bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endogenous hydrogen sulfide decreases Rho kinase and cGMP‐specific phosphodiesterase (PDE5) activity and induces relaxation of colonic smooth muscle (1110.3)
Authors: Nalli Ancy , Kendig Derek , Mahavadi Sunila , Grider John , Murthy Karnam 
Published: June 2021

H2S, synthesized endogenously from L‐cysteine via cystathionine‐γ‐lyase (CSE) and cystathionine‐β‐synthase (CBS), play a role in the regulation of gastrointestinal (GI) motility and secretion. Neither the expression of enzymes nor the mechanism of action of H2S in GI smooth muscle is fully known. Methods: Expression of CSE and CBS was analyzed by RT‐PCR and western blot. The effect of H2S on carbachol (CCh)‐induced Rho kinase activity and sustained contraction, and nitric oxide‐induced PDE5 activity, cGMP formation and muscle relaxation was examined using L‐cysteine (activator of CSE) or NaHS (H2S donor). Results: CSE, but not CBS was expressed in colonic smooth muscle cells of mouse, rabbit and human. CCh‐induced contraction in muscle strips and cells, and increased Rho kinase activity were inhibited by L‐cysteine and NaHS in a concentration‐dependent manner. Inhibition of Rho kinase activity and muscle contraction by L‐cysteine (but not NaHS) was blocked by CSE siRNA and CSE inhibitor, propargylglycine, respectively. L‐cysteine and NaHS also inhibited PDE5 activity, and augmented cGMP formation and muscle relaxation leading to increased (35%) colonic pellet propulsion. Conclusion: Activation of H2S‐producing CSE causes muscle relaxation via a dual mechanism involving inhibition of Rho kinase and PDE5 activity leading to downregulation of RhoA/Rho kinase pathway and upregulation of cGMP/PKG pathway.Grant Funding Source: DK‐28300
Effect of the bile acids unconjugated and conjugated, chenodeoxycholic acid and deoxycholic acid, on epithelial tight junction barrier function in a human colon carcinoma cell line, T84 (1113.3)
Authors: French Sydney , Framarin Nicole , Badar Ayesha , Akhtar Imran , Ao Mei , Domingue Jada , Rao Mrinalini , Sarathy Jayashree 
Published: June 2021

The human colon is exposed to cBA, and ucBA and excess BAs can increase fluid secretion and perturb the epithelial barrier. We showed in T84 cells (AJP 305: C447, ’13) that: a) BAs preferentially stimulate Cl‐ secretion from the basolateral surface (BLM); b) Overnight exposure (O/N) to proinflammatory cytokine (PiC), decreases transepithelial electrical resistance (TER) and then, luminal (AM) BA can stimulate Cl‐ secretion. We hypothesize that high AM [BAs] directly decrease TER, and this is enhanced by PiC. Confluent T84 cells (TER >1000 Ohm.cm2) were treated with 5‐500µM BAs for 0.5‐24h + PiCs (TNFα+IL‐1ß+IFNγ). Cell viability and toxicity were assessed by Accuri flow cytometry and lactate dehydrogenase respectively and barrier function by TER and dextran fluxes. Viability and cytotoxicity were dose and time dependent. UcBAs (500μM, O/N) had higher cytotoxicity (CDCA:28.6%; DCA:27.8%) than cBAs (TCDC:15.5%; TDC:13.3%). Physiological doses (5‐50μM) were less toxic (8‐10%). O/N PiC decreased TER by ~50% and 0.5mM CDCA (20’) decreased this further by 20%. AM, not BLM CDCA (0.5mM), increased (~2‐fold) 10‐kDa blue‐dextran AM to BLM flux over time (8h); PiC increased this further (+2‐fold). In summary, AM BAs alter epithelial barrier integrity allowing luminal BAs to access the BLM and this is enhanced by PiC. This will provide the basis for future studies on the role of BA‐induced diarrhea in inflammatory diseases.Grant Funding Source: Supported by COS, Benedictine U.
The role of protein synthesis in saturated fatty acid‐induced ER stress in H4IIE liver cells (1116.7)
Authors: Estrada Andrea , Peelor Frederick , Wang Dong , Wei Yuren , Hamilton Karyn , Miller Benjamin , Pagliassotti Michael 
Published: June 2021

Elevated fatty acids play a role in non‐alcoholic fatty liver disease (NAFLD). Disruption of endoplasmic reticulum (ER) homeostasis, or ER stress, is a characteristic feature of NAFLD. Elevated fatty acids, in particular saturated fatty acids, induce ER stress in numerous cell types including hepatocytes. However, the specific mechanisms that link fatty acids to ER stress in the liver are poorly understood. ER stress is the result of an imbalance between the protein load delivered to the ER from cellular protein synthesis, and the capacity of the ER to process, transport and/or degrade the client protein load. In this study, we examined the hypothesis that saturated fatty acid‐mediated ER stress resulted from increased cellular protein synthesis. H4IIE liver cells (n=3) were treated with palmitate (250µM), a saturated fatty acid that induces ER stress, oleate (250µM), an unsaturated fatty acid that does not induce ER stress, or a combination of both palmitate and oleate that does not induce ER stress. Protein synthesis was measured utilizing mass spectrometry based analysis of deuterated water incorporation into total cellular proteins and polysome profiling. Protein synthesis as determined by both techniques was not significantly different among groups. These data suggest that increased cellular protein synthesis does not mediate palmitate‐induced ER stress in H4IIE liver cells.Grant Funding Source: Supported by National Institutes of Health, DK‐072017, AG031829‐01
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
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
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
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
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)
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 &amp; 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.