bluuebunny/crossref_metadata_2025 huggingface dataset:
Exercise Reduces Proportions of Tumor Resident Myeloid‐Derived Suppressor Cells
Authors: Garritson Jacob , Haughian James , Pullen Nicholas , Hayward Reid
Published: June 2021

Myeloid‐derived suppressor cells (MDSCs) are a population of immature myeloid immune cells that promote tumor progression and metastasis by suppressing both innate and adaptive immune responses. Exercise modulates immune function and is generally thought to protect against tumor growth and metastasis, yet the mechanisms behind this protection are largely unknown.

PURPOSE

To examine how exercise impacts circulating and tissue resident lymphocyte and MDSC populations.

METHODS

Female mice were randomly assigned to treatment groups: exercise tumor (EX+TUM), sedentary tumor (SED+TUM), exercise (EX) or sedentary control (SED). EX groups underwent wheel running preconditioning for 6 weeks before tumor challenge. Following the 6‐week training period, animals in TUM groups were inoculated in the mammary fat pad with 1×104 4T1 mammary carcinoma cells and ran an additional 4‐weeks. Proportions of CD4+, CD8+, and CD335+ lymphocytes, as well as total MDSCs (CD11b+Ly6C+ Monocytic MDSCs (M‐MDSC) plus CD11b+Ly6G+ polymorphonuclear MDSCs (PMN‐MDSC)) were analyzed by flow cytometry in spleen, blood, and tumor.

RESULTS

No effect of exercise was observed in CD4+ or CD8+ lymphocyte populations. The 4T1 mammary tumors significantly expanded MDSC populations in both spleen and blood regardless of exercise treatment. This tumor‐induced MDSC expansion was associated with enlarged spleens in sedentary animals (SED+TUM: 0.62g ± 0.11g) whereas the spleens in exercised animals with tumors were smaller (EX+TUM: 0.44g ± 0.11g) and did not differ from controls (EX: 0.08g ± 0.01g and SED: 0.11g ± 0.01g). Although there was no change in tumor volumes or tumor weights, exercise significantly reduced proportions of tumor‐infiltrating MDSCs (EX+TUM: 4.1% ± 1.6% vs. SED+TUM: 15.9% ± 2.3%). Further, tumor‐bearing exercised animals maintained a higher proportion of CD335+ natural killer cells in their spleens (EX+TUM: 0.6% ± 0.3%) relative to sedentary animals (SED+TUM: 0.4% ± 0.1%), which showed significant splenic CD335+ depletion relative to EX (1.3% ± 0.6%) controls.

CONCLUSIONS

The observed reduction in tumor‐resident MDSCs suggests that exercise may improve antitumor immunity by decreasing MDSC‐related immune suppression in the tumor microenvironment and by preserving CD335+ lymphocyte populations. Exercise may reduce the severity of the aberrant myelopoiesis in tumor bearing animals as is evident by reduced splenomegaly in exercised animals.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vaping Condensate Exposure to in vitro Mouse Embryos
Authors: Carter Joseph William , Smith Kane William , Christine Catherine , Okimoto Richard , Swanson Jim
Published: June 2021

Objective

To clarify effects of electronic cigarette carrier fluid and high nicotine concentrations on embryonic development to reduce misconceptions about vaping during pregnancy.

Methods

With a vacuum, vapor condensate was collected from a glass‐bead‐filled, copper coil submerged in ice. Recovered condensate was injected into the intraperitoneal cavity of female mice prior to hormonally induced superovulation/mating. Daily injections of condensate allowed male and female gametes as well as embryos to be exposed to the condensate. With 3 injections per day, mice were divided into control (no injections) and 3 experimental groups. Experimental groups received: 1) vaped saline; 2) vaped carrier fluid (CF); 3) or vaped 1.2% nicotine/CF. Injected volume was 0.1 mL of fluid/dose. The basis for this injection volume was based on recent literature demonstrating how vaping for 5 minutes produces up to 7.9 ng of nicotine/mL +/− 0.9 ng of blood in humans [Farsalinos et al., 2015]. Mathematically, the predetermined volume of 0.1 mL/injection of our 1.2% nicotine/CF was diluted to 1% and resulted in a blood nicotine concentration of 8.2 ng/mL based on an average mouse weight of 25 g with a blood volume of approximately 1.7 mL.

Results

The results of our data were based on normal survival rate on a day‐6 embryo defined as embryo growth progressing to hatching or hatched blastocyst stage of development. Differences in embryo survival due to treatment were determined using ANOVA with a Least Significant Difference post hoc analysis using SPSS. Our results showed significant difference between the control group and 1.2 % nicotine group (P<0.007) as well as difference between the control group and carrier fluid group (P<0.049). The saline group was not significantly different from the control group.

Conclusions

These results indicate that vaping both CF and 1.2% nicotine/CF prove to be detrimental to normal embryo development, demonstrating the need for additional research into the potentially harmful effects of vaping CF, with or without nicotine.

