bluuebunny/crossref_metadata_2025 huggingface dataset:
Type 1 Induced Diabetes Abolishes Sex Differences in Proteinuria and Angiotensinogen Excretion in mRen2.Lewis Hypertensive Rats.
Authors: Gilliam Shea , Pirro Nancy , Cohen Jonathan , Chappell Mark , Yamaleyeva Liliya
Published: June 2021

The mRen2.Lewis strain exhibits sex differences such that males have higher blood pressure, proteinuria, and renal angiotensinogen (Aogen), as well as plasma and renal Ang II, but lower Ang‐(1–7). The present study examined whether these indices of cardiovascular disease remain in the adult mRen2 following induction of streptozotocin‐induced (STZ) diabetes at 15 weeks of age (65 mg/kg; single dose), and after ovariectomy (OVX, 11 weeks of age). In the STZ‐mRen2 after 4 weeks, proteinuria increased 8‐fold in males, but 20‐ and 30‐fold in intact and OVX females such that protein excretion was now similar among all groups. STZ increased Aogen excretion in males [3.5‐fold; 81 ± 13 to 292 ± 49 ng/mg creatinine], but to a greater extent in the intact [900‐fold; 0.3 ± 0.1 to 269 ± 109 ng/mg] and OVX females [200‐fold; 2.7 ± 0.7 to 475 ± 121 ng/mg]. Systolic blood pressures were not changed with STX in males [214 ± 6 vs. 218 ± 10 mmHg] or intact females [146 ± 6 vs. 164 ± 9 mmHg], but increased in the OVX group [162 ± 8 to 193 ± 3 mmHg]. However, plasma C‐reactive protein increased with STZ in intact [270 ±10 to 420 ± 17 μg/ml] and OVX females [216 ± 7 to 329 ± 18 μg/ml], but not males [246 ± 25 vs. 268 ± 14 μg/ml]. We conclude from these data that the renal protection afforded to the female mRen2 as compared to the males is abolished by STZ‐induced diabetes which may be independent of higher blood pressure. Funding: HL‐56973, HL‐51952.
Soluble Epoxide Hydrolase Inhibition (SEHi) and Thiazolidinedione (TZD) in combination provide greater renal injury protection in Spontaneously Hypertensive Obese (SHROB) Rats.
Authors: Shaw Sean , Cherian‐Shaw Mary , Imig John D
Published: June 2021

Epoxyeicosanoids have cardiovascular protective properties. SEHi increase epoxyeicosanoids and have therapeutic value in renal diseases. Rosiglitazone (RGZ) is a TZD used as an antidiabetic drug. RGZ and the SEHi, tAUCB, were used in this study to examine their actions to protect the kidney against injury in cardiometabolic syndrome SHROB rats. SHROB were treated with RGZ, tAUCB, tAUCB & RGZ for 4‐weeks and compared to WKY rats. Blood pressure was measured, kidneys processed for histology and urine analyzed. Blood pressure increased in SHROB rats (166±4 mmHg) compared to WKY (123±2 mmHg) and decreased in SHROB rats treated with RGZ (152±10 mmHg), tAUCB (137±7 mmHg), tAUCB & RGZ (152±10 mmHg). Proteinuria was increased in SHROB (370±35 mg/day) compared to WKY (29±2 mg/day). In SHROB treated with RGZ, tAUCB, tAUCB & RGZ proteinuria decreased by 38%, 38%, and 60%. Glomerular injury score in SHROB averaged 1.6±0.07 and was not decreased by RGZ or tAUCB treatment; however, tAUCB & RGZ combined significantly improved the glomerular injury score. Macrophage infiltration (CD68+) in the kidneys increased in SHROB rats vs. WKY (108±12 and 15±3 mm2). Combined tAUCB & RGZ demonstrated the greatest decrease in macrophage infiltration. These results indicate that even though SEHi or TZD have renal protective benefits when used individually, the tAUCB & RGZ combination is more beneficial in cardiometabolic syndrome SHROB rats.
In Situ Measurement of Oligomerization State of NBCe1‐A in Kidney Tissues using Spatial Fluorescence Intensity Fluctuation Analysis
Authors: Sergeev Mikhail , Godin Antoine G , Kao Liyo , Abuladze Natalia , Wiseman Paul W , Kurtz Ira
Published: June 2021

NBCe1‐A plays an important role in absorbing sodium bicarbonate across the basolateral membrane of the proximal tubule. We have previously showed that minimal functional unit of NBCe1‐A is a monomer, and based on in‐vitro biochemical studies in HEK293 cells, the oligomeric state of the cotransporter was shown to be predominantly dimeric with monomeric and higher oligomeric forms also present. In the present study, we developed an in situ measurement methodology to determine the oligomeric state of NBCe1‐A without requiring tissue disruption. We used fluorescent moment image analysis and spatial intensity distribution analysis (SpIDA) to study the oligomeric state of NBCe1‐A. Both methods allow for unbiased quantitative measurement of fluorescent particle densities and oligomerization states within individual images acquired with laser‐scanning microscopy. Initially we examined basal membranes of highly adherent CHO K1 cells expressing eGFP‐tagged NBCe1‐A, and showed that NBCe1‐A existed primarily in the monomeric state and negligibly in the dimeric state on the cell membrane. We immunolabeled NBCe1‐A in kidney tissues demonstrating the NBCe1‐A is present in dimeric and rarely in higher order oligomeric states. As a control, we compared the results obtained from LSM images of tissues of NBCe1‐A knockout mouse. These experiments demonstrate for the first time the in situ oligomeric state(s) of NBCe1‐A.
Inhibition of phosphatidylinositol 3‐kinase facilitates proton‐induced apoptosis of B lymphoma cells
Authors: Wang Li‐Hua , Wang Ning , Liang You‐You , Ma He‐Ping
Published: June 2021

