bluuebunny/crossref_metadata_2025 huggingface dataset:
Breast cancer derived from bone marrow after transplantation in an FVB mouse.
Authors: Guest Ian , Ilic Zoran , Ma Jun , Glinsky Gennadi , Sell Stewart
Published: June 2021

The hypothesis that the bone marrow contains cells that could give rise to breast cancer was tested by transplantation of bone marrow (BM) from male mice bearing a strong oncogene (polyoma middle T antigen gene, PyMT) under the control of a promoter activated in the mammary gland (mouse mammary tumor virus promoter, MMTV), into irradiated syngeneic female recipients, and following the recipient mice for development of breast cancer. Eight female FVB/N mice were irradiated with 900 to 1050 rads at 2 months of age and rescued by transplantation of 2 X 106 male BM cells from transgenic MMTV‐PyMT FVB/N donors. The transplanted mice developed a number of tumors by the age of 14 to 21 months, with a higher incidence and variation in types than 24 month old un‐irradiated female FVB/N controls as reported in the literature. One of the 8 recipient mice developed a diploid breast cancer, histologically indistinguishable from the primary breast cancers in MMTV‐PyMT mice. By fluorescent labeling over 70% of the breast cancer cells contained a Y‐chromosome. None of the other mice developed a breast cancer, but they did exhibit a number of other cancer types including squamous cell carcinoma, fibrosarcoma, lung and adrenal adenomas, and an Harderian gland adenocarcinoma. None of these cancers contained Y‐chromosomes, including a mixed cell cleaved lymphoma and an adrenal adenoma found in the same mouse that had the breast adenocarcinoma. This result provides proof of principle that breast cancer can arise from cells in the bone marrow. The presence of donor derived stromal cells in the other cancers raises the possibility that donor derived stroma might interact with recipient cells to cause cancer.
Histone methylation patterns in human breast cancer
Authors: Rivenbark Ashley Garrett , Coleman William B , Stahl Brian D
Published: June 2021

Histone post‐translational modifications regulate gene expression, and their misregulation is linked to cancer development. Histone methylation occurs on lysine residues, established by enzymes that regulate chromatin structure and gene activity. The ability of histone methylation to signal in chromatin depends on the lysine that is targeted and the methylation state (me1, me2, or me3) that is achieved. Methylation on histone H3 lysine 4 (H3K4) correlates with gene activity and the H3K4 methyltransferase MLL is translocated in hematological cancers. The H3K36 methyltransferase NSD1 is fused to NUP98 in leukemia and this histone mark is associated with active genes. The pattern of histone modification disruption in cancer has been largely unexplored. We examined by western blot the levels of H3K4me3 and H3K36me3 in breast cancer cell lines. We show that there are differential levels of histone methylation in several of the cancer cell lines when compared to a normal mammary epithelial cell line. BT549 and SUM102 cells exhibit low levels of H3K4me3 and increased levels of H3K36me3. These cell lines also have a defect in DNA methyltransferase activity resulting in increased levels of DNA methylation. Our data suggests that similar to DNA methylation, these histone marks are coupled to the pathology of breast cancer. Future studies will determine if histone methylation marks are clinically relevant for patient assessment.
The development of taste buds in two morphs of Astyanax mexicanus
Authors: Franz‐Odendaal Tamara Anne , Varatharasan Nirupa , Croll Roger P
Published: June 2021

The Mexican tetra, Astyanax mexicanus, is a fish that exists as two morphs of the same species; one sighted and one blind. Several regressive and constructive changes have been identified within blind tetra compared to their sighted ancestors. Of particular note here are the taste buds. Using immunohistochemical markers for receptor cells, basal cells and innervating axons (calretinin, serotonin, and zn‐12/tubulin), we have documented the distribution, number and development of taste buds on the upper and lower lips of the blind versus sighted Mexican tetra. We detected a difference in taste bud morphology between the two morphs as well as substantially more taste buds in blind versus sighted tetras. We show that as early as 5dpf blind tetra larvae have more taste buds on their lips than sighted tetras. The significance of this constructive evolutionary change in concert with the regressive change of losing eyesight will be discussed. This study is the first to document the timing of development of constructive changes which accompany the loss of eyes in the Mexican tetra and lays the foundation for future studies on gene expression patterns which mediate these changes. Research Support: NSERC, Canada

Grant Funding Source

NSERC, Canada
Three‐dimensional engineering of the nervous system
Authors: Berthod Francois
Published: June 2021

The nervous system is the target of various diseases or injuries that could lead to major handicaps or be life threatening. Innovative strategies using tissue‐engineered nerve conduits or in vitro models could help to understand and repair these disorders. To bridge the gap between nerve stumps after a peripheral nerve injury, we developed a tissue‐engineered neural tube to promote nerve repair. This living nerve conduit made of fibroblasts was prepared to serve as a nerve guide to enhance recovery of sensory and motor functions of the transected nerve.

The nervous system is also the target of life threatening neurodegenerative diseases such as the amyotrophic lateral sclerosis. We developed a tissue‐engineered model mimicking the spinal cord in vitro to better understand the pathogenesis of these disorders. Motor neurons were cultured on the top of a fibroblast‐populated sponge mimicking the connective tissue through which motor axons elongate in vivo. This model promoted axonal migration and the spontaneous formation of numerous thick myelin sheaths wrapping around motor fibers in presence of Schwann cells.

