bluuebunny/crossref_metadata_2025 huggingface dataset:
Providing structure checklists in the cadaver dissection laboratory: evaluation of outcomes and student satisfaction
Authors: Klump Kathryn Ellen , Thiessen Amy , Hill Molly , O'Donoghue Daniel , Halliday Nancy
Published: June 2021

167 first year medical students enrolled in a 7 week Human Structure course at The University of Oklahoma College of Medicine received a structure checklist for each of 3 dissections tracing branches of the abdominal aorta to supplied viscera. Students were instructed to record structures as identified by student, teaching assistant, or faculty. Measured outcomes included practical examination scores and self‐reported structure identification. A voluntary student satisfaction survey was made available following the final examination. The preponderance of the student response to structure lists in the gross anatomy lab was positive. Over 95% of respondents felt structure lists increased dissection efficiency. Students perceived an improvement in dissection quality and an increase in the number of structures they found on their own with implementation of lists. Test items from the structure lists scored higher on formative practical exams than items for which no structure list was provided. Our results, in concurrence with previously published data, support the use of structure lists to improve objectives‐driven learning outcomes and maximize efficiency in the gross anatomy lab.
Systems Approach to Digestive and Regulatory Processes through Exploration of Fasting and Postprandial Blood Glucose Levels
Authors: Hopper Mari K.
Published: June 2021

Digestive physiology laboratory exercises often explore regulation of enzyme action rather than systems physiology. This laboratory exercise explores the key concept of regulation of blood glucose, both fasting and postprandial, to provide a systems approach to regulatory and digestive function. Students enrolled in undergraduate Animal Physiology select to participate in either an oral glucose tolerance test (OGTT) or one of three meal treatments based on typical student breakfasts: 1) High Glycemic Load (HGL); 2) Moderate Glycemic Load (MGL); and 3) Low Glycemic Load (LGL). Caloric value of meals is 540 kcal. An Accucheck® glucometer is used to determine fasted, 30, 60, and 120 minute postprandial blood glucose levels. Fasted values range from 75 to 99 mg/dL. Postprandial glucose levels peak similarly for OGTT and HGL (151±7.1 and 145±4.7mg/dL) and remain higher than MGL and LGL over the two hour period. Between sampling, vibrant discussion covering such topics as glucose and cognitive function, insulin resistance, and fad diets occurs. Post lab assignment requires students to discuss importance of glucose homeostasis, graphically summarize findings, review literature to describe results in light of published data, and describe relationships between hyperglycemia and hyperinsulinemia and disease processes. Students evaluate this lab as highly effective and one of the top three experiences of the course. Support provided by the University of Southern Indiana Department of Biology
The “flipped classroom” (FC) as a way to increase face time for case studies and discussion
Authors: DeRuisseau Lara Roberts
Published: June 2021

The FC was initiated in an undergraduate Anatomy and Physiology course (54 students). Traditional lecture content was delivered via short (~20 min) e‐lectures using screen capture software (Camtasia and Echo360 Personal) and was accompanied with Ppt slides and review questions. E‐lectures and review questions were viewed and completed outside of class. The first 20 min of class was devoted to student discussion of review questions with their permanent teams; which were formed during the first week of the course. Students had the option to edit review answers prior to submitting the assignment for a participation grade. During the editing process the instructor answered individual team questions about the material. After the review question/edit period the instructor addressed the entire class while answering specific questions from students and further emphasizing more difficult concepts. On designated days, a case study was performed that helped with the integration of previously covered material. Answers to the case were completed and the following class period was used to review the case in detail. Five cases were utilized throughout the semester. The FC allowed ample time to complete case studies and address student misconceptions. The mean score for the first exam using the FC was higher than previous years (79.2 vs 74.3; p<0.01) suggesting increased student performance with this method of instruction.
Nutrition as a platform for reform in biochemistry education
Authors: Pogozelski Wendy Knapp , Feinman Richard D , Wood Richard
Published: June 2021

Reform in biochemistry education centers on 1) replacing rote memorization with a more analytical approach and 2) enabling students (especially future health practitioners) to understand nutritional therapies. We previously described how the glucose‐insulin axis can be used as a heuristic device in teaching metabolism (Pogozelski, et al 2005; Feinman and Makowske, 2003). We have further developed our approach to develop critical thinking skills and apply principles from nutrition and exercise physiology in our biochemistry classes. The need for this training is great, given the persistence of obesity and diabetes epidemics and the insufficient critical evaluation of some standard practices. Integration of nutrition with biochemistry increases student interest and helps empower physicians and researchers to make informed decisions.
Assessment of undergraduate writing in the sciences using eportfolios
Authors: Smith Sally Sommers , Lavalli Kari , Griffin Harry
Published: June 2021

Undergraduate biology students who are asked to write about their laboratory experiences frequently express confusion about their writing and concern that they will not be able to meet instructors’ expectations. Students may be ready to perform laboratory experiments in our undergraduate general biology course, but they are less able to communicate their findings clearly and persuasively in writing. We decided to investigate whether the opportunity to re‐write and re‐submit laboratory papers might improve the clarity and accuracy of student writing. We first identified student writing that fell short of expectations, and designed teaching tools that model good scientific writing for our students. We used detailed rubrics for expectations and grading, as well as student e‐portfolio postings to assess the differences between the first and the re‐written laboratory submissions. We also surveyed students to assess their confidence about writing for science courses. Student writing grades did improve markedly after reflection, e‐portfolio assessment, and re‐writing, but students’ confidence in their ability to write an excellent scientific paper remained low. Peer review of posters, however, improves the ability of students to recognize good scientific writing. We are currently assessing whether peer review, coupled with re‐writes, improves student writing projects in introductory biology.
Systematic analysis of the gene expression in the livers of nonalcoholic steatohepatitis: implications on potential biomarkers and molecular pathological mechanism
Authors: Zhang Yida , Baker Susan S , Baker Robert D , Zhu Ruixin , Zhu Lixin
Published: June 2021

