bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of Rosa canina Methanol Extract on Membrane Trafficking in Different Niemann‐Pick C1 Phenotypes
Authors: Wanes Dalanda , Al Aoua Sherin , Walters Friederike , Shammas Hadeel , Y. Naim Hassan
Published: May 2022

Niemann Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder that is caused by mutations in Niemann‐Pick C1 (NPC1) or Niemann‐Pick C2 (NPC2) genes ultimately leading to progressive neurological deterioration. 95% of NPC cases arise from mutations in the NPC1 gene. NPC1 protein mediates the intracellular cholesterol trafficking and plays a crucial role in maintaining lipid homeostasis. Mutations in NPC1 are associated with defective cholesterol mobilization and an accumulation of unesterified cholesterol and subsequently sphingomyelin and glycosphingolipids in the late endo‐lysosomes. N‐butyldeoxynojirimycin, known as Miglustat, is one of the approved therapeutic options. It is an inhibitor of sphingolipid synthesis and is used in substrate reduction therapy. Nevertheless, the efficiency of Miglustat varies among NPC patients depending on the type of the mutation and moreover its accompanied by several side effects, such as diarrhea. The focus of this study is to find an alternative therapy that presents less side effects. Rosa canina L. methanol extract (RCME) has been shown to improve the protein trafficking and maintain cholesterol homeostasis. Here, we used skin‐derived fibroblasts from patients harbouring homozygote or compound heterozygote mutations to assess the effects of RCME on the trafficking of NPC1 and cellular cholesterol contents as compared to wild type cells. For this purpose, the cells were treated for 24 h with RCME (100 µg/ml) with and without Miglustat (100 µM) and the trafficking of NPC1 between the endoplasmic reticulum (ER) and the Golgi was examined by endoglycosidase H treatment. This treatment, which discriminates between mannose‐rich N‐linked glycosylated proteins and mature complex glycosylated forms, demonstrated a significant enhancement of the trafficking of a NPC1 mutants between the ER and the Golgi upon RCME treatment as compared to Miglustat. This trafficking behaviour was observed with those NPC1 mutants that are not entirely blocked in the ER and exit the ER at a slow rate. Markedly, the high cholesterol levels measured in the patients’ fibroblasts are substantially reduced upon RCME treatment, while almost similar cholesterol levels were maintained in the wild type cells. Impaired cellular cholesterol trafficking is associated with partial distortion of the lipid membrane composition, particularly of the cholesterol‐ and sphingolipids‐enriched membrane microdomains or lipid rafts (LRs). Our data show that RCME and also Miglustat maintained a normal LRs distribution as assessed by the LR marker flotillin 2.

We conclude that RCME possess the capacity to reverse adverse effects caused by mutations in the NPC1gene including impaired NPC1 trafficking, elevated cholesterol levels and lipid raft alterations. All this together render the constituents of RCME, including quercetin, a promising agents in NPC therapy.
Comparative analysis of mitochondrial CRC in permeabilized cells and isolated cell mitochondria
Authors: Jang Sehwan , Chapa‐Dubocq Xavier R. , Fossati Silvia , Javadov Sabzali
Published: May 2022

Mitochondria undergo permeability transition (PT) resulting in the opening of the non‐selective PT pores (PTPs) in the inner mitochondrial membrane in response to energy and oxidative stresses associated with Ca2+ overload and ROS accumulation. The mitochondrial PTPs are permeable to ions and solutes with a molecular mass <1.5 kD that increases the colloidal osmotic pressure in the matrix leading to mitochondrial swelling. Calcium retention capacity (CRC) reflects the maximum amount of Ca2+ mitochondria can uptake to provoke the PTP opening. Quantification of CRC is important to study the effects of various pathological stimuli and the efficacy of pharmacological agents on the metabolism and function of mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the advantages/disadvantages of the fluorescent technique in isolated mitochondria vs. permeabilized cells. The cells were permeabilized using digitonin or saponin, and the CRC was assessed using the Ca2+‐sensitive fluorescence probe Calcium Green‐5N. Results demonstrated the interference of dye‐associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. The CRC of saponin‐permeabilized cells was higher than the CRC of digitonin‐permeabilized cells. In addition, the mitochondrial CRC of saponin‐permeabilized cells was higher than isolated mitochondria using the same number of cells. In conclusion, this study demonstrates that the fluorescent technique for CRC analysis in saponin‐permeabilized cells has more advantages than isolated mitochondria.
Effect of Lipocalin‐2 expression on mitochondrial function in renal tubular cells in chronic kidney disease
Authors: Marques Eloïse , Zablocki Aniela , Terzi Fabiola , Gallazzini Morgan
Published: May 2022

