bluuebunny/crossref_metadata_2025 huggingface dataset:
Transient Receptor Potential Melastatin 8 (TRPM8) Induced Relaxation of Pulmonary Artery is Mediated Through Inhibition of Store‐Operated Ca2+ Entry in Normoxic and Chronic Hypoxic Rats
Authors: Mu Yun‐Ping , Jiao Hai‐Xia , Lin Da‐Cen , Zheng Si‐Yi , Sham James , Lin Mo‐Jun
Published: June 2021

Pulmonary hypertension (PH) is characterized by profound vascular remodeling and alterations in Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Members of multiple transient receptor potential subfamilies (TRPC, TRPV, and TRPM) have been identified in pulmonary vascular tissue. Our previous studies showed that the expression and functions of the store‐operated TRPC1, receptor‐operated TRPC6, and the mechanosensitive TRPV4 channels are augmented in PASMCs during PH, and their upregulation contribute to PH development. In contrast, TRPM8 is down‐regulated in PASMCs of rats PH models, and activation of TRPM8 with agonists causes relaxation of pulmonary arteries (PAs)(Liu et al., Cell Physiol Biochem 2013, 31:892–904). However, the mechanism of TRPM8‐induced PA relaxation has not been determined. In this study, we examined the possible interactions of TRPM8 activation and store‐operated Ca2+ entry (SOCE) in PAs and PASMCs of normoxic and chronic hypoxic pulmonary hypertensive (CHPH) rats. We found that TRPM8 expression was down‐regulated in PAs and the TRPM8‐mediated cation entry was reduced significantly in the PASMCs of rats after 3 weeks of hypoxia (10% O2) exposure. Activation of TRPM8 with icilin (0.1–100 μM) caused concentration‐dependent relaxation of cyclopiazonic acid (CPA) and ET‐1 pre‐contracted endothelium‐denuded PAs in the presence of nifedipine. The vasorelaxant effect of icilin was abolished in the presence of the SOCE antagonist Gd3+ at submicromolar concentration. Icilin suppressed the CPA‐induced cation entry in PASMCs determined by the Mn2+ quenching technique and by Ca2+ transient measurement. The inhibitory effect of icilin on SOCE‐induced PA relaxation and Ca2+ entry were abolished by the TRPM8 antagonist AMTB, indicating that the effect was mediated specifically by TRPM8 channels. Moreover, TRPM8‐mediated inhibitory effects on CPA‐induced contraction in PAs and SOCE in PASMCs were significantly augmented in CHPH rats, probably related to the enhanced SOCE caused by PH. These results demonstrate for the first time that TRPM8 activation can cause relaxation of PA through inhibition of SOCE. Since SOCE plays many important roles in pulmonary vascular functions and is crucial for the vascular remodeling and the enhanced vasoconstriction in PH, the downregulation of TRPM8 expression and activity in PASMCs during PH may minimized the TRPM8‐dependent inhibition of SOCE, and allow unimpeded SOCE activity for PH development.

Support or Funding Information

Supported by grants NSFC31571179, NSFC31371165, NSF of Fujian Province 2015J01313, and Fujian Province Hundred Experts Award.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impact of Student's Prior Experience on the Design of Virtual Labs for Biomedical Courses
Authors: Silva Ricardo Alfonso Pena , Rivas Jose Oscar Mantilla , Rodriguez Daniel Francisco Santamaria , Montero Silvia Marcela Gomez , Akle Veronica
Published: June 2021

Laboratory practices facilitate deep learning in biomedical sciences, however, they require experience in laboratory techniques, financial resources and institutional infrastructure. This is why the use of “in silico” or virtual laboratories is a viable alternative to teach fundamental biomedical concepts to students of medicine and science. Our objective is to evaluate the impact of virtual laboratories on medical students.

Students of the third and fourth semesters of medicine used simvesel and simheart (Virtual Phisiology) simulators. We evaluated the perceptions and outcomes associated with the simulation of experiments related to concepts of the autonomic nervous system and cardiac mechanics.

Students from earlier semesters arrive with less prior knowledge than advanced students. However, both groups improve their mastery of the subject in similar magnitude. Although the instructions and tasks were the same for the groups, the students of third semester seem to have more problems with the manipulation of the simulator and show a slightly less positive experience.

After a second experience with the simulator, advanced students do not show changes in their already positive experience with the simulator. Third semester students, however, report a much higher functionality and usability with the simulator and a more favorable impact on their learning.

In conclusion, the use of virtual laboratories is a cost‐effective intervention to teach complex concepts of physiology and pharmacology. Students consider that these types of laboratories allow them to achieve deeper learning even though they may need to gain mastery in some elements of the simulators. Teachers should consider the importance of the students' experience when developing biomedical simulation activities.

