bluuebunny/crossref_metadata_2025 huggingface dataset:
New Rock Inhibitors Action Analysis in the Cytoskeleton and Cell Migration of Tumor Cell Line MDA‐MB 231
Authors: Guerra Fabiana Sélos , de Oliveira Ramon Guerra , Fraga Carlos Alberto Manssour , dos Santos Mermelstein Claudia , Fernandes Patricia Dias
Published: June 2021

Many patients with metastatic tumors have substantially worse survival in comparison with those patients with nonmetastatic tumors. The ability of cancer cells to undergo invasion and migration is a prerequisite for tumor metastasis and these events became targets for the development of new antimetastatic drugs. Rho kinases, ROCK1 and ROCK2, are key regulators of focal adhesion, actomyosin contraction, and thus cell motility. In this regard our group synthesized two sulfonylhydrazones, analogous to Fasudil (a classic Rho kinase inhibitor), named LASSBio 2020 and LASSBio 2065, differing each other by the presence of one methyl in LASSBio 2065. Therefore, the aim of the present work was to investigate the effects of these analogues against human breast carcinoma (MDA‐MB‐231) cell line migration and signalling pathways involved in in this phenomenon as well as cytoskeleton changes.

For the study, cells were incubated with LASSBio 2020 or LASSBio 2065 (0.1 to 30 μM) for 24 or 48 hours. The cell viability was evaluated by MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐difenltetrazolium) and plasma membrane integrity assay (lactate desidrogenase, LDH). Migration capacity of cells was evaluated using wound healing assay. Morphological analysis was carried out by phase‐contrast micrography, and analysed using Image J software. Actin structures were visualized by fluorescent phalloidin. Statistical analyses were performed by ANOVA with Newman‐Keuls post‐test (*p<0.05).

The results showed that none of analogues altered viability of MDA‐MB 231 cells. The effect of the treatment with 10 and 30 μM of LASSBio 2020 decreased cell migration in 30,38% and 32,7%, respectively, when compared to control. LASSBio 2065 decreased cell migration in 24,51% (10 μM) and 26,14% (30 μM), when compared to control. Control cells presented a sprawling appearance with extended cytoplasm and lamelipodios formation. Cells incubated with 10 μm of LASSBio 2020 and LASSBio 2065 presented area, height and width reduced by 40%, membranes with filopodia, protrusions and a fusiform appearance. The staining for F‐actin showed that the cell body became elongated, less spread and with a narrowing of the cell body shape (figure 1).

We can conclude that none of analogues (LASSBio 2020 and LASSBio 2065) reduced cell viability. However, the analogues reduced cell migration, altered cell morphology and interfered with formation of actin filaments. Taken together, our data can suggest that the fasudil analogues are a potential inhibitors of metastasis formation.

Support or Funding Information

Capes, FAPERJ and CNPq

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 small‐molecule inhibitor of the WNT/β‐catenin pathway show efficacy in treating diet induced alteration of glucose and lipid metabolism
Authors: Obianom Obinna , Yang Wei , Li Yingjun , Ai Yong , Xue Fengtian , Shu Yan
Published: June 2021

WNT/β‐catenin pathway is well studied for its role in cell differentiation and proliferation. The major effector of this pathway, β‐catenin, interacts with nuclear pool of co‐transcription factors T‐cell factor/lymphoid enhancer factor (TCF/LEF) to initiate transcription of WNT downstream genes that regulate different processes including glucose and lipid metabolism. Here, we screened a library of 90 compounds using a TCF/LEF responsive reporter assay. We identified a potent quinolinium compound, YW1128, which is able to stabilize Axin levels and decrease WNT signaling (IC50 = 8 nM). In vitro validation assays and in vivo efficacy characterization for this compound using a high fat diet C57BL/6J mouse model in contrast to a normal chow mouse model of the same background were carried out. Our western blot and immunocytochemistry results confirm the WNT inhibitory effects of YW1128 in HEK293 cells. Treatment of huh7 cells led to significant decrease in lipid accumulation shown by nile red staining. In mice fed high fat diet, YW1128 alleviated fatty liver disease and slightly improved glucose tolerance as assessed by haemotoxylin and eosin staining and intraperitoneal glucose tolerance tests, respectively. With no effect on toxicity markers, YW1128 significantly decreased serum cholesterol level in high fat diet mice group with no effect in the normal chow group. Treatment mice also had significant decrease in genes involved in glucose metabolism and fatty acid metabolism pathways along with YW1128 inhibitory effect in WNT signaling pathways. To conclude, we identified an novel WNT pathway inhibitor that show promising benefit for treating diseases associated with glucose and lipid metabolism through its effect on WNT signaling pathway.

