bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ceramide Containing Microparticles from Aged Stored Platelets Recapitulate Aspects of Murine Transfusion Related Acute Lung Injury
Authors: McVey Mark John , Maishan Mazharul , Lee Warren L. , Spring Chris , Semple John W. , Kuebler Wolfgang M. 
Published: June 2021

BACKGROUNDTransfusion‐related acute lung injury (TRALI) presents as chest infiltrates and hypoxia after 0.02–1% of transfusions with an associated mortality of 5–15%. Aged platelet products elicit TRALI via a mechanism invovling formation of the sphingolipid ceramide (CER) by acid sphingomyelinase (ASM), which causes lung injury in vivo and impairs pulmonary endothelial cell (EC) barrier integrity in vitro. Concomitantly, aged platelets form abundant microparticles (PMPs) which can shuttle nucleic acids, proteins, and ‐ importantly ‐ lipids, to target cells.OBJECTIVETo test the hypothesis that aged (5 days) but not fresh (1 day) platelets will generate PMP that mediate TRALI in a CER‐dependent manner.METHODSPMPs from male C57BL/6 wild type (WT) or Smpd1−/− (ASM knockout) platelets stored for 1 (fresh) or 5 (aged) days duration were isolated by differential centrifugation and filtration before enumeration (flow cytometry) and CER analysis (mass spectroscopy). Cultured human microvascular pulmonary ECs (passage 6–8) were incubated with PBS (sham) or 5×105 fresh or aged PMPs for 4 hours and transendothelial electrical resistance (TEER) was measured every 30 minutes. For TRALI induction, BALB/c male mice primed with 2 mg/kg intraperitoneal LPS two hours prior to intravenous (IV) infusion of 10 ml/kg PMPs (≈ 5×106) or saline (sham) and were assessed (6 hours later) by measuring lung tissue wet‐to‐dry (W/D) ratios, bronchoalveolar lavage fluid protein (BAL‐p) and lung tissue myeloperoxidase activity (MPO).RESULTSCompared with fresh WT PMP, aged WT PMPs were more numerous and CER‐enriched. In contrast, aged ASM KO PMPs were less numerous compared with fresh ASM KO PMP but contained similar CER content as WT aged PMPs. Aged (CER rich) but not fresh (CER poor) PMPs lead to reductions in TEER (< 2 hours) in vitro in keeping with EC loss of barrier integrity which persisted for > 4 hours. Reductions in TEER seen over 4 hours with aged PMPs was prevented with pre‐treatment of WT PMPs with ceramidase (0.75 μg/mL) prior to incubation with ECs. LPS primed mice receiving IV aged WT PMPs exhibited increased W/D (p < 0.05), MPO and BAL‐p (p<0.05) relative to mice receiving LPS priming in addition to saline, fresh PMPs or ASM KO aged PMPs.CONCLUSIONSCeramide formation in aged platelets contributes to PMP formation and impairs human EC barrier integrity resulting in TRALI. Removal or modification of aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.Support or Funding InformationThis work was jointly funded by Canadian Blood Services and the Canadian Institute of Health Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Sodium/Hydrogen Exchanger 2 (Slc9a2/NHE2) is Involved in the Differentiation of Colonic Intestinal Epithelial Cells
Authors: Nikolovska Katerina , Li Cao , Yu Yan , Yuan Zhenglin , Seidler Anna , Kini Archana , Yeruva Sunil , Singh Anurag Kumar , Riederer Brigitte , Seidler Ursula 
Published: June 2021

BackgroundBoth sodium/hydrogen exchanger 2 and 3 (NHE2 and NHE3) isoforms of the Slc9 family Na+/H+ exchangers are expressed in the luminal membrane of the intestinal enterocytes, but in contrast to the deletion of NHE3, the deletion of NHE2 does not result in diarrhea, hyperaldosteronism, or reduced survival in mice.Aim and methodsTo further analyze the physiological role of NHE2 in the intestinal epithelial cells, we fluorometrically measured the intracellular pH (pHi) along the colonic cryptal axis of NHE2−/− mice, and in the intestinal epithelial Caco 2Bbe (C2Bbe) after stable lentiviral sh‐mediated NHE2 silencing. RNA and protein expression were assessed by qPCR, Western analysis and immunohistochemistry, and proliferative, migratory, and functional features of colonic cells in vivo and in vitro were assessed by enzymatic and functional assays.ResultsSteady‐state pHI was significantly decreased in C2Bbe/shNHE2 compared to empty‐vector transfected C2Bbe cells. In the colonic crypts, the pHi in the cryptal base was significantly lower than in the surface