bluuebunny/crossref_metadata_2025 huggingface dataset:
Acute hypoxic hypoxia (HH) decreases active caspase‐3 (AC‐3) in fetal ovine hippocampus
Authors: Chang Eileen I-Ling , Wood Charles E.
Published: June 2021

HH activates fetal stress responses and may increase neuronal apoptosis. We hypothesized that HH will increase apoptotic markers, AC‐3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and that treatment with ketamine, an N‐Methyl‐D‐aspartate receptor antagonist, will reduce apoptosis in fetal hippocampus. Fetal sheep (125 d gestation) were chronically catheterized (n=3–5/group). Ketamine (3 mg/kg) was administered intravenously to the fetus 10 min prior to HH (30 min) stimulation or normoxia. Fetal brains were collected 1 hr after onset of HH for immunohistochemistry analysis with AC‐3 and TUNEL. Significant decreases in AC‐3 abundance were observed in both HH and ketamine+HH compared to normoxia animals (P<0.001, two‐way ANOVA). The group x treatment interaction was statistically significant among hippocampal regions (P<0.05, two‐way ANOVA). Ketamine did not have a statistically significant effect (HH vs ketamine+HH). We conclude that late gestation fetal hippocampus has high levels of AC‐3, that AC‐3 levels were attenuated 1 h after onset of HH, and that ketamine did not alter this response. We speculate that AC‐3 is involved in neurogenesis as well as apoptosis in the fetal brain hippocampus. Research supported by NIH grants HD33053 and DK076541.
Influence of Prior Online Course Experience on the Perceived Benefit of an Online Professional Development Course
Authors: Byse Miranda , Matyas Marsha Lakes
Published: June 2021

In 2011, the American Physiological Society (APS) offered an online professional development course entitled “Writing and Reviewing for Scientific Journals.” This 6‐week course was conducted exclusively online via the Blackboard Learn platform. Fifty percent of the students taking this course had previous online course experience and fifty percent did not. Therefore, this study sought to determine if students with previous online course experience found the online course easier, more useful, and more beneficial than students who did not have previous online course experience. To evaluate these questions, students were required to complete an anonymous post‐course survey consisting of Likert Scale questions and additional comment sections. Surprisingly, students without online course experience tended to find the online course more easy to use than students with online course experience. Additionally, although perceived usefulness of the course components tended to be the same between students from both groups, students with online course experience tended to rate their overall perceived skill strength after the course as higher than the students without online course experience. These finding suggests that prior online course experience may increase students’ perceived benefit of an online professional development course.
Binding of flavones to protein kinase c: a molecular modeling study for college undergraduates
Authors: Peek Mary Elizabeth , Duraj-Thatte Anna Maria , Bhatnagar Abhinav
Published: June 2021

A molecular modeling study was incorporated into the senior‐level Biochemistry Laboratory II course at Georgia Tech, with the hope of broader dissemination to other colleges and universities, as part of an educational “thread.” The molecular modeling experiment was developed to enable students to propose a structural basis for the activity of one of twenty different flavones by investigating the structural features of the flavone docked into Protein Kinase C, a key player in signal transduction. Experiments were conducted and data were analyzed using free software including AutoDock Vina, AutoDock Tools and Python Molecular Viewer. The experiment teaches students many basics of protein structure, builds on what they have learned in organic chemistry about bonding and functional group properties, and challenges them to think critically and creatively about connections between molecular interactions and biological activity. Moreover, students are exposed to real‐world approaches for investigating structure‐function relationships, which lays a great foundation for future careers in science, medicine and pharmaceutical research. Funding for this work was provided by the Division of Undergraduate Education of the National Science Foundation (TUES Grant #1044034; MEP).
Classic Experimentation and Working Models for Engaging and Inspiring Students
Authors: Giuliodori Mauricio , Lujan Heidi , DiCarlo Stephen
Published: June 2021

We developed a simple working model of the semilunar valve to highlight the mechanism of semilunar valve closure as a consequence of the decelerated systolic efflux. The model is inexpensive and simple to build, all that is required is two sections of tubing {one 10 cm long, 31 mm x 25 mm, and another 5 cm long, 25 mm x 19 mm (outside and inside diameter)}, and a long balloon (approx. 2.5 cm diameter). Instructions to build the model are provided. Once the model is fully assembled, place the model under a facet and run water through one end. Reverse ends and repeat the procedure. As you will discover, water will flow through the model in only one direction. A discussion of the physiological significance of one‐way valves in the heart will naturally evolve to their importance in veins (an often under‐discussed and appreciated regulatory mechanism). Building and testing the model with the assigned readings of the pioneering work of Giulio Ceradini was rewarding for the teacher and students. The activity encouraged in‐class discussion, collaborative problem‐solving, and inquiry‐based thinking. This activity also focused student learning on how to use scientific knowledge to solve important questions.
Prerecorded lecture content: Does this enhance student performance?
Authors: Osborne David L , Janssen Herbert F
Published: June 2021

In this day and age of computer‐based learning and instant access to all information, delivery of information to medical students has been significantly altered. The traditional lecture‐based class presentation has become less necessary. We have shown that many of the students are not even reading textbook‐based information, but are relying on computer‐based information delivery. We are currently delivering 24 textbooks to the students in an electronic format. The advantage of this electronic presentation of textbooks is that students can search textbook material like they do internet material. This allows the students to have vetted, accurate information on which to rely. The physiology portion of the curriculum at Paul Foster School of Medicine is delivered over a two‐year time frame. This presentation of material is integrated into the clinical presentation method of delivering basic science content in the context of clinical medicine. We have matriculated 3 classes: for the first two classes, physiology was delivered by lecture only. This past year all lecture material was prerecorded and class time has evolved into Q and A sessions involving deliberate practice of associated material. Anecdotally, students like the new format and in limited comparison they are performing about 10% better on exams. Our new format allows consistent content delivery with real time application.
Learning the cardiac cycle with a puzzle
Authors: Marcondes Fernanda Klein
Published: June 2021

