bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Botanical Viral Entry Inhibitors for SARS‐CoV‐2
Authors: Risener Caitlin J. , Woo Sunmin , Samarakoon Tharamangala , Caputo Marco , Edwards Emily , Zandi Keivan , Goh Shu Ling , Downs‐Bowen Jessica A. , Schinazi Raymond F. , Quave Cassandra L.
Published: May 2022

The examination of biodiversity across the world has historically been a critical part of drug development and led to the discovery of common medications for many medical issues including pain management, cancer, heart disease, and infections. During the SARS‐CoV‐2 pandemic, the use of natural supplements in the United States has increased. The efficacy of these natural products to prevent SARS‐CoV‐2 infection and the safety of their use remains unexplored; therefore, more research must be done to determine which supplements have antiviral properties. The Quave Natural Product Library (QNPL) is a collection of over 2,000 botanical and fungal extracts and includes the 40 most used natural supplements in the United States. Collection of the biological samples for the library requires field expeditions to areas throughout the world with high levels of biodiversity. Each of these extracts were tested in a SARS‐CoV‐2 pseudotyped virus system to determine which extracts inhibit viral entry, specifically the virus spike protein binding to host cells ACE2 receptors. Mammalian cell cytotoxicity assays were run in parallel. Evaluation of 1,887 extracts and 18 single compounds from the QNPL against SARS‐CoV‐2 identified. 317 extracts derived from 134 species across 76 families (1 lichen, 2 fungi, 73 plant families) exhibited ≥50% inhibition activity in the wild type spike pseudotyped model at 20 µg/mL. Of these bioactive extracts, 129 extracts derived from 95 plant species exhibited ≥85% inhibition activity and ≤15% cytotoxicity in the wild‐type model. Once these 129 extracts were identified, an interesting pattern emerged indicating many hits were from species that are known to be cardiotoxic due to rich composition of cardiac glycosides. For further selection and testing, we reviewed each extract and consulted the literature to eliminate extracts with those properties or similar compounds, which narrowed down our interest to 8 extracts. These extracts were further validated in a concentration‐response assay in a pseudotyped virus model. The EC50 values of the top 3 extracts were all under 10 µg/mL. These 3 extracts all exhibited activity (≥85% inhibition activity) in the wildtype and variant pseudotyped models. Testing in live SARS‐CoV‐2 confirmed antiviral activity from 2 of 3 extracts, Plant Aflowers and Plant B roots. Further chemical characterization of the major metabolites of these two hits was performed using MS/MS fragmentation data compared with the literature, in silico prediction, and web‐based databases. The results revealed phenylpropanoids, flavonoids, triterpenes, glycosidic terpenes, and fatty acids as the major chemical classes. The next steps of this study seek to identify and isolate purified bioactive compounds to further understand their role in SARS‐CoV‐2 inhibition.
Abnormal transporter and tight junction protein expression in environmental enteric dysfunction
Authors: Abtahi Shabnam , Sailer Anne , Sadiq Kamran , Iqbal Najeeha , Ahmed Kumail , Kabir Furqan , Ali S A. , Turner Jerrold R.
Published: May 2022

Environmental enteric dysfunction (EED), which is pervasive in severely underresourced regions with poor sanitation, is characterized by malabsorption and stunted growth and leads to irreversible deficits in physical and intellectual development. Previous studies have associated increased intestinal permeability with EED, but the molecular changes responsible for barrier loss and malabsorption have not been characterized. We sought to define expression of absorptive transport and tight junction proteins using quantitative morphometry of EED patient duodenal biopsies. Biopsies from Pakistani children with confirmed EED diagnoses were compared to those from age‐and sex‐matched North American healthy controls, celiac disease patients, and non‐celiac patients with villous atrophy or intraepithelial lymphocytosis. Claudin‐4 expression was increased in EED biopsies relative to all North American subject groups. Claudin‐4 expression was most increased within upper villous enterocytes, where it stained lateral membranes and, in EED, accumulated intracellularly. EED biopsies also demonstrated markedly increased occludin expression at tight junctions, lateral membranes, and within vesicular compartments. In contrast, expression of claudin‐2, claudin‐15, ZO‐1, JAM‐A, and E‐cadherin were similar in EED patients and controls. Villous expression of SGLT1, NHE3, and NaK‐ATPase, which drive absorption of glucose, Na+, and water, were all increased in EED. In contrast, PEPT1 expression was reduced in EED; expression of CFTR and sucrase isomaltase was unchanged. Increased expression of occludin and claudin‐4 were unexpected, as each of these increases tight junction barrier function in vitro. Further, the malabsorption that characterizes EED is at variance with increased SGLT1, NHE3, and NaK‐ATPase expression. Although further study is needed, it is possible that these changes represent unsuccessful adaptive responses intended to counteract barrier dysfunction and malabsorption. These data are paradoxical in that they demonstrate that, despite being characterized by malabsorption and barrier dysfunction, EED is associated with increased, rather than reduced, expression of barrier‐enhancing tight junction proteins or absorptive proteins. Further study is needed to fully understand the contributions of these alterations to the pathobiology of EED.
Enhanced Isolation of Single Myofibers in Flexor Digitorum Brevis Dissociation
Authors: Moore Logan , Awad Kamal , Mo Chenglin , Huang Jian , Wang Zhiyang , Brotto Leticia , Brotto Marco
Published: May 2022

Introduction

The isolation of single myofibers from the Flexor Digitorum Brevis (FBD) has been a well‐established model in skeletal muscle physiology research. The use of isolated single‐fibers has led to advancements in the understating of the excitation – contraction (E‐C) coupling mechanisms in skeletal muscle, calcium transients, the role of mitochondria, and much more. However, these experiments rely on the quality of the dissociation of myofibers and their attachment to the imaging dish which can be difficult and time exhaustive. The purpose of this study was to build upon previously established methodologies to improve the yield of intact‐alive myofibers in a time efficient manner.