Support or Funding Information

Funded by the National Institute of General Medical Sciences of the National Institutes of Health (NIH) under Award R01GM099742

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inositol trisphosphate dynamics underlie physiological calcium signals within the coronary artery endothelium
Authors: Francis Christopher M. , Knighten Jennifer M. , Taylor Mark S.
Published: June 2021

The arterial endothelium modulates smooth muscle tone in response to its local hemodynamic environment. This process is critical to normal vascular function, and is impaired by endothelial cell injury in coronary artery disease. Last year, we discovered a unique mode of calcium signaling of the porcine coronary artery endothelium, characterized by a pattern of continuous, cell‐wide calcium waves in the absence of agonist stimulation. Stimulation with substance P (10−13 to 10−6 ml/L) amplified this pattern to match dose‐dependent vessel relaxation responses. Here, we investigated the mechanism underlying the formation of this characteristic pattern of endothelial calcium waves with confocal microscopy, utilizing both pharmacological and computational approaches. We found that coronary endothelial cell calcium waves were mediated by inositol trisphosphate receptors (IP3R), which exhibited distinct intracellular isoform distributions. The distribution of the type 1 IP3R isoform corresponded to the area (33.9 ± 2.6 μm2) of propagating calcium waves, including perinuclear IP3R clustering at subcellular sites of constitutively elevated calcium. We utilized a computational model of calcium signaling that included this distinct IP3R distribution, and discovered that cell‐wide waves formed only if intracellular concentration gradients of inositol trisphosphate (IP3) were present along the longitudinal cell axis. Importantly, we found that production and diffusion of IP3 from a focal intracellular source was sufficient to initiate a calcium wave event with the same propagation velocity curve (mean: 6.08 ± 0.61 μm/s) of the experimentally‐observed wave. Furthermore, amplification of IP3 production in our model resulted in an increase of the frequency of elementary wave events which reflected the dose‐dependent expansion of calcium activity induced by substance P. These results indicate a fundamental role for IP3 dynamics as a physiological mediator of vascular responses, underlying both the initiation and expansion of unique endothelial calcium signal modalities.

Support or Funding Information

This work was supported by NIH grants: HL136869, HL085887, and S10RR027535.

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 Dopamine Receptor D3 Deficiency in Age‐dependent Left Ventricular Remodeling
Authors: Grilo Gabriel Araujo , Shaver Patti Rebecca , Stoffel Hamilton Joseph , Kennedy Kylie Ruthann , Morrow Caleb Anthony , Iyer Rugmani Padmanabhan , Clemens Stefan , de Castro Brás Lisandra Eunice
Published: June 2021

Aging is an independent risk factor for cardiovascular disease. Additionally, normal aging associates with a decrease on the levels of the neuromodulator dopamine (DA). While the role of DA in the pathophysiology of hypertension and in autonomic function is well‐established in young animals, much less is known on how DA levels and signaling affect cardiac aging. Decreased receptor expression/function of the D2‐like DA receptor family (DRD2 and DRD3) have been implicated in the development of renal and cardiac fibrosis. It is well established that myocardial aging is characterized by left ventricular (LV) fibrosis, which is a component of LV adverse remodeling. Accordingly, the goal of this study was to elucidate the effects of DRD3 deficiency in age‐dependent LV remodeling.

Methods

We used 3, 6, 12, and 18 month old (m.o.) wild type (WT) and DRD3 knockout (D3KO) male and female mice (n=6/sex/age/genotype). Cardiac function was assessed by serial echocardiography, and plasma and left ventricle were harvested to analyze molecular parameters of LV remodeling, particularly extracellular matrix composition.

Results

Echocardiography analysis demonstrated that D3KO mice have a continuous, age‐dependent, increase of the LV anterior wall (LVAW), compared to WT, in both genders, but more markedly in females. This suggests that female D3DKO may have age‐dependent hypertrophy. In addition, the interior diameter in diastole (LVIDd) of D3KO animals was consistently smaller than WT mice of the same age, particularly at 6 and 12 m.o. (Figure 1). Cardiac fibrosis increased in the older male animals (18 m.o.) compared to all other ages in both genotypes. Females, however, showed a distinct genotype‐dependent pattern, suggesting sex‐differences in collagen metabolism with age. Genes associated with cardiac remodeling, as Gsta1, Nrp2, Mmp12 and Vcam1 showed increased expression with aging in WT females, but not in D3KO. Male D3KO however, displayed increased Ccl2 and Ecm1 at 18 m.o. compared to all other ages and Casp1 (an inflammatory response initiator) was increased in D3KO with time.

Conclusion

Our data suggests that DRD3 function in the heart plays an important gender‐specific role in age‐dependent cardiac remodeling.

Support or Funding Information

We acknowledge support from American Heart Association (14SDG18860050) and the Research and Graduate School at East Carolina University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardiovascular Mathematics: Prevention, Promotion and Treatment
Authors: Calvachi Paola , Pizano Alejandro , Girón Felipe , Cordovez Juan Manuel
Published: June 2021

Introduction

Cardiovascular disease is the first cause of death in the world, being heart failure responsible for most of the hospital admissions. There is a strong relationship between heart failure and chronic renal disease, where kidney disease is one of the major contributors to cardiac damage and, likewise, heart disease promotes a progression of chronic kidney disease. Our main goal was to understand the dynamics of this vicious cycle in a person with heart failure and investigate mechanisms to prevent and treat this pathology.

Methods

To achieve this, a mathematical model was proposed based on fluid mechanics. We assumed the cardiovascular system as a circuit with two pumps (Right and Left ventricles) and 7 resistances: 4 that represent the cardiac valves (Tricuspid, Pulmonary, Mitral and Aortic), the systemic, renal and pulmonary capillaries. The model included compliances and resistances as parameters, and pressures, volume and flows were determined for each segment in the circulatory system. Parameters were determined according to anatomical and physiological properties found in the literature. The model was implemented in MATLAB (The MathWorks®, Inc.).

