bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Mitochondrial Fission Inhibitor Ameliorates DSS and DNBS Induced Murine Colitis
Authors: Goudie Luke J. L. , Mancini Nicole , Blote Karen R. , Wang Arthur , McKay Derek M. , Shearer Jane
Published: June 2021

Significance

Inflammatory bowel disease (IBD) encompasses a group of disorders that involve an exaggerated immune response to intestinal microbes. Maintenance of intestinal epithelial barrier function is highly dependent on normal mitochondrial networks. When the barrier is compromised, excessive pathological fission ensues via the enzyme dynamin related protein‐1 (DRP1) and its interaction with the receptor fission protein‐1 (Fis1), creating fragmented networks of mitochondria. Fragmented mitochondrial networks have impaired respiratory capacity, generate elevated reactive oxygen species and exacerbate inflammation

Objective

To determine whether a novel peptide, P110, can mitigate dextran sodium sulfate (DSS) and dinitrobenzene sulfonic acid (DNBS) induced colitis by inhibiting DRP1 and Fis1 mediated fission in murine models.

Design

Hypothesising that elevated mitochondrial fission occurs in DSS and DNBS colitis, male Balb/c mice were split into control (n=15), P110 (3 mg/kg/d, intraperitoneally)(n=8), DSS (5% (w/v), 5 days + 3 days water)(n=16) and DSS+P110 (n=16). For the DNBS protocol, male Balb/c mice were divided into control (n=8), DNBS (3 mg/kg/d, intraperitoneally)(n=11) and DNBS+P110 (3 mg/kg/d, intraperitoneally)(n=8). Experimental measures included: body mass, colon length, macroscopic disease score, cross‐sectional histopathology and colonic motility.

Results

Upon necropsy DSS and DNBS treated mice displayed the characteristic signs of colitis associated with these models. Disease was substantially less in both DSS+P110 and DNBS+P110 treated mice as gauged by: (i) percent weight loss (ii) macroscopic disease score and (iii) colon shortening (p<0.05). Functional improvements in colonic motility were seen in P110 treated mice when compared to DSS alone (p<0.05). Analysis of histopathology on H&E stained sections of mid‐colon revealed significant improvement in DNBS mice with P110 (p<0.05). P110 was also found to improve survival rate of mice, compared to DNBS alone.

Conclusions

Systemic administration of a selective inhibitor of mitochondrial fission (P110) reduced the severity of disease in two different, commonly used murine models of colitis. Future studies are required to define the mechanism of P110's action in terms of the target cell in IBD (e.g. epithelium, nerve, macrophage). We conclude that inhibition of DRP1 and Fis1 interaction provides a novel approach to mitigating gut barrier deterioration in IBD

Support or Funding Information

Crohn's and Colitis Canada. The authors wish to thank Daria Mochly‐Rosen for donating P110.

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 Gut Bacterial β‐Glucuronidase on Serum Bilirubin Levels
Authors: Stout Jacob , Hankins Michael , Stec David
Published: June 2021

The results of several large population studies have consistently demonstrated an inverse correlation between serum bilirubin levels and the development of cardiovascular and metabolic diseases. The goal of this study was to test the hypothesis that increased bacterial β‐Glucuronidase (β‐glu) activity can increase the levels of serum bilirubin by decreasing conjugation of bilirubin in the gut. Two strains of Lactobacillus acidophilus with different levels of β‐glu activity, NCK 56 (control) and NCK 2136 (constitutive overexpression), were grown and administered to mice once per day for five days. Each mouse was then administered 2 mL of fluid containing 500 million cells/ml via gastric gavage. Plasma samples were obtained prior to and after treatment. Serum bilirubin levels were measured using a specific assay kit via absorption at 700 nm. Serum bilirubin levels were similar in both groups prior to treatment averaging 2.3 +0.1 and 2.1 + 0.2 mg/dL in 56 and 2136 treated mice, respectively (n=3/group, p=0.8). Five day treatment of mice with control 56 L. acidophilus resulted in a significant decrease in serum bilirubin levels to 1.7 + 0.5 mg/dL (p<0.05), while treatment with β‐glu overexpressing L. acidophilus did not change serum bilirubin levels which averaged 2.3 + 0.3 mg/dL. Our results demonstrate that β‐Glu expression by modified L. acidophilus prevented the decrease in bilirubin that is seen upon administration of the control L. acidophilus bacterial strain. Overexpression β‐glu in a different host bacteria species with less reducing activity may provide a way to elevate serum bilirubin levels.

Support or Funding Information

Hearin Support Foundation, Medical Student Research Program, University of Mississippi Medical Center School of Medicine. National Heart, Lung, and Blood Institute, IP01HL‐051971. National Institute of General Medical Sciences, P20GM‐104357.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probiotic Lactobacilli Improves Intestinal Motility in Mice
Authors: CHANDRASEKHARAN BINDU , Saeedi Bejan , Alam M Ashfaqul , Neish Andrew S.
Published: June 2021

Rationale

Gastrointestinal (GI) motility is orchestrated by the enteric nervous system (ENS) organized into 2 main plexi‐sub mucosal and the myenteric plexi. Terminal differentiation of the ENS in the postnatal period is closely linked to the presence of microbiota.

Aim

The goal of this study was to examine if probiotics like Lactobacillus rhamnosus GG (LGG) can modulate pErk signaling in the ENS, b) regulate neuronal proliferation and/or differentiation, and thereby c) improve GI motility.