Methods and Materials

Euthanasia and experimental protocols were approved by the University of Texas at Arlington’s IACUC. Based upon previously established dissociation techniques (1–3), we modified the protocol as follows: the intact FDB was dissected out and placed in 2 mL of Ringer’s solution (142 mM NaCl2, 5 mM KCl, 1.8 mM MgCl2, 10 mM Hepes, 10 mM Glucose). Under the dissection microscope the whole muscle was separated into 4 muscle bundles; separated by the distal tendons. The muscle bundles were transferred to the digestion media (DMEM with glutamine, 2% sterile filtered fetal bovine serum (FBS), 0.1% gentamycin, and 4 mg ml‐1 Collagenase A). The muscle bundles were incubated for 30‐45 minutes at 37° C in 5% CO2. Following incubation, the bundles were transferred to 1mL of Ringer’s solution in a 24‐well plate and gently titrated with the cut end of a P1000 pipette tip. The solution was transferred to laminin coated glass bottom dishes and incubated for 15‐30 minutes for fiber attachment. Following fiber attachment, the fibers were either stained with the LIVE/DEAD™ Viability/Cytoxicity kit or loaded with Fura‐2 AM fluorescent dye for measurement of maximal calcium depolarization response induced with 100mM Potassium Chloride (KCl) using a PTI spectrophotometry system.

Results and Conclusion

We found that using an increased concentration of collagenase and a reduced time of digestion successfully increased the number of fibers in the field of vision.. Laminin coated glass bottom dishes produced quick and strong attachment, allowing for optimal stimulation conditions. The use of cell culture media with FBS protected the sarcolemma increasing the viability of the fibers. This is shown in both the LIVE/DEAD imaging and the response to KCl stimulation. This study demonstrates that the change of dissociation media and the decrease in dissociation time allow for protection of the myofiber for an increased yield and viability. This protocol will allow for improved experimental outcomes in which mature myofibers are subjected to electrical or chemical stimulations.

References

1. Calderón et al. JMRCMdoi: 10.1007/s10974‐009‐9181‐1.

2. Capote et al. JP doi: 10.1113/jphysiol.2004.081034.

3. Yi et al. JBC doi: 10.1074/jbc.M110.217711.
Inhibitory Effects of Major Cannabinoids and their Metabolites on Oxazepam Metabolism
Authors: Bardhi Keti , Watson Christy , Chen Gang , Lazarus Philip
Published: May 2022

Cannabis and cannabis‐derived products are increasingly used for recreational and medical purposes including anxiety and epilepsy and are often concurrently used with other types of conventional medications. The concomitant use of cannabis with other therapeutic drugs has been shown to increase the likelihood of deleterious drug‐drug interactions. Oxazepam is a well‐known benzodiazepine used in clinical practice as an anxiolytic, sedative, and anticonvulsant agent. Oxazepam is a racemic mixture of the S‐and R‐enantiomers and is primarily metabolized via glucuronidation in an enantiomeric‐specific manner, where S‐oxazepam is mainly glucuronidated by UDP‐glucuronosyltransferase (UGT) 2B15 and R‐oxazepam is glucuronidated primarily by UGT1A9 and UGT2B7. Recent comprehensive in vitrostudies using probe substrates have shown that the major cannabinoids Δ9‐ tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) can inhibit several of the primary hepatic and renal UGT enzymes. The objective of the present study was to evaluate the potential inhibitory effect of cannabinoids and their major circulating metabolites, 11‐hydroxy‐Δ9‐tetrahydrocannabinol (11‐OH‐THC), 11‐ nor‐9‐carboxy‐Δ9‐tetrahydrocannabinol (11‐COOH‐THC), and 11‐nor‐Δ9‐tetrahydrocannabinol‐carboxylic acid glucuronide (THC‐COOH‐Gluc) on the UGT enzymes involved in R,S‐oxazepam metabolism. We hypothesize that major cannabinoids and their metabolites will inhibit R,S‐oxazepam metabolism, leading to decreased clearance. To investigate potential drug‐drug interactions, cannabinoids and their major plasma metabolites were screened as inhibitors of R‐ and S‐oxazepam glucuronidation in human liver microsomes (HLM). In vitro inhibition screening assays with 10 μM or 100 μM of CBD and 11‐OH‐THC inhibited both R‐ and S‐oxazepam glucuronidation in HLM by up to 90%. The IC50 values 11‐OH‐THC and CBD inhibition of R‐oxazepam glucuronidation were 10.0 ± 7.8 μM and 7.0 ± 4.2 μM, respectively, in HLM; the respective IC50 values for the inhibition of S‐oxazepam glucuronidation by 11‐OH‐THC and CBD were 15.0 ± 6.4 μM and 52.2 ± 22.6 μM in HLM. These data indicate that co‐administration of oxazepam with cannabis may result in inhibition of oxazepam clearance, suggesting a strong potential for drug‐drug interactions in vivo.
Mechanism of Blood‐Heart‐Barrier Leakage: Implications for COVID‐19 induced Cardiovascular Injury
Authors: Singh Mahavir , Homme Rubens P. , George Akash K. , Zheng Yuting , Tyagi Suresh C.
Published: May 2022