Our previous studies have shown that phosphatidylinositol 3‐kinase (PI3K) promotes B lymphoma cell cycle progression by stimulating an intermediate‐conductance Ca2+‐activated K+ channel. To determine whether PI3K also participates in a pathway that accounts for defective apoptosis in B lymphoma cells, we used flow cytometry and confocal microscopy methods to evaluate apoptotic cells with the propidium iodide/annexin V double‐labeling technique. The data showed that B lymphoma cells were very resistant to acidic conditions. Significant intracellular acidification and apoptosis occurred only when extracellular pH was lower than 5. Inhibition of PI3K with LY‐294002 facilitated proton‐induced intracellular acidification and apoptosis. The effects of LY‐294002 on proton‐induced apoptosis and intracellular acidification were mimicked by bafilomycin, a potent inhibitor of vacuolar H+‐ATPase. Our results suggest that inhibition of PI3K facilitates proton‐induced apoptosis in B lymphoma cells probably by reducing H+‐ATPase‐mediated proton efflux. However, whether H+‐ATPase is mislocated in the plasma membrane and whether the mislocation is caused by elevated PI3K activity in B lymphoma cells remain to be further determined. This work is supported by Natural Science Foundation of Heilongjiang Province D2004‐03 to Li‐Hua Wang and Department of Health and Human Services, National Institutes of Health (NIH) Grant R01‐DK‐067110 to He‐Ping Ma
H11 Kinase/Hsp22‐mediated activation of the valosin‐containing protein promotes the expression of the inducible isoform of nitric oxide synthase in the heart
Authors: Lizano Paulo , Dai Huacheng , Depre Christophe
Published: June 2021

Expression of H11 kinase/Hsp22 (Hsp22) in the heart stimulates cell growth and survival, and activates the expression of the inducible nitric oxide synthase (iNOS) mediating ischemic preconditioning, the most powerful prophylaxis against cardiac cell death, through activation of the Akt pathway. Our goal was to elucidate the downstream effector by which Hsp22 and Akt increase iNOS expression. We hypothesized that such effector is the valosin‐containing protein (VCP), an Akt substrate activating the transcription factor NF‐kB by promoting the poly‐ubiquitination of the NF‐kB inhibitor, IkBa. Using two‐dimensional gel electrophoresis and mass spectrometry, and immunoprecipitation, we found that Hsp22 and Akt co‐localize and interact together with the valosin‐containing protein (VCP). This interaction predominated in the nuclear fraction of adult mouse cardiac myocytes, and it was markedly increased in a transgenic mouse with cardiac‐specific over‐expression of Hsp22. Adeno‐mediated over‐expression of VCP in isolated cardiomyocytes activated NF‐kB and dose‐dependently increased the expression of iNOS, which was abolished by the NF‐kB inhibitor SN50. Over‐expression of a dominant‐negative mutant of VCP lacking the domain of poly‐ubiquitination did not increase iNOS expression. Therefore, VCP mediates the expression of iNOS downstream Hsp22 and Akt through an NF‐kB‐dependent mechanism.
Skeletal Muscle as an Endocrine organ; the effect of TNF‐alpha
Authors: Lightfoot Adam Paul , Harrison Dawn , Kayani Anna , McArdle Francis , Griffiths Richard D , McArdle Anne
Published: June 2021

During chronic inflammation muscle is exposed to inflammatory cytokines; a key player in this is TNF‐α. Muscle has protective mechanisms to adapt to stress, via further expression of cytokines and Heat shock proteins (HSPs), which can be released into the extracellular environment and then involved in apocrine or paracrine signalling pathways. C2C12 myotubes in vitro treated with TNF‐α (25ng/ml) showed significant up‐regulation of 7 pro‐inflammatory genes by qPCR array analysis. Significant release of cytokines (IL‐6, MCP‐1, KC, IP‐10 & RANTES) was seen after 3 & 6 hours. In addition, TNF‐α up‐regulated intracellular levels of HSP60 & 70, with specific release of HSP60 at 3 hours. Treatment of C2C12 myotubes with recombinant HSP60 and 70 induced significant release of MCP‐1 and RANTES at 3 hours. Adult and old C57Bl6 mice treated with TNF‐α (7.5μg/kg) i.v. were subject to microdialysis perfusion of the gastrocnemius muscle to measure direct cytokine release from whole muscle. Adult mice showed a significant release of IL‐1β & MCP‐1, 1 hour post TNF‐α; old mice showed elevated basal levels of IL‐1β & MCP‐1, with a blunted response to TNF‐α. Data show TNF‐α induces an immune and stress response in muscle in vitro and HSPs have a paracrine signalling effect inducing release of cytokines. Whole muscle in vivo releases cytokines in response to TNF‐α treatment which is blunted in old mice. Supported by University of Liverpool.
Targeting of Pancreatic β‐Cells With High Specificity Using a Heterobivalent GLP‐1/Glibenclamide Ligand
Authors: Hart Nathaniel , Vagner Josef , Chung Woo Jin , Weber Craig , Limesand Sean W , De Silva Channa R , Lynch Ronald M
Published: June 2021

Pancreatic β‐cell dysfunction, and subsequent loss of β‐cell mass underlies the development of Diabetes Mellitus. The ability to monitor β‐cell mass in vivo is limited because the endocrine pancreas comprises less than 5% of the total pancreatic mass requiring highly specific β‐cell targeting agents; new targeting agents will require at least 100X higher specificity than previously tested agents. We previously demonstrated that multivalent ligands (MVL) composed of two different receptor binding moieties that crosslink multiple receptors, increase the specificity of binding to cells that express only the complimentary receptor pair. To test this approach for use in β‐cell specific targeting, we synthesized a ligand composed of Glucagon‐like Peptide 1 and Glibenclamide (Glb, Sulfonylurea Receptor 1ligand). Using rt‐PCR, expression of these surface receptors was demonstrated in a β‐cell line βTC3, while the ductal cell line PANC1 was shown to express only SUR1. The divalent GLP‐1/Glb binds βTC3 cells at concentrations at least 50 fold lower than required for binding to PANC1cells. The MVL rapidly is internalized from the cell surface (within 2–3 min) after binding via endocytosis. Our findings indicate that the GLP‐1/Glb MVL has increased affinity and specificity for β‐cells, and therefore provides a basis for developing high specificity targeting agents to monitor β‐cell mass.