Methods

The conventional (Conv) and germ free (GF) mice were gavaged with Hanks buffer (negative control), LGG or LGG ΩSpa C (adhesion mutant of LGG). The mice were sacrificed after 2 h, and the jejunum was cryofixed and immunostained for phospho‐Erk (p44/42 MAPK, mitogen activated protein kinase). Ileum was assayed for gene expressions of neuropeptides and Hand‐2 (transcription factor regulating neuronal differentiation) by real time polymerase chain reaction (qRT‐PCR). In addition, mice gavaged with LGG, were also intra peritoneally injected with a fluorescent hydro‐cy3 dye, and production of reactive oxygen species (ROS) was assessed in the longitudinal muscle myenteric plexus (LMMP) by confocal imaging after 2 h. The conventional mice were also gavaged daily with Hanks buffer or LGG or LGG ΩSpa C for 1 week, and motility was assessed from stool frequency (stool pellets per hr) and total gastrointestinal transit time (from red carmine dye assay).

Results

We observed that levels of pErk, Hand‐2, choline acetyl transferase (ChAT) and serotonin reuptake transporter (SERT) were significantly reduced in GF mice as compared to the Conv mice. LGG gavage resulted in enhanced phosphorylation of myenteric neuronal Erk at 2 h in both GF and Conv mice as observed from immunostaining and confocal microscopy. Further, LGG induced an upregulation of Hand‐2, and neuropeptides like ChAT and SERT in the ileum of both GF and Conv mice. The adhesion mutant LGGΩSpaC exhibited significantly reduced capacity to induce ROS, pErk, Hand‐2 and ChAT as compared to LGG in Conv mice. We observed that daily gavage of LGG, but not LGG ΩSpaC, for 1 week significantly improved gastrointestinal motility in mice as evident from motility assays.

Conclusions

Our study indicates LGG can induce neuronal differentiation via ROS‐pErk signaling, and thereby improve GI motility. Hence supplementation of probiotics in the diet is beneficial for people with gastrointestinal motility disorders.

Support or Funding Information

Support: R01AI064462, R01DK089763

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Profiling Mitochondrial Quality Control in Human Myotubes Derived from Severely Obese Non‐diabetic and Type 2 Diabetic Patients
Authors: Gundersen Anders , Park Sanghee , Dubis Gabe , Houmard Joseph , Zou Kai
Published: June 2021

Mitochondrial networks are maintained via integration of mitochondrial quality control processes, including biogenesis, fusion/fission dynamics and mitophagy. There is evidence to suggest that mitochondrial quality control plays a critical role in the regulation of mitochondrial function and glucose metabolism in skeletal muscle, and disruptions of these processes accompany the presence of skeletal muscle insulin resistance and type 2 diabetes (T2D). However, whether disruptions of mitochondrial quality control processes are retained at the muscle cell level is largely unknown. The purpose of this study was to determine whether the expression of proteins involved in mitochondrial quality control processes were dysregulated in primary human myotubes derived from type 2 diabetic patients. Primary human skeletal muscle cells were isolated from muscle biopsies obtained from eight severely obese, non‐diabetic subjects (BMI = 52.4 kg/m2, fasting glucose = 98.9 mg/dl) and eight severely obese, type 2 diabetics (BMI = 49 kg/m2, fasting glucose 132.3 mg/dl), and were differentiated to myotubes. On day 7 of differentiation, myotubes were harvested for immunoblot analysis to assess the expression of proteins involved in each of the four mitochondrial quality control processes. Mitochondrial fusion protein expression (Optic Atrophy Protein 1 (Opa1) and Mitofusin 1 (Mfn1)) were significantly lower in myotubes derived from type 2 diabetic patients in comparison to the BMI‐matched, non‐diabetic subjects (0.038 ± 0.003 vs. 0.024 ± 0.002 and 0.440 ± 0.042 vs. 0.320 ± 0.038, respectively, P < 0.05). In addition, expression of the mitophagic protein Parkin was significantly lower in primary myotubes derived from type 2 diabetic patients (0.0028 ± 0.0003 vs. 0.0018 ± 0.0002, P < 0.05). No significant differences were found in expression of other fusion or mitophagy proteins (Mitofusin 2 (Mfn2), or PTEN‐Induced Putative Kinase 1 (PINK1)), or proteins related to mitochondrial fission (dynamin related protein 1 (Drp1) and mitochondrial fission 1 protein (Fis1)), and biogenesis (PGC‐1α). These data suggest that mitochondrial quality control is dysregulated in primary myotubes derived from type 2 diabetic patients in a manner that favors fission over fusion.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TRPV4 channel deletion or pharmacological inhibition protects heart against adverse remodeling post‐myocardial infarction
Authors: Adapala Ravi kumar , Kanugula Anantha , Minasyan Ashot , Cappelli Holly , Paruchuri Sailaja , Meszaros J Gary , Thodeti Charles K.
Published: June 2021