Non‐alcoholic steatohepatitis (NASH) is a severe form of non‐alcoholic fatty liver disease (NAFLD). Taking advantage of well characterized microarray datasets of NASH livers, we performed a systematic analysis of potential biomarkers and possible pathological mechanism of NASH. CodeLink Human Whole Genome Bioarrays were analyzed to find differentially expressed genes (DEGs) between controls and NASH patients. Four methods were used to identify DEGs and the intersection of DEGs identified by these methods was subsequently used for downstream analysis. For biomarker prediction, rank aggregation was used to rank DEGs according to their significance of differential expression. Alcohol dehydrogenase 4 (ADH4) exhibited the highest rank suggesting the most significant differential expression between normal and disease condition. Together with the previous report demonstrating the association between ADH4 and the pathogenesis of NASH, our data suggest that ADH4 could be a potential biomarker for NASH. For molecular pathological mechanism analysis, two clusters of highly correlated annotation terms and genes in these terms were identified based on the intersection of DEGs. Then, pathways enriched with these genes were identified to construct the network. From this network, amino acid catabolism and urea cycle were implicated to play pivotal roles in the development of NASH.
S100A4 signaling in thyroid cancer
Authors: Medapati Manoj Reddy , Dahlmann Mathias , Ulrike Stein , Ghavami Saied , Hombach‐Klonisch Sabine
Published: June 2021

Thyroid cancer (TC) is one of the most frequently occurring cancers of endocrine glands in Canada and the United States. Most differentiated TC are curable, but some TCs develop a more aggressive phenotype with resulting metastasis and treatment failures. Our group has shown that relaxin‐like peptides promote TC cell tumor growth and migration, in part mediated via S100A4 which is known to promote metastasis and to confer poor prognosis in patients with TC. Several S100 proteins signal via receptor for advanced glycation end products (RAGE). This study investigates S100A4 signaling in promoting TC cell survival, growth and metastasis. TC cells were stimulated with S100A4 to determine cell motility, proliferation and apoptosis and to identify S100A4‐induced signaling pathways. Using PCR and immunohistochemistry expression of RAGE was detected in TC cells and in TC tissues, but not in normal thyroid tissues. RAGE pull‐down experiments demonstrated S100A4 to bind to RAGE in TC. ERK and JNK/SAPK signaling pathways were activated in TC cells upon S100A4 stimulation. We used RAGE blocking antibodies and diaphanous‐1 knock‐down to prevent RAGE‐mediated S100A4‐signaling in TC. S100A4‐induced ERK‐signaling appeared to be independent of RAGE in TC. We identified TLR4 expression in TC cells and demonstrate here that the effects of S100A4 are, in part, mediated via TLR4 in TC. This research is funded by NSERC of Canada and Dept of surgery research fund.

Grant Funding Source: n/a
Co‐distribution of Collagen Type IV and Laminin Around Sinusoids in Liver Fibrosis of Elderly Cadavers: Immunohistochemical Marker of Perisinusoidal Basement Membrane Formation
Authors: Mak Ki Mark , Chen Lucy L , Lee Ting‐Fang
Published: June 2021

Liver sinusoids are lined by a fenestrated endothelium that lacks a basement membrane (BM). Production of sinusoidal BM along with loss of fenestrae characterizes capillarization of sinusoids that is a significant pathology accompanying advanced fibrotic changes. Liver fibrosis is prevalent in elderly cadavers; however, BM formation in these liver samples has yet to be studied. Collagen type IV (col IV) and laminin are major BM proteins and their co‐distribution around sinusoids provides an immunohistochemical marker of BM formation. Here we examined sites of sinusoidal BM formation in elderly cadaveric livers with advanced septal fibrosis. We found co‐distribution of col IV and laminin in BM of bile ducts and blood vessels in portal tracts, providing a positive control. However, the thickened walls of fibrotic central veins were positive for col IV but not for laminin. Within the lobules, Col IV immunostaining of the sinusoidal lining appeared continuous and was panlobular. In contrast, laminin staining was patchy, occurring commonly in sinusoids close to fibrotic portal tracts and septa, less commonly in mid‐lobular and rarely in centrilobular areas. Consecutive sections revealed that laminin co‐distributed with col IV in these sinusoidal locations, thus marking the sites of perisinusoidal BM formation in the elderly fibrotic liver, possibly in association with the capillarization of sinusoids.
N‐sulfation of Cell Surface Heparan Sulfate Glycosaminoglycans is Critical for Podocyte‐Matrix Interactions
Authors: Sugar Terrel , McCarthy Deborah J. , Green Jonette , Orr A. Wayne , Esko Jeffrey D. , McCarthy Kevin J.
Published: June 2021

Podocytes adhere to the glomerular basement membrane via cell surface receptors which include heparan sulfate (HS) proteoglycans (PG). Podocytes unable to assemble HS glycosaminoglycan chains have defective cell attachment and spreading, and cytoskeletal disruption in vitro and in vivo. The current report explores the importance of heparan sulfation in regulating podocyte cell‐matrix interactions, using an immortalized podocyte cell line having the genomic deletion of N‐deacetylase/ N‐sulfotransferase 1 (NDST1), the enzyme responsible for N‐sulfation of HS. Compared to wild type podocytes, mutant podocytes attached, spread and migrated less efficiently in assays than control cells. In the process of cell‐matrix interaction, the mutant cells showed decreased clustering of syndecan 4, the cell surface HSPG involved in focal adhesion formation, and decreased recruitment of PKCα, α‐actinin 4, vinculin, and pFAK to focal adhesions. Cell surface integrin expression and integrin activation/signaling was significantly diminished in the mutant cells. These results serve to highlight the critical role of HS N‐sulfation in regulating the ability of podocytes to interact with the extracellular matrix.

