bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination and Characterization of Genes that Encode the Nicotinate Dehydrogenase and 6‐Hydroxynicotinate Dehydrogenase Complexes within the Nicotinic Acid Degradation Pathway by Bacillus Niacini
Authors: Haines Emilee R. , Semersky Zoë , Borgelt Luke , Snider Mark J.
Published: May 2022

N‐Heterocyclic aromatic compounds (NHACs) are constituents of many manufactured products such as pharmaceuticals, vitamins, and personal care products. Their widespread use has led to contamination of groundwater and soils, posing environmental concerns. Therefore, mechanisms of how NHACs are catabolized by bacteria have been studied to develop bioremediation strategies. Nicotinic acid (NA) is an example of an NHAC where its catabolism has been studied in a variety of microorganisms, such as Pseudomonas, Eubacterium,and Bacillus. While NA degradation has been elucidated in microorganisms such as P. putidaand E. barkeri,the majority of B. niacini’sNA catabolism pathway is relatively uncharacterized. Previous work identified a nicgene cluster that had been found to be upregulated in the presence of NA and 6‐hydroxynicotinic acid (6‐HNA), leading to the hypothesis that the set of protein subunits NicA1, NicA2, NicB1, NicB2 and CoxG are involved in the first two hydroxylation steps of B. niacini’sNA degradation pathway. These proteins are thought to form distinct multi‐subunit protein complexes with nicotinate dehydrogenase (NDH) and 6‐hydroxynicotinate dehydrogenase (6‐HNDH) activities. Due to the involvement of the molybdopterin cofactor present in the B subunits, recombinant forms of these enzymes have not shown activity when expressed and purified from E. coli. A vector specific for expressing the genes in Pseudomonas (pBBr) was engineered to contain the set of five B. niacini genes hypothesized to code for NA hydroxylating enzymes and was transformed into Pseudomonas fluorescens 1f2,a strain shown to be unable to degrade nicotinic acid naturally. Analysis of the media upon incubation of the Pf‐1f2 transformants with NA or 6‐HNA in minimal media by C‐18 chromatography (HPLC) showed rapid and complete hydroxylation of NA to 6‐HNA, as well as the hydroxylation of 6‐HNA to 2,6‐dihydroxynicotinic acid (2,6‐DHNA). This is the first evidence of activity observed from recombinant B. niacini“NicAB” protein complexes. However, the combination of gene‐encoded subunits that compose the NDH and 6‐HNDH complexes is currently unknown. Using deletion mutagenesis, specific genes cloned into the pBBr vector will be removed to recombinantly express variant complexes that lack a specific subunit to determine the substrate specificity and oligomeric structure of the complexes.
Implication of integrins in eptifibatide interference with platelet stimulation of ovarian cancer
Authors: Isingizwe Zitha Redempta , Kennedy Amy L. , Sjoelund Virginie , Benbrook Doris M.
Published: May 2022

Significance

A third of ovarian cancer patients have high platelet counts at diagnosis resulting in reduced survival compared to patients with low/normal platelet counts. Current studies suggest that platelets and ovarian cancer induce each other in a feed‐forward loop.

Objective and Hypothesis

The objectives were to: 1) evaluate differences in platelet counts and aggregation between healthy and newly‐diagnosed ovarian cancer patients; 2) develop an experimental model of the pathological interactions between platelets and ovarian cancer spheroids; and 3) identify pathways affected by these interactions and ways to intervene. Our hypothesis is that high platelet counts of ovarian cancer patients are associated with hypercoagulability, and inhibiting platelet aggregation reduces platelet‐mediated effects on ovarian cancer spheroids.

Materials and Methods

Complete blood count was evaluated between healthy controls and newly‐diagnosed ovarian cancer patients before treatment. A platelet aggregation assay was performed to assess platelet physiology between the two groups. The molecular mechanism of platelet effects on ovarian cancer spheroid properties was evaluated using mass spectrometry and bioinformatics analysis of a magnetic 3D cancer spheroid assay, in the presence and absence of eptifibatide, a potent platelet inhibitor.

Results

While ovarian cancer patients had significantly higher platelet counts than healthy controls, platelet aggregation profiles and parameters were similar between the two groups. Incubation of platelets with cancer spheroids altered spheroid size and density in an eptifibatide‐sensitive manner. Co‐incubation of platelets with ovarian cancer spheroids activated the LXR/RXR pathway, a known down‐stream effector of the integrin receptor that eptifibatide targets.

Conclusion

Platelet count does not predict platelet hypercoagulability in ovarian cancer patients. Pathological interaction between platelets and cancer cells can be mimicked in co‐culture, and eptifibatide inhibits these pathological interactions, suggesting that eptifibatide is a candidate drug for prevention of ovarian cancer and thrombosis in ovarian cancer patients. The LXR/RXR pathway is a likely mediator of the eptifibatide interference in platelet‐cancer interaction, and represents a candidate target for reducing platelet‐mediated adverse events in ovarian cancer patients.
USP13 deficiency aggravates cigarette smoke‐induced alveolar space enlargement through stabilization of TXNIP
Authors: He Jinshan , Gregory Alyssa D , Tran Kevin C , Tamaskar Arya S , Wei Jianxin , Zhao Jing , Zhao Yutong
Published: May 2022

