bluuebunny/crossref_metadata_2025 huggingface dataset:
Coilin Modulates Nuclear Organization by Promoting Protein SUMOylation
Authors: Lett Katheryn , Logan Madelyn , McLaurin Douglas , Hebert Michael
Published: May 2022

Coilin was first identified as the marker protein for Cajal Bodies (CBs). However, CBs are only abundant in neuronal, embryonic, and cancer cells, while primary cells have few CBs. Even in cells with CBs, 70% of coilin is nucleoplasmic, not found in CBs. CBs are subnuclear domains that function in small nuclear ribonucleoprotein (snRNP) biogenesis as well as production of telomerase RNP, expression and 3’end processing of small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and small CB‐associated RNAs (scaRNAs). The function of nucleoplasmic coilin is not well understood. We have previously shown that coilin promotes miRNA biogenesis by promoting phosphorylation of the Microprocessor component, DGCR8, at its S377 residue in both cells with and without CBs. We have since identified 7 additional serine residues of DGCR8 with decreased phosphorylation in coilin knockdown through phosphoproteomic analysis of HeLa cells. In addition to phosphorylation, the addition of a small ubiquitin‐like modifier (SUMO), or SUMOylation, of DGCR8 also increases its stability. Because of coilin’s role in the promotion of one post‐translational modification, here we aimed to test the hypothesis that coilin knockdown alters protein SUMOylation and results in altered nuclear organization. We conducted a series of siRNA knockdowns, Western blots, and immunoprecipitation experiments. Here we show, for the first time, that coilin knockdown results in global decrease of protein SUMOylation in cells with (HeLa) and without CBs (WI‐38), suggesting that regulation of protein SUMOylation may be a function of nucleoplasmic coilin. Specifically, we found that coilin knockdown results in decreased SUMOylation of DGCR8 and Speckled protein 100kDa (Sp100), a major component of the promyelocytic leukemia nuclear body (PML‐NB). Sp100 is vital to PML‐NB formation, and decreased SUMOylation of Sp100 decreases its stability. This information led us to the second part of our hypothesis. We used immunofluorescence and found that coilin knockdown results in decreased numbers of PML‐NBs per cell. To summarize, we have identified coilin as a regulator of protein phosphorylation, SUMOylation, and nuclear organization.
Tenofovir‐Diphosphate Formation by Creatine Kinase Brain‐Type is Diminished by Naturally Occurring Mutations In Vitro
Authors: Eberhard Colten , Bumpus Namandjé N.
Published: May 2022

Tenofovir (TFV) is a nucleotide reverse transcriptase inhibitor that is administered as a prodrug for use in both HIV treatment and prevention. TFV requires two sequential phosphorylation steps to form the pharmacologically active metabolite, tenofovir‐diphosphate (TFV‐DP). Previous studies have shown that creatine kinase muscle‐type (CKM) can catalyze the final phosphorylation step in colon tissue. Thus, to discover other TFV activating enzymes, we used CKM as a starting point and identified creatine kinase brain‐type (CKB) as a candidate TFV activating enzyme, as it is 80% homologous to CKM. To test the hypothesis that CKB can phosphorylate TFV in a manner similar to CKM, we performed in vitro activity assays by incubating recombinantly expressed CKB or CKM with reaction substrates, phosphocreatine and tenofovir‐monophosphate. TFV metabolites were detected using ultra‐high performance liquid chromatography tandem mass spectrometry (uHPLC/MS). Analysis of TFV‐DP levels revealed no significant difference between CKB and CKM catalyzed reactions. These data indicate that CKB may contribute to TFV activation in tissues where it is expressed, thus genetic variation in CKB may play a role in inter‐individual variability observed in TFV efficacy. To investigate the potential impact of genetic variation, fifteen naturally occurring missense mutations were chosen and mutant CKB enzymes were recombinantly expressed in E. coli. The purified enzymes were used in our in vitro activity assay and TFV metabolites were subsequently detected by uHPLC/MS. Eight mutations (C74S, R96P, S128R, R132H, R172P, R236Q, R292Q, and H296R) demonstrated a statistically significant reduction in the formation of TFV‐DP compared to that of wild‐type CKB. To gain insight into how these mutations disrupt enzymatic function, we exploited the reverse canonical reaction (ATP dephosphorylation) in an enzyme coupled system, from which Michaelis‐Menten curves were constructed and resulting kinetic parameters were used to calculate catalytic efficiencies. Five mutations (R96P, R132H, R236Q, R292Q, and H296R) resulted in catalytic efficiencies less than 20% of wild‐type, which can be attributed to increases in Km. Lastly, thermal stability was examined using differential scanning fluorimetry and showed four mutations (C74S, R96P, R172P, and H296P) had significantly lower melting temperatures than wild‐type. Additionally, seven mutations (C74S, R96P, R132H, R172G, R172P, R236Q, and H296P) displayed melting curves with double peaks or shouldering, suggesting local domain melting or dimer instability induced by the mutation. Together these data support our hypothesis that CKB contributes to TFV activation and indicates that naturally occurring mutations can diminish TFV‐DP formation, catalytic efficiency, and thermal stability in vitro.
Spironolactone prevents L‐type CaV1.2 potentiation and chronic vasoconstriction during diabetic hyperglycemia
Authors: Hong Junyoung , Martín‐Aragón Baudel Miguel , Reddy Gopyreddy R. , Nieves‐Cintrón Madeline , Navedo Manuel F.
Published: May 2022

