bluuebunny/crossref_metadata_2025 huggingface dataset:
Inorganic Nitrate Supplementation Improves Diastolic Function in Cancer Survivors treated with Anthracycline Chemotherapy
Authors: Parr Shannon , Banister Heather R , Caldwell Jacob , Lovoy Garrett M , Post Hunter K , Turpin Vanessa‐Rose G , Ade Carl J
Published: June 2021

Cancer survivors treated with anthracycline‐based chemotherapy have an increased risk of cardiotoxicity, resulting in the development of cardiac complications up to 10 years after chemotherapy cessation. Thus, cardiovascular events and heart failure are the second leading cause of morbidity and mortality in these women. Notably, the increased generation of reactive oxygen species relative to antioxidant defenses in the cardiomyocyte with anthracyclines results in lipid peroxidation and peroxynitrate formation, at the consequence of a decreased nitric oxide (NO) bioavailability, which over time results in ventricular systolic and diastolic dysfunction. Recently, orally ingested inorganic nitrates (NO3−), which increase NO bioavailability via the nitrate‐nitrite‐nitric oxide pathway and high antioxidant capacity, have shown to improve cardiac function in mice given anthracycline chemotherapy. Therefore, we hypothesized that inorganic NO3− administration would improve of left ventricular (LV) systolic and diastolic function, arterial stiffness, and ventricular‐arterial coupling in human cancer survivors with a history of anthracycline chemotherapy. Ten cancer survivors, 9 with breast cancer and 1 with lymphoma, participated in this randomized, double blind, crossover study comparing a week of daily doses NO3− rich beetroot juice with a nitrate‐depleted placebo. Standard and Tissue Doppler echocardiography were used to assess LV and carotid artery function during systole and diastole. NO3− supplementation significantly improved parameters of LV diastolic function, specifically strain rate in early filling (1.07±0.17 versus 0.80±0.29 1/s; P<0.5), early mitral septal wall annular velocity (0.07±0.02 versus 0.06±0.02 m/s; P=0.02, and mitral A‐wave velocity (0.65±0.23 versus 0.52±0.17 cm/s; P=0.009). There were no differences in LV systolic parameters; ejection fraction (P=0.2) ventricular‐arterial coupling (P =0.10), systolic strain rate (P =0.49). Arterial stiffness (P =0.38) was not different between conditions. These findings suggest that decrease in NO bioavailability may have a mechanistic role in determining LV diastolic function in cancer survivors treated with anthracycline‐based chemotherapy. This is critical information that can be used to developed therapeutic strategies targeting NO‐dependent changes in left ventricular function that occur following anthracycline chemotherapy.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cutaneous Microvascular Function in Hypertensive Adults is not Altered Following 16‐weeks of Pharmacological Antihypertensive Treatment
Authors: Dillon Gabrielle A , Greaney Jody L , Stanhewicz Anna E , Alexander Lacy M
Published: June 2021

Hypertension is characterized by pervasive microvascular endothelial dysfunction, resulting in a shift toward a proconstrictor vascular profile and contributing to increased cardiovascular morbidity and mortality. Cross‐sectional data suggest that pharmacologically lowering blood pressure (BP) is associated with improvements in microvascular function via endothelium‐dependent mechanisms. Well controlled longitudinal studies directly assessing mechanisms underlying microvascular dysfunction in hypertensive adults before and after a pharmacological antihypertensive intervention have not been performed. We tested the hypothesis that endothelium‐dependent dilation would be improved and adrenergic‐mediated vasoconstriction would be attenuated following 16‐weeks of pharmacological treatment to lower BP. Microvascular function was assessed before and after 16‐weeks of antihypertensive treatment (diuretic or angiotensin converting enzyme inhibitor) in eleven stage II hypertensive adults (9 men, 54±3 yrs, 29±1 kg/m2, resting BP 143±4/93±3 mmHg). Red blood cell flux (laser Doppler flowmetry) was measured during graded intradermal microdialysis perfusion of the endothelium‐dependent agonist acetylcholine (ACh; 10−10–10−1 M) and the adrenergic receptor agonist norepinephrine (NE, 10−12–10−2 M). Cutaneous vascular conductance (CVC; flux/mmHg) was normalized to and expressed as a percentage of either baseline or maximal conductance (28 mM sodium nitroprusside and heat to 43°C). Resting BP was reduced following treatment (143±4/93±3 pre vs. 105±4/68±2 mmHg post; p=0.003). Neither baseline (0.19±0.03 pre vs. 0.19±0.03 post flux/mmHg; p=0.97) nor maximal CVC (2.16±0.48 pre vs. 1.44±0.24 post flux/mmHg; p=0.24) were different following treatment. There was no difference in ACh‐induced vasodilation following antihypertensive treatment (logEC50: −4.28±0.48 pre vs. −3.15±4.68 post; p=0.83). Further, NE‐induced vasoconstriction was not different after treatment (logEC50: −4.85±1.23 pre vs. −6.28±0.68 post; p=0.65). Contrary to our hypothesis, these preliminary data suggest that a 16‐week pharmacological antihypertensive treatment to reduce BP does not improve cutaneous microvascular function in middle‐aged hypertensive adults.