Kidney cells metabolic status requires high abundance of mitochondria. It has been reported in chronic kidney disease (CKD) a loss of mitochondrial mass and function leading to reactive oxygen species (ROS) production and subsequent tubular cell death. Hence, mitochondrial dysfunction participates to CKD onset. Nevertheless, the cellular mechanisms inducing mitochondrial dysfunction in kidney cells are poorly understood. We have recently shown that in CKD, Lipocalin‐2 (Lcn2) expression is strongly induced in kidney cells leading to ROS production and apoptosis. However, the mechanisms by which Lcn2 triggers these phenomena are unknown. Considering that mitochondria can induce oxidative stress and apoptosis, we hypothesized that Lcn2 participates to CKD onset via an effect on mitochondrial dependent mechanisms. Using renal tubular cell lines overexpressing (OE) or knock‐down (KD) for Lcn2, we aimed at studying the effect of Lcn2 protein expression on mitochondrial function. First, we demonstrated that Lcn2 regulates mitochondrial dynamics, mass and function. Indeed, we observed by immunofluorescence and electron microscopy that kidney cells expressing Lcn2 present smaller and rounded mitochondria while Lcn2 KD induces mitochondrial elongation. Moreover, Lcn2 expression increases mitochondrial fission marker abundance and decreases fusion marker expression. Mitotracker staining and mtDNA content measurement experiments demonstrated that Lcn2 expression decreased mitochondrial mass while Lcn2 KD increased it. Furthermore, we observed that Lcn2 expression decrease mitochondrial membrane potential and accordingly increased Lactate production compared to cell KD for Lcn2. Using iron chelator or Lcn2 mutant unable to transport iron, we demonstrated that Lcn2 expression induces mitochondrial dysfunction independently of its iron transport effect. In addition, we observed that extracellular Lcn2 did not rescue Lcn2 effects on mitochondria while intracellular Lcn2 did. Finally, using a subtotal nephrectomy mice model, that recapitulates different features of human CKD, we demonstrated that Lcn2 invalidation by knock‐out in mice decreased the mitochondrial fission and the subsequent mitochondrial fragmentation and mass loss in kidney tubular cells normally observed during CKD onset in wild‐type mice.

Altogether, these results demonstrated that Lcn2 expression regulates mitochondrial dynamics, mass and function in kidney tubular cells. This suggests that Lcn2 driven mitochondrial dysfunction might by responsible at least in part of CKD onset.
Exploiting exercise and mTOR enhanced protein synthesis to augment mRNA vaccine translation
Authors: Bivona Joseph J. , Poynter Matthew E.
Published: May 2022

Exercise has been previously proposed as an adjuvant for traditional vaccines through mechanisms of lymph movement and skeletal muscle damage. However, the novel mRNA technology being used in COVID‐19 and other novel vaccines fundamentally changes how we should consider muscle’s active role in protein production and subsequent protective immunity. Following exercise, mammalian target of rapamycin (mTOR) activation drives total protein translation increases in skeletal muscle. We hypothesize that this 4‐24 hour window of opportunity can be exploited to augment exogenous mRNA translation. To evaluate the feasibility of this approach, we first interrogated mTOR using fully differentiated C2C12 mouse skeletal muscle myotubes that were transfected with mRNA encoding for enhanced green fluorescent protein (eGFP) or FLAG‐tagged Comirnaty SARS‐CoV‐2 Spike sequence in mTOR modulating conditions. Myotube transfection in the presence of rapamycin (Figure 1A) and resveratrol (Figure 1B) showed significant decreases in protein translation as measured by eGFP fluorescence, indicating that exogenous mRNA translation can be decreased pharmacologically by inhibiting the mTOR pathway. To simulate exercise enhanced protein synthesis, we treated myotubes with insulin‐like growth‐factor 1 (IGF‐1), as this protein is not only upregulated following exercise but also signals through an overlapping pathway with mechanoreceptors. Transfections with mRNA encoding for either eGFP or Comirnaty‐FLAG during IGF‐1 treatment showed a dose dependent increase in eGFP fluorescence (Figure 1C) and a significant increase in Comirnaty‐FLAG translation as measured by ELISA (Figure 1D). Current studies are investigating the use of electrical stimulation induced contraction on C2C12 myotubes prior to transfection as well as in vivo models of exercise and intramuscular mRNA injections. The implications of these results may impact future mRNA therapies to utilize exercise, or modulation of intracellular signaling pathways affected by exercise, as a method to improve vaccine‐induced exogenous protein production.
Investigating Landmodulin's EF Hands in Relation to Lanthanide Recycling Methods
Authors: Charville Lillian , Katz Iris , Reineck Anna , Suder Karen C. , Replogle Jessica B.
Published: May 2022

Rare earth elements (REEs) have distinct and varied electronic properties that have been applied for military and civilian use in electronics, such as cell phones and computer chip technologies. The recycling methods for REEs are complex and require a multitude of resources that prompt new studies to improve REEs separations. Lanmodulin (LanM), a protein that naturally bind to lanthanides, may provide a biological route for REE separation. Understanding the structure of LanM’s EF‐hands will explain how the lanthanides bond to them and lead to a greater understanding of how LanM can be used to recycle REEs to benefit our environment. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of EF1‐3 hands on lanmodulin. LanM has three tight binding EF‐hands, EF1 (D35‐E46), EF2 (D59‐E70), and EF3 (D84‐ E95), which are abundant in carboxylate side chains that bind the lanthanides. Lanthanide binding promotes a large conformational shift of the protein from a disorganized state to an organized state. The interior of the protein is a condensed helical bundle, and the majority of the exterior is composed of the EF hands, making bonding with lanthanides easier due to the EF hands being solely on the exterior. These three hands contain a higher number of coordination numbers (8‐9) compared to only 7 in Calmodulin (CaM), resulting in more specialized bonding properties. LanM also has a conserved proline between the first and second putative metal‐coordinating residues in each of the four EF hands, which is unique to this protein and may play a role in reducing affinity for calcium ions. In conclusion, analyzing the properties of the binding sites of EF1‐3 in LanM through 3D modeling can reveal insights on utilizing proteins as a method to extract and recycle REE in order to be environmentally cognizant.
Computational Approaches to Identify a Novel Binding Site on Estrogen Receptor α
Authors: Harris Myles , Proffett Destiny , Bean Monica , Alkohaif Reham , Fedraw Caela , Hanein Philopateer , Kim Dennis , Anbari Ahed , Ghantasala Vishnu , Livezey Mara R.
Published: May 2022

