bluuebunny/crossref_metadata_2025 huggingface dataset:
Curcumin Reverses Methylglyoxal Induced Apoptosis in RINm5F Cells and PC12 Cells in Hyperglycemic Conditions
Authors: Patangia Shaima Fazal , Mungre Sue
Published: June 2021

Hyperglycemia, a condition in which blood glucose level exceeds the normal level, is often observed in diabetes mellitus. Normally, the hormone insulin, secreted by β‐cells of the pancreas, regulates blood glucose level. However, in patients with diabetes type 2, hyperglycemia decreases beta cell mass, resulting in a deficiency of insulin. Methylglyoxal (MG), a carbonyl derivative of glucose, is believed to cause the deleterious effects observed in type‐2 diabetes, such as neuropathy. MG is known to cause oxidative stress in neural cells, leading to apoptosis. It was of interest to see whether MG showed a similar effect on pancreatic β‐cells. Also, curcumin, a compound present in turmeric is known to have anti‐oxidative and anti‐inflammatory properties. In this study, we determined the effect of MG and curcumin on the viability of RINm5F cells (rat pancreatic β‐cells). Cells were treated in the presence of 20μM and 40μM MG, with different concentrations of curcumin (2μM to 10μM). Preliminary results showed that MG treatment at 40 μM causes 50% cell death in 24 hours, due to apoptosis of cells. Curcumin alone induced cell proliferation. When curcumin was added along with MG, the effect of MG was alleviated. Similar results were observed earlier with neuronal PC12 cells. In addition, various spices such as cinnamon, black pepper and saffron were also tested and found to alleviate the effect of MG. We are currently studying the expression of Bax and Bcl2 in both RINm5F and PC12 cells in the presence of MG and curcumin. This study will help determine whether the loss of β‐cells seen in diabetes, could be prevented using natural products.

Support or Funding Information

McNair Scholars Program

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigating the Role of Trehalose Metabolism in Resistance to Abiotic Stress in the Filamentous Fungus Fusarium verticillioides
Authors: Oberlie Nicole Ruth , McMillan Sarah D. , Brown Daren W. , McQuade Kristi L.
Published: June 2021

Fusarium verticillioides is a maize pathogen that synthesizes toxic secondary metabolites called fumonisins, which can contaminate grain and cause disease in livestock and humans. We showed previously that an F. verticillioides strain (Δtps1) that lacks the gene coding for trehalose‐6‐phosphate (T6P) synthase produces no trehalose, has reduced pathogenicity, and produces significantly less fumonisins than wild type. Here, we characterize the effects of various abiotic stresses on spore viability and growth rates of the wild‐type and Δtps1 strains as well as strains expressing a catalytically inactive form of T6P synthase, which are also trehalose deficient. We show using a colony count assay that desiccation decreases the viability of spores from trehalose‐deficient strains significantly more than spores from the wild‐type strain. Likewise, oxidative stress‐inducing compounds reduce both the growth rate and maximum culture density of trehalose‐deficient strains more than the wild‐type strain. Live/dead fluorescence staining indicates that oxidative stress decreases the viability of trehalose‐deficient strains more significantly than wild type. Together, these data suggest that trehalose is critical for tolerance to desiccation and oxidative stress in F. verticillioides.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ESE‐1 Represses Formation of Tumor in vivo and Growth, Invasion and Migration of Human NSCLC Cells in vitro
Authors: Lou Zhiyuan , Lee Bok‐Soon , Ha Taekyu , Xu Yanrui , Kim Haeng‐Jun , Kim Chul‐Ho , Lee Seong‐Ho
Published: June 2021

