bluuebunny/crossref_metadata_2025 huggingface dataset:
Invitro insulinotropic actions of various extracts of Calosia argentea leaves
Authors: Ogunmodede Oluwaseyi Samson , Oseni Saheed Oluwasina , Adenmosun Olumide Olaseni , Mbagwu Smart Ikechukwu
Published: June 2021

This study investigated the effects of Calosia argentea obtained by different extraction methods on the insulin‐release and cytotoxicity on clonal pancreatic beta cells. Pulverized Calosia argentea leaves were sequentially extracted with dichloromethane, acetone, ethylacetate and water. Clonal pancreatic beta cells, BRIN‐BD11, were grown in an atmosphere of 5% CO2 and 95% air in RPMI‐1640. Insulin and LDH assays were carried out using Krebs Ringers Bicarbonate buffer. Insulin concentration was measured by radioimmunoassay and LDH release was assessed using a commercially available non‐radioactive assay kit (Promega, UK). Aqueous extraction of Calosia argentea produced the best yield (361 mg/20kg powder). Acute incubation of BRIN‐BD11 cells with different extracts of Calosia argentea produced a concentration‐dependent stimulation of insulin‐release at concentrations ≥10 μg/ml. The aqueous extract of Calosia argentea produced the best insulinotropic effect, with a maximum stimulation of 347% (P<0.001) of basal rate at 100μg/ml and the lowest stimulating concentration of 0.1 μg/ml. Dichloromethane and acetone extracts produced similar stimulatory effects (130% of basal rate, 100μg/ml, P<0.01). Insulin‐releasing effects of the four extract were not associated with beta cell cytotoxicity. The release of the cytosolic enzyme, lactate dehydrogenase (LDH), due to damaged plasma membrane was used as a marker of cytotoxicity in this study. Significant (P<0.001) release of the enzyme was observed in incubations involving DMSO (40% v/v). However, incubation with the different extracts of Calosia argentea produced no significant release of LDH. These results showed that extracts of Calosia argentea possess insulinotropic effects and encourage further studies to uncover the physiological mechanism involved.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
H2S Improves the Rate of Migration in Trophoblast Cells
Authors: GUPTA SUNIL KUMAR , MOCHAN SANKAT , SAXENA SHOBHIT , ARORA PALLAVI , RANI NEERJA , SK PALLAVI , SHARMA ARUNDAHTI , LUTHRA KALPANA , DWIVEDI SADANAND , BHATLA NEERJA , DHINGRA RENU
Published: June 2021

Introduction

The migration of trophoblast cells during the first trimester of pregnancy is crucial to placentation process. Defective migration of trophoblastic cells may affect the remodelling (pseduovasculogenesis) of spiral arteries resulting in Hypoxia/Reoxygenation (H/R) episodes. These repeated H/R events can leads to oxidative stress and release of anti‐angiogenic, anti‐inflammatory and pro‐oxidant factors in maternal circulation causing endothelial dysfunction resulting in adverse pregnancy outcomes like preeclampsia and fetal growth restriction. The anti‐inflammatory, pro‐angiogenic and pro‐migratory effect of H2S on endothelial cells have been documented in literature. So we hypothesized that H2S or its mimics (NaHS) may also affect the migration capacity of trophoblast cells during placentation.

Methods

The immortalized first trimester extravillous trophoblast cell line (HTR‐8/SVneo) were given various treatments like 50μM NaHS, Hypoxia/Reoxygenation [H/R (one and two cycles)] and Hypoxia/Reoxygenation supplemented with 50μM NaHS. The cell migration was assessed using wound scratch assay following these various exposures. The percentage wound area covered was assessed in all groups. The difference in wound covered area was statistically analysed. [ten different zone in triplicate (i.e. thirty) were used for calculating the wound areas]

Results

The percentage of wound covered area (rate of migration) increased in cells which received 50μM NaHS [control (32.8%), NaHS (67.5%)] whereas it reduced to 8.7% in Hypoxia/Reoxygenation one cycle exposed cells and more so (−18.9%) in the cells which received two cycles of Hypoxia/Reoxygenation. However the cells which received Hypoxia/Reoxygenation (one cycle and two cycles) and showed reduced migration, improved their rate of migration when supplemented with 50μM NaHS. [one cycle H/R (8.7%), one cycle H/R+NaHS (39.8) and two cycles H/R (−18.9%), two cycles H/R+NaHS (29.5%)]. The difference in wound covered area among various groups was statistically significant [(control vs NaHS; p<0.001, control vs one cycle H/R; p<0.001, control vs two cycle H/R; p<0.001, one cycle H/R vs one cycle H/R+NaHS; p <0.001, two cycle H/R vs two cycles H/R+NaHS; p<0.001 (one‐way ANOVA with Bonferroni correction)].

