bluuebunny/crossref_metadata_2025 huggingface dataset:
Follow Your Nose: Ontogeny of the Olfactory Duct in Primates
Authors: Haynes Laura K. , Franklin Katheryn P. , Smith Timothy D. , DeLeon Valerie B.
Published: May 2022

Most primates are characterized by orbital convergence, the displacement of the orbits toward the rostral midline. In platyrrhine primates and tarsiers, this convergence is so extreme that orbits meet in the midline at the apical interorbital septum. This produces a spatial separation of the anterior cranial fossa and the nasal cavity. The olfactory bulbs of the brain pass dorsal and then rostral to the orbits to reach the nasal cavity via a bony tube, the olfactory duct. Nerves are expected to constrain growth of musculoskeletal structures. We hypothesized that the network of olfactory nerves effectively tethers the olfactory bulbs to the mucosa of the nasal cavity, and the growth of facial structures in these primates results in rostral displacement of the olfactory bulbs through ontogeny. In addition, growth of the midface may be constrained by this functional relationship.

This comparative analysis was based on 3D reconstructions of cranial structures in newborns and adults from representative platyrrhine species (e.g., Aotus nancymaae and Cebuella pygmaea) and tarsiers (Tarsius syrichta). Spatial relationships of the olfactory duct were estimated using endocasts based on conventional microCT scans. In addition, diffusion iodine contrast‐enhanced CT (diceCT) images were used to study Aotus nancymaae at multiple stages of ontogeny. DiceCT allowed us to reconstruct the actual position of olfactory bulbs relative to the olfactory duct and the distribution of olfactory nerves in the nasal cavity. 3DSlicer and Blender software were used to render 3D models. These data confirm our hypothesis that the rostral projection of the olfactory bulbs relative to the frontal lobes of the brain increases through ontogeny. In addition, the paths of olfactory nerves are diverted around the convergent eyes. The ontogenetic relationships occurring in platyrrhine and tarsier olfactory ducts have evolutionary implications for biological variation in the primate skull.
High Fat Diet and High Fructose Diet‐fed Male and Female Liv‐ARKO Mice Displayed Impair Glucose‐Stimulated Insulin Secretion
Authors: Falzarano Claire , Wilson Andre , Carr Kiana , Levey Josephine , Lofton Taylor , Andrisse Stanley
Published: May 2022

Insulin resistance affects up to a third of the US adult population and polycystic ovary syndrome (PCOS) affects up to 10% of reproductive‐age adult women. The liver plays an essential component in the metabolism of insulin and androgen signaling. Hyperandrogenism in females can also increase predisposition to insulin resistance. In our study, we placed female and male liver androgen receptor knock‐out mice (Liv‐ARKO) on high fructose diets (HFrD) and high fat diets (HFD) to determine if Liv‐ARKO mice demonstrated altered glucose‐stimulated insulin secretion on special diets compared to a control diet.

In this study, female and male Liv‐ARKO mice were placed on three distinct diets: Control (Research Diets Inc), High Fat (HFD), and High Fructose (HFrD). After one month and two months on these diets, Glucose‐Stimulated Insulin Secretion tests were performed: each group was injected with a 20% glucose solution at 2g of glucose/kg body mass and blood samples were taken via tail milking at 0 minutes, 15 minutes, and 30 minutes. Blood samples were immediately centrifuged and serum supernatant was extracted for further analysis via Sigma Insulin Assay.

In the current study, after two months, female mice on HFrD demonstrated lower basal insulin levels compared to female mice fed a control diet. Interestingly, mice on a HFD showed insulin levels that were lower than the basal levels of both control mice and HFrD mice. Furthermore, compared to the control mice, the HFD mice demonstrated lower insulin levels at both basal level and 30 minutes post‐glucose administration.

It was hypothesized that HFD and HFrD‐fed Liv‐ARKO mice would impair glucose‐stimulated insulin secretion, which our current study supports. These data suggest that diets high in fructose and fat both blunt insulin secretion following a meal, and to a greater extent in high fat diets. This dysfunction may ultimately play a role in the development of insulin resistance. More research is necessary in order to further investigate the effects of high fat and high fructose diets on glucose‐stimulated insulin secretion and consequent development of insulin resistance.
Cutaneous Microvascular Endothelial Function: Effects of Sex and Menopause Stage
Authors: Parr Shannon , Hammond Stephen , Sutterfield Shelbi L. , Ade Carl J. , Smith Joshua R.
Published: May 2022

Background

Endothelial dysfunction and increased arterial stiffness are both associated with increased risk of cardiovascular events and mortality.Vascular aging has been characterized by deteriorating endothelial function accompanied by increasing vascular stiffness that occurs with increasing chronological age, with the onset of these changes varying by sex. Deteriorating large artery vascular function has been reported across the menopause transition in women, but it is unclear if this relationship extends to the cutaneous microvasculature, which may have critical implications for both thermal regulation and general cardiovascular health.

Purpose & Hypothesis

The primary purpose of this study was to determine the impact of sex and menopausal stage on cutaneous endothelial dependent (EDD) vasodilation. We hypothesized that cutaneous EDD vasodilation would decrease in women in a graded manner across the transition from pre, to peri, to postmenopause with differences compared to age matched men. Secondly, we hypothesized that there would be no differences in cutaneous EDD vasodilation between natural and surgical (i.e., hysterectomy) menopause.