In order to teach the phases of cardiac cycle for students from Dental School we developed a puzzle, with one table of 5 columns: 1. Phase of cardiac cycle, 2. Atrial state, 3. Ventricular state, 4. Atrio ventricular valves and 5. Pulmonary and aortic valves, painted in different colors: white, blue, green, yellow and pink, respectively. Figures of the phases of cardiac cycle and colored chips are also provided to be used in order to complete the table. White chips indicate the names of the cardiac cycle phases. Blue and green chips present the options relaxed or contracting. Open or closed is written in the yellow and pink chips. The students (in groups) are invited to analyze the figures and to complete the the table with the chips. During the activity the professors discuss with them why they choose each chip. Also the following questions are presented to the students during the activity: When and how do the cardiac valves open and close? How is the electrical stimulus transmitted through the heart during each phase? How do the gap junctions participate in the conduction of cardiac electrical stimulus? Which is the role of atrioventricular delay of action potential in the cardiac cycle? In recent years, using this puzzle, teachers have noticed that students more easily establish the relationship between the morphological characteristics of cardiac muscle with the cardiac cycle, compared with the lecture on the subject.
The Effect of Expanding vs. Uniform Retrieval Practice on the Retention of Selected Physiology Concepts
Authors: Dobson John
Published: June 2021

This study compared the recall of physiology concepts throughout 30 days of two different expanding schedules of retrieval and two different uniform schedules of retrieval. Participants (n = 250) first read 30 reproductive physiology and immunology concepts and were then repeatedly assessed, without feedback, according to one of the following four randomly assigned schedules: i) immediately after learning and again 9 and 19 days later (Uniform (1,10,20)), ii) 7, 14 and 21 days after learning (Uniform (8,15,22)), iii) immediately after learning and again 5 and 15 days later (Expanding (1,6,16)) and iv) 1, 6 and 16 days after learning (Expanding (2,7,17)). All participants completed a post‐test 29 days after learning the physiology concepts. Mean post‐test scores ± SE for the Uniform (1,10,20), Uniform (8,15,22), Expanding (1,6,16) and Expanding (2,7,17) groups were 36.15 ± 1.97, 32.31 ± 1.87, 45.80 ± 2.56, 39.71 ± 2.48, respectively. The Expanding (1,6,16) group scores were significantly higher than those in both uniform retrieval groups (ANOVA; F = 6.52, P = 0.00). Also, the combined mean of the two expanding retrieval conditions (42.57 ± 1.80) was significantly higher (F = 14.09, P = 0.00) than that of the two uniform conditions (34.10 ± 1.36). In summary, participants benefited more from expanding retrieval practice, particularly when the first recall assessment was administered immediately after learning.
Nontraditional case‐based instruction in physiology: Deconstructing the cryptophysiology of zombies
Authors: Harris Michael B
Published: June 2021

Modern teaching in physiology is greatly enhanced by case‐based instruction. It is challenging, however, to select cases that engage a wide audience. Without invoking the supernatural, general physiological principals are well illustrated by considering phenomena which could underlie the zombie state. Such treatments also include opportunities to formulate and test hypotheses, design experiments and interpret data to draw conclusions related to the physiology behind depictions of zombie behavior. Here I illustrate an exercise considering effective strategies of zombie defense which incorporates consideration of basic principles of metabolism, as well as respiratory and cardiovascular physiology. Such an exercise provides an opportunity to develop skills in analytical thinking and reflective judgment, and engages students interested in self preservation during the coming zombie apocalypse.
Breaking the tyranny of the textbook: a new approach to introductory biology
Authors: Rylaarsdam Robin Pals , Tischler Monica Lee , Kandra Kelly
Published: June 2021

Introductory textbooks in biological science introduce biological macromolecules in isolation, followed by a standard order of cellular processes. Thus, these subjects may be separated by weeks or even months. We reorganized our introductory biology course around biological themes in an attempt to improve comprehension and continuity. This non‐linear method also allowed the use of topical data from the primary literature in guided‐inquiry exercises, a goal that stretches student learning from memorization to analysis of knowledge. The reorganized course covered the same material as the previous course, as assessed by analysis of syllabi, lecture notes, and exams. Student mastery of the material was compared to previous semesters using course grades and common exam questions. A survey measured student attitudes toward the course. Guided interviews evaluated instructor opinion toward the new format. While preliminary analysis showed no significant difference in the % of students earning a C or better in the two course formats, significantly more students correctly answered analysis/critical thinking questions, such as human pedigree analysis with the new format. Anecdotal evidence suggests that students exhibited stronger data analysis skills in upper level courses. The course designers found this to be a rewarding yet challenging collaboration to improve introductory biology.
Load‐Carrying Lumbar Spine Kinematics in Active‐Duty Marines
Authors: Rodriguez-Soto Ana Elvira , Jensen Andrew , Mayfield Jarrott , Jaworski Rebecca , Frank Lawrence , Ward Samuel Richard , Kelly Karen
Published: June 2021

