bluuebunny/crossref_metadata_2025 huggingface dataset:
Finding cancerous anaplastic cells with image analysis
Authors: Nicol Kathleen , Plaskow Mark Edward , Barr Thomas , Billiter Dave
Published: June 2021

Objective

Advancing cancer research and location with image analysis

Method

The application was built using Microsoft technologies including Visual Basic 6.0, Visual Basic .NET, C++.NET, and SQLServer 2000‐2005. Key points of interest for anaplastic cells are stored in a database during the batching process, allowing for fast retrieval of areas for visual confirmation and analysis.

Results

87% of all anaplastic nuclei analyzed were located, counted, and highlighted for the using pathologist/reviewer.

Conclusion

Lack of differentiation in cell nuclei is a constant marker for malignancy. With technology, programming, and image analysis breakthroughs such diffusion of nuclei is able to be located for pathologists across cases, studies, slide libraries, etc. using digital pathology. Our team's pixel analysis and experience makes possible the location of nuclei, cytoplasm, and connective tissue areas within digitized slides for measuring, highlighting, counting, etc. Pathologists are able to get electronic decision support in rapidly finding certain features for malignant cancerous tumors.
Promoter methylation of P15(INK4B) gene is possibly associated with parvovirus B19 infection in adult acute leukemias
Authors: Serin Mehmet Sami , Yalcin Atilla , Emekdas Gurol , Tiftik Naci , Aslan Gonul , Eskandari Gulcin , Tezcan Seda
Published: June 2021

Myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) are clonal stem cell diseases. Pathogenesis of these diseases, has not been clarified definitely. DNA methylation is the main epigenetic modification frequenly seen in humans. Inactivation of the genes that regulate cell growth, differentiation and apoptosis may play a role in cancer pathogenesis. P15(INK4B) and its functional homologue, P16(INK4A) inhibit cell cycle through cyclin‐dependent kinases 4 and 6 and regulate G1 to S transition. In this study, we examined P15(INK4B) gene promoter methylation in 13 patients with MDS and 32 patients with acute leukemia (26 AML, 6 ALL) by methylation spesific‐PCR. We have also examined its possible association with parvovirus B19 infection by PCR. P15(INK4B) methylation frequency was higher in acute leukemia patients than that of non‐malignant patients. When the patients with MDS were included, no difference was found between these groups regarding the methylation status. The possible association between P15(INK4B)methylation and parvovirus B19 infection was observed in acute leukemia patients. To our knowledge, this is the first report showing the possible association between P15(INK4B) promoter methylation and parvovirus B19 infection in adult acute leukemia. This study was supported by Mersin University BAP TF DTB AY 2004‐3.
The ontogeny of mRNA expression of the genes from the SHFM3 critical region in chicken
Authors: Chryst‐Ladd Megan , Maze Jennifer , Schwartz Charles E
Published: June 2021

The split‐hand/split‐foot malformation (SHFM) is a congenital limb malformation that is characterized by absence or reduction of the central digital rays of the hands and feet. At one of the six well established loci, SHFM3 at chromosome 10q24, tandem duplications have been associated with the SHFM phenotype in humans; the smallest reported includes the genes POLL, BTRC, DPCD, and a portion of FBXW4. SUFU, a gene near the duplicated region, has been shown to be overexpressed in SHFM3 patients. RNA collected from chicken embryo limb buds (E2‐6), limbs (E7‐13), and wings (E14‐15) was analyzed for expression of the homologs of these genes by RT‐PCR. Poll expression was present in limb RNA at stages E2‐E15, although barely detectable. Expression of fbxw4 was detected in stages E3 through E15. Both sufu and dpcd expression was detected at E2, with no expression at E3, and the intensity of the bands varied for E4‐15. These results suggest that these four genes are expressed in the limb during development.

To better understand how the expression of these genes correlates to the stages of limb development, E3‐15 cDNA was analyzed using quantitative PCR (qPCR) by comparing their expression to that of a control gene. Early results indicate the timing of the expression of these genes correlates to the end of digit formation on the chicken limb. Future studies will investigate the localization of these genes in the developing chicken limb.
Homo schlubicus: the end product of 6 million years of useless parts tagging along
Authors: Laitman Jeffrey Todd , Smith Timothy D
Published: June 2021

Over the last 6 million years our hominid lineage has morphed into our supposedly majestic species, Homo sapiens. A fine‐tuned brain, carefully honed vocal tract, and distinctive musculoskeletal frame have positioned us as the lord of our planet. Yet our regal corpus has brought with it a host of odd, often useless, sometimes dangerous, baggage. These include reductions (vestiges, such as the coccyx), useless leftovers (atavisms, such as supernumery teeth) and odd byproducts (spandrels, such as the cavities of paranasal sinuses). Less often considered are longitudinal hangers‐on (which could be termed "longitudinal vestiges") that remain as we age, living, as it were, past the original "warranty" of our species. Indeed, the nexus of longevity and lifestyle yields an array of parts that marginally work: ears that can not hear, eyes that fail to see, knees that do not bend, taste‐buds that can not taste, and parts that leak, squirt and swell when we least want (can anyone say "prostate"?). The end product is a species whose sapient nature may be within, but whose corporeal being has become ungainly, un‐functional and overall unattractive, or to borrow the more descriptive term from informal Yiddish, we've become an unfortunate "new" species of the 21st century: Homo schlubicus
Aging imposes limitations on coronary vessel adaptations.
Authors: Tomanek Robert Joseph
Published: June 2021

