bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of a C‐terminal Electrostatic Interaction Domain on KCC3 Function
Authors: Flores‐Carrillo Alejandrina , Mercado‐Rodríguez Diana , Mercado Adriana , de los Heros Paola
Published: May 2022

Background

The K:Cl cotransporters (KCCs) are key players to maintain cellular volume, mediate epithelial transport and intracellular chloride concentration, and regulate GABA neurotransmission. Under isotonic conditions, KCCs are normally inhibited due to the phosphorylation of residues located within the cytoplasmic domains. On the contrary, dephosphorylation of those residues in response to hypotonic cell swelling stimulates transport activity. Dysfunction of these proteins and the processes involved in their regulation have been associated with various human diseases by alteration of ionic homeostasis mainly in the kidney and the brain. Based on the cryo‐EM structures recently reported of human KCCs, revealing structural determinants for regulation in both N‐ and C‐terminal domains, in the present study we analyzed the effect of a structural domain located at the C‐ terminal involved in electrostatic interactions. It has been stated that the side chains of residues as Gln (Q), Arg (R) and Lys (K) tend to form hydrogen bonds promoting the stabilization of tertiary and quaternary protein structures. However, whether the phosphorylation status is modified after the electrostatic disruption is unknown.

Methods

Xenopus laevis oocytes and HEK 293 cells were either microinjected or transfected with KCC3a wild type (WT) or C‐terminal mutants, Q905A, Q905/K934A (2M), Q905/K934/R937A (3M) and Q905/K934/R937/K938A (4M), key residues involved in electrostatic interactions and located within the C‐terminal domain of the cotransporter. The phosphorylation status of KCC3a in response to isotonic and hypotonic conditions was evaluated by immunoblot analysis of three key regulatory phosphorylation sites (pSer96, pThr991 and pThr1048). In parallel, phosphorylation status of SPAK/OSR1 activating regulatory site pSer373/pSer325 was analyzed under the same conditions.

Results

Cell swelling‐induced dephosphorylation is not observed on C‐terminal electrostatic interaction domain mutants compared to WT KCC3 in X. laevis oocytes and HEK cells. All three regulatory‐phosphorylation sites in KCC3a mutants remained phosphorylated under hypotonic conditions where dephosphorylation is required for the protein to become active. These results suggest that the disruption of the hydrogen bonds could disrupt the proper folding of the protein and thus interfere with the regulatory dephosphorylation/phosphorylation signaling pathway which is known to activate the transporter after cell swelling.

Conclusions

Our results show that the four residues in the electrostatic interaction domain of the C‐termini are involved in KCC3a response to hypotonic conditions affecting the phosphorylation status of the cotransporter. The side chains of these residues seem to be important in maintaining tertiary conformation given their ability to form hydrogen bonds and thus, the disruption of their interactions affects the cotransporter ability to respond to osmotic and cell volume changes.
Inflammatory cytokines analysis in C57BL/6 mouse skin after dermal exposure with chemical threat agent phosgene oxime
Authors: madadgar omid , Singh Satyendra , Monson Anna , Goswami Dinesh , Anantharam Poojya , Croutch Claire , Tewari‐Singh Neera
Published: May 2022

Phosgene Oxime (dichloroform oxime; CX), an urticant categorized as a vesicating agent, is a potential chemical threat agent. Its exposure causes rapid and painful dermal injury and systemic toxic effects. CX exposure can result in enzyme inactivation, corrosive injury, and cell death with rapid tissue destruction, and induce the recruitment of immune cells. However, the mechanism of CX‐induced skin toxicity and inflammation is not known. Studies in our lab have shown that CX exposure causes mast cell degranulation and release of inflammatory mediators like COX‐2, MMP‐9, myeloperoxidase, and an inflammatory response in the skin. In the present study, we analyzed the inflammatory cytokine profile in male and female C57BL/6 mouse skin following dermal CX exposure (neat CX for either 0.5 or 1.0 min using two 12 mm vapor caps on the dorsal skin at MRIGlobal). Cytokine array analyses showed an increase in the pro‐inflammatory cytokines IL‐6 (Interleukin 6) and TNF‐α (Tumor Necrosis Factor Alpha) in both male and female mice at 24h post CX‐exposure. KC (neutrophil chemoattractant), MCP‐1 (Monocyte chemoattractant), MCSF (Macrophage colony‐stimulating factor) and RANTES (T cell, monocyte attractant) increased in both sexes at day 3 post CX‐exposure, showing involvement of immune cells in the CX‐induced injury. IL‐13, an allergic inflammation and several diseases’ mediator was found to be increased at later time point in female mice and at 2 hours after 1.0 min CX exposure in male mice. IL‐4 and IL‐5 levels decreased at 24 h but were increased at day 3 post 0.5 min CX exposure. Altogether, our results show immune cell infiltration with changes in inflammatory cytokines. Similar Inflammatory cytokine changes from CX exposure were observed in male and female mice; however, slightly different patterns were evident in some cytokines in female mice that related to the observed difference in the inflammatory response and somewhat faster dermal wound healing. Further molecular analysis is underway to determine the signaling pathways involved in CX‐induced dermal inflammation and toxicity for identifying the therapeutic targets.
Efavirenz Biotransformation in Brain Microsomes and Neural Cells
Authors: Wheeler Abigail , Bumpus Namandjé
Published: May 2022