Deubiquitinating enzymes regulate cellular function through deubiquitination and stabilization of substrate proteins. We have shown that USP13 deficiency promotes lung inflammation. Cigarette smoke (CS) causes lung connective tissue disruption and oxidative stress in lung cells, resulting in the development of emphysema; however, the role of USP13 in the CS exposure‐induced emphysema has not been well studied. USP13‐deficient mice and wild type mice were exposed to 4 unfiltered cigarettes for 5 days each week, over the course of 6 months. While emphysema was not observed in wild type mice, CS exposure increased γ‐H2AX levels in lung tissues, suggesting a model of mild smoke exposure was established. Under the mild CS exposure condition, USP13‐deficient mice show significant increases in alveolar CL, indicating that USP13‐deficient mice are susceptible to CS‐induced alveolar enlargement. We have shown that USP13 knockout reduced fibronectin expression in lungs. Further, we found that collagen levels were reduced in USP13‐siRNA‐transfected lung fibroblast cells. It is possible that a loss of connective tissue function contributes to alveolar enlargement in USP13‐deficient mice. Oxidative stress is a hallmark of CS‐induced emphysema. We investigated the role of USP13 in the expression of an oxidative stress markers, named thioredoxin interacting protein (TXNIP), which modulates cellular redox states and NLRP3 inflammasome activity. TXNIP expression levels have been shown to be decreased in smokers compared with non‐smokers. TXNIP is unstable in the presence of protein synthesis inhibitor cycloheximide (CHX). Pretreatment with proteasome inhibitor, MG‐132, attenuated TXNIP degradation, suggesting that TXNIP degradation is mediated by the proteasome. Knockout or knockdown of USP13 in lungs or lung fibroblast cells significantly reduced TXNIP levels. TXNIP degradation is increased in USP13 knockdown cells. Further, the reduction of TXNIP was observed in USP13‐inhibitor‐treated mouse lung epithelial cells. This study provides evidence showing that USP13 deficiency plays a role in alveolar space enlargement.
Novel HM12, A Selective Inhibitor of L‐/T‐type Calcium Channels, Averts Doxorubicin Cardiotoxicity in Male Wistar Rats
Authors: Kale Oluwafemi E. , Gündüz Miyase G. , Awodele Olufunsho , Osonuga Olubunmi , Ekor Martins
Published: May 2022

Doxorubicin is a cytotoxic anthracycline anticancer drug that is limited by its cardiotoxic action. This study sought to investigate the cardiopreventive potential of HM12, a 1,4‐dihydropyridine (DHP) derivative (CCB) against doxorubicin induced cardiotoxicity in male Wistar rats. HM12 was synthesized as a DHP‐based hexahydroquinoline derivative (2‐[(2‐methylacryloyl)oxy]ethyl 4‐(3,5‐dibromo‐2‐hydro[1]xyphenyl)‐2,6,6‐trimethyl‐5‐oxo‐1,4,5,6,7,8‐hexahydroquinoline‐3‐carboxylate) according to the modified Hantzsch reaction via condensation reactions under microwave irradiation. HM12 structure was confirmed using infrared, proton, and carbon‐13 nuclear magnetic resonance, and mass spectra. The CCB activity was evaluated using whole‐cell patch‐clamp assays. Further, fourty‐eight adult male Wistar rats were divided into eight groups of 6/group and received distilled water saline; doxorubicin (DOX, 20 mg/kg, i.p.), HM12 lower (HM12LD) or upper (HM12UD) dose (5 or 20 mg/kg, p.o.), nifedipine (NFD, 20 mg/kg), and their combinations. All rats were sacrificed 24 h after the last administration and changes in blood pressure (BP), heart rate (HR), biochemical, oxidative stress indices, and heart histology were assessed. DOX cardiotoxicity produced spontaneous increased (p<0.05) BP (baseline 106.5/84 mmHg (day 1) versus 170/173.5 mm/Hg (59.6/72%, day 6) and 173.5/167 mmHg (98.8/69.1%, day 7) with altered HR (baseline 99 beats per minutes versus 167.5 bpm (69.1%, day 7). Also, DOX‐treated rats showed a significant increase in tumor necrosis factor‐α (TNF‐α), c‐reactive protein (CRP), and lactate dehydrogenase (LDH). This was accompanied by an increase (p<0.05) in cardiac malondialdehyde (MDAc) and decreased reduced glutathione (GSHc) levels, and activities of catalase (CATc) and superoxide dismutase (SODc). However, HM12UD + DOX had changes BP (baseline 106.5/96 mmHg (day 1) versus 181/179.5 mmHg (70.1/87%, day 6) versus 135/127 mmHg (26.8/32.3%, day 7) with HR (baseline 93.5 beats per minutes versus 164.5 bpm (75.9%, day 6) to 127 bpm (35.8%, day 7). Also, HM12UD was effective against IL‐6, CRP, LDH but not TNF‐α. Further, HM12UD significantly reversed DOX‐induced MDAc by elevating antioxidants GSHc levels as well as activities of CATc and SODs in the treated animals. Additionally, HM12UD averted histopathological changes observed in the rat heart due to DOX intoxication. Overall, the present study conferred new evidence on the protective effects CCBs, in particular, HM12 on DOX‐induced cardiotoxicity, oxidative stress, tumor biomarkers, and cardiac histological changes in rats.
Angiogenic role of lung pericytes in experimental viral lung injury
Authors: Hung Chi , Altemeier William
Published: May 2022

Lung pericytes are perivascular stromal cells that surround microvascular endothelial cells. While their roles in fibrogenesis have been explored, their angiogenic functions in experimental lung injury and repair have not been characterized. RNAseq analysis of primary lung pericytes activated ex vivoreveals upregulation of novel angiogenic pathways. One gene of interest we identified is angiopoietin‐L4 (Angptl4). Stromal cells, particularly pericytes, are the primary sources of angiopoietin‐L4 in the lung. ANGPTL4 has been shown to regulate angiogenesis in cancer biology and retinal diseases. We have shown that lung pericyte‐derived ANGPTL4 promotes network formation and monolayer permeability in endothelial cells in vitro. We hypothesize that knockdown of ANGPTL4 in vivo is protective in acute viral lung injury by promoting endothelial integrity and decreasing microvascular leak during injury and resolution. Transgenic mice with inducible genetic deletion of Angptl4 and littermate controls were inoculated with mouse‐adapted human influenza virus (A/PR/8). Mice lacking Angptl4 showed decreased measures of lung injury and protein leak during the acute and resolution phases of lung injury. Future directions include identification of ANGPTL4 signaling pathways and cellular targets in the lung, and characterization of the mechanisms by which pericyte‐derived ANGPTL4 leads to persistence of endothelial dysfunction or activation in viral lung injury.
Interactions of TUG‐UBL1 and Insulin‐Implications for Glucose Uptake
Authors: Kohout Megan , Gentile Lisa
Published: May 2022