Lung cancer is the leading cause of cancer mortality in the United State due to poor prognosis. Identification of effective molecular target is essential for lung cancer prevention and therapy. Epithelial Specific ETS‐1 (ESE‐1) is a transcription factor that belongs to ETS superfamily and associated with development of several types of cancer. However, the significance of ESE‐1 in lung cancer remains unanswered. The objective of current study was to investigate if ESE‐1 expression influences tumorigenic and metastatic activity of human non‐small cell lung cancer (NSCLC). Overexpression of ESE‐1 repressed the anchorage‐independent growth of human NSCLC cells and led to an increase of G1 arrest and apoptosis. ESE‐1 expression repressed invasion and migration of human NSCLC cells. Xenograft study indicated that ESE‐1 expression inhibited formation and development of tumor. ESE‐1 is a downstream target of TGF‐β‐stimulated epithelial mesenchymal transition (EMT). Our findings indicate that ESE‐1 serves as a tumor repressor in human NSCLC.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dysregulation of the Tumor Suppressor Tip60 and ATM Signaling Paradigm by the HIV Transactivator of Transcription
Authors: Simenauer Ari , Cota‐Gomez Adela
Published: June 2021

Despite the longevity afforded by modern anti‐retroviral therapy, long‐term HIV infection increases the risk for pulmonary co‐morbidities associated with cell cycle dysregulation including lung cancer and pulmonary arterial hypertension. As such, we postulate that select viral factors, in particular the HIV transactivator of transcription (Tat), interfere with cellular processes involving tumor suppressors and other cell cycle regulators during chronic infection. Tat is well known to inhibit the activity of the tumor suppressor Tat Interacting Protein 60 kDa (Tip60), an acetyltransferase critical in the progression to cell cycle arrest and/or apoptosis. In response to stressors such as DNA damage and oxidative stress, Tip60 activates the cell cycle master regulator kinase Ataxia Telangiectasia Mutated (ATM) via acetylation, initiating a signaling cascade that culminates in the activation of a DNA damage response resulting in both short and long term cellular arrest. Thus, we hypothesize that inhibition of Tip60 by Tat interferes with cellular arrest in responses to these stressors and results in abnormal proliferation. We addressed the hypothesis using HeLa cells stably transfected to express Tat (HeLa‐TatIII). Utilizing an ATP based luminescent cell viability assay, we observed accelerated cell proliferation in HeLa‐TatIII compared to its HeLa wild‐type counterpart. Furthermore, immunoprecipitation experiments in HeLa‐WT cells confirmed a physical interaction between Tat and Tip60. Acetylation by Tip60 results in autophosphorylation of ATM at ser1981, and this phosphorylation event is a canonical marker for ATM activation. As such, we determined the phosphorylation state of ATM ser1981 after treatment with doxorubicin in HeLa‐WT and HeLa‐TatIII cells. We found that, following treatment with doxorubicin, ATM phosphorylation at ser1981 was severely attenuated in HeLa‐TatIII cells compared to HeLa‐WTs, suggesting a role for Tat in the inhibition of ATM activation. Taken together, these data support the role of the HIV Tat protein in dysregulation of cell cycle arrest via ATM signaling pathways. Whether HIV promotes cellular proliferation in co‐morbidities such as lung cancer and pulmonary arterial hypertension via these pathways remains to be addressed.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polyunsaturated Fatty Acid Desaturase‐Mediated NAD+ Recycling Permits Ongoing Glycolysis and Cell Proliferation
Authors: Kim Wondong , Deik Amy , Florez Jose C. , Jacobs Suzanne B.R. , Clish Clary B. , Rhee Eugene P.
Published: June 2021

The delta‐5 desaturase (D5D) and delta‐6 desaturase (D6D), encoded by FADS1 and FADS2 respectively, are required for the synthesis of highly unsaturated fatty acids (HUFAs). Genetic variants associated with FADS1 and FADS2 expression are associated with HUFA content in circulating lipids, as well as fasting glucose, height and weight, and risk of colon and laryngeal cancer. Why genetic variation in HUFA synthesis has such a broad impact on human metabolic and proliferative phenotypes is unknown. Here, we show that HUFA synthesis is a mechanism for cytosolic NAD+ recycling that permits ongoing glycolysis. Consistent with this, lowering the cytosolic NAD+/NADH ratio via inhibition of mitochondrial respiration increased D5D and D6D activity in vitro and in vivo. Conversely, increasing the cytosolic NAD+/NADH ratio via expression of an NADH oxidase or activation of lactate dehydrogenase decreased D5D and D6D activity. Increasing D5D and D6D expression increased cytosolic NAD+/NADH, whereas reduced expression or inhibition of D5D and D6D decreased cytosolic NAD+/NADH and increased lactate production. Inhibition of D5D and D6D also reduced cell proliferation, an effect that was reversed by boosting cytosolic NAD+ but not with HUFA end‐product supplementation. Finally, we show that the type 2 diabetes risk haplotype in SLC16A11, which reduces pyruvate transport, also increases D5D and D6D activity, demonstrating the relevance of our findings to human disease. These results outline a novel link between glycolysis and HUFA desaturation mediated by cytosolic NAD+, and provide new insight on how D5D and D6D activity may impact glycemic, growth, and cancer traits in humans.