The purpose of this study was to measure kinematic changes in the lumbar spines of Marines carrying loads. We hypothesized that loads would increase lumbar lordosis and disc compression. Sagittal T2 MR images of the lumbar spine were acquired on a 0.6T vertical MRI scanner in 39 active‐duty Marines. Each Marine was scanned without load (BL) and after 45 min of standing with 50lbs of load (WL). Custom software was used to measure whole spine angles, intervertebral angles, and regional disc heights. Paired t‐tests were used to identify significant differences between tasks (α = 0.05). The overall position of the spine was significantly more horizontal at WL (71.6±6.8°) compared to BL (84.2±4.6°). Lumbar lordosis was reduced during WL (46.0±9.0°) compared with BL (58.2±9.1°). Sagittal intervertebral angles were significantly increased in L1L2 (BL=3.8±2.2°, WL=5.2±2.2°) and reduced in L4L5 (BL=10.2±2.7°, WL=6.9±4.5°) and L5S1 (BL=9.7±3.0°, WL=4.3±2.7°). Significant disc compression was evident anteriorly at L4L5 (BL=13.1±1.8mm, WL=12.5±2.4mm) and L5S1 (BL=12.4±2.9mm, WL=11.0±2.8mm) and reduced in the posterior region at L4L5 (BL=8.6±1.1mm, WL=9.1±1.4mm). Load carrying induced lumbar spine flexion, anterior disc compression and posterior disc distraction. These findings suggest a spine posture aimed to realign the center of mass of the soldier and load carrying system, but it may also increase lumbar spine susceptibility to shear‐load induced injuries.

Grant Funding Source: US ARMY RDECOM N6311610MP00182
BILATERAL ABDUCTOR POLLICIS LONGUS MUSCLE DUPLICATION
Authors: BYIRINGIRO Jean Claude , GASHEGU Julien , NYUNDO Martin
Published: June 2021

The abductor pollicis longus (APL) muscle has been largely reported to be a subject of numerous variations affecting the origin, the number of bellies and tendons, and the patterns of tendons insertions. We report a bilateral APL muscle duplication as a variation encountered during dissections of the forearm and hand of an adult female cadaver. The APL muscle presented with two bellies and each belly had a distinct tendon. The main belly originated as usual from the posterior aspect of the lateral border of the ulna, the interosseous membrane and the middle third of the radius while the additional belly was arising from a retinaculum‐like fascia formation attached at the lateral border of the radius and covering the extensor carpi radialis longus and brevis and both tendons inserted as usual on the base of the first metacarpal bone. The tendon from the additional belly was the most laterally inserted and therefore thought to be the main abductor, whereas the tendon from the main belly was thought to be the best extensor of the trapezo‐metacarpal joint. The presence of additional APL tendons has been regarded as a determinant in the development of De Quervain's stenosing tendovaginitis and the awareness of the existence of such anatomical variations may be important for reconstructive surgery as a potential source of transplant material.
Aortic depressor nerve differences between Wistar and Wistar‐Kyoto rats
Authors: de Aguiar Mendes Vânia Alice , de Amorim Milena Menezes , da Silva Letícia Oliveira Neri , Salgado Helio Cesar , Fazan Valéria Paula Sassoli
Published: June 2021

Previous reports from our laboratory [1, 2, 3] showed differences on the morphology of the aortic depressor nerve (ADN) between normotensive Wistar‐Kyoto (WKY) and spontaneously hypertensive rats (SHR). The aim of the present study was to investigate if there are morphological and morphometric differences on the AND of two different normotensive rats strains. Twelve Wistar rats (WR) and 13 WKY had their left ADN fixed in 2,5% glutaraldehyde. Semithin cross sections of proximal and distal segments of the ADN were analyzed by light microscopy and a morphometric study of the nerve fascicles and myelinated fibers was performed. Comparison between proximal and distal segments of the two strains revealed a longitudinal symmetry. Comparison between strains revealed that, despite the fact that WR are heavier (body weight = 339 ± 17g for WR and 277 ± 54 for WKY) than WKY, ADN fascicles and myelinated fibers are larger in WKY. Myelinated fiber distributions were unimodal in both segments for both strains, with peak in 2,5 μm but, there is a higher percentage of large fibers (> 4 μm) in WKY nerves. Average values of myelinated fibers morphometric parameters are higher in WKY due to a larger number of fibers bigger than 4 μm.

Grant Funding Source : FAPESP, CNPq, CAPES and FAEPA
Role of LEK1 in Cardiac Disease and Function
Authors: Miller Paul Myron , Dees Ellen , Bader David
Published: June 2021

In our lab, we have developed global as well as heart‐specific CENPF−/−(LEK1) mouse models. Utilizing our heart‐specific model, we have found that LEK1 mutation leads to a myriad of problems including: an overall decrease in intercalated disc number, significant fibrosis, erratic ECG patterns, progressive dilated cardiomyopathy, and a 20% chance of sudden death. To date, the molecular mechanism underlying LEK1 involvement in these processes remains unknown. Although LEK1 is a very large and multi‐functional protein, one common functional theme has emerged in the literature: the interaction of LEK1 with the microtubule cytoskeleton. Previous studies have identified an important role for LEK1 in microtubule nucleation at the centrosome as well regulation of vesicular transport and cell coupling. Here, using CENPF−/−mouse embryonic fibroblasts, we show that directionally persistent migration as well as overall microtubule network organization is disrupted. Our overall hypothesis is that cytoskeletal defects mediated by LEK1 mutation result in developmental, morphogenic, and functional abnormalities in the heart.