Conclusion

These results imply that H2S may promote the migration of trophoblastic cells, which might then lead to adequate conversion of spiral arteries into wider diameter low resistance vessels having good placental perfusion. In future the role of H2S on cellular and molecular factors responsible for cell migration needs to explored.

IESC/T‐467/23.12.2014

Support or Funding Information

F.8‐397/A‐397/2015/Rs

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oral Administration of Olive Oil may Exacerbate Imiquimod‐Induced Skin Inflammation in Mice
Authors: ROMANA‐SOUZA BRUNA , DONATO‐TRANCOSO ALINE , MONTE‐ALTO‐COSTA ANDRÉA
Published: June 2021

Psoriasis is a chronic inflammatory disease characterized by thick and erythematosus cutaneous plates, which leads to patient disfiguration and incapacitation and does not have cure. Oral administration of functional foods with antioxidant and anti‐inflammatory proprieties, as olive oil, could have beneficial effects in development of psoriasis. Thus, our hypothesis is that olive oil may attenuate inflammatory response and oxidative damage induced by psoriasis decreasing the development of psoriasiform plates. This study investigated if oral administration of olive oil could attenuate imiquimod‐induced skin inflammation in mice. Experiments with animals were approved by the Ethical Committee for Animal Use of State University of Rio de Janeiro (CEUA/045‐046/2016). To induce the psoriasis, dorsum skin of adult male BALB/c mice was topically treated with cream containing 5% of imiquimod for 6 days. The dorsum skin of control animals was treated with vaseline. The imiquimod‐induced skin inflammation is macroscopically, microscopically and molecularly to resemble psoriasiform plates in human. Concomitant to imiquimod or vaseline administration, some imiquimod or control‐treated animals were orally treated with 1.5g/kg of olive oil or water. The formation of erythema, scales and thickness in psoriasiform plates was macroscopically evaluated during all experiment. After 7 days of the beginning of imiquimod administration, animals' dorsum was collected and formalin‐fixed, and sections were stained with hematoxylin‐eosin to measure the thickness of epidermis. Olive oil did not alter macroscopically the formation of scattered red and scaly plaques induced by imiquimod in skin dorsum of mice compared to control (vaseline+water) after 7 days. However, olive oil potentiated the increase in the epidermis thickness induced by imiquimod when compared to control (vaseline+water) after 7 days. In conclusion, oral administration of olive oil may exacerbate the development of imiquimod‐induced skin inflammation in mice.

Support or Funding Information

CNPq (402582/2016‐0) and FAPERJ (E‐26/203.192/2017)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intermittent systemic hypoxia – A possible way to improved regeneration
Authors: Larsen Mikkel Christian , Elmengaard Brian , Pedersen Michael , Foldager Casper Bindzus
Published: June 2021

Background

Hypoxia affects the transcription of genes which controls and regulates cytokines with secondary promotion of angiogenesis and cell proliferation; a prerequisite tissue regeneration. We hypothesize that a prolonged exposure to intermittent systemic hypoxia (ISH) can initiate a systemic response, including release of proangiogenic factors, that will accelerate and improve bone‐tendon regeneration. In this study, the hypothesis is challenged in a rotator cuff rat model. This model was chosen because the rotator cuff surgeries pose orthopedic issues with a high frequency of undesirable outcome, making it relevant for investigation.

Materials and methods

Forty‐two 17‐week old male Sprague Dawley rats were randomly divided into an ISH group and a control group. All rats received unilateral supraspinatus surgery, which is a well‐established animal model. The ISH group was exposed to air containing 13.5% oxygen for one hour every morning and every night (responding to ISH) for 2 weeks. The controls were not exposed to hypoxia (21% oxygen). The rats were euthanized and evaluated though blood samples and histology after 2 and 4 weeks.

Results

Preliminary histology data indicates an improved regeneration in the ISH group.

After the two‐weeks of ISH exposure the test group shows significantly increased EPO levels compared to the controls (p=0.007). No significant differences were observed at 4 weeks postoperatively. TNF‐alfa levels were highest in the ISH group after 2 weeks (p= 0.026) but lowest after 4 weeks (p=0.028).

Cytokine array data showed multiple other significant difference at both time‐points. No difference in weight development was observed after 2 and 4 weeks.

Conclusion

At present, no final conclusion regarding primary outcome (histology: Tendon integrity) can be made. Preliminary data indicates an improved regeneration in the ISH group. ISH creates a systemic response altering the expression of EPO, TNF‐alfa and multiple other cytokines. Data from histology describing tendon integrity are in progress and are expected to reveal whether the proven systemic response is resulting in an improved regeneration as hypothesized.

