bluuebunny/crossref_metadata_2025 huggingface dataset:
Sympathetic transduction of blood pressure during graded lower body negative pressure
Authors: Nardone Massimo , Katerberg Carlin , Teixeira André L. , Millar Philip J.
Published: May 2022

Introduction

Cross‐sectional work has demonstrated that participants with high resting muscle sympathetic nerve activity (MSNA) have lower resting sympathetic transduction of blood pressure (BP), and a larger proportion of sympathetic bursts firing above the BP set‐point (or mean BP). However, to date, most of these observations have been evaluated between participants with different levels of resting MSNA. The objective of the current study is to evaluate the influence of acute elevations in MSNA on sympathetic transduction of BP in young healthy adults using graded lower body negative pressure (LBNP). We hypothesize that sympathetic transduction will decrease during acute elevations in MSNA due to an increased proportion of MSNA bursts firing above the BP set‐point.

Methods

Ten (1 female) young healthy participants underwent 8 minutes of quiet rest, followed by a graded LBNP protocol consisting of 8‐minute stages at ‐10, ‐20, and ‐30mmHg. Beat‐to‐beat heart rate and BP were quantified using single‐lead electrocardiography and finger photoplethysmography, respectively, while MSNA was quantified using fibular nerve microneurography. Sympathetic transduction was quantified in the time (signal‐averaging) and frequency (MSNA‐diastolic BP low frequency [LF: 0.04‐0.15 Hz] transfer function magnitude) domains. Tertiles of diastolic BP were determined for each participant, and the proportion of MSNA bursts firings within each BP tertile were quantified.

Results

MSNA burst frequency increased progressively during graded LBNP (from 15±7 to 17±7, 22±7, and 27±9 bursts/min; P<0.01), while the proportion of MSNA bursts firing in the highest BP tertile remained stable (19±7, 18±9, 19±9, 19±7 %; P=0.72). Sympathetic transduction of BP was unchanged during LBNP (from 2.1±1.1 to 2.3±1.5, 2.1±1.2, and 2.0±1.1 mmHg; P=0.51), while the BP responses to non‐burst cardiac cycles decreased progressively (from ‐0.7±0.5 to ‐0.9±0.5, ‐1.0±0.4, and ‐1.4±0.7 mmHg; P<0.01). The difference between burst and non‐burst cardiac cycles (from 2.8±1.4 to 3.2±1.9, 3.1±1.5, and 3.4±1.3 mmHg; P=0.26) and the MSNA‐diastolic BP transfer function magnitude (from 0.14±0.09 to 0.14±0.06, 0.14±0.06, and 0.13±0.07 au/mmHg; P=0.40) were also unchanged during LBNP. Lastly, the change in the proportion of MSNA bursts firing in the largest BP tertile was associated with the change in signal‐averaged sympathetic transduction across each stage of LBNP (r= ‐0.79, ‐0.78, ‐0.81; P≤0.01).

Conclusion

Sympathetic transduction of diastolic BP is unchanged during graded LBNP due to the heterogenous inter‐individual responses in the gating of bursts to diastolic BP. The proportion of bursts firing at high BPs are associated with changes in sympathetic transduction of BP. The determinants of gating MSNA burst occurrence by the arterial baroreflex and its capacity for modulation warrant further study.
AKAP‐mediated regulation of extracellular matrix secretion in cardiac fibrosis
Authors: Paterek Aleksandra , Delaunay Marion , Kaiser Simon , Gautschi Ivan , Diviani Dario
Published: May 2022

Cardiac fibroblasts represent the most abundant cell type in the myocardium and provide structural support through controlled proliferation and extracellular matrix (ECM) turnover. In response to chronic pressure overload, quiescent resident cardiac fibroblasts undergo transdifferentiation to myofibroblasts. These activated fibroblasts display increased ECM synthetic capacity and secrete large amounts of collagens. Excessive deposition of ECM results in massive interstitial fibrosis, which decreases myocardial compliance and affects electrical coupling between cardiomyocytes, causing diastolic dysfunctions and arrhythmias. In this context, deciphering the molecular mechanisms favouring increased ECM secretion and deposition in stressed hearts might be the key for the development of targeted approaches inhibiting excessive myocardial fibrosis and dysfunction.

A‐kinase anchoring proteins (AKAPs) are molecular scaffolds that act as signal organizers. By recruiting and coordinating the activity of signalling enzymes in space and time they control physiological and pathological responses in cardiac cells. In this respect, our current findings suggest that AKAPs directly regulate the activity of the pathway that controls intracellular ECM protein transport and secretion in myofibroblasts. We are currently evaluating the use of molecular disruptors of selected AKAP complexes to reduce collagen secretion. These disruptors will be tested for their ability to prevent fibrosis and myocardial dysfunction in response to pressure overload.
Glial cell‐Derived, but Not Neuron‐Derived, Extracellular Vesicles May Serve as Novel Biomarkers of Acute Sport‐Related Concussion
Authors: Rath Meghan , Sayoc Jacqueline , Burns Karlee , McDevitt Jane , Fan Xiaoxuan , Tierney Ryan , Wu Jingwei , Park Joon Y.
Published: May 2022

Extracellular vesicles (EVs) are released by cells of the central nervous system as a result of injury, including sport‐related concussion (SRC). Neurons, astrocytes, and oligodendrocytes have been shown to release EVs following brain injury and during pathological processes. The OBJECTIVE of this study was to evaluate circulating glial cell‐derived and neuronal‐derived EVs as novel biomarkers for acute SRC.

