bluuebunny/crossref_metadata_2025 huggingface dataset:
The Cholinergic Drug Galantamine Ameliorates Acute Respiratory Distress Syndrome in Mice
Authors: Falvey Aidan , Palandira Santhoshi P. , Chang Eric H. , Consolim‐Colombo Fernanda M. , Tracey Kevin J. , Pavlov Valentin A.
Published: May 2022

Acute respiratory distress syndrome (ARDS) is a life‐threatening form of acute lung injury and respiratory failure. The pandemic of SARS‐CoV‐2 has resulted in over 5 million deaths worldwide and many of those deaths have been attributed to complications associated with ARDS. No efficient pharmacological treatments for ARDS are currently available. In addition to lung injury and pulmonary inflammation, patients with ARDS have increased systemic inflammation. Previous insights have highlighted the role of cholinergic signalling in the vagus nerve‐based inflammatory reflex in controlling inflammation (Annu Rev Immunol, 2018, 36:783). Cholinergic anti‐inflammatory signalling can be activated by galantamine ‐ a centrally acting acetylcholinesterase inhibitor that is also clinically approved (for the treatment of Alzheimer’s disease). Here we examined the efficacy of galantamine in attenuating ARDS severity and inflammation in mice. We used a recently developed model of direct acute lung injury and ARDS, in which intratracheal (i.t.) hydrochloric acid (0.1N HCl, 2ml/kg) instillation is combined with LPS (10 mg/kg, i.t.) instillation 24h later in anesthetized mice. Male, 12‐week‐old C57BL/6 mice (n=7‐9), were subjected to this two hit ARDS model and treated 30 mins prior to each insult with galantamine (4 mg/kg, i.p.) or vehicle. Mice were euthanized 6h after LPS instillation and blood and bronchoalveolar lavage (BAL) fluid were collected. Galantamine significantly lowered the levels of BAL myeloperoxidase – an indicator of the accumulation of activated neutrophils in the airspaces of the lung (p = 0.0021) and the BAL total protein ‐ an indicator of alveolar permeability (p = 0.0076), compared with vehicle‐treated mice. BAL pro‐inflammatory cytokine levels were also reduced by galantamine, compared with vehicle‐treated mice – TNF (p = 0.0274), IL‐6 (p = 0.0009), and IL‐1β (p = 0.0380). In addition, treatment with galantamine significantly lowered the levels of serum pro‐inflammatory cytokines ‐ TNF (p = 0.0015), IL‐6 (p = 0.0089), and IL‐1β (p = 0.0149). In conclusion, galantamine reduced the severity of ARDS in mice and alleviated the local and systemic inflammatory responses. These results are of interest for further preclinical and clinical development of galantamine in the treatment of ARDS.
Oxidative Stress Biomarkers in Patients with End Stage Renal Disease
Authors: Sharma Rishima , Sridharan Divya , Bansal Vinod , Hoppensteadt Debra , Siddiqui Fakiha , Iqbal Omer , Laddu Atul , Fareed Jawed
Published: May 2022

Introduction

CKD5 is the final stage of chronic kidney disease in which the patient has end‐stage renal disease (ESRD) and their kidneys are about to fail. One of the larger factors that contributes to the progression of renal failure is oxidative stress. Oxidative stress occurs when there’s an imbalance between free radicals and antioxidants in the body. DNA, proteins, and lipids can become damaged as a result of this imbalance. Stable derivatives of oxidants are used as biomarkers of oxidative stress. When renal damage occurs, likely due to oxidative stress, the levels of biomarkers will likely be more elevated in ESRD patients compared to control samples. L‐FABP is a cytoplasmic protein involved in facilitating and transporting long‐chain polyunsaturated fatty acids and is very significant in many research experiments related to CKD5. The purpose of this research project is to explore the relationship between oxidative stress biomarkers in ESRD patients and how the presence of ESRD contributes to the concentration of these biomarkers in patients. Hypothesis: The levels of oxidative stress biomarkers in ESRD patients will be elevated in comparison to the concentrations of them in control patients. Materials and

Methods

Blood samples were collected from CKD‐5 patients and stored in sodium citrate tubes and centrifuged at 3000 rpm to separate the plasma. Normal human plasma (NHP) was obtained from a commercial vendor. The CKD‐5 and NHP were used to profile the biomarkers through ELISA kits. The statistical and correlation analysis was done through PRISM GraphPad software and IBM SPSS. Multiple statistical analysis tests were run to determine the correlation among the biomarkers and whether the data was statistically significant. Correlation analysis, Mann‐Whitney U tests, normal distribution, skewness tests, and quartile analysis tests were done through PRISM Graphpad Software and IBM SPSS.

Results

The results are calculated in terms of group means. L‐FABP showed statistically significantly higher values in ESRD patients compared to control patients. L‐FABP was found to be 106599.4 pg/ml in ESRD v.s. 5116.33 pg/ml in NHP. PAI‐1a was found to elevated in ESRD (17.9 ng/ml) compared to NHP (10.47 ng/ml), while NO also showed higher values in ESRD (34.74 umol/L) compared to NHP (13.79 umol/L). There was a weak correlation between the biomarkers, but ESRD patients showed highly elevated levels of L‐FABP in comparison to NO and PAI‐1.