Funding: 5 T32HL007751‐17, Training Grant in Mechanisms of Vascular Disease

Grant Funding Source : NIH
Interleukin‐8 Regulation by Histone Deacetylase Inhibitors and Bortezomib in Human Macrophages
Authors: Sanacora Shannon Marie , Ramaswami Sitharam , Chang Tzu-Pei , Manna Subrata , Singha Bipradeb , Vancurova Ivana
Published: June 2021

Interleukin‐8 (IL‐8) is a proinflammatory chemokine that plays a major role in inflammatory responses, as well as in tumor‐associated angiogenesis and tumor progression. Macrophages at the sites of tissue injury and cancer development are the major source of IL‐8. The transcriptional regulation of IL‐8 is mediated by the transcription factor NFκB. However, our previous results suggested that different mechanisms regulate the NFκB‐dependent transcription of IL‐8 and other cytokines, such as TNF, IL‐1 and IL‐6, in human macrophages. The goal of this study was to investigate the transcriptional regulation of IL‐8 in stimulated U‐937 human macrophages. Using chromatin immunoprecipitation, we show that while the NFκB promoter sites of TNF, IL‐1 and IL‐6 are occupied by p65 NFκB acetylated on K310, IL‐8 promoter is occupied by non‐acetylated p65. The histone deacetylase (HDAC) inhibitor, trichostatin A, enhances IL‐8, but not TNF transcription in stimulated macrophages, indicating that p65 K310 acetylation increases IL‐8 transcription. The proteasome inhibitor bortezomib, which down‐regulates HDACs expression, also increased IL‐8 expression in macrophages. Since HDAC inhibitors and bortezomib have emerged as promising new anti‐cancer therapies, understanding the mechanisms how they increase IL‐8 expression in macrophages may contribute to the development of more effective therapies.

Funded by: NIH grants GM079581 and AI085497 to I.V.
Exercise translocates RIP140 to the cytoplasm in human skeletal muscle
Authors: Sällstedt Eva-karin Sara , Ydfors Mia , Norrbom Jessica , Sundberg Carl Johan
Published: June 2021

In response to exercise, muscles adapt with a multitude of gene expression changes and protein modifications. One of the most profound changes is an increased mitochondrial biogenesis. The co‐repressor RIP140 interacts with several nuclear receptors and is recruited by transcription factors and co‐activators such as PGC‐1α and NRF‐1. Therefore, RIP140 may take part in the regulation of mitochondrial biogenesis.

Purpose

To investigate 1. if an acute bout of exercise reduces the inhibitory action of RIP140 through translocation from the nucleus to the cytoplasm and 2. the temporal resolution of RIP140 mRNA expression before and up to 24h after exercise.

Methods

Healthy men (n=4) and women (n=3) performed 1h of cycling exercise at 70 % of VO2max. Skeletal muscle biopsies were obtained before and after exercise (pre, 30′, 2h, 6h and 24h).

Results

RIP140 protein abundance increased in the cytoplasmic fraction 24h after exercise (n=3). RIP140 mRNA expression (n=7) was significantly increased 6h after exercise and returned to baseline values at 24h.

Conclusion

An acute bout of exercise appears to translocate RIP140 protein from the nucleus to the cytoplasm. This would decrease the inhibitory action of RIP140 on nuclear genes encoding mitochondrial proteins and might therefore be part of the adaptive mitochondrial response seen with training.
A means to an end: a role for phosphorylation in the degradation of Pax3
Authors: Loupe Jacob M , Miller Patrick J , Iyengar Aditi S , Hollenbach Andrew D
Published: June 2021

Pax3 is a transcription factor necessary for regulating the expression of genes crucial in early muscle development. Upon myogenic differentiation, Pax3 protein levels diminish due to increased proteasomal degradation. This degradation is signaled by Pax3 monoubiquitination by Taf1, a subunit of the TFIID general transcription complex. Our lab demonstrated that Pax3 is phosphorylated at only three sites present in the domain responsible for mediating protein‐protein interactions (Serines 201, 205, and 209). Unpublished data from this laboratory demonstrates that phosphorylation of Pax3 affects its ability to interact with known Pax3‐regulatory proteins, suggesting that phosphorylation is a potential mechanism to control Pax3 biological activity. Of the three identified sites, Ser201 is the only phosphorylation event present on Pax3 at the time of early differentiation when we begin to observe the degradation of Pax3. We show through immunofluorescence that as differentiation progresses, Pax3 phosphorylated at Ser201 localizes to the nucleolus, a known site of nuclear proteasomal degradation. Additionally, we perform in vitro interaction studies with GST‐Pax3 phosphomutants incubated with mouse primary myoblast total cell extract and show for the first time that phosphorylation at Ser201 promotes the ability of Pax3 to interact with Taf1 in differentiated myoblasts. (Funding: NCI R01 CA138656)
Cross talk in the regulation of miR‐192 by p53: An amplification loop in the pathogenesis of Diabetic Nephropathy
Authors: Deshpande Supriya , Kato Mitsuo , Putta Sumanth , Lanting Linda , Natarajan Rama
Published: June 2021