METHODS

Circulating EVs containing astrocyte‐specific glial fibrillary acidic protein (GFAP+EV), oligodendrocyte‐specific myelin oligodendrocyte glycoprotein (MOG+EV), and neuron specific enolase (NSE+EV) were analyzed in contact‐sport athletes with or without SRC (18‐29 yo, Control: n = 10, SRC: n = 7). Sodium citrate‐treated blood samples were obtained from athletes with SRC within 48‐hours of injury and from control athletes free of SRC for more than one year. Samples were double‐centrifuged to obtain platelet‐poor plasma and stored at ‐80°C until analyzed. Quantification of EVs was performed using a spectral flow cytometer. The study was approved by Temple University’s IRB, and all athletes provided written informed consent. Shapiro‐Wilk tests were used to determine if concentrations were normally distributed. Independent t‐tests were used to compare group means if samples showed normal distribution. Mann‐Whitney U tests were conducted if samples were not normally distributed. For correlational analysis, Pearson’s correlational coefficient was used for samples with normal distribution, while Spearman’s rank was used for nonnormally distributed samples. The Eta coefficient was used to assess correlations between gender and EV concentrations.

RESULTS

We found that plasma concentrations of GFAP+EVs and MOG+EVs were significantly elevated in athletes with SRC within 48 hours of injury (GFAP+EVs t‐test: t = 5.77, p < 0.0001; MOG+EVs Mann‐Whitney U = 2.00, p = 0.0004) compared to athlete controls. Plasma concentrations of NSE+EVs were not significantly different between athletes with acute SRC and athlete controls (t‐test: t = 0.20, p = 0.85). None of the EV concentrations were significantly correlated with history of prior SRC, age, or gender.

CONCLUSION

. Glial cell‐derived EV concentrations were significantly higher among athletes with acute SRC compared to controls, while neuron‐derived EVs did not differ. Our results suggest that analysis of brain cell‐specific EVs may shed new light on a way to leverage EV‐based biomarker discovery for SRC.
Biochemical Characterization of the Sexually Dimorphic Helicases DDX3X and DDX3Y
Authors: Owens Michael C. , Yanas Amber , Shen Hui , Zhang Celia , Liu Kathy F.
Published: May 2022

DEAD‐box helicases play a crucial role in the metabolism of cellular RNAs. One key member of this family is the helicase DDX3X, which is encoded on the X chromosome. Recently, the Y chromosome‐encoded homolog of DDX3X, DDX3Y, has been shown to be expressed at the protein level in several tissues, such as in the heart and in nervous tissue. Herein, we provide the first measurements of the catalytic activity of DDX3Y as it compares to DDX3X. Using both continuous and single time point ATPase measurements, we found DDX3Y to be a less active ATPase than DDX3X. Furthermore, using truncated forms of both proteins, we find that the intrinsically disordered regions (IDRs) of both proteins affect their ATPase activities to different degrees. Because the ATPase activity of DEAD‐box proteins is known to be RNA‐triggered, we also assayed the RNA binding propensity of these proteins. We found that, in agreement with our ATPase data, DDX3Y binds double stranded RNA less tightly than DDX3X. These findings may prove important to human health and disease, given that nearly half the population has a Y chromosome and thus expresses some amount of DDX3Y protein.
The scaffold protein Shoc2 controls ERK1/2‐driven neural crest development by balancing the expression of extracellular matrix components
Authors: Norcross Rebecca G. , Abdelmoti Lina , Landestoy Daileen , Tussey Olivia , Galperin Emilia
Published: May 2022

Dysregulation of the extracellular‐signal‐regulated kinase 1/2 (ERK1/2) pathway results in common developmental syndromes – RASopathies. Our lab studies a RASopathy called Noonan syndrome with loose anagen hair (NSLAH). This RASopathy is caused by mutations in a critical regulator of the ERK1/2 pathway, Shoc2. Patients harboring shoc2mutations have structural deficiencies common among RASopathies (e.g., altered pigmentation, craniofacial defects, valve disorders). Many NSLAH features could involve defects in the development of neural crest (NC).

Zebrafish provide an excellent system for studying neural crest development because of their transparency and highly conserved molecular pathways. Therefore, we utilized the zebrafish model to study the role of Shoc2‐mediated signals in NC development. We found that the loss of Shoc2 affects the expression of the transcription factors prdm1a, pax7, and foxd3‐ all required for the neural plate border formation in zebrafish. The loss of Shoc2 also led to the irregular expression of sox10, the global regulator of NC formation. Moreover, embryos depleted of Shoc2 displayed altered expression of dlx2a, a key transcription factor for the maintenance of cranial NCCs and their migration. We observed that loss of Shoc2 impaired the expression of the transcription factor inducing commitment to cartilage fate, promoting the activation of cartilage‐specific genes in the forebrain and the pharyngeal arch regions, and regulating chondrocyte stacking, sox9a. Moreover, the loss of the Shoc2‐ERK1/2 axis hindered the terminal differentiation of NCCs and resulted in the reduced number of NCC‐derived Schwann cells and pigment cells, including iridophores. Finally, our global assessment of the transcription programs affected by the loss of the Shoc2‐ERK1/2 signals revealed striking differences in the expression of a significant number of extracellular matrix proteins and proteoglycans, including collagen2aI, and aggrecans a and b ‐ all of which are critical for chondrocyte differentiation and maturation.

