bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Premolar Crown Sizes are Highly Variable Even in the Same Mouth, with Strong Crown Size Correlations among Premolars and All Molars Except the Third Molar
Authors: Khan Md Huzzatul Islam , Marchiori Denver F. , Boughner Julia C.
Published: May 2022

The Inhibitory Cascade Model (ICM) predicts in mice that a larger permanent first molar (M1) results in a smaller second molar (M2) and an even smaller third molar (M3). Counter to the ICM, our lab found in contemporary humans that instead of M1 size, only M2 size predicts M3 size. Our lab also identified in humans 13 different molar size ratio patterns instead of one classic ICM pattern (M1>M2>M3). Further, other work in nonhuman primates reported a Premolar Molar Module (PMM) where sizes of the fourth premolar (P4) and all three molars are highly correlated. However, premolar:molar size correlation in contemporary humans is yet to be explored. This exploration can help define how the development of these two different classes of teeth influence each other’s sizes. In humans, M1 initiates near birth, followed ~24 months later by P3 and P4 (almost simultaneously with each other), then ~12 months later by M2, and ~6 years later by M3. Our first aim was to define variation in premolar size ratio pattern. We hypothesized that all three mathematically possible premolar size ratio patterns (P3=P4, P3>P4, P3M2>M3). We hypothesized that absolute sizes as well as size ratios of earlier initiating teeth will predict the absolute sizes and size ratios of later initiating teeth. Maximum mesiodistal lengths of P3, P4, M1, M2, and M3 crowns were measured in millimeters to represent absolute tooth sizes using retrospective Cone Beam Computed Tomography scans of 91 dental patients (54 female, 37 male) aged 13‐23 years. A frequency analysis revealed all three mathematically possible premolar size ratio patterns in our sample (P3>P4: 49.83%, P3<P4: 34.13%, and P3=P4: 16.04%, n=293 oral quadrants). Also, 57% (n=49) of the subjects with data collected from all four quadrants showed all three premolar size ratio patterns within the same mouth. A linear mixed model analysis (95% confidence level, alpha = 0.05) showed that P3 and P4 predicted each other’s crown size, as well as that of M2, but not M3. M1 size predicts P4 size only. In sum, we found that premolar size ratio patterns are highly variable even within the same oral cavity. We also found strong correlations among P3, P4 and M2 sizes, and between M1 and P4 sizes. Our results found variability in premolar size ratio patterns that is similar to the variability that we previously found in molar size ratio patterns. Our study in contemporary humans does not support the ICM prediction of a single classic tooth size ratio pattern. However, our study outcomes supported the PMM findings with the exception that P4 was not correlated with M3.
Inhibition of Morphine Metabolism by Major Cannabinoids and their Metabolites
Authors: Coates Shelby M. , Watson Christy J. , Chen Gang , Lazarus Philip
Published: May 2022

Opioids including morphine are mu‐opioid receptor (MOR) agonists that suppress excitatory neurotransmitter release, hyperpolarize neuronal cells, and decrease overall excitability, resulting in the desired analgesic effect. As a result of their over‐prescription, the still‐growing opioid epidemic has devastated many communities, with many individuals often using them in combination with other drugs including cannabis. Morphine is primarily metabolized by the phase II enzyme UDP‐glucuronosyltransferase (UGT) 2B7 to an inactive metabolite, morphine‐3‐glucuronide (M3G), and an active metabolite, morphine‐6‐glucuronide (M6G), both of which are extensively excreted into the urine. Previous studies in our lab have shown that major cannabis constituents including Δ9‐tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) inhibit several major UGT enzymes. To examine whether cannabinoids or their major metabolites inhibit morphine glucuronidation by UGT2B7, screening assays were performed in pooled human liver microsomes (HLM) with the major cannabinoids and THC 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‐COO‐Gluc). IC50 values were then determined for those cannabinoids that exhibited at least 50% inhibition at 100 µM final concentration. THC, 11‐OH‐THC, THC‐COO‐Gluc, and CBD all exhibited greater than 50% inhibition of both M3G and M6G formation at either 10 or 100 µM. The IC50 values for THC, 11‐OH‐THC, THC‐COO‐Gluc, and CBD against M3G formation were determined to be 27 ± 9.97 µM, 5.97 ± 2.31 µM, 15.58 ± 12.28 µM, and 15.66 ± 19.98 µM, respectively. The IC50 values for THC, 11‐OH‐THC, THC‐COO‐Gluc, and CBD against M6G formation were 14.49 ± 4.03 µM, 6.26 ± 0.76 µM, 13.22 ± 5.28 µM, and 8.12 ± 9.64 µM, respectively. To confirm results from these inhibition screenings, M3G and M6G formation were further examined using microsomal fractions from HEK293 cells overexpressing recombinant UGT2B7. THC, 11‐OH‐THC, THC‐COO‐Gluc, and CBD all exhibited >50% inhibition of both M3G and M6G formation at either 10 or 100 µM in the recombinant enzyme system. These data suggest that several cannabinoids and their metabolites inhibit morphine metabolism, and that the co‐use of these agents may lead to adverse drug events in humans.
miR‐133a Mitigates Ferroptosis by Attenuating Fatty Acid Metabolism in the Diabetic Heart
Authors: Kambis Tyler N. , Kar Sumit , Mishra Paras K.
Published: May 2022