The 3D engineering of the peripheral and the central nervous systems by tissue engineering could help to better understand the pathogenesis of neurodegenerative diseases, to find curative treatments and to repair peripheral nerve damage or even spinal cord injuries.
Genetic pathway adaptation and eye development in the Iberian Mole
Authors: Collinson Jon Martin
Published: June 2021

We have studied eye development in the Iberian mole Talpa occidentalis, and present the first molecular embryological study of the eyes of any fossorial mammal. Although permanently closed, the eye potentially retains function, with transparent cornea and lens, and a fully differentiated retina that projects to an optic chiasm. In other model vertebrates, genetic pathways controlling lens development robustly compartmentalise the lens into a simple, undifferentiated, proliferative anterior epithelium, and quiescent, anuclear, terminally differentiated posterior lens fibres. These pathways are not as robust in the mole, and lead to loss of the anterior epithelial phenotype and only partial differentiation of the lens fibres, which continue to express 'epithelial' genes. FOXE3‐positive lens fibres continue to proliferate within the posterior compartment of the embryonic lens, and the undifferentiated status of the anterior epithelial cells was compromised: most of them undergo apoptosis. Furthermore, beta‐crystallin and PROX1 expression patterns are abnormal and our data suggest that genes encoding beta‐crystallins are not directly regulated by PAX6, c‐MAF and PROX1 in the Iberian mole, as they are in other model vertebrates. Paradigms of genetic regulatory networks developed in other vertebrates therefore appear not to hold true for the Iberian mole.
Environmental Changes Impair Stem and Progenitor Cell Function and Contribute to Cardiovascular Aging
Authors: Ballard Victoria
Published: June 2021

Cardiac stem and progenitor cells have been identified in the heart, bone marrow and other organs and have been shown to contribute to cardiovascular repair via both direct replacement and paracrine signaling that promotes local regeneration. These cells are maintained through adulthood and are present even in older adults. This finding is notable in light of the reduced capacity for repair and regeneration in older tissues. This apparent dichotomy can be reconciled through an appreciation of the age‐associated changes in the microenvironmental pathways that govern adult stem cell plasticity and function. Besides intrinsic changes to the cells themselves that are associated with aging, including DNA damage and loss of telomeres, changes in the microenvironment of the cell, both in the stem cell niche and at the site of injury, may impact the regenerative ability of the aging heart. We have identified a number of environmental pathways that become dysregulated over time and may underlie the impairments in cardiac stem and progenitor cell function with increasing age. It is hypothesized that identification of mechanisms to reverse these age‐associated changes may lead to the development of novel therapeutic strategies to enhance stem‐ and progenitor cell‐mediated repair and promote cardiovascular health in older individuals.
Chromatin signatures in multipotent hematopoietic stem cells indicate fate of bivalent genes during differentiation
Authors: Zhao Keji
Published: June 2021

Histone modifications are implicated in the maintenance and differentiation of stem cells. We analyzed genome‐wide changes of multiple histone modifications during differentiation of multipotent hematopoietic stem/progenitor cells (HSCs/HPCs) into erythrocyte precursor cells. We find that several monomethylations associate with critical enhancers of differentiation genes prior to their activation and are correlated with their basal level expression, suggesting that they are involved in the maintenance of the activation potential required for differentiation. The bivalent genes that lose H3K27me3 after differentiation are associated with increased levels of H2A.Z, H3K4me1, H3K9me1, H4K20me1 and Pol II in HSCs/HPCs, suggesting that the fate of bivalent genes during differentiation is already programmed by chromatin modifications at the HSC/HPC stage.
Alternative Splicing in the Mammalian Nervous System
Authors: Blencowe Benjamin J.
Published: June 2021

Alternative splicing is widely considered to be a major mechanism underlying the evolution of increased cellular and functional complexity in vertebrate species and is especially prevalent in the mammalian nervous system. Microarray profiling and, more recently, high‐throughput sequencing has resulted in the identification of a myriad of nervous system‐regulated exons. Many of these are located in widely expressed genes that have critical nervous system‐specific functions. Our research is currently focusing on elucidating the cis‐acting "code" and corresponding trans‐acting factors responsible for the regulation of these exons, and their specific roles in nervous system formation and function. In collaboration with the group of Brendan Frey (Dept. of Electrical and Computer Engineering, University of Toronto) we have developed a new machine learning algorithm that can predict nervous system and other tissue‐regulated alternative splicing patterns from sequence features alone. Using other genome‐wide strategies as well as focused experimental methods we are identifying and characterizing trans‐acting factors that link to specific elements of the cis‐regulatory code. A new trans‐acting factor emerging from one of the screens is the neural‐specific SR‐related protein of 100 kDa (nSR100). This protein is vertebrate‐lineage‐specific and functions as a coactivator to regulate ~10‐12% of nervous‐system specific exons via C/U‐rich motifs concentrated in flanking intron sequences. Knockdown of this protein disrupts the regulation of a network of alternative exons associated with neuronal differentiation and nervous system development.
Mining the receptorome: an efficient approach for target discovery and validation
Authors: Roth Bryan L
Published: June 2021

In this talk I will demonstrate the power of screening drugs and drug‐like compounds in a massively parallel fashion against the GPCR‐ome‐‐the most abundant family in the receptorome (see Armbruster and Roth, JBC 2005 for review). In this talk I will show how this procedure is highly suited to discovering the molecular target(s) responsible for drug actions and side‐effects in vivo.
Cancer and Aging in DNA repair deficiency: cause and treatment
Authors: Niedernhofer Laura J.
Published: June 2021