Support or Funding Information

This work was supported by NIH U01 DK060990 and the Extramural Grant Program of Satellite Health Care, a not‐for‐profit renal care provider.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thioesterase superfamily member 2 (Them2) Regulates Fatty Acid Partitioning Between Oxidative and Secretory Pathways in the Liver
Authors: Alves‐Bezerra Michele , Li Yingxia , Cohen David E.
Published: June 2021

Them2 (synonym: Acyl‐CoA thioesterase 13) is a mitochondria‐associated long‐chain acyl‐CoA thioesterase that is abundant in the liver and oxidative tissues. Evidence from whole‐body knockout mice suggests that Them2 prevents diet‐induced hepatosteatosis and improves hepatic glucose homeostasis. In cultured hepatocytes, Them2 promotes fatty acid oxidation and decreases lipogenesis. However, because it also suppresses thermogenesis in brown adipose tissue by limiting fatty acid oxidation, the true contribution to fatty acid metabolism of Them2 in the liver remains unclear. In order to evaluate the role of Them2 in liver to fatty acid metabolism, we created Them2 liver‐specific knockout (L‐Them2−/−) mice, which were fed chow or high fat diet for 12 weeks. L‐Them2−/− mice exhibited similar weight gain, body mass composition and glucose homeostasis in comparison to control animals. Whereas the rates of fatty acid uptake, de novo fatty acid synthesis, and hepatic concentrations of free fatty acids were unchanged, hepatic fatty acid oxidation was increased by 30% in chow‐fed L‐Them2−/− mice. In the absence of changes in steady concentrations of hepatic triglyceride and glycerolipid synthetic rates, a reduction in plasma triglyceride concentrations was observed upon hepatic Them2 ablation (38 and 43% decrease in chow and high fat fed, respectively). This was associated with a reduction in plasma VLDL‐triglyceride concentrations (29 and 9% in chow and high fat fed mice, respectively). Chow fed L‐Them2−/− mice also exhibited a 26% reduction in plasma apoB100 concentrations and a marked (70%) reduction in hepatic VLDL‐triglyceride secretion rates. Taken together, these data suggest that hepatic Them2 functions to direct fatty acids towards triglyceride incorporation into VLDL particles for secretion and away from mitochondrial β‐oxidation, thereby sustaining the export of lipids from the liver to the plasma and peripheral tissues. We speculate that Them2 in the liver plays a key role in determining the metabolic fates of fatty acids in response to nutritional status and metabolic demands.

Support or Funding Information

This work was supported by NIH R37 DK048873 and R01 DK056626 to D.E.C. M.A.‐B. was the recipient of a NASH Fatty Liver Disease Postdoctoral Research Fellowship Award from the American Liver Foundation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lysozyme over‐expression during nerve injury excites Aδ & C fibres in a fibre specific manner to incite neuropathic pain
Authors: SUROLIA AVADHESHA , Yadav Saurabh
Published: June 2021