region, and the acidic zone along the crypt axis was significantly longer in colonic crypts from NHE2−/− mice. NHE2−/− colonic crypts were elongated with a much longer proliferation zone but with less proliferating cells per crypt area. Cell proliferation in C2Bbe/shNHE2 cells was reduced compared to empty‐vector transfected C2Bbe. The expression and activity of alkaline phosphatase (an enterocyte differentiation marker), was significantly reduced in NHE2−/− colonic mucosa and in C2Bbe/shNHE2 cells. However, the number of goblet cells and mucin 2 (Muc2) expression was increased in the NHE2−/− compared to the WT colon, accompanied by formation of a thicker mucus layer. The shift from absorptive to secretory differentiation program was accompanied by a decrease in Hes1 expression, as a downstream transcription factor of Notch signaling that supports the development of absorptive enterocytes.ConclusionsThe results suggest that NHE2 expression and/or function is activated when the intestinal cells emerge from the stem cell niche, and that this is essential for the establishment of the pHi gradient along the colonic crypt axis. Its activity facilitates enterocyte proliferation and differentiation along the crypt axis.Support or Funding InformationVolkswagen Foundation (VW‐Vorab), SFB621/C9, Se460/9‐4 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inducible intestinal epithelial cell‐specific NHE3 knockout causes diarrhea and more alkaline luminal content
Authors: Xue Jianxiang , Valdez Alexandria , Rieg Jessica Dominguez , Rieg Timo 
Published: June 2021

The Na+/H+ exchanger isoform 3 (NHE3) has an important role in intestinal and renal Na+ uptake, acid‐base regulation, and composition of the gut microbiota. Reduced intestinal NHE3 activity and/or expression were found in congenital Na+ diarrhea and inflammatory bowel disease. Previously, no viable intestinal epithelial cell‐specific NHE3 knockout model has been generated; whole body NHE3 knockout mice show 70% mortality and non‐inducible intestinal epithelial cell‐specific NHE3 knockout mice show 100% postnatal mortality. Therefore, we generated a tamoxifen‐inducible intestinal epithelial‐specific NHE3 knockout mouse (NHE3IEC‐Ko; see abstract #1519). Two weeks after tamoxifen administration, NHE3IEC‐Ko mice show ~2‐fold increase in intestinal permeability, 4 hours after oral gavage of FITC‐Dextran, compared to control mice (P<0.05). The small intestine, cecum, and colon were markedly dilated in NHE3IEC‐Ko versus control mice, which was associated with a more than 2‐fold increase in total intestinal wet weight (4.9±0.5 vs 2.2±0.1 g, P<0.05, n=4 and 8, respectively) and ~1.5‐fold increase in fecal water content (55±2 vs 81±4 %, P<0.05). Small intestinal and colonic flush pH were 0.5 pH and 0.8 pH units more alkaline, respectively, in NHE3IEC‐Ko compared to control mice (both P<0.05). Small intestinal length was not different between genotypes (36±3 vs 39±1 cm, NS), but colon length was increased in NHE3IEC‐Ko compared to control mice (8.8±0.3 vs 7.1±0.2 cm, P<0.05). Although there was not an increase in intestinal length, the proximal small intestine of NHE3IEC‐Ko mice had longer villi (493±4 vs 392±19 μm, P<0.05) but similar crypt depths compared to control mice (89±4 vs 79±3 μm, NS). No differences in villus length (159±11 vs 139±7 μm, NS) or crypt depth (58±2 vs 58±2 μm, NS) were observed in the distal small intestine between genotypes. In conclusion, this novel intestinal epithelial cell‐specific NHE3 knockout model is a useful tool for studying inflammatory bowel disease, and the role of intestinal pH on the microbiome as well as intestinal proliferation and apoptosis.Support or Funding InformationNIH 1R01DK110621, APS STRIDE Undergraduate Summer Research Fellowship 1R25HL115473‐01.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Silencing of NHE2 Enhances Migratory Speed in Colonic Epithelial Cells
Authors: Nikolovska Katerina , Cao Li , Yu Yan , Yuan Zhenglin , Seidler Anna , Yeruva Sunil , Singh Anurag Kumar , Riederer Brigitte , Seidler Ursula 
Published: June 2021