Vascular complications are major causes of morbidity and mortality in diabetic patients. Recently, our group identified a novel purinergic signaling pathway involving the P2Y11/AKAP5/AC5/PKA/CaV1.2 protein network in arterial myocytes. This purinergic complex is activated by the release of nucleotides (e.g. ATP) to the extracellular space in response to hyperglycemic conditions, which promotes L‐type CaV1.2 potentiation and vasoconstriction. Pannexin 1 (Panx1) is a channel that mediates ATP efflux, thus inducing purinergic signaling activation. However, whether Panx1 forms part of the novel purinergic signaling complex that regulates L‐type CaV1.2 activity and vascular reactivity during diabetic hyperglycemia is unknown. Here, we find that Panx1 is in complex with P2Y11, AKAP5, AC5, PKA and CaV1.2 in arterial myocytes. This protein complex is strengthened upon elevated glucose (HG) treatment. However, the Panx1 inhibitor spironolactone prevented the HG‐induced strengthening of the complex. Spironolactone also blocked cAMP production, L‐type CaV1.2 potentiation and sustained vasoconstriction in response to elevated glucose. Consistent with a role for Panx1 upstream of P2Y11, spironolactone failed to prevent HG‐induced vasoconstriction upon addition of the specific P2Y11 agonist NF546. Altogether, these data suggest that Panx1 is part of the P2Y11/AKAP5/AC5/PKA/CaV1.2 signaling module in arterial myocytes. This Panx1‐led complex modulates L‐type CaV1.2 activity and vascular reactivity in response to diabetic hyperglycemic conditions. Thus, Panx1 could be a new therapeutic target to treat vascular complications during diabetes.
Hypoxic Preconditioning Attenuates Ischemia‐Reperfusion Injury in Older Adults
Authors: Stray‐Gundersen Sten , Massoudian Sahar D. , Wojan Frank , Tanaka Hirofumi , Lalande Sophie
Published: May 2022

Sudden restoration of blood flow to an ischemic vessel paradoxically damages endothelial cells. In young healthy adults, ischemic preconditioning, caused by repeated periods of brief ischemia induced by local cuff inflation prior to reperfusion, attenuates endothelial dysfunction following an ischemia‐reperfusion injury. However, ischemic preconditioning does not consistently protect against ischemia‐reperfusion injury in older adults. Intermittent systemic hypoxemia, induced via brief bouts of breathing low levels of oxygen, attenuates endothelial dysfunction following an ischemia‐reperfusion injury in young healthy adults. Therefore, the aim of the present study was to determine whether intermittent hypoxia protects against ischemia‐reperfusion injury in older adults. Eleven older adults (4 women, age: 57±9 years, height: 173±8 cm, body weight: 76±13 kg) visited the laboratory on two separate occasions. Endothelium‐dependent vasodilation was assessed by brachial artery flow‐mediated dilation using a semiautomated diagnostic ultrasound system before and after 20 minutes of blood flow occlusion to induce an ischemia‐reperfusion injury. Blood flow occlusion was preceded by either intermittent hypoxia (IH), consisting of three 4‐min hypoxic cycles at a targeted arterial oxygen saturation of 80% interspersed with 4‐min room air cycles, or intermittent normoxia (IN), consisting of three 4‐min normoxic cycles separated by 4‐min room air cycles. Intermittent hypoxia resulted in an arterial oxygen saturation of 80±2%, which corresponded to oxygen levels of 11.6±1.0%. When preceded by intermittent normoxia, blood flow occlusion reduced flow‐mediated dilation by 4.0±2.6% (6.4±1.7 to 2.4±1.8%). In contrast, flow‐mediated dilation was reduced by 1.8±1.5% when blood flow occlusion was preceded by intermittent hypoxia (5.6±1.8 to 3.7±2.4%: p=0.05). Intermittent hypoxia increased cardiac output (5.2±1.5 to 5.7±1.8 L·min‐1, p=0.03) mainly due to an increase in heart rate (61±12 to 69±10 bpm, p<0.01). In conclusion, hypoxic preconditioning attenuated the reduction in flow‐mediated dilation induced by blood flow occlusion in older adults. Thus, exposure to intermittent hypoxia represents a potential strategy to protect against ischemia‐reperfusion injury in populations at risk for ischemic events.
Stabilization of Meta‐I Rhodopsin Conformation by a Nanobody
Authors: Salom David , Wu Arum , Sander Christopher L. , Pardon Els , Steyaert Jan , Kiser Philip D. , Palczewski Krzysztof
Published: May 2022

Binding of an extracellular signaling molecule to a G protein‐coupled receptor (GPCR) results in G protein activation, which in turn triggers the production of second messengers. Although many advances have been achieved in understanding GPCR activation, the early steps of GPCR of this process are still poorly understood. In the case of rhodopsin, the paradigmatic member of class A GPCRs, the endogenous ligand is retinal which is covalently bound to K296 through a Schiff base. The activation of rhodopsin is triggered by the absorption of a photon and the isomerization of 11‐cis‐retinal to all‐trans‐retinal. When rhodopsin in ground state (λmax= 500 nm) is photoactivated, it progresses through short‐lived intermediates leading to the Meta‐I state (λmax= ~478 nm), which then establishes an equilibrium with Meta‐II (λmax= 380 nm), the G‐protein binding state.

We have developed a llama antibody (nanobody Nb2) that binds to bovine rhodopsin and stabilizes its early activation intermediate Meta‐I. Here we present the crystal structures of Nb2 in complex with ground‐state bovine rhodopsin, as well as Nb2 in complex with apo‐rhodopsin (opsin). The binding site of Nb2 includes rhodopsin’s N‐terminus and extracellular loops ECL2 and ECL3. Whereas rhodopsin’s structure in the ground‐state rhodopsin/Nb2 complex is virtually identical to that of rhodopsin alone, Nb2 binding induces a dramatic structural change in opsin, trapping it in a conformation similar to that of ground‐state rhodopsin. In a similar way, when Nb2 binds in solution to photoactivated, deprotonated rhodopsin (Meta‐II) it shifts the equilibrium towards a protonated Schiff base state corresponding to the Meta‐I conformation. Finally, co‐expression of Nb2 and P23H mutant rhodopsin in HEK293 cell lines is able to rescue the phenotype of misfolding and degradation in vitro, suggesting a therapeutic potential of this nanobody.

