bluuebunny/crossref_metadata_2025 huggingface dataset:
Changes in GDNF protein content of skeletal muscle following involuntary exercise are long lived
Authors: Kinnell Kyle R. , Czarnopys Monica J. , Spitsbergen John M.
Published: June 2021

Glial‐cell line derived neurotrophic factor (GDNF) is a neurotrophic factor produced by skeletal muscle. GDNF has been shown to be potent survival factor for motor neurons. We have previously shown that GDNF content in skeletal muscle is altered following exercise and GDNF levels 24 hours after the last bout of exercise are significantly different than those measured immediately after exercise. We sought to determine how long after exercise do GDNF protein levels remain altered. We removed extensor digitorum longus (EDL) and Soleus (SOL), 24 hr, 72 hr and 1 week following the last bout of involuntary exercise. Muscles were processed for GDNF protein content using enzyme linked immunosorbant assay. Contralateral EDL and SOL were removed at the same time points for immunohistochemical analysis. The results showed that 72 hr following exercise there was a significant increase of GDNF protein content in SOL and a significant decrease in EDL. The results suggest exercise causes a relatively long‐lived effect on GDNF protein content and that expression of GDNF protein in type I skeletal muscle maybe regulated differently that that in type II skeletal muscle.

This work was supported by NIH Grant 1R15AG022908‐01A2, MSU‐KCMS, and Western Michigan University.
Effects on dendritically located H‐current on synaptic integration: a mathematical model
Authors: Arencibia‐Albite Francisco Mariano , Marcano Mariano , Jimenéz‐Rivera Carlos
Published: June 2021

The h‐current (Ih) is a cation current that improves cell excitability in three interrelated ways: it helps to determine the resting potential and firing frequency; it reduces the amplitude and duration of hyperpolarizing inputs; and it generates rhythmic cell activity when coupled to other voltage dependent currents. However, the role of Ih in the excitability of nerve cells may deviate from the functions just mentioned. Dendritically located h‐channels, for example, may obstruct synaptic integration and thus reduced cell excitability. In this work we use a simple mathematical model to study how synaptic integration is affected by the presence of Ih in the dendrite of a neuron that consists of two compartments: the soma and one dendrite. The mathematical model simulates the effect of the activation of a synaptic conductance on the firing rate. Model simulations were conducted for a base case (none of the compartments contain Ih) and two cases where Ih was placed at the soma or at the dendrite but not in both compartments. The simulation results clearly show that the effect of Ih on cell firing rate is dependent on its location in the cell membrane. In particular, dendritically located Ih decreases cell firing rate. Therefore, a negative shift of the voltage dependence of dendritically located Ih may increase cell excitability whereas a positive shift may decrease it. This is consistent with experimental data.
Contribution of intracellular structures into static and relaxation properties of papillary muscles
Authors: Smoluk Leonid , Kobelev Alexandr , Lookin Oleg , Balakin Alexandr , Protsenko Yuri
Published: June 2021

Differentiating the role of extracellular matrix and intracellular structures of myocardium in the total mechanical response of passive muscles now takes attention of researchers due to recently investigated nonlinear features of giant intracellular protein titin. We assume here that the variety of spatial structures on a fascicule level can be represented by 2D‐model consisting of inclined, longitudinal and transversal elastic springs and damping elements. The titin block (consisting of N=10 times repeated rhombic structure) is inserted in the bulk of parallelogram‐shaped fascicule block. Now we can calculate steady state stress‐strain curve, hysteresis loop, and stress relaxation under step‐wise stretching in the model with or without titin block.

It was found that intracellular structures are the main sources of non‐linearity of stress‐strain relation. We have superimposed stress‐strain relations obtained for papillary muscle in normal conditions and after application of NaOH which disrupted intracellular structures of the muscle (Fig. 1A). The experimental protocol we used is shown on Fig. 1B (normal conditions) and after treatment of NaOH solution (Fig. 1C).

Studying the difference in normal case and after treating with NaOH, we can evaluate the partial contribution of intracellular structures into passive mechanics of papillary muscles.

This work is supported by RFBR URALS Grant #04‐04‐961091.
Functional and molecular partitioning of the brain provides neuroprotection to cardiorespiratory nuclei in ground squirrels during hibernation
Authors: Hengen Keith B. , Johnson Stephen M. , Carey Hannah V. , Behan Mary
Published: June 2021

Torpor and hibernation are among the most extreme examples of energy conservation and CNS plasticity in endotherms. In torpor the respiratory rate of the 13‐lined ground squirrel (Spermophilus tridecemlineatus) decreases 98%, and heart rate drops to 1% of the aroused state. While dendrites of cortical neurons retract during torpor, corresponding to a cessation of neuronal activity, cardiorespiratory function is maintained. We are investigating molecular and electrophysiological properties of neurons in the cortex and brainstem of ground squirrels during the annual cycle that may underlie this partitioning. Data indicate that cortex is significantly remodeled upon entrance into and arousal from torpor, whereas the brainstem, which contains cardiorespiratory nuclei, remains static. qRT‐PCR showed significant increases in mRNA for several GABA‐A receptor subunits as well as synaptophysin in cortex during interbout arousals. No such changes were seen in brainstem. Ongoing experiments using intracellular recordings and dye injection will determine functional components of this partitioning at a cellular level. We speculate that cardiorespiratory nuclei remain active during torpor and are protected from the morphological and functional remodeling associated with hibernation by modulation of GABA receptors. DARPA W81XWH‐05‐02‐0016 (HC), NIH AG18760 (MB) and NIH T32 GM007507.
The effect of progesteron on brain malondialdehyde and gluthatione levels in the experimental epilepsy in mice
Authors: Pence Sadrettin , Erkutlu Ibrahim , Alptekin Mehmet , Kurtul Naciye , Pence Halime Haným
Published: June 2021

Objective:

The aim of the present investigation was to determine the effects of progesterone on malondialdehyde and gluthatione levels in the different brain regions after PTZ‐induced epilepsy in the mice.

