bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantification of Learning Gains in a Science CURE: Leveraging Learning Objectives to Substantiate and Validate the Benefits of Experiential Education
Authors: Keating Rachel , Saco Vertiz Luzcarime , Manly Victor , Sastry Akshata , De Santanu , Sikora Arthur K.
Published: May 2022

Course‐based Undergraduate Research Experiences (CURE) aim to provide college students with hands‐on experiences to develop research skills and complex thinking. Development of these abilities is invaluable as students undertake further education and begin critical professions. Regardless of eventual career aspirations, CUREs make it possible for every student to experience the enchantment of science in its authentic form. Performing research serves to galvanize inquiring minds that serve as role models for future generations of scientists. Science, Technology, Engineering and Math (STEM) disciplines as a whole suffer from high rates of student attrition. Notably, students from underrepresented groups enter STEM disciplines at similar rates and with similar interest levels as White students, but do not persist at the same rates. CUREs have proven to be an indispensable model to expose every student, regardless of race, socioeconomic status or family obligations, to the process of science first hand. While many CURE curricula are freely available online, faculty are hesitant to adopt this new style of teaching. Using established anticipated learning outcomes (ALO) designed specifically for the Biochemistry Authentic Student Inquiry Laboratory (BASIL) CURE, this study aims to gauge student learning objective mastery quantitatively. Likert scale‐based analysis is employed to evaluate the level of content mastery student responses demonstrate. Assignment questions were designed to correspond to the most critical learning objectives, as identified by BASIL faculty. Researchers spent significant effort designing and refining laboratory assignment questions that correspond to and can reliability and consistently to gage mastery of the ALOs.

Data of student mastery spanning 4 semesters will be discussed. Students showed significant learning gains in all four semesters/modalities. Greater mastery of bioinformatic ALOs during remote learning shows quality education can be enhanced and improved by technology. The student’s mastery of wet‐lab ALOs coincided with our findings that lab courses need enhanced strategies to teach critical STEM lab‐research skills in an online setting. Novel assessment and instruction strategies targeted to learning mastery gaps identified as part of this work will pave the way for wider CURE adoption. Ultimately, serving as a platform to expose every undergraduate student to vital STEM research experiences.
Proper cell‐to‐cell communication relies on signal coordination, and signal coordination relies on transcription fidelity
Authors: Zhang Shu
Published: May 2022

Cryptic transcription is the process by which transcripts are initiated at intragenic promoters. Emerging studies indicate that transcription fidelity relies on the suppression of cryptic transcription, and this process is fine tuned in concert with signaling proteins and transcription regulators. The yeast gene STE11 is particularly important in dictating signal coordination and is well known to undergo cryptic transcription. The product of this gene, Ste11, is a protein kinase that is upstream of multiple MAP kinases, including one that promotes mating (Fus3) and another needed for adaptation to osmotic stress (Hog1). Both pathways are conserved in humans, but yeast has advantages that include unequalled genetic tractability.

Our hypothesis is that cells exposed to different and simultaneous external stimuli undergo adaptations that include cryptic transcription. In particular, we propose that yeast exposed to mating pheromone and osmotic stress prioritize the osmosensing pathway. We propose further that Ste11 coordinates both mating and osmosensing pathways by 1) cryptic transcription and expression of the gene and 2) phosphorylation, activation or inactivation of the MAPKs Fus3 and Hog1. We directly measured Ste11 cryptic transcripts and protein products after stimulation of both pathways. We also monitored phosphorylation and transcriptional outputs of the MAPKs in yeast strains that increase or decrease cryptic transcription of STE11, and upon stimulation of one or both pathways.

Our data indicate that cryptic Ste11 is promoted when the mating pathway is active. Furthermore, cryptic transcription elevated the osmosensing pathway and prolonged Hog1 activity. Our data indicate that cryptic transcription regulates MAPK signaling and suggest that cells use cryptic transcription as one of the strategies to coordinate responses to multiple stimuli.
Infection During Pregnancy Downregulates the Expression of Amino Acid Transporters in Rat and Human Placentas
Authors: McColl Eliza R. , Piquette‐Miller Micheline
Published: May 2022

Maternal immune activation (MIA) resulting from infection during pregnancy increases the chance of offspring having a neurodevelopmental disorder such as autism or schizophrenia. However, the mechanism underlying this association is unknown. Placental transport of amino acids from maternal to fetal circulation is essential for proper fetal neurodevelopment. We hypothesized that MIA could alter the expression of placental amino acid transporters, thereby altering fetal exposure to the amino acids required for proper brain development. Thus, the objective of this study was to examine whether MIA impacts the expression of amino acid transporters in the placentas of rats and humans. MIA was induced in pregnant rats through the administration of the viral mimetic poly(I:C) on gestational day 14. Placental and fetal tissues were collected 24 and 48 hours later. Human placentas isolated at term from pregnancies complicated with either chorioamnionitis (n = 17) or an unidentified active infection causing flu‐like symptoms (n = 6) were compared to healthy gestational age‐matched controls (n = 18). Protein expression of amino acid transporters in rat or human placentas, as well as fetal rat brains, was determined using immunoblotting. Levels of free amino acids in fetal rat brains were also quantified using reverse‐phase HPLC. In rats, poly(I:C) significantly downregulated the amino acid transporters ASCT1 and EAAT2 in the placenta, with a trend towards decreased SNAT2. SNAT5, EAAT1, and GLYT1 were also significantly reduced in fetal brains from poly(I:C)‐treated dams. Furthermore, the levels of multiple amino acids were significantly altered in fetal brains of poly(I:C)‐treated rats. In human placentas, ASCT1 was significantly downregulated in active infection cases, but not in chorioamnionitis cases. A pronounced trend of decreased SNAT2 expression was also seen in active infection cases, but this did not reach significance. To conclude, MIA decreases the expression of amino acid transporters in the placentas of rats and is associated with altered levels of free amino acids in fetal rat brains. Moreover, decreased expression of amino acid transporters is also seen in human placentas obtained from pregnancies with active infection. As amino acids are required for proper neurodevelopment, these changes in transport could provide a link between MIA and neurodevelopmental disorders.
Synergy of Nrf2‐Activating Electrophiles in Combination with the Nrf2‐Inhibitor KI696: A Model of Mechanistic Basis for Synergy
Authors: Allender Amanda , Eggler Aimee
Published: May 2022

