bluuebunny/crossref_metadata_2025 huggingface dataset:
Dedicator of cytokinesis 2 (DOCK2) mediates chronic high‐fat and high‐fructose diet induced lung injury
Authors: Guo Xia , Adeyanju Oluwaseun , Sunil Christudas , Huang Steven K. , Chen Shi‐You , Tucker Torry A. , Idell Steven , Qian Guoqing
Published: May 2022

Obesity is involved in the pathogenesis of multiple lung diseases. However, little is known about the effects of chronic high‐fat and high‐fructose (HFHF) diet‐induced obesity on lung inflammatory/injury. We have reported that dedicator of cytokinesis 2 (DOCK2) is important for high‐fat diet (HFD)‐induced obesity and adipose tissue inflammation. DOCK2 deficient mice were protected from HFD induced body weight gain, insulin resistance, and increased proinflammatory cytokines in the adipose tissue and peripheral circulation. However, it remains elusive whether DOCK2 plays a role in lung injury associated with chronic HFHF diet‐induced obesity. In this study, we showed that chronic HFHF diet (20 weeks) induced lung inflammatory infiltration and collagen expression in the wild‐type (WT) C57BL/6 mice. Macrophage marker CD68 and monocyte chemoattractant protein‐1 (MCP‐1) expression were notably increased in the lungs of WT mice fed a HFHF diet. Further, HFHF diet increased lung DOCK2 expression that co‐localized with fibroblast‐specific protein 1, suggesting a potential role of DOCK2 in regulating proinflammatory phenotype of lung fibroblasts. Conversely, DOCK2 deficiency attenuated lung inflammation and fibrosis induced by chronic HFHF diet. In primary human lung fibroblast, TNF‐α and IL‐1β induced DOCK2 expression concurrent with MCP‐1, IL‐6, and matrix metalloproteinase‐2. DOCK2 knockdown also suppressed TNF‐α induced increase of these inflammatory mediators. Taken together, these findings suggest a previously unrecognized role of DOCK2 in mediating lung inflammation and fibrosis in chronic HFHF diet caused obesity.
Role of medullary cardiorespiratory neurons on the maintenance of altered sleep function in heart failure
Authors: Toledo Camilo , Schwarz Karla G. , Diaz‐Jara Esteban , Pereyra Katherine , Las Heras Alexandra , Diaz Hugo , Del Rio Rodrigo
Published: May 2022

Sleep‐disordered breathing (SDB) and sleep fragmentation (SF) (i.e. repetitive short interruptions of sleep) are frequently reported in heart failure (HF) patients. Interestingly, SDB and SF has been both closely associated with the generation of breathing disorders. Importantly, our previous work showed that partial ablation of pre‐sympathetic catecholaminergic neurons (C1) located at the rostral ventrolateral medulla (RVLM) results in marked improvements in breathing disorders in rats with HF. However, the contribution of RVLM‐C1 neurons on sleep‐related disorders in the setting of cardiac failure has not been previously studied. Therefore, we aimed to determine the contribution of RVLM‐C1 neurons on sleep architecture in HF. Adult male Sprague‐Dawley rats (250 g) underwent volume overload to induce HF. After four weeks, anti‐dopamine β‐hydroxylase‐saporin (DβH–SAP: 2.5ng/150nl; or saline) was used to selectively destroy RVLM‐C1 neurons (‐12.36 mm AP from bregma; ± 2.2 mm ML; 8.5 mm DV). Breathing function was determined by unrestrained whole‐body plethysmograph and sleep function/sleep stages were assessed in electroencephalogram and electromyogram recordings obtained for chronic implanted leads. Compared to Sham rats, HF animals displayed no differences in the total time spent in rapid eye movement (REM) and non‐rapid eye movement (nREM) sleep stages. However, duration of each nREM epoch was significantly (p<0.05) lower in HF rats (3.4 ± 0.3 vs. 2.2 ± 0.2 min, Sham vs. HF, respectively). Accordingly, HF rats showed an increased number of nREM epochs compared to Sham animals (12.0 ± 2 vs. 17 ± 2 events/h, Sham vs. HF, respectively). Interestingly, HF rats displayed loss of ventilatory stability compared to Sham rats only at nREM sleep (Irregularity score, nREM: 12.1 ± 2.0 vs. 15.9 ± 1.8%; REM: 22.9 ± 2.0 vs 24.1 ± 3.2%; Sham vs. HF, respectively). Furthermore, the incidence of sigh‐triggered arousal events during nREM sleep was increased in HF rats. Conversely, sleep probability was decreased by ~35% in HF animals compared to Sham animals. Remarkably, ablation of RVLM‐C1 neurons with DβH–SAP in HF improved nREM breathing regularity and restores normal nREM sleep duration (3.4 ± 0.3 vs. 3.1 ± 0.3 min, Sham vs. HF+DβH–SAP, respectively). In addition, DβH–SAP treatment in HF decreased sigh‐triggered arousals by ~45% being the outcome a reduction in SF in HF rats that underwent C1 ablation. Our data shows that irregular breathing is closely linked to altered sleep in HF rats and support a role for RVLM‐C1 neurons on sleep regulation in the setting of HF.
RNA‐Seq and Recurrence Risk Testing for Breast Cancer: Implications for Patient Education
Authors: Soneral Paula
Published: May 2022

