bluuebunny/crossref_metadata_2025 huggingface dataset:
Renal Pathophysiological Changes During Chronic Metabolic Acidosis Associated Increased Blood Pressure
Authors: Aryal Dinesh , Jackson Keith E.
Published: May 2022

Activation of renal angiotensin II works synergistically with renal superoxides to promote an increase in blood pressure during chronic acidotic conditions has been previously delineated. Uncontrolled high blood pressure instigates renal pathophysiological function and eventual end organ damage is evident. This study aims to investigate the renal consequences of increased blood pressure provoked during chronic metabolic acidosis (CMA). It was a challenging prospect, as both hypertension and chronic metabolic acidosis are two different pathological conditions and can exist independent of each other. Renal functions in rats grouped in 3 groups; i) Control (nonacidotic and normotensive), ii) CMA (acidotic and hypertensive), iii) CMA+captopril+tempol (acidotic and normotensive) were critically assessed by measuring Glomerular Filtration Rate (GFR), Fractional excretion of sodium (FENa), urine flow rate, weight of the kidneys and albuminuria. Picro‐sirius staining of the cortical sections was performed to examine the collagen content as an index of fibrosis. The results demonstrated significant attenuation of GFR in the CMA group compared to the control group. However, GFR in captopril+tempol treated acidotic rats is significantly increased compared to the CMA groups, which suggested that the reduced GFR in CMA group is because the rats are hypertensive. Conversely, the FENa was significantly increased in the CMA group compared to the control but the captopril+tempol treated group showed a significant decrease in FENa compared to the CMA group, this implied that hypertension but not acidosis is the cause of increased FENa which increases blood flow to the kidney potentially leading to the pressure natriuresis and could potentially lead to kidney damage. The urinary albumin excretion was also significantly elevated in acidotic rats as compared to control but the captopril+tempol treated group showed a significant reduction in albuminuria, which revealed the acidosis‐induced hypertension is contributing for impaired renal infiltration and augmenting the albuminuria. The morphological analysis of renal changes using Picro‐sirius staining revealed significant structural damage in the glomeruli of chronically acidotic rats. However, when these rats were treated with captopril and tempol combined, the glomeruli were intact and a reduction in fibrosis was observed. Overall, our results suggested the renal consequences of acidosis induced hypertension can lead to eventual end organ damage which is independent to the function of acidosis. Hence, intrarenal Ang II and superoxide release in the kidney could be a potential target to reduce the blood pressure, albuminuria, and protection of renal function and delayed the onset of end‐stage renal disease associated with acidosis evoked hypertension.
Obese Adipose Tissue‐Derived Extracellular Vesicles increase Colorectal Cancer Cell proliferation and glycolysis
Authors: Haque Parsa , Souza Rhonda F. , Paris Shere L. , Theiss Arianne
Published: May 2022

Colorectal cancer (CRC) is the second deadliest cancer worldwide and obesity is an established risk factor for both men and women. However, we still have limited knowledge regarding the molecular mechanisms that support obesity‐induced CRC. Obesity substantially modifies the adipose tissue (AT) and increases secretion of various factors including nanometer‐sized extracellular vesicles (EVs). EVs are lipid bilayer vesicles released from most types of cells and contain lipids, proteins, microRNAs and other cytoplasmic components of the parent cell. Considering their ability to travel to distant sites and deliver cargo to recipient cells, EVs could act as intercellular mediators that alter the tumorigenic potential of recipient cancer cells. Therefore, we hypothesized that obese AT secreted EVs carry cargo that enhances tumorigenicity of CRC cells. Here, we aimed to determine the functional and metabolic changes induced by obese human AT‐secreted EVs on human CRC cell lines. Visceral AT tissue from patients with obesity (BMI>35) undergoing bariatric surgery and non‐obese patients (BMI<30) undergoing anti‐reflux surgery was cultured ex vivo in EV‐free DMEM‐H/Ham’s F12 for 48 hrs. Conditioned media was collected and EVs were isolated by ultracentrifugation. EV size and concentration were measured through Nanoparticle Tracking Analysis (NTA). EV protein cargo was characterized by unbiased proteomics analysis using LC‐MS. Human CRC cell lines HCT‐116 and HT‐29 were treated with 10^7 EV/ml for 24 or 48 hrs and proliferation and mitochondrial/glycolytic ATP production were measured by CCK‐8 assay or Seahorse XF ATP Rate Assay, respectively. NTA reported our EV preps were on average 120 nm in size, suggesting we isolated exosomes and/or small microvesicles. Using KEGG Pathway Database and Panther Protein Classification Software, 25% of EV proteins secreted from human AT were associated with metabolism, with 13% of total metabolic proteins in EVs associating with glycolysis. Obese EVs exhibited increased expression of glycolytic enzymes pyruvate kinase, phosphoglycerate kinase 1, and G3P dehydrogenase. Obese EVs increased proliferation and glycolytic ATP production in CRC cell lines, both serving as indicators of cancer aggressiveness. Taken together, our results suggest that obese adipose EVs mediate the tumorigenic potential of CRC cells. Our study is significant since it aims to identify druggable pathways which support tumorigenesis in obese CRC patients.
Regulation of Adipocyte Metabolic Homeostasis by SR‐BI and PCPE2
Authors: Knaack Darcy A. , Sorci‐Thomas Mary G. , Thomas Michael J. , Chen Yiliang , Sahoo Daisy
Published: May 2022

Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity. Adipocytes are also highly dynamic cells that perform many key metabolic processes including lipid and glucose metabolism. Recent collaborative studies demonstrated that SR‐BI function may require the extracellular matrix protein procollagen C‐endopeptidase enhancer 2 (PCPE2) to regulate cholesterol transport in hepatocytes and adipocytes. However, the roles of SR‐BI and PCPE2 in other adipocyte metabolic processes and whether they maintain their cooperative partnership for these functions remains unknown. This knowledge gap prompted our novel hypothesis that PCPE2 facilitates SR‐BI’s ability to regulate metabolic processes in adipocytes. To test our hypothesis, we have optimized an adipocyte model system where mesenchymal stem cells isolated from the outer ears of wild‐type (WT), SR‐BI knockout (SR‐BI‐/‐), and PCPE2‐/‐ mice are differentiated into adipocyte‐like cells. We validated our adipocyte model system by demonstrating increased Oil‐Red‐O staining, mRNA expression of adipogenesis markers, and adiponectin secretion over a 9‐day post‐differentiation period for all three genotypes. This model system was then used to test the roles of SR‐BI and PCPE2 on various metabolic functions, including glucose, fatty acid, and mitochondrial metabolism. Preliminary data suggest that loss of SR‐BI and PCPE2 impaired glucose uptake, despite observing elevated levels of GLUT4 at the cell surface. PCPE2‐/‐ adipocytes also showed increased response to insulin through AKT signaling, which may be responsible for the increased expression of GLUT4 at the cell surface. However, we observed that loss of SR‐BI or PCPE2 had no effect on transport of palmitic acid in adipocytes, despite there being elevated expression of fatty acid transporters (FABP4, CD36). Adipocytes lacking SR‐BI or PCPE2 showed an inverse response to insulin compared to WT adipocytes when measuring phosphorylation status of proteins responsible for mediating fatty acid synthesis (AMPK, ACC). These data suggest that although SR‐BI and PCPE2 do not appear to mediate uptake of palmitic acid, they may have altered lipid metabolism. Lastly, adipocytes lacking SR‐BI or PCPE2 exhibit changes in mitochondrial mass and morphology, but their role in maintaining healthy mitochondria remains inconclusive. Altogether, these data suggest that SR‐BI and PCPE2 appear to have independent roles in various steps of adipocyte metabolism, but their required partnership remains unclear.
Effect of SGLT2 Inhibition on the Development of Salt‐sensitive Hypertension, RAAS, and Sodium Channels and Transporters in Dahl SS Rats
Authors: Kravtsova Olha , Bohovyk Ruslan , Levchenko Vladislav , Rieg Timo , Palygin Oleg , Staruschenko Alexander
Published: May 2022

Hypertension is common among patients with type 2 diabetes mellitus (T2DM). The combination of diabetes and hypertension is associated with high morbidity and mortality because of an increased risk of cardiovascular and renal complications. Effective control of blood glucose levels and blood pressure is essential to prevent the development and progression of diabetic nephropathy in T2DM. Sodium‐Glucose Cotransporter‐2 (SGLT2) inhibitors are the new highly effective drugs for the treatment of T2DM and some cardiovascular diseases. Despite all the remarkable benefits on cardiovascular and renal systems, the molecular mechanisms of how SGLT2 inhibition affects water and electrolyte balance are incompletely understood. We hypothesized that SGLT2 inhibition alters blood pressure via affecting Renin‐Angiotensin‐Aldosterone System (RAAS), sodium channels and transporters. Dahl Salt‐Sensitive (SS) rat, a model for salt‐induced hypertension, was used to test this hypothesis in a hypertensive, non‐diabetic environment. We found that administration of dapagliflozin at a concentration of 2 mg/kg/day via drinking water during 3 weeks blunts the development of salt‐induced hypertension with no changes in the heart rate. Mean arterial pressure in the dapagliflozin treated group compared to the vehicle‐treated group during high salt (4% NaCl) challenge was significantly lower (136 ± 4 vs. 157 ± 6 mmHg; P<0.0001 on 21st day of the experiment). SGLT2 inhibition caused a significant diuretic effect (67 ± 6 vs. 40 ± 4 ml/day, P<0.001 on the 21st day of the experiment). Glucose excretion was dramatically increased in inhibitor‐treated rats compared to vehicle‐treated (747 ± 44 vs. 12 ± 2 mg/dL, P<0.0001 on the 21st day). We also observed increased Na+/creatinine ratios (138 ± 10 vs. 110 ± 6, P<0.004 on the 21st day) and Cl‐/creatinine ratios (133 ± 9 vs. 108 ± 6, P<0.03 on the 21st day). To define potential mechanisms, we first measured levels of hormones involved in the RAAS by using LC‐MS/MS. Despite effects on Na+/creatinine and Cl‐/creatinine ratios, dapagliflozin treatment did not affect RAAS metabolites. Subsequently, we assessed the effects of SGLT2 inhibition on renal Na+ channels and transporters using patch‐clamp, Western blotting, and RT‐PCR. Neither mRNA nor protein expressions levels of all tested Na+ transporters (SGLT2, SGLT1, NHE3, NKCC2, NCC, and α‐, β‐, γ‐ ENaC subunits) were different between groups. Electrophysiological studies did not reveal any difference of SGLT2 inhibition on the conductance and activity of ENaC. Therefore, our data suggest that SGLT2 inhibition blunts the development of salt‐induced hypertension, causes glucosuria and natriuresis but does not affect RAAS and expression and activity of Na+ channels and transporters in a model of non‐diabetic, salt‐sensitive hypertension.
The development of an intestinal microphysiological system to model oral drug disposition
Authors: Arian Christopher M. , Kelly Edward J. , Thummel Kenneth E.
Published: May 2022

