bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Recurrent Hypoglycaemia on the Activation of Insulin‐Responsive Medullary and Spinal Neurons Controlling Adrenaline Release
Authors: Llewellyn‐Smith Ida J. , Senthilkumaran Manjula , Verberne Anthony J. M. , Bobrovskaya Larisa
Published: June 2021

Recurrent episodes of low glucose availability (hypoglycaemia) lead to diminished adrenaline responses to subsequent bouts of hypoglycaemia, a condition known as hypoglycaemia‐associated autonomic failure (HAAF). To determine if any alteration in the activation of insulin‐responsive adrenergic neurons of the medulla oblongata and/or sympathetic preganglionic neurons (SPN) and/or adrenergic chromaffin cells of the adrenal medulla contribute to HAAF, we gave three groups of rats intraperitoneal injections of insulin (10 IU/kg, HAAF) or saline (Control) on 3 consecutive days or saline for 2 consecutive days and insulin on day 3 (Acute). Rats were fasted overnight prior to the last injection. Tail vein blood glucose was measured before injection and at 30 min on all days, and at 120 min on days 1 and 2 and at 90 min on day 3 at which time the rats were perfused with 4% formaldehyde. Coronal sections of perfused medulla (1:4 series) from the spinomedullary junction to mid‐facial nucleus, longitudinal sections of the spinal cord (1:2 series) containing segments T5–T8, T9–T11 and T12‐L2 and sections of adrenal gland (1:4 series) were immunoperoxidase stained to show Fos‐immunoreactivity (IR) with a black reaction product plus phenylethanolamine N‐methyltransferase (PNMT) or choline acetyltransferase (ChAT)‐immunoreactivity with a brown reaction product. At 90 min on day 3, blood glucose was significantly lowered from 5.5 ± 0.2 mmol/L and 5.9 ± 0.1 mmol/L to 1.4 ± 0.1 mmol/L and 1.3 ± 0.1 mmol/L (mean ± SEM, n=6–12) in the acute and HAAF groups, respectively. In the adrenal gland, nearly all PNMT‐IR chromaffin cells had Fos‐IR nuclei in both acute and HAAF groups. In response to both single and recurrent hypoglycaemia, many of the ChAT‐IR SPN in the intermediolateral cell column (IML) of thoracic segment T9–T10 had Fos‐IR nuclei. In HAAF rats that had recurrent hypoglycaemia, an average of 52.8 ± 4 PNMT‐IR C1 neurons of the RVLM had Fos‐IR nuclei which was not statistically different from rats that had single hypoglycaemia, in which 55 ± 6.2 PNMT‐IR C1 neurons in the RVLM had Fos‐IR nuclei. The proportion of Fos‐IR C2, C3 and vagal motor neurons did not appear to be different between the rats that had single hypoglycaemia and rats that had recurrent hypoglycaemia. These findings indicate that the activation of the main components of the medullo‐adrenal circuitry that control adrenaline release, including brainstem adrenergic neurons, SPN and adrenergic chromaffin cells, is unaffected by two episodes of antecedent hypoglycaemia. Thus, the primary reason for the defective adrenaline response in our model most likely lies within the adrenal gland.

Support or Funding Information

Diabetes Australia Research Trust

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Placental Ischemia leads to Postpartum Cerebral Inflammation and Edema in Rats
Authors: Clayton Ahsia M. , Shao Qingmei , Paauw Nina D. , Granger Joey P. , Warrington Junie Paula
Published: June 2021