Ischemic heart disease (IHD) is the major underlying cause of myocardial infarction (MI), scarring, and hypertrophy leading to heart failure. Cardiac remodeling following myocardial infarction involves scar formation by synthesis and reorganization of ECM which is mediated by myofibroblasts. We have previously shown that the mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid channel 4) regulates cardiac fibroblast differentiation into myofibroblasts via integration of soluble and mechanical signaling. However, the physiological or translational significance of TRPV4 in cardiac remodeling following MI is unknown. To determine this, we have subjected WT and TRPV4KO mice to MI (permanent LAD ligation). 2D‐echocardiography revealed that the cardiac function (ejection fraction and fractional shortening) is preserved post‐MI in TRPV4KO mice compared to WT mice. Further, we found reduced fibrosis at infarcted and remote zones in TRPV4KO‐MI hearts compared to WT‐MI and sham hearts. Furthermore, TRPV4KO hearts exhibited decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) post‐MI compared to WT hearts. To explore the translational significance of these findings, in separate experiments, we have given an orally active TRPV4 antagonist GSK2193874, immediately after MI surgery and followed for 5 weeks. Cardiac function analysis revealed that both ejection fraction and fractional shortening were preserved in GSK2193874‐treated WT mice compared to either WT or vehicle treated mice. Our results thus suggest that targeting TRPV4 protects the heart from myocardial infarction‐induced damage by preserving cardiac structure and function via reduced myocyte apoptosis, diminished fibrosis and increased revascularization, and identifies TRPV4 as a novel therapeutic target for heart failure.

Support or Funding Information

NIH (1RO1HL119705), NIH (1R15CA202847‐01).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Chronically Inhaled Nicotine on Cardiac Function
Authors: Oakes Joshua , Fuchs Robert , Basting Tyler M. , Lobell Thomas D. , Gilpin Nicholas W. , Lazartigues Eric , Yue Xinping , Gardner Jason D.
Published: June 2021

Nicotine is the addictive chemical in tobacco‐related products, such as cigarettes, water pipes, e‐cigs, and chewing tobacco. While the effects of smoke from cigarettes and cigars are well known, the rise in popularity of smokeless electronic cigarettes (e‐cigs) brings new questions about the effects of nicotine itself on the development of cardiovascular disease. These “smoke‐less” products have grown in popularity due to reduced costs, increased social acceptance, and a belief that e‐cigs are a less dangerous alternative to smoking, but very little is known about the cardiac effects of chronically inhaled nicotine.

Thus the goal of our study is to investigate the effects of chronic nicotine exposure on cardiac function.

C57BL/6 female mice aged 20 weeks were used in this study. Mice were exposed to nicotine vapors using a nicotine exposure chamber, in which home cages are placed in sealed chamber that provide control of air flow, chronicity, and nicotine vapor dose. Mice are exposed for 12 hours/day corresponding with the dark cycle during which they are active. Female mice exposed to nicotine vapor for five weeks showed a significant increased mean arterial blood pressure from baseline, (94.3 ± 1.1 mm Hg at baseline vs. 102.4 ± .1.8 mm Hg at five weeks; p<0.001). In addition, after five weeks nicotine exposure females were found to have decreased cardiac output (18.41 ± 2.23 ml/min for air exposed mice vs. 16.17 ± 2.08 ml/min for nicotine exposed mice; p<0.05) as well as thinner posterior walls at diastole (0.71 ± 0.1 mm for air exposed mice vs. 0.64 ± 0.1 mm for nicotine exposed mice; p<0.05).

These findings suggest that nicotine inhalation alters cardiovascular function, leading to increased mean arterial blood pressure, reduced cardiac output, and thinned cardiac walls. This data indicates that nicotine may play a role in cardiac functional and structural decline. Potential future directions include investigating modulators of blood pressure, such as the renin‐angiotensin system, and measuring expression levels of pathways known to adversely regulate cardiomyocyte size, such as Atrogin‐1 and MuRF‐1, following nicotine exposure.

Support or Funding Information

NIH NHLBI R01 HL135635

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Eight weeks of slow deep breathing training alters cardiorespiratory function and improves functional exercise capacity in chronic heart failure patients
Authors: Lewis Robert , Seo Yaewon , Hackfort Bryan T. , Pozehl Bunny , Schultz Harold D.
Published: June 2021

Patients with chronic heart failure (CHF) display autonomic imbalance characterized by excessive sympathoexcitation and reduced vagal outflow. Respiratory sinus arrhythmia (RSA) and baroreflex sensitivity (BRS) are often used as non‐invasive indices of autonomic function. Slow deep breathing (SDB) has been shown to modulate RSA and BRS. In this preliminary study, we examined the effects of SDB in CHF patients (n=4) during a baseline condition of spontaneous breathing (5 minutes), during SDB (5 minutes) controlled at 6 breaths/minute, and during a post‐SDB condition of spontaneous breathing (5 minutes). The patients then underwent an SDB training protocol, in which SDB was practiced at least 5 days/week and with the duration of practice incrementally increasing from 10 min/day at week 0 up to 30 minutes/day by week 8. Comparisons in cardiorespiratory function were made between each of the conditions (i.e., baseline, SDB, and post‐SDB) and between each time point (i.e., week 0 and week 8). We hypothesized that acute SDB would transiently decrease blood pressure, increase BRS, and increase RSA compared to the baseline and post‐SDB conditions. We also predicted that 8 wks of SDB training would decrease baseline blood pressure, increase BRS, and increase RSA compared to week 0. SDB produced a statistically significant (p=0.01) reduction in systolic pressure after 8 wks (114 ± 5.4 mmHg) compared to week 0 (132 ± 12 mmHg). Further, 8 wks of SDB training appears to have altered acute blood pressure responses to SDB. During SDB at week 0, systolic pressure did not differ from baseline spontaneous breathing. However, after 8 wks of SDB training, patients showed a 5.4% reduction in systolic pressure to acute SDB compared to baseline spontaneous breathing (p=0.02). The decrease in systolic pressure was not correlated with the type or dose of antihypertensive drugs. Acute SDB also produced a transient increase in BRS of ~25% from the baseline condition with no differences between week 0 and week 8. RSA increased during acute SDB compared to baseline (p=0.001) but did not change after 8 weeks of SDB training. Further, a six‐minute walk test was performed to assess functional exercise capacity, and the patients with the greatest impairment at baseline showed improvements in the six‐minute walk test after 8wks of SDB. While the data are preliminary, the results suggest that SDB training improves exercise capacity and produces a stable reduction in systolic pressure and transient change in non‐invasive measures of autonomic function in CHF patients.