The intestine has important gate‐keeping functions that can profoundly impact the systemic blood exposure of orally administered drugs. Over the past decade, there has been growing effort to develop advanced in vitro tissue models that better predict in vivo DMET processes. One such novel model is the microphysiological system (MPS), which provides a more physiologically and anatomically relevant epithelial cell culture platform than traditional, static cell culture models. The MPS incorporates flow of media down luminal and abluminal channels, with accompanying shear forces that have been shown to be beneficial for multicellular viability and function. In this study, we cultured the immortalized cell line, LS180, in the Microfluidic ChipShop’s “Fluidic 480” MPS. LS180 cells have demonstrated high metabolic activity, particularly for CYP3A substrates, but display poor performance for monolayer permeability studies due to a lack of tight junction formation and uncertain transporter function. We assessed CYP3A metabolic activity, cell monolayer integrity, and global RNA expression for LS180 cells cultured in the MPS versus traditional TranswellTM culture. To measure CYP3A activity, we incubated LS180 cells cultured MPS and TranswellTM with the CYP3A probe substrate, midazolam, and monitored total product formation and parent midazolam appearance in the basolateral media of the two culturing platforms (with static or dynamic flow). LS180 cells cultured in MPS display a 2‐3‐fold greater midazolam extraction ratio (ER) compared to LS180 cells cultured in TranswellTM. We then compared cell monolayer integrity in the two culture platforms by measuring the apparent permeability (Papp) of the paracellular transport probe, atenolol. LS180 cells cultured in MPS display a 3‐fold lower Papp for atenolol than LS180 cells cultured in TranswellTM, indicating improved barrier integrity and tight junction formation for LS180 cells cultured in MPS. Using RNA‐seq, we compared global RNA expression for LS180 cells cultured in MPS versus TranswellTM. There were minimal expression differences for phase I and II metabolic enzymes between the two groups. Interestingly, transporter expression differed greatly between the two groups, with some transporters displaying greater expression in MPS compared to TranswellTM (e.g., P‐gp, MRP‐2) while others displayed a decrease in expression (e.g., OATP2B1, ENT1). Further testing is required to assess whether the changes in transporter RNA expression leads to changes in transporter protein abundance and localization. The sum of these results suggest that culturing LS180 cells in a MPS improves their performance and ability to predict the disposition of orally administered drugs.
Butyrate supplementation rescues sex‐specific deficits in respiratory plasticity and mean arterial pressure in adult offspring exposed to gestational intermittent hypoxia
Authors: Mickelson Carly R. , Kobitter Emma P. , Meza Armand L. , Song Ruolin , Mishra Jay S. , McCann Erin C. , Radcliff Abigail B. , Hill Kelsea K. , Kumar Sathish , Watters Jyoti J. , Baker Tracy L.
Published: May 2022

Sleep disordered breathing (SDB) in pregnancy is increasing in parallel with the obesity epidemic, and is linked to adverse perinatal outcomes in the offspring. Consequences of maternal SDB during pregnancy on the adult offspring are not well‐understood. Preliminary data indicate that adult male, but not female, offspring of dams exposed to intermittent hypoxia during gestation (GIH ‐ gestational intermittent hypoxia), a rat model of SDB during pregnancy, have elevated mean arterial pressure (MAP), increased neuroinflammation, inflammation‐induced impairment of homeostatic plasticity in the respiratory control system, and gut dysbiosis. Specifically, adult male GIH offspring have decreased relative abundance of gut bacteria that produce butyrate, a short‐chain fatty acid that has potent anti‐inflammatory properties and regulates immune function in the central nervous system. Since literature suggests that gut dysbiosis, neuroinflammation, and hypertension exist in a complicated vicious cycle, we treated male GIH offspring with tributyrin, a butyrate prodrug. Prior to tributyrin treatment, MAP in male GIH offspring was 96±3 mmHg, versus 76±1 mmHg in control rats receiving gestational intermittent normoxia (GNX). Similarly, male GIH offspring exhibited deficits in respiratory plasticity in response to recurrent central apneas (3.6±6.8% plasticity), when compared to GNX offspring (59.3±4.8% plasticity). GIH offspring exhibited evidence of elevated neuroinflammation in brain regions underlying cardiorespiratory control (e.g., IL‐1β expression 1.6±0.2‐fold higher than GNX controls (1.0±0.2)). Following three weeks of tributyrin treatment, MAP had normalized (80±2 mmHg), neuroinflammation was reduced to control levels, and the ability to express respiratory neuroplasticity was rescued (78±30%). These data indicate that gut dysbiosis may contribute to sex‐specific impairments induced by maternal exposure to intermittent hypoxia during gestation that can be rescued in adulthood by supplementing with butyrate.
Citric acid cycle metabolites regulate phosphatidate phosphatase activity from the oleaginous yeast Yarrowia lipolytica
Authors: Pasham Sagar , Fakas Stylianos
Published: May 2022