Methods

Premenopausal, (n=8, 44±5y), perimenopausal (n=6, 51±2y), and postmenopausal women (n=9, 60±7y) as well as men age matched to the postmenopausal group (n=6, 59±7y) volunteered for this study. Cutaneous EDD vasodilation was measured by local iontophoresis of Acetylcholine (Ach) administration into the forearm vasculature. Red blood cell flux was determined by laser Doppler flowmetry at baseline and after 200µL of Ach. CVC was calculated as the quotient of red blood cell flux and mean arterial pressure and expressed as the percentage increased from baseline (CVC%). Differences were determined using a 1‐way ANOVA. Data are presented as mean ± standard error.

Results

Forearm microvascular CVC% was significantly different among groups (P=0.01) after Ach administration. Post hoc analyses revealed an attenuated CVC% response in postmenopausal women (691±150%; P=0.02) and men (733±127%; P=0.05) compared to premenopausal women (1398±247%). There were no differences detected between peri‐ and premenopausal women (1290±140% vs. 1398±247%; P=0.95). Further, we performed a subanalysis in our postmenopausal group which included surgical (n=3) and natural (n=8) postmenopausal women. No differences were present between surgical and natural postmenopausal women (P=0.87), while both the surgical (646±163%; P=0.08) and natural (713±221%; P=0.05) postmenopausal women appeared to have decreased CVC responses compared to premenopausal women (1398±247%).

Conclusions

Our findings indicate that cutaneous EDD vasodilation decreases from pre‐ to postmenopause in healthy women. Our data suggests that decreases in microvascular function agrees with reported decrements in large vessel vascular function associated with the menopause transition.
Knockout of fatty acid amide hydrolase (FAAH) gene attenuates cisplatin‐induced nephrotoxicity in mice
Authors: Chen Chaoling , Wang Weili , Ritter Joseph K. , Lichtman Aron H. , Gewirtz David A. , Li Ningjun
Published: May 2022

Cisplatin, a platinum‐containing drug, is widely used in cancer therapy for a variety of solid tumors. However, acute kidney injury (AKI) is the primary dose‐limiting toxicity of cisplatin treatment, which often requires discontinuation of the treatment. Recently, the endocannabinoid system including anandamide (AEA) and 2‐arachidonoylglycerol (2AG) and their cannabinoid receptors (CBs) has been documented to play important roles in renal function and participate in different kidney diseases. However, little is known about roles of their metabolic enzymes in kidney damages, including cisplatin nephrotoxicity. Inhibition of FAAH, the primary enzyme for the degradation of AEA, has been reported to elicit anti‐inflammatory effects. The current study tested the hypothesis that inhibition of FAAH attenuates cisplatin‐induced acute kidney injury. Male wild type C57BL6 (WT) and Faah−/−mice were subjected to a single dose of intraperitoneal injection of cisplatin (30 mg/kg) or saline as vehicle and sacrificed 72 hours later. In comparison with WT‐vehicle mice, Faah−/− mice showed pronounced reduction of blood urea nitrogen (BUN) (mean: 23.8 ±1.24, 156.6±15.2, and 81.8±12.25 mg/dL in WT‐vehicle, WT‐cisplatin, and Faah−/−‐cisplatin mice, respectively, p<0.001) and plasma creatinine level (mean: 1.18 ± 0.12, 3.30 ± 0.46, and 1.45±0.46 mg/dL in WT‐vehicle, WT‐ cisplatin, and Faah−/−‐ cisplatin mice, respectively, p<0.01) after cisplatin‐induced nephrotoxicity. Semi‐quantitative histologic analysis by Periodic acid–Schiff (PAS) staining demonstrated that cisplatin‐induced kidney injuries were significantly attenuated in Faah−/− mice especially in tubular structure (tubular injury score: 0 in WT‐vehicle, 3.6 in WT‐ cisplatin, and 2.3 in Faah−/−‐ cisplatin mice, respectively, p<0.001). Western blot analysis revealed that in comparison with WT‐vehicle mice, WT‐cisplatin mice showed significant upregulation of kidney injury molecule‐1 (KIM‐1), which was remarkably reduced in Faah−/−mice (1.6‐fold versus 0.8‐fold compared to the WT‐vehicle group, p<0.05). These data suggest that inactivation of FAAH prevents nephrotoxicity caused by cisplatin treatment and that targeting FAAH may provide a novel strategy to mitigate cisplatin‐induced nephrotoxicity.
Dysfunctional Nrf2‐Keap1 redox signaling in the RVLM is linked to obesity‐induced sympathoexcitation
Authors: Monteiro Raisa , Sivasubramanian Mahesh Kumar , Balasubramanian Priya , Subramanian Madhan
Published: May 2022