Our understanding of structural and functional changes occurring during the lifespan are limited by the nearly exclusive use of young experimental animals. Studies that have utilized middle‐aged and senescent animals have shown that many systems and organs undergo age‐related changes. Evidence from my laboratory supports the hypothesis that coronary angiogenesis and arteriogenesis are limited by age. These limitations are due, at least in part, to lower expression of growth factors in response to stress, i.e. myocardial infarction and exercise training. Data from other laboratories point to age‐associated endothelial dysfunction, which includes changes in endothelial nitric oxide, endothelin‐1 and angiotensin II. Limitations in bone marrow‐derived endothelial precursor cells and enhanced ROS production are two other topics receiving attention in the last few years. This review focuses on the structural and functional changes in the coronary vasculature which are 1) present under baseline conditions and 2) revealed when acute or chronic stress are imposed and adaptations are required. The relationship of disease states and aging will also be reviewed.
Evidence that Farnesoid X Receptor ligand through SF‐1 responsive element inhibits aromatase expression in Leydig tumor cells.
Authors: Andò Sebastiano , Malivindi Rocco , Rizza Pietro , Gu Guowei , Panza Salvatore , Catalano Stefania
Published: June 2021

The Farnesoid X Receptor (FXR) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors, that heterodimerizes with the Retinoid X Receptor (RXR).

FXR, normally present in the liver and in the gastrointestinal tract, has been characterized as a bile acid receptor, but it is also expressed in nonenterohepatic tissues, including kidneys, adrenal gland, heart, vascular tissue, thymus, ovary, spleen, breast and testis.

In this study, we investigated how transactivation of FXR by a specific ligand, chenodeoxycholic acid (CDCA), may modulate aromatase expression in R2C Leydig tumor cells.

Upon prolonged exposure to CDCA we observed a strong decrease of aromatase expression, in terms of protein content, as revealed by immunocitochemistry and western‐blotting analysis, mRNA and aromatase enzymatic activity.

Transient transfection experiment, using vector containing rat aromatase promoter PII, evidenced that CDCA reduced basal aromatase promoter activity. Specific mutagenesis studies, EMSA and ChIP assay, revealed that these inhibitory effects were due to FXR binding to SF‐1 responsive element present in the aromatase promoter region.

Our findings address how FXR ligands may perspectively represent potential pharmacological tools to inhibit local estrogen production sustaining Leydig cell tumor growth and progression
Dynamic interactions between herpes simplex virus and amyloid precursor protein: a novel disease mechanism
Authors: Cheng Shibin , Novikov Aleksey , Ferland Paulette , Bearer Elaine L.
Published: June 2021

Herpes simplex virus (HSV) is a neurotropic virus that causes either a chronic infection cycling between active and latent infection, or a rapidly progressive lethal encephalitis. The consequences of chronicity in the nervous system have been little studied, but may, like HPV and HIV, lead to deleterious effects in the long term. HSV travels inside neuronal processes to set up latency in the neuronal nucleus. Despite its world‐wide prevalence, little is known about the molecular mechanisms of HSV transport. We recently reported that HSV is physically associated with amyloid precursor protein (APP), the major component of senile plaques, which encodes a receptor for the anterograde transport machinery. Thus, HSV may exploit APP to promote its transport. Here we show that HSV infection alters the expression and distribution of cellular APP. In cells synchronously infected, GFP‐labeled HSV capsids (90.2±5.2%, n= 930) co‐localized with APP. By live confocal imaging, GFP‐capsids and APP‐mRFP travel together inside cells. Association with APP‐vesicles increased ability of capsids to move and their average instantaneous velocity (from 0.3 to 0.4 µm/s). Our results reveal a dynamic interaction between HSV particles and cellular APP that influences the transport and distribution of APP, suggesting a potential link between HSV infection and amyloid deposition. Supported by NINDS NS046810.
Toll‐like receptor 9 (TLR9) expression is correlated to progression of esophageal squamous cell carcinoma
Authors: Kauppila Joonas Henrikki , Takala Heikki , Selander Katri , Karttunen Tuomo , Saarnio Juha , Lehenkari Petri
Published: June 2021

Toll‐like receptors (TLR) identify a wide variety of microbial ligands and endogenous DNA fragments from apoptotic cells. TLR9 is located in endosomes and it recognises CpG‐DNA. TLR9 activation induces cell invasion in a model of breast cancer, suggesting a role for infections or tissue lysis in the pathophysiology behind cancer invasion. In this study we describe an expression pattern of TLR9 in esophagus samples varying from esophageal squamous cell dysplasia to squamous cell carcinoma. We have found strong positive staining in dysplastic samples which is differently organised to that of the healthy controls. Our data clearly indicates that TLR9‐expression, in addition of its role in immunity, is a marker of differentiation, proliferation or invasiveness in esophagus carcinoma sequence. This interpretion is supported by strong basal expression of TLR9 in the esophageal epithelium, where the stem cells are thought to reside, and weakening of the expression towards the lumen, where peeling off of the epithelia takes place. Also in the neoplastic changes, the epithelial expression of TLR9 becomes very prominent. This new staining might enable specific early recognition of malignant transformation and be a basis for new kind of understanding in the mucosal immunology and cancer. This study was supported by Finnish Medical Foundation and Oulu University Hospital grants.
Using Anatomy to lure underrepresented WV high school youths to health science careers
Authors: Klinkhachorn Penprapa S.
Published: June 2021

