bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of Dietary Intakes and Supplement Use in Elite Paralympic Athletes
Authors: Madden Robyn Frances , Shearer Jane , Parnell Jill
Published: June 2021

The Paralympic Games are highly competitive and athletes need to maximize performance, one crucial aspect of which is nutrition. Dietary intakes and supplement use in high‐performance, athletes with physical disabilities remain largely unexplored and specialized recommendations are lacking. Consequently, these athletes may be at risk for nutritional deficiencies and supplement misuse. The aim of this study is to assess nutritional intakes, dietary supplementation patterns, and sources of dietary supplement information in Canadian athletes with physical disabilities. Male (n=18) and female (n=22) athletes were recruited from various Paralympic sports across Canada through sporting organizations, coaches, and social media. Males had significantly greater total energy intakes than females (male=2092 kcal/day; female=1602 kcal/day, p=0.013). Males had greater protein intakes based on body weight than females (p=0.0017), while fat and carbohydrate intakes were similar in both genders. Females consumed a significantly greater percentage of their total calories from sugar as compared to males (p=0.049). Athletes' intakes met or exceeded the majority of recommended daily allowances (RDA) for vitamins and minerals, with the exception of vitamin D, vitamin E, and magnesium. Males did not meet the RDA for folate and vitamin A, while females did not meet the RDA for iron and calcium. Athletes did not meet the AI for pantothenic acid and potassium. Females had significantly lower percent of the recommendations for calcium (p=0.031), selenium (p=0.016), and iron (p<0.001) as compared to males. Supplement use is high in this population; 100% of males and 91% of females reported supplement use within the past three months. Males most regularly used sports bars, protein powder, and sport drinks, while females most regularly used vitamin D, protein powder, and fatty acids. Significant differences were noted in branched chain amino acids, as males were more likely to use this supplement (p=0.008). The top three reasons for taking supplements were “stay healthy,” “increase energy,” and “medical.” Sources of dietary supplements were reported as dietician/nutritionist, medical physician (doctor), and athletic trainer. In conclusion, elite Paralympic athletes tend to consume diets that are adequate in most macro‐ and micronutrients with a high usage of dietary supplements. Future studies should evaluate impairment levels on energy and macronutrient intakes to work towards recommendations and to adequately educate and support these athletes.

Support or Funding Information

Funded by a Mount Royal University Innovation Grant (J.P.) and the National Science and Engineering Research Council of Canada (J.S.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Soluble Guanylate Cyclase Stimulators and Activators Attenuate Placental Production of sFlt‐1
Authors: Bakrania Bhavisha A. , Travis Adam B. , Granger Joey P.
Published: June 2021

Preeclampsia (PE) is a pregnancy specific disorder associated with maternal hypertension due to widespread endothelial dysfunction. Placental ischemia leads to increases in the circulating anti‐angiogenic factor, sFlt‐1 and a PE‐like phenotype. PE and chronic sFlt‐1 excess is also associated with depletion of nitric oxide (NO), which, during normal pregnancy, binds to soluble guanylate cyclase (sGC), and synthesizes cGMP, to facilitate vasodilation.. In fact, studies in reduced uterine perfusion pressure (RUPP) model of placental ischemia demonstrate that restoring the angiogenic imbalance, administration of placental growth factor or VEGF abolishes hypertension. A novel drug, sGC stimulators and activators, which increase sGC activity independently of NO, are currently in clinical trials for the treatment of hypertension, however, whether this drug could be beneficial during PE is not known. We tested the hypothesis that sGC stimulators (Riociguat, Sigma‐Aldrich) and activators (Cinaciguat, Sigma‐Aldrich) attenuate sFlt‐1 production by the placenta. Placentas were isolated from normal pregnant rats on gestational day 19. Villous bundles were excised and plated in 24‐well plate coated with Matrix Matrigel Basement Membrane. Increasing concentrations of each the sGC stimulator and activator (0.1 μM, 1 μM, 10 μM, 30 μM) were added to the media for treatments. The explants were then exposed to a hypoxic environment (1% oxygen) to determine whether sFlt‐1 production, directly by the placenta, is attenuated by sGC stimulators and activators in a dose‐dependent manner. Interestingly, each of the doses administered significantly reduced sFlt‐1 production in response to hypoxia compared to the untreated group. However, the 30 μM sGC activator treatment produced the most profound effect (Untreated, n=6, 4699±942 pg/mL; 30 μM sGC stimulator‐treated, n=6, 3647±1385 pg/mL, 30 μM sGC activator‐treated, n=6, 3247±1402 pg/mL; P<0.01). The results of this study demonstrate that increasing sGC activity attenuates sFlt‐1 production from the placenta and could improve the angiogenic imbalance seen in this condition. In conclusion, these findings suggest there is a therapeutic potential for treating preeclampsia with sGC activators

Support or Funding Information

Funding source: NIH HL51971

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 Effects of Oral Contraceptive Use on Ergoreflex control of Blood Pressure and Heart Rate during Steady‐State Cycling in Healthy, Young Women
Authors: Chantigian Daniel , Asirvatham‐Jeyraj Ninitha , Keller‐Ross Manda
Published: June 2021

Background

Ergoreflex, activation of group III/IV skeletal muscle afferents, is an important regulator of mean arterial pressure (MAP) and heart rate (HR) during exercise. Oral Contraceptive (OC) use in women has been shown to increase resting BP, but the effect of OC use on the ergoreflex is unclear.

