bluuebunny/crossref_metadata_2025 huggingface dataset:
Combination of the anti‐apoptotic and pro‐apoptotic microRNAs expression and the anti‐apoptotic protein XIAP with serum CA19‐9 in the diagnosis of pancreatic adenocarcinoma
Authors: de Assis Cirino Mucio Luiz , Neto Fermino Sanches Lizarte , Filiputti Vitor Barioni , Fadel Patricia Ciciliato , da Cunha Tirapelli Daniela Pretti , Kemp Rafael , Sankarankutty Ajith Kumar , dos Santos José Sebastião
Published: June 2021

Introduction

Pancreatic adenocarcinoma (PA) remains one of the most aggressive malignant tumors of the digestive tract, with a five‐year survival rate of 5%. Although early detection offers the best chance to cure pancreatic adenocarcinoma, early stage disease presents little or no symptoms. The most commonly used marker for early detection of AP is serum CA19‐9, which has a sensitivity of 70% to 80%, however, with a specificity less than 50%. MiRNAs, endogenous small RNAs of approximately 17–25 nucleotides, present an altered expression in various cancers, suggesting its close relationship to disease processes, with the possibility of being a molecular biomarker for diagnosis and prognosis of cancer.

Objectives

To evaluate the effect of the combination of the association of anti‐apoptotic microRNAs: miR‐15a and miR‐16 and pro‐apoptotic microRNAs: miR‐21, miR‐221 and miR‐222 expression profile and the protein XIAP with serum CA19‐9 in diagnosis of diagnosis of pancreatic adenocarcinoma.

Patients and Methods

We studied 23 samples of PA and 20 as control samples: 13 of normal pancreatic tissue adjacent to the tumor (PPT), collected during pancreatic resections and 7 normal pancreas (NP) from deceased donors or patients who have undergone pancreatectomy affected by trauma. CA19‐9 was measured in serum and checked for expression of the miRNAs and XIAP in PA, PPT and NP through amplification by real‐time quantitative PCR (RT‐PCR). The usefulness of miRNAs and XIAP in the diagnosis of PA, alone or associated with CA19‐9, were analyzed by the construction of ROC curves.

Results

It was observed significant differences in the expression of miR‐21 in the comparison between the PA and NP groups (p<0.05). However, there was no significant difference in the expression of the protein XIAP between groups, as well as the expression of miR‐21 between groups NP and PPT. The combination of miR‐21 with CA19‐9 was more effective to discriminate the PA and NP (AUC‐ROC: 0.94; sensitivity: 78%, specificity: 99%) when compared with the assessments isolated.

Conclusion

The expression of miR‐21 was demonstrated to be effective in discriminating between PA and NP and the association between miR‐21 and CA19‐9 increased the sensitivity and accuracy in the diagnosis of PA in the sample studied.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gamma Delta T Cells and Its Interactions with Extracellular Vesicles during Malaria
Authors: Mbagwu Smart , Filgueira Luis , Walch Michael , Mantel Pierre‐Yves
Published: June 2021

Background and Objective

Extracellular vesicles (EVs) have been described as microparticles produced by different cells in the body with the capacity of mediating cell‐cell communication. A clear mechanism of how various immune cells function during malaria infection is still undergoing investigations. With various states and roles of gamma delta T cells reported, its role in human cerebral malaria has not yet been defined as a study has suggested that it is essential in the development of murine cerebral malaria through its production of interferon gamma and tumor necrosis factor. However, there is currently no report on interactions between EVs derived from Plasmodium falciparum infected red blood cells (iRC‐EVs) and human gamma delta T cells. Our aim therefore, was to investigate the interactions between iRBC‐EVs and gamma delta T cells and the resulting immunological responses that could play a role in the pathogenesis of cerebral malaria.

Method

By using Ultracentrifugation, EVs derived from Plasmodium falciparum infected red blood cell (iRBC‐EVs) culture were isolated and purified. The iRBC‐EVs were characterized by western blot, Fast protein liquid chromatography and electron microscopy. Gamma delta T cells were generated from peripheral blood mononuclear cells(PBMCs) isolated from human blood and stimulated with Plasmodium falciparum culture supernatant and IL‐2 for 14 days. The generated gamma delta T cells were treated with the iRBC‐EVs and monitored for uptake, proliferation and activation using confocal microscopy and flow cytometry.