Material and Methods:

Total 80 mice have been separated into 8 groups consisting of 10 mice. The groups were the sham, control, PTZ, PTZ plus progesterone, five dose progesterone, single dose progesterone, kindling, and kindling plus progesterone groups. In all groups brains were divided into 5 parts as right hemisphere, left hemisphere, right brain stem, left brain stem and cerebellum under microscopy. Malondialdehyde and gluthatione levels were measured spectrophotometrically.

Results:

MDA levels of 3rd and 7th groups in all brain regions were significantly higher than those of the other groups (P<0.05). While highest values were found in brain stems, lowest values were in the cerebral hemispheres. Moderate elevation was seen in the cerebellum compared to the sham group. MDA levels of in all brain regions decreased markedly in the groups who were given progesterone treatment compared to the control group (P<0.05), but no statistically significant increase was found in all other groups. Gluthatione levels of the groups with progesterone were significantly higher than those of the other groups (P<0.05). In addition, Gluthatione levels ere significantly lower in both 3rd and 7th groups in all brain regions.

Conclusion:

We demonstrated that progesterone improves experimental epilepsy by decreasing MDA and increasing gluthatione levels in both the brain hemispheres and brainstem structures.
Structural requirements for the electrogenicity of the electrogenic Na‐HCO3 cotransporter NBCe1
Authors: Chen Li‐Ming , Boron Walter F
Published: June 2021

Na/HCO3 cotransporters, including the electrogenic NBCe1 and the electroneutral NBCn1, belong to SLC4 family. The NBCe1‐A splicing variant plays a key role in HCO3 reabsorption by renal proximal tubules. NBCe1 and NBCn1 have 13 putative transmembrane (TM) segments and a large extracellular loop between TM5 and 6 (“the Loop”). Choi et al (J Physiol, 578:131, 2007) previously demonstrated that the electrogenecity of NBCe1‐A requires the interaction between TM1‐5 and TM6‐13, but does not require the cytoplasmic N terminus (Nt), the Loop, or the cytoplasmic C terminus (Ct). To understand better the structural basis of electrogenecity of NBCe1, we constructed a series of chimeras of human NBCe1‐A and NBCn1‐X (i.e., lacking cassette B in the Ct), starting with one consisting of Nt∼Loop of NBCe1‐A spliced to TM6∼Ct of NBCn1. We exposed Xenopus oocytes expressing chimeras or wild‐type NBCe1‐A to 1.5% CO2/10 mM HCO3 (pH 7.5), and monitored pHi and Vm. We found that this first chimera exhibits the Na conductance characteristic of NBCn1, but no HCO3 transport. Restoring TM6‐7 of NBCe1‐A restored some electrogenic HCO3 transport (i.e., CO2/HCO3 elicited a slight hyperpolarization and pHi recovery). The chimera in which we restored TM6‐9 of NBCe1‐A functions as a typical NBCe1‐A. We conclude that the putative TM6‐9 of NBCe1 (vs NBCn1) is very important, but TM10‐13 of NBCe1 is not essential, for the electrogenecity of NBCe1‐A.
Determination of ions that are actively transported across porcine vas deferens epithelia
Authors: Devlin Sarah Beth , Schultz Bruce D
Published: June 2021

An appropriate fluid environment is required for sperm development, storage and activation, although the underlying mechanisms to account for electrolyte secretion into the male reproductive duct are not fully defined. Radiotracer flux experiments were conducted to test for unidirectional and net Na+, Cl−, and K+ transport across epithelial cells that were isolated from porcine vas deferens and grown in culture. Epithelial monolayers were exposed to the corticosteroid hormone dexamethasone for 72 hours before being mounted in modified Ussing flux Chambers for ion transport analysis. The apical and basolateral aspects of the epithelia were separately exposed, in paired monolayers, to tracer amounts of 22Na+, 36Cl−, or 86Rb+. Sampling was conducted over twenty minute periods in four cumulative conditions: basal, amiloride, forskolin, and bumetanide. Samples were analyzed using scintillation fluorimetry. Results indicate that amiloride sensitive Na+ absorption is present at the outset of sampling. Subsequently, forskolin was shown to stimulate bumetanide sensitive Cl− secretion across these epithelia. No net 86Rb+ flux was observed, which indicates no net K+ movement. Na+ and Cl− flux may not fully account for the observed short circuit current. Thus, additional studies are required to test for the contribution of HCO3− to stimulated secretion, which may contribute to sperm activation.
Caffeine enhances sodium influx and sodium‐linked fluid transport in respiratory epithelium
Authors: Kapur Arvinder Kaur , Carlton David P
Published: June 2021