Support or Funding Information

Universidad de los Andes, Medical School and “Innovations in Education” Initiative of Conecta‐Te.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EMG Comparison between Sixth Grade Students
Authors: Rascon Jozelyn , Pena Francisco J. , Ramirez Juliana A. , Pena Marcelo , Trujillo Elizabeth , Melendez Gladys , Lazcano Cameron , Morales Acuna Francisco J. , Gurovich Benjamin , Valencia Carolina , Gurovich Alvaro N.
Published: June 2021

Physiology Understanding (PhUn) week is the K‐12 outreach program of the American Physiological Society to enhance STEM education. During a PhUn week activity, we seek to observed differences between boys and girls in the electromyography (EMG) of the biceps brachii. An EMG signal is the electrical manifestations of the neuromuscular activity, or action potential, in response to muscle contractions. The signals wavelengths are recorded as a mean voltage or peak‐to‐peak (PP) time. EMG could be affected by the anatomical and physiological characteristics of the muscle, the nervous system, as well as features of the instrumentation being utilized to analyze them. Seventeen middle school students (7 boys and 10 girls), between the ages of 12–14, were ask to voluntarily participate in PhUn week. A Biopac MP40 system was used to acquire the EMG data by examining motor recruitment and skeletal muscle fatigue. The EMG values were recorded on both dominant and non‐dominant arms while holding a 10lb weight at a 45‐degree elbow flexion, for a duration of 2 seconds. Thereafter the same procedure was performed with a 20lb weight and held until skeletal muscle fatigue was reached. The results showed that, overall, there was significant differences between dominant and non‐dominant arms only in P‐P at 10lb (1.4±2.1 vs. 0.9±1.2 ms, respectively, p<0.05). There were no differences between dominant and non‐dominant arms in any EMG signal in boys or girls. However, there was a significant difference between boys and girls when comparing all the P‐P times for 10 and 20lb in dominant and non‐dominant arms showing a higher muscle fiber recruitment in boys. The evidence suggest that the dominant arm tends to have a higher activation of muscle fibers, as opposed to the non‐dominant, and that more muscle fibers are recruited in boys than girls, when performing the same task.

Support or Funding Information

This work was partially supported by the National Institute of General Medicine Sciences of the National Institute of Health under linked Awards Numbers RL5GM118969, TL4GM118971, and UL1GM118970.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
INNOVATIONS IN HIGH SCHOOL PHYSIOLOGY: THE ARDUINO HEART RATE L.E.D MONITOR
Authors: Anjur Sowmya
Published: June 2021

Physiology and Disease (PAD) is a Biology elective at the Illinois Mathematics and Science Academy, Aurora, Illinois, which is a residential magnate school for students gifted in math and science. My PAD class is based upon student centered learning which has proven to be the best way to challenge students to take responsibility for their own learning. Students spend the most time on the cardiovascular unit, wherein they measure their heart rates, lung capacity and blood pressure before, during and after exercise, correlating their data to their understanding of neuronal controls. Students also build a working model of the mechanical pumping of the human heart. Although several such opportunities are provided in this unit for hands‐on experiences in the laboratory and in the classroom, the intrinsic conduction system of the heart has yet to be modeled. Students normally measure their heart rate using carotid pulse or radial pulse while exercising. Digital heart rate monitors are expensive and not very conducive to re‐use. Based on my previous experience with the mechanical heart models, requiring students to build models seems to correlate better with their improved understanding of the material. This prompted the creation of the arduino heart rate L.E.D. monitor project. Another factor that strengthened the necessity of an innovative lab that used their programming skills to model the electrical system of the heart, was the mandatory computer science requirement for high school graduation. For the arduino project, students are provided with Arduino UNO or compatible boards, Grove base shields, Grove ear clip heart rate sensors, Grove universal 4‐pin cables and LED and 330 Ohm resistors. They are provided with instructions on how to complete building their project, and are allowed to modify the instructions provided. At the end of this project, students are required to write a reflection on their work, correlating the working of the model to their understanding of the intrinsic conduction system of the heart. At this time, students in my classes are currently working on their heart rate models and I plan to collect their feedback for this project along with any suggestions for improvement or modification. Future research is planned to correlate student understanding to modeling activities in the classroom, based on their work and that of upcoming classes in the course. The main purpose of this assessment is for students to engage their creative skills and take responsibility for their own learning. The evaluation of this assessment includes appraisal of the quality, accuracy and creativity of their monitors and the accuracy of their reflective correlations with the electrical circuit of the human heart. It is hoped to continue building upon this non‐traditional method of modeling for other physiology concepts next semester, to further enhance student learning.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Who Donates the Body to Science? The Profile of the Donor in Southern Brazil
Authors: da Rocha Andrea Oxley , Maues João Lins , Macedo Mateus Belmonte , Thomaz Gabriela Dalla Giacomassa Rocha , Okabayashi Caio Seiti Mestre , Picanço João Marcelo Astolfi , de Moraes Maria Paula Oliveira
Published: June 2021

Objective

Human bodies are considered essential for teaching and research purposes involving the health sciences. To have a good understanding of the body structure and function, it is necessary throughout the graduation to grant opportunities for the study of anatomy directly from actual bodies. Therefore, the Body Donation Program at UFCSPA (BDP) was developed to fulfill the need of the university for material to the teaching of anatomy. However, it is still necessary to expand the understanding regarding body donation to the population, so that the project can be effectively implemented. To achieve this objective, this study aims to identify the demographic characteristics of the people registered in the program as an alternative to develop marketing strategies focused of the potential donors.

