bluuebunny/crossref_metadata_2025 huggingface dataset:
Addressing Race, Ethnicity, and Structural Inequality in Pharmacology Education and Assessment
Authors: Blumer Joe B. , Szarek John L. , Lee Michael W. , Zaveri Naunihal T. , Schneid Stephen D. , Kruidering Marieke , Quesnelle Kelly M.
Published: May 2022

OBJECTIVE

Social determinants of health, including race, ethnicity, and gender, represent important factors in health care delivery and patient outcomes, and contribute to health disparities. Pharmacology, as an integrated discipline, lies at the nexus between foundational science and clinical practice, providing a platform for educators to discuss the impact of social determinants on patient outcomes. Our goal is to create a framework that provides guidance for topics in pharmacotherapeutics for educators, learners, and providers to use in promoting antiracism in medical curricula.

METHODS

Through iterative discussions, we developed a conceptual framework that includes topics taught across medical schools, such as drug metabolism, pharmacogenomics, adverse drug reactions, prescribing guidelines, factors affecting medication adherence, pain management, and therapeutic outcomes. Under each topic, we describe how social constructs such as race, ethnicity, and gender can impact pharmacology and therapeutic outcomes.

RESULTS

Literature reports show that some topics in pharmacology education have already established cultural competency and should be adopted widely by all pharmacology educators, e.g., using more appropriate terminology for drug reactions such as vancomycin‐infusion reaction. Other topics are less established, so a more nuanced discussion with students is required. Society guidelines for managing diseases such as hypertension include prescribing recommendations based on race, with which learners are expected to be familiar as they enter clinical training but are actively being debated. Similarly, inter‐ethnic differences in drug metabolism are often reported as a function of genetic polymorphisms without considering non‐genetic factors.

CONCLUSIONS

We have developed a conceptual framework for promoting antiracism in pharmacology education by categorizing topics based on those where cultural competencies are clearly established and those where a more nuanced discussion is required with learners, as well as identifying and reducing bias in assessment. These are generalizable across health professions in which pharmacology education is a discipline in the curriculum.
Multi‐omics Sample Preparation Workflow for Proteins and DNA Using the Reversible Protein Tag ProMTag
Authors: Biedka Stephanie , Minden Jonathan S. , Lucas Amber
Published: May 2022

Sample preparation is a crucial first step for both genomics and proteomics workflows. Removal of contaminants such as salts, detergents, and other biologics while maintaining high yields of the desired product is key to reproducible and informative results from these analyses. More and more frequently, these ‐omics technologies are being used in tandem to gain deeper insights into biological processes. However, multi‐omics sample preparation remains tedious and usually requires many steps in multiple different sample preparation workflows.

In this study, we present a new multi‐omics workflow for the simultaneous preparation of DNA and protein samples from a single starting cell lysate. We accomplished this using the ProMTag reversible click chemistry technology that allows for reversible modification of the surface of proteins. Using ProMTag we were able to tag proteins in a cell lysate, bind them to ProMTag capture resin, and then precipitate nucleic acids so they also stay with the resin. With the nucleic acids and proteins bound to the resin, we were then able to wash away detergents, salts, and other contaminants. We then eluted the nucleic acids by resolubilizing in a nucleic acid elution buffer. We then were able to reverse the ProMTag by adding the protein elution buffer and eluted the sample in a mass spectrometry (MS) compatible buffer ready for proteomic analysis. Using this workflow we got yields >75% for protein and >90% for DNA. Gel electrophoresis showed a genomic DNA band free of degradation and the 260/280 absorption ratio indicated a pure DNA sample. Whole genome sequencing and MS proteomics analysis were performed and compared to traditional separate sample preparation workflows for DNA and proteins. This work establishes a new, high yield, reproducible workflow for the simultaneous preparation of DNA and proteins for genomics and proteomic analysis from a single starting sample.
Deletion of Sf3b4 in Neural Crest Cells Encapsulates the Variable Expressivity of Craniofacial Abnormalities Observed in Nager and Rodriguez Syndrome Patients
Authors: Kumar Shruti , Alam Sabrina , Beauchamp Marie‐Claude , Majewski Jacek , Jerome‐Majewska Loydie
Published: May 2022

Recently, both Nager syndrome and Rodriguez syndrome patients were identified to carry rare mutations in SF3B4. Both disorders affect the face as well as the limb including both hands and feet. Nager syndrome patients survive and exhibit smaller jaw bones, smaller cheek bones, cleft palate, hearing problems as well as downward slanted eyelids. However, Rodriguez syndrome is much more severe, and patients often die before or soon after birth. We hypothesized that craniofacial abnormalities associated with reduced SF3B4 levels is due to tissue‐specific expression and requirement of this gene in neural crest cells. To generate a conditional mutant mouse line for Sf3b4, we used CRISPR/Cas9 to insert LoxP sequences in intron 1 and 3 of this gene. To test our hypothesis, we mated Sf3b4conditional mutant mice to Wnt1‐Cre2 transgenic mice to delete exons 2 and 3 specifically in neural crest cells. Heterozygous mutant embryos from these matings were normal. Sf3b4L/L;Wnt1‐Cre2 embryos have brain and craniofacial abnormalities, including abnormal forebrain, midbrain and hindbrain, hypoplasia of the maxillomandibular region and midline cleft. Majority of these embryos die before birth and the few mutants that are born are indistinguishable from their wildtype littermates. Additionally, we generated Sf3b4L/‐;Wnt1‐Cre2 mutants with homozygous deletion of Sf3b4 in neural crest cells and heterozygous deletion of Sf3b4 in the body. These mutant embryos also had unfused frontonasal prominence and unfused and severely hypoplastic mandibular region. Sf3b4L/‐;Wnt1‐Cre2begin to die at E13.5 and none survive to birth. Thus, neural crest specific deletion of Sf3b4 in mice is able to model the craniofacial abnormalities presented in patients as well as capture the variable expressivity observed. Our model can further be used to uncover the splicing and molecular events regulated by SF3B4 during craniofacial development.
Glutathione Regulates Endothelial Cell Function and VEGFR2 Signaling
Authors: Rashdan Nabil A. , Mackay Ryan P. , Delgadillo Luisa F. , Pattillo Christopher B.
Published: May 2022