Supported by: Juvenile Diabetes Research Foundation, and the Arizona Biomedical Research Commission
Organic osmolytes and their role in adaptation of European eel Anguilla anguilla to high salinity environments.
Authors: Kalujnaia Svetlana , Osborne C. J. , Cramb G.
Published: June 2021

The European eel maintains strict osmoregulation of body fluids while migrating between freshwater (FW) and sea water (SW) environments. Microarray studies on salinity adaptation in the eel highlighted genes responsible for the transport of a variety of solutes known to act as osmolytes in mammals. SMIT1 and HMIT genes, implicated in myo‐inositol transport, were isolated, cloned and sequenced. Following these initial studies, other genes involved in osmolyte transport and regulation, including the taurine transporter (TauT) and TonEBP, were cloned and sequenced, and their role in salinity adaptation was investigated. Q‐PCR was used to determine gene expression in the major osmoregulatory tissues (intestine, kidney and gill) at 6 hours, 2, 7 days and 5 months after transfer of eels to SW. In the intestine, SMIT1 was up‐regulated following 2 days SW transfer, however increases in TonEBP expression were only found 5 months after salinity transfer. In the kidney, HMIT, TauT and TonEBP mRNAs were up‐regulated 7 days after fish acclimation to salinity. No changes in the expression of any of these genes were observed in the gill. These studies have revealed differential tissue expression of key osmolyte transporters and the TonEBP transcription factor in the eel during salinity acclimation indicating potential roles in the long term adaptation of eels to the SW environment.
Lowering NaCl accelerates degradation of TonEBP/OREBP mRNA
Authors: Izumi Yuiricho , Gallazzini Morgan , Ferraris Joan D , Burg Maurice B
Published: June 2021

Not only does hypertonicity increase the mRNA and protein abundance of the osmoprotective transcription factor, TonEBP/OREBP, but hypotonicity decreases them. The mechanism of the hypotonicity‐induced decrease is poorly understood. We examined this question by measuring the time course of tonicity‐induced increases and decreases of TonEBP/OREBP mRNA abundance, using real time RT‐PCR of extracts from HEK293 cells. Acute increase of osmolality from 300 to 500 mosmol/kg raises TonEBP/OREBP mRNA by a maximum of 2.8‐fold within 8 hours, and it remains 1.7‐fold higher after the cells are adapted to 500 mosmol/kg through several passages. The half life of TonEBP/OREBP mRNA (measured after addition of actinomycin D) is 24 hr both in HEK293 cells adapted to 500 mosmol/kg and in cells left at 300 mosmol/kg. However, decreasing osmolality from 500 to 300 mosmol/kg lowers TonEBP/OREBP mRNA abundance by 60% within 4 hours, which is much too rapid to be explained by decreased transcription alone. We conclude that lowering NaCl accelerates degradation of TonEBP/OREBP mRNA, reducing the time for adaptation to the new condition. Supported by IRP NHLBI
Quantification of Renal Albumin Filtration and FcRn‐Mediated Transcytosis via 2‐Photon Microscopy
Authors: Sandoval Ruben M , Wagner Mark C , Campos‐Bilderback Silvia B , Wean Sarah E , Molitoris Bruce A
Published: June 2021

The kidney filters and reclaims both ions and macromolecules of various size. Endocytosis is known to play a key role in protein reabsorption by proximal tubule cells (PTC) yet the fate of endocytosed molecules remains unknown. We have utilized and maximized intravital 2‐photon microscopy to optimize the signal from filtered Texas Red – rat serum albumin (TR‐RSA) and follow its endocytosis and intracellular trafficking in PTC compared with fluorescent dextrans and beta‐2‐microglobulin (B2M). Studies in Munich‐Wistar rats (MW), that have surface glomeruli, under optimal conditions using 12‐bit non‐descanned detectors gave background corrected glomerular sieving coefficient (GSC) values of 0.030 +/−0.005. Studies conducted in fasted vs fed MW rats revealed a drop in the GSC to 0.011 ± 0.005 in fasted rats. Endocytosis of RSA by proximal tubules (PT) consistently occurred within minutes after i.v. infusion followed by rapid and prolonged transcytosis of TR‐RSA via both intracellular vesicles and tubular projections at the basolateral‐interstitial junction of PT's. This was not true for filtered fluorescent dextrans or B2M. FcRn was visualized in the apical membrane of PTC with indirect IF and PT: apical membrane preparations were Western blot positive of FcRn. These studies indicate there is physiologic regulation of glomerular permeability and that FcRn mediates the transcytosis of albumin within PTC.
Adenosine monophosphate kinase (AMPK) promotes calcium signaling in lung endothelial barrier repair
Authors: Creighton Judy , Sayner Sarah , Alexeyev Misha , Insel Paul
Published: June 2021