In summary, we have developed a nanobody that traps photoactivated rhodopsin in the elusive Meta‐I state; in addition, Nb2 stabilizes a misfolding‐prone mutant of rhodopsin. Nb2 might also be a useful tool to facilitate the crystallization of wild type and mutants of rhodopsin in different conformational states bound to different ligands. Current efforts include solving the crystal structure of the Meta‐I rhodopsin/nanobody complex.
Lysine Deacetylases and Demethylases: Early Epigenetic Regulators of Intermittent Hypoxia induced Sympathetic Activation and Blood Pressure
Authors: Wang Ning , Peng Ying‐JIe , Su Xiaoyu , Prabhakar Nanduri R. , Nanduri Jayasri
Published: May 2022

Intermittent hypoxia (IH) is a hallmark manifestation of obstructive sleep apnea (OSA). Long term IH (LT‐IH) triggers epigenetic reprogramming of the redox state involving DNA hypermethylation leading to persistent sympathetic activation and hypertension. Present study examined whether IH also activates epigenetic mechanism(s) other than DNA methylation. Lysine modifications of histones is another major epigenetic mechanism associated with gene regulation. Interplay between lysine deacetylases (HDACs) and demethylases (KDMs) differentially regulate gene expression. Lysine acetylation of histone opens up the chromatin thereby facilitating binding of transcription factors. On the other hand, KDMs regulate transcription by demethylating histone lysines which activate gene expression. Here we report that exposure of rat pheochromocytoma (PC)‐12 cells to IH in vitro exhibit reduced HDAC5 and increased KDM6B enzyme activity leading to increased HIF‐1α stability and transcriptional activity. Treatment of rats with Trichostatin A (TSA), an inhibitor of HDACs increased HIF‐1 dependent NADPH oxidase (NOX)‐4 transcription, in adrenal medullae (AM), resulting in elevated plasma catecholamines and blood pressure. Treating IH exposed rats with GSKJ4 an inhibitor of KDMs blocked HIF‐1 dependent transcriptional activation of NOX4, as well as absence of elevated plasma catecholamines and hypertension. These findings indicate a hitherto uncharacterized role of HDACs and KDMs as early epigenetic regulators in IH‐augmented sympathetic nerve activation and hypertension in rodent models of IH.
Galectin‐3 Regulates Oxidant Stress in Obesity
Authors: Padgett Caleb A. , Speese Andrew C. , Rosewater Cody L. , Corley Zachary L. , Mintz James D. , Fulton David J. , Stepp David W.
Published: May 2022

Objective

Investigate the mechanism by which overexpression of Galectin‐3 (GAL3) in the endothelium of obese mice contributes to excess reactive oxygen species (ROS) production and subsequent vascular dysfunction.

Methods

db/db mice, a well‐characterized model of human obesity, were crossed with mice lacking GAL3, a receptor for advanced glycation end products, to generate lean and obese mice with and without GAL3 deletion. Metabolic and cardiovascular endpoints were assessed by mRNA and protein expression in freshly isolated microvascular endothelial cells, microvascular reactivity assessed by pressure myography, and ROS production assessed by dihydroethidium (DHE) staining. Other previously characterized db/db colonies were utilized to further elucidate the mechanistic contributions of GAL3, including mice harboring deletions of NADPH Oxidase I (NOX1), Myostatin (MSTN), and Protein Tyrosine Phosphatase 1B (PTP1B).

Results

Deletion of GAL3 in obese mice had no observable effect on anatomical, metabolic, or plasma phenotype, except for normalizing levels of plasma ROS as evidenced by decrease in thiobarbituric acid reactive substances (TBARS). Obese mice exhibited profound endothelial dysfunction, which was rescued by deletion of GAL3. Our data demonstrate that NOX1, the predominant source of endothelial superoxide production, is significantly downregulated by GAL3 deletion, thereby restoring endothelial function and ameliorating endothelial ROS production in obesity. Furthermore, we demonstrate that GAL3‐mediated NOX1 overexpression is amenable to improvements in metabolic status, such as lowering blood glucose with metformin, improving glucose handling by augmenting muscle mass through deletion of MSTN, or improving insulin signaling through deletion of PTP1B.

Conclusion

Taken together, these data demonstrate that the overproduction of superoxide by endothelial NOX1 is regulated by the metabolic sensor GAL3 in obesity, leading to endothelial dysfunction and subsequent cardiovascular disease. This pathway presents an attractive target for therapeutic intervention to break the link between aberrant metabolism in obesity and its corresponding vascular pathologies.
Depolarized Mitochondrial Membrane Potential and Protection with Duroquinone in Isolated Perfused Lungs from Rats Exposed to Hyperoxia
Authors: Audi Said , Ganesh Swetha , Taheri Pardis , Zhang Xiao , Dash Ranjan , Clough Anne , Jacobs Elizabeth
Published: May 2022

Objective

Dissipation of mitochondrial membrane potential (Δψm) is a hallmark of mitochondrial dysfunction, which plays a key in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS). Our objective was to use a previously developed experimental‐computational approach to estimate tissue Δψm in isolated perfused lungs of rats exposed to hyperoxia as a rat model of human ARDS, and to evaluate the ability of the ubiquinone analog duroquinone (DQ) to reverse any hyperoxia‐induced depolarization of lung Δψm.