Although blood‐heart‐barrier (BHB) leakage is the hallmark of congestive (cardio‐pulmonary) heart failure (CHF), the primary cause of death in elderly, and during viral myocarditis resulting from the novel coronavirus such as the severe acute respiratory syndrome novel corona virus 2 (SARS‐CoV‐2) known as COVI‐19, the mechanism is unclear. The goal of this project is to determine the mechanism BHB in CHF. Endocardial endothelium (EE) is the BHB against leakage of blood from endocardium to the interstitium; however, this BHB is broken during CHF. Previous studies from our laboratory, and others have shown a robust activation of matrix metalloproteinase‐9 (MMP‐9) during CHF. MMP‐9 degrades connexins leading to EE dysfunction. We demonstrated juxtacrine coupling of EE with myocyte, and mitochondria (Mito) but how it works still remains at large. To test whether activation of MMP‐9 causes EE barrier dysfunction, we hypothesized that if that were the case then treatment with hydroxychloroquine (HCQ) could, in fact, inhibit MMP‐9, and thus preserve the EE barrier/juxtacrine signaling, and synchronous endothelial‐myocyte coupling. To determine this, CHF was created by aorta‐vena cava fistula (AVF) employing the mouse as a model system. The sham, and AVF mice were treated with HCQ. Cardiac hypertrophy, tissue remodeling‐induced mitochondrial‐myocyte, and endothelial‐myocyte contractions were measured. Microvascular leakage was measured using FITC‐albumin conjugate. The cardiac function was measured by echocardiography (Echo). Results suggest that MMP‐9 activation, endocardial endothelial leakage, endothelial‐myocyte (E‐M) uncoupling, dyssynchronous mitochondrial fusion‐fission (Mfn2/Drp1 ratio) and mito‐myocyte uncoupling in AVF heart failure were found to be rampant however, treatment with HCQ successfully mitigated some of the deleterious cardiac alterations during CHF. The findings have direct relevance to the gamut of cardiac manifestations, and the resultant phenotypes arising from the ongoing complications of COVID‐19 in human subjects.
The Impact of Early Life Exposure to Glyphosate
Authors: Wang Joshua , Hara Takeo , Soto Gloria , Sasaki Masaru , Karakasheva Tatiana , Muir Amanda
Published: May 2022

Eosinophilic esophagitis (EoE) is a chronic immune mediated disease that affects populations of all ages. It is characterized by eosinophilic inflammation of the esophageal mucosa, decreased epithelial integrity, and lamina propria fibrosis. Clinically, the symptoms of EoE can range from mild (dysphagia, abdominal pain, vomiting), to severe (food impaction, inability to thrive), to complete loss of esophageal function in extreme cases. This disease, first described in 1993, is rising in incidence and prevalence. Unfortunately, the reason for disease emergence is unknown and environmental triggers have been postulated as a potential source. The role of environmental toxins, specifically herbicides, has never been examined in the pathophysiology of atopic disease. Glyphosate, the active ingredient in Roundup, is the most used herbicide in the United States, and is frequently used as a desiccant prior to harvest. Residual amounts can be detected in harvested crops and downstream food products, potentially leading to a diet of chronic exposure. Herein we sought to determine the effects of early life glyphosate treatment and the consequences of exposure on the development of eosinophilic esophagitis and the maintenance of epithelial integrity.

We exposed mice to physiologic levels of glyphosate in utero and throughout their life cycle to simulate glyphosate consumption in the typical American diet. We then induced EoE in mice using our established protocol and evaluated epithelial integrity and inflammation. Interestingly, we found that chronic glyphosate treatment alone induced a 2‐fold increase in esophageal eosinophils over baseline, similar to mice undergoing EoE. Secondarily, we exposed primary human esophageal and intestinal organoids to short term glyphosate treatment. We observed no changes in the esophageal organoids. However, we found increased gene expression of mucin 2 (MUC2) in organoids derived from terminal ileum and proximal colon.

Taken together these results suggest that there is increased inflammation in the glyphosate treated mice and this may be due, in part, to a response generated in the small intestine by glyphosate exposure.
Expression of Neural Crest Specifiers During the Two Waves of Trunk NCC Migration in Turtle Embryos
Authors: Cebra‐Thomas Judith A. , Smith Matthew , Akbar Arpan , Reineberg Anamarie , Stoltzfus Jonathan
Published: May 2022