Methods

The study was approved by the Research Ethics Committee of UFCSPA (No. 721/2008). The data were collected using a specific form filled out by the donors or their family members at the time of registration. It included data from the time of the program's creation, in January 2008, to June 2016. All individuals who participated in the study completed and signed a consent form at the time of registration. The survey included 12 questions covering demographic, socioeconomic and health related aspects.

Results

Data from 414 donors were analyzed. It was verified that 60% of the donors were 60 or older, being the majority female (62.9%), single, divorced and widowed donors (70.9%), and 26% of the donors had at least one family member already registered in the database. Most of the registrants (89%) claimed to have religious beliefs. There was no prevalence of health science professionals or students of health sciences among the donors. It was found, however, that the generic profile of the donors is represented by educated individuals, with 93,8% of them having completed high school, and individuals defined as belonging to the middle class (40,4%). Most of the registrants heard about the BDP through the press.

Conclusion

Since the study had the objective to expand knowledges about the possibility of the body donation, the results can be useful to universities faced with the problem of acquiring bodies for academic purposes. It may be beneficial to consider implementing body donation programs, and those universities that already have an ongoing program should consider targeting their campaigns more precisely. Furthermore, a higher number of studies aiming the analysis of shared aspects among donors would be even more useful, since a better understanding of donor populations can be advantageous for recruitment campaigns.

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 Impact of Images on Multiple‐Choice Questions in Anatomy Examination Score in Nursing Students
Authors: Narnaware Raj
Published: June 2021

Anatomy examinations often have a key visual component with the inclusion of many images, and research shows that visualization can have a positive effect on learning (Vorstenbosch et al., 2013) while altering learner's cognitive load, memory and examination anxiety (Mayer RE, 2005). However, most of the studies on the effects of images on anatomy examinations are focused in medical, pre‐nursing, or pre‐health science students but have not yet been assessed in nursing students. To assess the effects of images on anatomy examination scores, quizzes and final examination containing multiple‐choice questions with greater or lesser anatomical images was assessed. Results show that inclusion of more anatomical images in a multiple‐choice anatomy quiz (quizzes #1 and 2, section BN01) significantly (P<0.01) increased their percent class average compared to a similar quiz with fewer images (Quiz #1 and 2, section BN02). In quiz#3, reducing the number of images by 50% in anatomy section BN01 resulted in a significant decrease (P<0.05) in their percent class average compared to an image‐rich quiz in section BN02. However, the final anatomy examination containing an equal number of images in both anatomy sections (BN01 & BN02) did not alter their percent class average. Interestingly, the percent score of individual questions from the examinations with images was significantly higher than the same questions without images. In conclusion, the results from this study indicate that image inclusion in anatomy examinations can have a positive impact on recall of anatomical knowledge and may provide a hint to an answer in anatomy examinations in nursing students.

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.
How Students Choose E‐learning Resources ‐ The Importance of Convenience and Familiarity
Authors: Hortsch Michael
Published: June 2021

Electronic learning technologies are popular with today's generation of students. However, how they choose their favored resources from the plethora of available e‐learning tools has not been investigated in much detail and therefore is not sufficiently understood. A popular electronic self‐assessment resource was offered in three different formats to students participating in two histology courses at the University of Michigan (an introductory histology summer course for first year dental students and a full‐term histology course for graduate and undergraduate students). The SecondLook™ Histology review tool was available to them in the form of PowerPoint files, as an interactive website, and as a mobile smartphone and computer tablet app. Identical in content, each format had its specific advantages and disadvantages, specifically the types of compatible devices and access limitations. After the conclusion of both courses, students were surveyed about their format choices and reasons for selecting their favorite format, as well as usage patterns. The overall survey participation rate for both courses was 91.8%. Only one out of 156 participating students reported not having used the SecondLook™ resource. The majority of students (over 54%) tried only one resource format, with PowerPoint being the most popular final choice (58%). Although the interactive website and mobile app offered several user‐friendly features not available with the PowerPoint files, they each only garnered around 20% final popularity. Ease of access and familiarity with the PowerPoint software were given most often as reasons for students' choice of resource format. The availability of and preference for specific devices (laptops, smartphones and computer tablets) and the flexibility of time and place to use the resource were also named as factors in the final format selection. The availability of mobile learning resources encouraged some students to use the tool in environments with many distractions which may interfere with learning (commuting, gym etc.). It also encouraged some students to forgo other learning opportunities, specifically textbooks, the course website, and other supplementary learning resources. In conclusion, my results suggest that today's students are less motivated to actively seek out novel learning resources and formats than commonly believed and instead often select familiar and convenient approaches and study tools.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Combination of a Variety of Technological Tools in the Anatomy Classroom.
Authors: Stashenko Vetaley , Walker Candice
Published: June 2021