3,3‐bis(4‐hydroxyphenyl)‐7‐methyl‐1,3,dihydro‐2H‐indol‐2‐one (BHPI) is a biomodulator of Estrogen Receptor alpha (ERα) that targets ERα positive cancer cells by activating the unfolded protein response (UPR). BHPI induces strong and sustained activation of this pathway, eventually leading to necrotic cell death. While much is known about how BHPI triggers the UPR leading to necrotic cell death, it is not known how BHPI binds to its putative molecular target, ERα. In an effort to identify the binding site of BHPI on ERα, molecular docking studies in AutoDock Vina were utilized. This work uncovered a novel binding site that is not targeted by classical ligands, such as estrogen and tamoxifen.
Intermittent Hypoxia Attenuates and Reverses Epinephrine‐Induced Changes in Gene Expression in Endothelial Cells
Authors: Cetin‐Atalay Rengul , Meliton Angelo Y. , Sun Kaitlyn A. , Woods Parker S. , Peng Ying‐Jie , Fang Yun , Hamanaka Robert B. , Prabhakar Nanduri R. , Mutlu Gokhan M.
Published: May 2022

Background

Obstructive sleep apnea (OSA) is an independent risk factor for cardiovascular disease; however, the mechanisms are not completely known. OSA‐induced endothelial cell (EC) dysfunction and activation, which precede the development of cardiovascular disease may play a role, but how OSA causes EC dysfunction are not well‐understood. Intermittent hypoxia (IH) and sympathetic activation leading to elevated catecholamines (i.e., epinephrine) are hallmarks of OSA. We have recently demonstrated that IH causes EC activation not directly but via the IH‐induced sympathetic activation and catecholamines (i.e., epinephrine). The objective of this study was to investigate the effect of IH and epinephrine on EC gene expression in an unbiased fashion. We hypothesized that IH and epinephrine cause differential expression of genes in ECs contributing to EC dysfunction in OSA.

Methods

We exposed primary human aortic ECs to normoxia or IH (alternating between 5% O2 for 2 minutes and 20% O2 for 5 minutes) in the presence or absence of epinephrine (10μM) for 60 cycles and isolated RNA for sequencing at the end of exposure.

Results

Epinephrine induced significantly higher number of differentially expressed genes (DEGs) compared to IH alone. The majority (92%) of DEGs identified under IH were also seen in response to epinephrine (Figure 1). These common DEGs were enriched in leukocyte migration, extracellular matrix organization, cell adhesion and blood vessel development. IH‐related genes were also enriched in NOD‐like receptor signaling pathway, lipid and atherosclerosis, cytokine‐cytokine receptor interaction and chemokine signaling pathways, whereas epinephrine related genes were enriched in Ras signaling, MAPK signaling, PI3K‐Akt signaling and Phospholipase D signaling pathways as well as cytokine‐cytokine receptor interaction. Combining IH with epinephrine had significant impact on epinephrine‐induced gene expression under normoxic conditions and attenuated and reversed the epinephrine‐induced changes on gene expression.

Conclusions

The effect of epinephrine on gene expression is greater than IH alone in terms of the number of genes and their level of expression. The majority of genes that are differentially expressed in response to IH were also regulated by epinephrine resulting in activation of similar biologic processes by IH and epinephrine. We also identified new biologic and signaling pathways affected by IH and epinephrine alone and in combination. Further investigation of these IH‐ and epinephrine‐regulated genes and pathways will increase our understanding of the mechanisms by which OSA causes EC dysfunction and cardiovascular disease.
Obesity‐induced and LDL‐mediated NK Cell Function Loss is Accompanied by Proteasome‐dependent induction of Lysophagy in an at‐risk population of African American Women
Authors: Baumer Yvonne , Baez Andrew S. , Gray Veronica C. , Gutierrez‐Huerta Cristhian A. , Mitchell Valerie M. , Collins Billy S. , Singh Komudi , Powell‐Wiley Tiffany M.
Published: May 2022

Obesity and related chronic diseases (i.e. cardiovascular disease (CVD) and cancer) disproportionately affect African Americans (AA), shortening life expectancy and impacting quality of life. Life‐long exposure to adverse social determinants of health (SDoH) have been linked to obesity, warranting intensified research to further elucidate the ‘biology of adversity’ or the biologic consequences of adverse social conditions. Evidence suggests that exposure to chronic psychological and environmental stressors as SDoH leads to immune cell dysfunction, particularly for Natural Killer (NK) cells. Therefore, we examined changes in NK cell function in AA women, a group at high risk of Class III obesity (BMI ≥40 kg/m2). We have previously shown that NK cell degranulation is impaired in AA women with Class III obesity accompanied by a decrease in IFNg and TNFa, a loss in function driven by plasma LDL levels (b=‐0.35, p=0.03). A similar phenotype of dysfunctional NK cells could be achieved by overnight LDL treatment (50mg/dl) of healthy donor NK cells. To further determine the underlying signaling pathways, we employed proteomics of control and LDL treated NK cells. Here we identified 18 statistically significant regulated proteins (p<0.05), and 23 proteins approaching significance (p<0.07). Signaling pathway analysis revealed endolysosomal function, actin cytoskeleton regulation, and regulation of innate immune responses as significant pathways. Several proteins associated with autophagy and ubiquitination were also identified. This is of particular importance as LDL treatment of various cell types has been related to damaging lysosomes and a subsequent potential induction of lysophagy. Therefore, we investigated the impact of LDL on NK cell autophagy through western blot analysis of LC3 and p62 and found that enhanced autophagy is present in LDL treated NK cells. By employing the proteasome inhibitor MG‐132, we also found that the LDL‐induced increase in autophagy appears to be dependent on the activation of the 20S proteasome and subsequent ubiquitination of lysosomal proteins which results in depletion of functional lysosomes through lysophagy and is accompanied by NK cell function loss. In summary, we show a novel pathway potentially explaining obesity‐related NK cell function loss as a result of adverse SDoH in a population at highest risk for obesity‐related diseases like CVD and cancer. In the future, more research is needed to further understand the ‘biology of adversity’ and identifying targets for tailored interventions, ultimately reducing health inequities in chronic disease outcomes.
Carboxylesterase 1 (Ces1) Releases Oxylipins From Oxidized Triacylglycerols: Examination of Its Substrate Selectivity
Authors: Ross Matthew K. , Phillips Maggie , Borazjani Abdolsamad
Published: May 2022

