bluuebunny/crossref_metadata_2025 huggingface dataset:
Spontaneous maturation of human colonic organoids (1119.2)
Authors: Rodrigues Ely , Slobbe Lynn , Schultz Michael , Butt Grant
Published: June 2021

The intestinal epithelium is a complex structure consisting of distinct cell types derived from stem cells residing in the crypt base. Here we investigated the spontaneous maturation of organoids grown from human colonic crypts. Crypts were isolated by Ca2+ chelation from proximal colonic biopsies and transferred to Matrigel and growth media. The organoids were grown for 15 days and the gross appearance, ultrastructure, histology of the organoids and gene expression of proliferation, differentiation, maturation and stem cell activity markers analysed at day 4 and 15 by qPCR. At day 4, 71±7% of the organoids were thin walled spheroids, while at day 15 there was a mixture of large convoluted organoids (51±10%) and spheroids. At day 4 the organoids consisted of thin undifferentiated cells, whereas at day 15 the convoluted organoids consisted of differentiated columnar epithelial cells. Associated with this there was an increase (p < 0.0001) in expression of mature enterocyte and goblet cell differentiation marker, KRT20, a decrease (p<0.001) in CAECAM7, an indicator of undifferentiated cells, and an increase (p <0.01) in expression of CFTR, an anion channel expressed in colonic crypt epithelial cells. Although the cells appear to have differentiated they were still capable of proliferation, as indicated by the lack of a change in expression of MKi67, a marker of proliferation, and increased levels of the intestinal stem cell markers LGR5 (p< 0.0001) and OLFM4 (p<0.05). These findings suggest that over 15 days, cultured human intestinal colonic organoids form a secretory epithelium resembling that found in the colonic crypts.

Grant Funding Source: Supported by University of Otago Research Grant
Hydrogen sulfide promotes mitochondrial biogenesis through PPARγ activation in intra‐cerebral administration of homocysteine (1126.18)
Authors: Kamat Pardip , Kalani Anuradha , Tyagi Suresh , Tyagi Neetu
Published: June 2021

Previously we have shown that homocysteine (Hcy) induced oxidative stress, mitophagy and cerebrovascular dysfunction in mouse brain, which was mitigated by H2S (hydrogen sulfide) treatment. Further, Hcy‐induced oxidative stress affecting mitochondrial biogenesis and the protective mechanism implies by H2S are still unclear. The regulation of mitochondrial biogenesis primarily occurs by the transcription factors peroxisome proliferator‐activated receptor gamma (PPARγ), PGC‐1α, nuclear respiratory factor 2 (NRF2), and controlled through signaling (AKT) pathways during stress. Therefore, we hypothesize that H2S can ameliorate Hcy‐induced oxidative stress affecting mitochondrial biogenesis through PPARγ activation and AKT signaling. To test this hypothesis we employed 8‐10 weeks old male wild type (WT) mice: 1) WT+aCSF, 2) WT+ Hcy (0.5µmol) intra‐cerebral (I.C., once prior to NaHS treatment), 3) WT+Hcy +NaHS (NaHS precursor of H2S, 30 µmol/kg, body weight; i.p. for 7 days after Hcy treatment), and 4) WT+NaHS alone. We observed that Hcy led to down regulation of PPARγ through AKT signaling. The results also suggest down regulation of NRF2 which is implicated in mitochondrial biogenesis failure and apoptosis (Caspase‐3 and Caspase‐9) through alternation of mitochondrial markers (Triose Phosphate, NOX, PHB1, HSP60, COX‐4, Cyt‐p450, DRP‐1, MFN‐2, ATP) and DNA damage. Interestingly, H2S ameliorates all these events and endorses mitochondrial biogenesis. These findings demonstrate that Hcy‐induced mitochondrial biogenesis in part by PPARγ activation, AKT and NRF2 involvements, which is potentially mitigated through H2S treatment.

Grant Funding Source: HL107640
Neuronal networks on the nano alumina templates (1127.4)
Authors: Islam Asiful , Menon Latika
Published: June 2021

The hippocampus neuron cells were cultured on the nano‐alumina substrates. Several adhesion protein molecules e.g. Poly D Lysine (PDL), collagen (ECL) and leminin were applied as an interface in between the Al2O3 nano‐templates and the neuronal network before culturing the cells on top of them. The neuronal tissues were mechanically dissociated and cultured on the alumina nano template surfaces and incubated in an incubator. The cells grew on the top of the templates for 24 days. The well‐defined neuronal networks observed on the surface of the Al2O3‐nano templates. For control study, we also cultured neurons on the glass‐slip surfaces. This study revealed that the neurons can grow on any nano‐scale surfaces as well as on the standard cover slip if the proper growing environment will be provided.
Behavioral activation treatment increases parasympathetic activity in veterans (1130.23)
Authors: Reyes Levy , McAndrew Lisa , Ray Kathleen , Blatt Melissa , Ghobreal Bemin , Acosta Amanda , Serrador Jorge
Published: June 2021

Behavioral Activation is an effective treatment for depression and other psychological disorders that works by increasing patients’ preferred daily activities, particularly social engagement. Improving social engagement may improve autonomic function by buffering the impact of stress. We tested if Behavioral Activation improves the autonomic function of deployed Veterans. Veterans (n=5) participated in 12 weekly group sessions where they were encouraged to increase daily behaviors including social engagement. During weeks 1, 4, 8 and 12 autonomic function testing was performed including transcranial Doppler in the middle (MCA) and anterior cerebral artery (ACA) as well as blood pressure during several tests (sit to stand, hyper/ hypocapnia, cold presser and paced auditory serial addition test). All Veterans who started the intervention completed the treatment. Our results show an increase in high‐frequency heart rate variability, an autonomic indicator of parasympathetic activity (Week 1; 660K ± 147K, Week 12; 1390K ± 153K). In addition, mean blood pressure decreased over the same time period, although non‐significantly (Week1; 88.4 ± 3.0, Week 12; 83.2 ± 5.3 mmHg). However, no significant reduction of low‐frequency systolic blood pressure or heart rate variance was found, suggesting no change in sympathetic outflow. Our findings, while preliminary, suggest this behavioral intervention focused on improving social engagement may improve autonomic function. Further 100% of participants who started the treatment were adherent. Our preliminary data suggests that Behavioral Activation may be a promising intervention to improve autonomic dysfunction among Veterans.

