bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequential Model of Teaching in Anatomy Lab Aims to Improve Student Learning
Authors: Dugan Lauren , Thomas Kyle , Scali Frank , Hassan Sherif S.
Published: May 2022

Introduction

At the California University of Science and Medicine, School of Medicine, the anatomy department has developed a sequential model of teaching in which anatomical concepts are gradually introduced and integrated with increasingly complex modalities. This teaching pedagogy aims to establish strong anatomical foundations to equip students with the critical thinking skills to identify landmarks on prosected cadaveric specimens and radiographic imaging.

Methods

Anatomy labs are divided into virtual and in‐person components. These lab sessions have been developed to teach anatomical structures to students in an explicitly stepwise progression. During virtual anatomy sessions, students are initially taught to draw simple line drawings of anatomical structures. Students progress from drawing anatomical structures to studying more complex, professional illustrations of those same structures, such as those in Netter's Atlas of Human Anatomy. Following exposure to illustrations, students are introduced to three‐dimensional images of the structures using virtual anatomy software. During independent in‐person lab sessions, students apply knowledge acquired from each of these prior learning modalities to identify structures on prosected specimens. Lastly, students implement critical thinking skills to distinguish anatomical landmarks and pathological changes in radiographic images.

Results

This systematic progression of teaching aims to allow students to engage in higher‐level thinking in which they apply newly acquired knowledge to increasingly more complex material. By doing so, the following learning outcomes of each anatomy session can be successfully achieved:

Identifying anatomical structures on plastinated specimens

Identifying anatomical structures in various imaging modalities, such as X‐rays, CT, and MRI

Discerning pathological structures from healthy structures in various imaging modalities

Prospective data collection through course evaluation forms will be analyzed to assess the sequential teaching model's efficacy.

Conclusion & Significance

Using the sequential model of teaching in anatomy lab can effectively help medical students understand, rather than memorize, anatomy. The systematic scaffolding of anatomical concepts with increasingly complex material, progressing from line drawings to imaging interpretation, encourages critical thinking and reinforces learning. We expect these skills will prepare students for the challenges presented in their clinical years and beyond.
Phenotype‐specific signatures of gut microbiome, resistome, virulome, and metabolome associated with childhood airway allergies
Authors: Su Shih‐Chi , Chang Lun‐Ching , Chiu Chih‐Yung
Published: May 2022

Perturbation of gut dysbiosis has been linked to childhood allergic diseases. However, the association of fecal microbiome and metabolite profiles with specific phenotypes is poorly understood. To address this, we performed shotgun sequencing of intestinal microbiome and untargeted metabolomics profiling of fecal samples in a cohort of children mite‐sensitized airway allergies (25 with rhinitis and 31 with asthma) and 28 non‐allergic healthy controls. An integrative analysis of stool metagenomic and metabolomic profiles associated with IgE reactions in childhood allergic rhinitis and asthma was conducted. We noted a decrease in the number and abundance of gut microbiome‐encoded carbohydrate‐active enzyme (CAZyme) genes, accompanied with a reduction of within‐sample species richness (α diversity), in the asthmatic gut microbiome but not in that from allergic rhinitis. Such loss of CAZymes was consistent with the observation that decreased levels of butyrate and fumarate (a metabolic precursor of propionate) were detected in the fecal samples of asthmatics. In addition, genes related to rifamycin resistance were significantly enriched in fecal samples of cases with asthma, suggesting that altered resistome in gut microbiota may contribute to respiratory health in asthmatics. Further analysis of gut virulome identified differentially abundant virulence functions in asthmatic microbiome, such as enrichments for fibrinogen binding protein, magnesium uptake, and Ebp pili genes, and depletions for flagella‐mediated motility genes. Moreover, we demonstrated an inverse correlation between the abundance of Galactobacillus timonensis and serum IgE levels of the host. Our dual‐omics analyses reveal several microbial gene functions and species‐level clades derived from gut dysbiosis in robust associations with fecal metabolites and disease phenotypes, which may be of etiological and diagnostic implications in childhood airway allergies.
A novel compound AD021 attenuates renal fibrosis by inhibiting transforming growth factor‐β1 in renal proximal tubule epithelial cells
Authors: Poolsri Wanangkan , Jutabha Promsuk , Chavasiri Warinthorn , Muanprasat Chatchai
Published: May 2022

Renal fibrogenesis is the common final pathway to all renal injuries including injury from diabetes that consequently leads to end‐stage renal disease (ESRD). Currently, there are no potential therapeutic drugs that are both safe and effective to prevent the progression of diabetic nephropathy (DN) patients and eventually ESRD. Transforming growth factor‐β1 (TGF‐β1) as the major player of tubulointerstitial fibrosis induction in diabetic kidneys. Moreover, inhibition of the TGF‐β1 pathway is one of the most effective renal fibrosis therapies in DN. Therefore, we aimed to identify the novel anti‐fibrotic compound using TGF‐β1‐induced fibrosis cells. Renal proximal tubule epithelial cells (RPTEC) were used to evaluate the anti‐fibrotic effect of the natural compounds. Our laboratory has natural and synthetic compounds libraries and some of them have been reported to have beneficial effects in several diseases, but the anti‐fibrotic effect has never been studied. The picro‐sirius red staining and spectrophotometric analysis were used for screening of 335 compounds. The active compound, AD021, effectively inhibited TGF‐β1‐ induced fibrosis in RPTEC cells with IC50 of 11 μM. Profibrotic protein expression levels including fibronectin and α‐smooth muscle actin were decreased in AD021 co‐treatment with TGF‐β1 in a concentration‐dependent manner. Collectively, our findings demonstrated that this novel anti‐fibrotic agent has potential for the development of novel renal fibrosis treatment for DN patients.
How Acinetobacter baumanniiControls Antibiotic Resistance through Phosphorylation of the BfmR Global Regulator
Authors: Hernandez Gabrielle , Raustad Nicole , Iinishi Akira , Geisinger Edward
Published: May 2022