Support or Funding Information

LUCOM Center for Research

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Study of the Articular Disk of the Temporomandibular Joint in Human Ageing: Preliminary Macroscopic Analysis
Authors: Jacomo Alfredo Luiz , de Souza Cerioni Tales Gabriel , Frascino Alexandre Viana , Akamatsu Flavia Emi , Hojaij Flavio Carneiro
Published: June 2021

The processes of human ageing involve morphological alterations of the articular structures with consequent functional impairment. There are few reports on temporomandibular joint (TMJ) changes inherent in human ageing processes. The present study aims to morphologically study the anatomical alterations of articular discs of TMJs of 4 human cadavers, males older than 50 years, previously frozen. The specimens were obtained by exposing the joint capsule followed by an incision in the joint eminence of the temporal bone and dissection of the articular ligaments, thus exposing the articular disc. The disk was delicately manipulated by means of Dietrich tweezers for the separations of the structures inserted therein. The discs presented a mean of 24.63 mm and an average spine thickness of 4.59 mm. Perforations were observed in 3 articular discs (37.5%) compatible with the reports in the literature, and 5‐part tapering (62.5%). Further investigations are needed to assess TMJ morphological alterations associated to human ageing.

Support or Funding Information

Faculty of Medicine, University of São Paulo ‐ FMUSP

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Osteocyte morphology in the human craniofacial skeleton
Authors: Guerrero Kristi , Dechow Paul C , McGee‐Lawrence Meghan , Yu Kanglun , McIntosh Leslie Pryor
Published: June 2021

Osteocyte size and shape has been shown to vary by anatomical region, variation which reflects differences in osteocyte sensitivity to strain. Variation in osteocyte morphology should therefore be considered when interpreting primate craniofacial adaptations. Very little is known about how osteocyte shape and size vary in the human craniofacial skeleton. For this study, osteocyte size and shape in the human mandibular condyle was analyzed and compared to osteocyte size and shape in the human supraorbital and zygomatic regions from 2 individuals. A multiphoton confocal microscope was used to collect 3‐dimensional images from FITC stained human mandibular condyles and compared to existing data for the supraorbital and zygomatic regions from 2 individuals. Imaris analysis of osteocyte cell bodies show that osteocytes in the supraorbital region are significantly larger than in the zygoma and condyle (p≤0.001). Shape of osteocytes was measured and analyzed by creating a bounding box around 15–20 osteonal osteocytes per individual and comparing ellipsoid axis lengths a, b, and c. ANOVA testing found that osteocyte shape variation was non‐significant between the three locations. Osteocytes in all three cranial locations are mostly oblate in shape (disc‐like; where c>a). These preliminary results suggest that osteocyte morphology may be similar across the craniofacial skeleton; differences in osteocyte size may indicate metabolic differences across craniofacial regions.

Support or Funding Information

NSF BCS 0725126

NSF CMMI 1727949

Gail Dillard Faculty Enrichment Fund, ABAC Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of ZBP1 in the Neuroinflammatory Response in Glia Cell Models of Parkinson's Disease
Authors: Nease Alyssa , Malovic Emir , Kondru Naveen , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Kanthasamy Anumantha
Published: June 2021

The deposition of α‐synuclein inclusions known as Lewy bodies and the loss of dopaminergic neurons are hallmark pathological changes contributing to the motor and nonmotor symptoms of Parkinson's disease. The concomitant neuroinflammation, characterized by activated microglia and astroglia and the secretion of pro‐inflammatory cytokines, is known to trigger neurodegenerative processes in PD. Necroptosis is a mechanism of cell death that can be less inflammatory than pyroptosis by initiating cell death before the release of pro‐inflammatory cytokines. The innate immune sensor, Z‐DNA binding protein 1 (ZBP1), plays a role in necroptosis by activating downstream kinases leading to the formation of a cationic pore in the membrane. Recently, we have shown that α‐synuclein can act as the primer and trigger for pyroptosis. In the present study, we characterized ZBP1's role in the aggregated α‐synuclein‐induced neuroinflammatory response using the mouse microglial cell line (MMC), primary mouse microglia (PMG), and the human astrocytoma, U373 cells. After treating MMCs, PMGs, and U373s with aggregated α‐synuclein, we observed a decease in ZBP1 using Western blot analysis and this decrease was observed at 24 hours but not at earlier time points. We then used small interfering RNA (siRNA) to knock down ZBP1 in MMCs and used Luminex multiplex cytokine assay to measure the levels of secreted pro‐inflammatory cytokines in the media. This analysis revealed an increase in interleukin (IL)‐1β, IL‐6, IL‐17A, tumor necrosis factor α (TNF‐α), and interferon‐γ (IFN‐γ) in the media. Our data suggest that loss of ZBP1 in glial cells skews them towards more potent pro‐inflammatory signaling pathways known to be activated by aggregated alpha‐synuclein. Studies are underway to characterize the regulatory role of ZBP1 in α‐synuclein‐induced necroptosis and pyroptosis in glia cells during α‐synuclein neurotoxicity.