Conclusion

These results suggest that impaired renal function and kidney damage contribute to the marked increase of L‐FABP in ESRD patients. Moreover, the increase in PAI‐1 and NO suggest the upregulation of oxidative stress and hemostatic deficit.
Uric Acid and Genetic Polymorphisms in the SLC2A9 and Xanthine Oxidase Genes in Patients with Type 2 Diabetes Mellitus in Jordan: A Cross‐sectional Study
Authors: Al‐Azzam Nosayba , Elsalem Lina , Abumousa Balqis
Published: May 2022

Type 2 diabetes mellitus (T2DM) is rapidly increasing in prevalence. T2DM is defined by elevated blood glucose levels that occur as a result of cellular resistance to insulin action. Prediabetes, or poor glucose tolerance, is a pre‐diabetes condition. Prediabetes is reversible, and active intervention programs involving dietary and lifestyle changes may help prevent prediabetes from progressing to type 2 diabetes. A few studies found a favorable correlation between increased serum uric acid levels and type 2 diabetes, whereas others found no correlation or even a negative correlation. Jordan never put the aforementioned relationship under investigation. Our research focuses on comparing the serum uric acid levels in Jordan's non‐diabetic, prediabetic, and diabetic population groups (study IRB# 313/2020). Increased uric acid levels could be due to increased urate synthesis or decreased urate excretion. In humans, xanthine oxidase, a cytoplasmic enzyme, catalyzes the final two steps of purine breakdown. The oxidation of hypoxanthine to xanthine and then to uric acid is catalyzed by xanthine oxidase. The SLC2A9 gene encodes a membrane protein that is expressed in proximal renal tubular cells and functions in the excretion of urate. Numerous single nucleotide polymorphisms (SNPs) in the xanthine oxidase and SLC2A9 genes may affect the degree of expression or activity of their protein products. The connection between certain SNPs in the xanthine oxidase or SLC2A9 genes and T2DM has not been investigated in Jordan. Therefore, we aimed to study certain SNPs of the aforementioned genes in our study population and correlate the genetic analysis with the uric acid levels and the disease progression. Our results reveal the correlation between serum uric acid levels and T2DM and demonstrates if serum urate levels and SNPs affecting its synthesis or excretion may serve as early indicators of type 2 diabetes in Jordan.
Metal Substrate Translocation and Transport Mechanism of the Sinorhizobium melilotiP1B‐5‐type ATPase Nia Revealed by In‐vitro Transport Assays in Proteoliposomes
Authors: Abeyrathna Nisansala S. , Abeyrathna Sameera S. , Meloni Gabriele
Published: May 2022

P1B‐type ATPases are a class of transmembrane transporter proteins responsible for metal substrate translocation, and export across cellular membranes. These ion pumps are classified into seven sub‐families (P1B‐1‐P1B‐7‐type) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub‐family’s substrate selectivity. The P1B‐5‐type sub‐family is widely found in nitrogen fixing bacteria, such as Sinorhizobium meliloti,which form a unique symbiotic relationship with legumes. This symbiotic relationship is highly dependent on the nutrition trafficking between the two species, including a tight control of transition metal homeostasis. The P1B‐5‐type ATPase Nia from S. Meliloti is recognized as a putative iron and nickel transporter, and besides possessing the typical P‐type ATPase domains and topology, it features of a C‐terminal hemerythrin‐like domain containing a di‐iron binding site involved in oxygen sensing, as proposed by in‐vivo studies. However, the substrate transport across lipid bi‐layers, the overall mechanism for cargo translocation and the transport kinetic parameters remain elusive due to lack of molecular tools to study putative substrate transport across membranes in real‐time.

In this study, we coupled metal‐stimulated ATPase activity assays with an experimental platform based on multiple fluorescence sensor probes, to study substrate transport mechanism and translocation kinetics in real‐time with Nia reconstituted in a native‐like environment. We expressed and purified Nia in detergent micelles and subsequently incorporated it in artificial small unilamellar lipid bi‐layer vesicles (proteoliposomes) which allow to study the substrate specificity and transport mechanism in real‐time. The proteoliposomes were used as an in‐vitrotool to determine the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn‐on florescence signal upon substrate ion binding and translocation.

The study revealed that Fe2+, Ni2+ and Co2+are actively translocated by Nia and the kinetic parameter analysis demonstrated that Fe2+ is the preferential Nia substrate. In parallel, by encapsulating in the proteoliposome lumen pH and membrane sensitive fluorescent probes we are also revealing the chemistry underlying the mechanism of transport and potential ions involved as counter‐transported cargos in P1B‐5‐type ATPase catalytic cycle, with the aim of revealing unique molecular features for this subclass of transmembrane metal pumps.
Structural Insights into Phospholipase Cε
Authors: Garland‐Kuntz Elisabeth , Samassekou Kadidia , Fisher Isaac , Muralidharan Kaushik , Everly Amanda , Emmerson Lauren , Reyes Jasmine , Erramilli Satchal , Kossiakoff Anthony , Lyon Angeline
Published: May 2022