Phosphatidate phosphatase (PAP) catalyzes the conversion of phosphatidate (PA) to diacylglycerol (DAG) in a reaction that depends on Mg2+. This reaction is critical for de novolipid accumulation because it provides the DAG needed for the biosynthesis of triacylglycerol (TAG). Lipid accumulation is triggered by the downregulation of the citric acid cycle, which results in the exit of citrate from the mitochondria to the cytosol. In the cytosol, citrate is converted to oxaloacetate and acetyl‐CoA which is channeled to fatty acid biosynthesis. Oxaloacetate is converted to malate by malate dehydrogenase and malate to pyruvate by malic enzyme. Also, citrate stimulates the activity of acetyl‐CoA carboxylase, which catalyzes the rate‐limiting step in fatty acid biosynthesis. In this work, we examined the effect of citric acid cycle metabolites on PAP activity in cell extracts prepared from wild type cells and cells that lack PAH1 (i.e., pah1Δ). The cells were grown for 96 h on high glycerol media that induce lipid accumulation, and cell extracts were prepared. Control PAP assays done in the presence of Mg2+showed that the pah1Δ mutation resulted in a 95% decrease in PAP activity, indicating that PAH1 encoded for almost all PAP activity. The effects of citrate, malate, and pyruvate were examined at concentrations ranging from 0.1 mM to 5 mM. In wild type cell extracts, citrate (1 mM), malate (0.1 mM), and pyruvate (2.5 mM) caused a 220%, 217%, and 154% increase in PAP activity, respectively. In contrast, the addition of these acids to cell extracts prepared from pah1Δ cells did not affect PAP activity. The stimulatory effect of these organic acids on PAP activity could provide a direct link between fatty acid biosynthesis and DAG synthesis by Pah1. The buildup of citrate and its metabolites in the cytosol could induce PAP activity to direct the lipid biosynthetic pathway towards the synthesis of DAG. This regulation, combined with the stimulation of acetyl‐CoA carboxylase by citrate, could contribute to the induction in TAG synthesis typically observed during lipogenesis.
Obesity exacerbates SARS‐CoV‐2 infection in K18‐hACE2 mice
Authors: Berdasco Clara , Whitehead Anna , Qin Xuebin , Yue Xinping , Lazartigues Eric
Published: May 2022

Hypertension and obesity are considered risk factors for severe coronavirus disease 2019 (COVID‐19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). The aim of the current study was to examine whether hypertension and obesity alter SARS‐CoV‐2 infection, the development of the disease and possible underlying mechanisms, using the K18‐hACE2 transgenic mice.

Three groups of male K18‐hACE2 mice (5 mice/group, 5‐month‐old) were infected intranasally with SARS‐CoV‐2 (strain USA_WA1/2020, 104 PFU in 100 µl). Prior to infection, one group of mice was fed a hypercaloric diet (HCD, high fat high sucrose diet) for 3 months to model obesity; the second group of mice, on regular diet (RD), was infused with Angiotensin (Ang)‐II (600 ng/kg/min) for 3 weeks to model hypertension; and the third group of mice on RD served as control. Body weight was monitored daily post infection (dpi), and all mice were sacrificed at 7 dpi. RNA was extracted from kidney, heart, and lung, and RT‐qPCR was performed to measure tissue viral load and cytokine expression. Tissue damage was assessed on formalin‐fixed and paraffin‐embedded sections.

The three groups of mice suffered similar weight loss following infection (~15% by 7 dpi). Compared to control and hypertensive groups, in which the viral loads were similar across the tissues examined (lung, heart, and kidney), viral load in the heart of obese mice was significantly higher than in the lung and the kidney (Heart: 7.26 ±0.2 vs. Lung: 3.75 ±0.2 vs. Kidney: 2.48 ±0.4, expressed as Log10 of viral copy number per 100 ng RNA, p<0.05, two‐way ANOVA). In addition, compared to control mice, obese mice showed increased expression of CXCL1/KC in both kidney (HCD: 1.98 ±0.3 vs. RD: 0.74 ±0.1, p<0.05) and lung (HCD: 7.05 ±1.1 vs. RD: 2.39 ±0.3, p<0.05), and increased expression of IL‐17 in the lung (HCD: 1.44 ±0.1 vs. RD: 0.92 ±0.1, p<0.05). Furthermore, obese mice showed the highest expression of IL‐6 (HCD: 3.96 ±0.9 vs. Ang‐II: 1.1 ±0.1 vs. RD: 1.56 ±0.4, p<0.05) and TNFα (HCD: 14.08 ±0.6 vs. Ang‐II: 7.14 ±0.8 vs. RD: 6.03 ±1.13 mRNA expression level, p<0.05, one‐way ANOVA) in the lung among the three groups. Finally, the obese mice showed the highest lung infiltration (HCD: 15.6 ±1.4 vs. Ang‐II: 8.1 ±2.2 vs. RD: 6.38 ±1.3 infiltrated area/total area in %, p<0.05, one‐way ANOVA).

Our study suggests that obesity significantly increases tissue inflammation following SARS‐CoV‐2 infection, with the heart at particular risk of higher SARS‐CoV‐2 infection; in contrast, acute hypertension induced by Ang‐II infusion does not significantly alter SARS‐CoV‐2 infection or COVID‐19 disease course.
The Extracellular Chaperone Clusterin in Aβ and Non‐Aβ Cerebral Amyloidoses
Authors: Rostagno Agueda , Calero Miguel , Holton Janice , Revesz Tamas , Lashley Tammaryn , Ghiso Jorge
Published: May 2022

Clusterin, a multifunctional glycoprotein also known as apolipoprotein J, has been implicated in several physiological processes and is an important contributor to many pathological conditions, including Alzheimer’s disease (AD) in which genetic variants and plasma levels of the apolipoprotein were identified as risk factors for the disorder. To investigate whether clusterin exerted a more general role in the pathophysiology of cerebral amyloidoses, we turned to two unrelated hereditary conditions, familial British and Danish dementias (FBD and FDD), which share striking neuropathological similarities with AD, including neurofibrillary degeneration in limbic areas as well as parenchymal and vascular amyloid deposition. Two different 4 kDa molecules – ABri in FBD and ADan in FDD – totally unrelated to the 4 kDa Alzheimer’s Aβ, are the main constituents of the amyloid deposits in these forms of cerebral amyloidoses. Mirroring findings in AD, a panel of immunohistochemical studies demonstrated clusterin co‐localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri‐ and ADan‐binding plasma protein. Solid‐phase enzyme‐linked immunosorbent assays highlighted a specific saturable binding of clusterin to ABri and ADan. Non‐linear regression analysis of the data revealed Kd values within the same low nanomolar range as those demonstrated for the clusterin‐Aβ interaction. Consistent with its reported chaperone activity, clusterin showed a modulatory effect on ABri, ADan, and Aβ aggregation/fibrillization properties evaluated using thioflavin T, a dye that displays enhanced fluorescence upon binding fibrillar and protofibrillar amyloid conformations. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms – all known pathogenic features of protein folding disorders, including AD, FBD, and FDD – suggests the likelihood of a more complex role and a translational potential of clusterin in the amelioration/prevention of these pathogenic mechanisms.
Exosome‐Mediated Release of NLRP3 Inflammasome Products from Podocytes to Trigger Glomerular Inflammatory Response During Obesity
Authors: Li Guangbi , Huang Dandan , Zou Yao , Li Ningjun , Ritter Joseph , Li Pin‐Lan
Published: May 2022