Support or Funding Information

NIH R01HL093238

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immune Challenge During Reproduction Results in Tissue Specific and Intensity‐Dependent Responses for Mitochondrial Stress
Authors: Parry Hailey A , Josefson Chloe , Taylor Halie A , Andreasen Victoria , Park Noel R , Hood Wendy R , Kavazis Andreas N
Published: June 2021

Life‐history theory suggests that tradeoffs can occur between self‐maintance and an animal's capacity to allocate resources to reproduction. This effect is thought to be exacerbated when a female is faced with an immune challenge during a reproductive event. Ultimately, such tradeoffs are thought to curtail lifespan, but the bioenergetic underpinnings of this effect are not well understood. We determined the extent of any effects in mitochondrial function and oxidative damage in female mice when exposed to an immune challenge (keyhole limpet haemocyanin) during reproduction. Female ICR mice were paired with a male until just before giving birth and randomly assigned to one of three groups. Female mice in the control group (LC) were injected with saline 2 days after parturition, and then sacrificed 1 week after weaning. In the lactating immune challenge group (LI), females were treated similarly except they were injected with KLH. Finally, in the pregnant‐lactating immune challenge group (PLI), females were treated the same as LI group, except the male was left in the box with the female until 3 days post‐partum, so the female became pregnant again while lactating. The second litter was born within 3 days of weaning, immediately removed, and females were sacrificed 1 week later. Mitochondrial function and reactive oxygen species (ROS) emission were determined in the liver and hindlimb skeletal muscle. Antioxidant protein levels and oxidative damage markers were also analyzed in these tissues. A one‐way ANOVA was performed on all measures, with a Tukey's post hoc test to determine group differences. No significant differences were observed in liver or hindlimb skeletal muscle mitochondrial coupling as measured by the respiratory control ratio (RCR) for both complex I and complex II (p>0.05). Isolated mitochondrial ROS emission in the hindlimb skeletal muscle was significantly lower in LI compared to LC (p=0.041), but no differences were observed in the liver. Additionally, skeletal muscle PGC1‐α was significantly reduced in LI mice compared to LC (p=0.01). In skeletal muscle the antioxidant GPX was significantly increased in the PLI animals compared to LI animals (p=0.022). Lastly, liver SOD1 was significantly higher in LC compared to PLI (p=0.039). The opposite response was observed in HSP70, in which PLI had significantly increased expression compared to LC (p=0.018). In conclusion, females that experienced an immune challenge during reproduction did not appear to experience a decline in mitochondrial respiratory performance but a drop in PGC1‐a in skeletal muscle that could contribute to a reduction in future performance associated with a reduction in mitochondrial density.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Physiological Role of Inter‐Organ Network between Gastrointestine and Skeletal Muscle on the Regulation of Skeletal Muscle Volume
Authors: Goto Katsumasa , Aoshima Megumi , Suzuki Takashi , Kanda Yusuke , Inagaki Riko , Akiyama Kensuke , Yokoyama Shingo
Published: June 2021

Several inter‐organ networks have been proposed. In general, gastrointestinal hormone gastric inhibitory polypeptide (GIP), which is synthesized in and secreted from K cells, regulates nutrient absorption via inhibition of gastric contraction and acid secretion. GIP receptor On the other hand, GIPR expresses in not only a gastrointestinal tract but also β cells in the pancreas. Since GIP also modulates glucose metabolism via insulin synthesis and secretion, GIP is also a member of incretin. Recently, the expression of GIP is also confirmed in skeletal muscle. However, there is no evidence for the inter‐organ network between gastrointestine and skeletal muscle. In the present study, we investigated a physiological role of the inter‐organ network between gastrointestine and skeletal muscle via GIP. GIP stimulates myogenic differentiation of C2C12 cells. Expression of GIPR was observed in C2C12 myoblasts and myotubes. Knockdown of GIPR induced the down‐regulation of Pax7 in C2C12 myoblasts. In addition, GIPR‐knockdown‐associated depression of myotube formation of C2C12 cells were observed. On the other hand, GIPR‐knockdown stimulated proliferation of C2C12 myoblasts. Therefore, GIP‐GIPR intracellular signal(s) might play a role in the regulation of skeletal muscle volume via the mediation of myogenic differentiative potential.

Support or Funding Information

This study was supported, in part, by Grants‐in‐Aid for Challenging Exploratory Research (16K13022), and Grants‐in‐Aid for Scientific Research (17K01762, 18H03160) from the Japan Society for the Promotion of Science, the Descente Sports Foundation, the All Japan Coffee Association, the Science Research Promotion Fund from Promotion and Mutual Aid Corporation for Private Schools of Japan, and the research grants from Graduate School of Health Sciences, Toyohashi SOZO University.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of TSSK6 Activating Co‐chaperone (TSACC) in Skeletal Muscle
Authors: Tello Tala Shourbagi , Deluna Jonnaly , Waddell David
Published: June 2021