Under physiological and pathological conditions angiogenesis progresses in an idealized window of reactive oxygen species. Mice heterozygous for the Glutamate cysteine ligase modifier subunit knockout have augmented ischemic remodeling. During occlusion the ensuing ischemic milieu and accompanying vascular changes regulate the activity of vascular endothelial receptor 2 (VEGFR2). We hypothesized that cellular GSH concentration regulates endothelial cell function via altered VEGFR2 signaling.

Methods

To test this hypothesis, we first preformed mouse aortic endothelial cell (MAEC) outgrowth assays on aortic segments dissected from mice homozygous (KO) and heterozygous (Het) for GCLm knockout as well as wildtype controls (WT). VEGF stimulated chemotaxis was determined using the under‐agarose migration assay. Primary human aortic endothelial cells (HAEC) were purchased from Lonza and cultured following suppliers’ recomendations. Phospho‐Y1175 VEGFR2/total VEGFR2 and phospho‐Y416 proto‐oncogene c‐Src (Src)/total Src were determined by western blotting.

Results

Outgrowth area in the Het group was significantly larger than WT and KO at day 7 (2.15 fold, n=4, P<0.001). Het MAECs were 1.25 fold faster (n=4 p<0.001) while KO MAECs were 0.90 fold slower than WT (n=3, p<0.05) migrating towards VEGF‐A. Concurrently, we inhibited glutathione reductase in HAECs using 2‐acetylamino‐3‐[4‐(2‐acetylamino‐2‐carboxyethylsulfanylthiocarbonylamino)phenylthiocarbamoylsulfanyl]propionic acid (2‐AAPA). 2‐AAPA (75 µM) alone didn’t significantly activate VEGFR2. However, 2‐AAPA (75 µM) in combination with H2O2 (100 µM) induced a 5.5 fold (p<0.01, n=4) increase in the relative amount of active VEGFR2 compared to vehicle treated HAECs. 2‐AAPA both with and without H2O2 induced a significant increase in active Src, 5.3 and 9.0 fold (p<0.05, p<0.01, n=6) respectively. This effect was attenuated by the addition of Dithiothreitol (1mM). Furthermore, Src kinase inhibitor 1 (SKI) (200 nM) attenuated the effect of 2‐AAPA + H2O2 on VEGFR2. While the VEGFR2 kinase inhibitor SU1948 inhibited activation of VEGFR2, it had no significant effect on the 2‐AAPA + H2O2 mediated activation of Src. Glutathione S‐transferase P1 (GSTP1) catalyzes the enzymatic glutathionylation of proteins, The GSTP1 inhibitor TLK199 (50 µM) abrogated the effects of 2‐AAPA + H2O2 on VEGFR2.

Conclusion

Combined these data suggest Src mediates the 2‐AAPA + H2O2 activation of VEGFR2. And that glutathionylation is necessary for this effect, co‐immunoprecipitation experiments are underway to determine the glutathionylation status of Src in our system and its effect on the interaction of Src and VEGFR2.
A Closed Loop Feature Detection Platform for Automated Neonate Cardio‐Respiratory Measurements and Data Analysis
Authors: Ward Christopher S. , Aissi Eunice , Patel Dipak , Ruiz Brandon , Garcia Acosta Mariana , Ray Russell S.
Published: May 2022

Background

Sudden Infant Death Syndrome is thought to partly result from unseen brain abnormalities affecting cardio‐respiratory function, but for which no clear genetic or environmental mechanisms are known. Mouse genetic and exposure models present an opportunity to uncover molecular and environmental mechanisms. However, measuring cardio‐respiratory function in neonate mice is expensive, difficult, and inefficient. One major challenge is the time needed to carry out such measurements. The neonate autoresuscitation assay, consisting of repeated anoxic exposures followed by recovery, requires the full attention of an observer for the multi‐hour duration of a single‐subject assay, limiting throughput. To address this, we developed a closed‐loop feature detection platform for automated neonate cardio‐respiratory measurements.

Methods

Our platform design consists of:1) a pneumotachograph face‐mask for precise respiratory measurements. 2) a micro‐controller automated bell‐housing gas switching system for rapid induction of respiratory challenges. 3) a micro‐computer‐based data‐acquisition system with real‐time feature detection and outputs for controlling gas exposure. 4) a data analysis suite that assists with study recordkeeping and provides a facile method to extract key outcome measures from acquired data.

Results

Gas challenges are administered via the rotating bell housing system. A python program detects waveform features in real‐time, such as apnea and bradycardia. Upon apnea detection, the system switches to a rescue gas. Resumption of normal breathing and heart rate can also be detected and incorporated into criteria for automated initiation of the next anoxic exposure trial. Data is stored for later offline automated analysis using the data analysis suite.