Efavirenz (EFV) is a non‐nucleoside reverse transcriptase inhibitor that is metabolized by both the cytochromes P450 and UDP‐glucuronosyltransferases (UGTs). EFV is one of several anti‐HIV medications that can cross the blood‐brain barrier and is essential to combatting the viral reservoir in the brain that can eventually lead to HIV‐associated neurocognitive disorder (HAND) in nearly 50% of patients. EFV and 8‐hydroxyefavirenz (8‐OHEFV), the primary P450‐dependent metabolite of EFV, have both been linked to neurological adverse events and neurotoxicity. However, very little is known about the fate of EFV in the brain, particularly how it’s metabolized or the precise mechanism of neurotoxicity. To investigate EFV biotransformation in the brain, we first performed microsomal metabolism assays with brain microsomes obtained from mice, cynomolgus macaques, and humans. EFV was incubated with the brain microsomes and NADPH to characterize P450‐dependent metabolism, while EFV or one of its P450‐dependent metabolites, 8‐OHEFV or 8‐,14‐dihydroxyefavirenz (8,14‐diOHEFV), was incubated with brain microsomes and UDPGA to investigate UGT‐catalyzed metabolism. Ultra‐high‐performance liquid chromatography tandem high resolution mass spectrometry (UHPLC‐HRMS) was employed to detect metabolites. Formation of 8‐OHEFV was observed in all three species, as was glucuronidation of 8‐OHEFV and 8,14‐diOHEFV. Interestingly, direct glucuronidation of EFV was only found in the cynomolgus macaque brain microsomes. When comparing brain microsome metabolism to liver microsome metabolism, we see that the liver has a greater capacity to transform EFV and its P450‐dependent metabolites. We tested cell‐type specific metabolism using primary cortical neurons, striatal neurons, astrocytes, and microglia from mice. Cells were cultured and treated with EFV, 8‐OHEFV, or 8,14‐diOHEFV for approximately 24 hours, with DMSO as a vehicle control. In the microglia and astrocytes, we observed P450‐mediated formation of 8‐OHEFV in the EFV‐treated cells. In the cortical neurons, striatal neurons, and astrocytes, both 8‐OHEFV and 8,14‐diOHEFV were glucuronidated. These results shed additional light on the microsome data, indicating which cell‐types may be contributing to EFV metabolism. In male mice dosed with EFV for 5 days in drinking water, UHPLC‐HRMS analysis of brain tissue homogenate and serum revealed 8‐OHEFV in brain tissue in conjunction with serum concentrations in the range of 15‐20 ng/mL. EFV and other metabolites were below the limit of detection in both brain tissue and serum. These data indicate that 8‐OHEFV has the potential to be formed and/or further metabolized in mouse brain. Additionally, inter‐species differences in EFV glucuronidation were observed in brain microsomes. The characterization of EFV metabolism in the brain improves our understanding of tissue‐specific EFV disposition as well as brain glucuronidation more broadly.
Use Of Weighted Gene Coexpression Network Analysis To Identify Connectivity Between Gut And Brain Gene Expression
Authors: Khan Tasnin , Hatami Asa , Zhu Chunni , Kawaguchi Riki , Joshi Swapna , Chen Han , Hoffman Jill , Law Ivy K. M. , Rankin Carl R. , John Varghese , Geschwind Daniel , Pothoulakis Charalabos , Videlock Elizabeth J.
Published: May 2022

Background

Alterations in the gut brain axis are being recognized as a pathogenic factor for an increasing number of diseases, including neurodegenerative diseases such as Parkinson Disease (PD). The extent to which gene expression profiling in the gut and the brain is co‐regulated is not well understood. Weighted gene coexpression network analysis (WGCNA) uses unbiased hierarchical clustering to reduce gene expression profiling data to modules of highly correlated genes.

Hypothesis

Gut‐brain connectivity will be strongest for modules related to physiologic mechanisms with a systemic component, such as immune reactivity.

Methods

WGNCA networks were constructed for distal colon and striatum RNA seq data from mice overexpressing human wild type alpha synuclein (ASO, n=18) and wild type (WT, n=16) mice at 1 and 3 months. Mice with matched colon and striatum data (n=10/6 ASO/WT) were included in this analysis. Linear regression identified associations between colon and striatum modules. For this analysis, we controlled for both age and genotype as genotype‐ and age‐associated modules could be correlated due to these covariates alone. Colon‐striatum intermodular connectivity was visualized in Cytoscape. Overrepresented gene ontology (GO) terms in WGCNA modules were determined using the hypergeometric function in the GOstats package in R. Enrichment for gene signatures from single cell sequencing data was determined using the hypergeometric test against cell type signatures from the Panglao and Cellmarker databases.

Results

Selected associations are shown in Figure 1. There are strong correlations between colon and striatum modules that are enriched for terms related to the immune response. Modules most clearly related to the immune response are highlighted in yellow, but several other modules are also closely related to the immune response. For example, there is an association between the striatum module enriched for endothelial cells of the blood brain barrier, and the colon module enriched for goblet cells which produce the mucus layer of the epithelium, a key component of the innate immune system.

Conclusions

Through analysis of matched colon and striatum samples, we can see correlations between gene expression in the colon and striatum. Most modules in this gut‐brain network have some relevance to the immune system which is not unexpected. These results demonstrate the feasibility of “profiling” the gut‐brain axis. A larger sample size would permit evaluation of disease‐related changes in this profile.
Metabolomic responses in brain endothelial cells (CLU‐512‐hCMEC/D) to Angiotensin II and Angiotensin 1‐7
Authors: Pena‐Silva Ricardo A. , Ana Sierra , Cala‐Molina Monica P.
Published: May 2022

Metabolomics is the discipline focused on the study of the dynamics, composition, interactions and responses to changes in metabolites in cells, tissues and biological fluids. Vasoactive peptides such as angiotensins cause multiple metabolic changes in vascular tissues, however these changes have not been characterized in detail. Angiotensin 1‐7 is a peptide derived from angiotensin II through the action of Angiotensin Converting Enzyme type II. This exploratory study seeks to understand the metabolic responses of a human cerebral microvascular endothelial endothelial cell line to stimulation with vasoactive peptides, in vitro (Angiotensin II, Angiotensin 1‐7).

