bluuebunny/crossref_metadata_2025 huggingface dataset:
Elimination of Senescent Cells Attenuates Obesity‐Induced Increase in Sympathetic Nerve Activity
Authors: Sivasubramanian Mahesh Kumar , Monteiro Raisa , Balasubramanian Priya , Subramanian Madhan
Published: May 2022

Obesity is a major risk factor for the development of cardiovascular diseases like hypertension. It is estimated that obesity accounts for approximately 65‐78% of hypertension in adults. Accumulating evidence suggests that increase in sympathetic nerve activity (SNA) plays a crucial role in the pathophysiology of obesity‐induced hypertension. Previous studies from our lab have shown that obesity induces cellular senescence at the level of the rostral ventrolateral medulla (RVLM), which is responsible for basal and reflex control of sympathetic activity associated with cardiovascular function. Cellular senescence is a state of permanent, irreversible cell cycle arrest in proliferative cells. Senescent cells acquire senescence‐associated secretory phenotype (SASP) and secrete proinflammatory cytokines, chemokines and proteases. These SASP factors could serve as a potential source of neuroinflammation. Based on the previous studies, our hypothesis is that obesity‐induced senescence in the RVLM contributes to neuroinflammation and increases in SNA leading to the development of hypertension. To address this hypothesis, we eliminated senescent cells using a pharmacological approach (senolytic drug) and investigated its effect on sympathoexcitation and hypertension in obesity. Two‐month‐old male C57BL/6J mice were fed a high‐fat diet (HFD; 60% fat) or a chow diet (Controls; 10% fat) for 4‐6 months. After 4‐6 months of feeding, the two groups were again subdivided into two subgroups: Vehicle and a senolytic drug combination of Dasatinib+Quercetin (D+Q). So, the four different groups are Control + Vehicle, Controls + (D+Q), HFD + Vehicle, and HFD + (D+Q). Dasatinib (5 mg/kg) plus Quercetin (50 mg/kg) was prepared in a diluted solution comprising 10% ethanol, 30% polyethylene glycol 400, and 60% Phosal 50 PG. The intervention (Vehicle or D+Q) was performed for 3 consecutive days and for 2 cycles with a 2‐week interval between cycles. Hemodynamic parameters (Blood pressure & Heart rate) and SNA were analyzed by radiotelemetry. As expected, the low frequency/high frequency heart rate variability (LF/HF HRV) ratio which is an indicator of SNA was significantly higher in HFD animals, which was reversed by senolytic drug treatment with D+Q. No changes were observed in the blood pressure. These results suggest that obesity‐induced cellular senescence causes sympathoexcitation which was reversed by the removal of senescent cells using senolytics. Future studies will investigate the mechanistic role of cellular senescence at the level of RVLM in obesity‐induced sympathoexcitation.
Pregnancy‐induced physiological cardiac hypertrophy regulates the expression of perilipin isoforms
Authors: Soñanez‐Organis Jose G. , Verduzuco‐Avila Denisse G. , Padila‐Borquez Diana L. , Godoy‐Lugo Jose A. , Rosas‐Rodríguez Jesús A. , Soto‐Duarte Angel O. , Ruiz‐Castillo Enrico A. , Ortiz Rudy M.
Published: May 2022

Perilipins (PLINs) isoforms are proteins associated to surface of intracelular lipid droplets (LDs) that participate in the regulation of lipid metabolism and storage during hypertrophic cardiomyopathies. PLINs can associate to the PPAR coactivators peroxisome proliferator‐activated receptor gamma coactivator 1‐alpha (PGC‐1α) and Sirtuin 1 (SIRT1) to regulate the expression of genes involved in lipid metabolism. Pregnancy‐induced physiological cardiac hypertrophy is a reversible process in which there is a change in lipid metabolism. However, there are a lack attention of the participation of PLINs isoforms in response to pregnancy‐induced cardiac hypertrophy. In this work, we quantified the PLINs isoforms and PGC‐1α mRNAs and proteins level, along with the transcriptional activities de PPARs family, triacylglyceride (TAG) and cholesterol/ester cholesterol in left ventricle of rats before, during, and after pregnancy. Expressions of mRNAs for PLIN1, PLIN2 and PLIN5 increased 9.5‐ and 7.4‐fold, 27.5‐ and 17.7‐fold, and 8‐ and 2.4‐fold 2 during pregnancy and postpartum, respectively, while PLIN3 did not change and PLIN4 was not detected. In contrast, the protein content of PLIN2 increased 1.5‐fold during postpartum, and PLIN5 increased 1.3‐ and 1.5‐ fold during pregnancy and postpartum, respectively, while PLIN1 did not change. Expression of mRNA and protein content for PGC‐1α increased 17.7 and 11.26‐fold and 1.5‐ and 1.2‐ fold during pregnancy and postpartum, respectively. The activity of PPARγ increased 1.6‐fold during pregnancy and decreased to basal levels postpartum. The concentration of TAG and Cholesterol/Cholesteryl ester increased 1.5‐ and 1.4‐ fold in the pregnant group compared to the non‐pregnant group, and returned to basal levels at postpartum, respectively. The results demonstrate that pregnancy‐induced, physiological cardiac hypertrophy activates the expression of genes involved in lipid storage suggesting that the shift in cardiac metabolism is mediated by the activation of PPARγ/PGC‐1α.
Yeast Bax Inhibitor (Bxi1p/Ybh3p) is a Calcium Channel in E. coli
Authors: Aguiar Julia D. , Crago Madelyn , Mullin James , Kalhorn John , Mello Nicholas , Raffa Amanda , Strakosha Aleksander , Austriaco Nicanor
Published: May 2022