Together, our data suggest that the Shoc2‐ERK1/2 signaling axis is critical for the development, specification/differentiation, and morphogenesis of the NC‐derived tissues. Our study is the first to define that Shoc2‐mediated ERK1/2 signals regulate NC development. Future inquiries into the mechanisms by which Shoc2 controls embryogenesis will significantly advance our understanding of the mechanisms underlying pathological causes of NSLAH and, ultimately, other RASopathies.
Passive Heating after Heat‐ and Exercise‐Induced Collapse in Mice Induces a Severe Exertional Heat Stroke Phenotype that More Accurately Recapitulates Human Cases
Authors: Henderson Chloe G. , Plamper Mark L. , Mayer Thomas A. , Ward Jermaine A. , Ward Shauna M. , Leon Lisa R. , Oki Kentaro
Published: May 2022

Development of a mouse exertional heat stroke (EHS) model that more accurately recapitulates human EHS cases is essential in characterizing the pathophysiology of the condition and determining potential treatment options for heat stroke sequelae in humans. Previously developed murine heat illness models were either done passively [with or without anesthesia] or had an exertional component with little to no mortality and rapid recovery of mice following removal from the high‐temperature environment, effectively modeling a less severe heat illness, exertional heat injury (EHI). Accordingly, we examined whether leaving mice undisturbed in their environment following heat illness collapse results in an EHS model with a level of mortality that more accurately recapitulates the human condition. Trained mice were placed in a forced‐running wheel that gradually increased from low‐to‐moderate speed (3.0 to 8.5 m/min, increasing 0.5 m/min every 10 minutes). Ambient temperature was set at 25°C for Exercise Controls (ExC) and 37.5°C for EHI and EHS specimens while humidity was set at 30‐70% for all conditions. EHS and EHI mice ran in the wheels until losing consciousness; EHI mice were immediately returned to their home cage (25°C) while EHS mice and a conscious ExC counterpart were left undisturbed in their respective chambers for an additional 20 minutes before being returned to their home cages. Overall, higher mortality rates were observed in the EHS mice (14/63) compared to EHI (0/54) and ExC (0/52) mice. A subset of the mice were sacrificed at 48 hours following the exercise/heat bout (n=10‐12/group); in these mice, student’s T‐tests indicated that serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was elevated in EHS vs. ExC while AST was elevated in EHS vs. EHI, suggesting EHS mice exhibited the most hepatic damage. We have developed a protocol to more accurately model human EHS in mice. Our working hypothesis is that heat 1) in the ambient environment and 2) produced by exertion lead to increased core body temperatures in mice that initiate tissue / organ damage. Inhibiting heat dissipation by leaving mice undisturbed in the warm environment exacerbates tissue / organ damage, resulting in a systemic inflammatory response and mortality in some cases.
Uterine Fibroids ‐ Why Aren't We Talking About Them?
Authors: Wallace Kedra , Bouldin Sheila
Published: May 2022

Problem

Uterine fibroids (UFs) are smooth muscle tumors that are located in the uterus. Even though these tumors are mostly benign, they are the most common gynecological disorder; estimated to affect 70‐80% of women by the age of 50. Black women are disproportionately affected by UFs as UFs appear earlier in black women, the symptoms are often more severe and black women often wait longer to receive treatment and have more surgical complications relative to non‐black women. The epidemiology for UFs is unclear, however being of black race, hypertensive, obese, and pre‐menopausal increases the risk of developing UFs.

Data

Data from the COMPARE‐UF (Comparing Options for Management: Patient‐centered Results for UF) multi‐site observational registry indicated that black women with UFs who received hysterectomy’s (uterus removal) for treatment had a lower health related quality of life (HRQoL) compared to white women receiving the same procedure. In a separate analysis from this same population we also published data indicating that black women with UFs were more likely to have a surgery that was not a hysterectomy, unless they were treated in Mississippi (MS). We have previously published data from patients treated for UFs in MS and have reported that relative to women with abnormal uterine bleeding but no UF’s, women with UFs had significantly more hypertension but no change in body mass index. We also reported that circulating factors associated with hypertension (ET‐1 and sFlt‐1) were significantly increased among women with UF's relative to women with abnormal uterine bleeding.

Rationale

As MS ranks among the highest in the nation for hypertension, and black people per capita the likelihood for an increased prevalence of UFs is high. The combination of these studies and our own studies indicate that black women with UFs who reside in the rural South may be more affected and have lower HRQoL to black women with UFs who live in more urban areas. While the medical and surgical options are limited for UFs, data is beginning to emerge indicating geographical differences that elude to a different problem.