We have developed an Anatomy and Physiology course with highly effective uses of the latest advanced technological tools, which are essential to student's engagement, success and development of critical thinking abilities and the expansion of the classroom outside the college Campus. The presentation will discuss strategies, which we have implemented for the process of the selection of the text book, course software and hardware resulting in the improvement of grades, retention, and completion and mastery of the subject matter. The combination of visual approaches (text and digital) with the normally anatomically correct projected cadaver and specimens allows students to interact with the subject study in a 3D environment anytime and anywhere with customized interactive assessments that maintain the continuity of learning between lectures and the laboratory. Additionally this allows students to integrate basic scientific knowledge with clinical (personal) applications. Student's experiences and survey results will also be shared. Steps of our implementation process will also be discussed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Augmented Reality Glasses in Central Venous Line Placement Simulations
Authors: Thomas Jonathan , Huang Cynthia Y. , Alismail Abdullah , Cohen Avi , Almutairi Waleed , Daher Noha , Terry Michael , Tan Laren
Published: June 2021

Central venous lines are used to administer medicines, nutrients, and blood products to the heart via the large internal jugular vein. This technology is used when medications cannot be administered orally or injected through smaller veins. The procedure involves a high level of precision and there is little room for error. The goal of this research is to investigate the feasibility of using augmented reality (AR) glasses in central line simulation by novice operators and comparing its efficacy to standard central line simulation/teaching. With the Brother AiRScouter augmented reality glasses, the learner controls the timing of each step and receives real‐time information from the AR glasses. A grader documented the time to AR glasses placement, time to venipuncture, number of attempts, and completion of a pre‐determined checklist. This study used a prospective randomized enrollment of 32 novice operators. Subjects were randomized on a 1:1 basis to either a simulation using the augmented reality glasses or a simulation using only conventional instruction. There was a total of 32 subjects participating in this study. Mean age of participants was 39.8±7.8 years; 5 Females and 27 Males; 11 Physicians, 19 Respiratory Therapists, and 2 Sleep Technologists. The mean time for AR placement in the AR group was 71 ± 43 secs and time to IJ cannulation was 316 ± 112 sec. There was no significant difference in median (min, max) time (seconds) to IJ cannulation for those who were in the AR group and those who were not (339 (130, 550) vs 287 (35, 475), p=0.09), respectively. There was also no significant median difference between the two groups in total procedure time (524 (329, 792 vs 469 (198, 781), p=0.29), respectively. Our results are consistent with the idea that AR‐simulation of central venous catheters is feasible and efficacious for novice operators. AR technology is likely to be generally applicable to both procedural and fundamental medical education.

Support or Funding Information

Brother Augmented Reality glasses were provided by Brother‐USA. Tom & Vi Zapara School of Business at La Sierra University supported this study.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immersive Technologies to Augment Anatomy Education
Authors: Malone Erica R. , Pine Michelle , Seo Jinsil Hwaryoung , Smith Brian , Brunner Michael , Cook Margaret , Leal Steven , Bai Zhikun
Published: June 2021

Correctly defining the locations and positional relationships between anatomical structures requires learners to complete complex spatial mental transformations. Learners must ultimately be able to visualize structures and their relationships in order to apply anatomical concepts in practice. However, no group of students is homogeneous. Because students have varying levels of innate spatial ability, it is necessary to develop learning tools that provide scaffolding for individuals across this spectrum. To that end our multidisciplinary team is utilizing virtual reality technologies to address this need. Anatomy Builder VR is designed to encourage learning anatomy through exploration of 3Dimensional virtual skeletons. Learners start in a hub room and then choose to build either a human or canine skeleton. Novice learners start with introductory, guided lessons that demonstrate the following: 1) the use of directional nomenclature, 2) correct anatomical orientations, 3) basic movements of the joints of the thoracic limb, and 4) the proper names of each bone. Each lesson is designed with targeted learning outcomes and allows for exploration, practice of concepts, and assessment with immediate feedback. The application also has a sandbox component where learners can assemble skeletons with minimal guidance, and a game room that allows users to test their knowledge of osteology in a low stakes and fun environment. Our goal is to harness the unique features of immersive technology to help learners create their own accurate mental representations of the features and relationships of structures within the body. Overall feedback during interaction has been positive and the next phase of the study is to recruit undergraduate students and begin assessing the impact of using Anatomy Builder VR on visual‐spatial understanding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Real‐time Ultrasound Imaging to Enhance Students' Learning of Anatomical Knowledge in Physical Therapy Education
Authors: Day Leslie , Markowski Alycia , Watkins Maureen
Published: June 2021