Support or Funding Information

NIH PO1‐HL062222‐17

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Salt Diet Induces a Rapid Increase in Blood Pressure and Mortality in the Ren−/− Dahl SS Rats
Authors: Ilatovskaya Daria , Levchenko Vladislav , Zietara Adrian , Spires Denisha R. , Staruschenko Alexander
Published: June 2021

Renin‐angiotensin system (RAS) is an important regulator of fluid balance and blood pressure (BP), demonstrated by the widely employed use of angiotensin receptor and/or ACE inhibitors to control hypertension in humans. Here we used the renin knockout (Ren−/−) rats generated on the Dahl Salt‐Sensitive (SS) rat background to address the question of BP development in the absence of renin.

Ren−/− rats exhibit polyuria, lower body weight and mean arterial pressure (MAP) (65.9 ± 2.1 in Ren−/− rats vs 121.9 ± 1.9 mmHg in the wild type (WT) littermates). Baseline glomerular filtration rate (GFR) is also lower in the Ren−/− rats compared to WT littermates (0.54 ± 0.04 vs 0.19 ± 0.03 mL/min/100 g of body weight). To test the role of renin in the BP regulation during the development of salt‐induced hypertension, Ren−/− rats and their WT littermates were kept on 0.4% NaCl diet since weaning (normal salt, NS), and then challenged with sodium deficient (SD, 0.01% NaCl) or high salt (HS, 4% NaCl) diets. BP was recorded via telemetry, kidney function was evaluated by GFR measurements, and diuresis was assessed in metabolic cages.

After 10 days of a HS dietary challenge, 24 hr urinary output, sodium excretion, GFR and BP were elevated in the WT littermates, as expected. After a HS diet was introduced to Ren−/− rats there was a very rapid raise in MAP in the Ren−/− rats (ΔMAP was 60.4 ± 7.1 mmHg over a 5‐day period, compared to 5.0 ± 0.4 mmHg in the WT littermates). In the Ren−/− group, HS diet caused mortality. Neither of Ren−/− rats survived more than 13 days (8 days if animals were instrumented with a telemetry device), which was not observed in the WT littermates. Additionally, Ren−/− rats fed a HS diet had an increase in diuresis and sodium excretion during the first 2–4 days, which was blunted before the terminal point. Following an initial increase, a dramatic decline in GFR was also observed in these rats. Furthermore, before the endpoint there was a spike in plasma sodium level (up to 180 mMol/L) in the Ren−/− animals challenged with a HS diet.

SD diet did not significantly affect MAP or survival in either group within 12 days of the observations. All SD diet fed animals showed reduced urinary output compared to rats fed a NS diet; however, no difference was found in electrolyte excretion between groups fed a SD diet. GFR was not affected in either group fed a SD chow.

Therefore, BP in the Ren−/− rat increases in response to a HS diet, which suggests an involvement of the non‐renin components in this mechanism. Furthermore, we demonstrated that Ren−/− animals are able maintain homeostasis when challenged a SD diet. These data open new avenues to understanding the role of RAS in SS hypertension.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Ischemic Preconditioning on Oxygen Uptake and Extraction Kinetics During Exercise in Normoxia and Hypoxia
Authors: Wiggins Chad C. , Constantini Keren , Paris Hunter , Mickleborough Timothy , Chapman Robert
Published: June 2021

Background

Exercise tolerance is strongly related the physiological components of the primary (PC) and slow components (SC) of oxygen uptake. Exercise tolerance is reduced in clinical populations and during acute exposure to altitude by factors affecting skeletal muscle oxygenation. The practice of ischemic preconditioning has been shown to improve exercise tolerance or performance in a variety of different testing methods, but the mechanisms behind the improvements in exercise tolerance following IPC are still unclear.

Methods

Thirteen men (age= 24 ± 4 years, V̇O2max= 63.1 ± 5.1 ml·kg−1·min−1) completed four randomized trials of each combination of hypoxia (FIO2 = 0.16) or normoxia (FIO2 = 0.21) with a resting pre‐exercise IPC protocol (4 × 5 min at 220 mmHg) or SHAM procedure. Following the IPC/SHAM protocol subjects completed two constant load cycling bouts at power outputs corresponding to 1) 15% below the normoxic gas exchange threshold (CL #1) and 2) 85% of normoxic V̇O2max (CL #2). Breath‐by‐breath V̇O2, oxyhemoglobin saturation, and skeletal muscle oxygenation/extraction (measured via near‐infrared spectroscopy) were continuously monitored throughout the trials. V̇O2 and NIRS kinetics were quantified using a single (for CL#1) or double (for CL#2) exponential term equation with independent time delays for the V̇O2/NIRS primary and slow components.

