bluuebunny/crossref_metadata_2025 huggingface dataset:
ALPL, A Novel Marker Of Neutrophil Activation In Response To Obesity
Authors: Pan Yue , Choi Justin , Shi Huidong , Su Shaoyong , Wang Xiaoling
Published: June 2021

Low‐grade systemic inflammation accompanies obesity and etiologically contributes to obesity‐induced cardiovascular disease (CVD). Neutrophils represent the most abundant type of leukocytes in humans and neutrophil activation is a fundamental process in the inflammatory response. Growing evidence supports that neutrophil is most likely to be the target peripheral leukocyte subtype initiating the inflammatory cascade in response to obesity. However, few studies have systematically assessed the molecular changes of neutrophils in response to obesity. In this study, a hypothesis‐free OMIC approach (i.e. the discovery phase) and a target approach (i.e. the validation phase) were used to identify obesity related neutrophil activation markers and their roles on CVD risks. In the discovery phase, genome wide DNA methylation, RNA‐sequencing and quantitative proteomics were obtained from the purified neutrophils of 12 obese cases and 12 lean controls. Significant difference was found for one gene, alkaline phosphatase, liver/bone/kidney (ALPL), across 3 OMIC platforms. In the validation phase, the expression levels of ALPL in leukocytes were obtained from 81 obese cases and 83 lean controls. The ALPL expression levels were significantly higher in obese compared with lean subjects (p=0.021). Within obese population, we observed that ALPL expression level showed significantly positive association with CVD risk factors including systolic blood pressure (BP) (p=0.032), diastolic BP (p=0.011) and mean arterial pressure (p=0.008), as well as borderline significance with fasting insulin (p=0.059). This study identified one novel marker of neutrophil activation in response to obesity and provided further evidence that obesity induced change in ALPL expression is associated with CVD risk factors.

Support or Funding Information

NHLBI HL105689

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Concerted Molecular Clock Control of Myogenic Precursor Properties Promotes Skeletal Muscle Growth and Remodeling
Authors: Ma Ke , Chatterjee Somik , Lee Jeongkyong , Yechoor Vijay
Published: June 2021

Circadian clock imparts temporal regulation on diverse physiological processes. We and others recently demonstrated that the clock circuit plays an important role in muscle growth and repair for maintenance of muscle mass, raising the possibility of targeting this machinery for muscle‐wasting disease therapies. Currently, the mechanisms underlying clock regulation of muscle growth processes are largely unknown. Here we show that the antagonistic actions of the essential clock transcriptional activator, Brain and Muscle Ant‐like 1 (Bmal1), and the clock repressor protein, Rev‐erbα, coordinately modulate myogenic precursor cell (MPC) differentiation and proliferation properties to facilitate muscle growth and remodeling. We were the first to report a novel function of Bmal1 in promoting MPC myogenic differentiation required for skeletal muscle regenerative repair. Current studies reveal that the clock transcription repressor Rev‐erbα suppresses myocyte development and its genetic loss of function promotes regenerative myogenesis, through its negative transcription control of target genes in the Wnt signaling pathway. Furthermore, we show that Bmal1 promotes, whereas Reverbα inhibits MPC proliferative behavior in vitro and upon muscle regeneration, mediated by their respective transcriptional regulations of cell cycle regulators. Notably, our work uncovers that mechanical stretch and exercise‐associated signals induce clock gene expression, suggesting their roles in entraining the cell‐autonomous muscle clock oscillators to physiological stimuli. Collectively, our studies reveal concerted functions of the muscle‐resident clock machinery in orchestrating MPC properties to contribute to muscle growth and consequently mass maintenance in accordance with myogenic cues. Elucidation of this temporal control in skeletal muscle tissue remodeling may offer novel therapeutic strategies to combat muscle‐wasting associated with aging, cancer or chronic diseases.

Support or Funding Information

This project is supported by grants American Heart Association 17GRNT33370012, Muscular Dystrophy Association 381294, and NIH DK 1R01DK112794 to KM.

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 comparison of protective effects between electrical and pulsed‐magnetic stimulation in the deep layer of skeletal muscles under disuse condition
Authors: Ono Kohei , Nakanishi Ryosuke , Matsumoto Tomohiro , Takuwa Miho , Hirabayashi Takumi , Ikeji Takuya , Tanaka Minoru , Maeshige Noriaki , Kondo Hiroyo , Fujino Hidemi
Published: June 2021

