bluuebunny/crossref_metadata_2025 huggingface dataset:
Training scientists to become effective educators
Authors: Pajoohesh‐Ganji Ahdeah , Blanchard Tracy , Herrmann Debra
Published: May 2022

Research‐intensive faculty are often asked to teach occasional didactic sessions to students at different learning levels, with very little guidance in evidence‐based teaching methods. Although they are teaching students and staff in the laboratory setting on a daily basis, they have likely received little, if any, formal training in pedagogy but are expected to be able to teach effectively. To introduce research‐intensive faculty to different essential tools in teaching, we designed 5 “Scientists as Educators” hybrid sessions with the goal of helping these faculty members become more effective educators. The topics included: 1) writing learning objectives, 2) instructional engagement and assessment, 3) instructional design, 4) multiple choice question writing, and 5) balancing research and teaching. Each hybrid session consisted of a brief online component, including a video and application activity, followed by an hour in‐person session. The in‐person sessions, which were also open via Zoom, were accessible to participants in collaborating institutes, designed to delve within the topics, and allowed for the application of concepts introduced in the online modules. Findings from semi‐structured exit interviews of participants regarding the effectiveness of the sessions, how the sessions helped them in their teaching, and ideas for additional topics that should be included in future sessions will be presented. Our goal for these sessions is to introduce research‐intensive faculty to educational concepts and increase teaching effectiveness throughout our university.
Craniofacial Bone Mineral Density at Muscle Attachment Sites in Mice with Osteogenesis Imperfecta
Authors: Murabito Lauren , Miller Courtney A. , McBride Alexandra H. , Organ Jason M. , Menegaz Rachel
Published: May 2022

Osteogenesis imperfecta (OI) is a genetic disease commonly caused by mutations in the COL1A1 or COL1A2 gene affecting the production of type I collagen, a major component of bone that determines the toughness of bone, or its resistance to fracture. Thus, patients with OI present with brittle bones and higher rates of skeletal fractures. Additionally, defects in the collagen framework of developing bones impair their biomineralization, further compromising bone mechanical properties. Previous work in our lab investigated BMD at cranial locations which experience compressive forces during feeding (e.g. joints). Muscles also contribute to cranial growth through tensile forces exerted on bone during contraction, but the relationship between muscle function and bone quality in OI is unclear. The aim of this study is to quantify the BMD in a mouse model of OI, with a focus on skeletal sites associated with cranial musculature. We hypothesize that the mice with OI will have lower BMD in skeletal areas associated with muscle attachment compared to their unaffected littermates.

To test our hypothesis, we used the homozygous recessive OI murine (OIM) mouse (B6C3Fe a/a‐Col1a2oim/J), a model for the severe type III OI seen in humans. Adult (16 weeks old) OIM mice (n=11) and wildtype (WT, n=13) littermates were CT scanned, and BMD was measured using the Bruker CTAnalyzer software in 7 regions of interest encompassing muscle attachment sites on the mandible, zygomatic arch, and parietal bone. For each region, BMD values were compared between genotypes by a Kruskal‐Wallis test.

OI mice had significantly lower BMD at the coronoid process (p=0.007), parietal bone (p=0.031), and posterior mylohyoid line (p=0.007) compared to WT mice. No significant differences were found in BMD at the gonial angle or zygomatic arch. This suggests a disproportionate weakness of the temporalis and mylohyoid muscles in mice with OI, but not in the masseter muscle. While type I collagen defects impact the structural organization of skeletal muscle, it is possible that masseter muscle function is preserved in OIM mice because of its key roles in rodent feeding and postural control of the jaw. The imbalance between temporalis and masseter muscles may contribute to the prevalence of dental malocclusions in human patients with type III OI.

The findings of this study support further exploration of specific muscle groups (e.g. jaw adductors/temporalis, hyoid muscles/mylohyoid) and efforts to improve their functions during feeding, as a way of recovering facial growth and preventing dental malocclusions in OI. A better appreciation of muscle‐bone interactions in the skull and their effect on bone quality can potentially be used to inform oro‐motor therapies to improve feeding function, muscle strength, and facial bone quality for patients with OI.
Assessment by Multi‐Distance Hyperspectral NIRS of Changes in the Oxidation State of Cytochrome C Oxidase (oxCCO) to Carotid Artery Compressions
Authors: Shoemaker Leena , Suwalski Marianne , Milej Daniel , Shoemaker J. K. , Chui Jason , St. Lawrence Keith
Published: May 2022

Introduction

The occlusion of the common carotid artery (CCA) during carotid endarterectomy (CEA) is associated with a risk of cerebral ischemia due to inadequate collateral blood flow1. By monitoring tissue oxygen saturation (StO2), near‐infrared spectroscopy (NIRS) is frequently employed during CEA2 to determine sufficient oxygen delivery. However, StO2 is only a proxy of oxygen metabolism and an unreliable marker of ischemic injury. In contrast, cytochrome c oxidase (oxCCO) is an enzyme in the mitochondrial electron transport chain and therefore a specific marker of aerobic metabolism3. Thus, the aim of this study was to assess the ability of a multi‐distance hyperspectral (hs) NIRS to monitor changes in StO2 and oxCCO during CCA compressions in healthy participants. We hypothesized that changes in oxCCO concentration would be greater in the brain (i.e., long source‐detector distance, SDD) than in the extracerebral tissue (i.e., short SDD), and this difference would be larger than that of StO2.