Support or Funding Information

(NIH grants NS100090, NS088206, ES026892)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Manipulating Cellular Trafficking Positively Affects Syn‐TEF Function in Human Tissue
Authors: Rider Sam , Brandon Christopher J , Ali Asfa , Ansari Aseem Z
Published: June 2021

Synthetic transcription elongation factors (Syn‐TEFs) bind to the minor groove of DNA and recruit transcription machinery. Syn‐TEFs can provide therapeutic relief for transcriptionally defective disorders such as Friedreich's ataxia, a neurodegenerative disease resulting from a 90% loss in Frataxin (FXN) expression. Syn‐TEF1 restores FXN expression in Friedreich's ataxia patient cells. However, the mechanism of cellular entry has yet to be characterized. From our observations, only a fraction of the compound is available to act in the nucleus. Fluorescently tagged Syn‐TEF analogues aggregate into bright puncta in the cytoplasm of imaged cells. Using confocal microscopy and reverse transcription quantitative polymerase chain reaction (RT‐qPCR), we assessed conditions of puncta formation and tested means of resolving or preventing the aggregation of Syn‐TEFs into puncta in human cells. We have found that manipulating cellular uptake and trafficking can improve nuclear localization of fluorescent Syn‐TEF analogues and change Syn‐TEF1 effective concentrations. Understanding the mechanisms of uptake for Syn‐TEFs and their fluorescent analogues will provide insight into how Syn‐TEFs affect gene expression in Friedreich's ataxia and related disorders.

Support or Funding Information

This project was supported by NIH NINDS grant R01NS108376 and the Clinical and Translational Science Award (CTSA) program through the National Center for Advancing Translational Sciences (NCATS), grant UL1TR002373. S.R. was also supported by the Trewartha Senior Thesis Research Grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gut Microbiota‐Mediated Colonic Metabolism of Triclosan Contributes to its Proinflammatory Effects
Authors: Sanidad Katherine Zabala , Yang Jun , Zhao Ermin , Zhang Jianan , Yang Haixia , Xiao Hang , Zhang Guodong
Published: June 2021

Triclosan (TCS) is an antimicrobial ingredient used in thousands of consumer products. Because of its widespread use, TCS causes ubiquitous contamination in the environment and is one of the top‐ten pollutants found in rivers in the United States. We have recently shown that exposure to TCS, at doses relevant to human exposure, can enhance colonic inflammation and colitis‐associated colon tumorigenesis in mice, suggesting that TCS could be a risk factor for these diseases. We also showed that the promoting effect of TCS on colonic inflammation requires gut microbiota since TCS failed to promote colonic inflammation in germ‐free mice. However, the functional roles of gut microbiota in the proinflammatory effects of TCS are unknown; in addition, the specific gut bacteria involved are also unknown. In this study, we examined the metabolism of TCS in C57BL/6 mice and the role of the gut microbiota in TCS metabolism in the colon using LC/MS‐MS analysis. Our results show that in the small intestine of mice treated with TCS via the diet there were elevated levels of TCS‐glucuronide (TCS‐G) and TCS‐sulfate (TCS‐S) while in the colon there were elevated levels of free‐form TCS. However, when the mice were treated with antibiotics, to deplete gut microbiota, prior and during exposure to TCS, there were elevated levels of TCS‐G and TCS‐S in the colon instead of free‐form TCS. This suggests that gut microbiota are responsible for the deconjugation of TCS‐G and TCS‐S to free‐form TCS in the colon. Using in vitro fermentation, we were able to identify specific bacteria responsible for the deconjugation of TCS‐conjugates to free‐form TCS. In addition, we then exposed MC38 mouse colon cancer cells to TCS, TCS‐G, or TCS‐S at a dose mimicking their levels in the colon. Our results showed that TCS, not TCS‐G or TCS‐S, increased the expression of the proinflammatory cytokines and chemokines. In summary, our results suggest that the gut microbiota are responsible for the metabolism of TCS in the colon which contributes to the proinflammatory effects of TCS. Our results also suggest that TCS could exaggerate disease development via gut microbiota and suggests potential adverse effects of antimicrobials on gut health. Further studies are needed to characterize the impact of TCS on gut diseases.

Support or Funding Information

This research is supported by a new faculty start‐up funding from UMass‐Amherst (to G.Z.).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Compensatory Evolution within Viral RNA Structure: The Potential Role of Stable Intermediates in RNA Structural Heterogeneity
Authors: Magalis Brittany Rife , Tolbert Blanton S , Salemi Marco , Kosakovsky Pond Sergei L
Published: June 2021

Fast evolving viruses, such as human immunodeficiency virus (HIV), exist as a heterogeneous population of genotypic variants, each potentially with its own distinct RNA structural variant. Owing to the inherently compact nature of the viral genome, it is not unreasonable to believe that viruses such as HIV have adapted to take advantage of both sequence and structure heterogeneity, posing issues for RNA‐mediated drug targeting. Evolutionary analysis of compensatory mutations, using a variety of phylogenetic methods, is often used to evaluate RNA structure preservation and function. One such method is based on the identification of co‐varying sites along a single branch within the corresponding phylogeny of sequences. Despite the power of this method when applied to slower evolving organisms (e.g., vertebrate ribosomal RNA), viral genomic regions containing well‐characterized structure, such as the HIV 5’ untranslated region, consistently present very little evidence of co‐variation in the face of high sequence heterogeneity. We hypothesize that allowing for co‐variation to occur over multiple, neighboring branches would increase the robustness of this method in identifying biologically relevant RNA structures and that non‐covarying nucleotide substitutions that occur along the connecting internal branches act to stabilize structures within a population minority. We propose to integrate this approach into a Bayesian graphical network (BGN) framework for identification of larger (>2) networks of co‐evolving sites potentially acting to maintain the predominant RNA structure. Additional investigation using this method and experimental analyses are ongoing to uncover the role of the putative intermediate, minority structures in RNA structural heterogeneity, specifically within the host.