The rostral ventrolateral medulla (RVLM) is an important region in the brainstem responsible for the tonic and reflex control of sympathetic nerve activity (SNA). Pathological changes at the level of RVLM like oxidative stress has been previously associated with sympathetic nervous system overactivity in obesity. Nuclear factor erythroid 2‐related factor 2 (Nrf2) is a transcription factor that provides cytoprotection against oxidative stress by regulating the expression of antioxidant and anti‐inflammatory genes. Nrf2 bound to Kelch‐like ECH‐associated protein 1 (Keap1) in the cytoplasm is inactive and undergoes proteasomal degradation under normal conditions. In response to oxidative stress, Nrf2 dissociates from Keap1, translocates to the nucleus, and forms a complex with the antioxidant response elements (AREs) and small musculoaponeurotic fibrosarcoma protein (smaf) leading to the transcription of multiple antioxidant genes. Previous studies from our lab have shown that increased oxidative stress is associated with Nrf2 dysfunction leading to decreased expression of Nrf2 antioxidant target enzymes in the RVLM of obese mice. The molecular mechanisms of Keap1 in Nrf2 dysfunction and the phenotype of cells undergoing Nrf2 dysfunction at the level of the RVLM in obese animals are unclear. To address these questions, we performed immunofluorescence in 20µm RVLM brainstem sections of C57BL/6J mice fed with a high‐fat diet (60kcal% fat) and a chow diet (10kcal% fat) for a period of 16 weeks to investigate the expression of Nrf2 and Keap1. Immunofluorescence showed a higher expression of Keap1 and lower expression of Nrf2 in the RVLM of obese mice when compared with their lean counterparts. To determine the type of cells undergoing Nrf2 dysfunction, an in‐vitro cell culture model was developed with human brainstem astrocytes. We hypothesized that brainstem astrocytes are more susceptible to Nrf2 dysfunction mediated oxidative stress. In this experiment, hydrogen peroxide (H2O2) was used to mimic diet‐induced oxidative stress. Cultured human brainstem astrocytes were treated with H2O2 at the dose rate of 300μM for 2 hrs. Nrf2 and Keap1 levels were assessed by gene expression analysis at the end of 5 days. Data was analyzed by unpaired student's t‐test and a p‐value less than 0.05 was considered statistically significant. The results showed that H2O2 treatment significantly increased the gene expression levels of Keap1 in human brainstem astrocytes with a trend towards a decrease in the expression of Nrf2. Our results suggest that in obesity, Nrf2‐Keap1 dysfunction could occur in astrocytes leading to decreased antioxidant defenses and increased oxidative stress in the RVLM which in turn can cause sympathoexcitation. Future in vivogene transfer studies will investigate the mechanistic role of Nrf2‐Keap1 pathway in obesity‐induced sympathoexcitation.
Integrated redox sensitivity of vascular Kv1 channels
Authors: Raph Sean M. , Dwenger Marc M. , Nystoriak Matthew A.
Published: May 2022

Voltage‐gated potassium (Kv) channels in vascular smooth muscle are responsive to a range of molecular signals that link local metabolic demand to blood supply. We recently found that elevation of local lactate:pyruvate, reflective of enhanced tissue oxygen demand and consequent changes in smooth muscle cytosolic NADH:NAD+, potentiate native Kv1 activity in a manner that is dependent on pore complex‐associated Kvβ proteins (i.e., Kvβ1 and Kvβ2). Considering that these proteins also participate in the regulation of Kv function in response to oxidants, we tested the hypothesis that Kv1‐dependent vasoreactivity to the endogenous dilator hydrogen peroxide (H2O2) is modified by intracellular NAD(H) redox status. Application of H2O2 to preconstricted (100 nM U46619, 80 mmHg intravascular pressure) mesenteric arteries from mice resulted in concentration‐dependent vasodilation (0.1‐10 μΜ). However, the magnitude of vasodilation in response to 10 μM H2O2 was significantly enhanced when applied in the presence of 10 mM L‐lactate (10 μΜ Η2Ο2: 16 ± 9%; 10 mM L‐lactate + 10 μM H2O2: 80 ± 15% dilation), but not in the presence of 10 mM pyruvate (~11% H2O2‐induced constriction). Consistent with direct effects of pyridine nucleotide redox state on oxidant‐induced Kv potentiation, increases in single Kv1 channel open probability in excised membrane patches from isolated vascular smooth muscle cells in response to 10 μM H2O2 were significantly enhanced in the presence of 1 mM NADH. Based on these results, we examined the role of the intracellular Kvβ subunit complex in redox‐modified vasodilation in response to H2O2. Arteries isolated from double transgenic mice with doxycycline‐inducible increases in smooth muscle Kvβ1:Kvβ2 protein ratio exhibited a complete loss of L‐lactate‐induced enhancement of vasodilation to H2O2. Together, our results suggest that the Kvβ subunit complex in vascular smooth muscle may integrate multiple redox signals to enable tight control over Kv1 gating, vascular tone, and blood flow upon dynamic changes in metabolic demand.
Wavelet Based Machine Learning Approaches Towards Precision Medicine in Diabetes Mellitus
Authors: Shankar Adeethyia , Chang Stephanie , Wang Xiaodi , Zhao Yongzhong
Published: May 2022

It is estimated that 422 million people around the world have diabetes mellitus (DM)—a devastating, complex, and highly heterogeneous disease—requesting better interventions based on disease subtyping. In this research, we utilize the discrete wavelet transform (DWT) to decompose and denoise DM data. Using DWT, we enhance heart rate variability (HRV) based DM diagnosis, data visualization of the disparities in Human Microbiome Project (HMP) data (gut bacteria, metabolomics, proteomics, RNA sequencing, targeted proteomics, and transcriptomics data) using demographic features, and insulin resistance prediction. We also attempt to forecast continuous glucose monitoring (CGM) ahead by 90 minutes because CGM is unable to provide real‐time blood glucose measurements. We achieve 91.9% diagnosis accuracy for Type 1 DM using Random Forest on data transformed with DWT, holding the potential for usage in clinics. In addition, our DWT‐based t‐SNE and UMAP explorative analysis of HMP data support subtypes of prediabetic patients stratified by sex, race, and age. Moreover, DWT‐based transformations provide multi‐view clustering that any other methods would not provide on metabolomics, proteomics, RNA sequencing, targeted proteomics, and transcriptomics data and outperform those without DWT. Taken together, DWT‐based machine learning approaches enable a fine resolution of subtyping DM towards precision medicine.
Craniofacial Defects Persist in Eftud2 mutant mice with P53 Deletion
Authors: Beauchamp Marie‐Claude , Boucher Alexia , Dong Yanchen , Majewska Loydie J.
Published: May 2022

