bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher Order Thinking is Required in an Anatomy and Physiology Course Utilizing the Flipped Model
Authors: DeRuisseau Lara , Binion Brooke , Willman Terri Anne , Bidwell Allen
Published: June 2021

The flipped classroom was utilized in a two‐semester Anatomy and Physiology course enrolling 50‐80 students with pre‐requisites at a small, Liberal Arts College. With the flipped model, students watched 20‐minute lectures 2 days/week that were recorded via screen capture and accompanied by an outline, power point slides and review questions. During class time, the instructor went over common misconceptions, asked questions requiring higher order thinking and 5 case studies were performed each semester. To determine if the students exposed to the flipped model vs. traditional acquired enhanced higher order thinking skills, exam questions were analyzed via Blooms Taxonomy. To this end, the Blooming Biology Tool was used (Crowe, Dirks, Wenderoth, 2008) to compare assessments across two pedagogies taught by the same instructor where exams scores remained similar. In the flipped classroom vs. traditional, 30% vs. 62% of questions were on the knowledge level, requiring information recall only. Thirty‐eight percent vs. 28% of assessment questions were rated as comprehension in the flipped model vs. traditional, requiring understanding of concepts. Application level questions that required prediction of an outcome in a new scenario were 27% vs. 10%; flipped vs. traditional. The flipped model had the remaining questions as analysis and synthesis, while no questions in the traditional model reached this level. These data demonstrate the level of critical thinking assessed with this particular flipped model is higher compared to the traditional lecture format.

Funding: Echo360 Newcomer's grant to LRD.
How Confident Are You‐ Using Knowledge Confidence to Learn Anatomy
Authors: Nelson Larissa , Webster Samuel
Published: June 2021

The development of new and innovative methods to engage students in learning anatomy is a topic of great discussion. A new approach was developed using knowledge confidence as a tool to help teaching and learning. Sixty year 2 students carried out a short spot test on material covered in year 1. In addition to writing answers, students were asked to rate how confident they were on the answers. Answer sheets remained anonymous to reduce the psychological impact of the test. Upon completion, students were shown the answers and were given the opportunity to reflect on their confidence in their knowledge.

Feedback from students was generally positive and the consensus was that they would like more similar sessions to be introduced in anatomy teaching. The students found it interesting that they often did not trust their own knowledge and did not have confidence in their abilities. In a few cases, students were surprised to find that answers that they were confident in were not always correct. Providing students with this opportunity proved to be a successful method of teaching that allowed students to gauge their own knowledge whilst identifying gaps and encouraging memory recall. Adding confidence into the equation made students reflect on their own thought processes and gave them the opportunity to consider how they react under exam situations and implications of this on their overall attainment and the wider clinical setting.
Dissonance among learning modality interest and achievement in anatomical instruction
Authors: Alsup Kathleen , Derstine Mia , Fox Glenn
Published: June 2021

As creativity and technology offer more choices for anatomical instruction and while instructional hours decrease (Cottam, 1999), curricula can be made more efficient through incorporating effective modalities and tools for learning. To test the effectiveness among competing modalities for learning the human forearm, we assessed a wide array of adjunct instructional methodologies (prosections, plastinated specimens, plastic models, 3D atlas, atlas images, and videos) and their effects on student learning. Participants were randomized into one of six groups to investigate a list of learning objectives focusing on human forearm anatomy utilizing only their assigned modality and selected atlas images. Learning was assessed both quantitatively and qualitatively. Results indicate a positive correlation between interest and engagement with knowledge retention.

Cottam WW. 1999. Adequacy of medical school gross anatomy education as perceived by certain postgraduate residency programs and anatomy course directors. Clinical Anatomy. 12(2): 55‐65.
Knowing Your Limits: How a Time Constraint May Impact Spatial Reasoning
Authors: Roach Victoria , Kryklywy James , Mitchell Derek , Wilson Tim
Published: June 2021

We set out to evaluate how spatial ability (SA) relates to temporal and spatial patterns in average fixation duration (AFD). We hypothesized that SA would be negatively correlated with AFD. Additionally, we asserted that SA would be positively related to AFD in the spatially salient regions of interest (ROIs) of structures presented on a spatial test. The SA test was timed and based on the line drawings of Shepherd and Metzler. Individuals chose whether block pairs were rotations (same), or mirrored (different) images. Eye tracking revealed a significant relationship between AFD and SA (r = ‐.92*, n=10, p<.0001). This suggests that high SA individuals are able to discern and attend to the spatially salient features required to interpret spatial structures more rapidly than low scorers. Through the creation of spatial ROIs, the relationship between SA and AFD in different areas of the images (Bend, Tail, Straight or Empty) was revealed. When the proportional AFD was related to SA, two relationships were found; a negative relationship between the Tail AFD and SA (r=‐.65*, n=10, p=.042), and a positive relationship between the Straight AFD and SA (r=.66*, n=10, p=.037). Thus, low SA individuals dwell on the tail regions, using tail position to solve the question, while the high SA individuals quickly observe the tails, and store the information in their working memory for reference, as they proceed through the test.
Exploring the Inner Working of Anatomy Learning: An Interdisciplinary Approach
Authors: Eastwood Jennifer , Maltese Adam , Deleeuw Josh , Danish Joshua , Goldstone Robert , Motz Benjamin
Published: June 2021