Inherited mutations in genes required for DNA repair or the cellular response to DNA damage lead to a variety of diseases characterized by accelerated aging and/or an increased risk of cancer. Studies in mouse models of these diseases revealed a strong correlation in the molecular mechanisms and pathogenesis between these progeroid syndromes and natural aging. This provides evidence that DNA damage can play a causal role in aging. We created mice that express 10% of the normal level of ERCC1‐XPF endonuclease, an enzyme that participates in nucleotide excision repair, interstrand crosslink repair and double‐strand break repair. Beginning at 8 wks of life, the mice show spontaneous and progressive symptoms associated with aging including neurodegeneration, muscle wasting, loss of vision and hearing, urinary incontinence, epidermal atrophy, bone marrow failure, decreased liver and kidney function, osteoporosis and intervertebral disc degeneration. In contrast, mice expressing 30% of the normal level of ERCC1‐XPF have a very high incidence of cancer. These mice offer a model system for identifying the mechanism by which DNA damage can drive degenerative changes associated with aging as well as cancer. Currently, we are treating the mice with stem cells, radical scavengers and inhibitors of apoptosis to determine the underlying mechanism of how DNA damage promotes aging and age‐related diseases.
Regulation of Histone Modification and Transcription by the Yeast Paf1 Complex
Authors: Arndt Karen , Warner Marcie H. , Crisucci Elia M. , Piro Anthony S. , Davis Christopher
Published: June 2021

The Paf1 complex associates with RNA pol II and couples histone modification to transcription elongation. The Rtf1 subunit of the complex is essential for H2B K123 ubiquitylation and H3 K4 and K79 methylation. We have identified a conserved histone modification domain (HMD) in Rtf1 that is essential for these modifications. Specific amino acid substitutions in the HMD cause differential effects on H2B ubiquitylation and H3 K4 methylation, indicating that the functions of Rtf1 in directing these marks are genetically separable. Interestingly, expression of the isolated HMD in yeast cells is sufficient to promote histone modification. Microarray experiments have revealed broad effects of the Paf1 complex on transcription patterns, including many genes where the Paf1 complex functions as a repressor. Northern analyses demonstrated that Paf1 and Ctr9, and to a lesser extent Rtf1, are required for repression of the model gene ARG1. Analysis of ARG1 expression in single and double mutant strains indicated that Paf1 directs repression through a pathway that operates in parallel to known ARG1 regulators. The Paf1 complex localizes to ARG1 in both repressing and activating conditions, suggesting that it directly regulates ARG1 transcription. Replacement of the ARG1 coding sequence with that of a heterologous gene revealed that the Paf1 complex functions through the promoter or early transcribed region of ARG1. Our results suggest that the Paf1 complex inhibits transcription of ARG1 and potentially other genes by establishing a repressive chromatin structure at the promoters or 5' ends of these genes. Funding source: NIH.
STRATEGIES FOR SUBSTRATE RECOGNITION BY THE AAA+ UNFOLDASES AND PROTEASES
Authors: Baker Tania A.
Published: June 2021

ClpX and ClpA are AAA+ ATPases that specifically recognize and then actively unfold protein substrates. Each enzymes can act alone to disassemble protein complexes, or in a complex with the ClpP peptidase to form an ATP‐dependent protease. We are focusing on elucidating mechanisms used by these enzymes to select their substrate proteins. Some proteins are recognized via short peptide tags that bind directly to the enzyme's pore, whereas other proteins are recognized initially by interacting with the distinct peptide‐binding sites in the N‐terminal domains of the enzymes. Adaptor proteins also regulate the recognition of substrates by ClpX and ClpA. Structural, proteomic, and mechanistic studies are providing insight into the mechanisms of action of these adaptor proteins, as well as their biological impact.
Pegylated interferon‐alpha targets beta‐catenin by inducing its nuclear export
Authors: Thompson Michael D , Monga Satdarshan S
Published: June 2021

Treatment with Peg‐IFN, a first line therapy for chronic HCV infection, reduces the development of hepatocellular carcinoma (HCC). Furthermore, activation of the Wnt pathway is observed in a significant subset of HCC. We explored the possibility that Peg‐IFN may act through inhibition of Wnt signaling. To examine Peg‐IFN's effects in vivo, mice overexpressing β‐catenin were exposed to DEN and treated with Peg‐IFN (70,000 U) for six weeks. Treatment with Peg‐IFN decreased nuclear β‐catenin compared to saline treated and decreased Ki‐67 positive hepatocytes. No change in the transcription of β‐catenin was evident suggesting the effect to be through a post‐translational mechanism. We began in vitro studies to verify this finding and explore the mechanism of reduced Wnt signaling. Treatment of human hepatoma cells (HepG2 & Hep3B) with Peg‐IFN led to a decrease in the active form of β‐catenin. Likewise, β‐catenin dependent transcriptional activity was decreased by up to 50% as measured by TOPflash luciferase reporter assay. Real‐time PCR showed an increase in two negative regulators of Wnt signaling, Dkk1 and RanBP3. Overexpression of RanBP3 in HepG2 cells significantly decreased β‐catenin transcriptional activity. Thus Peg‐IFN effectively downregulates Wnt/β‐catenin signaling at least partly through increased expression of RanBP3, which is an attractive means of therapeutically inhibiting mutated‐β ‐catenin.
Unsuspected infection of laboratory animals with human adenovirus‐36 compromises experimental findings
Authors: Atkinson Richard L. , He Jia , Abbott David H. , Whigham Leah D.
Published: June 2021