BackgroundThe maintenance of epithelial function and barrier integrity is achieved by continuous renewal of the colonic epithelium through proliferation, migration and differentiation. Sodium hydrogen exchanger 2 (NHE2) is highly expressed in the colonic epithelium, where it is involved in Na+/H+ exchange, water absorption and pHi regulation. However, NHE2 deficiency in mice does not result in diarrheal phenotype. NHE2 is expressed in the cryptal region, where colonocytes exit the stem cell niche and migrate toward the surface.Aim and methodsTo study the role of NHE2 in colonocyte migration, we silenced NHE2 in the self‐differentiating Caco 2Bbe (C2Bbe) colonic cell line and studied the migration of cells using wound scratch assay. To analyze colonocyte migration during self‐renewal, NHE2−/− and WT mice were pulse‐labeled with bromodeoxyuridine (BrdU) and sacrificed at different time points. Sections taken from identical colonic segments were studied immunohistochemically.ResultsThe rate of colonocyte migration, defined by the occurrence of BrdU‐positive cells along the crypt‐villus axis was significantly higher in the colon of NHE2−/− mice compared to the control littermates. This was associated with a significant reduction in E‐cadherin and ZO‐1 expression in the basal parts of the crypts, pointing to alterations of the adhesion and tight junction formation of the colonocytes during the early stages of differentiation. Additionally, increased mRNA expression of β‐catenin was detected in isolated NHE2−/− colonic crypts compared to the control. To substantiate these findings, C2Bbe cells were stably transfected with shRNA, generating a cell line with ~70 % downregulated expression of NHE2. A significant increase of the migration rate, but reduced cell proliferation was observed in the NHE2 knock‐down compared to the mock transfected cells. This was accompanied with a significant decrease of E‐cadherin, but enhanced β‐catenin expression.ConclusionsOur results show that NHE2 is involved in the processes of colonocyte proliferation and migration along the crypt axis. Alterations in the cell adhesion formation, the Wnt/β‐catenin signaling pathway and disturbed formation of the E‐cadherin/β‐catenin complex, possibly induced by a lower pHi in NHE2‐deficient colonocytes, may be responsible for the increase in colonocyte migratory speed.Support or Funding InformationVolkswagen Foundation (VW‐Vorab), SFB621/C9, Se460/9‐4 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ACID SENSING ION CHANNEL‐2 LOCALIZES WITH α‐ AND γ‐, BUT NOT β‐ENAC SUBUNITS IN CILIATED AIRWAYS
Authors: Flores‐Delgado Guillermo , Quinton Paul M. 
Published: June 2021

The epithelial sodium channel (ENaC) and Acid‐sensing ion channels (ASIC) are members of a superfamily of channels that play a significant role in transepithelial cation transport. The protein structure of the subunits that form these channels share great similarity, with two transmembrane segments (TM1 and TM2) and a large extracellular component. ENaC subunits (α, β, γ and δ) have been identified in airways. However, the stoichiometry of the subunits that composed the ENaC channel remains elusive. ASIC2 expression has been reported in ciliated cells of trachea and nasal septum. Previous studies suggested that ENaC subunits and ASIC may form a functional hybrid of non‐selective cation channels (1–4). Herein, we hypothesized that ASIC2 is expressed in porcine airways and Na+ transport can be modulated by hybrid channels composed of ASIC2 and ENaC subunits. We obtained small pieces of peripheral porcine lung tissue of approximately 0.5 cm3 and pieces of trachea of approximately 0.5 cm2 that were dissected and immediately snap‐frozen at −80°C. Serial tissue sections (7 μm thick) containing cross‐sections of small airways (diameters 0.5 – 1 mm) were used for immunohistochemistry. We used polyclonal antibodies directed to ASIC2, α‐, β‐ and γ‐subunits of ENaC as well as monoclonal antibodies directed to β‐Tubulin and ZO1, the tight junction protein. Confocal microscopic analysis demonstrated that in distal airways ASIC2 and ENaC subunits were mainly localized in airway ciliated cells. ASIC2 was found distributed at the base of the multicilia, with a distribution similar to that observed for α‐ and γ‐ENaC subunits. In contrast, a strong signal for β‐ENaC subunit was observed along the cilia in a restricted group of airway ciliated cells. In proximal airways, ASIC2 signal was also identified at the base of the multicilia of ciliated cells, similar to the distribution of α‐ and γ‐ENaC subunits. Our data suggest that ASIC2 could combine with α‐ and γ‐ENaC subunits to form hybrids of non‐selective cation channels as previously indicated (1–4), and participate in mucociliary clearance, regulation of airway surface liquid volume and composition.Support or Funding InformationSupported by the Cystic Fibrosis Research Inc., and The Nancy Olmsted Trust for Pediatric Pulmonology.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Knockout of Kcnj16 (Kir5.1) in Dahl Salt‐Sensitive Rats Produces Seizure Phenotype