Rapid advances in RNA­seq technology and bioinformatic analysis of gene expression has normalized the use of precision diagnostics and recurrence risk testing for breast cancer patients. While these diagnostic approaches have numerous benefits, patients often lack the scientific literacy to evaluate data models ‐ and their uncertainties ‐ for enhancing their health decision‐making. To increase access to scientific knowledge for stakeholders, we designed an educational module that evaluates the validity of RNA‐seq gene expression as a tool for subtyping and determining the prognostic signature of a human breast tumor. Using a crowdsourced approach in the context of an undergraduate course, we recreated the workflow of prominent precision oncology companies ‐ we prepared a whole transcriptome library from a single breast cancer patient, and used multiplex sequencing on twelve uniquely barcoded Illumina preparations. Datasets obtained from the sequencing were subjected to quality control analysis using the Green Line of DNA Subway (FAST QC, FAST X, Kallisto, Sleuth algorithms). Log­ transformed gene expression ratios were used to determine the molecular subtype of the tumor. To calculate the recurrence risk, the Oncotype DX model was used, including 16 cancer genes and 5 reference genes. Results from the proof‐of‐concept experiment indicated significant overexpression of ER and PR genes (p<0.01) and concomitant underexpression of HER2 and Ki67 genes (p<0.01), consistent with a Luminal A tumor subtype. The Oncotype DX score was >25, suggesting high risk for 10 year distant recurrence (95% CI). Further exploration of the model indicated that normalization to aberrantly expressed reference genes can greatly skew the recurrence risk calculation, highlighting limits to the predictive power of this tool. Taken together, our findings illustrate the validity of RNA‐seq‐based tumor subtyping through an educational module, and underscores the importance of equitable access to genomics education as a form of patient self‐empowerment and decision‐making. We discuss the future implications of this work for education and policy.
Evidence of Mitochondrial Impairment in a Mouse Model of Down Syndrome
Authors: Anyaoku Cynthia , DeRuisseau Lara R. , Brandauer Josef
Published: May 2022

Introduction

Down Syndrome (DS) is typically the result of triplication of the 21st chromosome. DS causes widespread developmental and physical changes to the body. One commonly observed physical change of DS is muscle weakness, which can contribute to difficulty in executing activities of daily living.

Nicotinamide adenine dinucleotide (NAD) is an important determinant of skeletal muscle function and mitochondrial activity. Currently, it is unknown whether muscle weakness in those with DS is at least partially related to an imbalance of NAD or a lack of mitochondrial NAD usage. Nicotinamide phosphoribosyl transferase (Nampt) catalyzes the rate‐limiting step in the mammalian NAD recycling pathway, while SIRT3 is an NAD‐dependent protein deacetylase which regulates mitochondrial content and activity. We hypothesized the expression of these proteins would be decreased in Ts65Dn animals, an established mouse model of DS, relative to wild‐type (WT) animals.

Methods

Diaphragm and left ventricle samples from 12‐ or 18‐month‐old male Ts65Dn mice and WT animals were pulverized and homogenized in ice‐cold lysis buffer. Total lysate protein concentration was determined by Bradford assay. Samples were resolved in SDS gels, transferred to nitrocellulose membranes, and blotted for Nampt and SIRT3. Lysates were also separately analyzed for citrate synthase activity.

Results

Both Nampt (p = 0.02) and SIRT3 (p = 0.002) expression levels were significantly higher in WT than Ts65Dn ventricles at 12 months. In addition, SIRT3 levels were higher in diaphragm of 12‐month‐old WT animals (p = 0.02). Citrate synthase levels were similar in all tissues.

Conclusion

Our findings of reduced Nampt protein suggest that reduced NAD recycling takes place in DS muscle tissue despite similar observed mitochondrial (citrate synthase) activity. These results suggest a role of diminished peripheral mitochondrial function and ATP synthesis in Down Syndrome muscle.
Association of Mouse Vulnerability to Alzheimer’s Disease Pathology with Mutations of Gnb5: A Modulator of G‐protein Coupled Receptor Signaling
Authors: Fikse Emma N. , Zhang Jianhua , Pandey Mritunjay , Smerin Glenda , Awe Adam , Lue Nicole , Kittock Claire , Chen Weiping , Yang Yanqin , Adikaram Poorni , Jacob Nirmal , Greenfest‐Allen Emily , Thomas Rachel , Bomeny Laura , Wang Xiaowen , Li Yulong , Simonds William F.
Published: May 2022

The prevalence and devastating effects of Alzheimer’s Disease (AD) and lack of effective treatments underscore the need to determine genes and molecular pathways with pathophysiologic relevance to develop targeted therapies. Prior unpublished bioinformatic analysis by our laboratory of genomic and brain transcript expression data from subjects with and without AD has identified hundreds of potential AD causative genes, one being GNB5, which encodes the protein Gβ5. Gβ5 is a divergent member of the G‐protein β subunit family and is primarily expressed in neuronal tissues. It is known to stabilize members of the R7‐Regulator of G‐protein Signaling (RGS) subfamily that exert GTPase Accelerating Protein (GAP) activities on Gαi/o proteins during G‐protein coupled receptor (GPCR) signaling. Homozygous lack of Gβ5 causes significant neuronal and behavioral impairments in both mice and humans; however, the impact of GNB5 heterozygosity requires further examination. For this project, we demonstrated through fear conditioning behavioral tasks involving hippocampus and amygdala function that learning and memory deficits existed in mice heterozygous for Gnb5. In addition, the potential relevance of GNB5 in AD pathogenesis was confirmed in an APP/PSEN1 double transgenic AD mouse model in which missing even one copy of the Gnb5 gene enhanced amyloid plaque and neurofibrillary tangle formations in multiple brain regions of mice including, the entorhinal cortex, hippocampus, and frontal cortex. These results were confirmed by traditional immunohistochemistry methods as well as immunostaining of transparent mouse brains cleared using the clear, unobstructed brain/body imaging cocktails and computational analysis (CUBIC) method. We further demonstrated that the exacerbated AD pathology may be caused, at least in part, by the increased vulnerability of heterozygous GNB5 mouse neurons to the toxicity of Aβ42 peptide oligomers which have been linked to beta‐amyloid plaque formation. Our data suggest the involvement of GNB5 regulated GPCR signaling in the progression of AD pathology and illustrates a potential new therapeutic target for attenuation of cellular pathology associated with AD.
Sex‐ and region‐specific consequences of chronic nicotine in the murine hippocampus during fear extinction
Authors: Keady Jack V. , Turner Jill
Published: May 2022