Human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is the founding member of the evolutionary conserved TMBIM superfamily of proteins that share sequence homology within the transmembrane Bax inhibitor‐containing motif (TMBIM). Mechanistically, BI‐1/TMBIM6 and all the other mammalian TMBIM proteins appear to be involved in the maintenance of calcium homeostasis, and the crystal structure of a bacterial TMBIM protein, BsYetJ, suggests that the protein is a pH‐sensitive calcium leak. The budding yeast, Saccharomyces cerevisiae, has a single TMBIM family member (YNL305C) named Bxi1p/Ybh3p. To determine the function Bxi1p/Ybh3p, we overexpressed Bxi1p‐GFP in E. coli to interrogate its putative calcium channel function. We show that bacterial cells expressing Bxi1p‐GFP are more permeable to calcium than controls. Our data suggests that yeast Bax inhibitor (Bxi1p) is a calcium channel in E. coli, lending support to our proposal that Bxi1p is a bona fide member of the TMBIM family of proteins. We use our bacterial system to show that gadolinium is an inhibitor of Bxi1p in vivo, suggesting a path forward to identifying other small‐molecular inhibitors of this clinically‐important and highly conserved superfamily of proteins. Finally, parallel experiments revealed that the human Bax Inhibitor‐1 (HsBI‐1/TMBIM6) is also a calcium channel in bacteria that can be inhibited by gadolinium.
Activation of group II metabotropic glutamate (mGlu2/3) receptors regulates excitatory synaptic plasticity in the nucleus accumbens: Implications for the treatment of schizophrenia
Authors: Dogra Shalini , Conn P. Jeffrey
Published: May 2022

Accumulating clinical evidence suggests that glutamatergic dysfunction may contribute to the pathogenesis of schizophrenia. Several studies have shown beneficial effects of group II metabotropic glutamate (mGlu2/3) receptor agonists in rodents modeling schizophrenia symptomatology. However, the mechanisms through which mGlu2/3 receptor agonists modulate glutamatergic neurotransmission in the nucleus accumbens (NAc) and schizophrenia‐like symptoms are still not clear. Therefore, we performed a series of experiments to evaluate the role of mGlu2/3 receptors in regulating glutamatergic neurotransmission in the NAc. Using ex vivo whole‐cell patch‐clamp electrophysiology, we showed that bath application of mGlu2/3 receptor agonist, LY379268 (100 nM), induces long‐term depression (LTD) of excitatory neurotransmission onto D1 medium spiny neurons (MSNs) in the NAc. Further, we evaluated the expression of mGlu2/3‐LTD over the glutamatergic inputs from the midline nuclei of the thalamus (mThal) and basolateral amygdala (BLA) to the NAc. Interestingly, group II mGlu receptor agonist, LY379268, induces LTD of mThal‐NAc projections, but not BLA‐NAc projections, to D1 MSNs. Further, to test the hypothesis that mGlu2/3 receptor activation can normalize glutamatergic neurotransmission in the NAc of mouse models of schizophrenia, we first measured spontaneous excitatory postsynaptic currents (sEPSCs) onto D1 MSNs of NAc in the slices prepared from the mice treated with either saline or MK801 (0.2 mg/kg; s.c., 30 minutes). We found that acute treatment with MK801 enhances the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) onto D1 MSNs. Subsequently, we examined the effect of group II agonist on MK801‐induced dysregulation of excitatory neurotransmission in the NAc. Excitingly, bath application of group II agonist, LY379268 (100 nM), reversed the MK801‐induced changes in sEPSCs in the NAc. Collectively, these data provide an evidence that activation of mGlu2/3 receptors can rescue schizophrenia‐like pathophysiology in the NAc.
Sleuthing for the Enzymatic Activity of an Essential Trypanosoma bruceiHaloacid Dehalogenase
Authors: Mahoney Emily M. , Padykula Derrick E. , Palenchar Jennifer B.
Published: May 2022

Trypanosomes are eukaryotic parasites that can infect a variety of hosts and result in multiple diseases for which therapeutics are lacking. Trypanosoma brucei contains two putative haloacid dehalogenases, or HADs, that were acquired by the parasites from bacteria through horizontal gene transfer. One of these, HAD32, is essential in their life cycle. Both trypanosome HADs are uncharacterized. Exploring the function of HAD32 may hold therapeutic potential. We seek to find the function of the trypanosome HADs, starting with their enzymatic activity. Recombinant His10‐TbHAD32 was overexpressed in bacteria and purified to approximate homogeneity. The HAD superfamily of enzymes utilizes a wide range of substrates. TbHAD32 has a sequence motif associated with phosphatase activity within the HAD superfamily. We screened several potential substrates for the protein and found that Trypanosoma brucei HAD32 is a phosphatase. His10‐TbHAD32 has a catalytic efficiency of 65.73 +/‐ 12.44 mM‐1 min‐1 when utilizing para‐nitrophenol phosphate (PNPP) as a substrate. With PNPP as substrate, we determined that Mg2+ is the optimal metal required for His10‐TbHAD32 phosphatase activity. With broad activity in hand, we have identified three additional and potentially biologically relevant compounds that His10‐TbHAD32 utilizes as substrates and have determined the kinetic parameters of the enzyme with these compounds. These activities give us a solid foundation upon which to build hypotheses of enzyme function in the parasite.
Are Surface Models Obtained Using Different Methodologies Comparable?
Authors: Russell C. Kinley
Published: May 2022

Surface models are used to quantify differences in form in a variety of anatomical analyses. Different methods for obtaining surface models are used across studies and multiple methodologies may be used within a single study. The number of methodologies available for the collection of surface data has multiplied in recent years. Though this presents an opportunity, it also presents a potential problem. Are surface models obtained using different methodologies comparable? Here I quantify surface distances between aligned surface models obtained using a blue light scanner and photogrammetry.