PLC (phospholipase C) enzymes hydrolyze phosphatidylinositol lipids to generate inositol phosphates (IPx) and diacylglycerol (DAG). These second messengers stimulate intracellular calcium release and protein kinase C activation. PLCε is the most recently identified member of the PLC family, and is activated downstream of G protein‐coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) through direct interactions with G proteins. In the cardiovascular system, PLCε is necessary for normal cardiac contractility, and its dysregulation contributes to cardiac hypertrophy and heart failure. PLCε shares a highly conserved core that is observed in other PLC subfamilies, and contains a unique N‐terminal CDC25 domain and two C‐terminal Ras association (RA) domains. However, structural insights into the full‐length protein, or catalytically active fragments that contain two or more regulatory domains, have been lacking. Using a combination of structural and functional studies, we previously showed that the PH domain and first two EF hands are dynamic in solution, providing an explanation as to why high‐resolution structures have been elusive. We are now pursuing high‐resolution structural studies of larger PLCε variants and the full‐length enzyme using single particle cryo‐electron microscopy (cryo‐EM). To facilitate this process, we are utilizing domain‐specific fab antibodies as fiduciary markers and to stabilize this conformationally heterogeneous protein. Understanding the structure of PLCε will be critical in identifying sites that can be targeted by small molecule chemical probes, ultimately validating this enzyme as a therapeutic target in cardiovascular disease.
Prolonged diaphragm activation using closed loop optogenetic stimulation
Authors: Benevides Ethan S. , Sunshine Michael D. , Rana Sabhya , Fuller David D.
Published: May 2022

Diaphragm dysfunction in individuals with late stage neuromuscular disease and cervical spinal cord injury is a substantial problem, often resulting in respiratory insufficiencies and ultimately respiratory failure. Therefore, there is a need to develop new methods that can improve diaphragm function in these clinical populations. Here we used an optogenetic method of activating diaphragm myofibers to assess the feasibility of prolonged photostimulation of the diaphragm. Our hypothesis was that closed loop photostimulation can pace the diaphragm over a prolonged period (1 hour) without a decrease in evoked response magnitude. Wild type mice (C57/bl6, n= 5) received an injection of AAV9‐CAG‐ChR2‐mVenus (34 µl, titer: 1.8x1013 vg/ml) to the intrapleural space between the lung and chest wall to target the diaphragm. After an 8‐18 week incubation period, diaphragm EMG and respiratory airflow were measured under isoflurane anesthesia. A light stimulus was delivered to the inferior diaphragm surface using a high‐power, 472 nm, micro‐LED. Photostimulation was delivered in a closed loop fashion using the onset of the inspiratory EMG burst recorded in intact hemi‐diaphragm to trigger the stimulus. The photostimulation paradigm consisted of a 250 ms, ramping/decrementing train of light pulses (pulse duration = 0.1 ms, interpulse interval = 0.1 ms). After a 10‐minute baseline recording, a unilateral right phrenicotomy was performed to paralyze the right diaphragm. The photostimulation paradigm immediately restored EMG activity to the paralyzed hemi‐diaphragm. Further, the magnitude of the evoked diaphragm EMG bursts stayed consistent over the sixty‐minute period of closed loop photostimulation, and there was no evidence of a decrement in EMG activation. Post‐mortem histology confirmed mVenus expression in diaphragm myofibers. We conclude that stimulation of the diaphragm using optogenetics is effective for up to one hour. This is a first demonstration of prolonged photostimulation of the diaphragm and provides evidence to support the feasibility of this method for the treatment of chronic diaphragm paralysis.
Pyruvate kinase M1 regulates butyrate metabolism in cancerous colonocytes
Authors: Park Bohye , Kim Ji Yeon , Riffey Olivia F. , Bruckbauer Antje , McLoughlin James , Bettaieb Ahmed , Donohoe Dallas R.
Published: May 2022

Colorectal cancer (CRC) cells shift metabolism toward aerobic glycolysis and away from using oxidative substrates such as butyrate. Butyrate is a short‐chain fatty acid produced by resident bacteria in the colon and serves as the primary energy source for non‐cancerous colonocytes. Pyruvate kinase M1/2 (PKM) is an enzyme that catalyzes the last step in glycolysis, which converts phosphoenolpyruvate to pyruvate, and produces ATP. M1 and M2 are alternatively spliced isoforms of the Pkm gene. The PKM1 isoform promotes oxidative metabolism, whereas PKM2 enhances aerobic glycolysis. We hypothesize that the PKM isoforms are involved in the shift away from butyrate oxidation towards glycolysis in CRC cells. Here, we find that PKM2 is increased and PKM1 is decreased in human colorectal carcinomas as compared to non‐cancerous tissue. To test whether PKM1/2 alter colonocyte metabolism, we created a knockdown of PKM2 and PKM1 in CRC cells to analyze how butyrate oxidation and glycolysis would be impacted. We report that butyrate oxidation in CRC cells is regulated by PKM1 levels, not by PKM2. Decreased butyrate oxidation observed through knockdown of PKM1 and PKM2 is rescued through re‐addition of PKM1. Diminished PKM1 lowered mitochondrial basal respiration and decreased mitochondrial spare capacity. We demonstrate that PKM1 inhibits hypoxia‐inducible factor‐1 alpha (HIF1α) and suppresses glycolysis. These data suggest that reduced PKM1 is, in part, responsible for increased glycolysis and diminished butyrate oxidation in CRC cells.
Structure activity relationship of 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as CD73 inhibitors
Authors: Jacobson Kenneth A. , Scortichini Mirko , Idris Riham M. , Moschütz Susanne , Keim Antje , Salmaso Veronica A. , Dobelmann Clemens , Oliva Paola A. , Losenkova Karolina , Irjala Heikki , Vaittinen Samuli , Sandholm Jouko , Yegutkin Gennady G. , Sträter Norbert , Junker Anna , Müller Christa E.
Published: May 2022