In the Doctor of Physical Therapy program, students complete an anatomy course where they are expected to learn proper identification of anatomical structures and develop didactic manual skills to properly palpate those structures. For many students, the connection between what they view in the cadaveric laboratory and surface anatomy can be challenging. The purpose of this study was to use real‐time ultrasound imaging (RTUI) technology to enable students to visualize real‐time anatomy for the accuracy of their palpation skills. Subjects consisted of two consecutive years of students in the first semester of a Doctorate of Physical Therapy program taking gross anatomy (n=91; n= 102). The fifteen‐week gross anatomy course consisted of a lecture, prosected cadaver and a separate surface anatomy laboratories. Students were allocated into a control or experimental group based on laboratory sections. The standard surface anatomy laboratory education (control) included visual demonstration of palpation technique for the collateral ligaments of the knee and ankle, followed by verbal feedback from instructors while students were practicing. The experimental group also received visual feedback utilizing a GE Healthcare LOQIQ‐S7 Ultrasound Imaging Machine during identification of knee ligaments, but not during the identification of ankle ligaments. Four weeks following the intervention, all students' palpation performances of the knee and ankle ligaments were assessed for time and accuracy. In addition, students completed a six‐question survey assessing their perceived confidence in their palpation skills. Comparison of traditional and technology enhanced learning both within subject (knee v. ankle ligaments) and between subjects (control v. experimental) was analyzed for effectiveness on student's skill acquisition and confidence. The use of RTUI during laboratory teaching improved students' learning and accuracy in palpation of knee ligaments. The implementation of an emerging technology (RTUI) with traditional laboratory techniques may assist with student's transference of anatomical knowledge in cadaveric specimens to surface anatomy, which may have a long‐term impact on clinical skills.

Support or Funding Information

The development of this technology assisted instructional program is funded by Northeastern University Provost Instructional Development 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.
Surface Projection of Cubital Tunnel: Cadaver Study
Authors: Khan Mustafa A. , Khan Tasrif Alam , Khan Mahwish Iqbal , Kanwar Kiran , Liu Howe , Lovely Rehana Sultana
Published: June 2021

Cubital tunnel syndrome (CTS) involves the compression or stretching of the ulnar nerve within the cubital tunnel. Two possible sites of compression are the sulci nervi ulnaris and the tendinous arch of the flexor carpi ulnaris (FCU) muscle where the two heads (ulnar and humeral) join. Accurate identification of these locations is important for developing differential diagnoses and providing appropriate intervention for CTS. While the sulci nervi ulnaris can be palpated at the posterior surface of the medial epicondyle, locating the tendinous arch of the FCU can be difficult. This study was conducted to help clinicians identify the location of the FCU tendinous arch by using an average thumb width. Twenty cadavers (10 males and 10 females, ages 56–97 years) were dissected for analysis. The midpoint of a straight line between the olecranon (OE) and medial epicondyle (ME) was identified and the perpendicular distance from the OE‐ME line midpoint to the FCU tendinous arch was measured. These measurements were then converted to thumb interphalangeal joint widths. A mean thumb width of 17.94 ± 3.9 mm was used from a previous study. The distance between the midpoint of the OL‐ME line to the tendinous arch was found to be 1.14 thumb widths or 20.50 ± 1.3 mm. Based on this information, clinicians may be able to approximate where the two heads of the FCU join by palpating one thumb width distally from the OL‐ME line midpoint.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Error‐Prone DNA Polymerase IV preserves the memory of its interaction with RecA
Authors: Tashjian Tommy F. , Lin Ida , Nguyen Brian H. , Godoy Veronica G.
Published: June 2021

DNA Polymerase IV (DinB) is a highly conserved, error‐prone DNA polymerase (DNAP) expressed as part of Escherichia coli's DNA damage response. This polymerase is well known for its ability to perform translesion synthesis, or to bypass DNA damage on the template strand that would otherwise cause lethal replication fork stalling. As a consequence of this ability to accommodate DNA lesions, DinB is highly mutagenic compared to high fidelity DNAPs (100‐fold less accurate than replicative DNAP III). Despite its potential for mutagenesis, this enzyme is present at high concentrations (~250 nM) even in the absence of DNA damage; approximately 8‐fold higher than the replicative DNAP III. Upon induction of the DNA damage response, DinB is the most abundant DNAP in the cell (~2500 nM). DinB mutagenic potential and high intracellular concentration suggests that its activity must be strictly regulated. Indeed, it has been shown that DinB's activity is regulated by protein‐protein interactions.

In E. coli DinB is known to interact with the cells' highly conserved main recombinase, RecA. It has been shown that this interaction increases DinB's fidelity on damaged templates in vitro. In addition, our previous work identified a variant of DinB, DinB(C66A), which contains a single amino acid substitution in the DinB family highly conserved C66 residue. We previously showed that DinB(C66A) has altered binding to RecA and does not benefit from an increase in fidelity upon interaction with RecA.