Methods

We performed single cultures of the cell line (CLU‐512‐hCMEC/D), which was treated with Ang II, Ang 1‐7 or vehicle. Cells and culture medium were sampled before and after treatments. The samples were subjected to the process of extraction of metabolites with organic solvents. Each treatment was prepared in three biological replicates and was followed by metabolite extraction. Determination of metabolic profiles was performed by multiplatform, non‐targeted, metabolomic analysis, using liquid chromatography (LC) coupled to high resolution mass spectrometry (HRMS). To determine the differences in metabolic profiles between groups and to select statistically significant metabolites, multivariate statistical analysis was performed.

Results

We found 122 metabolites that varied between groups. In general, Ang II‐induced changes were similar to those found for Ang 1‐7. In particular, we found a reduction in the levels of several types of lipids including some bile salts, sphingolipids, glycerophospholipids and fatty acids.

This study is our first step in characterizing methods to study the impact of vasoactive peptides and inflammatory molecules on the blood‐brain barrier metabolome. Studying the modulation of the vascular tissue metabolome and endothelial cells could enhance our understanding of the effect of angiotensin 1‐7 and ACE2 in pathologies such as COVID‐19.
Obesity elicits a sex‐specific attenuation of the daily rhythm in ventilation in mice chronically fed a high‐fat diet
Authors: Jones Aaron A. , Framnes‐DeBoer Sarah N. , Arble Deanna M.
Published: May 2022

To efficiently deal with changes in nutrient availability across the 24‐hr day, animals have evolved daily rhythms in energy metabolism and, consequently, a daily rhythm in ventilation to mediate metabolic gas exchange. Daily ventilatory rhythms have been implicated in a variety of respiratory diseases such as sleep apnea, due to time‐specific occurrence of symptoms. However, it is unknown how obese physiology and intermittent hypoxia (IH), two hallmarks of sleep apnea, influence the daily rhythm in ventilation. Here, we use a dietary mouse model of obesity to determine the extent to which ventilation rhythms are influenced by obese physiology and IH. Using whole‐body plethysmography, we found that obese mice chronically‐fed a high‐fat diet exhibit a dampened minute ventilation rhythm and decreased mean ventilation during the night‐time. The obesity‐associated reduction in minute ventilation was sex‐specific and resulted in part from a failure of female mice to increase minute ventilation in proportion to weight gain. Moreover, we found that 5 days of daytime IH exposure minimally affected the daily rhythm in minute ventilation. We conclude that the daily rhythm in minute ventilation is attenuated by obesity in a sex‐specific manner but largely unaffected by intermittent hypoxia. Taken together, these results have clinical implications for obesity‐associated respiratory diseases such as obstructive sleep apnea.
A Novel En Bloc Circulatory System Dissection: A Teaching Tool for Systems‐Based Medical School Curricula
Authors: Gustilo Katherine S. , Stabio Maureen , Goldberg Chelsea , Lohman Bonfiglio Chelsea
Published: May 2022

Introduction and Objective

With the transition to integrative, systems‐based anatomical instruction in medical school curricula, the use of traditional, regionally‐based, cadaveric dissection has decreased while the use of systems‐based prosections has increased. Recent studies have demonstrated the value of such prosections for the nervous system through an en bloc extraction of the central nervous system. However, comparable approaches for the circulatory system have not been reported, and there are currently no dissection instructions for an en bloc circulatory system extraction that do not require polymer injection into vessels prior to embalming. Thus, the goal of this project was to prosect, extract, and preserve the circulatory system en bloc for long‐term use in a systems‐based integrated medical curriculum.

Materials and Methods

One male cadaveric donor without documented vascular disease or pathology was used to extract the following structures in a single interconnected piece: the heart, more than 350 named arteries, 10 superficial veins, 21 veins of the portal system, the liver, and at least 27 major veins of the caval system. Vascular structures were carefully isolated from the surrounding tissues in each region of the body over the course of approximately 250 hours by a single dissector. Muscles, nerves and organ tissues were cleared from the vessels using common dissection tools, including scissors, forceps, probes, and scalpels. Extraction of vessels from bones required use of chisels, rib cutters, and an autopsy electric saw. Best approaches, challenges, and time‐saving strategies were recorded.

Results & Conclusion

The circulatory system was successfully extracted from the limbs, torso and head in a single piece after embalming without the need for advanced polymer injection. A detailed dissection guide with instructions and images was developed, so this dissection could be replicated. The cadaveric specimen will be plastinated in approximate anatomical position with small tags placed on all named vessels to allow for easy identification of each structure and long‐term use.