Support or Funding Information

Funding: NIH‐NIGMS U01 GM110749

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
On the Spectrum with T‐box Brain Protein 1: A Transcription Factor Protein Critical for Normal Neural Development
Authors: Andrews Lin
Published: June 2021

The Independent School MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function relationships and applications of T‐box brain protein 1 (TBR1). The overall function of TBR1 is to act as a transcription factor protein. Located in the nucleus, this protein aids in vertebrate embryonic brain and neural development, specifically acting as an essential component in cerebral cortex development. TBR1 has the same general structure as other transcription factor protein homologs of the T‐box family, which all share a common DNA‐binding motif (the T‐box) and can form homodimers. Structurally, TBR1 has been studied to show how specific residues in its active site could lead to autism spectrum disorders (among other neurological diseases) if it does not align properly with DNA during transcription. Within the T‐box domain at Position 271, a tryptophan sidechain is located in the center of the motif that stabilizes the domain through contact with surrounding hydrophobic amino acids and ensures that DNA‐binding residues are correctly positioned. At Position 374, an asparagine residue also stabilizes the domain and ensures that DNA‐binding sites are positioned correctly by forming hydrogen bonds with its surrounding residues. Studies suggest that positively‐charged lysine amino acids that are close to the negatively‐charged phosphate backbone of DNA, such as those at Position 288 and Position 389, are critical for the binding of the protein to the minor groove of DNA. Additionally, TBR1 has been shown to act as either a repressor or activator in gene regulation of several important neurological cascades. Therefore, while not all the mechanisms are understood and many of its applications are still theoretical at this time, TBR1 has been shown to be involved with attention and memory. When this transcription factor protein malfunctions, its deregulation has been linked to Parkinson's disease and Alzheimer's disease. It has also been linked to several autism‐related disorder genes and a small percentage of unrelated sporadic autism disorders. Finally, TBR1 may be linked to language and speech disorders and synesthesia, though more research must be conducted to understand its exact function in these areas.

Support or Funding Information

Supported and Funded by The Independent School; Wichita, KS 67207

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural Insights into Phospholipase Cɛ Function
Authors: Rugema Ngango Yvon , Prahalad Keshav , Pearson Sean Patrick , O'Neill Hannah , Mbongo William , Garland‐Kuntz Elisabeth , Sieng Monita , Lyon Angeline M
Published: June 2021

Phospholipase Cɛ (PLCɛ) is a member of the PLC family of enzymes that hydrolyze phosphatidylinositol lipids downstream of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). PLCɛ is unique among the PLC superfamily as it contains an N‐terminal CDC25 domain, which has guanine nucleotide exchange factor (GEF) activity towards small G proteins Rap1A, and two C‐terminal Ras association (RA) domains. The best characterized pathway leading to PLCɛ activation is mediated through β‐adrenergic receptor (βARs) signaling. Stimulation of these receptors culminates in the activation of the small GTPase, Rap1A, which binds directly to PLCɛ and translocates the complex to the Golgi. There, PLCɛ hydrolyzes phosphatidylinol‐4‐phosphate (PI4P). Prolonged activation of this pathway results in increased expression of hypertrophy‐related genes. However, how the structural basis of PLCɛ activity under basal conditions and upon activation by Rap1A are unknown. Towards this end, we have obtained the first high‐resolution insights into a catalytically active PLCɛ variant. These studies, together with biochemical analysis of the intramolecular contacts observed in the structure, provide the first molecular insights into this enzyme.

Support or Funding Information

This research is supported by American Heart Association Scientist Development Grant 16SDG29920017 and NIH 1R01HL141076‐01 to A.M.L

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MppP: The Beginning of L‐End (Synthesis)
Authors: Tiffany Karen , Amundson Jacob , Boeselager Amy , Bote Kayla , Burgarino Michael , Burns Riley , Ertl Ellie , Fairchild Elliott , Fueger Kaitlyn , Garey Simon , Going Julia , Ketelhohn Amelia , Kubiak Katelyn , Kunz Josslyne , Langholz Andrew , Levy Sara , Novack Maya , Phelps Katelyn , Remington Erika , Schoenberg Spencer , Tiffany Benjamin , Silvaggi Nicholas
Published: June 2021

