bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of acetaminophen treatment on endocrine pathways in placenta: Proteomics investigation in JEG‐3 cell model
Authors: Naji‐Talakar Siavosh , Kruger Laken , Prasad Bhagwat
Published: May 2022

Acetaminophen (APAP) is the most used medication for pain and fever in pregnancy, but recent studies have elucidated in uteroAPAP as an endocrine disruptor associated with neurocognitive disorders. The placenta is a vital endocrine organ and disruption in metabolic pathways have been associated with adverse fetal neurodevelopment such as attention deficit hyperactivity disorder (ADHD) and autism. The aim of this study was to investigate the effect of APAP treatment on androgen pathways in JEG‐3 placental cells.

JEG‐3 cells were seeded in 6 well plates at 100,000 cells per well and treated with the vehicle control (DMSO) and 0.1, 0.3, and 0.9 mM APAP for 72 hours, in triplicate. Cells were harvested and samples were digested with trypsin. Global proteomics data were acquired using a Q‐Exactive HF Quadrupole‐Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA) followed by data processing by MaxQuant (version 1.6.8.0) using the Andromeda search engine. Unique and significantly different (upregulated or downregulated) proteins were identified. Label‐free quantification (LFQ) intensities of sex‐steroid pathway proteins were compared across control and treatment groups.

Proteomics data analysis detected 2338 – 2560 total proteins with 1953 proteins in common between all treatments. The vehicle control group had 88 unique proteins and APAP treatment groups ranging from 0.1, 0.3, and 0.9 mM resulted 62, 55, and 157 unique proteins, respectively. Treatment with APAP upregulated ~200 proteins and downregulated ~40 proteins representing critical metabolic processes. APAP treatment elevated the level of AKR1B1 by at least 63% across all treatment groups. There was no change in the levels of SOAT1, CYP19A1, and STS in any treatment group. Interestingly, ABCG2 was uniquely detected in the lower APAP treatments.

Sex steroid metabolism plays a significant in utero role in brain and reproductive development of a fetus. Determining how APAP disrupts endocrine pathways is critical in managing pain treatment guidelines in pregnant women. This is the first study investigating the effect the APAP on sex steroid related proteome in placental cells, which when validated by our metabolic and activity assays, will provide a critical mechanistic information towards mitigating adverse effects of APAP.
Protein‐DNA Interactomes of NKX2‐5 and TBX5 Mutants Identified in Congenital Heart Defects
Authors: Carrasquillo‐Dones Emmanuel , Cesar Brenda , Barreriro Adriana , LeMoine Kathryn , Rodriguez Jose A.
Published: May 2022

Congenital Heart Defects (CHDs) are the most common birth defect from children to adults in the United States. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, sometimes leading to embryonic death. Cardiac Transcription Factors (cardiac TFs) such as NKX2‐5 and TBX5 play a critical role in developmental stages and physiological processes of the heart by regulating gene expression through DNA‐protein interactions. Genetic analyses have identified various non‐synonymous mutations within the homeodomain (HD) DNA‐binding domain (DBD) of NKX2‐5 and the (Tbox) DBD of TBX5 in associated with CHD. We hypothesize that non‐synonymous mutations in cardiac TFs DBD will alter their DNA‐binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we studied how the cardiac TFs DNA‐protein interactomes are affected by non‐synonymous mutations found in CHD patients. We expressed and purified the DBD of NKX2‐5 (HD), TBX5 (Tbox), also NKX2‐5 and TBX5 non‐synonymous mutations (A148E, E154G, R161P, T178M, Q181H, R190C, Y191C), and (I54T, M74V, G80R, I101F, R113K, R237Q, R237W) respectively. Functional analysis using Electrophoretic Mobility Shift Assay (EMSA) confirms HD and Tbox binding to cognate DNA binding sites. DNA‐binding activity as assessed by EMSA, shows retention of DNA activity in the case of NKX2‐5 and TBX5 mutants A148E, R161P, Y191C and I54T, R113K respectively. Mutants T178M, Q181H, R190C, M74V, G80R, I101F, R237Q, R237W showed complete loss in DNA binding activity towards their specific cognate sites. We measured the comprehensive protein‐DNA interactomes of NKX2‐5 and TBX5 non‐synonymous mutants using High‐Throughput Systematic Evolution of Ligands and Exponential Enrichment (SELEX‐seq) to identify changes in DNA targets between wild‐type and mutant TFs. Mutants A148E, R161P and Y191C bind to similar binding sites as NKX2‐5 (HD). Interestingly, mutant Y191C showed an altered DNA binding site significantly different from NKX2‐5 (HD). Mutants I54T, R113K bind to similar binding sites as TBX5 (Tbox). Mutants E154G, T178M, Q181H, R190C, M74V, G80R, I101F, R237Q and R237W showed no significant enrichment for specific DNA binding sites. Integrative analysis with available NKX2‐5 and TBX5 genomic binding sites data (ChIP‐seq), human and mouse cardiac enhancers and promoters catalogued in the literature will facilitate the identification of putative genes mutant cardiac TFs are regulating and how different are from their wild‐type TFs.
Interactive Effects of Thermal and Salinity Stress Cause Cell Cycle Arrest in the Nile Tilapia
Authors: Buckley Bradley A. , Palmer Rachel M.
Published: May 2022