Human lysozyme is one of the most extensively studied proteins yet its exact physiological functions still remain a mystery. Here, we highlight a hitherto unrecognized role of lysozyme in neuropathic pain and neuronal excitability. Injection of lysozyme alone in healthy rats leads to heightened sensitivity in them towards both mechanical and thermal pain stimuli. Also, in rat models of neuropathic pain, lysozyme is over – expressed and its down – regulation by siRNA or inhibition of its activity by its disaccharide inhibitor ‐ chitobiose relieves pain confirming a causal role of lysozyme in neuropathic pain. Moreover, as the catalytically incompetent lysozyme or its chemically inhibited counterparts fail to provoke pain indicate that it deploys its catalytic site for eliciting pain. Bath application of lysozyme remarkably increased both the spontaneous excitatory potentials and currents (sEPSPs & sEPSCs) and the amplitudes of afferent mediated evoked excitatory synaptic inputs (eFPSPs & eEPSCs) in Aδ fibres in a fibre specific manner in the dorsal horn neurons. It also increased paired pulsed ratio only in the C‐fibres. When complexed with chitobiose it does not lead to any change in neuronal excitability. Thus, lysozyme excites neurons by deploying its catalytic region in a fibre specific manner. Further, we show that it deploys its catalytic site to recruit Annexin A2 to transduce TLR4→NFkβ signaling pathway which may culminate in the fusion of neurotransmitter vesicles at the synapse to manifest neuronal excitability and eventually pain. Thus, glycan inhibitors of lysozyme and antagonists of the pathway recruited by it to inflict pain pave not only the way for novel approaches for dissecting the molecular mechanism of neuropathic pain but also for treating this unmet medical need efficaciously.

Support or Funding Information

Council of Scientific and Industrial Research, India to AS

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Glycomics Studies on Nematodes Elucidate Conserved Functional Epitopes and Biosynthetic Pathways
Authors: Yan Shi , Vanbeselaere Jorick , Jin Chunsheng , Joachim Anja , Paschinger Katharina , Wilson Iain B. H.
Published: June 2021

Nematodes constitute a large number of different species on earth and some are disease‐causing parasites of mammals. Glycoproteins synthesised by nematodes are decorated with a different set of glycan structures as compared to the ones produced by mammals. During the course of parasite‐host interactions, nematode glycans could trigger or modulate host immune responses. To better understand the conserved N‐glycosylation machinery in nematodes, we use the free‐living Caenorhabditis elegans as a model; double/triple mutants were generated to investigate the function of genes which are involved in N‐glycan biosynthesis. N‐glycans of C. elegans as well as parasitic nematodes were released, either enzymatically or chemically, fluorescently labelled and analysed in a great detail using off‐line HPLC‐MALDI‐TOF‐MS in combination with exo‐glycosidases and chemical treatments. Furthermore, a number of fucosyltransferases (FUTs), hexosaminidases and N‐acetylglucosyltransferases were cloned and recombinantly expressed so that their enzymatic activities were studied in vitro by glycan remodelling. A range of conserved functional epitopes on N‐glycans were characterised in C. elegans wild‐type and mutant strains and were also identified in parasitic species, such as Oesophagostomum dentatum and Haemonchus contortus; this includes different types of Gal‐Fuc modifications of the chitobiose core and phosphorylcholine substitution on the N‐acetylhexosamine residues of the antennae. The functions of three core‐modifying FUTs were elucidated by in vitro and glycomic analyses. Moreover, the rice PNGase A or hydrazine released more N‐glycans with complex core modifications, which are not observed before using traditional PNGase F and almond PNGase A. These data expand our knowledge on nematode N‐glycosylation, particularly the plasticity in producing rare structures when a biosynthetic pathway is disturbed. Identification of nematode‐specific glyco‐epitopes would facilitate the design of novel strategies for diagnosing parasitic infections and aid engineering of glyco‐vaccines in the future.

Support or Funding Information

The Austrian Fonds zur Förderung der wissenschaftlichen Forschung (FWF; grants P30021 to S.Y., P21946 and P25058 to K.P. and P23922 and P29466 to I.B.H.W.)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dissecting difference in heterologous protein secretion titer by Type III secretion system between strains of Salmonella enterica
Authors: Wong Han Teng , Tullman‐Ercek Danielle
Published: June 2021

Type III secretion systems (T3SS) are complex macromolecular machines found in Gram‐negative bacteria. These bacteria utilize T3SS to facilitate microbe‐host interactions by translocating proteins, known as effectors, into the host cells. This provides an interesting system for engineering the selective translocation of proteins out of the bacterial cell. Secretion strategies have been useful in exporting proteins of biotechnological interest in systems such as yeast, insect cells and mammalian cells. Moreover, bacteria fermentations are faster and cheaper.