TGF‐β activates microRNA (miR) amplification circuits that include miR‐192 in mouse mesangial cells (MMCs) which regulate downstream events leading to diabetic nephropathy (DN). miR‐192 can downregulate Zeb2 and increase collagen expression and fibrosis. p53 regulates miR‐192 expression in cancer cells. Here we examined whether a cross talk between miR‐192 and p53 in MMCs can result in their mutually increased expression and amplification of downstream pathways related to DN. Primary WT, p53‐knockout (KO) and miR‐192‐KO MMCs were used to analyze effects of p53 and miR‐192 on each other. Kidney samples from diabetic, and locked‐nucleic‐acid (LNA) modified anti‐miR‐192 oligonucleotides treated diabetic mice were analyzed for p53 levels. p53‐KO MMCs showed lower expression of miR‐192, collagen and p21 but higher levels of Zeb2. Exogenous p53 increased miR‐192 promoter activity in p53KO MMCs. TGF‐β induced miR‐192 expression was diminished in p53‐KO MMCs. miR‐192‐KO MMCs had decreased p53 protein expression. miR‐192 mimics or Zeb2 siRNA increased p53 promoter activity and miR‐192 mimics increased p53 protein levels in WT MMCs. Increased p53 staining was observed in kidney glomeruli of diabetic mice relative to controls and this was attenuated by LNA‐anti‐ miR‐192. These results suggest that the reciprocal regulation of miR‐192 and p53 can amplify pathogenic signals in chronic kidney diseases such as DN. Source of research support ‐ NIH DK081705 and NIH DK058191
Integrative genome‐wide analysis reveals cooperative regulation of alternative splicing by hnRNP proteins
Authors: Huelga Stephanie C , Vu Anthony Q , Arnold Justin D , Liang Tiffany Y , Donohue John Paul , Shiue Lily , Hoon Shawn , Brenner Sydney , Ares Manuel , Yeo Gene W
Published: June 2021

Understanding how RNA binding proteins control the splicing code is fundamental to human biology and disease. Here we present a comprehensive study to elucidate how heterogeneous nuclear ribonucleoparticle (hnRNP) proteins, among the most abundant RNA binding proteins, coordinate to regulate alternative pre‐mRNA splicing (AS) in human cells. Using splicing‐sensitive microarrays, cross‐linking and immunoprecipitation coupled with high‐throughput sequencing, and cDNA sequencing, we find that more than half of all AS events are regulated by multiple hnRNP proteins, and that some combinations of hnRNP proteins exhibit significant synergy, whereas others act antagonistically. Our analyses reveals position‐dependent RNA splicing maps, in vivo consensus binding sites, a surprising level of cross‐ and auto‐regulation among hnRNP proteins, and the coordinated regulation by hnRNP proteins of dozens of other RNA binding proteins and genes associated with cancer. Our findings define an unprecedented degree of complexity and compensatory relationships among hnRNP proteins and their splicing targets that likely confer robustness to cells.
Structural investigations of the Get4/Get5/Sgt2 complex
Authors: Chartron Justin William , Clemons William M
Published: June 2021

Tail‐anchored (TA) proteins are classified by a single transmembrane helix at the extreme carboxyl terminus. In yeast, the Guided Entry of Tail‐anchored proteins (GET) pathway facilitates the post‐translational targeting of TA proteins to the endoplasmic reticulum (ER). A complex consisting of Get4, Get5, Sgt2 and chaperones delivers ER destined substrates to Get3, which then targets the substrate to the ER membrane. We have structurally characterized the Get4/Get5/Sgt2 complex. Get4 is an alpha‐helical repeat protein that directly binds Get3. Get4 and Get5 form an obligate dimer mediated by the amino terminal domain of Get5, and this heterodimer forms a higher order dimer mediated by the carboxyl terminal domain of Get5. We have determined structures of the Get5 homodimerization domain by crystallography and solution NMR and found it is a novel, high‐affinity oligomerization motif. Sgt2 contains a tetratricopeptide repeat (TPR) domain that binds to a wide variety of heat shock cognate (HSC) protein families. A crystal structure of the TPR domain with an HSC‐analog provides an explanation for this low specificity. Sgt2 also contains an amino terminal homodimerization domain that additionally binds a ubiquitin‐like domain within Get5. We utilize bioSAXS to combine high‐resolution structural data, generating a model of the Get4/Get5/Sgt2 complex in solution.
Immuno‐enrichment of the novel B‐raf target FAM129B from rat lung tissue
Authors: Holdman Erin Anne , Anderson Tanner , Todhunter Ryan , Riedinger John , Muhonen Wallace , Shabb John
Published: June 2021

The protein FAM129B is a recently discovered downstream target of the B‐Raf‐MEK‐ERK signaling pathway in melanoma cells. The ERK‐dependent phosphorylation of FAM129B is essential to melanoma cell invasion. FAM129B is localized to the plasma membrane in its less phosphorylated state, but becomes cytosolic upon ERK‐dependent phosphorylation. FAM129B is also expressed in normal tissue. Here we show its expression in rat lung. We hypothesize that FAM129B's subcellular location is mediated by protein‐protein interaction and that this interaction is dependent on its phosphorylation state. Polyclonal anti‐FAM129B antisera directed against three unique peptide sequences were tested to determine their efficacies in immuno‐enriching endogenous FAM129B in rat lung. Protein A Sepharose‐coupled antibodies were incubated with rat lung lysate. Specifically bound proteins were eluted and characterized by Western Blotting and silver staining. The region of the gel corresponding to FAM129B was subjected to trypsin digestion. Peptides were analyzed by MALDI TOF/TOF mass spectrometry (MS). Spectra were searched against a rat database and matched to FAM129B to give 40% sequence coverage. FAM129B was selectively enriched with two of the three antibodies. Further studies will use MS to identify candidate interacting partners of FAM129B. Supported by North Dakota INBRE (NCRR P20 RR016471); and UND School of Medicine.
Acid‐base chemical mechanism of the dihydrodipicolinate synthase from Escherichia coli
Authors: Bruxvoort Christina G. , Karsten William E. , Chooback Lilian
Published: June 2021