The bones of the plastron, the ventral portion of the turtle shell, develop through intramembranous ossification, in the same manner as facial bones, suggesting they are also produced by neural crest cells (NCCs). Previous work has demonstrated the existence of a unique second migration of NCCs away from the neural tube of turtle (Trachemys scripta) embryos; these migrate ventrally, appear to be skeletogenic and may contribute to the bones of the plastron. The goal of this project is to examine the expression of neural crest specifiers in the premigratory NCCs located in the neural tube and in migrating NCCs using immunofluorescence and whole mount in situ hybridization. During the switch‐over period, the neural crest specifiers Sox9 and Sox10 are both expressed in premigratory NCCs. However, their expression diverges in migrating NCCs; the cells that retain Sox10 are located within the major migratory stream in the rostral somite leading to the production of the dorsal root ganglion and other peripheral nerves, consistent with a neurogenic cell fate. By contrast, cells that retain Sox9 appear to take a novel migratory pathway through the caudal portion of the somite, and are excluded from the dorsal root ganglia, consistent with a non‐neurogenic and perhaps skeletogenic cell fate. We are currently examining the expression of transcription factors that have been found to be preferentially expressed in either trunk NCCs or cranial NCCs in the well‐characterized chicken (G. gallus) system. Elucidating the molecular control of NCC specification in the unique population of trunk NCCs would further our understanding of its role in the development of the turtle shell.
Influences of Housing Environment on Functional State of the Body after Action of Ulcerogenic Stressor
Authors: Filaretova Ludmila , Zhuikova Svetlana , Yarushkina Natalia , Komkova Olga , Morozova Olga , Zenko Mikhail , Baranova Ksenia , Rybnikova Elena
Published: May 2022

Here we studied influences of housing environment on functional state of the body after action of ulcerogenic stressor. The effects of ulcerogenic stressor on the gastric mucosa, the hypothalamic‐pituitary‐adrenocortical (HPA) axis reactivity and glucocorticoid receptors in hippocampus, behavior and somatic pain sensitivity were compared in rats kept in enriched environment, social isolation, and standard laboratory conditions (control group). The rats aged 30 days were divided into groups and placed in standard cages 6 rats per cage (control) or 1 rats per cage (isolation) or in enriched cages 6 rats per cage (enriched environment). One month later, all rats (after 24 h fasting) were stressed by cold restraint (10˚C) for 3 h. In 3 h one half of rats in each group were decapitated to examine the gastric mucosa. Another half of rats were subjected to behavior tests (open field and elevated plus maze) and somatic pain sensitivity test (hot plate) as well as the HPA axis reactivity testing by plasma corticosterone levels induced by mild procedural stressor. Glucocorticoid receptor expression was estimated in hippocampus by immunohistochemistry. Body weight gradually increased in all groups during 1 month before exposure of stressor, however, in the isolated rats, the weight gain was greater than that in enriched and control groups. In all groups cold restraint caused the gastric erosion, the mean areas of which were not significantly differed. In behavior tests, the animals of isolated group were approximately equally divided into vulnerable (showing depressive‐like reactions in the open field) and resistant (not differed from the non‐stressed control) to ulcerogenic stress (active subgroup). All isolated rats showed a reduced level of exploratory activity in the elevated plus maze. Rats from enriched environment showed greater motor and exploratory activity, as well as lower anxiety level compared to those in isolated group. Isolated rats were most sensitive to action of pain, whereas rats kept in enriched environment were less sensitive to pain exposure. In the isolated group, the weakest corticosterone release among all groups was observed in response to mild procedural stress. Low HPA stress‐reactivity of isolated rats was combined with enhanced expression of glucocorticoid receptors in the hippocampus indicating a sensitization of the mechanisms of negative glucocorticoid feedback following prolonged social isolation. Thus, social isolation is a stressor leading to weight gain, a decrease in stress reactivity accompanied by an increase of glucocorticoid receptor expression in the hippocampus, a decrease of exploratory activity and increased of somatic pain sensitivity whereas enriched environment exerts beneficial action on functional state of the body: increases motor and exploratory activity, decreases anxiety and somatic pain sensitivity.
Endothelial Cell Sprouting in Coronary Collateral Growth
Authors: Juguilon Cody , Jamaiyar Anurag , Wan Weiguo , Gadd James , Enrick Molly , Wang Yang , Clark Alyssa , Chilian William , Yin Liya
Published: May 2022

Rationale

– Coronary collateral growth is a natural bypass for ischemic heart diseases. It offers tremendous therapeutic benefit, but the process of coronary collateral growth is incompletely understood due to limited preclinical murine models that would enable interrogation of its mechanisms and processes via genetic modification and lineage tracing. Understanding the processes by which coronary collaterals develop can unlock new therapeutic strategies for ischemic heart disease.

Objective

– To develop a murine model of coronary collateral growth by repetitive ischemia (RI) and investigate the process of coronary collateral growth in adult hearts by lineage tracing.

Methods and Results

– A murine model of coronary collateral growth was developed using short episodes of repetitive ischemia. Repetitive ischemia resulted in robust collateral growth in adult mouse hearts, validated by high‐resolution micro‐computed tomography. ROSA mT/mG reporter mice with Apln‐CreERT2 transgene were used for lineage tracing of sprouting endothelial cells to determine cellular contributions to coronary collateral growth. Two‐photon and confocal microscopy imaging revealed RI‐induced coronary collateral growth initiated from sprouting Apelin+GFP+endothelial cells. Newly formed capillaries in the collateral‐dependent zone expanded in diameter upon RI stimulation and arterialized with smooth muscle cell recruitment to form mature coronary collateral arteries. Notably, pre‐existing coronary arteries and arterioles were not Apelin+ GFP+, and all Apelin+endothelial cells arose from sprouting capillaries. HIF1α, VEGFR2, CXCR4, MCP‐1, and JAG1 signaling in the collateral‐dependent zone were also upregulated during coronary collateral growth.