Grant Funding Source: NIDDK RO1‐DK07786
Vesicular glutamate transporter 1‐immunoreactive sensory neurons in the rat intrinsic cardiac ganglia
Authors: Wang Ting , Zhang Zijia , Miller Kenneth E.
Published: June 2021

Sensory neurons that reside in intrinsic cardiac ganglia (ICG) have an important role in monitoring the cardiac milieu for regional cardiac dynamic control. We recently described sensory neurons in ICG that project to the cardiac ventricular wall and currently are evaluating their neurochemical phenotype. Since many primary sensory neurons are glutamatergic, we hypothesized that ICG sensory neurons are glutamatergic, utilizing vesicular glutamate transporter 1 (VGLUT1) to package glutamate into synaptic vesicles. In the present study, the rat ventricle wall was injected (retro‐diaphragmatic, 8μl) with the retrograde tracer wheat germ agglutinin‐horseradish peroxidase (WGA‐HRP). After 72h, rats were euthanized and transcardially perfused with fixative. ICG were processed for immunohistochemistry studies. Our results demonstrated that: (1) VGLUT1‐immunoreactive (ir) neurons were present in ICG; (2) Some VGLUT1‐ir neurons were labeled from the ventricles with WGA‐HRP. Our previous studies indicated that sensory ICG neurons contain glutaminase, the synthetic enzyme for glutamate. These and the current results indicate that ICG sensory neurons are glutamatergic in neurochemical phenotype. Further investigation of VGLUTs during cardiac physiological and pathological conditions will help in deciphering the function of glutamate as a neurotransmitter in the ICG. Supported by NIH AR47410 (KEM).

Grant Funding Source: NIH AR47410
Human embryonic serial anatomy : 3D evolutionnary posterior fossa venous system
Authors: Braun Marc , Tonnelet Romain , Micard Emilien , Labrousse Marc
Published: June 2021

Introduction

Since the mid 1950's, very little work has been done to contribute to a better understanding of venous vasculature developpement during embryogenesis. We present a new analysis method with multiplanar reformation process based on microscopic sections. This présentation focus on the brain stem arachnoïdo‐pial venous anastomoses evolutive morphology.

Material and methods

High résolution numerised microscopic continuous sections (10 to 20 μ) from Carnegie stage XIX to XXIII human embryo were used. Specific image treatment was the following : binarisation‐thresholding‐labellisation, rigid shifting, median filtration (cubic matrix) (resolution 10μm/pixel) ; DICOM format images are exported for post‐treatment process. Angiographic rendering was obtained thanks to the Eosine/hematoxylin red cell fixation within the vessels.

Results

At first step, we confirmed the embryonnic status relatively to the Carnegie classification in assessing the cochlear canal orientation. The evolutive morphology of the brain stem primitive network is presented ; at stage XIX, the initial longitudinal veins are separated from the caudal plexus. During the next stages, we demonstrate how they connected to transverse pial anastomoses. The next period (i.e the early and late post‐embryonic period) show these intrinsic pial veins lying between the arteries and the brain surface leading to a progressive simplication pattern. During the stages XX to XXIII, we present the changing morphology of the trijeminal ganglion venous plexus. These organisations are compared to the Padget and Streeter works.

Conclusion

We present the preliminary work of the brain stem venous embryogenesis using 3D and angiographic rendering at the early post‐embryonic stages.
Is TMED2 essential in the chorion for normal interaction between the allantois and the chorion in mice?
Authors: Hou Wenyang (Dominic) , Majewska Loydie
Published: June 2021

During vesicular transport between the endoplasmic reticulum and the Golgi, members of the transmembrane emp24 domain (TMED) protein family form hetero‐oligomeric complexes that facilitate protein cargo transportation and secretion. In our laboratory, we are studying the function of one member of the TMED protein family, TMED2, in mouse placental labyrinth development. Formation of the mouse labyrinth layer requires proper interactions between two extraembryonic tissues, the allantois and the chorion; and is essential for nutrition, waste, as well as hormone exchange between fetal and maternal circulation. We have shown that Tmed2 is expressed in both allantois and chorion and is required for normal labyrinth layer formation. We hypothesized that TMED2 is essential in the chorion or allantois for normal interaction between the allantois and chorion‐a critical step in placental labyrinth layer development. To test this hypothesis, we have generated an ex‐vivo allantois and chorion recombination model. In our model, we recapitulated the early events of labyrinth layer development: chorioallantoic attachment, fusion of the mesothelium and allantois, and chorionic trophoblast differentiation. We used in situ hybridization and immunohistochemistry to confirm the chorioallantoic attachment event and to monitor development of labyrinth layer in the chimeric explants. We will then use combinations of wild type and Tmed2 null chorion and allantois in these ex‐vivo cultures to follow branching morphogenesis in the chorion. Our work will provide insight into the contribution of placental‐specific vesicular transport by TMED2 to labyrinth layer morphogenesis. Ultimately we will identify novel mechanisms that may be implicated in the prediction and treatment of placental diseases such as EPL and IUGR.
Heart valve substitute fabricated from silk protein, collagen, and poly‐glycerol sebacate has enhanced endothelial cell growth and reduced thrombogenicity
Authors: Wagner William D , Wang Rui , Levi‐Polyachenko Nicole
Published: June 2021