Human adenovirus‐36 (Ad‐36) affects biochemical functions and induces obesity in chickens, mice, rats, monkeys, and humans. The viral E4orf1 gene directly affects lipogenic enzymes to produce adiposity. Ad‐36 infection alters lipid & glucose metabolism, gene expression, and body composition. Serum antibodies (AB) assayed by serum neutralization (SN) are specific evidence of infection. We determined prevalence of unsuspected (natural) Ad‐36 infection in multiple laboratory species and report how experimental data are affected. We obtained serum from 40 rats (Harlan Labs, IN); 15 rhesus monkeys in the Aging Study, Wisconsin National Primate Res Ctr; 8 rabbits from US Biological (MA) & 6 cows (culture media serum). We fed rats a 36% or 48% kcal fat diet and measured wt gain and body fat. 25% of rats arrived at the lab with unsuspected Ad‐36 infection. Hi fat feeding induced greater wt gain (36.6 vs 9.6 g, p<.02) and 51% more body fat (82 vs 54 g, p<.02) in infected vs uninfected rats. 100% of monkeys became Ad‐36+ over the 7 yr study; all gained wt and lowered serum cholesterol (p<.05). 50% of rabbits were Ad‐36+ on arrival. Antibodies were present in 4 of 6 bovine sera and interfered in the SN assay.

Conclusions

Unsuspected Ad‐36 infection occurs in lab animals from suppliers and in university vivariums. Laboratory animals should be tested for Ad‐36 status before experiments are performed as Ad‐36 may compromise outcomes.
Using HPLC‐Mass Spectrometry to Teach Proteomics Concepts Using Problem‐Based Techniques
Authors: Short Michael Thomas , Short Anne Caitlin , Vankempen Rachel , Burnatowska‐Hledin Maria
Published: June 2021

Practical instruction of proteomics concepts was provided using high‐performance liquid chromatography coupled with a mass selective detection system (HPLC‐MS) with simulated protein digests. Purchased peptides prepared at concentrations above the instrument's limit of sensitivity were used for mass spectral identification to simulate fingerprinting of protein digests. Students were given mixtures of dipeptides as unknowns. The parent ion patterns of the dipeptides were calculated by the students using peptide calculator websites as part of their pre‐laboratory preparation. Following instruction on the use of the HPLC‐MS instrument, the unknown mixtures were analyzed and the dipeptides in the unknown mixtures were identified. In addition to this experiment, purchased chicken egg white lysozyme was alkylated with iodoacetamide, digested with trypsin, and analyzed using the HPLC‐MS. Key tryptic peptides were identified in the chromatogram using the HPLC‐MS coupled with information that was generated using the FindPept tool [1]. We have demonstrated that complex concepts can be taught experimentally in the undergraduate biochemistry laboratory using a problem‐based approach.
Developing Professional Skills Through a Graduate Enrichment Program
Authors: Schrott Lisa M
Published: June 2021

To complement the coursework and research training of the graduate students in our Pharmacology department, we have instituted a Graduate Enrichment Program. The series for first year students helps incoming students make a smooth transition to graduate school and provide them with academic and social support as they progress through their first year. Topics include ethics, research rotations, coursework, networking opportunities, and qualifying examinations. The second series is open to all students in the department. The goal of this program is to hone students' academic skills and professional development. Graduate student survival skill sessions include statistical reviews, ethics workshops, manuscript writing strategies, writing an IACUC, and presentation skills. Topics within the professional development area have included manuscript review, writing a CV, developing leadership skills, choosing a post‐doc, effective networking, careers in industry, and careers at teaching‐intensive colleges. Since we are in a medical school and have limited teaching opportunities, we have also incorporated sessions on becoming an effective teacher. These have included sessions on cognitive learning styles, using technology, and facilitating discussions. The sessions are led by department faculty, as well as faculty from other local colleges.
CEA‐CAM‐1 positive stem cells in the cerebral cortex of the adult rat
Authors: Clarke Emily Rae
Published: June 2021

Carcinoembryonic antigen‐cell adhesion molecule‐1 (CEA‐CAM‐1) positive stem cells have been isolated from the skeletal muscle of adult mammals, including humans. The current study was undertaken to determine the location of these cells with respect to the cerebral cortex of the adult rat. Adult rats were euthanized following the guidelines of Mercer University's IACUC. The heads were fixed by immersion fixation, the brains isolated, cryosectioned and stained with an antibody diagnostic for adult pluripotential stem cells, i.e. CEA‐CAM‐1. CEA‐CAM‐1 positive stem cells were located as a diffuse population of cells within both the white matter and gray matter of the cerebral cortex, as well as within the blood vessels and connective tissues of the pia mater. The results demonstrate that this primitive stem cell population is native to the adult rat brain. Studies are ongoing to address their functional significance in neuronal injury and repair. Funding was provided by LM & HO Young Estate Trust and Dragonfly Foundation.
Increase in primitive stem cell numbers in equine peripheral blood following stress
Authors: McCommon G , Lochner F , Samples O , Lochner D , Lochner D , Hixson D , Young H E
Published: June 2021