Adenosine in the tumor microenvironment acts on A2A and A2B adenosine receptors (ARs) on immune cells (T cells, dendritic cells, NK cells, macrophages and neutrophils) to prevent their activation. Therefore, a means of lowering extracellular adenosine would be beneficial in cancer immunotherapy either as a co‐therapy or monotherapy. Inhibitors of CD73 (ecto‐5’‐nucleotidase, the main extracellular adenosine‐forming enzyme) are already in clinical trials for cancer immunotherapy. Many purine (ADP) analogues have been shown to inhibit this enzyme, but we have explored pyrimidine (UDP/CDP) analogues. We recently reported N4‐substituted 3‐methylcytidine‐5’‐α,β‐methylenediphosphates as potent CD73 inhibitors. We now expand the structure activity relationship (SAR) of pyrimidine nucleotides as human CD73 inhibitors, focusing mainly on cytidine functionalization at the N4 position. 4‐Chloro (MRS4598 16, Ki 0.673 nM) and 4‐iodo (MRS4620 18, Ki 0.436 nM) substitution of the N4‐benzyloxy group increased inhibitory potency Ki by ~20‐fold. Primary alkylamine derivatives coupled through a p‐amido group and varying methylene chain length (24and 25) were functionalized congeners, for subsequent conjugation to carrier or reporter moieties. hCD73 X‐ray structures with two of the newly synthesized inhibitors indicated a ribose ring conformational adaptation, and the benzyloxyimino group (E configuration) binds to the same region (between C‐terminal and N‐terminal domains) as N4‐benzyl groups in potent adenine‐derived inhibitors. Molecular dynamics simulation identified stabilizing interactions and predicted conformational diversity around the N4‐benzyloxy moiety. Thus, by structure‐guided substitution of the N4‐benzyloxy moiety, we have greatly enhanced their inhibitory potency and added functionality enabling molecular probes. Their potential as anticancer drugs was confirmed by blocking CD73 activity in human head and neck squamous cell carcinoma (HNSCC) and palatine tonsils in situ.
F11 dorsal root ganglion nociceptor neurons release calcitonin gene related peptide in response to pathogen associated molecular patterns
Authors: Aguilar Diane , Zyuzin Katie , Millet Nicolas , Benefield Lauren , Iacovino Michelina , Swidergall Marc , Jendzjowsky Nicholas
Published: May 2022

The immune and nervous systems co‐evolved to direct and mount coordinated behaviors, reflexes and immune responses to protect the host. In the neuro‐immune paradigm sensory nociceptor neurons, located in multiple peripheral ganglia, have been shown to be sensitive to pattern associated molecular patterns (PAMPs). Upon stimulation, these sensory nociceptors release the neurotransmitter calcitonin gene related peptide (CGRP) which acts as a cytokine to assist with the coordination of the innate immune response. However, it remains unclear which PAMPs are sensed by these sensory c‐fiber nerves and what specific substances are released in response to the specific PAMPs. Additionally, how long does this stimulation by PAMPs last? These are critical knowledge gaps in our understanding of how sensory neurons shape the innate immune response. To answer these questions, we cultured immortalized F11 dorsal root ganglion (DRG) neurons and exposed them to lipopolysaccharide (LPS 500 ng/mL, Gram‐negative PAMP), lipoteichoic acid (LTA 50 μg/mL, Gram‐positive PAMP), tumor necrosis factor alpha (TNFα 500 ng/mL, inflammatory cytokine) and capsaicin (1 μM, known C‐fiber agonist, serving as a positive control). The exposures to the stimuli lasted for 0.5, 1, 2, 4 or 8 hours. The experiments were run in triplicate on three separate days where a control condition was run on each day for each time point. We measured the release of CGRP with ELISA, known to be released by DRG neurons in response to LPS. We hypothesized that CGRP would be released early on as neural excitation occurs quickly. All conditions and time points were normalized to the average of respective controls for specific time points on respective days. In contrast to our hypothesis, no condition increased CGRP concentration at 0.5 hours. However, at 1‐hour LPS, LTA, TNFα and capsaicin increased CGRP release significantly (p<0.05). At 2 hours, LPS and LTA retained their neural stimulation as CGRP was still increased in comparison to controls (P<0.01). Stimulation subsided at 4 and 8 hours as CGRP concentration returned close to control conditions at respective time points. Our data demonstrate that Gram‐positive and Gram‐negative PAMPs stimulate the release of a known neurotransmitter from sensory nociceptor cells. Additional experiments with fungal PAMPs will be run as well as identifying the complete secrotome of the neuronal culture in response to a variety of PAMPs. These data coincide with the notion that neurons are stimulated early during infection by PAMPs and inflammation directly, as indicated by TNFα stimulation, in order to quickly assist with the innate immune response.
Effector Or Substrate: Characterizing Molecular Features of 6‐HNA That Govern Enhanced Rates of NADH Oxidation and Hydroxylation by 6‐Hydroxynicotinate‐3‐Monooxygenase (NicC)
Authors: Brown Joel K. , Snider Mark J.
Published: May 2022