Grant Funding Source: Supported by: Department of Veteran Affairs and The Office of Public Health
Intraductal applications of lidocaine attenuate the severity of post‐ERCP acute pancreatitis (1131.2)
Authors: Skolka Michael , Babic Tanja , Travagli R. Alberto
Published: June 2021

Endoscopic retrograde cholangiopancreatography (ERCP) is a procedure used to identify biliary duct obstruction. 5‐10% of patients, however, develop acute pancreatitis (AP). AP is a severe disorder of the exocrine pancreas marked by acinar inflammation and a rise in plasma amylase levels. Pancreatic secretions, including exocrine secretions (PES) are modulated by the vagus nerve, and microinjections of group II mGluR agonist APDC in the dorsal motor nucleus of the vagus (DMV) increase PES. In AP, however, DMV neurons are less sensitive to APDC. The aim was to develop a rat model of post ERCP‐pancreatitis to test the hypothesis that intraductal application of lidocaine prior to ERCP attenuates AP. The femoral vein and pancreatic duct of male SD rats were cannulated to collect whole blood sample and PES (at 10 minutes intervals: 30min baseline, treatment, and 90min post‐treatment), respectively. Similar to what occurs in ERCP, mechanical stimulation of the papilla of Vater increased plasma amylase and reduced the PES response to DMV microinjections of APDC (100pmoles/60nl; N=6). Injections of 200μL of lidocaine (N=5) and/or contrast dye (N=3, 3) into the pancreatic duct prior to papilla stimulation prevented the rise in plasma amylase and restored the APDC‐induced increase in PES. Intraductal lidocaine prior to ERCP may be a useful prophylactic treatment to lower the incidence of post‐ERCP AP.

Group
Amylase (U/L)
Percent Change (%) PES to APDC

Sham (n=3)
52.9 ± 16.7
33.4 ± 7.7

Papilla stimulation (n=6)
1012.2 ± 353.5
8.6 ± 6.4

Lidocaine (n=3)
263.0 ± 124.4
38.8 ± 7.7

Papilla stim. + Lidocaine (n=5)
235.1 ± 33.2
38.9 ± 8.1

Contrast dye (n=3)
155.6 ± 65.4
33.9 ± 2.9

Contrast dye + Lidocaine (n=3)
148.2 ± 46.4
27.9 ± 3.0

Grant Funding Source: APS Undergraduate Summer Research Fellowship & NSF‐1049618
Nox4 contribution to H2O2 but not in superoxide production in response to increased tubular flow in the mTAL of Dahl salt‐sensitive rats (1134.3)
Authors: Zheleznova Nadezhda , Yang Chun , Cowley Allen
Published: June 2021

Studies were carried out to determine the relative contribution of Nox4 in the production of H2O2 vs. superoxide (O2‐) in the renal medullary thick ascending limb of Henle (mTAL) of Dahl salt‐sensitive (SS) rats. We have previously presented that Nox4 is the major source of H2O2 production in mTAL in response to increased luminal flow. Specifically, knock out of Nox4 in SS rats (SSNox4‐/‐) resulted in significant reduction of intracellular H2O2 production compared to SS. In the present study, using fluorescence microscopy (Eth/DHE ratio), we examined the contribution of Nox4 to O2‐ production in isolated microperfused mTAL in response to an increase of luminal flow (from 5 to 20 nl/min) in SS and SSNox4‐/‐ rats fed a 4% NaCl diet for 3 days. NADPH oxidase activity in outer medullary tissue was determined with DHE fluorescence assay. Results: 1) O2‐ production increased significantly and to the same extent in mTAL of both SS and SSNox4‐/‐ rats. 2) No differences were observed in NADPH activity between the two strains. We conclude that Nox4 contributes only to the production of intracellular H2O2 and not O2‐ in response to increased mTAL luminal flow in SS rats fed a high salt diet.
Mass spectrometric quantification of prostaglandin D2 levels in choroid plexus, cerebrospinal fluid and rostral ventrolateral medulla link PGD2 signaling to the development of neurogenic hypertension (1140.10)
Authors: Asirvatham‐Jeyaraj Ninitha , Jones A Daniel , Fink Gregory
Published: June 2021

Roles of Prostaglandin (PG) D2 signaling in blood pressure (BP) control are obscure. PGD2 is synthesized from precursor arachidonic acid and PGH2 by the action of enzyme lipocalin‐prostaglandin D synthase (L‐PGDS). Choroid plexus lining the third ventricle (3VCP) and cerebrospinal fluid (CSF) from AngII‐salt hypertensive (AngII‐HS‐HTN) rats express high mRNA and protein levels of L‐PGDS. Blocking the L‐PGDS enzyme centrally with its antagonist AT56 attenuates AngII‐salt‐HTN development. So we hypothesized that increased PGD2 synthesis in the brain is central to AngII‐salt mediated increase in BP.

Method: Two sets of experiments were performed. In one set of high salt (HS, 2%) fed rats, vehicle or AngII (150 ng/kg/min, sc) was infused. In another set of HS fed rats, L‐PGDS was first blocked chronically with different doses (nmol‐h‐1, sc) of AT56 (0.6,6,23) and then AngII was infused. From both studies, brain tissue and CSF were collected on day 4 of AngII infusion for ultra performance liquid chromatography‐tandem mass spectrometric analysis of substrate arachidonic acid (AA) and metabolites PGD2, PGJ2, delta12‐PGJ2 and PGE2.

Result: HS and AngII compared to vehicle controls (p<0.05) increased the levels of substrate AA in 3VCP. HS and AngII significantly increased the metabolite PGD2 in 3VCP, CSF and rostral ventrolateral medulla (RVLM) compared to vehicle controls. This increase in PGD2 was inhibited in the RVLM of rats treated with AT56 at doses (6 and 23 nmol‐h‐1, sc) that were previously observed to attenuate AngII‐HS‐HTN. There was with no change in the levels of metabolites PGJ2, delta12‐PGJ2 and PGE2 with HS and AngII treatment.