Skeletal muscle atrophy is a physiological condition that is caused by a variety of conditions, including immobilization, denervation, spinal cord injury and aging and results in decreased muscle size and strength. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated gastrocnemius muscle from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that TSSK6 Activating Co‐chaperone (TSACC) is expressed in skeletal muscle and is significantly induced in response to denervation. To confirm that TSACC is expressed in muscle, the predicted TSACC cDNA was cloned from cultured muscle cells, as was a novel TSACC splice variant that contains an additional exon. Quantitative PCR (qPCR) was then used to assess TSACC expression levels of the full‐length and novel splice variant in proliferating and differentiated muscle cells. The results demonstrate that full‐length TSACC expression levels are relatively low in proliferating myoblasts but show significantly elevated expression in differentiated myotubes, whereas the novel splice variant is expressed relatively evenly in proliferating and differentiated muscle cells. In addition, characterization of the transcriptional regulation of TSACC was assessed by fusing fragments of the proximal promoter located immediately upstream of the start of transcription with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells in combination with myogenic regulatory factor (MRF) expression plasmids, which resulted in significant activation of reporter gene activity. Interestingly, there are several predicted E‐box elements in the proximal promoter region of the TSACC gene further suggesting that TSACC may be regulated by muscle‐specific transcription factors. Finally, to determine a possible role for TSACC in skeletal muscle, sub‐cellular localization was determined by fusing the cDNA with GFP and expressing the fusion protein in cultured muscle cells. The discovery that TSACC is induced in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle atrophy and may eventually lead to the identification of new therapeutic targets for the treatment of muscle atrophy.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of the Regulation and Function of Family with Sequence Similarity 83D (Fam83d) in Skeletal Muscle
Authors: Kavanaugh Jack , Sanchez Victoria , Cooper Lisa , Waddell David
Published: June 2021

Skeletal muscle atrophy is a serious condition that can arise due to aging, cancer, corticosteroid exposure, and denervation. To better characterize the molecular genetic events of neurogenic atrophy, a previous study isolated mouse gastrocnemius muscle from mice at 3 and 14 days following sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was then analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Fam83d is expressed in skeletal muscle and is significantly induced in response to denervation. Quantitative PCR (qPCR) analysis revealed that Fam83d is more highly expressed in proliferating myoblasts compared to differentiated myotubes. Characterization of the transcriptional regulation of Fam83d was analyzed by cloning fragments of the proximal promoter region located immediately upstream from the start of transcription and fusing them with a reporter gene. The reporter plasmids were then transfected into C2C12 mouse muscle cells alone or in combination with myogenic regulatory factor expression plasmids, resulting in a differential effect on reporter activity. Analysis of the promoter of Fam83d revealed conserved E‐box elements in the proximal regulatory region, which are known myogenic regulatory factor (MRF) binding sites. To assess where Fam83d is localized in the cell, we fused Fam83d with green fluorescent protein (GFP), transfected the Fam83d‐GFP expression plasmid into C2C12 mouse myoblasts cells, visualized by confocal microscopy, and observed that Fam83d is localized exclusively to the cytoplasm. It has recently been determined that Fam83d contains a putative phospholipase domain, is mis‐regulated in several different cancer types, and may modulate MAP Kinase signaling. Therefore, Fam83d was ectopically expressed in cultured muscle cells and markers of muscle cell differentiation and MAP Kinase signaling were analyzed. Fam83d overexpression resulted in significantly repressed levels of myosin heavy chain and myogenin expression, while levels of phosphorylated ERK were dramatically repressed. The discovery that Fam83d is expressed in skeletal muscle combined with the observation that Fam83d, a potential modulator of MAP Kinase signaling, is induced in response to neurogenic atrophy helps further our understanding of the molecular and cellular events of skeletal muscle wasting.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of Ovarian Tumor Protease Domain Containing 1 (OTUD1) in Skeletal Muscle
Authors: Wortherly Keri , Waddell David
Published: June 2021

Skeletal muscle atrophy is a physiological condition that is caused by an array of conditions, including immobilization, denervation, spinal cord injury and corticosteroid use and results in decreased muscle size and strength. The molecular genetic events of neurogenic atrophy were analyzed in a previous study using gastrocnemius muscle isolated from mice following 3 days and 14 days of sciatic nerve denervation. The gene expression profile in the denervated muscle tissue was analyzed by microarray and compared to control muscle tissue to identify novel neurogenic atrophy‐induced genes. The microarray data revealed for the first time that Ovarian Tumor Protease Domain OTUD1 is expressed in skeletal muscle and is differentially regulated in response to denervation. OTUD1 is a deubiquitinase that has been linked to TGF‐β signaling in cancer via deubiquitination of Smad7. To confirm that OTUD1 is expressed in muscle, quantitative PCR (qPCR) was used to assess OTUD1 expression levels in both proliferating and differentiated muscle cells and the results demonstrate that expression levels appear to remain relatively constant in proliferating myoblasts and differentiated myotubes. Furthermore, Western blot analysis supported the qPCR data and showed that OTUD1 protein levels remain relatively constant as cultured myoblasts differentiate to myotubes. The discovery that OTUD1 is differentially regulated in response to neurogenic atrophy helps further our understanding of the molecular genetic events of muscle wasting and may eventually lead to the identification of new therapeutic targets for the treatment and prevention of atrophy.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification and Characterization of 1700029J07RIK, a Novel Gene Expressed in Skeletal Muscle
Authors: Lynch Sarah , Waddell David
Published: June 2021