Proposed Solution

Here in MS, we have proposed a patient navigator program (EMPOWER‐UF) to help educate women on the different treatment options available and to help women navigate the health care system. It is our belief that the combination of a patient navigator and a patience decision aid, adapted for women with UFs, could increase health literacy and improve the shared decision making process. The combination of these events will lead to an increased satisfaction in decision making regarding UF treatment choice and an increase in HRQoL.
Structural and Biophysical Mechanisms Driving Differential Hormone Response in Steroid Receptors
Authors: Khan Sabab H. , Braet Sean M. , Koehler Stephen J. , Elacqua Elizabeth , Anand Ganesh S. , Okafor Denise
Published: May 2022

Steroid receptors (SRs) belong to the superfamily of ligand‐activated transcription factors that regulate a myriad of biological processes upon binding of cholesterol‐derived hormones. A poorly understood aspect of this transcriptional regulation is how diverse steroid hormones modulate the ligand binding domain (LBD) of steroid receptors to achieve selective transcriptional outcomes. Here we investigate hormone specificity in the ancestral steroid receptor 2 (ancSR2) which is transcriptionally activated by 3‐ketosteroid hormones and unresponsive to 3‐hydroxylated, A‐ring aromatized steroids (i.e., estrogens). Previous work uncovered evolutionarily conserved ligand sensing‐residues in the binding pocket of LBD which also enables hormone discrimination. Specifically, the L42 (Helix3)‐M75 (Helix5) interaction was predicted to enable discrimination between 3‐ketosteroids and estrogens. To further probe the role of these ligand sensors in mediating differential hormone responses, we mutated M75 to modulate the Helix3‐Helix5 interaction and determined how transcriptional function, protein structure and dynamics are impacted. Specifically, we have combined site‐directed mutagenesis with biophysical experiments, cell‐based assays, and molecular dynamics (MD) simulations. While our secondary structure measurements reveal minor impacts on structure, MD simulations reveal that M75 mutations modulate the status of the H3‐H5 interaction, subsequently affecting ancSR2 conformations in a hormone‐dependent manner. Additionally, hydrogen deuterium exchange‐mass spectrometry (HDX‐MS) measurements reveal conformational effects at regions distant from the mutation site that may modulate ancSR2 inter‐residue interaction networks for differential hormone response. The differential hormone response is supported in luciferase reporter assays as well as ligand binding assays. These studies will provide biophysical and structural insight into how steroid receptors achieve hormone‐specific transcriptional responses.
Transitioning CUREs to Undergraduate Research Experiences
Authors: Hernandez Sebastian , Pieczynski Jay
Published: May 2022

Inquiry based and/or course‐based undergraduate research experiences (CUREs) have been repeatedly demonstrated as high impact practices in biology education for a host of factors including increased student engagement, retention, and building a general interest in science. When deployed in entry‐level courses, CUREs also have the benefit to cultivate strong working relationships between student and mentor, allowing the faculty member to recruit interested students to continue the project beyond the scope of the course. In the Fall of 2019, we updated our curriculum to incorporate molecular/cell biology CUREs into 100‐level courses with the goal of increased student engagement and research interest. Here we describe the process of successfully recruiting students and the transition from their previous strictly coursed‐based research experience to that of an independent research experience. Numerous challenges and changes occurred between the boundaries of the classroom and the research lab. Through our experience, we hope to provide a template for both students and faculty to consider using CUREs as a mechanism to recruit and train student researchers over the duration of their undergraduate careers.
Functional biodynamics theory and algorithms for inhibitor or activator effectors and their interactions, by computer simulation bioinformatics for translational medicine
Authors: Chou Ting‐Chao
Published: May 2022

The conventional bottom‐to‐up approach of R&D is usually: aims, design, findings, data analysis, statistics, and conclusions or suggestive hypothesis. The experimental findings need to fitting empirical curves, formula, or model. The alternative nonconventional, top‐to‐down approach,shown here, is the mathematical system analysis to derive the unified general pharmacodynamics theory/equation/algorithm (PD) based on mass‐action law (MAL)for automated computer simulation. Thus, the approach allows using small number of dose‐data–points to fit the unified MAL‐PD theory/algorithm, for the efficient, cost‐effective automated computer simulation to achieve quantitative/ indexed bioinformatics (BI) diagnostic simulations and conclusions. The median‐effect equation (MEE) or its plot (MEP)is the unified general theory of the MAL fordose‐effect biodynamics/pharmacodynamics (BD/PD), where “dose” can be drugs, biologicals, hormones, modulators, infecting agents, radiation, or UV; and the ”effect” can be inhibitors, activators, in cells, tissues, organs, diseases, animals or in clinical trials. The MAL algorithms also leads to the derivation of the general combination index equation (CIE) with its Fa‐CI plot and Isobologram for BI, that allow the quantification of Synergism (CI<1), Additive effect (CI=1) and Antagonism (CI>1) at different‐effect, or different effect levels. The unique features of MAL‐PD approach are: (A) All terms of the MEE and CIEof the MAL‐PD equations are “dimensionless relativity ratio”,therefore, MAL‐theory is validregardless of units, mode or mechanism of actions of effector(s), and the target systems. (B) The MEE with its MEP, linearizes dose‐effect curves into straight lines,and thereby MEP leads to the “Two Dose‐Data‐Points Minimum Theory”,where the 3rd point is dose zero, and the 4th dose‐point is Dm, which is the universal reference‐point and dynamic‐order common‐link. Thus, particularly useful in animal studies or clinical trials which rarely tolerated over 3 (or more) “dose‐data points” with acceptable dose‐rage and dose density; (C)MAL theory/algorithm allows the samegeneral MAL‐principle to bridge between the basic in vitro/animal studies and the clinical trials. In conclusion, the MAL‐BD/PD/BI/CI approach/theory/algorithm/method serves as the general largest‐common‐denominator forsimplifying the complexity and diversity of biological systems, with automated simulations. This MAL “top to down” innovation provides a new avenue forbiomedical R&D, and for drug evaluations, which is simple, quantitative, efficient and cost‐effective.
19F‐NMR shows that active site aromatic residues in CYP121 of Mycobacterial tuberculosisplay a dual role in substrate interaction and protein structure
Authors: Campomizzi Christopher S. , Estrada D. Fernando F.
Published: May 2022