Reduced utero‐placental perfusion results in placental ischemia, an initiating event in the pathophysiology of preeclampsia, a hypertensive disorder of pregnancy. There is evidence that women with a history of preeclampsia have increased risk of mortality from Alzheimer's disease, stroke, and cerebrovascular complications years after delivery; however, the underlying mechanisms are not known. Additionally, pre‐clinical studies demonstrate that during pregnancy, placental ischemia, induced by reducing uterine perfusion pressure (RUPP), leads to cerebral edema and increased blood‐brain barrier (BBB) permeability; however, it is not known whether these complications persist after delivery. In this study, we tested the hypothesis that placental ischemia contributes to cerebrovascular abnormalities postpartum. On gestational day 14, time‐pregnant Sprague Dawley rats underwent sham or RUPP surgery (n = 5 per group) and brains were collected 2 months postpartum. Cerebral edema, assessed by measuring water content, was observed in the posterior cortex following RUPP with no significant changes in the anterior cerebrum, hippocampus, or striatum. Using a rat cytokine/chemokine multi‐plex kit, we found a shift towards a pro‐inflammatory environment in the posterior cortex with increases in IL‐17, IL‐1α, and IL‐1β. In the hippocampus, where brain water content was unchanged, IL‐4 increased and IL‐18 decreased following RUPP. Western blot analysis showed no changes in the astrocyte marker, Glial Fibrillary Acidic Protein (GFAP); however, the microglia marker, ionized calcium binding adaptor molecule (Iba1) tended to increase in the hippocampus of RUPP‐exposed rats (p=0.05). There was no change in hippocampal or posterior cortical expression of the blood‐brain barrier associated proteins, Claudin‐1, zonular occludens (ZO‐1), or Aquaporin‐4 (AQP4); however, posterior cortical occludin was significantly reduced in RUPP‐exposed rats. These results suggest that at 2 months postpartum, a pro‐inflammatory environment in the posterior cortex is associated with tissue edema. Our results support the hypothesis that placental ischemia leads to sustained inflammation and reduced blood‐brain barrier integrity that predispose exposed moms to edema in brain regions important for learning and memory. Whether these findings are exacerbated with increased time after delivery is an area of active investigation.

Support or Funding Information

NIH K99/R00HL129192, COBRE Pilot Grant, P20GM104357, P01HL051971, R25HL121042

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 Effect of Flutamide on Spatial Working Memory and Serum Estrogen Levels in Female Rats
Authors: Pletsch James D. , Hershkowitz Richie , Mueller‐Luckey Georgia , Niepoetter Paige , Sanchez Alejandra , Rahn Hailey , Gopalan Chaya
Published: June 2021

In the absence of testosterone, the brain, by default, differentiates into a female brain. Sex differentiation of the rat brain occurs during the perinatal period (four days prior to and four days after birth) during which time both estrogen and androgen receptors are expressed in the female brain. However, the role of androgen receptors in the differentiation of the female brain during the perinatal period is unknown. Our purpose of this study was to examine the effects of blockade of androgen receptors on circulating estrogen levels and spatial working memory. Four timed‐pregnant Long Evans rats were divided into two groups: one group (N=2) received flutamide during perinatal period whereas the other group (N=2) served as control receiving vehicle (95% Olive oil‐5% ethanol). Female pups from the flutamide group (N=8) continued to receive flutamide whereas the pups from the control group received vehicle for the additional four days (N=15). Prior to performing Radial Arm Maze test to assess their spatial working memory, adult animals were placed on a 60 % food restriction to bring their body weights close to 85% of their original weight. spatial working memory testing involved a three‐day habituation, three‐day training, and one testing day. During the habituation period, all eight arms of the Radial Arm Maze contained food. During the training and the testing periods, however, four of the eight arms contained food and the other four did not. Any‐maze software linked to a video‐tracking device tracked the amount of time each animal spent in the arms with or without food. Flutamide‐treated animals spent more time in the arms with food compared to the control group suggesting greater choice accuracy or modestly improved spatial working memory (p= 0.06). At the end of the behavioral study, animals were sacrificed and trunk blood was collected to measure serum estrogen levels using ELISA. Flutamide‐treated animals showed an increase in serum estrogen levels (p<0.05) implying a relationship between improved spatial working memory and increased levels of estrogen. Thus, this study suggests that the blockade of androgen receptors during perinatal period increases serum estrogen levels and marginally improves spatial working memory in female rats.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Measurement of Maximum Tongue Protrusion Force (MTPF) In Healthy Normal Adults
Authors: KIM JA YOUNG , Hegland Karen , Vann William , Davenport Paul W.
Published: June 2021