Oxidative stress, which contributes to many chronic diseases such as diabetes and cancer, can be mitigated through the production of antioxidant proteins, controlled by the Nrf2 transcription factor. Combination drug treatments in disease therapy hold promise for their increased potency and therapeutic window as compared to single drug treatments. In this work, we tested combinations of three clinically relevant Nrf2 activating electrophiles (bardoxolone methyl, Tecfidera, and sulforaphane) with the Nrf2 inhibitor KI696 to see if there was a more than additive effect on ARE expression. Under basal conditions, Nrf2 is bound by Keap1 in the cytoplasm of the cell, and targeted for ubiquitination and degradation. When Keap1 C151 is modified by these electrophiles, Nrf2 escapes repression and localizes to the nucleus to bind to cognate antioxidant response element (ARE) sequences, thereby upregulating expression of antioxidant genes. KI696 binds to the Nrf2‐binding site on Keap1, allowing for accumulation of Nrf2 and subsequent ARE‐driven expression. Through an ARE‐reporter luminescence assay, we show that these three electrophiles each synergistically upregulate ARE‐driven expression with KI696 in a statistically significant manner, based on our Dose Equivalence/Zero Interaction (DE/ZI) method (Repash et al., Frontiers in Pharmacology, 2021). The combination of these clinically‐relevant electrophiles and KI696 are a model for mechanistic investigations of synergy and identification of targets other than Keap1 cysteines.
Acute Fasting Decreases Tolerance to Lower Body Negative Pressure
Authors: Gonzalez Joshua E. , Cooke William H.
Published: May 2022

Potential health benefits of an acute fast include reductions in blood pressure and increases in vagal cardiac control. These purported health benefits could put fasted humans at risk for cardiovascular collapse when exposed to central hypovolemia. Intense lower body negative pressure (LBNP) induces reproducible and reversible central hypovolemia. The purpose of this study was to investigate the influence of an acute 24‐hour fast on tolerance to severe LBNP. We tested the hypothesis that fasting would reduce tolerance to severe LBNP. Ours was a randomized, crossover design with an LBNP tolerance test occurring 3‐hrs postprandial (fed) and 24‐hrs postprandial (fast). A standardized meal was provided for both conditions. Criteria for termination of LBNP was; 1) a drop in systolic pressure of 15 mmHg or greater for 10 s and/or 2) sudden bradycardia. Eighteen young (23±0.7 yrs), normotensive, non‐obese (25±0.8 BMI), participants were brought to presyncope. We measured blood glucose (GLU), ketones (β‐OHB), and triglycerides (TRG) to confirm a successful fast. With subjects in a supine position, we recorded the ECG, beat‐to‐beat arterial pressure (finger plethysmography), muscle sympathetic nerve activity (MSNA; N=7), and forearm blood flow via venous occlusion plethysmography (VOP; N=12). Following a 5‐min baseline, LBNP was increased in 15 mmHg increments every 5 min in a stepwise manner until onset of presyncope. LBNP levels eliciting presyncope were denoted as 100% tolerance, data were assessed relative to this normalized maximal tolerance by expressing LBNP as 80, 40, and 0% (baseline) of maximal tolerance. Data are expressed as means ± SE. P‐values ≤ 0.05 were considered statistically significant and indicated by an (*). Changes in TRG, GLU and β‐OHB with fasting were consistent with expectations: TRG and GLU decreased (TRG; 130±16 fed vs. 78±9* mg/dL fast) (GLU; 100±3 fed vs. 81±2* mg/dL fast), and β‐OHB increased (β‐OHB; 0.12±.04 fed vs. 0.47±.11* mmol/L fast). Our participants were similarly hydrated, measured via urine specific gravity, (1.0168±.002 fed vs. 1.0122±.003 a.u. fast) and weight stable (77.8±3.6 fed vs. 76.9±3.4 kg fast) between conditions. Tolerance to central hypovolemia was decreased by ~10% in the fasted condition measured via total duration of negative pressure (1370±89 fed vs. 1229±94* s fast). At each normalized time point, heart rate and MSNA increased similarly in both conditions. Additionally, systolic blood pressure and stroke volume decreased similarly in both conditions. Both forearm blood flow (FBF) and forearm vascular resistance (FVR) are expressed as a percent change from 0 to 100%. ΔFBF significantly decreased in the fed condition at 100% (‐45.8±4.8 fed vs. ‐23.5±8.1* % fast) and ΔFVR increased in the fed condition at 100% (141.6±34.5 fed vs. 67.1±24.8* % fast). Our results suggest that acute fasting reduces tolerance to central hypovolemia by blunting increases in peripheral vascular resistance, and could lead to earlier decompensation when compared to the normal, fed state.
Brain Structural and Functional Connectivity in Collegiate Endurance Athletes
Authors: Tarumi Takashi , Fukuie Marina , Ohyama‐Byun Keigo , Maeda Seiji , Sugawara Jun
Published: May 2022

High‐intensity endurance training elicits profound cardiopulmonary adaptations, but its impact on the central nervous system, particularly the brain neuronal connectivity, remains unclear.