Conclusion: Increased PGD2 synthesis and signaling in the 3VCP, CSF and RVLM mediate development of AngII‐salt HTN.

AA‐3VCP(ng/region ± SE)
PGD2‐3VCP(ng/region ± SE)
PGD2‐RVLM(ng/region ± SE)
PGD2‐CSF (pg/ml ± SE)

HS Vehicle
333 ± 80
0.8 ± 0.4
0.3 ± 0.1
193 ± 39

HS AngII
3057 ± 1163
10.3 ± 4
9 ± 1
418 ± 51

Grant Funding Source: RO1 HL 076312 and RCO HL 060363
SULT1B1 dimerization provides the enzyme with increased thermal integrity and a higher affinity for cofactor PAPS (1141.11)
Authors: Tibbs Zachary , Falany Charles
Published: June 2021

Cytosolic sulfotransferases (SULTs) catalyze the conjugation of a multitude of compounds ranging from drugs and xenobiotics to environmental toxins. Though highly conserved, the twelve human SULT isoforms exhibit an array of substrate specificities that are difficult to predict. We aim to predict substrate specificity in silico with high accuracy, as it would be a more time and cost‐effective method of ligand screening. We must first understand the mechanism by which SULTs coordinate cofactor (PAPS) binding, substrate binding, PAP release, and product release. In general, SULTs contain 3 highly conserved groups of amino acid residues that are essential for either catalysis, PAPS binding, or dimerization. Though it is highly conserved in mammals, and proximal to the critical PAPS binding domain, the dimerization domain (DD) has not been linked to any kinetic function. To investigate functional aspects of the DD in vitro, we first bacterially expressed and purified WT hSULT1B1 and a DD point mutant (K266L). The mutant’s monomerization status was verified via gel filtration. Both WT and monomeric K266L‐SULT1B1 fully retained sulfation activity after incubation at 37°C for 90 minutes. However, upon incubation at 42°C, monomeric SULT1B1 lost over 90% of its enzymatic activity after 20 minutes, while dimeric SULT1B1 retained nearly 75% of its activity, suggesting the monomer is less thermo‐stable. The location of the DD suggests that it may also play a role in PAPS binding. The Km of PAPS for the monomer was 5. 2 µM while WT hSULT1B1 exhibited a Km of 1.34 µM as shown by a 1‐naphthol sulfation assay, while 1‐naphthol Km’s were unaltered. In a similar fashion, changes in protein intrinsic fluorescence showed that the WT Kd for PAPS was 332 nM while the monomer was 478 nM. Our data suggest that the dimerization of SULT1B1, and likely other SULTs, contributes to the thermal stability of the enzyme, while contributing to a higher affinity for PAPS binding.

Grant Funding Source: NIH grant R01 GM38953 to CNF
Targeting GUCY2C for anti‐obesity pharmacotherapy (1142.12)
Authors: Kim Gilbert , Lin Jieru , Snook Adam , Li Peng , Aing Amanda , Schulz Stephanie , Waldman Scott
Published: June 2021

Obesity is a global pandemic, with ominous trends in related morbidities, mortalities, and medical expenditures. Current therapies have proven limited in efficacy or burdened with safety concerns. The discovery of a new gut‐brain endocrine axis regulating appetite through the transmembrane receptor guanylyl cyclase C (GUCY2C) is a welcome opportunity for advancing clinical care. We aimed to define the hypothalamic organization and signaling of neuronal GUCY2C. Moreover, we assessed the integrity of the GUCY2C axis in the setting of obesity. Microdissection studies mapped GUCY2C expression and activity to key centers of central appetite control. Furthermore, mouse models of diet‐induced obesity suggest that obesity leads to intestinal GUCY2C hormone loss, which may contribute to appetite dysregulation. Thus, GUCY2C hormone supplementation to reconstitute GUCY2C signaling is promising, particularly given the recent regulatory approval for the GUCY2C ligand linaclotide in clinical use.
Effects of baicalin on Akt /TBC1D1 phosphorylation in mouse skeletal muscle (1142.13)
Authors: Liu Hua , Lv Hongmei , Xi Youli
Published: June 2021

Insulin resistance is a common pathophysiological basis of type 2 diabetes. Related studies have confirmed that high lipid environment can decrease insulin sensitivity in liver, fat and skeletal muscle. TBC1D1 was recently discovered as a member of the TBC1 Rab‐GTPase family, which involved in the regulation of glucose transport and highly expressed in skeletal muscle.The phosphorylation of TBC1D1 may play a key role in regulating insulin‐stimulated glucose transport. TBC1D1 Thr590 is match for the Akt recognition motifs. Akt may activate its downstream substrate TBC1D1 Thr590 phosphorylation, and trigger glucose transporter protein GLUT4 translocation to cell surface. Baicalin is a natural compound extracted from scutellaria root, which is a kind of flavonoids. It has been reported that baicalin can show improvement diabetic rats, and also increase the glucose uptake in fat cells. However ,the effect of baicalin on skeletal muscle less reported. The aim of our study was to identify whether the beneficial effects of baicalin are related to regulate the phosphorylation of Akt and TBC1D1 in skeletal muscle. Our study results showed that baicalin can improve blood lipid, reduce the fasting blood glucose and postprandial glucose tolerance, and simultaneously increase Akt and TBC1D1 phosphorylation in skeletal muscle. These data demonstrate for the first time that intervention with baicalin may improve insulin sensitivity in skeletal muscle, which is related in part to regulate Akt /TBC1D1 signaling pathway.
Combined treatment with diazepam and MK‐801 provide enhanced protection from tetramethylenedisulfotetramine‐induced seizures and lethality (1143.14)
Authors: Shakarjian Michael , Ali Mahil , Heck Diane , Velisek Jana , Velisek Libor
Published: June 2021