The Health Science & Technology Academy (HSTA) was created in 1994 at WVU as a community‐campus partnership. The goal is to expose these students to health science and technology, to further their interest in college, and to pursue as careers in WV. Programs are Fun with Science, Forensic Science, & Biomedical Summer Institute. HSTA eligible students are African American, financially disadvantaged, living in rural areas, or the first in their family to go to college. Anatomy is one of ten biomed groups offered. Activities are designed with student's age in mind to keep it fun, engaging, and interactive. Props are used to stimulate critical thinking skills. Students learn how to check the vital signs and perform physical exams. A gross lab tour offers a glimpse of how college students learn. Jeopardy is used as a tool to test what they learn in a fun game format. They learn a lot from their group inquiry (95%), and from visiting the gross lab (94%). We influence their decision to go to college (75%), to choose health careers (84%), and they are satisfied with the overall program (82%). 97% of them go to college vs. 56% of WV students. In conclusion, HSTA is very successful in providing opportunities for these youths and increases the number of health science professionals in WV, that otherwise may have been lost through the cracks. Supported in part by NIH‐NCCR # 10009490.28.1003654R
Production of instructional videos to use in anatomical dissection courses
Authors: Oliver Peter D , Gibson Edward , Seamster Matthew , McClugage Sam , Sedlmayr Jayc , Cork John , Sarphie TG , Pelias Marilyn , Swartz William
Published: June 2021

Faculty members at LSUHSC teach anatomy dissection courses to dental, medical, allied health and nursing students. Our department now operates an anatomy media studio equipped with lighting, cameras, sound equipment, and editing software. The aim of our program is to produce high quality videos to demonstrate various aspects of human anatomy. An arcing camera boom has been constructed to help render a three dimensional view in the recorded videos. A series of video clips would include osteology, surface anatomy, and a short demonstration of how to execute the dissection exercise to be conducted in the laboratory that day. Numerous large LCD screens have been installed in the anatomy lab to present the videos. For example, we have begun a sequence on the thorax, which includes an introduction to the bony thorax, surface landmarks, and dissection instructions using a cadaver to demonstrate how to remove the chest plate, lungs and heart. Last summer, dental students were involved in the dissections, recording, and editing of the material. The students have responded favorably to our efforts and we plan to expand our program and to involve students in the summer months when they are available between the first and second years of their respective professional schools.
Performance tasks as active‐learning tools for non‐science majors
Authors: Furge Laura Lowe
Published: June 2021

The goal of this project was to introduce students to critical thinking and problem solving in an area relevant to a course theme by use of a CLA‐like performance task (CLA is "Collegiate Learning Assessment", a program being widely used for assessment of value added education). The project described here relates to human papilloma virus (HPV) vaccines. The task sets up a real‐life scenario for a context for the assignment (in this case, a factitious health initiative that might include an HPV vaccine). After reviewing provided documents from various sources, students were required to answer questions relating to use of the vaccine and to how the vaccine might be funded using evidence to support their argument. The in‐class portion was followed by an out‐of‐class, collaborative assignment on the same topic. The project was designed to enhance interest in science, social issues, ethics, and disease. In addition, the project stresses the importance of assessment, of cooperative learning, of theme‐based learning, of building critical thinking skills, and of problem‐based learning. Student evaluation is also presented. (Supported by Teagle Foundation and Hutchcroft Funds of Kalamazoo College).
Isoflurane opens mitochondrial KATP channel and enhances ROS production in human embryonic stem cells derived cardiomyocytes
Authors: Sepac Ana , Sedlic Filip , Si‐Tayeb Karim , Bienengraeber Martin , Park Frank , Lough John W , Duncan Stephen A , Bosnjak Zeljko J
Published: June 2021

Transplantation of cardiomyocytes derived from human embryonic stem cells (CM‐hESCs) is promising tool for recovery of impaired cardiac function. We investigated whether cardioprotective agent isoflurane, elicits cardioprotective mechanisms in CM‐hESCs, which may suggest its role in protection of CM‐hESCs. CM‐hESCs were obtained by differentiation from H1 hESC line. Beating areas indicated the presence of CM‐hESCs which was confirmed by immunostaining for cardiac specific proteins, cardiac troponin I and titin. Living CM‐hESCs were labeled with eGFP under transcriptional control of the ventricular myosine light chain promoter, introduced into the cells using the lentiviral vectors. Once dissociated, eGFP‐positive cells were detected using confocal microscopy. Isoflurane depolarized mitochondria, as measured by an increase in TMRE fluorescence (110±1% of baseline) via opening of mitochondrial K channels (mitoKATP), since the channel inhibitor, 5‐hydroxydecanoate, attenuated the effect (103±1% of baseline), and diazoxide, mitoKATP opener mimicked the effect of isoflurane (109±1% of baseline). Isoflurane enhanced reactive oxygen species (ROS) production, measured by increase in DHE fluorescence (112±3% of baseline). In conclusion, isoflurane enhances ROS production and opens mitoKATP, two important mediators of the preconditioning signaling cascade.