Results and Conclusion

We found out that gamma delta T cells are capable of taking up iRBC‐EVs and were proliferating on co‐incubation with iRBC‐EVs and Plasmodium falciparum culture supernatant. In conclusion, our results show that there is an influence of iRBC‐EVs on gamma delta T cells which would form a basis for further investigations on the immunological responses mediated by these cells towards the development of cerebral malaria.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bite morphology of erythrocytes with G6PD deficiency is or is not a reliable parameter for forensic identification
Authors: Cusimano Maurizio
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is the most common enzyme deficiency in humans, affecting 400 million people worldwide. It has a high prevalence in persons of African descent. It is inherited as an X‐linked recessive disorder. G6PD deficiency is polymorphic, with more than 300 variants. 50 crime scenes were analyzed with microscopic examination of blood stains. The morphology of erythrocytes after controlled rehydration was examined under a microscope with May‐Grunwald Giemsa staining. In the 50 samples the RBC diameter, the morphology, the presence of Heinz's bodies and the presence of Bite erythrocytes were evaluated. On the samples the G6PD was analyzed. In 7 cases, typical changes in G6PD deficiency were identified. In the analyzed cases, DNA analysis was performed on other tests in the same scene. The result of the study was that in the 7 cases identified, these were black subjects. the conclusion is that the evaluation of the erythrocyte morphology is a reliable test (p <0.05) for the identification of black race of subjects without identity in the absence of other evidence.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Character displacement in two New World monkey genera, Cebus and Alouatta
Authors: Villamil Catalina I
Published: June 2021

As a result of recent fossil and genetic finds, paleoanthropologists have realized that hominin evolution is characterized by substantial geographical and temporal overlaps among species, as well as pervasive hybridization. However, the role of these interactions in our evolution is not well understood. One evolutionary process, character displacement, may have shaped the long‐term effects of these encounters. Character displacement occurs when hybridization is detrimental to survival, so that natural selection favors divergence between populations, typically in traits with reproductive or ecological functions such as species recognition or diet. However, divergence occurs only among the overlapping and potentially hybridizing populations, so that the two species are more different in areas where they overlap than in areas where they do not. This process has recently been suggested as a factor in the divergence of Paranthropus and Homo around 2–3 Ma, particularly in regards to their dentitions. However, character displacement in the primate skeleton has not been well characterized generally. Here, I assess character displacement in three platyrrhine groups that potentially hybridize in the wild: Alouatta palliata (N=38) and A. pigra (N=35), A. guariba (N=16) and A. caraya (N=31), and Cebus libidinosus (N=36) and C. nigritus (N=41). I collected 46 linear measurements on the skull and performed t‐Tests between overlapping and non‐overlapping groups. In all three groups, I found multiple traits that vary in ways consistent with character displacement. These traits are primarily facial and mandibular features that are potentially linked to diet. I also found that Cebus males and females display more consistent divergence patterns compared to Alouatta, suggesting that sex‐specific selection pressures play a role. These results indicate that character displacement directly affects the primate skeleton. They also provide a guideline of divergence patterns that could be identified in hominin fossils to determine whether character displacement has occurred.

Support or Funding Information

This project was supported by a grant for a scholarly project from Dickinson College.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Newly‐discovered craniomandibular remains preserve the first known incisors of the extinct Dominican monkey Antillothrix bernensis
Authors: Klukkert Zachary S , Halenary‐Price Lauren B , Almonte Juan , Shevchuk Oleg , Lehman Phillip , Cooke Siobhán B
Published: June 2021

Antillothrix bernensis, an extinct platyrrhine primate from the Dominican Republic, has been under intensive study following a series of new finds from two flooded cave sites. To date, three nearly complete crania and one mandible have been described, but none of them preserve the anterior dentition. This study reports the first discovery of Antillothrix incisor teeth from two crania and three mandibles from a new flooded cave site in the Altagracia region of the Dominican Republic. The new fossils provide valuable data on the diet and phyletic affinities of Antillothrix.

The craniodental traits of Antillothrix suggest a phyletic assignment within the family Pitheciidae due to similarities with the sclerocarpic harvester, Callicebus. Platyrrhine sclerocarpic harvesters are hard‐skinned‐fruit specialists that apply their anterior teeth to break down the outer husks of these foods to access the pulp and/or seeds within. Two hallmarks of this strategy are specialized anterior teeth and a vertically deep jaw that distributes stresses resulting from loads at the incisors and canine teeth during ingestion. Previously described Antillothrix material resembles Callicebus by sharing relatively short canine teeth and posteriorly expanded rami in their mandibles. Upper incisors in the new fossils are broad and spatulate but relatively small and short, more similar to those of atelids than sclerocarpic harvesters. The lower incisor crowns are also relatively short and lack the signature mesiodistally‐narrow shape found in pitheciids.

In sum, the newly discovered fossils illustrate an anterior dental battery that is inconsistent with sclerocarpic harvesting or close affiliation with the extant pitheciids despite other similarities in the jaw and molar teeth. These results expand upon the conclusions from molar crown morphology – Antillothrix was a generalized frugivore that likely fed on soft and/or small fruits without intensive ingestive steps. The lack of derived features in the dentition accords with previous interpretations of various aspects of Antillothrix cranial morphology, e.g., small relative brain size and a vertically oriented nuchal plane, as being primitive for pitheciids or even for platyrrhines more generally. Altogether the craniodental traits of this enigmatic primate continues to preclude direct analogy with any extant platyrrhine.