Objective

Determine if OC use causes an exaggerated MAP and HR response with ergoreflex activation in young, healthy women.

Methods

Twelve healthy women (mean ± SD; 24±2 yrs, 7 taking OCs) attended 2 experimental sessions; one during early follicular (low estrogen, days 1–4) and one during ovulation (high estrogen, days 10–14). Each session included 1) 3 minutes of light‐intensity steady‐state cycling (60 W at 65–70 rpm) and 2) 3 randomized, subsystolic circulatory occlusions applied bilaterally to the proximal thigh at 20, 60, and 100 mmHg of pressure to increase activation of group III/IV skeletal muscle afferents (ergoreflex). Occlusions lasted 2 min and were separated by 5 min of deflation with 2 min of recovery at the end (rest). MAP and HR were continuously monitored via non‐invasive blood pressure (Nexfin, Edwards Lifesciences, Irvine, CA) and a 3‐lead ECG (AD Instruments, Colorado Springs, CO), respectively.

Results

Data did not differ significantly between women in the follicular or ovulatory phases; thus, the ovulatory phase data was used for comparisons. HR increased from rest to exercise more with women in the ovulatory phase (68±14 to 130±12 bpm) as compared with women taking OCs (69±14 to 112±10 bpm, exercise *OC interaction: p=0.02). However, HR increased from baseline exercise with vascular occlusion (condition: p<0.01) similarly in women in the ovulatory phase and women taking OCs (20 mmHg: 3±9% vs. 1±2%, 60 mmHg: 4±3% vs 8±4%, and 100 mmHg: 9±17% vs. 20±8% respectively, condition*OC interaction: p=0.14). MAP increased from rest to exercise similarly in women in the ovulatory phase (83±8 to 92±14 mmHg) as compared with women taking OCs (88±7 to 92±8 mmHg, exercise*OC interaction: p=0.08). MAP increased from baseline exercise with vascular occlusion (condition: p=0.01) similarly in women in the ovulatory phase as compared with women taking OCs (20 mmHg: 7±19% vs. 2±8%, 60 mmHg: 15±16% vs. 2±9%, and 100 mmHg: 8±18% vs. 28±19%, condition*OC interaction: p=0.20).

Conclusion

Previous studies have implicated that blood pressure tends to be greater with OC use, this study suggests that OCs do not cause an exaggerated increase in MAP during exercise or activation of group III/IV afferents. OCs attenuate the HR response to exercise, but does not appear to affect the HR response to group III/IV afferent activation.

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 response of vagal motoneurons to brainstem oxytocin stimulation depends on sex and stress levels.
Authors: Jiang Yanyan , Zimmermann Julia , Travagli R. Alberto
Published: June 2021

Stress maladaptation plays a pivotal role in the development and exacerbation of functional gastrointestinal disorders (FGID), including impaired gastric emptying and decreased gastric motility, both of which are more prevalent in women. The prototypical anti‐stress hormone, oxytocin (OXY), has been shown to modulate GI tone and motility via vagal pathways. We have shown previously that in naïve male rats, OXY modulates the critical GABAergic synapse projecting to gastric‐projecting preganglionic vagal (DMV) neurons only after pharmacological increase of cAMP levels by forskolin or CRF pretreatment, suggesting a stress‐induced plasticity of brainstem vagal neurocircuits.

The aim of this study was to test the hypothesis that the response of vagal motoneurons to brainstem oxytocin application depends on sex and stress adaptation or resilience. Age‐matched male and female SD rats were subdivided in the following groups: i) control; ii) chronic repetitive homotypic (restraint) stress, rats in this group show adaptation to stress; iii) chronic repetitive heterotypic (restraint, forced swim, water avoidance and cold) stress, rats in this group do not show adaptation to stress. Female rats were further subdivided according to their estrogen levels (high: pro‐estrus and estrus; low: meta‐ and di‐estrus). Following 5‐days of stress, whole cell patch clamp recordings were made from thin brainstem slices to examine GABAergic miniature inhibitory postsynaptic currents (mIPSCs) in medial, presumably gastric‐projecting, DMV neurons.

Our results, see table, indicate that 1) in male rats OXY modulated inhibitory neurotransmission only in stressed rats; 2) in female rats, OXY modulated inhibitory neurotransmission independently of estrogen or stress load. 3) OXY effects on mIPSC frequency, however, is different when rats are exposed to heterotypic stress load. Taken together, our data support the hypothesis that the response of vagal motoneurons to brainstem oxytocin stimulation depends on sex and stress levels and suggest that, independently of estrogen or stress, female rats have a response similar to that of stressed males.

Support or Funding Information

NIH DK 55530

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Brain‐Heart Coupling Response during Auditory Startle
Authors: Wang Xiaoni , Luan Yewei , Dang Shijie , Li Mengjun , Zhang Jianbao
Published: June 2021

Objective

Previous clinical observations have revealed the possible relation between auditory startle stimulation and susceptibility to ventricular fibrillation, which cause 10% of sudden deaths without structural abnormalities of the heart. The objective of the study was to investigate the impact of auditory startle on time‐varying autonomic reactivity and cerebral‐cardiovascular coupling.