Support or Funding Information

The study was funded by an AUFF NOVA grant. There are no financial interestsin the project.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
3D Analysis of Primate Neck Anatomy using Contrast‐Enhanced CT Imaging, Fascicle‐Tracking Algorithms, and Muscle Mechanics
Authors: McGechie Faye , Sullivan Samuel , Middleton Kevin M , Holliday Casey M , Nalley Thierra K , Grider‐Potter Neysa , Ward Carol V
Published: June 2021

The functional anatomy of the neck is a major component of an animal's postural and locomotor repertoire, yet remains poorly understood in part due to the anatomical complexity of the cervical musculoskeletal system. Here we use a new methodology to visualize and quantify aspects of muscle architecture, as well as to explore 3D muscle mechanics of the head and neck of a Philippine tarsier (Carlito syrichta). First, the specimen was contrast stained and microCT‐scanned (0.043mm3 voxels). From these data, individual neck muscles were segmented using Avizo software. We tracked individual muscle fascicles based on grayscale values and fascicle diameter in Avizo Xfiber. Outputs from Xfiber (fascicle length, pennation angle, and muscle volume) were used to calculate estimates of physiological cross‐sectional area (PCSA) in the intrinsic epaxial neck muscles. We combined PCSA values with resultant muscle force vectors to estimate muscle moments about joints for flexion, extension, lateral flexion and rotation about each cervical intervertebral joint. Results demonstrate that the moment arms for muscles used in head and neck rotation like obliquus capitis inferior are larger in the tarsier relative to body size when compared to humans. This implies that an increase in rotational movements in this species is accomplished by modifying lines of action instead of increasing muscle force. This workflow provides a more comprehensive characterization of musculoskeletal functional capacity in a complex system. Not only does it provide novel insight into the neck of this unusual primate, but provides the basis to compare primates with different positional behaviors.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Superficial Neck Vasculature of the American Black Bear and its Implications for Intramuscular Tranquilization
Authors: Reed James A , Kilham Benjamin , McNutt Ellison J , Crosswait Alexandra G. , Voegele Georgina M.
Published: June 2021

Many ursid management protocols list the neck region as a target site for intramuscular (IM) delivery of tranquilizer; due to the large muscle mass and relatively thin subcutaneous fat layer. However, the superficial neck vasculature in American black bears has not been well studied. We find that the vasculature resembles that of the giant panda (Davis, 1964). The occurrence of several, potentially tranquilizer dosage related, fatalities in local bears prompted a further exploration of the region. Three bears were dissected with the permission of the New Hampshire Department of Fish and Game: one adult male, one adult female, and one juvenile female. Dissection revealed a large external jugular vein on the anterior surface of the neck, running superficial to the musculature; in the middle of the prescribed tranquilization site. This is contrary to available information of the region, cautioning to avoid only the inferior portion of the neck to avoid the carotid sheath and subclavian vessels. The presence of this vessel creates inherent danger in using this area as a site for the delivery of IM tranquilization, as the dosage would rapidly lower blood pressure and respiratory rate if accidentally delivered intravenously, and should be avoided if possible. This study identified safe boundaries for IM tranquilization to update animal management practices in New England.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New Imaging Approaches Enable Visualization of 3D Musculoskeletal Anatomy of African White‐bellied Pangolin
Authors: Verhulst Conner , Aitken‐Palmer Copper , Holliday Casey
Published: June 2021

Pangolins are critically endangered mammals whose anatomy and life history present numerous challenges to veterinary and conservation efforts. Among their unique features is their elongated tongue and associated skeletal tissues which extend from the mediastinum into the right flank of the abdominal cavity. Although some basic radiological and internal anatomy of the pangolin hyolingual apparatus is known, new details on the tongue, throat and head are needed to better guide surgical approaches and new imaging data in general are needed to better understand the morphology of these species and their functional and evolutionary biology. Using a combination of diffusible iodine contrast‐enhanced computed tomography (diceCT), 3D modeling, and 3D muscle tractography approaches we investigated the muscle architecture of the hyolingual apparatus of one specimen of white‐bellied pangolins (Phataginis tricuspis). We show that the tongues of pangolins are composed of bony xiphisternal rods that support a large tongue enshrouded by helically‐oriented muscle fibers and stiffened by a dense, partially‐calcified lyssa. The orientation of these muscles suggest they propel the tongue while curling the xiphisternal elements rostrally followed by retraction through hypothesized elastic mechanisms. In addition, we provide a 3D model of pangolin skeletal anatomy and other anatomical resources. Using 3D muscle tractography and other imaging and computational tools, we can better understand the muscle architecture and morphology that contributes to these animals' feeding functions, behavior and our understanding of their biology.