Significance and Implication

This prosection is a novel approach to meet the needs of changing medical school curricula by illustrating the complexity and continuity of the circulatory system as a whole system. The 3D display will allow students to access a real, anatomical model that comprehensively demonstrates the majority of important, named vessels, providing the necessary context to augment 2D images, vascular diagrams, and atlases. Subsequent investigations will evaluate the educational value of this model in a flipped‐classroom approach. Future plans also include surface scanning and 3D‐printing the model to reproduce a less time‐consuming replica. This reimagination of traditional dissection techniques has generated new avenues for illustrating the human body, even in an established field such as gross anatomy.
Glutamine Counteracts Glucose‐Mediated Human Endothelial Cell Dysfunction
Authors: Durante William , Peyton Kelly J.
Published: May 2022

Vascular disease is the major cause of morbidity and mortality in diabetes mellitus. While the etiology of vascular disease is multifactorial, endothelial cell (EC) dysfunction is a hallmark of diabetes‐associated vascular disease. Substantial evidence indicates that high glucose (HG) concentrations trigger EC dysfunction through the production of reactive oxygen species (ROS). Recently, studies from our laboratory and others identified the conditionally essential amino acid glutamine as a critical regulator of oxidative stress and EC phenotype. In the present study, we investigated the effect of HG concentrations on ECs and determined if glutamine influences the biological actions of HG in these cells. Treatment of human umbilical vein ECs with glucose (5.6‐25mM) resulted in a concentration‐ and time‐dependent increase in the production of reactive oxygen species (ROS), whereas mannitol (an osmotic control) minimally affected ROS formation. HG concentrations (25mM) also evoked a time‐dependent decrease in EC viability that was paralleled by an increase in apoptosis, as reflected by a rise in annexin V binding and caspase‐3 activity. Notably, the administration of glutamine (500μM) inhibits HG‐mediated endothelial ROS production and improves EC viability in response to HG levels. Glutamine also induces the expression of heme oxygenase‐1 (HO‐1) in ECs. However, pharmacological inhibition of HO‐1 activity with tin protoporphyrin‐IX (10μM) reversed the antioxidant and cytoprotective effect of glutamine in HG‐treated ECs. In conclusion, the present study found that glutamine counteracts HG concentration‐mediated ROS generation and EC death. Moreover, it demonstrates that the induction of HO‐1 by glutamine contributes to the beneficial actions of the amino acid and identifies glutamine as a critical regulator of EC survival in diabetes.
Change in Metabolomic Profile Associated with an Average Increase in Plain Water Intake of >+1 L/day, Sustained over 4 Weeks, in Healthy Young Men with Initial Total Water Intake below 2 L/day
Authors: Stookey Jodi , Paulweber Bernhard , Lang Florian , Ritter Markus
Published: May 2022

Cells adapt to chronic extracellular hypotonicity by altering metabolism, such that cell‐level chronic disease risk factors are reduced. Corresponding effects of sustained whole‐body hydration are hypothesized but remain to be confirmed and translated into population‐level chronic disease prevention.

This analysis aimed to 1) describe change in urine and serum metabolomic profiles associated with change in hydration, induced by four weeks of sustained higher intake of drinking water, 2) check if features recognized as key osmolytes in specific tissues changed in urine and/or serum, and 3) identify which metabolic pathways might potentially be impacted by sustained hypotonic hydration.

Adapt Study [Stookey JD et al., Physiol Rep 2013;1(5)] data from for 4 healthy, normal weight men (20‐25 years) who transitioned from not meeting to meeting 3 osmolality criteria (urine:<800mmol/kg, serum:<295mmol/kg and saliva:<100mmol/kg) over 4 weeks of >+1L/d intake of drinking water were used for this analysis. Untargeted metabolomic assays were performed by the UC Davis West Coast Metabolomic Center on first‐morning urine collected after overnight food and water restriction and urine (t+60min) and serum (t+90min) collected after a 750 ml bolus of drinking water. Metaboanalyst 5.0 was used to compare Week 1 vs Week 6 metabolomic profiles, using heatmap, OPLS‐DA, fold‐change, and pathway analyses.

In heatmap and OPLS‐DA plots, metabolomic profiles appeared more similar in Week 6.

Over 350 metabolic features decreased by 2‐fold or more relative to creatinine for at least 3 out of 4 participants in first‐morning and post‐bolus urine. Over 200 features increased by 2‐fold or more relative to creatinine in serum. A wide variety of known compounds changed by 2‐fold or more in at least one specimen type, including alcohols, monosaccharides, disaccharides, sugar acids, polyols, azoline, lactone, nucleosides, phenols, purines, pyridines, pyrimidines, fatty acids, cholesterol, glycerolipids, amino acids, carboxylic acids, phosphate, and urea. Based on hypergeometric test p‐value <0.05 or pathway impact factor >0.2, sustained +1L/d of drinking water was associated with concurrent changes in carbohydrate, protein, lipid, and micronutrient metabolism, a ‘reverse’ Warburg‐like pattern favoring carbohydrate oxidation via the TCA cycle in Week 6.

The results warrant further work to leverage cell hydration effects for chronic disease prevention.
Endothelial inwardly rectifying potassium channels regulates receptor‐mediated vasodilation via PI3K/Akt/eNOS signal pathway
Authors: Ahn Sang Joon , Fancher Ibra S. , Phillips Shane A. , Levitan Irena
Published: May 2022