Electrical stimulation (ES) fails to prevent the muscle atrophy in the deep layer of extremities and trunk because ES cannot easily pass through the adipose tissue to reach the deep muscles. Pulsed‐magnetic stimulation (MS) might reach to the deep muscles. Therefore, the aim of this study was to compare the effects of ES and MS on the deep muscle atrophy in rat calf muscles. Rats were randomly divided into the following groups: control, hindlimb unloading (HU), HU plus ES and HU plus MS. Transcutaneous ES and MS was carried out for calf muscles at 20% of the supramaximal stimulus, once a day, for 2 weeks during unloading. Unloading resulted in a decrease of muscle mass and muscle fiber cross‐sectional area and an increase of MuRF‐1 expression level in the gastrocnemius and soleus muscles. HU plus ES and HU plus MS groups were confirmed that the relative muscle ratio and fiber cross‐sectional area were higher, and the level of MuRF‐1 expression was lower in the gastrocnemius muscle compared with HU group. However, electrical stimulation failed to prevent the decrease of relative muscle ratio and fiber cross‐sectional area and the increase of MuRF‐1 expression level in the soleus muscle. In addition, pulsed‐magnetic stimulation increased the relative muscle ratio and fiber cross‐sectional area and decreased the MuRF‐1 expression level in the soleus muscle compared with electrical stimulation. These results indicate that pulsed‐magnetic stimulation is more effective in the prevention of deep muscle atrophy than electrical stimulation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of heat stress treatment and leucine supplementation on age‐related muscle loss in mice
Authors: Kakigi Ryo , Goto Ayumi , Yoshihara Toshinori , Tsuzuki Takamasa , Naito Hisashi
Published: June 2021

Acute heat stress treatment activates mammalian target of rapamycin (mTOR) signaling in skeletal muscle, leading to increase skeletal muscle protein synthesis and skeletal muscle mass. Leucine intake also activates mTOR signaling and stimulates protein synthesis. This study investigated the effects of heat stress treatment and leucine supplementation on sarcopenia (muscle loss with aging) in mice. C57BL/6J male mice (14 or 98 weeks old) were assigned randomly to young control (YC, N=6), old control (OC, n=9), heat stress (OH, n=7), leucine supplementation (OL, n=7), and heat stress and leucine supplementation (OLH) groups. Mice in OH and OLH groups were exposed to hot environment chamber (41ºC) for 40 min twice per week. Mice in OL and OLH groups were given ad libitum access to leucine‐supplemented water (114mM leucine). After 12‐week intervention, there were no differences in body weight and grip strength among groups. Body fat percentage in OLH group was significantly lower than that in OC group (29.0 ± 6.4 % vs. 39.1 ± 8.5 %, p < 0.05). The vastus lateralis muscle weight was significantly lower in OC, OH and OL groups compared with that of YC group, but the muscle weight in OLH group was significantly greater than that in OC group (191.9 ± 12.8 mg vs. 167.1 ± 10.1 mg, p < 0.05). Heat stress treatment induced the expression of heat shock protein 72 (HSP72) in muscle of OH and OLH groups (~5 fold, p < 0.05). There were no differences in the muscle mTOR signaling (the phosphorylation of mTOR, p70s6k, S6 and 4E‐BP1) between groups under overnight fasting condition. On the other hand, the expression of L‐type amino acid transporter 1 (LAT1) of muscle was significantly increased in OC, OH and OL groups compared with YC group (~3 fold, p < 0.05), but there were no difference between YC and OLH group. Our current observations show that the combination of heat stress treatment and leucine supplementation appears to be clinically beneficial for age‐related muscle loss. In addition, normal expression of LAT1 in muscle might contribute to suppression of muscle loss.

Support or Funding Information

This work was supported by JSPS KAKENHI Grant Number JP16K16565.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Skeletal Muscle Gene Expression in High Fat Diet‐fed LKB1 Knockout Mice.
Authors: Cheung Eric C.K. , Chen Ting , Hill Jonathon T. , Thomson David M.
Published: June 2021

The development of obesity leads to many alterations in skeletal muscle metabolism. Liver kinase B1 (LKB1) is well‐known to regulate skeletal muscle function and metabolism through its phosphorylation and regulation of AMPK activated protein kinase (AMPK) family members. LKB1 and AMPK signaling is known to affect gene expression in skeletal muscle through regulation of transcription factors and epigenetic regulation. The purpose of this study was to determine how the lack of LKB1 affects gene expression in skeletal muscle under high fat diet‐induced obesity (DIO). To test this, skeletal muscle specific LKB1 knockout (skmLKB1‐KO) and littermate LKB1‐floxed (WT) mice were fed high fat diet (HFD) or a standard chow diet (SCD) for 14 weeks. RNA sequencing and RT‐PCR was performed using RNA that was isolated from quadriceps muscles. Western blotting was performed using quadriceps homogenates. Gene ontology (GO) analysis showed 549 terms that were significantly affected by genotype in high fat fed mice. When ranked by p‐value, those GO terms with the highest level of significance were mitochondria/respiration‐related and oxidative stress‐related genes in the skmLKB1‐KO vs. WT muscles. Sixty‐nine genes were differentially expressed when comparing skmLKB1‐KO and WT muscles from HFD fed mice. The most under‐expressed of these in the skmLKB1‐KO vs. WT mice was dickkopf‐related protein 3 (DKK3), a regulator of the Wnt pathway. RT‐PCR confirmed the low level of DKK3 expression in skmLKB1‐KO muscles, but also suggested a potential upregulation by HFD feeding. Western blot analysis of DKK3 likewise showed increased expression in muscles from HFD vs. SCD mice. Intriguingly, when the mice were separated into HFD‐induced obesity resistant and obesity‐prone subgroups, DKK3 was only upregulated by HFD in the obesity resistant animals, suggesting a potential protective role against the development of obesity. In conclusion, skmLKB1‐KO under high‐fat diet conditions is associated with altered mitochondrial and oxidation‐reduction related gene expression, and DKK3 expression is greatly reduced in skmLKB1‐KO muscles.