In our lab, we make use of the SPI‐1 T3SS in Salmonella enterica serovar Typhimurium for protein export. Because the Salmonella SPI‐1 is not an essential metabolic pathway, we can harness it for heterologous protein secretion without affecting cell viability. Using secretion assays based on western blotting and a split GFP system, we were able to determine that the DW01 strain secretes ~20% more proteins than the LT2 strain.

In order to determine the genetic basis for the differences in secretion titer, we sequenced the genomes of both strains using next generation sequencing to determine single nucleotide variants and indels unique to DW01. Some of the mutations were then further verified by Sanger sequencing. The alleles for each locus are then exhaustively swapped in both strain using the recombineering method established by the Court lab. This allows us to determine the contribution of each alleles at every locus to the increased secretion observed in DW01. Other phenotypic differences are also characterized.

Through this work, we also identify genes outside of the SPI‐1 pathogenicity island that can modulate the heterologous protein secretion capability of S. enterica. Taken together, these experiments provide us with more tools to further increase the titer and achieve the industrial standards of >1g/L of protein.

Support or Funding Information

H.T.W. is funded by A*STAR. L.B. is funded by NSF GRFP. This work is funded by NSF CBET grant number 1706125.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oroxylin A prevents cardiac hypertrophy and dysfunction as a modulator of mitochondrial protein acetylation
Authors: Aguilar‐Sáenz Alejandro , Treviño‐Saldaña Niria , Oropeza‐Almazán Yuriana , García‐Rivas Gerardo
Published: June 2021

Cardiac hypertrophy and remodeling are both well‐known consequences mediated by increased levels of Angiotensin II (Ang II) as a compensatory mechanism in non‐ischemic HF. In this sense, mitochondrial protein hyper‐acetylation and ROS production that induce mitochondrial dysfunction have previously been associated with this condition. Oroxylin A (OA) is a methylated flavone found on the root of Scutellaria baicalensis known for its antioxidant properties, and as a modulator of mitochondrial deacetylase activity. Nevertheless, OA effects have not been explored in cardiac remodeling mediated by exacerbated ROS production and mitochondrial hyper‐acetylation. Therefore, we explore the potential cardioprotective effect of OA in an in vitro model of cardiac hypertrophy induced by Ang II treatment in H9c2 myocytes. We found that OA reverted completely the Ang II mediated hypertrophic and cell death effect. Mitochondrial acetylation profile and sirtuin3 expression analyzed by western blot showed that Ang II promotes ~2‐fold increase in protein acetylation; however, OA pre‐treatment abrogates Ang II‐induced hyper‐acetylation in a dose‐dependent fashion. Remarkably, we found that the OA pre‐treatment prevents mitochondrial dysfunction by decreasing significantly permeability transition susceptibility and membrane potential depolarization. These results were consistent with a decrease in apoptosis, determined by caspase 9 and 3,7 activity. Overall, these findings suggest that OA promotes sirtuin3 activation, targeting pivot mitochondrial proteins. Accordingly, we evaluated the cardioprotective effect of OA in an in vivo model of HF induced by AngII+L‐NAME. In this model, we found an increased mitochondrial acetylation in the myocardium and found that OA was capable of prevent cardiac hypertrophy and dysfunction. Taken together, our results suggest that OA is a novel strategy to treat cardiac dysfunction in HF.