Genioglossus (GG) is the primary lingual muscle responsible for tongue protrusion, which is important for speech, swallow and breathing. Contraction of the GG plays an important role in maintaining upper airway patency, preventing posterior tongue displacement and upper airway closure. GG electromyogram studies report GG tongue muscle activity increases with increased negative pressure in the upper airway. We propose that tongue protrusive strength and tone may be related to upper airway patency, and when protrusive strength is reduced, individuals are at higher risk of developing sleep apnea, or speech/swallow disorders. However, in order to test that hypothesis, it is necessary to determine a normal range for tongue protrusion strength. We designed a device to indirectly measure GG strength by measuring tongue protrusion force. The device is a piston in a cylinder with a force transducer at the base of the piston. The cylinder is held by the front teeth, and the tongue extends 0.5–1 cm beyond the teeth to touch the piston. The goal of the current study was to determine normative values of maximum tongue protrusion force (MTPF) in healthy young adults, using the new device. We hypothesized that MTPF would be greater in males than females. One hundred and one healthy adults (male: 23, female: 78) participated in this study. Inclusion criteria were age 18 or above, no self‐reported history of mouth or tongue disease/surgery, and no self‐reported sleep apnea. Their self‐reported sex, age, and smoking history were recorded; height and weight were measured. The subjects pushed their tongue forward against the device's piston (protrusion) as hard as possible for 2–5 seconds and MTPF was recorded in Newtons (N). Subjects performed 5–10 MTPF efforts. The MTPF for all efforts were averaged for each subject. All subjects were able to perform the MTPF task and no adverse responses were reported. The average MTPF for all subjects was 15.4 N (SD ± 3.8), with a range of 8–29. The male average MTPF was higher than female (17.8 N, SD ± 3.7 versus 14.7 N, SD ± 3.5; p = .001). There was no significant difference for age between males and females; males had significantly greater height and weight. The results demonstrate our novel device can effectively measure tongue protrusive force in health young adults. As well, this study provides normative values for MTPF, and identified significant tongue protrusion strength differences between males and females.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PTEN suppresses epigenetic modulation of ERG transcription factor to maintain endothelial lineage and vascular integrity
Authors: Vijay Avin B. R. , Anwar Mumtaz , Joshi Jagdish Chandra , Mohammad Tauseef , Mohammad Farazuddin , Yue Lili , Mehta Dolly
Published: June 2021

Homeostatic replenishment of endothelial cells (ECs) after injury is required for the efficient formation of a stable endothelial lineage and to prevent long‐lasting lung vascular injury such as protein‐rich edema in the interstitial tissue, a hallmark of tissue inflammation. However, mechanisms maintaining EC lineage remains unclear. Phosphatase and tensin homologue (PTEN), a tumor suppressor, is well‐known to function in cell membrane to negatively regulate PIP3 levels required for angiogenesis and endothelial cell proliferation and thereby may have role in regulating EC fate. Thus, we induced the conditional deletion of PTEN in ECs of adult mice using tamoxifen to address the hypothesis that PTEN regulates EC lineage. Intriguingly, we found that EC‐specific loss of PTEN led to intractable vascular injury basally due to marked reduction in EC number in the lung. FACS analysis showed that these ECs acquired fibroblast‐like phenotype. RNA seq analysis of PTEN depleted ECs showed PTEN was required for maintaining ECs genes including VE‐cadherin and PECAM. PTEN maintained ECs genes by activating ETS (E26 Transformation Specific) family member transcription factor, ERG. We show that this function of PTEN occur in nucleus since loss of PTEN increased the activity of epigenetic modulating gene, HDAC1 leading to upregulation of non‐EC genes and thereby converting these ECs into fibroblasts like cells. Inhibition of HDAC1 activity by velproic acid and trichostatin rescued ERG expression and transcription of VE‐cadherin in PTEN depleted cells. Thus, we identify a novel role of PTEN in suppressing HDAC1 activity enabling thereby ERG transcription of EC genes leading to EC generation and lung‐vascular homeostasis.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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

BACKGROUND

Transfusion‐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.

OBJECTIVE

To test the hypothesis that aged (5 days) but not fresh (1 day) platelets will generate PMP that mediate TRALI in a CER‐dependent manner.

METHODS

PMPs 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).

RESULTS

Compared 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.

CONCLUSIONS

Ceramide 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 Information

This 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

Background

Both 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 methods

To 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.

Results

Steady‐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.

Conclusions

The 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 Information

Volkswagen 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 Information

NIH 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

Background

The 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 methods

To 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.

Results

The 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.

Conclusions

Our 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 Information

Volkswagen 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 Information

Supported 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

Purpose

SLC4A11 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.

Methods

Corneal 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.

Results

KO 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.

Conclusion

We conclude that Slc4a11 protects cells from Gln and NH3 induced toxicity.