Support or Funding Information

This work was funded by a BYU Mentoring Environment Grant and Graduate Research Fellowship.

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 Student‐Centered, Active Learning Approach to Teaching Spinal Cord Anatomy
Authors: Nassrallah Zeinab , Frankfurt Maya , Hill Robert V.
Published: June 2021

Purpose

The purpose of this project was to employ and preliminarily evaluate the effectiveness of small group, active learning among second‐year medical students studying the anatomy of the spinal cord.

Methods

We presented spinal cord anatomy in our “Human Condition” course for two cohorts of second‐year medical students. In 2015, and years prior, the material was incorporated into large group sessions and presented using slides in a large lecture hall. In 2017, we reorganized this learning activity into a more student‐centered, small group laboratory session. The interactive learning approach emphasized group work and higher‐order thinking to apply knowledge gained from assigned readings. In addition, students were encouraged to correlate anatomical features of prosected human spinal cord specimens with histological sections. After working together to solve the prompts posed by the faculty member, the students applied the teach‐back method to explain their answers and rationales to their facilitator. Facilitators used Socratic questioning to guide the discussions and to promote higher‐order thinking. Course examinations from both years included one question about spinal cord anatomy relevant to the material covered in both sessions. We compared scores from both years to evaluate the effectiveness of these learning approaches.

Results

Students who participated in the student‐centered laboratory approach demonstrated a modest increase (3%) in overall performance on the spinal cord examination question. The transition was faculty‐intensive, requiring both an increase in the number of trained facilitators and multiple iterations of the same station.

Conclusions

The student‐centered, active learning approach was associated with a modest increase in student performance on the course examination. From our preliminary evaluation, we observed no detrimental effects from shifting the large group session to small group lab stations; indeed, our evaluation suggests at least a modest benefit. The use of spinal cord specimens underscores the importance of donated anatomical material for medical education. These specimens were integral to the learning activity, as students applied their observations of gross features to their decision‐making in solving prompts relevant to spinal cord histology. Additionally, the small group laboratory approach was an efficient use of curricular time. We aim to continue to study the effectiveness of this learning approach, ideally migrating other large group sessions into the laboratory whenever possible.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
BODY PAINTING PROMOTES SIGNIFICANT LEARNING IN TEACHING HUMAN ANATOMY
Authors: Barreto João Erivan Façanha , Lima Maria Lucianny Barbosa , Freitas Francisco Orlando Rafael , Pimenta Helder Binda , Gusmão Jonas Nogueira Ferreira Maciel , Silva Bruno Frota Amora Amora , Cerqueira Gilberto Santos
Published: June 2021

Body painting is an important tool in the process of teaching and learning anatomy in undergraduate courses, being a valuable tool in the construction of knowledge mainly in places with corpse acquisition deficiencies for the anatomical study. This technique has been seen as a method to improve learning in the teaching of anatomy, since it is not restricted only to the organ expressed in the drawing, but also its topographic location of the organs. Based on these premises the objective of this work was to investigate if body painting promotes significant learning of human anatomy. For this, a descriptive exploratory study with a quantitative approach was carried out through the application of a semistructured questionnaire with 80 students from three health sciences courses during two consecutive semesters. The data were statistically analyzed using the square test and the Kruskal Wallis test using the Graph Pad Prism version 6.0 software. The data presented a value of p <0.05. The values obtained for each group were expressed in median ± e.p. It was found that there was no statistically significant difference in age. It was found that 95% of the students preferred teaching with body painting while 5% preferred the traditional one. Regarding the adequacy of the learning objectives 3.08 ± 0.6337 (score from 0 to 5) stated that the objectives were achieved. It was observed that 100% of students consider painting promote meaningful learning. When analyzing the consideration of the interviewees about the importance of bodypaint for the construction of knowledge, an expressive result was observed regarding the use of this methodology, being approved by 97% of the students. It can be observed that body paint has great potential as a tool for teaching anatomy, since it stimulates and promotes the learning process, revealing as a positive and useful educational experience for these subjects. Thus, it is important to incorporate this technique as a routine teaching method, in view of the satisfactory results observed in the study and the provided educational aid.