The nucleotide‐binding oligomerization domain‐like receptor containing pyrin domain 3 (NLRP3) inflammasome products such as IL‐1β, IL‐18, and HMGB1 are critical factors to trigger glomerular inflammatory responses under different pathological conditions. The present study was designed to test a hypothesis that during obesity exosome release serves as the molecular mechanism mediating the release and action of NLRP3 inflammasome products from podocytes thereby leading to glomerular inflammation and sclerosis. By confocal microscopy and immunohistochemistry, NLRP3 inflammasome activation and associated T cell infiltration were found significantly enhanced in glomeruli of obese mice receiving high‐fat diet (HFD). Nanoparticle tracking analysis showed that podocyte‐derived exosomes containing NLRP3 inflammasome products remarkably increased in urine of these mice. By structured illumination microscopy, we observed the inhibition of lysosome‐MVB interaction by visfatin, a pro‐inflammatory adipokine, in podocytes isolated from mice on the normal diet, which was accompanied by enhanced exosome release. The HFD‐induced NLRP3 inflammasome activation and exosome release in mouse glomeruli and visfatin‐induced impairment of lysosome‐MVB interaction in primary cultures of podocytes were attenuated by Smpd1 gene knockout but amplified by podocyte‐specific Smpd1 gene overexpression (Smpd1 is the gene code of mouse acid sphingomyelinase). Correspondingly, HFD‐induced podocyte injury, glomerulosclerosis, and proteinuria were enhanced in mice with podocyte‐specific Smpd1 gene overexpression but reduced in Smpd1 gene knockout mice. Taken together, our results suggest that the release of exosomes containing NLRP3 inflammasome products from podocytes is critically implicated in the triggering of glomerular inflammatory response leading to glomerular sclerosis during obesity. The acid sphingomyelinase‐ceramide signaling pathway plays a key role in this exosome‐mediated release of NLRP3 inflammasome products.
Effects of Phosphorylation of ERK2 Kinase on the Properties of Active Site Amino Acids
Authors: Sun Joie , Trumble J. Max , Barnsley Kelly K. , Ondrechen Mary Jo
Published: May 2022

ERK2 is a mitogen‐activated protein kinase and is also called MAPK1. ERK2 is activated by phosphorylation at the side chains of two amino acids, Threonine 183 (T183) and Tyrosine 185 (Y185). Activation is accompanied by a conformational change. Upon activation, ERK2 enters the cell nucleus, where it carries signals involved in the regulation of cell proliferation, differentiation, and apoptosis. X‐ray crystal structures are available for both the phosphorylated (active) and unphosphorylated (inactive) conformations. Using computed electrical potential functions from numerical solutions to the Poisson‐Boltzmann equations and analysis with Partial Optimum Order Likelihood (POOL), electrostatic properties of the active site amino acids are calculated for both conformations. Changes in the proton transfer properties of active site amino acids D147, K149, and D165 are predicted. While structural and dynamic changes are known to be part of the activation of ERK2, the present results suggest that interactions with the phosphorylated amino acids also facilitate enzymatic activity. These electrostatic properties give insight into how phosphorylation controls the catalytic properties of ERK2.
Ribosomal quality control in repeat‐associated non‐AUG translation of GC rich repeats
Authors: Tseng Yi‐Ju , Todd Peter K.
Published: May 2022

Nucleotide repeat expansions cause multiple neurodegenerative disorders including C9orf72‐associated amyotrophic lateral sclerosis and frontotemporal dementia (C9 ALS/FTD) and Fragile X‐associated tremor/ataxia syndrome (FXTAS). C9 ALS/FTD results from a GGGGCC (G4C2) hexanucleotide repeat expansions within an intron of C9orf72while FXTAS is caused by CGG repeat expansions in the 5’UTR of FMR1. These repeat‐containing RNAs elicit toxicity at least in part by triggering repeat‐associated non‐AUG (RAN) translation, a non‐canonical initiation process that generates toxic proteins from GC rich repeats. As repetitive RNA elements form strong RNA secondary structures might impact translational elongation, we explored the impact of ribosome‐associated quality control (RQC) pathways on RAN translation. RQC rescues stalled ribosomes and prevents translation of aberrant transcripts, with specific roles for both the mammalian nuclear export mediator factor (NEMF, homologous of Rqc2 in yeast) and the E3 ubiquitin ligase Listerin. Here we show that depletion of NEMF markedly increases the production of RAN products from both G4C2 and CGG repeats. This effect appears to be mediated post transcriptionally and does not involve the nuclear‐cytoplasmic transport functions of the protein. Ongoing studies are characterizing how loss of Listerin impacts RAN translation and repeat RNA stability while exploring whether NEMF meditated CAT‐tailing of RAN translation products impacts their turnover and toxicity.
Macula densa cells control glomerular immune cell homing
Authors: Shroff Urvi Nikhil , Gyarmati Georgina , Izuhara Audrey , Deepak Sachin , Peti‐Peterdi Janos
Published: May 2022