Triacylglycerols (TAGs) are the most quantitatively important storage form of fat and are found primarily in cytoplasmic lipid droplets within cells. TAGs are broken down to its component free fatty acids by lipolytic enzymes when fuel reserves are required. However, when TAGs possess large quantities of polyunsaturated fatty acids (PUFAs) they are prone to nonenzymatic oxidation reactions, leading to formation of oxylipins (i.e., oxidized forms of fatty acids) that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. Although it is known that CES1 is a bona fideTAG hydrolase, it is unclear which specific fatty acids are preferentially released during lipolysis. Moreover, to better understand the biochemical function of CES1 in macrophages, we need to determine its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets. Thus, the goal of this study is to systematically identify those oxidized fatty acids that are liberated from oxTAGs by CES1, because their release activates signaling pathways important for the development of lipid‐driven inflammation. Gaining this knowledge will fill data gaps that exist between CES1 and the lipid‐sensing nuclear receptors, PPARγ and LXRα, that are abundantly expressed in macrophages and are important drivers of lipid metabolism and inflammation. Oxidized forms of triarachidonoylglycerol (oxTAG C20:4) and trilinoleoylglycerol (oxTAG C18:2) – containing physiologically relevant levels of oxidized PUFAs (<5 mol%) – were incubated with recombinant CES1, or Pseudomonaslipase (a positive control), to assess the release of oxylipins and non‐oxidized arachidonic acid (AA) and linoleic acid (LA), which were quantified by LC‐MS. CES1 was shown to efficiently metabolize oxTAG C20:4 and oxTAG C18:2, yielding several regioisomers of hydroxyeicosatetraenoic acid (5‐, 12‐, and 15‐HETE) and hydroxyoctadecadienoic acid (9‐ and 13‐HODE), respectively. The CES1‐catalyzed cumulative release of HODEs was faster than that of HETEs from the respective oxTAGs (7 pmol HODEs/min vs. 1 pmol HETEs/min). Thus, for oxTAGs, hydroxy fatty acids derived from LA are preferred by CES1 to those derived from AA. A similar trend was noted for the release of non‐oxidized PUFAs from the respective oxTAGs (470 pmol LA/min vs. 83 pmol AA/min). CES1 also preferentially liberated 5‐HETE over 12‐HETE and 15‐HETE from oxTAG C20:4, whereas no differences were noted between 9‐HODE and 13‐HODE. This study indicates that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs. It further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators that can regulate inflammatory activities of immune cells.
Phosphorylation‐Dependent Endocytosis of ROMK Limits Urinary Potassium Loss
Authors: Al‐Qusairi Lama , Kim Boyoung , Zapf Ava M. , Woodward Owen M. , Welling Paul A.
Published: May 2022

ROMK channels in the aldosterone‐sensitive distal nephron are tightly regulated to maintain potassium balance. In response to potassium restriction, ROMK channels are internalized from the apical membrane, safeguarding against extensive potassium loss. Previously, we found the clathrin adaptor protein, ARH, directly binds to ROMK to stimulate endocytosis (Fang et al, JCI ‘09). WNK signaling pathway has been implicated in activating ROMK endocytosis, but the mechanism is not clear, and physiological significance of ARH‐mediated ROMK regulation remains uncertain. To tackle these questions, we explored how WNK kinases activate ARH and compared the homeostatic response to dietary potassium restriction in wild type and ARH KO mice.

In vitro phosphorylation studies revealed ARH is phosphorylated by ERK, which in turn is activated by L‐WNK1 or WNK4. Examination of the phosphorylation cascade revealed that dietary potassium restriction increases phosphorylation of ERK and ARH‐S14 in the kidney, coincident with phospho‐activation of the WNKs, and reduction of ROMK apical surface expression. We found phosphorylation of ARH at S14 prevents its degradation, stimulates its membrane association, and this enhances ROMK endocytosis. ARH KO mice exhibit an increase in glycosylated ROMK in the renal cortex without changes in the abundance of the core‐glycosylated or unglycosylated forms, consistent with inhibition of post‐endocytic degradation. In dietary potassium deficiency, ARH KO mice are unable to reduce ROMK protein to the same extent as WT mice, and thus exhibit excessive urinary potassium loss compared to WT mice.

These studies identify ARH as a downstream effector of the WNK/ERK pathway that stimulates ROMK endocytosis in potassium deficiency.
Investigating the function of a unique DNA ligase (LIGK) in bdelloid rotifers
Authors: Schurko Andrew M. , Booth Halie J.
Published: May 2022