Results

The mathematical model showed how the renal system is integrated with the cardiovascular pump. The model demonstrated that physicians have a short time period of action in order to preserve the renal function in patients with cardiovascular diseases, and also emphasize in a way to prevent the progression from heart disease to renal failure. We were able to compare the pressure‐volume curves for disease and healthy states and establish the main challenges for the circulatory system under these conditions.

Conclusions

The mathematical model was helpful to understand the normal and pathological functioning of the cardiovascular and renal systems. In particular, our ability to keep track of total blood volume gave insight into the relationship between renal and cardiac malfunction. This approach could be the mainstay for health prevention, promotion and opportune treatment in cardiorenal syndrome.

Support or Funding Information

Research Group in Mathematical and Computational Biology (BIOMAC), Department of Biomedical Engineering, Universidad de los Andes, Colombia.

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 Mouse Heart Attack Research Tool (mHART) 1.0 Database
Authors: DeLeon‐Pennell Kristine Y. , Ma Yonggang , Yabluchanskiy Andriy , Flynn Elizabeth , Halade Ganesh V. , De Castro Bras Lisandra , Lindsey Merry L.
Published: June 2021

Background

The generation of Big Data has enabled systems‐level dissections into the mechanisms of cardiovascular pathology. Integration of dozens of variables across a variety of platforms and across laboratories fosters discoveries through multidisciplinary investigations, decreased costs, and elimination of unnecessary redundancy in research efforts. Moreover, Big Data brings speed, allowing more rapid and improved decision making. The Mouse Heart Attack Research Tool (mHART) harnesses a large dataset of over 10 years of experiments from a single site, for use by investigators to generate novel hypotheses, uncover hidden relationships, and identify new predictive markers of progressive cardiac remodeling following myocardial infarction (MI) in mice.

Methods and Results

We designed the mHART database to incorporate our own data, with future plans to integrate community participation. We generated genomic, proteomic, physiological, biochemical, and cellular outputs from plasma and left ventricles obtained from post‐MI and no MI (naïve) control groups. We included both male and female mice ranging in age from 3 to 36 months old. After variable collection, data underwent quality assessment to clean the data (i.e. to eliminate technical errors), check for completeness, remove duplicates, and define terms. Combined, mHART 1.0 contains >888,000 data points and includes results from >2,000 unique mice. Database performance was tested and an example applied to illustrate how this database can be used.

Conclusion

The efforts reported here establish the first version of the mHART database.

Support or Funding Information

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Vasopressin on Intrinsic Cardiac Activity in Conscious Wistar Rats.
Authors: da Silva Daniel Santana , Cafarchio Eduardo Mazuco , do Vale Bárbara , Antonio Bruno Brito , Venancio Daniel Paulino , de Souza Janaina Sena , Pino Jessica Monteiro Volejnik , Giannocco Gisele , Sato Monica Akemi
Published: June 2021

Background

The effects of vasopressin on cardiac chronotropism are controversial in different species.

Objective

To investigate if vasopressin can influence the cardiac intrinsic activity in conscious Wistar rats.

Methods

All the procedures of this study were approved by the Animal ethics committee (protocol #02/2016). Adult male Wistar rats underwent to a cannulation of the femoral artery and vein for mean arterial pressure (MAP) and heart rate (HR) recordings and drug infusions, respectively, in conscious rats, 24 h after the surgical procedures. After baseline recordings, i.v. atropine 2 mg/kg and i.v. metoprolol 1.7 mg/kg were injected with 5 min interval. After 10 min, i.v. vasopressin 0.0625 mg/mL/5 min (N=6) or saline (1 mL/5 min) (N=6) were infused and the variables were recorded for additional 10 min. Gene and protein expression of V1a, V1b and V2 subtype receptors for AVP were also carried in the right atrium by qPCR and Western Blotting, respectively. The AVP receptor subtypes were also labeled by immunofluorescence technique in the right atrium.

Results

Vasopressin evoked bradycardic (−51±5 bpm) and pressor responses (39±6 mmHg) compared to baseline post‐autonomic blockade (130 ±8 mmHg and 406±16 bpm). Saline did not change HR (1±4 bpm) and MAP (−3±1 mmHg) compared to baseline after autonomic blockade (120±5 mmHg and 411±52 bpm). All subtypes of AVP receptors were expressed in the right atrium by qPCR, Western Blotting and immunofluorescence.

Conclusion

The bradycardia elicited by vasopressin after cardiac autonomic blockade suggests that vasopressin can affect the cardiac intrinsic activity binding to the receptors in the right atrium.