Background/hypothesis of the study

Myocardial lipid accumulation occurs prior to the development of cardiac dysfunction in patients with diabetes mellitus (DM). miR‐133a prevents DM‐induced myocardial lipid deposition and is decreased in the DM heart, however its cardioprotective mechanisms remain unknown. Ferroptotic cell death which results from an increase in lipid peroxidation combined with a failure of lipid‐antioxidant mechanisms in the presence of iron, is upregulated in the DM heart. Thus, we hypothesize that overexpression of miR‐133a protects against ferroptotic cell death via inhibition of lipid accumulation, leading to decreased lipid peroxidation in the DM heart. We developed a novel Akita/miR‐133aTg (Ak/Tg) mouse model by crossbreeding DM Akita (Ak) with cardiac specific miR‐133a Tg (Tg) to elucidate how miR‐133a regulates ferroptotic cell death in the DM heart.

Methods and Results

Deep sequencing analysis revealed that miR‐133a activated fatty acid oxidation in the DM heart (p‐value=4.59E‐11). Protein levels of transcription factor Peroxisome proliferator‐activated receptor gamma (PPARG) (WT: 0.195±0.012, Ak: 0.23±0.012, Ak/Tg: 0.173±0.009, Tg: 0.16±0.012) and its coactivator Peroxisome proliferator‐activated receptor‐gamma coactivator (PGC‐1α) (WT: 0.162±0.006, Ak: 0.224±0.012, Ak/Tg: 0.149±0.011, Tg: 0.145±0.010). Furthermore, citrate synthase activity was increased in the presence of miR‐133a (WT: 0.793±0.043, Ak: 0.735±0.026, Ak/Tg: 0.994±0.055, Tg: 0.929±0.018), implying an increase of lipid clearance. Protein levels of glutathione peroxidase (GPX4), an antioxidant that inhibits the production of peroxidized lipids, were increased by the presence of miR‐133a (WT: 0.038±0.003, Ak: 0.024±0.002, Ak/Tg: 0.040±0.004, Tg: 0.030±0.006). miR‐133a also decreased levels of DM‐induced peroxidized lipids (WT: 203.9±12.69, Ak: 231.9±7.252, Ak/Tg: 209.8±8.885, Tg: 251.9±14.26).

Conclusions

These data support that miR‐133a decreases myocardial lipid deposition via increased lipid metabolism while simultaneously increasing levels of lipid antioxidant GPX4, leading to lower levels of peroxidized lipids resulting in the mitigation of ferroptotic cell death in the DM heart.
Student Outcomes Differ Based on Length of a Course‐based Undergraduate Research Experience (CURE)
Authors: Bell Jessica K. , DeChenne‐Peters Sue Ellen , Rakus John , Parente Amy , Mans Tamara L. , Eddy Rebecca , Galport Nicole , Koletar Courtney , Provost Joseph J. , Bell John E.
Published: May 2022

Course‐based Undergraduate Research Experiences (CUREs) offer an inclusive means to engage students in the scientific process and enhance student learning gains and persistence in STEM. To realize the benefits of CURE implementation, is there a minimum length of CURE? We studied the learning and attitudinal outcomes of more than 1,000 undergraduate students across the United States involved in chemistry, biochemistry, and molecular biology CUREs using malate dehydrogenase as the model system. There were three conditions, complete semester CURE (cCURE), modular CURE of about six weeks in length embedded into a laboratory course (mCURE), and no CURE (control). We also looked at the impact these conditions had on students who are persons excluded [from STEM] due to ethnicity or race (PEER). We hypothesized that the longer the students spent in a CURE the better their outcomes. We measured student outcomes using several validated measures and compared the conditions using ANOVA, ANCOVA, and chi‐square analyses. We found cCURE students had higher experimental design learning and STEM support than control students. We found cCURE>mCURE>control for student’s report of their interest in conducting research in the future, support for STEM students, and their interest in a STEM career. Students in the cCURE and control had higher positive attitudes about scientific research than students in the mCURE. There were no differences between conditions for negative attitudes towards science, science literacy, and beliefs about learning science. We found PEER students reporting the same results as White/Asian students, except for their interest in conducting research: PEER students in mCUREs had higher interest than their White/Asian counterparts in conducting research. We conclude that the overall pattern was for students in the cCURE condition to have better learning and attitudinal outcomes than students in the mCURE and control conditions. Additionally, PEER student outcomes in CURE conditions were similar to their White/Asian counterparts. These results should encourage faculty to use CUREs in laboratory courses to improve all students’ outcomes and indicate that even a short CURE embedded within a traditional laboratory course can benefit students.
Renal‐derived human sPRR increases blood pressure in female but not male mice
Authors: Arthur Gertrude , Poupeau Audrey , Nichols Kellea , Leachman Jacqueline , Loria Analia , Osborn Jeffrey , Yiannikouris Frederique
Published: May 2022

Previous studies showed that soluble prorenin receptor (sPRR) plays an important role in blood pressure regulation. In rodent models, sPRR contributes to AngII production by increasing renin activity and systolic blood pressure (SBP). Moreover, reducing sPRR release into plasma decreased blood pressure and attenuated angiotensin II (AngII) induced hypertension. However, there is a gap of knowledge concerning the functional role of locally produced sPRR from the kidney in blood pressure regulation. Additionally, the role of human sPRR in blood pressure regulation has not been delineated. Therefore, we evaluated the renal derived human sPRR role in SBP control.