Authors: Manis Anna D. , Hodges Matthew R. , Pavlov Tengis S. , Staruschenko Alexander , Palygin Oleg 
Published: June 2021

Kir5.1 is a member of an inwardly rectifying potassium (Kir) channel family notably present in the kidney and brain. We previously established that a knockout rat model of Kcnj16 (gene encoding Kir5.1) on a Dahl salt‐sensitive background (SSKcnj16−/−) exhibits a severe cardiovascular phenotype (Palygin et al., JCI Insight, 2017). Mutations in ion channel genes can alter neuronal excitability and mutations in Kir genes have been linked to seizure disorders in humans. Since Kir5.1 is known to be expressed in the brain and may contribute to neuronal membrane potential and spatial potassium buffering, we hypothesized that in addition to cardiovascular and renal dysfunction, neurological phenotypes, including seizures, would be prevalent in the SSKcnj16−/−rats. We found that SSKcnj16−/− rats (but not control SS rats) experience tonic‐clonic seizures when exposed to a 10 kHz tone (86 dB for 2 min), whereas other frequencies (0.1 or 1 kHz) did not elicit seizures. When exposed to the seizure‐inducing acoustic stimulus once/day for 10 days, we noted that SSKcnj16−/− rats experienced a seizure in response to 92% of stimuli. SSKcnj16−/− rats also showed spontaneous mortality within hours after a stimulus with a survival rate of 67% (N=21) for the duration of the 10 day protocol. Control rats (N=10) had no incidence of seizure or death during the 10 days of acoustic stimulation. We found no difference among male and female SSKcnj16−/− rats, where both sexes had a similar audiogenic seizure response and reduced survival. Behavioral tests including a modified Irwin screen and open field test revealed that before and after seizure stimulation SSKcnj16−/− rats tended to have altered activity levels, gait, piloerection, and respiration rates. Analysis of blood electrolytes revealed critically low serum potassium levels in SSKcnj16−/− compared to SS controls, a difference that appeared to be exacerbated by repeated seizures. We hypothesize that seizures may be intensifying potassium imbalance and disrupting the homeostasis that is particularly essential for excitable cells such as neurons. We conclude that knockout of Kir5.1 leads to distinct neurological phenotypes including a seizure disorder and subsequent spontaneous death, which may have relevance to channelopathies associated with seizure disorders in humans.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Slc4a11 Facilitates Glutamine Metabolism, Mitochondrial Function and ROS Prevention
Authors: Ogando Diego Gabriel , Choi Moon‐Jung , Kim Edward Taeyoon , Zhang Wenlin , Li Shimin , Bonanno Joseph Aurelio 
Published: June 2021

PurposeSLC4A11 codes for an electrogenic NH3‐sensitive H+ transporter. Slc4a11−/− mice show significant oxidative damage within corneal endothelial cells and an alteration of glutamine metabolism. Here, we asked if NH3 derived from glutamine (Gln) catabolism induces oxidative stress and whether Slc4a11 is protective.MethodsCorneal endothelial tissue from Slc4a11−/− mice was evaluated for oxidative stress (MitoSox, protein nitrosylation and OHdGuanine), mitochondrial morphology (MitoTracker Green) and autophagy (LC3B). Conditionally immortalized cell lines of Slc4a11−/− and WT mouse corneal endothelial cells (MCEC) expressing temperature sensitive Large T antigen were generated. In MCEC, cell death was quantified by AnV‐FITC + PI, mitochondrial anion superoxide (O2−) by MitoSox, mitochondrial membrane potential (MMP) measured by TMRE, and ATP was determined by luciferin‐luciferase assay. NH3 production was measured by colorimetric assay. Relative glucose and glutamine flux was quantified by GC‐MS.ResultsKO corneal endothelium from 12 and 40 week old mice show greater production of mitochondrial