Methods

Startle reflexes in the study were induced by auditory stimulation of 100 db white noise lasting 100 ms. 25 healthy male subjects were recruited. Electrocardiogram (ECG), beat‐to‐beat blood pressure and electroencephalography (EEG) were measured simultaneously and continuously during baseline and startle task. Further, RR intervals (RRI), systolic and diastolic blood pressure (SBP, DBP) and mean magnitude of auditory‐evoked alpha and beta potential oscillations were calculated. Continuous wavelet transformation of RRI was used to describe the time‐variant patterns of autonomic neural activities. Pearson correlation and cross‐correlation were used to estimate the coupling between evoked oscillation and RRI, as well as between RRI and SBP.

Results

Acute response of auditory startle was characterized by instant heart rate (HR) (0–1.5 s) decrease and subsequent (1.5–4 s) increase, tardive SBP elevating (3.5–7 s) and recovery (>7 s), accompanied with acute auditory‐evoked alpha and beta potential oscillations. Magnitude of evoked alpha oscillations in electrode FP1 and FP2 coupled with decreased HR in 0–1.5 s and increased HR (1.5–4 s) coupled with evoked beta oscillations magnitude in FT8, T8 and TP8. Moreover, directional influence from RRI to SBP was disrupted while baroreflex was enhanced in startle reflex. Therefore, it can be indicated that auditory startle evoked excitation of cochlear nerve is transmitted ascendingly into brainstem and affects cardiovascular system through three pathways: 1) inducing the descending activation of spinal sympathetic centers; 2) a further auditory radiation of ventral medial prefrontal cortex (vMPFC), which is related to parasympathetic activation; and 3) anterior and posterior temporal lobe, which is the cardiovascular sympathetic center. The excitement of three pathways above occurs almost simultaneously. However, the fact that parasympathetic nerves exert their effects on cardiovascular system more rapidly than sympathetic nerves accounts for the earlier decrease and subsequent increase of HR. Coupling of RRI and SBP predominantly contributes to HR and SBP changes after 5 s.

Conclusion

The results of the study confirmed the connection of cortex and cardiovascular system. Acute startle related cardiovascular responses is coupled with the startle evoked EEG oscillations while subsequent responses may be induced by RRI and BP coupling.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Consequences of postnatal myo‐inositol supplementation on respiratory control and nucleus tractus solitarius (nTS) neurons
Authors: Mayer Catherine A. , Difiore Juliann M. , MacFarlane Peter M.
Published: June 2021

Myo‐inositol is a naturally occurring sugar derivative formed endogenously from the breakdown of glucose; it is also found in a variety of foods including breast milk and is commercially available as a nutritional supplement. Serum levels of myo‐inositol in infants decrease naturally over the first few postnatal weeks; prior studies in preterm infants have demonstrated improved pulmonary function, reduced incidence of respiratory distress, and increased survival rates following myo‐inositol supplementation. In support of a potential beneficial effect of myo‐inositol supplementation on respiratory control, we have shown previously that myo‐inositol treatment over the first 2 postnatal weeks enhances the acute hypoxic ventilatory response (HVR). In the present study, we tested the hypothesis that the enhanced HVR following myo‐inositol treatment would be associated with increased excitability of neurons within the area of brainstem chemoafferent integration, the nucleus tractus solitarius (nTS). Mouse pups received subcutaneous injections of myo‐inositol (80 mg/kg/day) for the first two postnatal weeks. Whole cell patch clamp recordings were then used to examine the excitatory properties of nTS neurons monosynaptically connected to the solitary tract. Compared to saline treated mice, myo‐inositol exposure increased the rheobase and the failure rate of nTS neurons, and decreased the paired pulse ratio following 20 Hz stimulation. Spontaneous activity (EPSC frequency, area, rise time, decay time), and basic neuronal properties (membrane resistance, capacitance, resting membrane potential) did not differ between groups. In conclusion, and contrary to our hypothesis, the enhanced HVR following postnatal myo‐inositol treatment was associated with decreased excitability of nTS neurons. We speculate the reduced nTS neuron excitability may be a compensatory response to increased chemoafferent (presumably the carotid body) inputs during hypoxia. These data may be important to our understanding of how myo‐inositol supplementation could influence development and function of brainstem respiratory neural control regions and may have implications for the pathophysiology of various respiratory morbidities in preterm infants.

Support or Funding Information

The Gerber Foundation

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chemoreflex Responses to LPS Exposure During a Critical Window of Development in the in situ Arterially Perfused Working Heart Brainstem Preparation
Authors: Kola Gjinovefa , Chadha Priyal , Mayer Catherine A. , Dick Thomas E. , Dutschmann Mathias , Lewis Stephen J. , MacFarlane Peter M.
Published: June 2021