Human sPRR‐Myc‐tag transgenic mice were bred with mice expressing Hoxb7/Cre to selectively express human sPRR in the collecting duct (RHsPRR). RHsPRR and control (CTL) male and female mice were fed a standard diet for 10 months (n=8‐11/group). Body weight and urine flow rate were examined and SBP measured by radiotelemetry. Western blot analysis depicted the presence of human sPRR‐Myc‐tag (28 KDa) in the cortex and medulla of RHsPRR male mice which validated the humanized mouse model.

Male mice have higher body weights than female mice but renal derived human sPRR did not influence body weights (M: 34.1±0.8 and 32.9±0.8g, F: 28.4±0.8 and 29.6±0.5g, P<0.05). Circulating sPRR was unchanged between male and female mice (M: 3995±643 and 4342±500pg/ml, F: 3479±194 and 3948±238pg/ml). SBP increased significantly in female RHsPRR mice compared to CTL (F: 118.7±2 and 127.2±3 mmHg, P<0.05) but not in male mice (M: 123.8±2 and 119.9±6 mmHg). The SBP response to losartan, an AT1R antagonist, was unchanged in females but was impaired in males (M: ‐7.6±1.2 and ‐5.5±0.8 mmHg, F: ‐6.5±1.2 and ‐7.9±1.5 mmHg, P<0.05). Renal derived human sPRR significantly increased AT1R gene expression in females but not in males (M: 1.8±0.6 and 1.2±0.3 2‐ΔΔCT, F: 1.6±0.5 and 3.8±0.9 2‐ΔΔCT, P<0.05). Moreover, renal derived human sPRR decreased the gene expression of renin and angiotensinogen in female mice (Renin: 1.5±0.5 and 0.4±0.1 2‐ΔΔCT, Angiotensinogen: F: 0.9±0.1 and 0.6±0.1 2‐ΔΔCT, P<0.05) suggesting a negative feedback loop involving Angiotensin II‐AT1R pathway. These effects were not observed in males (Renin: 3.3±1.2 and 4.1±1.2 2‐ΔΔCT; Angiotensinogen: M: 1.4±0.4 and 1.4±0.4 2‐ΔΔCT).

Overall, our data suggest that renal‐derived human sPRR increases blood pressure in female mice likely via the activation of renal AT1R expression. In contrast, renal‐derived human sPRR expression impaired the blood pressure response to Losartan in male mice. In conclusion, our data support renal derived human sPRR as a promising therapeutic target in hypertension in humans.
The Effect of Potassium on the Structural Flexibility of β‐amylase2
Authors: Jaconski Sophia C. , Chandrasekharan Nithesh , Monroe Jonathan , Berndsen Christopher
Published: May 2022

Starch is an important compound in the lives of both plants and humans, with roles in human nutrition and industry such as paper making. β‐amylases are a class of proteins that break down starch in plants to produce maltose. My focus of study is β‐amylase2 (BAM2); one of the β‐amylase genes Arabidopsis thaliana. While the physiological function of BAM2 is unclear, we know three key things about its properties: 1) BAM2 has a tetrameric structure, 2) the enzyme exhibits sigmoidal kinetics through secondary binding sites, and 3) potassium salts are required for catalytic activity. We investigated the molecular basis for potassium stimulating activity in BAM2. Using molecular dynamics, we simulated the effect of potassium on BAM2 finding that potassium increased the flexibility of the enzyme. We are purifying BAM2 wild type and BAM2 with point substitutions which we propose alter the effect of potassium activity. Using these enzyme variants, we compared the structural stability of the enzymes in the presence of potassium, lithium, and sodium using Differential Scanning Fluorimetry (DSF) and Circular Dichroism (CD). Results from DSF showed that the melting temperature decreased with increasing salt concentration, which we interpret as an increase in structural flexibility of BAM2. The CD results support this trend as the melting temperature for BAM2 in the presence of lithium is higher than that of BAM2 in the presence of potassium. These data support a mechanism where potassium ions enhance the flexibility of BAM2.
OTUB1: A Key Player in the Adaptive Cardiac Hypertrophy
Authors: Wang Wenjuan , Li Jie , Zou Jianqiu , Ayala Juan , Liu Jinbao , Sun Shao‐Cong , Su Huabo
Published: May 2022

Deubiquitinases (DUBs), a family enzymes that reverse the process of ubiquitination, participate in diverse cellular processes through regulating protein degradation, intracellular trafficking, cell signaling, and protein‐protein interaction. The functional importance of DUBs in cardiac pathophysiology remains poorly defined. Herein, we report a critical role of OTUB1, one of the most highly expressed DUBs in the heart, in adaptive cardiac remodeling. Analysis of RNA sequencing data revealed that OTUB1 is upregulated in the failing human hearts. OTUB1 is also upregulated and activated in cardiomyocytes (CMs) treated with adrenergic agonists. OTUB1‐deficient CMs were resistant to phenylephrine (PE) ‐ and isoproterenol‐induced CM hypertrophy as characterized by attenuated cell enlargement, decreased fetal gene activation, and diminished protein synthesis. In vivo, CM‐specific deletion of OTUB1 in the developing mouse heart caused dilated cardiomyopathy without a prior development of compensatory cardiac hypertrophy, eventually leading to heart failure and premature lethality of mice by 6 months of age. The severe cardiac phenotype is accompanied by lipid accumulation, ER dilatation, prevalent autophagic vesicles containing ribosomes, as well as dysregulated expression of metabolic and ribosome genes. Delivery of wild‐type OTUB1, but not the enzymatic dead mutant, via AAV9 to OTUB1‐deficient hearts attenuated cardiac dysfunction and prolonged the lifespan of mice. On the molecular level, OTUB1 deficiency blunted mTOR signaling in cultured CMs and mouse hearts. Our findings indicate that OTUB1 is indispensable for the development of adaptive cardiac hypertrophy, possibly through modulating mTOR signaling.
Evaluating Cybersickness in Virtual 3D Models for Anatomy Learning
Authors: Ives Rowan , Lohit Simran , Touliopoulos Evangelea , Reis Isabella R. , Chan Sachi , Sinha Sakshi , Nehru Amit , DeYoung Veronica , Mitchell Josh P. , Brewer‐Deluce Danielle , Sonnadara Ranil , Wainman Bruce C.
Published: May 2022