Methods

Rats were exposed to hyperoxia (>95% O2) or normoxia (room air) for 48 hrs, after which lungs were excised and connected to a ventilation‐perfusion system. The experimental protocol consisted of measuring the concentration of the fluorescent dye rhodamine 6G (R6G) during three single‐pass phases: loading, washing, and uncoupling, in which the lungs were perfused with and without R6G, and with the mitochondrial uncoupler FCCP, respectively. For normoxic lungs, the protocol was repeated with 1) rotenone (mitochondrial complex I inhibitor), 2) rotenone and either DQ or its vehicle (DMSO), and 3) rotenone, glutathione (GSH), and either DQ or DMSO added to the perfusate. Hyperoxic lungs were studied with and without DQ and GSH added to the perfusate. A physiologically‐based pharmacokinetic (PBPK) computational model was used to estimate lung Δψm from R6G data.

Results

Figure 1 shows that for hyperoxic lungs, R6G venous effluent concentrations tended to be higher during the loading phase, but significantly lower during the uncoupling phase compared to normoxic lungs, consistent with less uptake of R6G by hyperoxic lungs due to partial depolarization of Δψm. Figure 1 also shows that adding DQ to perfusate during the three phases almost completely reversed differences in R6G concentrations between normoxic and hyperoxic lungs. The PBPK computational model analysis of the R6G data show that exposure to hyperoxia depolarized lung tissue Δψm from ‐140 ± 9 (SE, n = 5) mV to ‐107 ± 9 (n = 5) mV, and complex I inhibition depolarized lung Δψm from ‐140 ± 9 (n = 5) to ‐57 ± 2 (n = 4) mV. Computational model analysis results also demonstrate the efficacy of DQ to fully reverse both rotenone‐induced and hyperoxia‐induced lung Δψm depolarization, consistent with the ability of the lungs to reduce DQ to its hydroquinone form (DQH2), which in turn bypasses mitochondrial complexes I and II to reduce mitochondrial complex III and restores Δψm.

Conclusions

This study demonstrates hyperoxia‐induced Δψm depolarization in isolated perfused lungs, and the utility of this approach for assessing the impact of potential therapies such as exogenous quinones that target mitochondria in isolated perfused lungs.
Towards the discovery of virulence and survival factors in Fusobacterium using host‐mimicking DNA methylation to transcend genetic barriers
Authors: Nguyen Tam T. D. , Umana Ariana , Sanders Blake , Slade Daniel
Published: May 2022

Fusobacterium nucleatum (Fn) is an anaerobic, Gram‐negative, oral bacterium that disseminates from the mouth, and contributes to preterm birth, tissue infections, and acceleration of multiple cancers including colorectal and pancreatic. It is well‐established that most Fusobacteriumexhibit genetic recalcitrance, mainly owing to robust restriction modification systems. These systems allow bacteria to distinguish self from non‐self DNA via two enzymatic activities: a restriction endonuclease and a modification methyltransferase. Though the association of Fnin diseases is well‐established, the majority of our experimental work stems from the strain F. nucleatum 23726 because it is genetically tractable. To enable the study of Fn clinical isolates that are currently genetically intractable, here we show that we are able to fool host restriction modification systems that protect the bacteria against the introduction of foreign DNA by using FnDNA methyltransferases to protect our gene knockout plasmids against nuclease digestion. This genetic breakthrough has enabled the first mutants in F. nucleatum 25586, a feat that was never accomplished in decades of trying. Furthermore, we created a deletion library of genes predicted to be involved in host cell invasion and bacterial intracellular survival, which is enabling detailed mechanistic studies of these virulence proteins in Fn‐driven disease. Our results demonstrate the simplicity and convenience of this host‐mimicking approach, which enables circumvention of innate restriction modification barriers ‐ the most common underlying cause of bacterial genetic intractability. We anticipate in future applications our system could be expanded to study novel genetic elements of which are fundamental to the pathophysiology of a wide range of relevant Fusobacterium species.
A plasma membrane‐localized polycystin‐1/polycystin‐2 complex in endothelial cells elicits vasodilation
Authors: Mackay Charles , Floen Miranda , Leo M. D. , Hasan Raquibul , Fernandez‐Pena Carlos , Singh Purnima , Malik Kafait U. , Jaggar Jonathan H.
Published: May 2022

Polycystin‐1 (PC‐1, PKD1), a receptor‐like protein expressed by the Pkd1 gene, is present in a wide variety of cell types, but its cellular location, signaling mechanisms and physiological functions are poorly understood. Here, by studying tamoxifen‐inducible, endothelial cell (EC)‐specific Pkd1 knockout (Pkd1 ecKO) mice, we show that flow activates PC‐1‐mediated, Ca2+‐dependent cation currents in ECs. EC‐specific PC‐1 knockout attenuates flow‐mediated arterial hyperpolarization and vasodilation. PC‐1‐dependent vasodilation occurs over the entire functional shear stress range and via the activation of nitric oxide synthase (NOS) and small‐and intermediate‐conductance Ca2+‐activated K+ (SK/IK) channels. EC‐specific PC‐1 knockout increases systemic blood pressure without altering kidney anatomy. PC‐1 coimmunoprecipitates with polycystin‐2 (PC‐2, PKD2), a TRP polycystin channel, and clusters of both proteins locate in nanoscale proximity in the EC plasma membrane. Knockout of either PC‐1 or PC‐2 (Pkd2 ecKO mice) abolishes surface clusters of both PC‐1 and PC‐2 in ECs. Single knockout of PC‐1 or PC‐2 or double knockout of PC‐1 and PC‐2 (Pkd1/Pkd2 ecKO mice) similarly attenuates flow‐mediated vasodilation. Flow stimulates non‐selective cation currents in ECs that are similarly inhibited by either PC‐1 or PC‐2 knockout or by interference peptides corresponding to the C‐terminus coiled‐coil domains present in PC‐1 or PC‐2. In summary, we show that PC‐1 regulates arterial contractility and demonstrate that this occurs through the formation of an interdependent signaling complex with PC‐2 in endothelial cells. Flow stimulates PC‐1/PC‐2 clusters in the EC plasma membrane, leading to Ca2+ influx, NOS and SK/IK channel activation, vasodilation and a reduction in blood pressure.
The Effect of Spared Nerve Injury on Ethanol Reward Seeking and Reinstatement
Authors: Nothem Mitch A. , Barker Jacqueline M.
Published: May 2022

