bluuebunny/crossref_metadata_2025_split huggingface dataset:
MK2 Phosphorylates Caspase‐3, Facilitates Nuclear Translocation of Caspase 3, and Regulates Apoptosis
Authors: Del Rosario Othello Lance , Suresh Karthik , Shimoda Larissa , Rane Madhavi , Damarla Mahendra 
Published: May 2022

BackgroundApoptosis is a necessary and key pathologic feature in the development of acute lung injury (ALI). Previous research has identified MAPK Activated Protein Kinase 2 (MK2) as a potential regulator in the development of apoptosis, by facilitating the nuclear translocation of active caspase‐3, the executioner of apoptosis. Specifically, loss of MK2 results in cytoplasmic sequestration of active caspase‐3. Since MK2 is a kinase, we hypothesize that MK2 phosphorylates caspase‐3 leading to its nuclear translocation and ultimately executing apoptosis.MethodsCaspase‐3 phosphorylation was determined by 2‐D gel electrophoresis with and without phosphatases, in addition to direct protein kinase assays using radiolabelled phosphate. Caspase‐3 localization was determined by performing nuclear and cytoplasmic extractions, and subsequent measurement of caspase‐3 activity. Apoptosis was determined by flow cytometry. NCI‐H23 (non‐small cell cancer, NSLC) cells were used as a model due to their ability to resist apoptosis, as well as their ability to sequester active‐caspase‐3 in the cytoplasm.Results2‐D gel electrophoresis showed that the p‐19 fragment of cleaved caspase‐3, the fragment that translocates to the nucleus, has a shift in isoelectric focus that is absent with the loss of MK2. In addition, 2‐D gel analyses of lung homogenates from WT mice exposed to LPS and treated with phosphatase have similar isoelectric foci for caspase 3 as lung homogenates from MK2‐/‐ mice exposed to LPS. Furthermore, phosphor‐imaging shows that MK2 directly phosphorylates caspase‐3. Interestingly, immunoblots of NCI‐H23 cells (which sequester active‐caspase‐3 in the cytoplasm) have lower MK2 expression than the small‐cell lung cancer cell line, H446. Flow cytometry with DAPI staining demonstrated increased apoptosis following etoposide treatment in cells over‐expressing MK2 compared to control transfections. Over‐expression of MK2 led to an increase in nuclear caspase‐3 activity. Additionally, overexpression of nuclear localization sequence (NLS) tagged caspase 3 led to increased etoposide induced apoptosis compared to WT caspase 3 or control transfections.ConclusionCombined these results strongly suggest that MK2 phosphorylates caspase‐3, which may facilitate the nuclear translocation of caspase‐3, thereby promoting the execution of apoptosis. Further work is needed to understand the mechanisms by which MK2 regulates the trafficking of active caspase‐3.
Doxorubicin Induces Necroptosis in Young Mice
Authors: Imaralu Omonzejie , Singla Dinender 
Published: May 2022

Despite its potency as a chemotherapeutic drug, the prevalence of cardiotoxicity resulting from doxorubicin treatment in childhood survivors of cancer is a major concern. While the occurrence of apoptosis has been well ascertained in doxorubicin induced cardiotoxicity (DIC) by several studies; however, the presence of necroptosis in young DIC remains unknown. We hypothesize that doxorubicin will induce necroptotic cell death and adverse cardiac remodeling in young mice. Therefore, in this study we investigated the possible occurrence of necroptotic cell death in DIC in young mice in‐vivo to delineate underlying pathological mechanism.C57BL6 mice (6 + 2 weeks of age) were divided into two groups: Control (mice were given 0.9% saline) and Dox (drug was administered at 2.5mg/kg body weight (BW) for six i.p injection, with a cumulative dose of 15mg/kg BW). At day 14, heart function was examined using echocardiography, mice were euthanized. Heart tissues were collected and analyzed for necroptosis, adverse cardiac remodeling using immunohistochemistry, and histology. Upon analysis, we observed that Dox treatment significantly (p<0.05) increased the expression of necroptotic markers [Receptor interacting Serine/Threonine kinase 1 and 3 (RIPK1, RIPK3) and Mixed Lineage kinase domain‐Like (MLKL)] as confirmed by immunohistochemistry. Furthermore, histological analysis showed adverse cardiac remodeling with significant increase (p<0.05) in cardiac hypertrophy and fibrosis in Dox group compared to control. Moreover, a significant decrease (p<0.05) in heart function was observed in Dox treated animals compared to control. In conclusion the results of our study suggest that doxorubicin, induces necroptotic cell death which further leads to adverse cardiac remodeling in Dox treated young mice.
Metabolic effect of orchidectomy on C57BL6/J mice exposed to intermittent hypoxia
Authors: Ganouna‐Cohen Gauthier , Blachot Minassian Britanny , Marcouiller François , Joseph Vincent 
Published: May 2022