CYP121 is an essential cytochrome P450 enzyme (CYP) from Mycobacterium tuberculosis.CYP121 mediates an atypical phenol‐coupling reaction that converts cyclodityrosine (cYY) into mycocylosin. Our lab has previously employed a 19F nuclear magnetic resonance (NMR) label, 3‐bromo‐1,1,1‐trifluoroacetone (BTFA), to study dynamics of the FG‐loop, a region connecting the F and G‐helices. In other CYPs, the FG‐loop region has been implicated in active site access and substrate recognition. The 19F‐NMR spectrum for the FG‐loop in CYP121 is heterogeneous, suggesting that in solution there are multiple conformations. Upon titration of cYY, the signal converges to a single peak representing a bound state. Interestingly, this labeling site exists approximately 20 angstroms away from the active site heme, where ligand binding is typically measured in CYPs by UV‐visible spectroscopy. Crystal structures show an invariable protein structure with ligands clustered near the FG‐helices, away from the heme.

Here we investigate the specificity of this interaction site by expanding our use of 19F labeling. Supplementing cells with 5‐fluoroindole (5‐FI) during recombinant protein expression, 19F was incorporated into each tryptophan residue of CYP121. This method of labeling allowed for four tryptophan residues to be monitored, which span the entire protein structure. Notably, two residues, Trp‐182 and Trp‐380, are located adjacent to the CYP121 homodimer interface. The 19F‐NMR spectrum contains three distinct signals, two of which are equal in intensity, and a third which has been determined to be two overlapping resonances. Addition of cYY causes a shift of equilibrium in the spectra, with the overlapped resonances resolving and resulting in four peaks of roughly equal area and intensity. These results suggest that only the active site residue Trp‐182 is affected by the addition of cYY. Using this labeling scheme, we also tested alanine substitutions of Trp‐182 and another active site aromatic residue, Phe‐168. 19F‐NMR spectra from these mutants indicate a general perturbation of the structure, with only one Trp residue left unaffected. Since Trp‐182 extends into a hydrophobic pocket of CYP121 (suggesting a structural role), additional substitutions were made using tyrosine and leucine side chains. The conservative W182Y mutant yielded similar disorder as seen when replaced with alanine, while W182L results a partially restored spectrum. Interestingly, both mutants bind substrate in a similar manner as wild‐type when measured by UV‐visible spectroscopy, although with weakened affinity.

These findings indicate that while Phe‐168 and Trp‐182 are involved in substrate interaction, they also play a role in the fold of the protein, with mutagenesis causing long range changes in structure.
The benefits of incorporating active learning into online, asynchronous coursework in dental physiology
Authors: Metz Cynthia J. , Metz Michael J.
Published: May 2022

The COVID‐19 pandemic required a shift to online learning that posed particular challenges for the education of dental students. Basic science courses were presented in an online, asynchronous format to facilitate small group rotations through preclinical work. The success of active learning was previously examined in a Dental Physiology course, and it was desired to reassess those methods in an online environment. It was unclear whether the loss of the collaborative opportunities during the active learning sessions would negatively impact student perceptions or performance. In the asynchronous, online environment, students were unable to easily share their ideas with their classmates or ask questions of the lecturer in real time. The 120 students enrolled in the first‐year Dental Physiology course were provided with pre‐recorded sections of content containing learning activities, and the effectiveness was compared with sections of traditional lecture recordings. On surveys, dental students had more positive perceptions of online recordings that incorporated active learning strategies, with higher perceived effectiveness of the lecturer, ability to pay attention, motivation to study, and confidence with the material. This was similar to the previous study, in which face‐2‐face (F2F) active learning was more positively reviewed than traditional lectures. Compared with these past sections, the online cohort expressed similarly positive perceptions of the active learning sections, but the online traditional lectures were perceived to be less effective than F2F lectures. Despite the differences in student perceptions, unit exam scores were similar for traditional didactic lectures in both online and F2F settings (P=1.000, one‐way ANOVA). In the online setting, active learning exam scores were 8.13% higher than traditional lecture recordings (P<0.001, one‐way ANOVA and Bonferroni post‐hoc test). However, the previous F2F active learning produced significantly higher scores than online active learning on unit exams (P=0.04). While active learning strategies continue to be effective, further research is needed to optimize these methods and engage students in online coursework. The results suggest the importance of active and collaborative learning opportunities in the education of students in physiology coursework.
The Role of Heat Shock Proteins in Amyloid Body Reversal
Authors: Kirk Chloe C. , Bokros Michael , Balukoff Nathan , Theodoridis Phaedra , Moore Taylor , Grunfeld Alex , Rech Tondin Arthur , Marshall Maddison , Lee Stephen
Published: May 2022