Support or Funding Information

This work is supported by an Explorers Club Exploration Fund grant to ZSK.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution and Morphology of the Tetrapod Brachial Plexus
Authors: Meers Mason B. , Cook Rebecca M.
Published: June 2021

The brachial plexus is formed by interconnected spinal nerves that give rise to branches which innervate the muscles associated with the pectoral limb. Historically, the utility of this anatomical structure has largely been associated with practical medical and veterinary interests, although recently the utility of the plexus in understanding mammalian evolution has been explored. In this study, we examine published and novel dissections of the brachial plexus across a broad range of tetrapod taxa for common traits using character optimization, sometimes known as trait mapping. Each of the major clades of tetrapods is included in the analysis, as are major clades of mammals following from published records. Our results indicate that the overall dorso‐ventral patterning of the brachial plexus is a highly conserved trait, while other features of seem to characterize large clades. Autapomorphies are common throughout the plexus. The traits that characterize large clades may be indicative of developmental or functional constraints on brachial plexus morphology.

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 PROPOSED CLASSIFICATION OF RETROMANDIBULAR AND EXTERNAL JUGULAR VENOUS VARIANTS – TWO CASE REPORTS WITH REVIEW OF LITERATURE AND CLINICAL IMPLICATIONS
Authors: Sanyal Sanjoy
Published: June 2021

Typically the retro‐mandibular vein contributes to formation of common facial and external jugular veins, via its anterior and posterior divisions. However, cervico‐facial venous variants are rather common.

The cervico‐facial veins were studied in two successive dissections. In both cases the retromandibular vein was undivided. The first had external jugular vein draining into subclavian vein, but no common facial vein. The second had no external jugular vein, but had common facial vein draining into internal jugular vein.

The first variant was characterized as Posterior Dominant Retromandibular External Jugular Vein with no Common Facial Vein. The second variant was characterized as Anterior Dominant Retromandibular Common Facial Vein with no External Jugular Vein. Each was sub‐classified as Typical or Atypical based on its final drainage pattern.

Cervico‐facial venous variants often vary from case to case and side to side. Since these veins are used for clinical and surgical interventions, accurate preoperative classification of variations may preclude intra‐operative surprises and consequent complications.

Support or Funding Information

Not applicable

New proposed classification of retromandibular external jugular and common facial venous variants*

RMV Characterization
Divisions of Retromandibular Vein (RMV)
External Jugular Vein (EJV)
Common Facial Vein (CFV)

Typical Retromandibular Vein (RMV)
Divisions present

Anterior Division: Unites with Facial Vein to form CFV

Posterior Division: Unites with Posterior Auricular Vein to form EJV

EJV present

Drains into Subclavian Vein***

CFV present

Drains into Internal Jugular Vein (IJV)***

Variant Retromandibular Vein (RMV)
NO Division

Posterior Auricular Vein unites with main RMV, but may drain elsewhere

**Posterior Dominant: Whole RMV goes toward Posterior Triangle of Neck superficial to Sternocleidomastoid Muscle as External Jugular Vein (EJV)
Typical:

EJV present

Drains into Subclavian Vein

Atypical: Drains elsewhere
Typical:

CFV ABSENT

Original Facial Vein drains into IJV

Atypical: Drains elsewhere

**Anterior Dominant: Whole RMV goes toward Anterior Triangle of Neck and forms Common Facial Vein (CFV)
Typical

EJV ABSENT

Atypical: Other variation(s)
Typical

CFV present

Drains into IJV

Atypical: Drains elsewhere

This Characterization of variant Retromandibular Vein is proposed for adoption in clinical practice

Anterior/Posterior Dominance of Undivided RMV is the linchpin of the Classification because of its clinical significance

Copyright © Sanjoy Sanyal 2018–2019

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anatomical variants of the aortic arch using CT angiography
Authors: Quiroga‐Garza Alejandro , Tapia‐Nañez Mariana , Guzman‐Lopez Santos , Elizondo‐Omaña Rodrigo Enrique
Published: June 2021

population. Variants have been widely studied in different populations, with a general prevalence between 5% and 35% however, data in Latino population is limited. The purpose of our study was to determine the prevalence of variants in the ramification of the aortic arch in our population

Materials and methods

A retrospective, observational, comparative and transversal study was performed. A sample size calculation was made, with a result of 202 individuals. Using computed tomography angiography (CTA), the number of arteries that originated within the limits of the aortic arch was quantified; the branching pattern, the most cranial vertebral level of the aortic arch, luminal diameters of the proximal, middle, and distal segments were recorded. The diameter of each branch originated in the arch of the aorta was also evaluated.