Dihydrodipicolinate synthase (DHDPS) catalyzes the formation of dihydrodipicolinate from pyruvate and L‐aspartate‐β‐semialdehyde (ASA). The enzyme catalyzes the first committed step for the biosynthesis of L‐lysine in bacteria and plants. The enzyme from Escherichia coli is feedback inhibited by lysine, the end‐product of the pathway. Although numerous x‐ray structures have been solved for DHDPS from a number of organisms, few mechanistic and kinetic studies have been done on the enzyme. We have undertaken a study of the pH dependence of the kinetic parameters of DHDPS in order to elucidate the acid‐base chemical mechanism of the enzyme. The kinetic mechanism is ping pong with pyruvate binding to free enzyme. An active site lysine ε‐amino group attacks the carbonyl of pyruvate and forms a Schiff base intermediate. The loss of a proton from this intermediate leads to the formation of an enamine intermediate, with the loss of the proton accounting for the irreversible step and the ping pong kinetic mechanism. ASA binds to the enzyme:enamine covalent intermediate. The V/Et pH profile displays a bell shaped curve with a pKa of 7 on the acid side and a pKa of 8.6 on the basic side of the profile. The V/KASAEt pH profile is also bell shaped with pKa values of 6.5 and 8.8 on the acid and basic sides, respectively. The pKa on the acid side of the V/KASA profile likely represents the pK of the enamine intermediate and the pK on the basic side may represent the amino group of ASA. The V/KpyruvateEt pH profile displays a pKa on the basic side that likely represents the ε‐amino group of the active site lysine. Work supported by OK‐INBRE.
Substrate tolerance of acyl‐CoA ligases
Authors: Go Maybelle Darlene Kho , Tai Ai Tee , Yew Wen Shan
Published: June 2021

Acyl‐CoA ligases catalyze the activation of a carboxylic acid to an acyl‐CoA ester in a two‐step reaction via an adenylate intermediate. CoA esters are precursors in various biochemical processes such as fatty acid synthesis and polyketide synthesis. These enzymes are also known to have a remarkably broad substrate tolerance.

Five acyl‐CoA ligases were studied – cinnamate:CoA ligase and phenylacetate:CoA ligase from Streptomyces coelicolor, benzoate:CoA ligase from Rhodopseudomonas palustris, malonate:CoA ligase from Rhizobium trifolii, and 4‐coumarate:CoA ligase from Nicotina tabacum. All enzymes were expressed in Escherichia coli and purified to homogeneity in a single step using Ni2+‐chelating chromatography. Using the purified enzyme, various derivatives of cinnamic acid, 3‐phenylpropanoic acid, and benzoic acid were used to screen for a possible conversion of the various aromatic carboxylic acids to their corresponding acyl‐CoA esters. Aliphatic carboxylic acids were also screened as possible substrates. This study found that these enzymes were able to catalyze the CoA esterification of various aliphatic and aromatic carboxylic acids, highlighting the potential use of acyl‐CoA ligases as biosynthetic enzymes; the generated carboxyl‐CoA esters can be used as precursors in the biosynthesis of various secondary metabolites.

This project was supported by grants from the Ministry of Education, Singapore.
Heterologous expression of human phosphodiesterase 3A affects calcineurin‐mediated calcium homeostasis in yeast
Authors: Rhee Dong Keun , Hockman Steven Craig , Manganiello Vincent C.
Published: June 2021

S. cerevisiae easily adapts itself in altered environments by changing gene expression of stress‐related genes. PDEs control cAMP in regulating the cell cycle, and are components of the RAS/cAMP pathway that mediates general stress responses in yeast. While PDE2, one of two PDE isoforms in yeast, is involved in a wide range of stress responses, PDE1 along with calcineurin, a Ca2+‐ and calmodulin‐dependent Ser‐Thr phosphatase, is necessary for cation tolerance. High exogenous concentration of cations activate calcineurin to induce cation resistance in yeast. To determine the functional relation between hPDE3A and yeast PDEs in abiotic stresses, we expressed hPDE3A in yeast and tested sensitivity to some cations and hydrogen peroxide (H2O2). Yeast cells which expressed hPDE3A was sensitive to low [Ca2+] and to high [Na+] or [Li+], but resistant to H2O2, indicating hPDE3A enhanced calcium sensitivity. Interestingly, treatment with cyclosporin A, a potent calcineurin inhibitor, removed the calcium sensitivity. In other words, the enhanced Ca2+ sensitivity associated with expression of hPDE3A was compromised by the inhibition of calcineurin. Expression of hPDE3A was associated with altered expression of several stress related genes. Our results suggest that hPDE3A might be implicated in the regulation of calcineurin‐mediated calcium homeostasis in yeast. Research support provided by NHLBI.
Endoglin is a key mediator of BMP2 mediated Adipogenesis
Authors: Reese Adam
Published: June 2021

About 1.5 million fractures occur annually due to osteoporosis, which affects nearly 20% of all women. Osteoporosis is the thinning of bone tissue and loss of bone density over time in which the accrual of fat within the bone results in decreased bone density. Bone turnover relies on a dynamic balance between bone forming cells (osteoblasts) and fat cells (adipocytes). Redirecting these fat cells would allow for new development in treatments for osteoporosis. Bone Morphogenetic Protein 2 (BMP2) initiates osteoblast and adipocyte differentiation. Casein Kinase 2 (CK2) interacts with the Bone Morphogenetic Protein receptor type Ia (BMPRIa) during BMP2 signaling and controls the key regulatory mechanism for osteogenesis; however the mechanism is yet to be determined. Endoglin, a TGF‐β binding protein, plays a part in BMP2 signaling by associating with BMPRIa, but the effect is unclear. Since CK2 release of BMPRIa induces endoglin expression, the effect of endoglin expression on stimulated C2C12 cells was investigated. Our data suggest that endoglin is up‐regulated during BMP2 stimulation and affects differentiation. We postulate that endoglin co‐localizes with the BMPRIa receptor to different membrane areas causing adipogenesis. Data analysis supports that endoglin induces adipogenesis and reduces mineralization through the DeltaC and PP domains. Research support provided by Charles Peter White Fellowship.
The function of SREBP‐1a in high sucrose diet‐mediated metabolic disease
Authors: Im Seung-Soon , Roqueta-Rivera Manuel , Osbrone Timothy F
Published: June 2021