Group A flavin‐dependent monooxygenases are involved in the degradation of aromatic compounds and the biosynthesis of natural products. These enzymes catalyze the regioselective incorporation of one oxygen atom from dioxygen into their substrates. Comparison of the mechanisms through which the enzymes in this class work could assist in future bioremediation efforts and aid in lowering the costs of synthesis of hydroxylated aromatic compounds. 6‐Hydroxynicotinate 3‐monooxygenase (NicC), a member of this class of enzymes, catalyzes the decarboxylative‐hydroxylation of 6‐hydroxynicotinic acid (6‐HNA) to 2,5‐dihydroxypyridine with concomitant oxidation of NADH. Like other enzymes in its class, the binding of 6‐HNA stimulates an increase in the rate of oxidation of NADH, however the structural determinants of 6‐HNA responsible for this are unknown. Here we show by kinetic analyses of NicC variants that NicC uses Arg184 to form an ion pair with the carboxylate of 6‐HNA to stabilize the ES complex. Once 6‐HNA is bound, an electronegative atom para to the carboxylate (the 6‐oxo group in 6‐HNA) strongly increases the rate of NADH oxidation, possibly by changing the hydrogen bonding network in the active site and allowing the FAD cofactor to swing to the “out orientation”, allowing for closer contact with NADH and therefore more efficient hydride transfer. Previous work showed that NicC is able to bind its substrates (6‐HNA and 5‐chloro‐6‐HNA) via a 2‐step mechanism, where deprotonation of the pyrindolic group of the substrate governs the second step. Molecules that bind and increase the rate of NADH oxidation without turning over into products are called effectors. Thus, effectors share a common molecular characteristic that prevents them from undergoing the coupled hydroxylative pathway in NicC. Through this we sought to determine the 6‐HNA analogues that act as effectors of NicC in attempt to determine how NicC distinguishes between substrates and effectors. 6‐Chloro‐nicotinic acid, 6‐amino‐nicotinic acid and 6‐methyl nicotinic acid were found to be effectors in NicC (Fig 1). These three molecules are not likely to be ionized by deprotonation, signifying that deprotonation of the substrate could govern the coupled and uncoupled hydroxylation pathways in NicC (Scheme 1). Thus, NicC’s mechanism of controlling NADH reduction of FAD appears to be distinct from the mechanism utilized by p‐hydroxybenzoic acid hydroxylase, the quintessential group A flavin dependent monooxygenase.
Use of a Short, In‐Class, Free Response Activity to Assess Student Understanding of Core Concepts of the Cell Membrane in an Undergraduate Physiology Course
Authors: Walton Kristen L. W.
Published: May 2022

Students in an upper division, stand‐alone animal physiology course at Missouri Western State University are required to have completed a freshman‐level introductory cell biology course and a sophomore‐level genetics course as prerequisites. These prerequisite courses include discussion of the eukaryotic cell membrane and basic properties and functions of the membrane. However, many students remember the basic information about membrane structure but have difficulty relating that structure to membrane function and related topics in our physiology course, including vesicular transport, primary and secondary active transport, osmosis, and current flow across the membrane. To gain a better understanding of what students recall about the cell membrane before covering membrane structure and function in class, students were given an open‐ended prompt, “What do you know about the structure and function of the animal cell membrane?” The students were allowed to work in small groups or alone, and could generate their response as drawings, written statements, or both. They were given 10 minutes to construct a response. These student responses were analyzed using the conceptual framework for the “cell membrane” core concept recently published by Michael and Modell (Adv Physiol Educ 2019). This activity was repeated 2 weeks after finishing discussing of the cell membrane. Prior to covering this content in class, 100% (19 out of 19) submissions included a drawing or description of the cell membrane as a phospholipid bilayer, and 84% of submissions included integral and peripheral membrane proteins. Similar percentages of students included these concepts in the post‐coverage assessment. However, other components of the “cell membrane” conceptual framework were included much less frequently or not at all prior to covering the content in class, but improved dramatically afterwards. In the pre‐assessment, only 11% of responses included solute concentration gradients or selective permeability, while 70% of responses in the post‐assessment included this concepts. In the pre‐assessment less than 10% mentioned vesicular transport, receptors, or channel proteins; after covering the content in class, these topics were included by 15%, 45%, and 35% of student responses, respectively. This quick activity provides valuable information about what students recall from prior coursework and what concepts need to be revisited before moving into more detailed discussion of cell membrane function. When used in addition to objective exam or assignment questions, this free‐response activity can also provide a complementary view of student understanding of core concepts of the cell membrane.
Emerging from the Pandemic: Q‐Method Analysis of the Return to Normalcy in Undergraduate Anatomy
Authors: Metla Sai Gayathri , Akhtar‐Danesh Noori , Saini Jessica , Bayer Ilana , Wainman Bruce C.
Published: May 2022

Introduction

As a result of the Covid‐19 pandemic, anatomy education has had to adopt online modes of delivery. Previous research conducted on student views towards an online undergraduate anatomy and physiology course revealed areas of both strong preference (e.g., asynchronous lectures) and strong dislike (e.g., virtual specimens) within the online format. As restrictions are lessened and students transition back into in‐person learning, there exists the unique opportunity to examine the views of a consistent cohort of students towards virtual and in‐person modes of course delivery.

Objective

The current study seeks to compare the views of a single cohort of students experiencing both the online and in‐person anatomy and physiology course. To avoid the considerable drawbacks of course evaluations relying on Likert scales and traditional qualitative analysis, Q‐methodology is being used to measure the different perspectives of students between the two teaching modalities.

Method

Q‐methodology will be used to assess opinions of students enrolled in the same undergraduate introductory anatomy and physiology course.A list of 41 opinion‐based statements regarding anatomy education was compiled. Students will sort the statements in a quasi‐normal grid based on their degree of agreement with the statements. The rankings will then undergo a by‐person factor analysis which categorizes students with shared perceptions into groups allowing educators to better understand and respond to the needs of students. Students will have the opportunity to respond to the survey at two points: at the end of the Fall 2021 semester after a primarily online mode of delivery, and at the end of the Winter 2022 semester following a primarily in person mode of delivery.