There is an evolving threat to our healthcare system—the rise of antibiotic‐resistant bacteria, such as Acinetobacter baumannii, and the depletion of our few useful antibiotics. These bacteria have developed an ability to fight off increasing numbers of antibiotics used to treat hospitalized patients. To combat these infections, new solutions must be discovered to target the bacteria’s ability to resist drugs and restore the efficacy of our arsenal of antibiotics. One possible therapeutic target is the BfmRS two‐protein signaling system, which activates many genes that determine antibiotic resistance and virulence in A. baumannii. The transcription factor, BfmR, is key to this gene regulation pathway. How the activity of BfmR is controlled is unknown. We hypothesize that a phosphorylation signal activates BfmR, allowing it to form an oligomeric state that binds to and enhances expression of target genes. To test this hypothesis, we developed in vitrophosphorylation conditions using purified BfmR and phosphoramidate, a small molecule phosphodonor. We used phosphate‐affinity polyacrylamide gel electrophoresis to determine that phosphorylation of the purified protein was highly efficient. Size exclusion chromatography analysis of BfmR using high‐performance liquid chromatography (HPLC) was consistent with dimerization of the protein upon phosphorylation. Next steps will include evaluating the binding affinity of the phosphorylated protein to a series of target DNA sequences associated with resistance and virulence genes, chosen from chromatin immunoprecipitation sequencing (ChiP‐Seq) data showing conserved response elements. We will employ electrophoretic mobility shift assays (EMSAs) to analyze how phosphorylation affects BfmR binding to these sequences. The information we uncover on the regulatory mechanisms of BfmR can be used to guide the development of inhibitors that counteract A. baumannii’s resistance to essential antibiotics.
The role of nutrient‐responsive hippocampal neurons in feeding
Authors: Yang Mingxin , De Lartigue Guillaume
Published: May 2022

Feeding behavior is controlled by hippocampal‐dependent learning and memory that influence decisions about where, when and what to eat. However, the neuronal subtypes and stimuli that recruit these neurons remain poorly understood.

Here we leverage the unique strength of the recently‐developed Targeted Recombination in Activated Populations (TRAP2) mouse model, which provides cell type‐unbiased genetic access to neuronal populations based on their activation by defined and time‐constrained stimuli. Surprisingly, after comparing neuronal activity to separate post‐ingestive stimuli within the same animal, we identified distinct hippocampal subpopulations that respond to either fat or sugar. We validated a protocol for selective ablation of the sugar‐activated hippocampal neurons using viral mediated caspase in the TRAP2 mice. Using an automated feeding system, we found that these sugar‐ablated mice significantly reduced their chow and high‐fat diet intake compared to control littermates, and exhibited impaired food location memory in a sugar‐driven food cup task. No group differences were observed in the ability to learn a flavor‐nutrient conditioning paradigm.

Overall, these results demonstrate that the hippocampus is attuned to specific macronutrients rather than the feeding state, allowing highly‐refined appetitive learning and memory in response to context‐specific feeding cues.
Role of cannabinoids and vitamin E analogues in macrophages foam cells formation
Authors: Zingg Jean‐Marc , Li Mengrui , Bertoncini‐Silva Caroline , Daunert Sylvia
Published: May 2022

Recently, macrophages foam cells have gained attention in cases of electronic‐cigarette or vaping product use‐associated lung injury (EVALI). An increased number of foam cells was detected in bronchoalveolar lavages and lungs after inhaling e‐cigarette smoke from products containing cannabinoids and the vitamin E analogue, alpha‐tocopherol acetate (αTA), that was present as vape‐cartridge additive. Increased levels of intact αTA are reportedly detected in EVALI indicating that conversion of αTA to αT may less occur in lungs upon inhaling vapors from e‐cigarettes containing αTA.

Hypothesis

Whereas the molecular mechanisms by which macrophages foam cells are formed after vaping are unclear, it appears possible that cannabinoid receptors play a role, as lung‐resident macrophages express cannabinoid receptors. Dysregulation of cannabinoid receptors by αTA and cannabinoids may induce the CD36 scavenger receptor/fatty acids transporter (CD36/FAT) and/or reduce the ATP binding cassette transporters A1 and G1 (ABCA1/G1) expression leading to lipid accumulation. Therefore, we analyzed the regulatory effects on lipid homeostasis of natural alpha‐tocopherol (RRR‐αT), natural RRR‐αTA (αTAn) and synthetic racemic all‐rac‐αTA (αTAr) with and without co‐treatment with cannabinoids (cannabinol (CBD), Δ9‐tetrahydrocannabinol (THC)) in THP‐1 monocytes and macrophages (differentiated for 48 h with 100 ng/ml phorbol‐12‐myristate‐13‐acetate (PMA)).