Proximal tibiae from ten individuals were mounted to a rotating stand and scanned using an Artec Space Spider; surface models were automatically reconstructed using the real‐time fusion setting in Artec Studio 16. The same tibiae were photographed from 65 angles using a Canon Coolpix camera set to the macro setting. Photographs for each individual were masked and aligned to build a surface mesh using Agisoft Metashape. Artec scanner and photogrammetry surface models for each individual were imported into Avizo Lite. The photogrammetry model for each individual was scaled and aligned to the Artec model by iteratively minimizing surface distance between the models. Once surface alignment reached a stable configuration, surface distances from the Artec model to the photogrammetry model were calculated. Surface distances were also calculated between aligned pairs of individuals obtained using the Artec scanner.

Surface distances between different individuals obtained using the same methodology were 73 times larger than those between models of the same individual obtained using different methodologies (p<0.001). Mean surface distance between models of the same individual was 0.020 mm and mean surface distance between models of different individuals was 1.477 mm. While surface distances exceeded 0.5 mm for approximately 75% of vertices in between individual comparisons, surface distances never exceeded 0.5 mm in comparisons of Artec scanner and photogrammetry derived models. These results suggest that surface models obtained using different methodologies are comparable, at least in this case.
Macronutrient Signals for Adaptive Modulation of Intestinal Digestive Enzymes in Two Omnivorous Galliforms
Authors: Oguchi Yushi , Rolle Momoko , Mai Don , Caviedes‐Vidal Enrique , Karasov William H.
Published: May 2022

Most birds studied to date (15 out of 16 species) have shown the ability to adaptively modulate at least one small intestinal digestive enzyme activity to match the composition of macronutrient levels in their diet. Limited work on Galloanserae (waterfowl, quails, and chickens) suggested that this clade may have the mechanisms to modulate intestinal disaccharidase but not peptidase activity (6 out of 7 species). We tested this hypothesis in immature (70‐72 d) northern bobwhites (Colinus virginianus) and juvenile (20 d) and adult (230 d) domestic chickens (Gallus gallus domesticus) by raising them on 3 semisynthetic diets varying in starch, protein, and lipid composition for 7‐9 d (bobwhites) or 15 d (chickens). Birds were then euthanized, intestinal tissue harvested, and enzyme activities measured in tissue homogenates from proximal, medial and distal small intestine. We found that (1) disaccharidase (maltase and sucrase) activities were induced by dietary starch in both juvenile and adult chickens but not in northern bobwhites; (2) aminopeptidase‐N activities were induced by dietary protein in both bobwhites and juvenile and adult chickens; and (3) disaccharidase activities were suppressed by dietary lipid in both bobwhites and juvenile and adult chickens. Generally, diet effects were similar all along the intestine or most pronounced in the medial intestine (chicken disaccharidases). In chickens, age did not affect the diet‐driven modulation pattern except in sucrase activity – modulation was found in the distal but not in the proximal intestine in juveniles and the reverse was found in adults. We conclude that there is no general pattern with respect to which nutrient classes of digestive enzymes are adaptively modulated in the small intestine of Galloanserae, and that the modulation of each enzyme in species in this clade likely relates to its respective digestive ecology. On the other hand, the suppressive effect of lipid on the intestinal disaccharidases appears to be present in all vertebrates (birds, mammals, and fish) tested to date, although the biological significance of this effect is not yet understood. We will present a summary of adaptive intestinal digestive enzyme modulation patterns of all birds thus far studied, arranged in a cladogram.
Novel Insights into Regulation of GPCR Signaling by GRKs and β‐arrestins
Authors: Crecelius Joseph M. , Benzow Sara , Marchese Adriano
Published: May 2022

G protein‐coupled receptor (GPCR) signaling is tightly regulated by GPCR kinases (GRKs) and β‐arrestins 1 & 2 (βarr1/2). Following agonist stimulation, GRKs and βarr1/2 are important for returning cells to their physiological resting states by acting in tandem to promote receptor desensitization by reducing the ability of receptors to couple with G proteins and by targeting active GPCRs for endocytosis. In addition, GRKs and arrestins are also known to instigate non‐canonical signaling by scaffolding and activating various effector molecules. Although these functions of βarr1/2 are generalizable to all GPCRs, they can diverge dependent upon the ligand activating the same GPCR and the dominant function can vary between GPCRs. Although inherent properties of the GPCR likely specify these discrete functions, the determinants remain poorly understood. To address this, we are using the chemokine receptor CXCR5 as a model GPCR. CXCR5, and its endogenous ligand, CXCL13, have been linked to diseases ranging from cancer, chronic inflammation, and cardiovascular disease, among others, and yet very little is known about the regulation of CXCR5 signaling. To investigate this, we used CRISPR/Cas9 gene edited βarr1/2 knockout and matched parental HEK293 cells in bioluminescence resonance energy transfer (BRET), receptor trafficking, and signaling assays. Our data show a dose‐dependent increase in the BRET response following CXCL13 stimulation between βarr1/2‐GFP and CXCR5‐Rluc8, suggesting that βarr1/2 is recruited to activated CXCR5 at the plasma membrane. A selective inhibitor of GRK2/3, but not a non‐selective PKC inhibitor, reduced βarr1/2 recruitment to CXCR5 following CXCL13 stimulation. Mutation of potential phosphorylation sites within the carboxy‐terminus of CXCR5 did not impact CXCL13‐stimulated endocytosis, although βarr1/2 recruitment was impaired. Remarkably, CXCL13‐stimulated CXCR5 endocytosis was not impacted in βarr1/2 knockout HEK293 cells, while CXCL13‐stimulated ERK‐1/2 phosphorylation was reduced compared to parental HEK293 cells. Based on these data we hypothesize that CXCR5 signaling is regulated through a complex process that involves GRKs andβ‐arrestins via conventional and non‐conventional mechanisms.To explore this further, in future experiments we will identify the receptor determinants mediating GRK‐phosphorylation and βarr1/2 recruitment to CXCR5 and their contributions to desensitization, endocytosis, and signaling. Overall, we expect to learn how receptor phosphorylation contributes to the divergent functions of βarr1/2 via CXCR5. This research will help illuminate novel mechanisms governing GPCR regulation by GRKs and arrestins, which may translate into better therapeutics for diseases involving GPCR signaling.
Assessment of Sialic Acid Levels in an In Vitro Model of Muscle Atrophy
Authors: Poynter Megan N. , Crowe Kelly E.
Published: May 2022