Alcohol use disorder (AUD) and chronic pain are complex and debilitating conditions that are difficult to treat, lead to enormous economic costs, and excessive suffering. The difficulty in developing new treatments for AUD is partially due to the lack of consideration given to the wide array of factors that drive alcohol intake. Clinical data suggest that both men and women with chronic pain are more likely to develop alcohol use disorder and report using alcohol to deal with chronic pain. Additionally, it has been shown that self‐report of chronic pain correlates with alcohol relapse after a period of abstinence. These data led to the hypothesis that neural mechanisms that contribute to alcohol related behaviors are different in individuals that are also experiencing chronic pain. To test this hypothesis, we used a conditioned place preference (CPP) paradigm to model ethanol seeking in male and female rodents that had undergone either a spared nerve injury (SNI) model of chronic pain or a sham surgery. To determine a dose of ethanol that may be selectively rewarding to injured animals, we first tested the analgesic effects of multiple doses of ethanol on pain‐related behaviors in SNI and Sham mice. The higher doses of 1.0g/kg and 2.0g/kg (i.p.) of ethanol were anti‐allodynic in SNI mice and analgesic in sham mice. However, we found that the lower dose 0.5 g/kg of ethanol was able to fully reverse mechanical hypersensitivity in SNI animals, while not influencing mechanical thresholds in sham mice. Thus, we conditioned mice using 0.5g/kg of ethanol. We found this dose induced a modest conditioned place preference for the ethanol‐paired chamber in both SNI and Sham male mice. Mice then underwent extinction training for 1 week, during which time animals were exposed to the CPP apparatus without receiving any ethanol. To investigate relapse‐related behavior, we then tested whether painful stimulation would reinstate ethanol seeking behavior. We found that threshold hindpaw stimulation was able to reinstate ethanol seeking behavior in SNI, but not sham animals. This suggests that animals in chronic pain may associate alcohol with pain relief or that chronic pain animals may be more sensitive to stress induced reinstatement when pain is the stressor. We also tested the effect of drug induced reinstatement and found no differences between SNI and Sham mice, leading us to believe that differences found in pain induced reinstatement were selective to that modality. Ongoing work is aimed at determining the effect of ethanol on weight bearing behavior as well as the effect of chronic pain on ethanol seeking and relapse‐related behavior in female mice.
Mice Exposed to Chronic low‐dose Ethanol Exhibit Reduced Sensitivity to Changes in Magnitude of Reward
Authors: Curran‐Alfaro Christina M. , Bryant Kathleen G. , Barker Jacqueline M.
Published: May 2022

While approximately 2 billion individuals consume alcohol worldwide, the majority of these individuals to not exhibit drinking patterns consistent with alcohol use disorders (AUDs). Efforts have been made in investigating and characterizing the adverse effects on learning, memory and reward seeking with individuals afflicted with AUDs in a pursuit of therapeutic advancements. However, sub‐diagnostic alcohol effects have been understudied, despite the large number of low‐dose alcohol drinkers and the potential for low dose alcohol to impact both brain and behavior.

Data from our lab has shown that low‐dose alcohol exposure enhances reward motivation in male, but not female, mice, but the mechanism of this is effect is unclear. Thus, we investigated the effects of low‐dose alcohol exposure on sensitivity to change in reward magnitude. A group of adult male C57BL6J mice were trained to self‐administer a 20 ul sucrose reward and received either a low‐dose ethanol (0.5g/kg) or saline injection 1 hour after training. To assess motivation, we used a progressive ratio (PR) task in which the effort required to earn the sucrose reward was increased linearly. Mice received multiple counterbalanced PR sessions in which the progressive schedule remained the same, but the reward magnitude was changed from 20ul (baseline) to 35ul, 25ul, 15ul or 5ul in counterbalanced sessions.

Consistent with sensitivity to changes in reward value, saline control mice reduced or increased responding when reward volume was reduced or increased, respectively. Similar to saline controls, chronic low‐dose ethanol exposed mice increased their responding when reward volumes were increased compared to the 20ul baseline. However, ethanol exposed mice did not reduce responding to small reductions in reward magnitude. This observation in low‐dose ethanol mice suggests either a failure to detect reductions in reward magnitude or a dissociation between reduced reward magnitude and behavioral response. This lack of strategizing behavior to maintain effort‐to‐reward balance by ethanol exposed mice may imply that chronic low‐dose ethanol exposed mice may perceive greater reward value than controls even at reduced volume and implicates even low‐dose ethanol as a contributor to aberrant reward processing.
Structure–Activity Relationships of a Negative Allosteric Modulator of the D3 Dopamine Receptor and Investigation of its Binding Site
Authors: Moritz Amy E. , Madaras Nora S. , Inbody Laura R. , Kelley Amber M. , Gandhi Disha M. , Akano Emmanuel , Lee Kuo H. , Free R. B. , Hu Xin , Southall Noel T. , Ferrer Marc , Shi Lei , Frankowski Kevin J. , Sibley David R.
Published: May 2022