The synthetic rodenticide, tetramethylenedisulfotetramine (TMDT), is a persistent and lethal GABAA Cl‐ channel antagonist. Although banned, TMDT is clandestinely produced, remains popular in Mainland China, and is the cause of numerous unintentional and deliberate poisonings worldwide. TMDT has features that make it a likely tool for terrorist activity. We sought to characterize the TMDT syndrome in C57BL/6 mice and explore the best modalities for treatment. At 0.4 mg/kg ip, TMDT produced neurotoxicity consisting of symptoms of twitches, clonic seizures and tonic‐clonic seizures, the latter often ending in death of the subject upon occurrence of status epilepticus. Upon ip administration, the benzodiazepine, diazepam (DZP), effectively prevented all of TMDT symptoms, whereas the NMDA receptor antagonist, dizocilpine (MK‐801), prevented tonic‐clonic seizures more effectively than the less severe, clonic seizures. While both agents protected mice from the short‐term lethal effects of TMDT, MK‐801 better protected animals from delayed death. We hypothesized that although TMDT initiates its toxic action at GABA receptors, NMDA receptors play a key role in the persistent excitotoxic actions of the agent. We examined the effectiveness of joint treatment with both DZP and MK‐801. By applying isobolographic analysis to the data, we demonstrate that a binary mixture of DZP and MK‐801 displays synergy toward tonic‐clonic seizures and 24 hr lethality prevention. Although simultaneous treatment did not result in effective suppression of clonic seizures, DZP administration given 10 min prior to MK‐801 significantly reduced these seizures and improved overall outcome. In summary, TMDT is a potent neurotoxin and potential terrorist weapon. A sequential combination treatment with DZP and MK‐801 is superior to individual therapy with, or simultaneous administration of these two agents.

Grant Funding Source: Supported by CURE, NIH NS072966 and NIH U54AR055073
Effect of acute and chronic organophosphate pesticide exposure on neuronal viability (1143.4)
Authors: Garcia Juan , Garcia Genevieve , Ahn Brianna , Meuwissen Sarah , Jimenez Andrew , Fradinger Erica
Published: June 2021

Organophosphate pesticides irreversibly and semi‐permanently inhibit acetylcholinesterase, the enzyme responsible for the degradation of the neurotransmitter acetylcholine. Therefore, inhibition of acetylcholinesterase by organophosphate pesticides causes hyperstimulation of the cholinergic nervous system affecting the development and function of neurons in the CNS and PNS. This study seeks to understand the acute effects of organophosphate pesticides, including chlorpyrifos, on differentiating PC‐12 cells, and the effects of long‐term exposure on differentiated PC‐12 cell viability and susceptibility to neurotoxic agents. A dose‐dependent increase in cell death was observed for PC‐12 cells exposed to chlorpyrifos during differentiation. Additionally, results indicated that differentiating PC‐12 cells were most sensitive to chlorpyrifos at mid to late stages of differentiation when the neurons were beginning to form synapses. At earlier stages, chlorpyrifos had little effect on cell survival. Long‐term exposure to a sub‐lethal concentration of chlorpyrifos caused a decrease in neuronal viability, and influenced their susceptibility to H2O2‐induced oxidative damage and Aβ42‐induced damage. Together these data demonstrate that organophosphate exposure adversely affects PC‐12 cell survival and long‐term exposure alters their susceptibility to agents involved in neurodegenerative disease.
Activation of Nrf2 plays a role in the S‐equol‐mediated attenuation of oxidative stress‐induced endothelial cell injury (1146.3)
Authors: Zhang Ting , Mi Mantian , Liang Xinyu , Shi Linying
Published: June 2021

Objective To study the effect of S‐equol on the activation of Nrf2/ARE signaling pathway in human umbilical vein endothelial cells (HUVECs).

Method HUVECs were used in this study. Dual luciferase reporter gene assay was used to determine the effect of S‐equol on Nrf2‐ARE‐Luc activity. Total and nuclear protein were extracted, western blot assay was used to determine the effect of S‐equol on Nrf2 and its downstream antioxidant gene HO‐1 protein expression. Real‐time PCR assay was used to detect the effect of S‐equol on Nrf2 and HO‐1 mRNA levels. Cells were pretreated with Znpp, the specific antagonist of HO‐1 and Nrf2‐siRNA, CCK‐8 assay was used to determine the effect of S‐equol on cell viability under oxidative stress.

Results S‐equol (1 nM, 10 nM and 100 nM) dose‐dependently increased the activity of Nrf2‐ARE‐Luc. S‐equol (100 nM) up‐regulated Nrf2 and HO‐1 protein levels, which in a dose and time‐dependent in HO‐1 but no in Nrf2. S‐equol treatment induced Nrf2 nuclear translocation. S‐equol almost had no efffect on Nrf2 mRNA level, but a dose‐dependent upregulation of HO‐1 mRNA levels after treatment with S‐equol. Pretreatment of Znpp, the specific blocker of HO‐1 and Nrf2‐siRNA attenuated the protective effect of S‐equol under oxidative stress in HUVECs.

Conclusion Physiological concentration (nM) of S‐equol activated Nrf2/ARE signaling pathway, the main antioxidant mechanism, in HUVECs, which plays a role in attenuation of S‐equol in oxidative stress‐induced cell injury.