The sterol regulatory element binding proteins (SREBPs) activate expression of key genes required for lipid metabolism and regulation. We established a line of mice that selectively reduces expression of SREBP‐1a (SREBP‐1aDF) leaving expression of the overlapping SREBP‐1c and unlinked SREBP‐2 transcripts unaffected. Our current study demonstrates that SREBP‐1a is involved in high sucrose diet‐mediated metabolic disease. We show SREBP‐1aDF mice become glucose intolerant and insulin resistant on high sucrose. Both GLUT4 protein and mRNA expression are decreased in muscle and adipose tissue of high sucrose fed SREBP‐1aDF mice. However, SREBP‐1aDF mice prevent high sucrose dependent hepatic steatosis even though mutant mice induce insulin resistance. We further find SREBP‐1aDF mice also reduce serum free fatty acid on high sucrose diet. High sucrose diet does not activate adipose triglyceride lipase and hormone‐sensitive lipase mRNA expression in adipose tissue from SREBP‐1aDF mice. Moreover, expression of tumor necrosis factor‐a is decreased in high sucrose fed mutant adipose tissue compared to wild‐type mice. Tumor necrosis factor‐a has known to be a regulator of lipolysis in adipose tissue. Those data indicate adipose lipolysis is reduced due to decrease of tumor necrosis factor‐a in SREBP‐1aDF mice, resulting in decreased serum free fatty acid. In a previous study, we already demonstrated that fatty acid oxidation was increased in SREBP‐1aDF mice due to reduction of acetyl‐coA carboxylase 2 in liver. Thus, those data suggest that SREBP‐1aDF mice are more susceptible to high sucrose diet‐mediated insulin resistance owing to decreases of both GLUT4 protein and mRNA expression. In addition, SREBP‐1aDF mice are protected from hepatic steatosis on high sucrose diet through altered lipolysis and fatty acid oxidation.
Rgs16 is a pancreatic reporter of chronic hyperglycemia in diabetes
Authors: Ocal Ozhan , Asterholm Ingrid W. , Brekken Rolf A. , Cleaver Ondine , Scherer Philipp E. , Wilkie Thomas M.
Published: June 2021

Diabetes mellitus is a collection of metabolic diseases characterized by chronic hyperglycemia. Type 1 diabetes results from insulin insufficiency due to autoimmune dependent depletion of insulin producing beta cells, whereas type 2 diabetes occurs as a consequence of somatic cell insulin resistance. G‐protein coupled receptors (GPCR) represent the largest non‐antibiotic drug targets and dozens of them are expressed in beta cells. Regulator of G‐protein Signaling (RGS) proteins are feedback modulators of GPCRs. Their expression can be induced by GPCR or cross‐talk pathways, indicating when and where GPCR signaling occurs. Our studies with Rgs16::GFP transgenic mice showed Rgs16 expression in embryonic pancreas progenitors, differentiating endocrine cells, and postnatal vessel and ductal associated cells (VDAC). Pancreatic Rgs16::GFP was absent in normal glycemic adults, but was reactivated by diabetic conditions and pancreatic ductal adenocarcinoma (PDAC). In all tests with type 1 diabetic models of streptozotocin (STZ) treatment and PANIC‐ATTAC mice and type 2 diabetic models of ob/ob mice and diet induced obesity, Rgs16::GFP expression initiated in islets and VDAC after at least 6 days of persistent hyperglycemia. STZ induced Rgs16::GFP expression was reduced by lowering blood glucose levels with insulin administration. Diabetic Rgs16::GFP induction required the metabolic transcription factor ChREBP, as STZ treated ChREBP KO mice lacked Rgs16::GFP expression. Our results suggest that Rgs16::GFP responds to GPCR signals relayed from a “chronic hyperglycemia” sensor which stimulates islets and ductal progenitor cells. We determined the GPCR profile of PDAC culture cells that we plan to use, together with diabetic Rgs16::GFP reporter mice, to identify ligands that stimulate beta cell expansion without promoting cancer.
Interference with platelet‐derived growth factor‐induced activation of receptor tyrosine kinase and downstream signaling pathways by compound C
Authors: Yeo Eui-Ju , Kim Go-Eun , Kwon Hyun Jin , Jeong Meong-Sook , Lee Yoon-Taek
Published: June 2021