Methods

We modified an in‐house built hsNIRS4 to include a second channel for acquiring data at two SDD (1 and 3 cm) to detect potential changes in the extracerebral tissue. Data from 10 young, healthy participants was acquired continuously during one 30‐second unilateral digital CCA compression, performed ipsilateral to the location of the optic probes. In addition, cerebral blood flow index was determined using diffuse correlation spectroscopy and mean arterial blood pressure by finger photoplethysmography.

Results

During CCA compression, the change in oxCCO at 3 cm vs. 1 cm (0.4 ± 0.3 µM vs. 0.06 ± 0.1 µM, p = 0.027) was approximately 50% greater than the change in StO2 at 3 cm vs. 1cm (‐4.0 ± 2.2% vs. 1.2 ± 0.7% p = 0.007). The corresponding decrease in cerebral blood flow was 57 ± 14% (p < 0.001 vs. baseline). Mean arterial blood pressure increased 4 ± 1 mmHg (p < 0.001 vs. baseline).

Discussion

A larger difference between the two distances was observed for oxCCO than StO2, likely due to the greater concentration of mitochondria in the brain compared to extracerebral tissues. Collecting multi‐distance hsNIRS demonstrated the enhanced sensitivity of oxCCO to the brain. Lastly, this study demonstrates the utility of momentary unilateral CCA compressions as a fast, easy, and strong perturbation for transiently altering cerebral inflow and metabolism.

References

(1) Aceto, Eur. J. Anestesiol.(2020); (2) Yu, Cochrane Database Syst. Rev.(2014); (3) Bale, J. Biomed. Opt. (2020); (4) Rajaram, Brain Sci (2020).
Discerning the role of IP3R1 activation by Gq/G11 signaling in murine lymphatic collecting vessel pacemaking
Authors: Zawieja Scott D. , Li Min , Castorena Jorge , Drumm Ben , Davis Michael
Published: May 2022

Collecting lymphatic vessels (cLVs) exhibit spontaneous contractions with a pressure‐dependent frequency that increases with elevations in intraluminal pressure. Our recent work posits a putative role for lymphatic muscle cells (LMCs) as the pacemaker cell that regulates this pressure‐dependent phenomenon through activation of the Ca2+‐activated chloride channel anoctamin 1 (Ano1) in an IP3R1‐dependent process. However, the specific mechanisms underlying IP3R1 activation and SR Ca2+ release in lymphatic muscle cells is unknown. We tested the hypothesis that cLV pressure‐dependent pacemaking by the IP3R1‐Ano1 axis is downstream of the canonical Gq/G11‐PLC‐β signaling pathway. To this end, we crossed mice with the smooth muscle‐specific tamoxifen‐inducible Cre, MYH11CreERT2, to the fluorescent reporter ROSAmT/mG and sorted GFP+ LMCs from an isolated murine inguinal‐axillary (IngAx) lymphatic cLVs. Murine lymphatic muscle cell expression of Gq and G11 were assessed via RT‐PCR. We crossed MYH11CreERT2 mice with IP3R1fl/fl or G11KO/KO‐Gqfl/fl mice to achieve IP3R1ismKO (MYH11CreERT2‐IP3R1fl/fl) and double G11KO‐GqismKO (MYH11CreERT2‐ G11KO/KO‐Gqfl/fl) mice. We tested the function of the IngAx cLVs isolated from these mice, as well as their respective heterozygous WT/KO and fl/fl controls, using isolated vessel isobaric myography. Ca2+ imaging using IP3R1ismKO‐GCaMP6f mice, and membrane potential recordings using sharp electrodes. IngAx cLVs from G11KO‐GqismKO mice displayed a significant reduction in contraction frequency and vessel tone, however a pressure‐dependent increase in frequency was observed. Similarly, IngAx cLVs isolated from IP3R1ismKO mice had significantly lower vessel tone, a significant reduction in contraction frequency at all pressures tested, and a significantly higher contraction amplitude. Critically, contraction frequency in IngAx cLVs isolated from IP3R1ismKO mice did not increase with pressure in contrast to IngAx cLVs isolated from G11KO‐GqismKO mice and wildtype mice. In wildtype (WT) MYH11CreERT2‐GCaMP6f controls, Ca2+ puffs and/or Ca2+ waves were observed in the majority of LMCs during the diastolic period, intervening the bright global Ca2+ flashes indicative of Ca2+ entry through L‐type Ca2+ channels during the LMC action potential (AP). These local diastolic Ca2+ transients (CaTs) persisted in the presence of nifedipine, and the frequency and spatial spread of CaTs increased with pressure. Diastolic CaTs were largely absent in LMCs of IP3R1ismKO mice, and their diastolic depolarization recapitulated the lower rate observed in previous studies of Ano1smKO cLVs. However, APs recorded in IP3R1ismKO significantly increased AP plateau duration that corresponds with an increased Ca2+ flash duration. We conclude that the Gq/G11 dependent signaling is involved in pressure‐dependent pacemaking in murine cLVs, particularly at low pressure, but their deletion is insufficient to reproduce the IP3R1ismKO phenotype.
Leave it to the Learners … Student Perspectives on the Effectiveness of Prosection and Dissection
Authors: Dyre Lillian , Langley Natalie , Quillen Jaxon , Zhang Nan
Published: May 2022

Studies have shown that prosection and dissection facilitate similar academic outcomes and promote meaningful learning in anatomy laboratory courses. While dissection is the mainstay of many graduate level anatomy curricula, financial and other constraints have increased the use of prosection. This research evaluates student preference for studying different body regions and accomplishing learning outcomes related to content knowledge, teamwork, career preparation, humanistic learning, and wellness.