Support or Funding Information

Financial support: FAPESP, CNPq and NEPAS‐FMABC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EVALUATION OF MINERAL BONE DISEASE IN PATIENTS WITH ACUTE KIDNEY INJURY
Authors: Morales Armando , Lanzoni Luís Gustavo , Silveira Cylmara Gargalak Aziz , Grabulosa Caren Cristina , Pinto Walter Pereira , Tinucci Taís , Moysés Rosa Maria Affonso , Quinto Beata Marie Redublo , Batista Marcelo Costa , Dalboni Maria Aparecida
Published: June 2021

Introduction

Patients with end‐stage renal disease (ESRD) develop various kinds of abnormalities in bone and mineral metabolism, widely known mineral bone disease (MBD). The MBD is one of the main cause of vascular calcification (VC) and mortality in this patients. This abnormalities are a consequence of the glomerular filtration rate declined and retention of a large number of compounds which under normal conditions are excreted by healthy kidneys. In the last years, Fibroblast Growth Factor 23 (FGF‐23) a phosphaturic hormone, Sclerostin (SOST) (a protein that inhibit bone formation ) and inflammation markers have been described as a important axis of the physiopathology of MBD and VC in ESRD. The early detection of MBD can reduced the VC and its complications. However, patients with acute kidney injury (AKI) which abruptly lose kidney function; have not been yet investigated to MBD. Thus, the objective of this study was to investigate markers of MBD in AKI patients.

Material and Methods

We included 77 severe patients without AKI and 83 severe AKI patients (ageing‐ 77 (60–84) all admitted to the Intensive Care Unit. Patients with AKI were characterized by AKIN criteria that used a 0.3 mg/dL increase in serum creatinine within seven days of admission in the hospital. Serum markers of MBD and inflammation investigated were: FGF‐23, SOST, Interleukin‐6 (IL‐6), Tumor Necrosis Factor Alpha (TNF‐α) and Interleukin‐10 (IL‐10) (by enzyme‐linked immunosorbent assay (ELISA). Results are expressed as median. Mann‐Whitney test was used to analyze the groups.

Results

All AKI patients presented serum creatinine >3.1 mg/dL (reference value 0.8 – 1.3 mg/dL). We observed increased serum levels of SOST (pg/mL) [AKI 1.74 (1.74–46.40) vs. No‐AKI 1.74 (1.74–1.74); p=0.009)], IL‐6 (pg/mL) [AKI 21 (11–32) vs. No‐AKI 12 (7–19); p<0.001], TNF‐α (pg/mL) [AKI 8.2 (6.7–10.5) vs. No‐AKI 7.0 (6.2–7.9); p<0.001], IL‐10 (pg/mL) [AKI 1.1 (0.8–1.6) vs. No‐AKI 0.8 (0.7–1.0); p<0.001]. We did not observe difference in FGF‐23 (pg/mL) [AKI 48.3 (1.5–141.3) vs. No‐AKI 48.0 (1.5–188.5); p=0.35].

Conclusion

AKI patients presented higher inflammation state characterized by increased serum levels of IL‐6, TNF‐α and IL‐10. Additionally, as SOST was higher in AKI patients, is possible used it as a marker of early bone disease in this population. In summary, this is the first study that detected high levels of SOST in AKI patients.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cepharanthine improves glycemic status and slows the progression of renal and vascular injury in streptozotocin‐induced diabetic rats
Authors: Samra Yara A. , Katary Mohamed A. , El‐Shishtawy Mamdouh M. , Essa Laila A. , Baban Babak , Elmarakby Ahmed A.
Published: June 2021

We have previously demonstrated that Cepharanthine improves glycemic status and reduces renal injury in streptozotocin‐induced diabetic rats via antioxidant and anti‐inflammatory mechanisms. However, the hypoglycemic mechanism of Cepharanthine remains unclear. We hypothesize that Cepharanthine reduces blood glucose in steptozotocin‐induced diabetic rats via restoring pancreatic β‐cells mass and insulin secretion. Diabetes was induced by a single i.p. injection of streptozotocin (50 mg/kg) in male Sprague Dawley rats. Control and diabetic rats were treated with Cepharanthine (10 mg/kg/day, i.p.) for six weeks (n=6–8). Cepharanthine treatment significantly reduced blood glucose levels and increased plasma insulin levels in diabetic rats (blood glucose was 290± 67 in Cepharanthine treated diabetic rats vs. 550±17 mg/dl in non‐treated diabetic rats, P<0.05). The percentage of TUNEL positive cells was significantly elevated in the pancreas of diabetic rats and these changes was reduced by Cepharanthine treatment (P<0.05). Immunohistochemcial analysis revealed a decrease in pancreatic islet‐specific glucose‐6‐phosphatase catalytic subunit related protein (IGRP) and an increase in pancreatic and duodenal homeobox 1 (PDX‐1) expression levels in Cepharanthine treated vs. non treated diabetic rats (P<0.05). Cepharanthine also improved vascular endothelial function as it significantly attenuated the impairment in aortic ring relaxation to acetylcholine in diabetic rats. Moreover, Cepharanthine reduced markers of renal injury and inflammation and slowed the progression of renal fibrosis as evidenced by the reduction in levels of albuminuria, podocalyxin excretion, renal ICAM‐1 and TGF‐β in diabetic rats. The reduction in renal inflammation and injury was associated with decreased in renal caspase 3 expression in Cepharanthine treated diabetic rats. These data suggest that Cepharanthine halts the progression of streptozotocin‐induced diabetic complications in rats via reduced pancreatic β‐cells apoptosis and increased β‐cells maturation and subsequently insulin release probably via the regulation of the PDX‐1 and IGRP transcription factors of pancreatic β‐cells.