Results

Quantification of lipids in macrophages indicated that more lipids accumulate with αT, less with αTAn and αTAr, whereas control alpha‐tocopheryl phosphate (αTP) clearly inhibited. Co‐treatment with THC increased lipid accumulation with αT, αTAn as well as with CBD, whereas αTP again clearly inhibited. In THP‐1 monocytes, CD36 scavenger receptor was stimulated with αTAn and THC and with co‐treatment with THC and αTAn or CBD. In THP‐1 macrophages, CD36 was only up‐regulated by CBD and co‐treatment with αTAn or αTAr reduced it. In THP‐1 monocytes, cell cycle progression was only affected by CBD (increased G1, decreased S) and co‐treatment with αTAn and αTAr restored it. In macrophages, no significant toxicity was measured with lactate dehydrogenase (LDH) release assay by these treatments at 24 h, 48 h and 72 h at αTA concentrations below 50 μM.

Although the mechanisms by which intact αTA may affect lungs in cases of EVALI are still unclear it may disrupt the pulmonary surfactant, act as linactant, and/or dysregulate lipid homeostasis and contribute to formation of macrophages foam cells and inflammation. Lipid accumulation in monocytes/macrophages and foam cells formation were modulated by cannabinoids and αTA but the involvement of lipid homeostasis genes such as CD36/FAT and ABCA1/G1 remains to be further elucidated. A detailed analysis of the molecular mechanisms by which foam cells are formed will not only be important to understand EVALI but also for atherosclerosis and non‐alcoholic steatohepatitis (NASH).
The Effects of DNA Topoisomerase 1 Alteration in Herpes Simplex Virus Type 1 Infections
Authors: Esenwein Raegen M. , Dembowski Jill A.
Published: May 2022

Herpes simplex virus type 1 (HSV‐1) is a universal human pathogen that infects nearly 70% of the population. In addition to producing its own viral factors, HSV‐1 utilizes numerous host proteins to facilitate its infection cycle. One host protein of particular interest, found to specifically interact with the HSV‐1 genome throughout replication, is human topoisomerase 1 (Top1). In human cells, Top1 is an enzyme required for normal growth and development. Cellular Top1 ensures proper transcription and DNA replication by creating single stranded nicks in DNA to relieve topological stress and allow for relaxation of supercoiled DNA. Based on its role in cellular processes, we believe that Top1 aids in the regulation of viral transcription and/or replication. We therefore hypothesize that altering Top1 expression in HSV‐1 infected cells will decrease viral yield by not allowing for an efficient infection cycle. To alter Top1 expression, the commercial inhibitor, camptothecin (CPT) was used. Our studies revealed the novel observation that Top1 inhibition via CPT does reduce viral yield. Currently, an inducible Top1 shRNA knockdown cell line is being created as another method of alteration. In the future, we will determine if viral transcription or DNA replication are altered after Top1 inhibition or knockdown. These studies will further pinpoint the role of Top1 in HSV‐1 infection.
Development of a Nonhuman Primate Model of Resistance to Punishment of Ethanol Choice
Authors: Czoty Paul W. , Stinson Benjamin T.
Published: May 2022

Several of the diagnostic criteria for alcohol use disorder (AUD) reflect the fact that some people continue to drink despite negative consequences. That is, vulnerable individuals are resistant to punishment. A better understanding of the behavioral and neurobiological factors that underlie this characteristic will lead to better preventive and treatment approaches to AUD. The objective of this study was to extend rodent studies by developing and validating a model of resistance to punishment of alcohol drinking in nonhuman primates. To increase translational relevance, the study was conducted in the context of an ethanol‐water choice procedure. Each day two 800‐ml bottles were hung on the monkey’s home cage for three hours, one containing a 4% ethanol solution and the other containing water. The primary dependent variable was ethanol choice (ml of ethanol consumed divided by total ml consumed x 100). When choice was stable (±10% of the 3‐day mean with no upward or downward trend), several manipulations were made. Experiment 1 characterized choice of several concentrations of ethanol (0%‐8%) versus water by substituting other ethanol concentrations for the 4% solution until choice again stabilized. The relationship between the available ethanol concentration and ethanol choice was characterized by an inverted U‐shaped function. In Experiment 2, the alternative (water) was manipulated to further validate the procedure as a model of choice. For monkeys in whom choice of the 4% ethanol solution was relatively low (<30%), the bitter tastant quinine (0.01‐1.0 g/L) was added to the water solution. For monkeys with higher choice of 4% ethanol (>70%), sucrose (1.0‐5.0%) was added to the water solution. These manipulations predictably increased and decreased, respectively, choice of the 4% ethanol solution. Finally, in Experiment 3, consumption of the 4% ethanol solution was punished by adding quinine (0.01‐5.6 g/L) to the ethanol solution. Increasing quinine concentrations decreased ethanol choice and ethanol intake in all monkeys without decreasing the total volume of fluid consumed. Individual differences were observed in the potency of quinine which can be interpreted as individual differences in sensitivity to punishment. These studies established and validated a novel nonhuman primate model of punishment of ethanol choice which can be used to characterize factors that underlie susceptibility versus resistance to punishing effects of adverse consequences of alcohol drinking.
Investigation of structural factors controlling loop dynamics in acyl protein thioesterases
Authors: Johnson R. J. , Hossain Asif , Ratz Elizabeth , Harris William , Weiss William
Published: May 2022