PURPOSE

To compare the structural and functional brain connectivity between endurance athletes and age‐ and sex‐matched sedentary adults.

METHODS

Fifteen long‐distance male runners (20±1 years) in the university track‐and‐field team and 16 sedentary male students (21±2 years) underwent brain imaging with a 3‐tesla MRI system. Structural brain connectivity was quantified by fractional anisotropy (FA) calculated from diffusion tensor images and analyzed by tract‐of‐interest (TOI) and whole‐brain voxelwise statistics. Functional brain connectivity was assessed by seed‐based network analysis of the resting‐state functional MRI data. Brain volume and cortical thickness were also assessed by T1‐weighted images.

RESULTS

Endurance athletes had the maximal oxygen uptake of 69.5±3.1 ml/kg/min that is higher than 90 percentiles based on the American College of Sports Medicine guideline. TOI analysis exhibited that FA in the anterior thalamic radiation was significantly higher in the athlete group than the sedentary group (0.421±0.019 vs. 0.397±0.025, p=0.007). Furthermore, voxelwise analysis showed that athletes had a significant elevation of FA across the white matter, including the anterior portions of the corpus callosum, internal capsule, and corona radiata. Within the default mode network, athletes had stronger functional connectivity between the posterior cingulate and the right frontal pole than sedentary participants (p=0.0419 with false discovery rate correction). Regional brain volume and cortical thickness were similar between the athlete and sedentary groups (p>0.05).

CONCLUSION

Our findings suggest that high‐intensity endurance training is associated greater brain structural and functional connectivity in young men.
The design and development of cardio‐physiology domino as an interactive media to improve student’s learning achievements
Authors: Irfannuddin Muhammad , Zulissetiana Eka Febri , Santoso Budi , Susilawati Susilawati , Alexander Mailan , Aminuddin Muhammad
Published: May 2022

Physiology is a complex course that summarizes various aspects of basic medical science. Physiology is integrative, and requires logical causal thinking. Therefore, the innovation of interactive learning methods centered on learners is needed. An interactive physiology domino cards has been developed. The inventors focus on the topics of the heart system.

As inspired by dominoes, a set of 28 cardio‐physiology cards (CPC) contains 56 statements has been developed. The development was conducted through 16 steps of 4D model. Stands of define, design, development, and dissemination provided by Thiagarajan (1974). Physical prototype has been ready and has been tested to measure knowledge and practically level among medical students. Students were divided into experiments and controls. The experiment group (n=32) was conducting learning through dominoes in addition to lectures. The control group (n=31) was following learning through lectures and open questions independent tasks.

The MCQ‐test showed that the domino group had a better level of knowledge than the controls (p<0.001). The average n‐gain of the domino group was 68.3±7.1, higher than the control group of 54.4±6.7. The Likert’s scale of practicality feedback from experiment group gave a score between near to firmly agree (4.5±0.6).

Cardio‐physiology domino was effective to improve student outcomes. The domino also practical as a fun media of learning. Currently, the prototype is being developed in the form of a web‐based computer program with a JavaScript programming language supported by HTML and CSS.
Genetic Deletion of the Prostaglandin EP4 Receptor in the Kidney Tubule of Adult Male Mice Has Mild Impact on Kidney Water Handling
Authors: Esteva‐Font Cristina , Geurts Frank , Hoorn Ewout J. , Fenton Robert A.
Published: May 2022

Prostaglandin E2 (PGE2) is an essential lipid mediator modulating several aspects of renal function. PGE2 acts via four PGE2 receptors, EP1‐EP4. EP4 is abundant in the kidney glomerulus, where it plays a role in renal hemodynamics. EP4 is also expressed in the collecting duct, and constitutive deletion of EP4 from this segment in mice suggests it plays a role in urine concentration. To assess the tubular role of EP4 in adult mice we generated a mouse model with doxycycline‐dependent deletion of EP4 along the renal tubule (Pax8Cre system) and assessed their renal phenotype regarding water handling. Two weeks after treatment with doxycycline, EP4 mRNA expression was reduced by >80% in the renal medulla (RT‐q‐PCR) of knockout mice compared to control mice. Under basal conditions, there were no significant differences between genotypes in food and water intake, bodyweight or plasma biochemistries. EP4 knockout males showed a small increase in basal urinary output and a decreased osmolality, concomitant with lower urinary Na+, K+, Cl‐, urea and creatinine concentrations. There were no differences between genotypes in their renal handling of water during water deprivation for 14 hours, or in their response to the vasopressin V2 receptor agonist dDAVP. In a water overloading test only the EP4 knockout females showed an increased urinary output after 1 hour of the water overload. In conclusion, EP4 in the renal tubule has a mild role in renal water handling. This new model provides a novel tool for assessing the role of EP4 in other aspects of kidney function or disease independently of potential developmental abnormalities or systemic effects.
Opioid inhibition of projection‐specific, respiratory‐related pontomedullary neurons
Authors: Bateman Jordan T. , Levitt Erica S.
Published: May 2022

