bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of sex‐specific endothelial cell actin fiber orientation (675.2)
Authors: Stockard Kevin , Bingaman Susan , Huxley Virginia
Published: June 2021

Cardiovascular disease with subsequent heart failure is the leading cause of morbidity and mortality in the Western world. Sex disparities with regard to cardiovascular function at the cellular level have received minimal attention in either macro‐ or micro‐ vasculature. We hypothesized that structural differences exist between male and female endothelial cells (EC) isolated and cultured from three vessel types: aorta (macrovessel) and mesenteric and skeletal muscle microvessels. Atomic force microscopy was used to image all cell types for both sexes. Raw deflection images, height profiles, and color maps revealed sex differences in actin fiber orientation amongst all cell types. Profiles of frequency vs. fiber direction were also computed and graphed. Dominant actin fractal dimensions (DAFD) of each cell type were calculated. The DAFD was consistently larger in females (F) than in males (M): 53% larger in aorta; 39% larger in mesentery; and 171% larger in skeletal muscle vessel. These differences indicate that the overall actin fiber orientation of EC from F converges in one direction more uniformly than from M, consistent with a more organized arrangement of fibers in F EC. Knowledge of sex similarities and differences of microvascular EC phenotype may yield clues as to their structural properties (e.g. elastic moduli). These properties may translate to characteristics of the intact vessel and could serve as a starting point to understand sex differences in cardiovascular outcomes.

Grant Funding Source: Supported by: DK095501, HCO6RR17353, & the MU Pulmonary/MPP Partnership
In vivo and ex vivo dysfunction of neurovascular coupling in a mouse model of subarachnoid hemorrhage (676.3)
Authors: Koide Masayo , Dunn Kathryn , Bulkeley Evelyn , Nelson Mark , Wellman George
Published: June 2021

Neurovascular coupling (NVC) represents activity‐dependent focal increases in cerebral blood flow (CBF) crucial for the enhanced delivery of nutrients to maintain brain function in regions of high metabolic demand. We have previously reported inversion of NVC, with neuronal activation causing vasoconstriction rather than vasodilation, in brain slices from subarachnoid hemorrhage (SAH) model rats (Koide et al, 2012). Here, we examined ex vivo NVC, in vivo functional hyperemia and sensory motor function using a mouse endovascular perforation SAH model. In brain slices, astroctyic endfoot Ca2+ and adjoining parenchymal arteriolar diameter were measured using two‐photon and infrared‐differential interference contrast microscopy. Neuronal activation caused increased endfoot Ca2+ that was followed by an inverted neurovascular response (i.e. vasoconstriction rather than vasodilation) in ~ 80% of brain slices from day 1 and day 2 SAH mice and ~ 30% of slices from SAH day 4 mice. In vivo functional hyperemia (whisker stimulation‐induced CBF increases) measured by laser Doppler flowmetry in SAH day 1 mice was also significantly attenuated. Consistent with impaired NVC, SAH mice showed a decreased ability to perform sensory motor tasks. These data demonstrate dysfunction of neurovascular coupling occurs both ex vivo and in vivo following SAH, which may contribute to the development of neuronal deficits.

Grant Funding Source: Supported by Totman Research Trust, P. Martin Endowment, NIH P01HL095488, P30RR032135 & P30GM103498.
The sarcoplasmic reticulum enhances voltage‐gated Ca2+ influx in resistance arteries from ovariectomized mice (680.19)
Authors: Hill Brent , Fernando Charmain
Published: June 2021

Estrogen attenuates Ca2+ entry into arterial smooth muscle cells via its inhibition and/or downregulation of voltage‐gated Ca2+ channels (VGCCs). The purpose of this study is to determine the effect of the sarcoplasmic reticulum (SR) on VGCC function in ovariectomized (OVX) mice. Female mice (C57BL/6) underwent an ovariectomy or sham (SHAM) surgery at 8 wks of age. At 12 wks of age the mice were sacrificed. Compared to SHAM mice, the OVX mice had a 50% decrease in plasma E2 and a 7‐fold reduction in uteri weight. Smooth muscle cells were isolated from the mesenteric arteries and loaded with the ratiometric calcium indicator, fura‐2AM, to conduct fluorescent imaging on these cells. There was an enhanced fura‐2 ratio in cells from OVX mice exposed to the selective VGCC agonist, FPL64176 (1x10‐6 M; p=0.03). However, when the SR was Ca2+ depleted (using the ryanodine receptor agonist, caffeine, and the SR Ca2+‐ATPase pump inhibitor, thapsigargin) before exposing the cells to FPL64176 there was no difference (p=0.40) in the fura‐2 ratio between OVX and SHAM mice. These data suggest that the SR enhances the VGCC‐mediated entry of Ca2+ into smooth muscle cells in mice with low circulating plasma E2 levels. Overall, the decline of circulating estrogen in women can enhance Ca2+ entry into arterial cells. Long‐term this may lead to an elevation in arterial tone in resistance arteries.