Support or Funding Information

The work was supported by bridge funding grant from Augusta University, Augusta, Georgia to Dr. Ahmed A. Elmarakby

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary Nitrate Enhances Peak Power in Aged Murine Diaphragm
Authors: Kelley Rachel C. , Kumar Ravi A. , Hahn Dongwoo , Ferreira Leonardo F.
Published: June 2021

Inspiratory muscle (diaphragm) function declines with age. Aging‐related diaphragm atrophy and/or dysfunction likely contribute to impaired airway clearance and exercise intolerance in the elderly. The goal of our current study was to determine the therapeutic potential of dietary nitrate supplementation in aged diaphragm. Aging decreases the availability of nitric oxide (NO), but supplementation with NO precursors (e.g., sodium nitrate, beetroot juice, arginine) may augment NO production. Previous studies have shown that dietary nitrate may improve skeletal muscle contractile efficiency, mitochondrial function, and anabolism by enhancing NO's vasodilatory and/or cell signaling effects. In this study, old (n=14, 24 months old) mice received sodium nitrate (NaNO3, 1 mM) in drinking water or vehicle (deionized H2O) ad libitum for a total of 14 days. After treatment, diaphragm function was evaluated using isolated muscle bundles. Dietary nitrate supplementation did not result in improvements in maximal isometric force (control: 20.5 ± 2.9 vs NaNO3: 22 ± 3.4 N/cm2), twitch force (control: 3 ± 0.5 vs NaNO3: 3.6 ± 0.6 N/cm2), time‐to‐peak tension (control: 16.7 ± 0.5 vs NaNO3: 15.3 ± 1.7 ms), ½ relaxation time (control: 14.1 ± 1.8 vs NaNO3: 14.3 ± 2 ms), or maximal shortening velocity (control: 7 ± 3.1 vs NaNO3: 8.1 ± 1.2 Lo/s). However, dietary nitrate supplementation did significantly increase peak power from 161.4 ± 32.3 W/kg in controls to 224.8 ± 35.5 W/kg in supplemented mice (p < 0.05). Ventilation during exercise as well as non‐ventilatory behaviors needed for airway clearance, such as coughing and sneezing, require rapid, forceful contractions of the diaphragm. Therefore, our results suggest that supplementation with dietary nitrate may increase peak power and thus be of benefit in aging populations.

Support or Funding Information

NIA 1R03AG04040001A1

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Electric stimulation alters intercellular stress within the epithelial monolayer
Authors: Cho Youngbin , Son Minjeong , Ko Ung Hyun , Jeong Hyuntae , Shin Jennifer Hyunjong
Published: June 2021

Introduction

The directed migration of cell plays a pivotal role in physiological processes such as development, wound healing, vasculogenesis, and cancer metastasis. Electric field (EF) is one of the prevailing guidance cues in vivo, which promotes directional migration in both single and collective cells. Numerous studies have studied the detailed biochemical mechanism of electrotaxis, directional guidance by EF, both in vivo and in vitro. However, these studies mainly focused on a single cell behavior. Since the collective cells are feeling severe confinement due to their neighboring cells, not only biochemical but also physical analysis is essential to understand the mechanism of coordinated cellular guidance by EF. Here, we aimed to investigate how the cellular physical forces are altered in response to externally applied direct current electric field (dcEF). We established the EF stimulating platform combined with polyacrylamide gel for the simultaneous measurement of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy (MSM).

Materials and Methods

In this study, we developed cell observable platform, equipped with agar bridge and steinberg's solution to apply dcEF. To analyze cellular forces, we combined 0.5 μm fluorescence bead embedded poly‐acrylamide hydrogel within the chamber. We micropatterned HacaT cell‐line using PDMS stencil on the poly‐acrylamide gel before stimulating dcEF. After 1 day maturation of HacaT monolayer, we stimulated 0.5 V/cm dcEF continuously over the experiment. Traction field was calculated from the measured displacement field of fluorescent beads embedded within a hydrogel of 3 kPa stiffness. Gel displacement was quantified at each experimental time point, and a reference image was obtained after cell detachment. Traction was obtained by the numerical procedure from Fourier‐transform‐traction‐microscopy (FTTM). Based on the traction field, we calculated intercellular stress using monolayer stress microscopy (MSM).

Results and discussion

We observed that the EF stimulation induced the rapid response of collective migration toward the anode. This guided migration was followed by the reorientation of individual cells to perpendicular axis against the EF direction. Interestingly, enhancement of intercellular stress component perpendicular to the EF preceded the coordinated alignment of cellular orientation within the epithelial monolayer. These results suggest that the EF‐induced formation of anisotropic intercellular stress leads the reorientation of individual epithelial cells within the bulk of collective monolayer.

Support or Funding Information

This work was supported by the National Research Foundation of Korea Grant funded by the Korean Government (NRF‐2013S1A2A2035518, NRF‐2016K2A9A2A08003761)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inclusion of Dairy Cheese In An 8‐day Controlled Dietary Intervention Prevents Sodium‐Induced Endothelial Dysfunction in the Cutaneous Microcirculation of Healthy Older Adults
Authors: Alba Billie K. , Stanhewicz Anna E. , Bruno Richard S. , Kenney W. Larry , Alexander Lacy M.
Published: June 2021