The goal of this study was to test the interactive effects of sub‐lethal thermal stress and environmental salinity variation on a tropical freshwater species, the Nile tilapia, Oreochromis niloticus. This economically important species is vulnerable to increased morbidity at low temperatures, a condition sometimes termed “Winter Stress Syndrome”. It is possible that in addition to extreme weather events, saltwater intrusion into freshwater systems presents physiological challenges to this species. To test this hypothesis, Nile tilapia were exposed to a variety of treatments (two temperatures: 21°C & 14°C, three salinities: 0ppt, 16ppt, 34ppt) for 1‐h. Following exposures, flow cytometry was used to assess cell cycle staging in the spleen and liver. Specifically, we were testing the synergistic effects of temperature and salinity stress on cell cycle arrest and apoptosis, both metrics of sub‐lethal stress and a switch away from growth and towards stress responses. Both liver and spleen responded to temperature and salinity stress by inducing cell cycle arrest in a tissue‐specific manner. Apoptosis was also detected in the spleen. These findings support our hypothesis that the combination of thermal and salinity stress have deleterious impacts on the health of this important global source of protein.
Perception of Pharmacology Instruction and Confidence in Independent Prescription Among Osteopathic Medical Graduating Seniors
Authors: Eldeeb Khalil
Published: May 2022

The prescription of medicines is one of the most critical acts performed by physicians. With millions of drugs prescribed every year, poor prescribing may potentially impact patient safety and healthcare costs. Pharmacology curriculum varies in length, content, instruction methods, and assessment even within similar health‐related programs. Doctor of Osteopathic Medicine (DO) students' perceptions of these variations and their effects on their confidence to independently prescribe is not thoroughly studied.

Methods

This study reviewed the DO graduating seniors' evaluation of time devoted to clinical pharmacology and therapeutic management instruction and how confident they are to write safe and indicated orders and prescribe therapies or interventions in various settings without direct supervision. This study reviewed and analyzed a secondary database of the graduating seniors' survey published by the American Association of Colleges of Osteopathic Medicine from 2012‐2013 to 2020‐2021.

Results

The graduating seniors reported that the time devoted to clinical pharmacology instruction as appropriate increased from 78% in 2012/2013 to 87% in 2020/2021. On the other hand, no change in the percentage of graduating seniors who reported that the time devoted to therapeutic management instruction was appropriate in 2012/2013 and 2020/2021. The percentage of graduating seniors who feel very confident and somewhat confident to prescribe therapies or interventions in various settings without direct supervision was below 70% between 2012/213 and 2018/2019. Over the last two academic years, 2019/2020 and 2020/201, the percentage increased to 93% and 77%, respectively.

Conclusion

While most of the DO graduating seniors reported that the time devoted to clinical pharmacology and therapeutic management instructions as appropriate, their current confidence level to prescribe independently might highlight the need to update the pharmacology curriculum to emphasize the development of prescription skills.
NMDA receptor modulation in protracted ethanol withdrawal
Authors: Foster Mercedes , Christian Daniel , Giles Jennifer
Published: May 2022

Functional dysregulation of the glutamate system during withdrawal from chronic drug exposure is a primary driver of drug craving and relapse. Animal models of psychostimulant use have demonstrated dynamic alterations in NMDA receptor function and expression that contribute to drug seeking behavior. These changes begin as early as 5 days following cessation of drug use are persistently expressed into long term withdrawal (>60d). Evidence of NMDA receptor dependent changes during short term withdrawal (24h) suggest that similar mechanisms may drive drug craving and relapse behaviors following chronic exposure to ethanol. To this end, we investigated NMDA receptor protein and surface expression during protracted withdrawal from chronic intermittent ethanol (CIE) exposure using western blot. We focused on the basolateral amygdala (BLA), as glutamatergic signaling in this region is robustly modulated by short term (24h) withdrawal from CIE (10d, 12hr/day) and regulates anxiety‐like behavior expressed during withdrawal. Adolescent male rats were exposed to repeated cycles of CIE (12hr/day, 4d on/3d off, 3 cycles) and brain tissue was collected during protracted withdrawal. Optimization of experimental procedures is currently being conducted and analysis of total and membrane surface expression will be analyzed by surface biotinylation for NMDAR subunits (GluN1, GluN2B, GluN3). Cell surface biotinylation will be used to isolate cell surface membrane proteins to detect and quantify expression of NMDAR subunits. We expect to identify an increase in membrane surface expression for NMDAR subunits, in agreement with functional data obtained in the laboratory.
Cortisol Receptor Sensitivity as a Risk Factor for Depression
Authors: Michielli Michela , Cohen Brian
Published: May 2022

In 2020, the World Health Organization reported over 264 million people across the world were suffering from depression. Studies have demonstrated that one source of depression is a hormonal imbalance involved in the stress response. Cortisol is a stress hormone regulated by the Hypothalamic‐Anterior‐Pituitary (HPA) Axis. Its effects on the stress response and other metabolic activities in the body are exerted through the glucocorticoid and mineralocorticoid receptors (GR and MR respectively).

Our research has examined mutations known as single‐nucleotide‐polymorphisms (SNPs) relating to cortisol‐receptor sensitivity and the behavior of cortisol in the body to investigate the link between cortisol activity and depression. The relationship between SNPs relating to receptor sensitivity and depression in patients will be established by analysis of SNPs in coordination with data from psychological inventory assessments collected for each patient. Patient samples and clinical information was collected from adult patients diagnosed with depression by collaborating physicians. Quantitative polymerase chain reaction (qPCR) was used for genotyping. Analysis of the data yielded no significant correlation between SNPs relating to GR or MR and depression as scored by the Beck Depression Inventory (BDI). However one SNP in GR (rs33389) and one SNP on the 11β‐hydroxysteroid dehydrogenase type 1 gene (rs12086634) correlated with scores on the Mindful Attention Awareness Scale and State Trait Anxiety scale, respectively.