Cytosolic calcium [Ca2+]i signaling is a multi‐step process responsible for regulating many cellular events, including barrier disruption in endothelium. In parallel with, but considered separate from its barrier disruptive effects, [Ca2+]i activates AMPK. Although best known for its role as a metabolic sensor, AMPK also functions as a protective feedback mechanism that strengthens protein‐protein interactions and cell‐cell contact. In recent studies, we find that AMPK promotes a calcium entry mechanism in pulmonary microvascular endothelial cells (PMVECs). AMPK inhibition (using pharmacological or shRNA approaches) attenuated calcium entry induced by the inflammatory mimetic thapsigargin, decreased barrier resistance, and blunted PMVEC wound resealing. Immunochemistry and cell membrane fractionation studies revealed PMVECs express the AMPK alpha 1 subunit that localized to caveolin 1 enriched lipid rafts at cell‐cell borders. These data thus identify AMPK as a previously unappreciated contributor to the pulmonary endothelial injury response. (Supported by HL102296 and HL007261).
Female gender and estrous cycle‐dependent estrogen regulation of intestinal secretion and KCNQ1 channel function via membrane estrogen receptors
Authors: O'Mahony Fiona , Thomas Warren , Harvey Brian J.
Published: June 2021

Estradiol (E2) produces a female‐gender specific anti‐secretory response in rat distal colon which is mediated by rapid phosporylation (via PKCδ and PKA) and inactivation of KCNQ1 channels (1). The anti‐secretory action of E2 is sustained although the activation of the signalling protein kinases is transient. E2 (10nM) caused a rapid (<5min) PKCδ dependent decrease in KCNQ1 channel current and a latent (>30min) reduction in KCNQ1 channel density at the basolateral membrane of colonic crypt cells. E2 decreased secretion during the estrus, metaestrus and diestrus stages with only weak inhibition in the proestrus stage. The expression levels of PKCδ and PKA fluctuated throughout the estrous cycle and correlated with the potency of the anti‐secretory effect of E2. The expression of PKCδ and PKA were up‐regulated by E2 at a transcriptional level via a PKCδ‐MAPK‐CREB regulated pathway indicating a genomic priming of the anti‐secretory response. PKCδ was activated by the membrane impermeant E2‐BSA and this response was inhibited by the ER antagonist ICI 182,780. The 66kDa ERα was present at the plasma membrane in female colonic crypts and in lower abundance in male tissue. The study demonstrates an interdependent relationship between both rapid and genomic E2 anti‐secretory responses mediated by ERα.

Funded by the Higher Education Authority of Ireland PRTLI NBIP Cycle 4.
Clonal Analysis of Myofibroblast Differentiation
Authors: Chukwuma Sarah , Vaughan Melville
Published: June 2021

The objective of this study was to observe the growth and transition of the fibroblast cell into a myofibroblast. Fibroblasts make up the cells that maintain the dermis of skin, and slowly transform into the intermediary stage of the protomyofibroblast and finally the myofibroblast. We have observed cultured cells from tissue containing a mixed population of fibroblasts and myofibroblasts. Myofibroblasts are evident in normal wound healing due to the contraction effect of stress fibers. Alpha‐smooth muscle actin is an identifying protein found in stress fibers of these highly contractile cells that participate in normal wound healing. Protomyofibroblasts are made up of the necessary stress fibers that contain beta and gamma‐cytoplasmic actins, yet lack alpha‐smooth muscle actin to become a complete myofibroblast. After making an informative conclusion that fibroblasts are not genetically programmed to become myofibroblasts based on earlier experiments of isolating single cells and growing clonal populations, we wanted to observe this transition phase and determine the factors affecting the completion of this transition and inhibiting the protomyofibroblasts from becoming myofibroblasts. CT4hT cells were cultivated to isolate the protomyofibroblast cells, the phenotype is then induced with TGF‐beta, a transforming growth factor, and then grown in clonal populations.

Grant Funding Source: NSF SureStep/UCO Research & Grants
Virtual pelvic anatomy simulator improved medical student comprehension of pelvic anatomy
Authors: Hassinger James Peyton , Dozois Eric J , Holubar Stefan D , Pawlina Wojciech , Pendlimari Rajesh , Fidler Jeff L , Holmes David R , Robb Richard A
Published: June 2021

Given curricular time constraints, the complex relationships of pelvic viscera can be difficult for students to master. We developed an interactive 3‐dimensional virtual pelvic model using high‐resolution 3‐Telsa magnetic resonance and 64‐slice computed tomographic datasets of a healthy male. The aim of this study was to determine if use of this curricular adjunct was associated with improved comprehension of pelvic anatomy as measured by a 33 item test. The software was distributed to 47 first year medical students during their 3‐day pelvic anatomy curriculum. Forty students (85%) completed the test. Students used the software for a median of 15 (interquartile range [IQR] 10 – 22) minutes; total minutes studying pelvic anatomy was 240 (IQR 125 – 525) minutes. Minutes of software use was strongly associated with the total study time (p<0.0001). On univariate analysis, pelvic anatomy test scores were associated with both minutes of software use (p=0.05) and total study time (p=0.04). On multivariate analysis both software use (p=0.12) and total study time (p=0.08) trended towards significant association with test scores. Most students used the software alongside books to aid in initial spatial orientation to the pelvis. Virtual anatomy software can be a valuable, effective adjunct to traditional anatomy education. Source of funding: Mayo Foundation for Medical Education and Research.