Supported by HL034708
Comparative expression and transcriptional networks of human‐mouse syntenic genes during osteoblastic differentiation of bone marrow mesenchymal stem cells
Authors: Dernowsek Janaina Andréa , Fontana Vanessa , Evangelista Adriane F , Junta Cristina M , Marques Márcia M C , Macedo Claudia , Simões Belinda P , Prado Karina F B , Passos Geraldo A S
Published: June 2021

Osteoblasts are cells differentiate from mesenchymal precursors in a complex process regulated by transcription factors that regulate the expression of key genes. Many of these genes present human‐mouse synteny, i.e, they share sequence identity and the same physical distribution in chromosomal regions. Our aim in this study was to compare the transcriptional profiling and network interactions of syntenic genes during the osteogenesis in vitro. The mesenchymal stem cell (MSC) were collected from of the bone marrow of adult healthy donors or of 4 week‐old mice. SC cells were cultured in α‐MEM medium with fetal bovine serum. After expansion, cells were transferred to differentiation medium containing. Total RNA was prepared from undifferentiated and differentiated MSC after 24 and 48 h and 7 days. Fluorescent Cy3/Cy5 cDNA probes from total RNA of these cells were generated and hybridized with a cDNA microarray. The gene expression profiling was analyzed by hierarchical clustering and to reconstruct the regulatory transcriptional networks, we used the GeneNetwork program. A set of 227 syntenic genes were identified from which 108 were comodulated, i.e, genes presenting the same expression profiling. Gene profiling, although useful for characterizing the osteogenesis and identifying the expression signatures of syntenic genes. The GeneNetwork program was a useful tool allowing the detection of seven syntenic gene nodes (SHMT1, CACNB3, CASP8, GFM2, XPC, SYNGR2 and SDF2), which establish interactions with other genes. These are then considered as candidate genes controlling the osteogenesis.

Grant Funding Source

FAPESP, CNPQ, FAEPA
Mouse Latissimus Dorsi as a model system for evaluating tissue engineered skeletal muscle
Authors: Machingal Masood A. , Vishwajit Sonia , Bishwokarma Bimjhana , Zhao Weixin , Yoo James J , Christ George J.
Published: June 2021

The inability to engineer clinically relevant functional muscle tissues in vitro remains a major obstacle to the development of successful procedures for skeletal muscle (SKM) replacement in vivo. The goal of these studies is to further develop the technologies required for the creation of skeletal muscle biomimetics that can be used clinically for restoration of muscle function in vivo. As a first step in this direction we have examined the rodent latissimus dorsi (LD) as a model system for testing functional replacement of native SKM with tissue engineered SKM constructs (TE‐SKM). Porcine bladder acellular matrix was prepared and rat muscle precursor cells were seeded onto the matrix and preconditioned in a custom designed, computer controlled cyclic strain bioreactor for one week prior to subcutaneous implantation in nu/nu mice onto LD for 1 month. The size of the TE‐SKM was 15 mm X 3mm X 0.35 mm; dimensions similar to that of the native mouse LD. TE‐SKM isolated after one month implantation showed a maximal isometric tetanic response of 42.9 mN/mm2 which was 26 % of that of native LD isolated from contra lateral side, but 50‐ and 3500‐fold greater than that previously observed for TE‐SKM. To our knowledge, this is the largest specific force yet recorded for TE‐SKM on a decellularized matrix. In short, the LD appears to be an attractive model for further studies of functional SKM replacement with TE‐SKM constructs.
Imaging of Cellular Epiblastic and Sub‐Epiblastic ECM Movements in Avian Primitive Streak Stage Embryos Elucidate Morphogenetic Mechanisms
Authors: Zamir Evan , Rongish Brenda , Little Charles
Published: June 2021

A century of scientific articles has advanced the concept that embryonic cells move ("migrate") in an autonomous fashion. In contrast, the (convective) motion of the extracellular matrix (ECM) has been largely ignored. We developed imaging methods that can measure the extent embryonic cells move ("crawl") relative to the ECM. We have applied these techniques to studying the movements of dorsal/ventral (epiblast/mesoderm) tissues in early‐stage quail embryos. Our time‐lapse data show that epiblastic cells largely move in concert with a sub‐epiblastic extracellular matrix in Stage 2‐3 primitive streak quail embryos. In other words, there is little cellular motion relative to the extracellular matrix scaffold ‐ both components move together as a tissue. The extracellular matrix displacements exhibit bilateral vortical motion, convergence to the midline and extension along the presumptive vertebral axis ‐ all patterns previously attributed solely to cellular "migration". Our time‐resolved data pose new challenges for understanding how extracellular chemical (morphogen) gradients, widely hypothesized to guide cellular trajectories at early gastrulation stages, are maintained in this dynamic extracellular environment. In general, we hope that our new techniques and findings will encourage others in the field to be more rigorous in defining and quantifying cell migration in the embryo.
The effect of early Bone Morphogenetic Protein (BMP) activation on craniofacial development
Authors: Chong H. Jonathan , Marcucio Ralph
Published: June 2021

Bone morphogenetic proteins (BMPs) are a family of growth factors that play important roles in craniofacial development. Inhibition of BMP signaling in the head mesenchyme of early stage chick embryos leads to craniofacial defects. The aim of this study was to activate the BMP pathway and investigate effects on craniofacial development. Activation of the BMP pathway was achieved via placement of BMP‐2‐soaked beads in the head mesenchyme or injection of DF‐1 fibroblasts transfected with an avian retrovirus expressing BMP2 or BMP4 (RCAS‐BMP2 and RCAS‐BMP4, respectively). Placement of BMP‐2‐soaked beads led to significant defects affecting the brain, eyes, face, and jaw structures. Phenotype severity appeared to be dose‐dependent, and TUNEL analysis revealed an increase in cell death compared to controls. Injection of DF‐1 cells transfected with RCAS‐BMP2 or RCAS‐BMP4 yielded similar results. In conjunction with previous studies examining BMP inhibition in the head mesenchyme, these data suggest that a delicate balance in the degree of BMP signaling regulates normal craniofacial morphogenesis.

