bluuebunny/crossref_metadata_2025_split huggingface dataset:
An Essential Fringe‐Like Protein in the Plasmodium falciparum Asexual Life Cycle
Authors: Villegas Alejandra , Fierro Manuel Antonio , Muralidharan Vasant 
Published: April 2020

Malaria is a deadly disease caused by the apicomplexan parasite Plasmodium. Plasmodium asexual replication occurs in the red blood cell (RBC) and is what causes clinical symptoms of disease. In the RBC, Plasmodium moves through 3 developmental stages ending with schizogony and creating 16–32 daughter merozoites. At the end of schizogony, merozoites egress the host cell by breaking out of two membranes, the parasitophorous vacuole membrane and the RBC membrane. Schizogony and successful egress allows for exponential Plasmodium replication and RBC infection. Our lab recently identified a B3GLCT‐like protein in the Plasmodium ER (PfB3ER) that interacts with an essential ER chaperone and whose expression profile is similar to that of proteins required for egress. B3GLCT‐like proteins are known to act as glucosyltransferases and work in concert with protein O‐fucosyltransferases (POFUT2) to modify thrombospondin‐like repeats in mammalian cells. Surprisingly, the P. falciparum POFUT2 has been shown to be non‐essential while PfB3ER is predicted to be essential in the asexual stages. Based on PfB3ER homology to the mammalian B3GLCT and its predicted expression profile, we hypothesize that the O‐glycosylation function of PfB3ER is required for efficient egress of P. falciparum from RBCs. To test this, we have employed CRISPR/Cas9 gene editing to HA‐tag PfB3ER and create conditional mutants utilizing the TetR‐DOZI‐aptamer system. We show that PfB3ER localizes to the ER, unlike previous published results that suggest that PfB3ER is exported to the host RBC, and that PfB3ER is primarily expressed during schizogony. Further, we show that PfB3ER is essential for the asexual replication of P. falciparum. Future studies will focus on the specific stage of the intraerythrocytic lifecycle that are affected by PfB3ER knockdown and test the requirement of its glycosyltransferase activity. Together, these data have uncovered an essential function for a putative glycosyltransferase in the intraerythrocytic lifecycle of P. falciparum.Support or Funding Information
R01 AI130139/AI/NIAID NIH HHS/United States
cAMP Biosensors with Restricted Subcellular Localization Reveal Persistent Signaling Changes Following Antidepressant Withdrawal
Authors: Senese Nicolas B. , Rasenick Mark M. 
Published: April 2020

Many patients experience a constellation of side effects following termination of antidepressant therapy. Collectively these effects are known as the antidepressant discontinuation syndrome. All currently prescribed antidepressant medications can cause this syndrome, regardless of primary mechanism of action. This suggests that some shared mechanism, independent of these drugs’ known binding targets, may contribute to the discontinuation syndrome.Lipid rafts are membrane microdomains with distinct signaling characteristics and protein expression compared to other membrane regions. Previous work has shown that chronic, but not acute, treatment with traditional antidepressants induces translocation of Gs out of lipid raft microdomains. In contrast, rapid acting antidepressants such as ketamine cause Gs translocation following acute exposure. This promotes Gs signaling in general, likely due to increased coupling to downstream effectors such as adenylyl cyclase.Using a lipid raft restricted cAMP biosensor we show that perturbations of Gs signaling persist for over 24 hr after antidepressant withdrawal in an in vitro model system. These signaling changes are variable between different subcellular microdomains, and between different cell types. In general, signaling changes in lipid rafts are more persistent than changes observed in non‐raft membrane regions, or in the cytoplasm. Effects are compared between c6 glioma, SK‐N‐SH neuronal, and HEK‐293 kidney derived cell lines.We also show that changes in lipid raft cAMP signaling are reflected by persistent reductions in lipid raft Gs expression following drug withdrawal. Protein expression is quantified following membrane isolation and sequential detergent extraction to separate lipid raft from non‐raft membranes.The observed antidepressant effects occur in model systems lacking the primary target for these drugs (e.g. SERT and escitalopram), and as such may represent a novel mechanism by which antidepressants regulate cellular activity. Understanding these residual antidepressant effects may aid in the development of novel antidepressants without an associated discontinuation syndrome, and in the discovery of new strategies to mitigate these persistent effects following drug withdrawal.Support or Funding InformationSupported by: VA Merit BX001149, NIH T32 MH067631 and NIH R01 AT009169
Neuregulin‐4 Limits Pro‐Inflammatory Cytokine Production in Macrophages
Authors: Schumacher Michael A. , Dennis Isabella , Robinson Cache , Frey Mark R. 
Published: April 2020