We describe a novel approach to examining the relationship between human anatomy instruction and student learning through a collaboration among neuroscientists, cognitive scientists, and science educators. The project applies electroencephalograph (EEG) technology, which has been used to study memory encoding effects in a laboratory setting, to a naturalistic educational setting. Undergraduate students viewed five 3‐minute lecture segments while wearing wireless EEG headsets. Videos were designed to exemplify typical anatomy lectures, where aspects of textbook images were shown and annotated and narrations provided explanations. Learning was assessed using a pre‐post test of basic concepts that could be pinpointed to a particular timeframe during instruction. Items missed on the pre‐test were considered learned if those items were answered correctly on the post‐test. Five‐second‐wide epochs were identified marking the video segments in which test items were addressed. For 23 complete participant data sets, EEG data were examined corresponding to epochs in which content was identified as learned (41%) or not learned (39%) according to the pre and post tests. Preliminary findings suggest that the use of portable EEG devices to study memory effects may be effectively extended to naturalistic environments, such as a lecture setting. Further work to refine the technique will allow us to examine the effects of various educational methods on student learning, particularly in the discipline of anatomy, which relies heavily upon learning vocabulary, facts, and spatial relationships.
Analysis of the Quality, Themes, and Reliability of Faculty vs. Student Feedback Following Student Group Presentations in a Medical School Curriculum
Authors: Melton Dresden , Edmondson Anna , Nichols Carol
Published: June 2021

Giving & receiving feedback is a cornerstone of medical education. To practice these skills, first year medical students evaluated small group clinical case presentations given by peers. Faculty also evaluated the presentations. The evaluation rubric included descriptive statements, rating scales, & a comment section. The goals of the study were to compare faculty & peer evaluations & to determine if male & female students evaluated peers differently. We hypothesized that peers would give higher scores & fewer constructive comments than faculty & that we would see a difference in feedback based on sex. To analyze peer & faculty feedback, >3,400 comments were sorted into themes under six domains. Peer & faculty feedback themes were similar, focusing on presentation skills & content. Most peer feedback was positive (76%), while faculty provided more constructive narrative feedback (74%). Numeric peer ratings were significantly higher than faculty ratingsfor all domains (p<0.05). Contrary to our hypothesis, there were no differences in themes or numeric ratings between male & female peer evaluators. However, females were more likely to leave comments (62% vs. 55%), but, males were more likely to leave constructively critical comments. Overall, feedback themes were similar among students & faculty, peer numeric ratings were significantly higher, & the percentage of constructive feedback from faculty was greater.
Are Clinical Vignette Questions Harder Than Traditional Questions in Gross Anatomy Course?
Authors: Lu Yuefeng , Conway Marianne , Sinning Allan , Lynch James , Lehman Michael
Published: June 2021

The introduction of clinically relevant materials into Gross Anatomy education has created the need for these changes to be reflected in assessment of anatomy knowledge in the form of clinical vignette questions (CVQs). Although there is widespread support for more integration of clinical scenarios throughout the new basic science curricula, little is known about how first year medical students handle the seemingly more complicated CVQ format. Thus, it is incumbent upon us as anatomists and educators to evaluate the pros and cons about CVQs using other than anecdotal evidences. The purpose of this study is to compare the scores between CVQs and well‐matched traditional questions in a summer pre‐matriculation program. 24 students who had already been accepted into either medical or dental school participated in a six‐week‐long summer gross anatomy program that consisted of two blocks covering the back & upper limb and head & neck accompanied by cadaveric dissection. Both lab and written tests were given for each section. The latter consisted of 50 multiple‐choice questions with 25 CVQs and 25 traditional questions. Our data indicated that students didn't adapt well to the CVQ format in the first exam (P<0.05, n=24). However, results from the second test demonstrated an opposite but statistically insignificant trend between the CVQs and traditional questions (n=22). The current study suggests that students can quickly learn how to answer questions with clinical scenarios after practice. Although a survey indicated that students perceived CVQs as more difficult than traditional questions (n=22), results of the second test indicated that CVQs were not harder than similar traditional questions.
Ultrasound – an essential part of the anatomy educator toolkit?
Authors: Royer Danielle
Published: June 2021

Ultrasound (US) is increasingly taught in the medical curriculum, often concurrent with gross anatomy. A needs assessment was conducted to determine whether US should be part of the anatomy educator toolkit. A 24 question online survey was distributed to faculty in anatomy Master's and PhD programs to determine the current extent of US training and perceptions of US. Respondents represent over half of anatomy programs in the USA. The majority of anatomists surveyed have little/no experience with US. Results reveal that many grad programs provide training in medical imaging, but only a minority includes training in US specifically and less require teaching US. Further work is needed to assess US curricula across programs. Responses to five‐point Likert items show notable trends: most anatomists highly value US to reinforce anatomical concepts and clinical correlates; most consider US useful and marketable; some think US should be required to teach medical students. Integrating US into anatomy training may reinforce concepts and relationships, while providing future anatomists with a foundation in a modern skill, allowing them to meet the changing needs of medical students. For US to become an essential part of the anatomy educator toolkit, anatomy‐centered US curricula emphasizing normal anatomy and tissues, major relationships, and basic scanning skills, should be developed and integrated into anatomy programs.
Google Glass: Integrating Ultrasound with Anatomy
Authors: Lee Vaughan , Bollinger Emily , Shoujaa Adham
Published: June 2021