We find that DinB undergoes a detectable conformational change upon interaction with RecA in vitro. Our evidence also suggests that the DinB interaction with RecA is short lived. Remarkably the conformational change in DinB, which is effected by RecA, persists after RecA protein is removed by chromatography. In addition, DinB becomes more susceptible to proteolysis. We also find that the variant DinB(C66A), which does not show increased fidelity upon RecA‐interaction, either does not undergo the conformational change, or it is unable to stably maintain said conformational change. These data suggest that DinB recalls previous interaction with RecA, which in turn affect the fidelity of DinB beyond the time of interaction with RecA. The interplay between DinB and RecA represents a clever mechanism by which RecA is recycled and available for its many other cellular functions while at the same time DinB fidelity is elevated preventing high mutagenic burden despite DinB's high intracellular concentrations.

Support or Funding Information

Research funded by National Institute of General Medical Sciences (RO1GM088230 to VG) and Northeastern University Office of the Provost (Graduate Dissertation Research Grant to TT).

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 of Eukaryotic CMG Helicase at a Replication Fork and Implications for Replisome Architecture and Origin Initiation
Authors: Yuan Zuanning , Georgescu Roxana , Lin Bai , de Luna Almeida Santos Ruda , Zhang Dan , Yurieva Olga , O'Donnell Michael , Li Huilin
Published: June 2021

Helicases are a class of motor proteins that move directionally along a nucleic acid phosphodiester backbone and separate two annealed nucleic acid strands by using ATP hydrolysis. Helicases exist in all living organisms. Based on their shared sequence motifs, they are classified into 6 classes in which superfamilies 1 and 2 are not ring‐shaped structure and superfamilies 3–6 are ring‐forming helicases. In eukaryotes, DNA helicase CMG consists of the Mcm2–7 hexameric ring along with five accessory factors. The Mcm2–7 heterohexamer, like all hexameric helicases, is shaped like a ring with two tiers referred to as an NTD tier (N‐terminal domain) and a CTD tier (C‐terminal domain); the CTD tier contains the ATP motors. In principle, either tier could translocate ahead of the other during movement on DNA. By using single‐particle cryo‐EM of an active CMG on the forked DNA with a dual biotin‐streptavidin block, we solved the first structure of helicase in complex with a DNA fork. The DNA duplex stem penetrates into the central channel of the NTD tier and the unwound leading ssDNA traverses the channel through the NTD tier into the CTD motor tier, 5′‐3′ through CMG. Therefore, during translocation the CTD motor is behind the NTD ring that is pushed ahead to face the fork, opposite the currently accepted polarity. The polarity of the NTD ahead of the CTD places the leading Polɛ below CMG and Polα‐primase at the top of CMG, above the unwinding point of the replication fork. The DNA direction requires that the Mcm rings must first pass one another when the Mcm2–7 double hexamer come apart. Interestingly, the new NTD‐to‐CTD polarity of translocation reveals an unforeseen quality control mechanism that each Mcm2–7 is fully converted from encircling dsDNA to ssDNA before they can depart from the origin. We propose this architecture of two motors head to head may generate energy that underlies initial melting at the origin.

Support or Funding Information

This work was funded by NIH Grants GM111472 (to H.L.) and GM115809 (to M.E.O.); the Van Andel Research Institute (H.L); and the Howard Hughes Medical Institute (M.E.O.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FOXC1 is Over‐expressed and is More Stable in Triple Negative/Basal‐Like Breast Cancer
Authors: Elian Fahed A. , McMullen Todd PW. , Brindley David N. , Walter Michael A.
Published: June 2021

Rapidly accumulating evidence implicates forkhead box C1 (FOXC1) in Triple Negative Breast Cancer (TNBC), particularly in Basal‐Like Breast cancer (BLBC). Recently additional studies have demonstrating that FOXC1 is also a major player in hepatocellular carcinoma (HCC), endometrial cancer, Hodgkin's lymphoma (HL), non‐Hodgkin's lymphoma (NHL), and others. The FOXC1 gene encodes a transcription factor that is crucial to mesodermal, neural crest, and ocular development. Loss of function mutations in FOXC1 have been shown to cause autosomal dominantly inherited Axenfeld‐Rieger's Syndrome (ARS), a developmental disorder associated in eye anomalies and glaucoma. Interestingly, while FOXC1 missense mutations that cause ARS reduce FOXC1 activity, increased FOXC1 function now appears to be often linked to more aggressive cancer phenotypes in TN/BL‐BC, HCC, HL, and NHL.