Current studies focus on SNPs involved in the regulation of GR and MR activity that are not directly found in those receptors such as the FKBP5 GR co‐regulator protein. Investigating other non‐GR and MR SNPs could provide alternate explanations for cortisol sensitivity beyond the receptors themselves. In analyzing genetic differences relating to cortisol sensitivity in patients clinically diagnosed with depression, we aim to determine if an imbalance in an individual’s response to cortisol is linked to depression. If such a relationship can be established, this can provide physicians with a screening tool to better inform them of a patient’s risk factors for depression.
Mapping of GPCR‐p38 Signaling Reveals A Spatiotemporal Key for Regulating Vascular Inflammation
Authors: Burton Jeremy J. , Okalova Jennifer , Grimsey Neil J.
Published: May 2022

Vascular dysfunction and inflammation drives the onset and progression of many diseases including acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). The mitogen‐activated protein kinase (MAPK) p38 is a master regulator of inflammation and vascular function, yet despite extensive and ongoing studies, p38‐targeted therapeutics have failed. Their failure is in part due to the ubiquitous expression and dichotomous role of p38 in physiological and pathological signaling. Defining selective pathological p38 signaling pathways represents a novel therapeutic strategy to treat many diseases. Our prior studies demonstrate that G‐protein coupled receptor (GPCR) stimulation activates an understudied atypical p38 pathway. Atypical p38 activity selectively induces pathological p38 signaling, via direct interaction between p38 and an essential adaptor protein TAB1, inducing p38 autophosphorylation. We hypothesized that a spatial bias in activated p38 differentially drives pathological vascular dysfunction. To test this, we developed a Forster’s Resonance Energy Transfer (FRET) biosensors platform to spatially map subcellular p38 activity. We demonstrate that proinflammatory G protein‐coupled receptor (GPCR)‐mediated p38 activity is rapidly trafficked away from the plasma membrane, primarily occurring in the cytosol and more specifically the endosomal compartment, peaking at 15 minutes post‐stimulation. Interestingly, these signaling dynamics are altered when receptor internalization is inhibited; where p38 activity is shifted into the nucleus, decreasing kinase activity at the cytoplasm and endosome. This shift towards a nuclear bias mirroring p38 signaling dynamics seen with classical MKK3/6‐mediated signaling, which occurs primarily in the nucleus after osmotic stress. These data were validated with endogenous GPCRs in primary endothelial cells. Our preliminary data suggest that endosomal/cytosolically biased p38 signaling drives pathological atypical p38 signaling in vascular disease, regulated by a spatial axis at the endosome. In concurrent studies, we have also developed an array of inhibitory intrabodies that specifically block the critical p38‐TAB1 interaction in atypical p38 signaling. Preliminary data suggests that these first generation intrabodies effectively block atypical p38 signaling and in conjunction with our biosensor platform will reveal how this endosomal axis drives vascular inflammation. Studies are ongoing to explore their capacity to block pathological signaling in vitro, and in future acute lung injury studies.
Uncovering the Perceptions of Grades in Dentistry Students – How Grades are Impacting Learning & Clinical Development
Authors: Yuen Jenna J. , Horne Andrew , Beveridge Tyler S. , McLean Sarah
Published: May 2022

Introduction

Grades in healthcare professional schools may be doing more harm than good. In fact, educators and researchers suggest grades have lasting negative impacts on professional development and the ability to navigate clinical settings (i.e., impairs self‐regulated learning, interferes with developing patient‐care competencies, and decreases levels of empathy). Although some studies have incorporated grade perceptions into their investigations, we lack research dedicated to primarily understanding the student’s perception of grades, and whether they think grades help or hinder the quality of their clinical learning.

Aims

The first aim of this study is to identify the most prevalent grade perceptions among dentistry students. The second aim is to explore the underlying contributing factors and to determine how dentistry students’ grade perceptions are influencing their learning and clinical development.

Methods

To address the first aim, we distributed an online survey to dentistry students at the Schulich School of Medicine & Dentistry. To address our second aim, we conducted virtual semi‐structured interviews and a focus group on the same population of dentistry students. Survey results guided and informed interview/focus group discussions. Qualitative data was analyzed through an inductive thematic process.

Results

Fourteen grade perception themes were identified. The three most frequent perceptions were grades as a tool for ranking students, an evaluation tool to test knowledge and comprehension, and a feedback tool on comprehension. Seventy students participated in the survey; 70% agreed that grades guided their learning/studying strategies; 91% agreed grades reflected their ability to memorize content; 85% did not think grades accurately represented their practical skills; and 89% agreed that grades determined their acceptance into specialty/residency programs.

Nine students participated in the interviews/focus group. Emerging topics included the importance of grades during specialty/residency applications, the disconnect between grades on a 0‐100% scale and clinical development, and mixed opinions about grades facilitating learning. Characterization of dentistry students’ perceptions towards grades in a clinical learning environment is ongoing.

Conclusion

Dentistry students commonly perceive grades as a ranking tool because of its central role in specialty/residency applications. While some note that grades help their learning, the majority of students agree that grades reflect memorization of content. Our data also suggest students do not find grades on the 0‐100% scale helpful in their clinical development because it does not indicate whether professionally acceptable standards are met. Findings obtained from our study brings students’ voices to the forefront of the ongoing discussion about grades, and may provide important considerations for future healthcare professional curricula.
Preservation of Right Ventricular Contractility After Transient Phenylephrine‐Induced Pressure Overload in Swine
Authors: Konecny Filip , Weil Brian R.
Published: May 2022

Objective

We have previously demonstrated that a transient phenylephrine (PE)‐induced rise in systemic arterial blood pressure leads to reversible left ventricular (LV) systolic dysfunction in the absence of ischemia (i.e., “stretch‐induced stunning”). However, the extent to which this phenomenon occurs in the right ventricle (RV) is unclear. Accordingly, the present study was designed to assess changes in pulmonary artery (PA) pressure and RV function during and after systemic administration of PE in swine.