Conclusions

The system offers a relatively inexpensive approach for automated high throughput neonate cardio‐respiratory assessment for facile screening to yield important clues in developmental pathophysiology.
Deglycosylation Does Not Affect the Cytotoxic Action of Adenoviral Interferon α‐Induced Bystander Proteins
Authors: Wang Desuo , Zhang Xiaoqiao , Benedict William F.
Published: May 2022

We previously reported an indirect effect of adenoviral interferon α (Ad‐IFNα) that is mediated by secreting bystander proteins (BSPs) and causing cytotoxic responses in both interferon α (IFNα)‐sensitive and –resistant cancer cells. Here, we investigated whether glycosylation of BSPs is necessary for the inhibition of IFNα‐resistant human bladder cancer cell line (KU7). The condition medium (CM) from the normal human urothelial cell line, TERT‐NHUC, was collected as described previously and incubated with and without N‐glycosidase F at 37°C (overnight). The growth inhibition of KU7 was compared between the control and experimental conditions. The proliferation of KU7 cells treated with interferon a protein (Intron A) is comparable to the cells treated with control TERT‐NHUC‐CM (without Ad‐IFNα infection) after 72 hr incubation. The growth of KU7 was significantly inhibited by the CM from Ad‐IFNα–infected TERT‐NHUC culture. N‐glycosidase F incubation effectively deglycosylated the BSPs in the CM (Ad‐IFNα TERT‐NHUC) as demonstrated by the band shifting in the PAGE gel verification. The deglycosylated CM (Ad‐IFNα TERT‐NHUC) produced a same degree of inhibition on the proliferation of KU7 cells (Fig 1). In conclusion, the cytotoxic effects of BSPs is independent of its status of glycosylation.
Exploring new bacterial‐fungal interactions: the role of mannan degradation in Streptococci growth
Authors: Ticer Taylor , Fultz Robert , Glover Janiece , Engevik Melinda A.
Published: May 2022

Background

Streptococci colonize multiple sites of the human body, including the oral cavity, nasopharynx, skin, respiratory, genitourinary and gastrointestinal tracts. At these various body sites, Streptococci not only interact with other microbes, but also with fungal organisms, such as Candida and Saccharomyces species. Several studies have identified an interaction between Streptococci and Candida species, but the details of these interactions are still being discovered and it is unclear if Candidaspecies can provide a nutrient source to support the growth of Streptococci. Candida and Saccharomyces species are covered by a polymer of mannose known as mannan. Mannan makes up the outermost layer of the fungal cell wall. A few gut microbes have been found to possess glycosyl hydrolases that degrade mannan, but the mannan‐degrading capacity of Streptococci has yet to be examined.

Hypothesis

We hypothesized that Streptococci that possess mannan‐degrading glycosyl hydrolases can enzymatically cleave mannose residues. We speculated that freed mannose residues could serve as a carbon source to promote Streptococci growth.

Methods & Results

Using the Carbohydrate‐Active enZYme Database (CAZY) and Integrated Microbial Genomes (IMG) database, we analyzed the ability of 90 Streptococci genomes to transport and utilize mannose and to degrade diverse mannose‐linkages found on mannan. Our computational analysis identified that most Streptococci were able to transport mannose and drive glycolysis, but <50% of Streptococci harbored the glycosyl hydrolases required for mannan degradation. To confirm the ability of select Streptococci to degrade mannan, we grew 6 representative Streptococci in a chemically defined media lacking glucose supplemented with mannose, yeast extract or purified mannan isolated from Candida and Saccharomyces strains. Growth curve analysis revealed that all Streptococcus strains could use mannose to support growth. However, the ability to use yeast extract and mannan was strain specific. S. salivariusand S. agalactiae, which did not possess mannan‐degrading glycosyl hydrolases, could not use yeast extract or mannan to enhance their growth. In contrast, we found that S. mitis, S. parasanguinis, S. sanguinis, and S. pyogenes readily used yeast extract and isolated mannan for growth. Incubation with heat‐killed C. albicans confirmed the ability of mannan‐degrading strains to use intact mannan as a nutrient source.

Conclusions

Our data indicates that select Streptococci with mannan degrading glycosyl hydrolases are capable of degrading fungal mannans and harvesting mannose for energy. These findings reveal a previously undescribed aspect of Streptococcal‐Candida interactions.
Cryptococcus neoformans capsule regrowth experiments reveal dynamics of enlargement and architecture
Authors: Jacobs Ella A.
Published: May 2022

The polysaccharide capsule of fungal pathogen Cryptococcus neoformans is a critical virulence factor that has historically evaded characterization. Polysaccharides remain attached to the cell as capsular polysaccharide (CPS) or are shed into the surroundings in the form of exopolysaccharide (EPS). While a great deal of study has been done examining the properties of EPS, far less is known about CPS. In this work, we detail the development of sonication, French Press, and glucanex digestion to both remove the capsule and produce ‘capsule ghosts.’ The ability of sonication to strip cells of capsular material while maintaining survival allowed us to explore of capsule regrowth dynamics, immune modulation, as well as NMR characterization of removed material. Development of these new methodologies will allow for exciting future work exploring cell wall capsule dynamics and improving the characterization of CPS.
Long‐term Gut Microbiota Depletion Exacerbates Hypertension and Renal Injury in an Experimental Model of Autoimmune Disease
Authors: Taylor Erin , Case Clinton T. , Reese Jayla‐Danielle , Kalusche William , Jones Katie
Published: May 2022