Excess dietary sodium is associated with impaired vascular function and increased cardiovascular mortality. In controlled feeding studies, the inclusion of 3 daily servings of any dairy product improves blood pressure and measures of vascular health. Few studies have examined the inclusion of individual dairy products that contain both the beneficial bioactive dairy proteins and sodium. We recently demonstrated that single‐meal cheese consumption ameliorates acute sodium‐induced impairments in nitric oxide‐dependent vasodilation. However, controlled feeding studies examining the vasoprotective role of dairy cheese on sodium‐induced microvascular dysfunction are lacking. We hypothesized that (1) endothelium‐dependent vasodilation would be impaired by a high‐sodium diet compared to a low sodium diet, and (2) that a high‐cheese diet would preserve endothelium‐dependent vasodilation that is otherwise impaired by a high‐sodium diet. In a randomized, crossover study design, seven healthy older adults (63 ± 3 years; salt‐resistant: <10 mmHg increase in mean arterial pressure, 24 hour monitoring) with normal to elevated blood pressure completed 4 separate 8‐day controlled dietary interventions: 1) low‐sodium (LNa: 1500 mg sodium) diet devoid of dairy products, 2) low‐sodium high‐cheese diet (LNaC: 1500 mg sodium, 6 oz/d cheese), 3) high‐sodium (HNa: 5500 mg sodium) diet devoid of dairy products, and 4) high‐sodium high‐cheese diet (HNaC: 5500 mg sodium, 6 oz/d cheese). At the end of each dietary intervention, subjects participated in an intradermal microdialysis protocol in which one microdialysis fiber was inserted into the forearm skin for measurement of red cell flux (laser Doppler flowmetry) during local infusion of the endothelium‐dependent agonist acetylcholine (Ach; 10−12 to 10−1 M). Data were expressed as a percentage of maximum cutaneous vascular conductance (%CVCmax; 28 mM sodium nitroprusside, 43°C). Ach‐induced vasodilation was significantly attenuated during the HNa diet compared to the LNa diet (LNa: −5.06±0.20 M vs HNa: −3.22±0.63 M logEC50: p < 0.05). Conversely, Ach‐induced dilation during either the LNaC or HNaC diet was not different from the LNa diet (p > 0.05). These results demonstrate that increased dietary sodium intake impairs endothelium‐dependent dilation whereas the inclusion of cheese prevents sodium‐induced endothelial dysfunction in salt‐insensitive older adults.

Support or Funding Information

Supported by National Dairy Council

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of basolateral ClC‐K2‐dependent Cl‐ reabsorption in collecting duct intercalated cells by dietary electrolyte intake and Ang II
Authors: Zaika Oleg , Tomilin Viktor N. , Pochynyuk Oleh M.
Published: June 2021

Hypertension is the major clinically relevant problem, which commonly stems from excessive renal salt retention. Both basic and clinical studies show that elevated dietary Na+ can promote volume expansion and increased blood pressure only when accompanied with high Cl−. The collecting duct (CD) is the only tubular segment with capacity to separately reabsorb Na+ and Cl−. Here, principal cells perform electrogenic Na+ reabsorption via the epithelial Na+ channel (ENaC), whereas activity of the basolateral ClC‐K2 chloride channel mediates trans‐cellular Clmovement in electrically uncoupled intercalated cells. In contrast to ENaC, little is known about dietary and hormonal means controlling ClC‐K2 activity in the CD. Using patch clamp electrophysiology at both single channel and macroscopic levels in freshly isolated CDs, we found that ClC‐K2 single channel activity and macroscopic Cl− conductance in intercalated cells are inversely related to dietary Cl−, but not K+ intake. Furthermore, stimulation of mineralocorticoid receptors with deoxycorticosterone acetate (DOCA) failed to affect ClC‐K2 activity arguing against a role of aldosterone in regulation of ClC‐K2 by dietary Cl−. In contrast, Angiotensin II (Ang II) exerts a set of stimulatory actions by acutely increasing ClC‐K2 single channel open probability, ClC‐K2/b‐dependent Cl− conductance in ICs and on a longer timescale promotes ClC‐K2/b trafficking to the basolateral membrane of ICs in freshly isolated CDs implicating its potential role during variations in dietary Cl− intake. Inhibition of AT1 receptors (AT1R) with losartan precluded regulation of ClC‐K2 by Ang II. Moreover, ClC‐K2 abundance was significantly reduced in mice lacking AT1R. Overall, our results point to a critical role of dietary Cl− in controlling ClC‐K2 activity in the CD likely in an Ang II‐AT1R dependent manner. We propose that this mechanism could be instrumental for the discrete CD responses to hypovolemia (when Ang II is high) and hyperkalemia (Ang II is low).

Support or Funding Information

This research was supported by NIH‐NIDDK DK095029 (to O. P.), AHA 17GRNT33660488 (to O. P.), and ASN Ben J. Lipps Research Fellowship (to V. T.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metformin Blunts Exercise‐Induced Improvements in Skeletal Muscle Mitochondrial Respiration Independent of Changes in Mitochondrial Biogenesis
Authors: Konopka Adam R. , Castor William M. , Reid Justin , Schoenberg Hayden , Laurin Jamie , Wolff Christopher , Hamilton Karyn , Miller Benjamin
Published: June 2021