The ability of cells to adapt to a wide variety of stress conditions plays a critical role in various physiological and pathological settings, including development, cancer and neurological disorders. We recently reported the surprising discovery of stress‐induced low complexity noncoding RNA derived from stimuli‐specific loci of the ribosomal intergenic spacer (rIGSRNA); an enigmatic region of the human genome historically dismissed as “junk” DNA. We showed that low complexity rIGSRNA activate a physiological amyloidogenic program that converts the nucleolus into Amyloid Bodies: a molecular prison of immobilized proteins in an amyloid‐like state. This conserved post‐translational regulatory pathway enables cells to rapidly and reversibly store an array of endogenous proteins in Amyloid‐bodies and enter quiescence in response to severe environmental insults. While many membrane‐less compartments have been described as liquid‐like (e.g., stress granules, P‐bodies, germ cell granules), our discovery of Amyloid‐bodies provided evidence of an amyloidogenic process that can physiologically transition biological matter to a solid‐like state. The ability of mammalian cells to efficiently dissemble Amyloid‐bodies raises a fundamental question: Are mammalian cell disaggregases involved in Amyloid‐body disassembly? Here, we show that Amyloid‐bodies undergo a multi‐step process solid‐to‐liquid‐phase transition to release sequestered proteins and restore nucleolar functions. An RNAi screened identified key heat shock proteins (hsps) that remodel Amyloid‐bodies back to the liquid nucleoli on stress termination. The composition of mammalian disaggregases differs considerably from non‐metazoans and those involved in disassembly of pathological amyloids in vitro. Activation of mammalian disaggregases and Amyloid‐body disassembly is dependent on ATP concentration. Conceptually, this work identifies metazoan disaggregases challenging the widely accepted paradigm that the amyloid state is irreversible in mammalian cells. The data also provides alternative insights into pathogenic amyloids by examining their disassembly in cellular systems.
Microautophagy catabolizes lipid droplets in liver hepatocytes via the small GTPase Rab5 and is connected to alcoholic fatty liver
Authors: Rozeveld Cody N. , Mehall Bridget , Casey Carol , McNiven Mark , Schott Micah
Published: May 2022

Introduction and Objective

Alcohol consumption has increased dramatically in recent years, representing a significant challenge to human health in metabolic disease. The leading cause of alcohol‐related death is alcohol‐associated liver disease (AALD), which is present at early stages (i.e. steatosis) in 90% of heavy drinkers. During early AALD, liver hepatocytes accumulate large stores of triglycerides in the form of lipid droplets (LDs). Under normal conditions, degradative lysosomes form direct contacts with LDs and catabolize lipid via microlipophagy, a specialized autophagic pathway utilizing the lipid‐degrading enzyme lysosomal acid lipase (LAL). However, the trafficking mechanisms that govern LD‐lysosome contacts during microlipophagy are poorly understood. Several promising candidates exist within the Rab family of small GTPases, which are well‐known to direct the trafficking of intracellular vesicles and LDs. Moreover, previous studies suggest that Rab proteins play central roles in LD catabolism. Thus, the OBJECTIVE of this study was to investigate novel Rab proteins that might be impaired by EtOH leading to altered LD dynamics and microautophagy.

Materials and Methods

To this end, we utilized RNAi gene silencing, subcellular fractionation, Western blot analysis, and high‐resolution microscopy to define Rabs that might target LDs to lysosomes during microlipophagy. To mimic alcoholic fatty liver, cellular and animal models of alcohol consumption were also used. Changes in LD‐lysosome interaction were measured by quantitative confocal microscopy following pharmacological inhibition of LAL by LAListat.

Results

First, confocal microscopy revealed that GFP‐Rab5, an early endosomal Rab GTPase, localizes to hepatocellular LDs consistent with proteomic and Western blot analyses showing abundant levels of Rab5 in biochemically isolated LDs. Importantly, siRNA knockdown of Rab5 in cultured hepatocytes showed substantial increases in total LD area (2‐fold) and LD number (1.6‐fold), and also blocked the catabolism of LDs in lipid‐loaded cells. Finally, Rab5 knockdown nearly completely abolished microlipophagy as measured by fluorescent LD accumulation in lysosomes following LAListat treatment. The dissociation of LDs from lysosomes in Rab5 knockdown cells was strikingly similar to the defect observed in EtOH‐fed hepatocytes. Together, these findings suggest that Rab5 is a key mediator of targeting LDs to the lysosome compartment for catabolism and may be a target of EtOH insult.

Significance/Implication

This study sheds light on new molecular mechanisms of microlipophagy that are affected by chronic alcohol consumption. These mechanisms may represent promising avenues for future therapeutic development toward mitigating hepatic steatosis and slowing the progression of AALD.
Characterizing Murine Diabetic Glucose Metabolism Under Conditions of Attenuated Renal Glucose Reabsorption
Authors: Hotait Zahraa S. , Shepard Blythe D.
Published: May 2022

