation of Clinically‐Based Ultrasound Workshops in a 1st Year Medical Anatomy Course
Authors: Nydam Randall , Patel Akash , Finch Charles
Published: June 2021

Ultrasound (US) is quickly becoming an increasingly common tool in point‐of‐care medical practice (commonly called “the new stethoscope”). To better prepare our students for this environment, the Arizona College of Osteopathic Medicine (AZCOM) has developed and is implementing a 4‐year, longitudinal, integrated, US curricular experience. The initial phase (1st year courses) includes a major US component in gross anatomy. The gross anatomy series is two regional, dissection‐based courses with eight exam units (four per course) over two quarters. The US component to the gross courses includes increased use of US images in lectures as well as eight, clinically‐based, hands‐on US workshops; one for each exam unit. Modifications in the lecture schedule made inclusions of the workshops credit‐hour neutral. The exam units and associated US workshops (in italics) are: Back/Scapular—lumbar puncture; Upper Extremity—carpal tunnel syndrome; Thorax—4‐view cardiac assessment; Abdominal—biliary colic; Posterior Abdominal wall/Pelvis/Perineum—AAA, FAST; Lower Extremity—plantar fasciitis, calcaneal tendonitis, tarsal tunnel syndrome; Head and Neck I—ocular and thyroid exams; Head and Neck II—carotid stenosis. The US workshops are designed for students to visualize the relevant anatomy of a common, point‐of‐care clinical scenario on living subjects. Student assessment is in the form of oral “check‐out” quizzes in the workshops and US‐based questions in the written and practical portions of the unit exams. Overall performance on US questions in the first four exams was below the exam averages (77% v. 83%), but with a marked upward trajectory of the US question performance (69%, 62%, 76%, 89%). Practical exam questions showed a similar trajectory, but with lower averages. A Likert scale survey—first of a series as part of an ongoing four‐year study—was given at the end of the first quarter (4 exam units) to assess student opinions regarding US in the anatomy course. The non‐incentivized response rate was 146/279. Students overwhelmingly (90+%) feel US training is an important part of the curriculum that should begin as early as possible. For each workshop a strong majority agreed that it improved their comprehension of both the anatomy of the region scanned (87%, 92, 91, 92) and the regional clinical concerns (91%, 94, 93, 93). A majority of students also agreed that the US workshops led them to do some form of further research of the clinical concerns (65%, 64%, 74%, 71%). These preliminary results are very encouraging. Another survey will be taken at the end of the winter quarter, but current indications are that the utilization of US workshops in the AZCOM gross anatomy course has been a valuable addition.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neural Basis of Spatial Ability and Anatomical Concepts: An fMRI pilot study
Authors: Smith Theodore C. , O'Loughlin Valerie , Newman Sharlene
Published: June 2021

First‐time anatomy students struggle with organizing massive amounts of material, as well as demonstrating the necessary spatial ability skills to be successful. Instruction of undergraduate students can be quite challenging due to students' lack of metacognition and unfamiliarity with anatomical concepts. Functional magnetic resonance imaging (fMRI) can bypass this lack of metacognition and examine students' neural organization of anatomical knowledge and their neural basis of spatial ability. In this project, a group of undergraduate anatomy students (n= 53) completed a series of cognitive tasks. A simple linear regression was then performed to predict final course grade based on students' scores on cognitive tasks as well as examine the similarities between common cognitive tasks. Gender was not a predictor of mental rotation score (F(1, 51)= 3.230, p = .078), with an R2 of .06. A second cohort of undergraduate students (n=2) and graduate students (n=2) completed a Shepard‐Metzler (1971) Mental Rotation task and an anatomical concepts task under fMRI. The undergraduate and graduate cohorts' cortical activation during the tasks was compared using statistical parametric mapping (SPM). This work demonstrates the importance of spatial ability in the undergraduate anatomy setting and builds a neural foundation for understanding how anatomy learners organize anatomical concepts. This works also illustrates how the collaboration of educational researchers, cognitive scientists, imaging specialists and anatomical educators can provide diverse techniques and data for the betterment of anatomical and medical education.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EphB Kinases expressed in osteoblastic stem cell niches regulate bone growth
Authors: Kamath Rajay A. D. , Benson M Douglas
Published: June 2021