Google Glass is wearable technology that can supplement instruction for procedures and skills. Google Glass was used to create online instructional modules to conduct ultrasound scans of the hepatobiliary system. Ultrasound imaging is an important tool for physicians at the point of care that requires a working knowledge and understanding of anatomy. TTUHSC/SOM incorporates ultrasound in undergraduate medical education, integrating these skills with anatomical dissections to provide a method of visualizing anatomy in a clinical context. Ultrasound sessions were integrated with dissection sessions of the wrist, neck and liver. These exercises were assessed with five point pre‐ and post‐tests for each session and average student performance increased (p<0.0001) from pre‐ to post‐test; wrist (2.71/4.20), neck (3.20/4.73), and liver (2.89/4.45). Student opinion was assessed by survey on a five‐point Likert scale. Improved understanding of anatomy scored 3.52 and students rated the Google Glass modules significantly higher (3.51, p<0.01) than the previous modules (3.13). Students were neutral regarding their perceived confidence to utilize ultrasound on patients but indicated a desire for better skills assessment and more deliberate practice with ultrasound, which could be enhanced using Google Glass for individual instruction. This project was funded by the TTUHSC/SOM.
Adaptation of an internet‐based DICOM viewer for use with the Medical Imaging Resource Center – Teaching File System (MIRC‐TFS)
Authors: Carter Yasmin , McKenzie Adam , Costa John , Burbridge Brent
Published: June 2021

DICOM is the standard file format for medical imaging; however, storage, transmission and viewing of these images over the internet for teaching and learning purposes outside of the clinical setting presents a challenge as they do not lend themselves well to rapid display and interrogation due to their atypical file format. The aim of this project was to utilize the web technology standard HTML 5 offers to deploy a DICOM viewer that functions well, directly in the user's browser, without the need for complex client side‐applications, application switching, or user login requirements. To achieve this we have adapted the open source HTML 5 DICOM viewer, Oviyam, for integration with the Radiological Society of North America's Medical Imaging Resource Center – Teaching File System (MIRC‐TFS) on a virtual server to facilitate access to DICOM objects for teaching and learning. For learners a plugin was developed to transform the DICOM images in MIRC‐TFS into JPEG format resulting in a reduction in file size allowing users to view the images more rapidly while supporting higher user traffic for viewing images concurrently, as frequently occurs with large class sizes. In addition, a second plugin was developed for BlackBoard Learn that allows instructors to browse the MIRC‐TFS teaching cases and insert them alongside their other course materials. The tool set and image viewing capabilities of Oviyam were enhanced to include annotation tools allowing students to create marked up versions of the images for evaluation of their knowledge. The deployment of this solution has significantly facilitated the interaction of teachers and learners with DICOM based images for use in their academic environment.
Is Skull Shape Related to Aggressive Displays in Pinnipeds?
Authors: Dewar Eric , Crocker Carly , Bauchiero Ann , Livingstone John Paul
Published: June 2021

Some pinniped species display aggression as male‐male vocal displays only. In highly‐aggressive species, outright combat is waged between males, and in other cases aggression can occur between males and females of the same species. Are these differences in behavior manifested in skeletal dimorphism? To answer this, we compared species' aggressive behaviors with skull proportions measured from photographs of museum specimens, including 26 species of seals (n = 143) and five species of fur seals and sea lions (n = 39).

We predicted that there would be a difference in skull shape between males of highly‐aggressive and less‐aggressive species, and also between males and females of species with male‐male combat. On average, the highly‐aggressive species did have relatively wider zygomatic arches (p < 0.001), likely to allow the emplacement of large jaw‐closing muscles, but no difference in relative facial length between these two groups (p = 0.14). Most highly‐aggressive males also had substantial nuchal crests to allow fierce rear‐and‐slam behaviors. Males that were more aggressive also had wider zygomatic arches than less‐aggressive females (p < 0.001). We found a characteristic shape for “less‐aggressive” species, but the skull proportions of highly‐aggressive species were not uniform. We found no difference in skull shape for species in which both sexes displayed similar aggressive levels. From these trends, we were able to reconstruct the relative aggression of several extinct pinnipeds.
Hoxd11 Influences Growth Plate Organization in the Mammalian Pisiform
Authors: Kjosness Kelsey , Hines Jasmine , Reno Philip
Published: June 2021