The anti‐hyperglycemic agent Metformin and exercise independently prevent the progression from prediabetes to overt type two diabetes (T2D) and are both implicated in healthspan extension. However, emerging evidence indicates the addition of Metformin to exercise may inhibit the health benefits of exercise. The purpose of the current study was to test the hypothesis that Metformin blunts the positive adaptations to exercise by inhibiting skeletal muscle mitochondrial fractional protein synthesis rates (FSR) and respiration. Older adults (n=53, 62±5 yrs) with at least one risk factor for T2D were randomized in a double‐blinded fashion to consume Metformin or placebo (1500–2000 mg/day) during 12‐weeks of aerobic exercise training (AET). VO2max and skeletal muscle mitochondrial respiration were assessed before (PRE) and after (POST) the 12‐week intervention. Skeletal muscle biopsy samples were obtained 48 hrs after the last exercise bout and 36 hrs after the last metformin dose. High‐resolution respirometry was performed on permeabilized skeletal muscle fibers to assess submaximal and maximal oxidative phosphorylation (OXPHOS; P) primarily linked to complex‐I (CIP), fatty acid oxidation (CIP&FAO), and complex II (CI+IIP&FAO). Subsequent addition of FCCP determined maximal uncoupled electron transport system (ETS) capacity. During the last four weeks of the intervention, participants consumed the stable isotope deuterium oxide (2H2O) to measure cumulative FSR (%/day) in a mitochondrial enriched skeletal muscle fraction. Compared to placebo, Metformin tended (P=0.10) to attenuate the increase in VO2max after 12 weeks of AET (Metformin: 0.06±0.02 vs. Placebo: 0.13±0.03 L/min). Congruent with these findings, Metformin abrogated (P<0.05) the exercise‐mediated increase in submaximal mitochondrial respiration, CIp, CI+IIP, and CI+IIP&FAO after AET (Metformin: −0.7 to 2.6 vs Placebo: 5.3 to 22.8 pmol/s/mg tissue). Unlike the effect of Metformin on mitochondrial respiration, mitochondrial protein synthesis rates assessed during AET were not different between Metformin and placebo (1.06±0.05 vs. 1.00±0.04 %/day; P=0.38). Here we demonstrate that metformin tended to diminish the improvement in VO2max and inhibited the increase in skeletal muscle mitochondrial respiration after AET in older adults at risk for T2D. However, the effects on mitochondrial respiration do not appear to be related to changes in mitochondrial biogenesis. Future research is warranted to determine mechanisms by which metformin influences skeletal muscle mitochondrial respiration and impacts the health benefits of exercise.

Support or Funding Information

This study was supported by the National Dairy Council

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
CHARACTERISATION OF NF‐KB ACTIVATION IN REGENERATING FIBRES OF OLD MICE
Authors: Owen Euan David , Pollock Natalie , Jackson Malcolm Joseph , Vasilaki Aphrodite , McArdle Anne
Published: June 2021

Sarcopenia is the age‐related loss of muscle mass and function. In human studies a decrease of up to 50% in muscle mass and function is observed from the ages of 25 to 70 years and this contributes significantly to frailty and loss of independence in the ageing population. An increase in pro‐inflammatory cytokines is associated with many age‐related conditions including sarcopenia (particularly TNF‐alpha and IL‐6). Many inflammatory processes are under the direct influence of the NF‐κB pathway and a chronic activation of NF‐κB has been shown in muscles from old mice and in accelerated models of muscle ageing (eg. Vasilaki et al, 2006). Additional studies using immunofluorescent antibody staining demonstrated the presence of p65 (a subunit of NF‐κB) only in muscle fibres containing centrally located nuclei, suggesting that NF‐kB was only active in regenerating muscle fibres.

The aim of this study was to determine the extent and nature of activation of NF‐kB during regeneration in a well established mouse model of muscle degeneration and regeneration using the myotoxin, barium chloride (BaCl‐2‐). BaCl‐2‐ was injected into the extensor digitorum longus (EDL) muscles of adult (8 months) and old (22 month) wild‐type (WT) C57BL/6 mice. At defined time points the muscles were quickly excised, weighed and frozen in OCT for transverse sectioning. Changes in muscle morphology were observed through haemotoxylin and eosin staining while fluorescently labelled antibodies against p65 targeted the cellular localization of NF‐κB.

Centrally located nuclei were present in the majority of the fibres at 14, 28 and 60 days post BaCl2 injection. Nuclear translocation of p65 was seen 14 and 28 days post BaCl2 injection indicating activation of the canonical NF‐κB pathway in regenerating fibres.

The results of these experiments reveal an association between muscle fibre regeneration and activation of the canonical NF‐κB pathway. Further studies will determine the effect of activation of NF‐kB during regeneration on inflammatory gene expression and compare with activation in ageing models.