Caffeine minimizes the time on assisted ventilation in premature infants, possibly as a result of an effect on central respiratory drive. Alternatively, we considered that caffeine improves outcome as a result of improved lung water balance. To test this notion, we studied the effect of caffeine on ion transport in cultured lung cells. We treated an immortal lung epithelial cell line (A549) and distal lung epithelial cells from fetal sheep (FDLE) with caffeine (10−5 M) for 24 h and measured the accumulation of 22Na through pathways that are inhibited by amiloride. Caffeine increased the fraction of amiloride‐sensitive Na uptake by 25% in both cell lines (A549: control 214 ± 51 v caffeine 282 ± 133; FDLE: control 126 ± 29 v caffeine 150 ± 48 nmol/mg protein/5 min, P < 0.05). To study the associated change in fluid movement, we grew the cells on a semi‐permeable surface and measured the change in protein concentration in the supernatant as an index of fluid movement across the monolayer. Caffeine treatment as above increased fluid clearance by 30% (A549: control 106 ± 29 v caffeine 137 ± 26; FDLE: control 79 ± 29 v caffeine 106 ± 30 μL/24h, P < 0.05), an effect that was inhibited in the presence of amiloride (A549: control 61 ± 24 v caffeine 69 ± 25; FDLE: control 103 ± 14 v caffeine 109 ± 16 μL/24h). Our results indicate caffeine increases transepithelial sodium reabsorption and fluid movement through amiloride‐sensitive pathways. We speculate that the beneficial effect of caffeine on respiratory function in the premature lung may be, at least in part, a result of an improvement in lung water balance.
CONTINUOUS MEASURES OF BREATHING AND PHASE ANGLE IN CONSCIOUS CYNOMOLGUS MONKEYS
Authors: Curran Aidan , Burton Josh , Skeans Susan , Landers Devon , O'Brien Shannon , Chapman Richard
Published: June 2021

We evaluated an inductance plethysmography (IP) system in 3 allergic cynomolgus monkeys for measurement of breathing and phase angle (Φ) as a surrogate for airway resistance. Initial studies in anesthetized, intubated animals compared a standard pneumotachograph measure of ventilation with IP to estimate the accuracy of the measurement. Data showed excellent agreement between IP and the standard approach. 24 hours of baseline unanesthetized breathing was then recorded. After 24 hours, monkeys were re‐anesthetized, exposed to an aerosolized ascaris suum antigen and immediately returned to their cages. A subsequent 24 hours of recording provided data from each animal during and following a bronchoconstrictor response. Data indicate that ventilatory pattern is distinctly altered for a period of at least 60 minutes post challenge at which point breathing recovers to normal values. These data agree with our AUC measures in anesthetized animals after antigen exposure. The Φ for each breath was also calculated. During bronchoconstriction, a distinct different angle was measurable. Upon recovery, Φ returned to normal and remained stable, regardless breathing pattern. We conclude that this system is a useful tool to measure breathing in unencumbered, conscious animals for a long period and may be useful in evaluating bronchoconstrictor events.
Pulmonary gas exchange following rapid intravenous saline infusion
Authors: Prisk G. Kim , Olfert I. Mark , Arai Tatsuya J , Hinds Richard M , Huang Kun Lun , Wagner Harrieth E , Wagner Peter D , Hopkins Susan R
Published: June 2021

Rapid infusion of i.v. saline, a model of pulmonary interstitial edema, alters the distribution of pulmonary perfusion, raises pulmonary capillary blood volume, and increases bronchial wall thickness in humans. We hypothesized infusion would disrupt pulmonary gas exchange by altering VA/Q matching. Seven males (26±3 years, FEV1 110±16% pred.) performed spirometry and had the distribution of VA/Q measured using MIGET, before and after 20ml/kg i.v. of normal saline delivered in ~30 minutes. Infusion increased thoracic fluid content by 12% (P < 0.0001), left FVC unchanged, but reduced expiratory flows (e.g. FEF25–75 fell from 5.1±0.4 to 4.1±0.4 l/sec, P < 0.05). However, infusion left the distribution of VA/Q unchanged; log SDV 0.40±0.03 pre, 0.38±0.04 post, NS; logSDQ, 0.38±0.03 pre, 0.37±0.03 post, NS. There was no significant change in arterial PO2 (99.2±2.5 pre, 99.5±2.9 mmHg post, NS) but arterial PCO2 was decreased (38.7±0.6 pre, 37.1±1.1 mmHg post, P<0.05). Thus, infusion compressed small airways, and caused a mild degree of hyperventilation, but the matching of ventilation to perfusion was maintained, suggesting active control of blood flow to minimize the gas exchange consequences.
Using animations to learn Ca2+ signaling pathways in vascular endothelial cells
Authors: Liang Willmann , Pang Lori Yaoxian
Published: June 2021

The well being of the cardiovascular system depends largely on the health of endothelial cells (ECs). Pathophysiology of ECs, often associated with abnormal Ca2+ signaling characteristics, results in vasoactive secretions that lead to functional defects in cardiovascular disorders. Understanding the fundamentals of EC Ca2+ signaling is therefore crucial for research personnel in this field. Despite the wealth of literature describing various aspects of Ca2+ regulation in ECs, one could easily be overwhelmed by the complexity of the topic. Thus, we developed an animation series to help students and researchers learn the basic concepts of Ca2+ regulation in ECs. Graphics and animations were constructed using conventional, commercially available softwares. The animations depict major modes of EC Ca2+ transport systems and the various intracellular Ca2+ stores involved. Interactions between Ca2+‐storing compartments and the directional flow of Ca2+ are illustrated as well. Concise text description accompanies the animations for each concept. Compared to typical textbooks and journal articles, annotated animations would be more easily comprehensible and stimulating to the mind. As a result, key features of EC Ca2+ signaling could be conveyed to a novice more efficiently using the animations here.
Gross anatomy as the core for early clinical education
Authors: Fisk Robert , Hanna Jandy , Foster Robert , Nazar Andrea , Leech David
Published: June 2021