Methods

Closed‐chest propofol‐anesthetized swine (n=5) were instrumented with a Swan‐Ganz catheter and an admittance catheter for continuous measurement of PA pressure, RV pressure, and RV volume (ADV500; Transonic SciSense, Inc.). Ventricular end‐diastolic and end‐systolic volumes were assessed by multi‐detector computed tomography with iodinated contrast (1 mL/kg at 4 mL/s) before, during, and 30‐minutes after transient pressure overload elicited by a 1‐hour intravenous infusion of PE (18 mg/hour). Load‐independent RV systolic function was derived from RV pressure‐volume loops collected during inferior vena cava occlusion at baseline and 30 min after PE.

Results

PE elicited a significant rise in PA pressure (27±1/14±1 to 43±1/23±31mmHg; p<0.05) and RV pressure (25±2/3±1 to 44±2/8±2 mmHg; p<0.05) that normalized 30‐minutes after the end of the infusion period (PA: 28±1/11±1 mmHg; RV: 29±5/4±2 mmHg). This was accompanied by a significant increase in RV end‐diastolic and end‐systolic volumes and decrease in RV ejection fraction that tended to persist 30‐minutes after cessation of PE (Table; top panel). However, load‐dependent (dP/dtmax) and load‐independent (ESPVR and PRSW) parameters of RV systolic function were preserved after PE‐induced TPO (Table; bottom panel).

Conclusions

Despite causing a significant rise in RV preload volume and pressure, transient PE‐induced pressure overload is not associated with RV contractile dysfunction during the post‐infusion period. The absence of stretch‐induced stunning in the RV may be linked to inherent differences in compliance relative to the LV as well as a diminished pressor response to PE in the pulmonary vs. systemic circulation.
Quantitative analysis of straight length of the external ear
Authors: Mokhtar Abd‐Elmagid Ahmed Elsir , Elfaki Amani A. , Şahin Bünyamin , Medani Khalid , Ali Qurashi M. , Babikir Haydar E. , Nahir Mert , Demir Osman , Elmustafa Shamail D. A. , Mohammed Noor Abdulrahman
Published: May 2022

Introduction

Clinical anatomy of the external ear canal (EEC) has a significant role in the diagnosis of Otitis Media (OM) and other auditory system diseases. OM is one of the most common infections and major causes of morbidity in adults if the complications of the OM take place. In the developing and low‐income countries, the OM has a direct and indirect role in their life, because it’s one of the causes leading to doctors’ visits, and the indirect costs may affect the families and economy. EC measurements are needed in daily life, as an example; in the ears plugs manufacturing.

Method

This study was conducted by the Declaration of Helsinki, and approved by the research ethics committee at the Faculty of Medicine, University of Gezira, Sudan, this observational case‐control study. Informed consent was obtained from all‐volunteer participants enrolled in the study. From January 2015‐2018, temporal bone computed tomography (CT) images of volunteer patients and healthy subjects, were collected from Khartoum State. ImageJ software (V 1.52a), used in the measurement of EC; OsiriX image processing application for Mac (OsiriX Lite) to display images for the external ear area of the skull’s temporal bone, statistical analysis was performed by Statistical Package for the Social Sciences SPSS V.22, and statistical software suite developed by SAS Institute (The SAS System V.9). The level of significance was set at P‐value <0.05. Chi‐Square test was used and Receiver Operating Characteristic (ROC) analysis was applied to determine the power of straight length (SL) in predicting patient.

Results

In the cases the total number is 140 (patient 70 and healthy 70), with a range of 18–40 years, 52.9 % (n=74) of the patients were females and 47.1% (n=66) are males. The distribution of quantitative variables shows that the mean of the straight length (SL) is ±34.13, Standard Deviation (SD) ± 5.83, and the minimum and maximum SL are 11.33 and 47.00, respectively. Distribution of quantitative variables by a case in SL, shown that the patients mean ±32.69, SD ±6.58; and the healthy mean ±35.58 SD ±4.66, t ±3.106 and the P‐value <0.003. Distribution of SL by Gender shown that in male mean ±34.89, SD ±6.22, female mean ±33.45, SD ±5.42. Distribution of SL by Side shown that left side means ±34.14, and SD ±5.66, right side mean ± 34.12, and SD ±6.04.

Conclusion

If the straight length (SL) value is equal to 31.33 or less, the case sample is classified as an otitis media patient. There is a highly significant difference in the SL between male patients and healthy a p‐value <0.061 and between females’ patients and healthy as a p‐value<0.016. and significance differences comparing the patients and healthy groups with the gender, result shown that t =2.909 in males and females t=2,456.
The activation of C1 neurons during REM sleep is dependent on the arterial baroreflex
Authors: Souza George M. , Stornetta Daniel S. , Abbott Stephen B.
Published: May 2022

Introduction

Natural sleep‐wake patterns are associated with changes in sympathetic nerve activity (SNA) controlling cardiovascular function. The mechanisms underlying the effects of sleep‐wake state on SNA are not well understood. C1 neurons in the rostral ventrolateral medulla regulate cardiovascular function through SNA and mediate the sympathetic arterial baroreflex. In this study we determine the effect of sleep‐wake transitions on the activity of C1 neurons and investigate the contribution of the arterial baroreflex to these effects.