Skeletal muscle atrophy is the loss of muscle mass that results from a wide range of conditions including denervation, corticosteroid exposure, and aging. 1700029J07RIK is a putative novel gene suggested to be involved in skeletal muscle atrophy. The 1700029J07RIK gene was found to be induced following denervation in mice and is differentially expressed in Muscle RING Finger 1 (MuRF1) knockout mice compared to wild‐type mice. A previous microarray analysis of muscle tissue isolated from wild‐type control mice and MuRF1 knockout mice revealed that the expression of the 1700029J07RIK gene is unchanged at 3 days post‐denervation, while expression is significantly induced in wild‐type mice at 14 days post denervation but remains unchanged in the MuRF1‐null animals. It has been well established that muscle cells go through a distinct developmental timeline in which they proliferate as single cell myoblasts and then differentiate and fuse into multinucleated myotubes. Quantitative PCR was used to confirm that the 1700029J07RIK gene is expressed in proliferating cells and increases in expression as muscle cells differentiate. To characterize the transcriptional regulation of 1700029J07RIK, promoter fragments were cloned into a reporter plasmid, transfected into muscle cells and found to have significant transcriptional activity, while ectopic expression of myogenic regulatory factors appear to modulate the activity of the 1700029J07RIK reporter genes. In addition, the 1700029J07RIK cDNA was cloned from muscle cells and fused with green fluorescent protein and visualized by confocal fluorescent microscopy revealing an interesting perinuclear and cytoplasmic localization of the 1700029J07RIK protein in muscle cells. Finally, it was observed that overexpression of 1700029J07RIK resulted in inhibition of muscle cell differentiation and attenuation of the ERK branch of the MAP Kinase signaling pathway. While there is virtually nothing known about this novel gene in the context of skeletal muscle, the results presented here suggest 1700029J07RIK may play a role in muscle cell differentiation and in the skeletal muscle atrophy cascade.

Support or Funding Information

The work was support by University of North Florida Transformational Learning Opportunity grants and a University of North Florida Foundation Board Grant to D.W.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skeletal Muscle Mitochondrial Function in Goto‐Kakizaki Rat Model of Type 2 Diabetes
Authors: Lewis Matthew T , Kasper Jonathan D , Bazil Jason N , Frisbee Jefferson C , Meyer Ronald A , Wiseman Robert W
Published: June 2021

Type 2 diabetes (T2D) presents with significant comorbidities and reported exercise intolerance affecting over 30 million people in the US alone. Aerobic metabolism is the predominant pathway for fueling exercise and utilizing glucose, therefore mitochondrial oxidative phosphorylation (MOP) is important in diabetes and the future of diabetic interventions. However, studies in T2D have conflicting reports regarding the functional status of the mitochondrial pool suggesting deficits are an inherent cause of the disease but a comprehensive study has yet to be done. The Goto‐Kakizaki (GK) rat model of T2D was used to measure both mitochondrial function and density in vitro and in vivo over a broad range of metabolic challenges. Mitochondrial densities were similar in red (RG) and white gastrocnemius (WG) muscles of the GK rat (Cyt c oxidase, RG: 22.2 ± 1.6 versus 23.3 ± 1.7 μmol/min/g, WG: 10.8 ± 1.1 versus 12.1 ± 0.9 μmol/min/g; n=7–8). Mitochondria isolated from the muscles of GK rats and their Wistar controls showed no difference in mitochondrial respiration in vitro as measured by high resolution respirometry. Mitochondrial function in vivo, measured by 31Phosphorus magnetic resonance spectroscopy, was similar between GK and Wistar controls determined from phosphocreatine dynamics reaching similar exercise steady states at submaximal workloads with no difference in recovery time constant (72 ± 6 s GK vs. 71 ± 2 s Wistar). However, at exercise intensities beyond what can be sustained aerobically, the GK rat exhibited bioenergetic deficits suggesting something other than mitochondrial function is responsible at these workloads. The current work demonstrates normal mitochondrial function in a non‐obese non‐sedentary rat model of T2D in vitro and in vivo and no change in mitochondrial content. Taken together these results show when considering both density and function of mitochondria no deficit in MOP is present in the GK T2D rat model and may not be in T2D patients but these studies remain to be done.

Support or Funding Information

NIH DK 095210, NIH R00HL121160, CIHR R5521A02

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic Dexamethasone Exposure Induced Mucosal Injuries by Activating Epithelial Apoptosis and Inflammatory Response in The Colon of Goats
Authors: Cai Liuping
Published: June 2021

Background

Chronic stress has a profound effect on health in both animals and humans. Chronic stress can influence the level of glucocorticoid. Dexamethasone (Dex), a synthetic glucocorticoid, is used to induce chronic stress. However, the aim of the present study was to investigate the effect of chronic stress caused by long term injection of Dex on changes on colonic epithelium in goats.