Acyl protein thioesterases catalyze the depalmitoylation of key signaling proteins attached to the plasma membrane. Recently, human APTs were shown to use a two‐step catalytic mechanism involving initial membrane binding of an exposed flexible loop followed by substrate binding and cleavage. This two‐step catalytic mechanism is likely conserved as a bacterial homologue of human APTs is also regulated by the large‐scale movement of an essential dynamic loop upon membrane binding. However, the causes for the movement of this dynamic loop in APTs are unknown. Herein, we investigated factors that trigger loop movement in human and bacterial APTs. In particular, APTs were assayed with inhibitors structurally similar to long chain lipids and to membrane‐mimicking surfactants to determine if these factors induce loop movement. Loop movement was detected by measuring the change in intrinsic tryptophan fluorescence of a central tryptophan on the dynamic loop before and after exposure to these conditions. Although inhibitors and substrate mimics induced minor changes in loop dynamics, larger shifts were measured with various membrane‐mimicking surfactants, suggesting that APT loop dynamics are mainly regulated by proximity to the plasma membrane. Ultimately, understanding the factors controlling APT function could provide novel mechanisms for controlling cell signaling.
Partially Conserved Motifs Support an Alternating Access Model for the Human Mitochondrial NAD+ Transporter
Authors: Goyal Shivansh , Cambronne Xiaolu A.
Published: May 2022

SLC25A51 resides in the inner mitochondrial membrane and it is required for cellular respiration and the mitochondrial production of ATP. As a member of the mitochondrial carrier family (MCF), it controls NAD+ concentrations in the mitochondrial matrix by directly importing the oxidized dinucleotide. Nevertheless, the mechanism by which SLC25A51 transports NAD+ across the inner mitochondrial membrane is not known, and its primary sequence lacks key residues that have been otherwise attributed to shared mechanisms of MCF transporters. To investigate this discrepancy, we used Molecular Dynamics simulations to characterize both the outward‐open conformation of SLC25A51 where NAD+ is expected to bind and the inward‐open conformation. We tested predictions from the simulations using a genetically‐encoded fluorescent biosensor that can directly monitor changes in free NAD+ concentrations in the mitochondrial matrix.

We found that SLC25A51 lacks a three‐fold symmetry of its pore that is a characteristic of many MCF transporters. Instead, the core of SLC25A51 adopts an oblong and flattened shape. We predict this may be critical for the recognition and discrimination of the oxidized dinucleotide from other structurally related molecules, including its reduced form. A similar asymmetry was observed at the salt‐bridge network on the matrix side, produced by the asymmetric formation of a single salt‐bridge between E139 and K236 that linked helices 3 and 5. This ionic interaction was consistently observed throughout the simulations and mutation of either residue ablated SLC25A51 activity. We identified three cardiolipin binding sites on the membrane‐facing exterior of SLC25A51 that permitted cardiolipin to bridge interactions between the helical chains. We showed that these sites impact SLC25A51 function when mutated. Our simulations further predicted an energetically‐comparable single salt‐bridge on the cytoplasmic side of the transporter that could stabilize a complementary matrix‐open state. Mutation of the predicted cytoplasmic salt bridge between K198 and E291, as well as introduction of an ectopic salt bridge to imbalance the potential energetics of transport, diminished SLC25A51 activity. Together, the data indicate that although SLC25A51 lacks may conserved residues compared to other MCF family members, it functions through an alternating access mechanism and its activity depends on cardiolipin.
RXFP2 Small Molecule Agonists: Potential Therapeutics for Osteoporosis
Authors: Esteban‐Lopez Maria , Wilson Kenneth , Myhr Courtney , Kaftanovskaya Elena , Henderson Mark , Southall Noel , Hu Xin , Barnaeva Elena , Ferrer Marc , Kim David , Ye Wenjuan , Morello Roy , Agoulnik Irina , Marugan Juan , Agoulnik Alexander
Published: May 2022

Osteoporosis is a chronic bone disease characterized by decreased bone mass and increased risk of developing fractures, predominantly observed in the elderly. Osteoporosis affects approximately 10 million people in the US, and the number is expected to increase exponentially as the elderly population continues to grow. The pathophysiological cause of the disease is a decrease in the activity of the bone‐forming cells (osteoblasts) that alters bone remodeling in favor of bone resorption, leading to a decrease in bone mass. Recent studies identified the G protein‐coupled receptor (GPCR) for insulin‐like 3 peptide (INSL3), relaxin family peptide receptor 2 (RXFP2), as an attractive target for the treatment of osteoporosis. The goal of this study is to develop small molecule agonists of RXFP2, expressed in osteoblast cells, to promote bone growth and counteract the deleterious effects of osteoporosis. Currently, the most effective available treatment for osteoporosis is an expensive hormone therapy that requires daily injections. We aim to create drugs that are stable and can be delivered orally. Several low molecular weight compounds were identified as agonists of the RXFP2 receptor using a high‐throughput screen of the NCATS small molecule library. The screening assay measured cAMP response in RXFP2‐transfected HEK293T cells. An extensive structure‐activity relationship campaign resulted in highly potent and efficient full RXFP2 agonists. The selectivity and specificity of the compounds for human and mouse RXFP2 was shown using orthogonal cAMP assays, counter‐screening against the related relaxin receptor RXFP1, and a GPCRome screen using the PRESTO‐Tango assay. The drug candidates were further tested in primary human osteoblasts to demonstrate that they promote mineralization by measuring hydroxyapatite deposition in the cell matrix. We showed that the compounds had low cytotoxicity in various cell types. Using a series of RXFP2/RXFP1 chimeric receptors, we established that the compounds are allosteric agonists of the RXFP2 receptor and identified the GPCR transmembrane domains as the specific region for compound interaction. The RXFP2 agonist with the highest activity in vitro was selected for pharmacokinetics (PK) profiling in mice, showing optimal oral bioavailability and bone exposure. An efficacy study using 2‐month‐old WT female mice treated orally with 10 mg/kg of compound 3 times a week for 8 weeks showed a significant increase of the vertebral trabecular number and thickness by micro‐CT analysis compared to vehicle treated controls. We expect that the further characterization of these compounds may lead to the development of a new class of cost‐effective drugs for the treatment of osteoporosis and other diseases associated with bone loss.
Muscle Sympathetic Nerve Activity and Cerebrovascular Responses to Mental Stress
Authors: Eisenmann Nicole A. , Pearson Andrew G. , Howery Anna J. , Corkery Adam T. , Miller Kathleen B. , Barnes Jill N.
Published: May 2022