Infections caused by antibiotic‐resistant pathogens like MRSA are serious problems that require the development of new antibiotics. The non‐proteinogenic amino acid L‐enduracididine (L‐End) is looked at as a solution, as it could be used to synthesize new versions of L‐End‐containing antibiotics and improve drug efficacy of antibiotics not normally containing L‐End. However, L‐End is difficult to synthesize and is not commercially available. The enzyme MppP, an L‐arginine deaminase/hydroxylase from Streptomyces wadayamensis, is involved in L‐Arg biosynthesis. MppP is a pyridoxal 5′‐phosphate (PLP)‐dependent oxidase catalyzing the oxidation of L‐Arg to 2‐oxo‐4‐hydroxy‐guanidinovaleric acid, which continues along the pathway to produce L‐End. Four electrons are transferred to L‐Arg from molecular oxygen via the PLP cofactor; water contributes the hydroxyl and ketone oxygen atoms of the product. MppP functions as a homodimer. Each 376‐residue protomer contains a large domain, primarily composed of beta sheets, and a smaller domain consisting of a mix of alpha helices and beta sheets. The active site, containing PLP and the catalytic Lys221, is located between these two domains. The N‐terminal portion of the protein is not ordered unless substrate or product is bound in the active site, and this region appears to be critical for catalysis. The Cedarburg SMART (Students Modeling A Research Topic) Team has designed a model of MppP using 3D printing technology to investigate MppP structure‐function relationships. Studying the structure and function of MppP can elucidate the mechanism of L‐End biosynthesis, making possible the commercial production of L‐End for use in new MRSA‐fighting antibiotics.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Isolation of Synergistic Natural Products Targeting Metabolic Dysfunction in Pediatric Pre‐B Cell Acute Lymphoblastic Leukemia Using High‐Throughput Screening and Metabolomics
Authors: Collins Meghan E , Sweeney Shannon R. , Tiziani Stefano
Published: June 2021

High‐throughput screening was used to isolate synergistic compounds from a library of 136 natural products that exhibited negligible effects on non‐malignant cell lines. Three groups were screened including four primary cell lines isolated from pediatric patients with pre‐B ALL (acute lymphoblastic leukemia), five pediatric immortalized B‐ALL cell lines, and three non‐malignant cell lines. A total of 9 compounds were selected from the primary round of screening, these exhibited an inhibition average of at least 20% in the primary B‐ALL cell lines with a consistent trend in the immortalized B‐ALL cell lines. Of these compounds, parthenolide had the most negligible effect on the non‐malignant cell lines, the highest efficacy across the all B‐ALL cell lines, and was therefore selected as the top candidate. The remaining treatments were grouped as toxic or selective depending on if the average inhibition of the non‐malignant cell lines was greater than 50%. A total of 42 combinations were evaluated for each treatment pair in the second round of screening; the Chou‐Talalay and BLISS models were utilized to determine synergism. Based on both models, parthenolide was observed to have the highest and most consistent synergistic relationship with shikonin across all pre‐B ALL cell lines while showing no toxicity in the non‐malignant cell lines. Untargeted metabolomics and metabolic flux analysis using isotopically labeled glutamine and glucose was performed to analyze the impact of the combinatorial treatment on pediatric pre‐B cell acute lymphoblastic leukemia metabolism.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Bioactive molecules isolated from olive pomace extract protect murine cortex neurons from NMDA‐mediated cell death
Authors: Franchi Alice , Pedrazzi Marco , Casazza Alberto Alessandro , Millo Enrico , Damonte Gianluca , Salis Annalisa , Patrone Mauro , De Tullio Roberta , Sparatore Bianca , Perego Patrizia , Averna Monica
Published: June 2021

Here we demonstrate that olive pomace extracts contain bioactive molecules able to prevent primary neuron death induced by altered calcium homeostasis.

We have observed previously that partially purified extracts counteracted calcium induced cell damages on human neuroblastoma (SK‐N‐BE) and mouse brain endothelioma (bEnd5) cell lines operating through the regulation of the cell dynamics that involve the calcium ions.

It is well known that changes in [Ca2+]i are crucial events during signal transduction processes involved in several cellular physiological functions. Moreover, even a limited dysregulation in intracellular Ca2+ homeostasis is related to several acute and chronic diseases including neurodegenerations. In this context, we have now tested the effect of the bioactive molecules, detected in olive pomace, on primary cell cultures of murine cortex neurons, used as a cellular model. Accordingly, in order to induce a cytotoxic influx of Ca2+ through opening of the NMDA receptor, we treated neurons s with NMDA (N‐Methyl‐D‐Aspartic‐Acid) for 24 hour. When this cell treatment was carried out in the presence of preparations of the bioactive molecules, the effect of NMDA, evaluated as percentage of cell death, was significantly reduced. Moreover, following cell exposure to NMDA in the absence or the presence of the bioactive molecules, the [Ca2+]i was measured using different alternative techniques. The results obtained confirmed that these bioactive preparations act by molecular mechanisms that prevent the alteration of the intracellular calcium homeostasis mediated by NMDA. In conclusion, these bioactive molecules protect murine cortex neurons by reducing the toxic effects of [Ca2+]i, operated by the NMDA receptor. Further investigations are in progress to understand if the bioactive olive pomace derived molecules act directly on this receptor or on pathways activated downstream its opening. At present, the issue we give the highest priority is to identify the molecular nature of the relevant bioactive compounds. To solve this question we are carrying out mass spectrometry analyses on differently purified extracts.