Bdelloid rotifers are a class of microscopic invertebrates with an extraordinary DNA repair system that allows individuals to recover from DNA damage induced by desiccation or ionizing radiation. While this DNA repair system provides a potential means for surviving harsh conditions, the mechanism for DNA repair is not understood. Previous work determined that the gene encoding DNA ligase K (LIGK) is upregulated in the bdelloid Adineta vagafollowing exposure to ionizing radiation, suggesting a role for the LIGK protein in DNA repair. The objective of this project was to explore the role of LIGK during DNA repair in bdelloid rotifers by examining phylogenetic relationship of LIGK and other DNA ligases, and by identifying phenotypic effects when LIGKgene expression is repressed. Bioinformatics analyses were first done to uncover the evolutionary history of LIGK.Homologs of LIGKwere found sporadically in prokaryotic and eukaryotic lineages, but the gene is absent in related monogonont rotifers and most model organisms. The domain structure of LIGK proteins was distinct from canonical DNA ligases I, III and IV, consistent with a less well understood function for LIGK. Next, RNA interference (RNAi) and CRISPR/Cas9 genome editing were used to inhibit gene expression of LIGK. We first attempted using RNAi to silence LIGKexpression. We created an E. coli feeder strain that expressed a double stranded RNA copy of LIGK,and this was fed to rotifers at various concentrations and lengths of time. However, real‐time PCR failed to show strong evidence for a gene knockdown. We then used CRISPR/Cas9 genome editing to mutate and inactivate LIGK. We generated a single guide RNA (sgRNA) and used an in vitro Cas9 cleavage assay to demonstrate that sgRNA/Cas9 complexes cut the target sequence. We then designed a single‐stranded oligodeoxynucleotide (ssODN) template for homology‐directed repair that would introduce stop codons in the LIGKcoding sequence. Rotifer embryos were electroporated with sgRNA/Cas9 complexes and the ssODN. Then, individuals were screened by PCR using mutation‐specific primers which revealed evidence of genome editing and introduction of the desired mutation. To assess the effects of LIGKinactivation on DNA repair, we did desiccation recovery assays on RNAi‐ and CRISPR‐treated rotifers. If LIGKhas a role in DNA repair, then a lower recovery rate for treated rotifers compared to untreated or wild‐type rotifers would be expected. Overall, bioinformatics analysis and gene expression analyses are helping to understand the function of this unusual DNA ligase in bdelloid rotifers, which will broaden our understand of DNA repair beyond the typical model organisms.
Sexual Dimorphic Regulation of MicroRNAs Alters Sodium Transport in the Kidney Distal Nephron
Authors: Farrell Corinne E. , Liu Xiaoning , Ozbaki Yagan Nejla , Mutchler Stephanie , Bodnar Andrew J. , Ho Jacqueline , Butterworth Michael
Published: May 2022

Background

Hypertension affects more than one billion people worldwide. A key regulator of blood pressure homeostasis is aldosterone, the final signaling hormone of the renin‐angiotensin‐aldosterone‐signaling (RAAS) system. Aldosterone increases sodium (Na+) transport in the kidney distal nephron to regulate blood volume. Premenopausal women are less likely to develop hypertension than age‐matched men, due to both lower aldosterone levels and estrogen signaling. We previously demonstrated that aldosterone alters the expression of microRNAs (miRs) in collecting duct epithelial cells to modulate the Na+ transport response to aldosterone. However, the sex‐specific regulation of miRs and role of estrogen to alter miR expression in the kidney distal nephron has not been explored. This study investigated the hypothesis that estrogen alters aldosterone signaling in the distal nephron by regulating the expression of miRs in collecting duct epithelia.

Methods

Primary cortical collecting duct (CCD) cells derived from male and female mice as well as the mCCD‐cl1 cell line were cultured on permeable filter supports to measure Na+ transport by short‐circuit current recordings in Ussing chambers. Cells were incubated with estrogen for time and dose responses to determine the impact on aldosterone stimulation and Na+ transport. RT‐qPCR quantified miR and mRNA expression and western blot analysis quantified protein expression after aldosterone and estrogen stimulation. In vivo regulation of miRs by aldosterone was confirmed using freshly isolated CCD cells from male and female mice placed on low Na+ diets.

Results

A sex‐specific upregulation of the miR‐17~92 cluster was observed in female mice placed on a low‐Na+ diet to stimulate aldosterone release. MiRs‐19a&b were also upregulated in mCCD cells stimulated with estrogen. Estrogen pretreatment blunted aldosterone stimulation, by targeting the serum and glucocorticoid induced kinase (SGK1) mRNA. Luciferase assays demonstrated that miRs‐19a&b bind to the 3’‐UTR of SGK1. Overexpression of miR‐19 in mCCD cells using miR mimics significantly inhibited aldosterone stimulation of Na+ transport. Conversely, aldosterone stimulation was greater in mCCD cells transfected with a miR‐19 inhibitor (antagomir).

Conclusion

The miR‐17~92 cluster is regulated by aldosterone and estrogen. Mir‐19 targets SGK1 and may account for estrogen’s inhibition of aldosterone signaling. This miR cluster may be responsible, in part, for the observed sex‐specific differences in aldosterone signaling. Ongoing studies aim to determine if altering the expression of miR‐17~92 disrupts RAAS signaling in the kidney in vivo.
Structural Differences in SARS‐Cov‐2 Spike Proteins and their Effects
Authors: Cetin Sena
Published: May 2022

COVID‐19 is a highly infectious disease that mainly affects the human respiratory system. Coronavirus spike glycoproteins assist in entry and infection of cells by binding to the human ACE2 receptor. Because of this, they are the main target for antibodies and inhibitors (Walls et al., 2020).