When an increase in class size at our school demanded curricular integration, we amalgamated gross anatomy with the clinical skills and OPP courses. The central feature that brings the courses into integration is the anatomy dissection schedule. All other teaching in the 3 courses follows this calendar. In addition to the integrating effect of the schedule, there is common usage of the same anatomy illustrations, plastinated specimens, and original videos (e.g., locomotor sequences). We have also developed crossover and team teaching between the courses, written assignments based on clinical findings in the cadavers, and required case presentations, all of which include active clinician participation. Finally, our anatomy exam includes some questions from the related courses, which count in both. The result is that gross anatomy now forms the core knowledge base for the two clinical courses, with learning in all 3 reinforcing common educational goals. Anatomy also provides many resources for early clinical learning. Survey indicates that students generally appreciate this integration, particularly in the lab. Such integration is very satisfying to the faculty, but it can be accomplished only by a common appreciation of specific educational goals and a good deal of give‐and‐take.
Dyad Pedagogy: Broadening the Learning Agenda in Anatomy with an Eye toward Practice
Authors: Márquez Samuel , Sherman Lloyd R
Published: June 2021

Dyad Pedagogy (DP), a powerful method for acquiring and integrating anatomical knowledge, takes the student beyond the classroom into the workplace. DP has been effectively used in anatomy at SUNY Downstate Medical Center. Sherman and Márquez reported on its positive effect on student performance in 2006 and 2007. Its use in an anatomy course on cadaveric dissection comprising PA and PT students, is reported here. Firstly, dyads were arranged by the instructor, with specific activities thematically and serially organized in accordance with the course objectives. The traditional course was upgraded with new components: written summaries, pre‐ and post‐tests, dissection projects and clinical set‐ups designed to broaden the scope of learning in three dimensions all at once: cognitive, affective, and psychomotor. Evaluation metrics showed dyads functioning at the higher cognitive, affective and psychomotor levels of the Blooms Taxonomies of Learning ‐ most impressive being the levels of student workplace skills: working together, data‐gathering, integration and synthesis, problem‐solving: all behaviors essential to the workplace. By broadening the learning experience to include workplace knowledge and skills, the course ensures that students will achieve robust anatomical knowledge with the added capacity to put that knowledge into practice beyond the classroom, as they pursue their career goals.
Addressing the challenge of learning cranial nerves in a medical school curriculum and a proposed solution
Authors: Homer Heather , Rasmussen Isaac R , Lowe Garrett C , Liddle David G , Foreman K Bo , Morton David A
Published: June 2021

Historically, cranial nerves have been a problematic subject for medical students to learn. Although there are an abundance of illustrations and depictions available, a challenge medical students face is to understand systemically how each cranial nerve relates to one another. To help address this problem, a cranial nerve (CN) tutorial was created. We demonstrated all twelve pairs of cranial nerves together in one illustration using macromedia flash. The interactive program was posted on our anatomy website. The CN tutorial allowed students to explore individual cranial nerves on one diagram, as well as pathways, foramina, modalities, and regional innervation. In addition to these illustrations, students tested their knowledge tracing modalities, pathways, and quizzing autonomously within the CN template. As a result, a template was developed enabling students to independently study the cranial nerves in relation to each other. At the conclusion of the course, a short questionnaire was administered concerning the effectiveness of the cranial nerve tutorial. We received positive feedback.
The effect of intentional cranial deformation on frontal sinus development and morphology
Authors: Tessema Belachew , Lawson William , Schaefer Steven D , Marquez Samuel
Published: June 2021

Introduction: Intentional cranial deformation (ICD) has been documented to be a world‐wide phenomenon whose practice elicits considerable shape changes to the neurocranium during the early stages of postnatal development. Since the development of the frontal sinuses begins after early childhood, the application of ICD procedures should affect morphological growth trajectories in its anatomy. This qualitative and quantitative study examines the effects of ICD on the frontal sinus development and morphology.

Methods: Twenty intentionally deformed (ID) and twenty non‐deformed (ND) dry crania from distinct Bolivian and Peruvian indigenous populations were selected from the Division of Anthropology at the American Museum of Natural History. The crania were scanned using a hispeed helical CT scanner. Craniofacial linear measures, along with endocranial (EC) and frontal sinus volumetric determinations were obtained using a three dimensional (3‐D) volume rendering program.

Results: 3‐D reconstructed volumetric studies showed no statistical differences between the EC volume of ID and ND crania. Analysis of frontal angle showed statistically significant difference between ID and ND crania (p<0.001). Volumetric measurements of frontal sinus showed no statistically significant differences between ID and ND crania.

Conclusion: ICD procedures affect more readily the extrasplanchnocranial moiety as detected by external linear measurements of adult crania. However, the development of the frontal sinus seems not to be affected by these external changes. This study emphasizes the developmental potential of the paranasal sinuses which is not affected by structural changes within the neurocranium.
Carpal bones and Tarsal bones in Korean Chinese water deer (Hydropotes inermis argyropus, Heude 1884)
Authors: Ahn Dongchoon
Published: June 2021

Hydropotes inermis argyropus, Chinese water deer living in Korea, belongs to Order of Artiodactyla and Family of Cervidae. However, it was regarded as a primitive deer in its large laniary canine teeth and the absence of antlers. Carpal bone and tarsal bone are different in their number among animals even in Cervidae caused by absent or fusion between them. So, the shape and number of carpal bones and tarsal bones were used for the morphological feature in determining Cervidae affinities. We investigated the carpal bone and tarsal bones in skeletons in 11 H. inermis arygyropus. Their body weights were above 10 kg. 10 H. inermis arygyropus had 7 carpal bones that were consisted of radial carpal, intermediate carpal, ulnar carpal, and accessory carpal bone in proximal row, firs carpal bone, fused second and third carpal bones, and fourth carpal bone in distal row. This arrangement and articulation with antebrachium, metacarpal and each other was similar with other telemetacarpal type deer. 5 tarsal bones were shown in 10 animals. Calcaneus and talus consist of proximal row, the first tarsal, fused second and third tarsal bones, and fused centroquartal bone in distal row. The arrangement and number of tarsal bone are similar with other Cervidae except Pudu, Muntiacus, and Elaphodus. There is no sesamoid bone which was observed occasionally in small ruminants. One female had one more carpal bone and tarsal bone that might be considered as the fifth carpal bone and the fifth tarsal bone with regarding the location and references. These bones were so tiny with size of 2.0×1.5×1.0mm.