Conclusions

– We developed a murine model of RI‐induced coronary collateral growth, and our data show sprouting EC contribute to coronary collateral growth in adult hearts. Sprouting angiogenesis is shown for the first time to arterialize into mature blood vessels of larger diameter in response to repetitive ischemia in adult hearts.
Effect of Sulindac in an in vitro AD Model Under hypoxic conditions
Authors: Rivera Oscar , Chen Belinda , Prentice Howard , Weissbach Hebert , Allani Shailaja
Published: May 2022

Background

Alzheimer’s Disease (AD) continues to be the most prevalent form of senile dementia without any significant therapy. Oxidative damage and hypoxia are known to play a major role in the pathology of AD. Hypoxia increases production of ß amyloid (Aß) plaques and dysfunction in tau proteins which leads to neuronal death by increasing reactive oxidative species (ROS) formation. Furthermore, Aß derived from the proteolytic cleavage of β‐amyloid precursor protein (APP) acts as a biomarker of AD. It is known that in an APP transgenic mice model of AD exhibits oxidative damage suggesting that a possible therapeutic approach would be to protect neuronal cells against oxidative damage. Our laboratory has shown previously that the drug sulindac, a known non‐steroidal anti‐inflammatory drug (NSAID) can protect retinal cells and the heart against oxidative damage by initiating a preconditioning response, similar to ischemic preconditioning that is independent of its NSAID activity. The goal of the present studies is to investigate whether sulindac can protect APP‐transfected neuronal cells from oxidative damage under hypoxic conditions.

Methods

We established an AD neuronal cell line by overexpressing APP using the plasmid vector pCAX‐APP (Swe/Ind) in SHSY5Y neuroblastoma cell line. Cell proliferation was assayed in the AD cell model under hypoxic conditions using trypan blue, under normoxic and hypoxic conditions, with and without sulindac. APP overexpression in the transfected cells was analyzed via Western blot.

Results

Our results show that the plasmid DNA induced the expression of APP in transfected SHSY5Y cells compared to untransfected cells and the expression of APP was strongest at 24 h. We observed that the cell proliferation decreased in APP transfected cells in both hypoxic and normoxic conditions. Further, reduced cell proliferation was seen under hypoxic conditions compared to normoxic conditions indicating that hypoxic conditions further exacerbateAD pathology. Our findings in an in vitro AD model, suggest that sulindac has a potential therapeutic value to ameliorate AD pathology.
Prevalence of Wormian (Sutural) Bones in the Paris Catacombs Ossuary
Authors: Schmeidler Katherine T.
Published: May 2022

Wormian, or sutural bones, vary in number, size, and arrangement in humans and other vertebrates. While their occurrence has been noted in some genetic bone development disorders, Wormian bones are also found in the general population. Currently, there are not reliable data to estimate the frequency at which they are found in humans with no underlying disorder. Some anthropologists have attempted to assess Wormian bone frequency in different human populations hoping to find whether there are differences among peoples of different genetic lineage. However, the sample sizes in these studies are too small to be able to compare such highly variable structures. The data presented here examining 500 skulls in the ossuary within les catacombes de Paris has revealed that Wormian bone occur in about 85% of skulls examined. This contrasts with most published studies that report incidences in the 10‐20% range. Les catacombes de Parisare believed to contain remains mostly from the late 18th and early 19th centuries. While there may be bones from earlier times, it is likely that these are fragile and fragmentary, and thus almost certainly do not contribute to the accessible visible walls of bones. These results are compared to surveys of skulls in three Italian collections: Museo di Anatomia umanaat Luigi Rolando dell'Università di Torino, Museo di Antropologiaat Università degli Studi di Padova,and Museo di Storia Naturale Sezione di Antropologia e Etnologia di Firenze, and in the only two known medieval English church ossuaries, in Hythe and Rothwell. The Italian collections represent Europeans, most if not all from late 19th and early 20th centuries, and the English skulls are presumed to be from the 13th and14th centuries. Wormian bones were found slightly more frequently in les catacombes de Paris than the other sites, but in each as well as all taken together, the incidence is significantly higher than is commonly reported for skulls from other parts of the world. The sample size of each of these studies individually, as well as in composite, is a very large. This large sample size further increases the credibility of these data. These findings expand the data set for comparison of Wormian bones from different parts of Europe and to peoples from other regions of the world, thus serving the greater community by providing a large baseline against which smaller sample groups may be compared. Additionally, the photographic evidence collected in this project may be used to support further research in human anatomical variation.
Use of Labeled Prosections for Self‐Guided Learning in Head & Neck Anatomy: Comparison to Traditional Instructional Approaches
Authors: Mears David
Published: May 2022

Aim

The purpose of this study was to evaluate the effectiveness of a self‐guided learning approach for head and neck anatomy using faculty‐prepared, labeled cadaveric prosections.