Results

A total of 220 individuals were sampled, of which 52% (n 114) were men. The mean age was 55 years. The classical branching was present in 77.7%, followed by a common carotid trunk and left subclavian pattern (13.6%), the classic branching adding the left vertebral artery (7.3%), and others (1.5%). Highest vertebral was T3 in 32.3%, between T2–T3 in 26.8%, and between T3–T4 in 23.2%. Mean luminal diameter were 92.10±12.35, 76.75±10.32, and 73.85±12.27mm2 in the proximal, middle, and distal segments, respectively. Statistical differences were found between men and women in the middle and distal portions.

Conclusion

Our population showed an anatomical variation of the typical branching pattern of the aortic arch in 22.3% (n 49). The most cranial point of the aortic arch is found between T2 and T4 in 82.3% of the cases.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Side and Sexual Dimorphism of the Anterior Crest of the Mandibular Fossa
Authors: Teaff Noah James , Zdilla Mathew
Published: June 2021

The temporomandibular joint (TMJ) is a complex and constantly moving part of the human body with over 2000 movements a day. Because of the joint's complex movements, symptomatic temporomandibular dysfunction (TMD) occurs in 5–12% of the population. Prosthetics may be used to treat TMD; yet, with potential anatomic variation, prosthetics may suffer from not being specific enough to a patient. Identifying and categorizing anatomical variations may result in improved treatment. This study analyzed the anterior crest of the mandibular fossa by landmarking 40 equidistant points from the most medial to lateral aspects of the crest, on both the left and right sides of the crania. The points were used to compare both side and sexual dimorphism of the fossa's anterior margin. Principle component analysis showed that 84.0 % of morphological variance is accounted for by the first two principle components (PC1= 66.4% PC2= 17.6%). Canonical variate analysis showed a significant difference between the sexes and sides (Side: Mahalanobis distance= 2.5399, p<0.0001) (Sex: Mahalanobis distance= 1.7350, p<0.0001). The information assessed from this study provides insight with regard to side and sexual dimorphism of the anterior crest of the mandibular fossa. The results of this study may aid in creating a TMJ prosthetic with a higher success rate than the stock models available today.

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 Contour of the Glenoid Fossa: Variance in Concavity
Authors: French Evan Dean , Alfaro‐Gomez Uriel , Guzman‐Lopez Santos , Elizondo‐Omaña Rodrigo , Quiroga‐Garza Alejandro , Zdilla Matthew J
Published: June 2021

The glenohumeral joint, by nature of its freedom in range of motion, is particularly vulnerable to injury. Though many aspects of the joint, such as rotator cuff musculature and the glenoid labrum, have been assessed, there is a paucity of data regarding the variation in the contour of the glenoid fossa, the bony support for the humeral head. Therefore, this study applied geometric morphometric techniques to demonstrate the variance in the contours of glenoid fossae. A total of 76 shoulder CT scans, with planes oriented through the glenoid cavity, coronally, were assessed. Principle component analysis explained 80.1% of the cumulative variance in the glenoid fossa shape with the first two principle components (PC1 = 65.7%; PC2 = 14.4%). The first principle component revealed the shape variance to range from a pronounced concave shape to a minimally concave, nearly linear, shape. The results of this study suggest that, varied concavity of the glenoid cavity might influence both the range of motion and the structural integrity of the glenohumeral joint.

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 Extensor Pollicis et Indicis Communis Muscle: A Rare Muscular Variation
Authors: Souder Kendall S , Petrone Ashley B , Lambert H. Wayne , Zdilla Matthew J
Published: June 2021

Extension of the index finger and thumb are typically independent movements that are not tethered by muscular attachment. This report demonstrates a case of an extensor pollicis et indicis communis found during the dissection of an 84 year‐old female body donor. The extensor pollicis et indicis communis is a rare muscular variation that originates in the posterior compartment of the forearm, traverses the fourth extensor compartment of the extensor retinaculum, and then bifurcates in the dorsum of the hand to insert at both the thumb and index finger. The action of this muscle is to extend the index and thumb simultaneously in addition to performing thumb reposition. Further, this variation is unique in that it would antagonize and potentially impede the act of pinching as well as opposition of the thumb— both functions that are well‐developed in humans.

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 Multi‐Year Quantitative Analysis of Semispinalis Capitis Morphological Variations in the Neck
Authors: Bee Mary Tracy , Ciavaglia Marco , Elturk Adam , Aljerdi Ibrahim , Mehrabi Negar
Published: June 2021

The semispinalis capitis muscle is located in the posterior neck of humans. This muscle allows us to extend our head and neck. The semispinalis capitis muscle is also known as the biventer cervicis muscle. The majority of anatomy textbooks and atlases depict this muscle as containing two bellies of muscle with a linear intervening white tendinous band that neatly separates the muscle into upper and lower parts. We dissected the posterior neck and upper back of 48 cadavers with a total of 96 individual muscles in order to isolate this muscle and gain better clarification of this muscle's appearance. We found that this muscle does not appear as commonly depicted in most anatomy books. Instead we found that its intervening tendon is not linearly horizontal. Its tendon consists of multiple different parts that are commonly disconnected and lie either superior or inferior to each other in three different categories. The three main classifications include two uniform vertical bands, one vertical with one curved band, and an other group. We found that 56.9 percent falls in the two uniform vertical band classification, 23.2 percent falls in the one vertical one curved band classification, and 19.9 percent falls in the other classification. We recommend that artists change the way in which this muscle is drawn in anatomical textbooks.