The second week of postnatal life represents a critical window of brainstem cardio‐respiratory neural development in rats. We showed previously that endotoxin (lipopolysaccharide, LPS) exposure during this critical period of development (specifically at P10 days of age) attenuated the acute hypoxic ventilatory response and increased brainstem cytokine mRNA expression; these effects of LPS were also associated with an unexpected increase in mortality. These data suggest there may be a critical window of development during which the (cardio)‐respiratory control system exhibits a heightened vulnerability to inflammatory challenges. The potential impact of LPS exposure on neural control of cardio‐respiratory function during this critical window of development is unknown. We used the in situ arterially perfused working heart brainstem preparation to investigate how LPS affects central nervous control of respiratory and autonomic function in P10 rats. Two hours after an intraperitoneal injection of LPS (0.1mg/kg, n=24), rats were surgically prepared for chemoreceptor mediated sympathoexcitation by measuring phrenic, vagal, and thoracic sympathetic nerve responses to an intra‐arterially injection of sodium cyanide (75 μl, 0.03% NaCN). Rats were also cannulated for measurements of heart rate and arterial/perfusion pressure. Compared to saline treated controls, LPS decreased baseline phrenic nerve burst frequency by 27% and increased vagal nerve activity by 76% (peak amplitude). Baseline thoracic sympathetic nerve activity, heart rate and arterial perfusion pressure were similar between treatment groups. Although NaCN increased respiratory frequency and vagal nerve activity in both groups, the frequency response was attenuated by 35% in LPS treated rats. The magnitude of the thoracic sympathetic nerve and bradycardic response to NaCN was not affected by LPS treatment. These data indicate that LPS depresses baseline respiratory network function and impairs vital respiratory response to arterial chemoreceptor activation. In conclusion, the present data suggest that inflammatory challenges during a critical period of development trigger potentially fatal instabilities in respiratory network function.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aspects of Respiratory Control in the Dystrophin‐deficient mdx Mouse
Authors: Burns David P. , O'Driscoll Eabha , Edge Deirdre , Lucking Eric F. , O'Halloran Ken D.
Published: June 2021

Duchenne muscular dystrophy (DMD) is a fatal neuromuscular disease characterised by skeletal muscle weakness which extends to the respiratory musculature. Although deficits in the diaphragm muscle of dystrophin‐deficient mdx mice are well described, there is a paucity of information pertaining to central control of breathing in dystrophinopathies. We have previously reported hypoventilation in mdx mice with reserved capacity to increase ventilation in response to chemoactivation (Burns et al., 2017). Systemic and muscle inflammation are reported in mdx mice and human DMD. We sought to examine aspects of respiratory system control in the mdx mouse model of DMD including electrical activity of the diaphragm muscle and markers of inflammation in putative phrenic motor nuclei.

Young adult (8 week old) wild‐type (WT) and mdx mice were studied. Inspiratory (Ti) and expiratory (Te) durations were assessed in conscious WT and mdx mice using whole‐body plethysmograpghy. Diaphragm EMG activity was assessed in WT and mdx mice using fine concentric needle electrodes in anaesthetised spontaneously breathing mice. Basal EMG and responsiveness to chemoactivation were determined. Diaphragm muscle function was examined ex vivo. Cervical spinal cord (C3–C5 region) segments of WT and mdx mice were examined for immunohistochemical and gene expression analyses.

Poincaré analysis of Ti and Te revealed no evidence of perturbed respiratory rhythm in young mdx mice based on short‐ and long‐term variability of breathing. Diaphragm muscle force was severely depressed in mdx diaphragm within the frequency range of both ventilatory and airway protective behaviours. Mdx diaphragm had potentiated EMG activity during chemoactivation compared to WT mice. Dystrophin mRNA is expressed in the C3–C5 region of WT spinal cords and expression is significantly reduced in mdx mice, with no indication of altered inflammatory gene expression. Putative phrenic motornuclei in mdx mice had unaltered expression of microglia and astrocytes compared to WT.

Our study revealed a potentiated neural drive to breathe in mdx mice during chemoactivation suggesting compensatory neuroplasticity facilitating respiratory motor output. Although systemic and muscle inflammation are present in mdx mice, there is no evidence of neuroinflammation in the cervical spinal cord (C3–C5) of young mdx mice. If neuroplasticity is a feature of DMD, it may be possible to boost motor facilitation of breathing through safe interventions and thus preserve or limit deficiencies in respiratory capacity.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Activation of Respiratory Muscles during Lower Thoracic High Frequency Spinal Cord Stimulation (HF‐SCS)
Authors: Kowalski Krzysztof E. , Romaniuk J Richard , Pawlowski Gary , DiMarco Anthony F.
Published: June 2021

Objective

Lower thoracic HF‐SCS (500 Hz, 1 mA) applied via a single stimulating electrode located on the dorsal surface of the spinal cord at the T9 level results in large positive airway pressures consistent with marked expiratory muscle activation. The purpose of the present study was to evaluate the effects of lower thoracic HF‐SCS on the ventral surface at this spinal level.

Methods

In 4 anesthetized dogs, HF‐SCS was applied via a stimulating electrode (4 mm disc) positioned on the ventral surface of the spinal cord at the T9 level. Airway pressure generation was monitored before and following spinal sections at the C8 (to eliminate potential diaphragm activation) and subsequently at the T6 (to eliminate potential intercostal muscle activation) levels.

Results

HF‐SCS (500 Hz, 1 mA) on the ventral surface, results in inspiratory airway pressure generation of 40 ± 3 cmH2O. Following complete C8 spinal section, HF‐SCS results in a significant reduction in negative airway pressure generation to 14 ± 1 cmH2O (p<0.05). Subsequent section at the T6 level, HF‐SCS results in a positive airway pressure of 10±1 cmH2O (p<0.05 when compared to before either section).