BackgroundPro‐inflammatory macrophages are critical mediators of intestinal inflammation. Over‐aggressive responses from these cells, including elevated cytokine release and prolonged persistence in tissue, may contribute to inflammatory bowel disease. We have previously found that the ErbB4 receptor tyrosine kinase is induced on macrophages by pro‐inflammatory stimulation, and that treatment with the ErbB4‐specific ligand neuregulin‐4 (NRG4) reduces macrophage numbers through apoptosis and ameliorates colitis. This suggests that macrophage‐expressed ErbB4 limits colonic inflammation, but the impact of NRG4/ErbB4 on cytokine regulation in these cells remains untested.
We hypothesized that ErbB4 inhibits pro‐inflammatory cytokine production in macrophages.MethodsTo test the role of ErbB4 in regulating cytokine expression, we generated bone‐marrow derived macrophages from mice lacking ErbB4 in cells of myeloid lineage (LysMCre/ErbB4FF) or sex‐matched littermate controls, and activated with IFN□/LPS (pro‐inflammatory M1) or IL‐4 (anti‐inflammatory M2). To test the role of NRG4, we treated activated cells with recombinant ligand at 100 ng/ml for 24 hours, harvesting cells before the onset of NRG4‐induced apoptosis to compare functional effects on cytokine expression. RNA was collected and analyzed for cytokines by qPCR.ResultsPro‐inflammatory M1 activation of macrophages induced NRG4 expression 11‐fold (p=0.01). M1 macrophages from LysMCre/ErbB4FF mice expressed elevated levels of TNF (32% higher, p=0.02) and CXCL1 (69% higher, p=0.01) versus M1 macrophages from littermate controls. No differences in expression were seen in anti‐inflammatory M2 macrophages. NRG4 treatment led to reduced TNF (31% lower, p=0.005) and IL‐1β (16% lower, p=0.01) in M1 macrophages from wild‐type mice.Summary/ConclusionNRG4 reduces pro‐inflammatory cytokine production in macrophages through its receptor, ErbB4. This work suggests that ErbB4 signaling in macrophages drives an anti‐inflammatory feedback loop to limit colonic inflammation.Support or Funding InformationNIH 2R01DK095004‐06A1 (Frey); Crohn's and Colitis Foundation (Frey and Schumacher)
Novel roles of PFKFB3 in mediating endothelial dysfunction in obesity
Authors: Batori Robert Karoly , Bordan Zsuzsanna , do Nascimento Thiago Bruder , Padgett Caleb , Huo Yuqing , Stepp David , de Chantemele Eric Belin , Fulton David 
Published: April 2020

Obesity is a major and growing health problem in the US with over 93 million afflicted. Although, obesity itself is not considered a disease, it is a strong risk factor for cardiovascular disease, in large part due to an altered metabolic state. The relationship between metabolic dysfunction and impaired cardiovascular health remain incompletely understood.One of the earliest consequences of cardiovascular dysfunction in obesity is the loss of endothelial function and impaired nitric oxide (NO) signaling. NO is best known as the mediator of endothelium‐dependent relaxation, but it is also vital for maintaining blood vessel homeostasis through inhibition of leukocyte adhesion and smooth muscle cell proliferation. In blood vessels, NO is synthesized by endothelial nitric oxide synthase (eNOS) and dysregulation of eNOS has been shown to occur through changes in its expression and post‐translational phosphorylation. The biological actions of NO can be further compromised by rapid inactivation by reactive oxygen species (ROS) such as superoxide that are generated by NADPH oxidases (NOX) as well as numerous other enzyme systems.Our laboratory has previously shown that an imbalance between NO and superoxide‐generating pathways is responsible for the loss of endothelial function in obesity. However, the signals coordinating the loss of NO and increased superoxide with altered metabolism remain poorly defined.In our studies we have found that expression of the glycolytic enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐biphosphatase 3 (PFKFB3) is significantly increased in endothelial cells from obese animals. PFKFB3 is responsible for fructose‐2,6‐bisphosphate generation in vascular cells which is a potent allosteric activator of phosphofructokinase‐1 (PFK‐1), one of the rate‐limiting enzymes of glycolysis. We have also found that adenovirus‐mediated expression of PFKFB3 within the endothelium of mouse blood vessels is accompanied by the loss of endothelium‐dependent relaxation.In addition to its effects on blood vessels, overexpression of PFKFB3 in cultured cells resulted in decreased NO production as a result of increased eNOS phosphorylation on the well‐known inhibitory PKC phosphorylation site, threonine 495 (T495). Furthermore, PFKFB3 overexpression blunted Akt‐S473 phosphorylation, reducing stimulus‐dependent phosphorylation of eNOS at S1177. In addition to its negative effect on NO signaling overexpression of PFKFB3 increased the activity and mRNA level of NOX1, a major contributor to endothelial dysfunction in obesity.These results demonstrate a novel functional relationship between endothelial cell metabolism, nitric oxide and ROS production that may contribute to endothelial dysfunction in obesity.Support or Funding Information1R01HL147159‐01
Determining the Role of Obesity and Muscarinic Signaling in Asthma
Authors: Bruggink Stephanie , Kentch Kyle , Kronenfeld Jason , Renquist Benjamin 
Published: April 2020