Muscular atrophy is a loss of muscle tissue that can occur due to inactivity or underlying disease processes. Studies have shown that muscle pathology may be linked to levels of sialic acid (SA), an essential glycan found capping extracellular glycan chains of muscle cells. This is particularly evident in GNE myopathy, a disease of muscle wasting in which the causative genetic mutation is linked to decreased SA production. However, the relationship between muscle atrophy and sialylation is poorly understood. Here, we have assessed SA levels in an in vitro model of atrophy in which C2C12 mouse myotubes were treated with dexamethasone. We have utilized lectins, glycan‐binding proteins, to probe linkage‐specific effects of atrophy on sialylation in this in vitro model. This work can help to elucidate the relationship between atrophy and muscle sialylation, and may inform future therapeutic development.
Mass Spectrometry Based Subcellular Coenzyme Analysis
Authors: Snyder Nathaniel W. , Bostwick Anna , Pepper Hannah , Trefely Sophie
Published: May 2022

Metabolism in eukaryotes relies on compartmentalization of processes between sub‐cellular compartments. Our objective was to develop, test, and apply methods that can quantitatively measure families of metabolites within distinct sub‐cellular compartments in eukaryotic cells. We created Stable Isotope Labeling of Essential nutrients in Cell culture ‐ Subcellular Fractionation (SILEC‐SF) with the essential precursors of the major cellular coenzymes, Coenzyme A and NAD to incorporate a 13C,15N‐label into the families of each coenzyme present within cells. Using multiple fractionation techniques coupled to liquid chromatography‐high resolution mass spectrometry we quantify distinct cytoplasmic, mitochondrial, and nuclear pools within eukaryotic cells. We successfully applied these methods to cells and human tissue demonstrating distinct compartmental metabolic changes by pathway in genetic models of compartmentalized metabolism, in adipocyte differentiation and in changing oxygen tension. This confirmed orthogonal measurements of subcellular metabolism but revealed unexpected localizations and enrichments of certain metabolite pools.
Optimization of expression and purification of SUMO‐tagged LL‐37 peptides
Authors: Himmelberger Julie , Rush Coby , Drauch Jocelyn , Harakal Bekkah , Nguyen Tram
Published: May 2022

Antimicrobial peptides (AMPs) are a class of naturally occurring biomolecules found in all multicellular organisms, and are used as a first line of defense for plants and animals. AMPs have been found to cause bacterial membrane rupture and in some cases they also inhibit intracellular function, in both cases resulting in bacterial lysis and death. A broad collection of AMPs have been discovered that can selectively target bacteria, yeasts, fungi, viruses, and even cancer cells. For example, AMPs that target bacteria are typically positively charged, and therefore interact via electrostatic interactions with negatively charged bacterial membranes. Cathelicidins, are small, cationic, amphiphilic AMPs found in many mammalian species. Cathelicidins exibit a broad range of antimicrobial activity against fungi, bacteria, and enveloped viruses, allowing for innate immunity in the skin. LL‐37 is the sole member of the human cathelicidins. Numerous studies have investigated the structure and function of LL‐37, in an effort to develop antimicrobial therapeutics based on its core structure. Many of these potential therapeutics rely on automated peptide synthesis. We seek to contribute to the understanding of the LL‐37 peptide structure and function by further investigating key residues which are required for antimicrobial activity, and optimize the expression of recombinant LL‐37‐based peptides in E.coli. A structure‐based design strategy informed the construction of a series of mutant LL‐37 peptides, that were generated using site‐directed mutagenesis. The following mutants were investigated: a series of single amino acid substitutions, G14P, K12R, K18R, K25R, addition of a hydrophobic tail (LLAA) and hydrophilic tail (LRQA). Cloning was achieved using the ThermoFisher Champion pET SUMO Protein Expression System, and sequences were confirmed using Sanger sequencing. The 11‐kD SUMO (small ubiquitin‐related modifier) on the C‐terminus of the recombinant protein enhances the expression and solubility of the LL‐37 peptide in E. coli. The His‐tagged, LL‐37‐SUMO peptides are expressed in BL21(DE3) E. coli, and following removal of the SUMO and His tags, ongoing studies examining the activity of the recombinant peptides against gram‐positive and gram‐negative pathogens continues.
Cypin binds to fully polymerized microtubules and tubulin heterodimers via distinct domains
Authors: Lange Keith R. , Fenner Madeleine R. , Sweet Eric S. , Firestein Bonnie L.
Published: May 2022