In the United States 90% of people experience at least one event in their lifetime that meets the DMS‐5 criteria for a traumatic event, but only 8.3% of Americans develop Post‐Traumatic Stress Disorder (PTSD). While severity and frequency of trauma impact the development of PTSD, there is an increasing emphasis on biological factors explaining why only a subset present with PTSD. We know sex is a major risk factor for PTSD, with women being twice as likely to develop PTSD than men. Additionally, people suffering from PTSD are twice as likely to smoke daily than healthy controls. However, while nicotine dependence is positively correlated with PTSD symptoms in men, this is not true in women, suggesting sex divergence in the neurobiological response to trauma and the resulting increased susceptibility to nicotine use disorder. The hippocampus has a well‐defined regiospecific role in both contextual memory and affective responding, the dorsal hippocampus (DHIPP) and ventral hippocampus (VHIPP) respectively. This places the hippocampus as a nexus point for both memory and emotion, suggesting these subregions are potential targets for mechanistic evaluation of comorbid PTSD and nicotine dependence. In these studies, we chronically treated male and female 7 to 8‐week‐old mice with either saline or intermittent nicotine (18 mg/kg/day) via osmotic minipumps (Alzet 1002 model). On the twelfth day of treatment, the subjects were trained in a fear conditioning paradigm with two CS‐US pairings (CS 89 dB tone and US 0.35 mA shock), then underwent 6 contextual fear extinction sessions. Following behavioral testing, the DHIPP and VHIPP were collected and assessed for genomic, transcriptomic, and proteomic changes. Preliminary results from these experiments suggest that the behavioral sex differences observed likely relate to differences in transcriptional control mechanisms. Further examination of treatment by sex effects in extinction behavior and the genomic and functional alterations underpinning these effects are ongoing.
Physiologically Based Pharmacokinetic (PBPK) Modelling to Predict Breast Milk Exposure of Valproic Acid
Authors: Nauwelaerts Nina , Bouillon Thomas , Deferm Neel , Lammens Bart , Annaert Pieter
Published: May 2022

Breastfeeding has many positive effects on the health and well‐being of mother and baby. During lactation, a pharmacological therapy may be required. Currently there is a knowledge gap regarding the safety of medicines during lactation, which may lead to unnecessary cessation of breastfeeding or incur unknown risks to the child when continuing breastfeeding while on medication. The Innovative Medicines Initiative (IMI) ConcePTION aims to address this and other issues related to medication use during pregnancy and lactation.

The objective of this study performed in the IMI ConcePTION framework was to explore the utility of Physiologically‐Based Pharmacokinetic (PBPK) modelling in the generation of information regarding the safety of medicines (e.g. valproic acid, VPA) during breastfeeding.

To achieve this goal, PK‐Sim® (v 9.1) (Open Systems Pharmacology) was used to first develop a reference PBPK model for VPA in adults using 7 training datasets. The median fold errors of the Area Under the Curve (AUC) and maximum plasma concentration (Cmax) for the 17 validation datasets were 0.89 (range: 0.74‐0.134) and 0.90 (range: 0.60‐1.36), indicating a good predictive performance. In a next step, this PBPK model was extended with a breast compartment (including a subcompartment representing the breast milk) according to the work from Dallmann et al. (2018). Transfer of medicines between the milk and the intracellular compartment was parametrized with a clearance for secretion to milk (Clsec), and a clearance for reuptake (Clre). These clearance values were determined based on physicochemical properties using the empirical model from Koshimichi et al. (2011). Clsec and Clre were 235.05 and 1452.78 mL/h. The PBPK model for lactating women was used to predict exposure of VPA in plasma and human breast milk. The simulated milk‐to‐plasma (M/P) ratio was 0.016, similar to reported M/P ratio’s (range: 0.007‐0.25). Finally, the daily infant dose was calculated based on the simulated concentration in the milk and used as input for an infant PBPK model. The relative infant dose was 1.4 %. Average steady‐state infant plasma concentration was estimated to be 0.83 mg/L (relative infant exposure: 1.2 %), compared to a wide range of observations starting from undetectable concentrations up to 41 mg/L.