Support or Funding Information

NSF IOS 1457319, NSF EAR 163 1684, Missouri Research Council, University of Missouri Department of Pathology and Anatomical Sciences

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CT Imaging of the Internal Anatomy of Sloth Claws
Authors: Grass Andy
Published: June 2021

One of the most immediately noticeable peculiarities about sloths, among many, are their large, curved claws. Indeed, the name Megalonyx, a giant ground sloth from North America named by Thomas Jefferson, means “great claw.” Sloths have used their claws for a variety of purposes, including suspension from tree branches in modern sloths, hooking branches to pull food closer while eating, to gigantic burrows that have been attributed to some genera of ground sloths. Large claws may also have contributed to the evolution of the pedolateral foot orientation in some ground sloths, where the fifth metatarsal is the primary weight bearing bone, and the dorsal surface of the foot faces laterally. As such, a sloth's claws are and were integral to their existence.

For this study sloth claws were scanned in a Micro CT at the American Museum of Natural History, and these scans were segmented to view their internal anatomy. Both manus and pes claws from both genera of modern sloths, Bradypus and Choloepus, were scanned, as well as manus and pes unguals from the ground sloths Megalonyx jeffersonii and Paramylodon harlani, including specimens from the Tarkio Valley M. jeffersonii from the University of Iowa. Two of the ground sloth claws were broken on the ends, which revealed holes on the distal surface. It was unclear if these holes were natural, or the result of taphonomy or tools. The CT scan revealed that these holes are in fact passages for blood vessels, and lead to discrete canals in the interior of the claw. These canals originate from external foramina on the lateral sides of the claws, which are next to very clear external vessel impressions. These two lateral canals come together to form one central canal, which then branches again more distally. Bradypus is considered to be basal to all other sloths, including the other modern sloth, Choloepus. Despite this phylogenetic distance, all of the sloths showed similar patterns of vascular canal branching. There tended to be more vessels on the volar side of the claws, perhaps to supply areas that are more used for touch and pressure sensation. It will be interesting to see if anteaters and armadillos, which also have large claws but use them for more strenuous digging activities than modern sloths, also show similar vasculature patterns and structure.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Rare Case of a Five‐Branched Aortic Arch Exhibiting a Retroesophageal Right Subclavian Artery and an Accessory Left Vertebral Artery
Authors: Granite Guinevere , Meshida Keiko , Jones Shiloh
Published: June 2021

Head and neck vascular variations are common in human beings, but often go undetected. They are generally asymptomatic. Awareness of such anatomical variations is clinically important for surgeons and interventional radiologists as they may pose risk for iatrogenic complications or even the potential for unanticipated fatalities.

Although aortic arch variations are relatively common in human fetuses, variations of its branches, i.e. the brachiocephalic trunk, the subclavian arteries, the vertebral arteries, and the common carotid arteries, are observed and/or described variably and fairly infrequently during human cadaveric dissection.

When aberrant subclavian arteries are described in adults, they frequently involve the right side and are usually retroesophageal. In such cases, the right brachiocephalic trunk is absent. The following four branches of the aortic arch found most commonly then are: a left subclavian artery, an aberrant left vertebral artery, and right and left common carotid arteries. During cadaveric dissection of a 76 year‐old White male, we observed an additional fifth branch: an accessory left vertebral artery. A combination of five such aortic arch vessels in a single individual may be rare.

Disclaimer: The opinions or assertions contained herein are the private ones of the author/speaker and are not to be construed as official or reflecting the views of the Department of Defense, the Uniformed Services University of the Health Sciences or any other agency of the U.S. Government.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tri‐lobed left lung with corresponding additional secondary lobar bronchi: A case report on anatomic variation
Authors: Nguyen Alan Thinh , Tran Deanna , Volin Joshua , Quach Duyen , Koslakiewicz Sarah , Ezeokoli Ekene , Barremkala Malli
Published: June 2021

Fissures of the lung separate bronchopulmonary segments to form detachment planes for distention upon respiration and localization of pulmonary lesions. Knowledge of variations in lung fissures is necessary for locating lobes of the lung to exactly interpret radiographs, computed tomography scans, and thorough planning of surgical procedures. 24 pairs of lungs obtained from routine dissection of adult formalin fixed cadavers used for first‐year regional anatomy course at Oakland University William Beaumont School of Medicine were used in this study. There was a total of 12 male and 12 female cadavers with an average age of 82 years. The lungs were observed and photographed for presence or absence of fissures and lobes. We report a case of a tri‐lobed left lung with a complete horizontal fissure inferior to the left root of the lung, and each lobe features its own independent secondary lobar bronchus. The corresponding right lung features normal fissures and lobes. 8 additional right lobes had incomplete accessory fissures measuring <5cm in length with greatest depth of 2cm. 6 additional left lobes had incomplete accessory (4) and horizontal (2) fissures of <4cm in length with greatest depth of 2cm. Collectively, our results demonstrate a lung variation for awareness and for interpretation of radiological images and locating the bronchopulmonary segments in lobectomies, bronchoscopies, and segmental resection.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Circle of Willis Variation: Implications for Aneurysm, Atherosclerosis, and Stroke
Authors: Klinkhachorn Penprapa , Kumar Anil , Ruiz Kristina , Umstot Samuel , Ritz Brianna , Nestor Nicholas , Zdilla Matthew
Published: June 2021