IntroductionSleep apnea (SA) is characterized by obstructions of the airways or cessation of breathing during sleep leading to intermittent hypoxia (IH). IH is a source of oxidative stress and inflammation altering the functions of pancreas, liver, muscles and adipose tissue. As a consequence, sleep apnea patients have a high prevalence of insulin resistance and glucose intolerance which can lead to type 2 diabetes. In mice, exposures to IH induce adipose tissue inflammation, liver injury and worsen the pancreatic β‐cell injury in a model of diabetes. While these effects are associated with insulin resistance, glucose tolerance is improved, notably via an enhanced glucose uptake by skeletal muscles. In men, sleep apnea and diabetes are also characterized by low testosterone levels. Interestingly, testosterone levels are positively corelated with insulin sensitivity in human, and low testosterone levels can be associated with inflammation, oxidative stress and insulin resistance. Therefore, we tested the hypothesis that testosterone modulates the metabolic alterations induced by IH in male mice.MethodWe used intact (Sham) or orchidectomized (ORX) C57BL/6J male mice exposed for 14 days to IH (12h / day, 10 cycles / h, 6% nadir oxygen) or normoxia (Nx). The animals were weighed, and we assessed body composition in terms of lean and fat mass, as well as fluid content by using a small animal MRI scanner before the surgery, before IH or Nx exposures and at the end of the protocol. The day after the last IH session, mice were fasted for 6h, we measured fasting glycaemia and insulinemia then we performed a glucose tolerance test.ResultsIn Sham mice, IH decreased bodyweight and fat mass. This was associated with a decrease of fasting glycaemia, insulinemia and HOMA‐IR (index of insulin resistance), and improved glucose sensitivity. ORX decreased bodyweight and lean tissue mass, associated with lower fasting glycemia, insulinemia, HOMA‐IR and improved whole body glucose sensitivity. ORX mice exposed to IH had a decrease of both fat and lean tissue mass leading to further reduction of body weight. However, there was no additive effects of ORX and ORX IH for fasting glycaemia, insulinemia, HOMA‐IR and glucose sensitivity.ConclusionIH and ORX respectively reduce fat and lean tissue mass, and these effects are additive when both stimuli are applied simultaneously. Contrastingly, there are no additive effects of IH and ORX on the metabolic parameters reported here.
Changes in oxygen and carbon dioxide in umbilical cord arterial blood before and after the first breath of a newborn to explore the mechanisms of respiratory regulation
Authors: Shi Chao , Sun Xingguo 
Published: May 2022

The fetus has no actual respiration, and the newborn begins to breathe after birth. We assume that the first breath dominantly generated by hypoxia. In this study, the changes and lowest limit of blood oxygen partial pressure of umbilical artery (PuaO2) after chemoreceptor were analyzed to explore the mechanism of neonatal spontaneous breathing.MethodsWith signed consent form by all fetal parents before birth, 14 newborns successfully completed the umbilical artery or vein catheterization and drawn blood according to the heartbeat. All blood samples analyzed by blood gas analyzer, calculated and analyzed the similarities and differences between umbilical vein(Puv) and umbilical artery(Pua).ResultsAlthough we completed 14 newborns, there were only 9 cases of umbilical artery samples and 8 cases of umbilical vein samples were collected. Only 3 cases collected both Pua and Puv blood samples at the same tim (see serial paper II). PuaO2 in gradually decreased with time (heartbeat frequency), until Pua contracted after spontaneous breathing produced about 8‐10 heartbeats, and then could not get enough blood samples. Only 3 newborns were able to take blood samples after spontaneous breathing for 8‐10 heartbeats, and PuaO2 jumped to 93.8,137.0 and 93.8 respectively The mean value of PuaO2 was 25.94±6.79 (range 18.04‐37.55)mmHg, the highest value was 32.41±11.97 (range 23.0‐61.0) mmHg, and the lowest value was 22.18±6.35 (range 14.0‐33.6)mmHg. Although PuvO2 decreased gradually with time (heartbeat) too, most of them also showed the tendency of alternately rising and falling with the regularity of mother’s respiration. The mean value of PuvO2 was 50.35±21.35 (range 32.56‐100.73)mmHg, the highest value was 90.34±8.44 (range 43.4‐153.0)mmHg, and the lowest value was 36.96±14.90 (range 24.8‐73.8)mmHg, although.ConclusionPuaO2 decreases gradually with time before spontaneous breathing after the delivered fetus as a newborn, and it induces the first inhalation to start spontaneous breathing when it reaches the threshold of triggering breathing.
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.
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.
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

BackgroundObstructive 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.MethodsWe 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.ResultsEpinephrine 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.ConclusionsThe 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.
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.
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

BackgroundHypertension 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.MethodsPrimary 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.ResultsA 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).ConclusionThe 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.