The gut microbiota plays an indispensable role in the development and maintenance of both the innate and adaptive immune systems. Alterations in the gut microbiota, or gut dysbiosis, is associated with many autoimmune disorders, including those without gastrointestinal manifestations, such as rheumatoid arthritis and systemic lupus erythematosus (SLE). SLE is a multisystem autoimmune disorder that primarily affects women and is characterized by high rates of hypertension, renal injury, and cardiovascular disease. Recent studies in several animal models have also posited a possible causative role of the gut dysbiosis in the development of hypertension. In the present study, we hypothesized that the gut dysbiosis in SLE may play a role in the development of immune system dysfunction and hypertension in an experimental model of SLE, the NZBWF1 mouse. To test this hypothesis, we treated 18‐week‐old female control (NZW, n=14) and SLE (NZBWF1, n=14) mice with broad spectrum antibiotics (Abx: 0.4 g/L ampicillin, 0.4 g/L neomycin sulfate, 0.4 g/L metronidazole, and 0.2 g/L vancomycin) in drinking water for 16 weeks. Additional groups of control and SLE mice received normal drinking water (control, n=14; SLE, n=14) over the course of the study. At the conclusion of the treatment, we confirmed successful gut microbiome depletion in the feces of control and SLE mice by qPCR of 16S rRNA. Mean arterial pressure (MAP; mmHg), measured in conscious mice by carotid artery catheter, was higher in SLE mice than in control mice (110±4 mmHg control vs. 131±6 mmHg SLE, p<0.01). Unexpectedly, treatment with antibiotics further increased blood pressure in SLE mice (SLE‐Abx: 151±4 mmHg, p<0.01 vs. SLE), but had no impact on blood pressure in control animals (110±4 mmHg control vs. 106±4 mmHg control‐Abx, p=0.91). Urinary albumin excretion, a marker of renal injury, was assessed by ELISA at the conclusion of the study. Albumin excretion was increased in SLE mice (0.22±0.08 mg/day control vs. 25.1±15.7 mg/day SLE, p=0.08), and urinary albumin remained elevated in SLE mice administered antibiotics (43.6±17.3 mg/day, p=0.68 vs. SLE‐Abx). Additionally, renal immune cell infiltration was assessed using flow cytometry. SLE mice had significantly higher levels of renal CD4+ T cells as compared to control mice (0.46±0.04% control vs. 0.84±0.1% SLE, p<0.05), but treatment with antibiotics elevated renal CD4+ T cells further (2.9±0.5%, p<0.001 vs. SLE). An additional pilot study (n=7‐9 mice/group) of short‐term antibiotic treatment from 26‐32 weeks of age had no impact on blood pressure in SLE mice (120±4 mmHg SLE vs. 122±3 mmHg SLE‐Abx, p=0.97). These data suggest that a long‐term loss of the microbiome in SLE actually further exacerbate disease progression and the development of hypertension.
Potential presynaptic effects of opioids on medullary 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 can lead to respiratory depression and eventually death. Opioids can alter the frequency and magnitude of inspiratory motor drive by binding to mu‐opioid receptors on inspiratory neurons located in several areas of the brainstem. Lalley (Am J Physiol: Regul. 285:R1287‐1304, 2003) suggested that some of the effects of fentanyl on bulbospinal inspiratory neuron activity were presynaptic. We tested the feasibility of pre‐ vs post‐synaptic effects of opioids with an integrate‐and‐fire computational neural biomechanical model of the brainstem respiratory network. Our previous simulations utilizing this model involved separately reducing conductance at groups of synapses that excited inspiratory‐augmenting (I‐Aug) or inspiratory‐decrementing (I‐Dec) neuron populations to simulate opioid‐mediated decreases in the presynaptic excitability of excitatory axons to these neurons from inspiratory driver neurons (I‐driver). We found that the presynaptic hypothesis was best supported by a mechanism involving depression of the excitatory synapse between I‐driver and I‐decrementing neurons. The current modeling investigation extends that work by focusing on three inspiratory neuron populations: I‐Aug, I‐Dec, and late inspiratory (late‐I). We separately decreased the conductance of each excitatory synapse to examine how these changes would affect the respiratory pattern. Simulations revealed that systematically decreasing the conductance of synapses from the I‐Aug neuron population to late‐I and I‐ Aug bulbospinal populations by 50% resulted in a decrease in frequency, duration, and amplitude of inspiratory drive. Decreasing the conductance of excitatory synapses from I‐Dec neurons to I‐Aug, and late‐I populations had a limited effect on inspiratory activity. Similar results were observed when the conductance of excitatory synapses was reduced from the late‐I population to its I‐Dec and I‐Aug targets. Overall, these results indicate that a plausible mechanism for respiratory suppression by opioids could include depression of excitatory synaptic actions between selected medullary inspiratory neuron populations.
Very Low‐Density Lipoprotein (VLDL) Signals Through Sphingosine‐1‐Phosphate Receptor 1, Upon Binding to VLDL Receptor, to Induce Aldosterone Synthesis and Secretion in Human Adrenocortical Cells
Authors: Spaulding Shinjini C. , Choudhary Vivek , Choudhary Mrunal , Chen Xunsheng , Bollag Wendy B.
Published: May 2022