Support or Funding Information

UFC

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Exploring Molecular Mechanisms of Stress Susceptibility After Traumatic Brain Injury Using Drosophila as a Model Organism
Authors: García‐Rivera Lizmarie , Saikumar Janani , Bonini Nancy
Published: June 2021

Traumatic Brain Injury (TBI) is a global public health concern that affects individuals across all age groups. Although mortality rates have decreased with improved primary care, TBI patients commonly face neurocognitive deficits, psychological health issues, and impaired mobility. TBI occurs when an external force causes the brain to collide against the skull. This leads to immediate damage known as primary injury. The subsequent secondary injury, which is less understood, involves cellular and molecular cascades that promote cell death, and ultimately, neurodegeneration. Because secondary injury is a delayed effect of TBI, current research aims to understand the processes involved, including mechanisms of activation and regulation, to develop therapeutic interventions. This study aims to explore secondary injury mechanisms by replicating TBI in a simpler organism, Drosophila melanogaster, and challenging the subjects with known cellular stressors. Susceptibility to new stress can possibly indicate which cellular and molecular pathways are being altered as a result of the trauma. Flies were subjected to TBI and allowed to recover, and then treated with one of two stressors, heat shock or starvation, and survival was evaluated over time. For both experiments, we expected to see lower survival of TBI flies in comparison to the sham group because of damage caused by primary injury and subsequent secondary injury mechanisms. Remarkably, the opposite effect was observed; TBI subjects were more resistant to both heat shock and starvation. We hypothesized that TBI triggered some response that temporarily protected flies from these stressors. To study this further, we measured HSP70 and phosphorylated eIF2α levels after TBI, proteins indicative of Heat Shock Response and Unfolded Protein Response (UPR), respectively. Western blots were normalized to tubulin expression levels, quantified using ImageJ, and analyzed with two‐way ANOVA. Results for HSP70 showed a significant difference (p< 0.0001 ****) between the TBI and sham fly groups only at the 24 hour timepoint, while results for phospho‐eIF2α showed no significant difference at any of the timepoints post‐injury. These findings suggest that HSP70 was upregulated following TBI, a phenomenon also observed after human TBI, and because phospho‐eIF2α showed no change, the PERK pathway of the UPR is not involved in post‐trauma response. These studies and further research will help provide insight into the molecular mechanisms that underlie secondary injury, identify mechanisms that may contribute to cell recovery, and highlight how these can be enhanced to delay, or even avoid, degenerative changes to the brain.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Association of the Metabolomics Footprint, Body Fat and Serum Levels of Leptin, Ghrelin and Adiponectin In Shift Workers. Pilot Study
Authors: Gordillo‐Moscoso Antonio Augusto , Bujaidar‐Jonguitud Fernanda , Almendra‐Pegueros Rafael , De Leon‐Diaz de Leon Edgar , Flores‐Ramirez Rogelio , Rodriguez‐Aguilar Maribel , Jimenez‐Gonzalez Carlos , Medina Ursula
Published: June 2021

Obesity prevalence in Mexico is higher than in other countries (70%). Cross‐sectional studies with rotation shifts have observed that sleep disruption affects dietary intake and hormonal regulation (Adiponectin/Leptin/Ghrelin). Proposed mechanism is an increase on expression of fatty acid transport molecules, which would induce weight gain and adiposity, and increase the risk of developing obesity. Metabolomics analysis could help us to know if metabolic changes of lipid products have susceptibility to contribute to hormonal alterations and accumulation of fat mass. Our main objective was to determine if there was an association of metabolomics footprint, body fat and serum levels of leptin, ghrelin and adiponectin in workers with one week shift rotation schedule. We designed a prospective cohort, internal pilot study (n=30). Participants: shift workers (14‐day follow‐up) both sex (20–60 years old), work scheduled morning and night, and agree to participate by signing informed consent duly explained. We collect anthropometric data, dietary intake, physical activity and serum's metabolomics to establish the differences in the metabolomic footprint and identify the most important variables of the groups, by a principal component analysis (PCA), random forest and partial least squares (PLS) in R statics software. Serum concentrations of leptin, ghrelin and adiponectin will be determined by immunoassay. Results: We have observed 15 subjects, 9 woman and 6 man, main age 30.2 ± 6.5, 3 ± 2.6 years working in that schedule. In the nocturnal shift, we found weight gain (71.3vs70.8kg), increased intake of carbs, lipids and water; and more sedentary hours with statistical significances (table 1). We identify 5 principal components (45% variance explicated), the more representative metabolites were cyclobutane, cycloheptane and 1Iodo‐2methyl; the first two are low when the subjects are in the morning shift and increase when they rotate to the night shift, inverse case as for the compound 1Iodo‐ 2methyl. Conclusions: The metabolites increased in the night shift could be related to the metabolic pathway of lipogenesis, product of a higher carbs intake during the night and more hours of sedentary lifestyle, finally showing gain of fat mass in the morning shift.