Grant Funding Source: Supported by The National Natural Science Foundation of China (81102129)
Metabolomics reveals 1‐methylnicotinamide and nicotinamide N‐methyltransferase as crucial biomarkers in microRNA‐1291‐altered pancreatic carcinoma cell metabolism (1147.1)
Authors: Bi Huichang , Pan Yu‐Zhuo , Gonzalez Frank , Yu Aiming
Published: June 2021

Our recent studies have demonstrated that microRNA miR‐1291 may play an important role in the regulation of tumorigenesis of pancreatic cancer cells. To understand the underlying mechanisms, we employed a LC/MS‐based metabolomics approach to determine the impact of miR‐1291 on pancreatic carcinoma cell metabolism. Our data showed that the miR‐1291 stably transfected and control PANC‐1 cells are readily segregated based upon their metabolomic profiles. A number of significantly altered cellular metabolites were identified, including 1‐methylnicotinamide (MNA), taurine, L‐carnitine, isobutyryl‐carnitine, isovaleryl‐carnitine and hydroxybutyryl‐carnitine. Among them, MNA was altered to the greatest extent, and the increase in cellular MNA was associated with a sharply elevated expression of nicotinamide N‐methyltransferase (NNMT). In addition, NNMT mRNA expression was inversely related to the size of PANC‐1 derived xenograft tumors. Our results suggest that MNA and NNMT are possible biomarkers in miR‐1291‐altered pancreatic carcinoma cell metabolism. The findings may help identify key metabolic pathways in PANC‐1 cells perturbed by miR‐1291 and understand their involvement in pancreatic tumorigenesis.
Using “short snorters” to document aero medical milestones in World War II: development, testing and evaluation of oxygen equipment for the USAAF (1149.3)
Authors: Dean Jay
Published: June 2021

“Short Snorters” are banknotes (often $1 bills) that have been signed by the participants of a significant event, such as a trip, important event or, as in the case of aero medical physiologists in 1942, conquering the 40,000 ft barrier in flight for the first time with the newest pressure breathing (PB) oxygen equipment. During Nov 1942, Dr. (Major) Randy Lovelace (Chief, Wright Field Aero Med Lab, Dayton, OH) and an aircrew from Boeing’s Flight Test Unit flew to 42,900 ft in a B‐17E Flying Fortress using the PB oxygen mask prototype. The 3‐man crew spent a total time at or above 40,000 ft of 1 hr 40 min. They performed favorably using PB: Major Lovelace “…was able to exercise with vigor at all altitudes above 40,000 ft…” while “…the pilots devoted their full efforts without any hesitation to the operation of the controls.” Their original goal of 47,000 ft was abandoned when the B‐17E’s supercharger turbo exploded at 42,900 ft, limiting further ascent, to which “…the emergency was attended to quickly and with alacrity by the pilots.” They concluded that “…the new PB oxygen equipment makes it possible for the first time to extend the current engineering test program of the B‐17 series well up into the 40,000 to 50,000 feet range of altitudes… without awaiting the development and production of pressure cabins.” The short snorter they signed that day is entitled “W.R. Lovelace 40,000 Footer”. Short snorter rules state that if, at a later date, a signee of the $1 bill approaches the holder of the bill and demands to see it, but the holder cannot produce the banknote, then they have to pay the signee $1 or else buy them a “short snort” of liquor. Randy Lovelace’s collection of short snorters is used here as the framework for reviewing the wartime development of demand and PB demand oxygen equipment by physiologists of the Wright Field Aero Med Lab for the USAAF.
Cytometric characterization and free radical production of two different mitochondrial populations from human placenta (1153.13)
Authors: Ramirez Velez Robinson
Published: June 2021

PURPOSE: To study the metabolic function from two mitochondrial populations described as heavy (HM) and light mitochondria (LM), present in the human placenta.

METHODS. Placentas were collected within 15 min of delivery. Mitochondrial fractions were isolated from human placental tissue by differential centrifugation. Mitochondrial oxygen uptake, superoxide levels and hydrogen peroxide were measured to determine oxygen metabolism. Flow‐cytometry assays were carried out to measure mitochondrial membrane potential (αΨm). A spectrophotometric assay was used to measure mitochondrial respiratory complexes and levels of cytochrome P‐450.

RESULTS. The oxygen uptake showed that a better respiratory control (3.7±0.3) was observed in the HM, as compared with the LM (2.2±0.3, p<0.01). HM produced lower levels of hydrogen peroxide than LM (0.41±0.6 vs. 0.63±0.20 nmol/min.mg protein, p<0.01). Quantification of the superoxide anion levels as percentage values of MitoSox r. f. i was 78% higher in HM than in LM populations. Similar results were obtained during the evaluation of the αΨm. The activity of complex I‐III in the HM and LM was 55.2±1.5 and 76.2±3.0 nmol cytc/min.mg protein, respectively, (p<0.05). Complex II‐III activity in the HM and LM was 9.5±0.5 and 5.8±0.4 nmol cytc/min.mg proteins respectively. Evaluation of complex IV activity (cytochrome c oxidase) showed values of 20.9±1.1 in LM and 26.7±0.7 nmol cyt c/min.mg protein in HM. The LM from term human placenta, during active steroidogenesis, showed levels of 0.13±0.02 nmol/mg protein vs. HM were quite low 0.027±0.009 nmol/mg protein (cytochrome P‐450).

CONCLUSIONS. These results were associated with higher levels of hydrogen peroxide produced in the LM. Increased levels of cytochrome P‐450scc in the LM were consistent with a higher steroidogenic activity present in this mitochondrial fraction.
Measurement of leg fluid volume using bioelectrical impedance (1156.14)
Authors: Singh Bhajan , Yadollahi Azadeh , Bradley T
Published: June 2021

Background: Fluid volume (FV) of body segments can be measured non‐invasively and continuously using bioelectrical impedance (BI). Electrical impedance of the cell membrane is high at low frequencies suggesting that BI using low frequencies will detect mainly extracellular fluid (ECF). The objective of this study was to determine relationships between leg FV measured using BI and changes in leg volume (LV) calculated geometrically.

Methods: We measured FV between the knee and ankle using BI at low frequencies (50 & 100 KHz, Biopac) and leg circumferences at the knee, ankle and widest part of the calf in both legs of 7 healthy subjects (5 men, age 50.7±7.1 yrs, BMI 23.6±2.8 kg/m2). Measurements were made every 30 min while sitting for 4 hrs. LV was calculated geometrically by assuming a shape of a truncated cone. We also measured FV between the left knee and ankle every hour using a Xitron device that uses multiple frequencies up to 1 MHz to distinguish between ECF and intracellular fluid volumes. Paired t‐tests were used to compare changes (Δ) in Biopac FV (FV‐B) with ΔLV and Xitron measurements of ECF (ECF‐X) and total FV (TFV‐X).