Proper branching of neuronal dendrites is crucial for healthy brain function. We previously reported that cypin, a guanine deaminase, binds to tubulin heterodimers via its collapsin response mediator protein (CRMP) homology domain (amino acids 350‐403), promoting microtubule assembly in a cell‐free system. This increased microtubule assembly results in increased dendrite number and branching. Here, we ask how cypin alters microtubules in neurons. We found that overexpression of cypin increases the number of and decreases the spacing between microtubules. We also observed that overexpression of cypin increases microtubule polymerization as evidenced by increased movement of end‐binding protein 3 (EB3) comets. To determine whether cypin binds polymerized microtubules in addition to tubulin heterodimers, we performed a series of biochemical and computational experiments. We found that cypin binds to fully formed microtubules but does not prefer microtubule ends or shafts. In addition, preliminary protein‐protein docking strategies suggest that cypin binds to microtubules through several unreported residues near the N‐terminal end of cypin, which form a surface‐exposed loop with several bulky residues. We used structure‐based approaches through docking analyses of “straight” polymerized microtubules and of “curved” soluble tubulin heterodimers and found that cypin binds free heterodimers though its CRMP homology domain along with a small amino acid region near the N‐terminus (residues 43‐63). Molecular dynamics data also suggest that the region of residues 43‐63 is highly flexible and may bind protein partners. Our results suggest that cypin regulates the microtubule cytoskeleton by promoting assembly and stabilizing microtubules.
Metabolic Regulation of Quiescence and Antibiotic Tolerance in Uropathogenic Escherichia coli
Authors: Morrison Josiah J. , DiBiasio Eric C. , Camberg Jodi L.
Published: May 2022

Urinary tract infections (UTIs) pose a global public health threat. Uropathogenic Escherichia coli (UPEC) cause the majority of UTIs and a quarter of infections are recurrent within six months. This may be attributed to UPEC entering a non‐proliferative, or quiescent state that allows some cell populations to evade the host immune response and survive exposure to antibiotics, thus causing reinfection upon cessation of antibiotic treatment. A significant proportion of UPEC isolates, including the classic endemic UPEC lineage CFT073, are unable to proliferate at low cell density in vitro with glucose as the sole carbon source, but proliferate robustly at high cell density. Quiescence at low cell density is inhibited by external cues, such as peptidoglycan fragments, L‐Lysine, L‐Methionine, and succinate, which induce proliferation of quiescent cells and lead to a rapid increase in cell number. Here, we show that quiescent CFT073 cells are tolerant to antibiotics in vitro; however, proliferative cells are susceptible to antibiotics. Furthermore, we show that succinate fails to induce proliferation of cells deleted for sucCD, which encode enzymes that interconvert succinate and succinyl‐CoA in the tricarboxylic acid cycle. This suggests that succinyl‐CoA is limiting in the cell during quiescence and that quiescent cells may have lower metabolic activity. We demonstrate that increased levels of SucAB, which synthesize succinyl‐CoA from alpha ketoglutarate, prevent quiescence and induce proliferation. These results are consistent with a model in which quiescent UPEC cells exhibit reduced metabolism with limited succinyl‐CoA, and this underlies the antibiotic tolerance exhibited during UPEC quiescence in vitro.
Potential Renoprotective Effects of Chronic Remote Ischemic Conditioning in a Rodent Model of Sleep Apnea
Authors: Marcus Noah J. , Madigan Benjamin G. , Harbeck Katherine A. , Gerritts Raina M. , Savage Kalie , Kious Kiefer W. , Schwartz Kelsey S. , Lang James A. , Clayton Sarah C.
Published: May 2022

Sleep apnea (SA) is a chronic condition characterized by recurrent cessation of airflow during sleep. Episodes of SA lead to hemodynamic perturbations, neurohormonal activation, and repeated cycles of hypoxia/reoxygenation. SA is independently associated with chronic kidney disease however the underlying mechanisms are incompletely understood. It is theorized that oxidative stress, inflammation, and activation of pro‐fibrotic signaling in the kidney may underlie this association. Remote ischemic conditioning (RIC) is the application of circulatory occlusion to an extremity to induce transient ischemia. This ischemia stimulates a cascade of signals which spread throughout the body via neural and humoral pathways and stimulates adaptive changes in distant organs that aid in resisting future ischemic insults. There is evidence that remote ischemic conditioning has protective effects in the kidney in acute applications, however it has never been investigated in the context of treatment for chronic disease. We hypothesized that application of RIC in a rodent model of SA (chronic intermittent hypoxia, CIH) would have beneficial effects on regulation of renal hemodynamics and would attenuate inflammation, oxidative stress, and pro‐fibrotic signaling. Adult male Sprague Dawley rats were exposed to CIH or sham for 14 days; two sub‐groups (n=6 per group) received RIC every 48 hours over the final 7 days. Renal perfusion and PO2 were measured at baseline and in response to hypoxia. Cortical tissue was assessed for gene expression related to inflammation, oxidative stress, and fibrosis using qRT‐PCR. CIH resulted in baseline reductions in renal blood flow and PO2 relative to sham in addition to greater hypoxia‐evoked reductions in these measures(p>0.05). No differences were observed in renal blood flow/perfusion or PO2 between RIC‐AIR and RIC‐CIH groups (p>0.05). Cortical expression of IL‐6, IL‐1β, CuZn‐SOD, and Collagen III were increased in CIH vs. sham (p<0.05), whereas no differences in expression where observed between RIC‐AIR and RIC‐CIH groups (p>0.05). These results suggest RIC may confer a salutary effect on regulation of renal hemodynamics and biochemical changes associated with exposure to CIH.
Exercise‐induced Bronchoconstriction does not Change the Configuration of the Maximal Expiratory Flow‐Volume Curve in Adults with Asthma
Authors: Luu Peter , Klimenko Oksana , Klansky Andrew , Noggle Nathan , Petrics Greg , Haverkamp Hans C.
Published: May 2022