We have investigated the mechanism(s) by which FOXC1 activity is increased in BLBC. Samples were obtained from TNBC tumors, Triple Positive Breast Cancer (TPBC) tumors or from breast tissue from normal patients. Using quantitative PCR, we found that FOXC1 was significantly over‐expressed in TNBC patients as compared to controls. In contrast, FOXC1 mRNA was significantly less expressed in TPBC samples as compared to either control or TNBC samples. FOXC1 protein half‐life was significantly longer in the TNBC/BLBC cell lines (HS 587T and BT 549) compared to FOXC1 protein's half‐life in HeLa cells. Studies of FOXC1 Copy‐number variation (CNV) in TNBC/BLBC cell lines reveals that cell lines that have higher levels of FOXC1 protein (HS 587T, BT‐549) have extra copies of FOXC1. This contrasts with a cell line with lower expression of FOXC1 protein, MDA‐MB‐231, which may be explained by a deletion of FOXC1 in this TNBC cell line. Our results suggest that increased FOXC1 function in TNBC/BLBC results from over‐expression of FOXC1 in tumors of TNBC patients that appears to be the result of changes to FOXC1 stability and amplification of FOXC1 copy number. We predict that understanding the mechanism(s) underlying FOXC1's activation in cancer could aid in designing improved therapies for TNBC patients.

Support or Funding Information

Verag Donation to MAW

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Receptor‐Mediated Delivery of CRISPR‐Cas9 Endonuclease for Cell Type Specific Gene Editing
Authors: Roy Marc , Rouet Romain , Thuma Benjamin , Lintner Nathanael , Rubitski David , Finley James , Wisniewska Hanna , Mendonsa Rima , de Oñate Lorena , Hirsh Ariana , Barrón Joan Compte , McLellan Thomas , Bellenger Justin , Feng Xidong , Varghese Alison , Chrunyk Boris , Borzilleri Kris , Hesp Kevin , Zhou Kaihong , Ma Nannan , Tu Meihua , Wilson Ross , Dullea Robert , McClure Kim , Liras Spiros , Mascitti Vincent , Doudna Jennifer
Published: June 2021

CRISPR‐Cas RNA‐guided endonucleases hold great promise for disrupting or correcting genomic sequences through site‐specific DNA cleavage and repair. However, the lack of methods for cell‐ and tissue‐selective delivery currently limits both research and clinical uses of these enzymes. Here we report the design and in vitro evaluation of S. pyogenes Cas9 proteins harboring asialoglycoprotein receptor ligands (ASGPrL). In particular, we demonstrate that the resulting ribonucleoproteins (Cas9‐ASGPrL RNP) can be engineered to be preferentially internalized into cells expressing the corresponding receptor on their surface. Live cell imaging studies are consistent with a receptor‐mediated endocytosis mechanism of active uptake of the RNP. When uptake occurred in the presence of a peptide with endosomolytic properties, we observed receptor‐facilitated and cell‐type specific gene editing that did not rely on electroporation or the use of transfection reagents. This approach provides a framework for applications to selective gene editing in the liver and offers a potentially general mechanism for cell‐, tissue‐, or organ‐targeted gene editing by receptor‐mediated delivery of genome‐editing enzymes.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
JNK‐mediated eIF4E phosphorylation and signaling in fibrotic functions of lung‐resident mesenchymal cells (MCs)
Authors: Walker Natalie Maureen , Lama Vibha N.
Published: June 2021

INTRO

We have recently demonstrated that cap‐dependent translation regulated through 4E‐BP1/eIF4E signaling plays a critical role in regulating fibrotic functions of activated mesenchymal cells, specifically collagen I expression. Fibrotic MC signaling to 4EBP1 is resistant to rapamycin, and resistance to TOR inhibitors has also been described in cancer. This underscores the need for better therapeutic options targeting aberrant translation, and further understanding of upstream signaling mechanisms regulating translational control will be key. Here we investigate mechanisms driving cap‐dependent translation upstream of eIF4E Serine 209 phosphorylation in lung MCs.

Materials/methods

Lung‐resident mesenchymal cells were isolated from Bronchoalveolar lavage fluid of lung transplant recipients with and without fibrosis by plastic adherence. MCs were treated for 2 hours with 10uM SP600125 (JNK inhibitor), U0126 (MEK inhibitor), SB202190 (p38inhibitor), or the MNK kinase inhibitors CGP57380 and eFT‐508. Constitutively active JNK1 (MKK7‐JNK1 fusion) was shuttled into plentiLOX‐IRES‐puro and packaged into lenti‐viral particles. Non‐fibrotic MCs were infected for 48 hours, lysed and protein expression was analyzed by western blot.

RESULTS

Fibrotic MCs isolated from patients with Bronchiolitis obliterans had significantly higher cap‐dependent translation compared to non‐fibrotic MCs when measured utilizing luciferase reporter assay (n=7; p<0.01). Fibrotic MCs had significantly higher phosphorylation of eIF4E at serine 209 compared to non‐fibrotic MCs when baseline expression was measured by western blotting (n=8; p<0.01), suggesting elevated translational activity. MCs treated with rapamycin (250 nM) for 2 hours demonstrated increased phosphorylation of eIF4E (S209), which was sensitive to MNK kinase inhibition. Treatment of fibrotic MCs with JNK MAPkinase inhibitor SP600125 or the MNK inhibitors eFT‐508 and CGP57380 led to a significant decrease in collagen I expression and eIF4E (S209) phosphorylation, while MEK and p38 inhibitors did not. Expression of constitutively active JNK led to a significant increase in phosphorylated eIF4E (S209) in non‐fibrotic MCs, implicating JNK activation as key to causing eIF4E phosphorylation and potentially increased cap‐dependent translation.