Findings: Relative to ΔLV, ΔFV‐B was lower in the first 2 hours of sitting (ΔLV 188.7±31.5, ΔFV‐B 79.8.9±121.3 ml, p<0.0001) but similar between 2 and 4 hours (ΔLV 68.8±45.7 ml, ΔFV‐B 56.2±22.2 ml, p=0.50). In the left leg, all estimates of ΔFV‐B were consistent with ΔECF‐X and lower than ΔTFV‐X and ΔLV (at 4 hrs, ΔFV‐B 0.06±0.01 L, ΔECF‐X 0.06±0.01 L, ΔTFV‐X 0.08±0.07 L, ΔLV 0.14±0.04 L).

Conclusions: FV measured using Biopac at low frequencies seems to quantify changes in ECF volume. Expansion of ECF volume accounts for only about half the volume expansion below the knees during the first 2 hours of sitting but accounts for most of it between 2 and 4 hours.
Effect of Paeotang on TNF‐α‐induced vascular inflammation in human umbilical vein endothelial cells (1158.4)
Authors: Kim Dae Hwan , Lee Yun Jung , Yoon Jung Joo , Lee So Min , Kang Dae Gill , Lee Ho Sub
Published: June 2021

Vascular inflammation is an important event in the development of vascular disease such as tumor progression and atherosclerosis. Paeotang, a formula composed of eight herbs, has been used for extravasated blood in Korean medicine. In this study, the inhibitory effects of Paeotang on TNF‐α‐induced vascular inflammation process in human umbilical vein endothelial cells (HUVEC). Pretreatment with Paeotang decreased TNF‐α‐induced adhesion HL‐60 monocytic cells, as well as mRNA and protein expression of intracellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and endothelial cell selectin (E‐selectin). Paeotang also dose‐dependently inhibited TNF‐α‐induced matrix metalloproteinase‐2 and ‐9 expression. Furthermore, Paeotang significantly decreased TNF‐α‐induced intracellular reactive oxygen species (ROS) production. The western blot and immunofluorescence analysis showed that Paeotang suppressed the translocation of p65 NF‐κB to the nucleus. In addition Paeotang inhibited the TNF‐α‐induced degradation of IκB‐α, and inhibitor of NF‐κB, by inhibiting the phosphorylation of IκB‐α. These results suggest that Paeotang may be a therapetic agent in vascular inflammation through inhibition of ROS/NF‐κB pathway.
mTOR regulation of trophoblast transcriptome and mitochondrial respiration (1159.1)
Authors: Rosario Fredrick , Cox Laura , Lange Kenneth , Muralimanoharan Subha , Myatt Leslie , Powell Theresa , Jansson Thomas
Published: June 2021

The role of mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2 in regulating the function of the syncytiotrophoblast, the placental transporting epithelium, is largely unknown. We hypothesized that silencing of mTORC1 in primary human trophoblast (PHT) cells (a) down‐regulates key genes involved in oxidative phosphorylation and ribosome synthesis and (b) inhibits mitochondrial respiration. We silenced raptor (mTORC1 inhibition), rictor (mTORC2 inhibition) or deptor (mTORC1/2 activation) in cultured term PHT cells and performed transcriptome profiling and measured mitochondrial respiration. Inhibition of mTORC1 resulted in a highly coordinated transcriptional response including downregulation of genes in networks related to oxidative phosphorylation, ribosome and lipid biosynthesis whereas inhibition of mTORC2 had no effect on these pathways. Inhibition of mTORC1, but not mTORC2, decreased mitochondrial basal/maximal respiration, ATP coupling, reserve capacity and proton leak, whereas activation of mTORC1/2 had the opposite effect. In conclusion, mTORC1 and 2 differentially regulates the trophoblast transcriptome and mTORC1 is a positive regulator of mitochondrial respiratory function. Placental mTOR signaling is inhibited in fetal growth restriction, which may impair mitochondrial respiration and contribute to placental insufficiency in this pregnancy complication.

Grant Funding Source: NIH HD068370
Mcl‐1 and VDAC interaction promotes mitochondrial Ca2+ uptake and ROS production (1159.12)
Authors: Huang Huiya , Li Chi , White Carl
Published: June 2021

The overexpression of Mcl‐1 protein has been implicated in a wide range of cancer cells and is associated with anti‐apoptotic function and cancer migration and invasion. In the current study the role of Mcl‐1 and voltage‐dependent anion channel (VDAC) interaction in regulating mitochondrial Ca2+ ([Ca2+]mito) and ROS production has been studied. In mouse embryonic fibroblasts (MEF), Mcl‐1 knock‐out caused decreased [Ca2+]mito uptake and mitochondrial ROS production compared to wild‐type cells. Application of two peptides, VDAC‐N‐terminal and L14‐15 shown to disrupt Mcl‐1‐VDAC interaction reduced [Ca2+]mito uptake and ROS levels in the WT and Bax/Bak double knock‐out MEF cells. Similar results were observed in lung cancer cell lines A549 and H1299 which were shown to express high level of Mcl‐1. The presence of N‐terminal and L14‐15 peptides reduced [Ca2+]mito uptake and ROS levels in control cells but were without effect on Mcl‐1 knock‐down cells. In permeabilized A549 cells, higher level of [Ca2+]mito is correlated with a higher ROS level. When given an exogenous [Ca2+] that produced a similar level of [Ca2+]mito, no difference in mitochondrial ROS were observed between control and Mcl‐1 knock‐down cells. Mcl‐1 knock‐down and the presence of the two peptides inhibit cancer cell migration and proliferation as indicated by scratch assay and CyQUANT® Cell Proliferation Assay, respectively, consistent with the role of ROS in regulating migration and proliferation. However, in cancer cell line H460 shown to have low level of Mcl‐1 expression, neither Mcl‐1 knock‐down nor peptides produced a significant effect on mitochondrial ROS level. Taken together, these result suggest that Mcl‐1 and VDAC interaction facilitates [Ca2+]mito uptake, leading to increased ROS production, migration and proliferation in lung cancer cells.