Substantial evidence indicates that the membrane‐bound ephrins and their Eph tyrosine kinase receptors regulate bone growth and homeostasis. Yet, little is known about which ligand/receptor pairs are involved or their mechanistic roles in bone biology. We observed expression of the EphB2 and EphB3 kinases in calvarial sutures and of EphB3 in the proliferative zones of long bone metaphases. As both of these sites are reported niches for osteoblastic stem cells, we hypothesized that ablation of these Ephs would result in dysregulated bone growth. We examined EphB1/2/3 triple knockout mice and wild type controls at 10–12 weeks of age for phenotypes associated with loss of these receptors, using plain x‐ray and three‐dimensional micro‐CT analyses. EphB1 was included because it was observed in the periosteum, a known source of osteoblasts. X‐rays of triple knockout mice showed increased cortical thickness in long bones with fewer metaphyseal trabeculae compared to wild type controls, especially in the tibiae. Triple mutant calvariae appeared normally patterned, but had markedly increased radiolucency in the frontal bones, suggesting hypomineralization in that area. Micro‐CT analysis of triple mutant femora confirmed their increased cortical bone volume compared to controls, as well as increased cortical density and an increase in metaphyseal trabecular thickness. Despite the plain x‐ray presentation of the frontal bones, triple mutant calvariae showed an overall increased bone volume/total volume (BV/TV) ratio compared to control calvariae. These data indicate that Ephs B1, B2, and B3 regulate bone growth. Ongoing studies will identify the contribution of each individual EphB to bone development and healing.

Support or Funding Information

NIH/NIDCR R01DE022804

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

The pronator muscles of the upper limb, pronator teres (PT) and pronator quadratus (PQ), work together to rotate the radius around the ulna along an axis parallel to the central bands of the interosseous membrane while stabilizing the involved joints. Anatomical variations that affect the rotational efficiency of the PT have been described. Previously, we have described an aberrant PQ with a possible flexor function, and also defined 4 types of anatomical variations in PQ, based on the number of bellies present, and whether it occurred unilaterally or bilaterally, suggesting that PQ is more variable than appreciated. The PT typically has 2 distinct heads of origin (ulnar and humeral) and inserts onto the lateral radius. However, several anatomical variations have been reported, including the number of heads, locations of origin and insertion points, anomalous fiber orientations, and the presence of fibrous bridges and/or arches. In the current study, we sought to determine if there are patterns of anatomical variations in the PT, and whether any such variations occurred in relationship to those of the PQ. Sixteen forearms from 8 embalmed cadavers (4F,4M ages 64–93, mean 82) were dissected, and the PT and PQ muscles observed for muscle shape, fascicle orientation, and attachment points. PT and PQ heads was described, and PT tendon length, width, and distance from the radial head and styloid process measured. Our data revealed that the PT existed as only a single muscle head (humeral), bilaterally, in 25% of the cadavers examined, while exhibiting the traditional two muscle heads (humeral, ulnar) bilaterally in 75% of cadavers. Interestingly, the PT variant with a single humeral head occurred in conjunction with Type II PQ muscles bilaterally (i.e., the presence of 2 muscle bellies). Of the six cadavers with both humeral and ulnar heads of PT, 33.3% had an occurred with Type I PQ (1 head, bilateral), 33.3% with Type II PQ (2 heads, unilateral), and 33.3% with Type III PQ (2–3 heads, bilaterally). Our data suggests that there may be a relationship between the patterns of anatomical variations between PT and PQ muscles, which may provide new insight into the biomechanical relationship between these 2 muscles. The inclusion of additional cadaveric specimens, as well as quantitative comparisons between PT and PQ muscles, including cross sectional area, volume, and line of action, will help to elucidate the potential relationship between PT and PQ muscles.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hind Limb Myology of the Kinkajou (Potos flavus)
Authors: Walker Allison M. , Ahrens Heather E.
Published: June 2021