Support or Funding Information

The authors acknowledge grants from National Council for Science and Technology‐National Problems (#2015‐1340) Table 1. Descriptive statistics variable analysis

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
In‐Vitro Evaluation of the Antidiabetic Effects of Coffee as a Natural Inhibitor of Alpha‐Glucosidase
Authors: Day Xiara , Hill Jenna , Obih John‐Clifford Agbasi , Tolessa Wajira , Obih Patience O
Published: June 2021

Diabetes mellitus is the seventh leading cause of death in the U.S., and affects over 29 million people. Type 1 diabetes, Type 2 diabetes, and gestational diabetes are the three prominent types. It is a progressive disease, often associated with numerous complications, including, but not limited to, retinopathy, neuropathy, and nephropathy. In 2012, the costs of diabetes exceeded 245 billion dollars. Oral hypoglycemic medications, insulin, and other injectable medications, have been widely used to control Type 2 diabetes, but have undesirable side effects and are expensive. Some agents control postprandial blood glucose levels through inhibition of α‐glucosidase, which delays the cleaving of polysaccharides, preventing the resulting monosaccharides from entering the blood stream. Examples of α‐glucosidase inhibitors are acarbose and miglitol. However, they both have undesirable side effects. Recent studies have pointed toward Phytotherapy, the use of medicinal plants or herbs which have little or no side effects, to treat hyperglycemia. This study investigates the hypoglycemic effects of coffee. Some studies have proposed that coffee may suppress postprandial hyperglycemia. We hypothesized that coffee reduces hyperglycemia by inhibiting alpha‐glucosidase activity. To determine this effect, serial dilutions of 200 mg/mL aqueous coffee extract was mixed with a 0.2 M phosphate buffer and 5 mM p‐Nitrophenyl α‐D‐glucopyranoside in a 96 well plate. The assay quantifying the α‐glucosidase inhibition activity of coffee was performed in‐vitro and measured using a spectrophotometer. Acarbose was used as the positive control of α‐glucosidase inhibition. The IC50 was evaluated, as it is the concentration of the extract required to inhibit 50% of α‐glucosidase activity. From our observation, coffee extract did not inhibit alpha glucosidase at the doses used. More investigations are needed to optimize the approach in assaying the α‐glucosidase inhibition of coffee as well as identifying other mechanisms of action.

Support or Funding Information

Center of Excellence (COE) and BUILD program at Xavier 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.
Titanium Dioxide TiO2 Induces Loss of Proliferative Capability Related to Ultrastructural Changes in Human Mesenchymal Stem Cells from Wharton Jelly
Authors: Lugo‐Martínez Gabriela , Gutierrez‐Iglesias Gisela Camelia , Parra‐Barrera Alberto , Calzada‐Mendoza Claudia Camelia , Ramos‐Godínez Maria Del Pilar , Cáceres‐Cortés Julio Roberto
Published: June 2021

Introduction

Titanium dioxide is one of the most widely used mineral pigments in the world in the field of paints, cosmetics, sunscreens, plastics, paper, food, etc. It has been reported that TiO2 at different nanometric scales is considered toxic, depending on the concentration and type of cell that is exposed. TiO2 nanoparticles (TiO2‐NPs) have the ability to cross biological barriers. In vivo models, it cross the blood‐brain barrier and also it could to cross the fetoplacental barrier, although it is unknown if they are capable to internalize in sites called niches of mesenchymal stem cells, which relevant cells sources due to the importance in regeneration of various tissues. The internalization of nanometric TiO2 particles on Wharton's jelly hMSC remains unknown.

Objective

Evaluate the toxic effect and internalization of TiO2 nanoparticles in hMSCs from Wharton's Jelly.

Materials and Methods

Wharton gelatin hMSC were cultured and were exposed to three different concentrations (500, 62.5 and 4 μg/mL) of anatase TiO2‐NPs. After 2, 5 and 7 days of exposure, viability tests were performed by alamar blue assay for quantify the toxic effect of TiO2. To identify the location of TiO2‐NPs and morphological changes in the hMSCs, various preparations with different TiO2 concentration were observed by transmission microscopy.

Results

The results showed that the cellular viability of hMSC decreased significantly at days 2, 5 and 7 of exposure, reduction of viability 40%, 30% and 30% respectively (fig 1). Transmission microscopy showed that the hMSCs had morphological changes and, more specifically, that the TiO2‐NPs took a perinuclear distribution as they accumulated inside the cell, forming a characteristic halo (fig 2). The images showed the internalization of NPs through vesicles, which led to the loss in the continuity of the membrane and the formation of intracytoplasmic spaces representing cellular structural damage in different cellular organelles.

Conclusions

This results are strong evidences that have to be considered in the regulation of the use of nanotechnology in different products containing TiO2 without this affecting public health. Sanitary regulation is suggested in the use of TiO2‐NPs to prevent the health condition as it has been done in different countries.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differentiation of the anti‐coagulant effects of Activated Protein C and Recombinant Thrombomodulin as studied in whole blood and retrieved plasma
Authors: Siddiqui Fakiha , Laddu Rajan , Hoppensteadt Debra , Fareed Jawed
Published: June 2021

Introduction

Activated protein C (APC) mediates its anti‐coagulant effects by digesting Factor Va & Factor VIIa, whereas recombinant thrombomodulin (rTM) produces its effects by complexing with thrombin and mediating the formation of APC. Both agents have been developed for clinical indications such as Sepsis associated coagulopathy. The purpose of this study is to compare the anti‐coagulant effects of these two agents in deidentified patients whole blood and retrieved plasma.