Grant Funding Source: Supported by NIGMM of the NIH, Grant #P20 GM103429‐11
Interaction of ApoE genotype, antioxidants and exercise on brain function (684.12)
Authors: Chaudhari Kiran , Wong Jessica , Vann Philip , Sumien Nathalie
Published: June 2021

Increased risk of developing Alzheimer’s disease (AD) has been associated with the presence of the ε4 allele of apolipoprotein E (ApoE). To reduce brain dysfunction, a healthy lifestyle, such as daily exercise and healthy eating habits, is often recommended. Though both interventions have shown promising results individually, it remains unknown whether their concurrent implementation will lead to a synergistic/additive effect or an antagonistic interaction nullifying their beneficial effects in the context of ApoE genotype and AD risk. Our study aimed at determining the nature of the interaction between a moderate exercise regimen and antioxidant supplementation on functional outcomes in a mouse model of increased AD risk. Male and female mice (12‐13 month old), expressing the human ApoE3 or E4, were placed under one of the treatments: Sedentary/Control diet (SedCon), Sedentary /Antioxidant‐rich diet (Vitamins E and C; SedEC), Exercise/Control diet (ExCon), Exercise/ Antioxidant‐rich diet (ExEC), for 8 weeks prior to and throughout behavioral testing. The ApoE3 mice performed better than the ApoE4 ones on a test for coordinated running. Only the ExEC treatment improved the performance of the mice, however only of the male ApoE3. In a test for spatial learning and memory, all treatment improved the performance of the ApoE4 mice but not the ApoE3. In the active avoidance test, Ex and EXEC treatments improved performance in ApoE3 mice during acquisition and improved cognitive flexibility but did not affect the performance of the ApoE4 mice. These preliminary data indicate that genotype and sex may be critical determinants in the functional outcomes of interventions such as exercise and antioxidants. There also was no evidence of either a synergistic or antagonistic interaction between the two treatments.
Anti‐asthmatic drug pranlukast attenuates cyst progression in an MDCK cyst model (690.4)
Authors: Pathomthongtaweechai Nutthapoom , Chatsudthipong Varanuj , Muanprasat Chatchai
Published: June 2021

Renal cyst enlargement requires two pathophysiological circumstances including CFTR‐mediated enormous chloride secretion and mTOR signaling‐promoted aberrant renal cell proliferation. These two processes are negatively controlled by AMP‐activated protein kinase (AMPK), an energy sensor. Preliminary data has shown that a cysteinyl leukotriene 1 receptor antagonist montelukast reduced forskolin‐induced Madine Darby canine kidney (MDCK) cyst growth and formation. Here, we asked whether pranlukast, another drug in the same class, is a potential drug candidate for the treatment of polycystic kidney disease (PKD). Consistently, pranlukast (50 µM) inhibited MDCK cyst growth and formation by 50 and 80%, respectively. As analyzed by short‐circuit current measurement, pranlukast (50 µM, 24h) inhibited cAMP‐activated chloride secretion by 50%. Using bromodeoxyuridine (BrdU) incorporation assay, pranlukast reduced MDCK cell proliferation by 60%. Inhibition of both chloride secretion and cell proliferation by pranlukast was reversed by compound C, an inhibitor of AMPK. In addition, pranlukast enhanced AMPK activation. Our findings imdicate that pranlukast retards MDCK cyst progression by inhibiting cAMP‐induced chloride secretion and renal cell proliferation in an AMPK‐dependent manner. Pranlukast therefore represents a potential drug candidate for PKD treatment.
A mathematical model for epithelial cell wound healing as a diffusion problem (694.6)
Authors: Ogawa Katherine , Nguyen Crystal , Nepomuceno Helene , Hasfura‐Buenaga J. Roberto , Balreira E. Cabral , King Jonathan
Published: June 2021

Epithelial cells respond to a damaged cell layer by undergoing physiological transitions required for migration and wound closure. We hypothesized that a diffusion model can explain the behavior of this response. Confluent monolayers of Madin‐Darby Canine Kidney (MDCK) cells were used for wounding assays and time‐lapse imaged on a Nikon A1R laser scanning confocal microscope with live cell capability. A diffusion model was developed with a source term for proliferation. A further two‐step process was considered, where after an initial diffusion, the process shifts to a linear progression of movement. A MATLAB code was used to fit the model parameters using the finite‐difference method to determine the best fitting proliferation and diffusion coefficients by minimizing the sum of square errors. Analyses of the model indicated that following a time delay, the diffusion process transitions into a constant rate migration process. This two‐step process provided a better fit than a diffusion process. Future research will aim to associate the diffusion coefficient to the role of key tight junction proteins and the significance of the two‐step process in relation to epithelial cell migration.

Grant Funding Source: Supported in part by NSF‐MRI #1229702 and NSF Award #0926702.
L‐type Ca2+ channel current is controlled by ROS regulated by glucose‐6‐phosphate dehydrogenase and mitochondrial respiratory chain complex I (698.2)
Authors: Ochi Rikuo , Chettimada Sukrutha , Wolin Michael , Gupte Sachin
Published: June 2021

H2O2 moderately inhibits L‐type Ca2+ channel current (ICa,L) and dithiothreitol (DTT), a reducing agent, reverses ICa,L inhibition in rat aorta‐derived A7r5 cells. This suggests that oxidation of thiol on Cav1.2 protein by H2O2, at least partly, contributes to the inhibition of ICa,L. Furthermore, the whole‐cell ICa,L recorded with 10 mM‐Ba2+ as a charge carrier and evoked by a depolarization step repetitively at 1/20 s are completely suppressed by ebselen (3 μM). This suggests that S‐glutathionylation of CaV1.2 might also be potentially involved in the H2O2‐induced inhibition. 6‐Aminonicotinamide (6‐AN; 1 mM), an inhibitor of glucose‐6‐phosphate dehydrogenase (G6PD) that blocks reduction of NADP+ to NADPH and GSSG to GSH and O2‐ production from NADPH‐oxidase, inhibited ICa,L by 18% in 5 min. Rotenone (Rot), an inhibitor of mitochondrial complex I that reduces O2‐ in a tissue specific manner, rapidly but transiently augmented ICa,L amplitude in a dose‐dependent manner (10 nM‐10 μM), and increased ICa,L up to 30% at 10 μM. But, it subsequently accelerated the time course of inactivation. The 6‐AN‐induced inhibition of ICa,L suggests that NADPH generated by G6PD contributes to sustain normal ICa,L by reducing S‐glutathionylation induced by H2O2. While, Rot‐induced reduction of mitochondrial H2O2 potentially removes its basal inhibitory action on CaV1.2 and this increases ICa,L in the presence of G6PD‐derived NADPH. In conclusion, our results indicate that basal ICa,L magnitude is regulated by extra‐mitochondrial and mitochondrial redox.
Nitroglycerin but not the new NO donor RuBPY phosphorylates eNOS‐Ser1177 (700.4)
Authors: Paulo Michele , Grando Marcella , Vercesi Juliana , da Silva Roberto , Bendhack Lusiane
Published: June 2021