Kinkajous (Carnivora: Procyonidae) spend most of their time in the canopy, exhibiting highly arboreal and frugivorous behaviors. Associated with navigating their arboreal environment, kinkajous possess complete hind‐foot reversal. A limited number of studies present detailed examination of the musculature in mammals capable of hind‐foot reversal. We describe and present muscle maps of the hind limb of a fresh‐frozen male Potos flavus. P. flavus resembles the arboreal red panda (Ailurus fulgens), sharing similar gluteal and caudal leg muscle morphology and attachments. Additional observations include a shared proximal tendon for the m. biceps femoris and m. abductor cruris caudalis, presence of the m. femorococcygeus inserting on the distal half of the caudal femur, presence of a femoral head of m. semimembranosus, and fusion of m. fibularis longus with brevis midway along their length. Comparison of the myology of the kinkajou with descriptions of A. fulgens and other members of Procyonidae indicate that the hind limb musculature is generally conserved across taxa.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Type IV Collagenase Inhibitor, BiPS, Alters Matrix Metalloproteinase 9 Expression in Sulfur Mustard Exposed Mouse Skin
Authors: Chang Yoke‐Chen , Hahn Rita A. , Svoboda Kathy K. , Gordon Marion K. , Gerecke Donald R.
Published: June 2021

Sulfur mustard (bis‐2‐chloroethylsulfide, SM), is a chemical warfare agent that penetrates the skin rapidly causing extensive blisters and constitutes a potentially serious threat to military and civilian populations. While its mechanisms of toxic action are yet poorly defined, animal models demonstrate disruption of the skin architecture at the dermal‐epidermal junction (DEJ). Collagen degradation in the basement membrane zone by matrix metalloproteinases (MMPs) further exacerbates vesication post SM exposure. The type IV collagenase (MMP9 and MMP2) may mediate the disruption of type IV collagen (Col IV), a main component of the basement membrane zone (BMZ) in skin wounds post SM exposure. N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide (BiPS) was tested as a highly selective MMP2/MMP9 inhibitor. The study aimed to evaluate the therapeutic effectiveness of topically delivered BiPS to protect against SM induced skin injury. We performed a time course study (24, 72, and 168 hrs post‐SM exposure) and a compound evaluation study (BiPS as a single pretreatment using the same time points post SM exposure) on mouse ear skin to determine improvement using histopathologic changes, MMP9 gene expression, and basement membrane protein expression as markers. SM induced characteristic skin structural damage including edema, inflammatory cell infiltration, and epidermal hyperplasia. MMP9 mRNA expression increased with time. Dual immunofluorescent studies using Col IV α1 and MMP9 antibodies showed BMZ breakdown of Col IV in the basement membrane and apparent MMP9 expression in the epidermis and the adjacent dermal matrix of the SM injured skin. In the compound evaluation study, the BiPS pretreatment group demonstrated significant reduction in edema and improvement in overall tissue structure by 72 hr post‐SM exposure. mRNA expression of MMP9 was significantly downregulated in the BiPS treated group by 168 hr post exposure. Immunofluorescent studies showed that significant reduction of MMP9 expression in the skin sections for all time points; the basement membrane appeared improved as indicated by a continuous (without disruption) expression of Col IV in the BMZ; which was similar to the unexposed control skin. These results suggest that BiPS may modulate MMP9 expression which may in turn improve basement membrane integrity after skin exposure to SM. BiPS, the MMP2/MMP9 inhibitor II, may present as a potential therapeutic countermeasure against SM induced skin injury.