Results

There was a significant effect of inspirate on deoxyhemoglobin time constant (τ) during CL#1 (Hypoxia: 5.58 ± 2.82 s vs. Normoxia: 8.01 ± 4.71s, P < 0.05) with no effect of IPC treatment (P = 0.84). During CL #2, there was a significant effect of inspirate on V̇O2 primary amplitude (PC) (Normoxia: 3.13 ± 0.36 L/min vs. Hypoxia: 2.92 ± 0.39 L/min, P < 0.05). IPC did preserve microvascular oxygen extraction during high‐intensity cycling (CL #2) in Hypoxia (IPC: 21.3 ± 11.0 μM vs. SHAM: 19.9 ± 9.9 μM, P < 0.05), but did not affect any of the NIRS or V̇O2 parameters in Normoxia.

Conclusion

Oxygen uptake and peripheral oxygen delivery are compromised during high‐intensity exercise in hypoxia. Ischemic preconditioning prior to high‐intensity bouts is effective at mitigating the declines in microvascular oxygen extraction seen during exercise in hypoxia, however the rates (τ) of microvascular extraction and oxygen uptake are not altered following treatment with IPC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Incorporating Technology in Anatomy Education to Align with Educational Outcomes
Authors: McBride Jennifer
Published: June 2021

Over the past few decades, computer‐based educational technologies such as multi‐touch screen interfaces, software‐based applications and now consumer grade augmented, virtual and mixed reality devices have been introduced in the anatomical sciences. Unfortunately, methodologies to evaluate the impact of these computer‐based learning interventions on undergraduate medical students' acquisition of knowledge remains deficient. In addition, the level of mental effort or cognitive load placed on the learner with these learning interventions is a source of rich debate. During this presentation, a brief review of current data regarding the use of these computer‐based educational tools, as well as essential elements to consider prior to the incorporation of technology in the educational environment will be discussed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inspiratory Resistive (R) Load Endurance Testing in Normal Healthy Adults: Load compensation, effort sensation and affective perception.
Authors: Davenport Paul Wesley , Davenport Matthew Paul , Feinstein Justin , Khalsa Sahib , von Leupoldt Andreas
Published: June 2021

Increased respiratory load is a key feature in various respiratory diseases, accompanied by ventilatory compensation, cognitive awareness and affective perception. Loaded breathing can occur from minutes to days requiring the individual to modulate their breathing pattern to maintain alveolar ventilation, minimize work of breathing and minimize aversive load sensations. Increased inspiratory load is associated with cognitive sensation of the load magnitude and affective escape behavior to reduce the aversive perception of loaded breathing. During exacerbations of lung diseases, an individual often must compensate for increased loads that exceed their ability to sustain alveolar ventilation. Yet it is difficult to assess the capacity of an individual to adapt to an increased respiratory load. The aim of this study was to determine the ventilatory and perceptual endurance response to a high magnitude inspiratory R load applied for 10 minutes. It was hypothesized that some individuals would be able to complete the task and other individuals would terminate the task early. It was further hypothesized that early termination of the task would be due to increased aversive affective awareness. Healthy participants were tested while breathing through a non‐rebreathing circuit with an inspiratory load R=50 cmH20/L/s. The individual respired with no‐load for 1 minute, then the load was applied for 10 minutes, followed by 1 minute of no‐load breathing. The subject was instructed that they could terminate the task at any time by removing the mouthpiece. Subjects estimated their perceived load intensity (MEInt) and unpleasantness (MEUn) every minute. Fear (MEF) was estimated before and after the load trial. Two of six subjects terminated the trial early. The ventilatory load pattern for all subjects was a load dependent increased maximum inspiratory pressure, decreased airflow, increased inspiratory duration and unchanged expiratory duration, increased total breath time, increased inspiratory volume, unchanged minute ventilation, PCO2 and PO2. The MEInt increased with the onset of the load, was constant throughout the task and was the same for individuals that completed and stopped the task. MEUn increased with the load onset with greater MEUn for subjects that stopped early. The individuals that stopped early had equal MEF at task onset but much greater MEF when the task stopped. The results suggest that inspiratory R50 endurance capacity is decreased in individuals with greater negative affective responses to high magnitude loaded breathing.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ATP in the Lateral Hypothalamus/Perifornical area (LH/PFA) Increases CO2 Chemoreflex.
Authors: Dias Mirela Barros , da Silva Eliandra Nunes , Gargaglioni Luciane Helena , de Anchieta de Castro Horta‐Junior Jose
Published: June 2021

The Lateral Hypothalamus/Perifornical area (LH/PFA) is a chemosensitive site and participates in the CO2 ventilatoy response in a vigilance‐state‐dependent manner. Studies provide evidence of a physiological role for ATP in central chemoreception. We tested the hypothesis that ATP acting on LH/PFA contributes to the CO2 ventilatory response. To this end, ventilation (VE) was recorded in unanesthetized rats by whole body plethysmograph, together with EEG and EMG. We dialyzed into the LH/PFA vehicle and then α,β‐methyleneATP (selective P2X receptor agonist; 10 mM) or PPADS (purinergic antagonist; 1 mM), and studied its effects on VE in air and hypercapnia (7% CO2) during the light and dark period of the diurnal cycle. During wakefulness, but not during sleep, α,β‐methyleneATP microdialysis augmented the hypercapnic ventilatory response by 25% during the dark‐active period (2288 ± 80 mL/kg/min) compared with its vehicle (1786 ± 161 mL/kg/min) (P< 0.01), but not during the light. This effect was due to an increased tidal volume. No effect was observed in normocapnia or in a separate group of animals who had the microdialysis probe misplaced (peri‐LH/PFA). PPADS did not change the ventilatory parameters in all groups. These results suggest that ATP exerts, in the LH/PFA, an excitatory modulation in the hypercapnic chemoreflex during wakefulness in the dark‐active period of the diurnal cycle.