The typical mammalian pisiform is rod‐shaped, forms from two centers of ossification, and contains an organized growth plate at the palmar end. The pisiform is the only carpal to form a growth plate; however, humans possess a short pisiform that forms from a single ossification center and lacks a growth plate. In the mouse, Hoxa11 and Hoxd11 mutations lead to abnormal shortening of this bone, and variable fusion between bones in the proximal carpal row. We have previously observed that Hoxd11 expression is present in tissues surrounding the developing pisiform in embryonic mice. This study seeks to examine the structure of shortened pisiforms in Hoxd11 mutant mice during development and determine if the growth plate is affected by deletion of one or both Hoxd11 alleles. We conducted histological analyses of the carpus in juvenile mice, comparing wild type (+/+) to heterozygotes (+/‐) and homozygotes (‐/‐) for a Hoxd11 deletion. Pisiforms from +/+ and +/‐ mice have organized resting, columnar, and hypertrophic cartilaginous zones, while ‐/‐ mice have disorganized cartilage where the growth plate typically forms. We also observe fusion of the pisiform and triquetral in ‐/‐ mice, consistent with observations in previous studies. These results support the role of Hox genes in growth plate specification, and indicate that presence of Hoxd11 expression around the developing pisiform may signal formation of its growth plate. Additionally, these data may help to explain the developmental processes underlying evolutionary differences within the piso‐triquetral complex among apes and humans, including the loss of a growth plate and substantial shortening of the human pisiform. This research is funded by the Hill Fellowship awarded by the Department of Anthropology at Penn State.
A new hypothesis: Were Neanderthals susceptible to high rates of otitis media?
Authors: Pagano Anthony , Bluestone Charles , Marquez Samuel , Laitman Jeffrey
Published: June 2021

Otitis media (OM) is among the most common pediatric clinical conditions worldwide but few have considered the importance of the cartilaginous Eustachian tube (CET) and OM over human evolution. This study is the first to attempt reconstruction of CET morphology among Neanderthals and mid‐Pleistocene hominins (mPh). Neanderthals were represented by Gibraltar Forbes' Quarry 1, Monte Circeo 1, and Saccopastore 1 crania. Mid‐Pleistocene Homo was represented by Kabwe 1, Petralona 1, Steinheim 1, and Atapuerca 5 crania. These were analyzed alongside a geographically diverse human growth series spanning from newborns (n=28) to adults (n=185). Measurement was done via a microscribe digitizer and CET dimensions were calculated among three‐dimensional landmark coordinates spanning the osseous CET orifice to points along the medial pterygoid plate. All coordinate data were scaled over centroid size, allowing direct comparison of interlandmark distances. Results indicated that Neanderthals possessed longer CET's (P<0.05) than adult modern humans and mPh and that their choanae are obtusely oriented, resembling human infants. This suggests that anatomical conditions promoting OM among modern human infants and young children likely persisted over longer spans of Neanderthal development, increasing morbidity and impacting their ability to compete with sympatric modern humans.
Notoginsenoside R1 Promotes SGC7901 Apoptosis by Up‐regulating the ProNGF Signaling Pathway
Authors: Yang jinwei , Ma wei , Du bin , Cheng jingru , Guo jianhui , Li liyan
Published: June 2021

Objective

To investigate the affect of Notoginsenoside R1 on human gastric cancer cells (SGC7901) and its relationship with precursor nerve growth factor (proNGF) signal pathway.

Methods

SGC‐7901 was cultured in vitro and randomly divided into normal, control and experimental groups, and then, each group was divided into 24, 48 and 72 h groups. The experimental group cells were co‐cultured with Notoginsenoside R1 at 2 mg/mL, but saline in control groups, and normal groups had no other treatment. After that,3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay was used to detect the cell activity and terminal dexynucleotidyl transferase(TdT)‐mediated dUTP nick end labeling (TUNEL) was used to detect the apoptosis of the cells. Immunofluorescence was used to locate proNGF, p75 and Sortilin expression, and the proNGF, p75 and Sortilin mRNA was detected by Real time Polymerase Chain Reaction (PCR), and Western Blotting was used to detect the protein expression.

Results

1, SGC7901 was successfully cultured. 2, Cell viability in experimental groups was decreased compared with normal or control groups, but the apoptosis was increased. 3, proNGF, p75 and Sortilin mRNA or protein were up‐regulated in experimental groups.

Conclusion

Notoginsenoside R1 can decrease the biological activity and increase the apoptosis of SGC7901, and the mechanism may be by increasing the expression of proNGF apoptosis signal pathway.

* Corresponding author: Jian‐hui Guo (guojianhuikm@163.com) and Li‐yan Li (kmliyanl@163.com)
Extended Cocaine Access Results in a Distinct Epigenetic Alterations to the Homer2 Gene in the Dorsal Medial Prefrontal Cortex
Authors: Ploense Kyle , Carr Amanda , Baker‐Andresen Danay , Li Xiang , Sun Yi , Bredy Timothy , Kippin Tod
Published: June 2021

DNA methylation is a key determinant of gene expression and is implicated in neuroplasticity relevant to addiction. Here, we examine DNA methylation, hydroxymethylation, binding of the transcription factor p300, and mRNA expression within the dorsal medial prefrontal cortex (dmPFC) following limited cocaine self‐administration (1h), prolonged cocaine self‐administration (6h), and saline self‐administration (1h). Rats were fitted with IV catheters and allowed to lever press for saline or cocaine (0.25mg/kg/infusion) in the different access conditions for 20 days. 6h rats exhibited escalation in cocaine intake over the course of training, and had a distinct epigenetic profile and mRNA expression pattern for the Homer2 gene. qPCR analyses revealed an increase in Homer2 mRNA levels in the dmPFC for 6h rats only. The changes in mRNA were accompanied by lower DNA methylation, increased DNA hydroxymethylation, and decreased p300 binding in the Homer2 promoter; these 3 epi‐marks in the promoter allotted for an open transcriptional state in the 6h rats that was not present in the 1h or saline rats that allowed for increased Homer2 mRNA transcription.
Diminished Ang‐2 Expression and Microvascular Density in COX‐2 Knock‐out Mouse Cerebral Cortex in Moderate Hypobaric Hypoxia
Authors: Benderro Girriso , Xu Kui , LaManna Joseph
Published: June 2021