First‐year medical students (n=51) who completed a hybrid prosection and dissection course completed a post‐course survey about their perceptions of the effectiveness of each lab learning modality. Response options included prosection, dissection, no preference (equally effective), and no preference (equally ineffective). A one‐way ANOVA was used to evaluate performance differences on course assessments between students in the hybrid course and students in past years who participated in a dissection based course.

The response rate was 51%. Respondents preferred dissection for learning the back (62%), thorax (68%), abdomen (54%), shoulder, axilla, and arm (61.5%), and hip, thigh, and leg (46%). Prosection was favored for the pelvis (perineum 81%, cavity 69%), foot (58%), hand (46%), and head/neck (64%). Students favored dissection for gaining hands‐on skills relevant to surgical practice, appreciating anatomical variation, becoming more comfortable handling the human body, engaging in self‐directed learning, and fostering teamwork. Respondents felt prosection provided more time for independent study and wellness activities. Prosection and dissection were rated equally effective for developing a medical vocabulary, applying anatomical knowledge to clinical conditions, and gaining a 3D appreciation of the body. A hybrid prosection and dissection course was preferred by 73% of respondents, compared to 4% who ranked prosection alone as their preference; no students ranked dissection alone as their first choice. There were no statistically significant differences in academic performance related to teaching methods (F(3,195) = 2.65, p=0.34) between students in the hybrid course and students in previous dissection only courses.

Students prefer prosection for learning more spatially complex and intricate regions requiring time‐consuming and skilled dissection. However, learners recognize distinct benefits of dissection for acquiring various technical and non‐technical skills. Hybrid courses should capitalize on the strengths of each method, aligning instructional methods with outcomes and incorporating learner preferences to achieve meaningful, lasting learning.
The function of β3‐adrenergic receptors in resistance‐sized arteries
Authors: Choi Young D. , Reinert Angela , Bulley Simon
Published: May 2022

Beta‐adrenergic receptors, including the β1, β2, and β3‐adrenergic receptors, are a component of the adrenergic nervous system, with significant expression identified in cardiac, airway smooth muscle, and adipose tissue, respectively. An elevated stimulation of the beta‐adrenergic system has been linked to a variety of cardiovascular complications, including hypertension, heart failure, and cardiac myocyte injury. Upregulation of the β3‐adrenergic receptor is observed in patients with heart failure, and it has been proposed that β3‐adrenergic receptor agonists may potentially be utilized therapeutically to treat the disease. However, little is understood about the function of β3‐adrenergic receptors in systemic resistance‐sized arteries that regulate blood pressure, and whether their expression is altered in cardiovascular diseases, such as hypertension. Simple Western indicates that β3‐adrenergic receptor expression is elevated in resistance‐sized mesenteric and hindlimb arteries isolated from hypertensive C57BL/6J mice, compared to controls. Immunofluorescence of en face mesenteric artery segments reveals expression of β3‐adrenergic receptors in both vascular smooth muscle cells (VSMCs) and endothelial cells (ECs). The β3‐adrenergic receptor agonists mirabegron and L‐755,507 cause vasodilation in cannulated 2nd‐ and 3rd‐order mesenteric artery segments after they have developed myogenic tone at physiological pressure (80 mmHg). These data suggest that β3‐adrenergic receptors regulate arterial tone in systemic resistance‐sized arteries and receptor activation may attenuate elevated vasoconstriction observed in hypertension.
Enhanced expression of lectin‐like oxidized LDL receptor‐1 is implicated in the sodium arsenite‐induced increase in foam cells from murine macrophages
Authors: Hossain Ekhtear , Tasnim Homyra , Rosby Raphyel , Rogers Bryan T. , D’auvernge Oswald
Published: May 2022

Arsenic is a naturally occurring toxic element that is present in the air, food, soil, and water of many countries, including the U.S. The U.S. Geological Survey reported that about 44 million people in the U.S. use private wells for water and estimated that over 2 million of those people drink from wells with high concentrations (>10 ug/L) of arsenic. Epidemiological studies have shown that chronic arsenic exposure is a significant risk factor for cardiovascular diseases, including atherosclerosis. However, the cellular and molecular mechanisms connecting arsenic exposure to atherosclerosis are not known. It has been established that Macrophage‐derived, lipid‐loaded, foam cells are a key component of atherosclerotic plaques and are essential for their formation. Furthermore scavenger receptors, such as lectin‐like oxLDL receptor 1 (LOX‐1), CD36, and scavenger receptor type A (SR‐A), regulate the lipid uptake of macrophages and are likely key to the transformation of these cells to foam cells. Our previous study showed that in mouse aortic endothelial cells, arsenic exposure stimulated the uptake of oxidized low‐density lipoprotein (oxLDL) by upregulating the expression of LOX‐1. To see if LOX‐1 might play a key role in foam cell formation, in this study, we used Oil‐red‐O assay to show that sodium arsenite (SA) exposure dose‐dependently enhanced the uptake of oxLDL in murine RAW 264.7 macrophages with a commensurate increase in foam cell count. In addition, while we showed that SA treatment caused a significant increase in both LOX‐1 mRNA and protein levels, only slight or no significant increases were observed in CD36 or SR‐A mRNA expression. Our results indicate that in arsenic induced atherosclerosis, LOX‐1 may play a critical and perhaps unique role among scavenger receptors in the production of foam cells. Further studies, including in vivo and in vitro, are warranted to verify and clarify the role of Lox‐1 in a disease model and to identify other cells and components of the disease pathway.
The Role of Vitamin D and Platelet Aggregation in Patients with Hypertension
Authors: Vuthaluru Ashita R. , Goyal Manjeet K.
Published: May 2022

Introduction

Platelet aggregation is seen in many inflammatory conditions and in the pathogenesis of chronic conditions like hypertension,atherosclerosis, diabetes mellitus. It is seen to be associated with complications of these diseases and also has a considerable influence on the morbidity and mortality. Vitamin D improves endothelial function and causes reduction of platelet aggregation. This study aims to determine the role of Vitamin D in platelet aggregation and its effects in reducing the morbidity and mortality in patients with hypertension.