Hypertension is prevalent in 46% of the US adult population with 40% of cases being attributable to obesity. Obese individuals have high serum levels of very low‐density lipoprotein (VLDL) and sphingosine‐1‐phosphate (S1P). VLDL and S1P have been shown to stimulate aldosterone production in multiple zona glomerulosa cell models, with aldosterone thought to be the link between hypertension and obesity. S1P is transported in blood bound to lipoproteins such as VLDL, low‐density lipoprotein (LDL), and high‐density lipoprotein (HDL); the VLDL particle contains the highest S1P levels. S1P in HDL has been shown to promote interactions between the scavenger receptor class B, type I (SR‐BI) and S1P receptor 1 (S1PR1). We hypothesized that like HDL, VLDL will signal through S1PRs, upon binding to SR‐BI; therefore, VLDL‐induced aldosterone secretion will be inhibited by S1PR and SR‐BI antagonists. Human adrenocortical (HAC15) cells were treated with VLDL and/or an S1PR1 antagonist (Ex26) or an anti‐SR‐BI blocking antibody for 24 h. Steroidogenic gene expression and aldosterone secretion were monitored by qRT‐PCR and radioimmunoassay, respectively. Ex26 significantly inhibited VLDL‐induced increases in CYP11B2 (22‐fold) and StAR (1.5‐fold) expression and aldosterone secretion (5‐fold) by 43%, 10%, and 36%, respectively. Unexpectedly, the anti‐SR‐BI antibody enhanced VLDL‐induced increases in CYP11B2 (29‐fold) and NR4A1 (2.2‐fold) expression by 17% and 33%, respectively; no effect was observed on VLDL‐induced increases in StAR and NR4A2 expression. However, when the VLDL receptor was blocked with recombinant human LDL receptor related protein associated protein 1, VLDL‐induced increases in CYP11B2 (2.8‐fold) and NR4A2 (5.3‐fold) expression were significantly reduced by 42% and 67%, respectively. Our results, therefore, indicate that VLDL signals through S1PR1, upon binding to the VLDL receptor and not SR‐BI, to induce aldosterone synthesis and secretion. Our study warrants further investigation into VLDL‐induced steroidogenic signaling pathways which may lead to the identification of novel therapeutic targets like S1PR1 to potentially treat obesity‐associated hypertension.
Effect of Short‐Term and Long‐Term Exposure to Sucrose on Protein Expression and Oxidative Modification of Glutathione‐S‐Transferase Mu 1 (GSTM1) in Liver of Wild Type Mice
Authors: Boyd‐Kimball Debra , Chojnacki Elise , Diaz‐Palma Santino , Graham Shelby , McMillen Andrew , Scheib Brett , Trenka Benjamin B. , Metzler Alaina , Gonczy Katelyn , Paxton Ashlyn B.
Published: May 2022

High dietary sugar intake is an environmental risk factor for the development of metabolic syndrome (MS), type 2 diabetes (T2D), and non‐alcoholic fatty liver disease (NAFLD). All three of these conditions are associated with oxidative stress and a high sugar diet has been shown to affect the protein expression and activity of antioxidant and detoxification enzymes including glutathione‐S‐transferase (GST). In this study, the effect of short‐term (ST) 2‐month and long‐term (LT) 8‐month sucrose exposure on the protein expression and oxidative modification of GST mu 1 (GSTM1) in liver of wild type mice was assessed. It was hypothesized that sucrose exposure would result in a decrease in GSTM1 protein expression and an increase in oxidative modification of GSTM1 as indexed by protein‐bound 4‐hydroxy‐2‐trans‐nonenal (HNE). It was determined that the protein expression of GSTM1 was significantly increased in the ST sucrose exposed mice, but significantly decreased in the LT control mice compared to the ST control mice. Additionally, GSTM1 was found to be significantly oxidatively modified by protein‐bound HNE in the LT sucrose exposed mice compared to the ST control mice, the ST sucrose exposed mice, and the LT control mice. These results suggest that a high sugar diet may impair the antioxidant status of the liver by oxidatively modifying detoxification enzymes such as GSTM1.
Studying the Impact of Annonaceous Acetogenin, A Prominent Component in Graviola Leaves, on the Growth of Glioblastoma Cancer Cells
Authors: Nusair Nisreen , Yoseph Hana
Published: May 2022

Cancer is the second most common cause of death in the United States. In 2018, for every 100,000 people, 436 new cancer cases were reported and 149 people died of cancer. This research focuses on studying the impact of natural chemical compounds extracted from healthful food products, such as plant herbs on modulating cancer metabolism to potentially use these compounds as cancer therapeutic agents.

This study investigates the effects of the natural chemical compound, Annonaceous Acetogenin (ACGs), that is a prominent component in the graviola plant leaves on the growth of glioblastoma cancer cells, U87MG. It is hypothesized that ACGs can reduce the growth of glioblastoma cancer cells.

The presence of ACGs in the extract sample has been verified using the Matrix‐Assisted Laser Desorption/Ionization Time of Flight, also known as MALDI‐TOF. Then, extract samples with different concentrations of ACGs have been prepared and applied to glioblastoma cancer cells. Using the light microscope, images have shown that ACGs has significantly reduced the proliferation of glioblastoma cancer cells, when compared to the control samples. To confirm the results that have been observed in the images, a cell proliferation assay has been performed to detect the number of viable cells. In wells of a 96‐well plate, different concentrations of ACGs have been added to glioblastoma cancer cell samples. The absorbances at 490 nm were recorded using an ELISA plate reader. The results indicate a linear response between the cell number and absorbance at 490nm. The data suggests that as the amount of ACGs added to the cancer cells increases, the cell number (absorbance value) decreases.