Support or Funding Information

FAPESP

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gene Ontology Analysis of and Subtyping of Breast Tumors by RNA‐Seq and BiNGO
Authors: Soneral Paula , Skorseth Claire
Published: June 2021

Breast cancer, the second deadliest among women, kills about 40,000 individuals worldwide per year. Such lethality is due, in part, to the inherent heterogeneity of breast cancer disease, posing challenges for clinicians to accurately diagnose and subtype tumors. Recently, RNA‐seq technology has made it possible to subtype and classify tumors using gene expression profiling. In this experiment, we validated the efficacy of using RNA‐seq gene expression technology as a diagnostic tool for breast tumor subtyping in the context of an undergraduate course. Publicly available datasets from the Sequence Read Archive (SRA) of MCF‐7 breast cancer cells were subjected to quality control analysis using the Green Line of DNA Subway (Tuxedo Pipeline, FAST‐X, FAST‐QC, Tophat, and CuffDiff algorithms). Log‐transformed gene expression ratios were subjected to the BiNGO gene ontology algorithm. Significant over‐expression of genes for metabolic and cellular processes pathways included cell death and cell division (p<0.01 Binomial). Significantly down‐regulated pathways included those in development (organ development, etc), regulation (stimulus response, inflammatory response, etc), and signaling (cellular communication, etc; p<0.01 Binomial). Furthermore, using previously established biomarker genes, we subtyped the cells as basal‐like ‐ showing little change in expression for Estrogen Receptor (ER), Her2/neu (HER), or Ki‐67 biomarkers, but a significant over‐expression of the progesterone receptor (q = .0031). Using the Mammaprint suite of biomarkers we diagnosed a highly progressed tumor, wherein 60% and 73% of the Mammaprint mid‐hallmark genes and 38% late hallmarks were differentially expressed. This analysis was corroborated by previous datasets from DNA microarrays. Taken together, our findings suggest that RNA‐seq sequence data can be a valid approach for subtyping human breast cancers, and determining progression of disease.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of the Effects of SULT2A1 Genetic Polymorphisms on the Sulfation of Dehydroepiandrosterone by Human Cytosolic Sulfotransferase SULT2A1
Authors: Abunnaja Maryam , Liu Ming‐Cheh
Published: June 2021

The cytosolic sulfotransferase (SULT) SULT2A1 is known to mediate the sulfation of dehydroepiandrosterone (DHEA) as well as many other sterols and steroids. The present study was designed to investigate how genetic polymorphisms of the human SULT2A1 gene may affect the sulfation of DHEA. Online databases were comprehensively searched to identify human SULT2A1 single nucleotide polymorphisms (SNPs). Of the 98 SULT2A1 non‐synonymous coding SNPs identified, seven were selected for further investigation. Site‐directed mutagenesis was used to generate cDNAs encoding these seven SULT2A1 allozymes, which were expressed in BL21 E. coli cells and purified by glutathione‐Sepharose affinity chromatography. Enzymatic assays revealed that purified SULT2A1 allozymes displayed differential sulfating activity toward DHEA. Kinetic analyses showed further differential catalytic efficiency and substrate affinity of the SULT2A1 allozymes, in comparison with wild‐type SULT2A1. These findings provided useful information concerning the effects of genetic polymorphisms on the sulfating activity of SULT2A1 allozymes.

Support or Funding Information

This work was supported in part by a grant from National Institutes of Health (Grant # R03HD071146).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Binding of 4‐hydroxy‐2‐nonenal Decrease Non‐enzymatic Methemoglobin Reducing Capacity
Authors: Wilson Parker , Mowery Colin , Ramanathan Ranith
Published: June 2021