While type 1 (T1DM) and type 2 (T2DM) diabetes mellitus differ in etiology, both are metabolic disorders characterized by chronic hyperglycemia; if left untreated, this can lead to serious complications including renal failure. Glucoregulation is critical to meeting tissues’ energy needs without exceeding adaptive glucose levels and involves several metabolic pathways including gluconeogenesis (occurring in the kidney and liver) and glycolysis, which are primarily regulated through certain rate‐limiting enzymes involved in the committed steps of each pathway. Beyond its metabolic contributions, the kidney also mediates glucoregulation through the process of glucose reabsorption in the proximal tubules (PT); crucial symporters known as sodium glucose cotransporters (SGLTs) move glucose into PT epithelial cells to facilitate its return to the bloodstream rather than excretion in the urine. Inhibiting renal glucose reabsorption via the modulation of SGLTs has been linked to improved diabetic outcomes, yet the impacts of this intervention on whole body glucose metabolism have not been fully characterized. Thus, we aimed to determine if SGLT modulation alters expression of rate‐limiting glucometabolic enzymes under conditions of T1DM and T2DM. Firstly, we compared renal and hepatic tissues isolated from nondiabetic and type 1 diabetic Olfactory Receptor 1393 (Olfr1393) knockout (KO) and wild type mice; Olfr1393 is a GPCR that has been previously demonstrated to impact localization of glucose transporters, and Olfr1393 KO mice exhibit improved diabetic phenotypes (Shepard et al., 2019; Schiazza et al., 2021). Secondly, hepatic tissues from T2DM TallyHo (TH) mice fed a milk fat diet with or without the SGLT2 inhibitor Empagliflozin were analyzed. Initial analysis of qPCR and Western blot data from each condition reveals diabetic differences in glucometabolic enzyme expression within each group; under conditions of T1DM, gluconeogenesis (via expression of glucose 6‐phosphatase) appears to be decreased in both the kidney and liver. In addition, we noted reciprocal regulation of glycolytic enzymes PKM 1 and 2; in the kidney, T1DM decreases PKM1 expression but upregulates PKM2. However, the data collectively suggest that SGLT modulation does not alter renal or hepatic glucose metabolism. Further in vivo exploration may elucidate patterns of similar or differential glucoregulation that will contribute to our current understanding of the body’s dynamic glucometabolic profile in the context of diabetes and the efficacy and whole body impacts of applying SGLT inhibition in the treatment of these disorders.
Experience in the application of remote anesthesia in Callorhinus ursinus
Authors: Nikitina Anastasia , Gaponova Viktoriya , Bukina Lidiya , Trushkin Vyacheslav , Burkanov Vladimir
Published: May 2022

Marine mammals often become entangled in various objects of anthropogenic origin floating on the surface of the water. Plastic, polyethylene, synthetic packing straps, and fishing gear threaten marine mammals in the water and on land. The study was carried out on 29 specimens of Callorhinus ursinus. We used a combined Zoletil 100 and Medetin 1% for remote sedation of injured Callorhinus ursinus to release foreign objects.

Results

the first signs of anesthesia were recorded 3‐5 minutes after drug delivery. The difficulty of movement, coordination disorder, weakness were noted. After 5‐15 minutes, the animals lay down but reacted to touch, and their eyes were open. After 15‐20 minutes, the animals lay motionless, practically not reacting to external stimuli. Their eyes were in a half‐closed state. In this state, it is possible to carry out manipulations to remove foreign objects from the animal. The depth of narcotic sleep varied depending on the dose: mild sedation developed at Medetin 1% 0.02‐0.03 mg/kg and Zoletil 100 0.45‐0.65 mg/kg. The animals were lying down, we could approach them, but they reacted to touches and loud sounds. The eyes were open, and it was dangerous to carry out any manipulations with such animals. The seals did not fall into a deep sleep; after 30‐60 minutes without the use of antagonists, they began to move, but there was a slight impairment of coordination of movements for 1‐2 hours. Moderate sedation was observed at a dose of 0.03‐0.05 mg / kg Medetin 1% and 0.5‐0.9 mg / kg Zoletil 100. The animals lay motionless; sometimes, there was a slight twitching of their fins, whiskers, or ears during manipulation of slight pain. Eyes half‐closed. It is possible to carry out small short‐term manipulations with the animal to remove a foreign object without strong painful sensations. After 30‐60 minutes, the seals wiggled their flippers and tried to raise their heads. They began to move independently after 1‐1.5 hours. Slight impairment of coordination was noted 1.5‐2 hours after full awakening. At a dose of 0.05‐0.09 mg/kg Medetin 1% and 0.8‐2.0 mg/kg Zoletil 100, the animals lay motionless and did not react to external stimuli, even during painful manipulations or movement. This sedation is deep, suitable for painful procedures to remove foreign objects with deep skin lesions. Without the use of antagonists, such animals woke up after 1‐1.5 hours. They began to move their flippers, mustache, move around after 2.5‐3 hours. Determining the optimal doses of drugs for remote anesthesia of marine mammals is essential, both for the safe release of foreign objects from the animals and also for the people who perform this work.
Phrenic Motor Neuron Fractalkine Expression After Intermittent Hypoxia and Spinal Cord Injury
Authors: Burrowes Kayla A. , Michaelson Alysha L. , Seven Yasin B. , Mitchell Gordon S.
Published: May 2022