Dopamine receptors (DARs) are G protein‐coupled receptors (GPCRs) that regulate diverse physiological functions and are involved in the treatment and/or etiology of many neuropsychiatric disorders including schizophrenia and substance use disorder (SUD). DARs are classified as either D1‐like (D1R and D5R) or D2‐like (D2R, D3R, and D4R) based on structural homology and pharmacological profiles. Antagonists of D2‐like DARs are currently used in the therapies for many neuropsychiatric disorders. D3R‐selective antagonists have the potential to be better therapeutics for schizophrenia or SUD as they could attenuate psychotic or drug craving symptoms without the motor side effects frequently produced by D2R‐preferring antagonists. Unfortunately, discovery of subtype‐selective compounds for the D3R and D2R has been challenging due to high sequence homology within the orthosteric binding sites of DARs. However, compounds that modulate receptor activities through interactions with less conserved allosteric sites have the potential to be highly selective. To find highly selective allosteric antagonists of the D3R, we screened the NIH Molecular Libraries Program 400,000+ small molecule library with a D3R‐mediated β‐arrestin recruitment assay. We found that one compound, MLS6357, was selective for the D3R over the D2R and D4R in several functional outputs including β‐arrestin recruitment and G‐protein activation. Radioligand binding and functional assays using closely related GPCRs revealed that MLS6357 has very limited cross‐reactivity with other GPCRs. Additionally, Schild‐type functional assays showed that MLS6357 acts as a purely non‐competitive negative allosteric modulator (NAM) of the D3R. We synthesized and characterized >60 analogs of MLS6357 using iterative medicinal chemistry approaches, which revealed structure–activity relationships and enabled further optimization of the scaffold. These efforts produced analogs that are 10‐fold and 30‐fold more potent than the parent compound in D3R‐mediated β‐arrestin recruitment and G‐protein activation assays, respectively. Moreover, some analogs appear to display functional selectivity for inhibition of G‐protein activation versus inhibition of β‐arrestin recruitment, and vice versa, and some also display inverse agonist activity. To identify the allosteric binding site for the MLS6357 scaffold on the D3R, we utilized various D3R/D2R chimeras, receptor mutants, and molecular modeling techniques to reveal and characterize receptor regions necessary for compound efficacy. Further refinement of the binding pocket for MLS6357 will inform future medicinal chemistry efforts. Ultimately, this novel scaffold may be of benefit as a pharmacological probe or therapeutic lead for D3R‐related pathophysiology.
Critical residues necessary for folding of a carboxypeptidase in the absence of a prodomain
Authors: Domecillo Masy , Shin Erica , Latour Donn M. , Lyons Peter J.
Published: May 2022

The structure and functions of proteins are highly reliant on their folding. Molecular chaperones assist with this process, and these chaperones may occur within the protein itself. Carboxypeptidase A1 (CPA1) is an enzyme that contains a prodomain that has been previously inferred to be an intra‐molecular chaperone; however, the discovery of a related enzyme lacking a prodomain, carboxypeptidase O (CPO), questions the necessity of the prodomain for folding. The amino acid sequence of CPO was analyzed to find residues that were conserved and unique to CPO when compared with CPA1 and related carboxypeptidases. Four such residues were identified and found through site‐directed mutagenesis and western blotting to be necessary for expression of CPO. In preparation for transferring these CPO residues to CPA1 by site‐directed mutagenesis to examine their effect on CPA1 expression, PCR was used to insert an HA tag at the C‐terminus of CPA1. Transfection of this plasmid encoding HA‐tagged CPA1 into HEK293T cells resulted in expression of the protein, as determined by western blotting, and detection of enzymatic activity. Subsequent deletion of the prodomain, transferring of unique CPO residues to CPA1, and analysis of expression is ongoing. These studies will help us to determine the mechanism by which CPO folds in the absence of a prodomain, and so further our understanding of protein stability and folding.
The effects of excess salt consumption on contextual fear generalization in mice
Authors: Kassis Sarah K. , Weber Brady L. , Ford Matthew T. , Beaver Jasmin N. , Anello Anna E. , Gilman Lee T.
Published: May 2022

Excess salt (NaCl) consumption is prevalent worldwide. Most Americans consume more than twice the daily recommended amount of sodium (Na; 2300mg). Excessive salt intake is a risk factor for cardiovascular pathology such as hypertension. Anxiety is often comorbid with cardiovascular diseases. Research has been done to examine the relationship between salt consumption and cardiovascular and other related peripheral diseases. However, far less research has been done to examine the relationship between salt consumption and brain health or anxiety. To examine this, mice were used because their salt intake and stress exposure can be rigorously controlled, unlike in humans. Specifically, we focused on fear generalization, a key characteristic of anxiety symptoms involving contextually inappropriate fear of situations not currently present. We hypothesized that mice consuming a diet with excess salt would exhibit higher levels of fear generalization relative to mice consuming a low salt diet. Adult (9 wk old) male and female C57BL/6J mice were given either a low (0.4% NaCl, w/w) or high (4.0% NaCl) salt diet ad libitum. Mice underwent contextual fear training 2 or 6 wks after commencing diet manipulation. Testing for contextually appropriate (fear expression) and contextually inappropriate (fear generalization) fear behavior occurred either 2 days (2 and 6 wk groups) or 4 wks (6 wk group) after training. Regardless of diet condition, mice of both sexes acquired contextual fear similarly when trained at the 2 wk timepoint, and exhibited equivalent fear expression and contextual discrimination when tested 2 days later. Similarly, and as expected, male mice consuming a low salt diet expressed normal levels of fear generalization at 4 wks. Contrary to our hypothesis, however, male mice consuming a high salt diet did not exhibit fear generalization at 4 wks. Female tests of 4 wk fear generalization, and both sexes in the 6 wk group being tested after 2 days, are ongoing. Thus far, our findings indicate that male mice consuming a high salt diet do not generalize their contextual fear after a standard time delay (4 wks), or alternatively display enhanced contextual discrimination at the 4 wk timepoint post‐training. Our findings suggest intake of salt, a non‐caloric dietary component, can have a significant effect on a measure directly related to anxiety symptoms. Future experiments will evaluate the contribution of neuroinflammation to these behavioral changes, as well as investigate cued fear responses in both sexes.
Differential Behavior of 4E‐BP1 and 4E‐BP2 Under ER Stress
Authors: Gobbooru Shruthi , Low Woon‐Kai
Published: May 2022