Arterial variations within the Circle of Willis, especially hypoplastic arteries, are linked to intracranial aneurysm, atherosclerosis, and stroke; therefore, the study of Circle of Willis variants are of clinical importance. In a sample of 116 cadavers, the arteries comprising the Circle of Willis were directly observed and assessed for hypoplasia, hyperplasia, aplasia, duplication, triplication, and plexiform structure. A total of 53 different arterial patterns of the Circle of Willis were identified. The most common vascular pattern was one with no noteworthy variation of the arteries, which occurred in 34.48% (40:116) of the sample. The second most common pattern was a right hyperplastic posterior communicating artery, with concomitant right hypoplastic P1 and right hyperplastic P2 vessels which occurred in 3.45% (4:116) of cadavers. The most common variation of an individual artery, regardless of concomitant variations, was a hyperplastic right posterior communicating artery, which occurred in 19.8% (23:116) of the sample. Similarly, a right hyperplastic P2, right hyperplastic posterior communicating artery, left hypoplastic P1, and left hyperplastic posterior communicating artery each occurred in 18.1% (21:116) of cadavers. A duplicated anterior communicating artery was observed in 12.9% (15:116) of cadavers. Aplasia was only observed in the posterior communicating artery (left‐sided: 7.8%; 9:116, right‐sided: 6.0%; 7:116). The results of this study provide a clearer understanding of the variant vascular patterns of the Circle of Willis that create predisposition for aneurysm, atherosclerosis, and stroke.

Support or Funding Information

West Virginia University, Oman Medical College and West Liberty University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Supernumerary coronary artery. Case report
Authors: Zahoi Delia Elena , Sztika Dorina , Enache Alexandra , Daescu Ecaterina
Published: June 2021

During the routine dissection of a 60‐year‐old male within our university's Department of Anatomy, we identified a peculiar aspect of the coronary blood supply.

The heart is normally supplied by two coronary arteries, however in our case, we identified a supernumerary coronary artery. It arose from the aorta, through a separate ostium, anterior of the right coronary artery's point of origin. This third artery descended diagonally along the anterior surface of the right ventricle. At the heart's apex, it then continued for approximately 2cm along the posterior interventricular sulcus.

The right marginal branch took a diagonal descending path to the middle of the posterior interventricular sulcus, close to the terminal point of the third artery. At the same time, the right coronary artery was shortened, and only descended to the first third of the posterior interventricular sulcus.

The left coronary artery presented with two branches: the circumflex branch, and a common trunk for the anterior interventricular branch and the left marginal branch. The anterior interventricular branch was shortened, only descending two thirds of the way along the anterior interventricular sulcus. Both it and the left marginal branch were partially covered by myocardial bridges. The circumflex branch terminated near the posterior interventricular sulcus through two ventricular branches.

The study and knowledge of coronary variants is critical in identifying potential collateral paths, of which a supernumerary coronary artery is a good example.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Utilizing Inquiry Based eLearning Modules for Pre‐Lecture Content Delivery in a Molecular Biology Course at an Australian University to Facilitate Active Learning in the Lecture Theatre
Authors: Kuit Tracey , Fildes Karen , Treweek Teresa , Moscova Michelle
Published: June 2021

Currently in many large universities and colleges, the delivery of content in lectures is still largely based on a passive stand and deliver model. This method of lecture delivery means that the lectures are not utilised as a platform for student discussion and deep engagement with key concepts. This could negatively impact student learning and the development of conceptual understanding, especially in content heavy subjects such as biochemistry and molecular biology. Although teaching methods based on how adults learn have been well described, they can be perceived as logistically difficult to apply. With internal university funding, we have developed an alternative model of content delivery through the use of interactive technology for pre‐lecture preparation. The molecular biology in the modules is presented via a medical case, which gives the students a real life context with which to explore key concepts. Formative assessment with immediate feedback is embedded into the modules. The online modules are followed by a face‐to‐face lecture using active inquiry‐based learning, creating space for discussion to build on core concepts previously explored online. Fifty‐two second‐year graduate medical and 291 first‐year molecular biology students were surveyed to evaluate students' perception of the module. A knowledge‐based quiz was also given to the 291 molecular biology students one week after the lecture to assess short‐term knowledge retention. Results show 75–100% of students felt that the module was easy to understand; 60–89% found it engaging and wanted similar modules available across a variety of topics and 60–65% felt well prepared for class. Molecular biology students who did the module and attended the lecture did significantly better on the knowledge quiz compared to students who either attended the lecture or completed the online module alone. Our study supports the notion that the use of interactive online modules to prepare students for active learning lectures creates a transformation from passive to active learning experiences where students are active participants in their own learning.