Acute intermittent hypoxia (AIH) elicits spinal respiratory motor plasticity, and has emerged as a promising therapeutic approach to restore lost respiratory (and non‐respiratory) motor function following cervical spinal cord injury (cSCI). Conversely, most individuals with cSCI experience sleep disordered breathing, generating chronic intermittent hypoxia (CIH) at intensities sufficient to trigger pathology. Since glia play a critical role in regulating neuroplasticity, they may play a key role in differentiating responses to therapeutic (AIH) versus pathogenic (CIH) intermittent hypoxia. Motor neuron‐microglial interactions regulate AIH‐induced phrenic motor plasticity. Specifically, phrenic motor neuron fractalkine, a chemokine unique to neurons released during hypoxia activates its high affinity receptor, CX3CR1—which is unique to microglia in the CNS. In response, microglia release ATP/adenosine, inhibiting AIH‐induced phrenic motor plasticity. Thus, if intermittent hypoxia or cSCI impact phrenic motor neuron fractalkine expression, they may directly impact plasticity and the potential of therapeutic AIH. In this study, we tested the hypothesis that phrenic motor neuron fractalkine expression is attenuated by daily AIH (but not CIH) in spinally intact rats, but not in rats with cSCI. Fractalkine was assessed in phrenic motor neurons of male rats with/without C2 hemisection (12 weeks post‐injury) exposed to 28 days of: 1) normoxia; 2) daily AIH (10, 5‐min episodes per day, 10.5% O2; 5‐min intervals); and 3) moderate CIH (2‐min 10.5% O2 episodes; 2‐min normoxic intervals; 8 hr/day). Fourteen days before hemisection, intrapleural Cholera toxin B injections were made to retrogradely label phrenic motor neurons. Fractalkine expression was assessed in 40 mm sections using a custom MATLAB algorithm to assess immunofluorescence optical density within labeled phrenic motor neurons. Spinally intact and injured rats have differential responses to daily AIH vs CIH. Whereas injured rats had no change in phrenic motor neuron fractalkine expression after daily AIH or CIH, fractalkine expression was marginally reduced in intact rats following daily AIH (p = 0.07; cohen’s d= 132%), but not CIH (p = 0.88; cohen’s d= 23%). Thus, daily (therapeutic) AIH may decrease motor neuron fractalkine and microglial adenosine release, thereby enhancing moderate AIH‐induced phrenic motor plasticity.
Electrostatic Fingerprints of Catalytically Active Amino Acids in Enzymes
Authors: Ondrechen Mary Jo , Iyengar Suhasini M. , Barnsley Kelly K. , Xu Rholee , Prystupa Aleksandr
Published: May 2022

The computed electrostatic and proton transfer properties are studied for 20 enzymes that represent all six major EC classes and a variety of different folds. The properties of aspartate, glutamate, and lysine residues that have been previously experimentally determined to be catalytically active are reported. All catalytic aspartate and glutamate residues studied here are strongly coupled to at least one other aspartate or glutamate residue and often to multiple other carboxylate residues with intrinsic pKa differences less than 1 pH unit. Sometimes these catalytic acidic residues are also coupled to a histidine residue, such that the intrinsic pKa of the acidic residue is higher than that of the histidine. All catalytic lysine residues studied here are strongly coupled to tyrosine or cysteine residues, wherein the intrinsic pKa of the anion‐forming residue is higher than that of the lysine. Some catalytic lysines are also coupled to other lysines with intrinsic pKa differences within 1 pH unit. Some evidence of the possible types of interactions that facilitate nucleophilicity is discussed. The interactions reported here provide important clues about how side chain functional groups that are weak Br⊘nsted acids or bases for the free amino acid in solution can become strong acids, bases or nucleophiles in the enzymatic environment. Such interactions are important considerations in enzyme design.
Carotenoids in Orange Carrots Mitigate Against Non‐alcoholic Fatty Liver Disease Progression
Authors: Balbuena Emilio , Cheng Junrui , Eroglu Abdulkerim
Published: May 2022

Background

Carotenoids are naturally occurring compounds found in brightly colored fruits and vegetables. Increased consumption of these dietary sources may alleviate the harmful effects of the high‐fat diet. They can be used as co‐adjuvant against metabolic syndrome (MetS). Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of MetS characterized by the extensive lipid deposition in the liver tissue without significant alcohol intake. Mechanistically, NAFLD results from imbalanced hepatic lipid homeostasis stemming from elevated hepatic triglyceride (TG) uptake, upregulated fatty acid (FA) synthesis, or decreased β‐oxidation.

Objective

The purpose of the study was to illustrate the impact of carotenoid‐rich whole foods on obesity‐associated NAFLD progression.

Methods

Male C57BL/6J mice (n = 48) were randomized to one of four experimental diets for 15 weeks (n=12 animals/diet): Low‐fat diet (LFD, 10% calories from fat), high‐fat diet (HFD, 60% calories from fat), HFD with 20% white carrot powders (HFD+WC), and HFD with 20% orange carrot powders (HFD+OC). The concentrations of α‐carotene and β‐carotene in carrot powders, diet pellets, serum, liver, and feces were detected by analytical assays. Tissue histology and TG content analysis were used to depict the extent of hepatic steatosis. Western blot and qPCR were utilized to observe trends of fatty synthesis and β‐oxidation.

Results

The HFD+OC group reduced weight gain significantly compared to the HFD group and efficiently decreased hepatic lipid accumulation based on the histological and TG analysis. We observed minimal down‐regulation of FA synthesis proteins in the HFD+OC group as depicted by Western blot and qPCR. On the other hand, the HFD+OC treatment significantly enhanced β‐oxidation proteins, possibly by promoting master regulators of hepatic lipid metabolism (i.e., p‐AMPK, PGC‐1α, PPARα).

Conclusions

Carotenoids in orange carrots may decrease the severity of hepatic steatosis in NAFLD by partial inhibition of fatty acid synthesis and increased activity of β‐oxidation.