These results suggest that H. inermis arygyropus could be a good deer to study on the limb skeleton in phylogenetic research.

This study was supported by Korea Research Foundation
Grading system for osteophytic changes in zygapophysial (Z) joints following induced segmental hypomobility in the rat
Authors: Cramer Gregory D. , Henderson Charles N.R. , Pocius Judy D. , Little Joshua W. , Grieve Thomas J. , Daley Clover J.
Published: June 2021

Osteophytic changes (OCs) have been identified on the cephalad articular processes (CAPs) of hypomobile zygapophysial (Z) joints of rats. Our objective was to refine the method of grading OCs for use in a large scale study. Thirty‐eight Z joint CAPs (left and right L4, L5, L6, and S1) of control and experimental rats were assessed. The experimental animals had undergone 8 weeks of fixation, using an established, IACUC approved, rat model, in which 3 contiguous segments (L4, L5, L6) were fixed with a specially engineered, surgically implanted, vertebral fixation device. The spines of all animals had been prepared for dissection microscopy. The CAPs were graded by 2 observers using specific OC criteria developed for this study. The lateral, cephalad, and dorsal surfaces of each CAP were systematically evaluated. Each observer graded the spines on 2 separate occasions, separated by 30 days. Intra‐ and inter‐observer reliability of grading was assessed using kappa coefficients. Results: Intra‐examiner reliability was 0.62. Inter‐examiner reliability was 0.64. We conclude that good reliability was achieved using these methods. Future studies will use a consensus procedure for segments scored differently by the two observers.

Support: NIH/NCCAM (Grant #s 1R21 AT00784 and U19 AT002006).
PTH Receptor Responds To Shear Stress Perturbation of Plasma Membrane
Authors: Chachisvilis Mirianas , Frangos John A , Zhang Yan‐Liang
Published: June 2021

The precise molecular mechanisms by which bone cells transduce mechanical stimulus into intracellular biochemical response have not been established yet. Here we show that mechanical perturbation of the plasma membrane leads to ligand‐independent conformational transitions in G protein coupled receptor (GPCR) such as parathyroid hormone type 1 receptor (PTH1r). By using picosecond time‐resolved fluorescence microscopy and GPCR conformation‐sensitive fluorescence energy transfer (FRET) we found that stimulation of MC3T3 cells with fluid shear stress or membrane fluidizing agent leads to a significant changes in conformational equilibrium of PTH1r in MC3T3 cells. The PTH1r conformational dynamics was detected by monitoring redistribution of GPCRs between inactive and active conformations in a single cell under fluid shear stress in real time using genetically engineered PTH1r containing intramolecular FRET pair. Our data demonstrate that changes in cell membrane tension and membrane fluidity affect conformational dynamics of PTH1r. Therefore we suggest that PTH1r is involved in mediating primary mechanochemical signal transduction in MC3T3 cells. Furthermore we show that conformational transitions induced by mechanical stress are different from those induced by its ligand PTH(1‐34).

This research was supported by the NIH grant HL086943.
Non‐cytoplasmic localization of a Dps protein from Deinococcus radiodurans
Authors: Reon Brian , Bhattacharyya Gargi , Grove Anne
Published: June 2021

Deinococcus radiodurans, a Gram‐positive eubacterium is widely recognized for its extreme resistance to various DNA‐damaging conditions, such as desiccation and ionizing and ultraviolet radiation. D. radiodurans is one of only a few bacteria which encode two different DNA protection during starvation (Dps) proteins. We report here a comparison of both Dps homologs from D. radiodurans. Dps‐2 was found to bind to DNA with a much lower affinity than Dps‐1 and both have the ability to oxidize Fe2+. Dps‐2 protects DNA from degradation by reactive oxygen species (ROS) where as Dps‐1 cannot. Dps‐1 was shown to condense DNA in vivo while Dps‐2 was incapable of doing so. In vivo localization assays showed that unlike all previously characterized Dps proteins, Dps‐2 is non‐cytoplasmically localized. While Dps‐1 may serve a function in iron homeostasis, the function of Dps‐2 may be to protect against ROS‐mediated hydroxyl radical damage. Together these proteins may contribute to D. radiodurans ability to withstand such extreme environments.
Maintenance and dynamic of the chromatin structure in Trypanosoma cruzi
Authors: Flawiá Mirtha María , Alonso Guillermo D. , Meyer Cristian G. , Vilchez Larrea Salomé C. , Fassolari Matías , Torres Héctor N. , Fernández Villamil Silvia H.
Published: June 2021

Dynamic structural changes on the chromatin occur throughout the cell cycle. These changes range from local ones, necessary for transcriptional regulation and DNA damage repair machinery accessibility, to global changes necessary for chromosome segregation. We demonstrated that the poly(ADP‐ribose)polymerase from T. cruzi (TcPARP) is strongly activated by nicked DNA and attaches polymers of ADP‐ribose (PAR) to itself and to T. cruzi histones. We assayed several DNA damaging agents that trigger different DNA repair mechanisms and confirmed an increase in PAR synthesis in every case. However, when the induced damage led to cell death, the amount of PAR detected decreased. To study in depth the chromatin remodeling we cloned two T. cruzi HATs (TcHAT1 and TcElp3). TcElp3 failed to rescue a thermosensitive mutant yeast strain. We also analyzed the expression and localization of TcHAT1 and TcElp3 by using specific antibodies. We also analyzed the effect of HDACs inhibitors Trichostatin A (TSA) and sodium butyrate on epimastigotes. When parasites were treated with 50 nM TSA strong proliferation inhibition was observed and a sub G1 peak appeared on DNA histogram when analyzed by flow cytometry. This evidence not only supports a physiological relevance of chromatin remodeling in Trypanosoma cruzi but also opens new insights in drug therapy against Chagas′ disease.