Results

The results show that chronic stress increased the number of TUNEL‐apoptosis‐positive cells, upregulated caspase‐3 enzyme activity, and decreased the proteins expression of cell proliferation markers proliferating cell nuclear antigen (PCNA) and Cyclin D2 (CCND2) in the colonic epithelium. The gene expression of claudin‐1, claudin‐4 and ZO‐1 were upregulated by Dex (P < 0.05). But claudin‐4 protein expression was increased by Dex (P < 0.05). The gene expression of cell cycle such as CDKs (cyclin‐dependent kinases), CCND1, and CCND2 were all increased by Dex (P < 0.05). The gene expression of inflammtory cytokines interlukin‐10 (IL‐10), interlukin‐1β (IL‐1β), inducible nitric oxide synthase 2 (iNOS2), and inflammatory‐related transcriptional factors mitogen‐activated protein kinase (MAPK), signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NFκB) were all upregulated by Dex (P < 0.05). The protein expression of NFκB and Toll‐like receptors (TLR4) were increased by Dex (P < 0.05). Moreover, the results showed that the methylation levels of total DNA and TLR4, caspase‐3 promoter were significantly decreased in the colonic epithelium compared to control group.

Conclusions

These results indicated that long‐term injection of a low dosage of Dex caused damage to the colon epithelium and inhibition of cell proliferation, and result in the activation of the inflammatory response and decreased methylation of the total DNA.

Support or Funding Information

1. National Nature Science Foundation of China (project no.31572433)

2. The Program for New Century Excellent Talents in University (NCET‐13‐0862) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Surface Functionalization of Nanocrystalline and Nanostructured Diamond to Form Interface with Newborn and Adult Neurons
Authors: Krusek Jan , Dittert Ivan , Smejkalova Tereza , Korinek Miloslav , Gottftriedova Kristyna , Freislebenova Hana , Neuhoferova Eva , Taylor Andrew , Mortet Vincent , Petrak Vaclav , Benson Veronika , Petrakova Vladimira
Published: June 2021

Boron doped diamonds (BDD) are a promising material for fabrication of neuroelectrodes and formation of neurointerfaces. Surface properties of BDD thin films can be adjusted by variation of grain sizes, patterning, lithography techniques and coating by different biopolymers to further promote performance of neuroelectrode and enhance the neuron adhesion.

We used 3T3 fibroblasts and rat hippocampal neurons (newborn and adult) to estimate planar and nanostructured BDD surface biocompatibility. Optical microscopy, lactate dehydrogenase assay and calcium imaging were used to check cell adhesion and longterm survival.

3T3 fibroblasts are able to grow on BDD without any coating. No signs of cytotoxicity and changes in cell cycle were found. Adhesion and longterm survival of newborn rat hippocampal neurons were observed only on poly‐L‐lysine coated planar and nanostructured BDD surface. Calcium imaging of longterm cultures of newborn neurons grown on poly‐L‐lysine coated planar and nanostructured BDD showed functionality and synaptic activity of hippocampal neurons. Nanodiamond surface without poly‐L‐lysine coating do not support adhesion of newborn neurons.

Adult rat hippocampal neurons preferred poly‐D‐lysine coated planar nanodiamonds but were able to grow on nanostructured diamonds without any coating.

Support or Funding Information

This work was supported by Czech Science Foundation grant 17–15319S.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Onset of Exercise‐induced Microvascular Hyperpermeability is Delayed in Diabetic Skeletal Muscle: In Vivo Imaging Using Two‐Photon Laser Scanning Microscopy
Authors: Hotta Kazuki , Shimotsu Rie , Masamoto Kazuto , Yagishita Kazuyoshi , Behnke Brad J , Poole David C , Kano Yutaka
Published: June 2021

To investigate whether Type I diabetes (DIA) alters the temporal characteristics of muscle microvascular function and fiber damage incurred by eccentric contraction (ECC)‐induced muscle injury, male Wistar rats (n=53, 10–14 wks old) were randomly assigned to control (CON) or Streptozocin (45 mg/kg, i.p. DIA) groups. Four weeks later (blood glucose, 354±46 mg/dL), the tibialis anterior (TA) muscle of 1 leg was injured by 200 times ECC. Microvascular permeability was assessed by in vivo imaging using two‐photon laser scanning microscopy on 1, 3, 7 and 14 days post‐ECC. The TA microcirculation was visualized after intravenous infusion of fluorescent dye; extravasation of which was quantified to assess microvascular permeability. CON and ECC TA muscles were dissected and frozen after in vivo imaging, and serial cryosections were co‐stained with vascular endothelial growth factor A (VEGFA) antibody and hematoxylin‐eosin. Compared with CON rats, non‐ECC TA microvascular permeability was slightly but significantly enhanced in DIA with higher expression of VEGFA in myocytes. In CON rats, compared with non‐ECC TA, injured myocytes and microvascular hyper‐permeability were observed simultaneously on 1 and 3 days (injured myocyte 48, 73%, extravasation 262, 266 μm3×106xmin, 1, 3 days post‐ECC, respectively) and were repaired by 7 days after ECC (no injured myocytes, regenerated myocyte 29%, extravasation 101 μm3×106xmin). DIA TA exhibited delayed muscle injury onset and microvascular hyper‐permeability from 1 to between 3 and 7 days (injured 8, 81, 35%, extravasation 33, 183, 180 μm3×106xmin, 1, 3, 7 days post‐ECC, respectively). Regenerated myocyte was observed between 7 and 14 days post‐ECC (regenerated 47, 23%, 7, 14 days, respectively, extravasation 105 μm3×106xmin, 14 days post‐ECC). Regenerated cell size was smaller in DIA compared to CON (3214 vs. 1377 μm2, CON vs. DIA 14 days post‐ECC, P<0.01). Percentage of VEGFA‐positive myocytes to injured myocytes was increased in both CON (28%, 43%, 1 and 3 days post‐ECC, respectively) and DIA (44%, 43%, 65%, 1, 3, 7 days post‐ECC) rats with a temporal profile corresponding to that of increased dye extravasation. These results suggest that DIA retards the onset of microvascular hyper‐permeability after ECC thereby delaying skeletal muscle injury and recovery. VEGF may regulate microvascular permeability in both CON and DIA skeletal muscle and therefore play a commanding role in post‐ECC damage and regeneration.