Support or Funding Information

ES005022, T32ES007148, and NIAMS U54AR055073

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing PTGER4 as a Target Gene of Autism Protein E6AP
Authors: Amadei Celeste , Alessandri Michael , Nawaz Zafar
Published: June 2021

Background

Autism spectrum disorder (ASD) has been found to be associated with duplication or triplication of the UBE3A gene, which encodes for E6‐associated protein (E6AP, an E3 ubiquitin ligase and transcriptional coactivator). Mice with three copies of UBE3A exhibit core ASD features and humans with Dup15q share many symptoms with ASD. However, the few E6AP ubiquitination substrates found do not explain ASD pathology. Our lab has identified and characterized E6AP as a coactivator of steroid hormone receptor signaling. Estrogens (E2) affect learning, memory and many other brain processes via estrogen receptors, which are transcription factors.

Objectives

This study tests the hypothesis that deregulation of E6AP‐mediated steroid hormone receptor transcriptional signaling in the brain leads to the development of ASD. The project aims are to identify steroid hormone‐dependent E6AP target genes in neurons and to study the role of these target genes in the pathogenesis of ASD. Identification of new molecular pathways that are transcriptionally regulated by E6AP will broaden our understanding of ASD.

Methods

Potential E6AP target genes were identified by microarray of MCF‐7 breast cancer cells. Cells from the mouse neuroblastoma Neuro2a cell line were cultured. Cells were transfected with E6AP or had E6AP knocked down by siRNA and then were treated with physiologically relevant doses of estrogen, the estrogen receptor antagonist tamoxifen, or vehicle. Assays included western blot, co‐immunoprecipitation, qRT‐PCR, and microscopy.

Results

1) E6‐AP and ER colocalize in mouse HPC neurons. 2) E6‐AP and ERα translocate to the nucleus of Neuro2a (N2a) cells upon E2 treatment. 3) E6‐AP and ERα complex in N2a. 4) The learning and memory gene for prostaglandin E receptor 4, PTGER4, is an E6AP‐dependent target gene that is downregulated in the presence of E6AP or estrogen.

Conclusions

We have identified a memory and learning gene that is regulated by E6AP and E2‐dependent: PTGER4. This is evidence that PTGER4 may be altered in ASD, leading to learning and memory symptoms. PTGER4 allows phosphorylation of glycogen synthase kinase 3 (GSK3). GSK3 has a large role in apoptosis and has been implicated in neuropsychiatric disorders such as Alzheimer's disease and bipolar disorder. Given that GSK3 is amyloidogenic and ASD patients exhibit increased beta amyloid deposition, increased E6AP leading to decreased PTGER4 may lead to decreased inhibitory phosphorylation of GSK3. In a case study, the GSK3 inhibitor ketamine actually improved an adult ASD patient's symptoms, supporting this theory. Further experiments will be necessary to confirm these promising findings.

Support or Funding Information

University of Miami
Regulation of OPN expression in MDA‐MB‐435: role of genistein
Authors: Das Bidyadhar , Khongsti Kitboklang
Published: June 2021

Breast cancer is the most common cause of cancer‐related mortality in women worldwide and metastatic spread is the leading cause of breast cancer deaths. Osteopontin (OPN) plays a functional role in normal mammary gland development, progression of breast cancer and in malignancy of breast cancer. High expression of OPN is observed in high‐grade metastatic malignant human gliomas and in the highly invasive and metastatic human breast cancer cell lines (e.g. MDA‐MB‐435). Soy isoflavone genistein, a tyrosine kinase inhibitor and agonist of estrogen receptor‐β (ERβ), is known to have antitumoural properties and also induce apoptotic cell death of ERα‐negative breast cancer cells via p53‐dependent pathway. The isoflavone has been reported to exert antiproliferative activity in some breast cancer cell lines; however, its role with respect to OPN expression is not well understood. Therefore, to address this, the expression level of OPN was investigated upon genistein treatment in MDA‐MB‐435. The cells were treated with different concentrations of genistein for different time intervals; the expression of OPN, the signaling pathways and the promoter regions involved in the regulation of OPN were studied. Genistein displayed an inhibitory effect on OPN protein and mRNA levels in a dose dependent manner and treatment with 50 μM genistein for 24 h showed to be promising. Analysis of the signaling pathways showed no activation of Akt/PKB pathway, no alteration in p‐MEK½ expression whilst an increase in the expression of p‐ERK½ was observed in genistein treated group. These results indicate that genistein acts as an anti‐metastatic agent by inhibiting OPN expression through the activation of p‐ERK½ in MDA‐MB‐435 cells. The transcription factors responsible for this event are being studied in details.