Introduction

At the onset of the COVID‐19 pandemic, when in‐person group learning activities were suspended for public health reasons, medical schools worldwide were forced to develop innovative ways to teach anatomy without traditional student‐led dissections. One approach that we implemented for head and neck anatomy at USU was to display faculty‐prepared, fully‐labeled prosections in the anatomy lab, and allow students to come in individually to learn from them. This self‐guided approach was positively received by students as a COVID‐19 contingency (spring 2020), so we decided to use a modified version of this strategy for some of the head and neck anatomy labs even after the local pandemic conditions had allowed in‐person dissection to resume (spring 2021). In this study, we quantitatively compared learning outcomes from self‐guided learning, with outcomes from two traditional instructional methods: student‐led dissection and faculty‐guided prosection demonstration.

Approach/Methods

For the 2021 Head and Neck Anatomy block, six cadaver laboratories were taught by traditional dissection (teams of four students with faculty preceptors present) and six were taught using labeled prosections. The prosections were put on display along with a detailed key to guide the students’ learning of the structures and key relationships. For the lab session, students in groups of four were assigned times to view the displays (5 minutes per specimen), with faculty on hand to answer questions. After the session, specimens were made available 24/7 for students’ independent review. Student performance on practical exam questions was compared for labs taught with labeled prosections versus the same labs taught by traditional dissection or faculty‐guided prosection demonstration from a prior year of the course (2019), using ANOVA and student’s t‐tests.

Results

Learner satisfaction with the self‐guided prosections was high, with 87% of students reporting that they found this instructional method to be helpful for learning anatomy. There were no significant differences in student exam performance for labs taught via labeled prosections compared to traditional dissection or faculty‐guided prosection demonstration.

Discussion

We found that self‐guided learning from labeled prosections was an effective, well‐received instructional method for head and neck anatomy. Implementing this approach for 6 of the 12 laboratory sessions in the Head and Neck Anatomy block resulted in a 25% decrease in curricular contact hours, and a 33% decrease in faculty time required to staff the laboratory sessions, with no detriment to learning outcomes. This approach could easily be extended to other aspects of the Gross Anatomy curriculum.
Blood Storage Solutions Effecting Advanced Glycated End Products (AGEs) N‐CEL and N‐CML on Red Blood Cell Membranes
Authors: Skrajewski Lauren , Geiger Morgan , Jacobs Monica , Spence Dana
Published: May 2022

Blood transfusions are critical, often lifesaving, procedures that result in approximately 10.5 million transfusions per year in the United States. Donated red blood cells (RBCs) is stored for a maximum of 42 days in FDA approved storage solutions. These storage solutions contain high levels of glucose to promote the longevity of the cells donated. However, AS‐1 storage solution contains 111 mM glucose, which is about 20 times higher than normal blood concentrations (5‐6 mM). Hyperglycemia is often associated with diabetes (7‐9 mM glucose), and prolonged hyperglycemic conditions are known to cause various blood flow‐related complications (retinopathy, neuropathy, nephropathy, cardiovascular disease). There is evidence of adverse RBCs changes in hyperglycemic conditions during the time over which storage would occur. Specifically, increased glycation is linked to increased amounts of advanced glycated end products (AGEs).

AGEs are formed from the glycation process, as higher glucose conditions produce a spontaneous non‐enzymatic reaction of free reducing sugars with free amino groups that form Amadori products, which undergo irreversible changes that lead to the formation of AGEs. These compounds promote oxidative stress and inflammation and have been linked to various chronic diseases, often linked with diabetes. RBCs release the necessary ATP for proper blood circulation, and there is previous evidence showing the high glucose storage decreases ATP release over 36 days. Also, there is a consistent ATP levels for the AS‐1N (5 mM glucose) storage solution over 36 days which are higher than the hyperglycemic storage solution.

Decreased ATP release during storage may be linked to hyperglycemic storage conditions increasing AGE biomarkers on the RBC membrane. Two of these AGE adducts of carboxymethyl lysine (N‐CEL and N‐CML) are used to quantify glycation on the RBC membrane by studying adduct concentration changes during storage. Two solutions were prepared (AS‐1 and AS‐1N) and used for storage for 36 days, with aliquots of RBCs removed every 4‐7 days for analysis. The amount of irreversible glycation products, thiols, and total lysine groups were determined to evaluate the amount of modified species over time. LC‐MS was used to quantify these modified N‐CEL and N‐CML adducts on the RBCs using internal standards. Overall, our results probe the hyperglycemic conditions on RBCs and examine the effects of increased glycation on cellular release of components from these stored cells.
A mechanism of regulation for the ubiquitin‐sensing kinase TNK1
Authors: Vaughan Alec J. , Egbert Christina , Chan Yin , Lopez Tania , Ashworth Spencer , McCormack Katherine , Andersen Joshua
Published: May 2022