Support or Funding Information

NIH RO1 EY008834

This 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.
CTNNA1 is Expressed in U937 Cells and is Regulated by miR9
Authors: Patel Nikhil , Rabquer Bradley James
Published: June 2021

Rheumatoid arthritis (RA), a chronic, auto‐immune, inflammatory disorder characterized by progressive joint destruction, affects 0.3–1% of people worldwide and is a major cause of disabilities. Monocyte (MN) trafficking into inflamed joints is critical to the pathogenesis of RA. Micro RNAs (miRNAs) are approximately 22‐nucleotide long single‐stranded, noncoding RNA which bind to mRNA, thus making them act as gene expression regulators. miRNAs bind to complementary sites on the 3′‐end of target mRNAs and either block their translation or enhance their degradation, resulting in downregulation of certain genes. Previous studies in our lab have identified a gene of interest called CTNNA1, as prior research has suggested miR9 to be a suitable binding match. CTNNA1 plays a role in several pathological processes, including muscle contraction and tumor metastasis. It's hypothesized that miR9 binds to CTNNA1 and that miR9 will mediate its degradation.

Methods and Results

U937 monocytes were cultured, mRNA was isolated, and cDNA was prepared. During this, the DNA sequence of CTNNA1 in humans was analyzed so primers could be designed to bind to the predicted 3′ UTR binding sites of miR9. PCR was then performed to yield the isolated, predicted binding site, and then base pair size was confirmed via electrophoresis gels. The gene product was then cut with restriction enzymes Sac I and Sal I, ligated into the pmirGLO vector, and was transformed into E. coli cells and plated for incubation. Selected E. coli colonies expressing the gene were grown and incubated in overnight cultures.

Conclusion

CTNNA1 is expressed by U937 cells and it's 3′ UTR has been cloned into pmirGLO for mir9 binding assessment.

Support or Funding Information

Albion College Biology Department and the Foundation for Undergraduate Research, Scholarly, and Creative Activity

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 IMPC: A Global Scientific Infrastructure for Understanding the Role of Genes in Complex Traits
Authors: Muñoz‐Fuentes Violeta , Meehan Terrence F. , Lloyd KC Kent , Mallon Ann‐Marie , Smedley Damian , Parkinson Helen
Published: June 2021

The International Mouse Phenotyping Consortium (IMPC) is building a functional catalogue of the mammalian genome by producing and phenotyping a knockout mouse strain for every protein‐coding gene. To date, over 4,000 knockout mouse lines, many for poorly understood genes, have been characterized and made available to the research community in a coordinated effort involving more than a dozen global research centers and dedicated publicly‐available online resources. Using a standardized adult phenotyping pipeline, centers measure each mouse for more than 250 phenotypic parameters including metabolite content in blood, hearing capacity, and skeletal malformations. In addition, over 1,000 embryonic lethal mouse lines are analyzed in a specialized embryonic development pipeline that uses high‐resolution 3D imaging. All data is quality controlled and analyzed by a dedicated informatics consortium and all abnormal phenotypes automatically compared to clinical features of human disease populations to identify robust mouse models of disease and inform efforts in precision medicine. We will present our discoveries into the wide‐ranging sexual dimorphism of phenotypic traits, the latest findings in metabolism, hearing and aging and provide examples of how researchers are using IMPC resources to identify causative alleles within GWAS loci. The plethora of new genetic disease models as well as the basic and translational knowledge that has arisen from our analysis has recently earned the IMPC recognition by the G7 for being the first global research infrastructure for the life sciences.

Support or Funding Information

NIH Common Fund:U54H G006370, U54H G006364

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Coding and Noncoding Variants for Human CKD Using Novel Strategies
Authors: Prokop Jeremy W. , Yeo Nan C. , Ottmann Christian , Link Brian A. , Mendenhall Eric M. , Freedman Barry I. , Lazar Jozef , Jacob Howard J.
Published: June 2021