Support or Funding Information

This work was partially supported by Xignus Research Foundation as well endowed Chair in Cardiology/Grupo de Enfoque Medicina Cardiovascular Tec de Monterrey, CONACYT Grant CB‐256577, Fronteras de la Ciencias 0682 (Gerardo García‐Rivas), and Red Temática Farmoquímicos del CONACYT.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expression of Fusion Proteins in Different Subpopulations of Cardiac Mitochondria
Authors: Guzmán‐Hernández Roberto , Parodi‐Rullán Rebecca M. , Chapa‐Dubocq Xavier , Jang Sehwan , Javadov Sabzali
Published: June 2021

Background

Mitochondrial dynamics including fission and fusion, along with mitophagy, play a vital role in the maintenance of the structural and functional integrity and number of mitochondria in the cell. These processes work to sustain the mitochondrial quality in the cell, in order to ensure optimal functioning of the cell. Mitochondrial fission and fusion are mediated by several proteins; including dynamin‐related 1 and fission protein 1 for fission and, optic atrophy type 1 (OPA1) and mitofusins 1 (Mfn1) and 2 (Mfn2) for fusion. Fusion proteins (OPA1, Mfn1 and Mfn2) promote fusion of two mitochondria into one, thereby eliminating dysfunctional mitochondria and thus, maintaining their normal morphology, content, and function, as well as the quality of the mitochondria in different subcellular compartments. The mitochondria in cardiomyocytes can be categorized in subpopulations based on their structural and physiological differences: subsarcolemmal mitochondria (SSM), which are found underneath the sarcolemma; interfibrillar mitochondria (IFM), localized between the myofibrils; and perinuclear mitochondria, which reside near the nuclear poles. Connexin‐43, a transmembrane protein responsible for the formation of gap junctions, can be used to differentiate between the SSM and IFM. In this project, we aim to study the role of mitochondrial fusion proteins between these two populations of mitochondria.

Hypothesis

Expression of fusion proteins are different between the SSM and IFM in intact rat hearts.

Methods

Mitochondria were isolated from Sprague‐Dawley male rat hearts (n=6). Mitochondrial proteins were visualized by SDS‐PAGE followed by western blot analysis using antibodies against OPA1, Mfn1, and Mfn2.

Results

The level of connexin‐43 was 95% (P<0.001) lower in IFM compared to SSM. Analysis of fusion proteins revealed that IFM contained 10% (P<0.47), 88% (P<0.001) and 24% (P<0.31) less OPA‐1, Mfn1 and Mfn2, respectively, when compared to SSM. Short and long isoforms of OPA‐1 were 70% (P< 0.002) and 32% (P<0.05) lower in IFM compared to SSM.

Conclusion

Lower expression of fusion proteins in IFM can explain differences in functional parameters between two mitochondrial subpopulations in the heart.

Support or Funding Information

Supported by the NHLBI NIH grants SC1HL118669 (S.J.) and 1 R25 HL115473‐01 (R.G‐H.).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Inhibition of choline acetyltransferase activity abrogates the growth of lung adenocarcinoma patients who are exposed to tobacco smoke
Authors: Akers Austin T. , Brown Kathleen C. , Colclough Kate W. , Nolan Nicholas A. , Friedman Jamie R. , Miles Sarah L. , Bow Eric W. , Rimoldi John M. , Hardman Elaine W. , Dasgupta Piyali
Published: June 2021

The pathophysiological characteristics of lung adenocarcinoma (LAC) in smokers is divergent from LAC in non‐smokers. One of the distinctive features of LAC in smokers is that the disease is relatively resistant to targeted molecular therapies. For example, targeted therapeutic agents like EGFR‐inhibitors (erlotinib and gefitinib) are highly effective in LACs in non‐smokers. However, these agents display much lower anti‐tumor activity in LACs in patients who are active smokers. Similarly, lung cancer patients (who are active smokers) show a lower response to chemotherapy than those who are non‐smokers. However, the majority of LAC patients are smokers. This underlines the need to identify viable drug targets for LAC therapy in patients who are exposed to cigarette smoke. A survey of literature reveals that nicotine (the addictive component of cigarette smoke) accelerates the growth of lung cancers, as well as confers resistance to chemotherapy. One of the mechanisms underlying the mitogenic activity of nicotine is that it promotes the production of the neurotransmitter acetylcholine (ACh) from LAC cells. ACh is known to be an autocrine growth factor for LAC cells and is synthesized by the enzyme choline acetyltransferase (ChAT). The present study investigates the feasibility of ChAT as a molecular target for LAC in smokers. We find that ChAT levels are upregulated in human LAC cell lines and tissues in analogous to their smoking history. Finally, the ChAT inhibitor BW813U causes robust apoptosis in human LAC cells. The magnitude of BW813U‐induced apoptosis is similar across LAC cell lines (irrespective of smoking history); however, the concentration of BW813U which causes apoptosis is lower in LAC cell lines belonging to heavy smokers. The anti‐tumor activity of BW813U is also observed in H838 cells (belonging to an 80 pack‐year smoker) xenografted on athymic mice. Our studies show that antagonists of ChAT like BW813U may have therapeutic applications in the majority of the population of LAC who are smokers.