PBPK modelling was successfully used to predict the transfer of VPA into the human breast milk, and to estimate subsequent infant exposure via breastfeeding. Based on ongoing application to 11 other medicines, a generic workflow for PBPK‐based breast milk transfer of medicines has been developed. Current efforts to determine the in vitro permeability of VPA across the blood‐milk barrier may obviate the current need for (sparse) milk concentrations. This study illustrated the potential of PBPK modelling as a tool to fill the knowledge gap regarding the exposure to medicines in lactating women and their infants.
Phenobarbital induces SLC13A5 expression through activation of PXR but not CAR in human primary hepatocytes
Authors: Li Zhihui , Li Linhao , Heyward Scott , Men Shuaiqian , Xu Meishu , Sueyoshi Tatsuya , Wang Hongbing
Published: May 2022

Phenobarbital (PB), a widely used anti‐epileptic drug, is known to upregulate the expression of numerous drug‐metabolizing enzymes and transporters in the liver primarily via activation of the constitutive androstane receptor (CAR, NR1I3). The solute carrier family 13 member 5 (SLC13A5), a sodium‐coupled citrate transporter, plays an important role in intracellular citrate homeostasis that is associated with a number of metabolic syndromes and neurological disorders. Here, we show that PB markedly elevates the expression of SLC13A5 through a pregnane X receptor (PXR)‐dependent but CAR‐independent signaling pathway. In human primary hepatocytes, the mRNA and protein expression of SLC13A5 was robustly induced by PB treatment, while genetic knockdown or pharmacological inhibition of PXR significantly attenuated this induction. Utilizing genetically modified HepaRG cells, we found that PB induces SLC13A5 expression in both wild type (WT) and CAR‐knockout HepaRG cells, whereas such induction was fully abolished in the PXR‐knockout HepaRG cells. Mechanistically, we identified and functionally characterized three enhancer modules located upstream of the transcription start site or introns of the SLC13A5 gene that are associated with the regulation of PXR‐mediated SLC13A5 induction. Moreover, metformin, a deactivator of PXR, dramatically suppressed PB‐mediated induction of hepatic SLC13A5 as well as its activation of the SLC13A5 luciferase reporter activity via PXR. Collectively, these data reveal PB as a potent inducer of SLC13A5 through the activation of PXR but not CAR in human primary hepatocytes.
Effects of cannabidiol (CBD), ∆9‐tetrahydrocannabinol (THC), and CBD/THC mixtures on fentanyl self‐administration in a food drug choice procedure in rhesus monkeys
Authors: Carey Lawrence M. , Maguire David R. , France Charles P.
Published: May 2022

The use of cannabinoids as adjuvants to opioid therapies to mitigate the problematic side effects of opioids or to decrease the doses of opioids necessary to relieve pain is an area of intense interest. In rhesus monkeys, ∆9‐tetrahydrocannabinol (THC) enhances the acute antinociceptive effects of opioids. However, THC does not enhance other effects of opioids including physical dependence, ventilatory depression, reinforcing effects, or discriminative stimulus effects. Evidence from rodent studies suggest other phytocannabinoids like cannabidiol (CBD) might decrease the reinforcing effects of several classes of drugs of abuse, including opioids. The present studies sought to assess the impact of THC, CBD, and THC/CBD mixtures on i.v. fentanyl self‐administration in rhesus monkeys (n=4) in a food drug choice procedure. Experimental sessions consisted of 2 forced trials followed by up to 30 choice trials where animals had the opportunity to respond on one lever for a sucrose pellet and another for an i.v. infusion (fentanyl or vehicle). Fentanyl dose was adjusted for each animal to find the largest dose that occasioned <20% drug choice (0.0001 mg/kg/infusion), and the smallest dose that elicited >80% drug choice (0.001 or 0.0032 mg/kg/infusion). CBD (10‐17.8 mg/kg), THC (0.032‐1 mg/kg), and THC/CBD mixtures (dose ratios of 1:10 and 1:32) were delivered i.v. 15 minutes prior to testing sessions. CBD or THC alone did not affect choice for either dose of fentanyl. THC/CBD mixtures similarly did not produce an increase in choice for the small dose of fentanyl, or a decrease for the large dose of fentanyl. The present studies suggest that phytocannabinoids such as THC or CBD, alone or in combination, do not enhance or diminish the rewarding effects of fentanyl. These results add to the body of evidence that phytocannabinoids might be a safe option to enhance the antinociceptive effects of opioids without enhancing their rewarding properties.
Effect of pH Dependent Substrate Inhibition on Glutamate Dehydrogenase Kinetics
Authors: Desrochers Andrea , Slade Kristen
Published: May 2022

Glutamate dehydrogenase (GDH) serves as the main regulated enzyme linking protein and carbohydrate metabolism. This enzyme converts the amino acid glutamate to the TCA intermediate, α‐ketoglutarate. While dysregulation of GDH is linked to hyperinsulinism and other diseases, the kinetics this enzyme of have only been analyzed under dilution conditions. However, cells are more crowded than these buffer solutions frequently used to collect enzyme kinetics data because their cytoplasm contains high concentrations of proteins, carbohydrates, and other macromolecules. Evidence has shown that this macromolecular crowding slow diffusion rates and changes protein conformations, which alters the kinetics of enzymes. Scientists often mimic the crowded environment of a cell using large, synthetic polymers, such as dextran, a glucose polymer, which excludes volume to other biological molecules in the same solution. While several studies have characterized these excluded volume effects on enzyme kinetics, this is the first study to analyze the effects of crowding on enzyme inhibition. Investigating how inhibitors like GTP and activators like leucine effect the kinetics of GDH will provide a deeper understanding of the regulation of this enzyme inside out bodies. Furthermore, saturating amounts of glutamate results in substrate inhibition of GDH by forming a “dead end complex” comprised of glutamate, NADH, and GDH at low pH values. To understand the effects of crowding on substrate inhibition, GDH Michaelis Menten kinetics were analyzed in the presence and absence of dextran at four pH values. These assays were repeated with glucose, the monomer of dextran as a control to separate chemical effects from excluded volume effects caused by the large size of dextran. The presence of dextran resulted in more substrate inhibition than in the presence of glucose and this inhibition that was observed at higher pH values than in buffer alone. Leucine, a known activator of GDH, operates by destabilizing the dead‐end complex. While the presence of leucine decreases substrate inhibition in dilute conditions, this compound was unable to active GDH in the presence of dextran. Overall, these results suggest that crowding promotes substrate inhibition by stabilizing the dead‐end complex of GDH which is a stronger interaction than leucine dissociation of the dead‐end complex. Therefore, macromolecular crowding in a cell could provide an additional level of regulation on GDH inside cells.
Transcriptional Characterization Suggests Accelerated Epithelial Differentiation in Trisomy 21 Lungs
Authors: Danopoulos Soula , Bhattacharya Soumyaroop , Cherry Caroline , Deutsch Gail , Mariani Thomas , Al Alam Denise
Published: May 2022