Support or Funding Information

This work was supported by Japan Society for the Promotion of Science KAKENHI Grants JP17K13185, 16700450, 18700526, and 23650403.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exosomes derived from adipose tissue stem cells regenerate ischemic myocardium following intravenous administration
Authors: Wang Min , Xu Jie , Liu Min , Wang YiGang , Ashraf Muhammad , Xu Meifeng
Published: June 2021

Objective

Exosomes (EXO) are nanosized bi‐lipid‐layered vesicles that contain proteins, mRNAs, and miRs. Increased evidence indicates that EXO play an important role in intercellular communication and cardiac repair. They have long circulating half‐life and cellular effects. EXO can be administered intravenously (iv) without risk of microvasculature entrapment. Our previous studies indicated that mesenchymal stem cells (MSCs) isolated from adipose tissue (AD‐MSCs) proliferated faster and secreted more EXO compared to those derived from bone marrow (BM‐MSCs). In this study, we investigated if intravenous administration of AD‐EXO can improve cardiac function and regenerate ischemic myocardium via native cardiomyocytes (CMs) protection.

Methods and Results

MSCs were harvested from adipose tissue of 6–8 weeks old C57BL/6 mice. EXO were collected from MSC conditional medium by ultracentrifugation, and were characterized by morphology and surface markers. Mouse myocardial infarction (MI) was created by ligation of left anterior descending (LAD) coronary artery. EXO (50 μg) were injected through tail vein every two days for a total of 2 or 4 times, respectively. Primary CMs were obtained from ventricles of 2‐day‐old rats. Ischemic injury was mimicked by exposing CMs to hypoxic culture (1% O2, 5%CO2, and 94% N2). We found that 1) Left ventricular function following LAD ligation was significantly improved and infarction size was significantly reduced in mice injected with AD‐EXO (50 μg) for a total of 4 times. 2) Distribution of AD‐EXO in heart was clearly observed following injection of pre‐labeled EXO with PKH26. 3) AD‐EXO significantly protected CMs against hypoxia induced injury.

Conclusions

EXO derived from AD‐MSCs can be successfully delivered to myocardium following intravenous administration, which results in improved cardiac function and reduced infarction size in mouse MI model.

Support or Funding Information

This work was supported by NIH Grants HL114654, and HL140962 (M. Xu).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Atg16L1 Knockout Induces Insulin Resistance through Proteasomal IRS1 Degradation, Mediated by the Induction of ER Stress
Authors: Frendo‐Cumbo Scott , Jaldin‐Fincati Javier , Brumell John H. , Klip Amira
Published: June 2021

Insulin resistance is a defining feature of type 2 diabetes, yet our understanding of the progression and development of insulin resistance is incomplete. Recently, deficient autophagy, a bulk degradation pathway, was associated with the induction of insulin resistance, although the causative mechanism remains unknown. We sought to investigate the underlying signals responsible for how deficient autophagy induces insulin resistance. We report that knockout of an essential autophagy protein, Autophagy Related 16L1 (Atg16L1), in mouse embryonic fibroblasts (MEFs) markedly decreases insulin and insulin‐like growth factor 1 (IGF1)‐stimulated Akt recruitment to the cellular membrane and phosphorylation on S473 and T308, as well as cortical actin remodelling. Atg16L1 KO did not adversely affect insulin receptor (IR) or IGF1 receptor content, cell surface IR localization or 125I‐insulin binding. However, Atg16L1 KO MEFs and Atg16L1 depleted HeLa cells displayed reduced protein content of Insulin Receptor Substrates (IRS1 and IRS2), pivotal proximal signals in insulin signaling known to be dysregulated in insulin resistant and diabetic states. Corroborating these findings, visceral adipose tissue of high fat‐fed mice displayed lower Atg16L1 mRNA and IRS1 protein content. Expression of IRS1myc recovered insulin‐stimulated Akt phosphorylation in KO MEFs. Treatment of KO MEFs with the proteasome inhibitor MG132restored IRS1 protein content and insulin‐stimulated Akt phosphorylation. Moreover, ER stress, which induces insulin resistance at the level of IRS1, was upregulated in Atg16L1 KO MEFs. Alleviating ER stress with the chemical chaperone phenylbutyrate increased IRS1 protein expression and insulin stimulated Akt phosphorylation similar to MG132. Together, we show that Atg16L1 KO induces insulin and IGF1 resistance through the induction of ER stress, leading to targeted proteasomal degradation of IRS1.