Circulating and kidney resident immune cells preferentially home in and around the glomerulus compared to other vascular beds in both normal and inflammatory states, suggesting the presence of unique antimicrobial mechanisms in the glomerular microcirculation that protect the kidney filter. However, the underlying cell and molecular mechanisms are largely unknown. Macula densa (MD) cells are localized at the glomerular vascular entrance as a unique juxtaglomerular cell type and are known to control glomerular hemodynamics, and more recently, glomerular tissue remodeling. This study addressed the hypothesis that MD cells control glomerular immune cell homing by the release of paracrine‐acting pro‐inflammatory factors including opsonins, cytokines, and lymphokines. Bulk and single cell RNA sequencing and transcriptomic analysis of MD vs. control cells isolated from MD‐GFP mouse kidneys identified the high MD‐enrichment of several pathways and genes in the inflammatory response and cellular infiltration by leukocytes (Lcn2, Fga, Fgg, Casp4, Ptgs2, Ptges, Anxa1, Spns2, Spp1, Clu, Ccn1, Cxcl14, Mif). Mice with MD‐specific gain‐of‐function of mTOR (MD‐mTORgof) were developed by intercrossing nNOS/Cre and TSC2/fl mice, and feature globally increased MD cell protein synthesis. Immunoblotting of kidney cortex homogenates confirmed the significant increase in the expression of MD‐specific Ccn1, Cxcl14, Ptgs2, and Ptges in MD‐mTORgof vs. control mice. Intravital multiphoton imaging of the kidney cortex and endogenous circulating and kidney resident immune cell populations (labeled with anti‐CD44‐Alexa680 and F4/80‐Alexa488 antibodies) observed the significantly increased glomerular number, transit time, and migration of circulating CD44+ immune cells and tissue macrophages in MD‐mTORgof vs. control mice. NZM.2328 lupus mice featured significantly increased MD‐centric glomerular density and migration of CD8+ T cells. These results suggest the new role of MD cells and several MD‐derived paracrine‐acting molecular factors as important control mechanisms of glomerular inflammation in health and disease conditions. These newly identified MD mechanisms may be targeted in future anti‐inflammatory therapeutic strategies.
Increased Muscle Sympathetic Nerve Activity with Acute Hyperinsulinemia: Role of Insulin‐stimulated Peripheral Vasodilation and the Response of the Arterial Baroreflex
Authors: McMillan Neil J. , Soares Rogerio N. , Harper Jennifer L. , Shariffi Brian , Moreno‐Cabanas Alfonso , Curry Timothy B. , Manrique‐Acevedo Camila , Padilla Jaume , Limberg Jacqueline K.
Published: May 2022

Objective

Hyperinsulinemia elicits an increase sympathetic nervous system activity directed toward skeletal muscle (muscle sympathetic nerve activity, MSNA). The insulin‐mediated increase in MSNA is primarily attributed to its effect within the central nervous system. In addition to direct central effects, insulin also elicits peripheral vasodilation, which may further increase MSNA via arterial baroreflex‐mediated mechanisms in order to maintain blood pressure (BP). Herein we examined the contribution of insulin‐induced peripheral vasodilation and role for the arterial baroreflex in the MSNA response to hyperinsulinemia. We hypothesized that rescuing peripheral resistance with co‐infusion of the vasoconstrictor phenylephrine would attenuate the MSNA response to hyperinsulinemia. We further hypothesized the insulin‐mediated increase in MSNA would be recapitulated with another vasodilator (sodium nitroprusside, SNP).

Methods

In 33 healthy adults (28M/5F, 28±1 yrs, BMI 25.0±0.5 kg/m2) MSNA (microneurography) and BP (Finometer/brachial catheter) were measured, and total peripheral resistance (TPR, ModelFlow) was calculated at rest and during intravenous insulin (n=20) or SNP (n=13) infusion. A subset of participants receiving insulin (n=7) were co‐infused with phenylephrine. Spontaneous arterial baroreflex sensitivity was determined over 5‐min at baseline and during pharmacological intervention(s).

Results

Insulin infusion increased MSNA (p=0.05) and decreased TPR (p<0.05), with no effects on arterial baroreflex sensitivity or BP (p>0.05). Co‐infusion with phenylephrine returned MSNA and TPR to baseline, with no effect on baroreflex sensitivity (p>0.05). Similar to insulin, SNP increased MSNA (p<0.01) and decreased TPR (p<0.05), with no effect on baroreflex sensitivity (p>0.05).

Conclusions

Present findings reveal that acute hyperinsulinemia increases MSNA without a change in arterial baroreflex sensitivity in healthy men and women. Insulin‐mediated increases in MSNA are lost with co‐infusion of phenylephrine and can be recapitulated with infusion of SNP. Results support a dynamic interplay between central and peripheral mechanisms during hyperinsulinemia to increase MSNA and maintain BP.
The DNA repair endonuclease MutLalpha is activated by a novel mechanism that promotes action at a distance
Authors: Witte Scott J. , Rosa Isabella M. , Manhart Carol M.
Published: May 2022