Mental stress has been shown to elicit increases in arterial blood pressure, heart rate and cerebral blood flow, whereas the degree of sympathoexcitation induced by mental stress has been shown to be inconsistent. Previous studies have examined the relationship between limb vascular responsiveness and muscle sympathetic nerve activity (MSNA) during mental stress; however, this has not been studied in relation to the cerebral vasculature. The purpose of this study was to examine concurrent neural and cerebrovascular responses to mental stress. Thirteen healthy adults (men/women: 7/6; age = 28 ± 5 years; BMI = 25 ± 2 kg/m2) completed the Stroop Color Word Test. Beat‐to‐beat mean arterial pressure (MAP) using finger photoplethysmograpy, heart rate (HR) using a 3‐lead ECG, middle cerebral artery velocity (MCAv) using transcranial doppler ultrasound, and MSNA using microneurography were continuously recorded at baseline and in response to the Stroop Color Word Test. The response to the mental stress was recorded as the change from baseline. Linear regression was used to assess the influence of MSNA on cerebrovascular and hemodynamic variables. As expected, there were increases in HR (baseline = 58 ± 8 bpm vs. Stroop = 66 ± 10 bpm; p = 0.045) and MCAv (baseline = 58 ± 8 cm/s vs. Stroop = 69 ± 13 cm/s; p = 0.016) from baseline during the Stroop test. However, there was no change in MAP (baseline = 96 ± 16 mmHg vs. Stroop = 99 ± 17 mmHg; p > 0.05) or MSNA (baseline = 16 ± 6 bursts/min vs. Stroop = 17 ± 7 bursts/min; p > 0.05) from baseline during the Stroop test. Although the change in MAP was positively associated with the change in MCAv (r = 0.46, p = 0.025), there were no associations between the change in MSNA and the increase in MCAv, MAP, or HR in response to the Stroop test. In conclusion, our results demonstrate a disassociation between the MSNA, MAP and MCAv responses to mental stress. It is also possible that the temporal response to mental stress is different and future studies could examine the time course of the sympathetic and cerebrovascular responses.
Interaction of the Class A Scavenger Receptor with Multiple Myeloma Cells
Authors: Deshpande Shayu , Post Ginell R. , Post Steven R.
Published: May 2022

Tumor‐associated macrophages (TAMs) play a central role in regulating the immune response within the cancer microenvironment. TAMs express Class A Scavenger Receptors (SR‐A), which are pattern recognition receptors that bind a wide array of ligands. SR‐A expression by TAMs is often associated with an immune‐suppressive macrophage phenotype. Multiple myeloma (MM) is a malignant disease of the bone marrow characterized by the proliferation of differentiated plasma cells. The disease progresses from asymptomatic monoclonal gammopathy of undetermined significance (MGUS) to smoldering myeloma (SMM) and finally to symptomatic MM. In many solid tumors, the number of SR‐A‐expressing TAMs correlates with tumor aggressiveness and poor prognosis. Although SR‐A‐expressing macrophages are abundant in the bone marrow, the importance of SR‐A in the progression of MM is unknown. All patient samples for the study were collected under IRB approved protocol 260665. To determine if SR‐A may be important in MM, we utilized a multiplexed immunofluorescence (IF) approach to localize SR‐A‐expressing macrophages within the MM bone marrow microenvironment. Macrophages were detected using CD163 and SR‐A antibodies and plasma cells were identified using CD138 antibody. Multiplexed‐IF staining showed that SR‐A‐positive macrophages were localized near MM cells. This was especially evident in tumor‐rich regions where SR‐A‐expressing TAMs displayed a distinct branched morphology. SR‐A binding to ligand induces macrophage attachment and spreading. Thus, we hypothesized that the localization of SR‐A‐expressing TAMs near MM cells results from SR‐A binding to a ligand expressed on these cells. We tested this possibility using a flow cytometry‐based assay to measure the binding of a soluble SR‐A (sSR‐A) protein with MM cell lines and plasma cells obtained from bone marrow aspirates of MM patients. sSR‐A was able to bind to all six different MM cell lines that were examined. In bone marrow aspirates (n=15), sSR‐A bound to neoplastic plasma cells (CD138+CD45‐) but not to non‐plasma cells (CD45+). sSR‐A binding to neoplastic plasma cells was confirmed using enriched plasma cells isolated from bone marrow aspirates using anti‐CD138 magnetic beads. sSR‐A binding to plasma cells was abolished by the SR‐A ligand, fucoidan, demonstrating specificity. Together, our results demonstrate that SR‐A is expressed on TAMs within the MM microenvironment and binds to MM cells. SR‐A binding to MM cells may be a mechanism for regulating TAM localization and function in the tumor microenvironment.
Mass Spectrometry based Proteomics to Investigate and Characterize the Human Jumping Translocation Breakpoint (hJTB) Protein using Cancer Cell Lines
Authors: Jayathirtha Madhuri , Whitham Danielle , Channaveerappa Devika , Darie Costel
Published: May 2022