Interpreting the mechanism of genetic variants is one of the greatest challenges impeding the analysis of the rapidly increasing volume of patients' genomic data, particularly for rare variants that do not reach GWAS significance due to low population frequencies. There are 153 GWAS hits for altered kidney function due to commonly inherited genetic variants, yet minimal knowledge of the mechanism of these variations is known at the current time. Utilizing a computational strategy to filter 3,921 genetic variants that are found within linkage disequilibrium of the GWAS hits we have nominated 4 damaging protein coding variants and more importantly 30 noncoding variants predicted to alter transcription factor based gene regulation. One of these noncoding variants, rs17319721 associated with risk of chronic kidney disease (CKD), is found near the SHROOM3 gene and is thought to play a functional role in podocytes of the kidney. In this study we show this variant falls upstream of a novel transcriptional start site that results in a shorter SHROOM3 isoform lacking the PDZ domain. This variant disrupts binding of TCF7L2 in podocytes and the single variant (rs17319721) alters transcription levels of SHROOM3 as determined by variant mutation into the genome using CRISPR/Cas9 technology.

To begin deciphering the role of rare variants within human genomes we performed a full assessment of the ExAC database for >60,000 sequenced human exomes, revealing several coding variants in SHROOM3 that are likely to alter function, including a coding variant (P1244L) we show to have association to CKD in humans. We determined that P1244L attenuates the interaction of SHROOM3 with 14‐3‐3 binding and alters signaling through the Hippo pathway, a known mediator of CKD. Animal studies show a loss of function in zebrafish kidney due to the variant, resulting in increased glomerular permeability. These data demonstrate multiple new SHROOM3‐dependent genetic and molecular mechanisms that regulate CKD and demonstrate a novel analytical pipeline with broad application to systematically characterizing risk mechanisms of CKD and other complex diseases.

Support or Funding Information

Support for this study was from NIH‐K01ES025435 (to JWP), NIH‐R01HL069321 (to HJJ), NIH‐R01EY014167 (to BL), the Collaborative Research Centre 1093 through the Deutsche Forschungsgemeinschaft (DFG, to CO), and HudsonAlpha Institute for Biotechnology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In the absence of weight gain, survival rates of DF508‐CF female mice are increased by genistein diet.
Authors: Al‐Nakkash Layla , Fairbourn Nathan , Mylavarapu Charisma , Dbeis Ammer , Lord Ryan , Chandrashekar Archana , Bowman Taylor , Banayat Tatum , Hodges Craig
Published: June 2021

Mice homozygous for the most common disease‐associated mutation, DF508, are characterized with severe intestinal disease and require constant laxative treatment for survival. This pathology mimics the intestinal obstruction (meconium ileus) seen in some CF patients. Genistein is a naturally occurring isoflavone found in soy. Patch clamp studies have demonstrated that genistein increases the open probability of DF508 CFTR to wild‐type (WT) levels. We evaluated whether dietary genistein alone would eliminate the need for laxatives for survival of the DF508 CF mouse, thereby improving mortality rates. At age 21 days, we maintained male and female DF508 mice on three diet regimens for 45 days; normal diet, normal diet + Colyte or 600 mg genistein/kg diet, 600G. Survival rates and body weight data were determined. At the completion of the diet study, tissues were extracted, immediately frozen at −80ºC until use and evaluated later for protein expression and histological evaluation. Survival rates for each diet group were as follows; males fed normal diet = 38% (8/21 mice), males fed normal diet + Colyte = 83% (35/42 mice), males fed 600G = 60% (9/15 mice) and females fed normal diet = 47% (9/19 mice), females fed normal diet + Colyte 71% (27/38 mice), females fed 600G = 87% (13/15 mice). Body weight at the end of the diet study was greater in males fed 600G (21.96±0.68 g, n=14) versus those males on Colyte (18.86±0.64 g, n=8). There was no change in overall weight gain in the female groups. Jejunum villi length, crypt depth and wall thickness was comparable between diet groups. Total protein expression of jejunum Na+/K+‐ATPase and NKCC1 was comparable between groups. Total expression of jejunum GLUT2 and GLUT5 were unchanged, however SGLT1 expression was significantly increased 2‐fold in females fed 600G. We are currently exploring whether liver protein expression and serum metabolic markers are modified to better elucidate the mechanism of action of 600G to improve weight in males and to improve survival rate in females. We conclude that feeding DF508 female mice 600G genistein‐diet abolished the dependence upon laxatives for survival. These studies could have implications in reducing the sex‐dependent differences in the age of survival of CF female patients.

Support or Funding Information

Supported by Soy Health Research Program and MWU intramural funds (LA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.