Introduction

Cybersickness is an array of symptoms associated with exposure to three‐dimensional visualization technology (3DVT) environments, such as virtual reality (VR). It is thought that cybersickness is a type of motion sickness caused by a mismatch in sensory and vestibular input when using these modalities. Symptoms of cybersickness are often akin to those of traditional motion sickness, such as headache and nausea. In the literature, as many as 40‐60% of VR users report symptoms of cybersickness, though in our laboratory approximately 20% of users report symptoms of cybersickness. Our past research has shown that the physical environment is often preferred by students, and more effective for learning anatomy, compared to 3DVT environments. We hypothesize that the preference for, and effectiveness of, the physical environment over 3DVT may be a result of cybersickness due to the isolation of the learner from the physical environment. However, no direct measurements of cybersickness or comparisons between 3DVT environments in anatomy are available.

Methods

Our study involves comparisons of cybersickness during anatomy learning between three 3D modalities: a VR Oculus Quest 2TM headset, an autostereoscopic screen (AlioscopyTM), and a 3D‐printed physical model. Undergraduate students, with no formal anatomy training, will be randomized via a Latin square design to view one of three skeletal models (human hemipelvis, zygomatic bone, or calcaneus) in each of the modalities. Participants may not touch the model but may rotate it along the horizontal plane using an XboxTM controller. Participants will have four minutes to learn ten bony landmarks on the viewed model and will then take an untimed, recognition‐based test for landmarks learned before progressing onto the next modality. Cybersickness will be assessed following testing in each modality, via self‐reports on the simulation sickness questionnaire (SSQ).

Results

We hypothesise that reported cybersickness will be highest for the VR displayed on the head‐mounted display of the Oculus Quest 2TM which entirely covers the visual field, compared to learning through the AlioscopyTM screen or the physical model due to the relative lack of isolation of the learner from the physical environment in the last two treatment groups.

Conclusion

With the rapid development of 3DVT for use in anatomical education, cybersickness is an important issue in evaluating the quality of 3D modalities. The results of this study will allow educators and students to make informed decisions about the use of 3DVT in anatomy thus preventing uncomfortable physical symptoms associated with learning using 3DVT and improving learning as a result.
Eribulin Enhances Interferon Production by STING Agonists in Models of Triple‐Negative Breast Cancer
Authors: Takahashi‐Ruiz Leila , Fermaintt Charles S. , Chan Peter Y. , Liang Hui , Mooberry Susan L. , Risinger April L.
Published: May 2022

Triple‐negative breast cancer (TNBC) is a highly heterogenous disease that lacks effective targeted treatment options. However, TNBC typically has a higher mutational burden and greater degree of immunogenicity than other breast tumors, making immunotherapy a viable strategy for effective treatment of this disease. Strategies to improve the response of TNBC patients to immune checkpoint inhibitors include the upregulation of the cGAS‐STING innate immune sensing pathway. Multiple STING agonists are in clinical development and have shown promise for the treatment of TNBC as single agents and combined with checkpoint inhibitors. We recently demonstrated that eribulin, a microtubule destabilizer used in the treatment of TNBC, functions as an indirect STING agonist because it promotes the release of mitochondrial DNA into the cytoplasm (Mol Pharmacol 100: 309‐318, 2021). We further hypothesized that eribulin could enhance the expression of type I interferons in combination with direct STING agonists. Indeed, our results show that the microtubule destabilizer eribulin significantly enhances interferon expression induced by STING agonists with diverse mechanisms of action in human monocytes and TNBC cells. This phenotype is shared with other microtubule destabilizers, including vinorelbine, but not with the microtubule stabilizing agent paclitaxel, which is also used in the treatment of TNBC. The eribulin‐mediated enhancement of interferon expression induced by multiple pharmacological STING agonists, as well as HT‐DNA and cGAMP ligands, occurs within 6 hours and is mechanistically distinct from the previously reported eribulin‐mediated release of mitochondrial DNA into the cytoplasm. This response is dependent on TBK1 and associated with TBK1 phosphorylation but was not inhibited by the STING antagonist H‐151 or by inhibition of the JNK or JAK1/2 signaling pathways in TNBC cells. Ongoing in vivo studies are determining the effects of eribulin on the immunological response to TNBC tumors alone and in combination with pharmacological STING agonists and whether this improves antitumor efficacy. These studies will determine if eribulin, a chemotherapeutic agent already used in the treatment of TNBC, can increase the immunogenicity of TNBC tumors and inform on combination treatment of eribulin with STING agonists and potentially other immunotherapies.
Virtual Reality, Autostereoscopy, and Physical Models for Learning Anatomy: Performance Comparisons
Authors: Lohit Simran , Reis Isabella R. , Ives Rowan , Chan Sachi , Touliopoulos Evangelea , Sinha Sakshi , Nehru Amit , DeYoung Veronica , Mitchell Josh P. , Brewer‐Deluce Danielle , Sonnadara Ranil , Wainman Bruce C.
Published: May 2022