Methods

The activity of C1 neurons was measured using fiber photometry (calcium imaging) in freely behaving adult rats while monitoring the vigilance states. C1 neurons were targeted with GCaMP7s by microinjection of AAV1‐syn‐FLEX‐jGCaMP7s in the RVLM of Tyrosine Hydroxylase‐Cre transgenic rats, an optical fiber was implanted above the injection site, rats were fitted with a EEG/EMG head stage to monitor sleep‐wake state, and a radio telemetry probe to record BP. One month after the surgery, C1 neuron activity was recorded before and 7 days after sino‐aortic denervation to eliminate inputs from arterial baroreflex. Normalized fluorescence changes are expressed as Z‐score (mean ± SD) and t‐test was used to determine statistically significant differences.

Results

C1 activity was increased by acute hypotension induced by sodium nitroprusside (2.1 ± 0.5 vs. ‐0.92 ± 0.4, p<0.0001, n=5), indicating the barosensitive nature of the recorded cells. C1 activity increased during transitions from NREM to wake (‐0.60 ± 0.1 vs. 0.59 ± 0.2, p<0.0001, n=6) and decreased during transitions from wake to NREM (0.34 ± 0.2 vs. ‐0.32 ± 0.2, p<0.0001, n=6). C1 activity increased during transitions from NREM to REM sleep (‐0.74 ± 0.1 vs. 0.77 ± 0.1, p<0.0001, n=6) and decreased in transitions from REM to wake (0.58 ± 0.2 vs. ‐0.59 ± 0.2, p<0.0001, n=5). Baroreceptor denervation attenuates the increase in C1 neuron activity during REM sleep (0.4 ± 0.7 vs1.8 ± 0.4 Δ Z‐score, p=0.0054, n=4) and during wakefulness (0.59 ±0.6 vs 1.6 ± 0.4, p=0.0210, n=4) when compared to the increase before sino‐aortic denervation.

Conclusion

C1 neuron activity during sleep‐wake transitions mirrors the pattern of SNA. This observation indicates that C1 neurons contribute to changes in SNA associated with natural sleep‐wake patterns. Baroreceptor denervation attenuates the change in C1 activity observed during transitions to REM, indicating that the feedback from the arterial baroreflex contributes to the elevation in C1 activity in this sleep state.
Enhanced Senescent Cell Burden in the Quadriceps, Articular Cartilage and Periarticular Bone Accompanies Atrophy and Osteoarthritis Following ACL Injury in Mice
Authors: Dungan Cory M. , Thomas Nicholas T. , Brightwell Camille R. , Latham Christine M. , Noehren Brian , Fry Christopher S.
Published: May 2022

Protracted quadriceps weakness and atrophy after anterior cruciate ligament (ACL) injury result in poor knee mechanics and contribute to the development of posttraumatic osteoarthritis. The biological underpinnings of poor functional recovery and osteoarthritis are unknown, limiting the development of evidence‐based therapies. The identification of shared cellular pathology between skeletal muscle, bone and articular cartilage would offer powerful therapeutic targets to combat musculoskeletal degeneration; therefore, we sought to define the musculoskeletal cellular senescence burden as a cellular predictor of muscle atrophy and osteoarthritis severity. We hypothesized that cellular senescence would be associated with greater muscle atrophy and osteoarthritis severity. Male and female C57BL/6 mice between 4 and 6 months of age underwent unilateral ACL transection surgery (ACLT), and then quadriceps and whole knees (healthy contralateral and ACLT) were collected at 7, 14 and 28d post‐ACLT. Quadriceps fiber size was determined using immunohistochemistry (IHC), and osteoarthritis severity was assessed with Safranin O staining via Osteoarthritis Research Society International (OARSI) scoring. Additionally, cellular senescence was determined by quantification of senescence‐associated β‐galactosidase (SA β‐Gal) and p16Ink4a (p16) positive cells using IHC. SA β‐Gal expression was greatest at 7 days post‐ACLT (ACLT: 18.6±4.3 vs. Healthy: 3.3±2.6 cells/mm2, p<0.05) but remained elevated through 28 days post‐ACLT. Quadriceps senescent cell density occurred alongside atrophy (7 days: ‐21%, 14 days: ‐16%, 28 days: ‐13%, all p<0.05 vs healthy contralateral). p16+ cell density was elevated within the articular cartilage (ACLT: 32.0±5.8 vs. Healthy: 10.0±2.5 cells/mm2, p<0.05) and trabecular bone (ACLT: 435.5±130.6 vs. Healthy: 119.4±8.6 cells/mm2, p=0.08) at 28 days post‐ACLT. OARSI scoring demonstrated cartilage erosion at 28 days post‐ACLT (ACLT: 2.9±1.0 vs Healthy: 0.4±0.4 OARSI score, p<0.05). These findings support an elevated cellular senescence burden within muscle, bone and cartilage following ACL injury associated with musculoskeletal deficits and osteoarthritis. Future research should focus on strategies to target senescent cells to enhance functional recovery and mitigate osteoarthritis following ACL injury.
The role of FIP200 in Phagophore formation during Autophagy
Authors: Shannon Daniel , Gruber Weston , Lohmueller Isaac , Hawryschuk Ethan , Medley Wyatt , Poetker Gavin , Diao JaiJie
Published: May 2022