Support or Funding Information

DBT (GoI) No. BT/253/NE/TBP/2011 dated May 07, 2012

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tributyltin Effects on Akt/Protein Kinase B and Ribosomal S6 Protein Phosphorylation
Authors: Hamza Nafisa , Boules Mariam , Whalen Margaret
Published: June 2021

The ERK1/2 and p38 mitogen activated protein kinase (MAPK) pathway have been shown to be activated by exposure to the environmental contaminant tributyltin (TBT). TBT is found to contaminate a number of food products that humans consume resulting in measureable levels in human blood (ranging as high as 261 nM) as well as other tissues. Recent studies have shown that TBT at certain concentrations increases the production of the pro‐inflammatory cytokine interleukin 1 beta (IL‐1β) from human lymphocytes without increasing the mRNA for IL‐1β. TBT‐induced production of IL‐1β appears to require the ERK1/2 and p38 pathways. However, it is possible that TBT may be activating other pathways such as Akt/protein kinase B (PKB) which could also contribute to its ability to increase IL‐1β production. Additionally, downstream substrates of both ERK1/2 and Akt/PKB can lead to the activation of protein synthesis in part by stimulating phosphorylation of the ribosomal S6 protein. Thus, the current study examines whether TBT is able to activate Akt/PKB and S6 phosphorylation as possible mechanisms for the TBT‐induced increases in IL‐1β production. Human lymphocytes were exposed to TBT for 10 minutes. Following the exposure the cells were lysed. The lysates were analyzed using western blot. Antibodies specific to the activated forms of Akt/PKB and S6 were used. Initial results indicate that exposure to TBT increases the activation of Akt/PKB and S6. Thus, it is possible that TBT is stimulating IL‐1β production by activating both MAPK and Akt/PKB signaling pathways leading to increased translation of IL‐1β.

Support or Funding Information

Supported by NIH grant U54CA163066 and 2T34GM007663

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LRP1 requires the I3 domain of hDlg for interaction with the motor protein KIF13B
Authors: Mills Joslyn E. , Hanada Toshihiko , Liscum Laura , Chishti Athar
Published: June 2021

KIF13B, a kinesin‐3 family motor, was identified by virtue of its biochemical interaction with human homologue of Drosophila discs large tumor suppressor (hDlg). Unlike its orthologue KIF13A, KIF13B contains a C‐terminal CAP‐Gly domain. Recently, the generation of a full‐length KIF13B null mouse model revealed a functional role in the endocytosis of LDL receptor related protein (LRP1) resulting in elevated serum cholesterol and plasma factor VIII activity. Here, we have developed two mouse models, a full‐length KIF13A null mouse and a mouse with a truncated KIF13B protein lacking its C‐terminal CAP‐Gly domain (KIF13ΔCG). To define the specific role of the CAP‐Gly domain of KIF13B in LRP1 endocytosis, we show that KIF13BΔCG mice exhibit higher levels of serum cholesterol consistent with the reduced uptake of [3H]‐LDL in KIF13BΔCG mouse embryonic fibroblasts. Alternatively, the plasma levels of factor VIII are not significantly different from wild type mice suggesting that the CAP‐Gly domain of KIF13B plays a role in lipoprotein endocytosis but not necessarily other functions of LRP1‐mediated endocytosis. We find that the truncation of KIF13B causes subcellular mislocalization of the protein and also leads to the mislocalization of LRP1, which likely contributes to the observed phenotype. To delineate the interactions of the LRP1 complex at the plasma membrane, we discovered that the cytoplasmic tail of LRP1 interacts with the alternatively spliced I3 domain of hDlg, which in turn interacts with KIF13B. Together, the LRP1‐hDlg‐KIF13B complex emerges as a novel regulator of LRP1‐mediated interactions with implications in metabolism, cell polarity, and development.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Dog, Cat, Guinea Pig, and Human IAPP toxicity in HeLa cells
Authors: Ledesma Litza Itzel
Published: June 2021