Conclusion

In contrast to dorsal stimulation, lower thoracic ventral HF‐SCS (500 Hz, 1 mA), at T9 results in activation of pathways with connections to both the phrenic and inspiratory intercostal motoneuron pools resulting in the generation of the large negative airway pressures and minimal expiratory muscle activation. Under the experimental conditions of this study, neuronal pathways with connections to the inspiratory and expiratory motoneuron pools are each limited anatomically to specific regions of the spinal cord. These anatomic differences allow for separate activation of the inspiratory and expiratory muscles to restore function.

Support or Funding Information

Support: Merit Review Award I01‐RX‐001488 from the U.S. Department of Veterans Affairs, Rehabilitation Research and Development Service.

Disclosure: Dr. DiMarco holds two U.S. patents for technology related to the content of this abstract: Method and Apparatus for Electrical Activation of the Expiratory Muscles to Restore Cough (5,999,855) and Bipolar Spinal Cord Stimulation to Activate the Expiratory Muscles to Restore Cough (8,751,004).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Mouse Model of a Human Mutation in NKCC1 Confirms its Mistargeting in Epithelia
Authors: Koumangoye Rainelli , Omer Salma , Delpire Eric
Published: June 2021

A 15‐year old patient with complete gastrointestinal and bladder failure, endocrine insufficiencies, and orthostatic intolerance carries a de novo mutation in SLC12A2, the gene encoding the Na‐K‐2Cl cotransporter‐1 or NKCC1. The 11 bp deletion leads to truncation of the cytosolic COOH‐terminal tail of the cotransporter making it non‐functional. Expression of the mutant transporter increases the amount of dimerization, suggesting the possibility that the mutant transporter might exert dominant‐negative effects on the wild‐type transporter. In polarized MDCK cells grown on permeable filters or in 3‐D culture cysts, most of the mutant transporter signal is observed on the apical membrane, in contrast to the basolateral expression of wild‐type cotransporter. The mutant transporter affects the formation of MDCK cysts, as we observed the presence of multiple lumens, as opposed to single lumen formation with wild‐type cells. The mutant transporter also causes trafficking of the wild‐type cotransporter to the apical membrane.

To assess the relevance of this observation in vivo, we created a mouse model that recapitulates the human mutation. Using CRISPR/cas9, a guide RNA that targets cas9 to exon 22 of the Slc12a2 gene, and a repair oligonucleotide that deletes 11 bp from the exon, we successfully generated a female founder animal that carried the designed mutant allele in addition to a non‐homologous end joining mutation in the second allele. After one backcross that segregated the two alleles, the line was further established by backcrossing for 2 additional generations in C57BL/6J. This mouse now carries the 11 bp deletion mutant allele and a wild‐type allele. We examined the expression of NKCC1 in the salivary gland and the intestine, two Cl− secreting epithelia where the wild‐type cotransporter is typically expressed on the basolateral membrane. In both tissues, intense apical staining was observed, confirming the data obtained with MDCK cells. Preliminary studies indicate that the mouse has no overt adverse phenotype, although the mouse lives in a pathogen‐free barrier facility and has not been challenged.

In conclusion, our data demonstrate that expression of a truncated Na‐K‐2Cl cotransporter causes mistargeting of the wild‐type cotransporter to the apical membrane of Cl− secreting epithelia and affects proper lumen formation in MDCK cysts. These observations may explain the multisystem dysfunction that is observed in the patient.

Support or Funding Information

Supported by NIH grants R21GM118944 and 1RO1DK093501.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phenotypic Differences Between Tissues and Sex Observed in Mice with Human Gout Causing ABCG2 Variant
Authors: Hoque Kazi Mirajul , Lewis Raychel M. , Woodward Owen M.
Published: June 2021

Hyperuricemia is a risk factor for gout and other complex human disorders. We have established in humans that ABCG2 codes a high‐capacity uric acid (UA) efflux transporter, that when dysfunctional causes hyperuricemia and significantly increases the risk for gout. Recent work has demonstrated ABCG2 mediated secretion is important for intestinal excretion of UA, so to better understand the intestinal contribution of ABCG2 dysfunction to hyperuricemia and gout, we created a new model where a Q140K (human Q141K) point mutation in the Abcg2 gene was made using CRISPR Cas9 gene editing techniques on a C57BL/6 mouse background. We employed both western blot and confocal microscopy to generate profiles of ABCG2 in both sexes and multiple tissues and assessed the differences of luminal UA excretion using mouse intestinal closed loop method. We observed in the wild type mice ABCG2 expression varies along the GI tract with the highest levels in the jejunum and ileum, and with low levels in the colon of both male and female mice. The localization of the wild type ABCG2 protein was exclusive to the brush border of villus cells and luminal membrane of the crypt cells. In contrast, one or two copies of the Q140K Abcg2 allele resulted in significant decreases (53% and 88% respectively) in total intestinal ABCG2 expression and apical membrane staining in villus cells. In female mice, the Q140K ABCG2 mutation resulted in little difference of ABCG2 expression, similar to the mostly unaltered expression of ABCG2 in the renal cortex of both male and female Q140K ABCG2 mice. Concomitant with the protein abundance defect, Q140K ABCG2 male mice had higher serum UA (3.31 ± 0.37 mg/dl) than WT mice (2.13 ± 0.14 mg/dl) and significantly reduced (−40%) acute UA flux in an intestinal loop assay. The reduction in the UA flux in the Q140K loop model was comparable to the reduction observed when the WT intestinal loop was treated with the ABCG2 inhibitor FTC (30%), suggesting the Q140K mutation approximates a complete loss of ABCG2 function in the small intestines. The sex dependence of the pathology of the Q140K mutation suggested that ABCG2 and UA secretion could be under hormonal control. In intestinal loop experiments we found FBS and more specifically angiotensin II modulated UA secretion. We conclude that (i) The Q141K ABCG2 mutation (Q140K in mouse) results in severe loss of ABCG2 mediated UA secretion in the small intestine (ii) sex determines the pathology and abundance of the mutant ABCG2 protein along the intestine and (iii) Small intestines may be the site of hormonal regulation of both ABCG2 and UA secretion.