Opioids depress breathing by inhibition of inter‐connected respiratory nuclei in the pons and medulla. We have previously shown that mu opioid receptor (MOR) agonists hyperpolarize a population of neurons in the dorsolateral pons due to activation of G protein‐mediated inwardly rectifying potassium conductance. However, we do not know the projection target of MOR‐expressing, dorsolateral pontine neurons, if they project at all. Here, we show that MOR‐expressing, dorsolateral pontine neurons project to core respiratory nuclei in the ventrolateral medulla, identifying projection‐related heterogeneity of dorsolateral pontine neurons vulnerable to opioid inhibition. First, retrograde DIO‐eGFP injections into the ventrolateral medulla of oprm1‐cre mice showed that dorsolateral pontine neurons project to the Bötzinger complex (BötC), pre‐Bötzinger complex (preBötC), and rostral ventral respiratory group (rVRG), and are distinct from calcitonin gene‐related peptide (CGRP)‐expressing parabrachial neurons. Second, we performed whole‐cell, voltage‐clamp recordings from dorsolateral pontine Kölliker‐Fuse neurons that were retrogradely labeled by fluorescent bead injections into the ventrolateral medulla of wild‐type mice. MOR‐mediated outward currents were identified in dorsolateral pontine neurons that project to the BötC, preBötC, and rVRG, confirming that these projection neurons are functionally inhibited by MORs and vulnerable to opioid inhibition. Together, these approaches demonstrate that MOR‐expressing, dorsolateral pontine neurons project to medullary respiratory nuclei and further unravel mechanisms of opioid‐induced respiratory depression.
Dopamine Receptor Antagonist‐Induced Elevation of the Compulsion Zone Results in Differential Effects on Cocaine Self‐Administration and Lever‐Pressing Behavior
Authors: Zinani Dakota B. , Norman Andrew B.
Published: May 2022

Dopamine receptor antagonists raise both the cocaine priming and satiety thresholds (the lower and upper limits of the compulsion zone, respectively), so antagonists will decrease the probability of cocaine priming self‐administration behavior. However, if priming occurs in the presence of dopamine receptor antagonists, then cocaine consumption will accelerate. The compulsion zone also explains the lever‐pressing after access to cocaine is terminated, but the effects of antagonists on the compulsion zone are unexplored. The compulsion zone theory predicts that antagonists will decrease the duration of unloading activity. Male rats were trained to self‐administer cocaine on an FR1 schedule. Upon lever‐pressing, a unit dose (3 umol/kg i.v.) was administered. The dopamine receptor antagonists, SCH23390 (10‐30 nmol/kg), eticlopride (10‐30 nmol/kg), or molindone (750‐3000 nmol/kg) were administered i.v. either while cocaine was accessible during the maintenance phase or following termination of access denoting the start of the unloading phase. The lever‐pressing activity was recorded during the unloading phase. Antagonists accelerated self‐administration during the maintenance phase due to the elevated satiety threshold, consistent with previous studies. Antagonists administered immediately prior to the unloading phase shortened the duration of lever‐pressing, and lever‐pressing began at a higher calculated cocaine level. This is consistent with the antagonists elevating the compulsion zone. The faster elimination of cocaine at these elevated levels results in cocaine levels transiting the compulsion zone more rapidly. The differential effect of dopamine receptor antagonists on lever‐pressing behavior may provide insights to the appropriate use of antagonists as medications for cocaine use disorder.
Using molecular modeling to identify potential anti‐tubulin compounds
Authors: Evans Blake A. , Cole Kathryn
Published: May 2022

In 2020 the Center for Disease Control and Prevention reported cancer as the second leading cause of death in the United States with over 598,000 cases. Therefore, the need for new innovative cancer drugs is ever‐growing. Tubulin, a protein crucial to cell dynamics, has been the target for many anti‐cancer drugs since the 1960’s. Most anti‐tubulin compounds to date, however, are complex molecules that have issues with solubility, toxicity, multi‐drug resistance, and low efficacy amongst various cancers. The severity of these problems highlights the need to develop a new generation of anti‐tubulin molecules.

Previously we synthesized a small vicinal diaryl heterocycle, PY‐407‐C, which showed potent antiproliferative activity against U‐937 cancer cells. Further investigations in vitrodetermined the mode of inhibition is by anti‐tubulin polymerization. Analysis of the known binding sites of tubulin led us to hypothesize PY‐407‐C binds in the tubulin colchicine‐binding site due the similarities of having vicinal diaryl groups. Initial molecular modeling studies were then carried out in AutoDock Vina which strongly supported our predictions. Most significantly, PY‐407‐C makes many hydrophobic and van der Waals interactions within a known hydrophobic pocket of the beta‐tubulin subunit congruent to colchicine, such as L242, L248, L252, L255, T314, V315, I318, N350, K352, and A354.