The Moeller Molecular Modeling Team in conjunction with Dr. Jiajie Diao at the University of Cincinnati Medical College used 3D modeling and printing technology to study the vital role of the focal adhesion kinase family interacting protein of 200 kD (FIP200) in the autophagy process. The cellular process of autophagy is enabled by formation of autophagosomes, a double membraned structure that isolates substrates designated by the cell to be eliminated by autophagy. FIP200 is a 200 kD subunit of the ULK1 complex which facilitates the formation of phagophores, the first step in autophagosome formation. An ULK1 protein kinase is necessary for the activation of the ULK1 complex during autophagosome formation. The positively charged “claw domain” area at the C‐terminal region of FIP200 interacts with p62, a receptor protein, which delivers ubiquitinated cargo proteins bound to LC3B, a central regulator for autophagosome formation. After the binding of these proteins, the autophagosome forms around the ubiquitinated cargo and continues to the next step of the autophagy process.
Diet Significantly Influences the Interleukin‐6 Response to Mouse Models of Peritoneal Sepsis
Authors: Klug Mikahla A. , Patrick‐Eisenberg Austin , Thomas Blake , Rose Christian , Houghton Jeffrey , Gigliotti Joseph C.
Published: May 2022

Introduction

Sepsis remains to be a tremendous health burden in the United States, affecting 1.7 million Americans and causing over 250,000 deaths each year. Previous work has shown high‐calorie diets to significantly alter the immune response and outcomes in animal models of sepsis; however, the diets used model overt metabolic dysfunction and not dietary patterns observed in a “typical” American. We developed a novel Americanized diet (AD) that incorporates several of the nutritional inadequacies reported in Americans and set out to determine how consumption of the AD influences immunological outcomes of peritoneal sepsis in mice.

Methodology

Male C57Bl/6 mice were used in accordance to protocols approved by the Institutional Animal Care and Use Committee at LUCOM and compliant with the Guide for the Care and Use of Laboratory Animals. Mice received ad libitum access to experimental diets [chow, AD, or a commercially available high‐calorie Western diet (WD)] for 8‐weeks and peritonitis was induced with cecal‐ligation and puncture (CLP) or intraperitoneal injection of a fecal slurry (fecal peritonitis, “FP”). For both CLP and FP, blood was collected 24‐hours later for quantification of circulating interleukin‐6 (IL6). Another group of mice were fed their assigned diets and the spleens cultured ex vivo in DMEM media with lipopolysaccharide (LPS, 10ug/mL) or saline for 3 hours. Tissue and supernatant were collected and frozen until assayed for IL6 and inflammatory gene expression using a commercially available PCR‐microarray. All data were analyzed using general linear model procedures in SPSS with Tukey’s post hoc test and group averages were considered statistically significant with a P‐value less than 0.05.

Results

Mice consuming AD and WD had ~3‐fold greater circulating IL6 following CLP (P=0.04) and FP (P<0.05) as compared to mice consuming chow. LPS stimulation of splenic tissue ex vivo caused a significant increase in secreted IL6 protein (P<0.001). Mice fed WD had the greatest IL6 concentration (425 pg/mL), which was greater than mice fed chow (163 pg/mL, P=0.04) but not mice fed AD (360 pg/mL, P=0.9). PCR‐array analysis confirmed the upregulation of IL6, with mice fed WD having double (P=0.03) the mRNA expression of splenic IL6 after LPS as compared to mice fed AD and chow. CCL12 expression was similarly upregulated in mice fed WD as compared to mice fed AD and chow (P=0.005).

Conclusion

Our data support the hypothesis that diet significantly influences the severity of animal models of sepsis. Consumption of a nutritionally inadequate AD caused a similar increase in circulating IL6 as compared to mice consuming a high calorie WD. However, the source of the additional circulating IL6 is unknown, ex vivo spleen stimulation studies with LPS did not report an increase in IL6 production in mice fed the AD. Future studies will determine if and how diet influences IL6 production in other tissues.
Domain Analysis of Arabidopsis Thaliana Sis1 Orthologs in Yeast Prion Propagation
Authors: Bhavsar Harshil S. , Corpus Bridget A. , Hines Justin K.
Published: May 2022

Protein misfolding is associated with many common neurological diseases such as Alzheimer’s disease and Parkinson’s disease. A hallmark of these neurodegenerative diseases is protein aggregation in the form of amyloids. Amyloids are also the structural basis of at least some prions. Amyloid‐based prions are infectious, self‐propagating protein conformers that cause normally‐folded protein to adopt a similar misfolded conformation and aggregate to form amyloid. This amyloid‐based prion propagation is observed in the baker’s yeast Saccharomyces cerevisiae. In S. cerevisiae, chaperone proteins are also involved in the propagation of prions within cell populations. One such chaperone, the J‐protein Sis1, is required for propagating all amyloid‐based yeast prions, including the prions [PSI+] and [RNQ+]. Sis1 is a multi‐domain protein and numerous experiments have shown domain‐specific requirements for the propagation of distinct prions. We previously identified six functional orthologs of Sis1 in the model plant Arabidopsis thalianaand showed that these six paralogous proteins differ in their ability to maintain distinct prions when complementing a deletion of SIS1 in yeast; however, the combination of domains needed to propagate each prion is unknown. Therefore, this study focuses on testing the individual prion‐propagating properties of various A. thalianaSis1 constructs to elucidate the domain combinations of each ortholog necessary to propagate specific prions using biochemical assays and yeast plasmid shuffling. Preliminary results indicate the primacy of the glycine‐rich region of the Sis1 protein. Additionally they indicate that, although naturally occurring as dimers, these proteins may function as well or better as monomers in prion propagation, consistent with previous work on Sis1 itself. The long‐term goal of the investigation is to decipher specific structural characteristics, and amino acid sequences, that give rise to prion‐specific propagating functions in chaperone proteins.
The Role of Butyrate in Paclitaxel and Morphine Induced Peripheral Hypersensitivity
Authors: Jessup Donald K. , Akbarali Hamid I.
Published: May 2022