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 anatomical variant of the pudendal neurovascular bundle: Description of its course and potential clinical importance
Authors: Pagano Anthony Santino , Titunick Michelle B. , Metzger Keith , Marquez Samuel
Published: June 2021

Anatomical variations in the course of the pudendal nerve have been implicated in clinical conditions related to neuralgia and nerve compression. A transgluteal approach has been advocated previously for surgical access to the pudendal nerve as it enters the lesser sciatic foramen. However, as with any surgical approach, one must be aware of anatomical variants that complicate treatment. We performed 20 gluteal dissections on 10 cadavers at the Hackensack‐Meridian School of Medicine at Seton Hall University and an additional 48 gluteal dissections on 24 cadavers at the SUNY Downstate College of Medicine for a total of 68 pudendal nerves. Results confirmed that most individuals possessed a pudendal neurovascular bundle lying directly over the ischial spine. None of the cadavers exhibited the transligamentous route (through the sacrospinous ligament) reported previously in the clinical literature. However, one individual out of 68 exhibited a rare anatomical variation of a pudendal bundle lying over the sacrotuberous ligament. This same individual exhibited the normal anatomical configuration on the contralateral side. Such a configuration could complicate a surgical approach when, lying over the sacrotuberous ligament, it may be mistaken for a secondary or tertiary branch of the inferior gluteal neurovascular bundle. Despite the rare nature of the anatomical variant found in this study, its presence should be documented to reduce the chances of injury to a patient undergoing pudendal release surgery. Further work should be done to increase sample size and better reflect the true occurrence of this variant among asymptomatic individuals. Additionally, further information is needed to document the clinical impact of this variant on the patient population.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Journal Club 2.0: A reproducible, high yield, self‐propagating method of training students to analyze primary literature via student‐to‐student training
Authors: McGraw Kyle , Lange Jenna , Meike Elise , van der Rijn Eli , Celli Amanda , Stein Sydney , Putman Claire , Present Adam , De Luke
Published: June 2021

Individuals are asked to make high‐impact decisions that become the world we live in, informing the treatment of addiction, gun violence, bioethics laws, and global climate change. Decisions of this magnitude should be made with evidence from primary source research. Yet few methods effectively teach this skill to high school students. Here we describe a highly coordinated system of 7 teams of students that train others with little scientific training or presentation skill to present research topics that are relevant and controversial. These presenters have little to no scientific or presentation training. The teams are capable of training one presenter a week over the course of a year. The system is self‐propagating and can be maintained with minimal faculty involvement. Our data indicate that a single faculty member can engage a team of 12 that in turn engages a student body of 30–70 students weekly. Further details will be shared at www.project80.org

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Innovative Biochemistry Course Redesign to Integrate Basic and Clinical Sciences
Authors: Nusair Nisreen
Published: June 2021

The Biochemistry course is redesigned to include both service learning and clinical experiences. This innovative approach promotes the integration of basic and clinical sciences in the pre‐professional science curriculum. The redesigned course includes the following components:

(1) Clinical case studies, that are complementary to the course material, have been implemented. Group discussions of case studies, pre‐case questions and post‐case reflections have motivated students and enhanced their learning, material retention, and academic success. (2) Students have volunteered in a medical center and a community clinic to reinforce both service learning and clinical experiences. (3) In a written report and an oral presentation, each student has described this volunteering experience and has also addressed examples that correlate between basic and clinical sciences. The students pointed that this experience has opened their eyes to multiple health issues. As future healthcare providers, students believe they should be proactive and make a difference through providing sustainable health system change that will result in better health outcomes. (4) Students have presented posters in the annual college research symposium. Then, these posters are displayed in the community clinic to serve as a tool to educate the patients, patient's families, and the community on preventing, managing and improving various illnesses.

The goals of the novel course redesign along with its impact on students learning outcomes and development of their professional and graduate capabilities have been met. This course redesign has offered a significant change in class dynamics, promoted application of learned materials, and encouraged social interactions among students and students with their instructor and the community.

Support or Funding Information

None

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adding Spice to the Lab with BASIL: Combining Wet‐Lab Protein Biochemistry with Computational Analysis to Analyze Proteins of Unknown Function
Authors: Pikaart Michael
Published: June 2021

Most undergraduates studying biochemistry and molecular biology get their broadest exposure to wet‐lab techniques in protein and nucleic acid chemistry (and, increasingly, computer/visualization) in their upper‐level laboratory courses. Students in these classes are juniors and seniors with well‐defined career goals. Many will have already had a research background in a traditional one‐to‐one (or one‐to‐few) research mentoring setting, for example a summer research program – an experience known to increase student learning and persistence in the sciences. At the same time, extended full‐time PI‐directed research is limited in the number of students served, and can even present a barrier. To broaden the impact of teaching through research, many practitioners have adopted the CURE, or Course‐based Undergraduate Research Experience, approach.