Mechanical or collagen based porcine valves currently used to replace diseased or nonfunctional valves are subject to thrombosis, material failure, or inadequate in situ remodeling. In this study a new electrospun nanofiber composite was fabricated from mechanically robust silk fibroin, (F), Type I collagen (C), and the synthetic elastic polymer, poly (glycerol‐sebacate) (P). Materials fabricated with different weight ratios of F:C:P had an elastic modulus between 2.8–4.1 Mpa; tensile stresses from 1.1–1.5 Mpa and strains between 41–44% which were similar to values reported for native heart valve. In vitro degradation demonstrated <0.3% mass loss per week with no change in nanofiber diameter. In culture electrospun nanofibers mats of FCP compared to structurally similar C nanofibers had greater human umbilical vein endothelial cell growth and by confocal microscopy demonstrated tight intercellular junctions. Using human platelet rich plasma reduced platelet numbers were observed for FCP (3440 ± 302) (mean ± SEM, platelets/cm2)mats compared to C (5268 ± 848) and collagen gels (27,549 ± 698). By SEM, platelets appeared significantly less activated on FCP. Materials preseeded with cells had platelet adhesion of 555 ± 137, 1588 ± 41, and 12311 ± 208 respectfully for FCP, C, and collagen gels. The findings suggest FCP may be a superior material for heart valve replacement.
Raman spectroscopic signature of the cardiovascular commitment and cardiac phenotypes of mouse embryonic stem cells differentiated towards the cardiovascular lineage
Authors: Brauchle Eva , Linder Sandra , Knopf Anne , Schenke‐Layland Katja
Published: June 2021

Raman spectroscopy is a powerful optical technology that can be used to generate biochemical profiles of living cells. In this study, Raman spectroscopy was employed as a non‐invasive approach for the characterization of murine embryonic stem cells (mESCs) and their cardiac derivatives, such as cardiac progenitor cells (CPCs) and cardiomyocytes (CMs). mESCs were differentiated towards the cardiovascular fate yielding CPCs as well as spontaneously beating CMs. Raman spectra of the in vitro‐differentiated cells were compared to spectra generated from isolated fetal and adult murine CMs. All Raman spectra displayed a unique pattern for all cardiovascular phenotypes. Interestingly, the Raman spectra of spontaneously beating mESC‐derived CMs and CMs isolated from murine fetal hearts showed similar spectral patterns; however, both were distinct when compared to the spectra of adult CMs. Raman spectra of CPCs also exhibited similarities to those of CMs, whereas non‐CPCs did not.

To conclude, we identified the presence of a Raman spectral signature that helps predicting cardiovascular lineage commitment in early‐differentiated pluripotent stem cells.

Grant Funding Source: : Fraunhofer‐Gesellschaft (Attract 692263), the BMBF (0316059), the DFG (SCHE 701/7–1), the Ministry of Science, Research and the Arts of Baden‐Württemberg (33–729.55–3/214) and the Landesstiftung Baden Württemberg (PLS‐SPII/18)
The Role of Nitric Oxide in Cardiopulmonary Endurance
Authors: Adar Tony , Baral Sumit , Márquez Samuel
Published: June 2021

Nitric Oxide (NO) has been intensely investigated in various physiological as well as pathophysiological processes in humans but has received its greatest attention for its vasodilator properties. Since the nose and paranasal sinuses have been implicated as the sole producers of NO in the upper respiratory system, NO may be regarded as an aerocrine hormone produced in the nasal complex and transported to a distal site of action with every inhalation by enhancing pulmonary oxygen uptake via local vasodilation, thus facilitating athletic endurance. Our study examines fractional NO concentration (FeNO) response in expired air during exercise (Ex). A strict three step Ex protocol was followed in which FeNO measurements were obtained in 18 healthy human volunteers prior to Ex (rest stage (RS)), during a strenuous 200 w ergometer cycle and 5 minutes into the recovery period. FeNO measurements were performed according to ERS/ATS recommendations with an electrochemical sensor (Niox Mino Aerocrine Stockholm, Sweden). Results showed an overall decrease in average FeNO levels from resting stage to endurance phase of Ex (16.4 to 12.6 ppb p< .005) with levels increasing towards the original RS value 5 minutes into the recovery phase (12.6 to 14.1 ppb p< .005). Further investigation may shed light as to the exact mechanism of NO recruitment by the lower respiratory tract in maximizing cardiopulmonary endurance.