Grant Funding Source: Source of funding: Mayo Foundation for Medical Education and Research.
Cadaver programs at the undergraduate level: student‐led dissection with a focus on exploring pathologies and anatomical variations
Authors: Flynn Vicki , Blackmon Ian , Darkow Andrew , Nekouian Natalie , Schulte Katherine , Potterfield April Collins
Published: June 2021

At Westminster College, an undergraduate institution, a cadaveric gross anatomical dissection was completed using the thirteenth edition of Grant's Dissector. The objectives of this dissection were to determine possible indicated health conditions, pathologies, and anatomical variations present in a single cadaver. Anatomical variations in the cadaver included renal, pelvic, coronary, porta hepatic, and transverse cervical vasculature, as well as variations of rib structure. In addition to anatomical variations, pathologies were studied by comparing findings to anatomical standards. Pathologies included pleural adhesion of the left lung, bronchopulmonary lymph nodes with anthracotic pigmentation, and calcification of the abdominal aorta. The analyses of pathologies were conducted to include histopathology of pulmonary, vascular, and cardiovascular tissue, as well as a calcium analysis of the abdominal aorta. These pathological test results assisted in confirming the indicated health condition of the cadaver. Overall, students gained an understanding and appreciation of standard human anatomy, pathologies, and anatomical variations through this exploration.
Diffusion of Innovations: Use of Mobile Technology for Point‐of‐Contact Clinical Anatomy Learning Resources
Authors: Trelease Robert
Published: June 2021

Scholarly work in anatomical sciences education has emphasized a crucial foundational role for introductory courses in gross anatomy in preclinical medical education. Despite successes of anatomy courses in preclinical curricula, a need remains for reinforcing fundamental knowledge with clinically relevant detail in clerkships and residencies. Clinical tasks and restrictive schedules typically leave little time for formal instruction. However, diffusion of innovations in education has provided new resources that may help address these challenges. Widespread self‐adoption of smartphones and mobile computing devices now equips students with "wearable" personal technology useful for self‐learning in point‐of‐contact clinical settings. The author designed Surgical Anatomy PRN to be a mobile anatomy learning resource for surgical clerkships and residencies. Initially programmed with XHTML, descriptive text, and anatomical images for personal digital assistants (PDAs), code and data were also usable on the more capable Web browsers of smartphones and personal computers. Chapter information was assembled for over two dozen of the most common diseases and clinical conditions encountered in UCLA general surgery clerkships. Surgical Anatomy PRN is currently being published for clerkships and residences as a combined, printed pocket book, Web‐based online resource, and iPhone ebook application.
Development of a Scientist‐Educator Program
Authors: Bader David , Dalley A F , Norden J J , Nanney L B , Pettepher C C , Richmond A W , Wente S , Chalkley R
Published: June 2021

Training programs in basic medical research and education have become increasingly specialized in recent years. The number of active basic research scientists with a comprehensive understanding of human biology, and specifically human anatomy, is rapidly decreasing. In response to this documented trend, Vanderbilt University initiated a Scientist‐Educator Program in 2005 to provide a coordinated curriculum where post‐doctoral fellows could obtain a cutting‐edge research experience coupled to in‐depth training in medical education initially focused on a gross anatomy curriculum. Now established, this three/four year program has expanded to include training fellows in neuroscience and will begin to explore training in other areas of need in medical education. Building upon the Program's documented strengths, the leadership team seeks to grow and establish itself as a nationally‐recognized leader in this important area of biomedical science. We will present our curriculum, databases on recruitment, fellow placement, and future plans.
Digital photography versus anatomical illustration: Which method is more accurate?
Authors: Reidenberg Joy S. , Asch Noah , Laitman Jeffrey T.
Published: June 2021

Has the digital revolution provided advances in generating more accurate images compared to hand‐drawn illustrations? In this study, we compared the accuracy of hand‐drawn illustrations versus digital photographs. A dolphin skull was both drawn and photographed from a lateral view, and the resulting images were scaled and superimposed over each other on a lateral photograph of the dolphin's whole head. Results show that both methodologies introduced morphological distortions during the reduction of a 3‐D object to a 2‐D image. Variability occurred in perspective, focus, and lighting. Artistic renditions incorporated perspective distortions when multiple views were summed into one image. Digital images revealed perspective variability related to the angle between the object and the camera. Variations in the range of focus were related to depth of field, which was fixed during camera shutter depression. However, the human eye accommodated to a range of focal points at variable distances, thus enabling the artist to draw all points in focus. The angle and amount of lighting increased depth perception, but obscured regions in shadow unless artificially lit by a flash. Digital manipulations of lighting also affected brightness/contrast or color saturation/hue. In sum, each method provided advantages that enhanced perception, but simultaneously created artifacts that distorted reality.
Using gross anatomy as a predictor of academic success in a post baccalaureate program
Authors: Norton Neil S , Jergenson Margaret A , Barritt Laura C , Ayers Frank J
Published: June 2021

Many Post‐Baccalaureate programs have been designed to aid students interested in a career in medicine. Over the past decade, significant interest in a career in dentistry has grown as applications in the United States have doubled. Unfortunately, the growth in Post‐Baccalaureate programs for pre‐dental students has lagged behind their medical counterparts. To help those applicants, our institution established a Post‐Baccalaureate program which is designed to help strengthen their academic abilities. As part of the program, in the summer prior to their matriculation into dental school, the students are required to take a two‐week intensive course in gross anatomy. In this course, students are given the same lectures and laboratory dissection that they would receive during their regular year dental school gross anatomy course. The students are required to take a written exam and practical exam at the end of the course. We have observed that success in the Gross Anatomy Post‐Baccalaureate course serves as a good predictor of future academic success in dental school. Additionally, it helped successfully enhance the students' test‐taking skills and prepared them for the rigors of dental school.
How Dental Students are taught the Anatomical Sciences: Survey results from North American basic science course directors
Authors: Lambert H Wayne , Gould Douglas J. , Burk Dorothy T. , Lee Lisa M. J. , Atsas Stavros , Hutchins Bob
Published: June 2021

Members of the Anatomical Sciences Section of the American Dental Education Association (ADEA) surveyed North American course directors to assess how undergraduate dental students are taught the anatomical sciences. Web‐based surveys were sent to faculty members charged with teaching anatomy, neuroanatomy, histology, and embryology in the US and Canada. The completed anatomy and neuroanatomy surveys received 100% and 98.5% response rates from the 67 dental schools, respectively. The ongoing embryology and histology survey has had 53 (79.1%) schools respond.