Materials and Methods

The study was conducted at a tertiary healthcare centre in 50 primary hypertensives. All subjects completed physical and cardiovascular system examination. Blood pressure recordings were done. A blood sample was taken from each patient. Platelet aggregation was estimated using chronologaggregometer after extracting platelet rich plasma using principle of light transmission. ADP and epinephrine were added to PRP to induce platelet aggregation. Serum Vitamin D levels were also measured. The values of platelet aggregation were correlated with serum Vitamin D levels in each subject.

Results

Platelet aggregation and Vitamin D levels were correlated and a negative correlation was found. Correlation coefficient of platelet aggregation to ADP with Vitamin D was ‐0.992 (p value<0.0001) and of platelet aggregation to epinephrine with Vitamin D was ‐0.838 (p value<0.0001).

Conclusion

The study determines that platelet aggregation is one of the physiological events that occurs in hypertension and may be one of the causative factors of complications like cerebrovascular accidents, ischemic heart diseases etc. Cases with lower serum Vitamin D levels showed increased platelet aggregation showing an inversely proportional relationship between platelet aggregation and serum Vitamin D levels. Hence, it can be concluded that supplementation of Vitamin D can be considered in patients with hypertension as a primary prophylactic measure for reducing thrombotic complications of hypertension and helps improve the morbidity and mortality.
Characterization of Hyperactive Mutant forms of the Renal Potassium Channel, ROMK, a Protein Linked to Hypertension
Authors: Nguyen Nga H. , Wang Xueqi , Chen Jingxin , Mackie Timothy D. , Kleyman Thomas R. , Sheng Shaohu , Brodsky Jeffrey L.
Published: May 2022

Hypertension affects one billion people worldwide and is the most common risk factor for cardiovascular disease. Despite the high heritability of hypertension, the relative contributions of known and as yet undiscovered genetic factors are not fully defined. However, amongst the regulators of hypertension are salt‐handling transporters in the kidney, one of which is the renal outer medullary K+ (ROMK) channel. Recent data from our labs indicate that unstable mutant forms of ROMK are prone to premature degradation, which give rise to Bartter syndrome Type II, a renal salt‐wasting disease. Intriguingly, heterozygous carriers of these same mutations are protected from hypertension. Therefore, we hypothesized that hyperactive, gain‐of‐function (GOF) mutations in ROMK that increase potassium flux will predispose individuals to hypertension. To test this hypothesis, we expressed ROMK mutants in yeast lacking its endogenous potassium transporters and then screened for potential GOF mutations that improve yeast growth on low K+. By utilizing both an unbiased genetic screen and a candidate‐based approach, we identified six GOF mutations, including one that exists in the NIH TOPMed human database. To assess how these mutations affect protein function and channel activity, we next performed cellular, biochemical, and electrophysiological assays in yeast, Xenopusoocytes, and human HEK293 cells. Data from these assays categorize the mutations into two main groups. In the first group, two mutations promote biosynthesis by stabilizing the proteins in the endoplasmic reticulum, thus promoting tetrameric channel assembly and increasing cell‐surface expression. In contrast, the second group reside in the PIP2‐binding pocket and significantly increase channel currents, yet protein synthesis is unaffected. Ongoing studies will elucidate how mutations in the PIP2 binding site allosterically affect channel function via patch‐clamp studies, and future efforts will be dedicated to developing murine models in which blood pressure regulation can be directly assessed. Ultimately, our findings will bring us one step closer to uncovering the complex genetics of hypertension as well as enhancing our understanding of how ROMK functions at the molecular level. Both efforts will aid in the development of new therapeutic strategies for hypertension.
Endothelial Sensitivity to Exercise‐Induced Shear Rate in Pre‐ and Post‐Menopausal Females and in Males
Authors: Debray Amélie , Ravanelli Nicholas , Gravel Hugo , Bartlett Audrey‐Ann , Usselman Charlotte W. , Bherer Louis , Gagnon Daniel
Published: May 2022

BACKGROUND

Post‐menopausal females appear resistant to the beneficial effects of exercise on endothelial function. Repeated increases in shear stress are considered important mediators of exercise‐induced improvements in endothelial function. It is therefore possible that endothelial sensitivity to shear stress is reduced in post‐menopausal females, thereby blunting endothelial adaptations to exercise training. The objective of this retrospective analysis was to test the hypothesis that post‐menopausal females exhibit a lower endothelial sensitivity to exercise‐induced shear rate relative to pre‐menopausal females and age‐matched males.

METHODS

Five pre‐ (48 ± 1 yrs) and fifteen post‐ (65 ± 10 yrs) menopausal females, and five males (67 ± 5 yrs) performed 4‐min rhythmic handgrip exercise bouts. Ten participants (6 post and 6 males) exercised at 20%, 40%, 60% and 80% of maximal voluntary contraction, and 15 participants (5 pre, 9 post and 2 males) exercised at 3 kg, 6 kg and 9 kg. The contraction/relaxation cycle was 2‐sec/3‐sec and each bout was separated by 10 min of rest. Brachial artery diameter and blood velocity were measured using high‐resolution ultrasound and analyzed using edge‐detection software. Endothelial sensitivity to exercise‐induced shear rate was quantified as the slope of the relationship between brachial artery dilation (% change from baseline) and shear rate (in s‐1).