Support or Funding Information

National Institute of Health RO1 grant (DK114091‐01) & American Hear Association grant: 14SDG18060004

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Tiliacora triandra (Colebr.) Diels Ameliorates Hepatic Steatosis by Inhibition of HMGR and Down‐regulation of FAT/CD36
Authors: Pasachan Tipthida , Ontawong Atcharaporn , Soodvilai Sunhapas , Duangjai Acharaporn , Pongchaidecha Anchalee , Amornlerdpison Doungporn , Srimaroeng Chutima
Published: June 2021

Background & Aims

Tiliacora triandra (Colebr.) Diels (TT) is a native plant of Southeast Asia widely used in northeastern Thai cuisines, and is known in Thai language as Yanang. High concentration of polyphenolic compounds found in TT aqueous extract (TTE) include epicatechin (EC), quercetin (QC), cyanidine (CD), and gallic acid (GA), respectively. TTE has shown various beneficial pharmacological effects such as anti‐inflammatory, anti‐cancer, anti‐aging, anti‐oxidant, anti‐hypercholesterolemia, and inhibition of intestinal cholesterol absorption. However, the effects of TTE on cholesterol synthesis and hepatic steatosis are limited. This study aimed to investigate the direct effects of TTE on cholesterol synthesis and hepatic lipid accumulation in hepatocellular carcinoma (HepG2) cells.

Methods

HepG2 cells at the cell density of 5×105 cells/well were treated with TTE, EC, and QC in a molar ratio for 24 hr. The cytotoxic effect of TTE was evaluated using MTT assay. Hepatic lipid accumulation was quantified by Nile red staining and detected by flow cytometry. The mRNA expression of genes involved in cholesterol synthesis, HMGR, and the fatty acid transporter, FAT/CD36, was also evaluated using quantitative real‐time PCR.

Results

TTE significantly reduced hepatic lipid accumulation up to the dose of 100 μg/ml similarly to that of EC and QC without any cytotoxic effect. This effect also corresponded to the reduction in HMGR and FAT/CD36 gene expressions, resulting in a significant decreased in de novo hepatic cholesterol synthesis and free fatty acid accumulation.

Conclusion

These findings suggest that TTE exhibits lipid‐lowering action by decreasing both hepatic cholesterol synthesis and lipid accumulation, leading to improved non‐alcoholic fatty liver. Thus, TTE could potentially be developed as nutraceutical product for prevention of progressive liver diseases in obesity, diabetes, or metabolic syndrome.

Support or Funding Information

This study is supported by the Faculty of Medicine Endowment fund (093/2560), Chiang Mai University, Research and Researcher for Industry (RRI) by Thailand Research Fund (TRF), (PHDI0009 to CS) and the NSTDA Chair Professor grant (the Fourth Grant) of the Crown Property Bureau Foundation and the National Science and Technology Development Agency to Professor Dr. Vatcharin Rukachaisirikul.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Enhanced expression of colonic non‐heme iron transporters divalent metal transporter‐1 (DMT‐1) and ferroportin (FPN) in patients with ulcerative colitis
Authors: Minor Emily , Kupec Justin , Nickerson Andrew , Rottgen Trey , Rajendran Vazhaikkurichi
Published: June 2021

Iron is an essential micronutrient that is involved in many different physiological processes, such as redox reactions of the electron transport chain, oxygen transport, and DNA synthesis. Additionally, iron is an important limiting factor for microbial growth. The tight regulation of whole body and cellular iron concentration is part of the innate immune system whereby the body can restrict the amount of available iron to help prevent the growth of pathogenic bacteria. This is crucial because microbial dysbiosis is detrimental to health and is thought to play a causative role in the development of ulcerative colitis (UC), a chronic inflammatory bowel disease (IBD) associated with bloody, mucous‐laden stools. Patients with ulcerative colitis (UC) frequently suffer from iron deficiency and may be prescribed oral iron supplementation, but there is fine line between trying to correct the iron deficiency and providing excess iron that can exacerbate disease activity and alter the microbial community. Most iron absorption occurs in the duodenum, but the colon is also known to contain non‐heme iron transporters. The purpose of this study was to characterize the location of iron transporters in normal human colon and determine whether their expression varied in patients with UC. RNA and protein were isolated from colonic biopsy specimens obtained during previously scheduled colonoscopies. We found a trend of decreasing mRNA and protein abundance from the cecum to the distal colon of DMT‐1 and FPN. Patients with ulcerative colitis had increased expression of colonic iron transporters, with the highest expression of those transporters found in patients with active disease activity. The upregulation of iron transporters in the colonic epithelium of patients with UC may have a two‐fold benefit: increased colonic iron absorption would help alleviate the iron deficiency frequently seen in patients with UC and it would limit the amount of available iron in the colonic lumen, thus reducing the growth of pathogenic bacteria.