Introduction

Traditional anatomical education relies on textbooks, physical models, and cadaveric specimens. Advances in technology have provided other display modalities including virtual reality (VR), and autostereoscopic displays. VR uses head‐mounted displays (HMDs) for an interactive experience and autostereoscopic screens, like AlioscopyTM, offer a stereoscopic but HMD‐free environment. Current research shows contradictory results on the efficacy of VR compared to physical models, and there appears to be no data on the efficacy of autostereoscopic displays in learning anatomy. The purpose of this study is to determine whether VR, AlioscopyTM, or physical models yield the best performance for anatomical education.

Methods

Students at McMaster University without prior anatomy training will learn nominal skeletal anatomy in three different modalities: VR (Oculus Quest 2TM), AlioscopyTM, or a physical 3D‐printed bone model. Each of the environments will be as identical as possible (i.e, the VR environment is a rendering of the exact room and set‐up used for testing the physical bone models). Participants will be randomized to one of three interventions where they will study ten bony landmarks on either the human hemipelvis, zygomatic bone, or calcaneus in a distinct modality. Participants will be seated and use an XboxTM controller to rotate the bone along the vertical axis of rotation. After four minutes, an untimed, recognition‐based test will be administered, where participants will be given a 3D‐printed bone identical to the one used in the learning phase, with randomized landmarks and word bank of landmarks learnt. Performance will be evaluated based on landmarks correctly identified on the recognition‐based test.

Results

Based on the current literature, we hypothesize that the physical models will be a superior learning environment compared to the VR environment. However, significant improvements in the HMD of the Oculus Quest 2 may have ameliorated previous issues with the VR environment. There is no data available on the efficacy of AlioscopyTM and while the absence of an HMD is appealing, its narrow viewing angle compared to the immersive VR environment may be problematic.

Conclusion

With the push to increase accessibility and decrease costs associated with anatomical education with the help of digital modalities, the findings from this study are critical to informing teaching practices, and technology use in anatomy education.
Oral administration of a novel, synthetic ketogenic compound elevates blood β‐hydroxybutyrate levels in mice in both food‐restricted and ab‐libitum conditions
Authors: Soliven Maricel A. , Williams Michael S. , Rogers Christopher Q. , D'Agostino Dominic P.
Published: May 2022

Elevated ketone levels have been shown to lead to improvement in diseased contexts including epilepsy, diabetes, obesity, cancer, Alzheimer’s, and Parkinson’s Disease. It can be difficult for many patients, especially children, to adhere to the strict and intense ketogenic diet necessary to achieve such elevation via dietary manipulation, hence why the field of metabolic therapy continues to explore the use of exogenous ketone supplementation. This preliminary pharmacodynamic study characterizes the effects of a glycerol acetoacetate tri‐ester on the blood β‐hydroxybutyrate levels in VmDk mice, a model often used in cancer studies in both fasted and non‐restricted states. Promising β‐hydroxybutyrate levels were observed in both conditions along with a hypoglycemic effect when fasted. No detrimental side effects have been observed. Future studies will include characterizing the compound's effects on metabolomics and cytokines.
Development of an in vitro bioassay to assess the regulation of immune genes in nontraditional model species
Authors: Bradshaw Sarah , Petitte James N. , Roberts Natalie , Koci Matthew D.
Published: May 2022

Three out of four emerging infectious diseases of humans originated in animals. The COVID‐19 pandemic highlights the importance of understanding how the immune systems of animals interact with viruses, bacteria, and parasites allowing them to serve as disease reservoirs. Unfortunately, the tools to study the immune system of most animal species are not at the level needed to investigate these interactions. Therefore, our laboratory is constructing a new reporter plasmid that will allow for the development of species‐specific transcription factor‐activated reporter cell lines. The initial proof of concept is focused on avian species. Birds are known reservoirs for zoonotic pathogens like influenza A viruses, West Nile virus, Salmonella, Campylobacter, among others. Understanding how the avian immune system responds to zoonotic pathogens is critical to understanding how they serve as reservoirs, and potentially developing novel ways to prevent disease in humans. Transcription factors regulating pro‐ and anti‐inflammatory genes were chosen and candidate binding sites conserved across multiple avian species were identified. These candidate transcriptional binding sites were inserted into a novel dual reporter plasmid and screened for their ability to control expression of green fluorescent protein. Plasmids containing validated binding sites will then be stably integrated into the genomes of different avian cell lines generating a transcription factor‐specific reporter cell line. These cell lines will then be used as a library to characterize the transcriptional response of various cell lineages following stimulation by different pathogens, pathogen pattern molecular patterns, and cytokines. Ultimately this platform will be expanded to other species, allowing us to compare the in vitro host response across different mammalian, avian, and teleost species. Shedding new light on the role the immune response plays in the pathogenicity of different zoonotic agents.
Identification of a direct role of CPSF6 and a key cis‐acting motif in promoting TRIM9 proximal polyA site usage
Authors: Herron Richard S. , Kunisky Alexander , Hwang Hun‐Way
Published: May 2022