Results

Data collection and analysis are anticipated to be completed in March of 2022.

Conclusion

We hypothesize that Q‐methodology will enable us to discover student views specifically relevant to their experience in both the online and in‐person format of the course. We predict that this study will help identify areas of strength in both virtual and in‐person learning environments. These findings will be useful in integrating the strengths of both learning environments when developing future course offerings.
Epac1 Regulates Cellular SUMOylation by Promoting The Formation of SUMO‐activating Nuclear Condensates
Authors: Yang Wenli , Robichaux William G. , Mei Fang , Lin Wei
Published: May 2022

Protein SUMOylation plays an essential role in maintaining cellular homeostasis when cells are under stress. However, how SUMOylation is regulated, and a molecular mechanism linking cellular stress to SUMOylation remains elusive. We hypothesized that cAMP, a major stress‐response second messenger, acts through exchange protein directly activated by cAMP (Epac1) to regulate cellular SUMOylation. Proteomic analysis reveals that Epac1 associated proteome is highly‐enriched with major components of the SUMOylation machinery and well‐known SUMO target proteins. Activation of Epac1 by intracellular cAMP in Human Umbilical Vein Endothelial Cells (HUVECs) promotes cellular SUMOylation and activates cellular SUMO‐activating enzyme E1 (AOS1/UBA2). Conversely, silencing Epac1 leads to a reduced cellular SUMOylation and SUMO E1 activation. However, in vitro SUMOylation assay using purified recombinant components of SUMOylation machinery shows that Epac1 does not directly activate SUMO E1 and SUMO‐conjugating enzyme E2 (UBC9). Moreover, Epac1‐mediated cellular SUMOylation is independent on its guanine nucleotide exchange activity, suggesting that Epac1 promotes cellular SUMOylation through an unconventional mechanism. Co‐immunofluorescence staining and Structured Illumination Microscopy (SIM) super‐resolution imaging analyses reveal that Epac1 activation promotes the formation of Epac1 nuclear condensates that co‐localize with nuclear UBA2/UBC9/SUMO2/3. The discovery of Epac1‐based biomolecular condensates is consistent with the facts that Epac1 contains intrinsically disordered regions (IDRs) and undergoes salt‐ and cAMP‐dependent phase separation (PS). Importantly, the formation of Epac1 nuclear condensates is required for Epac1‐mediated cellular SUMOylation, as a Δ (1‐148) Epac1 mutant incapable of PS and forming nuclear condensates fails to promote cellular SUMOylation. Furthermore, genetic knockout of Epac1 obliterates oxidized low‐density lipoprotein (oxLDL) induced cellular SUMOylation in macrophages, leading to suppression of foam cell formation. Our findings represent a major conceptual advance in our understanding of cell stress responses by providing a direct connection between protein SUMOylation and cAMP/Epac1 signaling pathway, two major cellular stress processes. The ability of cAMP to directly modulate the dynamics of cellular condensate provides the first experimental evidence that protein phase separation can be regulated by an endogenous ligand.
Upper Airway Dysfunction and Discoordination are Reversed by Isometric Tongue Exercise in a Novel Rodent Model of Hypoglossal Motor Neuron Loss
Authors: Nichols Nicole L. , Murphy Erika R. , Osman Kate , Smith Catherine L. , Thompson Rebecca , Kloepper Ashley , Haxton Chandler , Warncke Kristen , Lee Li , Ma Lixin , Lever Teresa E.
Published: May 2022

Motor neuron diseases (e.g., amyotrophic lateral sclerosis, pseudobulbar palsy, and Kennedy’s disease) result in life‐threatening alterations in upper airway function (i.e., swallowing and breathing) primarily due to degeneration of hypoglossal (XII) motor neurons, which leads to ventilator and/or feeding‐tube dependence. Despite its critical importance, upper airway function has seldom been studied in motor neuron diseases; thus, effective treatments remain to be discovered. Since genetic rodent models of motor neuron loss develop global symptoms (e.g., limb dysfunction, etc.), we have developed an inducible model of only XII motor neuron death in order to study targeted therapeutic interventions to enhance the functional capacity of spared XII motor neurons to improve functional outcomes. We have previously found that adult male rats intralingually injected with cholera toxin B conjugated to saporin (CTB‐SAP) have XII motor neuron loss and decreased XII motor output, degenerative changes in the XII nerve and genioglossus, and decreased tongue motility and swallowing rate. We hypothesize that deficits in upper airway function and coordination are reversed by using a translational, non‐invasive therapeutic strategy of tongue resistance exercise training in CTB‐SAP rats. To test our hypothesis, we intralingually injected adult male rats with CTB‐SAP or control (CTB unconjugated to SAP), and then studied the following parameters +/‐ isometric tongue exercise (n=3‐7/group): 1) swallowing function via videofluoroscopic swallow studies and force lickometer testing; 2) breathing function via whole‐body plethysmography; 3) XII motor output at baseline and in response to a maximal ventilatory challenge in anesthetized and ventilated rats via in vivo neurophysiology; 4) coordination of swallowing and breathing in anesthetized rats via evoked swallowing electrophysiological recordings; and 5) structural changes via magnetic resonance imaging. Thus far, our data suggests that tongue exercise in CTB‐SAP treated rats remarkably preserves tongue strength and motility and swallowing and breathing patterns/coordination, and reduces structural degeneration in the brainstem and tongue (p<0.05 vs. controls). In conclusion, tongue exercise appears to preserve upper airway function and coordination in the face of XII motor neuron degeneration. Studies are now underway to understand the underlying mechanism of tongue exercise‐induced neuroplasticity in spared XII motor neurons. If successful, this work will identify behavioral (tongue exercise) and molecular strategies for future translational studies to prolong and/or improve upper airway function and coordination in patients with motor neuron diseases to significantly improve their quality and duration of life.
Defining the role of RNA acetylation via a sequence‐specific RNA acetyltransferase
Authors: Nance Samantha R‐A , Meier Jordan L. , Chari Raj , Evans Christine N.
Published: May 2022