EFTUD2 is mutated in patients with mandibulofacial dysostosis with microcephaly (MFDM). We previously showed that homozygous deletion of Eftud2 in neural crest cells causes brain and craniofacial malformations, affecting the same precursors as in MFDM patients. This phenotype was partially rescued by inhibition of the P53‐pathway using pifithrin‐α. Herein, we specifically evaluated the role of P53 in craniofacial abnormalities in Eftud2 mutants by genetically removing both Eftud2 and Trp53 in the neural crest cells (Eftud2‐P53 double knock‐outs (DKOs). We found that removing one or two alleles of Trp53 did not improve survival or craniofacial development in Eftud2 mutants. However, cleaved caspase‐3 immunostaining revealed that the abnormal increase in cell death found in Eftud2 mutants at E10.5 was reduced in the neural tube of Eftud2‐P53 neural crest DKOs. This suggests that the craniofacial abnormalities found in Eftud2‐P53 neural crest DKOs mutants are not only caused by apoptosis. We next aim to evaluate if cell cycle arrest could contribute to the phenotype. FoxM1 is a transcription factor which is essential for G2 progression into mitosis and is regulated by P53. We found increased splicing of FoxM1 in RNAseq experiments from Eftud2 mutants. Interestingly, the expression of the FoxM1 target, Cdc25b, was decreased by removal of P53 in Eftud2mutants as was that of Cdkn1a, a P53‐target required for mitosis. Altogether, our data suggest that in the presence of P53, Eftud2 mutants neural crest cells undergo apoptosis, whereas in the absence of this protein, they undergo cell cycle arrest. Ultimately, mis‐splicing leads to malformation in both a P53‐dependent and independent manner, which both ultimately contribute to a loss of neural crest cells and their derivative structures.
Regulation of ATP‐Citrate Lyase During Lipogenesis in the Oleaginous Yeast Yarrowia lipolytica
Authors: Anche Varsha C. , Fakas Stylianos
Published: May 2022

ATP citrate lyase (ACL) catalyzes the ATP‐dependent conversion of citrate to the fatty acid precursor, acetyl‐CoA. ACL presence in yeasts has been associated with their ability to accumulate lipids (i.e., oleaginous phenotype), but little is known about the regulation of this enzyme in oleaginous yeasts. In the model oleaginous yeast Yarrowia lipolytica, ACL is a heterodimer comprised of a catalytic and a regulatory subunit, encoded by the ACL1 and ACL2 genes, respectively. From the earlier studies, it was shown that the loss of ACL1 resulted in lower lipid levels and altered fatty acid profiles. However, the regulation of ACL expression and activity during lipogenesis has not been studied. To better understand the role, ACL plays during lipogenesis in Y.lipolytica, we generated antibodies against its two subunits (i.e., Acl1 and Acl2). We also constructed strains that lack Acl2 (i.e., acl2Δ) and strains that overexpress Acl1 and Acl2 either alone or in combination. Preliminary experiments showed that the overexpression of Acl1 increased the protein levels of Acl2. We are currently analyzing the effects of Acl2 overexpression and the time‐dependent regulation of Acl1 and Acl2.
The GluN2A Subunit of the NMDA Receptor Modulates the Rate of Functional Maturation in Parvalbumin‐positive Interneurons
Authors: Camp Chad R. , Shapiro Lindsey , Vlachos Anna , Perszyk Riley E. , Shariatzadeh Nima , White Jacob , Sanchez Russell , Koh Sookyong , Escayg Andrew , McBain Christopher , Pelkey Kenneth , Traynelis Stephen F.
Published: May 2022

N‐methyl‐D‐aspartate receptors are glutamate‐gated, calcium‐permeable ion channels involved in a host of normal brain functions, including neuronal development. Traditionally, the GluN2A subunit, encoded by the GRIN2A gene, is thought to signal brain and circuit maturation, given its unique expression pattern where transcripts are low embryonically and then significantly increase during preadolescence in mice. Advances in whole exome sequencing have identified mutations in the GRIN2A gene to be causal to several neuropathological diseases, including epilepsy and intellectual delay. More specifically, 56% of GRIN2A mutations are loss‐of‐function, displaying diminished receptor function and/or surface expression, with Grin2a­‐/‐ mice have been reported as having signatures of epileptiform activity. However, little is understood regarding GluN2A’s impact on neurodevelopmental and how its absence may the GABAergic interneuron network. Our data show that Grin2a ‐/‐ mice contain 33% more parvalbumin+ interneurons in the CA1 region of the hippocampus during preadolescence and adulthood. This increase in GABAergic interneuron cell density appears to be unique to parvalbumin+ cells, as cholecystokinin+ and somatostatin+ interneurons are unchanged across Grin2a‐/‐ and wildtype mice. Despite an increased cell count, colocalization staining for parvalbumin+ synaptic densities and measures of parvalbumin cell‐specific tonic inhibition remains unchanged in preadolescence and increases as the animal ages, suggesting delayed maturation. In addition, parvalbumin+ cells from preadolescent Grin2a‐/‐ mice exhibit immature intrinsic and firing properties, such as an increased membrane time constant and input resistance, lower threshold for depolarization‐induced block, and longer action potential half‐widths than age‐matched WT controls, all of which become corrected in adulthood. The sum of these data suggest that the GluN2A subunit is involved in promoting cues for interneuron apoptosis during development, as well as influencing the rate of parvalbumin+ cell functional maturation. The ramifications of these abnormalities are still underexplored, but likely generate a vulnerability time window during preadolescence in which an epileptiform network can manifest. Viral strategies aimed at reintroduction of the GRIN2A gene during this critical time window could represent a viable gene therapy option for loss‐of‐function GRIN2A EE patients.
Foundations in Physiology: An Introductory Course Using the Core Concepts
Authors: Ball Karen L.
Published: May 2022