Supported by CONICET, FONCYT and UBA‐Argentina.
Interactions of HP1 with other chromosomal proteins
Authors: Mendez Deanna Lynn , Stephens Gena , Volpi Gina , Kim Daesung , Khorasanizadeh Sepideh , Elgin Sarah CR
Published: June 2021

To investigate the molecular basis of HP1a binding with a number of non‐histone chromosomal proteins in D. melanogaster, we are focusing on three examples: HP2, PIWI, and dAF10. Using co‐immunoprecipitation assays we demonstrate that an HP2 region containing an LCVKI sequence is responsible for binding to the chromo shadow domain (CSD) of HP1a. HP2 co‐precipitates with native HP1, but not with mutatants in HP1 that disrupt dimerization (I191E) or disrupt the PXVXL platform (W200A); mutation of the HP2 peptide at V2468E also abrogates binding. Using fluorescence polarization binding assays, we measure the observed dissociation constant of a partner peptide interacting with the HP1a‐CSD. We find that all three peptides bind with micromolar affinity and that the HP2 peptide binds the tightest followed by PIWI and the dAF10 peptides. Using 2D NMR spectroscopy we show that binding of any of the three peptides causes similar chemical shift perturbations in the portion of HP1a encompassing the CSD and CTE (C‐terminal extension) regions. Together our studies show that different short linear hydrophobic peptides can bind to the HP1a CSD‐CTE region, and potentially influence its localization in heterochromatin.

Supported by NIH GM068388‐18 to SCRE and GM064786 to SK.
Characterization of the specific role and activation of the High Mobility Group A1 (HMGA1) in colon cancer
Authors: Bush Bethany Marie , McKinney Kimberly Q , Sumter Takita Felder
Published: June 2021

The High Mobility Group A1 (HMGA1) proteins are a class of oncogenes whose specific role in colon and other cancers is currently emerging. Our analysis of the HMGA1 promoter region revealed several putative sites for beta‐catenin/tcf binding suggesting that HMGA1 may be activated by the adenomatous polyposis coli (Apc)/beta‐catenin pathway in colon cancer. To better understand the specific role of HMGA1 in colon carcinoma formation, we evaluated the expression of HMGA1 in the Apc/min colon cancer mouse model. Results of quantitative real‐time RT‐PCR (qRT‐PCR) show a 9‐fold increase over normal intestine in HMGA1 mRNA where Apc is truncated and in tumors. Consistent with qRT‐PCR data, HMGA1 protein levels were also significantly higher in Apc‐min intestinal tumors than those from normal mouse tissues. To determine if Apc/beta catenin signaling directly impacts HMGA1 expression, we tested the effect of Apc recovery on HMGA1 expression. Preliminary results indicate that zinc‐induced full‐length Apc expression suppresses HMGA1 levels and that truncations in Apc may activate HMGA1 to promote cancer initiation and progression. Collectively, these results provide the first line of evidence supporting the cooperation of truncated Apc and HMGA1 in colon cancer.

This project was supported by NIH grant P20 RR‐16461 in support of SC INBRE.
Synergistic regulation of the Runx2 P1 promoter in mesenchymal cells by a conserved HLH box and purine‐rich elements (GAY motifs)
Authors: zhang ying , Hassan Mohammad Q. , Montecino Martin , Hawse John R. , Spelsberg Thomas C. , Stein Janet L. , Lian Jane B. , van Wijnen Andre J. , Stein Gary S.
Published: June 2021

Transcriptional control of Runx2 gene expression through two alternative promoters (P1 and P2) is critical for the execution of its function as an osteogenic cell fate determining factor. Using genomic DNaseI footprinting and in vivo DNA binding assays, we identified a helix‐loop‐helix (HLH) motif that overlaps with a Runx motif in the P1 promoter. The HLH mutation decreases P1 transcription 10‐fold in mesenchymal cells, while mutation of the Runx site enhances transcription 2 fold. Although the HLH element is not cell‐type specific, its function may depend on promoter context. Promoter deletion analysis shows that a GA‐rich region (−200 to −128) positively regulates Runx2 P1 transcription and synergizes with the HLH motif. Examination of the −200/−128 region reveals two tandemly repeated GA‐rich elements (GAY motifs) that each contain recognition motifs for purine‐rich DNA binding factors (i.e., SP1 and KLF10/TGFâ Inducible Factor [TIEG]). Forced expression of SP1 or TIEG modulates Runx2 P1 promoter activity through the GAY motifs. Taken together, our data suggest that the Runx2 gene transcription is regulated by synergistic interactions at multiple conserved composite promoter elements to control Runx2 expression and skeletal development.
Post transcriptional regulation of human interleukin‐3 mRNA by the adenosine/uridine Rich element
Authors: Gonzalez Jose Antonio , Colon Daisy , Martinez Daviana , Mauras Kirla R. , Estrella Luis A. , Gonzalez Carlos I.
Published: June 2021