Stout et al. (Amer Surg. 73:1106‐1110, 2007) reported base‐line levels of primitive stem cells circulating in adult porcine peripheral blood, which increased dramatically after just 90 minutes of trauma. Ongoing studies have shown similar stem cells in adult equine blood and that base‐line levels varied according to breed of the horse. The current study was undertaken to determine whether these stem cells would increase in number in the peripheral blood following stress in the same horse. Stress was defined as moderate exercise, i.e., 10 minutes of cantering. Blood withdrawal followed the guidelines of FVSU‐IACUC. Adult horses had 8 ml of blood withdrawn by venipuncture immediately prior to and immediately after exercising. After gravity separation, the plasma was withdrawn and 15 microliters of plasma from each sample was mixed with 15 microliters of 0.4% Trypan blue. The resultant solution was mixed, placed onto a hemocytometer and photographed: blastomere‐like stem cells (BLSCs) were Trypan blue positive and < 2.0 microns in size; transitional‐BLSCs (Tr‐BLSCs) were both Trypan blue positive & negative and 2‐5 microns in size; and epiblast‐like stem cells (ELSCs) were Trypan blue negative and 6‐8 microns in size (Young and Black, Minerva Biotech 17:55‐63, 2005). This study demonstrates that moderate exercise will increase the level of these primitive pluripotent stem cells in the blood of adult equines.
Production of a nanocomposite for tissue repair application requiring viscoelasticity
Authors: LIU JIE , Argenta Louis , Morykwas Michael , Carroll David , Wagner William
Published: June 2021

The production of repair materials where an elastic component is required is lacking due to the difficulty of incorporating insoluble elastin. Poly (1, 8‐octanediol citrate) (POC), a synthetic, biodegradable, elastomeric, and hydrophilic material may be used as a substitute for the elastic fiber. To simulate extracellular matrix (ECM), five composite fibers were fabricated using electrospinning technology: Collagen Type I (I), I +Collagen Type III (III), I+POC, I+III+POC, and I+Versican+Decorin+POC. Fiber sizes in all samples ranged from 200nm to 600nm mimicking ECM fibers. By atomic force microscopy, a single fiber of I, I+III, I+POC, I+III+POC, and I+Versican+Decorin+POC had respectively a Young's modulus of 1.37±0.20 (mean ± SD) GPa, 1.21±0.14 GPa, 0.63±0.07 GPa, 0.44±0.12 GPa, 0.62±0.18 GPa. On the average, comparing samples with POC and without POC, elasticity increased 3.2 fold and maximum stress only decreased by 1.3 fold. Fibroblasts in culture had high affinity for all fibers and proliferated during the 7 days period of study. The study demonstrates the usefulness of POC in the production of an ECM‐like viscoelastic nanocomposite for tissue repair.

Grant Funding Source

Department of Plastic Surgery
Evidence that mesodermal migration does not occur during human gastrulation (DREM databases utilization # 2)
Authors: Gasser Raymond Frank
Published: June 2021

The objective was to determine in human embryos if the changing positions of mesodermal cells from the primitive (gastrulation) streak result from migration between the overlying epiblast and underlying hypoblast layers as described in embryology books or if their changing positions result from cell proliferation and changes in the size and shape (growth movements) of the embryo. Embryos rapidly change in size and shape during the period when mesodermal migration is supposed to occur. Reconstructions were made from digital images of serial sections of Carnegie specimens at representative periods during gastrulation (stages 6‐9). Magnifications were kept identical. The caudal end of the primitive streak was used as the reference point because it ends close to the relatively stationary connecting stalk. The reconstructions were superimposed revealing that the changing positions of the mesodermal cells result from growth movements of the whole embryo together with cell proliferation. Rather than mesodermal cells moving from one region to another (migration) the study showed that they remain in the same region and move as the entire embryo enlarges and changes shape. The study found that cells in all three layers proliferate rapidly with the conclusion that mesodermal cell migration during gastrulation between the epiblast and hypoblast layers is unnecessary and does not occur. Supported by NIH grant RO1 HD 37811.
Disruption of Development in the Intestinal Epithelium Leads to a Metabolic Syndrome in Adult Rats
Authors: Dlugosz Malgosia A. , Zong Haihong , Pessin Jeffery E. , Gad Ashraf , Larson Janet E. , Cohen J Craig
Published: June 2021

Several lines of evidence suggest that CFTR (the gene defective in cystic fibrosis) plays a role in proper development of the intestine. To analyze temporal aspects of CFTR requirement for post‐natal phenotype, we transiently suppressed CFTR expression in the intestine of rat fetuses using antisense in utero gene therapy.

Depending on the gestational age of the fetus, transient reduction of CFTR function resulted in phenotypes ranging from meconium ileus and subsequent death to obesity. Of particular interest are animals treated during day 17 (E17) of embryonic development. After birth, these animals display dysregulated production of hormones and neuropeptides responsible for promoting satiety, leading to increased food intake and excess body fat. We also observed significant peripheral tissue resistance to insulin, as indicated by clamp studies. Microarray analysis showed changes in gene expression in the intestine both before and after birth, and the results were confirmed by immunofluorescent staining. These data demonstrate that weight gain and insulin resistance in adults can result solely from subtle changes in gene expression during gestation

expression during intestinal development results in a metabolic syndrome in the adult animal.
VEGF Regulates Cervical Remodeling By Increasing Epithelial Folds During Pregnancy in Rodents
Authors: Palmer Jennifer , Scott Kelsey , Mowa Chishimba Nathan
Published: June 2021