This study aimed to verify the tolerance induced by RuBPY in rat cava vein and the role of NOS activation. We have analyzed the maximum relaxing effect (ME) induced by GNT and RuBP. In vitro tolerance was induced by 60 min‐exposure to 0.1 µM or 4 µM GTN or 0.1µM or 2µM RuBPY in the presence of L‐NAME, BH4 or tiron. The eNOS phosphorylation in the activation (Ser1177) and in the inactivation site (Thr495) was accessed by Western Blot. The procedures are according to the Ethics Committee. Differences were considered significant for P<0.05. Pre‐exposure for 60 min with 2µM and 10µM RuBPY reduced the ME from 92.7 to 48.0±2.3%, n=7 and 30.1±1.2%, n= 7, respectively. In tolerant vessels, tiron increased the ME induced by GTN from 75.3 to 60.0±4.1%, n=6 and RuBPY (73.0±3.4%, n=6). BH4 increased the ME induced by GTN to 67.4± 3.3%, n=7 and RuBPY to 65.0±2.3%, n=6. L‐NAME increased the ME induced by GNT to 70.3±3.7%, n=6, but it had no effect on RuBPY. Whereas RuBPY and GTN had phosphorylated eNOS in Thr495, only GTN phosphorylated eNOS‐Ser1177. Therefore, tolerance induced by NO donors RuBPY and GTN involves eNOS and O2_. GTN but not RuBPY interferes with the activation site of eNOS.

Supported by FAPESP and CNPq.
Differential regulations of ASICs and TRPV1 by zinc in rat bronchopulmonary sensory neurons (712.3)
Authors: Vysotskaya Zhanna , Moss Charles , Gu Qihai
Published: June 2021

Zinc, a divalent heavy metal ion and an essential trace element in body, functions as a signaling molecule that regulates a variety of neuronal functions. In this study, we examined the effect of zinc on two populations of acid sensitive ion channels, acid‐sensing ion channels (ASICs) and transient receptor potential vanilloid receptor‐1 (TRPV1), in rat bronchopulmonary sensory neurons. In our patch‐clamp recordings, ASIC currents were induced by a pH drop from 7.4 to 6.8 or 6.5 in the presence of capsazepine (10 µM), a specific TRPV1 antagonist. Our data showed that pretreatment with zinc (50 or 300 µM, 2 min) displayed different effects on the two distinct phenotypes of ASIC currents: a marked potentiation on ASIC channels with fast kinetics of activation and deactivation, or no significant effect on ASIC currents with slow activation and deactivation. On the other hand, pretreatment with zinc significantly inhibited the acid (pH 5.3 or 5.5)‐induced TRPV1 currents in these sensory neurons. The inhibition was effectively abolished by the intracellular application of a selective zinc chelator TPEN (25 µM) indicating that the inhibitory effect of zinc on TRPV1 channels depends on its intracellular binding to these channels. In summary, our study showed that zinc differentially regulates the activities of ASICs and TRPV1 channels in rat bronchopulmonary sensory neurons.

Grant Funding Source: Supported by funds from AHA, MEDCEN and MUSM
The effectiveness of utilizing pre‐lecture and interactive lecture models on student learning outcomes in undergraduate functional anatomy courses (719.10)
Authors: Domire Zachary , Rider Patrick
Published: June 2021

Functional anatomy is a challenging course that is required for many undergraduate kinesiology majors. Traditional lecture (TL) models have shown poor results with regards to quiz and exam performance. Pre‐lecture (PL) and interactive lecture (IL) models have shown positive results on class performance outcomes in other scientific disciplines. The objective of this investigation was to determine if utilizing an IL or a PL model would improve quiz and exam performance in undergraduate functional anatomy courses. Quiz and exam performance measures were analyzed from TL (n = 135), IL (n = 193) and PL (n = 22) course delivery models in several sections of a functional anatomy course. All quiz questions and 70% of exam questions were consistent across all sections of the course and used for comparison. A one‐way ANOVA analysis revealed significant differences in exam and final grades. Post‐hoc Student’s t‐tests revealed significant differences in exam and final grades between TL and PL and TL and IL groups, but no differences between PL and IL. See Table 1 for mean and standard deviation values for each group. Quizzes were completed open book therefore; not finding a difference between groups was not surprising. Overall, results indicate that IL and PL models are effective for teaching undergraduate functional anatomy courses and significantly improve student exam and course performance compared to TL.