RNA modifications play a critical role in protein abundance, biology, and disease. However, methods for testing the causal effects of individual RNA modifications remain in their infancy. Here we report the preliminary development of an epitranscriptome engineering strategy to manipulate RNA cytidine acetylation in living cells. Our approach fuses a eukaryotic cytidine acetyltransferase to a sequence‐specific RNA‐binding protein. First, we optimize the ectopic expression of these constructs in human embryonic kidney cells. Next, we develop and apply assays to assess guide‐dependent and independent acetylation of a model substrate. Finally, we demonstrate the dependence of modification on consensus sequence, guide/target proximity, and linker protein fusion geometry. These studies provide a foundation for site‐specific rescue of individual cytidine acetylation events in model organisms, and in so doing will help illuminate the function of RNA modifications in disease.
Effect of Maximal and Submaximal Muscle Activation on Corticospinal Excitability in Humans
Authors: Paish Alexander D. , Zero Alexander M. , Rice Charles L.
Published: May 2022

Introduction

Following short duration (5‐10s), high intensity (≥70%MVC) muscle activation, there is an enhancement of muscle contractile properties, termed post action potentiation (PAP). During PAP induced by voluntary or electrically stimulated conditioning contractions (CC) it has been reported that the corticospinal silent period (SP), assessed by transcranial magnetic stimulation (TMS), is elongated. Although a coexistence between the two occurs, the direct effect of PAP on corticospinal excitability has not been systematically evaluated to help determine whether a direct link exists. The purpose was to assess SP duration following potentiating and non‐potentiating voluntary and electrically stimulated contractions. We hypothesized that maximal and submaximal CCs inducing PAP and those not inducing PAP enhancement, respectively, will prolong SP durations albeit to a lesser degree at submaximal intensities.

Methods

Ten healthy young (three females; 18‐35y) individuals free of neurological issues to date have participated in the study. Subjects were seated with their right forearm pronated and their hand was fixed to a custom finger abduction force dynamometer. Monopolar surface electromyography signals were recorded from the first dorsal interosseous muscle (FDI) with self‐adhering Ag/AgCl electrodes. Peripheral stimulated twitches and tetani (50Hz) were evoked over the ulnar nerve at the wrist via a pair of small readjustable electrodes. Maximal M‐waves (M‐max) and baseline levels of (voluntary and stimulated) PAP and force were obtained for each subject. Electrically stimulated and voluntarily induced CCs of 10s were performed at ~95% MVC and ~35% MVC for a total of four contraction conditions. Before and after these contractions SP times were obtained during the plateau of ~25% MVC held for three seconds. To obtain SP times the TMS output was set to elicit a response ~75% of M‐max. The four conditions of voluntary or stimulated, maximal and submaximal CCs were pseudo‐randomized and repeated four times per subject. The nearest three values from each subject were averaged and compared as a normalized percent change from baseline.

Results

Following both maximal voluntary and stimulated CCs, mean twitch torque was enhanced similarly (PAP ~180%) with no PAP at the submaximal levels. Mean change in SP duration following maximal voluntary CCs was ~14% longer than control values, while maximal tetanic CCs similarly prolonged the SP by ~13%. Both voluntary and stimulated submaximal contractions prolonged SP duration similarly by ~5% and ~6%, respectively.

Conclusion

These findings indicate that corticospinal inhibition assessed as SP elongation is present not only when the muscle is enhanced under PAP but also occurs following contractions inducing no PAP. This suggests that the muscle activation required to induce PAP likely causes the observed corticospinal inhibition, not the enhanced muscle properties of PAP per se.
Lack of Xdh Leads to Alterations in Renin‐Angiotensin‐Aldosterone System and Kidney Injury
Authors: Dissanayake Lashodya V. , Zietara Adrian , Levchenko Vladislav , Palygin Oleg , Staruschenko Alexander
Published: May 2022