Materials & Method

Recombinant thrombomodulin (recomodulin) was obtained from Asahi kasei Tokyo, Japan. Plasma derived activated protein C was obtained from Hematologic technologies, Burlington Vermont, USA. Both agents were supplemented in 39 individual citrated whole blood collected from patient from clinical coagulation laboratories at a concentration of 2.5ug/ml. Whole blood was analyzed for PT and aPTT on a ST4 instrument (Stago Paris, France). Whole blood was centrifuge at 2500 g to contained plasma samples, which was analyzed for PT & aPTT. Saline supplemented individual blood samples were used as control. Individual blood and plasma results were compiled as group Mean ± SD and Percent change levels were calculated.

Results

In the whole blood studies, rTM produced a modest increase in the PT assay (2%), in contrast to APC which produced stronger response (36%). In the aPTT assay both agents produced stronger effects, rTM produced a 36% increase whereas APC produced a much stronger increase amounting to 172%. In retrieved plasma rTM produced slight increase in PT assay (2%) while APC produced strong response (20%). In the aPTT assay rTM produced strong effects with 13% however APC produced much stronger effects about 198%. These results were given in Table.

Conclusion

These results indicates that APC is a stronger anti‐coagulant then the rTM in both the whole blood and plasma system as measured by the PT and aPTT. Both agents produced stronger anti‐coagulant effects in the aPTT assay suggesting that the intrinsic coagulation pathway is strongly inhibited by these agents in comparison to the extrinsic pathway.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining the intracellular redox‐dependent protein signaling networks between cell‐junctions and focal adhesions during intestinal mucosal repair
Authors: Matthews Jason , Reedy April , Jones Rheinallt , Neish Andrew
Published: June 2021

Mucosal wounds and inflammation of the human intestine are hallmark features of intestinal bowel disease. It is the coordinated intestinal epithelial cell spreading and migration at the wound edge that facilitates mucosal wound closure in a process known as restitution. During restitution, intestinal epithelial cells spread and migrate as a sheet along focal adhesions, or points of basal cellular contact with the extracellular matrix. Cell‐to‐cell contacts of migrating cells are maintained by junction proteins. As restitution begins, epithelial cells produce reactive oxygen species (ROS) by NOX‐1 enzyme. Over time immune cells infiltrate the inflamed and damaged mucosa and increase the oxidative stress by depositing even larger quantities of ROS around and onto the damaged epithelium, leading to cysteine oxidation of a wide variety of cellular and extracellular proteins. As intestinal epithelial cells spread and migrate as a sheet to close the wound, the cellular junctions and the focal adhesions must somehow communicate through a cross‐talk that maintains directional migration and barrier properties. Little is known about the cross‐talk between cell junctions and focal adhesions, or how ROS regulates this mucosal repair program. We have previously reported redox‐dependent subcellular changes of the focal adhesion protein, Cas, localizing to cellular junctions within the epithelium at the edges of mucosal wounds. We hypothesize those redox‐dependent changes in protein signaling drive the mucosal repair program by cysteine‐oxidation and subsequent alteration in kinase/phosphatase activity that produces changes in cell function, and we have sought to elucidate the profile of this protein network. We show here that epithelial cell Cas localization is indeed dependent on oxidation by ROS generated by immune cells during mucosal repair, and provide evidence that similar redox conditions also regulate the cell border accumulation of AKT and FAK. Remarkably, pharmacological inhibition of FAK increased barrier function. Furthermore, we have employed a novel proteomics technique, called Isotope‐Coded Affinity Tag (ICAT) that quantifies the level of proteome‐wide cysteine oxidation in order to further define these signaling modules. Our analysis has revealed redox‐dependent protein signaling driven by MAPK/PAK2/TLN1/actin cytoskeleton interactions, which might regulate Cas, AKT and FAK localization to junctions during mucosal repair. Collectively, these data shed new light on the redox‐dependent junction/focal adhesion cross‐talk during intestinal restitution.