Rationale

Down Syndrome (DS), from trisomy 21 (T21), is the most common human chromosomal anomaly in the United States. Although DS can affect many organ systems, lung and heart disease are the leading causes of death. We recently reported that structural and molecular changes originate at the late pseudoglandular/early canalicular stages of development in T21 lungs. The initial, most striking, observation was dilatation of the terminal airways/acinar tubules. Thus, our goal was to better understand cellular heterogeneity at the transcriptional level that could be contributing to this phenotypic anomaly.

Methods

Single cell RNA sequencing (scRNAseq) was used to generate transcriptomic profiles of individual human lung cells in tissue obtained from T21 (n=5) and non‐T21 (n=4) prenatal lungs. Clustering, cluster marker identification, differential expression analysis, and UMAP representation was performed in Seurat. Cell populations were annotated using Toppfun. Spatial differences in cellular phenotypes were examined using immunofluorescent staining (IF) and fluorescent in situ hybridization (FISH).

Results

All the major cell lineages were identified in both the T21 and non‐T21 lungs, with mesenchymal cells representing the largest fraction (63%), followed by epithelial (16%), immune (13%), and endothelial (8%) cells. Each cluster displayed a relatively equal distribution of cells between T21 and non‐T21 (Figure 1). Having previously observed structural changes in the airways, we focused on re‐clustering the epithelial cell population. This resulted in 12 different epithelial sub‐clusters, with 4 distal bud clusters analyzed separately. We noted differentially expressed genes within each epithelial sub‐cluster between the T21 and non‐T21 samples. 332 genes were significantly different in T21 in one or more cell clusters at FDR < 0.05. SOX2 expression was significantly down in two of the distal bud clusters, which was confirmed by IF. Additionally, immunohistochemical staining showed a trend for an increase in the number of SCGB1A1 and FOXJ1 positive cells within the proximal airways of T21 lungs as compared to non‐T21. Simultaneously, multiple distal epithelial sub‐populations demonstrated significant increases in gene expression associated with surfactant synthesis in T21, with pathway analysis displaying the activation of surfactant metabolism. Our sequencing data demonstrated differentially increased SFTPC within the AT2 and distal bud populations, which was confirmed by FISH.

Conclusion

Our data demonstrate that T21 is associated with increased expression of markers of differentiation in the prenatal lung, which may be an indication of accelerated epithelial cell differentiation.
Treatment with the tellurium compound AS101 inhibits acute myeloid leukemia cells (AML) invasion and migration
Authors: Sredni Benjamin , Mendelson Moria , Sredni‐Kenigsbuch Dvora , Kalechman Yona
Published: May 2022

Adhesion molecules, such as integrins, enable acute myeloid leukemia (AML) cells, which travel in the bloodstream, to adhere to the vessels walls. This adhesion is a prerequisite for extra vascular leukemic cell infiltration. The penetrating cells reach vital organs, proliferate and damage the functionality of the tissue which aggravates the patient's condition. The Tellurium compound, AS101, is known for its ability to inhibit specific integrins by Oxidizing key cysteines within the integrins. In this study we found that AS101 can reduce the attachment of human U937 AML cell that expresses high levels of the a4b1 (VLA‐4) integrin to its ligands FN and VCAM‐1. furthermore, AS101 also inhibited the attachment of U937 to the HUVEC human endothelial cells, which express high levels of VCAM‐1 in the presents of TNF‐α. Exposure of U937 cells to AS101 inhibited the pFAK‐ pAKT‐ pGSK signaling, known to be involved in the penetration and migration of cells. AS101 is shown to inhibits the secretion of active MMP‐2 and MMP‐9, enabling cells to penetrate the ECM. Importantly, AS101 could prevent infiltration and migration of AML cells, provided they highly express VLA‐4. This was demonstrated by using patients' AML cells that express high or low levels of the VLA‐4 integrin. In vivo, AS101 prevents invasion of AML cells to the spleen and increase the survival of mice significantly when combined with the chemotherapy ara‐c. This result suggests that the AS101 compound may delay and even prevent the infiltration of the leukemic cells and by doing so, to increase the chances for recovery of the AML patients.
Structure‐activity Relation of Halogenated 2,5‐Dimethoxyamphetamines Compared to their α‑Desmethyl (2C) Analogues
Authors: Rudin Deborah , Luethi Dino , Hoener Marius C. , Liechti Matthias E.
Published: May 2022

Objective and hypothesis

4‐Substituted derivatives of 2,5‐dimethoxyamphetamine (referred to as DOX derivatives) exhibit psychedelic and euphoria‐inducing effects, mainly mediated by 5‐HT2A receptor activation; for this reason, they are used recreationally but also bear therapeutic potential. Studies in humans and animals revealed that 4‐substitution with halogens results in high clinical potency, which increased with increasing size of the halogen. We therefore hypothesized that increasing size of the halogen substituent results in increased affinity at the 5‐HT2A receptor. Hence, we investigated the interaction profiles of 4‐halogenated DOX derivatives with monoaminergic receptors and transporters, and the trace amine‐associated receptor (TAAR) 1 and compared the obtained values to already known data of their α‐desmethyl (2C) analogues. This will lead to a better understanding of the structure‐activity relationship and potential side effects of these compounds, which could lead to novel and more efficacious drug candidates for drug‐assisted psychotherapy.