Normal brain function is dependent on continuous and controlled oxygen delivery. Prolonged hypoxia leads to angiogenesis, suggesting a modulatory role for oxygen in expression of angiogenic factors that are responsible for brain microvascular adaptational plasticity. Angiopoetin‐2 (Ang‐2) is one of the critical molecules in brain microvascular remodeling in order to regulate cerebral oxygen homeostasis. The objective of this study was to determine effects of cyclooxygenase‐2 (COX‐2) knock‐out (KO) on Ang‐2 expression and microvascular remodeling in the mouse cerebral cortexduring prolonged moderate hypobaric hypoxia. Three month old male COX‐2 gene KO and wild type (WT) mice were kept in a 0.4 ATM (290 torr) hypobaric hypoxia chamber for up to 21 days. Normoxic littermates were kept in the same room outside the chamber. The mice were sacrificed and brain samples collected were either lysated for Western blot protein analysis or paraffin embedded for immunohistochemistry (IHC), to determine microvascular count. Compared to wild type mice, we observed attenuated Ang‐2 protein induction in COX‐2 KO mice in hypoxic conditions. Baseline capillary count in normoxia is similar in both WT and COX‐2 KO mice, but hypoxia induced angiogenesis (microvascular density) was recorded to be diminished in COX‐2 KO mice compared to wild type cohorts during prolonged hypoxic conditions. These results imply the importance of COX‐2 in expression of important vascular remodeling protein (Ang‐2), and brain microvascular angioplasticity to acclimatize to ambient oxygen availability.

This study was supported by National Institute of Health grant NS 38632.
Thymoquinone Protects Neurons and Astrocytes in Hippocampal Primary Cell Culture from Lead Toxicity
Authors: Rao Muddanna , Shivanandan Smitha
Published: June 2021

Thymoquinone (TQ) is the active constituent of Nigella sativa(NS) seeds. Quinines, of which TQ is the most abundant, accounts for most of the pharmacological properties of NS. TQ possesses anti‐inflammatory, antioxidant and neuroprotective properties. Present study was aimed to investigate the role of TQ in protection of newly born neurons and astrocytes against lead induced toxicity in hippocampal cell culture. Fetal hippocampal primary cultures were divided into four groups: i) Normal Control (NC, n=6) ‐ no treatment, ii) Lead only (LO, n=6)‐ media was replaced with media containing 20µM lead nitrate, iii) Thymoquinone TQ, n=6)‐ media containing 10µM TQ, iv) Lead + TQ, (L+TQ, n=6) ‐ media containing 20µM lead nitrate and 10µM TQ. Cell survival was analyzed by counting number of cells in randomly selected fields. Culture dishes were stained for doublecortin and GFAP immunostaining and the number of neurons and astrocytes were quantified. Data were analyzed with one way ANOVA. The LO cultures showed significantly decreased number of surviving cells compared to NC, TQ, and L+ TQ groups (20‐30% decrease, P<0.001). Cells in the LO group had fewer and shorter processes. The cell death count was significantly decreased (40%, P<0.001) in cultures treated with lead and TQ, compared to LO group. The cultures treated with L+TQ showed significantly increased number of doublecortin positive neurons (40%, P<0.001) and GFAP immunopositive astrocytes (50%, P<0.001) compared to LO group. These data suggest that TQ can counteract lead toxicity and enhance cell survival and differentiation into neurons and astrocytes in culture. (We acknowledge RCF, Faculty of medicine, Kuwait University (Grant No. SRUL02/13 technical support for confocal and phase contrast microscopy).
Intrauterine Growth Restriction Disrupts Nucleosome Positioning at a Growth Hormone Response Element in the Insulin‐like Growth Factor 1 Gene
Authors: McKnight Robert , Callaway Christopher , Yu Xing , Wiedmeier Julia , Lane Robert , Fung Camille
Published: June 2021

Intrauterine growth restriction (IUGR) increases the risk of adult onset diseases such as obesity and type II diabetes. Persistent downregulation of insulin‐like growth factor 1 (IGF1) has been implicated in these diseases. IUGR downregulates liver IGF1 expression in newborn and in day 21 (d21) mice. IGF1 expression at d21 is dependent on growth hormone (GH)‐mediated upregulation at multiple GH response elements (GHRE) including one containing tandem STAT5b binding sites within intron 2 (i2). We hypothesized that IUGR would disrupt prenatal epigenetic programming of the i2 GHRE and prevent postnatal i2 GHRE activation. IUGR mice were generated by thromboxane A2 analog infusion from gestation day 12.5 to term to elicit maternal hypertension. The GHRE chromatin state at the i2 GHRE was assessed by Na‐bisulfite sequencing for DNA methylation and GpC methyltransferase mapping for nucleosome positioning. In normal mice, the GHRE remains hypermethylated throughout gestation and begins to demethylate at d17. By d21 a single nucleosome is displaced. IUGR animals undergo similar DNA demethylation. However, we found two distinct nucleosome patterns at the d21 hepatic i2 GHRE. Some IUGR animals showed no nucleosome displacement, while in others, the nucleosome free region (NFR) was expanded on the 3′ side suggesting the NFR was misaligned. Evidence suggests different GHRE in IGF1 have unique functions and when tandem STAT5b sites are used, both must be functional for the enhancer to work. We speculate the altered NFR spacing could disrupt proper enhanceosome assembly and result in loss/altered GHRE activity.
EVALUATION OF REAL TIME – PCR AS A TOOL FOR DIAGNOSIS OF SCHISTOSOMA MANSONI IN A LOW TRANSMISSION AREA, MONTES CLAROS, MINAS GERAIS, BRAZIL.
Authors: Siqueira Liliane , Oliveira Edward , Gomes Luciana , Senra Souza Carolina , Rabello Ana , Zech Coelho Paulo Marcos
Published: June 2021