Grant Funding Source: SUNY Downstate Alumni Association
PE transformation is stimulated by AV cushion cells that stimulate fibrous ECM production in developing valves
Authors: Riley Ashlie Evans , Roberts Andrea , Junor Lorain , Potts Jay , Yost Michael , Goodwin Richard
Published: June 2021

The contribution of epicardially‐derived and their progenitors, proepicardial (PE) cells, to the development of the atrioventricular (AV) valves have been recently described in mouse linage tracing experiments. Results presented here indicate that PE cell epithelial to mesenchymal transformation (EMT) is dependent on the presence of AV cushions. Once in the AV cushions, PE cells appear to regulate the expression and deposition of fibrous extracellular matrix (ECM) proteins. Using a tubular, 3D culturing system, we show that AV cushions induce PE cells to undergo an EMT and migrate into AV cushions where they stimulate the expression and deposition of ECM proteins including tenascin C, periostin and collagen I, which are critical for proper valve function. It was tested if members of the TGFβ family of proteins were sufficient to stimulate PE EMT and ECM expression. Only the combination of TGFβ 1, 2 and 3, was found to regulate these processes. When PE cells were cultured on tube scaffolds containing TGFβ 1, 2 and 3 together, PE cell EMT increased compared to cultures with only a single TGFβ isoform. qRT‐PCR and confocal analysis found increased expression and localization of ECM molecules in these transformed cells. These results indicate all three isoforms of TGFβ are required for epicardial EMT, which appear to be necessary the critical expression and deposition of fibrous ECM proteins during valve development.
The evaluation of manual 2D/3D registration technology and its potential to deduce prosthetic wear in patients with metalon‐metal hip resurfacing prostheses
Authors: Chu Yan Yee , St. John Paul , Rudan John , Easteal Ronald A.
Published: June 2021

Metal‐on‐metal hip resurfacing arthroplasty (MoMHRA) has been a popular alternative treatment for young patients with hip osteoarthritis. Despite its advantages over total hip arthroplasty, MoMHRA remains controversial. Malpositioning of the metal components can result in abnormal levels of blood metal ions in the patient; and yet, post‐operative management using 2D x‐rays contain high variance leading to poor detection of prosthetic wear. The purpose of this study is to determine whether 2D/3D registration technology can generate accurate acetabular implant measurements; and, whether 3D data can correlate to metal ion counts to deduce wear. Virtual 3D pelvic models (n=72) and acetabular implants were manually superimposed over 2D x‐ray images according to anatomical landmarks to measure acetabular inclination and version angles. CT models were generated for validation. No significant difference was reported between 2D vs. 3D vs. CT data, suggesting measurements were similar to the results of the gold standard CT model; although 3D measurements were more precise compared to 2D. Furthermore, there was no significant correlation in either 2D or 3D measurements compared to metal ion levels, although a stronger trend is demonstrated in 3D measurements. The findings of this study are inconsistent with the reports in literature and so further investigation is required. Supported by Queen's Graduate Award.
Immature Neurons in the Temporal Cortex of the Aging Rhesus Monkey
Authors: Heyworth Nadine , Rosene Douglas L.
Published: June 2021

Adult neurogenesis is known to occur in specific regions of the brain including the Subgranular Zone (SGZ) of the hippocampal dentate gyrus (DG) and the Subventricular Zone (SVZ) of the lateral ventricle. The new neurons originating in these regions have shown to be necessary for specific cognitive functions. However, it remains unknown if adult‐generated neurons exist in other regions of the brain and contribute to cognitive processes. A novel population of cells expressing the immature neuronal marker, Doublecortin (DCX) has been identified in regions previously termed ‘non‐neurogenic’ including the cortex of cats, guinea pig, non‐human primate, and human. DCX+ cells co‐localize with the proliferative marker Bromodeoxyuridine (BrdU) in the cortex, but only at very low levels, suggesting cortical DCX+ cells with an extended maturation period. Preliminary work in our lab has identified DCX+ cells in the temporal cortex of rhesus monkeys. Tissue from behaviorally characterized young and old monkeys given BrdU for 21 days is being analyzed to quantify DCX+ cells in the cortex and the proportion that co‐localize with BrdU. Our objective is to use Stereologic analysis to quantify age‐related changes and the impact on cognitive function. The characterization of this unique population of cells is necessary to further investigate their role in cortical plasticity and aging. (Supported by R01AG021133 and P01AG000001)
The Claustrum: Puddles are Plumbs
Authors: Johnson John Irwin , Buchanan Kenneth , Fenske Brian Allen , Yalamarthy Ayesha
Published: June 2021

The claustrum is so named for its appearance as a thin layer of cells narrowly enclosed between the corpus striatum and the overlying insular cortex. However, in comparative study of a diversity of mammalian species we found a good portion of the claustrum forming “puddles” of tissue that appear to be spill out from the enclosed claustral lamina in various locations. In the domestic pig, a huge egg‐shaped mass projects back from the posterior edge of the lamina. In carnivores, a large pyramidal shaped mass of claustrum pours out the top of the lamina. In primates (humans included), a large globular puddle protrudes anterior and inferior from the lamina. The posterior portion of the carnivore pyramid mass has been shown in cats to be entirely an accessory of visual cortical function; and can be considered a visual lobe. All input to, and output from, this visual lobe comes from, and goes to, visual cortex. The other puddles may harbor other specialized connections and functions, placed around the laminar stem rather like fruits on a tree. Comprehensive morphological and physiological studies, modeled on those done in cats, are needed to resolve the question of the function of the claustrum. It may well be an accessory to all of mammalian cerebral cortices, with local elaborations related to behavioral specializations. Such studies may offer a major advance in understanding of function of the cerebral cortex.
Observations on the Shapes of Dendrites
Authors: WHITE EDWARD L
Published: June 2021