TUG‐UBL1 (Tether Containing UBX Domain for GLUT‐4 Ubiquitin Like 1) is a glucose transporter 4 (GLUT4) regulating protein which facilitates the release of GLUT4 transport vesicles when insulin stimulation is present. To understand the role of TUG‐UBL1 in glucose metabolism, especially for type two diabetic patients for whom increases in insulin concentration do not correlate with increased glucose uptake, we explored the research question of whether TUG‐UBL1 binds to insulin. Initially, models of full‐length TUG‐UBL1 (amino acids 1‐473), the GLUT4 binding domain of TUG‐UBL1 (amino acids 86‐337), and the insulin heterodimer were built using PHYRE2. All of these structures were further refined with DeepRefiner, and then validated using MolProbity. Including all‐atom contacts, full‐length TUG‐UBL1 scored in the 99th percentile, the GLUT4 binding domain of TUG‐UBL1 scored in the 97th percentile, and the insulin heterodimer scored in the 97th percentile. Insulin binding to both the full‐length TUG‐UBL1 domain and the GLUT4 binding domain were then explored with HawkDock. In both cases, favorable binding energies were observed with predictions of GLUT4 binding region of TUG‐UBL1 binding to insulin at ‐43.31 kcal/mol and full‐length TUG‐UBL1 binding to insulin at ‐32.31 kcal/mol, with predictions favoring insulin binding to the GLUT4 binding domain. To support these computational findings, the GLUT4 binding domain of TUG‐UBL1 is being over‐expressed and purified for use in intrinsic tryptophan fluorescence binding studies with insulin. The finding of TUG‐UBL1 binding to insulin are interesting in light of the potential implications for the care type two diabetic patients as binding characterization could lead to further mechanistic understanding of insulin resistance and possible therapeutic development.
Tumor Microenvironment Characterization using a Spatial Multiomic Assay to Simultaneously Profile Protein and Gene Expression in FFPE Tumors
Authors: Chew Jennifer , Uytingco Cedric , Anaparthy Naishitha , Chiang Jun , Galonska Christina , Ganapathy Karthik , Hatori Ryo , Hermes Alexander , Katiraee Layla , Katsori Anna‐Maria , Nitsch William , Roelli Patrick , Shuga Joe , Spalinkas Rapolas , Turkekul Mesruh , Veire Benton , Walker Dan , Sheldon Jyoti , Weisenfeld Neil , Williams Stephen , Bent Zachary , Stoeckius Marlon
Published: May 2022

The tumor microenvironment (TME) is composed of highly heterogeneous structures and cell types that dynamically influence and communicate with each other. The constant interaction between a tumor and its microenvironment plays a critical role in how the cancer develops, progresses, and responds to therapies. Traditionally, Hematoxylin and Eosin (H&E) and immunohistochemistry staining have been used to annotate and characterize tissues and associated pathologies. Recent single analyte approaches spatially interrogate targeted or transcriptome‐wide expression of RNA in tissue sections, while others capture phenotypes using a limited number of protein markers. However, for a more comprehensive understanding of the unique characteristics of cell types, cell states, and cell‐cell interactions within the TME, analysis of multiple analytes is necessary.

Here we demonstrate a novel, streamlined multiomic spatial assay that integrates histological staining and imaging with simultaneous transcriptome‐wide gene expression and highly multiplexed protein expression profiling from the same formalin‐fixed paraffin embedded (FFPE) tissue section. In short, tissue sections from archived FFPE samples were placed on slides containing arrayed capture oligos with unique positional barcodes. The H&E stained tissues were then imaged, followed by incubation with transcriptome‐wide probes and a high‐plex DNA‐barcoded antibody panel containing intra‐ and extracellular markers. Transcriptome probes and antibody‐barcodes were then spatially captured on the slide and converted into sequencing‐ready libraries. Our data analysis and interactive visualization software enable interrogation of all data layers (H&E morphology, RNA, protein) from the same tissue section.

We apply this method to simultaneously measure gene and protein expression within the TME of human breast cancer and melanoma FFPE samples using whole transcriptome probes and an immune‐oncology antibody panel. The data enables comparison and correlation of multiple analytes and their patterns within the same sample section. In addition, this simultaneous detection enables marker‐guided regional selection and differential gene expression analysis on the defined areas. Taken together, our data demonstrates that a spatially resolved, multiomic approach provides a more comprehensive understanding of cellular behavior in and around tumors, yielding new insights into disease progression, predictive biomarkers, drug response and resistance, and therapeutic development.
Regional Tongue Anatomy and Corresponding Kinematics During Chewing and Drinking in the Pig
Authors: Olson Rachel A. , Montuelle Stephane J. , Curtis Hannah , Chadwell Brad , Williams Susan H.
Published: May 2022