Utilizing these inhibitory docking studies, we hypothesize analogs that theoretically have stronger binding energies than colchicine or PY‐407‐C will also make stronger inhibitors. This allows for an efficient system where synthetic targets are first determined through computational studies, to which they would be synthesized and then tested for biological activity. More specifically, by analyzing interactions between our potential inhibitors and nearby residues, we can make informed decisions on which changes will lead to stronger binding. For example, this methodology led to the design of a new compound for synthesis containing a large hydrophobic propyl‐benzene substituent, which when simulated gave the highest binding affinity of compounds tested thus far.
Mechanistic Insights into Macrophages Regulation of Neutrophil Transendothelial Migration in Inflamed Mucosa
Authors: Ren Xingsheng , Urbanczyk Jessica M. , Sumagin Ronen
Published: May 2022

Rapid neutrophil (PMN) mobilization to sites of insult is critical for host defense and requires crossing of the vascular wall. PMN transendothelial migration (TEM) involves several well‐studied sequential adhesive interactions with vascular ECs, however what initiates or terminates this process is not well‐understood. Our findings identified a new mechanism where gut interstitial macrophages, which are rapidly recruited towards the vascular wall in response to inflammatory cues, were found to locally prime endothelial cells (ECs) responses to regulate PMN TEM. Using real‐time intravital microscopy (IVM) on lipopolysaccharides (LPS)‐inflamed intestines in anesthetized CX3CR1‐EGFP macrophage‐reporter mice, complemented by whole‐mount tissue imaging we demonstrate that macrophage presence was critical for the induction of PMN‐ECs adhesive interactions and subsequent PMN recruitment and accumulation in the intestinal mucosa. Anti CSFR‐1 antibody‐based macrophage depletion in the lamina propria and at the vessel wall significantly reduced PMN adhesion and TEM in inflamed intestines. We further observed that macrophages at the vessel wall localized specifically to regions of high ICAM‐1 intensity and their removal resulted in elimination of the ICAM‐1 “hot spots”, overall lowering the ECs ICAM‐1 expression. Mechanistically, using murine/human ECs‐macrophage and PMN co‐cultures we established that activated macrophages elevate PMN adhesion and TEM via TNFα‐dependent upregulation of ECs ICAM‐1. Antibody‐mediated neutralization of TNFα in macrophage co‐cultures with ECs and/or PMNs suppressed ICAM‐1 upregulation, decreasing PMN TEM. Further in vivo imaging studies of inflamed gut revealed high TNFα expression in macrophages and specific expression of TNFa receptor type II (TNFR II) but not type I in intestinal ECs. The use of bone marrow chimeras with TNFα knockout macrophages further confirmed the novel role of macrophages‐TNFα in regulating ECs adhesion molecule expression and PMN TEM. As such, our findings identify new, clinically relevant mechanism by which macrophages regulate PMN trafficking in inflamed mucosa.
Exaggerated Store‐Operated Calcium Entry: A Mechanism for High Glucose‐induced Podocyte Injury and Mitochondria Damage
Authors: Tao Yu , Chaudhari Sarika , Yazdizadeh Shotorbani Parisa , Ma Rong
Published: May 2022

Purpose and hypothesis

Podocyte injury induced by hyperglycemia is the key factor contributing to proteinuria and kidney dysfunction in diabetic nephropathy (DN). Accumulating evidence suggests that mitochondria dysfunction leads to DN‐associated podocyte injury. However, the mechanism of podocyte mitochondria injury by high glucose (HG) is poorly understood. Store‐operated calcium entry (SOCE) is essential for multiple cell functions in both excitable and non‐excitable cells. However, the role of this Ca2+ signaling in podocytopathy remains unknown. The present study was carried out to test the hypothesis that enhanced SOCE mediated HG‐induced podocyte injury and mitochondria damage.

Methods

All experiments were carried out using cultured immortalized human podocytes. The conventional whole‐cell patch‐clamp was performed to measure Orai1 channel currents. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and STIM1. Calcium imaging was used to evaluate SOCE. Confocal microscopy was used to visualize podocyte actin arrangement. Mitochondrial function was evaluated by measuring: 1) the mitochondria membrane potential (MMP) using TMRE fluorescence; 2) generation of mitochondria reactive oxygen species (ROS) using MitoSox Red Mitochondrial Superoxide Indicator; 3) ATP production using ATP assay kit. In addition, CRISPR‐CAS 9 approach was used to delete Orai1 in podocytes.

Results

Thapsigargin (TG, 1 µM), the activator of Orai1 channel induced robust inward currents which were blocked by La3+ (2 µM), an inhibitor of Orai1 channel. Ang II (1 µM) also evoked inward currents, which were significantly blunted by BTP2 (10 µM), an Orai1 channel blocker. Orai1 protein abundance significantly increased by HG (25 mM) treatment for the time periods ranging from 2 to 12 h. This HG induced Orai1 increase was dose‐dependent. Consistently, Ca2+ imaging experiment showed that HG treatment for 12 h significantly increased podocyte SOCE. Thus, the biochemical and functional data suggested an enhancement of SOCE by HG. Furthermore, HG treatment resulted in podocyte cytoskeleton rearrangement by formation of cortical F‐actin, an indication of podocyte injury. This HG response was significantly blunted by BTP2 and Orai1 CRISPR‐CAS 9 lentivirus. Moreover, HG treatment significantly decreased podocyte MMP, ATP production and increased mitochondrial ROS generation, all of which were significantly inhibited by BTP2. Furthermore, activation of SOCE by TG significantly increased mitochondrial ROS generation, which was prevented by pre‐treatment of BTP2.