RESULTS

Baseline diameter was greater in males (4.68 ± 0.55 mm) compared with post‐ (3.24 ± 0.53 mm, p<0.01) and pre‐ (3.55 ± 0.19 mm, p<0.01) menopausal females. The rhythmic handgrip protocols elicited net shear rates that ranged between 38 s‐1 and 392 s‐1. A positive correlation was observed between brachial artery dilation and net shear rate in males (r=0.66, p<0.01), and post‐menopausal females (r=0.38; p<0.01), but not in pre‐menopausal females (r=‐0.25, p=0.36). Endothelial sensitivity to exercise‐induced shear rate did not differ between pre‐ (0.037 ± 0.120 %/s‐1)and post‐ (0.069 ± 0.081 %/s‐1, p>0.99) menopausal females, between pre‐menopausal females (0.037 ± 0.120 %/s‐1)and males (0.027 ± 0.055 %/s‐1, p>0.99), or between post‐menopausal females (0.069 ± 0.081 %/s‐1) and males (0.027 ± 0.055 %/s‐1, p>0.99).

CONCLUSION

These results suggest that post‐menopausal females do not demonstrate a reduced endothelial sensitivity to exercise‐induced shear rate compared with pre‐menopausal females as well as with males of similar age.
Exercise mimicry: Characterization of nutraceutical agents that may contribute to mitochondrial homeostasis in skeletal muscle
Authors: Moradi Neushaw , Hood David A.
Published: May 2022

While it is well established that endurance exercise improves mitochondrial health in skeletal muscle, optimizing nutraceutical agents to mimic these metabolic achievements in the cell remains a challenge. Nonetheless, the application of exercise mimetics is a fruitful direction to pursue, as they may target and activate the same mechanisms that are upregulated with exercise administration alone. This is particularly useful under conditions where contractile activity is compromised due to muscle disuse, disease, or aging. The agents Sulforaphane (SFN) and Urolithin A (UroA) represent our preliminary candidates for antioxidation and mitophagy, respectively, for maintaining mitochondrial turnover and homeostasis. SFN is a powerful inducer of the Nrf‐2‐ARE pathway, a mechanism that upregulates cellular defences against oxidative stress. On the other hand, the metabolite, UroA, has developed a reputable role in regulating mitochondrial turnover via mitophagy. The purpose of this ongoing study is to characterize these nutraceutical agents both in their time‐ and dose‐dependent capacities to induce changes in protein content relative to the antioxidant and mitophagy pathways, in C2C12 myotubes. Differentiated muscle cells were treated with two concentrations of each nutraceutical for 4 h, 24 h, or 48 h. Immunoblot analysis was conducted to measure changes in protein content. SFN treatment after 4 h rendered a marked increase in the master regulator for antioxidation, and transcription factor, Nrf‐2, with no apparent changes in its negative regulator Keap‐1 at any given time point. Nuclear‐cytoplasmic fractions confirmed Nrf‐2 translocation to the nucleus following 4 h of treatment, likely representing the potent activation of antioxidant genes. This aligns with the upregulation of the downstream antioxidant markers HO‐1 and NQO1, showing 4‐5‐fold increases as early as 4 h and 24 h, respectively, and maintained after 48 h. Despite modest effects with Urolithin A, some significant changes took place under basal conditions. The upstream kinase phospho‐AMPK was activated by 2‐fold as early as 4 hr of treatment with UroA, which subsided by 24 and 48 h. Previous reports observed changes with the agent on autophagy markers, which includes the modest 1.3‐fold increases in p62 and LC3‐II after 48 h of treatment. However, no observable changes took place with respect to the mitophagy markers phospho‐ULK1, Parkin, or PINK1. Nonetheless, our preliminary results suggest that these agents may be suitable candidates as exercise mimetics. These data also set the stage for an examination of the synergistic effect of these nutraceuticals, in combination with contractile activity, on mitochondrial turnover and function.
The Differential Contribution of TRIM72/MG53 Protein Domains in Plasma Membrane Repair
Authors: Lopez Perez Miguel A. , Banford Kassidy K. , Weisleder Noah L.
Published: May 2022

Duchenne Muscular Dystrophy (DMD) is a fatal X‐linked genetic disease that is hallmarked by progressive muscle weakness and degeneration. DMD is the most common form of all the muscular dystrophies, affecting 1:5,000 live male births. DMD is caused by mutations in the dystrophin gene, which ablate the expression of dystrophin resulting in compromised structural integrity of the muscle cell plasma membrane, also known as the sarcolemma. There is still an unmet need for novel therapies that address the underlying molecular cause of the disease. Previous work has shown that the tripartite motif protein 72/mitsugumin 53 (TRIM72/MG53) is critical for an effective cell membrane repair response after injury. Our previous results demonstrated that overexpressing TRIM72/MG53 or exogenously delivering recombinant human MG53 protein (rhMG53) can increase membrane repair capacity in many different cell types and improve pathology in multiple animal models of muscular dystrophy. TRIM72/MG53 mediates this effect on membrane repair by binding phosphatidylserine (PS) at membrane injury sites. However, there is a poor understanding of the structural basis of PS binding by TRIM72/MG53 and how this contributes to the membrane repair effects of the protein when expressed endogenously or delivered exogenously. Here we aimed to address this knowledge gap through structure/function analysis. In these studies, we examined the mechanistic basis for MG53 binding to PS by conducting a systematic analysis of MG53 canonical protein domains. We generated a catalytic mutant of TRIM72/MG53 (hMG53(ΔE3)) to delineate its native E3 ubiquitin ligase enzymatic activity and its effect on membrane repair. To identify which protein domains are required for the effect on membrane repair, we generated a panel of nine deletion and fusion protein constructs of human TRIM72/MG53. We confirmed protein expression by transient transfection of human embryonic kidney (HEK293) cells and found that specific protein domains mediated the subcellular location of the protein mutant. Moreover, we evaluated ensemble membrane repair capacity by overexpressing our mutant panel in mouse neuroblastoma (N2a) cells and measuring lactate dehydrogenase (LDH) release after damage with ~500 micrometer glass beads. We found that hMG53(ΔE3) displays comparable subcellular localization to wild‐type hMG53 and improves membrane resealing when endogenously expressed in HEK293 cells or made available exogenously as recombinant protein. We observed that hMG53(ΔE3) can also bind phosphatidylserine (PS)‐coated beads. We found that at least two deletion constructs display enhanced membrane repair response comparable to full length TRIM72/MG53. Taken together, these data suggest that the therapeutic effect of TRIM72/MG53 on membrane repair involves specific protein domains but does not require its E3 ligase enzymatic activity.
Downregulation of BCRP (ABCG2) in Placenta of Rat Model of Preeclampsia
Authors: Dai Wanying , Piquette‐Miller Micheline
Published: May 2022