Human interleukin‐3 (IL‐3) is a cytokine that stimulates the growth and differentiation of early lymphoid stem cells and has been implicated in cancer. IL‐3 is a member of a class of transiently expressed mRNAs that harbor Adenosine/Uridine‐Rich Elements (ARE) in their 3′‐UTRs (untranslated region). These AREs play a role in post‐transcriptional control by altering mRNA stability and/or translation. The regulatory effects of AREs are often mediated by specific ARE‐binding proteins (ARE‐BPs). To understand how the AREs and ARE‐BPs in the 3′‐UTR of the IL‐3 mRNA regulate its expression, we constructed a series of firefly luciferase reporter chimeras harboring specific regions of the IL‐3 3′‐UTR. Transient transfection and in‐vitro translation assays of the luciferase‐IL‐3 3′UTR reporter constructs showed a significant reduction in luciferase activity. In addition electrophoretic mobility shift assays demonstrated the formation of several protein complexes in the IL‐3 3′‐UTR regions. Together, these results provide evidence for ARE‐dependent post‐transcriptional control of the human IL‐3 mRNA. JAGF is supported by FIPI‐UPR. This work is supported by grants to C.I.G. (KO1 HL‐04355‐05, GM008102‐3052, U54 CA96297‐03, AABRE‐P20 RR‐016470 from NCRR, RISE 2R25GM61151, PR‐LSAMP).
Template specificity studies of west nile virus RNA dependent RNA polymerase
Authors: Smith Addie , Bursavich Bradley , Temple Jeffrey , Doughty Michael
Published: June 2021

West Nile Virus is a virulent member of the Flaviviridae family of viruses. Infection occurs through viral transfer from mosquitoes to birds with some human and equine infections. Since the summer of 2002 there have been over 16,000 human cases with a mortality rate of 3.8%. The WNV genome contains approximately 11,000 base pairs encoding ten viral proteins, three of which are structural proteins (C, prM, and E), while the other seven are non‐structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS5 region is of particular interest to this project since it encodes the RNA dependent RNA polymerase (RdRp). It is believed that the polymerase uniquely replicates the RNA genome during the replication process. The coding region for RNA polymerase has been amplified via Polymerase Chain Reaction and cloned into an E. coli expression system. The enzyme has been partially purified by affinity chromatography and gel filtration chromatography and an assay for the enzyme activity has been developed. Initial experiments suggest that the enzyme prefers a polyribonucleotide template to an oligoribonucleotide template. Presented here are the results for template specificity studies for both homo‐ and heteroribonucleotides.
Identification of the DAG Lipase alpha‐1 and beta‐1 mRNA transcripts in Human Tissues.
Authors: liang yanbin , Li Chen , guzman Victor , Woodward David
Published: June 2021

Using human DAG lipase α and β specific RT‐PCR, we identified two alternatively spliced variants of human DAG lipase mRNAs, DAG lipase α–1 and β‐1, in human multiple tissues. The alternative splicing of DAG lipase α‐1 occurred at +206 (Gene bank: NM_006133), spliced out 212 bp (+206 to +418 bp) of the coding region, and encoded a 956 amino acid DAG lipase α‐1 protein. The N‐terminus of DAG lipase α–1 differed from that of DAG lipase α. The alternative splicing of DAG lipase β‐1 occurred at 360 (Gene bank: NM_139179), spliced out 552 bp (+360 to +912 bp) of the coding region, and encoded a 391 amino acid DAG lipase β‐1 protein with a 281 amino acid N‐terminal deletion. This study is the first to document alternative splicing of DAG lipase genes. We therefore studied DAG lipase α‐1 and DAG lipase β‐1 mRNA distribution in human tissues (heart, kidney, brain, lung, liver, spleen, and small intestine). DAG lipase α‐1 mRNA was highly expressed in heart, kidney, brain, liver, spleen, small intestine and smooth muscle, but not lung. DAG lipase β‐1 mRNA was highly expressed in kidney, brain, spleen, small intestine and smooth muscle, but not heart, liver, and lung. DAG lipase α‐1 and DAG lipase β‐1 were not found in rat and mouse tissues, indicating species specificity of the alternative splicing variants of DAG lipase mRNA expression. When recombinant DAG lipase α‐1 and DAG lipase β‐1 were expressed in HEK293/EBNA cells, DAG lipase α‐1 was present almost exclusively in membrane, whereas DAG lipase β‐1 was present in cytosolic fractions. The data suggest that the N‐terminus of DAG lipases may be important for this protein translocating to the cell membrane, and alternative splicing variants of DAG lipase α or β may play an unique role in the regulation of “on‐demand” 2‐AG production in human tissues.
Amyloid‐β degradation by insulin‐degrading enzyme is inhibited by nitric oxide
Authors: Cordes Crystal M , Hamel Frederick G
Published: June 2021

Amyloid beta (Aβ) accumulation is a crucial step in Alzheimer.s disease (AD) development. Insulin‐degrading enzyme (IDE) degrades Aβ. We examined the effect of nitric oxide on IDE‐mediated degradation of Aβ. Partially purified rat liver IDE was treated with NO donors S‐Nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) and sodium nitroprusside (SNP), or control compounds N‐Acetyl‐D‐penicillamine (NAP) and potassium ferricyanide (KFC). IDE activity was measured by trichloroacetic acid precipitation of 125I‐amyloid β. IDE cysteine nitrosylation was measured using the biotin switch assay. Degradation of Aβ was specifically mediated by IDE, as unlabeled insulin inhibited Aβ degradation with an approximate Km 10−7 M. SNP and SNAP inhibited IDE degradation of Aβ by 50% and 25% respectively. KFC and NAP had no effects. As a possible mechanism for the inhibitory effect on Aβ degradation, we found IDE to be nitrosylated by SNAP, indicating a new posttranslational modification of the enzyme. Aβ accumulation in the AD brain contributes to the disease progression and pathology, and IDE dysfunction has been implicated as a possible reason for this accumulation of Aβ. We have shown that increased NO levels, which have been observed in patients with AD, can decrease IDE enzymatic function, potentially resulting in increases in Aβ deposition in the brain and development of AD.