Type 2 diabetes is one of the leading causes of death in the U.S. Type 2 Diabetes is the most generic form of diabetes, where your body suffers from insulin resistance. Islet Amyloid Polypeptide (IAPP) is a regulatory peptide which inhibits glucagon and insulin secretion. The aggregation of IAPP is present in pancreatic islet amyloid deposits seen especially in Type 2 diabetes in humans and other mammalian species. Our objective was to see how the changes in IAPP sequence correlates to the toxicity of IAPP on mammalian cells. Here, we focus on dog, cat, and guinea pig IAPP. Molecular techniques such as MTT assay and LDH Cytotoxicity assay were used to test the effect animal IAPP has on HeLa cells. As well as to see the effect mixing human and animal IAPP has on the HeLa cells. MTT assay results showed that the animal IAPP volumes had no significant effect on mammalian cell viability while the human IAPP was consistently toxic to the cells. Cell viability percentages stayed relatively constant despite increasing IAPP volume. In comparison, human IAPP showed a decreasing cell viability trend with increasing IAPP volume. However, when dog IAPP and human IAPP were mixed, cell viability was increased. The same effect occurred with the mixing of cat IAPP and human IAPP. LDH Cytotoxicity assay results showed that percent toxicity increased with increasing IAPP volumes for human, cat and guinea pig. Dog IAPP cell toxicity stayed constant as IAPP volume increased. When dog IAPP and cat IAPP were separately mixed with human IAPP, a decrease in cell toxicity is seen. Mixing guinea pig IAPP with human IAPP had no effect on cell viability or cell toxicity. Further experiments are necessary to determine a correlation between these specific animal IAPPs and cell toxicity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure and Interaction Analysis of Human R7‐RGS/Gβ5/R7BP complexes
Authors: Adikaram Poorni R. , Zhang Jianhua , Pandey Mritunjay , Kittock Claire , Simonds William F.
Published: June 2021

The R7‐RGS/Gβ5 protein duplex functions as a GTPase‐activating protein (GAP) to facilitate the Gαi/o intrinsic GTPase activity. The R7‐RGS subfamily of RGS (Regulator of G protein Signaling) proteins comprises RGS6, RGS7, RGS9 and RGS11. The R7 family‐binding protein (R7BP) anchors the R7‐RGS/Gβ5 duplex to the cell membrane to enhance its GAP activity. Recently it was shown that knock‐out of R7BP in mice led to decreased acute and chronic itch sensation, indicating that R7BP has a central role in itch response. We are developing interfering peptides to block the R7BP and R7‐RGS/Gβ5 interactions as potential inhibitors of chronic itch. We demonstrate here the use of cross‐linking mass spectrometry (XL‐MS) to obtain structural clues about human R7‐RGS/Gβ5/R7BP triplexes. To this end, we first transfected plasmids encoding human RGS7, Gβ5 and R7BP into Expi293 cells and purified the protein triplex to 95% purity. The triplex was treated with the cleavable cross‐linking reagent DSSO (disuccinimidyl sulfoxide) at different protein:DSSO molar ratios and analyzed by mass spectrometry. The experiment was repeated using the human RGS9/Gβ5/R7BP triplex. Both experiments yielded peptides involved in the RGS7/9‐R7BP interaction, with one R7BP peptide common to both. We believe this peptide region to be an essential interaction surface on R7BP and an ideal target for interfering peptide design to block the R7‐RGS/R7BP interaction. Taken together, we demonstrate that XL‐MS is an effective way to shed light on structural features of the R7‐RGS/Gβ5/R7BP triplex to facilitate the development of novel anti‐itch drugs.