Lipid peroxidation can produce reactive primary and secondary oxidation products. Various studies have shown that secondary lipid oxidation products, such as 4‐hydroxy‐2‐nonenal (HNE), can bind with proteins and minimize their functional properties. Hemoglobin is the primary oxygen‐carrying protein and can exist in three different forms; oxyhemoglobin, deoxyhemoglobin, and methemoglobin. Oxyhemoglobin/deoxyhemoglobin can carry oxygen, while the oxidized form, methemoglobin, cannot. Physiologically, methemoglobin can be converted to deoxyhemoglobin by enzymatic and non‐enzymatic reduction. Studies have shown that HNE can preferentially bind with histidine and lysine residues within a protein by Michael addition; Hemoglobin has several histidine and lysine residues in alpha and beta structures. However, limited studies have determined the effects of HEN binding on non‐enzymatic methemoglobin reducing capacity. The overall goal of this study was to determine the effects of HNE on non‐enzymatic methemoglobin reducing capacity. Equine hemoglobin (0.1 mM) was pre‐incubated with HNE (0, 0.1, and 0.7 mM) at pH 7.4 and 37 °C for 5 hours to promote covalent binding of HNE to methemoglobin. Following pre‐incubation, methemoglobin was combined with methylene blue and EDTA. The non‐enzymatic reduction was initiated by the addition of NADH and the reduction of methemoglobin was monitored using a UV‐Vis spectrophotometer. Methemoglobin reduction was expressed as nanomoles of methemoglobin reduced per min. The experiment was replicated three times. Pre‐incubation of HNE decreased (p < 0.05) non‐enzymatic methemoglobin reducing capacity. There were concentration‐dependent effects of HNE on non‐enzymatic reduction. Methemoglobin reduction was decreased (p < 0.05) by 17% for 0.1 mM HNE compared with control methemoglobin, however, there was 60% decrease (p < 0.05) in non‐enzymatic methemoglobin reducing activity with 0.7 mM HNE. Lipid peroxidation in various physiological and pathological conditions can increase HNE level in tissue and plasma. The current study suggests that HNE can decrease non‐enzymatic hemoglobin reduction.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Subcellular Distribution of the Dopamine Transporter Phosphorylated on Threonine 53
Authors: Gilchrist Thomas J. , Gaarder Zachary S. , Hovde Moriah J. , Vaughan Roxanne A. , Foster James D.
Published: June 2021

Dopamine (DA) is a catecholamine neurotransmitter, which controls many physiological and psychological processes including reward, motivation, attention, and motor activity. Disruption of DA homeostasis has been implicated in a number of neurological diseases such as attention‐deficit hyperactivity disorder (ADHD), schizophrenia, depression, addiction, and Parkinson's disease. The dopamine transporter (DAT) is a plasma membrane protein that functions to move extracellular dopamine back into presynaptic neurons and is the primary means by which DA is cleared from the extraneuronal space. DAT is also a site of action for psychostimulant drugs such as cocaine and amphetamines, as well as therapeutic drugs such as methylphenidate and bupropion that are used to treat ADHD and depression respectively. Studying DAT function and its regulatory elements is crucial to better understand the pathophysiology of dopaminergic disorders, and to identify and improve treatments for these disorders. DAT activity is controlled by post translational modifications including ubiquitination, palmitoylation and phosphorylation. Phosphorylation occurs on two domains within the DAT N‐terminus: a protein kinase C (PKC) domain at the distal end containing serine 7 within a serine cluster and a proline‐directed mitogen activated protein kinase (MAPK) site at threonine 53 proximal to transmembrane domain 1. We have recently developed an antibody that specifically recognizes pT53 DAT and have utilized this tool to further investigate the functional importance of DAT T53 phosphorylation using fluorescent immunocytochemistry and confocal microscopy to determine subcellular localization of pT53 DAT in comparison with a pan antibody for total DAT (MAB16). Our preliminary results using two cell models (LLC‐PK1 and N27 neurons) expressing rat DAT suggest that pT53 DAT is found at the plasma membrane and extensively throughout the cytoplasm displaying a distinct and intense punctate pattern while fluorescence detected with the pan MAB16 antibody was most intense at the plasma membrane with some intracellular punctate staining. These initial studies create a foundation for additional studies that will include fluorescent subcellular markers and treatments that modulate DAT phosphorylation at threonine 53 which may further our understanding of the functional relevance of DAT phosphorylation at this site and provide potential insight for involvement of dysregulation of DAT phosphorylation in dopaminergic disorders.

Support or Funding Information

Support: National Institutes of Health grants DA 031991 (JDF), DA13147 and 5P20‐104360 (RAV), P30‐GM103329 (UND), P20‐GM12345 (UND)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of Mesenchymal Stem Cells on Qualitative and Quantitative Characteristics of Energy Transformation in Muscle Tissue of Lower Extremity in Ischemia.
Authors: Shypilova Inna , Gami Rakesh
Published: June 2021

Energy transformation required for the performance of multiple energy dependent functions of the body, i.e. mechanical, chemical, and electrical, is important part of cell activity. Energy dependent functions become slower at intracellular level due to decreased ATP by 10–15% and stops completely with 25–30% decrease in ATP in the cell. Adenylic nucleotide concentration measures the mismatch between the energy demand and energy production in the cell's mitochondrial oxidative phosphorylation.

A study was done with pubescent Wistar rats with body mass of 160–200 grams. The ischemia of the lower extremities was achieved by ligation of femoral artery. Bone marrow was taken from femoral, humoral and tibial bones. Cell culture was grown in Eagle medium IGLA MEM, enriched with L‐glutamine, 10% calf embryo serum with antibiotics added and 5 million of cell were injected intramuscularly in the experimental animals. The concentration of ATP, ADP and AMP in the gastrocnemius muscle measured by means of thin‐layer filtration on silica gel plates on 3rd, 5th, 7th, 14th, 21st and 28th day after injection of mesenchymal stem cells (MSC).

The concentration of ATP decreased significantly in the control group after ischemia in consecutive study on specified days. It has been proved that MSC ejection redistributes the pool of adenylic nucleotides towards the increased ATP biosynthesis, so that such parameters of energy metabolism as energy charge, energy potential, thermodynamic control of respiration are increased and the correlation between the components of adenylate kinase reaction is decreased.