The results of these surveys indicate, amongst other things, that: 1) reliance upon medical school faculty and facilities is high; 2) emphasis on clinical topics has increased; 3) a general trend for a decrease in student contact hours is ongoing; 4) the use of computer‐assisted instruction (CAI) tools has increased; 5) a disparity in the number of contact hours reported for anatomical science courses exists among institutions; 6) a pattern of increased use of integrated curricula among dental schools has emerged; and 7) the experience levels of faculty indicate a future need for young faculty competent at teaching in the anatomical sciences.
Mentoring women‐ strategies and success stories
Authors: Benore Marilee Ann
Published: June 2021

Retaining, supporting and promoting women are vital to research progress and science education. From grade to graduate school, from the bench to the boardroom, mentoring and role models can make critical differences to the achievements of women scientists. This seminar will highlight and summarize the strategies and programs that have proven successful in ensuring that women continue to choose, stay and excel in science. Examples include modes of mentoring such as mentornet, programs such as NSF ADVANCE and Women in Learning and Leadership (WILL), as well as organizations such as the Association for Women in Science (WISE).
CHANGES OF ANTIOXIDANT ACTIVITY AND PHENOLIC COMPOUNDS DURING FERMENTATION BY LACTIC ACID BACTERA
Authors: Jung Jihye , Shin Jung Hee , Yoo Sun Kyun
Published: June 2021

The rhizome of Angelica sinensis is one of the most important natural herbs used as a sedative or a tonic agent. Its ferulic acid, ligustilide, and phthalides of pharmacological effects include anti‐oxidative, anti‐inflammatory, and immunomodulatory activities. The object of this study was to evaluate the effect of fermented extract of Angelica sinensis on antioxidant activity. The extract was manufactured using hot‐water (105oC) and concentrated to 20%. Fermentation was conducted on pH 7.0, 30oC and 5% concentrated extract as conversion substrate. The mineral medium consisted of 3.0 g KH2PO4, 0.01 g FeSO4 · H2O, 0.01 g MnSO4 · 4H2O, 0.2 g MgSO4 · 7H2O, 0.01 g NaCl, and 0.05 g CaCO3 per 1 liter deionized water. With the cell free extract, the changes of total phenolic content, DPPH radical scavenging activity, total flavonoids, and total antioxidant activity were analyzed during the fermentation. The 104.0 GAE (gallic acid)/100 g of total phenolic compounds, 52.4 catechin mg/100 g, 82.1 % DPPH radical scavenging capacity, and 82.1 umol trolox/g were detected from the before fermentation. After 24 h fermentation, all four factors were increased feasibly to about 20 to 50%. This result suggests that the antioxidant activity of Angelica sinensis extract could be improved through microbial fermentation.
Identification of novel regulatory domains in the yeast Snf2 protein
Authors: Sen Payel , Ghosh Sujana , Pugh Frank , Bartholomew Blaine
Published: June 2021

The yeast SWI/SNF complex is a 1MDa multisubunit chromatin remodeler that uses ATP to alter chromatin structure for global transcription regulation. The ATPase‐helicase domain of the catalytic subunit Snf2 has been studied extensively but there has been little focus on regions outside this domain. This study identifies four domains outside the ATPase‐helicase domain that regulate catalytic activity, one of which also plays a role in targeting the complex for recruitment. Deletion of the first sixty amino acids (ΔN, 1–60) and the double AT‐hook (ΔAT, 1446–1530) decrease the rate of remodeling without much effect on ATP hydrolysis whereas deletion of spacer (1312–1444) and QLQ (239–527) show a severe defect in ATP hydrolysis. ΔN and ΔAT reduce remodeling efficiency by 13 and 29 fold respectively compared to WT, an extent that far exceeds their defect in ATP hydrolysis that is only 2.5 and 3.2 fold. Thus ΔN and ΔAT uncouple ATP hydrolysis from remodeling. The defects conferred by deletion of these domains are validated in vivo by growth assays and genome‐wide expression profiling. Biochemical characterization of complexes harboring point mutations in 9 amino acids conserved from yeast to humans in the spacer domain revealed moderate to severe remodeling defects. This study identifies novel, physiologically relevant domains and residues in Snf2 that regulate its targeting and catalytic function.

NIH (GM48413)
Chemical‐genomic dissection of the CTD code
Authors: Ansari Aseem Z
Published: June 2021

Sequential waves of modifications of the RNA polymerase II (Pol II) carboxyl‐terminal domain (CTD) coordinate the stage‐specific association and release of cellular machines that participate in RNA biogenesis. We examined the genome‐wide distributions of the “early” (phospho‐serine 5), “mid” (phospho‐serine 7) and “late” (phospho‐serine 2) marks of the CTD. We observed gene‐class specific patterns and found a surprising degree of co‐occurrence of the CTD marks. In particular, contrary to its role in 3′ processing of non‐coding RNA, the Ser7‐P marks are placed early and retained until transcription termination at all Pol II‐dependent genes. Chemical‐genomic analysis reveals that the promoter‐distal Ser7‐P marks are not remnants of early phosphorylation events, but are placed anew by Bur1, a CTD kinase that associates with the elongating Pol II. Consistent with the ability of Bur1 to facilitate transcription elongation, high levels of Ser7‐P are observed at highly transcribed genes. This pattern of Ser7‐P enrichment could serve as a shared signal for cellular machines that process functional non‐coding RNA and enhance transcription elongation through protein‐coding genes.
Structural Studies of ATP‐dependent chromatin remodeling
Authors: Leschziner Andres , Isaac Berith , Clapier Cedric R , Cairns Bradley R
Published: June 2021

ATP‐dependent chromatin remodeling complexes (“remodelers”) are large macromolecular assemblies that use ATP hydrolysis to non‐covalently alter the structure of nucleosomes. Remodelers, conserved from yeast to humans, can be classified into different families based on the identity of their ATPase subunit. The diversity in their composition beyond the ATPase is reflected in the variety of outcomes of which they are capable both in vitro and in vivo: sliding of nucleosomes along DNA, ejection of histone dimers or octamers and histone dimer exchange.