In conclusion, this research work suggests that ACGs, a prominent component in graviola leaves, can significantly reduce the growth of the glioblastoma cancer cells. Further investigations are necessary to examine how this natural chemical extract exhibits an anticancer effect and how it can potentially be used as a therapeutic agent against cancer.
Trabecular Bone Structural Variation of the Human Distal Tibia, Talus, and Calcaneus
Authors: DeMars Lily J. , Kuo Sharon , Saers Jaap P. , Stock Jay T. , Ryan Timothy M.
Published: May 2022

Based on the principles of bone functional adaptation, bone structure has been interpreted as reflective, at least in part, of the mechanical loading environment experienced by an individual during their lifetime. However, the extent to which reduced skeletal robusticity in modern humans over the last 10,000 years is the result of reduced activity and increasing reliance on agricultural subsistence strategies, rather than genetic or environmental factors known to influence skeletal phenotype, is not well understood. The objective of this study is to investigate how bone structure varies between human groups with distinct subsistence strategies, and thus different levels of physical activity and mechanical loading. Here we compare trabecular bone structure in the distal tibia, talus, and calcaneus of three archaeological groups from central and southern Illinois with varying levels of mobility from relatively low mobility mixed strategy agriculturalists to relatively high mobility hunter‐gatherers. High‐resolution CT image data of the distal tibia, talus, and calcaneus was used to quantify trabecular bone microarchitecture variables including bone volume fraction and degree of anisotropy. Bone structural variables are compared between subsistence groups using registered point clouds and Bayesian multilevel modeling to test whether bone structure differs between groups with different inferred mobility and activity levels, but similar population history and environmental context. Results indicate that variation in trabecular bone structure for the distal tibia, talus, and calcaneus support the hypothesis that more highly mobile subsistence groups have greater trabecular robusticity. Investigating the relationship between bone structure variation and differences in activity and mobility is important for understanding the role of behavioral, ecological, and biological factors in determining human skeletal phenotype.
Development and Implementation of an Online OSPE Test Bank Graded by Artificial Intelligence
Authors: Coelho Varun , Amoudi Lana , Segovia Gerald , Vejdani Pariya , Varambally Meghna , Wang Tracy , Ball Alexander , Bayer Ilana , Bernard Jason , Helli Peter , Mitchell Joshua , Pitt Courtney , Saraco Anthony , Walker O'Llenecia , Wainman Bruce
Published: May 2022

The development of online anatomy education has rapidly accelerated during the COVID‐19 pandemic. This shift to the online world has mainly been focused on the delivery of content, while testing anatomical knowledge has proven to be more challenging, particularly for objective structured practical exams (OSPEs, also known as “spot tests” or “practical exams”). Online resources for OSPEs are uncommon compared to the extensive banks of multiple‐choice questions available. Another issue is that whether virtual or in‐person, grading OSPEs is challenging and time consuming. Recent research in our laboratory has suggested that machine learning algorithms, using decision trees, can be trained to mark OSPEs with a >95% accuracy. Building on these findings, the goal of this project is to create a virtual OSPE bank, train the AI to grade OSPEs, and develop an application with automated AI grading to act as a resource for students studying anatomy and physiology. Currently, we have written over 120 OSPE question sets using images from the Bassett Collection, the UBC Neuroanatomy collection, and images developed at the Education Program in Anatomy at McMaster University. The questions and answers were initially developed by senior undergraduate students, with coaching from faculty and staff familiar with OSPE generation acting as experts. The questions were then collectively reviewed by the students before undergoing two independent reviews by the experts. After revision, a final blind review of the questions was undertaken by a third expert to ensure validity and accuracy. These questions are being made available on the undergraduate anatomy and physiology course learning management system (LMS), Avenue to Learn. On this LMS, students will be able to use the questions for OSPE practice and answers will be available in the traditional manner. The answers given by the students and graded by the faculty will be collected and analyzed to determine the difficulty and discrimination of the questions. The data and analyses will be used to refine additional OSPEs, as well as refine the AI marking tool for the virtual OSPE application. Our hypothesis is that by providing instant and accurate feedback on valid and accurate questions, course evaluations and performance on OSPEs will improve. The research group is actively searching for collaborators willing to generate and review additional OSPE questions to expand the question bank and improve the AI marking tool.
Multi‐omics Analysis of S. Pneumoniae Extracellular Vesicles
Authors: Biedka Stephanie , Eutsey Rory , Hiller Luisa , Lucas Amber
Published: May 2022

Extracellular vesicles (EVs) are cell‐derived nanoparticles that can serve as carriers of DNA, RNA, and protein. For the pathogen S. pneumoniae,which causes diseases such as pneumonia and meningitis, EVs can be used to transport these macromolecules to recipient cells, facilitating communication and signaling between cells. However, the overall composition of these S. pneumoniaeEVs is still unknown and how the macromolecules packaged within these EVs contribute to the pathogenicity of S. pneumoniaeremains a hot research topic. In this work, we have developed a novel multi‐omic workflow that isolates DNA, RNA and protein from a single S. pneumoniaeEV starting sample, enabling analysis of these macromolecules without the need for multiple sample preparation steps and allowing us to gain insights into the composition and functionality of S. pneumoniaeEVs.