Over the past 5 years, the Alma College Integrative Physiology and Health Science Department has noted a decrease in student success in the introductory human physiology course. A major element in variable performance is the increasing diversity of prior experience in students’ educational background, specifically as it relates to courses in STEM disciplines. Within the life science community, and more recently, the physiology community, use of “core concepts” in undergraduate education has been actively promoted as a means to improve student outcomes. To address the concern regarding student success, the department implemented a new “Foundations” course as a pre‐requisite to the Anatomy and Physiology sequence. This class replaced the previous pre‐requisite, Introduction to Cell Biology. We hypothesized that this approach would produce significant gains in student learning. The specific core concepts covered in the class were: causality, scientific reasoning, physics/chemistry, structure‐function, homeostasis, flow down gradients, cell membrane, energy, cell‐cell communication, and interdependence/integration. This set of concepts included and class design emphasized the 5 principles identified by physiology educators as most important for students to know.1 To assess learning, the Phys‐MAPS assessment tool was administered to students enrolled in IPH 227: Human Physiology (for students who had previously completed the Cell Biology pre‐requisite) and students enrolled in IPH 131: Foundations in Physiology (no pre‐requisite completed).2 The tests were taken during the first week of class and again in the final week of the term. Although average scores increased by the end of the semester, the improvement was not significant, regardless of the class assessed (IPH 227: 36.8+5.8 vs 39.2+5.9 total points; IPH 131: 33.3+5.6 vs 36.5+7.3 total points). Importantly, students completing the Foundations class performed as well as those students who had previously taken the Introduction to Cell Biology as represented in the IPH 131 post and IPH 227 pre data, respectively (36.5+7.3 vs. 36.8+5.8). Therefore, while students from the Foundations course performed as well as those enrolled in a traditional introductory cell biology course at the end of each course, use of the “core concepts” failed to result in the expected learning outcomes. As an initial offering, there were multiple challenges/limitations that will be discussed. Adjustments in course design, together with additional/alternative assessment methods, could have an impact on measured student learning. Details of course design, assessment, and limitations will be presented.
Convolutional Neural Network Analysis to Predict Expiratory Flow Limitation During Exercise in Adults with Asthma
Authors: Haverkamp Hans C. , Petrics Greg
Published: May 2022

OBJECTIVE and HYPOTHESIS

The objective of this study was to assess the potential for using well‐known convolutional neural network (CNN) designs to accurately identify the presence of expiratory flow‐limitation (EFL) during exercise in adult asthmatics. We hypothesized that a CNN model would accurately identify spontaneous exercise tidal flow‐volume loops (eFVL) that exceed maximal airflow from the maximal forced expiration (i.e., EFL).

METHODS and RESULTS

The expiratory portion of spontaneous eFVLs were placed within the pre‐exercise maximal volitional flow‐volume envelope. Inspiratory capacity maneuvers were performed to determine the correct lung volume for placement of the eFVLs. We assembled a collection of spontaneous eFVLs from one asthmatic subject consisting of an equal number of eFVLs that were flow‐limited and non‐flow‐limited. Subsequently, our overall approach followed standard deep learning practices. The eFVLs were manually labelled as EFL and non‐EFL. The sci‐kit learn train_test_split() method was used to split the eFVLs randomly into training and testing sets using a 67%/33% split. We trained both a one‐ (expired airflow only) and a two‐channel (expired volume and airflow) CNN model to predict the presence or absence of EFL as a response to an eFVL. The models were instantiated as sequential models using the TensorFlow 2.6.0 python API. After 100 training epochs on the training cases, the 1‐channel model was challenged to predict the labels of the previously unseen testing cases. The 1‐channel model achieved 93.3% accuracy at predicting the EFL/non‐EFL labels as a response to the eFVLs despite having not been trained on these data. Additionally, the 2‐channel model was 100% accurate on the unseen test cases. We then challenged the 2‐channel model on 45 unseen eFVLs from a different asthmatic subject; the eFVLs consisted of a balance of EFL and non‐EFL. The model performed poorly, accurately identifying only 57% of the eFVLs as EFL or non‐EFL. We then trained an additional 2‐channel model on a random sample of the combined data from the two subjects and challenged the model on the unseen eFVLs from the same two subjects. At 25 epochs and 100 epochs, the CNN was challenged with unseen testing subjects and achieved 88 and 92% accuracy, respectively, at distinguishing between EFL and non‐EFL.

CONCLUSIONS

These proof‐of‐concept findings provide strong evidence that neural network analyses hold promise as a novel method for identifying either the presence or absence of EFL. Given the current, tedious and nuanced methods for determining EFL, such artificial intelligence analyses hold promise for automating the analysis and identification of exercise ventilatory constraints.
Roleplay as a Tool in Anatomy Teaching
Authors: Campos Letícia C. , Morano Domingos A. C. M. S , Carioca Levi C. , da Silva José M. , De Pinho Francisco V. A. , Teixeira Cláudio S. , Silva Renata S. E. , Cerqueira Gilberto S.
Published: May 2022