Recent evidence implicates the gut microbiome in peripheral hypersensitivity in chronic opioid use models, and in chemotherapeutic‐induced hypersensitivity in mice. An important constituent of the commensal microbiome are bacterial species that ferment fibers through complex enzymatic pathways and produce short chain fatty acids, acetate, propionate, and butyrate. Butyrate is known to improve the integrity of the epithelium and enhance antimicrobial peptides thus preventing colonization by pathogenic bacteria, as well as having an established role in reducing inflammation through the inhibition of histone deacetylase. Based on this, we hypothesized that sodium butyrate can relieve peripheral hypersensitivity as well as reduce primary nociceptor hyperexcitability in experimental models of peripheral hypersensitivity. We used both paclitaxel (PAC) and morphine (MOR) animal models, which were organized into the following treatment groups: PAC (8mg/kg i.p q.d, total 4 injections) ± Sodium Butyrate (10mg/kg i.p b.i.d), MOR (escalating from 20mg/kg i.p b.i.d on day 1 to 80mg/kg i.p b.i.d on day 4) ± Sodium Butyrate (10mg/kg i.p b.i.d). PAC animals were assessed for peripheral cold hypersensitivity using an acetone evaporation assay. MOR‐treated animals were assayed using a hot‐plate test for opioid‐induced hyperalgesia. Primary nociceptors from L4‐S1 dorsal root ganglia were collected from these mice for whole‐cell patch clamp electrophysiological recordings to assess for enhanced excitability. PAC‐treated mice developed significant cold hypersensitivity 7 days post PAC, when compared with vehicle (average time engaging stimulated paw 2.0s vs 8.6s respectively). Butyrate ameliorated this paclitaxel‐induced hypersensitivity (average time reduced to 2.5s). Similarly, MOR induced thermal hypersensitivity (Hot‐plate latency of 14.8s vs 28.16s saline), which was also reversed by sodium butyrate (Hot‐plate latency of 24.84s). Whole‐cell patch clamp recordings revealed that PAC and MOR‐treated neurons fired a greater number of regenerating action potentials compared to respective controls (PAC: 4 vs 2; MOR: 3.8 vs 2.1), indicating enhanced excitability. In‐vivo butyrate treatment attenuated this enhanced excitability. Hysteresis plots of hyperexcitable cells revealed a depolarizing shift in the membrane potential of regenerating action potentials. Sodium butyrate treatment recovered this shift in membrane potential within the repetitive action potentials. Therefore, similar to the in‐vivo peripheral hypersensitivity, butyrate reversed enhanced excitability of PAC and MOR‐treated DRG neurons. These findings demonstrate that the short chain fatty acid, butyrate, a major metabolite of the gut microbiome plays an important role in preventing the development of drug‐induced peripheral hypersensitivity.
Evaluation of the HyFlex, Hybrid, and Asynchronous Online Teaching Modalities on Student Learning in Graduate Microbiology Coursework
Authors: Davie Jeremiah
Published: May 2022

The COVID‐19 Pandemic has impacted educational offerings and student learning at all instructional levels. In the USA, the uneven distribution of COVID‐19 cases and their sudden appearance has created a need for flexibility in available instructional modalities to account for students and/or faculty that may need to be quarantined or isolated due to COVID‐19 exposure or infection. This study sought to compare the implementation of the HyFlex (in‐person, asynchronously online, or synchronously online at the student’s discretion), Hybrid (asynchronously delivered, pre‐recorded lectures coupled with fixed, in‐person meetings), and fully asynchronously delivered microbiology coursework for graduate students. Comparison of individual exam scores and overall exam averages revealed that students instructed in the HyFlex and Hybrid modalities consistently scored similarly to, or exceeded in a statistically significant manner, the average exam scores earned by students in a traditional, in‐person lecture format. Comparisons of the individual exam or overall average exam grades observed in students instructed in HyFlex, Hybrid, and asynchronous online learning modalities revealed no statistically significant difference in student performance when the modalities were compared to one another. Successful instruction in the areas of molecular biology and biochemistry, namely cellular structure and physiology, microbial virulence mechanisms, molecular and cellular mechanisms of immune defense, microbial growth and metabolism, and the principles of mutation and recombination, was evaluated by measuring student performance on a pre‐selected set of identical examination items. Analysis of these Student Learning Outcomes data revealed largely similar levels of achievement independent of instructional modality. Collectively, these data demonstrate that graduate‐level microbiology instruction can be successfully implemented across a multitude of learning modalities each offered partly or entirely online. These findings are anticipated to be of value to faculty and administrators of institutions higher education concerned with continuity of instruction in general, and COVID‐19 contingency planning in particular.
Transcriptome and regulome signatures of multiple myeloma induced by bone marrow stromal cells
Authors: Dziadowicz Sebastian , Akhter Halima , Sharma Tulika , Hazlehurst Lori , Hu Gangqing
Published: May 2022