Grant Funding Source

NIH/NIDCR R01DE018234 to R.M. and HHMI Medical Research Training Fellowship to H.J.C.
Analysis of the effects of nickel, cobalt and ROS on Nodal signaling and axis specification in sea urchin embryos
Authors: Venuti Judith M. , Agca Cavit , Klein William H.
Published: June 2021

Nodal is asymmetrically expressed in the early sea urchin embryo and is the earliest indicator of the oral ectoderm territory. Nickel (Ni) treatment oralizes ectoderm and expands nodal expression, but the mechanism of Ni's action is unclear. In other systems, Ni generates reactive oxygen species (ROS) and stabilizes hypoxia inducible factors (HIFs). Since Cobalt (Co) has similar effects to Ni, we were interested in determining if Co similarly influenced sea urchin development. We show that, Co, like Ni oralizes ectoderm and upregulates nodal expression. Although neither metal induces nodal transcription precociously, both upregulate nodal expression after its initial activation. Because redox signaling is thought to be an important component of axis specification in sea urchin embryos we investigated the consequences of elevating ROS by other means. Both hypoxia and paraquat elevate ROS in vertebrate cells. However, hypoxia and paraquat treatment do not oralize sea urchin embryos or upregulate nodal significantly; instead, ectoderm specification is repressed while specification of other lineages is unaffected. Furthermore, an analysis of sea urchin HIFs suggests that Co and Ni are unlikely to influence ectoderm specification through the HIF pathway. Supported by the LSUHSC Enhancement Fund (JMV) and the Robert A.Welch Foundation (WHK).
Right‐to‐left shunt, right aortic arch and aberrant left subclavian artery presenting with thoracic outlet syndrome (TOS) and migraine: MRI, MRA, MRV and Transcranial Doppler (TCD)
Authors: Saxton Ernestina H. , Collins James D. , Ahn Samuel S. , Gelabert Hugh , Carnes Alfred
Published: June 2021

Patients with thoracic outlet syndrome have severe migraine with or without aura. There is an increased prevalence of right‐to‐left shunt, mainly patent foramen ovale (PFO) in migraineurs with aura and an increased prevalence of migraine in patients with PFO. Atrial septal defects may occur as well. Studies indicate that migraine is a relative risk factor for stroke. Cerebral embolism rarely may occur as a complication of TOS. A possible mechanism of stroke in both migraine and TOS may be passage of microemboli or toxic substances. Over 500 patients have presented with symptoms of migraine and a clinical diagnosis of TOS. We have also evaluated over 100 to assess cerebral blood flow and microembolic signals. Monitored multiplanar images with abduction external rotation and 2D Time of Flight MRA/MRV without contrast were acquired on a 1.5 Tesla GE Signa LX unit, 44 cm field of view, 521 x 256 matrix and saline water bags to enhance signal to noise ratio. A Portable Nicolet/Viasys Companion III Transcranial Doppler Unit with a 2MHZ transducer was used to interrogate the arterial system of the Circle of Willis. We present two patients with severe migraine and TOS who were found to have right aortic arch and aberrant left subclavian artery (Type II) on MRI/MRA/MRV and right to left shunt on transcranial Doppler (TCD). Neither patient has experienced an ischemic event and is treated with aspirin for primary prevention of stroke.
Observations by a middle‐aged university anatomy teacher and suggestions for curricular change
Authors: Darda David M
Published: June 2021

In general, as biology departments at four‐year colleges and universities have bolstered the curriculum in cell and molecular biology and emphasized the increasing importance of ecological study, the amount of curricular space for organismal level courses has decreased. In the anatomical realm, classical embryology and histology have been pushed aside to make room for other priorities. Each of the few remaining courses offers a limited view of anatomical science as a whole. Given that students struggle to make room for even a single anatomical course in their undergraduate program, they are left with an incomplete perspective on animal form and function and are underprepared for the rigors of medical school and other professional programs. In this presentation, I summarize initial data resulting from an informal survey I conducted among the four‐year institutions in Washington State and suggest how the undergraduate anatomy curriculum might be modified to meet student needs (both pre‐health career students and "regular" biology majors) given the constraints of staffing and time. While here I concentrate primarily on content, attention must also be paid to pedagogy and assessment.
Structuring a structures course around core competencies
Authors: Gregory Jeremy K , Camp Christopher L , Jacobs Sarah M , Chen Laura P , Alexander Cara J , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

Recent curricular redesign at Mayo Medical School integrated radiology into first‐year gross and developmental anatomy. The resulting six‐week (120 contact hours) Human Structure block teaches gross anatomy through dissection, radiologic imaging, and correlation with embryology. To further promote holistic physician‐development, refinement of the Human Structure course has been driven by the six core competencies of the Accreditation Council for Graduate Medical Education (ACGME): patient care, medical knowledge, practice‐based learning, interpersonal skills, professionalism, and systems‐based practice. We report here more than 20 educational interventions built into the Human Structure block that may serve as a model for incorporating the ACGME core competencies into early medical education. The block emphasizes clinically oriented anatomy, invites self‐ and peer‐evaluation, provides daily formative feedback through an audience response system, and employs team‐based learning. The course curriculum includes didactic briefing sessions and roles for students as teachers, leaders, and collaborators. Third‐year medical students serve as teaching assistants, enhancing their own knowledge of anatomy, bettering their skills as medical educators, and facilitating group communication and critiques. This work was supported by the Mayo Clinic Department of Anatomy.