In the U.S., one in thirteen individuals have been diagnosed with asthma. Obesity increases the incidence of asthma by 56%. The degree of obesity is associated with the severity of reduction in lung volume and lung volume can be restored with weight loss. Obesity also increases the risk of hospitalization by 460%. Although asthma and altered lung function is commonly assessed with spirometry in humans, plethysmography is recommended by the American Thoracic Society for assessing lung volume. The most common and effective method for assessing lung function in mice, forced ventilation, is terminal and limited to assessing lower airway function, preventing repeated measures. We aimed to develop a head‐out, variable pressure plethysmography system that would allow for repeated sensitive assessment of lung function and volume in lean and diet‐induced obese mice while mimicking the measures in human clinical practice. Preventing air leak from the chamber is vital for recording accurate measurements in a variable pressure plethysmography system. To eliminate air leak, we created an inflatable cuff that encompassed the mouse’s neck. This allowed us to repeatedly and accurately assess airway function in mice. To validate the head‐out, variable pressure plethysmography system, we used a series of models with altered muscarinic signaling. First, we assessed the response to a nebulized bronchoconstrictor, methacholine, establishing that methacholine resulted in the expected decreased breaths/minute, increased expiratory and inspiratory time, and decreased the rate of air flow at 50% of exhalation (EF50). To assess chronic muscarinic signaling, we used an adeno‐associated viral delivery to induce expression of a mutated constitutively active muscarinic 3 receptor (Q409L M3R) in airway smooth muscle, creating a model of chronic bronchoconstriction. In mice that expressed the Q490L M3R, we similarly found a decrease in breaths/minute and an increase in expiratory and inspiratory time at tidal breathing. Finally, we observed a decrease in breaths per minute at tidal breathing in muscarinic 3 receptor knock‐out mice. In our model of diet‐induced obesity we showed that obesity increased breaths per minute and decreased expiratory time at tidal breathing relative to that observed in lean mice. We have developed a reliable tool to repeatedly assess lung function in mice using the same clinically relevant measure applied in human asthmatics. Future research will apply this tool to better understand the mechanism by which obesity alters lung function and tidal breathing volume in mice.Support or Funding InformationABRC ADHS14‐082986ABRC ADHS17‐00002043T32 HL 007249
Early Life Stress Mediates Enhanced Aortic Vasorelaxation in Sv129 Mice
Authors: Kellum Cailin E. , Allan J. Miller , McPherson Kasi C. , Neder Thomas H. , Seifert Michael E. , Feig Daniel I. , Pollock Jennifer S. 
Published: April 2020