BACKGROUND

Recent studies in our laboratory demonstrated downregulation of breast cancer resistance protein (BCRP/ABCG2) in placenta obtained from women with preeclampsia (PE). Pregnant women with PE are often prescribed medications to manage disease progression and prevent adverse outcomes. Many of these drugs, as well as other clinically relevant therapeutics are substrates for BCRP. BCRP is highly expressed in placenta and plays an important role in preventing xenobiotics from crossing the placenta and entering the fetal compartment. Thus, the impact of PE and BCRP downregulation on fetal drug exposure needs to be examined. As studies in pregnant women are often not feasible, use of preclinical models is an important approach. Thus, our objective was to characterize transporter changes in an endotoxin rat model of PE to determine its utility and predictive value for future drug disposition studies. This frequently utilized PE model is associated with maternal immune activation and induction of PE‐like symptoms including high blood pressure, proteinuria and pathology which are characteristic of human disease.

METHODS

Pregnant rats were injected intraperitoneally with 10 μg/kg of bacterial endotoxin (LPS) on GD13, followed by 40 μg/kg LPS daily until GD 16. Controls were injected with sterile saline. Urine was collected daily. Dams were sacrificed on GD17 or GD18 for collection of maternal plasma, placentas, and fetuses (n=7‐8/group/day). Plasma levels of TNF‐α and IL‐6 were quantified by ELISA Kit. Tissues were analyzed for the expression of transporters by using qPCR and western blot. Urine was analyzed using creatinine colorimetric and protein assays.

RESULTS

Consistent with previous reports, the urinary ratio of protein/creatinine was significantly elevated in PE dams on GDs 16‐18, demonstrating proteinuria. As compared to controls, maternal plasma concentrations and placental mRNA levels of the pro‐inflammatory cytokines, TNF‐α and IL‐6, were significantly higher in the PE group on GD17 and GD18. The mRNA levels of BCRP was significantly decreased by 40‐60 % in placenta of PE dams on GD17 and GD18 along with a corresponding decrease in protein expression on GD18. Significantly lower mRNA levels of Abcb1a, Abcb1b and Slco2b1 were observed in the PE group. PE was also associated with significantly decreased transcript levels of Bcrp, Abcb1a and Abc1b in fetal brains.

CONCLUSION

Our data demonstrates that the endotoxin PE rat model exhibits proteinuria and immune activation consistent with human disease. Importantly, our observations of decreased placenta expression of BCRP in this PE model is consistent with findings in humans. This indicates that this preclinical model may serve as a useful translational tool to examine the impact of PE on the disposition and fetal exposure of BCRP drug substrates.
Characterization and application of a novel immortalized murine thick ascending limb (imTAL) cell line to investigate the role of myosin II motor proteins Myh9 and Myh10 in TAL cargo transport
Authors: Busselman Brook , Zimmerman Jenna , Otterpohl Karla , Mukherjee Malini , Chandrasekar Indra
Published: May 2022