Support or Funding Information

S.F.C was supported by an NSERC Alexander Graham Bell Canada Graduate Scholarship‐Doctoral (CGS D), as well as Restracomp and a 2018 Pricewaterhouse Coopers Student Bursary from the Hospital for Sick Children.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human Craniofacial Spliceosomopathies
Authors: Bernier Francois
Published: June 2021

Germline mutation of core components of the spliceosomes have been associated with a range of clinical disorders from isolated retinitis pigmentosa (RP) to developmental syndromes, while somatic mutations of spliceosome components are linked to cell cycle dysregulation and cancer. Recently a group of craniofacial and skeletal disorders caused by dominant mutations in spliceosomal genes has emerged. The first of these to be identified is mandibulofacial dysostosis type Guion‐Almeida (MFDGA), caused by haploinsufficiency of the U5 snRNP component EFTUD2. We recently identified haploinsufficiency of SF3B4 as the cause of Nager syndrome, an acrofacial dysostosis (AFD), although only in 60% of cases suggesting genetic heterogeneity. Mutation of SF3B4 was subsequently reported in AFD Rodriquez type. SF3B4 is part of the U2 snRNP of the major spliceosome. We also identified heterozygous mutations in a highly conserved regulatory exon of SNRPBas the cause of cerebro‐costo‐mandibular syndrome (CCMS). Our experimental data show that these mutations disturb highly conserved exonic splicing silencer sequences crucial to the regulation of the gene's expression. SNRPB encodes a component of the Sm complex of the spliceosome. Additional craniofacial spliceosomopathies result from mutations in splicing factors TXNL4A, RBM8A and CWC27. The frequent involvement of craniofacial structures invite questions on the sensitivity of craniofacial and in particular pharyngeal arch development to spliceosomal defects, and on the role of the spliceosome in the regulation of development. The report of a young patient with Nager syndrome with bilateral synchronous breast cancer raises the possibility of a link between germline mutation of splicing factors and cancer predisposition. We also have a patient with a pathogenic SNRPB mutation who is non‐penetrant for CCMS syndrome but was diagnosed with early onset breast cancer, further linking heritable splicing mutations with cancer predisposition.

Many of the craniofacial spliceosomopathies share overlapping features with Treacher‐Collins syndrome (TCS), a ribosomopathy. We are currently validating mutations in a ribosomal gene in two patients with typical Nager syndrome. This finding may provide unique insight into the overlapping roles of transcription and translation in craniofacial development. A combination of human genetics and model systems research will be required to further unravel the role of the spliceosome and the ribosome in craniofacial and skeletal development.

Support or Funding Information

Genome Canada, Alberta Children's Hospital Research Institute, Genome Alberta

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Alterations in Sirt1 Attenuate Host Defense to Pneumococcal Infection in Sickle Cell Disease
Authors: Bedi Brahmchetna , Kang Bum‐Yong , Hart C Michael , Sadikot Ruxana T
Published: June 2021

Sickle cell disease (SCD) is the most common inherited blood disorder in the United States. Bacterial pneumonia caused by Streptococcus pneumoniae, a gram‐positive pathogen, is a major contributor of morbidity and mortality in patients with SCD. Hence there is an urgent need to develop new strategies to enhance host defense for combating Pneumococcal infections in SCD. Hemolytic disorders such as SCD are characterized by the presence of large amounts of extracellular heme. Heme is a multifaceted contributor to cellular stress and a deregulator of cellular homeostasis, including host defense. We attempt to bridge the gap between dysregulation of cellular homeostasis and infection susceptibility caused by the presence of heme. Sirtuin1 (Sirt1), a protein deacetylase and an intracellular target of Resveratrol (RSV), has been shown to be attenuated in conditions of increased oxidative stress by heme. Heme induces the generation of reactive oxygen species (ROS) through the activation of NADPH oxidase. Human aortic endothelial cells (HAECs) treated with hem‐bound iron (hemin, 5 mM) showed a dose‐ and time‐dependent decrease in Sirt1 expression. This decrease in Sirt1 expression was concomitant with a significant increase in NAPDH oxidase (NOX4) expression. Increase in NOX4 expression was regulated by miR‐301b. Further, we found that depletion of Sirt1 in HAECs did not affect NOX4 expression, whereas knockdown in HAEC NOX4 significantly attenuated Sirt1 levels. These results indicate that NOX4 modulates Sirt1 expression. Total RNAs were isolated from lungs of sickle cell (SS) mice and littermate control (AA) mice at age 15–17 weeks. Levels of Sirt1 an NOX4 in lungs of SS mice were compared to AA mice using qRT‐PCR and Western blot analysis. Levels of Sirt1 were decreased in SS mouse lungs whereas NOX4 expression was increased, as compared with AA mouse lungs. The effect of hemin treatment on HAECs as well as silencing Sirt1 also significantly repressed the expression levels of anti‐microbial peptide (AMP), human beta defensin (hBD2), indicating alterations in host immunity. Treatment with RSV reversed these effects. RSV can be explored as a therapeutic modality to enhance host defense