Table 1: Group Means

Group
Mean Quiz Score %
Mean Exam Score %
Mean Final Course Score %

Traditional
84.1 ± 10.6
78.4 ± 11.2
81.0 ± 10.9

Interactive
84.2 ± 12.0
82.0 ± 11.6*
83.6 ± 11.8*

Pre‐lecture
85.9 ± 11.0
83.8 ± 6.9*
87.7 ± 7.0*

(*) denotes significant difference from traditional group (a < 0.05).
Racial disparities among willed‐body donors in human anatomy laboratories (721.6)
Authors: Fraser Patrick , Neuman Tanner , Reeves Rustin , Rosales Armando
Published: June 2021

A study of the racial background of willed‐body donors (WBD), most commonly referred to as cadavers, revealed a strikingly disproportionate distribution. Data were collected from state issued death certificates and donor forms from the University of North Texas Health Science Center (UNTHSC) Willed‐Body Program and analyzed for multiple variables. All statistical analyses were performed using Microsoft Excel™. Our analysis of the WBD (n=225) demonstrated racial proportions as 96.43%, 2.23%, 1.34%, 0.00% for Caucasian, African‐American, Latino, and all other races respectively. Similar trends are seen at medical schools across the Western Hemisphere. At one particular foreign medical school, where the indigenous population is primarily dark‐skinned, mostly Caucasian WBD are used in their anatomy laboratories. We also show that formal education (mean=13.07yrs) is correlated with WBD participation in the anatomy training of healthcare providers and other related professionals. The study of anatomy is a cornerstone of clinical knowledge. Human pathology is known to have differential expression across races. We conclude that many racial groups are excluded from anatomical studies, and as such, may translate to inappropriate understanding of anatomical nuances of races other than Caucasian. Further work is ongoing to elucidate the origins of the racial disparities.
A digital learning object for teaching human embryology: version 2 of Embryon (725.13)
Authors: Fredieu John , Kerbo Jennifer , Mulcahy Brendan , Bartel Carly , Fedor‐Garcia Maia , Patterson Kyle , Pasini Julianne
Published: June 2021

Learning human embryology is very important in the understanding of congenital anomalies, in the development of accurate diagnoses, and in the construction of effective treatment plans to address congenital anomalies. However, addressing concepts in human embryology can be difficult in medical and dental student populations with different learning styles and different levels of prior knowledge. Here, we have incorporated prior focus group, curricular implementation, and student learning data into an existing digital interactive educational module designed to teach human facial development to pre‐clinical medical and dental students. This second version addresses issues in interface, interactivity, and content of the initial learning object that were considered detrimental to learning during analyses of the initial version. In addition, active learning assessment has been incorporated into the learning object in the form of short quizzes and internal data tracking. Focus group analysis, limited implementation data, and learning assessment analysis are examined to determine the efficacy of the learning object in student learning and retention of human embryology content.
Developing a virtual microscopy laboratory manual to help teach histology in resource‐challenged African medical schools (725.14)
Authors: Anderson Matthew , Cotlin Laura , Anderson Peter
Published: June 2021

Background: Providing basic science practical laboratory experiences can be problematic especially for new medical schools and in resource challenged schools from less developed parts of the world. The microscopes and glass slide sets needed for robust laboratory sessions are often difficult to procure and to maintain. Virtual Microscopy (VM) can facilitate the teaching of histology, histopathology, microbiology, and other microscopy‐based subjects using a computer based delivery platform. We have developed a VM Histology Laboratory Manual that can be used by students as part of their histology curriculum in medical schools with minimal resources.

Design: The UAB histology teaching program has developed a histology laboratory manual that students use as a guide in our histology curriculum. We scanned the histology glass slide collection and incorporated links to these VM slide files into our histology laboratory manual. The PDF of the manual with the VM slide links was locked as a read‐only file and along with the VM slide files was loaded onto inexpensive 64 GB thumb drives. The thumb drives containing the laboratory manual and VM slides, along with the free VM slide viewer, can be used by medical students as a histology teaching resource even in the absence of internet access.

Conclusion: Repurposing existing teaching materials utilizing VM technology can provide an inexpensive user‐friendly option for resource challenged African medical schools.
Development of an interactive online educational resource for histology (725.15)
Authors: Yazdian Farimah
Published: June 2021

The Department of Biomedical and Molecular Sciences developed an online educational resource called Scalable Gross Anatomy and Histology Image Catalogue (SGAHIC). This resource is a comprehensive database of approximately 10,000 gross anatomical specimens and histology images. SGAHIC uses virtual microscopy technology, which serves to emulate a full range of traditional microscope functionality. The purpose of this initiative was to innovatively enhance the utility of SGAHIC histology, with focus on the facilitation of teaching and learning. Histology Tutorial, using virtual microscopy, is an interactive resource developed to assist students with identifying organs and their respective tissues at the microscopic level. The implementation of self‐assessment modules encompassing labeled images, identification, and short‐answer questions, emphasize theory underlying the histology. Students are able to make connections between structures and functions, allowing them to achieve a deeper level of understanding. It is also designed to provide students with an immediate reinforcement of knowledge, which supports the learning process. This enhancement of SGAHIC has been received positively by students studying histology as well as faculty teachers. It is our hope that Histology Tutorial will promote a learning‐centered environment, enabling students to reach beyond surface learning of histology.
Portable document format files showing the surface models of cadavers (725.9)
Authors: Chung Beom Sun , Chung Min Suk
Published: June 2021