Cells often arrange complex enzymatic reactions using organelles and phase separation. But for specific interactions with signal or enzymatic target sites, such as in DNA processing pathways, smaller but ordered high‐specificity structures can be used. We have only scratched the surface in identifying how protein structures signal across distances on DNA strands, but our work characterizing a new model suggests how signals can be relayed through protein complexes and illuminates the mechanism of an essential biological process of DNA mismatch repair. Using combinations of biochemistry, biophysics, and molecular biology, our work indicates that the DNA mismatch repair endonuclease MutLalpha is regulated by DNA topology to initiate DNA repair at a site distant from the DNA error. Failures in this process are linked to a hereditary cancer syndrome, Lynch Syndrome, that affects approximately one in every three hundred individuals. Our data aid in our understanding of DNA repair and how failures contribute to human cancers and genome instability. Our work also informs models for other pathways that require action at a distance as well as systems that use mismatch repair proteins. These include trinucleotide repeat expansions which are linked to human neurological disorders, recombination which is linking to genetic variation and stability, and somatic hypermutation which can utilize mismatches formed by activation‐induced cytosine deaminase to switch the class of immunoglobulins to adapt the immune system.
A Meta‐transcriptomic Analysis of Complicated Diverticulitis Tissue: The Role of Xenobiotics in the Gut
Authors: McMullen Brittney N. , Baker Samantha K.
Published: May 2022

Diverticulitis, a common inflammatory disease characterized by infected pouches in the intestinal wall, can become especially painful when complications, such as abscesses and fistulas, arise. Despite affecting over 58% of adults over the age of 60, not much is known how the gut microbiome may influence or change as a result. Herein we resected diseased and adjacent tissue from both complicated and uncomplicated diverticulitis patients, extracting DNA and RNA from all samples. 16S rRNA gene Illumina‐tag PCR was performed on all samples extracted, analysis revealing an abundance of sulfur oxidizing bacteria in complicated diseased tissue compared to their adjacent tissue counterpart. This discovery led us to perform microbial gene expression using shotgun meta‐transcriptomics analysis on these complicated diseased tissues and their adjacent counterpart tissue to confirm expression of sulfur oxidizing behavior. Instead, differential gene expression revealed a fascinating trend in the increased expression of microbial xenobiotic degradation pathways in diseased tissue. Specifically, an uptake in Cytochrome‐P450 and Glutathione S‐transferase was found. Detailing how these environmental toxin degradation enzymes could be activated or cause the excessive immune response notorious of complicated diverticulitis will have profound impacts of treatment of the disease, as well as our understanding of how xenobiotics affect human health.
NUT Carcinoma: Investigating Drug Synergy and the Production of Drug Resistance
Authors: Vaughan Carolyn
Published: May 2022

NUT Carcinoma (NC) is a rare cancer that results from the formation of an oncogenic fusion protein between a general bromodomain gene on chromosome 19 with NUT, which typically is only involved in genes for testes, on chromosome 15, causing high levels of undifferentiated cells to develop into malignant tumors. The average patient prognosis is between 6‐7 months with a 0% survival rate. In clinical applications, NC has a high chance of becoming resistant to treatments. Current clinical studies focus on the effects of single drugs on NC, yet there is more to be investigated for synergistic drug treatments. The purpose of this study is to investigate different drug combinations that could be of use clinically, as well as to create cell resistance in NC cell lines which will then be used to determine if resistant cells obtain collateral sensitivity or resistance to other chemotherapy drugs. Three separate cell lines (NC‐14, NC‐1015, and NC‐690) directly derived from NC patients (gifted from Dr. Christopher French, Brigham and Women’s Hospital, Boston) were evaluated for initial sensitivity to different chemotherapeutic drugs via GI50assays. Seven drugs were selected based on either clinical application in NC patients or use in cancers sharing similar mechanisms to NC including Mirdametinib, Molibresib, Venetoclax, Birabresib, ARV‐771, (+)‐JQ1, and Trametinib. Consistent results indicate that Mirdametinib, Trametinib, and Venetoclax do not appear to have an impact on any of the three NC cell lines. However, promising results for ARV‐771, (+)‐JQ1, and Birabresib for 1015 and 690 cell lines were obtained, with GI50values for both cells lines in the low nM range for all three drugs. Current, ongoing experiments include growing resistant 1015 and 690 cell lines to ARV‐771 and (+)‐JQ1, respectively, by initially dosing cells with GI10values from the collected data, as well as evaluating their sensitivity to combination treatments.
Cardiovascular and autonomic profiles in offspring of fructose fed parents: role of anti‐inflammatory cholinergic reflex and exercise training
Authors: da Silva Dias Danielle , dos Santos Camila P. , Araújo Amanda A. , Ferreira Maycon J. , Nascimento‐Filho Antonio V. , Bernardes Nathalia , Irigoyen Maria Cláudia , De Angelis Kátia
Published: May 2022

Aim

Considering that anti‐inflammatory cholinergic reflex has been implicated in cardiovascular disorder, and spleen seems to be a critical organ in this reflex, here we evaluated the impact of aerobic exercise training on cardiovascular and autonomic profiles in offspring of rats submitted to fructose overload, as well as, the role of the splenectomy in this condition.

Methods

Offspring of Wistar rats treated with fructose or placebo (water) were divided into 5 groups: Sedentary Control (OC), Sedentary Fructose (OF), Trained Fructose (OFT), Splenectomized Sedentary Fructose (OFE) and Splenectomized Trained Fructose (OFST). Fructose overload (10%) was started 30 days after weaning only for the parents and continued until the end of lactation. The offspring received standard feeding and were evaluated 30 days after weaning, at which time the spleen was removed (splenectomy) and the aerobic exercise training was realized on treadmill (1h/d, 5d/week, 4 weeks, moderate intensity). Blood pressure (BP) and heart rate (HR) signals were direct recorded, baroreflex sensitivity (BRS) was measured using increasing doses of phenylephrine and sodium nitroprusside. Oxidative stress was measured in cardiac tissue.