Grant Funding Source

None
Learning to lead: Student perceptions of team leaders in medical school
Authors: Chen Laura PeiHan , Jacobs Sarah M , Gregory Jeremy K , Alexander Cara J , Camp Christopher L , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

Early medical education courses provide opportunities for promoting leadership in future physicians. Our research sought to integrate explicit activities of teamwork and leadership into the first‐year gross anatomy course and assess student leaders through self reflection and team evaluation. Fifty first‐year medical students were assigned to three‐ or four‐member dissection teams with group leaders designated on a rotating basis. Teamwork was emphasized through daily group quizzes, oral patient presentations relating clinical diagnoses to anatomy, and extensive embryology presentations. Group leaders were responsible for leading the dissections after attending daily orientation sessions on the teaching cadaver. Team and self evaluations of group leaders were gathered with weekly electronic surveys. Preliminary results suggest that team leaders rate themselves lower than their teammates do with respect to altruism, accountability and responsibility. One explanation for this discrepancy is decreased self‐awareness of student leaders regarding their own performance. Further research may demonstrate whether increased communication between leaders and team members can improve leaders' awareness of their capabilities. Research support came from the Mayo Clinic Department of Anatomy.

Grant Funding Source

N/A
Determining optimal learning conditions for acquiring spatial 3D information using computer‐based anatomical reconstructions
Authors: Nguyen Ngan , Nelson Andrew J , Wilson Timothy D
Published: June 2021

Interactive stereoscopic 3D digital models of head and neck structures, generated from CT scans of a male cadaver, have been developed. The display and interactive features of the models will be used to determine optimal conditions for acquiring 3D information for individuals with different spatial abilities. A pre‐mental rotation test will be used to determine participants' spatial ability. Based on pre‐test results, participants will be assigned to either the high or low spatial ability group. Members of each group will be further divided into four subgroups based on four learning conditions. A stereo/high‐interactive group will study anatomy using stereoscopic models and will have active control over the pace and direction of the model. A non‐stereo/low‐interactive group will examine anatomy with 2D images and restricted interaction. A stereo/low‐interactive and non‐stereo/high‐interactive group will study the same anatomy in corresponding conditions. Learning will be assessed by 50 multiple‐choice questions that require mental manipulation of internalized 3D anatomical representations. The results of this study will aid the design and implementation of effective 3D computer visualizations to help students comprehend the spatial 3D organization of the human body, while accounting for individual differences in spatial abilities.

Grant Funding Source

Internal
PriA Participates in Nascent DNA Synthesis in Escherichia coli
Authors: Wang Tzu‐Chien V , Huang Helen
Published: June 2021

Replication of DNA is a semiconservative process, and is catalyzed by DNA polymerase in the 5' to 3' direction. Because of the antiparallel nature of a DNA double helix, replication of DNA cannot be continuous on both strands at a replication fork. A semi‐discontinuous model of DNA replication suggests that DNA synthesis is continuous on the leading strand, whereas synthesis is discontinuous on the lagging strand. On the other hand, a discontinuous model of DNA replication suggests that DNA synthesis is discontinuous on both the leading and lagging strands. Evidences obtained from the vast majority of in vivo studies are in favor of a discontinuous DNA replication model in E. coli. In this study, the participation of priA, B, C and rep genes in discontinuous DNA replication was examined by analyzing the size distribution of nascent DNA synthesized in wild‐type, lig‐7 and polA4113 genetic backgrounds. Inactivation of priA, but not priB, priC or rep, resulted in a significant increase of high molecular weight (HMW) DNA in the short pulse‐labeled DNA in the wild‐type lig+ polA+ strains. Inactivation of priA also produced a significant increase of HMW DNA in the nascent DNA synthesized in lig‐7 and polA4113 strains. These results indicate that PriA is involved in the discontinuous synthesis of nascent DNA.
Osmotic enhancement of DNA binding by glutamate: Klenow and Klentaq DNA binding studies
Authors: Deredge Daniel Joseph , Thompson Greg , Jiang Ke , Patel Shree , Baker John , LiCata Vince J.
Published: June 2021