Roleplay is a technique that allows students to explore realistic situations by interacting with others in a managed way, in order to develop experiences, skills, and test different strategies in a learning environment. In the teaching of clinical anatomy, the use of this teaching methodology as a tool is scarce. Thus, we implemented the use of this technique as a strategy in the teaching and learning process. The objective of this paper was to report our experience based on the use of the roleplay technique in the teaching and learning process of clinical anatomy. An exploratory, cross‐sectional study with a quantitative approach was carried out with 61 students from the anatomy discipline. To carry out the proposed activity, the students were divided into four groups, in which each one planned a clinical anatomy situation. To meet these objectives, Roleplay was structured in three parts: planning the implementation of the roleplay technique, application of roleplay, and debriefing. The results showed that roleplay can be used adequately in the teaching of clinical anatomy. It was observed that 98.1% of students approved the use of the technique in teaching anatomy. It was found that 95.1% of respondents reported that the roleplay achieved the learning goals, while 4.9% reported that the learning goals were not achieved. There was no statistically significant difference between the participants' ages (p>0.05). It was found that 86.9% of respondents would recommend the use of roleplay as a teaching methodology for the next semester. It was found that roleplay is a playful, versatile, and totally viable and feasible teaching methodology as a tool in the teaching and learning process of clinical anatomy. In addition, this training contributes to discussions and reflections in the teaching‐learning process, especially regarding the use of active teaching and learning strategies.
Urinary Potassium Excretion Inversely Correlates with Diastolic Blood Pressure Variability in Healthy Normotensive Adults
Authors: Axler Michael R. , Kaseman Kathryn E. , Kingham Anna L. , Lobene Andrea J. , Lennon Shannon L.
Published: May 2022

High blood pressure (BP) is a common risk factor for cardiovascular disease (CVD), the leading cause of death worldwide. Dietary intake is an important contributor to BP. Increased dietary sodium intake has been shown to increase BP, while evidence suggests dietary potassium decreases BP. BP variability (BPV) refers to spontaneous fluctuations in BP that if exaggerated, are considered a risk factor for CVD. Limited work in this area suggests a high sodium diet may relate to increased BPV while potassium may decrease BPV. The purpose of our study was to investigate whether dietary intake of sodium and potassium relates to BPV in healthy, normotensive subjects. We hypothesized that individuals with a higher sodium intake, as well as increased sodium to potassium excretion ratio, would exhibit a higher BPV while those with increased potassium intake would have a lower BPV. Healthy men and women between the ages of 18 and 45 were recruited to participate. Habitual sodium and potassium intake was quantified with 24‐hour urine samples. During this same time period, subjects wore an ambulatory BP monitor for 24 hours with measures taken every 20 minutes during the day and every 30 minutes at night. These data were used to determine short term BPV. BPV was quantified by calculating the average real variability (ARV) index. Pearson bivariate correlations were run to assess the relation between sodium, potassium, and the sodium to potassium excretion ratio and the ARV of both systolic and diastolic BP. Additionally, partial correlations were run to control for the effects of age and sex. Fifty‐six (20M/36W) subjects (27±1 year; BMI 23±0.4; BP 114±1/67±1 mmHg) participated. Twenty‐four‐hour urinary potassium excretion was inversely associated with the ARV of diastolic BP (r=‐0.33, p=0.02). When controlling for age and sex this relation remained (r=‐0.31, p=0.03). However, neither 24‐hour urinary sodium excretion or the sodium to potassium excretion ratio was associated with the ARV of systolic (r=‐0.25, p=0.07; r=‐0.10, p=0.48, respectively) or diastolic BP (r=‐0.12, p=0.41; r=0.11, p=0.43, respectively). In summary, in this cohort of healthy normotensive adults, a higher potassium intake was associated with a lower diastolic BPV; however, there was no relation with sodium or the sodium to potassium excretion ratio. More research is needed to better understand the effects of both sodium and the ratio of sodium to potassium intake on BPV in healthy normotensive men and women.
External Collaboration Results in Student Learning Gains and Positive STEM Attitudes in CUREs
Authors: Peterson Celeste N. , Callahan Kevin P. , Martinez‐Vaz Betsy M. , Huisinga Kathryn L. , Galport Nicole , Koletar Courtney , Eddy Rebecca , Provost Joseph J. , Bell Jessica J. , Bell Ellis J.
Published: May 2022

The implementation of Course‐Based Undergraduate Research Experiences (CUREs) has made it possible to expose large undergraduate populations to research experiences. For these research experiences to be authentic, they should reflect the increasing collaborative nature of research. While some CUREs have expanded with multiple schools across the nation, it is still unclear how a structured extramural collaboration between students and faculty from an outside institution affects student outcomes. In this study, we established three cohorts of students: 1) no‐CURE 2) single institution CURE (CURE) and 3) external collaborative CURE (ec‐CURE) and assessed academic and attitudinal outcomes. The ec‐CURE differs from a regular CURE in that students work with faculty member from an external institution to refine their hypothesis and discuss their data. The sharing of ideas, data and materials with an external faculty allowed students to experience a level of collaboration not typically found in an undergraduate setting. Students in the ec‐CURE had the greatest gains in experimental design, self‐reported course benefits, scientific skills and STEM importance. Importantly this study occurred in a diverse community of STEM disciplinary faculty from 2‐ and 4‐year institutions illustrating that exposing students to structured external collaboration is both feasible and beneficial to student learning.
Susceptibility of Hepatic ProgenitoInhibition by acetazolamide of reactivated carbonic anhydrase activity in a receptor protein tyrosine phosphatase γ (RPTPγ) mutantr Cells to Lipotoxicity as Contributor for Impaired Liver Regeneration and Disease Advancement In Pediatric Nonalcoholic Fatty Liver Disease (PNAFLD)
Authors: Viswanathan Preeti , Maisuradze luka , Jaber Fadi‐luc , Gupta Sanjeev
Published: May 2022

Background

In PNAFLD mechanisms underlying early onset and rapid disease progression have been unclear. Active liver growth in preadolescent children imparted by facultative progenitor cells would increase genome damage following oxidative stress and lipotoxicity, leading to DDR and further hepatic injury events. To reveal this we considered hTERT‐FH‐B hepatocytes expressing progenitor properties will be ideal for modeling growth‐related lipotoxicity and DDR of PNAFLD.