The conventional diagnosis of schistosomiasis mansoni is realized by parasitological approaches, especially by Kato‐Katz technique, however in situations of low parasite burden this technique loses sensitivity. Thus, the aim of this study was to evaluate the molecular assay, the Real Time‐PCR for diagnosis and assessment of cure of schistosomiasis. A population‐based study was carried out with 148 inhabitants from a low transmission area in Brazil. To establish the infection rate one stool sample was analyzed by the parasitological techniques Kato‐Katz (24 slides) and the Saline Gradient (1000mg). The Real Time‐PCR was performed using 1000 mg of the same stool sample. To establish the cure rate, the positive individuals were followed‐up 30, 90 and 180 days after the treatment. The prevalence rate obtained by the two parasitological techniques was 31.75% (47/148). The positivity rate was 29.0% (43/148) by Saline Gradient technique, 20.9% (31/148) by 24 K‐K slides, 30.4% (45/148) by Real Time‐PCR. In order to determine the cure rate of 47 positive individuals, they were followed‐up for 6 months, and the cure rates were 100% (39/39), 91.6% (33/36) and 78.4% (29/37), 30, 90 and 180 days after treatment. For the Real Time‐PCR the same cure rates were 100% (39/39), 83.3% (30/36) and 62.1% (23/37), respectively. The results obtained by Real Time‐PCR point to development of a promising diagnostic method and could represent an important step forward to support studies on epidemiology and cure control of the disease, especially when its cost to reduce.
ADAR1‐mediated RNA editing, a novel mechanism controlling phenotypic modulation of vascular smooth muscle cells
Authors: fei jia , Chen Shiyou
Published: June 2021

Vascular smooth muscle cell (VSMC) phenotypic modulation plays a critical role in the development and progression of several proliferative cardiovascular diseases such as atherosclerosis, hypertension, restenosis after angioplasty or bypass, diabetic vascular complications, and transplantation arteriopathy. A hallmark feature of the phenotypic modulation is the down‐regulation of SMC contractile genes. Platelet‐derived growth factor‐BB (PDGF‐BB), a well‐known stimulator of SMC phenotypic modulation, down‐regulates SMC gene expression via posttranscriptional regulation of the related genes. The post‐transcriptional mechanisms involved in SMC phenotypic gene expression, however, remain largely unknown. We found that the down‐regulation of SMC contractile genes is caused by abnormal RNA splicing of their precursor mRNAs (pre‐mRNAs). This abnormal pre‐mRNA editing is facilitated by adenosine deaminase acting on RNA (ADAR), which converts adenosines to inosines (A→I editing). PDGF‐BB induces ADAR1 while down‐regulating the expression of SMC myosin heavy chain (SMMHC) and calponin (CNN). Knockdown of ADAR1 by shRNA restores PDGF‐BB‐blocked SMMHC and CNN expression, demonstrating that ADAR1 plays an essential role in SMC phenotype modulation. In vivo animal studies show that SMMHC and CNN pre‐mRNA is accumulated while their mature mRNA is decreased in balloon‐injured rat carotid arteries. Moreover, ADAR1 is highly induced in media layer SMCs initially, and neointima SMCs subsequently following the injury. Our study suggests that ADAR1‐mediated RNA splicing may also play an important role in VSMC phenotypic modulation and vascular remodeling in vivo.
Identification of Determinants that Govern Processive and Efficient Selenocysteine (Sec) Incorporation
Authors: Shetty Sumangala , Copeland Paul
Published: June 2021

Selenoproteins include essential proteins and are unique as they incorporate selenocysteine (Sec), the 21st amino acid, in response to an stop codon (UGA). Thus selenoprotein expression is a challenge as it requires stop codon redefinition. To date, five essential factors for Sec incorporation have been identified. This includes the mRNA with an inframe UGA codon, a stem loop structure referred to as SECIS element located in the 3′UTR, a dedicated elongation factor (eEFSec), a SECIS binding protein (SBP2) and a unique Sec tRNA Sec‐tRNASec. However the mechanistic details of Sec incorporation still remains unclear. Among the known selenoproteins, Selenoprotein P (SEPP1) is unique mainly as it contains multiple UGA codons and possesses two SECIS elements in the 3′UTR. Thus, full length SEPP1 synthesis requires processive Sec incorporation which in vivo appears to be efficient. The focus of this research is to identify core determinants that govern processive and efficient Sec incorporation. Using 75Se labeling in vitro we demonstrate that selenium supplementation stimulates processive Sec incorporation by 4‐fold thus linking active Sec‐tRNASec synthesis with processive Sec incorporation. We also establish that while the SEPP1 mRNA is inherently processive for Sec incorporation, there exists an interplay between the coding region and the 3′UTR that governs the degree of processivity. Furthermore cellular transfection studies indicate RNA stability as a crucial determinant of efficiency in mammalian SEPP1 synthesis. Thus translation regulation primarily governs processive and efficient Sec incorporation via channeling of Sec incorporation components and also by recruiting novel cis elements.
Role of nuclear factor TDP‐43 in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia
Authors: Buratti Emanuele
Published: June 2021