Support or Funding Information

Department of Veterans Affairs 2I01BX001786 (RTS)

NIH HL133053 (BYK)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional Response to Intermittent Hypoxia in the Heart of Germ‐Free Mice
Authors: Zhou Dan , Hartley Iain , Poulsen Orit , Xue Jin , Haddad Gabriel G
Published: June 2021

Accumulating evidence has demonstrated that the gut microbiota plays a critical role in health and disease and regulates the organismal response to environmental stresses. For example, we have found that intermittent hypoxia/hypercapnia (IHH), a condition mimicking obstructive sleep apnea in humans, induced significant alterations in gut microbiome/metabolism and enhanced atherogenesis and progression in two atherosclerosis mouse models (i.e., the LDLR−/− and ApoE−/− mice). Indeed, with a joint microbiome and metabolome analysis, we have determined that IHH‐induced remarkable compositional changes in both microbial (>10%, most remarkably in Clostridia) and metabolites (>22%) in the gut of LDLR−/− mice, which are tightly correlated with a significantly enhanced atherosclerotic lesion formation in pulmonary arteries and aorta. In the current project, in order to further understand the role of the microbiome in the cardiovascular response to hypoxia and/or hypercapnia, we developed a set up that allows the treatment of experimental mice with different levels of O2 and/or CO2 in a germ‐free environment. Using this experimental set up, we studied the impact of the microbiome on transcriptional response to intermittent hypoxia (IH) in the left ventricle of mouse heart. We identified modest but significant changes in gene expression in both conventionally reared and germ‐free mice under IH condition. We found 52 significantly altered genes (19 up‐regulated and 33 down‐regulated) in the conventional mice and 76 significantly altered genes (24 upregulated and 52 down‐regulated) in the germ‐free mice. In addition, we found that these significantly altered genes play important roles in the development and function of cardiomyocytes and endothelial cells. Interestingly, there were only 15 genes (2 up‐regulated and 13 down‐regulated) that were commonly altered in both conventional and germ‐free mice, representing 28% changes in conventional mice and 20% changes in germ‐free mice, respectively. In summary, we discovered that IH induces distinct transcriptional alterations between conventional and germ‐free mice, which demonstrates that microbiome plays a critical role in the transcriptional response to IH in the left ventricle of mouse heart.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stress Tolerance and Protein O‐GlcNAc Regulation in Neuroblastoma Cells under Hypoglycemic Condition
Authors: Nagy Zsófia , Kátai Emese , Miseta Attila , Nagy Tamas
Published: June 2021

Alzheimer's disease is often referred to as type III diabetes since many studies demonstrated that glucose metabolism is significantly decreased in these patients. The exact mechanism how this metabolic change leads to Alzheimer's is still unclear. However Protein O‐linked N‐acetylglucosamine modification (O‐GlcNAc) may play a pivotal role in this process. O‐GlcNAc modification is a reversible post‐translational modification influenced by substrate availability (hexosamine pathway and UDP‐GlcNAc) which depends on glucose metabolism. Importantly, O‐GlcNAc and phosphorylation seems to be in balance on Tau‐protein, which may form neurofibrillary tangles if hyperphosphorylated. Despite of O‐GlcNAc's obvious connection to glucose metabolism, the level of O‐GlcNAc modification under hypoglycemic condition was not in the focus of research. Thus, the objective of this study was to investigate the relationship between O‐GlcNAc modification and Tau phosphorylation in a hypoglycemic cell culture model.

We cultured the neuroblastoma cell line SH‐SY5Y in various amount of glucose‐containing media (5.5, 3, 1.5, 1, 0.5, 0.25 mM). In the first set of experiments, the glucose concentrations were kept constant for 96 hours. In a second set of experiments, we let the cells run out of glucose. Cell viability was analyzed by flow cytometry and direct microscopic observation. O‐GlcNAc, Tau‐Ps199, total Tau levels were detected by Western‐blot.

We have found that SH‐SY5Y cells were resistant to very low level of glucose since the viability, cell morphology did not change significantly when compared to 5.5 mM glucose condition, as long as the media glucose was replenished to maintain the low, but steady level. However, when the cells were left to metabolize all of the glucose, the viability quickly deteriorated. O‐GlcNAc levels and P‐Tau levels were also steady, independent of the glucose concentration. In the second set of experiments, soon after glucose was completely depleted by the cells, O‐GlcNAc initially decreased then significantly elevated when detected with O‐GlcNAc specific antibody CTD110.6.

In conclusion, our data suggest that hypoglycemic glucose concentrations could be still sufficient for neuronal cells, however glucose fasting quickly leads to cellular stress and eventually, cell death. In the future, we plan to study whether nutrient supplements, such as beta‐hydroxy‐butirate or galactose might alleviate or delay the effect of the unavailable glucose.

Support or Funding Information

This work was funded by the Research Fund of the University of Pécs, Faculty of Medicine (AOK‐KA 2018) and the EU founded Hungarian project GINOP 2.3.2.‐15 (grant No.: 2.3.2.‐15‐2016‐00050).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.