The common assumption that dendrites are cylindrical is incorrect. Analyses of spiny stellate cell dendrites reconstructed from serial thin sections of mouse barrels indicates that these dendrites are flattened; the ratio of long to short axes in cross‐sectioned dendritic profiles is typically 2:1. Planes of flattening are unrelated to the orientation of dendritic segments within the brain, bear no relationship to the plane of section, and are unchanged for various estimates of section thickness. Thus, the flattened shape is not due to compression, sectioning artifact, or to inaccuracy of the reconstruction method. 2/3 of spines are emitted from the relatively narrow, ridge‐like regions of the flattened shafts, causing the dendritic cross‐section to resemble bitufted cell bodies that also are elongated along the poles that give rise to their major projections. Apical dendrites of pyramidal cells also exhibit a flattened morphology and preferential distribution of spines. The flattened shape may reflect the in vivo condition, or be an artifact of fixation. If the latter, flattening could be imagined to result from the shrinking of cell membrane around the cytoskeleton and/or from the collapse of particularly delicate cytoskeletal elements within certain dendritic compartments.
Successful computational prediction of active site and distal residues essential for function in DNA polymerase III alpha subunit
Authors: Parasuram Ramya , Beuning Penny J , Ondrechen Mary Jo
Published: June 2021

The process of DNA replication is carried out with high efficiency and accuracy by DNA polymerases. The replicative polymerase in E. coli is DNA pol III, which is a 10‐subunit complex that coordinates simultaneous replication on the leading and lagging strands. The 1160‐residue pol III alpha subunit copies DNA accurately, making one error per 105 nucleotide incorporations. The goal of this research is to determine the residues that contribute to this high fidelity and activity of the polymerase subunit. Homology modelingand the computational methods of Theoretical Microscopic Anomalous Titration Curve Shapes (THEMATICS) and Partial Order Optimum Likelihood (POOL)have been used to predict functionally important amino acid residues. Site‐directed mutagenesis and primer extension assays have been used to validate these predictions. This and previous work show that the top 11 residues predicted by POOL, including the three catalytic aspartates plus distal residues, are important for function. Fluorescence binding assays, thermal denaturation assays and steady‐state kinetic assays are being performed to better understand the contribution of these residues to the function of the polymerase. Work supported by NSF‐MCB‐0843603, NSF‐MCB‐0845033, NSF‐MCB‐1158176 and Research Corporation for Science Advancement.
Consistent mutations in Type II Topoismerases of Vibrio cholerae O1 and global reduction in fluoroquinolone susceptibility
Authors: Kumar Pramod , Bhatt Dharmendra Nath , Sharma Naresh Chandra , Jain Meenu , Mishra Deepak Kumar , Goel Ajay Kumar , Yadava Pramod Kumar
Published: June 2021

Vibrio cholerae O1 the epidemic strains causing devastating cholera harbors two related type II topoisomerases, gyrase and topoisomerase IV which alter DNA topology through a DNA cleavage complex. Fluoroquinolones stabilizing the DNA breakage‐reunion complex are effective WHO recommended antibiotics used in the treatment of severe cholera cases. A total of 200 Vibrio cholerae O1 strains (clinical and environmental) collected from different parts of India were subjected for MIC calculations for ciprofloxacin according to the CLSI, 2010 guidelines. The strains showed reduced susceptibility to ciprofloxacin (MIC 0.1 to 1 μg/ml). Two mutation in quinolone resistance determining region, 1 mutation in gyrA (83S>;I) and 1 mutation in parC (85S>;L) were found consistent in all strains. These amino acid substitutions are previously known to be associated with quinolone resistance in V. cholerae. Molecular modelling studies of mutant gyrase and topoisomerase IV revealed change in binding pocket for ciprofloxacin and would lead to alter holding over DNA cleavage complex. Such type of strains experiences the inhibitory dose of ciprofloxacin as sub‐inhibitory concentration, and could be the reason of longer duration of diarrhoea. The reduced susceptibility to ciprofloxacin was also noticed in the hybrid V. cholerae O1 strains from recent large cholera epidemics in Western Africa and Haiti. The study suggested that the circulating epidemic strains are consistently spreading globally. It hampers the outbreak control policies which can be a serious concern in future. The use of alternate chemotherapeutic agents stabilizing the transition complex of typeII topoisomerases with DNA breakage‐reunion should be of pressing importance.
Functional Characterization Of Dbf4 Interactions With DNA Replication And Checkpoint Factors
Authors: Jones Darryl R , Matthews Lindsay A , McConkey Brendan J , Guarné Alba , Duncker Bernard P
Published: June 2021

The Dbf4/Cdc7 kinase complex (DDK) is essential for the initiation of DNA replication, acting upon the MCM helicase complex to trigger the initiation of replication. DDK is also a target of the intra‐S phase checkpoint, important in preserving genomic stability. Dbf4, the regulatory subunit of this kinase complex, contains three regions that are highly conserved across eukaryotic species. Of these regions the amino terminal N motif adopts a modified BRCT fold that is crucial to maintain the interaction with the checkpoint kinase Rad53, while the M and C motifs are both involved with the interaction with Cdc7 and the Mcm2 component of the MCM helicase. Mutations to critical amino acids within either of the N or C motifs result in sensitivity to a variety of DNA damaging agents. Employing both bioinformatics and structural NMR based approaches we have identified a novel surface of the forkhead‐associated (FHA1) domain of Rad53 that mediates its interaction with Dbf4. Additionally, we have generated strains that combine Dbf4 motif C mutation with mutations in previously characterized checkpoint proteins in order to better define the checkpoint role of Dbf4. Together, these findings shed light on the mechanism by which Dbf4 aids in maintaining genome stability.