Support or Funding Information

R01: 10019896.1.1006856R

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Impairment of renal organic anion transport mediated by organic anion transporter 1 (Oat1) and 3 (Oat3) in acute myocardial ischemic and reperfusion injury in rats
Authors: Srimaroeng Chutima , Sirijariyawat Kungsadal , Ontawong Atcharaporn , Palee Siripong , Thummasorn Savitree , Maneechot Chayodom , Boonphang Oranit , Chatsudthipong Varanuj , Chattipakorn Nipon
Published: June 2021

Acute kidney injury (AKI) is frequently associated with acute myocardial infarction (AMI). Kidney plays a crucial role in the clearance of uremic toxins, produced from organ dysfunctions, mainly through organic anion transporters 1 (Oat1) and 3 (Oat3). This study aims to examine the effects of acute myocardial ischemia and ischemic/reperfusion (I/R) on renal transporter functions, and identify the mechanisms involved in the impairment of these transporters. Myocardial ischemia and I/R were induced in rats by either 30‐minute left anterior descending coronary artery occlusion or 30‐minute occlusion followed by 120‐minute of reperfusion, respectively. The renal hemodynamic parameters and renal transporter functions were subsequently determined along with renal biochemical markers. Results showed that myocardial ischemia led to reduction in renal blood flow, perfusion pressure, and kidney function by approximately 40%, while the reperfusion phase partially recovered renal hemodynamic parameters. Moreover, myocardial ischemia and I/R progressively induced renal oxidative stresses and mitochondria dysfunction, all of which directly deteriorated renal Oat1 and Oat3 functions. Thus, this study indicates that myocardial ischemia and I/R conditions leads to a progressively declined renal transport function, which could potentially worsen AKI. Renal Oat1 and Oat3 transporters, therefore, might be potential therapeutic targets for reversing such injury.

Support or Funding Information

This research was supported by the Thailand Research Fund (RSA5980009 to CS), Faculty of Medicine Endowment Fund, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of a new splice variant of large‐conductance Ca2+‐activated K+ (BK) channel α subunit from human chondrocyte
Authors: Suzuki Yoshiaki , Ohya Susumu , Yamamura Hisao , Giles Wayne R. , Imaizumi Yuji
Published: June 2021

Changes in intracellular calcium concentration ([Ca2+]i) modulate chondrogenesis, proliferation, cell death and baseline anabolic and catabolic activities in chondrocytes. Under pathophysiological conditions, like osteoarthritis (OA), histamine release from resident mast cells enhances the production of pro‐inflammatory mediators and the activities of matrix degrading enzymes. Specific patterns of [Ca2+]i changes are considered as essential components initiating and/or progressing the disease. In an established model cell‐line of the human chondrocyte, OUMS‐27 cells, large‐conductance Ca2+‐activated K+ (BK) channels are involved in the enhancement of histamine‐induced Ca2+ influx by the activation of Ca2+ release‐activated Ca2+ (CRAC) channels consisting of Orai1‐Orai2 heteromers and STIM1 via the modulation of membrane potential. We have identified a novel BKα splice variant (BKαΔe2) in OUMS‐27 cells. Although BKαΔe2 lacks exon2 that codes the intracellular S0–S1 linker (Glu127‐Leu180), significant expression was detected in several human tissues including chondrocytes of OA patients and in mouse tissues as well. BKαΔe2 channels are not expressed on plasma membrane (PM), but can traffic to PM after forming hetero‐tetramer units with wild‐type BKα (BKαWT). These hetero‐tetramers of BKα have a smaller single channel conductance and exhibit lower trafficking efficiency than BKαWT homo‐tetramers in a stoichiometry‐dependent manner. BKαΔe2 knockdown in OUMS‐27 chondrocytes increased BK current density, and augmented the responsiveness to histamine assayed as cyclooxygenase‐2 gene expression. These findings provide significant new evidence that BKαΔe2 can modulate cellular responses to physiological stimuli in human chondrocyte and contribute under pathophysiological conditions, such as OA.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Numbers, 26293021 and 15H01408 to Y.I., 26860059, 16H06215 and 16K15127 to Y.S., 16K08278 to H.Y. This work was also supported by a Grant‐in‐Aid from Takeda Science Foundation (to Y.S.) and Salt Science Research Foundation [Grant 1637] (to Y.S.). W.G. holds an Alberta Innovates‐Health Solutions Scientist Award, and CIHR funding.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Role for the Na‐K‐2Cl Cotransporter in Mitochondrial Respiration
Authors: Omer Salma , Delpire Eric
Published: June 2021