We recently demonstrated that endothelial inwardly rectifying potassium (Kir) channels play a key role in flow‐induced vasodilation (FIV) of resistance arteries via regulating nitric oxide (NO) production with Akt/eNOS pathway. However, the role of endothelial Kir channels in receptor‐mediated vasodilation remains unknown. Therefore, we tested the role of Kir channels in receptor‐mediated vasodilation of mesenteric arteries. A significant reduction in the vasodilation to bradykinin was observed in mesenteric arteries from Kir2.1+/‐ mice, which we have shown earlier to lack functional expression of Kir2.1 in endothelial cells, as compared to arteries from WT mice. Moreover, blocking Kir channels with barium, Ba2+, significantly reduced bradykinin‐induced vasodilation in WT but not in Kir2.1+/‐ mice. Inhibition of eNOS withN(G)‐Nitro‐L‐arginine methyl ester (L‐NAME), which is known to reduce bradykinin‐induced vasodilation in WT mice, also had no effect in Kir2.1+/‐ mice. However, the vasodilation was completely blocked in Kir2.1+/‐ mice with apamin, a well‐known inhibitor of small‐conductance calcium activated potassium (SK) channels. These findings indicate that endothelial Kir2.1 channels play a key role in receptor‐mediated NO production, an effect that is independent of SK channel‐dependent dilation. In mechanistic terms, we tested the activation of PI3K/Akt1/eNOS pathway known to be a major contributor of receptor‐mediated NO production and vasodilation. In the mesenteric arterial endothelial cells (ECs) derived from Kir deficient mouse, there was no significant activation of PI3K, Akt, and eNOS in response to bradykinin (10‐6 M), whereas the ECs from WT mice showed significant activation in 3 minutes after the application of bradykinin. The application of adenoviral construct overexpressing WT Kir2.1 resulted in the recovery of the activation of PI3K/Akt/eNOS in Kir deficient ECs, whereas no further increase of activation in WT ECs was observed. In addition, bradykinin‐induced NO production was significantly reduced in Kir deficient ECs compared to WT ECs, and it was recovered by overexpressing WT Kir2.1. Taken together, we conclude that Kir2.1 channels regulate bradykinin‐induced NO production in ECs through PI3K/Akt/eNOS pathway, similar to our observations regarding flow‐induced vasodilation.
Dynamic Changes in ABCC4 Protein‐Protein Interactions during PKA Signaling: Role of the ABCC4 PDZ Motif
Authors: Ranjit Sabina , Zhu Jingwen , Pitre Aaron , Wang Yao , Crawford Rebecca , Nourse Amanda , Li Yuxin , Pagala Vishwajeeth , Peng Junmin , Schuetz John D.
Published: May 2022

We recently demonstrated that a protein interaction between the PDZ motif of ABCC4 and a single PDZ‐domain‐containing protein, MPP1 had an important role in ABCC4 function in acute myeloid leukemia (Pitre et al. Nature Comm. 2017). To gain a better understanding into the role of the highly conserved ABCC4 PDZ‐motif, we performed live cell imaging using total internal reflectance fluorescence, TIRF, of ABCC4‐GFP expression constructs with or without the PDZ motif. The expression constructs were introduced into HEK293 cells where ABCC4 had been deleted by CRISPR‐Cas9 technology. ABCC4‐GFP was static at the membrane showing little movement, while deletion of the PDZ motif promoted rapid movement. Activation of PKA with forskolin and the phosphodiesterase inhibitor, iso‐butyl‐methyl xanthine increased intracellular cAMP and promoted membrane movement of ABCC4 harboring the PDZ motif. To get an unbiased understanding of the proteins interacting with ABCC4 both in cis‐ and trans‐, and under PKA activation conditions, we developed stable cell lines in the ABCC4‐null HEK293 cells. These stable cell lines harbored either an APEX2‐ABCC4 or an APEX2‐ABCC4 lacking a PDZ motif. APEX2‐derived ascorbate oxidase and biotin phenol, coupled with Avidin‐beads was used to interrogate the neighborhood of proteins within ~10 nm of ABCC4. We also developed stable cells harboring expression constructs for either Avi‐tagged ABCC4 or Avi‐ABCC4 without the PDZ motif to determine proteins that directly interacted with ABCC4 through the PDZ motif. Each Avi‐expression construct harbored a biotin ligase, thus enabling ABCC4 to be tagged by biotin and readily “pulled down” by avidin‐beads. The interacting proteins were identified by quantitative proteomics. Several PDZ specific interacting proteins were identified in these cell lines. By using analytical density gradient ultracentrifugation, we identified distinct PDZ specific ABCC4‐complexes that formed upon PKA activation. The actin‐binding cytoskeletal protein, Moesin (MSN) associated with ABCC4, requiring the PDZ‐motif. We used quantitative proteomics and APEX2 ABCC4 constructs to show that the neighborhood surrounding ABCC4 lacking the PDZ‐motif had fewer proteins, especially cytoskeletal proteins interacting with it. We propose that the ABCC4‐MSN interaction is important for tethering ABCC4 to the cytoskeletal actin network. Importantly, siRNA knockdown of MSN disrupted the association between ABCC4 and its interactors indicating the key role of MSN in the dynamic protein interactions occurring between ABCC4 and its interactors.
Electro‐metabolic signaling in heart: how local blood flow is regulated
Authors: Zhao Guiling , Lederer W. Jonathan
Published: May 2022