Some variants of SARS‐CoV‐2 have been classified as Variants of Concern (VOCs). One characteristic of these VOCs is an “increase in transmissibility or detrimental change in COVID‐19 epidemiology” (World Health Organization, 2021) as a result of mutations in the SARS‐CoV‐2 genome. By comparing the structures of the S proteins of SARS‐CoV‐2 (Pango lineage A) and its B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants using PyMOL, we studied the differences in the structure of the receptor binding domain (RBD) of these S proteins, explaining their increased binding affinity to the ACE2 receptor, and their consequent increased transmissibility. The structure of a protein is directly related to its function which is why we think that this investigation is crucial to understanding SARS‐CoV‐2 and its variants. We expect that these regions are important for studying possible impacts on vaccine effectiveness and specific drug targets for SARS‐CoV‐2.
Mice Exposed to Maternal Separation are Resilient to the Loss of Circadian Heart Rate Rhythm in Response to Chronic High Salt Diet
Authors: Ghuneim Sundus , Dalmasso Carolina , Leachman Jacqueline , Cincinelli Cole , Loria Analia S.
Published: May 2022

Early life stress (ELS) is associated with increases in body mass index and systolic blood pressure in adult life, leading to obesity and obesity‐induced hypertension, major risk factors for cardiovascular disease (CVD). Previously, we have shown that Maternal Separation and Early Weaning (MSEW), a model that mimics the effects of ELS in humans, increases blood pressure and sympathetic activation in obese male mice. We have also shown that MSEW males fed a low‐fat diet (LF) displayed increased water intake, urine excretion, potassium excretion, glomerular filtration rate (GFR), and renal cortex NHE3 expression compared to controls. The aim of this study was to further investigate the status of the water and electrolyte homeostasis (WEH) when we challenge mice exposed to MSEW with a chronic high‐salt diet (HS). C57BL/6J mice pups were separated from the dams from postnatal day (PD) 2 to PD 16 and weaned early on PD 17. Control (C) litters remained undisturbed with the dams and were weaned on PD 21. At weaning, mice were placed on a LF (10% Kcal from fat, Research Diets). After 18 weeks, mice were implanted with radiotelemeters to measure systolic blood pressure (SBP) and heart rate (HR). At 20 weeks, a baseline was recorded and then mice were placed on a LF+HS (10% Kcal from fat, 4% NaCl, Research Diets) for 6 weeks. Single mouse metabolic cages were used to measure water intake and urine excretion before and after LF+HS. GFR was measured using a transcutaneous technique. After 6 weeks, control and MSEW mice showed a similar response to LF+HS in water intake (3.84 ± 0.54 vs. 4.56 ± 1.01 ml), urine excretion (1.90 ± 0.93 vs. 2.63 ± 1.18 ml), and GFR (930.98 ± 41.30 vs. 975.97 ± 86.12 ml/min/100g BW). In addition, 6 weeks of LF+HS did not change 24‐hr SBP from baseline in control (125.5 ± 5.85 vs. 129.33 ± 6.0 mmHg) and MSEW mice (117.33 ± 9.55 vs. 126.67 ± 11.98 mmHg). Similarly, HR did not change in control (612.88 ± 19.51 vs. 651.67 ± 17.84 bpm) and MSEW mice (602.25 ± 15.27 vs. 575.83 ± 11.24 bpm). At baseline, both groups show normal circadian rhythm of blood pressure. However, LF+HS control males lack day‐night HR amplitude (day: 655 ± 14.50 vs. night: 647.67 ± 11.17 bpm, p=NS) while MSEW mice preserve this function (day: 557.67 ± 6.97 mmHg vs. night: 594 ± 5.51 bpm, p<0.05). These data indicate that MSEW male mice are resilient to loss of circadian HR rhythm in response to a chronic HS. Ongoing studies of renal sodium transporters expression may provide insights into the potential mechanisms by which MSEW male mice display this resilience.
Real Time Measurement of Hepatic β‐oxidation with Deuterium Magnetic Resonance in Murine Models on a High Fat Diet
Authors: Mcleod Marc , Ragavan Mukundan , Giacalone Anthony , Rushin Anna , Merritt Matthew E.
Published: May 2022

Non‐alcoholic fatty liver disease (NAFLD) is characterized by >5% hepatic fat by mass. As one of the most common liver diseases, with a prevalence of roughly 24% in the U.S. adult population, NAFLD related morbidities account for a striking ~300 billion dollars a year in healthcare costs for the U.S. alone [Shetty, A., & Syn, W. K. Federal practitioner: for the health care professionals of the VA, DoD, and PHS, (2019), 36(1), 14–19]. NAFLD, both in humans and murine models of the disease, has been associated with increased rates of hepatic β‐oxidation and reactive oxygen species production [Sunny, N. E., Parks, E. J., Browning, J. D., & Burgess, S. C. Cell metabolism, (2011). 14(6), 804–810]. These characteristics are thought to drive a pro‐inflammatory state that causes hepatic damage, and can transition to the much more serious malady nonalcoholic steatohepatitis. To directly quantify hepatic β‐oxidation as a potential biomarker of NAFLD disease development and progression, we are developing deuterium magnetic resonance (DMR) techniques to monitor oxidation of D15‐octanoate. In the perfused mouse liver, we demonstrate that DMR measures of HDO production, as a direct byproduct of β‐oxidation, estimate hepatic β‐oxidation rates within 5% of standard quantitation by mass spectrometry of fatty acid depletion from perfusate. In a dietary model of NAFLD (60% fat by kcal, HFD) we observed increased overall b‐oxidation, but when normalized to the total liver weight, the HFD liver oxidized less than the low‐fat diet control. By coupling DMR spectroscopy, gas chromatography‐mass spectrometry and metabolic modelling, we were able to identify increases in total ketogenesis and FADH2 oxidation in high fat diet perfused livers. When normalizing metabolic flux to liver protein, both octanoate and FADH2 oxidation were significantly lower in high fat diet livers. Increased total fatty acid oxidation and ketogenesis implicate activation of compensatory mechanisms by the liver during NAFLD to address higher levels of circulating lipids. Decreases in efficiency/relative oxidation indicate an accrual of hepatic damage that hinders the efficient clearance of excess lipids. These results demonstrate the utility of DMR as a sensitive measure for detecting changes in metabolic activity during NAFLD progression, and suggests that a clinical imaging paradigm might be feasible.
Inhibition of Nuclear Transport of Perilipin 5
Authors: Lewis Hope N. , Tansey John T.
Published: May 2022