Conclusion

Enhanced SOCE contributed to HG induced podocyte injury and mitochondrial dysfunction.
Development and validation of a novel UPLC‐MS/MS method for the simultaneous quantification of mycophenolic mofetil and its major metabolites in rat plasma and liver S9 hydrolysis assay in rats
Authors: Etim Imoh , Du Ting , Sunsong Robin , Siddiqui Nyma , Gao Song
Published: May 2022

A sensitive liquid chromatography‐tandem mass spectrometry method was developed and validated for the simultaneous quantification of mycophenolic mofetil (MMF) and its major metabolites, mycophenolic acid (MPA), mycophenolic acyl glucuronide (AcMPAG), and mycophenolic glucuronide (MPAG), in rat plasma. Upon administration, mycophenolic mofetil (MMF), a prodrug, undergoes extensive metabolism in the liver. Following hydrolyzation, the active metabolite MPA is produced. MPA can then be further metabolized to produce its glucuronide conjugated metabolites, AcMPAG and MPAG. This method can simultaneously determine both the parent compound and its major metabolites within biological samples. In addition, liver S9 fraction hydrolysis studies of MMF results in the production of MPA, demonstrating that the parent drug is first hydrolyzed within the liver. A novel UPLC method was developed to simultaneously quantify both compounds following S9 liver enzyme hydrolysis.

A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Separation was achieved using an Ultra Biphenyl 5µm column (100 × 2.1mm) with acetonitrile and 0.1% formic acid as the mobile phases. Analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. Additionally, to determine the hydrolysis rate, different concentrations of MMF were incubated with liver S9 fraction (0.5µg/ml final concentration) for 30 mins, after which the reaction was terminated using 6% formic acid in acetonitrile. Samples were prepared and MPA concentrations were determined in the reaction system using UPLC for detection and quantification.

The method was linear in the range of 9.77nM – 5000nM with correlation coefficient values > 0.99 for all components. The method has been shown to be reproducible, with intra‐ and inter‐day accuracy and precision ±12.3% of nominal values, for all analytes. The average extraction recovery rates for MMF, MPA, AcMPAG, and MPAG were 91.3%, 97.9%, 98.4%, and 90.5%, respectively. Matrix effect was in the acceptable range (<15%). The analytes in plasma were found to be stable under bench –top, freeze‐thaw, and storage (‐4°C) conditions. The metabolic studies showed that mycophenolic mofetil can be rapidly hydrolyzed by rat liver S9 fraction with Vmax and Km values of 0.66±0.78 nmol/min/µg and 80.06±123.1µM respectively.

This novel UPLC‐MS/MS method can comprehensively evaluate and quantify the concentration of both mycophenolic mofetil and its major metabolites, mycophenolic acid, mycophenolic acyl glucuronide, and mycophenolic glucuronide in biological samples. This method can be used as a tool to further investigate and understand the extensive metabolism of mycophenolic mofetil in clinical practice.
A novel ClpX degron targets the E. coliantitoxin MqsA for degradation by ClpXP
Authors: Vos Margaret R. , Piraino Benjamin , LaBreck Christopher J. , Rahmani Negar , Trebino Catherine E. , Peti Wolfgang , Camberg Jodi L. , Page Rebecca
Published: May 2022

It is well established that the antitoxins of toxin‐antitoxin (TA) systems are selectively degraded by bacterial proteases in response to stress. However, how distinct stressors result in the selective degradation of specific antitoxins remains unanswered. MqsRA is a TA system activated by stress, including oxidation. To understand how, at a molecular level, MqsA is targeted for degradation by bacterial proteases and to advance our understanding of how substrates are recognized by bacterial proteolytic systems, especially ClpXP, we studied the mechanism of MqsA recognition and degradation by the ClpXP protease. Here, we show that MqsA is efficiently degraded by ClpXP in the absence of zinc, and that both zinc and MqsR binding antagonize ClpXP mediated degradation. Further, using biomolecular NMR based chemical shift perturbation experiments, we show that folded, metal‐bound MqsA does not interact with the ClpX N‐domain (a small recognition domain that binds a subset of ClpXP substrates and adaptor proteins), but instead ClpXN interacts only with unfolded, metal‐free MqsA. This led to the identification of a cryptic N‐domain recognition sequence in unfolded MqsA that targets it for ClpXP degradation. Furthermore, we discovered this sequence is transplantable and sufficient to target a fusion protein for in vivo and in vitro degradation. This recognition sequence defines a novel ClpX degron, which differs in sequence from all previously described degrons. Finally, we present a model in which oxidative stress causes rapid cysteine oxidation of nascent, zinc free MqsA, and exposure of the degron for ClpXP mediated degradation.
Location Bias of G Protein‐Coupled Receptor Kinases Promotes Biased Signaling at CXCR3
Authors: Hicks Chloe N. , Eiger Dylan , Gardner Julia , Boldizsar Noelia , Honeycutt Cole , Choi Issac , Rajagopal Sudarshan
Published: May 2022