Glutamate (Glu−) is the major osmolyte accumulated in E.coli in response to external salt. In vitro, the 'glutamate effect' is primarily characterized by enhanced DNA binding when Glu− replaces Cl−. Some proteins also exhibit reduced ion release upon DNA binding in Glu−. Klenow polymerase (E.coli) exhibits both aspects of the 'glutamate effect'. Upon replacement of Cl− by Glu−, the ion release upon DNA binding is reduced by >50%. In addition, the apparent water release upon DNA binding is reduced by ~70% (150 waters) and proton linkage increases 5 fold. The decrease in apparent water release highlights the osmotic nature of the glutamate effect: Glu− and Cl− salts behave as ionic inhibitors of DNA binding but Glu− salts also behave as osmotic enhancers. While Klentaq's (T.aquaticus) DNA binding is enhanced in Glu−, its ionic and proton linkages are not altered. The osmotic enhancement is present for Klentaq polymerase but it is not as significant in the salt concentration range at which nanomolar Klentaq‐DNA binding occurs. E.coli DNA‐binding proteins might have evolved to bind tightly at higher salt concentrations in order to use osmotic enhancement of Glu− upon its intracellular accumulation. NSF.
Interaction Domains of Components of the ERα/Sp1 and C/EBPβ Complex Essential for Prolactin Receptor Transcription.
Authors: Kang Jung‐Hoon , Dong J. , Tsai‐Morris C. H. , Dufau M. L.
Published: June 2021

Transcription of the human prolactin receptor (PRLR) is governed by an estradiol (E2)/estrogen receptor α(ERα) mechanism independent of an estrogen response element. E2/ERα, through complex formation with Sp1 and C/EBPβ that associate to cognate elements, leads to coactivator assembly, H3/4 acetylation and recruitment of TFIIB and Pol II (Dong et. al. JBC, 2006). The ligand binding domain of ERα was required for interaction with C/EBPβ or Sp1, and E2 promoted this association. The zinc finger motifs of Sp1 (2nd/ 3rd) and the leucine zipper of C/EBPβ were identified as the domain of interaction of the individual transfactors with ERα. Complex formation of ERα/Sp1/C/EBPβ enhanced by E2 was observed upon incubation of transfactors with ERα‐GST. Although C/EBPβ associated strongly with ERα, marked reduction of C/EBPβ association was observed upon its co‐incubation with ERα and Sp1. In contrast, the association of C/EBPβ was enhanced in the presence of the preformed Sp1/ERα complex (sequential addition). This is consistent with previous ChIP studies showing tethering of the C/EBPβ to the complex through association with ERα. In addition, C/EBPβ was found to associate with the Sp1 zinc finger domain. These studies provide evidence for direct association of components of the complex essential for PRLR transcription through defined interaction domains of the individual proteins. Supported by the IRP, NICHD.
A synthetic‐lethal screen to identify non‐functional ribosomal RNA decay pathway factors in Saccharomyces cerevisiae
Authors: Connor Sarah W. , Levin Carly S. , LaRiviere Frederick J.
Published: June 2021

Nonfunctional rRNA Decay (NRD) is a quality control pathway in eukaryotes which eliminates nonfunctional ribosomes that have been assembled with mutant rRNAs. To investigate the mechanistic details of NRD, a synthetic‐lethal screen was developed to identify trans‐acting factors associated with this pathway. Galactose‐inducible plasmids encoding rRNA genes with mutations in functionally important regions of 18S and 25S rRNA, as well as a control plasmid with wildtype 18S and 25S rRNA genes, were transformed into Saccharomyces cerevisiae. Transformants were exposed to ultra‐violet (UV) radiation in order to induce random genomic DNA mutations. Mutations in genes required for NRD may result in a synthetic‐lethal phenotype when mutant rRNAs are expressed in these cells. To determine which cells acquired a synthetic lethal phenotype, UV‐exposed colonies were replica plated onto galactose to allow expression of the plasmid‐encoded mutant rRNAs. Thus far, we have isolated eight different S. cerevisiae colonies with the 18S mutant plasmid and ten colonies with the 25S mutant plasmid that are unable to grow on galactose after UV exposure. Phenotypic analysis, rRNA expression analysis, and complementation studies are currently underway to characterize the mutant strains and to determine the identity of the NRD factor mutations. This work is supported by the Dreyfus Foundation and the Jeffress Memorial Trust.
Effects of diminished protein synthesis on bone anabolic response to load in RPL29‐deficient mice
Authors: Sloofman Laura Gail , Chen David , Zhou Xiaozhou , Price Christopher , Novotny John E. , Wang Liyun , Kirn‐Safran Catherine B.
Published: June 2021

Deletion of a single ribosomal protein, RPL29, increases bone fragility due to diminished protein synthesis and poor tissue quality, suggesting an important link between skeletal tissue growth and efficient protein production. The goal of this study is to determine if reduced capacity to synthesize large volumes of proteins modifies bone anabolic response to mechanical load. To test this idea, tibiae of RPL29‐null mice and age‐matched wild type (WT) controls were loaded at 20% maximal load, 0.5 Hz for 50 cycles/day, with a five‐second rest period inserted between two cycles for two weeks.