Methods

We assessed the binding affinity of 2,5‐dimethoxyamphetamine and its halogenated derivatives (DOF, DOC, DOB, and DOI) at several human serotonin (5‐HT) receptor subtypes, adrenergic and dopaminergic receptors, the trace amine‐associated receptor (TAAR) 1, and monoamine transporters. We determined the binding affinity by displacement of radioactively labeled ligands by the DOX derivatives by liquid scintillation counting in cell membrane preparations from cells transfected with the respective human receptor or transporter. Moreover, we investigated the activation potency of the DOX derivatives at the 5‐HT2A and 5‐HT2B receptor by calcium mobilization and at the TAAR1 by cAMP accumulation in cells transfected with the respective human receptor.

Results

The 5‐HT2A receptor selectivity was substantially increased with increasing size of the halogen, with DOI displaying almost three orders of magnitude higher selectivity towards the 2A receptor subtype than DOF. All tested DOX derivatives were partial to full 5‐HT2A receptor agonists. The observed 5‐HT2 receptor interaction potency of the DOX derivatives is comparable to previously reported values of their 2C analogues, but with increased 5‐HT2A vs. 5‐HT1Aselectivity. None of the tested derivatives showed relevant affinity in the nanomolar range for TAAR1, α1A, α2A, D2, or monoamine transporters.

Conclusion

The increased 5‐HT2A vs. 5‐HT1A selectivity of halogenated DOX derivatives compared to their 2C analogues may explain their increased clinical potency. However, increased 5‐HT2A vs. 5‐HT1A selectivity may also increase the risk of seizures. Hence, while DOX derivatives display advantages as 5‐HT2A‐selective compounds for pharmacological research, for clinical applications they may be inferior to 2C derivatives due to their potentially increased risks for seizures.
Determining Antibiotic and Anti‐cancer Potential of Small Compounds
Authors: Manning Maegan , Mowery Patricia
Published: May 2022

Natural compounds are important leads for drug discovery. Staurosporine, which is produced by a Streptomyces species, has anti‐cancer properties but is too toxic for treatment. Analogs have been synthesized in the hopes of targeting cancer with less side effects.

The Pelkey research group has made staurosporine analogs which the Mowery lab tests for biology activity. Recently, one compound was found to be highly potent against cancer cells and induce death by binding tubulin. Tubulin is a eukaryotic protein that is necessary for cell division. Because bacteria have a homologous protein, Fts‐Z, we wanted to determine if compounds acted as antibiotics. In addition, the Pelkey group has synthesized a suite of new compounds that are isomeric to our previously most potent ones. We are testing these compounds for anti‐cancer activity to determine what structural parameters are necessary to induce cell death.
In Vivo Evaluation of Validated Targets from High Throughput Screening Models for Sulfur Mustard Ocular Injury
Authors: Ma Ki H. , Merriman Abigail F. , Ruff Albert L.
Published: May 2022

This work was conducted in an effort to develop medical countermeasures to treat exposure to toxicants that can be utilized in a terrorism or warfare scenario. Chemical injuries to the eyes are true ocular emergencies because of their high potential to inflict rapid and enduring tissue damage. The cornea and other ocular surface tissues are particularly susceptible to chemical exposures such as sulfur mustard (SM), and long‐term prognosis can be poor due to progressive corneal pathologies. Chronic and delayed complications include persistent corneal epithelial defects, dry eye, corneal opacification, and corneal neovascularization. Such long‐term complications can lead to permanent discomfort and vision deficits or blindness despite the best current treatment regimens. To identify novel therapeutic targets, we had carried out siRNA‐mediated high throughput screening (HTS) using human corneal epithelial cell models of SM exposure. Over 3000 genes identified as differentially regulated after SM ocular exposure by previous microarray studies were tested for their functions involved in cell viability and IL‐8 production. The current study transitioned eight validated targets from the HTS to a mouse model of SM‐induced ocular injury. Interestingly, four out of the eight targets, PSMA1, PSMA2, PSMD7 and FBXO5, were subunits of proteasomes or E3 ubiquitin ligase complexes. The ubiquitin‐proteasome pathway maintains protein homeostasis and therefore important cellular functions through the degradation of misfolded, redundant, and damaged proteins. Treatment with the proteasome inhibitor bortezomib in the first two weeks after SM ocular exposure greatly promoted the rate and number of corneal neovascularization, which is a key feature of a delayed injury phase. Such outcomes were also observed with treatment with another proteasome inhibitor, carfilzomib. In addition to proteasome subunits, GABRR1 (GABA type A receptor subunit rho‐1) was among the validated targets from the HTS. We learned that muscimol, a GABA type A receptor agonist, significantly decreased corneal neovascularization during 8‐week observation periods. Taken together, these outcomes demonstrated the effectiveness of our systemic approach to identify novel molecular signaling pathways implicated in toxicant‐induced ocular injuries and future studies for potential drugs. For example, our future studies include investigating proteasome‐mediated protein regulation of angiogenic factors in response to SM ocular exposure and testing the therapeutic potential of proteasome agonists. Some small molecule proteasome agonists such as chlorpromazine and imidazoline were identified in recent years and have been studied for their efficacy in treating different diseases such as neurodegeneration, cancer, and autoimmunity.
Identification of Signaling Pathways and Phase Separating Domains that Drive Cajal Body Formation
Authors: Logan Madelyn K. , McLaurin Douglas , Lett Katheryn , Hebert Michael
Published: May 2022