G protein‐coupled receptors (GPCRs) are the largest family of cell‐surface receptors and are known to canonically signal through two main effector proteins: G proteins and β‐arrestins. Biased agonism at GPCRs refers to the functional selectivity of a ligand towards certain signaling pathways over others. In addition to biased agonists, there are various other sources of bias that influence GPCR signaling, such as receptor and location bias, which affect the downstream pathways of receptor activation, leading to disproportionate levels of signaling between the G protein and β‐arrestin pathway. One of the mechanisms underlying this signaling bias is the phosphorylation barcode hypothesis which states that ligand induced receptor phosphorylation promotes differential engagement with downstream transducers. Across the entire GPCR superfamily, GPCR kinases (GRKs) are known to be primarily responsible for receptor phosphorylation. Of the seven known isoforms in the GRK family, GRK2, GRK3, GRK5, and GRK6 are expressed ubiquitously, whereas GRK1 and GRK7 are mainly found in the eye while GRK4 is predominantly located in the testes. Although there is supportive evidence demonstrating that differential phosphorylation promotes biased signaling, there is relatively little known about the role of GRKs in directing GPCR functional selectivity. To study the role of GRKs in biased signaling, we studied the GPCR CXCR3, a chemokine receptor primarily involved in leukocyte migration, which has three endogenous biased ligands, CXCL9, CXCL10, and CXCL11, and two synthetic biased ligands, VUF10661 and VUF11418. We found that, following ligand stimulation, GRK2 and GRK3 are recruited to CXCR3, with VUF10661 and CXCL11 being the most efficacious ligands. However, we also observed that GRK3 and GRK5 are able to traffic to endosomes following ligand stimulation with VUF10661, even though all ligands are able to promote receptor internalization. Given biased engagement of the GRKs observed at both the receptor and in endosomes, we next studied the functional consequences of ligand and location bias by creating GRK 2, 3, 5 and 6 constructs that are specifically targeted to the plasma membrane or the endosome. Using GRK2/3/5/6 KO cells, we determined that localization of the GRKs impacts receptor internalization, ERK activity, and transcription in a ligand specific manner. Together, we observed that the endogenous and synthetic ligands of CXCR3 demonstrate biased engagement of the GRKs at different subcellular locations and highlight the functional significance of these findings on downstream receptor signaling. These data provide evidence that the functional selectivity of biased signaling extends beyond β‐arrestins and G‐proteins to other effector proteins that interact with GPCRs.
A Biophysical Analysis on the Effect of Ligands on Fluorescently Active G‐quadruplex Structures
Authors: Bryan Chloe , Thomas Leona , Ajmera Riya , Jose Davis
Published: May 2022

The formation of G‐quadruplex (GQ), a non‐canonical nucleic acid secondary structure, can inhibit the elevated telomerase activity that is common in most cancers. The global structure and the thermal stability of the GQs are usually evaluated by spectroscopic methods and thermal denaturation properties. However, most of the biochemical processes involving GQs involve local conformational changes of GQs at the guanine tetrad (G4) level. Previously we developed a method to study the local conformations of individual G4 layers in GQs that uses 6‐methylisoxanthopterine (6MI), a Circular Dichroism (CD)‐active fluorescent base analogue of guanine. Using this method here we explore the local conformational changes of individual G4 layers in GQ‐drug interactions. Experiments were performed with human telomeric 22AG sequence (5′‐AGGGTTAGGGTTAGGGTTAGGG‐3′) where guanines at the positions 3,9,15 and 21 were site‐specifically replaced by 6MI monomers. The CD and fluorescence properties of the GQ structures with and without the ligands were characterized under various conditions. Thermal denaturation studies showed that properly positioned 6MI monomers and dimers in a GQ forming sequence can form stable GQs with CD‐active fluorescent G4 layers. The local conformation of individual fluorescent G4 layers in the GQ structure was then monitored by following the fluorescence intensity and circular dichroism changes of the incorporated probes. The results showed the interaction of ligands with the fluorescent G4 layer varied depending on the functional groups as well as the bulkiness of the ligand suggesting that there are multiple ways of G4‐ligand interaction.
GPCR signaling measurement and drug profiling with an automated live‐cell microscopy system
Authors: Posner Clara , Greenwald Eric , Bharath Ananya , Lyons Anne , Salmerón Cristina , Smitham Jane , Insel Paul A. , Zhang Jin
Published: May 2022

Time‐lapse microscopy combined with genetically encoded fluorescent biosensors is a powerful tool for quantitatively measuring live cell signaling dynamics with high spatial and temporal resolution in single cells, providing rich signaling information beyond population‐based biochemical assays and plate reading measurements. One major limitation, however, is the low experimental throughput. To overcome this limitation, we created a highly customizable MATLAB‐based platform called FALCOscope (Flexible Automated Liquid‐handling Combined Microscope) that coordinates between an OpenTrons liquid handler and a fluorescent microscope to automate drug stimulations, fluorescence imaging and single cell analysis. We demonstrated the feasibility of our approach by quantifying GPCR‐stimulated Protein Kinase A (PKA) activity dynamics and by measuring cAMP dynamics stimulated by a panel of 14 GPCR agonists and antagonists. We then characterized the cAMP dynamics induced by GPR68, a proton sensing GPCR, in response to extracellular pH changes in the presence of its positive allosteric modulator ogerin. We found that GPR68 induced cAMP responses are more transient for acidic pH’s than neutral pH’s, suggesting receptor desensitization at acidic pHs. We further identified that ogerin, in addition to enhancing the cAMP response mostly strongly for pH 7.0, also sustains the cAMP response for acidic pH’s, demonstrating the novel findings captured by live‐cell dynamics. In addition, we show that 100 µM ogerin also has off‐target effects on cAMP/PKA signaling, likely as a phosphodiesterase inhibitor. This FALCOscope system enables single‐cell dynamic measurement in high throughput, providing a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells as well as drug profiling and screening.
3D‐Printing Offers Dry Replication and New Insight into Unique Museum Specimens
Authors: Streicher Johannes , Reisinger Andreas , Warnung Lukas , Pahr Dieter
Published: May 2022