EVs from a S. pneumoniaeculture were isolated and the proteins in the EV homogenate were labeled with the bifunctional ProMTag. One end of the ProMTag forms a reversible, covalent bond with proteins. ProMTag’s other functional group is methyltetrazine, which forms an irreversible bond with trans‐Cyclooctene (TCO), allowing capture of EV proteins on TCO‐agarose beads. Organic solvent was then added to precipitate the nucleic acids in the EV homogenate. Nucleic acids were released by a series of washes and subsequently separated into DNA and RNA fractions by RNase or DNase treatment, respectively. Proteins were then released from the TCO‐agarose beads by reversing the ProMTag‐protein linkage, yielding proteins in their original, unmodified state ready for analysis. DNA and RNA were sequenced, and proteins were identified using mass spectrometry. Using this workflow, we were able to describe the composition of multiple macromolecules within S. pneumoniaeEVs and gain insights into how these macromolecules might facilitate communication and infection within the S. pneumoniaepopulation.
Induction of Autophagy in Motor Neurons with Lanthionine Ketenamine Analogs
Authors: Gransee Heather M. , Gonzalez Porras Maria A. , Denton Travis T. , Shen Dunxin , Sieck Gary C. , Mantilla Carlos B.
Published: May 2022

Autophagy is a cellular digestion process that contributes to cellular homeostasis by the elimination of proteins and damaged organelles. Impaired autophagy has been implicated in aging‐related disorders and in neurodegenerative diseases, including motor neuron disorders. Therefore, upregulation of autophagy may serve as a promising therapeutic approach. Lanthionine ketenamine (LK), an amino acid metabolite found in mammalian brain tissue at low concentrations, activates autophagy in rat glioma and human neuroblastoma cells in vitro, as well as in hippocampus and cortex after in vivo administration. With the overall goal of using LK to induce autophagy flux to prevent age‐related motor neuron dysfunction, we recently tested the effects of two lanthionine ketenamine phosphonates (LK‐Ps) and one lanthionine ketenamine ethyl (ester) phosphonate (LKE‐P) on autophagy modulation in a mouse motor neuron‐like hybrid cell line (NSC‐34). For fluorescence visualization of autophagy flux, NSC‐34 cells were transfected with mCherry‐GFP‐LC3 plasmid. Motor neurons treated with LK analogs displayed increased autophagy flux measured by the ratio of mCherry area (lysosomes) over GFP area (autophagosomes that have not fused with lysosomes) compared to control cells. There results were corroborated by increased LC3‐II/LC3‐I ratio as measured by protein expression using Western blot; and presence of autophagic vacuoles identified using transmission electron microscopy. In conclusion, LK analogs constitute a promising tool to induce autophagy flux in motor neurons, which may serve to prevent age‐related motor axon withdrawal and muscle denervation.
Response to Endurance Training Using Critical Speed: Influence of Genetic Background and Exercise Intensity
Authors: Nino Matthew , Kim Hyoseon , Massett Michael P.
Published: May 2022

Exercise is used as a therapeutic option to improve an individual's quality of life or as a method to attenuate the severity of cardiovascular, respiratory, or metabolic‐related diseases. However, changes in cardiorespiratory fitness in response to endurance training are often heterogenous with individuals being more or less responsive to a similar exercise protocol. Heterogeneity in responses to exercise training is observed in humans and rodents. Low responses to training might be mitigated by modifying the standard exercise training protocol by altering the frequency, intensity, or duration of training. Threshold‐based exercise training prescription, e.g., critical speed, has also been proposed as a means to reduce the variability of training responses. The aim of this study was to identify the effect of different exercise training intensities on changes in exercise capacity in two inbred strains of mice, NZW/LacJ (NZW) and FVB/NJ (FVB). NZW mice were selected because they respond poorly to moderate intensity endurance exercise whereas, FVB mice respond well to endurance training. We hypothesized that increasing the intensity of the training would reduce the number of low or non‐responding NZW mice. To test this hypothesis, female mice from each strain were assigned to one of three treadmill training groups based on critical speed (CS): 80% of their critical speed (CS80%), 90% of their critical speed (CS90%), or sedentary controls (SED). Exercise groups within each strain were volume matched and thus ran the same distance over the 6‐week training period. Pre‐training exercise capacity differed between NZW (30.5 ± 3.5 min) and FVB (36.1 ± 2.8 min) mice (P < 0.001). Critical speeds also differed between strains (NZW: 25.1 ± 2.4 m/min; FVB: 32.9 ± 1.6 m/min, P < 0.001). After training, both FVB exercise groups, CS80% (10.0 ± 4.9 min, P = 0.004) and CS90% (14.0 ± 7.7 min, P = 0.007), improved their endurance capacity. In contrast, NZW CS80% did not significantly improve after training (0.4 ± 4.6 min), but NZW CS90% improved significantly (3.6 ± 2.7 min, P = 0.02). Collectively, these data indicate that the high‐responder FVB strain can adapt to moderate‐intensity and high‐intensity endurance training. The low‐responder NZW strain only showed significant improvement after sufficient homeostatic stress was induced through high‐intensity endurance training. These data suggest that increasing exercise intensity can improve responses to exercise training in low‐ or non‐responding mice.
A machine‐learned microvasculature optimizes physiological insulin secretion in a vascularized pancreas‐chip
Authors: Tronolone James J. , Mathur Tanmay , Chaftari Christopher P. , Jain Abhishek
Published: May 2022

Microphysiological systems have gained increasing attention for their applications in drug discovery and testing, toxicity screening, and more recently disease modeling. However, microvascular networks are rarely incorporated into these devices despite being a major component of almost all tissues of the body. Of the reports integrating these networks, many use different quantification tools and fail to relate these quantifications to the biological function of the networks. Here, we standardized vasculature engineering by first generating 500 samples of microvascular networks‐on‐chips composed of different endothelial cell densities and sources, supporting cells, growth factors, and extracellular matrices. For each sample, 10 individual morphological metrics were generated using a peer‐reviewed, open‐source network quantification tool. Simultaneously, the mass transport of each sample was quantified using numerical simulation, resulting in a continuous value representing the biological function of each sample. Using machine learning, we then developed models using neural network regression methods that related the morphological metrics with the simulated biological function. Further, we identified which metrics most significantly impact the mass transport, as well as the integrable chip components that optimized mass transport and its related morphological metrics. Finally, we demonstrated how these optimizations lead to the vascularization of a pancreas microenvironment featuring insulin‐secreting islet cells. Further, vascularization rescues insulin secretion when pancreas‐chips are placed in hypoxia, demonstrating that the integration of microvascular networks plays a crucial role in the oxygenation and maintenance of a tissue‐specific microphysiological system.
Effects of ferric carboxymaltose on phosphate and calcium homeostasis in a murine model of iron‐deficiency anemia
Authors: Stevens Monica , Thomas Linto , Rieg Timo , Dominguez Rieg Jessica
Published: May 2022