It was recently discovered that popular PDF file enables users to approach the numerous surface models conveniently on Adobe Reader. Purpose of this study was to present PDF files including systematized surface models of human body as the beneficial contents. To achieve the purpose, 642 three‐dimensional surface models had been built from the sectioned images of a male cadaver. Two‐dimensional surface models including the original sectioned images were embedded into the stereoscopic surface models. The entire surface models were categorized into systems and then groups. The adjusted surface models were inserted to a PDF file, where relevant multimedia data were added. Fitting software packages were employed in accordance with these procedures. The finalized PDF file containing comprehensive data of a whole body could be explored in varying manners. We have further produced PDF files of male head and female pelvis from other subjects’ sectioned images. The PDF files, downloadable freely from the homepage (anatomy.co.kr), are expected to be used as a satisfactory self‐learning tool of anatomy. Raw data of the surface models can be extracted from the PDF files and employed for various simulations.
Arsenic inhibits DNA mismatch repair by altering PCNA function (735.9)
Authors: Kim Christine , Tong Dan , Ortega Janice , Li Guo‐Min , Gu Liya
Published: June 2021

Chronic exposure to arsenic is associated with the development of certain types of cancer, including lung and skin cancers. The exact mechanism by which arsenic induces tumorigenesis is unknown. Based on the fact that arsenic can generate reactive oxygen species to damage DNA and proteins, and that arsenic enhances expression of epidermal growth factor receptor (EGFR), which phosphorylates the proliferating cell nuclear antigen (PCNA), an important mismatch repair (MMR) component, we hypothesize that arsenic induces tumorigenesis is through its ability to inactivate the MMR system. We demonstrate here that exposure to arsenic indeed induces elevated EGFR expression and increased PCNA phosphorylation. Cells or cell extracts treated with arsenic are defective in MMR. Interestingly, the arsenic‐caused MMR deficiency can be reversed by PCNA, but not the PCNA preincubated with arsenic. We also found that the treatment of PCNA with arsenic changes PCNA conformation. These results suggest that arsenic inhibits MMR by directly altering the structure of PCNA, and by promoting PCNA phosphorylation through the activation of EGFR. Our work therefore reveals a novel mechanism by which arsenic induces carcinogenesis.
DNA‐PKcs activates the Chk2‐Brca1 pathway during mitosis to ensure chromosomal stability (736.4)
Authors: Chen Benjamin , Shang Zengfu
Published: June 2021

The catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs) is known to play a critical role in DNA double‐strand break repair. We previously reported that DNA‐PKcs is activated when cells enter mitosis and functions in mitotic spindle assembly and chromosome segregation. Here we report that DNA‐PKcs is the upstream regulator of the Chk2‐Brca1 pathway, which impacts microtubule dynamics, kinetochore attachment, and chromosomal segregation in mitosis. Downstream from Chk2, Brca1 promotes monoubiquitination of γ‐tubulin to inhibit microtubule nucleation and growth. We found that DNA‐PKcs is essential for mitotic Chk2 phosphorylation at Thr68. As in Chk2‐ and Brca1‐deficient cells, loss of DNA‐PKcs resulted in chromosome misalignment and lagging during anaphase due to elevation in microtubule dynamics. Importantly, these mitotic aberrations in DNA‐PKcs‐defective cells were alleviated by over‐expression of phosphomimetic Chk2 or Brca1 mutant proteins but not their wild‐type counterparts. Taken together, these results demonstrate that DNA‐PKcs regulates mitotic spindle organization and chromosomal instability via the Chk2‐Brca1 signaling pathway.

Grant Funding Source: NIH, CA166677
Physiological and pathophysiological stimuli alter endothelial cell autophagy (737.4)
Authors: Uzoigwe Emmanuella , Panneerseelan Leena , Li YouYou , Deeter Lance , Kunz David , Graham Timothy , Jionang Dapeu Elisee , Symons J David
Published: June 2021

Autophagy plays a central role in cellular quality control by destroying damaged or excess proteins, lipids, membranes, and organelles that accumulate in response to deviations from homeostasis. The existence and role of autophagy in endothelial cells (ECs) and blood vessels has not been established. We quantified the ratio of the membrane bound conjugate of microtubule‐associated protein light chain 3 (LC3‐II) to the cytosolic non‐lipidated conjugate LC3‐1 (LC3‐II:LC3‐I) or the protein normalizer GAPDH (LC3‐II:GAPDH) in ECs and blood vessels in response to a variety of cellular stressors. LC3‐II:LC3‐I or LC3‐II:GAPDH was elevated (p<0.05) (i) 450±6% (n=4) in ECs incubated for 2 h in amino acid (AA)‐deplete vs. AA‐replete media; (ii) 47±3% (n=3) in arteries from fasted (14 h) vs. fasted / refed (1 h) mice; (iii) 40±2% (n=3) in arteries from mice that completed acute exercise vs. sedentary controls; (iv) 38±1% in arteries from exercise‐trained vs. sedentary mice under basal conditions (n=2 per group); and was decreased (p<0.05) (v) 57±8% (n=4) in arteries from ~30 month‐old (i.e., old) vs. ~6 month‐old (i.e., young) mice. Further, indices of autophagy were elevated (p<0.05) 101±6% in ECs exposed to 3 h x 500 uM palmitate vs. vehicle (n=4), and 50±6% in arteries from obese vs. lean mice (n=4 per group). Thus, autophagy is altered in ECs and blood vessels in response to physiological (e.g., fasting, acute exercise, exercise training, aging) and pathophysiological (acute lipotoxicity, diet‐induced obesity) stimuli.
RNAi knockdown of CLN3P gives clues to it's function (738.2)
Authors: Dorminey Katherine , Hinderer Eugene , Krzysiak Amanda
Published: June 2021