Support or Funding Information

University of Wollongong Educational Strategies Development Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
XRT: A reproducible and scalable training system that produces students capable of conducting novel molecular biology research
Authors: Zarrinnegar Hanna , Adams Clay , Penchina Kaitlyn , Alonzo Amanda , Sweeney Robert , De Luke
Published: June 2021

Many high‐school science education programs do not train students to be independent researchers that make novel contributions to the scientific community, which is arguably the primary goal of a scientist. There are many possible explanations for this paradox. Due to high student/teacher ratios, teachers are unable to give the individualized attention necessary to cultivate a scientist from a student. Class schedule, student motivation, and diverse intellectual support are also huge barriers. As a result, most high‐school programs struggle to establish a system that allows for university and graduate‐level laboratory education of a significant number of students.

Here, we report a curriculum that trains students to design, conduct, manage, lead, and present novel research projects, but also a program that is self‐sustaining and scalable. The system produces a three‐tiered system of research students, each with an increasing set of laboratory and troubleshooting, goal‐setting, and project management skills. This management system is scaffolded. It can be completed at different speeds. These teams design projects that are novel and real. Trained teams come together and systematically create projects that can not only sustain themselves and pursue novel intellectual discovery, but also projects that are able to expand and adapt through multiple years and to a changing number of students, independent of staffing.

Support or Funding Information

This work was supported by The Nueva School.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“A Day in the Clinical Laboratory!” STEM Outreach with St. Louis High Schools
Authors: Mak Victoria , Le Austin , Tripathi Rohan , Ezekiel Uthayashanker
Published: June 2021

For the past three years, the Saint Louis University American Society for Biochemistry and Molecular Biology (SLU ASBMB) student chapter has planned and held “A Day in the Clinical Laboratory.” We selected 20 sophomore and junior students from local St. Louis high schools to learn about and perform different laboratory techniques in order to complete our designed case study. During the day, we had four lab stations that included hematology, urinalysis, blood bank, and chemistry. The laboratory techniques and case study were challenging in order to allow the students to think critically and to ensure interest in the study. The individual labs were all connected, and it showed how different clinical laboratories are done and how there is a need for collaboration between different departments in order to get a diagnosis for patients. Each group of students had 30 minutes at each station to ensure that they had enough time to complete the task and to ask any questions that they had. The students that came had an interest in science or medicine, and this program allowed them to get early exposure to research, science, technology, engineering, and mathematics fields. The event was free of charge and provided them the opportunity to see the importance in research and allowed them to network with different faculty mentors. Parents and teachers were also invited to attend, and student volunteers led a tour of the University campus facilities. High school students and their parents and teachers had the opportunity to speak with Saint Louis University Doisy College of Health Sciences Faculty on college application advising and additional opportunities available to local students interested in a scientific career. The event was run by SLU ASBMB students. At the end of the day, high school students were surveyed anonymously. All students gave positive feedback, and everyone expressed interest in attending another “A Day in the Clinical Laboratory” event.

Support or Funding Information

Financial support was received from the ASBMB Student Chapter Outreach Grant and the Saint Louis University Student Government Association.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Suppression of template switching in postreplication gaps via Uup and RadD proteins
Authors: Thimmesch Sarah Christine , Romero Zachary J , Wood Elizabeth A , Lovett Susan T , Cox Michael M
Published: June 2021

We are investigating the role of two bacterial proteins, RadD and Uup, in suppressing genomic stability. Uup is a UvrA‐like ABC family ATPase and RadD is a RecQ‐like SF2 family ATPase. Both of these proteins bind to branched DNAs. More specifically, RadD suppresses crossover events in the cell. The proteins resolve branched DNA intermediates that have arisen from template switching in post replication gaps. When a replication fork comes upon a lesion in the DNA, and a gap forms, the cell attempts to fill this gap and repair the DNA. One way of doing this is through template switching, a RecA‐independent process that creates recombination‐like branched DNA intermediates. These are the DNA intermediates at which RadD and Uup function. When genomic repeats exist in the chromosome, template switching sometimes results in mispairing that produces genomic deletions. Under normal growth conditions, with repeats in the chromosome, the deletion of Uup and RadD leads to the generation of chromosomal dimers that are not readily resolved. This experiment brings light to the functions of both proteins and recognizes the importance of genomic stability for cell viability. The results indicate that template switching happens often and that Uup and RadD play important roles in suppressing genomic stability.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Counting R‐loops: A Novel Quantification Method for Nuclear DNA:RNA Hybrids
Authors: Ramirez Prisila S , Ali SK Imran , Ashley Amanda K
Published: June 2021