Conclusion

These data demonstrate that cap‐dependent translation downstream of 4E‐BP1/eIF4E is elevated in fibrotic MCs and that eIF4E phosphorylation at serine 209 is sensitive to JNK/MNK inhibition. This suggests that the JNK/MNK signaling axis upstream of 4E‐BP1/eIF4E and hence translation is key to fibrotic functions of MCs during pathogenesis of fibrotic lung diseases, and a viable target for treating fibrosis.

Support or Funding Information

This abstract is funded by NIH grants: RO1F035722 and RO1F037975.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RpoS recovery from phosphate starvation
Authors: Hamdallah Issam Nihad , Majdalani Nadim , Tripathi Arti , Gottesman Susan
Published: June 2021

Acclimation of E. coli to environmental stress is dependent on changes in gene expression. RpoS is a sigma factor that helps the cell deal with a range of stresses. When cells are not under stress, RpoS is rapidly degraded; however, during stationary phase or under other stresses during growth, RpoS accumulates due to increased translation or decreased proteolysis. Under phosphate starvation, RpoS degradation is inhibited by a small antiadaptor protein, IraP. IraP achieves RpoS stabilization by blocking adaptor protein RssB from delivering RpoS to the ClpXP protease for degradation. While the pathway for RpoS stabilization under phosphate starvation is well defined, recovery from stress is less well understood. Western blot analysis indicates that when phosphate is introduced into phosphate‐deprived cultures, RpoS levels sharply decrease to barely detectable levels within five minutes of exposure, which strongly suggests that rapid degradation is restored when phosphate is restored to cultures. We are examining the mechanism behind this recovery process by monitoring the fate of IraP and control of the adaptor protein RssB in the presence of phosphate. We hope this study will help us better understand how the interplay of stress induction and recovery govern adaptations under nutrient starvation. The lessons learned from the recovery of IraP may also provide insight into the mechanism of recovery of the two other known antiadaptors, IraD and IraM.

Support or Funding Information

NIH Intramural Funding

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 a Protein‐Protein Interaction Between NIL‐16 and HDAC3
Authors: Dailey Leandrew A. , Baugh Keith Daniel , Fenster Steven Daniel
Published: June 2021

Neuronal Interleukin‐16 (NIL‐16) is the neuronal‐specific splice isoform of the interleukin‐16 (IL16) gene. NIL‐16 shows restricted expression to the hippocampus and cerebellum: brain regions involved in learning and memory. NIL‐16 shares an identical C‐terminal protein‐coding sequence with pro‐IL‐16; the shorter, immune‐specific splice isoform that is the pro‐inflammatory precursor to the cytokine IL‐16. The mutual amino acid sequence between pro‐IL‐16 and NIL‐16 contains three PDZ protein interaction domains (PDZ‐1, PDZ‐2 and PDZ‐2). In addition to serving as the precursor to IL‐16, pro‐IL‐16 regulates immune cell proliferation through a PDZ‐2 mediated‐interaction with the nuclear‐associated protein HDAC3. Emerging evidence indicates that HDAC3 plays a critical role in regulating fundamental mechanisms involved in brain development and mechanisms of learning and memory. Here, we hypothesize that like pro‐IL‐16, a similar protein‐protein interaction exists between NIL‐16 and HDAC3 suggesting a possible role in mediating important processes in neurons. To test this hypothesis, we conducted pulldown assays with mouse cerebellar brain extract using fusion proteins coding for the PDZ‐2 domain of NIL‐16 and the PDZ‐interacting motif of HDAC3. Our results indicate that we can affinity purify HDAC3 from cerebellar extract with the PDZ‐2 domain of NIL‐16. Likewise, we also affinity purified NIL‐16 with the PDZ‐interacting motif of HDAC3. Additionally, using immunofluorescent microscopy, we show overlapping distribution of NIL‐16 and HDAC3 expressed in COS‐7 cells and in cultured cerebellar granule neurons. Finally, we performed co‐immunoprecipitation assays with cerebellar extract demonstrating preliminary evidence that NIL‐16 and HDAC3 form an in vivo protein complex in neurons.

Support or Funding Information

Research in this study was funded by NIH‐Maximizing Access to Research Careers Undergraduate Student Training in Academic Research (MARC U*STAR) award number T34GM092711, start‐up funds provided to Dr. Steven Fenster (Fort Lewis College), and funds received from SSNAP (Science Scholars the Native American Path) by SACNAS (Dr. Les Sommerville, Fort Lewis College).

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