Background

Several indices of airflow and volume derived from the maximal expiratory flow volume (MEFV) curve are used to identify and diagnose the severity of respiratory disease. In addition to the objective measures of expiratory airflow and volume, the shape of the MEFV curve is thought to provide information on pulmonary (patho)physiology. In the asthmatic, the MEFV curve is often convex relative to the volume axis (“scooped”). This “scooped” curve is thought to indicate heterogeneous airway emptying during the forced exhalation. The effect of exercise‐induced airway narrowing on the shape of the MEFV curve is not known. The purpose of this study was to determine the effect of exercise‐induced bronchoconstriction (EIB) on the shape of the MEFV curve in asthmatic adults. We hypothesized that the slope‐ratio (SR) index, and thus the overall shape of the MEFV curve would not be affected by EIB.

Methods

Adult male and female asthmatics (n=9; age, 26.8 ± 8.4 yrs; BMI, 27.4 ± 4.3 kg/m2) and non‐asthmatics (n=9; age, 25.4 ± 7.8 yrs; BMI, 21.9 ± 2.0 kg/m2) completed a high‐intensity cycle ergometry bout for six minutes at 85% of peak power. Maximal forced exhalations (MFE) were performed before exercise. Following exercise cessation, subjects remained on the ergometer and performed a MFE every two‐minutes for a total 20 minutes. In each MEFV curve, the SR index was calculated in 1% volume increments between 20% and 80% of the vital capacity. To calculate SR, the tangent and chord slope were generated at each point of interest, and the tangent slope was divided by the chord slope.

Results

Pulmonary function at baseline was lower in asthmatics compared to the control group (FEV1% predicted, 84.9 ± 11.0 vs. 96.5 ± 12.2% in asthmatics vs. control, respectively; P<0.05). In asthmatic subjects, post‐exercise nadir FEV1 decreased by 29.1 ± 13.7% from baseline (3.40 ± 0.74 to 2.47 ± 0.7 L for baseline and nadir, respectively; P<0.001), whereas FEV1 did not change after exercise in control subjects (3.84 ± 0.9 to 3.70 ± 0.8 L for baseline and nadir, respectively). At all time points, the average SR between 20 and 80% of vital capacity was significantly larger in asthmatic than control subjects (1.49 ± 0.02 vs. 1.23 ± 0.02 [SD] for asthma vs. control; P<0.005). However, in both control and asthmatic subjects, post‐exercise SR was not different from baseline at any time point. In asthmatic subjects, average SR between 20 and 80% of vital capacity was 1.49 ± 0.13 at baseline and averaged 1.49 ± 0.02 across all post‐exercise time points (range = 1.48 to 1.53).

Conclusion

In this group of adults with asthma, EIB did not influence the SR index, indicating no change to the overall shape of the MEFV curve. Thus, these findings suggest that inhomogeneities in airway caliber and lung emptying are not magnified during an episode of EIB.
Fractionated Lichen Parmelia vagansExtract Exhibits Two distinct Antiproliferative Activities Against Human Cancer Cells
Authors: Bondarenko Vladimir , Kelley Laura , Argyle Daniel
Published: May 2022

Introduction

Approximately one quarter of all prescription drugs contain active ingredients of plant origins. Lichens have been historically used to treat a multitude of ailments, ranging from headaches during the Middle Ages to dressing wounds before colonial times and much of the potency of lichen relies on the secondary metabolites they produce. The purpose of this project is to analyze antiproliferative properties of the secondary metabolites of lichen Parmelia vagans.

Methods

Lichen extracts were prepared using different organic solvents and further fractionated either by preparative TLC or by reverse‐phase chromatography. We assessed the antiproliferative activity of the fraction using human cancer cell lines. The same number of cells of different cell lines were plated into a 96‐well plate and allowed to adhere overnight. Next day, different dilutions of the enriched fractions were added to the adhered cells and the plate was placed in CO2 incubator for 24 hours. By the end of the incubation time a picture of each well was taken to assess cell confluency and morphology followed by Resazurin assay to analyze the effect of the lichen extract on cell growth and proliferation. After Resazurin assay, the cells in each well were treated with trypsin, suspended in growth medium and counted using Bio‐Rad cell counter to evaluate a percent of live cells.

Results

Assessment of antiproliferative activity of the TLC and reverse‐phase chromatography fractions revealed two separate peaks of inhibitory activity against cancer cell. The fraction obtained from P.vagans extract significantly suppress (30% ‐ 80%) the growth of human lung carcinoma A549. We also found that cancer cells treated by the lichen extracts results in their morphological changes that resemble an activation of apoptosis.