Cajal bodies (CBs) are membraneless subnuclear domains that contribute to the biogenesis and maturation of ribonucleoproteins (RNPs) which take part in fundamental cellular activities such as translation, pre‐mRNA splicing, and telomere maintenance. Partitioning of CBs occurs through liquid‐liquid phase separation (LLPS) where the CB marker protein, coilin, is thought to scaffold the CB. However, the formation of CBs and the regulation of CB number has not been fully elucidated. Disease states such as cancer and preeclampsia have altered CB expression, thus additional insight into CB condensation would provide an important cellular model system that could be used to investigate these and other diseases. We hypothesize that the post‐translational modification (PTM) of coilin regulates CB formation and number by influencing the LLPS of coilin via residues in its intrinsically disordered region (IDR). Here we show, for the first time, a cycloheximide inducible model of CB formation in fibroblasts without increasing coilin concentration. We are also able to induce CB formation by inhibiting kinase GSK3β with indirubin, as well as abolish CB formation by inhibiting phosphatases PP1 and PP2A with okadaic acid. Lastly, we have identified two phase‐separating domains in coilin’s IDR using the phase separation predicting software ParSe. Deletion of each domain prevents CB formation in HeLa cells but does not prevent coilin’s self‐interaction. Collectively, these data will be able to help us understand the role of coilin’s scaffolding capabilities via LLPS mediated CB condensation, as well as provide an important cellular model system that can be used to investigate CB functionality in disease states where CB formation is dysregulated.
The Effect of Perceived Classroom Fear Tactics on STEM Students
Authors: Forshee Lance , Jefferies Tanner , Mraz‐Craig Jennifer
Published: May 2022

In today's education system fear is a common and complex problem found at all levels. It is unknown what effect fear has, specifically, on STEM (Science, Technology, Engineering, or Math students) students at the college level. The objective of this study is to determine what students perceive as Fear Tactics and which specific Fear Tactics had a positive, motivating effect, versus a negative, demotivating, effect on the STEM students. The researchers performed semi‐structured interviews (n=51) using inductive coding to identify specific student perceived Fear Tactics and sorted them into three different categories on why the Fear Tactics worked: Motivation, Guidance, and Fear of Failure. The researchers identified three categories of positively motivating Fear Tactics (Motivating, Provided Guidance, and Addressed Fear of Failure) and three categories of negative Fear Tactics (Good luck passing, Amount of study time, and Heavily weighted assignments). Specific tactics such as “Good luck passing” were indicated by students to be a harmful tactic and “This is going to be hard/this is serious” were indicated by students to be a helpful tactic. By accumulating a specific list of harmful Fear Tactics we can help eliminate these tactics in the classroom and replace them with positive tactics to help create a positive learning environment with the goal of reducing attrition in STEM fields.
Clinical Communication Skills Training in Third‐ and Fourth‐Year Medical Students: A Qualitative Study
Authors: Veazey Kathryn , Robertson Ellen M.
Published: May 2022

Healthcare professionals require strong clinical communication skills (CCS), yet many healthcare workers demonstrate discrepancies between their self‐rated competency in communication skills and patient satisfaction. These communication skill deficiencies may cause increased stress and anxiety in healthcare professionals, which in turn adversely affects patient compliance and long‐term health outcomes. According to the Transaction Model of Communication, communication can be shaped by social, relational, and cultural contexts. This study sought to identify the factors that influence how medical students gained CCS and how those factors evolved over the course of the medical school experience. Semi‐structured interviews of third‐ and fourth‐year medical students were conducted at the University of Mississippi Medical Center. Qualitative responses underwent deductive thematic analysis using the Transaction Model of Communication with iterative open coding to identity and name themes. Results indicated that students felt there were profound differences in CCS training between the preclinical versus clinical years. They suggested preclinical CCS training, while valuable, often falls short in preparing them for the challenges of navigating real patient care. Medical students also recognized the value of CCS for safe and effective healthcare practice, but strongly advocated for changes to the current CCS curriculum, including earlier exposure to real patients, increased cultural awareness training, explicit telehealth training, and increased opportunities to practice “delivering bad news”, even in simulated settings. The findings of this study have provided new insights into the individual medical student experience, which speak to the experiences of medical students and practitioners throughout the global healthcare field. This study’s findings may be used to inform curricular changes such as increased training on select topics and earlier access to real patient experiences, which may improve students’ confidence and competency in their clinical communication abilities, thereby improving patient care.
TFAP2 Paralogs Regulate Alx Genes During Midface Cranial Neural Crest Development
Authors: Nguyen Tim , Mitchell Jennyfer , Kiel Michaela , Williams Trevor , Nichols James , Van Otterloo Eric
Published: May 2022