TNK1 is a poorly understood non‐receptor tyrosine kinase that by mutation can be converted into an oncogenic driver. While little is known about the normal function of TNK1, it possesses a C‐terminal ubiquitin binding domain (UBA) that interacts with high affinity to multiple poly‐ubiquitin linkages. To our knowledge, this ability to directly interact with poly‐ubiquitin makes TNK1 unique across the human kinome. Our recent data suggest that the UBA domain of TNK1 homes the kinase to phase‐separated condensates of poly‐ubiquitinated proteins where TNK1 becomes fully active. We hypothesize that the interaction between the TNK1 UBA and poly‐ubiquitin at these ubiquitin‐rich condensates tethers the kinase to its substrates, which are involved in condensate biology and cell survival signaling. These findings led us to question how the interaction between TNK1 and ubiquitin is regulated. We have focused on a phosphorylation at Y661 within the TNK1 UBA domain, which has been identified in high throughput PTM mass spectrometry (phosphosite.org) but is of unknown function. Here we show that intrinsic TNK1 kinase activity is required for phosphorylation of Y661, suggesting that it may be an autophosphorylation. Furthermore, our preliminary data suggest that a phosphomimic mutation at Y661 leads to an increase in ubiquitin binding, suggesting that this phosphorylation may enhance the affinity of the TNK1 UBA for ubiquitin. Together, our data suggest a feed‐forward mechanism of TNK1 regulation in which activation of TNK1 kinase activity primes the UBA for binding ubiquitin via phosphorylation at Y661. In turn, the UBA domain helps cluster active TNK1 at ubiquitin‐rich condensates where TNK1 is brought into proximity to its substrates.
Defining the role of endotoxemia during heat stress in oxidative skeletal muscle
Authors: Rudolph Tori E. , Harold Kylene , Opgenorth Julie , Baumgard Lance H. , Selsby Joshua T.
Published: May 2022

Heat stress (HS) is a multifaceted stress, negatively impacting the skeletal muscle, reproductive, and gastrointestinal systems, among others. Endotoxemia occurs during HS and originates from structural changes to the gastrointestinal system, making it permissive for leakage of typically impermeable bacterial lipopolysaccharides (LPS). In this investigation, to better understand the complexities of HS‐mediated changes in skeletal muscle we aimed to identify contributions of endotoxemia to changes in skeletal muscle that occur during HS. Pigs (44 ± 2.6 kg) were assigned to 1 of 2 treatment groups: control (CON; 2mL saline injection; n=6) or LPS (40 µg/ kg BW; Escherichia coli O55:B5; n=6). Injections were given intramuscularly in the mid‐gluteal/ superficial gluteal muscles. All pigs were individually housed with restricted access to food and water 2 h prior to and throughout the 6 h experimental period. Pigs were euthanized 6 h post injection, and the oxidative (STR) portion of the semitendinosus muscle was collected for analysis. Injection with LPS decreased total circulating Ca2+ (30%, p=0.04), but increased skeletal muscle Ca2+ content (55%, p=0.03). Within STR, LPS decreased SERCA2a (28%, p=0.032) and Phospholamban (PLB; 59%, p=0.05) protein abundance but increased PLB bound to SERCA2 (100%, p=0.052), which antagonizes SERCA2a function. These data suggest decreased capacity of Ca2+ clearance, which can cause alterations in mitochondrial membrane potential, as mitochondria serve as secondary Ca2+ storage reservoirs. The change in mitochondrial membrane potential caused by the influx of Ca2+ can alter proteins associated with mitochondrial clearance via mitophagy. Consistent with this notion, LPS treatment decreased Parkin (52%, p=0.0002) however, increased BNIP3L protein abundance (25%, p=0.005), which may support a PINK1/Parkin‐independent mode of mitophagy. As a result, markers of mitochondrial abundance were decreased (NDUFB8 – 43%, p=0.0035; COX IV – 39%, p=0.0358; SOD2 – 44%, p=0.009) by LPS. Notably, these outcomes are distinct from previous reports in skeletal muscle following 6 h of HS indicating endotoxemia, alone, is not sufficient to drive changes associated with HS in skeletal muscle.
Endothelial Mineralocorticoid Receptors Constrain Arteriogenesis and Perfusion Recovery Following Chronic Arterial Ligation in a Sex‐specific Manner
Authors: Brown Scott M. , Meuth Alex I. , Aragonez Christian G. , Bailey Chastidy A. , Ruff Margot L. , Chandrasekar Bysani , Martinez‐Lemus Luis A. , Grisanti Laurel , Jaffe Iris Z. , Bender Shawn B.
Published: May 2022

Vascular occlusive diseases are major contributors to cardiovascular morbidity and mortality. Prior work has implicated mineralocorticoid receptor (MR) signaling in perfusion recovery following arterial occlusion, however, cell‐specific MR signaling in the associated angiogenic and arteriogenic responses has not been elucidated. Further, accumulating evidence has revealed sex‐specific involvement of MR signaling in a number of cardiovascular disease states. Accordingly, we examined the hypothesis that MR signaling, specifically endothelial cell (EC) MR signaling, attenuates vascular compensation and perfusion recovery following femoral artery excision and that this effect would be more pronounced in females. Unilateral femoral artery ligation and excision was performed in male and female C57BL/6J mice treated with vehicle or the MR antagonist spironolactone (Spiro; 20 mg/kg/d, sc) as well as in male and female MR Intact and EC MR knockout (KO) mice. Serial assessment of hindlimb perfusion for 28 days following surgery revealed enhanced perfusion recovery following Spiro treatment in female, but not male, mice. Interestingly, Spiro increased gastrocnemius capillary density in both the ischemic and sham limb in males and females. Following perfusion fixation, collateral remodeling was assessed by micro‐computed tomography revealing enhanced collateral expansion following Spiro treatment in female, but not male, mice. Additional mechanistic experiments similarly revealed increased perfusion recovery and enhanced collateral expansion in female, not male, EC MR KO mice independent of capillary density. At 28 days post‐surgery, distribution analysis revealed larger overall arteriolar diameters only in the collateral zone of EC MR KO females. Further analysis revealed enhanced early (7 days post‐surgery) collateral expansion in male and female EC MR KO mice. In female, but not male, EC MR KO mice this was associated with increased CD68+ macrophages and CD206+ ‘M2’‐like macrophages in the collateral zone. Together, these data demonstrate that EC MR signaling constrains early arteriogenic collateralization following arterial excision in both sexes and constrains long‐term collateralization only in females involving limitation of pro‐arteriogenic CD206+ ‘M2’‐like macrophages in the collateral zone. To our knowledge, this is the first report of a role for EC MR signaling in arteriogenesis and highlights potential use of MR blockers in the treatment of vascular occlusive events.
Examining the Impact of Cadaver‐Based Anatomy Laboratory Experiences on Medical Students Through a Thematic Analysis of their Written Reflections
Authors: Bellas Brooke , Guo Iris , Rauch Magnus , Ventura Nicole M. , McWatt Sean C.
Published: May 2022