Funding: Veterans Affairs Merit Review
The 599‐611 loop in thimet oligopeptidase modulates substrate selection via hydrogen bonding
Authors: Song Michelle M. , Elmore Donald E. , Sigman Jeffrey A. , Bruce Lisa A. , Pabon Amanda , Glucksman Marc J. , Wolfson Adele J.
Published: June 2021

Thimet oligopeptidase (TOP) is a metalloenzyme with a broad specificity for small bioactive neuropeptides. We have previously shown that flexibility in a glycine‐rich loop region comprised of residues 599‐611 modulates substrate selection and catalysis. Computer simulations reveal that mutations of Gly to Ala in the loop lead to decreased flexibility in this region of the enzyme proximal to the active site. Molecular dynamics simulations further indicate that the Gly to Ala mutations’ effect upon catalytic activity towards different substrates is due to a variation in the degree of hydrogen bonding between the substrates and catalytic residues, specifically Tyr609 and Tyr612, which are implicated in transition state stabilization. The mutants and wild type enzyme exhibit similar inhibitor binding affinity, and the substrate cleavage sites are the same for both mutant and wild‐type enzymes, indicating that neither binding affinity nor substrate alignment are affected by the mutations. We conclude that flexibility in the loop confers a selectivity to a variety of neuropeptide substrates by altering the level of hydrogen bonding to a given peptide during catalysis.
Neuronal Restrictive Silencing Factor Suppresses Key Players in Breast Cancer Metastasis
Authors: Wagoner Matthew P , Schoenike Barry , Dang Christina , Lurie Louis , Gunsalus K.T.W. , Roopra Avtar
Published: June 2021

Neuronal Restrictive Silencing Factor (NRSF) is expressed in non‐neuronal tissues throughout the body where it curbs the inappropriate expression of neuronal genes. Recent reports have implicated NRSF as an initiating factor in many brain breast and colon cancers by stimulating cell growth and survival pathways; however which NRSF target genes are responsible for oncogenesis is still unknown. Using databases of known and potential NRSF binding sites in the genome, in coordination with chromatin immunoprecipitation assays, we have identified several positive regulators of the phosphatidylinositol 3 kinase (PI3K) pathway as potential NRSF regulated genes. Knockdown of NRSF in breast epithelial cell lines results in their increased mRNA and proteins levels, as well as PI3K pathway sensitivity. Importantly, we found that many, but not all, cellular functions downstream of the PI3K pathway have also been altered, including cellular metabolism and other survival signaling pathways. Our work aims to assess the importance of these genes and their metabolic consequences in the oncogenic transformation caused by loss of NRSF in epithelial cells.
Inhibiting EMT via glycolytic inhibition: therapeutic implications
Authors: Gunsalus Kearney T.W. , Dang Ngocly T. , Schoenike Barry , Wagoner Matthew P. , Roopra Avtar
Published: June 2021

Cancer cells are characterized by a heightened rate of glycolysis, a phenomenon known as the “Warburg Effect.” This increase in anaerobic respiration is one manifestation of the metabolic rigidity of transformed cells. Given that most mammalian cells have some ability to adapt to alterations in energy source, this lack of metabolic plasticity seen in cancers represents an attractive therapeutic target. Ninety percent of breast cancer mortalities can be attributed to metastasis, rather than the primary tumor per se. We therefore investigated the impact of targeting cancer cell metabolism using an in vitro model of metastasis. We find that although the absence of glycolysis has little effect on the growth and survival of cultured mammary epithelial cells in vitro, glycolytic inhibition had a potent impact on the epithelial to mesenchymal transition (EMT) normally induced by TGF‐β. Investigations into the molecular mechanisms behind the requirement for glycolysis during EMT suggest a role for the tumor suppressor NRSF and its metabolism‐sensing corepressor CtBP in regulating expression of pro‐metastatic genes such as SNAIL, SLUG and TWIST.
Investigating the Expression of Inducible Nitric Oxide Synthase in BV‐2 Microglial Cells: Role of Protein Kinase C in the Expression
Authors: Lawwell Leanne , Moore D. Blaine , Stevens‐Truss Regina
Published: June 2021

Although the production of high levels of nitric oxide by activated microglial cells has been associated with the Aβ plaques of Alzheimer's disease patients, the exact mechanism by which plaque formation leads to nitric oxide upregulation is not fully understood. This study was done to investigate the molecular mechanisms involved in expression of inducible nitric oxide synthase (iNOS) in BV‐2 cells. BV‐2 cells were treated with several activators believed to affect iNOS expression, specifically PMA, LPS, and γ‐INF, and levels of nitrite (measured using the Griess assay) and iNOS protein (measured by Western analysis) were measured. The role of Protein Kinase C (PKC) in the expression of iNOS was assessed by treating the cells with several PKC inhibitors (Ro‐31‐8220, Go6976, and Go6983) prior to activation. Nitrite and iNOS protein levels were again measured. The data showed a direct correlation between the levels of nitrite and iNOS protein following every treatment. As expected, LPS and γ‐INF increased iNOS expression and corresponding nitrite levels, and additionally were synergistic when used in combination in these cells. PMA showed no activation of iNOS when used alone. However, when used in combination with either LPS or γ‐INF, it produced separate effects. In combination with LPS, LPS activity was reduced; however, with γ‐INF activity was enhanced. Each of the PKC inhibitors differently affected nitrite production and iNOS expression observed with the various treatments, suggesting a role for different PKC isoforms in this action in the cell. These results demonstrate that PKC is involved in the expression of iNOS and that expression is regulated via different cellular pathways.