Chewing and drinking are fundamental oral behaviors for nutrient ingestion in mammals. They consist of coordinated tongue and jaw movements to ingest, transport, and swallow. However, differences in bolus properties and intraoral management suggest the potential for differences in the movements and deformations performed by the tongue throughout the cycle. Additionally, regional heterogeneity of muscle fascicle orientation is observed throughout the body of the tongue which likely imposes different constraints on the capabilities of different regions to move and deform. The goal of this study is to determine how regional differences in tongue movements and deformations during chewing and drinking in pigs relate to their corresponding muscle fascicle anatomy. Muscle fascicle orientations were quantified using diffusible iodine contrast‐enhanced computed tomography (diceCT) scans relative to the sagittal plane. Cycle parameters and corresponding regional tongue deformations were analyzed during chewing and drinking using X‐ray Reconstruction of Moving Morphology (XROMM) with additional markers implanted in the soft tissues of the tongue (Ohio University Institutional Animal Care and Use Committee: protocol #12‐U‐009). Examination of muscle fascicles highlights clear regional differences in their orientations. XROMM results demonstrate differences in kinematic parameters between chewing and drinking cycles, with drinking cycles exhibiting a smaller amount of jaw movement for all degrees of freedom used during both behaviors: Tx (condylar protraction‐retraction), Rz (jaw pitch), and Ry (jaw yaw). Tongue protraction‐retraction was greater during chewing than drinking. Additionally, during both behaviors, a near‐constant total tongue length was observed, but regional contributions to total tongue length varied significantly between behaviors, with higher magnitudes of length deformations observed during chewing. This work demonstrates that different regions of the tongue do move and deform in different ways, and that they vary between chewing and drinking behaviors. The observed deformations also correspond to the underlying muscle fascicle anatomy as revealed by diceCT.
Using targeted LC‐MS/MS‐based metabolomics to measure a broad constellation of bile acids/salts in disorders of human health
Authors: Horvath Thomas D. , Haidacher Sigmund J. , Hoch Kathleen M. , Engevik Melinda A. , Haag Anthony M. , Engevik Amy C.
Published: May 2022

Background

Bile is an important liver secretion which aids the digestion of fats in the intestine. Bile acids are the major organic solute component of bile and are synthesized from cholesterol in the liver and stored in the gallbladder. After secretion in the intestine, bile acids are re‐absorbed in the distal small intestine (ileum). Imbalances in the reabsorption of bile acids can lead to bile acid diarrhea when bile acid malabsorption occurs. Conversely, when there is an increase in bile acid absorption hepatic disorders such as nonalcoholic fatty liver disease can arise. A wide array of distinct bile acid structures have been identified in vertebrates. Developing and refining experimental tools to identify the role of bile acids in health and disease will enhance our ability to treat complex disorders impacting multiple organ systems.

Methods & Results

We have expanded our existing reverse‐phase liquid chromatography‐tandem mass spectrometry (LC‐MS/MS)‐based targeted metabolomics method to accommodate a simple‐to‐use standards kit that includes reference standards for 16 bile acids/salts and their corresponding deuterated isotopologs as internal standards. LC‐MS/MS method enhancements include scheduled Selected‐Reaction Monitoring (sSRM) to reduce instrument duty cycle, and carefully tailored chromatographic conditions to resolve the following groups of structurally similar or isobaric bile acids/salts: i) Glycocholic Acid (GCA) and Taurocholic Acid (TCA); ii) Lithocholic Acid (LCA), Glycolithocholic Acid (GLCA), and Taurolithocholic Acid (TLCA); iii) isobaric Cholic Acid (CA) and beta‐Muricholic Acid (b‐MCA); iv) isobaric Ursodeoxycholic Acid (UDCA), Chenodeoxycholic Acid (CDCA), and Deoxycholic Acid (DCA); v) isobaric Glycoursodeoxycholic Acid (GUDCA), Glycochenodeoxycholic Acid (GCDCA), and Glycodeoxycholic Acid (GDCA); and vi) isobaric Tauroursodeoxycholic Acid (TUDCA), Taurochenodeoxycholic Acid (TCDCA), and Taurodeoxycholic Acid (TDCA). Our tissue processing and metabolite extraction procedures have been optimized for hepatic and fecal tissues. Individuals with inactivating mutations in the molecular motor, Myosin Vb, have severe diarrhea and often present with liver cholestasis. We used an animal model of loss of Myosin Vb to determine alterations in bile acid composition in the distal small intestine and liver. Whole segments of the ileum and liver were processed in methanol and analyzed by LC‐MS/MS.

Conclusion

We have developed a targeted metabolomics method to accurately identify and quantify bile acids by LC‐MS/MS and this technique can be used to investigate changes in bile acid composition in gastrointestinal disorders.
Evaluating the Effects of Chondroitin Sulfate and Hyaluronic Acid in Modifying the Properties of Collagen‐Based Matrices
Authors: Cortes‐Medina Marcos G. , Bushman Andrew , Beshay Peter , Adorno Jonathan J. , Menyhert Miles M. , Hildebrand Riley , Avendano Alex , Song Jonathan W.
Published: May 2022

The extracellular matrix (ECM) enveloping cells in living tissue is comprised of signaling and structural support molecules. The ECM is vital to maintenance of normal tissue as it controls cell signaling and is a semiporous barrier to interstitial fluid flow. Two important types of ECM molecules are fibrillar collagen (e.g. type I) and glycosaminoglycans (GAGs). In certain solid tumors, hyaluronic acid (HA) and chondroitin sulfate (CS) are two types of GAG molecules that are known to be unusually abundant. These GAGs are negatively charged with the propensity to imbibe aqueous fluid and promote tissue swelling. While many studies have described the effect each molecule has on shaping ECM properties, studies describing how the CS and HA interact with each other are much less common. This study uses an integrated approach to characterize the effects of HA and CS on fluid transport (hydraulic permeability), stiffness (indentation modulus), collagen polymerization kinetics (turbidity), and matrix microarchitecture (pore size and fiber radius). Utilizing a microfluidic approach to study hydraulic permeability observed that the addition of GAGs decreased convective fluid transport. Indenting the hydrogels with a macroscale indenter demonstrated no changes in stiffness when adding CS, while HA, and CS/HA increased hydrogel stiffness. Analysis of confocal reflectance microscopy images showed increases in both pore size and fiber radius with the addition of both GAG molecules. Finally, turbidity measurements concluded that adding GAGs to a collagen gel accelerated its polymerization, with the change in fiber radius being the primary determinant in enhanced fibril formation. This study provides additional evidence that GAGs play an important role in defining physical properties of the ECM and demonstrates further ways into controllably modifying collagen based hydrogels via the addition of native ECM molecules.
Effects of a Treadmill Workstation on Coagulation Potential
Authors: DeJonge Sydney R. , Del Pozzi Andrew , Nagelkirk Paul R.
Published: May 2022