Support or Funding Information

Funded by the National Institutes of Health (NIA).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
New insights into a decerebrate feline model of swallow‐breathing coordination
Authors: Horton Kofi‐Kermit , Segers Lauren , Nuding Sarah , O'Connor Russell , Alencar Pierina , Davenport Paul , Lindsey Bruce , Morris Kendall , Gestreau Christian
Published: June 2021

In the medulla, the swallow and respiratory central pattern generators (CPGs) interact to coordinate swallow initiation and permit swallow execution during the expiratory phase of breathing. Disruption of swallow‐breathing coordination can result in dysphagia, which is a major cause of morbidity and mortality in many neuro‐muscular diseases, especially stroke. Despite extensive study, the precise mechanisms of swallow‐breathing coordination are not known. We tested the hypothesis that both central inspiratory activity and vagal feedback from the lungs are key elements for effective swallow‐breathing coordination. Swallows were triggered by electrical stimulation of the superior laryngeal nerves (SLN) or by injection of water into the pharyngeal cavity in decerebrate, paralyzed and artificially ventilated cats while motor activities were recorded from phrenic, hypoglossal, lumbar and vagal nerves. Mechanical ventilation was either triggered by central inspiratory activity or set independently from the phrenic activity to alter the timing of bronchopulmonary vagal afferent input and allow analysis of its influence on swallow‐breathing coordination. We observed two types of swallows that produced opposite effects on central respiratory‐rhythm resetting across all conditions: post‐Inspiratory type swallows disrupted central I activity without affecting expiration, whereas expiratory type swallows prolonged expiration without affecting central inspiratory activity. Repetitive swallows observed during apnea reset the E2 phase of central respiration and produced facilitation of swallow motor output. Moreover, swallow initiation was negatively modulated by vagal feedback and was reset by lung inflation. Collectively, these findings support a reciprocal inhibition between central inspiration and the swallow CPG and they are consistent with a role for pulmonary stretch receptors in the coordination between brainstem CPGs for breathing and swallowing. They may also prove relevant for healthcare management of dysphagia.

Support or Funding Information

OT2OD023854‐01, HL109025

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Membrane curvature and pore formation induced by antimicrobial peptides
Authors: Lee Hwan Kyu
Published: June 2021

Amphiphilic peptides of different lengths were simulated with lipid bilayers composed of 1,2‐ dimyristoyl‐sn‐glycero‐3‐phosphocholine (DMPC) and 1‐myristoyl‐2‐hydroxy‐sn‐glycero‐3‐ phosphocholine (lysoMPC) at different ratios. Simulations of lipid bilayers without peptides show that the bilayers with more lysoMPC become more disordered and thinner. Amphiphilic peptides added to this simulation do not insert into the DMPC bilayer at low peptide/lipid ratio (P/L ≤ 1/50), while they do insert to the DMPC/lysoMPC bilayer and form a toroidal pore even at such a low P/L ratio, where the pore edge is surrounded by lysoMPC rather than by DMPC. In particular, upon pore formation, peptides migrate toward the edge of a pore and become tilted, showing transmembrane alignment regardless of the peptide length, in qualitative agreement with experiments. This pore formation occurs more frequently in larger bilayers that allow greater curvature, indicating that bilayer curvature is important for pore formation. These indicate that the addition of lysoMPC induces the thinner bilayer with greater curvature, and thus the bilayer with lysoMPC can be more easily penetrated by peptides, leading to the formation of a toroidal pore stabilized by peptides and lysoMPC. These findings help explain experimental observations of the effect of the inverted cone‐shaped lyso‐lipid on pore formation and peptide orientation, and also support the experimental suggestion regarding the formation of an iris‐like ring of helices lining a toroidal pore.

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 Hydrophobic Domain of ENV9 is a Lipid Droplet Localization Signal in Saccharomyces cerevisiae
Authors: Ricci Anthony , Valencia Sara , Siddiqah Ikha , Manandhar Surya , Gharakhanian Editte
Published: June 2021

Lipid droplets (LDs) are dynamic organelles involved in the storage of neutral lipids for membrane biogenesis and metabolism. Malfunction of LDs has been associated with diabetes, obesity, and cancer, whereas proper organelle and cell function is mediated through cellular trafficking. Therefore, uncovering the mechanism by which proteins that are necessary for LD function are transported is essential for understanding the development of these diseases. We have found Saccharomyces cerevisiae Env9 to be a novel oxidoreductase that is conserved and involved in LD morphology. It contains a hydrophobic domain (HD) sequence (aa241‐265) that we have previously reported as necessary for Env9 localization to the LD membrane. For a sequence to be categorized to a specific pathway it needs to be demonstrated as both necessary and sufficient. We hypothesize that the HD is a LD localization signal and is therefore sufficient to direct proteins to the LD. Here, we are testing if the HD sequence is sufficient to direct a reporter protein to the LD membrane that typically would not localize to the LD. The protein we chose is Env7, a protein kinase that normally anchors to the lysosomal vacuole by palmitoylation of a triple cysteine stretch at its N‐terminus. A new plasmid construct was engineered where the Env9 HD sequence was integrated into GFP tagged Env7 (C13‐15S) plasmid. Cells expressing ENV7 (C13‐15S)‐HD‐GFP were investigated using confocal microscopy to confirm localization of Env7. The HD of Env9 was seen to mediate the localization of Env7 (C13‐15S) to the LD membrane. Thus, the HD is sufficient for localization and is therefore a LD targeting signal. Biochemical analysis is being done to further confirm membrane association of Env7 (C13‐15S) to the LD. Thus far, this research could provide a better insight into LD protein trafficking mechanisms.

Support or Funding Information

This work was supported by NIH AREA #R15 GM85794‐02 to EG and by NSF‐MRI grant DBI0722757 for confocal microscopy. As well as, BUILD grants given by National Institute of General Medical Sciences of the National Institutes of Health under Award Numbers; 8UL1GM118979‐02; 8TL4GM118980‐02; 8RL5GM118978‐02

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