Cadaver dissection has long been considered the gold standard pedagogical approach in anatomical education for its ability to teach students not only anatomy knowledge, but also non‐traditional discipline‐independent skills that are pertinent to professional training programs in healthcare. With the onset of the COVID‐19 pandemic in 2020, many medical schools were forced to adopt remote or blended teaching formats, reducing students’ dissection time in the anatomy laboratory. Some have raised concerns about how having limited interaction with body donors in anatomical education may dehumanize medical students’ learning experiences and alter their learning outcomes, but there is a paucity of research in this area. To examine how reduced exposure to cadavers during the COVID‐19 pandemic may impact the student experience, a pre‐pandemic assessment must first be made. Halfway through the first year of medical school, McGill University medical students consider the meaning of their anatomy laboratory experience and how it has contributed to their future career through a written reflective assignment. The present study aimed to describe how students reflected on their laboratory experience and its importance in their training as both individuals and future healthcare professionals, prior to the onset of COVID‐19. Reflections written by the McGill University medical class of 2023, whose time in the anatomy laboratory was not limited by the pandemic, were analyzed through the six‐step framework to thematic analysis by Braun and Clarke (2006). Three undergraduate research students (BB, MR, IG) inductively coded students’ reflections (n = 134) using NVivo software to identify preliminary themes. Themes were reviewed, defined, and named through discussion with the entire research team (all listed authors). Three central themes were identified: (1) philosophical reflections, (2) perceived outcomes of the anatomy laboratory and (3) reflections on the body donor or body donation. Notable sub‐themes included: (1.1) reflections on the body and soul, (2.1) reflections on the current medical curriculum and (3.1) the dichotomy of objectification and personification during student‐donor interaction. The presence of these themes and example quotes drawn from the reflections confirm that donor‐dissection not only teaches students anatomical knowledge but enables them to realize the importance of dissection to the medical curriculum, consider the gift that is body donation, and explore philosophical ideas of life and death. A thematic analysis of the reflections written by McGill University’s 2024 medical class, who were subject to a blended learning format during the COVID‐19 pandemic, is currently underway to determine the degree to which decreased laboratory exposure influenced the students’ reflections on their laboratory experience.
Evolution and Development of the Interparietal in Dolphins and Other Toothed Whales (Odontocetes)
Authors: Roston Rachel , Boessenecker Robert , Geisler Jonathan
Published: May 2022

Mammal skulls of diverse shapes and sizes exhibit a skull roof composed of the same bones with a similar topological arrangement: bilateral frontal and parietal bones meet at the midline, and in many cases, an interparietal bone lies posterior to the parietals. Despite this conserved skull roof arrangement, the interparietal has been a puzzling bone in comparative embryology and evolutionary morphology due to its mixed developmental origins and its variable patterns of fusion with neighboring bones, resulting in mixed reports of presence and absence in different taxa. The skull roof of dolphins, in contrast to other mammals, features a prominent interparietal that separates the parietal bones so that the parietals do not meet at the midline. While this unusual configuration has been described in individual specimens, the evolutionary developmental patterns that led to the dolphin arrangement are largely unknown. To understand how this unique arrangement arose, we surveyed presence, absence, morphology, position, and developmental origins among early odontocete fossils and extant odontocete fetuses. Among extant odontocetes, delphinoids all have multi‐element interparietal bones: one comprising the traditional “interparietal” between the paired parietal bones, and two that fuse posteriorly to the supraoccipital. However, extant physeteroids lack an identifiable interparietal, possibly resulting from the fusion of interparietal elements to the supraoccipital during early development. Thus, in physeteroids, the parietals meet at the midline as in other mammals. Olympicetus, an Oligocene stem odontocete, exhibits several well‐preserved multi‐element interparietals, including one underneath and at the midline of its paired parietal bones. However, other Oligocene stem odontocetes from South Carolina revealed no identifiable interparietal at multiple growth stages, which may have resulted from fusion of interparietal elements to other skull bones. These new data suggest that fusion patterns of the multiple elements of the interparietal are highly variable among odontocetes and support the hypothesis that the presence of a large interparietal separating the parietals is a uniquely delphinoid feature.