Introduction

Approximately 2.7 million blood clot‐induced events occur each year, including most strokes and heart attacks. An upright, seated position reduces blood flow, promotes blood pooling, viscosity, and may active the coagulation system. Thus, occupational environments that include prolonged periods of seated work may contribute to the formation of a thrombus. Use of an active workstation may beneficially impact thrombolytic potential.

Purpose

To observe the effects of low intensity walking at a treadmill workstation on changes in coagulation potential compared to use of a traditional, sedentary workstation.

Methods

Five apparently healthy college‐aged students (n= 4 men, 1 woman; 22 ± 2 yrs; 27.3 ± 6.2 kg/m2) completed the same series of mental tasks at a sedentary workstation and while walking one mile‐per‐hour at a treadmill workstation. Blood samples were collected at baseline and immediately after completion of tasks and analyzed for the following: prothrombin time (PT), active partial thromboplastin time (APTT), factor VII (FVII), factor VIII (FVIII), and fibrinogen (FGN).

Results

The walking condition elicited greater responses in FVII compared to the sedentary condition at baseline. Additionally, there was a non‐significant trend observed for FVIII. All data presented in Table 1.

Conclusion

Low intensity walking at a treadmill workstation can attenuate prothrombotic changes that are associated with completing tasks at a sedentary workstation.
Rab11A forms a complex with Interferon Regulatory Factor 6 in recycling endosomes
Authors: Antiguas Angelo , Dunnwald Martine
Published: May 2022

Interferon Regulatory Factor 6 (IRF6) is a transcription factor essential for regulating keratinocyte migration, proliferation and differentiation. Structurally, the IRF6 protein includes a DNA binding domain (DBD), a protein binding domain (PBD), and a serine rich autoinhibitory region (SRR). Historically, IRF6 has been assumed to play its role by regulating gene expression, however growing evidence suggest that some of its functions may be dependent on protein‐protein interactions. Although the effect of such interactions on the regulated proteins remains to be elucidated, some groups have proposed a protective role for IRF6 preventing protein degradation. Alternatively, we hypothesize that IRF6 may also play a scaffolding function regulating the subcellular localization of effector proteins. We recently demonstrated that IRF6 is required for the delivery of E‐cadherin to the plasma membrane. More specifically, we found that recycling of E‐cadherin was defective in absence of IRF6. Interestingly, the total E‐cadherin levels were not altered in the absence of IRF6, and under culture conditions used to test keratinocyte adhesion behavior, IRF6 was not detected in the nucleus. To explore the molecular mechanism used by IRF6 to regulate E‐cadherin at the plasma membrane, we investigated whether IRF6 colocalized with NME1 and NME2, two proteins known to internalize E‐cadherin towards recycling endosomes and regulate the secretory pathway, respectively. We found that in our experimental conditions NME2 does not colocalize with IRF6. However, by immunofluorescence, IRF6 and NME1 colocalize and their mutual coimmunoprecipitation suggest they are part of the same complex. Because NME1 is necessary for E‐cadherin recycling, we hypothesized that IRF6‐NME1 regulates recycling endosome function. To test our hypothesis, we evaluated the interaction between E‐cadherin, IRF6 and Rab11A, a required promoter of recycling endosome. Our results show that Rab11A colocalizes with all these proteins in recycling endosomes, and that IRF6, Rab11A and NME1 coimmunoprecipitated with each other, suggesting that they belong to the same complex. Overall, our results suggest that IRF6 participate in a complex with Rab11A and NME1, potentially modulating recycling endosome function and promoting the recyling of Ecadherin.
Investigating Variant Surface Glycoprotein Dynamics under Inhibition of Fatty Acid Synthesis
Authors: Poudyal Nava R. , Paul Kimberly S.
Published: May 2022

Trypanosoma brucei is a hemoflagellate that causes African Sleeping Sickness in humans and nagana in cattle. The T. brucei surface is coated with ten million identical copies of single Variant Surface Glycoprotein (VSG), one of ~2500 VSGs encoded in the genome. VSG helps T. brucei evade host immune response by two mechanisms: antigenic variation (changing the surface coat) and endocytosis (internalizing immune complexes, recycling the VSG while degrading the immune components). For both strategies, the VSG glycosylphosphatidylinositol (GPI) membrane anchor and its two myristates are thought to play a key role in VSG mobility and trafficking. The role of fatty acid synthesis (FAS) in VSG function is unknown, but it is likely needed to maintain VSG GPI‐anchor myristoylation. To reduce FAS, we use RNAi‐mediated depletion of Acetyl‐CoA Carboxylase (ACC), the first enzyme in the FAS pathway. We found that ACC RNAi resulted in a significant 30‐40% reduction in both receptor‐mediated and fluid‐phase endocytosis. To determine the effect of ACC RNAi on VSG dynamics, we assessed VSG half‐life by surface biotinylation and streptavidin blotting. We found that ACC RNAi resulted in ~40% decrease in surface VSG half‐life (p<0.05). Currently, we are examining two possible mechanisms leading to increased VSG turnover upon ACC RNAi: shedding in the media or intracellular degradation. Our preliminary data rules out degradation by proteasome or lysosome and reveals that ACC RNAi altered shedding of multiple surface proteins, including VSG. For the future, we will use proteomics to identify and quantify the proteins shed in the media, and we will investigate the mechanisms of their release.
aPC/PAR1 confers endothelial anti‐apoptotic activity via a discrete β‐arrestin‐2 mediated SphK1‐S1PR1‐Akt signaling axis
Authors: Orduna‐Castillo Lennis B. , Molinar‐Inglis Olivia , Nicholas Dequina , Cisneros‐Aguirre Metztli , Patwardhan Anand , Grimsey Neil , Lawson Mark A. , Patel Hemal , Trejo JoAnn
Published: May 2022