A patient recruited by the undiagnosed disease program (UDP) at the NIH suffers from multi organ failure and deficits in energy homeostasis. The multi organ failure consists of complete gastrointestinal and bladder dysfunction, as well as thyroid, parathyroid, adrenal, and pancreatic deficiencies. The deficits in energy homeostasis were evidenced in lab tests conducted on the patient's muscle and liver biopsies, where there was an abnormal increase in glycogen content and mitochondrial DNA copy number. Whole exome sequencing showed that the patient expresses a de novo mutation in an allele of the Slc12a2 gene encoding the Na‐K‐2Cl cotransporter‐1 (NKCC1). Previous studies in the laboratory demonstrated that the 11 base pair deletion causing a truncation of the carboxyl‐terminus, leads to a non‐functional transporter. We have also demonstrated that the UDP patient's fibroblasts displayed reduced total NKCC1‐mediated K+ influx. Additionally, trafficking studies in transfected HEK293 cells expressing the NKCC1 truncation mutation have revealed that this mutated protein dimerizes with wild‐type (WT) NKCC1 and is trafficked to the plasma membrane along with WT NKCC1.

To address a possible role of the cotransporter in metabolism, I utilized fibroblasts isolated from the patient and from healthy controls and performed mitochondrial respiration stress tests. I showed that mitochondrial respiration and ATP production was significantly higher in the patient's fibroblasts. Because the cells from the patient might carry additional mutations, I transfected MDCK cells with EGFP‐tagged wild‐type or mutant transporter, sorted the cells, and tested them. Cells expressing the mutant cotransporter also demonstrated higher mitochondrial respiration, compared to cells expressing wild‐type EGFP‐NKCC1.

To further confirm the role of NKCC1 in mitochondrial respiration, I subjected control MDCK cells to bumetanide, a NKCC1 specific inhibitor. MDCK cells treated with 20 mM bumetanide for 20 min also demonstrated an increase in mitochondrial respiration. To further assess whether this effect in mitochondrial respiration is NKCC1 specific, I subjected bumetanide to two stable lines of MDCK cells (LK‐A3 and LK‐C1) that carry a distinct mutation in NKCC1, resulting in a non‐functional transporter. There was no change in mitochondrial respiration levels in these cells, which indicated that the rise in mitochondrial respiration levels in wild‐type cell is NKCC1 specific. These data reveal a novel role for NKCC1 in mitochondrial respiration. Future work will address the mechanism by which NKCC1 activity affects mitochondrial function.

Support or Funding Information

Supported by NIH grants R21GM118944 and RO1DK093501

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Orai1‐mediated store‐operated Ca2+ entry by a novel compound RP4010 for anti‐proliferative activity against esophagus squamous cell carcinoma
Authors: Chang Yan , Cui Chaochu , Zhang Xiaoli , Penmetsa Kumar , Viswanadha Srikant , Fu Liwu , Pan Zui
Published: June 2021

Targeting Orai1‐mediated store‐operated Ca2+ entry by a novel compound RP4010 for anti‐proliferative activity against esophagus squamous cell carcinoma

The University of Texas System STARs Award

Esophageal cancer is the 6th leading cause of cancer mortality worldwide with poor prognosis and 5‐year survival rate as low as 18%. It is an unmet need to develop more effective chemotherapeutic drugs for esophageal cancer patients. We previously reported that the expression of Orai1, a Ca2+ channel, was elevated in esophageal squamous cell carcinoma (ESCC), a major form of esophageal cancer, and the high expression is associated with poor survival rate in patients. Orai1‐mediated store‐operated calcium entry (SOCE) has been demonstrated to contribute to cancer cell proliferation, migration and invasion in ESCC and other cancers. Here we evaluated the anti‐cancer effect of a novel Orai1 channel inhibitor, named RP4010, in cultured ESCC cells and xenograft animals.

RP4010 is a novel, oral inhibitor of Orai1 channel currently in Phase I/IB clinical development. Compared to commonly used Orai1 channel blockers (such as BTP‐2), RP4010 is more potent to block Orai1‐mediated SOCE and cell proliferation in ESCC cells. The IC50 of RP4010 in cell viability analysis were calculated as 1.2, 1.4, 1.4 and 5.5 μM for KYSE‐30, KYSE‐70, KYSE‐150 and KYSE‐790, respectively while IC50 for BTP‐2 was 17.7 – 17.8 μM for these cells. Treatment of RP4010 (10 μM) inhibited intracellular Ca2+ oscillations, halted Ca2+‐dependent nuclear translocation of NF‐κB, and resulted in cell cycles arrested at G0/G1 phase. Interestingly, RP4010 did not induce significant cell death even at rather high concentrations. In vivo study revealed that the mice can tolerate daily I.P. injection of 20 mg/kg RP4010 without body weight loss or noticeable adverse effects up to 4 weeks. At this dose, RP4010 could significantly reduce tumor growth compared to vehicle treated control group.

In summary, these data demonstrated that R4010 is a novel potent inhibitor for Orai1‐meidated SOCE. It has potential as a new kind of targeted compound blocking Orai1‐mediated intracellular Ca2+ signaling in ESCC treatment.

Support or Funding Information

National Cancer Institute and The University of Texas System STARs Award

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