Support or Funding Information

Funding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Is HPV Vaccination Secure Protection of Women Without or With Cytologic Cancer Screening
Authors: Markovic Olivera , Markovic Nenad
Published: June 2021

Today, million of girls around the world receive vaccination against HPV hoping vaccines will protect them from the risk of getting cervical cancer if having sex with HPV positive partners.

But, is it true? And, if it is true, how long the protection lasts?

This dilemma has controversial documented data. Therefore, manufacturer of HPV vaccines and health care providers in charge of cervical cancer prevention, advertised, via guidelines, testing for antibody titer and for cytopathological changes of cervical specimen – similar to Pap test. The open dilemma is about scheduling: from 1‐to 5‐year periods between screening. Many studies have been published and supported with statistical data, but, indeed, this is only a guessing game.

The Pap test was introduced as screening for lesions which can be developed into cervical cancer and the suspect lesions were recommended for colposcopy/biopsy/histology – finally to early removal of those lesions. This removal of lesions, not the Pap test alone, has interrupted the natural history of cervical cancer. The definition “cancer is unstoppable growth of malignant cells,” was replaced with “no lesion – no growth of cancer.”

HPV vaccine protection is specific for few strains of HPV virus which have been found in developed cervical cancers. Cervical cancer has a much wider palette of risk factors including other HPV strains, and other promoters of malignancy [3]. Because of that, the cytopathologic screening must be kept regularly active. This fact returns us to the old concerns: Pap test staining has inherited 20% false negative readings. The recommended MEDYKO composite biomarker [1–3] consists of metabolic biomarker, “ME” (increasing sensitivity to include BCC category), “DY” dysplastic changes defining the level of clinical diagnosis, and “KO” (koilocytes, HPV disease indicating worse prognosis).

All three markers are visible on one slide reducing false negative results of Pap test, and false positives of HPV infection testing. All are amenable for digital communication between POC and remote medical centers. Further, the specimen can be obtained at home with the Self‐sampling Kit what increases the outreach and opens the opportunity for annual testing.

The concept has been tested, confirmed, but still, waits for mass applications.

Conclusion

Cytologicl testing is still necessary for HPV vaccinated women, but Pap tests needs improvement as MEDYKO™ described above, annual home specimen sampling, collection of specimens in scattered POC, evaluation in remote specialist centers and early, the same day, intervention for removal of lesions if necessary.

Support or Funding Information

No external support

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chronic cadmium exposure reduces the dependence of MCF7 cells on ERa for cell growth
Authors: Bloomfield Mathew , Louie Maggie
Published: June 2021