Obesity, type II diabetes mellitus, and non‐alcoholic fatty liver disease are all conditions related to aberrant lipid storage in the organism. Nearly all cells have the capacity to store neutral lipids in lipid storage droplets, organelles that regulate the storage of their hydrophobic contents. All lipid storage droplets are coated with at least one member of the perilipin family of proteins. These proteins serve as a substrate of protein kinase A, act as cofactors for lipases, are implicated in lipid trafficking, and have been shown to act as transcriptional coactivators. Perilipin 5 is expressed in tissues including oxidative muscle and fasted liver and has been shown to play important roles in neutral lipid metabolism in these tissues and metabolic states. The mechanism through which perilipin 5 enters the nucleus is not known. Using a cultured cell model expressing either perilipin 5 or a perilipin 5 3X‐FLAG fusion protein, assayed with immunoblotting and immunofluorescence microscopy we have observed protein kinase A dependent phosphorylation of perilipin 5 and translocation of perilipin 5 to the cytosol and nucleus. There are five known mechanisms for large molecules to gain entry to the nucleus through the nuclear pore. We have used multiple compounds to attempt to block entry of perilipin 5. Preliminary results indicate that the synthetic steroid mifepristone, but not other inhibitors, block entry, implicating the importin α/β1 nuclear transport pathway in this process. These data indicate that the biological function of perilipin 5 can be at least partially blocked pharmacologically using commercially available drugs. This presents an interesting potential for further examination and treatment of metabolic diseases.
Analysis of Anti‐CRISPR in relation to structurally similar proteins
Authors: Ostapchuk Nikita
Published: May 2022

Anti‐CRISPR(Acr) serves as a control element in CRISPR genome editing processes. However there are many other applications that require control outside of said usage. The development of any new protein structures is a time consuming process, especially if the new protein needs to have a specific function in relation to other proteins. Fortunately there are many ways to gather data about the protein structure and interactions, including physical experiments and computer simulations. By analyzing the structural similarities between Acr and other proteins we may be able to more efficiently design protein blockers.
Chronic Pain State Mediates Development of Hippocampal and Renal Inflammatory Responses
Authors: Foltz Jeffery B. , Babich Victor , Duong Brittany , Winter Lori , Di Sole Francesca , Duric Vanja
Published: May 2022

Chronic pain and related stress have been previously linked to the development of mood disorders and dysfunction of peripheral organs such as the kidney. While the underlying neurophysiological mechanisms remain elusive, here we examined the effects of chronic pain on activation of immune‐inflammatory responses in the brain and kidney. Male rats were exposed to chronic inflammatory pain for up to 42 days [multiple injections of Freund’s adjuvant (CFA) into the hind paw], which produced a state of chronic allodynia and enhanced behavioral emotionality. Biochemical analysis of the hippocampus, a limbic region that regulates mood and stress responses, showed that CFA evoked increases in expression of ionized calcium binding adaptor molecule 1 (IBA1) and NLRP3 inflammasome proteins, known markers of microglial activation and neuroinflammatory responses, respectively. An increase in the neutrophil gelatinase‐associated lipocalin (NGAL) and IL‐18 are recognized as early diagnostic injury and inflammatory biomarkers of kidney disease. NLRP3 is implicated in the pathogenesis of kidney diseases by regulation of renal inflammation. Analysis via immunocytochemistry demonstrated that CFA also evoked significant increases in NGAL, IL‐18 and NLRP3 protein levels in the renal glomeruli and tubules, suggesting that chronic pain and related stress effects induce renal inflammation and possibly a reduction in kidney function. Together, these findings provide new evidence to support a mechanistical understanding of a bidirectional relationship between chronic pain‐related stress and kidney dysfunction. Further understanding of this relationship could contribute to the identification of novel treatment strategies to diminish both mental health and renal physiological consequences of chronic pain.
The Structure of Tau Proteins and Its Role in Neurodegenerative Disease
Authors: Pidara Ishani , Yamada Koyuki
Published: May 2022

Abundant in the nerve cells of the central nervous system, tau proteins primarily serve to help maintain the stabilization of microtubules in axons (Michalicova et al., 2020). These microtubules are essential for the rigid structure of the cell and allow for intracellular transport of nutrients and other molecules (Parker et al., 2014). Tau proteins, using polymerization, regulate microtubule stability by binding between the tubulin heterodimers, which form the microtubules. Irregular interaction between tau proteins and microtubules can lead to associated neurodegenerative diseases called tauopathies. While there are many tauopathies, such as frontotemporal dementia with parkinsonism‐17 and Pick disease, Alzheimer’s is one of the most prevalent and studied diseases associated with tau (Gao et al., 2018). Tau protein aggression causes neurofibrillary tangles that deteriorate the microtubules’ structure, thus eventually blocking the synaptic communication between neurons (NIH National Institute on Aging, 2017). Over the past few years, numerous research has been conducted for potential tau immunotherapies, raising hopes for a possible solution (Congdon & Sigurdsson, 2018). Microtubule‐stabilizing drugs may also be instrumental in easing neurodegenerative diseases (Kadavath et al., 2015). We will, therefore, investigate the relationship between tau proteins and the development of these diseases.
Enzymatic Coupling of a Classical Luciferase Assay for the Quantitative Monitoring of the Protein‐Free ADP‐ribose
Authors: Kasson Samuel P. , Kim In‐Kwon
Published: May 2022