Methods/Results

The FH‐B cells were derived from late‐gestation liver with doubling time of 24h, including 20‐30% in S phase, which was similar to pH3 histone +ve proliferating liver cell fractions in 5‐8‐yr old healthy children. Moreover, FH‐B cells express hepatobiliary markers (G6P, Glycogen, GGT, DPPIV) along with progenitor markers (SOX9, FOXA2, Cx43, EpCAM) and P450 profiles indicating hepatic immaturity. To induce lipotoxicity in cultured cells 50 μM palmitic acid (PA) was added with and without oxidized collagen matrix or 2,5‐10 ng/ml TNFa. This dose‐dependently caused oxidative stress (dihydrorhodamine dye for ROS) and lower mitochondrial membrane potential (MMP, JC‐1 dye) in 1‐4h and onset of steatosis (Oil red O or Bodipy staining) and decreased cell viability (MTT assay) in 18‐24h. The contribution of ATM signaling during lipotoxicity was apparent from pNBS1 accumulation, which marks DNA strand break sites. Moreover, lipotoxicity and DNA damage greatly increased in presence of ATM‐specific kinase inhibitor, KU‐60019. Additionally, ATM insufficiency exacerbated cell growth‐arrest through downstream p53‐dependent p21 activation, which increased polyploidy indicated by Hoechst dye‐stained nuclear DNA content analysis, and inhibited cell number increases in growth assays over few days. To test therapeutic feasibility, we studied obeticholic acid (OCA) and found this drug decreased ATM insufficiency‐related exacerbation of lipotoxicity. The improvements included steatosis, ROS, MMP. DDR, cell viability, as well as less growth‐arrest or polyploid accumulations and continued cycling.

Conclusions

The successful modeling of PNAFLD in hTERT‐FH‐B cells establishes the critical susceptibility of actively growing facultative progenitor hepatocytes to mitochondrial dysfunction and impaired DDR related to ATM insufficiency. The potential of OBC in improving lipotoxic outcomes will be relevant for PNAFLD. These pathophysiology‐based models offer opportunities to advance therapy in PNAFLD.
Plausible presynaptic effects of opioids on pontine inspiratory circuits: New insights from a joint neural biomechanical model
Authors: Olsen Wendy L. , Morris Kendall F. , Bolser Donald C.
Published: May 2022

Administration of opioids significantly effects tidal volume, respiratory rate, and blunts CO2 responsiveness. The precise neural mechanisms that determine the respiratory depressant effects of opioids are controversial. One hypothesis is that mu‐opioid agonists hyperpolarize neurons located in the pons and this action prolongs inspiratory cycle duration. We tested the plausibility of this hypothesis by employing a neuromechanical model of the respiratory control system. We targeted medullary inspiratory neuron axon terminals that influence 4 neuronal populations within the pons that had an inspiratory component to their discharge (inspiratory (I), expiratory/inspiratory (EI), rostral inspiratory/expiratory (rIE), and caudal inspiratory/expiratory (cIE)) neurons). We systematically decreased the conductance of these synapses by 25, 50, 75 and 100 percent and monitored the effect on the respiratory motor pattern. Simulations revealed that decreasing the conductance of synapses from medullary inspiratory neurons to neuronal populations in the pons altered the breathing pattern. In general, a reduction in synaptic conductance from medullary inspiratory neurons to each of the 4 pontine neuron populations decreased their excitability. Decreasing the synaptic conductance from the medullary inspiratory decrementing (I‐Dec) population to the: 1) I pons population resulted in a decrease in phrenic burst frequency and a decrease in the Inspiratory‐Driver (I‐Driver) population excitability, 2) EI pons population resulted in a decrease in I pons excitability and phrenic neurogram magnitude, 3) rIE population did not reveal significant changes in the breathing pattern from control, and 4) cIE population resulted a decrease in I‐Driver population excitability, phrenic neurogram amplitude and phrenic burst frequency. Decreasing conductance of synapses from the inspiratory augmenting (I‐Aug) population to the I pons, cIE, or rIE populations resulted in decreases in phrenic neurogram magnitude. Our simulation data support the plausibility of presynaptic effects of opioids within the pontine respiratory group that induce respiratory depression. Further, these presynaptic effects can induce selective alterations in inspiratory drive and/or breathing cycle timing.
Identification Of Compounds In Melaleuca AlternifoliaOil With Bacteriostatic Activity In SalmonellaTyphimurium
Authors: Roeder Peyton , Harvey Pamela A.
Published: May 2022