Support or Funding Information

National Institute of Diabetes and Digestive and Kidney Diseases Intramural Funding, NIH

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Kinetics, Mechanism, and Reaction Pathway of a Biodesulfurizing Enzyme from Rhodococcus erythropolis, Dibenzothiophene Monooxygenase
Authors: Jirde Samatar Ali , Mendez Gilberto , Gonzalez‐Osorio Liliana , Johnson Kellee Joanne Eberle , Palfey Bruce , Vey Jessica
Published: June 2021

Fossil fuel combustion is a major source of sulfur dioxide, dangerous to respiratory health and the environment. Sulfur removal from fossil fuels eases these effects, however, the current method, hydrodesulfurization, requires costly high temperatures and pressures. The bacterial species Rhodococcus erythropolis uses a four‐step enzymatic process, known as biodesulfurization, to remove sulfur in fuel compounds. The first enzyme in the pathway, dibenzothiophene monooxygenase (DszC), uses reduced flavin and molecular oxygen to generate a flavin intermediate that oxygenates its substrate, dibenzothiophene, to dibenzothiophene sulfoxide, then dibenzothiophene sulfone. Our objectives are to understand DszC's enzymatic mechanism and steps of the catalytic pathway to engineer DszC to accept various organosulfur compounds and increase catalytic efficiency. Towards our goals, we have purified active DszC; determined steady‐state kinetic parameters of DszC as a function of dibenzothiophene, dibenzothiophene sulfoxide, and flavin substrates; performed steady‐state experiments to describe DszC's enzymatic mechanism; and used transient‐state kinetics to ascertain the effects of dibenzothiophene and age times on flavin binding. Using affinity chromatography, we purified DszC at 0.5 gram/liter of culture in its native tetrameric state. Reverse‐phase HPLC experiments showed DszC had faster product turnover using dibenzothiophene sulfoxide as substrate versus dibenzothiophene (11.8 × 10−3 s−1 vs. 4.84 × 10−3 s−1). HPLC assays also demonstrated that having equimolar flavin and DszC resulted in a faster turnover rate than using excess flavin (4.84 × 10−3 s−1 vs. 1.49 × 10−3 s−1). Varying flavin and dibenzothiophene concentrations under steady‐state conditions indicated DszC likely forms a ternary complex with the flavin intermediate and dibenzothiophene to facilitate oxygenation. Using stopped‐flow spectrophotometry, we observed fast flavin binding (<1 second) and that having equimolar dibenzothiophene and DszC increased intermediate formation rates. Dibenzothiophene pre‐incubation with DszC, however, decreased intermediate formation rates, suggesting reduced flavin is the initial substrate to bind to DszC. These results provided us kinetic and mechanistic insights towards understanding the full catalytic pathway of DszC to support our goals of engineering a robust and efficient DszC enzyme for industrial applications.

Support or Funding Information

National Institute of Health (SCORE 5SC2AI109500)

Research Corporation CCSA 22672

MARC/RISE Program (NIH Grant Number 2R25GM063787‐10)

CSUPERB New Investigator Grant

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphoproteomic Analysis Identifies Dynamic Regulation of Caveolin‐1 Phosphorylation and Caveolae Formation by mTORC2 in Bladder Cancer Cells
Authors: Hau Andrew , Gupta Sounak , Leivo Mariah , Zhou Weidong , Hodge Alex , Wulfkuhle Julie , Conkright Brian , Bhuvaneshwar Krithika , Rao Shruti , Madhavan Subha , Nakashima Kazufumi , Petricoin Emanuel
Published: June 2021