Early life stress (ELS) is an under‐appreciated risk factor for cardiovascular disease (CVD). In humans, adverse childhood experiences (ACEs) or ELS is recognized as inducing behavioral or emotional stress. We reported that young adults exposed to ACEs display enhanced vascular stiffness, increased peripheral resistance, and elevated plasma ET‐1. We hypothesized that vascular endothelial‐derived ET‐1 is associated with vascular dysfunction after exposure to ELS. We hypothesized that endothelial‐derived ET‐1 is linked to vascular dysfunction in ELS. To test this hypothesis, we utilized vascular endothelial‐specific ET‐1 knockout (VEETKO) and flox control mice exposed to normal rearing (NR) or maternal separation with early weaning (MSEW). Briefly, MSEW involves maternal separation for 4h/day (postnatal (PD) 2 to 5), 8h/day (PD6 to 16), and weaned at PD17. NR litters were weaned at PD21. Male and female adult NR and MSEW, flox control (n=3–5) and VEETKO (n=5) mice were used to study acetylcholine (Ach)‐ and sodium nitroprusside (SNP)‐mediated aortic vasorelaxation. Interestingly, aorta from MSEW flox mice showed significantly greater maximal Ach‐mediated vasorelaxation compared to NR flox mice (Flox‐control 74.7% SEM 4.15, Flox‐MSEW 93.7% SEM 1.63, VEETKO‐control 78.0% SEM 6.81, VEETKO‐MSEW 86.9% SEM 2.05; p=0.0023). EC50 responses to Ach were not significantly different (Flox‐NR −6.22 SEM 0.330, Flox‐MSEW −6.88 SEM 0.124, VEETKO‐NR −6.70 SEM 0.144, VEETKO‐MSEW −6.88 SEM 0.268; p=0.066). All responses to SNP were similar. The VEETKO and flox control mice are on a Sv129 background. We previously reported that C57Bl/6J mice exposed to MSEW displayed endothelial dysfunction when compared to NR. Thus, these data suggest that exposure to MSEW has a different mechanistic response to the MSEW protocol with different strains of mice. Future studies will include behavioral studies to determine whether the MSEW protocol in Sv129 mice is distinct from the pro‐anxiety response reported in C57Bl/6J mice.Support or Funding InformationFunding K01‐DK‐105038 KAH, RO1 HL136936 JSP and DMPPROmoTE R25 DK 112731
Delayed delivery of metformin promotes functional recovery in a model of neonatal stroke
Authors: Livingston Jessica , Gilbert Emily , Syeda Tasfia , Christie Taryn , Morshead Cindi 
Published: April 2020

Hypoxia‐ischemia (HI) is one of the leading causes of neonatal death and long‐term disability. Currently, the only standard treatment for HI is therapeutic hypothermia, which affords only modest benefit. The drug metformin has been shown to have pleiotropic effects in the brain, including activation of endogenous neural precursor pools. Importantly, administration of metformin leads to improved functional improvements following neonatal HI. Our previous work has demonstrated that metformin treatment beginning 24 hours after HI injury on post‐natal day (P8), and lasting for 1 week leads to attenuation of motor deficits at P22 and activation of endogenous neural precursor cells (NPCs) in the subventricular zone (SVZ). NPCs expanded in number, migrated into the parenchyma and differentiated into neurons and glia. Improved cognitive outcomes in young adults was also observed with extended metformin treatment in HI injured mice. Given these promising outcomes, and to improve the clinical application of the therapeutic intervention, we asked if delayed metformin treatment would be equally effective at improving functional outcomes. Male and female C57/BL6 mice were subjected to HI (or a sham procedure) at P8. Metformin (or vehicle) treatment began on P15 and lasted 4 weeks. Motor and cognitive functions were assessed across time using various behavioural assays (cylinder, foot fault, and puzzle box). Tissue was collected at P28 and 63 and analyzed for changes in inflammation, proliferation and differentiation. We found that delaying metformin treatment was effective at promoting motor and cognitive recovery. Treatment led to a decrease in inflammation after injury, as seen by a reduction in microglia, and an increase in oligodendrocytes in the motor cortex. Hence, our findings demonstrate that a clinically relevant delayed metformin treatment paradigm has therapeutic potential following neonatal HI.Support or Funding InformationCIHR, Medicine by Design
Mouse Models Expressing Mutant CUL3 Provide Insight into the Mechanism of Familial Hyperkalemic Hypertension (FHHT) and Reveal Additional Functional Defects
Authors: McCormick James A. , Blatt Phillip J. , Sharma Avika , Ferdaus Mohammed Z. 
Published: April 2020