TDP‐43 is one of the major components of the neuronal and glial inclusions observed in several neurodegenerative diseases such as ALS and FTLD. These characteristic aggregates are a “landmark” of the disease but their role in the pathogenesis is still obscure. At the moment, in fact, very little is known regarding the biological processes that underlie TDP‐43 aggregation and, most importantly, its potential consequences on cellular metabolism.

In our laboratory, aggregate formation has been recently assessed using a novel system capable of inducing TDP‐43 aggregation in experimental cell lines and primary neuronal cultures. Our experiments show that in cell lines and primary rat neuronal cultures the introduction of tandem repeats carrying the 331‐369 Q/N region from TDP‐43 can trigger the formation of phosphorylated and ubiquitinated aggregates that recapitulate many of the characteristics observed in patients (Figure 1A and 1B). We have recently better characterized their formation with regards to: (i) the ability of other regions of TDP‐43 to influence aggregate formation (ii) the composition of the aggregates (iii) the effects of these aggregates on cellular pre‐mRNA splicing regulation (iv) the physiological consequences of inducing these aggregates in Drosophila.

In addition, we have recently started to engineer a wide range of Drosophila models to determine which of the major hnRNP proteins can induce neurodegeneration. For the moment, we have already gathered evidence that the interplay between the Drosophila homologues of TDP‐43 and hnRNP A/B can be critical for neurodegeneration. The final aim of this work is to determine how hnRNP proteins can generally contribute positively or negatively to the action of TDP‐43 in mediating neurodegeneration.
Investigation of how the Saccharomyces cerevisiae Lsm2‐8 proteins bind to the 3´ end of U6 snRNA
Authors: Liao Hong hong , Montemayor Eric , Hardin John , Kondo Yasushi , Nagai Kiyoshi , Brow David , Butcher Samuel
Published: June 2021

Pre‐mRNA splicing is a key step in eukaryotic gene expression that is catalyzed by the spliceosome. The spliceosome contains 5 RNA‐protein complexes called small nuclear ribonucleoprotein particles (U1, U2, U4, U5 and U6 snRNPs). Assembly of the spliceosome from these snRNPs involves numerous complex and dynamic RNA‐protein interactions. The U6 snRNP is comprised of the U6 small nuclear RNA (snRNA) bound to proteins Prp24 and the Lsm2‐8 heteroheptameric ring, the latter of which binds to the 3′ end of U6. Previous research in our laboratory determined the crystal structure of a large fragment of U6 snRNA bound to the four RNA recognition motifs (RRMs) in Prp24. As an extension of that work, we are now investigating the interaction of the Lsm2‐8 ring complex with the 3′ ‐end of U6, using native polyacrylamide gel electrophoresis (PAGE). Previously published data indicated that the 3′ end of yeast U6 snRNA contains either a free 3′ hydroxyl or 3′ phosphate group, whereas most other organisms have a 2′,3′ cyclic phosphate at the end of U6 snRNA. Our preliminary native PAGE data demonstrate that yeast U6 with a free 3′ hydroxyl end has a significantly higher binding affinity for the yeast Lsm2‐8 complex than the same RNA with a cyclic phosphate. We hypothesize that a free 3′ hydroxyl group may be important for high affinity binding of the Lsm2‐8 complex and subsequent crystallization of the yeast U6 snRNP. Attempts to crystallize the complete U6 snRNP are currently underway.
Structure and Function of the HTLV‐1 pro‐pol Frameshift Site
Authors: Chadeayne Devon , Davis Summer , GreyEyes Shawn , Knewitz Allison , Stelmaszek Jordan , Mouzakis Kathryn
Published: June 2021

Human T‐cell leukemia virus type I (HTLV‐1) is a human retrovirus. Replication of retroviruses, such as HTLV, is dependent upon synthesis of viral structural and enzymatic proteins. Translation of HTLV's enzymatic proteins depends upon two ‐1 programmed ribosomal frameshifts. While ‐1 programmed ribosomal frameshifting and the slippery sequences of the HTLV‐1 gag‐pro and pro‐pol frameshift sites have been established, the frameshift efficiencies and the structures within these sites have not been determined. Each frameshift site includes a heptanucleotide slippery sequence followed by a downstream RNA structure. In HTLV‐1's pro‐pol frameshift site, a pseudoknot structure is predicted downstream of the UUUAAAC slippery sequence. We hypothesize that this pseudoknot structure contributes significantly to frameshift efficiency. To test this hypothesis, we designed three variant frameshift sites, each of which disrupts a helix important to the pseudoknot structure. We report the in vitro frameshift efficiencies for the wild‐type and three variant HTLV pro‐pol frameshift sites. These efficiencies were measured using an in vitro dual‐luciferase frameshift assay. Preliminary data suggest that the pseudoknot structure is important to frameshifting.
Construction of a Tryptophan‐less P‐glycoprotein by Directed Evolution for Mapping Drug Binding Sites
Authors: Swartz Douglas , Urbatsch Ina
Published: June 2021