How local blood flow is controlled in heart has proved a mystery after decades of extensive investigation by many research groups. Multiple metabolic products and factors have been suggested to affect coronary blood flow and to contribute its regulation. Nevertheless, how this blood flow is regulated and shown to be responsive to metabolic need remains unclear. Our new observations provided compelling evidence for electro‐metabolic signaling (Zhao et al. PNAS 2020) that originates in ventricular myocytes and is conducted to capillary endothelial cells and then to contractile smooth muscle cells and pericytes. This hypothesis posits that the metabolic sensors are the contractile myocytes themselves and the information distribution system is an electrical network composed of the same capillaries that distribute the oxygen‐rich blood. The “valves” that open to permit more blood to flow to the needy myocytes or close to slow the flow are contractile smooth muscle cells on the end‐arterioles and also the contractile pericytes attached to the capillary endothelial cells and end‐arterioles. Each of these critical elements communicate with the other electrically in a functioning EMS communication system because gap junctions (or the equivalent) connect them. As cardiac myocytes carry out pressure‐volume work, they consume ATP and produce elevated ADP. This leads to a decline in [ATP] and an increase in [ADP], actions that lead to the opening of KATP channels which are hugely abundant in the cardiac myocytes and which drive EMS. This produces a time‐averaged hyperpolarization of the cardiac action potential (AP) which produces a low‐pass filtered hyperpolarization in the neighboring EMS network. To confirm and extend our hypothesis and observations, additional studies have been conducted on perfused and pressurized mouse cardiac papillary muscle preparations to investigate how signals from cardiac myocytes are sent through the neighboring capillaries to the contractile elements. Specifically, gap junction permeable dye (lucifer yellow, LY), was used to trace the intercellular conduction pathway and cdh5‐GCaMP8 mice were used to evaluate the effect of myocyte voltage on capillary endothelial cells function. Optogenetic experiments with channel‐rhodopsin was also used to evaluate the intercellular signals from capillary endothelial cells to pericytes, vascular smooth muscle cells and cardiac myocytes. Our results show that when LY was injected into the cardiac myocytes it was also observed in the neighboring capillaries. Hyperpolarization of cardiac myocyte was also shown to hyperpolarize capillary endothelial cells. These investigations provide stunning and compelling support for electro‐metabolic signaling and lay the foundation for advanced investigations of its dynamic role in health and disease.
Endosomal b2‐adrenergic receptor controls nuclear cAMP signal in learning and memory via sequestration of phosphodiesterase 4D
Authors: Martinez Joseph M. , Shen Ao , Xu Bing , Jovanovic Aleksandra , Xiang Yang K.
Published: May 2022

Learning and memory‐related behaviors depend on GPCR signaling to the nucleus to induce Immediate early gene (IEG) expression. However, the molecular mechanisms mediating this process remain unclear. We have previously identified a subpopulation of β2AR which is preferentially phosphorylated by GRK sites and internalized to the endosome upon stimulation. Here, we demonstrate that GRK phosphorylated endosomal β2Adrenergic Receptor (β2AR) indirectly facilitates nuclear cAMP signaling through sequestration of a PDE4D/β‐arrestin complex. Combining subcellular cAMP biosensors and transgenic mice, we found deleting GRK phosphorylation sites on β2AR (GRKD) blocked agonist‐induced receptor endocytosis and nuclear cAMP signaling in hippocampal neurons. This resulted in impaired PKA‐mediated IEGs expression in vitro,reduced learning induced IEG expression in vivo, and impaired long‐term memory in a Morris water maze. Mechanistically, while β2AR stimulation promoted β‐arrestin‐dependent recruitment of PDE4D5, inhibition of β‐arrestin‐PDE4D5 association alone prevented β2AR‐induced increases in nuclear cAMP signaling without affecting receptor endocytosis. Furthermore, direct PDE4 inhibition was sufficient to rescue the β2AR‐induced nuclear cAMP signal in GRKD neurons and ameliorate the long‐term memory deficits in GRKD mice. This work indicates that the sequestration of the β‐arrestin/PDE4D complex by the GRK‐phosphorylated β2AR critically controls the receptor‐induced nuclear cAMP signals. This constitutes a novel mechanism by which GPCR activation promotes nuclear cAMP signaling through endocytosis dependent relocalization of PDE4D isoforms, IEG expression, and learning and memory‐related behaviors.
Generation and characterization of thick ascending limb specific knockout of NHE3 mouse model
Authors: Xue Jianxiang , Thomas Linto , Fenton Robert A. , Dominguez Rieg Jessica A. , Rieg Timo
Published: May 2022

The Na+/H+ exchanger 3 (NHE3) mediates Na+ (re)absorption in the intestine and the kidney, thereby playing a key role in fluid homeostasis and blood pressure (BP) regulation. NHE3 is expressed in the luminal membrane of the renal proximal tubule, where the majority of renal Na+ reabsorption (60% to 75%) occurs. Whole body NHE3 knockout mice exhibit hypovolemia, hypotension, diarrhea, metabolic acidosis and renal Na+ wasting. Study in kidney‐specific (proximal tubule [PT] and thick ascending limb [TAL]) NHE3 knockout mice has reported a rather mild phenotype in terms of Na+ homeostasis and no phenotype for acid‐base homeostasis (Kidney International; 2017, 92:397‐414). NHE3 is also substantially expressed in the TAL and our data point to a novel role in this nephron segment for urinary concentrating ability. To determine the physiological role of NHE3 in the TAL for salt homeostasis, urinary concentrating ability and BP regulation, we generated a novel tamoxifen (Tam)‐inducible TAL‐specific NHE3 knockout (NHE3TAL‐KO) mice by crossing floxed NHE3 (Control, Con) with UmodCreERT2 mice. Immunofluorescent staining showed that application of Tam (3 days, 67 mg/kg via oral gavage) selectively deleted NHE3 from TAL but not PT. Before Tam, in Con and NHE3TAL‐KO mice had comparable food (4.9±0.1 vs 5.3±0.2 g/(day*30g)) and water intake (5.6±0.3 vs 5.9±0.4 g/(day*30g)), urinary osmolality (2435±107.4 vs 2220±111.9 mOsm/kg), urinary pH (6.02±0.06 vs 6.02±0.06), urinary Na+/creatinine (46.3±6.2 vs 52.0±5.2 mmol/mmol) and K+/creatinine (109.0±9.5 vs 97.2±9.2 mmol/mmol). Two weeks after Tam, compared to Con mice, NHE3TAL‐KO mice showed lower urinary osmolality (2224±90.1 vs 1826±92.8 mOsm/kg, P<0.01) associated with a strong tendency of higher water intake (5.3±0.2 vs 6.1±0.3 g/(day*30g), P=0.08). However, no difference was found in urinary pH (6.07±0.05 vs 6.17±0.05, NS), urinary Na+/creatinine (37.4±4.6 vs 40.9±4.5 mmol/mmol, NS) and K+/creatinine (80.9±5.7 vs 94.1±6.8 mmol/mmol, NS). In conclusion, we successfully generated TAL‐specific NHE3 knockout mouse model. This mouse model will be used to study the isolated role of NHE3 in the TAL regarding to salt homeostasis, urinary concentrating ability and BP regulation to distinguish the contribution of TAL from PT.
Spatial Compartmentation of mTORC1 Signaling at the Mitochondria
Authors: Sahan Ayse Z. , Zhong Yanghao , Zhou Xin , Schmitt Danielle L. , Zhang Jin
Published: May 2022