Human JTB (hJTB) is a gene located on the human chromosome 1 at q21 which is involved in the unbalanced translocation in various types of cancer. JTB protein is ubiquitously present in normal cells and is found to be overexpressed in various types of cancer including prostate and breast cancer. Hence this protein could be a biomarker for tumor malignancies and a potential target for their treatment. However, the biological function and the pathway through which this protein causes increased cell growth and proliferation is not entirely clear. Investigation and comparison of the proteomes of cells with upregulated and downregulated JTB can be a good approach to understand the function of the protein and also its contribution to tumorigenesis. In this study, MCF7 breast cancer cell lines were transfected with the sense orientation of the JTB cDNA in HA, His and FLAG tagged CMV expression vector as well as with shRNA plasmids. Proteins extracted from transient and stable transfected cells were separated using SDS‐PAGE. The expression of JTB was confirmed by western blotting technique. In gel digested peptides were analyzed by a Nano Acquity UPLC coupled with QTOF Xevo G2 Mass Spectrometer. Data processing was done using Mascot 2.4 server and Scaffold 4.1 software. We found several proteins that were dysregulated. Furthermore, we will do Immunoprecipitation to look at JTB protein interacting partners. These studies could help us elucidate the mechanism through which JTB induces cell proliferation and test the JTB protein as a potential drug target for malignancies with overexpression of the protein.
Non‐canonical Recruitment of PKA Catalytic Subunits to RIα‐driven Biomolecular Condensates
Authors: Hardy Julia C. , Zhang Jason Z. , Pool Emily H. , Choi Jaclyn L. , Taylor Susan S. , Zhuo Xin , Zhang Jin
Published: May 2022

A regulatory subunit of cAMP‐dependent protein kinase (PKA), RIα, was recently shown to undergo liquid‐liquid phase separation (LLPS), which is critical for cAMP compartmentation. These RIα biomolecular condensates form upon cAMP production, contain high levels of cAMP and active PKA catalytic (PKAcat) subunits, buffer increases in cAMP concentration, and suppress tumorigenic phenotypes. Through mutagenesis studies of key residues within different domains of RIα, we found that cAMP binding to both CNB‐A and B domains is required to stimulate RIα LLPS and that the dimerization via both the dimerization/docking (D/D) domain and the N3A motif of the CNB‐A is required for RIα LLPS. Our data suggests that the inhibitory sequence (IS), which binds and inhibits the catalytic subunit, is also involved in the regulation of RIα LLPS as well as recruitment of the catalytic subunit to the RIα puncta, likely through a non‐canonical interaction. RIα mutants carrying point mutations (G98S and A99S) in the IS showed enhanced RIα LLPS but weakened R:C complex and decreased capability to recruit PKAcat to RIα puncta. Intriguingly, these mutants generated puncta that did not disassemble when cAMP levels decreased, whereas puncta from wildtype RIα showed complete disassembly, suggesting that the IS motif impacts the dynamics of RIα LLPS. Our study led to a further understanding of the stimulation, formation, PKAcat recruitment, and disassembly of PKA biomolecular condensates, which may lead to novel therapeutic approaches for diseases driven by a loss of PKA RIα LLPS, such as fibrolamellar carcinoma.
Modular Cloning of MUC1 Recombinant Antibodies by Assembly of Synthetic Domain Genes
Authors: Villegas Carina A. , Bedolla Yazmine , Brooks Cory L.
Published: May 2022

Antibody immunotherapy has emerged as a constructive way to fight cancer. New immunotherapies require the identification of a tumor‐specific antigen. The protein mucin 1 (MUC1) has long been recognized as an important specific antigen and a potential immunotherapy target. In healthy human tissues, MUC1 is responsible for protecting the epithelial lining. However, in cancer cells, MUC1 displays truncated glycosylation, revealing tumor‐specific epitopes. Recombinant antibodies are preferred for therapeutic development, and in some cases, it is desirable to produce different antibody formats, including intact IgG or Fab fragments. Using the MUC1 specific antibody 4H5 as a model, we have developed a modular cloning approach that can rapidly produce different antibody modalities to facilitate the rapid production of both IgG and Fab antibody formats. The 4H5 variable domains were PCR amplified to contain 20 base‐pair (bp) overlaps with the pcDNA3.1 vector on the 5’ end, and 20 bp overlaps to either the human CH1 or Ck domains on the 3’end of the genes. To produce the Fab, a His‐tag and a 20 base‐pair (bp) overlap to the vector was added to the 3’ end of a human CH1 domain gene sequence. For IgG production, human CH1‐CH2‐CH3 domains were PCR amplified, and the same vector overlap sequence was added to the 3’ end of the sequence. Finally, a 20 bp overlap to the vector was added to the 3’ end of a human Ck domain. The PCR products were purified, and the constructs were assembled using the NEBuilder®HiFi DNA Assembly Cloning Kit. DNA sequencing confirmed the successful assembly of the coding sequence, and the 4H5 Fab fragments and IgG were produced by transient transfection in CHO cells. The antibodies (Fab and IgG) were purified by affinity chromatography and binding to recombinant MUC1 antigens confirmed by ELISA. The cloning method presented here simultaneously produces multiple antibody formats. This approach will permit rapid assembly and screening of different antibody formats and isotypes for therapeutic antibody discovery and analysis.
Identification of a Pseudosubstrate Stimulatory Motif Uncovers Novel Roles of Cdc14 Phosphatase in Fungal Cell Wall Integrity
Authors: Milholland Kedric L. , Naughton Noelle H. , DeMarco Andrew G. , Baker Kortany M. , Briggs Scott D. , Correa‐Bordes Jaime , Hall Mark C.
Published: May 2022