Endothelial dysfunction is associated with vascular disease and results in disruption of endothelial barrier function and increased sensitivity to apoptosis. Currently, there are limited treatments for improving endothelial dysfunction. Activated Protein C (aPC), a promising therapeutic, signals via protease‐activated receptor‐1 (PAR1) and mediates several cytoprotective responses including endothelial barrier stabilization and anti‐apoptotic responses. We showed that aPC‐activated PAR1 signals preferentially via b‐arrestin‐2 (b‐arr2) and dishevelled‐2 (Dvl2) scaffolds rather than G proteins to promote Rac1 activation and barrier protection. However, the signaling pathways utilized by aPC/PAR1 to mediate anti‐apoptotic activities are not known. APC/PAR1 cytoprotective responses also require co‐receptors, however it is not clear how co‐receptors impact different aPC/PAR1 signaling pathways to drive distinct cytoprotective responses. The objective of this study is to determine the function of coreceptors on aPC/PAR1‐induced cytoprotective responses in endothelial cells. We hypothesize that different co‐receptors distinctly regulate specific APC/PAR1 cytoprotective responses by b‐arr2 recruitment and biased signaling. Here we define a novel β‐arr2‐mediated sphingosine kinase‐1 (SphK1)‐sphingosine‐1‐phosphate receptor‐1 (S1PR1)‐Akt signaling axis that confers aPC/PAR1‐mediated protection against cell death. Using human cultured endothelial cells, we found that endogenous PAR1 and S1PR1 co‐exist in caveolin‐1(Cav1)‐rich microdomains and S1PR1 co‐association with Cav1 is increased by aPC activation of PAR1. Our study further shows that aPC stimulates b‐arr2‐dependent SphK1 activation independent of Dvl2 and is required for transactivation of S1PR1‐Akt signaling and protection against cell death. While aPC/PAR1‐induced extracellular signal‐regulated kinase 1/2 (ERK1/2) activation is also dependent on b‐arr2, neither SphK1 nor S1PR1 are integrated into the ERK1/2 pathway. Finally, aPC activation of PAR1‐b‐arr2 mediated protection against apoptosis is dependent on Cav1, the principal structural protein of endothelial caveolae. These studies reveal that different aPC/PAR1 cytoprotective responses are mediated by discrete b‐arr2‐driven signaling pathways in caveolae.
The effect of Aldolase C reflects the neurotransmitter secretion and PPARγ signaling in Glioblastoma
Authors: Chang Yu‐Chan , Hsiao Michael
Published: May 2022

Glioblastoma (GBM) are high‐grade gliomas with aggressive progression, poor survival rate, and insufficient therapeutic options. According to molecular characteristics, GBM can be divided into proneural, classical, and mesenchymal subtypes. This study identified that one of the glycolytic enzymes, aldolase C (ALDOC), with the prognostic value could reflect these subtypes' survival rate. In GBM, ALDOC has hypermethylation and loss of expression in tumors, especially mesenchymal subtypes. Interestingly, glycolysis is not affected in the metabolic event, but some brain neurotransmitters (Serotonin) and sugars (inositol) are affected. We confirmed that Serotonin could significantly promote the migration/invasion ability of GBM cells when ALDOC is hypermethylated/loss of function. Mechanistically, Serotonin is excreted to the peripheral area through 5‐hydroxytryptamine receptors (HTRs) family members. We further recruited HTR inhibitors or selective serotonin reuptake inhibitors to reverse such phenotypes. However, due to Serotonin regulating some physiological functions, they may not be appropriate for use. Instead, we analyzed the ALDOC‐based transcriptomics profile and confirmed that the expression of PPARγ is similar to ALDOC in GBM cells. Treating PPARγ agonists can regulate the production of Serotonin and reduce the metastatic state of GBM cells. Additionally, we have observed that the combination of PPARγ agonist and Temozolomide has a synergistic effect that can decrease tumorigenicity and in situbrain tumors. Histological staining demonstrated that the ALDOC and PPARγ signals in the combined group could be rescued. The downstream candidates of PPARγ combined with ALDOC can be applied to the prognostic value of patient survival and various clinicopathological factors. Combining all evidence, we claim that we should pay attention to the importance of the ALDOC‐PPARγ axis and regard it as a novel strategy against GBM.
A Potential Link Between Negative Affective Responsivity to Daily Stress and Blood Pressure Reactivity to Acute Stress in College‐Aged Adults
Authors: Darling Ashley M. , Mogle Jacqueline , Fadel Paul J. , Saunders Erika F. , Almeida David M. , Greaney Jody L.
Published: May 2022