The ultimate aim is to achieve definitive conclusions about the potential practical applications for these natural molecules in their purified form. Indeed, providing comprehensive information about the structural and functional aspects of these molecules is crucial to propose new possible strategies for therapeutic interventions in neurodegenerations.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acetoacetate Enhances the Cytotoxicity of Anti‐tumor Agents on MCF‐7 Breast Cancer Cells Without Itself Inducing Cell Death
Authors: Lin Bo , Miller Anna I , Fine Eugene J , Feinman Richard , Pincus Matthew J
Published: June 2021

The potential for enhancing the effect of drugs, radiation or other modalities is one of the potential benefits of ketogenic diets for cancer therapy. Cancer cells grown in culture provide a model for the phenomenon. Acetoacetate (AcAc), one of the ketone bodies, has been shown to inhibit several cancer lines in culture [1] but is without effect on normal fibroblast controls [1]. Here we show that AcAc also increased the sensitivity of a breast cancer cell line, MCF‐7, to the peptide, PNC‐27 that is known to kill cancer cells but that does not affect normal cells [2,3].

MCF‐7 cells were grown in high (25 mM) or low (10 mM) glucose medium and incubated for 48 hours with or without several doses of AcAc. Consistent with previous studies, MCF‐7 cells were viable but proliferation was inhibited by AcAc in a dose‐dependent manner as indicated by cell number.

Cell killing induced by 50 μg/m of PNC‐27 increased 6‐fold in the presence of 10 mM AcAc compared to its absence, and increased 12‐fold at 20 mM AcAc. Similar results were obtained at higher doses of peptide (such as 100 – 200 μg/mL). Thus, AcAc enhances the killing rate of this peptide on cancer cells while it does not itself induce cell death and does not affect the growth of normal cells.

If this effect carries over to ketogenic diets, combination of chemotherapeutic agents with a ketogenic diet may result in the ability to provide more reliable outcomes and allow lower doses of anti‐cancer chemo‐ and radio‐therapeutic modalities in order to reduce toxicity and side‐effects.

Support or Funding Information

Supported in. part by Nutrition & Metabolism Society and the Research Foundation of SUNY

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The polymorphic region an interleukin B1 is highly associated with type 1 diabetes in Saudis
Authors: Hamza Muaawia Ahmed
Published: June 2021

Type 1 diabetes (T1D) is an auto‐immune disease mediated by T lymphocytes that lead to insulin secreting islet B cell destruction. T1D is a multifactorial disease with strong genetic component. Pancreatic sample from patients with T1D showed mononuclear cell infiltration in islets, include mainly B cells and T cells that required for disease development.

In this study, we genotyped a group of Single Nucleotide Polymorphisms (SNPs) that located at IL1B1 (rs16944), IL1A (rs1800587), IL1B (rs1143634), IL1R (rs2234650) and IL1RN (rs315952) cytokines and cytokine receptors to investigate a possible association between these genetic markers and T1D.

Sequence‐specific forward and reverse primers and two TaqMan® MGB probes with dyes (VIC/FAM) were used to detect allele 1 and allele 2 of each SNP. 150 patients with T1D and 150 normal healthy individuals with no history of auto‐immune disease were recruited. The genotype models for each SNP (codominant, dominant and recessive traits) were derived using the SNPStat software, Fisher exact test and logistic regression methods were used to compare patient and control results. The dominant model of rs16944 (G/G vs G/A‐A/A)) was found highly significant in patient than in control (41.3% vs 14.6%, p value <0.0001) and considered as a risk factor for T1D development and individuals having this genotype are 4 fold susceptible to develop the disease. No other significant association was detected.

In conclusion, rs16944G/G model is considered positively associated with T1D.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Metabolomic and Lipidomic Characterization of Oxalobacter formigenes to Define Intestinal Oxalate Secretion
Authors: Chamberlain Casey A , Hatch Marguerite , Garrett Timothy J
Published: June 2021

Kidney stone disease afflicts 1 in 11 people in the U.S. and burdens the economy with an annual treatment cost of over $10 billion. Nearly 80% of kidney stones are composed of calcium oxalate (CaOx) and form when humans are exposed to oxalate through the diet and endogenous glyoxylate metabolism in the liver. Recently, significant clinical interest has turned to the intestinal microbiome, specifically a class of “oxalotrophic” bacteria that degrade dietary oxalate in the intestine. These bacteria reduce the amount of oxalate absorbed by the body, lowering circulating and urinary oxalate, both which directly influence CaOx stone formation. One species in particular, Oxalobacter formigenes (Oxf), solely utilizes oxalate as an energy source and has the unique ability among oxalotrophs to transport endogenous oxalate from the bloodstream into the intestine to increase oxalate availability, allowing it to feed on both metabolic and dietary oxalate. The potential for Oxf as a probiotic therapy for kidney stone disease is currently being evaluated by numerous institutions. Compelling evidence demonstrates that Oxf produces some form of secreted bioactive compound to regulate oxalate transport proteins in the intestinal wall to bring oxalate across the epithelium. This compound, if identified, could potentially serve as a future therapy for kidney stone formation. This study seeks to identify this secreted factor through an Ultra High Performance Liquid Chromatography‐Mass Spectrometry‐based characterization and comparison of the metabolome and lipidome of Oxf with other oxalotrophs that cannot initiate oxalate secretion, the rationale being that the bioactive compound(s) responsible for this function should be uniquely expressed by Oxf. We also present the first characterization of the Oxf metabolome and lipidome by comparing the human strain HC1 and the wild rat strain OxWR, both which have been shown to induce net intestinal oxalate secretion across native intestinal epithelial tissue. It is essential to elucidate the molecular nature of the Oxf‐host relationship to bring this bacterium and its metabolic products to the clinic as a novel approach to kidney stone disease.