eIF4E‐binding proteins (4E‐BPs) are a family of translational repressors, when unphosphorylated or hypo‐phosphorylated, can bind to eIF4E and inhibit eukaryotic cap‐dependent translation initiation. The 3 4E‐BPs (4E‐BP1, 4E‐BP2 and 4E‐BP3) are not isoforms but are often regarded alike in terms of inhibiting translation initiation. They are not clearly differentiated in terms of function. Not only do they originate from different genes on different chromosomes, but they also have distinct mRNA structure. We hypothesized a potential for their differential regulation under cell stress. During cell stress, 4E‐BPs need to be upregulated to stop protein synthesis and conserve cellular energy. 4E‐BP1 promoter has CARE (C/Ebp‐ATF Response Element) in its promoter region, that helps it respond to ER stress in an ATF4‐dependent manner. 4E‐BP2 on the other hand, does not have any CARE elements in its promoter, hence ideally should remain unchanged under ER stress. Gene expression was observed through qPCR and protein levels were studied using western blotting. We observed a nearly 4‐fold increase in 4E‐BP1 mRNA levels under ER stress in sync with ATF4 mRNA levels, whereas 4E‐BP2 mRNA levels didn’t change significantly. 4E‐BP1 protein levels increased under ER stress whereas 4E‐BP2 protein levels decreased under ER stress. We observed selective proteasomal degradation of 4E‐BP2 under ER stress, which recovered when treated with proteasome inhibitor MG132. The reason behind this selective degradation needs to be further elucidated. These findings shed a new light on the behavior of 4E‐BP2 under cell stress which has been, until now, treated synonymous to 4E‐BP1 in the cellular landscape.
Alteration in MUC2 and OCLN Protein Expression in Lung Epithelial Cells After E‐Cigarette Chemical Exposure
Authors: Huynh Thien‐Huong K. , Daft Joseph
Published: May 2022

Electronic cigarettes (e‐cigarettes) have become increasingly popular, especially among young adults, and are marketed as a safer and healthier alternative to traditional tobacco cigarettes (t‐cigarettes). There has not been sufficient time since their introduction into the US market in 2006 for long‐term studies looking at the possible side effects of e‐cigarettes. However, various short‐term studies have pointed to possible health risks. To further investigate these effects, this study looked at the mucus membrane and tight junction disruption in A549 cells by focusing on the alteration of mucin 2 (MUC2) protein and occludin (OCLN) protein expression after exposure to formaldehyde (one chemical in e‐cigarettes). Immunohistochemistry was used to visualize the expression of these proteins. As hypothesized, an alteration in MUC2 and OCLN protein expression was found after exposure to the formaldehyde. For the 12‐hour treatment, a shift in MUC2 expression from nuclear to cytoplasmic and a downregulation of OCLN expression were observed. For the 24‐hour treatment, an upregulation in both MUC2 and OCLN expression around the cell nuclei was observed.
Examining the role of Drp1 in age‐related microvascular dysfunction
Authors: Gutierrez Huerta Cristhian A. , Hader Shelby N. , Beare Jason E. , Tracy Evan , Astbury Katherine , Jacobs Elizabeth R. , LeBlanc Amanda J. , Gutterman David D. , Beyer Andreas M.
Published: May 2022

Introduction

Advanced age is a well‐documented risk factor for coronary microvascular dysfunction. Recent evidence has underscored that advanced age is associated with a shift toward greater mitochondrial fission rather than mitochondrial fusion. Therefore, understanding key regulators of the balance of mitochondrial fission/fusion may help to identify the contribution of mitochondrial dynamics on microvascular endothelial function. DRP1 is a pro‐fission factor that has been linked to several disease manifestations including advanced age and cardiovascular disease (CVD).

Methods

Coronary arteries were obtained from young control rats (YC, 15‐20 weeks) and old control rats (OC, 95‐100 weeks) and expression of DRP1 was assessed by immunofluorescence (IF). A novel rat model crossing flox‐stop DRP1‐GFP rats and VECAD‐Cre expressing rats was generated to assess the vascular and cardiac effects of endothelial‐specific DRP1 overexpression. Endothelial vascular function was determined by endothelial‐dependent flow‐mediated dilation while cardiac function was determined by echocardiography at 20 weeks old and compared to wildtype control group.

Results

Data from IF comparing coronary arteries from YC and OC show that OC had higher DRP1 expression compared to YC (1.42 to 1.00, p=0.01). Max dilation measurements, taken at 20‐weeks of age, showed that DRP1‐overexpressing rats was decreased when compared to wildtype (WT) control rats (31.4 to 60.1, p=0.009). Echocardiography revealed that DRP1‐overexpressing rats showed a trend towards a decrease in coronary flow reserve and increased HR in response to a dobutamine stressor when compared to WT. Ejection fraction between the two groups remained largely preserved.