Grant Funding Source: American Heart Association
Akt signaling mediates the anabolic action of sympathetic innervation in brown adipose tissue from cold‐exposed rats (1161.6)
Authors: Przygodda Franciele , Buzelle Samyra , Garófalo Maria , Carmo Lilian , Navegantes Luiz Carlos , Kettelhut Isis
Published: June 2021

Previous studies from our lab have demonstrated that sympathetic nervous system (SNS) exerts an anabolic effect on muscle protein metabolism. However, the physiological role of SNS in the maintenance of brown adipose tissue (BAT) mass, the main site of organism heat production, is unclear. Thus, our goal was to investigate the adrenergic mechanisms controlling BAT protein metabolism. For this, adult Wistar rats were submitted to surgical sympathetic hemidenervation (DEN) of BAT. Six days later, rats were exposed to cold (4oC) for 2 days. The phosphorylation levels of proteins involved in the control of proteolytic pathways and protein synthesis were analyzed by Western blotting. BAT noradrenaline was measured by HPLC. Cold‐exposed animals exhibited an increase in BAT noradrenaline content and in the levels of total (2‐fold) and mitochondrial (60%) proteins. These effects were associated with an up‐regulation in the phosphorylation levels of AktSer (70%), GSK3α (30%) and 4E‐BP1 (2‐fold), which are key mediators involved in protein synthesis. Furthermore, BAT from cold‐exposed animals also showed an increase in the phosphorylation levels of the transcription factor Foxo1, which could inhibit the expression of proteolytic genes. All these effects were abolished in cold‐exposed rats submitted to DEN. In summary, the data suggest that SNS plays an important role in cold‐induced increase in total and mitochondrial proteins in BAT and these effects may be partially mediated by downstream targets of AKT such as FoxO1, GSK3a and 4E‐BP1.

Grant Funding Source: Supported by CNPq and FAPESP (13/17111‐0; 12/24524‐6).
High fat feeding does not impair muscle growth in MuRF1 KO mice (1163.17)
Authors: Baehr Leslie , Bodine Sue
Published: June 2021

Obesity is rapidly becoming a global epidemic and is associated with a number of chronic diseases including type II diabetes, metabolic syndrome, and cardiovascular disease. In skeletal muscle, chronic exposure to high levels of fatty acids can decrease oxidative capacity and increase endoplasmic reticulum (ER) stress, both of which could contribute to an attenuation of load‐induced growth. MuRF1 is an E3 ubiquitin ligase that is associated with muscle atrophy. MuRF1 null (KO) mice have been shown to spare muscle mass under atrophy conditions, and more recently we have shown that MuRF1 KO mice exhibit a superior ability to hypertrophy with age, suggesting that MuRF1 plays a role in the acquisition of anabolic resistance. Thus, the aim of this study was to determine if MuRF1 KO mice develop anabolic resistance in response to a high fat diet. Beginning at six weeks of age, male WT and MuRF1 KO mice were divided into two diet groups: mice fed a high fat diet (45% fat, HFD) and mice fed a normal chow diet (10% fat, LFD). At 20 weeks, a subset of mice from each diet group was subjected to bilateral functional overload (FO) for 14 days. WT HFD mice had significantly less hypertrophy than WT LFD mice (31% vs. 76% growth), while the MuRF1 KO HFD mice showed no growth impairment (76% growth). Expression of ER stress markers were highest in the WT HFD mice. Interestingly, MuRF1 KO mice on a HFD appeared to accumulate less intramuscular fat compared to the HFD WT mice. These results suggest that the accumulation of intramuscular fat and an increase in ER stress may be responsible for the development of anabolic resistance in mice fed a HFD.
ABSTRACT IS WITHDRAWN. (1164.8)
Authors: Kirby Tyler , Fry Christopher , Jackson Janna , Chaillou Thomas , Peterson Charlotte , McCarthy John
Published: June 2021

We previously reported that hypertrophy of skeletal muscle can occur independent of satellite cell fusion; however, the compensatory mechanism allowing this growth to occur remains unknown. mTOR signaling and total protein content were unaffected by the loss of satellite cell fusion, arguing against a compensatory mechanism involving translation. Alternatively, total RNA content was maintained even though DNA accrual was blocked by the absence of satellite cell fusion. Based on this finding, we postulated that in the absence of satellite cell fusion, myonuclei would increase their transcriptional output in order to maintain RNA levels and thus muscle growth. Preliminary data revealed that RNA:DNA ratio and RNA content per myonucleus was greater in satellite cell‐depleted muscle, which was accompanied by an increase in myonuclear size. Though the significance of this observation remains to be determined, we hypothesize the change in myonuclear shape is indicative of an increase in transcriptional output, specifically ribosomal RNA. Consistent with this idea, we measured under hypertrophic conditions a difference in 28S and 18S content as well as pre‐47S transcript levels in satellite cell‐depleted muscle relative to muscle with a full complement of satellite cells. Thus, it appears myonuclei are able to compensate for the loss of satellite cell fusion during hypertrophy by increasing ribosomal biogenesis.

Grant Funding Source: Supported by R01AR060701
Hemodynamic responses to electrical stimulation of carotid sinus in conscious rats (1169.15)
Authors: Castania Jaci , Silva Carlos Alberto , Souza Gean , Dias Daniel , Salgado Helio , Fazan Rubens
Published: June 2021

Carotid sinus (CS) is an important site of arterial baroreceptors and its electrical stimulation has been used to treat patients with resistant hypertension. This study evaluated hemodynamic responses to electrical stimulation of CS in normal conscious rats. Rats (N=5) had left carotid sinus (CS) carefully isolated, equipped with a pair of electrodes and insulated with a layer of Kwik sil. A catheter was introduced into abdominal aorta throughout laparotomy and exteriorized in the back of the neck, together with the CS electrodes. After two days, conscious rats, under continuous recording of AP and HR, had their CS electrically stimulated, during 20 s, with square pulses of 2 ms, 1 mA and 15, 30, 60 and 90 Hz. Rats showed a basal arterial pressure (AP) and heart rate (HR) of 108±3 mmHg and 353±9 bpm. The recorded voltage of the 1 mA pulses allowed the calculation of carotid sinus resistance that ranged from 4.3 to 7.2 kΩ. A fall in mean AP (Δ=19±2, 28±7, 31±4 and 39±4 mmHg) was observed 5‐9 s after the onset of carotid sinus stimulation with 15, 30, 60 and 90 Hz, respectively. A slight and non‐statistically significant fall in HR (9‐35 bpm) was also observed immediately (1‐3 s) after the onset of CS stimulation. In contrast to electrical stimulation of aortic nerve in conscious rats, currently performed in our lab, CS stimulation affected mainly vascular resistance, once only a slight and non‐significant effect was seem in HR.