This research was supported through grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR)
Localization to the Nuclear Pore Complex is Required to Prevent Trinucleotide Repeat Instability
Authors: Su Xiaofeng , Dion Vincent , Gasser Susan , Freudenreich Catherine
Published: June 2021

Trinucleotide repeat expansions, such as CAG repeat expansions, cause more than 40 neurological disorders. CAG repeats are prone to break, and repair of the breaks can cause expansions. In yeast, some persistent DNA breaks are localized to the nuclear periphery which influences the break repair. We integrated an expanded CAG repeat into a yeast artificial chromosome (YAC) to evaluate the CAG repeat fragility and instability (contraction and expansion) in various nuclear periphery mutation backgrounds, as well as used fluorescent microscopy to study the localization of a GFP (green fluorescent protein)‐tagged CAG repeat in the nucleus over the cell cycle. Our results show that the Nup84 nuclear pore complex and its associated SUMO targeting ubiquitin ligase (STUbL), Slx5/Slx8, are crucial to maintain CAG repeat stability and prevent fragility. The repeat instability caused by deletion of Nup84 is dependent on the homologous recombination (HR) DNA break repair. Moreover, we found that expanded CAG‐70 and ‐130 repeats are more likely than a control to localize to the nuclear periphery during S‐phase. In conclusion, our study indicates that expanded CAG repeats localize to the nuclear periphery during S‐phase to promote proper repair in the presence of Nup84, which stabilizes CAG repeats by preventing aberrant HR repair. This study is funded by Tufts Department of Biology.
Structural and functional studies of the histone chaperone Hif1, a component of the Hat1 chromatin assembly complex
Authors: Bagley Michael J. , Dutnall Robert N.
Published: June 2021

The Hat1 chromatin assembly complex contains the Hat1 histone acetyltransferase and two Hat1‐interacting factors: Hat2 and Hif1. The Hat1 complex specifically acetylates histone H4 and is part of a pathway that deposits newly synthesized histone H3 and histone H4 onto DNA during chromatin assembly. In yeast, Hat1 complexes contribute to gene regulation and DNA repair. Hat1, Hat2 and Hif1 are each highly conserved and form similar complexes in organisms ranging from yeast to humans. We are using biochemical and biophysical methods to study the structure and function of the Hif1 protein from yeast with the goal of understanding how it contributes to the activity of the Hat1 chromatin assembly complex. Hif1 contains four tetratricopeptide (TPR) sequence motifs, binds histones and has histone chaperone activity. Bacterially expressed yeast Hif1 is an oligomer, forming dimers and possibly tetrameric assemblies, and interacts with histones. A truncation mutant consisting of only the TPR sequence motifs of Hif1 is also an oligomer and retains histone binding activity. We are currently testing further truncation mutants to delineate the Hif1 region(s) responsible for oligomer formation and histone binding.
Pyrimidine dimer glycosylase mediated repair of ultravioletinduced mtDNA damage
Authors: Calkins Marcus J , Lloyd R. Stephen , McCullough Amanda K
Published: June 2021

Since mitochondria are known to leave cyclobutane pyrimidine dimers (CPDs) unrepaired after ultraviolet light exposure, we introduced a mitochondria‐targeted pyrimidine glycosylase (MTS‐pdg) into keratinocytes and thereby initiated mtDNA repair after ultraviolet exposure. We generated stable clonal cell lines with which we are testing whether persistent mtDNA damage may lead to aberant mitochondrial function or cycling. After UV‐B exposure, mtDNA is repaired by MTS‐pdg, and MTS‐pdg expression leads to alterations in cytokine production and mtDNA content after UV‐B exposure. These results confirm that mtDNA plays a role in cellular response to ultraviolet light and may provide clues as to the mechanism by which mitochondrial damage alters cellular processes.
Regulation of VEGF Expression in Hypoxic Pulmonary Artery Endothelial Cells (PAECs) by Base Excision DNA Repair (BER) Activity
Authors: Pastukh Viktor , Bardwell Gina , Pastukh Viktoriya , Gillespie Mark
Published: June 2021

Reactive species generated by peri‐nuclear mitochondria cause formation of the common base oxidation product 8‐oxoguanine (8‐ oxoG) in hypoxic response elements (HRE) of inducible genes (Al‐Mehdi et al., Sci Signaling, 2012). Repair 8‐oxoG by the BER pathway is initiated by the lesion‐specific DNA glycosylase OGG1. The specific mechanism whereby DNA repair interacts with transcription is still unknown. We tested the idea that modulation of OGG1 alters transcription complex formation on the VEGF HRE and VEGF mRNA expression. In cultured PAECs, we confirmed that hypoxia increased 8‐oxoG in the VEGF HRE. siRNA‐mediated knockdown and transgenic over‐expression of OGG1 increased and decreased, respectively, the proportion of VEGF HREs harboring 8‐oxoG. The hypoxia‐induced increase in HRE sequences associating with Hif‐1 was inhibited by OGG1 knockdown and augmented by OGG1 over‐expression. Hypoxia‐induced Vegf mRNA expression tracked Hif‐1 binding in cells with modulated OGG1 activity. These findings indicate that the BER pathway may be important for transcription and point to new mechanisms linking hypoxia to mutagenesis, aging and other oxidant‐related disorders.