Historically, mechanisms of erasers of ADP‐ribosylations have been understudied, primarily due to the lack of quantitative tools to selectively monitor specific activities of different ADP‐ribosylation reversal enzymes. Here, we developed a new NUDT5‐coupled AMP‐Glo (NCAG) assay to specifically monitor the protein‐free ADP‐ribose. We found that due to NUDT5's selectivity for protein‐free ADP‐ribose, but not protein‐bound poly‐ and mono‐ADP‐ribosylations, protein‐free poly(ADP‐ribose) chains, or NAD+, it could be used in complex solutions to target protein‐free ADP‐ribose for quantitative detection. The NCAG assay functions through a series of enzymatic reactions starting with cleavage of ADP‐ribose to AMP, followed by conversion of AMP to ATP, and finally detection via classical luciferase assay. This NCAG assay can be used as a general platform to study the mechanisms of diverse ADP‐ribosylation reversal enzymes that release protein‐free ADP‐ribose as a product. Furthermore, using the NCAG assay to monitor changes in the protein‐free ADP‐ribose can grant additional insight into ADP‐ribose recycling and reactivity.
Dual role of low frequency magnetic field on neuroinflammation induced dendritic trimming and memory impairment on rat model of sporadic Alzheimer’s disease
Authors: Roy Avishek , Sharma Binney , Banerjee Jyotirmoy , Iyenger Soumya , Jain Suman
Published: May 2022

Gliosis and dendritic loss is reported to be secondary to the amyloid beta (Aß) and hyperphosphorylated tau (hp‐tau) production in Alzheimer’s disease(AD) brain. STZ administration is reported to induce sporadic AD. Previous clinical or preclinical studies on effect of rTMS treatment in AD patients or APP23 mice showed cognitive improvement alone or as adjuvant therapy along‐with amelioration of the glial aggravation in the hippocampus. However, studies on the effect of magnetic field stimulation on the gliosis related dendritic trimming in animal models of sporadic AD is elusive. Therefore, in the present study we have looked in to the effect of extremely low frequency magnetic field stimulation (ELF‐MF; 17.96µT, 50Hz, 2hr/day) for one month post‐injection of STZ (bilateral, 3mg/kg). Total 45 animals were (ethical no. 937/IAEC/2016) randomly divided into five groups: Control, Sham, AD, Sham+MF and AD+MF. After assessing retention of spatial and avoidance memory using Morris water maze and passive avoidance step down test on 30th day, animals were sacrificed and tissues processed for oxidative stress and immunofluorescence, Golgi‐Cox staining. Behavioural analysis revealed spatial memory improvement with no effect on the avoidance memory loss in AD+MF group as compared to AD group. We also found significant decrease in superoxide dismutase, catalase, GSH and increase in malon‐di‐aldehyde (MDA) in AD group. ELF‐MF treatment only reduced MDA level in hippocampus and frontal cortex. GFAP, IBA‐1 and hp‐tau (ser356) positive cells were found to be reduced in AD+MF group, as compared to all groups except Control. Sholl analysis revealed partial improvement of reduction in the number of intersection, bifurcation and length in basal and apical dendrite in the AD group after ELF‐MF treatment in CA3 layer. These findings suggests dual role of ELF‐MF exposure on STZ model of AD showing improvement of spatial memory by reducing the gliosis induced dendritic loss. However, inability to manage oxidative stress due to STZ, which may lead to avoidance memory deficit even after exposure period of one month.
Blockade of Trpm7 in the Carotid Body area attenuated intermittent hypoxia‐induced Hypertension
Authors: Shin Mi Kyung , Kim Lenise J. , Tang Wan‐Yee , Sun Boran , Cui Honggang , Polotsky Vsevolod Y.
Published: May 2022

RATIONALE

Obesity leads to high cardiovascular morbidity and mortality via multiple mechanisms including obstructive sleep apnea (OSA) and hypertension. Intermittent hypoxia (IH), a hallmark manifestation of OSA, is an established cause of hypertension. The carotid body (CB) has been implicated in the pathogenesis of hypertension in IH and obesity. We discovered that obesity‐induced hypertension is mediated via the transient receptor potential melastatin 7 (TRPM7) channel in the glomus cells of CB. Here, we investigate the relevance of TRPM7 signaling in CB for the pathogenesis of hypertension by IH. We developed a novel technique allowing continuous sustained release of the TRPM7 inhibitor FTY720 into the CB area using a hydrogel formulation with a single injection abolishing hypertension in obesity for 3 weeks. We hypothesize that IH causes hypertension acting on CB TRPM7.

METHODS

C57BL/6J male mice (n = 9) were implanted with telemetry in the left femoral artery for continuous blood pressure monitoring and exposed to IH for 5 days. IH was administered by decreasing O2 levels from 21% to 6%, 60 cycles/hr, during the light phase, 9AM ‐9PM. FTY720 hydrogel (n = 5) or control hydrogel (N = 4) were applied locally to the CB region. Blood pressure was recorded at baseline and during IH and the measurement was repeated 2 weeks after hydrogel application.

RESULT

IH increased mean arterial pressure from 103.8±1.2 mmHg to 114.3±1.4 mmHg on Day 1 of IH and to 113.0±0.6 mmHg on Day 5 of IH respectively. FTY720 hydrogel abolished IH‐induced hypertension by decreasing blood pressure to 106.6±1.6 mmHg (p<0.005) on Day 1 of IH and to 101.5±1.6 mmHg (p<0.001) on Day 5 of IH. In contrast, control hydrogel had no effect on IH‐induced hypertension.

CONCLUSION

Our study has shown that inhibition of TRPM7in the carotid bodies abolished IH‐induced hypertension.