O2−, oxidized DNA and protein, induction of autophagy, and alteration of mitochondrial morphology compared to WT. In WT MCEC, Gln (0.5 mM) increases [ATP] concomitant with increased mitochondrial O2−, and increased apoptosis (compared to Gluc only). In contrast, in KO MCEC Gln decreases [ATP], with significantly greater mitochondrial O2− and apoptosis (compared to WT) that can be rescued by MitoQ. Application of NH3 alone increased mitochondrial O2−, depolarized MMP and increased apoptosis significantly more in KO. Gln derived NH3 toxicity can be partially reduced by GLS1 inhibitors BPTES or CB839 and totally inhibited by supplementation with Dimethyl‐α‐Ketoglutarate (α‐KG) or by Cyclosporin A (inhibitor of the opening of the mitochondrial transition pore). In MCEC stained with TMRE, 2 population of were observed: a population of smaller cells with depolarized MMP (TMRE− cells) and a population of normal size cells with hyperpolarized MMP (TMRE+ cells). Even though Gln increases the percentage of TMRE− cells the remainder TMRE+ cells are hyperpolarized as expected as Gln fuels the TCA cycle. This hyperpolarization is much higher in KO and suggest that it is the cause of O2− production followed by PTP opening and then secondary MMP depolarization. Gln induced hyperpolarization and NH3 derived Gln are the cause of high O2− production in KO cells.ConclusionWe conclude that Slc4a11 protects cells from Gln and NH3 induced toxicity.Support or Funding InformationNIH RO1 EY008834This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Potassium Chloride Cotransporter‐3 (KCC3) in Motor Neurons
Authors: Schornak Cara C. , Delpire Eric 
Published: June 2021

The K‐Cl cotransporter KCC3 is an electroneutral membrane transporter of the SLC12A gene family. It uses the K+ gradient established by the Na+/K+ ATPase to efflux K+, Cl−, and obligatory water from cells. KCC3 loss‐of‐function (LOF) mutations have been found to cause the disease Agenesis of the Corpus Callosum with Peripheral Neuropathy (ACCPN). ACCPN patients suffer from intellectual disability, dysmorphic features, hallucinations, degrading ambulation, and a severe, progressive sensorimotor neuropathy. Previous literature has confirmed that both global KCC3 knock‐out mice and neuron‐specific knock‐out (KO) mice recapitulate the severe sensorimotor deficits seen in patients. Furthermore, mice with KCC3 deleted in a subset of sensory neurons (parvalbumin‐positive proprioceptive neurons) also show a locomotor deficit. Thus, KCC3 dysfunction in sensory neurons may be a key to ACCPN pathogenesis; however, it is unknown if KCC3 dysfunction in sensory neurons could account for the entire range of patient symptoms, that is, both sensory and motor neuropathy. From the spinal cord, motor neurons extend their axons to the periphery where they synapse onto skeletal muscles. Reciprocally, sensory neurons, specifically proprioceptive neurons, bring information from muscles back to the spinal cord, where they synapse onto motor neurons and interneurons. Therefore, KCC3 LOF directly in motor neurons, in sensory neurons, or in both types of neurons could be responsible for the ACCPN phenotype.To investigate the participation of motor neurons, we used a mouse line that expresses the CRE recombinase under the control of the HB9 promoter. HB9 is a transcription factor expressed during peak motor neuron development at mouse embryonic days E9.5–10.5. We crossed HB9‐CRE mice with a KCC3‐floxed line to generate mice with KCC3 deleted in motor neurons (HB9‐CRE x KCC3‐flox). We then subjected cohorts of HB9‐positive KCC3‐flox mice versus HB9‐negative KCC3‐flox mice to locomotor tests. The HB9‐positive KCC3‐flox mice did not show any deficits in the accelerated rotarod test, which assesses balance and overall locomotion performance, nor in the elevated balance beam task, which assesses finer movement coordination. Absence of a phenotype indicates that targeting KCC3 deletion in motor neurons does not contribute to the development of neuropathy. These data suggest that the motor phenotype observed in ACCPN patients might be secondary to the sensory deficit. Such an indirect effect is possible as motor neurons need proper sensory feedback from muscles to appropriately coordinate posture and movement.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.