Daily stress, defined as the routine challenges of day‐to‐day living and the unexpected small hassles that disrupt everyday life, is predictive of future cardiovascular disease (CVD)‐related health outcomes; however, the underlying mechanisms remain unclear. Given that heightened blood pressure (BP) reactivity to stress is also purported to increase future CVD risk, we examined a potential link between daily stress and acute stress‐induced increases in BP. We hypothesized that increased frequency of daily stressor exposures and greater negative emotional responsiveness to daily stress (i.e., negative affective responsivity) would be positively related to increased BP reactivity to acute stressors in the laboratory setting. Thirty adults (23±5 yrs; 21 female) completed the Daily Inventory of Stressful Events (DISE) interview every day for 8 consecutive days to quantify objective (e.g., frequency) and subjective appraisal characteristics (e.g., emotions) of naturally occurring daily stressors (e.g., stressful event at home/work). On DISE Day 8, mean arterial pressure (MAP; finger photoplethysmography) was measured during two distinct laboratory‐based acute stress stimuli: the cold pressor test (CPT; generalized sympathoexcitatory stimulus) and the Stroop Color Word Test (SCWT; cognitive stress stimulus). Daily stress exposure was calculated as the total number of stressors across the 8‐day protocol, and negative affective responsivity was calculated as the magnitude of the increase in negative affect on stressor exposure days (at least 1 stressor reported) compared to negative affect on non‐stressor exposure days (no stressors reported). BP reactivity was quantified as the peak acute stress‐induced increase in MAP to the CPT and SCWT. Participants experienced a total of 3±3 stressors (range: 0‐9) over the 8‐day interview timeframe. Negative affect was greater on stressor exposure days (0.68±0.50 a.u.) compared to non‐exposure days (0.37±0.36 a.u.; p<0.01). Both the CPT (∆21±7 mmHg) and the SCWT (∆9±6 mmHg) elicited increases in MAP (both p<0.05). The total number of stressors was not related to the peak increase in MAP during either the CPT (slope=‐0.10; r=0.04; p=0.85) or the SCWT (slope=‐0.43; r=19; p=0.31). Negative affective responsivity was positively related to the peak increase in MAP during the CPT (slope=12.41; r=0.45; p=0.03) but not the SCWT (slope=3.00; r=0.14, p=0.50). These preliminary data demonstrate that daily stressor exposure by itself is not related to BP reactivity to laboratory stressors in college‐aged adults. However, greater negative affective responsivity is associated with greater acute stress‐induced increases in BP during the CPT, but not the SCWT, perhaps suggesting that the link between daily stress processes and BP reactivity may be dependent on the stress‐responsive neurocircuitry being activated.
A Role for Inositol Pyrophosphates in ER Protein Translocation?
Authors: Molnar Abby , Keeney Jill
Published: May 2022

The purpose of this study is to identify proteins involved in endoplasmic reticulum to cytoplasm transport of the Gag protein of the yeast Saccharomyces cerevisiae Ty1 retroelement. Saccharomyces cerevisiae, or bakers/brewer’s yeast, exists as a model organism due to its quick growth time and easily manipulated genome. A multitude of yeast genes are also homologous to ones found in larger eukaryotes. During Ty1 transposition, Gag is translocated by the signal recognition protein (SRP) from the cytoplasm to the ER lumen where it adopts a stable conformation. The stabilized Gag protein is then translocated through an unknown mechanism to the cytoplasm where it aggregates to form virus‐like particles. Screening of a yeast deletion library identified SIW14 as a potential candidate gene involved in Gag translocation. The yeast deletion library was transformed with a Ty1 Gag‐Ura3 fusion protein on a LEU2 selectable plasmid. In wild type cells, the Gag‐Ura3 fusion is transported to the cytoplasm, thus yeast cells are unable to survive on media containing 5‐fluoroorotic acid (5‐FOA), as the URA3 gene converts 5‐FOA to the toxic product 5‐fluorouracil. Cells with an siw14 deletion are viable on 5‐FOA containing media, presumably sequestering the Ty1 Gag‐Ura3 fusion protein in the ER. Siw14p is an inositol phosphatase that hydrolyzes the beta phosphate on 5‐diphospho‐1,2,3,4,6‐pentakisphosphate (5PP‐IP5) to inositol hexaphosphate (IP6). Inositol pyrophosphates are energy‐rich signaling molecules and can act as biological messengers. Vip1p, Ddp1p, and Kcs1p are also proteins involved in the phosphorylation state of inositol pyrophosphates in yeast. inositol heptaphosphate (IP7) or inositol octaphosphate (IP8) are substrates for pyrophosphorylation of phosphorylated serine residues in proximity to acidic amino acids, such as aspartate and glutamate. No energy is expended during pyrophosphorylation; it is dependent on the presence of an Mg2+ divalent ion. We hypothesize that the deletion of a pyrophosphatase kinase results in the inability of an unknown transport protein from being pyrophopshoprylated to facilitate Gag transport across the ER. Strains harboring deletions of siw14, kcs1, vip1, and ddp1 will be assayed for Ty1 transposition. Fluorescence microscopy of deletion and wild type cells containing a fluorescently labeled Gag protein will be observed for Gag localization.
The Transcriptional Coregulatory Protein YAP1 Interacts with RNA Binding Protein NPM1
Authors: Dwead Abdulrahman M. , Al‐Mathkour Marwah M. , Cinar Bekir
Published: May 2022