Fungal pathogens are a growing threat to human health and global food security as they are becoming increasingly resistant to the few existing antifungal treatments. Cdc14 is a protein phosphatase primarily known for its essential role in controlling mitotic exit in the model yeast Saccharomyces cerevisiae. However, in other fungal species Cdc14 is non‐essential and the reasons for its strict conservation across Dikarya remain unclear. Recently, Cdc14 was shown to be required for host infection by several plant pathogenic fungi. Here, we show that Cdc14 is also required for virulence in the human pathogen, Candida albicans.These observations, coupled with our structural and biochemical knowledge of Cdc14 phosphatases, suggest it could be a useful target for antifungal development. In our search for differences between fungal and animal Cdc14 enzymes that could be exploited for antifungal design, we discovered an invariant motif in the otherwise disordered C‐terminal tail of fungal Cdc14 orthologs. The motif resembles the recognition sequence of optimal Cdc14 substrates. We provide evidence from molecular modeling and enzyme kinetic studies that this motif is a “pseudosubstrate” that binds the Cdc14 active site and functions to accelerate the rate‐limiting catalytic step, providing a plausible mechanism for dynamic control of intracellular Cdc14 activity. The turnover rate for Cdc14 lacking a functional pseudosubstrate motif is reduced by an order of magnitude compared to the wild‐type enzyme. Both S. cerevisiaeand C. albicanscells expressing Cdc14 variants with point mutations in this motif exhibit a specific and pronounced sensitivity to cell wall stresses, including treatment with echinocandin antifungal drugs that inhibit cell wall synthesis. Moreover, signaling through the cell wall integrity pathway is chronically elevated in these cells, indicative of a cell wall structural defect. Preliminary evidence in synchronized S. cerevisiaecultures with reduced Cdc14 activity suggests that cell wall integrity is compromised around the time of cytokinesis and septation, consistent with its known functions in these processes. We are currently testing if the pseudosubstrate motif is required for virulence of C. albicans.Our findings reveal 1) a novel contribution to catalysis of a structural element outside the conserved catalytic core of Cdc14 enzymes and 2) novel roles of Cdc14 in promoting cell wall integrity and pathogenesis. More detailed characterization of the mechanisms by which Cdc14 contributes to cell wall integrity and host infection in human pathogenic fungi should help assess its future value for antifungal development.
Acute Cannabidiol Administration Does Not Increase the Thermic Effect of Feeding in Men with Overweight and Obesity
Authors: Ewell Taylor R. , Abbotts Kieran S. S. , Butterklee Hannah M. , Bomar Matthew C. , Bell Christopher
Published: May 2022

The thermic effect of feeding (the increase in energy expenditure above resting metabolic rate following ingestion of food) is lower in adult humans with overweight and obesity compared to their lean counterparts. In animal models of obesity, the endocannabinoid system is upregulated and is thought to contribute to an unfavorable metabolic phenotype. The influence of cannabidiol, a non‐psychoactive component of Cannabis sativa L., on the thermogenic properties of the endocannabinoid system in humans is unclear. Accordingly, the purpose of the current study was to explore the hypothesis that acute administration of cannabidiol would increase the thermic effect of feeding. Metabolic rate, via indirect calorimetry, was determined at rest and over 4‐hours following ingestion of a liquid, mixed macronutrient meal (22% fat, 62% carbohydrate, 16% protein) equivalent to 40% of resting metabolism, in 11 adult men with body mass index greater than 25 kg/m2 (age: 26 ± 9 years; body mass: 93.7 ± 13.0 kg; body mass index: 29.8 ± 3.0 kg/m²; body fat: 29.4% ± 4.6% (mean ± SD)) on two separate occasions. Immediately following meal ingestion, participants were administered either 30 mg of cannabidiol or placebo, in a randomized, single‐blind crossover fashion. Resting metabolic rate did not differ between trials (1745 ± 311 vs. 1744 ± 288 kcal/day; P=0.98). Compared with rest, energy expenditure was increased after ingestion of the liquid meal (P<0.01) but was unaffected by cannabidiol (Time x Placebo vs. Cannabidiol: P = 0.21). The area under the thermic effect of feeding response curve was also unaffected by cannabidiol (57 ± 24 vs. 58 ± 16 kcal/min x min; P=0.92). Respiratory exchange ratio, an indicator of substrate utilization, was not different between trials at rest (0.81 ± 0.04 vs. 0.79 ± 0.04; P=0.09). Ingestion of the meal increased respiratory exchange ratio (P<0.01), indicative of increased reliance on carbohydrate oxidation, but again this response was unaffected by cannabidiol (Time x Placebo vs. Cannabidiol: P=0.08). These preliminary data suggest that a single 30 mg dose of cannabidiol does not influence the thermic effect of feeding, a significant contributor to total daily energy expenditure, in men with overweight or obesity. Cannabidiol has been purported to have multiple beneficial effects on physiological function, it appears that these effects may be independent of any influence on the thermic effect of feeding.
The Relation of Subjective and Objective Assessment of Sleep quality with Post Concussive Symptoms in Veterans
Authors: Pollin Kamila U. , Samuel Immanuel , Breneman Charity B. , Brewster Ryan , Reinhard Matthew , Costanzo Michelle E.
Published: May 2022

Sleep complaints are a significant and persistent health problem reported by military Veterans. Veterans with traumatic brain injury (TBI) experience a higher incidence of sleep disturbances, with mild TBI (mTBI) resulting in chronic sleep disruption post injury. However, the relation between sleep and mTBI history is still poorly understood.