Conclusion

Our data demonstrate that there may be a critical role for DRP1 within in vitro vasodilation that mimics a CVD pro‐fission phenotype. Future studies will be performed to further evaluate the role of DRP1 in response to other stressors well‐established in age‐dependent CVD such as hypertension.
Dietary nitrate supplementation attenuates the mechanoflex in older women with early menopause: A pilot study
Authors: Manabe Kazumasa , Yoo Jeung‐Ki , Takeda Ryosuke , Curtis Bryon , Roberts‐Reeves Monique , Fu Qi
Published: May 2022

Women who reached early menopause (EM, 40‐44 yrs) have a greater risk for cardiovascular disease than women with normal menopause (NM, 50‐54 yrs), which may be attributed to a longer duration of estrogen loss and a greater reduction in nitric oxide bioavailability in both the peripherals and center. Previous studies have shown that dietary nitrate supplementation improves cardiovascular function in older women, but how autonomic neural control changes in daily living, such as during exercise, has been involved is unclear. We examined the hypothesis that 2wks continuous use of dietary nitrate supplements would attenuate the exercise pressor reflex in older women with EM.

Six EM (65±4 [SD] yrs) and 8 NM (64±2 yrs) women participated in this randomized, placebo‐controlled, double‐blind, cross‐over pilot study. All subjects underwent 2 wks of beetroot juice (BRJ; 140 ml containing ~800 mg nitrate per day) or placebo (140 ml nitrate‐depleted BRJ, daily) intervention with ≥14 days of wash‐out. Before and after each intervention period, subjects performed SHG using the dominant hand at 40% of maximum voluntary contraction (MVC) until fatigue, followed by 2 min of PECA with an upper arm cuff inflated to 250 mmHg. Heart rate (HR; ECG), blood pressure (BP; finger photoplethysmography), muscle sympathetic nerve activity (MSNA; microneurography), and respiration (capnography) were continuously measured. In addition, 24‐h ambulatory BP standard deviation (SD) was monitored before and after each intervention (an indication of BP variability).

There were no differences in baseline HR, BP and MSNA, MVC, and the time to fatigue between the EM and NM groups before and after intervention (all p>0.05). At the first 30 s of SHG, the EM group showed significant decreases in MSNA burst frequency (DBF; ‐4.5±6.5 in EM vs. 2.5±6.2 bursts/min in NM, p=0.065) and burst incidence (DBI; ‐10.6±12.6 vs. 2.6±8.4 bursts/100 heartbeats, p=0.037) after BRJ intervention compared with the NM group (Figure1A and B). However, there were no differences between groups at fatiguing exercise and during PECA after the intervention. The interventional changes in 24‐h diastolic BP SD had a positive correlation with ΔMSNA BF (r=0.55, p=0.053) and BI (r=0.58, p=0.036) at the first 30 s of SHG in all subjects and EM individuals were in the left‐lower area (Figure2 A and B).

These results suggest that dietary nitrate supplementation may attenuate and mechanoreflex, but not the metaboreflex, in older women with EM during static hand grip, which has a relation with BP variability during ambulation. Certainly, these preliminary observations need to be verified in more older women in future studies.
Identifying Novel Signaling Mechanisms Downstream of Gq‐Coupled Receptors
Authors: Loomis Joseph F. , Chandan Naincy R. , Brody Matthew J. , Smrcka Alan V.
Published: May 2022

The G protein alpha subunit G□q is a critical mediator of cells’ response to a variety of external stimuli as a result of activation of Gq‐coupled GPCRs. G□q‐dependent signaling influences an array of physiological processes, including neurotransmission and vasoconstriction; additionally, dysregulated G□q‐dependent signaling has been firmly linked to several pathologies, including uveal melanoma and maladaptive cardiomyocyte hypertrophy. One established mechanism by which activated G□q subunits might promote these processes involves activation of phospholipase C beta isoforms (PLC□), which catalyze the hydrolysis of PI(4,5)P2 into inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG), ultimately leading to Ca2+ mobilization and protein kinase C activation. However, G□q subunits also signal independently of PLC□, through p63RhoGEF and Trio, for example.

To identify novel G□q targets that might be involved in Gq‐driven pathologies, we conducted a proximity biotinylation proteomic screen in HEK293A cells comparing wild‐type G□q and constitutively active G□q Q209L each fused to TurboID, a promiscuous biotin ligase. This approach enabled the high‐confidence identification of numerous proteins that were selectively enriched in cells expressing G□q‐Q209L‐TurboID compared to cells expressing G□q‐WT‐TurboID. These enriched proteins included known G□q interactors (PLC□, Trio, and GRK2); however, scattered among these known interactors were several proteins that have not been previously shown to interact with active G□q, including SPRED1, SMARCD3, YAP1, BCAS2, and HDAC9.

Our initial efforts have focused on SMARCD3, a regulatory component of the SWI/SNF chromatin remodeling complex. To test for SMARCD3 interactions with G□q, we expressed SMARCD3 in COS‐7 cells and found that SMARCD3 selectively inhibits G□q‐stimulated, but not G□□‐stimulated, PLC□ activity, indicating that SMARCD3 competes with PLC□ for access to active G□q. In addition, we expressed SMARCD3 in HEK293A cells and found that (1) SMARCD3 co‐immunoprecipitates with G□q and (2) interacts with G□q in a nanoluciferase fragment complementation assay.

SWI/SNF regulates gene transcription by modulating chromatin accessibility. Given that Gq signaling regulates gene expression in both physiological and pathological systems, we will assess the impact of SMARCD3 knockdown in cellular models of G□q‐driven transcription and cell function. Ultimately, we will expand this approach to test other top hits' ability to interact with and potentiate signaling downstream of active G□q. Through these endeavors, we aim to uncover novel G□q signaling mechanisms and inform therapeutic strategies for combatting pathologies marked by aberrant G□q signaling.