Support or Funding Information

This work was funded by the National Institutes of Health grant 2R01DK088892‐05A1.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Prostaglandin E2 signaling networks in T cells revealed through a systems approach
Authors: Lone Anna Mari , Giansanti Piero , Jørgensen Marthe Jøntvedt , Gjerga Enio , Dugourd Aurelien , Scholten Arjen , Saez‐Rodriguez Julio , Heck Albert , Tasken Kjetil
Published: June 2021

Prostaglandin E2 (PGE2) plays a crucial role in immune homeostasis, inflammation and cancer. Elevated in several cancers, PGE2 promotes tumor progression and an immunosuppressive tumor microenvironment, partly through its actions on T cells. Currently, we lack a comprehensive view of the signaling pathways and networks that PGE2 triggers through its four distinct receptors (EP1‐EP4) in different T cells subsets. Here, we use a two‐pronged approach based on phosphoproteomics and phosphoflow cytometry in combination with highly specific agonists and antagonists of EP1–4 to fully characterize PGE2 signaling in T cell subsets, including CD4, CD8 and Tregs. Phosphoproteomics revealed >1000 phosphosites regulated upon EP1–4 triggering, among them novel sites as well as sites with known biological function. Phosphosites identified were specifically regulated by one receptor, or by several, indicating potential receptor cross‐talk. Functional analyses demonstrated the involvement of PGE2 signaling in key immunological processes. Phosphorylation motif characterization and kinase predictions confirmed the involvement of kinases such as PKC, CDKs, CK2, MAPKs and PKA, and revealed signaling differences between CD4 and CD8 cells. Modeling of the MS data enabled visualization of predicted networks. Phosphoflow cytometry studies elucidated signaling kinetics, including differences in signal duration between EP2 and EP4 receptors, which both signal through Gαs. Cell type differences observed by phosphoflow mirrored the phosphoproteomics data, with the highest signaling levels observed in EP2‐stimulated CD8 cells and different T cell subtypes exhibiting distinct signaling pathways. Predicted kinases, notably PKA and CK2, were verified using kinase inhibitors, and further kinases involved in PGE2 signaling, including PI3K and Src, were also identified. Finally, we used logic‐based models to understand the organization and dynamic behavior of the networks. In sum, we present a comprehensive and detailed view of PGE2 signaling nodes, pathways and networks in T cells, thus improving the current understanding of PGE2's role in T cells and providing a solid foundation for targeted and hypothesis‐based investigations of PGE2 signaling and functions in T cells.

Support or Funding Information

Norwegian Cancer Society, Health South‐East, Research Council of Norway, EU‐project Prime‐XS.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Brown adipose tissue metabolism in arsenic environmental health and obesity
Authors: Ro Seung‐Hyun , Jang Yura , Bae Jiyoung
Published: June 2021

Arsenic is an element that occurs naturally in the environment. Humans are likely to be exposed to higher amount of arsenic through arsenic contaminated drinking water and consuming foods including fruits and vegetables, and the arsenic can accumulate in the human body at high concentrations. Previous research has shown that arsenic induces oxidative stress linked to metabolic diseases such as obesity and diabetes. In mice, arsenic exposure during embryonic development is linked to an early puberty and adult obesity. Recent findings of brown adipocytes, capable of dissipating energy as heat, in adult humans have promised new hope for obesity treatment and prevention. Therefore, understanding the pathophysiological role of brown adipocytes and uncoupling protein 1 (UCP1) regulation in thermogenesis can provide effective strategies against obesity. Here, we investigated the effects of arsenic on differentiation, mitochondrial respiration and thermogenesis in immortalized murine brown adipocytes. We observed that arsenic significantly reduced brown adipocyte differentiation in dose dependent manner. Arsenic suppressed mitochondrial respiration and biogenesis, leading to attenuated brown adipocytes specific thermogenesis functions including UCP1 in immortalized murine brown adipocytes. In mice studies, we confirmed the heavy arsenic accumulation in brown adipose tissue (BAT) and the suppression of gene expression levels of mitochondria specific markers after the oral administration of arsenic into mice at dose of 10 mg/kg/day for 9 days. Arsenic significantly suppressed the BAT thermogenesis marker UCP1 when exposed to cold temperature for 24 hr at the last day of oral gavage. These results reveal the underlying mechanism of how arsenic aggravates metabolic diseases such as obesity by suppressing brown adipocyte differentiation, mitochondria function and thermogenesis, which can be utilized for development of arsenic‐specific inhibitor drugs protecting essential physiological functions of BAT in humans.