Batten disease is a fatal neurodegenerative disease affecting children between the ages of five and ten years with an incidence of 2 to 4:100 000 live births in the United States and Canada. It is a type of lysosomal storage disease that causes an accumulation of lipofuscin in neuronal cells eventually leading to cellular degradation by normal physiological factors. The cause of Batten disease has been determined to be a 1.02 kb genomic deletion that mutates a gene known as CLN3, producing a mutant protein responsible for the pathology. The function this CLN3 protein has not been determined, although it has been predicted to be a transmembrane protein involved with lysosome trafficking. To aid future investigations pertaining to the pathology of Batten disease and in the development of therapies, we investigated the normal function of CLN3. Knockdown of CLN3 in HeLa cells was used to determine its role in autophagy. We determined that CLN3 has a role in cellular autophagy.
HSF‐1 modulates autophagy via transcriptional regulation of autophagy‐related genes in A549 cells (738.3)
Authors: Dokladny Karol , Moseley Pope
Published: June 2021

Autophagy is an evolutionary conserved catabolic process employed by eukaryotic cells in response to nutrients starvation, hypoxia, high temperature, and acute exercise. Autophagy, along with other cellular processes, is required for a normal cell function and is responsible for degrading dysfunctional cellular constituents in double‐membrane structures called autophagosomes upon fusion with lysosomes. Recently, we have shown that HSF‐1 knockdown with siRNA increased the LC3 lipidation associated with formation of autophagosomal organelles and augmented both starvation and rapamycin‐induced autophagy. The aim of the present study was to evaluate whether HSF‐1 inhibition by siRNA changes mRNA levels of autophagy related genes in A549 cells (adenocarcinomic human alveolar basal epithelial cells). A549 cells were treated with HSF‐1 siRNA for 48h, total RNA was collected, cDNA was generated, and the quantitative RT‐PCR was performed. Exposure of A549 cells to HSF‐1 siRNA resulted in an increase in LC3, beclin, and Atg12, but not Atg7 mRNA expression. These changes in mRNA expressions were correlated with protein expressions. We conclude that HSF‐1 acts as a negative regulator of autophagy related genes and inhibits autophagy under control conditions.

Grant Funding Source: UNM CTSA Grant UL1 TR000041 and UNM School of Medicine Research Allocation Committee Grant
Effect of dimerization of peroxiredoxin 6 on its enzymatic activities (739.4)
Authors: Zhou Suiping , Dodia Chandra , Sorokina Elena , Harper Sandra , Speicher David , Feinstein Sheldon , Fisher Aron
Published: June 2021

Peroxiredoxin 6 (Prdx6) is a unique non‐seleno 1‐Cys protein with both peroxidase and phospholipase A2 (PLA2) activities. It is highly expressed in the lung where it plays an important role in antioxidant defense and lung surfactant metabolism. Glutathionylation of Prdx6 mediated by πGST is required for its peroxidatic catalytic cycle while its PLA2 activity is enhanced by the presence of GSH. The crystal structure of human Prdx6 reveals a dimer with discrete domains for each activity in each monomer. The N‐terminal domain has a thioredoxin fold and the C‐terminal domain functions in dimerization. We hypothesize that dimerization of Prdx6 might play an important role in its enzymatic activities and have predicted that amino acid residues L145 and L148 are important for protein dimerization. By analytical ultracentrifugation, protein mutated at L145/148 unlike wild type did not dimerize. Peroxidase activity was dramatically reduced by mutation at either of these sites and abolished by double mutation. However, these mutations had no effect on PLA2 activity. Therefore, the dimerization of Prdx6 plays an important role for its peroxidase but not its PLA2 activity.

Grant Funding Source: Supported by NIH grants HL102016 and HL19737
Chaperone‐mediated autophagy is impaired in the lysosomal storage disorder cystinosis (740.2)
Authors: Napolitano Gennaro , Johnson Jennifer , Monfregola Jlenia , Rocca Celine , Cherqui Stephanie , Catz Sergio
Published: June 2021

Functional lysosomes are essential for autophagosome maturation and clearance of autophagic substrates. Accordingly, Lysosomal Storage Disorders (LSDs) have been associated with autophagy defects. Cell malfunction in the LSD Cystinosis is caused by the accumulation of the amino acid cystine in lysosomes due to genetic defects in the Ctns gene (cystinosin), a lysosomal transporter. Using EM and TIRFM microscopy, we showed that lysosomal number, size and trafficking was affected in Ctns‐/‐ cells. Although the number of autophagosomes was strongly increased in Ctns‐/‐ cells under resting conditions, fusion of autophagosomes with lysosomes, as well as starvation‐induced degradation of the autophagosome marker LC3‐II, was not affected in these cells, indicating that autophagic flux is increased but not impaired in cystinosis. Interestingly, the receptor for chaperone‐mediated autophagy (CMA) LAMP2a, the rate limiting step for CMA, was downregulated and mislocalized in Ctns‐/‐ cells and tissues. Lysosomal translocation and degradation of CMA substrates was defective in Ctns‐/‐ lysosomes, suggesting defective CMA in cystinosis. Reconstitution of Ctns‐/‐ cells with EGFP‐CTNS normalized macroautophagy and partially rescued LAMP2a expression but not localization suggesting underlying trafficking defects in Ctns‐/‐ cells. We propose that defects in CMA contribute to the pathogenesis of cystinosis.