Antibiotic‐resistant infections are projected to cause over 10 million deaths annually by 2050. Despite this growing global crisis, there is waning interest in antimicrobial development due to declining financial incentives and regulatory barriers. There exists a critical need for identification of novel antimicrobial agents. Nearly 75% of US FDA‐approved antibiotics are derived from natural sources. Both plant oils and extracts have been recognized for their potent antimicrobial properties in Gram‐positive and –negative bacteria. However, few investigators have thoroughly explored the effects of individual active compounds that compose these oils. Melaleuca alternifolia (tea tree) oil has been shown to exert broad‐spectrum antimicrobial effects in several bacterial species. We hypothesized that these properties are attributable to monoterpenoid compounds that can diffuse through and disrupt cell membranes. Seven of the most abundant monoterpenoids in Melaleuca alternifolia (tea tree) oil [ρ‐cymene, eucalyptol, (R)‐(+)‐limonene, linalool, terpinen‐4‐ol, α‐terpinene, and terpinolene] were tested individually against SalmonellaTyphimurium, a Gram‐negative bacterium with up to 70% of strains exhibiting resistance to one or more antibiotic. The Mueller‐Hinton agar well diffusion test was used to determine antimicrobial activity through measurement of zones of inhibition. The minimal inhibitory concentration (MIC) and half‐maximal inhibitor concentration (IC50) of the subset of compounds exhibiting antimicrobial effects (linalool, terpinen‐4‐ol, α‐terpinene, and terpinolene) were also determined. MIC and IC50 values were 0.78% ‐ 6.25% and 1.5% ‐ 50% vol/vol, respectively. Each compound exerted its effects through bacteriostatic mechanisms as determined by time‐kill assays. These data highlight a continued role for natural products in the search for novel antimicrobials and importantly, identify the chemical components of Melaleuca alternifolia oil that confer its antimicrobials effects.
PFKFB3 Mediated Changes in ROS/NO Balance Contribute to Endothelial Dysfunction in Obesity
Authors: Batori Robert K. , Bordan Zsuzsanna , Padgett Caleb , Atawia Reem , de Chantemele Eric Belin , Stepp David , Fulton David J.
Published: May 2022

Obesity is a strong risk factor for cardiovascular disease, in large part due to an altered metabolic state. One of the earliest consequences of cardiovascular dysfunction in obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. In blood vessels, NO is synthesized by endothelial nitric oxide synthase (eNOS). The biological actions of NO can be compromised by rapid inactivation by reactive oxygen species (ROS) such as superoxide that is mainly generated by NADPH oxidases (NOXs). However, the signals coordinating the increased superoxide production leading to loss of NO with altered metabolism remain unknown.

We have found that the expression of 6‐phosphofructo‐2‐kinase/fructose‐2, 6‐biphosphatase 3 (PFKFB3), which is a key glycolytic regulatory enzyme, is significantly increased in endothelial cells of obese animals. Overexpression of PFKFB3 within the endothelium of blood vessels is accompanied by the loss of vascular relaxation and in decreased NO production as a result of increased eNOS phosphorylation on the inhibitory T495 residue. PFKFB3 overexpression also blunted Akt‐S473 phosphorylation, reducing stimulus‐dependent phosphorylation and activation of eNOS at S1177. While PFKFB3 exerted negative effects on NO signaling, it increased the activity and mRNA levels of NOX1, a major contributor to endothelial dysfunction. Further, we have also found that pharmacological inhibition of NOX1 attenuates the decreased vasodilator responses in PFKFB3 overexpressing aortic rings.

These results demonstrate a novel functional relationship between endothelial metabolism, ROS and NO production that may contribute to endothelial dysfunction in obesity.
Inhibition of PLK‐1 by using a Small Molecule Inhibitor HMN‐214 is a Novel Therapeutic Approach for High‐risk Neuroblastoma
Authors: Chilamakuri Rameswari , Agarwal Saurabh
Published: May 2022

Polo‐like kinase1 (PLK‐1), a member of the polo‐like kinase family of serine/threonine protein kinases, plays an essential role in regulating the cell cycle. PLK‐1 has a strong relationship with numerous regulatory events progressing during G2/M transition, chromosomal segregation, spindle assembly maturation, and mitotic exit. In addition, PLK‐1 also regulates DNA damage response, DNA replication, transcription, translation, chromosomes dynamics, and checkpoint adoption. Elevated levels of PLK‐1 have been observed in a plethora of cancers, including neuroblastoma (NB). NB is the most prevalent solid tumor that develops during the early embryonic stage and accounts for 15% of pediatric cancer‐related deaths. Current cytotoxic treatment strategies for NB are limiting and failed to prevent relapse and metastasis. In the present study, we hypothesized that inhibition of cell cycle regulator PLK‐1 by a small molecule inhibitor HMN‐214 will inhibit NB growth. To determine the role of PLK‐1 in NB, we analyzed multiple NB patient datasets and observed that PLK‐1 expression is inversely correlated with overall patient survival. PLK‐1 also strongly correlates with MYCN amplification and overall NB disease progression in our patient dataset analysis. Further, we used six NB cell lines, including MYCN‐ amplified and ‐non‐amplified cell lines, to evaluate the anti‐proliferative effects of HMN‐214 in NB. Results showed that HMN‐214 significantly and in a dose‐dependent manner inhibits NB proliferation and colony formation capacity. Additionally, we found that HMN‐214 significantly induces apoptosis and blocks cell cycle progression at the G2‐M phase in treatment groups compared to controls. Our molecular analysis further revealed that HMN‐214 significantly inhibits the mRNA and protein levels of PLK‐1 and CDK1 in contrast to control treatments. Further, we developed a 3D spheroid tumor model for NB to mimic in vivo tumor growth and utilized it to determine the effects of HMN‐214 on NB tumor growth. Results showed that HMN‐214 significantly and in a dose‐dependent manner inhibits NB 3D spheroidal tumor growth by specifically inducing tumor cell death. Overall, our data highlights the role of PLK‐1 in the oncogenic progression of NB and showed the efficacy of HMN‐214 in inhibiting NB growth. Our future efforts will be directed towards elucidating the role of PLK‐1 in NB and developing effective therapeutic strategies incorporating PLK‐1 inhibitor.