Alternative polyadenylation (APA), an RNA processing mechanism that results in mRNA with distinct 3’ termini, is a rapidly expanding area of research that in recent studies has been linked to the mechanistic target of rapamycin (mTOR) signaling pathway, a key regulatory pathway in physiology and metabolism. Despite the recent implications of APA in mTOR signaling, the mechanistic link between mTOR signaling and APA remains poorly understood. We previously leveraged our cTag‐PAPERCLIP technique to generate a dataset of in vivo APA shifts following neuronal mTOR induction and identified TRIM9, an E3 ubiquitin ligase with a role in neurodevelopment, as a gene of interest. In this study, we further characterized the regulation of the mTOR‐induced TRIM9 APA shift observed in mouse neurons in vivo. Further study of the regulation of TRIM9 APA by the core protein complexes of the cleavage and polyadenylation (CP) machinery revealed CSPF6, a component of the CFIm complex, as essential for physiological regulation of TRIM9 isoforms, with loss of CPSF6 leading to an enrichment of the distal TRIM9 isoform. Additional study into the 3’UTR sequence elements of TRIM9 isoforms revealed multiple UGUA sequence motifs, the binding sequence element of the CFIm complex, upstream of the TRIM9 proximal polyA site (PAS). In order to identify the key sequence elements essential for CPSF6‐mediated regulation of the proximal TRIM9 PAS, we developed a RT‐qPCR PAS competition assay to quantify sequence‐mediated usage of PASs. Utilizing this assay, we assessed usage of the TRIM9 proximal PAS in both the absence and the presence of CPSF6. Additionally, we generated constructs containing mutated UGUA sequences in order to ascertain the importance of the UGUA motif to TRIM9 proximal PAS usage. We found that loss of CPSF6 leads to reduced usage of the TRIM9 proximal PAS. Furthermore, mutation of a twin UGUA motif (UGUACUGUA) lead to a reduction in TRIM9 proximal PAS usage. Our results demonstrate a direct role of CPSF6 and identify a key cis‐acting motif in promoting TRIM9 proximal PAS usage. Furthermore, our results also suggest a possible link between neurological disorders with mTOR pathway dysregulation (“mTORopathies”) and neurodevelopment through TRIM9.
Distributed latent dynamics underlie breathing control in the brainstem during normal breathing, OIRD, and gasping
Authors: Bush Nicholas E. , Quina Lely , Ramirez Jan‐Marino
Published: May 2022

The neural control of breathing involves the coordinated activity of diverse populations of medullary neurons distributed along the rostrocaudal aspect of the ventrolateral medulla. Here, we use high‐density electrophysiological recordings (Neuropixels) to record spiking activity simultaneously from hundreds of neurons across the ventral respiratory column (VRC, including the Bötzinger and pre‐Bötzinger complexes) in anesthetized, freely breathing mice. Across sequential recordings, we have recorded from over 13,000 medullary neurons. We couple this with an optogenetic tagging approach to identify ChAT, Dbx1, Vgat, and Vglut2 positive neurons; and reconstruct the three dimensional location of each recorded neuron. We find diverse patterns of respiratory‐related neural activity throughout the medulla that tile the breathing phase such that discrete functional cell classes cannot be quantitatively defined. Moreover, while certain genotypes are biased towards particular anatomical locations or functional activity patterns, cells active during all parts of the respiratory phase are found across the VRC, and across all genotypes. We use dimensionality reduction techniques to show that the neural populations governing breathing follow rotational trajectories on low dimensional neural manifolds. Analyses of these trajectories indicate that the termination of inspiration (“inspiratory‐off switch”) may represent an attractor in the low‐dimensional neural space. Further, during opioid induced respiratory depression (OIRD), the network reconfigures, but the low‐dimensional trajectories of the neural population are preserved. Thus, patterns of neural coactivity remain the same, while breathing is slowed. Finally, we expose animals to hypoxia which reconfigures the respiratory network into gasping. The low‐dimensional neural activity no longer follows rotational trajectories. Instead, gasping is a ballistic, non‐rotational dynamic mode strikingly different than eupnea or OIRD. The transition to gasping is represented in the low dimensional trajectories by a gradual sequestering of the neural activity away from normal eupneic rotations. Together these data suggest that the VRC functions as a distributed and well‐coordinated population. The eupneic cycle begins with the inspiratory off‐switch and is maintained by the reciprocal interactions between inspiratory and expiratory neurons. This reciprocity is lost as the network transitions into gasping, a rhythm governed by fundamentally different mechanisms.
Determining Whether Tightness of Substrate Binding in the Active Site of a Dimeric Taurocyamine Kinase Plays a Role in Inducing Negative Cooperativity
Authors: Beal Robby M. , Li Mingyuan , Fraga Dean , Snider Mark J.
Published: May 2022