How do remodelers remodel chromatin? How are different products generated? Are they the result of fundamentally different mechanisms or rather of the relative fine‐tuning of a common one? We are taking a structural approach to addressing these questions, using three‐dimensional electron microscopy (3D EM), a technique well suited to the study of large, conformationally flexible complexes.

Our model system is the remodeler RSC from S.cerevisiae, a ~ 1MDa 15‐subunit complex. We have obtained 3D reconstructions of RSC and have shown that it is conformationally flexible. We have proposed that a large cavity seen in the structure is the binding site for the nucleosome as it accounts for a number of biochemical observations. Our immediate goals are to map the location of RSC components as well as the actual binding site of the nucleosome in our reconstruction.
Genome wide mapping of histone methylation reveals a distinct epigenomic signature in human pluripotent stem cells
Authors: Hitchler Michael James , Veerappan Chendhore , Aman Joseph , Fox Victoria , Cuningham Cynthia , Van Den Berg David , Berman Benjamin , Pera Martin , Laird Peter , Rice Judd C
Published: June 2021

In human pluripotent stem cell cultures, differentiation‐associated genes are silenced but “poised” for expression by bivalent chromatin marks: the concurrent presence of activating (H3K4me3) and repressing (H3K27me3) histone modifications. Here we sought to map this bivalent state across the genome of a subset of heterogeneous cultured human embryonic stem cells; the fraction that expresses the highest amounts of pluripotency‐associated factors (hpESCs). To this end, ChIP‐Seq was performed for H3K4me3 and H3K27me3 in hpESCs and the heterogeneous culture from which they were purified. Consistent with previous reports, both populations displayed high levels of H3K27me3 at differentiation‐associated genes. While H3K4me3 was also abundant at differentiation‐associated genes in the heterogeneous culture, this histone modification was generally absent at these genes in hpESCs. Therefore, our findings indicate that hpESCs possess a distinct epigenomic signature that typically lacks bivalent domains and that the repression of differentiation‐associated genes is regulated primarily by Polycomb. Collectively, our results strongly suggest that bivalency represents a transitory state between pluripotent and lineage committed cells.
DNA Sequence Specificity of Alternatively Spliced TCF1E
Authors: Hoverter Nate Pasquale , Ting Ju Hui , Waterman Marian
Published: June 2021

The purpose of this study is to define the activities of a unique DNA binding domain called the C‐clamp, present in alternatively spliced TCF1E. All isoforms of TCF1 contain a sequence specific high mobility group (HMG) box that binds Wnt response elements to control target gene expression. To determine if the C‐clamp also has DNA sequence specificity, Cyclic Amplification and Selection of Targets (CASTing) was performed by isolation of TCF1E‐DNA complexes and progressively enriching high affinity binding sequences with PCR. CASTing revealed that the C‐clamp of TCF1E contains sequence specificity for 5′‐RCCG‐3′ elements independent of the HMG box. Inclusion of RCCG elements in TCF luciferase reporters increased the transcriptional output of TCF1E, validating the CASTing data. The recognition of RCCG elements by TCF1E may be important for its specific activation of target genes such as SP5 and LEF1, which contain RCCG elements adjacent to Wnt response elements in their promoters. The DNA binding activities of the C‐clamp are also important to the regulation of p21 expression. Expression of TCF1E in colon cancer cells caused a p21 dependent stall in cell growth, whereas expression of a non‐DNA binding C‐clamp mutant TCF1E did not upregulate p21 and did not stall cell growth. Thus, the DNA binding activities of the C‐clamp play an important role in determining the transcriptional output of TCF1E.
TREM‐2, an innate immune receptor, is dependent on C/EBPalpha and PU.1 transcription factors during the differentiation of hematopoietic stem cells to macrophages
Authors: MANCARELLI M.MICHELA , Derossi Charles , Torbett Bruce E
Published: June 2021

Triggering receptor on myeloid cells‐2 (TREM‐2) is a member of the innate immune signaling receptor TREM family, reportedly only expressed on macrophages, dendritic cells, microglia, but not on monocytes. TREM‐2 is necessary for regulation of macrophage activation, and loss of TREM‐2 results in a decrease of phagocytosis and an inability to control inflammatory mediators. Since little is known about TREM‐2 gene expression during human stem cell development to macrophages or granulocytes, we first determined that TREM‐2 was only expressed on fully differentiated macrophages. We further confirmed loss of TREM‐2 expression in macrophage colony forming receptor null mice, which cannot generate macrophages, thus demonstrating similar TREM‐2 expression patterns in human and mice. Studies of PU.1 restoration in PU.1 null cells demonstrated a requirement for PU.1 in TREM‐2 regulation. To determine the mechanisms for end‐stage myeloid lineage regulation, the human TREM‐2 promoter region was cloned, sequenced, and transcriptionally mapped and putative C/EBPalpha (cebpa) and pu.1 binding sites were identified. Functional studies showed requirements for both CEBPA and PU.1 for TREM‐2 activation. These findings define the mechanisms of lineage specific TREM‐2 regulation. We are now investigating the role of TREM‐2 in macrophage function during angiogenesis and inflammation.
Characterization of the small heat shock protein αB‐crystallin chaperone binding regions in human γD‐crystallin using single domain constructs of γD‐crystallin
Authors: Acosta‐Sampson Ligia , King Jonathan
Published: June 2021