Multiple myeloma (MM) is the second most common hematological cancer and remains incurable due to inevitable drug resistance. One contribution to drug resistance stems from the protective bone marrow (BM) microenvironment. MM cells undergo a substantial change in transcription program in the presence of bone marrow stromal cells (BMSCs) and develop de novo drug resistance. The de novo drug resistance occurs rapidly and is reversible, supporting a contribution from epigenetic mechanisms. However, as a critical component in transcriptional regulation, how chromatin landscape is transformed in MM and regulates transcriptional response to BMSCs remains elusive. To this end, we generated genome‐wide profiles for gene expression (transcriptome) and chromatin accessibility (regulome) of MM cells in a transwell coculture system, where some of the MM cells coexisted with BMSCs in the lower chamber and others resided in the upper transwell. The transcriptomes of the two populations of MM cells were similar but distinctive from those in monoculture. Genes increased in transcription were enriched IL6/JAK/STAT3 signaling pathway, unfolded protein stress, signatures of early plasma cells, and genes responsive to treatments of proteasome inhibitors. Similar to gene expression, the difference in regulomes was unremarkable between MM cells in the transwell and those in the lower chamber but both were substantially different from the monoculture. Chromatin accessibility increased at enhancers of genes linked to the promotion of de novo drug resistance and featured as unfavorable prognostic markers in MM. Lastly, integrative analysis with public ChIP‐seq data sets identified candidate transcription factors that contributed to the BMSC‐induced transformation of transcriptome and regulome of MM cells and are prominent targets for future studies.
A Metabolomics‐Based Screening System for UDP‐Dependent Glycosyltransferases Involved in Wallflower Specialized Metabolism
Authors: Brody Rebecca , Holland Cynthia
Published: May 2022

Cardiac glycosides are secondary metabolites that have evolved independently in at least 12 plant families to serve as an herbivore defense mechanism. While cardiac glycosides are widely used by humans as a treatment for heart arrhythmias and heart failure, their biosynthetic pathway remains unknown. Cardenolides are cardiac glycosides that have a 6/6/6/5 steroid core with a five‐membered lactone ring and one or more sugars. We used Erysimum cheiranthoides (wormseed wallflower) as a model system to identify UDP‐glycosyltransferases (UGTs) that add a sugar to the aglycone steroid core. We have identified candidate UGT genes based on shared homology with UGTs known to glycosylate steroids in other plants. Because many putative substrates are commercially unavailable, we have developed a novel semi‐targeted mass spectrometry screening system to identify the substrates of our candidate UGTs by incubating the purified proteins with plant extracts. This approach has allowed us to successfully identify the substrates of several candidate UGTs. Next, we will confirm the activity of the candidate UGTs by generating knockouts of the lead UGT gene candidates in E. cheiranthoides plants. The current system can then be used to identify additional genes of interest in the cardenolide biosynthetic pathway. Methods that we have developed for rapidly identifying substrates of UGTs involved in specialized metabolism may also be applied to other plant systems to allow for the characterization of enzymes involved in a diverse range of biosynthetic processes.
Adipose‐derived human soluble (pro)renin receptor causes resistance to Losartan treatment in high‐fat diet male and female mice
Authors: Nichols Kellea , Arthur Gertrude , Hinds Terry , Yiannikouris Frederique
Published: May 2022

Obesity, affecting more that 37% of the US, contributes to hypertension. Despite the use of one or more anti‐hypertensive treatments, 48% of the hypertensive population remains with resistant hypertension, which prompts the development for new therapeutic targets. We demonstrated that obesity increased the expression of prorenin receptor (PRR) in the adipose tissue and elevated plasma soluble PRR (sPRR). In addition, the infusion of mouse sPRR increased blood pressure in male mice fed high fat‐diet (HF); indicating that adipose‐derived sPRR could increase circulating sPRR and contribute to hypertension. However, there is a critical gap in the functional role of human sPRR in obesity‐hypertension. In this study, we aim to define whether adipose‐derived human sPRR contributes to obesity‐hypertension.

Human sPRR‐Myc‐tag transgenic mice were bred with mice expressing adiponectin/Cre to selectively express human sPRR in adipocytes (adi‐HsPRR). Adi‐HsPRR and control littermate (CTL) male and female mice were fed HF‐diet for 20 weeks (N=8‐15/group). Body weight was assessed weekly and body composition monthly. Blood pressure was measured by telemetry after 15 weeks of diet.

Adipose‐derived human sPRR did not significantly elevate body weight or fat mass (Male: CTL. 18.3±1.0g; adi‐HsPRR. 17.5±0.8g. Female: CTL. 15.6±1.5g; adi‐HsPRR. 11.9±1.3g; p>0.05). Systolic blood pressure (SBP) significantly increased in HF‐fed male and female mice. However, adipose‐derived human sPRR did not further elevate SBP (24h SBP. Male: CTL. 136.0±1.7 mmHg; adi‐HsPRR: 133.4±1.5 mmHg; Female: CTL. 131.9±2.8 mmHg; adi‐HsPRR: 130.6±3.1 mmHg; p>0.05). Surprisingly, the anti‐hypertensive effect of losartan (Los) to lower blood pressure was significantly reduced in adi‐HsPRR male and female mice (Male: CTL. ΔSBP: ‐12.1±1.5 ΔmmHg; adi‐HsPRR: ‐7.8±0.6 ΔmmHg; Female: CTL. ΔSBP: ‐13.4±1.1 ΔmmHg; adi‐HsPRR: ‐5.7±2.3 ΔmmHg; p<0.05). In 3T3‐L1 cells, sPRR significantly increased phosphorylation of ERK1/2, which was not completely blunted by Los indicating that human sPRR could act as a partial agonist of AT1R or activate ERK1/2 independently of AT1R. Our data suggests that adipose‐derived sPRR does not stimulate AT1R‐mediated contractility, instead impairs Los efficacy.