Grant Funding Source: Supported by CAPES, FAPESP, CNPq.
Angiotensin II enhances the endocytosis of proteins by podocytes in vivo ő a multiphoton microscopy study (1173.1)
Authors: Schiessl Ina Maria , Castrop Hayo
Published: June 2021

Glomerular filtration may trap plasma proteins within the filtration barrier. We assessed the hypothesis that podocytes clear these proteins by endocytosis. Experiments were performed in young Munich Wister Froemter rats using intravital multiphoton microscopy. Rhodamin B was injected i.v. to label plasma proteins. To assess the regulation of the endocytotic activity of podocytes, z‐stacks of glomeruli were acquired, and the volume of the dye‐containing vesicles within the podocytes was determined. At baseline, the total volume of Rhodamin B‐containing vesicles in podocytes averaged 5.8±3.2 µm3/glomerulus and remained stable over time. During the infusion of Ang II (80 ng/kg/min), podocytes rapidly accumulated Rhodamin B‐labeled proteins, and the volume of dye‐containing vesicles increased to 193±32 µm3/glomerulus (p=.001). This was blocked by the AT1 antagonist losartan (9.9 mg/kg). Similar results were obtained for Alexa 584‐labeled albumin. The endocytosis of the low molecular weight Texas Red dextrane (10 kDa) by podocytes also increased during the infusion of Ang II (from 240±47 to 394±55 µm3/glomerulus; p=.003), while the glomerular sieving coefficient of TxR dextrane remained unchanged. In summary, our data provide evidence that podocytes have endocytotic activity in vivo. The endocytosis is enhanced by Ang II, and this is only partially related to changes in the macromolecule filtration.
Role for histone deacetylases in regulating angiotensinogen expression mediated by the JAK‐STAT pathway by suppressor of cytokine signaling in renal proximal tubular cells (1173.2)
Authors: Satou Ryousuke , Bourgeois Camille , Navar L. Gabriel
Published: June 2021

Activated JAK‐STAT pathway stimulates SOCS expression, which inhibits JAK‐STAT pathway as a negative‐feedback system. Elevated SOCS limits AGT augmentation in PTC, suggesting that induction of SOCS elevation is an effective strategy to treat the renin‐angiotensin system (RAS) associated diseases. Thus, establishment of an approach to elevate SOCS without activation of the JAK‐STAT pathway is required. The aim of this study was to demonstrate that epigenetic modification by pharmacological treatments induces SOCS elevation leading to AGT suppression in PTC. Rat PTC were treated with sodium butyrate (NaB), a non‐selective HDAC inhibitor, for 6 hours. NaB induced SOCS1 (1.71 ± 0.04, ratio to control) and SOCS3 (1.67 ± 0.2) mRNA elevation in PTC. In the NaB‐treated cells, decreases in STAT3 phosphorylation levels (0.56 ± 0.02), AGT mRNA (0.19 ± 0.04) and protein (0.76 ± 0.04) levels were observed. PCI‐34051, an HDAC inhibitor exhibiting higher affinity to HDAC8 than other HDACs, also suppressed AGT expression. Inversely, Inhibition of class IIa HDACs by TMP269 increased AGT expression, indicating that class IIa HDACs include a repressor of AGT expression. These results suggest that an important role for HDACs, particularly HDAC8, in regulating AGT expression via the JAK‐STAT pathway in PTC. The findings will provide a more effective approach to treat RAS associated diseases by SOCS elevation.
Extracellular ADP‐ribose induces [Ca2+]i increase via purinergic pathway in pulmonary artery smooth muscle cells (1175.4)
Authors: Huang Chun , Hu Jinxing , Subedi Krishna , Lin Amanda , Sham James
Published: June 2021

ADP‐ribose (ADPR) is the product of NAD metabolism and cyclic ADPR hydrolysis by CD38, and poly(ADPR) degradation by poly(ADPR) glycohydrolase. It is recognized as a signaling molecule for the activation of the transient receptor potential melastatin 2 (TRPM2) channel and play important roles in H2O2 induced response. In the plasma membrane, CD38 catalytic site orients both extra‐ and intracellularly. Hence, ADPR can be generated and release outside of the cells. However, the role of extracellular ADPR is unclear. Recent studies suggest that ADPR can activate purinergic receptors in pancreaticβcells. Here we examined the Ca2+ mobilizing effect of extracellular ADPR in rat pulmonary artery smooth muscle cells (PASMCs). Application of ADPR (30 µM to 1 mM) elicited concentration dependent increase in [Ca2+]i of PASMCs. At 1 mM, ADPR induced Ca2+ response with a transient and a sustained phase. The sustained phase was abolished by the removal of extracellular Ca2+. Suramin (a non‐selective P2 receptor antagonist) attenuated ADPR‐induced sustain phase, and PPADS (a P2X receptor antagonist) inhibited the peak response. MRS2179 (a selective P2Y1 receptor inhibitor) had no effect on ADPR‐induced Ca2+ mobilization. The PLC inhibitor U73122, but not its inactive analog U73343, inhibited the ADPR‐induced response by 70%. U73122 plus PPADS further decreased the ADPR‐induced peak response. 2‐APB inhibited ADPR‐induced peak response but not the sustain phase. These data indicate that ADPR is an agonist of purinergic receptors, and it mobilizes [Ca2+]i via P2 receptor‐PLC‐IP3R calcium release pathway, but not through TRPM2‐mediated Ca2+ entry. (Supported by R01 HL071835 and R01 HL075134)