α‐crystallin, an ATP‐independent small heat shock protein, is one of the ubiquitous crystallins in vertebrate lenses. It is a polydisperse complex of ~ 800 kD consisting of two subunits (~20 kD) αA‐ and αB‐crystallin (αA‐ and αB‐Crys). Its chaperone activity involves suppressing aggregation by binding aggregation‐prone species. γD‐crystallin (γD‐Crys), a member of the βγ‐crystallin family, is localized in the lens nucleus. Aggregates isolated from mature‐onset cataracts contain damaged and misfolded forms of γD‐Crys. Human γD‐Crys refolds through the population of a stable, partially folded intermediate in vitro, but aggregation reactions compete with productive folding. Our previous work showed that HγD‐Crys substrate in γD—αB complexes resembles this folding intermediate, which has its N‐terminal domain unfolded and its C‐terminal domain folded. Using single domain constructs of HγD‐Crys, we showed that the C‐terminal domain construct (γD‐Ctd) aggregates upon refolding, while the N‐terminal domain construct (γD‐Ntd) does not aggregate under similar conditions. Unfolding and refolding γD‐Ctd and γD‐Ntd together at equal molar ratios resulted in γD‐Ctd recruiting γD‐Ntd into the aggregate. HαB‐Crys can suppress the aggregation of the γD‐Ctd and forms γD‐Ctd—αB complexes. These results provide further insight into how α‐crystallin interacts with aggregation‐prone substrates.
Novel phosducin‐like protein 1 interactions
Authors: Gray Amy , Howlett Alyson , Prince John , Ahn Natalie , Willardson Barry
Published: June 2021

Phosducin‐like protein 1 (PhLP1) has been recently shown tobe a co‐chaperone with the cytosolic chaperonin complex (CCT) in the folding and assembly of nascentG protein βγ subunit dimers and Gβ5‐RGS proteins. However, other possible functions for PhLP1 either as a co‐chaperone or otherwise are yet to be investigated. Using PhLP1 as bait, a co‐immunoprecipitation proteomics screen indicated that PhLP1 interacted with programmed cell death 5 protein (PDCD5) and hydroxysteroid (17β) dehydrogenase 4 (HSD17B4). PDCD5 is a known tumor suppressor which is down‐regulated in several tumor types and is found to translocate rapidly from the cytoplasm to the nucleus during the initial stages of apoptosis. HSD17B4 is involved in the beta‐oxidation pathway for fatty acids and is implicated in D‐bifunctional protein deficiency. Subsequent binding experiments have validated both the PhLP1‐PDCD5 and PhLP1‐HSD17B4 interactions and revealed that both proteins interacted specifically with PhLP1 and not other members of the phosducin family of proteins. Additional experiments have shown a strong interaction between PDCD5 and CCT which was inhibited in the presence of PhLP1. The current study addresses the questions raised by these interactions. Namely, do PDCD5 and HSD17B4 require the assistance of the PhLP1‐CCT co‐chaperone complex to fold properly or are the proteins interacting for other purposes?
The group II chaperonin Mm‐Cpn binds and refolds mutant H[&gamma]D Crystallin to a native‐like structure
Authors: Knee Kelly Marie , Goulet Daniel R , Moreau Kate , King Jonathan
Published: June 2021

The aggregation of damaged or misfolded proteins is associated with a number of human diseases, including Alzheimer's disease, Huntington's disease, and cataract. In this study, we investigate the ability of the Group II chaperonin from Methanococcus marapaludis, Mm‐Cpn, a homolog of the eukaryotic chaperonin TRiC, to bind and refold human γD crystallins. Crystallins are a family of structural proteins found in the lens of the human eye, and aggregation of these proteins is thought to be the cause of cataract. Mm‐Cpn interactions with both wild type HγD‐Crys, and damage and disease model mutant HγD‐Crys were evaluated. Solution turbidity studies indicate that Mm‐Cpn suppresses aggregation of both wild type and disease model mutant HγD‐Crys, and exhibits a greater affinity for the destabilized mutant HγD‐Crys. In addition, size exclusion chromatography and fluorescence spectroscopy show that Mm‐Cpn can refold HγD to a native like state, as well as form a long‐lived Mm‐Cpn/HγD complex with both the wild type and mutant HγD crystallins. This long‐lived complex may be ideal for imaging of the chaperonin/substrate complex by cryo‐EM or x‐ray crystallography. These data suggest that the Mm‐Cpn/HγD interaction may serve as a model for general chaperonin/substrate interaction, as well as provide a better understanding of the mechanism of binding and refolding by the Group II chaperonins.
Human αB‐crystallin binds to and suppresses the aggregation of human γD‐crystallin congenital cataract mutants
Authors: Moreau Kate , Sampson Ligia Acosta , King Jonathan
Published: June 2021

The eye lens is composed of fiber cells packed with crystallins up to several hundred mg/ml. α‐Crystallin, the chaperone system of the lens, is a small heat shock protein composed of αA and αB (HαB) subunits. γD‐Crystallin (HγD) is a monomeric, two‐domain protein found in the lens nucleus. It is synthesized in utero and must maintain its native fold for a lifetime. WT HγD exhibited a three‐state unfolding pathway with the N‐terminal domain (N‐td) unfolding first, followed by the C‐terminal domain (C‐td). The intermediate populated on this pathway had a folded C‐td and unfolded N‐td. Upon dilution from denaturant, aggregation competed with productive refolding. The HγD mutants V75D and I90F were destabilized in equilibrium unfolding/refolding experiments and this destabilization was dependent on the location of the mutation. Like WT, both mutants aggregated upon dilution from denaturant. HαB recognized and bound an intermediate species, suppressing WT HγD aggregation. HαB suppressed aggregation of both mutants and formed stable complexes with them. This demonstrated that HαB recognized and bound aggregation‐prone species of both WT and mutant HγD proteins. Because precursors to aggregation and cataract formation are not accessible in the lens, these results provide insights into the conformation of aggregation‐prone crystallin intermediates. This work was supported by NEI grant EY015834 to JK.