Conditional genetic inactivation of Myh9and Myh10, the genes which encode the heavy chains of nonmuscle myosin IIA and IIB, respectively, in adult murine renal epithelium leads to aberrant trafficking of the thick ascending limb‐specific GPI‐anchored glycoprotein uromodulin (UMOD) and its subcellular accumulation in cells of the thick ascending limb of Henle’s loop (Otterpohl et al. 2020). A novel immortalized murine thick ascending limb (imTAL) cell line harboring doxycycline inducible Myh9and Myh10conditional knockout (cKO) capability was established in our lab to facilitate long term in vitrostudies. We utilized biotinylated anti‐uromodulin antibody to isolate cells from collagenase digested Myh9&10cKO whole mouse kidneys. Characterization of these cells has revealed endogenous expression of TAL‐specific protein UMOD and NKCC2. RT‐qPCR detected mRNA expression of TAL specific proteins. We generated imTAL cells stably expressing GFP‐tagged UMOD using retroviral transduction. GFP‐UMOD expressing cells underwent 72‐hour doxycycline treatment and, along with untreated control cells, were fixed and analyzed via confocal microscope. While GFP‐UMOD is enriched along the plasma membrane in control cells, we observe accumulation of GFP‐UMOD within vesicular structures upon doxycycline‐induced loss of Myh9 and Myh10 proteins. We also utilized imTAL cells stably expressing an GFP‐tagged thermosensitive variant of vesicular stomatitis virus glycoprotein (VSV‐GtsO45‐GFP), which has been widely employed in studying the secretory pathway. The temperature sensitive nature of VSV‐GtsO45 enabled us to perform pulse‐chase type experiments, following its anterograde transport through the ER, Golgi, and ultimately to the plasma membrane. Following incubation at the restrictive temperature (40°C) overnight, cells were moved to permissive temperature (32°C) and followed until 45 minutes. VSV‐GtsO45‐GFP in control cells undergoes ER export immediately (5 minutes) after incubation at the permissive temperature and is transported along the secretory pathway, arriving at the plasma membrane 30‐40 minutes later. Inactivation of Myh9and Myh10 results in sequestration of VSV‐GtsO45‐GFP in perinuclear vesicular and tubular structures. Immunolocalization studies are being performed to identify the vesicular compartment(s) in which VSV‐GtsO45‐GFP is sequestered in the absence of Myh9 and Myh10 proteins. imTAL cells stably expressing NKCC2‐GFP are also being analyzed to assess the anterograde transport of NKCC2 in doxycycline‐treated cells. Taken together, we have established a novel, immortalized, inducible, Myh9&10 cKO murine TAL cell line to enable long term in vitro studies and have expanded the molecular toolbox to elucidate the mechanisms underlying NM2 motor mediated TAL cargo transport.
A Novel Curcumin Isomer, cis‐transCurcumin, Activates Adenosine Receptor Subtypes A1 And A3: Potential for Pain Therapy
Authors: Hamilton Luke J. , Vaughn Hilary , Pattabiraman Mahesh , Zhong Haizhen A. , Luedtke Brandon , Chandra Surabhi
Published: May 2022

A wide variety of plants produce compounds that contain the 4‐hydroxy‐3‐methoxybenzyl or “vanillyl” group. These vanilloid compounds include such well‐known food molecules as vanillin, capsaicin, gingerol, and curcumin. Vanilloid compounds have been widely studied for their medicinal properties, and one property that most of them have in common is the ability to induce analgesia. Studies have indicated multiple potential cellular mechanisms for this analgesic activity, including TRPV1 receptors, opioid receptors, and adenosine receptors (ARs). There are only a few studies on the interaction of vanilloid compounds with ARs, and to our knowledge no previous study has measured the binding affinity of any vanilloid compound with either of the two AR subtypes that are linked to the inhibitory Gαsubunit (subtypes A1and A3). Our study objective was to assess the binding affinity and receptor activation of a novel curcumin isomer, cis‐transcurcumin (CTCUR), at AR subtypes A1and A3. Since agonism of these subtypes is associated with analgesia, we hypothesized that CTCUR would be an agonist at AR subtypes A1 and A3. Two lines of Chinese hamster ovary cells were used, each transfected to overexpress one of the AR subtypes. Cell survival assays were performed to assess toxicity. Competitive binding assays and confocal microscopy were performed to measure binding affinity. Computer docking was also performed, to assess location of binding. Assays of intracellular cAMP levels were performed to measure receptor activation. Survival results indicate that CTCUR is not toxic to CHO cells at any of the concentrations tested (the highest being 100 μM). Competition assay results indicate that CTCUR binds to subtype A1 with a Ki of 306 nM and to subtype A3 with a Ki of 400 nM. Microscopy results confirm sub‐micromolar affinity for A1, but not for A3. Docking results confirm sub‐micromolar affinity for both subtypes, and furthermore indicate that CTCUR binds to the “toggle switch” domain of α helix 6 that is important for receptor activation. Intracellular cAMP results indicate that CTCUR acts as an agonist of ARs. Thus, we fail to reject our hypothesis. These results are of interest because they provide the first in vitro confirmation of the hypothesis that vanilloid compounds can activate ARs, in accordance with the results of previous studies in vivo. Vanilloid compounds that activate ARs are therefore worthy of further study, as a potential class of analgesic therapeutics.
A Novel Technique to Reduce Kidney Injury by Maintaining Na/K Pump Functions Using Electric Energy
Authors: Wang Lei , Chen Wei , Liang Pengfei , Wei Jin , Zhang Jie , Buggs Jacentha , Liu Ruisheng
Published: May 2022

Renal ischemia‐reperfusion injury is an important contributor to the development of delayed graft function, which is associated with higher rejection rates and worse long‐term outcomes of the allografts. One of the earliest impairments during ischemia is insufficient ATP supply‐induced Na/K pump dysfunction, which results in subsequent cellular damage. Consequently, strategies that preserve ATP levels or Na/K pump function may limit the extent of renal injury during ischemia‐reperfusion.

In this study, we developed a novel technique that applies an oscillating electrical field to first synchronize the pump molecules, and then modulate their pumping rates. We dubbed this technique as the synchronization modulation electric field. We present this novel technique of using electric energy to substitute ATPs in fueling and activating the pumps, thereby efficiently maintaining cellular functions under ATP insufficient conditions. We tested the effectiveness of this technique in two kinds of models of ischemic renal injury: an in situ renal ischemia‐reperfusion injury model (predominantly warm ischemia) and kidney transplantation rodent and swine models (predominantly cold ischemia).

Our results show that application of the specially designed electric field effectively delayed ATP depletion during ischemia and preserves Na/K pump activity, thereby alleviating kidney injury by about 45% (plasma creatinine of 1.17±0.04 and 1.97±0.06 mg/dL for electric field treated and untreated groups, respectively) and improving renal allograft function by over 50% compared with the controls.