R‐loops are three stranded nucleic acid structures that form during transcription; when the nascent strand of RNA invades the replication bubble, hybridizes with the DNA template strand and displaces the non‐template strand. For many years, these DNA:RNA hybrids were thought to only be byproducts of transcription. In recent years, however, it has been shown that R‐loops play many roles in diverse cellular processes, such as immunoglobulin class‐switching recombination, epigenetic regulation, and in DNA replication. R‐loop mis regulation can lead to DNA damage and genomic instability. Additionally, R‐loops have been shown to play a role in several neurodegenerative diseases. Although, our knowledge of R‐loops has grown in the last few years, detection and quantification of R‐loops can still be difficult and inaccurate. Here, we provide a novel method for quantification of nuclear R‐loops based on cellular fractionation and compare it with commonly used R‐loop quantification methods.

Support or Funding Information

Research was funded by HHMI Research Scholars program at NMSU, Cowboys for Cancer Research and the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103451.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of split Cas6 for inducible dimerization modules
Authors: Vontalge Emma J. , Ling Justin A. , Sashital Dipali G.
Published: June 2021

Cas6 endoribonucleases are essential for guide RNA biogenesis during CRISPR‐Cas (clustered regularly interspaced short palindromic repeats‐CRISPR associated) immunity, and have exquisite specificity for short structured RNAs that form in the repeating regions of CRISPR transcripts. The Cas6 protein fromThermus thermophilus contains two domains, an N‐terminal domain (NTD) containing the endoribonuclease active site, and an RNA‐binding C‐terminal domain (CTD). The modular nature of this protein makes it a potential platform for molecular scaffolding by splitting the two domains and inducing dimerization through RNA binding. Here we show that the Cas6 NTD and CTD can be split into two separate proteins, and that the domains retain their RNA binding and cleavage activity. Fusion of the domains to the C‐termini of cyan or yellow fluorescent protein (CFP or YFP) enabled measurement of dimerization in the absence or presence of RNA using fluorescence resonance energy transfer (FRET) in vitro. We find that wild‐type Cas6 NTD and CTD dimerize even in the absence of RNA. However, introduction of mutations in the interface between the two domains disrupts this interaction. The interface mutations do not disrupt RNA binding or cleavage, and addition of RNA restores dimerization of the two domains. In addition, we can measure the amount of dimerization between the two domains in vivo when the domains are expressed from two compatible vectors in Escherichia coli. These results demonstrate that split Cas6 can be developed into a molecular scaffolding platform affecting protein interactions that can be controlled through the addition of RNA.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Downregulation of SP‐R210L Alters the Macrophage Epigenetic Chromatin Landscape Associated with Changes in Transcription Factor Binding
Authors: Yau Eric , Chen Yan , Song Chunhua , Imamura Yuka , Chroneos Zissis
Published: June 2021

Alveolar macrophages are the first immune cells to come into contact with air‐borne pathogens such as influenza. Previous studies demonstrated that the surfactant protein A (SP‐A) receptor, SP‐R210, also known as MYO18A, regulates the immune functions of macrophages through the action of two isoforms; a long form SP‐R210L(L), and a short form SP‐R210S(S). Stable knockdown of L in RAW264.7 cells (abbreviated as DN) revealed alterations in mRNA expression of innate immune receptors and responsiveness to various inflammatory stimuli. To elucidate the intrinsic functions of the L isoform, the present study addressed the hypothesis that L modulates macrophage gene transcription through epigenetic modification of chromatin. We thus utilized chromatin immunoprecipitation and sequencing (ChIP‐seq) to determine the impact of L depletion on histone modification marks H3K4me3, H3K9me3, and H3K27me3 and binding of transcription factors PU.1 and NFκB subunit RelA, which regulate macrophage differentiationand inflammatory responses, respectively. Pulled‐down sequences were visualized on the UCSC genome browser to identify the spatial relation between enriched sequences. This analysis showed reduced PU.1 binding at genes associated with macrophage differentiation and intercellular communication between innate and adaptive immune cells, such as interleukin 18 (IL‐18) and colony‐stimulating factor receptors (CSF1R, CSF2RB) in DN cells. Additionally, differences in PU.1 binding were reflected in similar suppression of H3K4me3 marks in DN cells. Further analysis by ChIPpeakAnno and ChIPseeker and ingenuity pathway analysis, however, revealed enrichment in genes that regulate TNFα and interferon (IFN) signaling in DN cells. Collectively, these findings support the idea that the L isoform of SP‐R210 regulates chromatin architecture and accessibility of transcription factors at genes with discrete roles in macrophage differentiation and activation.