P‐glycoprotein (Pgp) is an ATP Binding Cassette (ABC) transporter that functions as a multidrug efflux pump, causing drug resistance in cancer and other diseases. Polyspecific drug transport is likely achieved by drugs binding within sub‐pockets of a central cavity formed by the transmembrane domains (TMDs). However, little is known about the nature or location of drug binding in Pgp. Our objective is to use site‐specific tryptophan (Trp) fluorescence to map drug‐protein interactions. This has not been previously applied to Pgp due to 11 native Trps that contribute to a high intrinsic fluorescence. We used directed evolution to remove the native Trps while maintaining protein expression and function. Two successive rounds of site saturation mutagenesis were used to replace the 8 TMD Trps with the 19 other amino acids in Pgp. The mutants were subjected to a stringent selection process in Saccharomyces cerevisiae, using well established Pgp functional assays. Surviving mutants were sequenced to identify the amino acids that could successfully replace Trp at each site. This culminated in the identification of a mutant that lacks all 8 TMD Trps, and exhibits similar protein expression and activity as wild‐type Pgp. It also has significantly reduced the intrinsic Trp fluorescence signal, making it suitable for re‐introduction of Trps in the TMD to measure drug binding. Work is ongoing to remove the 3 remaining Trps from this mutant to produce a fully Trp‐less Pgp. We expect that this mutant will be a versatile new tool for studying Pgp using site‐specific Trp fluorescence.
Differential Regulatory Response for the Δ9 Desaturase in Saccharomyces cerevisiae Based on Fatty Acid Species and Membrane‐Altering Conditions
Authors: Ochs Matthew , Schriemer Clara , McDonough Virginia
Published: June 2021

Unsaturated fatty acids (UFAs) regulate the expression of OLE1, the gene encoding the Δ9 desaturase. The transcription factors Mga2p and Spt23p are responsible for at least part of this regulation. In this study, it was found that the species of unsaturated fatty acids and environmental conditions that change membrane conditions affect regulation of OLE1. Yeast grown in 16:1Δ9, and 18:2Δ9,12 strongly down‐regulate transcription of OLE1, while 18:1Δ9 and 17:1Δ10 had a smaller effect in the wild‐type strain. As it does not appear that the regulation is responding in a consistent manner to either authentic end product, the presence of a single double bond, or carbon number, we hypothisized that the sensor may be responding to the fluidity of the membrane. In order to investigate this, growth temperature and growth in saturated or trans‐UFAs was examined, and found that while these conditions may have an effect on expression, it was not in the direction hypothesized by membrane conditions. Western blot analysis of processing of Mga2p and Spt23p showed that most Spt23p protein is in the inactive form when cells are grown in the 17:1Δ10, and 18:2Δ9,12, while other supplement conditions for Spt23p and all supplement conditions for Mga2p result in protein in both the active and the inactive form. These results indicate that several systems may be involved in regulation of OLE1 in response to UFA species and membrane conditions.
A study on Manduca sexta perilipin 2: cloning, developmental expression and characterization of the recombinant protein
Authors: Chen Xiao , Firdaus Sarah , Daniel Stuart , Howard Alisha , Soulages Jose , Arrese Estela
Published: June 2021

Animals accumulate excess energy as triglycerides (TG) in cytosolic organelles, the lipid droplets (LD), in which the lipid core is surrounded by phospholipids and a dynamic coat of proteins. The perilipins (PLIN) are a distinctive group of LD proteins involved in TG storage. PLIN 1‐2 occur in insects. PLIN2 is required for normal storage of TG in the fruit fly. To expand our knowledge on the function of PLIN2 in insects, we have cloned M. sexta PLIN2 and profiled the mRNA and protein levels in several TG‐storing tissues. PLIN2 was predominantly expressed in ovary where its abundance followed an inverse relationship with the content of TG. In the fat body PLIN2 was more abundant in the lipid‐mobilizing tissue of adult animals than in the lipid‐storing tissue of larval insects. In the larval fat body, PLIN2 increased at the beginning of the development when the tissue is storing lipids but declined at the end of the period when TG storage peaks. However, an increase of fat body PLIN2 that correlated with TG synthesis was observed during the re‐feeding of starved larval insects in which fat body lipids were depleted by starvation. In the midgut, PLIN2 expression correlated with TG synthesis. PLIN2 was exclusively localized to LDs. PLIN2 was a soluble protein with a folded conformation that binds to phospholipids. In agreement with the intracellular localization, lipid‐bound PLIN2 showed higher stability than the lipid‐free form. Our study suggests that MsPLIN2 is not critical for LD stability. PLIN2 seems to be required for the early deposition of lipids presumably stored in new LD. In this context, the high abundance of PLIN2 in the lipid mobilizing fat body suggests an involvement of PLIN2 in TG re‐synthesis.