RNA binding proteins (RBPs) regulate all aspects of RNA biogenesis from transcription, splicing, translation to degradation, and they have a critical role in cellular homeostasis and functional diversity. Recent studies have indicated that altered expressions of RBPs are associated with many human diseases ranging from neurologic disorders to cancer. The transcriptional coregulator yes‐associated protein 1 (YAP1), a critical nuclear effector of the mammalian Hippo pathway, regulates cell fate, cell contact, metabolism, and developmental processes. This study aims to demonstrate a link between YAP1 and nucleophosmin 1 (NPM1) protein. NPM1 is an RNA‐binding protein that regulates many cellular activities, including ribosome biogenesis, RNA processing, chromatin remodeling, DNA repair, and genomic stability. Using proteomics approaches, we identified NPM1 from YAP1 protein complexes of androgen‐responsive human cancer cells. Our proximity ligation assay demonstrated that YAP1 and NPM1 physically interacted with each other. The interaction between YAP1 and NPM1 occurred in cell nuclei and was regulated by androgen hormone signaling. In addition, our GST‐pulldown assay demonstrated that NPM1 formed a protein complex with the proline‐rich domain of YAP1. Furthermore, our enhanced RNA interactome capture (eRIC) assay showed that androgen also regulated the interaction of RBPs to polyA+ mRNA within the cell. Consistent with this observation, our eRIC assay combined with the mass spectrometry method enabled identifying distinct RBP patterns in human cancer cells that are genetically related but phenotypically different. These observations indicate that global alterations of RBPs under changing environmental conditions may have important roles in cellular physiology and disease biology.
Exogenous thyroxine increases GLUT4 translocation to the membrane in cardiomyocytes in insulin resistant OLETF rats Dora A Mendez1, Jose G. Soñanez‐Organis2, Guillermo Vasquez‐Anaya1, Daisuke Nakano3, Akira Nishiyama3, Rudy M Ortiz11Department of Molecular and Cell Biology, School of Natural Sciences, University of California, Merced, CA, USA 2Division of Science and Engineering, Department of Chemical Biological and Agropecuary Sciences, University of Sonora, Lazaro Cardenas 100, Colonia, Campus Navojoa, Francisco Villa, 85890 Navojoa, SON, Mexico 3Department of Pharmacology, Kagawa University Medical School, Kagawa, Japan
Authors: Mendez Dora A. , Soñanez‐Organis Jose G. , Vasquez‐Anaya Guillermo , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy M.
Published: May 2022

During insulin resistance the heart undergoes a metabolic shift in which fatty acids (FA) accounts for about 99% of the ATP production. This metabolic shift is indicative of impaired glucose metabolism. A shift in FA metabolism with impaired glucose tolerance can cause an increase in reactive oxygen species (ROS) and lipotoxicity ultimately leading to impaired mitochondrial function. Thyroid hormones (TH) may improve glucose intolerance by increasing glucose reabsorption and metabolism in peripheral tissues but its effects on cardiac tissue during insulin resistance is not well known. The glucose transport protein, GLUT4, is a downstream target of the TH response element (TRE) indicating a possible mechanism by which THs regulate glucose metabolism. Non‐classical TH signaling may also phosphorylate protein kinase B (Akt) and increase activity of phosphoinositide‐3‐kinase (PI3K), which are two key regulators in insulin‐mediated glucose uptake. Insulin resistant, Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exogenous thyroxine (T4) on glucose metabolism in cardiac tissue. Rats were assigned to four groups: 1) lean, Long Evans Tokushima Otsuka (LETO; n=6), 2) LETO + T4 (8 μg/100g BM/d × 5 wks; n=7), 3) untreated OLETF (n=6), and 4) OLETF + T4 (n=7). T4 increased GLUT4 gene expression by 85% (p<0.05) in OLETF and increased GLUT4 protein translocation to the membrane by 328% (p<0.05). Additionally, T4 increased phosphofructokinase‐1 (PFK‐1) gene expression, the rate limiting step in glycolysis, by 98% (p<0.05) in OLETF. T4 increased AKT and p‐AKT by 177% and 134% (p<0.05), respectively, in LETO, but not in OLETF. The results suggest that increased T4 has the potential to increase glucose reabsorption and metabolism in the heart of insulin resistant rats to help restore impaired substrate metabolism.
Understanding patient‐level immune response to COVID‐19 can inform treatment options
Authors: Prokop Jeremy W. , Frisch Austin W. , Hartog Nicholas , Bupp Caleb , Rajasekaran Surender
Published: May 2022

COVID‐19 is caused by an evolving pathogen that results in a disease that exhibits variability in progression and treatment response, highlighting the need for developing new and more precise therapies. Over the past two years, we have built a collection of tools to understand variability and mechanisms of COVID‐19 immune responses, including the SARS‐CoV‐2 evolutionary/structural dynamicome, curated lists of SARS‐CoV‐2 protein‐protein interactions (PPI), NCBI SRA data integrations for SARS‐CoV‐2 exposed cells, data from thousands of blood‐based RNA‐Seq analyzed, and created a clinical cohort of blood‐based RNA‐Seq of COVID‐19 and other critical illness pathologies. The dynamicome and PPI list elucidated several viral proteins that modulate the host immune response, including the nucleocapsid (N) role in suppressing nonsense‐mediated decay leading to rare disease phenotypes, ORF8 interaction with innate immune components, and the Papain‐like protease (NSP3) regulation of intracellular ISG15 interferon signaling. This stimulation in hospitalized COVID‐19 patients exerts three diverse systemic blood RNA‐Seq changes noted on five independent cohorts processed with a precision transcriptomics multi‐data analysis pipeline. The first was patients that are immunosuppressed, which showed blunted interferon response and often secondary viral activation, suggesting antiviral treatments and immune stimulation. The second patient group is those who go into cytokine storm, often seen with overly active ISG15 levels in peripheral blood, who benefit from immunosuppressive agents. The third group often has normal interferon activation but a triggered systemic process of NETosis in blood, activating neutrophils through controlled death to build DNA/histone nets to contain the virus. The third group likely requires specialized treatment strategies to prevent the NETosis from driving vascular dysfunction and COVID long hauler syndromes. These three groups resulting from varied pathologic mechanisms, showcase the need for different therapeutic approaches, and may explain the many failed clinical trials for COVID‐19. Thus, it would be beneficial to develop a deeper understanding of each patient’s immune response moving forward to optimize treatment and outcomes.