Iron deficiency anemia (IDA) can be caused by heavy uterine bleeding, inflammatory bowel disease, or chronic kidney disease. Ferric carboxymaltose (FCM) is an intravenous form of iron replacement therapy that has been shown clinically to induce severe hypophosphatemia. However, the mechanism by which this occurs is poorly understood. The objective of this study was to develop and characterize a mouse model that would allow investigating the effects of FCM on phosphate (Pi) and calcium (Ca2+) homeostasis. Female C57Bl/6J mice (n=40; 10/group) were randomized to either iron deficient (ID) diet (4 ppm iron) or control diet (48 ppm iron) for 4 weeks followed by intravenous bleeding (0.7% of body weight) 4 and 2 days prior to starting the study. Baseline blood and urine was collected for hematocrit, complete blood count, and serum and urine Pi and Ca2+ levels. Subsequently, mice were treated with a single IV injection of either vehicle (saline) or FCM (20 mg/kg) via retroorbital injection. One week later, terminal blood, urine, feces, liver, and spleen were collected. At baseline, compared with controls, ID mice had lower hematocrit (35±1% vs 39±1%; P<0.05) and microcytic hypochromic anemia. After 7 days, FCM‐treated ID mice had significantly higher hematocrit compared with vehicle‐treated ID mice (43±1% vs 30±1%; P<0.05). FCM treatment also significantly increased iron content in liver and spleen in control mice (18±1 vs 6±1 μmol/g and 36±2 vs 27±3 μmol/g, respectively; P<0.05) and ID mice (16±1 vs 1±0.1 μmol/g and 21±2 vs 6±1 μmol/g, respectively; P<0.05). At baseline, urinary Pi/creatinine ratios were significantly lower in ID mice compared with controls (30±5 vs 58±7 mmol/mmol; P<0.05); however, FCM treatment in ID mice did not affect urinary Pi/creatinine ratios after 7 days (31±6 vs 18±5 mmol/mmol; not significant). There were no differences between groups for serum Pi, erythrocyte Pi, or fecal Pi. Under baseline conditions, no differences in plasma Ca2+ or urinary Ca2+/creatinine ratios were observed between groups; however, FCM‐treated ID mice had significantly lower plasma Ca2+ levels after 7 days compared with ID mice at baseline (2.3±0.1 vs 2.6±0.1 mmol; P<0.05) and lower urinary Ca2+/creatinine ratios (0.1±0.1 vs 1.3±0.2 mmol/mmol; P<0.05). We speculate this may be due to increased PTH and FGF23 levels in FCM‐treated ID mice. In summary, our study demonstrates for the first time that FCM affects Pi and Ca2+ homeostasis in a murine model of IDA. Further studies are needed to better understand the mechanisms of FCM on mineral homeostasis as well as the driving factors for species differences.
A New Method to Self‐Associate Proteins Using a Cooperative DNA‐binding Protein
Authors: Shara Stephanie , Junker Matthew
Published: May 2022

Many cellular processes depend on proteins that must self‐associate into larger complexes in order to function. This includes membrane receptors that dimerize (form pairs) to transmit transmembrane signals and scaffolding proteins that assemble into structural filaments. This project is developing a new method to experimentally control protein self‐association in order to better study such processes. The method is based on the DNA‐binding protein HAP1 (heme activation protein 1). As an isolated fragment, the DNA binding region of HAP1 is a monomer in solution but binds cooperatively as a dimer to its DNA target site. Using recombinant DNA methods, an 11 kDa fragment of the HAP1 DNA‐binding region was fused to the 43 kDa maltose binding protein (MBP) to test if adding the HAP1 target site DNA could induce dimerization of the fused MBP. The HAP1 DNA‐binding fragment was modified to remove a protease‐sensitive site observed during recombinant expression. In addition, two different lengths of flexible linker were introduced between the MBP and HAP1 proteins to reduce potential steric interference among MBP, HAP1, and bound DNA. Binding of MBP‐HAP1 fusions to DNA was measured by fluorescence anisotropy using 24 base‐pair fluorescein‐labeled DNA duplexes. A titration of the MBP‐HAP1 fusion with 15 residue linker to a labeled target site showed strong binding with half‐maximal binding at 0.3 μM MBP‐HAP1. In addition, the titration was fit with a Hill constant of 2.0 ± 0.3 indicative of cooperative dimer formation on the DNA. Binding to a labeled non‐target DNA was much weaker with half‐maximal binding greater than 2 μM. Gel filtration chromatography of the protease‐sensitive MBP‐HAP1 fusion showed a DNA‐induced shift from monomer to a larger protein‐DNA complex containing at least two proteins per DNA. These tests are being repeated with the protease‐insensitive MBP‐HAP1 fusion. The ability of the HAP1 DNA‐binding region to induce the self‐association of MBP using an inexpensive DNA ligand should be generalizable to other proteins, giving this method wide utility.