Cooperative binding of ligands within multi‐subunit proteins is a mechanism that has evolved to enhance a protein’s function. Driven by conformational changes upon ligand binding in one subunit, the resulting change in affinity at a neighboring subunit can be enhanced (positive) or reduced (negative) depending on the mechanism. The structural features, both on the substrate and within the active site, that determine the nature of cooperativity remain largely unknown, particularly within the phosphagen kinases (PKs). PKs belong to a widely distributed enzyme family important for energy homeostasis, with isoforms that exist as monomers and multimers, with some, but not all, of the homodimers that have been studied exhibiting negative cooperativity. Here we investigated the structural determinants that elicit negative cooperativity of transition state analogue (TSA; TK–MgADP–NO3‐–taurocyamine) binding within the Arenicola Brasiliensis mitochondrial taurocyamine kinase (miTK), one member of the phosphagen kinases that catalyzes the interconversion of MgATP and MgADP using taurocyamine or phosphotaurocyamine as substrates. Isothermal titration of miTK with taurocyamine in the presence of MgADP and nitrate to form the quaternary TSA complex fits a two‐site binding model in which the first site forms a tighter complex than the second site (i.e. negative cooperativity). MiTK also shows activity with glycocyamine, although with reduced efficiency and weaker binding affinity. To determine whether this weakly bound substrate also elicits cooperativity we attempted to measure the wild‐type miTK’s affinity for glycocyamine in forming a TSAC. Results showed that this complex does not form appreciably and therefore the presence of negative cooperativity could not be elucidated. To further examine whether the tightness of binding or a specific structural feature of the substrate triggers cooperativity, the V71A active site variant of miTK that has shown to have 10x greater catalytic efficiency with glycocyamine compared to the wild‐type miTK, will be assessed for cooperative TSAC binding using taurocyamine and glycocyamine. Additional active site variants with more moderate changes in affinity for these substrates will also be examined for cooperativity to further discern if negative cooperativity can be induced at a certain catalytic efficiency threshold.
Minigames for Teaching the Cardiovascular System to Students of Human Anatomy
Authors: Naples Virginia , Sullivan Ian , LaBarbera Dean , Kluga Ben , Edwards Lucas , Freedman Reva
Published: May 2022

We are building a series of cell‐phone based minigames as a new approach to teaching cardiovascular concepts. The minigames belong to the first iteration of our intelligent textbook, Cardiovascular and Respiratory Anatomy Map (CRAM). They are part of a larger suite of games under a common umbrella that teach students the key concepts in human anatomy. Playing games engages student interest and enables students to learn at a lower stress level. Our goal is to enable students to master this material without getting distracted by different but related concepts in the cardiovascular system.

The purpose of this game is to demonstrate that oxygenated and deoxygenated blood flow patterns in the pulmonary circuit are different than in the systemic circuit. We ask students to learn the names of the arteries and veins that supply and drain the heart and lungs. In the first stage of the game, students just need to identify correctly which vessels contain oxygenated and deoxygenated blood and the direction of blood flow.

Students run this game on a cell phone or laptop. The screen shows the main organs involved in this process, namely the heart, the two lungs (one on each side of the screen), and a stick‐figure representation of the body. The organs are connected by the great vessels that transport blood to and from the heart and lungs. Students click on the arrows representing the vessels and color them red or blue. When they color a vessel incorrectly, the system responds with information to correct their misconception. For example, if the student colors the pulmonary arteries red, they are informed that the pulmonary arteries carry deoxygenated blood to the heart.

This game is the first of a series of games teaching students the fundamentals of cardiac anatomy and function. Different from other materials that teach cardiovascular function, the first game does not make students memorize the chambers of the heart and their relationship to the great vessels. We believe that separating this issue from understanding the higher level relationship between the organs reduces the initial cognitive load on students and streamlines their ability to attain mastery of the entire system. A typical human anatomy book requires 30–40 pages of text to cover the material we present in a series of minigames. This approach appeals to contemporary students who prefer to use their cell phones for learning and are reluctant to read the assigned text.

The system has two take‐home messages. In mammals, the pulmonary arteries are the exception to the rule that an artery always carries oxygenated blood because they receive deoxygenated blood from the heart. In addition, the lungs only provide oxygen to body tissues via the heart, which is a double‐circuit pump. In this game, students assimilate this information via active participation rather than by rote memorization.
Contribution of Genetic Background to Vascular Adaptation to Exercise Training Based on Critical Speed of Different Intensities
Authors: Kim Hyoseon , Nino Matthew , Massett Michael
Published: May 2022

An impaired endothelial function is a fundamental component of the pathogenesis of cardiovascular disease. In mice, exercise training is well‐known to improve cardiorespiratory fitness and endothelial function. However, the post exercise training effect on endothelial function varies among different strains of mice, suggesting that genetic background contributes to the heterogeneous effect of exercise on vascular health. NZW/LacJ (NZW) mice are known as relatively low responders to exercise training compared to high responder strains such as FVB/NJ (FVB). NZW mice also have relatively poor endothelial function in thoracic aorta when compared with other mouse strains. Therefore, we aimed to determine the contribution of genetic background to endothelial adaptation to exercise training in FVB and NZW mice. Vasoreactivity was assessed in femoral arteries from female mice from both strains (NZW and FVB) after 6 weeks of training using two different critical speed (CS) treadmill training intensities. Mice from each strain were randomly assigned to one of three groups: sedentary (SED), 80% of CS training (CS80), and 90% of CS (CS90) training. After training, endothelium‐dependent vasorelaxation to acetylcholine (ACh) and endothelium‐independent vasorelaxation to sodium nitroprusside (SNP) were measured as well as the vasocontractile responses to phenylephrine (PE) and potassium chloride (KCl). Maximal relaxation responses and IC50 for ACh and SNP were similar between NZW groups (SED, CS80, and CS90). In femoral arteries from FVB mice, there were no differences for maximal relaxation to ACh, but there was significant difference in IC50 for ACh‐induced vasorelaxation between SED vs. CS90 groups (p=0.05). Maximal responses to ACh were similar between NZW and FVB; however, IC50 was significantly lower in FVB compared with NZW, indicating that sensitivity to ACh is greater in arteries from FVB. There was a significant strain‐dependent difference for IC50 for SNP despite no strain differences for maximal responses. There was no effect of exercise or strain on contractile responses to PE and KCl, although the EC50 for KCl induced vasocontraction was higher (p=0.0329) in NZW. Treadmill exercise training at 80% or 90% CS did not contribute to improved vasomotor function in NZW while higher intensity exercise improved sensitivity to ACh in arteries from FVB. Genetic background did not significantly impact vasomotor function or endothelial responses to exercise training in FVB and NZW mice.
Course‐based Undergraduate Research Linked Across Two Core Biology Courses
Authors: Listenberger Laura , Walter Anne , Porterfield Jean , Bowers Lisa , Fruehling Sara , Kandl Kim
Published: May 2022