This presentation reports on BASIL (Biochemical Authentic Scientific Inquiry Laboratory), the result of a team of faculty who have worked to bring computational and wet‐lab protein science to the biochemistry teaching lab. Together, we have developed a protein biochemistry CURE to determine enzymatic function of proteins of unknown activity. With eleven modules available online (basiliuse.blogspot.com), the BASIL curriculum represents a one‐semester upper level biochemistry laboratory course; five of the modules are computational/structural, while six are wet‐lab protein expression, isolation, and analysis experiments. Because the computational tools may be more challenging for biochemistry faculty and students to adopt than typical bench methods, we have developed a suite of analysis tools available on a USB stick. This presentation summarizes the status of BASIL on multiple campuses and seeks to promote further adaptation of this CURE.

Support or Funding Information

NSF IUSE Awards 1709278, 1503710

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Extensive DNA Repeats Residing in Orthologs of PKD1 Correspond With Inactivating Mutagenesis and Polycystic Kidney Disease
Authors: Larson Erik , Zebolsky Aaron , Dewey John , Swayze Emma , Alvarado Gloria , Ellis Monica , Heuvel Gregory Vanden
Published: June 2021

Bi‐allelic inactivation of the PKD1 gene results in Autosomal Dominant Polycystic Kidney Disease (ADPKD), a common and lethal disorder. ADPKD is thought to follow a two‐hit pathway, where one inherited mutation in PKD1 is followed later by somatic inactivation of the other allele. Loss of PKD1 expression results in cysts, which are clonal in nature and can number in the thousands. The mechanisms are unclear, but human PKD1 appears prone to mutagenesis and inactivation. Interestingly, naturally occurring ADPKD is observed in humans, cats, and pigs, but not mice. We have therefore compared PKD1 gene sequences across species to determine if there are shared sequence or structural characteristics that could explain PKD1 instability. Despite conservation of PKD1 organization and coding across species, we find an over‐representation of G/C content and tandem repeats in PKD1 from species that are naturally prone to ADPKD. It is already established that guanine‐rich DNA triggers site‐specific genetic instability via the formation of G‐quadruplex (G4) DNA structures. Cytosine repeats also support structures called, i‐motifs, which have the potential to induce site‐specific genetic instability. We find that the human, feline, and porcine PKD1 genes are rich in G and C tandem repeats and G4 sequences, while murine Pkd1 is not. Circular dichroism spectroscopy demonstrates that structure formation occurs under physiological conditions. The possibility that G4 DNA contributes to PKD1 instability is further supported by the fact that cataloged somatic mutations are clustered near predicted G4 forming motifs. Our results identify G4 DNA and i‐motifs as structures that may form on both strands of the PKD1 gene, uncovering a molecular rationale to explain the instability of the gene and the emergence of cysts in ADPKD carriers.

Support or Funding Information

WMed Pilot Grant Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of thermal manipulation during embryogenesis and post‐hatch acute heat stress on the mRNA expression dynamics of cytokines in broiler chickens
Authors: Al‐Zghoul Moh'd Borhan
Published: June 2021

Heat stress significantly impacts chicken immunity and cytokine expression, but, the effects of embryonic thermal manipulation (TM) on cytokine expression in broiler chickens is unclear. The aim of the present study was to evaluate the effects of TM on the splenic mRNA expression dynamics of certain cytokines (IFN‐α, IFN‐β, IFN‐γ, IL‐4, IL‐8, IL‐15, IL‐16, IL‐17 and IL‐18) in broilers during subsequent exposure to acute heat stress (AHS). TM was performed by elevating the incubation temperature to 39°C at 65% relative humidity (RH) for 18 h daily during embryonic days (ED) 10 to 18. AHS was carried out by raising the temperature to 40°C for 7 h on post‐hatch day 28. At 0, 1, 3, 5, and 7 h of AHS, the spleen was collected from all groups to evaluate mRNA expression by relative‐quantitative real‐time RT‐PCR, and blood was collected to measure plasma levels of IL‐4, IL‐8, and IFN‐γ. At 0 h, TM led to a significant reduction in the basal mRNA level of IFN‐β and the plasma level of IFN‐γ and IL‐8. Moreover, AHS significantly decreased IFN‐β in control chicks, decreased IL‐4 in both TM and control chicks, and increased IFN‐γ and IL‐16 in TM chicks. IL‐8, IL‐15, IL‐17, IFN‐α, and IL‐18 expression significantly increased during AHS in both TM and control chicks, but expression dynamics was improved in TM chicks for all cytokines except IL‐17. AHS resulted in increased plasma IFN‐γ levels in TM chicks only, and it increased IL‐8 levels at 3 and 5 h of AHS in TM chicks but at 7 h in control chicks. Lastly, 3 h of AHS increased IL‐4 plasma levels in control chicks. The results of this study may indicate that TM has a long‐term effect on the expression dynamics of cytokines, especially during AHS, suggesting that TM affects post‐hatch immunity. Furthermore, this study may suggest that TM improves heat tolerance by increasing the expression of signaling proteins important to the wound‐healing process.