Support or Funding Information

Crohn's and Colitis Foundation, Career Development Award # 451678

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recombinant Fasciola hepatica Fatty Acid Binding Protein Downregulates Inflammatory Cytokines/Chemokines in a Mouse Model of Septic Shock and Shows a Wide Spectrum of Action
Authors: Rosado‐Franco Jose Javier , Benitez‐Ramos Marcos Javier , Ruiz‐Jiménez Caleb , Espino‐Hernández Ana Margarita
Published: June 2021

Sepsis is the leading cause of death from infections. It involves, both pathogen and host factors and it is characterized by an exacerbation of inflammatory cytokines and chemokines and organ dysfunction and failure. Recently, we reported that a recombinant Fasciola hepatica fatty acid binding protein, termed Fh15, is capable of suppressing the LPS induced expression of inflammatory cytokines such as IL‐1β and TNFα in murine bone marrow derived macrophages in vitro. We also demonstrated that such anti‐inflammatory effect is achieved by targeting specifically the CD14 co‐receptor, which results in a complete suppression of TLR4 activation and makes macrophages irresponsive to subsequent LPS‐stimuli. In the current study, we evaluated the capacity of Fh15 to suppress the TLR4‐activation in vitro, as well as its capacity to therapeutically suppress the cytokine storm in a mouse model of septic shock. We also tested the ability of Fh15 to suppress the activation of multiple TLRs in response to Gram‐positive and Gram‐negative whole bacteria extracts in vitro. For in vivo studies, we used female BALB/c mice that received a lethal intraperitoneal (i.p.) injection of LPS‐E. coli and 1h later received a single i.p. injection with 50μg Fh15. Animals were sacrificed 12h after the LPS challenge. For in vitro experiments, we used THP1‐CD14 cells, which were cultured with Fh15 prior to stimulation with optimized concentrations of 1×108 cells of Klebsiella pneumoniae and Enterococcus faecalis. Results demonstrated that, although Fh15 was unable to stimulate the secretion of any inflammatory cytokine, it significantly suppressed the levels of serum IFNγ (p=0.0425), TNFα (p=0.0008), IL‐1β (p=0.0021) in all animals exposed to lethal doses of LPS. Fh15 also significantly suppressed the levels in serum of two macrophage inflammatory proteins MIP‐1α (p=0.0027) and MIP‐1β (p<0.0001), two chemotactic chemokines that play relevant roles in the inflammatory process. Moreover, we also demonstrated that Fh15 inhibits the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti‐inflammatory drug in pre‐clinical studies in the near future.

Support or Funding Information

This study was supported by MBRS‐RISE R25GM061838‐13, and NIH grants G12MD007600 and 2P40OD012217.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A2A Receptors Are Highly Expressed in Young Patients with Luminal B and Triple Negative Breast Cancer Subtypes and Are Associated with a Higher Proliferate Index
Authors: Guimei Maha , Eladl Mohamed Ahmed , Tawfik Dalia Ra'a Ahmed , Habbal Fuad Wardan , Harrous Abdelkader , El‐Sherbiny Mohamed
Published: June 2021

Background and Objectives

Numerous Molecules in the tumor microenvironment orchestrate the body's immune response against tumor cells thereby affecting tumor cell growth and proliferation. Adenosine is produced in the hypoxic tumor milieu and by stimulating its receptors (A2AR), plays an important immunosuppressive role that helps tumor cells evade the body s immune mechanisms. Furthermore, stimulation of these receptors also has a profound role in promoting the tumor vasculature and contributing to tumor aggressiveness. The present study aimed at investigating A2AR expression in breast cancer tissue and its association with different clinicopathological parameters of the tumor.

Methods

This study was conducted on 30 formalin‐fixed paraffin‐embedded (FFPE) human breast cancer tissues. Sections were immunohistochemically stained with of A2AR antibodies. Clinical and pathological data were retrospectively obtained from the patients' records in the archives of the pathology department at Alexandria University, Egypt.

Results

A2ARs were expressed not only on the tumor‐infiltrating immune cells (TILs) but were also found on the tumor cells themselves in 73.3% (n=22) of breast cancer tumors. Their expression on the tumor cells was significantly higher in younger age patients (< 50 years) (88.2%) compared to older ones (54.5%) [p=0.044]. A2AR expression was also significantly associated with a Luminal B as well as triple‐negative tumors [p=0.028] compared to Luminal A and Her2‐enriched tumors. Patients expressing A2A receptors had a significantly higher expression of Progesterone receptors [P=0.031] compared to ER expression [P=0.098]. The majority of A2AR expressing tumors had a mitotic index score of 2 [p=0.013] as well as a significantly higher proliferative index (ki‐67 >20%) [p=0.018]. No association was observed between A2AR expression and tumor size, type, grade, Lymph node status, the percentage of TILs or patient survival after 2 years of follow‐up.