Discussion

Our findings indicate that from P.vagans produces secondary metabolites that slow cancer cells growth and proliferation. Future experiments will be focused on isolation and identification of the compounds from P.vagans that are responsible for the inhibition of cancer cells growth. Another important aspect of the future research is to identify the cellular target(s) of the antiproliferative compound(s) and its molecular mechanism of action. I summary, the lichen’s secondary metabolites may hold a vast medicinal potential and could be a viable source of a novel of anticancer drugs.
Effects of Intra‐arterial Fentanyl and Codeine on Medullary Respiratory Network Organization
Authors: Bolser Donald C. , Shen Tabitha , Olsen Wendy , Nuding Sarah , Segers Lauren S. , Rose Melanie J. , Valarizo Pierina , Wang Wei C. , Morris Kendall F.
Published: May 2022

The actions of mu‐opioid agonists to depress breathing are currently controversial, encompassing several proposed mechanisms that are focused on preferential effects in specific locations in the brainstem. We hypothesized that opioids have multiple actions at the respiratory network scale which include loss of synchrony and/or network dynamics. Experiments were conducted in 4 anesthetized, spontaneously breathing and 11 decerebrated, artificially ventilated, paralyzed and vagal intact cats. In spontaneously breathing animals, EMGs were recorded from diaphragm, parasternal, abdominal and upper airway muscles. In paralyzed animals, neurograms were recorded from phrenic and abdominal nerves. Fentanyl or codeine were administered via the vertebral artery (i.a.; fentanyl, 1‐10 ng/kg, codeine 1‐3000 μg/kg) while simultaneously recording from multiple medullary neurons. Analyses included assessment of autocorrelation histograms (ACH), significant features in cross correlation histograms (CCHs), and discharge identity (phenotype of firing frequency of a neuron during the breathing cycle). Qualitatively similar opioid‐induced changes in neuron firing rates, ACHs, CCHs, and discharge identity were observed during either fentanyl or codeine dose responses. Some neurons lost or gained excitability during the dose responses. ACHs of some neurons exhibited changes in spiking periodicity. Features in CCHs changed during administration of opioids including loss of some central or offset peaks. Detectability index (a metric of the magnitude of significant functional interactions) in some neuron pairs increased during opioid administration. Further, the discharge identity of many neurons changed during the dose responses with most of these changes occurring in neurons with tonic discharge during breathing. The extent of these changes strongly suggest that opioids induce significant changes in network organization including reduced synchrony and altered discharge identity of network members. These changes can occur at low doses of opioids that do not profoundly alter the breathing pattern.
Intestinal epithelial specific AhR deficient mice exhibit increased severity of experimentally induced chronic colitis
Authors: Qazi Aisha , Kumar Anoop , Sharma Anchal , Calzadilla Nathan , Weber Christopher R. , Mongan Kai , Saksena Seema , Dudeja Pradeep K. , Alrefai Waddah A. , Gill Ravinder K.
Published: May 2022

Inflammatory Bowel Diseases (IBD), including Crohn’s Disease (CD) and ulcerative colitis (UC), are associated with chronic inflammation of the gastrointestinal tract. While it is a multifactorial disease, recently the aryl hydrocarbon receptor (AhR) has been identified as a susceptibility gene for IBD. AhR is a ubiquitously expressed transcription factor and its activation results in xenobiotic detoxification by CYP1A1 and CYP1B1, as well as induction of anti‐inflammatory pathways such as IL‐10 and IL‐22. In the gut, AhR is vital in maintaining immune cell populations, proliferation, and motility. Previous studies utilizing constitutive knock out of AhR in IECs showed exacerbated inflammation in acute DSS model. How intestinal epithelial cell (IEC) AhR deficiency impacts chronic colitis resembling human IBD is not yet known. Here, we investigated the effect of removing of IEC AhR in an inducible manner utilizing DSS induced chronic colitis (resembling Th1/Th17 and Th2 infiltration as seen in CD patients) and elucidated underlying mechanisms.

Methods

AhRfl/fl x Villin‐CreERT mice received intraperitoneal injections of Tamoxifen (AhRΔIECKO) to induce knockout or the vehicle (WT) for 5 days. The mice were given water or 1.0% DSS for 5 weeks, alternating weekly with water. Distal colon tissue samples were taken for myeloperoxidase (MPO) assay, immunostaining, and histology to assess severity of inflammation. Additionally, distal colon mucosa samples were collected for mRNA and protein expression. Serum was used to assess levels of LPS through ELISA.

Results

AhRΔIECKOmice given DSS had increased severity of colitis as evidenced by i) a significant decrease in the colon length when compared to the WT given DSS; ii) an increase in mRNA expression of pro‐inflammatory cytokines, IFN‐ γ and IL‐1□; iii) an increase in MPO activity in the distal colon; iv) a significant increase in the spleen to body weight ratio; and v) a significant increase in histological scores. Interestingly, DSS treatment caused an increase in lipopolysaccharide (LPS) in the serum that was significantly higher in AhRΔIECKO as compared to WT indicative of increased paracellular permeability. There were no significant differences observed in the protein expression of Occludin, Claudin‐2, and ZO‐1 between AhRΔIECKO and WT DSS groups, however AhR KO mice given DSS had significantly increased phosphorylated ERK1/2 (pERK normalized to total ERK) levels which may contribute to the increased permeability and severity of inflammation.

Conclusion

Deletion of AhR from intestinal epithelial cells increases severity of chronic colitis associated with increased permeability possibly through ERK1/2 dependent mechanisms.
Preconditioning Exercise Protects Against Tumor‐Mediated Muscle Dysfunction in the Male Mouse
Authors: Parry Traci L. , Brantley Jason (TJ) , Tichy Louisa
Published: May 2022

Background

Cancer is associated with cardiac and skeletal muscle wasting. While exercise has been shown to be myoprotective, less is known about the most effective “dose” of exercise in terms of intensity, timing, and duration. Therefore, the purpose of this study was to determine the effects of preconditioning exercise – exercise completedJ before tumor bearing ‐ as a protective measure for tumor‐mediated muscle wasting.