This novel technique for preserving Na/K pump function may have therapeutic potential not only for ischemic kidney injury, but also for other diseases associated with dysfunction of Na/K pumps.
Obesity in Angiogenesis and Lung Regeneration
Authors: Hunyenyiwa Tendai , Kyi Priscilla , Hendee Kathryn , Matus Kienna , Mammoto Tadanori , Mammoto Akiko
Published: May 2022

Obesity is associated with impairment of wound healing and tissue regeneration. Angiogenesis, the formation of new blood capillaries, plays a key role in organ regeneration and repair. We and other groups have reported that inhibition of angiogenesis attenuates lung growth after unilateral pneumonectomy (PNX) and that obesity is accompanied by endothelial cell dysfunction. The aim of this project is to investigate the effects of obesity on post‐PNX lung vascular and alveolar morphogenesis. We have found that post‐PNX regenerative lung growth is inhibited in Lepob/ob obese mice compared to Lepob/+ mice. The levels of the major angiogenic factor, vascular endothelial growth factor (VEGF) are higher in the serum and the lung tissue collected from post‐PNX mice compared to those from sham‐operated control mice, while these effects are attenuated in post‐PNX Lepob/ob mice. The levels of adiponectin, one of the adipokines that exhibits pro‐angiogenic and vascular protective properties, increase in the remaining mouse lungs after unilateral PNX, while these effects are attenuated in Lepob/ob obese mice. Regenerative lung growth, vascular and alveolar morphogenesis, and VEGF levels in the post‐PNX mouse lungs are inhibited in adiponectin knockout mice. These results suggest that obesity inhibits post‐PNX regenerative lung growth through adiponectin‐VEGF signaling. Modulation of adiponectin‐VEGF signaling may be the efficient strategy to restore lung regeneration and repair in obese people.
IL32 overexpression is driven by DNA hypomethylation and contributes to an extracellular matrix (ECM) remodeling phenotype in EpCAM‐/CD49f‐enriched breast cancer cells
Authors: Benson Elayne M. , Wilson Megan A. , Gray Emma V. , Rizzo Paris L. , Ouzounova Maria , Korkaya Hasan , Shull Austin Y.
Published: May 2022

Basal‐like breast cancers typically correspond with increased enrichment of EpCAM‐/CD49f‐ cancer stem cells (CSC) and a propensity toward metastasis. However, the molecular mechanisms underlying these general characteristics are not well understood. To provide further insight concerning CSCs and their intrinsic metastatic mechanisms, we compared the 450K DNA methylation profile of EpCAM‐/CD49f‐ poor breast cancer cell lines to that of EpCAM‐/CD49f‐ enriched breast cancer cell lines. From our results, we were able to determine and highlight IL32 as a gene whose promoter is hypomethylated in EpCAM‐/CD49f‐ enriched cell lines. The hypomethylated IL32 promoter corresponded with increased IL32 expression in both cell lines and basal‐like breast cancer patients from The Cancer Genome Atlas (TCGA) database. Interestingly, increased IL32 expression preferentially occurred for the IL32‐beta transcript and corresponds with previous reports demonstrating that IL32‐beta is not secreted from the cell like other canonical interleukins and preferentially localizes to the mitochondria in breast cancer cells. Additionally, expression of the beta‐transcript could be suppressed when CSC‐enriched cells were treated with the BET‐bromodomain inhibitor JQ1. Because of this phenomenon, we sought to determine the effects of suppressing IL32 in the EpCAM‐/CD49f‐ enriched cell line SUM159PT via siRNA‐mediated knockdown and subsequent RNAseq differential expression analysis. From our results, we determined that transcripts involved in extracellular matrix (ECM) organization and collagen/integrin interaction were preferentially affected by IL32 silencing. Additionally, IL32 suppression decreased the invasiveness of SUM159PT based on an ECM‐matrix cell invasion assay. Collectively, our results reflect the notion that differential IL32 expression by promoter hypomethylation in breast CSCs plays a role in ECM remodeling for purposes of breast cancer cell invasion and metastasis.
Retinitis Pigmentosa Alleles, in PRPF31 and PRPF6, Effects on Pre‐Messenger RNA Splicing
Authors: Vargas Marina
Published: May 2022

Retinitis pigmentosa (RP) is a genetically heterogeneous group of retinal disorders that affect 1 in 4,000 individuals in the United States. Patients with RP suffer from progressive degeneration of the light‐sensitive photoreceptors in the retina, which eventually leads to severe vision loss. The autosomal dominant form of RP (adRP) has been linked to eight splicing‐related proteins that affect the accuracy and efficiency of pre‐mRNA splicing. Among these splicing proteins, PRPF31 and PRPF6 are essential to the formation of the pre‐spliceosome. The purpose of this study is to examine the mechanisms by which adRP mutations in PRPF31 and PRPF6 affect pre‐mRNA splicing and to distinguish if these effects are widespread or locally more severe to the retina. This was done by constructing adRP allele orthologs, found first in human adRP patients, into two model organisms, Saccharomyces cerevisiae, and Mus musculus cell lines. First, we utilized S.cerevisiae to examine these mutations' effects on cell growth indicating a possible splicing defect and then examined splicing through in vitro splicing assays. Second, a mouse retinal ganglion precursor‐like cell line was utilized to identify various mRNAs effects of adRP mutants on splicing function and its effects on the phenotypic expression for PRPF6. In order to understand the role of pre‐mRNA splicing in photoreceptor gene expression and function, we have identified photoreceptor genes of interest whose pre‐mRNA splicing can potentially be affected by mutations in PRPF6. This is done by analyzing these genes of interest through splicing assays and immunofluorescent assays. Overall, this project adds a unique approach to studying how adRP alleles affect pre‐messenger RNA splicing.