Purpose

The purpose of this study is to assess sleep using subjective (Pittsburgh Quality Sleep index; PSQI) and objective (actigraphy) measurements in Veterans with a history of mTBI using remote assessments.

Methods

Seven Veterans (7 Males; age: 54±7yrs, body mass index (BMI): 31±7 kg/m2 mean±SD) completed a telemedicine VA clinical evaluation and enrolled in the study. Participants completed web‐based versions of the PSQI to assess subjective sleep quality (range 0‐21) and the Neurobehavioral Symptom Inventory (NSI) to assess post concussive syndrome (PCS) symptoms (range 0‐88). Participants wore a wrist‐worn actigraph (wGT3X‐BT) over a 7‐day baseline period to measure total sleep time and sleep variability (standard deviation) from home. Data were analyzed using Pearson’s correlations.

Results

NSI scores are significantly correlated with global PSQI (R2=0.81, P=0.02) where higher NSI scores are associated with a greater PSQI total score. There is no relation between NSI score and sleep time (R2=0.22, P=0.34). There are trending relations between sleep time variability (R2=0.50, P=0.07) and sleep disturbances (R2=0.5, P=0.10; ex. Feeling too hot or too cold, having bad dreams) and NSI, such that those with higher NSI scores have greater sleep variability and sleep disturbances.

Conclusion

Home‐based assessments offer a promising means of monitoring sleep in Veterans. Our preliminary data suggests that subjective measurement of sleep relate to PCS symptoms, however trending results suggest that specific features of sleep quality may be affected by mTBI history. Remote evaluations are critical to understand sleep quality of Veterans to aid improvements in PCS symptoms following mTBI.
The Role of Carotid Body Chemoreceptor Afferents in Mediating Abnormal Ventilation in Acute Lung Injury
Authors: Kamra Kajal , Karpuk Nikolay , Adams Ryan , Schultz Harold , Wang Hanjun
Published: May 2022

Background‐

Acute lung injury (ALI) induces inflammation that disrupts the normal alveolar‐capillary endothelial barrier which impairs gas exchange to induce hypoxemia that reflexively increases respiration. Increased respiratory rate (RR) usually develops within a few hours to a few days after ALI. This disorder of acute respiratory failure affects approximately 200,000 new cases each year in the US alone and has high morbidity and mortality. The neural mechanisms underlying the respiratory dysfunction after ALI are not fully understood. The purpose of this study involved investigating the role of carotid body chemoreceptor afferents in mediating abnormal ventilation in ALI.

Methods and Results‐

ALI was induced in male SD rats (200‐250g) using a single intra‐tracheal injection of bleomycin (Bleo) (day 0) and respiratory variables‐ RR, TV (Tidal Volume), and MV (Minute Ventilation) in response to 10% hypoxia (10% O2, 0% CO2) and 5% hypercapnia (21% O2, 5% CO2) were measured weekly from W1‐W4 using whole‐ body plethysmography (WBP). MV at 21% O2 increased from baseline (W0) to W1 and W2 in bleo treated rats (p≤0.01 compared to saline treated sham rats). Changes in MV were due to a significant increase in RR while TV showed no changes. Chemoreflex was assessed by measuring the absolute difference between 21% O2 and hypoxia (10% O2, 0% CO2) & hypercapnia (21% O2, 5% CO2). Chemoreflex activation is a fundamental protective reflex that maintains oxygen homeostasis. Our data indicated a blunted chemoreflex at W1 and a sensitized chemoreflex in W3‐W4 during hypoxia and hypercapnia. We hypothesized that the blunted chemoreflex in W1 could be due to maximal pre‐activation of chemoreceptors, called the ‘ceiling effect’. To test this possibility, 90% hyperoxia (90% O2, 0% CO2) was given to bleo treated rats to inhibit the chemoreflex and our results showed no changes in RR, suggesting absence of the tonic activity at W1 post‐ALI.

Conclusions‐

These data suggest that chemoreflex activation in response to hypoxia and hypercapnia is blunted at acute stage of lung injury but became sensitized at the recovery stage.
Craniofacial morphology of a new mouse model of Down syndrome
Authors: Singh Nandini , Richtsmeier Joan T. , Reeves Roger
Published: May 2022

Down syndrome (DS) is a complex condition caused by triplication of human chromosome 21 (Hsa21). Several mouse models have been developed to study the genotypic and phenotypic outcomes of DS. The most extensively studied and used in DS research is Ts65Dn. More recently, the TcMAC21 ‐ a transchromosomic mouse strain with comparable gene dosage to Hsa21 ‐ was developed, and represents a more complete genetic mouse model of DS than Ts65Dn. While Ts65Dn has been the focus of several studies, relatively little is known about the craniofacial phenotype of TcMAC21 mice relative to the other DS mouse models. Here we conducted a quantitative study of the cranial morphology of TcMAC21 and Ts65Dn mice and their respective unaffected littermates. Comparative data comprised of three‐dimensional cranial measurements taken from micro‐computed tomography scans of the mouse models used in the study. Our results show that TcMAC21 exhibit similar patterns of craniofacial change to Ts65Dn, but are less severe in their DS‐related cranial manifestations than Ts65Dn. TcMAC21 also do not present with the broadening and general ‘globularity’ of the cranium, particularly the face, as seen in Ts65Dn. Since Ts65Dn and TcMAC21 carry similarly large but not the same numbers of genes at dosage imbalance, our findings reveal the complexity of potential gene interaction in the production of craniofacial phenotypes.