The pathogenesis of essential hypertension is complex and multifactorial. Studying Mendelian forms of hypertension like Familial Hyperkalemic Hypertension (FHHt) is a powerful approach to identify and understand pathways that may contribute. Cullin‐3 (Cul3) mutations cause the most severe form of FHHt. Cullin 3 (CUL3) along with a substrate adaptor and RING ligase forms a complex that ubiquitinates proteins for proteasomal degradation. In FHHt, mutant CUL3 (CUL3‐D9) activity impairs With‐No‐lysine [K] kinase 4 (WNK4) degradation, leading to increased phosphorylation and thus activation of the Na+ Cl− cotransporter (NCC) along the renal distal convoluted tubule (DCT). We previously reported that CUL3‐D9 degrades the WNK4 adaptor KLHL3 in vitro, and a recent study suggests this occurs in vivo. This depletion of KLHL3 could prevent WNK4 degradation, providing a mechanism by which CUL3‐D9 leads to FHHt. To confirm that CUL3‐D9 degrades KLHL3 in vivo and determine whether it has any other activities, we studied inducible renal epithelial‐specific Cul3 knock‐out (CUL3‐KO) mice and CUL3‐KO mice expressing CUL3‐D9 (CUL3‐KO/D9). Western blotting and immunofluorescence showed that CUL3‐KO mice had higher KLHL3 abundance compared with controls, while CUL3‐KO/D9 had similar KLHL3 abundance to controls. CUL3‐KO and CUL3‐KO/D9 mice also had higher WNK4 abundance compared with controls. These data suggest that KLHL3 is a substrate of wild‐type CUL3, and that CUL3‐D9 is an active form of CUL3 since it can prevent KLHL3 accumulation in CUL3‐KO mice. However, CUL3‐D9 cannot promote WNK4 degradation and did not rescue the chronic kidney disease CUL3‐KO phenotype, suggesting a generalized loss‐of‐function of CUL3‐D9‐containing ubiquitin ligase complexes. A model of FHHt, inducible renal epithelia‐specific heterozygous Cul3 mice with CUL3‐D9 expression (CUL3‐Het/D9), further confirmed this observation with significantly lower KLHL3 abundance compared with control and heterozygous Cul3 mice. To investigate effects of CUL3‐D9 on other CUL3 targets, we investigated the abundance of another CUL3 adaptor protein, Kelch‐like ECH Associated Protein 1 (Keap1), the adaptor for Nuclear factor erythroid 2‐related factor 2 (NRF2). Both CUL3‐KO/D9 and CUL3‐Het/D9 had similar Keap 1 abundance compared with controls, but NAD(P)H Quinone Dehydrogenase 1 (NQO1), a surrogate for NRF2 activity, was significantly higher. These data suggest that in addition to effects on KLHL3/WNK4, CUL3‐D9 forms a dysfunctional complex with Keap1, leading to increased NRF2 activity. In conclusion, we confirm that CUL3‐D9 degrades its adaptor KLHL3 in vivo resulting in WNK4 accumulation, and also show that CUL3‐D9 cannot form normally‐functioning complexes with other substrate adaptors.Support or Funding InformationNIH grants DK098141 and DK117903 to JAM, and AHA 17POST33670206 to MZF
EGFR signaling in breast cancer requires licensing from separate membrane nanodomains
Authors: Sugiyama Michael G. , Fairn Gregory D. , Antonescu Costin N. 
Published: April 2020

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with numerous roles in maintaining cell physiology; it is upregulated in many cancers, including breast cancer, where it contributes to tumor growth and metastasis. This feature of EGFR signaling in cancer has prompted the development of anti‐EGFR therapies. However, many cancers, while initially responsive to anti‐EGFR therapies, develop resistance to prolonged treatment, which imposes a significant challenge in applying anti‐EGFR therapies to the treatment of difficult to treat cancers such as triple negative breast cancer. Resistance to anti‐EGFR therapy is not a consequence of EGFR mutation, and instead it has been speculated that alterations in EGFR plasma membrane confinement within specialized membrane nanodomains contributes to aberrant signaling in cancer. We previously reported that EGFR activation of downstream Akt signaling requires clathrin nanodomains; the contribution of other nanodomains (flotillins, tetraspanins, caveolae, etc.) to EGFR signaling remains unknown. To probe EGFR signaling in membrane nanodomains we developed tools to track EGFR, EGF‐ligand, and various nanodomains at the single molecule level by dual‐colour, live‐cell TIRF microscopy coupled with MATLAB automated detection and particle analysis. Treatment of breast cancer cell lines with EGFR tyrosine kinase inhibitors (TKI) attenuated EGFR confinement within clathrin nanodomains and subsequent Akt signaling, but only in cell lines sensitive to the cytotoxic effects of the drugs. Interestingly, mobilization of EGFR to clathrin following stimulation with EGF‐ligand required PKC activation and confinement within non‐clathrin nanodomains, a feature that was altered in EGFR TKI‐resistant breast cancer cells. This two nanodomain model of EGFR activation resolves some longstanding, unexplained features of EGFR signaling, such the inability of the majority of EGFR to bind ligand and suggests that EGFR licensing by membrane nanodomains is a critical regulator of EGFR signaling that contributes to therapeutic resistance. Ongoing work will determine the mechanism that links EGFR licensing between membrane nanodomains, EGFR signaling, and drug resistance in breast cancer.Support or Funding InformationCIHR PJT‐156355 to GDF and CNA