Craniofacial anomalies are the 2nd leading structural malformation present at birth. Those in the midface, such as orofacial clefts and hypertelorism, can arise from mutations in the Transcription Factor Activating Protein 2 genes TFAP2A and TFAP2B (abbreviated as TFAP2). These mutations do not reduce their gene expression, but instead generate mutant proteins that disrupt the activity of other wild‐type and functionally redundant paralogs. Previous studies showed that both paralogs are co‐expressed in the embryonic midface, hinting at their cooperative role during face development. Yet, how TFAP2 regulate midface development at the cellular and molecular level is poorly understood. To this end, we leveraged CRE‐based approaches in mice to delete both Tfap2 members in the neural crest (NC), a cell lineage that gives rise to craniofacial bone and cartilage. We show that NC‐specific knockout of Tfap2 (Tfap2NCKO) results in a fully penetrant midface cleft compared to controls and single paralog mutants. Lineage tracing analyses in Tfap2NCKO embryos suggest that cranial NC migration into the embryonic face is not severely impaired. Corroborating this result, we find that Tfap2 deletion in either pre‐migratory or late‐migratory NC cells still results in a midface cleft. This suggests this phenotype stems from disrupted TFAP2 activity in post‐migratory NC cells. Moreover, when examining Tfap2NCKO craniofacial skeletons, we observe severe bone malformations and ectopic structures in the frontonasal regions when compared to controls. Single‐cell RNA sequencing analysis reveals that loss of Tfap2 results in reduced cranial NC cell numbers and dysregulated Alx1/3/4 (Alx) gene expression. This is supported by the literature, as the Tfap2NCKO midface clefts phenocopy those with loss of various Alxgene combinations. Our ongoing efforts aim to test how TFAP2 regulates Alx genes, if loss of Tfap2 compromises midface NC survival or proliferation, and whether Tfap2 and Alx genetically interact. In sum, we propose the model that TFAP2 regulates Alx gene expression in NC cells during midface development.
Identification of Fungus‐derived Compounds as a Novel CFTR inhibitor: Mechanism and Potential Anti‐diarrheal Application
Authors: Noitem Rattikarn , Pongkorpsakol Pawin , Changsen Chartchai , Rukachaisirikul Vatcharin , Muanprasat Chatchai
Published: May 2022

Secretory diarrhea, which has been considered as a health burden worldwide, could be alleviated by inhibiting fluid secretion. CFTR is considered to be a drug target for development of anti‐secretory agent. However, there is no currently FDA‐approval drug targeting CFTR. This study aimed to identify CFTR inhibitors from a library of fungal bioactive metabolites found in Thailand. Electrophysiological analyses were used for identifying the inhibitory effect of 40 compounds. Three active compounds with the best one being DHLV, which effectively inhibited CFTR‐mediated Clˉ secretion with low micromolar potency (IC50 ~1.8 μM) in T84 cells without effect on Na+‐K+ ATPase activity. In addition, DHLV inhibited Ca2+‐activated Clˉ channels stimulated by ATP. In vivo experiments showed that administration of DHLV by intraperitoneal route (2 mg/kg) and intraluminal (22 µg/kg or 20 µM) injections reduced cholera toxin‐induced intestinal fluid secretion in mice by ∼46.7% and ∼42.8%, respectively without affecting intestinal fluid absorption. This study indicates that the novel CFTR inhibitor, DHLV, is of potential utility in the treatment of secretory diarrhea.
The Effect of Antibiotics on Outer Membrane Vesicle Production
Authors: Dinu Milena A. , Michel Lea , Gaborski Thomas , Perdue Janai , Kasper Anna , Morehouse Ryan
Published: May 2022

Sepsis is a condition in which the body’s inflammatory response to an infection is overwhelming and unregulated. Sepsis‐related inflammation can lead to tissue damage, organ failure, and even death. Once diagnosed, most sepsis patients are put on a regimen of antibiotics, especially if a bacterial infection is the suspected cause of sepsis. We proposed that at least some antibiotics may enhance the release of potentially toxic and inflammatory molecules from bacteria. Specifically, we considered several clinically relevant antibiotics and their effect on the release of outer membrane vesicles (OMVs) from Gram‐negative Escherichia coli. We employed ultracentrifugation to purify the OMVs and Western blotting to quantify the OMVs released from E. coli. Preliminary results suggest that, using our methods, some antibiotics enhance the production of OMVs from E. coli.
Expression, Characterization, and Analysis of Type II Metacaspases from the Fungus Schizophyllum commune
Authors: Sadak Katherine , Fox Kristin M.
Published: May 2022

Programmed cell death (PCD), otherwise known as apoptosis, is implicit across all kingdoms of life especially in the processes of development, aging, reproduction, responses to stress, and maintaining homeostasis. In humans and other animals, apoptosis is mediated by caspases, proteases that cleave other proteins after Asp residues. Plants, fungi, and protozoa do not have caspases, but rather produce homologous proteins known as metascapases. In these organisms, metacaspases are the proteins primarily responsible for programmed cell death. Studying their mechanisms can provide insight into opportunistic fungal infections. In our research, Schizophyllum commune is the model organism for studying metacaspase proteolytic function in fungi. There are two types of metacaspases in S. commune with varying structures and organization (Type I and Type II). Both types contain a larger p20 catalytic region with a Cys‐His catalytic dyad similar to casapses, and a smaller p10 region with a conserved sequence. The Type I metacaspases cleave preferentially after Lys and Arg and are activated by calcium ions. The focus of this research project is the characterization and analysis of Type II metacaspases (ScMCA‐II) in S. commune. Type II metacaspases have a large linker region between the p20 and p10 subunits, and are hypothesized to be the chief enzyme involved in apoptosis. Previous literature has focused primarily on Type I metacaspases, making the studies of Type II metacaspase especially crucial. There are six Type II metacaspases that have been identified in the fungus S. commune (labelled ScMCAIIa‐f). Full‐length constructs of ScMCAIIa and ScMCAIIf are expressed at low levels in E. coli and expression is not improved by decreasing the growth temperature. Preliminary results with small amounts of partially‐purified protein suggest that S. commune Type II metacaspases are cleaved during expression and are active against the metacaspase substrate Z‐GGR‐AMC in both the presence and absence of calcium.