Uric acid (UA) homeostasis has been elucidated as a key player in chronic kidney disease and cardiovascular diseases, yet definitive mechanisms are unknown. Previous studies in rodents and humans have shown that increased levels of serum UA predict an activated intrarenal Renin‐Angiotensin‐Aldosterone System (RAAS). In hyperuricemic rats, nephropathy was reported with glomerular hypertrophy and interstitial inflammation accompanied by increased renal renin expression. To explore whether hypouricemia leads to RAAS changes and kidney damage, we utilized a novel rat model with genetic ablation of the Xdh gene in the Dahl salt‐sensitive rat background (SSXdh‐/‐). The Xdh gene encodes for the Xanthine Oxidoreductase enzyme group, which produces UA from xanthine and hypoxanthine. We have shown that the SSXdh‐/‐model is hypouricemic and has severe kidney damage. The SSXdh‐/‐ rats demonstrated significant kidney function decline (SSXdh+/+& SSXdh‐/‐respectively, diuresis: 2.7 ± 0.9 & 14.4 ± 5.1 ml; creatinine clearance: 0.51 ± 0.19 & 0.12 ± 0.04 ml/min; and plasma Na+: 136 ± 1.8 & 150 ± 3.7 mmol/l). Quantification of RAAS components was done using liquid chromatography‐tandem mass spectrometry. The comparison of plasma from SSXdh+/+vs SSXdh‐/‐rats revealed significantly lower levels of angiotensin I (650 ± 109, 178 ± 25 pmol/l), Ang II (583 ± 70, 171 ± 16 pmol/l), and renin (indirect measurements using Ang I + Ang II: 1233 ± 172, 349 ± 40 pmol/l). Aldosterone level was increased in SSXdh‐/‐rats (870 ± 115 vs. 4106 ± 1038 pmol/l). The aldosterone/ Ang II ratio was significantly increased in the homozygous rats suggesting enhanced adrenal function (2 ± 0.1, 25 ± 6.3). To further evaluate the contribution of RAAS and its downstream signaling mechanisms, we performed RNA‐Seq analysis on kidney cortex tissue, which showed a number of RAAS‐related genes being differentially expressed between the SSXdh+/+and SSXdh‐/‐ rats. In hypouricemic rats, there was an increase in expression levels of Ace, Ccl2, Fos, Map3k1, Ptger2,and Stat3,and a decrease of Agtr1a, Hsd11b1,and Renb. Therefore, our results show that lack of Xdh/UA leads to kidney injury and functional decline with substantial remodeling in RAAS.
Exposure to the Brominated Flame Retardant Hexabromocyclododecane Results in Systemic Changes in Polyunsaturated Fatty Acid Metabolism Within Phospholipids of Mice
Authors: Kramer Naomi E. , Barnett Lillie , Xu Hannah , Guo Tai , Wagner John , Cummings Brian
Published: May 2022

Brominated flame retardants are used in many household products to reduce flammability, but often leach into the surrounding environment over time. Hexabromocyclododecane (HBCD) is one brominated flame retardant detected in human blood across the world. HBCD exposure can result in neurological problems and altered lipid metabolism, but to date the two remain unlinked. As lipids constitute ~50% of brain dry weight, lipid metabolism plays a critical role in neuronal function and homeostasis. To determine the effect of HBCD exposure on brain lipid metabolism, young adult male mice were exposed to 1 mg/kg HBCD every 3 days for 28 days. HBCD exposure altered lipid abundance of 8 major lipid classes in the dorsal striatum, including fatty acids (FA), lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE), phosphatidylcholines (PC), phosphatidylethanolamines (PE) alkylphosphatidylethanolamines (PEO), phosphatidylethanolamine plasmalogens (PEP), and sphingomyelins (SM). The liver, as a major lipid metabolism hub, and blood were also examined to determine whether these alterations correlated to systemic changes in lipid metabolism. Although several lipid classes changed in the liver and blood, only PE was consistently altered between organs. Despite the lack of commonalities between lipid class alterations of differing organs, phospholipid fatty acid tails displayed a consistent enrichment of Polyunsaturated Fatty Acids (PUFAs) such as Arachidonic Acid (AA) and Docosahexaenoic Acid (DHA) across lipid classes and organs. Free AA was enriched in the blood as well as AA contained within phospholipids of the liver (in PE), blood (in LPC, PEO, and PEP), and dorsal striatum (in PE, PEO). Additionally, although free DHA was unchanged in the blood, DHA‐containing phospholipids were enriched in the liver (in PE), blood (in LPC, LPE), and dorsal striatum (in PE, PEO, PEP, PS). These studies indicate that HBCD can cause systemic alterations in PUFA metabolism. Further study is necessary to determine the mechanisms underlying the lipid alterations and their functional consequences.
A new therapeutic target for chronic pain: GPR171 agonism reduces chronic neuropathic and inflammatory pain
Authors: Ram Akila , Edwards Taylor M. , McCarty Ashley , Afrose Leela , McDermott Max , Bobeck Erin N.
Published: May 2022

Chronic pain is a debilitating condition leading to impaired quality of life but has few efficacious therapeutic options. The use of opioids to treat chronic pain is associated with well‐known drawbacks including drug dependence and addiction. The development of opioid alternatives to treat chronic pain is an urgent, yet unmet, need. Here we show the therapeutic potential of GPR171, a G‐Protein Coupled Receptor that was recently identified to regulate morphine antinociception. Previously shown that systemic administration of the GPR171 agonist, MS15203, enhances morphine antinociception in acute pain tests in mice. Here, we show that once‐daily systemic administration of MS15203 (10 mg/kg, i.p.) over the course of 1 week improved chronic pain outcomes in two different mouse pain models. Chemotherapy‐induced peripheral neuropathy (CIPN) was induced by injecting Paclitaxel (16 mg/kg cumulative) and inflammatory pain was induced by injecting Complete Freund’s Adjuvant (CFA) under the epidermis into the plantar surface of both hind paws (20 µl/paw). MS15203 improved mechanical allodynia caused by CIPN and reduced thermal hypersensitivity caused by CFA‐induced inflammatory pain. The reduction of chronic pain duration was observed only in male mice, but not in female mice, indicating a sex‐dependent effect at this particular dose of GPR171 agonist. Analysis of GPR171 protein levels in the midbrain Periaqueductal Gray showed that neuropathic pain decreased GPR171 receptor levels in males, but treatment by MS15203 rescued this decrease. Taken together, our findings show that GPR171 activity exhibits sexual dimorphism and can regulate multiple modalities of chronic pain.