Cervical remodeling is a physiological inflammatory process which is associated and possibly regulated by proliferating cervical epithelial. We have previously shown that levels of VEGF increase with advancing pregnancy in rodents (mice and rats). Here, we show that as VEGF increases, the folds in the surface epithelium of the cervix increase as pregnancy advances in untreated rodents, which we interpreted as increased epithelial growth. We also show that ovariectomized nonpregnant rodents treated with VEGF have increased epithelial folds versus control using SEM.. We conclude that VEGF plays a role in cervical remodeling as pregnancy progresses by an increase in epithelial proliferation.
Backwards displacement of the manubrium causes crimping of the great vessels in patients with thoracic outlet syndrome (TOS): MRI/MRA/MRV
Authors: Collins James D. , Saxton Ernestina H. , Ahn Samuel S. , Gelabert Hugh , Carnes Alfred
Published: June 2021

Rounding of the shoulders increases the slope of the first ribs and asymmetrically displaces the manubrium posteriorly. This compresses the retrosternal fat with the involuted thymus gland and the left brachiocephalic vein against the brachiocephalic trunk. This displaces the aorta posteriorly and crimps (like a water hose) the great vessels: right brachiocephalic vein with the superior vena cava, common carotid arteries, subclavian arteries with binding nerve roots and vertebral arteries. Bilateral MRI/MRA/MRV displays costoclavicular compression and crimping of the great vessels that trigger complaints of TOS: facial, back and upper and lower extremity pain; numbness, tingling and edema; visual blurring and "floaters"; syncope and headache (FASEB 2003; 17:A784). Monitored multiplanar images with abduction external rotation, 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. Two patients were selected, one following acute trauma, the other with repetitive strain injury. The second patient was misdiagnosed and underwent acromioclavicular joint surgery with a resultant unstable joint. Both patients developed laxity of the sling/erector muscles and round shoulders. Both displayed crimping of the great vessels and costoclavicular compression with TOS symptoms.
Evaluation of a comprehensive neuroanatomy website in a distributed medical curriculum
Authors: Krebs Claudia
Published: June 2021

A neuroanatomy website (www.neuroanatomy.ca) was created as a resource for all students and as a means of bridging the access to information and specimens gap between all sites in a distributed medical curriculum.

The website includes a web atlas with both photos and diagrams, an imaging section with MRI scans, a section with 3D MRI reconstructions, a stroke section and course specific sections such as a weekly quiz. Web 2.0 elements were incorporated such as a wiki and podcasts.

A survey was given to the students at the end of the lab block to evaluate the use and usefulness of the website.

85% of students used the website, both at home and during the lab sessions.

The sections used primarily were the atlas and the 3D reconstructions. This could be attributable to the uniqueness of the 3D section and the scarcity of prosections to study with. Both the stroke section and the quiz section also showed a good usage by the students. The wiki was poorly received, this might be due to perceived difficulties with the technology or the use of other, preferred means of communication by the class such as online message boards.

While most respondents found the website to be a useful tool during the lab sessions (54%), a smaller percentage found the website useful for other curricular objectives in the block (45%).

Overall the students rated the website as being a useful learning tool, it was easy to navigate and information was easy to find.
Anatomy students' initial perceptions about learning anatomy
Authors: Notebaert Andrew , Park Soonhye , Hand Brian
Published: June 2021

Knowledge about a student's approach to learning is essential to anatomy curricular changes. Understanding how students prefer to learn can help create anatomy courses that move away from rote memorization and minimal understanding of anatomy. We conducted a research study using interviews of both students and instructors and survey instruments to describe their perceptions of learning. Data were collected at the beginning of the course and at the end to determine if there are changes made during the length of the course. The initial student survey included several open ended questions aimed at identifying general perceptions of learning and how they thought they would learn anatomy. Fifty‐six initial surveys were completed from students in four different anatomy courses. Preliminary results indicate that students preferred either an apprenticeship or a practice model for general learning. Students indicated that they would learn anatomy by repetition/memorization, reading, attending lecture, and studying notes. Although anatomy students believed that they learned better in an active learning environment, many indicated that they perceived learning anatomy in passive and behaviorist approaches. Instructors should approach curriculum development around student ideas of ideal learning environments in order to move towards deeper understanding. This project was not funded.

Grant Funding Source

None
Comparative efficacy of group versus individual feedback regarding medical student professionalism in gross anatomy
Authors: Camp Christopher Lee , Gregory Jeremy K , Chen Laura P , Alexander Cara J , Jacobs Sarah M , Lachman Nirusha , Pawlina Wojciech
Published: June 2021

Professionalism is a core competency of gross anatomy and general medical school curricula requiring students to develop the skills of giving and receiving peer feedback. Our study evaluated the effectiveness of delivering feedback in a group setting compared to delivering it individually. The first year class of Mayo medical students (n=50) enrolled in gross anatomy (in dissection teams of 3 or 4) completed weekly anonymous peer‐evaluations of their team regarding multiple aspects of professionalism (integrity, responsibility, compassion, and peer respect). Professionalism scores were calculated using the quantitative values from these surveys. Students were also asked to comment on strengths and areas for improvement for each student. At the mid‐point of the course, peer comments and professionalism scores were shared with students in debriefing sessions, either individually or as a team. Preliminary analysis of pre‐ and post‐intervention professionalism scores indicates that students receiving feedback in a group setting are more likely to demonstrate higher scores on subsequent evaluations. Our findings suggest that providing feedback to first‐year medical students in a group setting is more beneficial for group dynamics and may improve professional attitudes and behaviors. This work was supported by the Mayo Clinic Department of Anatomy.