Grant Funding Source: Supported by Cystinosis Research Foundation and NIH
Short‐range and long‐range transcriptional repressors exhibit distinct and highly context‐specific chromatin signatures (741.2)
Authors: Arnosti David , Kok Kurtulus , Morsdorf David , Payankaulam Sandhya
Published: June 2021

Transcriptional repressors employ a variety of mechanisms to regulate eukaryotic gene expression, and research from the Drosophila embryo has highlighted two distinct functional classes of repressor. Short‐range repressors such as Knirps work locally to interfere with proximal transcription factors, and others, such as the HES family protein Hairy, dominantly repress transcription of an entire locus with multiple regulatory elements. Both classes of repressor interact with common corepressors such as the evolutionarily conserved C‐terminal binding protein CtBP and WD‐40 domain protein Groucho. The basis and importance of such complexity is unknown, however it is possible that these types of repressors may exert unique effects in a gene‐specific manner. Our previous studies have highlighted the very localized chromatin modifications induced by the Knirps short‐range repressor, contrasted with kbp‐wide alterations observed with Hairy, however these studies were limited to individual target genes, leaving open the question about gene specificity, and whether repressors exert the same effect on all loci, regardless of gene activity or activator inputs. We have now analyzed the impact of Knirps and Hairy repression on a genome‐wide basis. We observe strong indications of correlated chromatin modifications on a wide set of targets, for instance, both H3K4 and H3K27 acetylation are coordinately reduced at Hairy targets, showing changes over a range an average of ~2kbp (often up to 10 kbp). A subset of targeted genes lacks significant deacetylation marks, but reveals strong upregulation of H3K9 trimethylation, as well as increased occupancy of histone H1 on the gene body. Our initial analysis indicates that complex and diverse repression pathways may be tailored to specific gene contexts, permitting a uniform control of heterogeneously regulated genes.

Grant Funding Source: Supported by NIH GMS 056976
Kinetic and structural analysis of the group I catalytic ribozyme utilizing fluorescent probes (743.2)
Authors: Schumaker Alex , Rohlman Christopher
Published: June 2021

Group I introns are catalytic RNAs capable of performing a range of phospho‐transesterification reactions including self‐splicing and RNA cleavage. This study focuses on the Tetrahymena and Anabaena Group I ribozymes as models. This work describes the application of steady‐state gel based assays utilizing fluorescently labeled substrate or ribozymes to evaluate structure function relationships within the ribozymes. We report the initial results from the application of this technique to a study of variants of the P1 substrate‐binding domain of the Anabaena Group I ribozyme.
Identification and characterization of phosphorylated regions in the nonsense‐mediated mRNA decay protein Upf2 from S. cerevisiae (748.1)
Authors: Colon Edgardo , Lin Qisha , Lasalde Clarivel , Gonzalez Carlos
Published: June 2021

About 30% of all mutations causing human diseases generate mRNAs with premature termination codons (PTCs). These PTCs can be incorporated in the mRNA via nonsense or frameshift mutations, normal programmed rearrangements, or biosynthetic errors. As a result, these PTC‐harboring transcripts encode potentially non‐functional and/or toxic proteins that can lead to disease. To avoid the synthesis of these aberrant proteins, the nonsense‐mediated mRNA decay (NMD) pathway targets the PTC‐containing mRNAs for degradation. The core NMD proteins Upf1, Upf2, and Upf3 are evolutionary conserved from lower to higher eukaryotes. Upf2 is a phosphorylated protein in yeast and mammals but the precise role of this post‐translational modification remains elusive. Using tandem mass spectrometry analyses, twelve novel phosphorylation sites (S54, S55, T327, S424, T842, S868, T869, T872, S874, S1021, S1022 and S1023) were identified in Upf2 protein from S. cerevisiae. Furthermore, functional analysis revealed that a small amino‐terminal motif harboring at least two phosphorylated residues is important for NMD activity. Together, these results suggest that Upf2 phosphorylation is a conserved feature of NMD and provide a foundation for future studies to elucidate the functional relevance of Upf2 phosphorylation in the NMD surveillance pathway. Supported by U54 CA96297, PR‐LSAMP HRD‐0601843, and RISE 2R25GM61151.
Phosphorylation‐independent recruitment of 14‐3‐3 alters tristetraprolin function (748.3)
Authors: Kraemer Laura , Clement Sandra
Published: June 2021

Cell signaling pathways influence mRNA turnover rates through reversible post‐translational modifications that alter the activity of a number of mRNA binding proteins. Tristetraprolin (TTP) is a zinc‐finger RNA binding protein that binds to AU‐rich elements (AREs) in the 3’ untranslated regions (UTRs) of transiently expressed messages encoding cytokines and pro‐inflammatory proteins. Unphosphorylated TTP causes rapid mRNA turnover by recruiting mRNA decay enzymes, but phosphorylation of TTP at serines 52 and 178 by the p38 MAPK activated kinase MK2 prevents its ability to do so. Phosphorylation also leads to the recruitment of 14‐3‐3, is associated with alterations in the localization of TTP, and may protect TTP from dephosphorylation and destruction by the proteasome. However, because mutations that destroy 14‐3‐3 binding sites in TTP also prevent its phosphorylation, it is unclear whether 14‐3‐3 binding is required to inhibit TTP function. To better understand the role of 14‐3‐3 in the inhibition of TTP we replaced serines 52 and 178 in TTP via site‐directed mutagenesis with either a peptide sequence known to bind 14‐3‐3 independently of phosphorylation or with an altered version of this peptide sequence that does not. We tested the ability of these mutant forms of TTP to recruit 14‐3‐3 using co‐immunoprecipitation assays from transiently transfected Human Embryonic Kidney (HEK) 293T cell lysates. We observed that protein levels for mutant forms of TTP that failed to associate with 14‐3‐3 were consistently lower than versions of TTP that retained the ability to do so, supporting the hypothesis that 14‐3‐3 may protect TTP from destruction by the proteasome. We also observed that recruitment of 14‐3‐3 was sufficient to alter localization of TTP from the nucleus to the cytoplasm. Experiments are currently underway to determine whether 14‐3‐3 association is sufficient to alter the recruitment of mRNA decay enzymes and the protein phosphatase PP2A.
Mechanism of strong inhibition of HNF4α1 protein expression by its 5' UTR (749.7)
Authors: Guo Shangdong , Lu Hong
Published: June 2021