Methods

Male mice were randomly separated into four groups: sedentary non‐tumor bearing (SED+NT), sedentary tumor bearing (SED+T), treadmill exercise non‐tumor bearing (TM+NT), and treadmill exercise tumor bearing (TM+T). Mice underwent an 8‐week progressive treadmill exercise training protocol (TM) or remained sedentary (SED). At the end of the preconditioning treadmill training protocol, mice remained sedentary for 4 weeks. During this 4 week sedentary period, mice were implanted with tumor cells (T group; 1x105 LLC cells in flank) or remained non‐tumor (NT). To determine whether 8 weeks of exercise preconditioning could preserve cardiac and skeletal muscle function, mice underwent echocardiogram and grip strength analyses at baseline, 8‐week, and 12‐week time points.

Results

Tumor bearing resulted in significant muscle dysfunction. SED+T showed a significant decrease in fractional shortening (p<0.05) and grip strength (p<0.05) when compared to all other groups. This coincided with SED+T’s significantly smaller heart masses and skeletal masses (p<0.05 vs to all other groups). Interestingly, preconditioning exercise (exercise prior to tumor bearing) appeared to offer myoprotection since TM groups were not significantly different from SED+NT for fractional shortening and grip strength (p>0.05). This data was in step with preserved heart mass and skeletal muscle mass in TM+T (p>0.05 vs SED+NT).

Conclusion

These data indicate that exercise initiated prior to tumor bearing can protect the musculature from tumor‐mediated muscle dysfunction. This may be attributed to slowed muscle wasting as evidenced by maintenance of muscle mass in TM+T groups. These data make an important case for the long‐term benefits of an active lifestyle that last even after exercise is stopped (i.e., upon diagnosis and treatment). Such information is critical in elucidating the most effective “dose” of exercise for cancer survivors.
Keratinocyte‐associated Protein 3May Participate in the Stress Response to Impact Adiposity
Authors: Szalanczy Alexandria M. , Giorgio Gina , Goff Emily , Seshie Osborne , Grzybowski Michael , Klotz Jason , Roberts Mackenzie , Geurts Aron M. , Redei Eva E. , Solberg Woods Leah C.
Published: May 2022

Although obesity rates have been steadily climbing the last few decades, the genetics underlying the disease are poorly understood. Previous work in our lab identified Keratinocyte‐associated protein 3(Krtcap3) as a candidate gene for adiposity. To characterize the role of Krtcap3in adiposity, we developed an in vivowhole‐body knock‐out (KO) rat model of Krtcap3,hypothesizing that KO rats would have greater adiposity compared to wild‐type (WT) rats.

In our first in vivostudy, primarily occurring before the COVID‐19 shut down, we found that female KO rats have significantly increased food intake and fat mass compared to WT rats, supporting a role of Krtcap3in adiposity. We conducted a second study in 2020‐2021, when traffic in the vivarium was lighter due to COVID‐19, and were unable to replicate the initial differences in food intake or fat mass between WT and KO rats. Compared to Study 1, WT rats ate more and had a corresponding increase in adiposity, with no corresponding change in KO rats. We hypothesized that with decreased traffic the environment in Study 2 became less stressful for the WT rats, who then began to eat more. KO rats appeared to be resistant to the effects of chronic stress, and thus ate similar amounts between the two studies. Supporting this difference, measurements of corticosterone (CORT) in rats between Study 1 and Study 2 revealed that WT rats had increased CORT in Study 1 relative to Study 2, with no differences in CORT between the studies for the KO rats.

To determine if Krtcap3may be responsive to stress, we measured Krtcap3expression in key stress response tissues between unstressed rats and rats injected with a high dose of CORT (40 mg/kg). We found that Krtcap3is highly expressed in the pituitary gland, with expression significantly decreased following the CORT injection. Although expression of Krtcap3is very low in blood, we also saw a significant decrease in Krtcap3expression in the blood after the CORT injection. In contrast, Krtcap3has low expression in the adrenal glands, with no change after the CORT injection. These results indicate that Krtcap3may play a role in the negative feedback regulation of the HPA axis, potentially explaining the differences in food intake and fat mass between studies 1 and 2 discussed above. Future studies will seek to confirm that decreased Krtcap3expression is protective against chronic stress, and that this can impact feeding behavior to influence adiposity.
Identification and Characterization TSSK6 Activating Co‐chaperone (TSACC) in Skeletal Muscle
Authors: Shourbagi Tello Tala , Waddell David
Published: May 2022

Skeletal muscle atrophy is a physiological condition that is caused by a range of conditions, including immobilization, denervation, spinal cord injury, and aging. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated the gastrocnemius muscle from mice following 3 and 14 days of sciatic nerve transection. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that TSSK6 Activating Co‐chaperone (TSACC) is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that TSACC is expressed in muscle, the predicted TSACC cDNA was cloned from cultured muscle cells, as was a novel TSACC splice variant that contains an additional exon. Quantitative PCR (qPCR) was performed to assess TSACC expression levels of the full‐length and novel splice variants in proliferating and differentiated muscle cells. The results demonstrate that TSACC expression levels are relatively low in proliferating myoblasts but show significantly elevated expression in differentiated myotubes. In addition, characterization of the transcriptional regulation of TSACC was assessed by fusing fragments of the proximal promoter located immediately upstream of the start of transcription with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Interestingly, there are several predicted E‐box elements in the proximal promoter region of the TSACC gene further suggesting that TSACC may be regulated by muscle‐specific transcription factors. To identify a possible role for TSACC in skeletal muscle, sub‐cellular localization was assessed by fusing TSACC with GFP and expressing the fusion protein in cultured muscle cells, revealing that TSACC is localized predominantly to the cytoplasm. The discovery that TSACC is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.