Authentic mentored undergraduate research is known to improve student learning and engagement. Course‐based undergraduate research experiences (CUREs) allow more students to participate in research early in their academic careers. We are expanding the CURE concept by linking research projects across two distinct yet interdependent courses required for the Biology major, Cell Biology and Intermediate Genetics. Each course enrolls 130‐150 students per year and may be taken in either order. To support successful projects, we first mentored upper level students in the development of protocols and generation of strains that could be employed to explore metabolic strategies in the aquatic ciliate Tetrahymena thermophila. In a pilot of a linked CURE, students investigated whether exogenous addition of oleate, an unsaturated fatty acid, alters the phenotype of T. thermophila.Students in Cell Biology examined changes in growth rate, metabolism, and lipid storage using light and fluorescent microscopy, thin layer chromatography, and spectrophotometry. Students in Intermediate Genetics examined changes in gene expression with both qualitative and quantitative RT‐PCR. Students in our linked CUREs responded to their lab experience with enthusiasm for participation in novel research, and curiosity about the research results gathered in the other of the linked courses. Future offerings of linked CUREs will expand the number of students that engage with a year‐long research project using multiple approaches to address a single question. Importantly, this approach is adaptable to different questions and model organisms, providing a template for sustainable research opportunities.
Male and Female Mice Respond Differently to Short‐term Ketogenic Diet
Authors: Sprankle Kenyon W. , Knappenberger Mya A. , Locke Erica J. , Knapsack Kaylin , Vinovrski Madison F. , Kolwicz Stephen C.
Published: May 2022

The ketogenic diet (KD) is a high‐fat, low‐carbohydrate diet that results in the elevation of ketone bodies in the blood, known as ketosis. This metabolic consequence has been suggested as a method for treating neurological conditions, improving exercise performance, and facilitating weight loss. Since most research studies primarily use male populations, less information is available regarding potential sex differences in response to various interventions. Therefore, the purpose of this study was to explore the sex differences in physical, metabolic, and motor performance in mice fed the KD. Male (n=29) and female (n=26) C57BL/6 were randomly assigned to KD (90% fat, 1% carbohydrate) or chow (13% fat, 60% carbohydrate) group for 6 weeks. Body weight and food intake were tracked weekly. At baseline and every 2 weeks, the Rotarod performance test assessed motor coordination and activity levels in all mice. After 6 weeks, adipose tissue, quadriceps, and heart were weighed to observe changes in organ mass. Triglyceride (TG) was measured in the heart, liver, and quadriceps. Blood was drawn to measure changes in serum metabolite levels. Final body weights in Male‐KD were similar to Male‐chow; however, Female‐KD mice were significantly higher relative to Female‐chow. Although adipose tissue mass increased significantly in both KD groups, the increase was ~40% higher in Female‐KD. Quadriceps mass was lower in Male‐KD compared to Male‐chow (188±6 vs.169±5 mg, P<0.05) while not statistically different in Female‐KD relative to Female‐chow. In contrast, heart weight to tibia length ratios (HW:TL) were increased in Female‐KD compared to Female‐chow (5.08±0.06 vs.5.58±0.12, P<0.05) with no significant change in Male‐KD. The KD resulted in similar measures of ketosis, glucose intolerance, and hyperlipidemia in Male‐KD and Female‐KD. While cardiac TG content tended to increase in Male‐KD and Female‐KD to a similar extent (P<0.10), the increase in hepatic TG content was ~30% lower in Female‐KD (P<0.05 vs. Male‐KD). Although Rotarod performance was higher in Female‐KD compared to Male‐KD at week 2 (28.6±3.1 vs.15.2±1.6 sec, P<0.05), performance decreased in Female‐KD and increased in Male‐KD over the remaining weeks and was similar at week 6 (25.7±2.0 vs.20.7±2.0 sec). In summary, these data show sex specific differences in weight gain, adiposity, and muscle (i.e., cardiac and skeletal) mass changes in response to a short‐term KD. Moreover, the data suggest that male and female mice on the KD differ in time sensitive adaptation to ketosis and motor activity. Since both males and females increase adiposity, develop glucose intolerance, and hyperlipidemia, this study questions the viability of the KD as a potential tool for weight loss, and importantly, highlights sex differences in the adaptation to the KD.