Grant Funding Source

None
Teaching‐Learning Outcome of Gross Anatomy in the Presence of Teaching Assistants in a Caribbean Medical School
Authors: Senthilkumar Uma , Patel Vinit , Jansari Tanha
Published: June 2021

While it is generally believed that the teaching assistants (TAs) who work under the direction of faculty strengthens the organization of gross anatomy course, the contribution of TAs in the overall teaching‐learning outcome of gross anatomy courses in Caribbean medical schools is unknown. A structured questionnaire was given to first semester students who recently received the help from TAs in gross anatomy laboratory in St. Matthews University, Grand Cayman. Approximately 80 percent of students preferred TAs on each station during small group discussion to help comprehend the objectives as well wanted to continue to have TAs to pursue further study.
A slow ATP‐induced conformational change limits the rate of DNA binding but not the rate of β clamp binding by the Escherichia coli γ‐complex clamp loader
Authors: Thompson Jennifer Ann , Paschall Christopher O , Bloom Linda B
Published: June 2021

In the E. coli DNA replication system, the five‐subunit clamp loader, γ‐complex, loads the βsliding clamp onto DNA. The βclamp, a ring shaped dimer of identical subunits, tethers DNA polymerase III to DNA and enhances the efficiency of replication by increasing the processivity of DNA synthesis. In the presence of ATP, γ‐complex binds to βand DNA to form a ternary complex. Binding to primed template DNA triggers the clamp loader to hydrolyze ATP. Upon ATP hydrolysis, βis released onto DNA and γ‐complex dissociates from the complex. We have developed a βbinding fluorescence assay by covalently attaching a fluorophore, pyrene, onto a Cys residue (Q299C) ofβ. The fluorescence increases two‐fold when γ‐ complex binds β‐pyrene. Using this assay, the rates of association, kon of 3.5 x 107 M−1s−1 and dissociation, koff of 0.005 s−1 of γ‐complex binding to βwere determined in real time. In addition, the kinetics of γ‐ complex binding to DNA were measured in real time to compare to those for βbinding. When γ‐complex is pre‐incubated with ATP, the clamp loader can bind βor DNA with no preferred order. When there is no ATP pre‐incubation, γ‐complex must undergo a slow ATP‐induced conformational change before binding to DNA but does not have to undergo this change when binding toβ. Thus, there may be a kinetic preference for γ‐complex to bind βbefore DNA during the clamp loading reaction cycle.

This work was supported by NIH grant GM055596.
Analyzing DNA Replication Timing in the Human Genome
Authors: Taylor Christopher Michael , Karnani Neerja , Malhotra Ankit , Robins Gabriel , Dutta Anindya
Published: June 2021

The time of replication in the human genome is of interest due to its interaction with chromatin structure, gene density, and origin activity. We developed a method to profile this timing using DNA tiling microarrays. Areas of the genome were categorized to replicate in early S‐phase, mid S‐phase, late S‐phase, or to exhibit inter‐allelic variation in replication timing. Further, a continuous profile of replication timing was reconstructed for areas that did not exhibit inter‐allelic variation. The robustsness of the computational algorithm was tested on simulated positive control data and random negative control data. Early replicating regions showed strong positive correlation with activating histone methylation and acetylation marks and high gene density. Late replicating areas displayed strong positive correlation with a repressive histone marker, and low gene density. Interestingly, areas exhibiting inter‐allelic variation in replication timing showed positive correlations with both repressive and activating histone marks, consistent with the hypothesis that they represent areas where the two alleles are in different epigenetic environments. This work was supported by funding from the NIH.
Solution conformations of primer DNA shuttling between the polymerase and 3′‐5′ exonuclease sites of DNA polymerase I
Authors: Datta Kausiki , Johnson Neil P. , LiCata Vince J. , von Hippel Peter H.
Published: June 2021

E. coli DNA polymerase I serves as an excellent model for understanding structure‐function relationships in DNAP. To better understand the role of the intrinsic 3'→5' exonuclease (exo) activity of DNAP in controlling the fidelity of DNA synthesis we have examined the equilibrium distribution of the primer terminus between the exo and polymerase (pol) active sites during both template‐directed synthesis and misincorporation processes. By monitoring the low energy (>300 nm) circular dichroism and fluorescence of 2‐aminopurine dimer probes placed at various positions in Primer‐Template (P‐T) DNA constructs we have been able to confirm structural studies which showed that when the 3′‐end of the primer is bound at the exo site the three residues at primer terminus are significantly 'frayed' beyond the level characteristic of the ss‐DNA conformation. We have also shown that up to four bps of the ds‐DNA at the P‐T junction adopt an A‐form conformation when the primer end occupies the pol site, but not when it is bound at the exo site. This shows that the duplex DNA beyond the P‐T junction plays an important mechanistic role in guiding the primer end to the appropriate active site. In addition we show that even for matched P‐T DNA the primer terminus partitions between the two active sites, and that the fractional occupancy of the two sites depends on the stability of the bps at the P‐T junction and on the presence of divalent metal ions.
Interaction of the Glucocorticoid Receptor with the Chromatin Landscape
Authors: Hager Gordon Lee , Sabo Pete , Thurman Robert , Stamatoyannopoulos John A , Sung Myong‐Hee , Biddie Simon C , John Sam
Published: June 2021