Support or Funding Information

Deanship of Research/Jordan University of Science & Technology (Grant#: 128/2017).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Quantitative Analysis of p73 Gene Promoter Methylation
Authors: Nicolaci Angelo A , Carastro L. Michael
Published: June 2021

The p73 gene is a member of the p53 tumor suppressor gene family. The p73 gene contains a distal P1 promoter and a proximal P2 promoter. The P1 promoter is responsible for expression of the TAp73 protein isoform containing a transactivation domain (TA). The ΔNp73 protein isoform is expressed from the P2 promoter and lacks the N‐terminal TA domain. In this study, we analyzed six CpG islands in these p73 promoter regions. The purpose of this project was to quantitatively assess the degree of methylation at each CpG site in the six CpG islands. Three prostate cancer (PCa) cell lines (DU145, PC‐3, LNCaP) and non‐cancerous primary prostate cells were cultured, and cell pellets were collected. Cellular genomic DNAs were subjected to pyrosequencing analyses of five CpG islands (CpG islands #1–5) located in the P1 promoter region and one CpG island (CpG island #6) in the P2 promoter region. In total, the degree of methylation was determined for 241 individual CpG sites. Pyrosequencing analysis of CpG island #1 indicated 15 of 23 CpG sites were methylated >90% in all four cell types. Seven CpG sites had a methylation frequency range of 71.1% to 96.5%. The remaining CpG site methylation ranged between 60% to 68.8% across the four cell types studied. In CpG island #2, 9 of the 14 CpG sites were methylated >91.9% in all four cell types studied, and the 5 remaining CpG sites ranged from 70.3% to 95.6% methylation. In CpG island #3, the 37 CpG sites were methylated <6.4% in all cell types studied. In CpG island #4, none of the 112 CpG sites were methylated >64.7% in primary cells. Differential methylation was observed in the 112 CpG sites among the three PCa cell lines, ranging from 0.0% up to 91.8%. In CpG island #5, the 45 CpG sites in primary cells were methylated <43.19 %, but PCa cells displayed differential methylation values from 0.0% up to 92.7%. In CpG island #6, the 11 CpG sites were methylated <9.0% in primary cells, whereas, PCa cell lines were differentially methylated from 0.1% up to 98.1%. Of the six CpG islands analyzed, CpG island #3 had the lowest degree of methylation in all samples analyzed. CpG islands #1 and #2 had high degrees of CpG methylation in all four cell types. Differential methylation was observed in CpG island #4, CpG island #5, and CpG island #6. Generally, CpG sites were methylated to higher degrees in PCa cell lines compared to primary cells. Furthermore, the overall trend in high frequency CpG methylation decreased from DU145 to PC‐3 to LNCaP. We are currently performing chromatin immunoprecipitation (ChIP) assays in the differentially methylated regions in order to identify potentially important transcription factors.

Support or Funding Information

University of Tampa Department of Chemistry, Biochemistry & Physics Summer Research Fellowship Program

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bacterial Expression of Chimeric Escherichia coli and Trypanosoma brucei DNA Methyltransferases
Authors: Taber Cassandra Carol , Militello Kevin
Published: June 2021

Our laboratory is interested in DNA and RNA methylation in E. coli and T. brucei as little is known about this form of epigenetic regulation in microorganisms. One methyltransferase being studied at this time is a putative DNA methyltransferase (TbDmt) from Trypanosoma brucei. The exact function of TbDmt is unknown but the protein strongly resembles bacterial DNA methyltransferases such as DNA cytosine methyltransferase (EcDcm) from E. coli. To test our hypothesis that TbDmt is a DNA methyltransferase, we expressed TbDmt in bacteria and created two chimeric protein sequences switching the DNA binding domain and enzymatic domain of EcDcm and TbDmt. Exchanging the DNA binding domain and enzymatic domain of TbDmt with a known methyltransferase may help us discover the function of the enzyme and, if it is a methyltransferase, what DNA sequence is targeted for methylation. Plasmids were made containing the sequences for EcDcm, TbDmt, and both chimeric proteins where the genes are adjacent to the T5 promoter and lac operator. E. coli lacking a cytosine DNA methylation pathway were transformed with the plasmids and expression was induced with IPTG. All four proteins were produced at 20°C, but the proteins with the TbDmt DNA binding domain were less soluble than the other two. The plasmids were re‐isolated after three hours of growth in the presence of IPTG. The plasmids were then digested with eight restriction enzymes blocked by methylation. Each digestion was run on an agarose gel with DNA from an uninduced cell and unmethylated phage lambda DNA as controls. EcDcm methylated at its expected site, 5′CCWGG3′, as determined by resistance to PspGI. In addition, there is partial resistance to HpaII in cells containing EcDcm indicating methylation at 5′CCGG3′ or related sequences. We are currently testing restriction enzyme sensitivity in the samples containing full length TbDmt and chimeric TbDMTs. Further work will be done to improve the conditions in which the chimeric proteins are produced to enhance folding of the potential DNA methyltransferases. In summary, this work contributes to our limited knowledge of DNA methylation enzymes and targets in bacteria and protists.