The cortical bone microstructure and mechanical properties of the non‐loaded and loaded tibiae of both RPL29‐null and WT mice were analyzed using micro‐computed tomography (microCT) and three‐point‐bending tests, respectively. Results indicate that following loading minor changes occur at the structural and material property levels in both control and null tibiae. Ongoing studies are using dynamic histomorphometry to quantify differences in bone formation capacity between loaded and non‐loaded conditions. These studies will establish the importance of high volume protein synthesis for the regulation of bone formation. This work is supported by NIH P20 RR016458‐06 and an HHMI Undergraduate Science Education Grant.
Characterization of Mutations in Ribosomal Proteins L4 and L22 that confer Erythromycin Resistance in E. coli
Authors: Zaman Sephorah , Fitzpatrick Megan , Zengel Janice M , Lindahl Lasse
Published: June 2021

L4 and L22, proteins of the large ribosomal subunit, contain globular surface domains and elongated "tentacles" that reach into the core of the large subunit to form part of the lining of the peptide exit tunnel. To learn more about the roles of the tentacles of L4 and L22 in ribosomal assembly and function, we isolated and characterized erythromycin resistant mutants in E. coli. All of the mutants grew slower than the parent and all showed reduced in vivo rates of peptide elongation and increased levels of precursor 23S rRNA. Sucrose gradient sedimentation analysis also showed that large insertions in L4 and L22 result in accumulation of abnormal ribosomal subunits. In addition, structural studies using chemical modification followed by primer extension reveal changes in rRNA structure due to large insertion mutations in L4 and L22, particularly at A2450 and A2451, key nucleotides in the peptidyl transferease center. Further studies are underway to correlate structural changes in 23S rRNA with slower peptide elongation rates observed in mutant strains. These results highlight the important role of L4 and L22 in ribosome structure, function and assembly.
Purification of MBP Fused Mycobacterium Tuberculosis Proteins
Authors: Crenshaw Ezekiel Matthew David
Published: June 2021

Mycobacterium tuberculosis (Mtb) is a bacterium that causes tuberculosis (TB), a bacterial infection that mainly affects the respiratory system. The objective of the TB Structural Genomics Consortium is to solve the structure of Mtb proteins related to disease, with the aim of determining the function associated with those structures which could potentially lead to new treatments. In order to solve the structure of those proteins, highly purified proteins are needed which is achieved through protein purification. Protein purification is important to determine the characteristics of the function, structure, and interactions of the protein of interest. Our method of purifying proteins is with a Maltose Binding Protein (MBP) which increases expression level and the solubility of our protein of interest. The first step is to express the fusion protein with MBP which is accomplished through fermentation. The next step is to isolate the fusion protein through His‐tag affinity chromatography. We then cleave the fusion protein with TEV Protease. After the cleavage has taken place, we remove the MBP using MBP affinity chromatography and/or Gel Filtration Chromatography is needed. In this poster, we show the process of isolating two Mtb target proteins,Rv0472 and Rv3567, fused with MBP.
Aldehyde oxidase catalyzes nitrite reduction: An important nitric oxide producing pathway during ischemia
Authors: Li Haitao , Kundu Tapan Kumar , Zweier Jay L.
Published: June 2021

Aldehyde oxidase (AO) plays an important role in drug and xenobiotic metabolism. While AO has structural similarity to bacterial nitrite reductases, it is unknown if AO catalyzes nitrite reduction to NO. Furthermore, the mechanism, magnitude, and quantitative importance of AO‐mediated nitrite reduction have not been studied. To investigate this pathway and its quantitative importance, isolated enzymatic studies were performed. The kinetics and magnitude of AO dependent NO formation was characterized. Typical reducing substrates of AO such as aldehyde and NADH reduced nitrite to NO in the presence of AO. Reduced AO was the direct electron donor to nitrite with reduction occurring at its molybdenum site. Studies of NO generation at different pH values indicated that nitrite reduction occurs via an acid‐catalyzed mechanism. Kinetics of AO catalyzed nitrite reduction under anaerobic conditions followed Michaelis‐Menten kinetics with Vmax= 8.5 nmol/s.unit, Km= 2.7 mM with the flavin site electron donor NADH, and Vmax= 13.5 nmol/s.unit, Km= 3.3 mM with the molybdenum site electron donor cinnamaldehyde. pH values, oxygen pressure, and reducing substrate levels were important regulators of AO catalyzed NO generation. It was determined that during ischemia pulmonary or myocardial AO and nitrite levels are sufficient to result in NO generation comparable to maximal production by NOS.
The BTI1 Azoreductase Colorimetric and Fluorometric Reporter System
Authors: Johansson Hans E. , Johansson Mary K. , Wong Albert , Peterson Erik J. , Armstrong Eliana S. , Grant Richard E. , Reddington Mark V. , Cook Ronald M.
Published: June 2021

Reliable duplex and multiplex assays remain a challenge for current fluorescence reporter enzyme technology. To this end we have developed a system that takes advantage of pro‐fluorescent probes and a novel azoreductase.

The BTI1 azoreductase gene was cloned from a soil sample, optimized and used to bacterially express the protein. Biochemical characterization of the purified enzyme in solution revealed a tetramer carrying a non‐covalently bound flavin mononucleotide cofactor. BTI1 readily reduced an array of carboxylated and sulfonated azo dyes including methyl orange (DABSYL) and the Black Hole Quencher (BHQ(tm)) dyes. Enzyme kinetic parameters of the four‐hydride transfer reaction were consistent with the bi‐bi ping‐pong mechanism of related oxidoreductases. In our optimized colorimetric assay, the detection limit of the BTI1 azoreductase was >10 fold lower than that of beta‐galactosidase.

Reduction of the azo‐quenchers in pro‐fluorescent substrates containing BHQs and a range of fluorophores readily released fluorescence in an NADPH‐dependent manner with robust signal to noise ratios.

In short, by matching BTI azoreductase with pro‐fluorescent substrates we have created a simple, yet adaptable, reporter system that generates diverse fluorescence signals.

This research was supported in part by a grant from NIH to HEJ (R43/R44 GM076843).