Students dissection classes again and again reveal unique anatomical variations and pathologies, which are usually preserved and stored in fluid. Due to their unique nature and hazardous conservation fluids, they neither ever leave their display case again nor are handed over to students at the dissection lab.

Thus the present study aims at routinely generating solid, dry, and numerically unlimited 3D‐replica of such unique specimens.

We have selected a particularely severe case of Mönckebergs aortic valve sclerosis and a transcatheter aortic valve implantation (TAVI)‐treated valve to develop a standard scanning, segmentation and multicolor 3D‐printing protocol for soft and hard tissue specimens.

Subsequent to anatomical dissection of the appropriate valve segment from the ventricular outlet and the ascending aorta, scanning was performed on a Skyscan 1173 micro‐CT at a x‐y‐z resolution of 30µm. Soft tissue, calcified tissue, and the TAVI's wire cage were segmented separately using the software packages Slicer and Medtool. For 3D‐printing, the Prusa MK3S filament printer and the Prusa SL 1 resin printer were used.

The overall time expense for scanning (40 minutes), data processing and segmentation (about 16 hours) was around two to three working days per model. The 3D‐printing took from 5 hours (single‐color resin printing at a scale of 1:1) up to 4 days (triple‐color filament printing at a scale of 3:1).

The generated display models range from real size, opaque surface models to semi‐vitreous visualizations of the calcification pattern and the implanted TAVI.

Taken together, after a substantial time expenditure for initial data processing and modeling, with small time requirements not only hands on replica for teaching can be easily reproduced at different scales without limitation by specimen durability or chemical hazards. Additional impact is achieved by the ability to highlight and analyze internal structures in situ, making laborious, traditional whole mount staining and ‐clearing dispensable. In a next step, the adaption of colors and material characteristics will approximate the haptic experience to the real specimen.
Respiratory defects caused by mutations affecting the Endoplasmic Reticulum‐Mitochondria Encounter Structure (ERMES) can be rescued by the deletion of COQ11
Authors: Novales Noelle Alexa , Clarke Catherine
Published: May 2022

Coenzyme Q (CoQ) is an essential redox‐active lipid that plays a major role in the electron transport chain, driving mitochondrial ATP synthesis. Deficiency of CoQ causes a wide range of clinical deficiencies, highlighting the need to study the biosynthesis of this lipid to design therapeutics to treat these symptoms. In Saccharomyces cerevisiae, CoQ biosynthesis takes place exclusively in the mitochondrial matrix using a multi‐subunit protein‐lipid complex, the CoQ Synthome, that includes the polypeptides Coq3‐Coq9 and Coq11. A recently identified regulator of CoQ Synthome assembly and CoQ production is the ER‐mitochondria encounter structure (ERMES). ERMES is a tethering complex that bridges the ER and mitochondria, and the CoQ Synthome resides in specific membrane niches or domains directly adjacent to this complex. Loss of ERMES results in transcriptionally upregulated expression of COQ genes, yet inefficient synthesis of CoQ due to a destabilized CoQ Synthome. In this work, ERMESΔcoq11Δ mutants have been generated in an effort to correct this defect. Deletion of COQ11 has been shown to promote mitochondrial CoQ content, enhance CoQ Synthome stability, and rescue the respiratory deficiency of the coq10Δ mutant. We seek to investigate the functional roles of Coq11 and ERMES to better understand the regulation of CoQ biosynthesis and aid in the development of more effective therapeutics for diseases linked to CoQ deficiencies.
Elucidating the Effect of Point Mutations on Gamma B Crystallin Protein Interactions
Authors: Labbe Natalie , Michel Lea , Thurston George , Mills Jeffrey , Williams Zach , Hasselbeck Jimmy , Cirrincione Lucas
Published: May 2022

Cataracts form when crystallin proteins in the eye lens undergo phase separation or aggregation, thereby failing to maintain lens transparency. Mutations in the amino acid sequence of the crystallins can alter the structure and/or chemical properties of the proteins, resulting in increased inter‐protein interactions, increased aggregation, or changes to the proteins’ phase diagram, all of which can enhance cataract formation. Here, we describe our work on γB crystallin, the bovine homologue to human γD crystallin, and two site‐directed mutants of γB crystallin with known links to congenital cataracts. Using nuclear magnetic resonance spectroscopy, we aim to determine how inter‐protein interactions of bovine γB crystallin are altered by point mutations so that we can gain a better understanding of the mechanism behind cataract formation. Results from preliminary experiments suggest that the inter‐protein interactions of γB crystallin are highly dependent on environmental conditions, such as temperature, pH, and protein concentration, and studies are underway to compare those behaviors to the behavior of mutant proteins under similar conditions.