This study showed that the ability of MSC provide protective action on energy homeostasis by means of the synthesis of large amount of cytokines and growth factors which stimulate the synthesis of glycolytic and Krebs cycle enzymes, increase glucose utilisation and ATP synthesis. Thus, activation of ATP synthesis restores the level of ATP and high energy phosphate bonds.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational studies on HdeA and its pH‐Dependent Activation
Authors: Pacheco Sayuri , Ravinder Abrol
Published: June 2021

HdeA is a chaperone protein found in pathogenic gram‐negative bacteria such as E. coli that forms homodimers, provides bacterial stress response and acid resistance. HdeA is inactive in its dimeric form and becomes activated in low pH environments such as the mammalian stomach. In low pH environments it binds to other periplasmic proteins protecting them from aggregation and denaturation. Computational biophysical methods such as constant pH molecular dynamics in explicit and implicit solvent are utilized to couple protein dynamics and protonation states in order to reveal a plausible unfolding mechanism for HdeA. Our computational models are providing insight into the unfolding mechanisms for HdeA. The structural models have shown important interactions, some of which have not yet been observed in experimental structures, which can potentially stabilize the monomers at neutral pH while destabilizing and triggering partial unfolding in acidic pH conditions.

Support or Funding Information

Building Infrastructure Leading to Diversity (BUILD) # 5TL4GM118977

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Ghrelin sensitises colonic myenteric neurons to the neurostimulatory effects of glucagonlike peptide‐1 in Sprague Dawley and Wistar Kyoto rats.
Authors: O'Brien Rebecca , Buckley Maria M. , O'Malley Dervla
Published: June 2021

Irritable Bowel Syndrome (IBS) is a common functional bowel disorder affecting approximately 10–20% of the population with many experiencing post‐prandial exacerbation of symptoms. Although IBS pathophysiology remains elusive, dysfunctional endocrine signalling has been implicated. Ghrelin is an orexigenic hormone which peaks prior to food ingestion. Glucagon‐like peptide‐1 (GLP‐1) is secreted by L‐cells in the mucosa of the small and large intestine in response to the arrival of nutrients. Previous studies have shown that ghrelin directly primes L‐cells to secrete GLP‐1 and increases GLP‐1 release (Gagnon et al., 2014). The aims of this study were to investigate if ghrelin sensitises colonic enteric neurons for GLP‐1 mediated signalling in two animal models; Sprague Dawley (control) and Wistar Kyoto (stress model of IBS).

Immunofluorescence was used to stain for ghrelin and GLP‐1 receptors. Colonic motility was assessed in organ baths, using SD and WKY colonic segments, in response to a GLP‐1 mimetic before and after ghrelin stimulation.

Ghrelin and GLP‐1 receptors were expressed in discrete co‐localised clusters in myenteric neurons. GLP‐1 reduced contractile activity in both SD and WKY rat colons. However, this response was potentiated by prior exposure to ghrelin in both circular and longitudinal muscle in Sprague Dawley ((p=0.0116), (p=0.0093), respectively) and Wistar Kyoto ((p= 0.0129), (p=0.0006), respectively) rat colonic segments.

Ghrelin sensitises the colon to GLP‐1‐evoked contractile activity. Although similar mechanisms of action were evidence in both species, the baseline contractile activity was aberrant in WKY rats.

Support or Funding Information

Funded by UCC TRAP funding and the Department of Physiology, University College of Cork

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular Basis of Hypoxia‐Induced Excessive Erythrocytosis of High Altitude
Authors: Azad Priti , Haddad Gabriel
Published: June 2021

Rationale

Since polycythemia is a predominant trait in some high altitude dwellers (Chronic Mountain Sickness, CMS, or Monge's disease) but not others living at the same altitude in the Andes, we took advantage of this human “experiment in nature” and studied both populations (with CMS and without, non‐CMS).

Methods

In order to understand the molecular basis for polycythemia of high altitude, we generated a disease in‐the dish‐model by re‐programming fibroblasts from CMS and non‐CMS subjects. In the past, we have discovered that there are selective sweeps that seem to be statistically different between CMS and non‐CMS and hence contain candidate genes. In this study, by manipulating expression of these candidate genes (Eg. SENP1, ARID1B) in our in‐vitro model system, we delineate the molecular mechanism(s) of the CMS polycythemia..

Results

As compared to sea level controls who responded to hypoxia by increasing their RBCs modestly, CMS cells increased theirs remarkably (up to 60 fold) with a dose‐dependent response to graded hypoxia (1.5, 5, 10% O2). Non‐CMS cells showed a blunted increase in RBC production under hypoxia. In order to understand the genetic basis of these phenotypes, we knocked down SENP1 (a desumoylase) in CMS iPS cells using lentiviral constructs and observed a striking reduction (>90%) of the CMS excessive erythropoietic response to low O2. Similarly, by knocking down ARID1B in non‐CMS we observed that RBC formation increased significantly under hypoxia.

Conclusion

Our results demonstrate the presence of a regulatory system (SENP1 and ARID1B) that is critical in erythropoiesis and that is out of balance in the CMS subjects.

Further, by combining iPS technology and genomics, we believe that we will be able to determine a comprehensive model of the basis for hypoxia‐induced polycythemia.

Support or Funding Information

This study is funded by National Institutes of Health (NIH) grants (1P01HL098053 and 5P01HD32573) to G.G. Haddad

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