The mechanistic target of rapamycin complex 1 (mTORC1) senses diverse signals to regulate cell growth and metabolism. The complex is present at the plasma membrane, nucleus, lysosomes, and the outer mitochondrial membrane. Such spatial compartmentation has been suggested to enhance signaling efficiency and specificity. For instance, we recently discovered nuclear mTORC1 activity, which is distinctly regulated from the canonical lysosomal mTORC1 (Zhou et al., 2020). Previous studies have shown that mTOR is present at the outer mitochondrial membrane (OMM), but it is not clear whether mTORC1 is active at this location and what the functional consequences are. To investigate this, we targeted our FRET‐based mTORC1 activity reporter, TORCAR (Zhou et al., 2015), to the OMM and probed the subcellular activity of mTORC1. We found that platelet‐derived growth factor (PDGF) stimulation increases mTORC1 activity at the OMM in addition to at the lysosome and in the nucleus, whereas insulin specifically stimulates mTORC1 activity at the OMM without affecting the lysosomal and nuclear activities. We further dissected the regulation of mitochondrial mTORC1 activity and applied a novel approach of identifying new mTORC1 substrates. Elucidating the signaling events that lead to subcellular mTORC1 activity at mitochondria and its downstream functions will increase our understanding of the roles that mTORC1 may play in diseases associated with altered metabolism or mitochondrial dysfunction, such as diabetes and cancer.
Pyridoxine and cobalamin supplementation effect regarding cell death in a human glioblastoma cell line
Authors: Martínez‐Mendiola Carolina A. , Estrada Jose A. , Zapi‐Colín Luis Á. , Contreras Irazú
Published: May 2022

Glioblastoma multiforme is the most common and aggressive form of brain tumor. Its treatment is challenging despite the use of combined treatments including surgery, chemotherapy, and radiotherapy. Mean survival rates after 5 years are usually around 5%. Recent advances in cancer therapy have focused on the use of adjuvant strategies to improve clinical outcomes. Among these, nutritional interventions have shown significant potential for preventing tumor development and enhancing response to standard treatments, improving life quality and expectancy in cancer patients.

Vitamins are essential micronutrients required for cellular metabolism and homeostasis. B group vitamins are key for development of the nervous system, participating in de novo synthesis of nucleic acids, homocysteine and one‐carbon metabolism, as well as contributing to mitochondrial functions. B vitamins have been shown to possess significant properties relating to the prevention of tumor development in some types of cancer. However, their role in glioblastoma has not been determined.

The aim of this study is to determine the effect of B group vitamin supplementation on the survival of the human glioblastoma cells. B6 (pyridoxine) and B12 (cobalamin) vitamins were administered directly to cultured U87MG human glioblastoma cells at different concentrations and exposure times. A crystal violet colorimetric assay was used to evaluate cell viability after vitamin supplementation. The results show a strong dose‐dependent effect of pyridoxine supplementation on glioblastoma cell viability at 2,000‐4,000 µM concentrations. Viability also decreased at longer exposure times. In contrast, cobalamin supplementation at 1‐100 µM concentrations does not appear to have any effect on glioblastoma cell viability at any exposure time.

These data suggest that direct supplementation with pyridoxine, but not cobalamin, may affect glioblastoma cell viability. Additional experiments are underway to explore the molecular mechanisms responsible for the observed effects.
Combined Inhibition of the Ataxia‐telangiectasia mutated and Rad3‐related Pathway and the G9a Methyltransferase Synergize to Reduce Pancreatic Cancer Cell Growth
Authors: Rodrigues de Oliveira Daniela , Milech de Assuncao Thiago , Urrutia Guillermo , Lomberk Gwen
Published: May 2022

Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer‐related deaths in the United States with a devastating overall five‐year survival rate of only 10%. Consequently, there is an urgent need to identify innovative therapeutic strategies. PDAC exhibits chronic replication stress (RS), becoming reliant on a proficient Ataxia‐telangiectasia mutated and Rad3‐related (ATR) pathway to adapt to high RS levels in cells. This also presents an exploitable therapeutic vulnerability in tumors via strategies that enhance the endogenous RS to trigger replication catastrophe and cell death. Our laboratory is focused on utilizing epigenetic inhibitors in this context as novel therapeutic approaches. Recently, we have shown that combined inhibition of the major ATR effector, CHK1, and the G9a methyltransferase leads to replication catastrophe, providing an effective therapeutic approach for PDAC by priming replication failure through the epigenome rather than genomic damage. In the current study, we sought to investigate additional targets in the RS response pathway through direct ATR inhibition combined with G9a inhibition and perform mechanistic studies on DNA damage response checkpoints and cell cycle. We treated multiple PDAC cell lines with a combination of the ATR inhibitor, AZD6738, and either BRD4770 or UNC0642 to inhibit G9a at various concentrations to determine the effect on cell viability and cytotoxicity by live‐cell analysis. We find that the AZD6738‐BRD4770 and AZD6738‐UNC0642 combinations display synergistic effects to disrupt the RS response and inhibit PDAC growth. Overall, our work provides quantitative insights into a novel therapeutic opportunity for PDAC and expands the universality of a synergistic interaction resulting from simultaneous targeting of the ATR‐CHK1 RS pathway and the epigenomic regulator G9a.
Gaseotransmitters Modulate Inspiratory Drive from the Hypoglossal Nucleus
Authors: Browe Brigitte M. , Khan Shakil , Nanduri Jaysri , Prabhakar Nanduri R. , Garcia Alfredo
Published: May 2022

Gaseotransmitters, carbon monoxide (CO) and hydrogen sulfide (H2S), play an important role in maintaining proper function of cardio‐respiratory systems. Previous work has demonstrated that Heme Oxygenase‐2 knock out mice (HO‐2‐/‐) develop a sleep apnea phenotype characterized predominantly by obstructive apneas, which could be normalized by either treating HO‐2‐/‐ with L‐propargylglycine (L‐PAG) an inhibitor of cystathionine gamma‐lysase (CSE) and H2S synthesizing enzyme or in HO‐2+ CSE double null mice. Obstructive apneas are often caused by a loss in upper airway tone, we sought to examine how H2S and CO signaling impacts inspiratory activity from the preBötC and the hypoglossal nucleus (XIIn). We hypothesized that HO‐2 and CSE are involved in the regulation of inspiratory motor output from XIIn. Immunohistochemical analysis revealed many ChAT+ cells of XIIn are positive for HO‐2 and CSE. Biochemical analysis showed elevated H2S abundance in XIIn of HO‐2‐/‐mice. Electrophysiological recordings demonstrated transmission failure from preBötC to XIIn in response to NaHS, a H2S donor or Cr (III) Mesoporphyrin IX chloride (ChrMP459), an inhibitor of HO in brain slices from wild type mice. Furthermore, ChrMP459 reduced inspiratory drive currents received by XIIn neurons and suppressed excitability in XIIn neurons. Brain slices from HO‐2‐/‐mice showed: (1) a larger rate of transmission failure between preBötC and XIIn motor pools; and (2) smaller drive currents from individual XIIn neurons. Brain slices from HO‐2+ CSE double null mice displayed reduced transmission failures from preBötC to XIIn. Furthermore, L‐PAG mitigated transmission failures and increased the magnitude of inspiratory drive currents in XIIn neurons in HO‐2‐/‐mice. These findings demonstrate that HO‐2 and CSE regulate inspiratory drive from XIIn and suggest potential role for gaseotransmitters in regulating upper airway tone.
Differential Response of MED12 Downregulated Breast Cancer to Targeted Therapy
Authors: Kermet Sophia , Myneni Nishanth , Burleson Marieke
Published: May 2022

MED12 is a subunit of a large multi‐subunit protein complex called Mediator. Mediator plays critical roles in gene regulation by repressing or activating transcription through modulation of RNA polymerase II. The specific role of MED12 within Mediator is to regulate the activity of CDK8 kinase. Many studies in the last several years have uncovered that mutations within MED12 or downregulation of MED12 leads to dysregulation of CDK8 kinase activity which has many physiological implications, including promotion of tumorigenesis in a variety of different cancers. Specifically, MED12 mutations have been found in up to 67% of breast cancer patients. Although many strides have been made towards finding superior treatment strategies for breast cancer, there is still a critical need for uncovering targeted therapies for specific genetic subsets of breast cancer. Therefore, we are aiming to find a superior treatment strategy for breast cancer patients who carry MED12 mutations. Natural compounds are a promising treatment option since more than 50% of drugs currently available in the pharmaceutical industry are derived from natural sources. Consequently, we have employed a screen of a natural compound library to discover novel therapeutics for MED12 mutant breast cancer. Specifically, we generated a stable MED12 knockdown MCF‐7 cell line through the use lentiviral‐mediated shRNA. Since MED12 mutations abolish MED12 functions, the MED12 knockdown cell line should mimic a MED12 mutant setting. We first confirmed that the MED12 knockdown cell line exhibits enhanced proliferation through the employment of proliferation assays. Next, we performed a natural compound screen as assayed by MTT cell viability assays and found that MED12 knockdown cells are differentially affected by the compounds. Our results indicated that MED12 knockdown cells appear to be more resistant to treatment when compared to MED12 wildtype cells, although we did also uncover compounds that differentially targeted MED12 knockdown cells. We selected out the compounds that differentially targeted MED12 knockdown cells and subjected them to quantitative PCR analysis. MED12 has previously been shown to place a constraint on GLI3‐mediated SHH signaling, a finding that is of interest since SHH signaling is often hyperactivated in breast cancer. We therefore examined the expression of GLI3 target genes in our MED12 knockdown cell line that were either treated with control vehicle or selected natural compounds. Interestingly, we found that GLI3 signaling is upregulated in MED12 knockdown cells, and more importantly, the upregulation is drastically reversed in the compound‐treated cells. Overall, our results demonstrate that natural compounds could prove to be an effective targeted therapy for MED12 mutant breast cancer by targeting the GLI3‐mediated SHH signaling pathway.