Support or Funding Information

This work was supported by University of Nebraska‐Lincoln ARD/ORED/BIOC grants, Layman award, Nebraska Tobacco Settlement‐Biomedical research enhancement funds, and Nebraska Center for thePrevention of Obesity Diseases (NPOD) seed grant from NIH (P20GM104320) to S.R.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
C16‐Ceramide directly binds and activates p53 in response to cellular stress
Authors: Jeffries Kristen , Krupenko Natalia
Published: June 2021

Ceramide is a key bioeffector in cellular adaptation to stress, and increased ceramide generation often functions as a pro‐apoptotic signal. Another key mediator of apoptosis, the p53 tumor suppressor protein, responds to diverse stress stimuli through its role as a transcription factor. We observed that folate withdrawal leads to CerS6 up‐regulation and C16‐ceramide accumulation in a p53‐dependent manner as a pro‐apoptotic cue, and hypothesized that p53 activation could result from the direct binding of C16‐ceramide to p53, which disrupts the protein interaction with its negative regulator, MDM2. Pulldown with biotinylated C16‐ceramide and membrane binding assays confirmed that p53 binds C16‐ceramide with high affinity and selectivity towards ceramide acyl chain length. In agreement, C16‐ceramide treatment of cultured cells disrupted the interaction between MDM2 and p53 in bimolecular fluorescence complementation experiments. The physiological relevance of this mechanism of p53 activation was demonstrated in serum starved cells where CerS6‐dependent C16‐ceramide generation mediated the stress response. Strikingly, the siRNA silencing of CerS6 prevented both p53 and p21 accumulation in response to serum starvation and partially rescued cells from G0/G1 arrest. These findings establish C16‐ceramide as the first physiologic metabolite activating p53 through the direct binding to the protein. Future experiments will determine the mechanism for the loading of C16‐ceramide onto p53 in cellular stress response.

Support or Funding Information

NIH CA193782‐01

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Cks2 localization and interaction with Cdk1 in mitotic cells of Xenopus laevis embryos
Authors: Lu Zhiye , Kim Yeaseul , Sandquist Joshua C.
Published: June 2021

Although mitotic regulation has been extensively studied, several of the mechanisms behind this process have yet to be fully understood. Our group is particularly interested in mechanisms regulating the timing of mitotic exit and how such mechanisms coordinate with other mitotic cues such as spindle orientation. Recent work in our lab has shown that spindle movements in epithelial cells of the developing frog embryo correlate with mitotic exit and perturbations in normal spindle movements lengthen metaphase. Further, these changes in mitotic progression are accompanied by increases in Cdk1 Tyr15 phosphorylation, specifically at cell junctions. We propose that the link between spindle movement and mitotic progression is an important part of the mechanisms influencing spindle orientation in the frog epithelium. Our current work examines the role of Cks2 in the aforementioned mechanism. Cks2 serves as a targeting subunit for the mitotic kinase Cdk1. Specifically, work in yeast suggests Cks2 may direct Cdk1 toward the anaphase‐promoting complex (APC) and interact with both complexes, facilitating the phosphorylation of Cdk1 and activation of the APC and thus help control mitotic exit. In this study, we characterize Cks2's mitotic function in Xenopus laevis. Our aim is to characterize Cks2 distribution throughout mitosis and determine whether perturbing Cks2 function alters mitotic progression in the gastrula‐stage epithelium. We hypothesize that if Cks2 influences Cdk1 function in mitosis, then the two proteins will exhibit similar localization patterns in mitotic cells. We visualized endogenous Cks2 and also expressed GFP‐tagged Cks2 in fixed epithelial tissues of Xenopus laevis embryos through confocal microscopy. Our early data show that Cks2 localization overlaps with that of a phosphorylated Cdk1(pY15) at cell junctions and overexpression of GFP‐tagged Cks2 results in increased levels of Cdk1‐pY15. In addition, the assay expressing a dominant negative version of Cks2 suggests that depletion of endogenous Cks2 activity may contribute to decreased Cdk1‐pY15 at cell junctions and increased cell area. Therefore, these data suggest a link between Cks2 and Cdk1 in mitotic regulation and are a first step toward a greater characterization of Cks2's role in regulating mitotic progression in vertebrate organisms.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Zyflamend Induces Apoptosis in Pancreatic Cancer Cells via Modulation of the JNK Pathway
Authors: Bettaieb Ahmed Bettaieb , Puckett Dexter , Chahed Samah , Whelan Jay
Published: June 2021

Pancreatic cancer is the fourth leading cause of death in the United States with over 53,670 new cases and 80% mortality in 2017. The overall prognosis and survival rate of this diagnosis are grim, and in the vast majority of cases, terminal. Current pharmacological therapies and treatments targeting pancreatic cancer have proven ineffective far too often. Therefore, there is an urgent need for alternative therapeutic approaches. Zyflamend, an anti‐inflammatory herbal compound, has proven effective in various in‐vitro and in‐vivo cancer platforms, shows promise. However, its effects on pancreatic cancer in particular, remain largely unexplored. The objective of this study is to investigate the effects of Zyflamend on pancreatic cancer cells survival and decipher the underlying molecular mechanisms. Pancreatic cancer cells were treated with Zyflamend and key signaling pathways involved in the survival and proliferation were investigated. Our studies show that Zyflamend treatment caused a dose‐dependent decrease in cell proliferation, consistent with an increase in apoptotic cell death and activation of the JNK pathway. Additionally, Zyflamend in conjunction with chemotherapeutic compounds exhibited a synergistic effect against pancreatic cancer. Inhibition of JNK abrogated the pro‐apoptotic effects of Zyflamend. These studies identify Zyflamend as a potential novel approach to treat pancreatic cancer via modulation of the JNK pathway.

Support or Funding Information

This work was supported by a grant from NIH/NIDDK R00DK100736 to AB

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