Supported by NIH.
Mechanistic studies of transcription initiation by the yeast mitochondrial RNA polymerase
Authors: Deshpande Aishwarya P , Patel Smita S
Published: June 2021

The yeast mitochondrial DNA contains multiple promoters that direct the synthesis of rRNAs, tRNAs, and the mRNAs of the ATP synthesis complex. These RNAs are transcribed by a distinct two‐component complex consisting of the core RNA polymerase, Rpo41, which is the catalytic subunit and an accessory factor, Mtf1, which is necessary for promoter opening and start site selection. Although, currently we have a basic understanding of the mechanism of open complex formation and the respective roles of Rpo41 and Mtf1, we lack a similar understanding of the initial transcription mechanism involving +1 and +2 NTP binding and promoter strength. Using synthetic promoters, we observed a range of transcriptional efficiency from various promoters. Using promoter mutants and fluorescence‐based assays, we show that these differences in transcriptional efficiencies stem from differences in downstream promoter melting, which go hand in hand with differential NTP binding affinities. We propose a model, where either Rpo41 or Mtf1 or both through an orchestration of interactions with the promoter region as well as the incoming nucleotides regulate the transcriptional efficiency of various promoters.

This work was supported by the NIH grant RM51966 to S.S.P
Transcriptional regulation of the sodium‐coupled neutral amino acid transporter (SNAT2) by 17β‐estradiol
Authors: Velazquez‐Villegas Laura , Ortiz Victor , Ordaz‐Rosado David , Garcia‐Becerra Rocio , Larrea Fernando , Torres Nimbe , Tovar Armando R
Published: June 2021

The SNAT2 amino acid transporter allows the entry of small neutral amino acids into the mammary gland to promote cell proliferation during gestation and milk protein synthesis during lactation. During pregnancy, 17β‐estradiol promotes the expression of this transporter by increasing SNAT2 promoter activity. The objective of this work was to elucidate the mechanism by which the 17β‐estradiol regulates the transcription of SNAT2. Computational analysis of the promoter region of SNAT2 showed the presence of a potential estrogen response element (ERE). Studies in HeLa cells using the luciferase reporter gene showed that the activity of the promoter region of SNAT2 increased with the co‐transfection of constructions with progressive deletions of SNAT2 promoter, and the plasmid that expresses the estrogen receptor alpha (ERα). The highest luciferase activity was found with the construct containing the ERE (40%), but 20% of activity remained in smaller constructions. EMSA and supershift assays showed that ERα binds to the ERE in SNAT2 promoter and this binding competed with ERα interaction to its consensus sequence. This binding was also demonstrated in vivo by ChIP assay where ERα bound to the promoter region of SNAT2 in the mammary gland of pregnant rats on days 5, 14 and 20 of gestation. We conclude that 17β‐estradiol increased SNAT2 promoter activity in the presence of ERα by a classic genomic pathway.
The JmjC Domain‐containing Regulator Gis1 Is Regulated by Heme and Oxygen
Authors: Cao Thai , Shah Ajit , Lal Sneha , Zhang Li
Published: June 2021

JmjC domain‐containing proteins have demethylase activity and can remove specific methyl groups on histones or other proteins. There are over 100 proteins in diverse organisms known to contain the JmjC domain. These proteins act by oxidizing the methyl group to yield formaldehyde and the unmodified lysine or arginine. Due to the requirement for oxygen for demethylase activity, these proteins can serve as oxygen sensors. The yeast Gis1 is a unique transcriptional regulator belonging to the JHDM3/JMJD2 subfamily of demethylases. Further, Gis1 is highly homologous to the mammalian JmjC domain‐containing JMJD2B protein that is likely to play important roles in histone demethylation, oxygen regulation and hormonal signaling. Recently, our studies showed that Gis1 activity is regulated by both heme and oxygen. Analysis of PDS‐LEU2‐lacZ reporter gene showed a significant increase in PDS reporter gene under high heme concentration. However, a decrease in the Gis1 activity was observed under hypoxia as compared to normoxia. Confocal fluorescent live cell imaging of Gis1 showed that over 95% of the Gis1 protein were nuclear in normoxic cells, while over 90% of Gis1 protein were retained in the cytosol in hypoxic cells. Time course characterization of relocalization of Gis1 showed that Gis1 relocalized in a significantly shorter time period in response to both hypoxia and reoxygenation.

Grants: Cecil H. and Ida Green Funds
Selective mRNA translation by eIF2 phosphorylation during ER stress
Authors: Wek Ronald C , Baird Thomas D , Palam Lakshmi Reddy
Published: June 2021

When the protein flux into the endoplasmic reticulum exceeds its folding capacity, a condition referred to as ER stress, there is an induction of translational and transcriptional gene expression designed to alleviate the underlying stress. This Unfolded Protein Response (UPR) features a global decrease in translation, which is facilitated by PERK phosphorylation of eIF2 (eIF2~P). Coincident with global translational control, eIF2~P directs the preferential translation of mRNAs induced by the UPR. In this study, we use sucrose gradient ultracentrifugation and a genome‐wide microarray approach to measure changes in translational efficiencies for individual mRNAs during ER stress. We find that while many transcripts are repressed during eIF2~P, some appear resistant to stress, while a subset is preferentially translated. ATF5 mRNA showed the greatest shift towards higher polysomes during ER stress. Translation of ATF5 is controlled by a mechanism of delayed ribosome reinitiation. Additionally, CHOP mRNA was shifted substantially towards the higher polysome fractions via a ribosomal bypass of an inhibitory uORF situated in the 5′‐leader of the mRNA. Lastly, we report novel mRNAs involved in protein turnover and synthesis and mRNA processing, which are preferentially translated. Identification of novel preferentially translated genes identifies new control schemes for key regulatory steps in the UPR.