HNF4α is a liver‐enriched master regulator down‐regulated in liver cancer and cirrhosis. Restoration of HNF4α expression can treat both liver cancer and liver fibrosis. However, lack of known activating ligand makes HNF4α “undruggable”. The purpose of this study was to elucidate the mechanism of translational regulation of HNF4α1, the adult‐predominant HNF4α isoform, so as to develop a novel approach to restore HNF4α1 expression for cancer therapy. The 5' UTR of both human and mouse HNF4α1 have potential G‐quadruplex (G4), a stable secondary structure known to inhibit transcription and translation. FRET assay of dual‐labeled oligo from human HNF4α1 5' UTR indicated the formation of G4. Luciferase reporter assay showed the wild‐type, but not G4‐mutated, 5' UTR of HNF4α1 strongly inhibited protein translation without affecting mRNA expression, which is further confirmed by Western blot analysis of protein expression of HNF4α1 in the presence/absence of its 5' UTR. In HEK 293 cells, protein expression of mouse Hnf4α1 was completely inhibited by its 5' UTR. Luciferase assays of the deletion/mutation reporter constructs of Hnf4α1 5' UTR suggest G4 motif and its adjacent stem loop may play the key role in suppressing Hnf4α1 protein expression. In summary, the 5' UTR of human and mouse HNF4α1 markedly inhibit HNF4α1 expression at translational levels, most likely due to interaction of G4 motif with stem loop within 5' UTR.

Grant Funding Source: NIH Grant CA143656
A role for the ribosome in deciding the fate of damaged RNA (752.10)
Authors: Zaher Hani
Published: June 2021

Fast and faithful translation of the cellular messenger RNAs is a defining feature of the ribosome and the translation factors. High‐accuracy protein synthesis ensures that errant proteins, which are more prone to misfold, are not made. On aberrant mRNAs, such as truncated ones and those either containing premature or lacking stop codons, a number of ribosome‐based quality control processes ensure that these RNAs are not translated and instead are targeted for degradation. While these mRNA‐surveillance mechanisms have received much attention during the past decade, curiously a different class of aberrant mRNAs has received little study. In particular, chemically‐damaged RNAs pose a significant hurdle to translational fidelity and efficiency. Of particular interest to us is oxidized RNA in the form of 8‐oxoguanine, which has been linked to a number of neurodegenerative diseases. The exact mechanism by which the cell recognizes the aberrant mRNAs and targets them for degradation remains elusive. To this end, we have begun to investigate the effects of oxidation on the decoding process using a high‐resolution‐in‐vitro translation system. Preliminary data suggests that 8‐oxoguanine is detrimental to the decoding process, for which we measure rates of peptide‐bond formation that are 4 to 5 orders of magnitude lower relative to undamaged RNA. We are currently investigating tRNA‐selection steps preceding the final step of peptidyl transfer in an attempt to understand the mechanism by which such a minor change to the nucleobase could have dramatic effects on the decoding process. We are also interested in studying the fate of the oxidized RNAs and the role of the ribosome in this process. Preliminary data is promising and suggests that the ribosome takes advantage of existent quality control processes to target these mRNAs for degradation.

Grant Funding Source: NIH
Low resolution structural determination of lipoprotein6.6: a membrane component of Borrelia burgdorferi (753.2)
Authors: Freeman David , Kumru Ozan , Middaugh C. , Joshi Sangeeta , Zückert Wolfram
Published: June 2021

Recent studies conducted on Borrelia burgdorferi, the causative agent of Lyme disease, have focused on lipoproteins. Lipoprotein 6.6 (Lp6.6) has been shown to play a vital role in the transmission from the Ixodes tick vector to the mammalian host. Lp6.6 is highly abundant in the periplasmic leaflet of the outer membrane and is part of a number of protein complexes. The objectives of this study were to purify a recombinant form of Lp6.6 and gain some insight as to the structure of Lp6.6 by using various low‐resolution spectroscopic techniques. A recombinant non‐lipidated form of Lp6.6 was produced in E. coli and purified using nickel affinity chromatography. Lp6.6 was then studied by far‐UV circular dichroism, dynamic light scattering, and extrinsic fluorescence spectroscopy. Circular dichroism measurements indicate Lp6.6 contains random coil (negative peak at ~200nm) and α‐helix (positive peak at ~190nm). Dynamic light scattering measurements suggests some aggregates are present after proteolytic removal of the 6xHis tag. Extrinsic fluorescence spectroscopy, using 8‐Anilino‐1‐naphthalenesulfonic acid as a probe for exposed hydrophobic moieties, showed an increase in fluorescence at ~45°C, suggesting a perturbation in tertiary structure. Taken together, Lp6.6 has structural components that may be a vital part of outer membrane protein complexes.