Although AMP‐activated protein kinase (AMPK) activation has been implicated in anti‐proliferative actions in various cell systems, we have observed that compound C, an inhibitor of AMPK, reduced the cell viability of human diploid fibroblasts (HDFs). Flow cytometry revealed blocking of cell cycle progression by compound C. Preincubation of HDFs with compound C abrogated PDGF‐induced Akt phosphorylation. Compound C did not affect Akt activity in vitro. Compound C completely inhibited PDGF‐induced phosphorylation of PDGF receptor‐β, which thereby could block downstream PDGF‐induced PI3K, Akt, and ERK1/2. Compound C did not affect EGF‐ and IGF‐induced receptor tyrosine phosphorylation and downstream signaling. In vitro receptor tyrosine kinase activity profiling revealed that IC50 for PDGFR was 5.07 μM for PDGFRβ, whereas IC50 for EGFR and IGF1R was over 100 μM. The inhibitory effect of compound C on PDGF receptor‐β and Akt phosphorylation was also observed in AMPKα1/α2‐knockout MEF cells, indicating that the inhibitory effect of compound C is independent of regulating the AMPK activity. These results suggest that the inhibitory effect of compound C on the PDGF‐BB‐induced proliferation of HDFs is mediated by blocking tyrosine kinase activity of PDGFRβ and downstream signaling pathways, which is independent of regulating AMPK activity [Research support: KRF # 2010‐000‐ 4564 and # 2011‐000‐5951].
Discrepancies in purified and cellular PKMζ inhibition profiles invalidate its proposed role as a mediator of memory
Authors: Schramm Cicely L , Wu-Zhang Alyssa X , Nabavi Sadegh , Xie Lei , Xie Li , Bourne Philip E , Malinow Roberto , Newton Alexandra C
Published: June 2021

Protein kinase M ζ (PKMζ, an alternative transcript of protein kinase C ζ (PKCζ) encoding only the catalytic domain) has been implicated in mediating long‐term potentiation (LTP) and memory. Evidence for a role of PKMζ in such processes has relied heavily on pharmacological approaches using chelerythrine and zeta inhibitory peptide (ZIP) as PKMζ inhibitors; staurosporine, a nonspecific kinase inhibitor, does not affect LTP or memory. We have shown that neither chelerythrine nor ZIP inhibits PKMζ in cultured cells or brain slices. In contrast, staurosporine blocks PKMζ activity through inhibition of its upstream kinase PDK1, suggesting that PKMζ is unlikely to mediate LTP or memory. Using bioinformatics approaches, we identified the protein scaffold p62 as a potential novel target of ZIP. This protein scaffold binds to the AMPA receptor, which plays a key role in synaptic plasticity and memory. Co‐immunoprecipitation studies reveal that ZIP disrupts the interaction of AMPA receptor subunits with p62. Our results suggest that ZIP may affect LTP and memory by modulating key protein interactions of the scaffold p62 rather than through inhibition of PKMζ activity. Support: NIH GM‐43154, NIH MH‐049159, Cure Alzheimer's Foundation, T32‐GM007752, NSF 0742551, and 5T32‐DK007233.
Biophotonic and bioenergetic phototherapy for treatment of antimicrobial resistant S aureus infection [ARSI]
Authors: Einstein George Przybyl , Kowalczewska Malgorzata , Tulp Orien L
Published: June 2021

UV phototherapy [UVPT] has been used as an adjunct therapy for microbial infections. The molecular mechanisms contributing to UVPT remain unclear but are proposed to invoke elements of both microbial and inflammatory immunogenic mechanisms. The effectiveness of the Einstein Matrix of Blood Irradiation [EMBI] UVPT was determined in the treatment of antibiotic resistant S aureus infection [ARSI] in type II diabetic patients. Biochemical and cellular markers of immunomodulation, hematopoetic, and glycemic measures were obtained following EMBI UVPT. EMBI was accomplished via establishment of sterile autotransfusion, during which patient blood was exposed to calibrated bouts of UVA and/or UVC irradiation [96 seconds, 1.944 MJ, quartz cuvette] before reinfusion. ARSI was established via antimicrobial therapeutic failure and antibiotic sensitivity studies prior to EMBI. The effect of in vitro blood irradiation was without effect on morphological or biochemical parameters of hematopoesis and glycemia. In contrast, EMBI was associated with dose‐dependent decreases in expression of pro‐inflammatory cytokines (IL‐1p, IFN‐y and TNF‐a) mRNA expression and increases in mRNA expression of anti‐inflammatory Il‐b activity in mononuclear cells. These results are consistent with UVC induced modulation of immune responses to ARSI, in concert with direct effects of UVPT on microbial viability. Institute Support.
Analysis of the Positively and Non‐Positively Selected Non‐Coding Sequences of Human Chromosome 16
Authors: Snedeker John , Tretina Kyle , Triplet Meredith , Lee David , Poirier Anne , Bragg Melissa , Pun Pattle , Hayward John , Leung Ross , Tsui Stephen
Published: June 2021

We propose the flanking sequences of the SNPs from the noncoding regions of chromosome 16 recently positively selected (Pos) exhibit regulatory processes different from sequences recently non‐positively selected (NonPos)(Nature 449.7164 (2007): 913–18). SNPs from the noncoding regions identified from UCSC Genome Browser Collection were screened using Haplotter in the Phase II HapMap Database found within all people groups. SNP flanking sequences from dbSNP build 131 with perfect matches were searched with the PATCH program of TRANSFAC database. Proximity Testing was done on these SNPs by analyzing their distances from nearest known genes. Of the 10537 and 18433 NonPos and Pos SNPs examined respectively, 74% and 76% of each are located at transcription factor related sites; 1.2% and 1.5% are unique. The proximity test rendered statistically significant data: NonPos SNPs are on average closer to genes than Pos SNPs at the same distance ranges. There are differences in the classes of regulatory functions between the two cohorts, (e.g. enhancers only in Unique Pos) bearing out our prediction. It seems reasonable to assume the Pos cohort should be closer to coding sequences. The result may prompt us to examine the appropriateness of using the chimp allele from pantro2 as ancestral allele in the selection algorithm. This work was supported by Wheaton College Alumni Association and Hong Kong Bioinformatics Center.