Support or Funding Information

NIH, 1R15AI133428‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Importance of Genomic RNA1 3′ CITE in Blackcurrant Reversion Nepovirus Protein Synthesis
Authors: Baquero Galvis Laura D. , Shields Elizabeth , Morrison Evan J. , Filbin‐Wong Megan E.
Published: June 2021

Canonical translation initiation and regulation involve end‐to‐end interactions between initiation factors bound to a 7‐methyl guanosine triphosphate cap at the 5′ end and at a poly(A) tail, at the 3′ end of the messenger RNA. This characteristic is also prevalent in many RNA viruses such as the Blackcurrant Reversion Nepovirus (BRV), which has two bipartite genomic RNAs (RNA1 and RNA2) that contain the 3′ poly(A) tail but lack the 5′ cap. The mechanism implemented by BRV during translation initiation is not clearly defined; however, in other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 3′ UTR binds translation initiation factors and delivers the machinery to the 5′ end via proposed RNA‐RNA kissing‐loop interactions. This non‐canonical (cap‐independent) mechanism is directly related to the primary, secondary and higher‐order RNA structure called a cap‐independent translation enhancer (CITE) in the 3′ UTR. Considering the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our goal is to determine the secondary and putative tertiary structure of the 3′ CITE found in the genomic RNA1 in BRV, and correlate the structure to translation initiation function. With this in mind, we identified a set of functionally‐deficient mutants using a luciferase reporter translation assay. We then probed our purified wild‐type and mutant RNAs with a series of covalent nucleobase and sugar backbone modifiers to identify the secondary (and potential tertiary) structure of each RNA. Linking the location of specific modifications to the efficiency of the translation process, our findings show that mutation of a conserved uracil within a highly stable G+C‐rich stem‐loop decreases translation by approximately 40%, but does not significantly alter the structure of the CITE RNA. However, mutating half of the stem to its Watson‐Crick complement significantly alters the RNA1 CITE structure and decreases translation by approximately 80%. From these results, we propose a model whereby the structure and stability of the stem is required to present the loop sequence (including the conserved U) for translation factor recruitment to the viral RNA. Additionally, this data indicates the locations and RNA structures within the CITE directly involved in translation machinery recruitment, allowing for a better understanding and comparison of the tendencies displayed by BRV as well as other positive‐sense RNA plant viruses.

Support or Funding Information

Colorado‐Wyoming Alliance for Minority Participation in STEM Research Grant MSU Denver College of Letters, Arts and Sciences Dean's Seed Grant MSU Denver Undergraduate Research Conference Student Grant

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermodynamic examination of magnesium ion binding in the M‐box core 2 4×4 internal loop
Authors: Hall Simone , Gilbertson Ellen , Grover Neena
Published: June 2021

RNA binds to magnesium ions through diffuse and site‐specific interactions. Diffuse binding of magnesium ions to the backbone occurs in all RNA and is critical for the stabilization of RNA structure. Magnesium ions also bind to specific sites in particular RNA structures, often with direct Mg2+‐RNA bond formation. In this study, Isothermal Titration Calorimetry (ITC) was used to experimentally measure the enthalpy, binding constants, and Gibbs free energy of diffuse and site‐specific binding of magnesium ions to the core 2 of the Salmonella Typhimurium M‐box riboswitch. This Mg2+ binding bacterial riboswitch upregulates magnesium transporting proteins when intracellular ion concentrations are above an acceptable level. Crystal structure of the M‐box riboswitch RNA shows eight magnesium ion binding sites clustered in three cores. The core 2 of the M‐box riboswitch is made up of a 4×4 adenine‐rich internal loop that binds magnesium ions at residues A63 and A80. RNA constructs were designed to mimic the wild type core 2 and were also modified at positions A63 and A80. A helix construct, with the internal loop deleted, was also utilized. A statistical model for RNA‐Mg2+ binding was used to fit the ITC data to determine the thermodynamic parameters of diffuse and site‐specific magnesium ion binding to the RNA. The models used to fit ITC for ion binding will be presented. In general, we find that the diffuse binding is spontaneous, endothermic and entropically driven with binding constants in the low millimolar range. Site‐specific binding showed micromolar binding constants.

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