Support or Funding Information

This work was supported by HL128746, The Children's Miracle Network, and the Department of Pediatrics, Penn State University College of Medicine

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Epigenetic regulating enzyme activity modification in natural cinnamon extract treated MCF‐7 breast cancer cells
Authors: Hall Seth , Hill Matthew , Almehmadi Seham , Aulthouse Amy , Kinder David , Stockert Amy
Published: June 2021

As research continues to hone in on different tumorigenesis mechanisms, epigenetic dysregulation continues to show promise as a major contributing factor in cancer pathology. There are a variety of mechanisms involved in epigenetic regulation, but it can occur most prominently through direct DNA methylation and demethylation by DNA methyl‐transferase (DNMT) enzymes. Dysregulation of this epigenetic modifying mechanism from exogenous stimuli can lead to alteration of DNA transcription and protein expression within cells causing mutations to develop that result in increased cell proliferation and metastasis. Dietary polyphenols are known exogenous factors that influence epigenetic change. In particular, cinnamon is a natural product that contains many of these natural polyphenols, and it has been shown to affect protein expression in MCF‐7 breast cancer cells. With our current research, we have shown that cinnamon causes changes in activity of DNMT enzymes in MCF‐7 breast cancer cells. Results from a real‐time PCR assay for DNMT have shown modified levels of expression and activity with both enzymes in vitro. It remains to be determined whether these changes promote or inhibit tumorigenesis effects within cells, but do demonstrate that natural polyphenols found within cinnamon can cause epigenetic modification in cells. Future research will determine the resultant changes in protein expression and proliferative or anti‐proliferative nature of the effects that could arise from these epigenetic modifications.

Support or Funding Information

Departments of Pharmaceutical and Biomedical Sciences and Biological and Allied Health Sciences, Ohio Northern University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Effects of 5‐Azacytidine on the Long‐term Stability of HLA Class I Upregulation
Authors: Hansen Adam , O'Donnell Robert W
Published: June 2021

The epigenetic modifiers, such as DNA methylation inhibitors and histone deacetylase inhibitors, 5‐Azacytidine (5‐AzaC) and Vorinostat, have been shown to increase human leukocyte antigen (HLA) expression. The expression of HLA is required for a T‐cell response to detect tumor cells. A lack of HLA expression allows tumor cells to escape immune detection. It has been previously shown that 5‐AzaC is able to upregulate HLA expression in the cell line MDA‐MB‐435. However, the long‐term stability of increased HLA expression, following a transient absence of 5‐AzaC, is not known. Two flasks of MDA‐MB‐435 cells, one being continually treated twice a week with 5‐AzaC at 0.1 μg/mL, and the other, an untreated control flask are being propagated for long periods of time. Periodically the cells are harvested and incubated with control antibodies or the experimental antibodies, Anti‐HLA‐ABC and analyzed via flow cytometry. Our experiments will help determine if the length of 5‐AzaC exposure modifies HLA expression in MDA‐MB‐435 cells. Experimentation on the role of 5‐AzaC is also planned to optimize both the overall upregulation of HLA expression, and the long‐term expression following the removal of 5‐AzaC. Understanding the long‐term stability of HLA upregulation may prove to be beneficial and applicable to cancer immunotherapy, an expanding field of improved cancer treatments.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of DNA Methylation Regulates Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene Expression
Authors: Kumar Prerna
Published: June 2021

Cardiac hormone atrial natriuretic peptide binds to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) and produces second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. The molecular and epigenetic mechanisms that contribute to the activation of Npr1 (coding for GC‐A/NPRA) gene expression are not well understood. The present study was aimed at understanding the mechanisms involved in DNA methylation inhibitor, 5‐Azacytidine (5‐aza)‐mediated Npr1 (coding for GC‐A/NPRA) gene transcription. The studies were carried out in rat thoracic aortic vascular smooth muscle cells (RTASMC), cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and treated with 5‐aza for 24 h. The MethPrimer search result showed the presence of three cytosine‐phosphate‐guanine (CpG) islands on the Npr1 full length promoter, namely island 1 (−886 to −752, 134 bp), island 2 (−310 to −58, 152 bp), and island 3 (−514 to +310, 464 bp) from the transcription start site. 5‐azacytidine significantly induced mRNA levels of Npr1 gene by 4‐fold and NPRA protein expression by 3‐fold in RTASMCs in a dose‐dependent manner. Treatment of cells with 5‐aza significantly (p<0.05) attenuated DNA methyltransferase 1 (DNMT1) protein expression in a dose‐dependent manner. However, there was no noticeable change in the DNMT3a and DNMT3b protein levels. There was a significant reduction in the DNMT activity in 5‐aza‐treated RTASMCs compared with vehicle–treated cells as quantified by EpiQuik DNMT activity assay. The present findings provide a novel regulatory mechanism for Npr1 gene transcription, an important player in the control of hypertension and cardiovascular homeostasis.

Support or Funding Information

This work was supported by an Institutional Development Award (IDeA) from the NIGMS and a partial support from Tulane Carol Lavin Bernick grant award.

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