The mammalian target of rapamycin (mTOR) and associated PI3K/AKT/mTOR signaling pathway is commonly upregulated in cancer, including bladder. We previously described that mTOR complex 2 (mTORC2) is a critical regulator of bladder cancer cell migration and invasion, but the precise mechanisms by which mTORC2 regulates these processes are poorly defined. Using a dual mass‐spectrometry (MS) and reverse‐phase protein array (RPPA) proteomics screening approach to reveal novel downstream phosphoprotein targets of mTORC2 in J82 and T24 bladder cancer cells, we identified 26 and 22 significantly altered biological functions putatively upregulated or down‐regulated, respectively, by mTORC2. Represented among these functions are phosphoproteins involved in regulating cell morphology, cellular assembly and organization, cell and focal adhesion, cytoskeletal rearrangement, cellular motility and others not previously linked to mTORC2 activity. Validation of a subset of these proteins revealed the ability of mTORC2 to regulate caveolin‐1 (CAV‐1) localization and dynamic phosphorylation of CAV‐1 on Y14. Regulation of CAV‐1 activity also altered the abundance of caveolae, which are specialized lipid raft invaginations of the plasma membrane that play a critical role in cell signaling pathways associated with cell and focal adhesion, and cell motility. Our results have demonstrated the utility of proteomics‐based analyses for novel elucidation of protein targets of mTORC2.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A mechanism for signal‐dependent IKKβ activation driven by molecular interactions with poly‐ubiquitin‐bound NEMO
Authors: Cohen Samantha , Shumate Kyle , Huxford Tom , Ghosh Gourisankar
Published: June 2021

The IκB kinase (IKK) complex is an enzyme that regulates the activation of the transcription factor NF‐κB in response to a diverse set of extracellular signals to control expression of many genes including those that mediate the cell survival program involving immune and inflammatory responses. IKK is composed of two catalytic subunits: IKK2/β and IKK1/α in addition to a non‐catalytic subunit known as the NF‐κB essential modulator (NEMO)/IKKγ. In this study, we focus on IKKβ which is the subunit primarily responsible for signal‐dependent activity in pro‐inflammatory signaling. Like many protein kinases, IKKβ activation is achieved through phosphorylation of two serine residues (S177 and S181 in IKKβ) located within its activation loop. However, the molecular details of how the activation loop serine residues undergo signal‐induced phosphorylation is still unknown. The x‐ray crystal structure and mutational analyses from our laboratory identified specific crystallographic interfaces (V‐shaped and KD‐KD interfaces) that are critical for IKKβ activation. Disruption of these interfaces reduced the ability of IKKβ to be activated in cells, suggesting the significance of protein‐protein contacts in activation. It is known that an N‐terminal segment of NEMO comprising the IKKβ binding domain (IKKBD) is responsible for a stable interaction with the C‐terminal NEMO binding domain (NBD) of IKKβ. Here, we propose a mechanistic model of IKKβ activation which invokes a transient second site interaction between the NEMO Ubiquitin binding in ABIN and NEMO (UBAN) domain and the IKKβ V‐shaped interface via a ubiquitin (Ub)‐chain. Engagement of poly‐Ub chain‐bound NEMO to IKKβ at this second site leads to conformational changes of the activation loop in an allosteric manner, resulting in the phosphorylation of S177 and S181. Cell transfection studies using V‐shaped interface mutants indicates that the second site of interaction critical for IKKβ activation coincides with the V‐shaped interface in a NEMO‐dependent manner. In vitro binding assays show that the NEMO UBAN (which lacks the IKKBD) binds to IKKβ (devoid of the NBD) in the presence of poly‐Ub. In vitro activation assays reveal that the activation loop is phosphorylated in an ATP, NEMO UBAN, and poly‐Ub‐dependent manner. We conclude that the molecular interactions between the V‐shaped interface of IKKβ and the poly‐Ub‐bound NEMO UBAN is responsible for signal‐dependent IKKβ activation loop phosphorylation. Our biochemical and cell‐based results give a detailed molecular understanding behind IKKβ activation in pro‐inflammatory signaling.

Support or Funding Information

This project was funded by an NIH R01 grant to Gourisankar Ghosh and supported by the California Metabolic Research Foundation at San Diego State University.

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