In model systems, nuclear receptors induce chromatin remodeling at regulatory elements. We have carried out a genome‐wide analysis of GR binding events, as well as a global characterization of DNaseI hypersensitive transitions associated with GR responsive genes. Our findings indicate that receptor/genome interactions are universally associated with local chromatin remodeling events. Furthermore, multiple remodeling systems are required for these events. The presence of these locally open chromatin regions is highly cell specific, and both GR binding and GR transcription regulation correlate well with the presence of these sites. These results indicate that the chromatin status of a specific gene locus in a given cell type is a critical determinant for the GR responsiveness of the locus.
The role of SWI/SNF in regulating smooth muscle differentiation
Authors: Herring B. Paul , Zhang Min , Zhou Jiliang
Published: June 2021

The myocardin related transcription factors (MRTFs) play a critical role in regulating serum response factor's (SRF) ability to drive smooth muscle differentiation. MRTFs can induce the transcription of smooth muscle‐specific genes in nonmuscle cells through a mechanism involving the induction of SRF binding to the promoters of these genes, within intact chromatin. The goal of our studies is to understand how MRTFs can facilitate SRF binding within an otherwise closed chromatin landscape. We found that MRTFs can recruit SWI/SNF ATP‐dependent chromatin remodeling complexes, to the promoters of smooth muscle‐specific genes thereby permitting high affinity binding of SRF. Expression of dominant negative Brg1 or knockdown of Brg1 attenuated expression of SRF dependent smooth muscle‐specific genes in primary cultures of smooth muscle cells. Immunoprecipitation assays revealed that Brg1, SRF and MRTFs form a complex in vivo and Brg1 directly binds MRTFs in vitro. Dominant negative Brg1 significantly attenuated the ability of MRTFs to increase SRF binding to the promoters of smooth muscle‐specific genes. Mice harboring a smooth muscle‐specific knockout of Brg1 were generated and preliminary analysis of these mice revealed defects in GI development, including a significantly shorter gut with attenuated contractility in Brg1 knockout mice. Supported by DK61130, an AHA fellowship to MZ and an AHA SDG to LZ.
Antiproliferative Effects of Novel Glycosylated 24‐kDa Human Growth Hormone on MCF‐7 Breast Cancer Cells
Authors: Haro Luis S. , Haro Elizabeth K. , Muñoz Jesus , Martinez Andrew O.
Published: June 2021

Human growth hormone (hGH) has been correlated with increased incidence of breast cancer, pointing to a need for development of hGH antagonists to block hGH‐dependent breast cancers. The objective of this research was to investigate the new untested concept that glycosylated 24‐kDa‐hGH would impede proliferation of human breast cancer cells. The hypothesis tested was that glycosylated 24‐kDa‐hGH would produce growth‐inhibitory effects in MCF‐7 human breast cancer cells.

Methods

To test this hypothesis, the number of viable breast cancer cells were quantified following a 72 hr incubation in nutrient‐rich media at 37 °C, in a humidified atmosphere of 95% air, 5% CO2, in the absence of GHs (vehicle control) or in the presence of either 22‐kDa hGH or glycosylated 24‐kDa‐hGH. One‐Way ANOVA followed by Student‐Neuman‐Keuls post‐hoc test assessed differences in breast cancer cell proliferation among the treatments. Results demonstrated that glycosylated 24‐kDa‐hGH significantly decreased the growth of human breast cancer cells compared to the 22‐kDa hGH and to the control group whereas 22‐kDa hGH stimulated their proliferation. Glycosylated 24‐kDa‐hGH acted as an antagonist of 22‐kDa hGH. The importance of this work is that it demonstrates that glycosylated 24‐kDa‐hGH possesses therapeutic potential as an anti‐neoplastic agent for combating human breast cancer growth in vivo.
Effect of large ribosomal subunit proteins on ribosome biogenesis and cell cycle
Authors: Thapa Mamata , Bommakanti Ananth , Lindahl Lasse , Zengel Janice
Published: June 2021

Ribosome biogenesis and cell cycle are coordinated processes. Recent studies in mammalian cell lines have showed that ribosome biogenesis is linked to tumorgenesis, where mutation or depletion of ribosomal factors, leads cancer cell proliferation. The yeast Saccharomyces cerevisae is a useful model organism for understanding the connections between ribosome biogenesis and cell cycle control. Only a handful of studies have been done and these have mainly focused on different transacting factors involved in ribosome biogenesis. Here we are focusing on the role of ribosomal proteins. Our lab has found that depleting the ribosomal protein Rpl4 results in defects in both rRNA processing and cell cycle with accumulation of di‐ and tri‐ budded cells. This discovery has led our lab to investigate other ribosomal proteins also have implications in these two cellular pathways. Depletion of ribosomal proteins Rpl7A, Rpl18A, Rpl37, Rpl40A in S. cerevisae also resulted in yeast cells with di‐ and tri‐ buds, unlike the wild type cells, which only produce a single bud at a time. To fully understand which stage of cell cycle is defective, fluorescent activated cell sorter and microscopy analysis are in progress. We are currently characterizing defects in rRNA processing and ribosomal subunit assembly during depletion of these ribosomal proteins.