Results

Systolic BP, Diastolic BP and Mean BP increase in OF compared to OC, OFT, OFE and OFST groups (OC: 105±1.3, OF: 112±1.6, OFT: 104±2.4, OFE: 103±3.1, OFST: 104±2 mmHg) HR reduced in the OFT compared to the OC group. Regarding BRS, bradycardic and tachycardic responses were impaired in OF compared to OC group; and OFE, OFT and OFST groups increased these parameters compared to OF group. In the antioxidant profile in cardiac tissue, the activities of catalase and superoxide dismutase were reduced in the OF group compared to the OC group, and increased in the OFE, OFT and OFST groups compared to the OF group. Protein oxidation was increased in the OF group compared to the OC, OFE, OFT and OFST groups (OC: 5.37±0.15, OF: 6.41±0.21, OFT: 4.93±0.3, OFE: 4.94±0.23, OFST: 4.66±0.46 nmol/mg protein). The redox balance of glutathione (GSH/GSSG) was decreased in the OF group compared to the OC and OFT groups.

Conclusion

These results demonstrate that fructose exposure in parents induced early changes in offspring BP, BRS and cardiac oxidative stress, that seems to be mediated, at least in part, by the anti‐inflammatory cholinergic reflex. Moreover exercise training seems to prevent/attenuate such dysfunctions.
Tumor‐Suppressor Role of the Caveolar α1‐Na/K‐ATPase Signalosome in NASH related Hepatocellular Carcinoma
Authors: Udoh Utibe‐Abasi S. , Banerjee Moumita , Rajan Pradeep K. , Sanabria Juan D. , Smith Gary , Nakafuku Yuto , Sodhi Komal , Pierre Sandrine V. , Xie Zijian , Shapiro Joseph I. , Sanabria Juan R.
Published: May 2022

Background

Hepatocellular Carcinoma (HCC) is the second and fastest‐growing cause of cancer‐ related mortality worldwide. In the Western countries, due to the epidemic of obesity, non‐alcoholic steatohepatitis (NASH) has become the major cause of HCC. Intriguingly, the molecular mechanisms that underlie the progression of NASH to HCC is still unclear.

Hypothesis

We hypothesized that, the growing uncoupled metabolism during NASH progression to ESLD and HCC manifested by lower cell oxi‐redox status and an apoptotic ‘switch’ activity, follows a dysregulation in the wild type Caveolar α1‐Na/K‐ATPase (NKA)/Src signalosome, promoting an amplification of the pSrc → PI3K→ Akt pathway, which mediates survivin overexpression driving local carcinogenesis.

Methods

Expression of Cav‐1/Smac‐Diablo/Survivin proteins was performed by confocal‐microscopy on immuno‐stained HCC cell lines (Hep3B and SNU475), and livers in both NASH and NASH‐HCC rodent models, and in humans. Apoptotic activity was assayed via TUNEL. Tumor burden in rodents’ livers were evaluated using ImageJ software on H&E‐stained liver tissues and fibrosis via trichrome staining. Furthermore, signaling pathway studies were explored in‐vitro guided by RNA sequencing. Selective blockage of Src‐p at its kinase domain was performed by administration of a synthetic peptide (pNaKtide). Significant differences among groups were established at p<0.05 using ANOVA/Turkey’s post hoc test.

Results

Blockage of Src‐p at the α1‐NKA promoted apoptosis of Human HCC cell lines and tumors of disease murine models in a dose dependent manner (p< 0.01). pNaKtide at IC50 drove the downregulation and upregulation of Survivin and SMAC expressions (p<0.05), respectively in HCC cell lines. In‐vivo, liver tumor burden and fibrosis were significantly lower in animals treated with pNaKtide vs non‐treated animals (p<0.01). Furthermore, Cav‐1 and Survivin expressions were significantly higher, while SMAC protein expression was significantly lower in livers from rodents with NASH/HCC vs animals treated with pNaKtide (p<0.01). Similar pattern of proteins expressions was noted in tumors from patients with NASH±HCC vs liver tissue from healthy subjects or subjects with liver metastases (p<0.05). In‐vitro, Src‐p at the α1‐NKA activates PI3K/Akt pathway with concomitant inhibition of the FoxO cascade, favoring cell division and primary liver carcinogenesis.

Conclusions

Caveolar Src‐phosphorylation at the α1‐Na/K‐ATPase regulated Survivin/SMAC expressions, leading to cellular “switch” from apoptosis to cell division via the activation of the PI3K/Akt pro‐survival pathway with concomitant inhibition of the FoxO cascade. Therefore, point target blockage may be explored as a treatment for tumor regression in HCC.
Find out if your protein is O‐GlcNAc modified: The O‐GlcNAc database
Authors: Olivier‐Van Stichelen Stephanie , Malard Florian , Berendt Rex , Wulff‐Fuentes Eugenia , Danner Laura
Published: May 2022

Post‐translational modifications (PTMs) are ubiquitous and essential for protein function and signaling, motivating the need for sustainable benefit and open models of web databases. Highly conserved O‐GlcNAcylation is a case example of one of the most recently discovered PTMs, investigated by a growing community. Historically, details about O‐GlcNAcylated proteins and sites were dispersed across literature and in non‐O‐GlcNAc‐focused, rapidly outdated or now defunct web databases. In a first effort to fill the gap, we recently published a human O‐GlcNAcome catalog with a basic web interface. Based on the enthusiasm generated by this first resource, we extended our O‐GlcNAcome catalog to include data from 42 distinct organisms and released the O‐GlcNAc Database v1.2. In this version, more than 14 500 O‐GlcNAcylated proteins and 11 000 O‐GlcNAcylation sites are referenced from the curation of 2200 publications. Here, we present the extensive features of the O‐GlcNAc Database, including the user‐friendly interface, back‐end and client–server interactions. Finally, this database system can be administrated with little to no programming skills and is meant to be an example of a useful, sustainable and cost‐efficient resource, which exclusively relies on free open‐source software elements (www.oglcnac.mcw.edu).