Conclusion

Our study confirmed that A2AR were strongly expressed on breast cancer tumor cells in younger patients particularly those of luminal B and triple negative subtypes. Their expression was associated with a significantly higher proliferative index suggesting a possible role of these receptors in imparting a more aggressive phenotype and that targeting these receptors could have a potential role in improving the outcome of these 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.
In vitro effect of 5G8‐specific antibodies on Giardia lamblia trophozoites
Authors: Rascon Raul , Dominguez Victor , Garzon Thania , Samaniego Brenda , Valdez Alejandra , Quintero Jael , Rascon Lucila , Velazquez Carlos
Published: June 2021

Giardiasis is one of the most common gastrointestinal diseases worldwide, caused by the protozoan parasite Giardia lamblia. Although several immunogenic proteins of the parasite have been identified, the capacity of inducing a protective humoral response has been scarcely evaluated. Our research group have identified and characterized the immunogenic 5G8 protein, a 70 kDa Variant‐specific surface protein (VSP). The administration of 5G8‐specific monoclonal antibodies (AcMo 5G8.B5) to infected animal decreased the parasite intestinal load. The aim of the present study was to evaluate the in vitro effect of 5G8 specific antibodies on G. lamblia trophozoites. We isolated 5G8 protein by passive elution from SDS‐PAGE (membrane protein enriched fraction of trophozoites lysates). C3H/HeJ mice were intraperitoneally immunized with isolated 5G8 protein to generate polyclonal antibodies. GS/M‐83‐H7 5G8 (+) trophozoites were incubated with monoclonal and polyclonal antibodies specific to the 5G8 protein, and their agglutination capacity was evaluated. After 24, 48 and 72 hours incubation, cell proliferation was determinated and the expression of 5G8 protein on trophozoites was evaluated by Flow Cytometry. Polyclonal antibodies recognized 97 % of GS/M‐83‐H7 5G8 (+) trophozoite population, and a single band of 70 kDa (SDS‐PAGE). The AcMo 5G8.B5 and polyclonal antibodies had the ability to agglutinate G. lamblia trophozoites in vitro after 60 min of antibody exposure. Antibody treatments inhibited the in vitro G. lamblia growth. Isotype control antibody and preimmunized serum showed no inhibition of trophozoites proliferation. Surface 5G8 protein expression did not change during antibody treatments. Altogether, our data suggest a possible in vitro cytostatic effect of 5G8 specific antibodies on G. lamblia trophozoites. These findings put forward the capacity of the 5G8 protein to induce a protective humoral response against the infection.

Support or Funding Information

CONACYT: CB‐2010‐01/155224

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 Myocardial Ca2+ Dynamics by the G Protein‐Coupled Estrogen Receptor
Authors: Whitcomb Victoria , Wauson Eric , Matnishian Vahe , Tran Quang‐Kim
Published: June 2021

The G protein‐coupled estrogen receptor (GPER) participates in many cardioprotective activities. However, the mechanisms of such activities are not known. We have begun to investigate the effects of alterations in GPER activity on myocardial signaling via the beta‐1 adrenergic receptor. In freshly isolated primary murine cardiomyocytes, β1AR agonist isoproterenol triggers robust Ca2+ signals that are oscillatory and dependent on extracellular entry. GPER activation dose‐dependently inhibits the isoproterenol‐induced Ca2+ signals. On the other hand, GPER antagonism exerts the opposite effects. Similar effects were observed in H9C2 cardiomyocytes. Mechanisms of these observations were examined on various components of myocardial Ca2+ signaling. The data indicate that GPER activity is involved in β1AR‐mediated Ca2+ signaling in the myocardium.

Support or Funding Information

AHA grant 15SDG25090279 (to E Wauson) and NIH Grant HL112184 (to Q‐K Tran)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synaptic mechanisms underlying the rapid‐acting antidepressant actions of ketamine
Authors: Zobel Allison , Pope Brock , Rehman Zarin , Herring Laura , Wauson Eric , Graves Lee , Duric Vanja , Yuan LiLian
Published: June 2021

Ketamine at sub‐anesthetic doses has shown promising results as a potent and fast‐acting antidepressant. Evidence from animal model studies suggests that the functional restoration by ketamine is associated with activation of MAPK and mTOR signaling cascades in the prefrontal cortex (PFC) within a few hours of ketamine administration, followed by a second wave of synaptic protein upregulation. Together, these molecular events lead to rapid synaptogenesis and reversal of neural atrophy. Furthermore, (2R, 6R)‐hydroxynorketamine (HNK) has recently been identified as the main active component of ketamine metabolism. HNK is believed to be responsible for the antidepressant actions of ketamine, but with minimal side effects, and potentially through NMDAR‐independent mechanism of action. However, critical gaps exist in understanding the exact molecular mechanisms of ketamine actions. Thus, it would be of great importance and interest to delineate the molecular mechanisms that translate mTOR activation into synaptogenesis‐based structural remodeling. Cofilin and its upstream pathways, ideally positioned to bridge the gap between mTOR activation and spine formation, have emerged from an unbiased kinome/phosphoproteome screening for targets of ketamine treatment. As the major cytoskeletal component of dendritic spines, actin controls spine morphology and formation through changes in its polymerization state. Actin polymerization is negatively regulated by activation of cofilin protein. Phospho‐antibody based assays and direct or indirect measurement of kinase activity are currently underway to validate those targets in acutely and chronically stressed animal models. We anticipate addressing the significance of cofilin phosphorylation and actin polymerization in antidepressant responses of ketamine and HNK, and whether those responses are modulated by other factors including stress and sex.

Support or Funding Information

NIH grant MH108043 and IOER funds

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