The global prevalence of breast cancer in women illustrates the importance of identifying factors that contribute to disease onset and progression. Endogenous and environmental agents that interact with estrogen receptor alpha (ERa) have been shown to play a role in breast cancer etiology. Evidence from epidemiological studies and animal models have suggested that cadmium, a heavy metal that can activate ERa, contributes to the development and progression of breast cancer. Additionally, our lab showed that chronic cadmium exposure altered the expression of several ERa‐responsive genes and increased the malignancy of MCF7 breast cancer cells. Although these studies support cadmium's function as a hormone disrupter, the role of ERa in cadmium‐induced breast cancer progression remains unclear. Therefore, we modulated the expression of ERa in MCF7 cells after chronic cadmium exposure (Cd7 and Cd12) in order to understand its role in cadmium‐induced cancer cell growth and gene expression. The doubling times of MCF7, Cd7, and Cd12 cells were increased as a result of ERa loss, but this effect was significantly less in the cadmium‐adapted cells suggesting chronic cadmium exposure reduces the dependence of MCF7 cells on ERa for cell growth. Furthermore, preliminary analysis showed the transcript levels of classical and non‐classical ERa‐regulated genes were reduced in MCF7 cells after transient and permanent modification of ERa expression, while the non‐classical targets were not as affected in Cd7 and Cd12 cells after ERa knockout indicating cadmium exposure may have altered the regulation of these genes. These results demonstrate that while ERa still contributes to the growth of MCF7 cells chronically exposed to cadmium, their dependency on ERa appears to have diminished. The relevant processes affected by chronic cadmium exposure remain unclear, but these may enable MCF7 cells to adapt to ERa loss and potentially provide a path to ERa independence as the cancer progresses.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Study of the Chemo‐sensitizing Effects of Thymoquinone, the Active Constituent of Negilla Sativa (Black Seed), to Nitrogen Mustards, Chlorambucil and Cyclophosphamide, in Human Melanoma Cell Lines
Authors: Alsulaimany Marwa , Kerr Stephen G.
Published: June 2021

Black seed is often used as a spice in food and as a preservative in many products. The main active constituent of black seed oil is thymoquinone (TQ) which constitutes 30–57% of the oil. Thymoquinone has shown varying therapeutic benefits in various disease states, including cancer. The aim of this study was to investigate the cytotoxic and chemo‐sensitizing potential of TQ and compare it to ethacrynic acid (EA), a known cytotoxic agent in sensitizing melanoma cells to the effects of the N‐mustards, chlorambucil (CHL) and cyclophosphamide (CP). Both TQ and EA have an α, β unsaturated carbonyl system which could act as potent Michael Acceptors to mediate the cytotoxicity. Studies also assessed the cytotoxic potential of cyclophosphamide(CP) and chlorambucil (CHL) alone and in combination with TQ. Approximately 1×104 cells/100μL/well of two melanoma cell lines, lightly pigmented SK‐MEL19 and pigmented SK‐MEL23 were seeded in 96‐well plates and incubated for 24h at 37C° and 5% CO2. The cells were then treated with TQ (25–500 μM), EA (25–200 μM), CHL (25–500 μM) and CP (25–500 μM), for 24h and 48h. Combination treatment included pretreatment of the cells with TQ (100 μM) for 24 h followed by further exposure to CHL for an additional 24 h. Similarly, cells were pretreated with EA (100 μM) for 24h, followed by further exposure to CHL for 24h. Colorimetric MTS (Abcam) cytotoxicity assays, per manufacturer's instructions, was then carried out to determine cell viability. The results indicated that TQ exerts its toxic effect in a concentration‐dependent manner in SKMEL19 cell lines at concentrations ≥75μM, while in SK‐MEL23 at concentrations ≥ 100 μM, relative to the control. In addition, EA reduced cells viability significantly in both cell lines at concentration of 100 μM and above. However, CP did not show any cytotoxic effect in both cell lines at 24h or 48h up to concentrations of 500 μM. In contrast, CHL reduced cell viability in both cell lines at high concentrations (250 and 500μM) within 24h. When the incubation time was increased to 48h, the cytotoxic effect of CHL in SK‐MEL19 was observed at concentrations ≥ 100 μM, while in SK‐MEL23 at concentrations ≥ 150 μM. Pre‐treatment with TQ (100 μM), as well as EA (100 μM) greatly enhanced the cytotoxic effect of a non‐toxic dose of CHL (100 μM) in both cell lines after 24h as compared to the effect of CHL alone. These studies demonstrate the potentiating effect of TQ on CHL cytotoxicity possibly by sensitizing the human melanoma cell lines to the chemotherapeutic agent.

Support or Funding Information

MA acknowledges SACM for financial support

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determination of 13 ginsenosides in diverse ginseng product by LC‐MS/MS
Authors: Cho Pil Joung , Gao Yan , Jo Jung Jae , Lee Sangkyu
Published: June 2021

Ginseng (Panax ginseng Meyer) has been used as traditional herbal drug in Asian countries. Ginsenosides, the ginseng saponins, are major components, have pharmacological and biological efficacy, including anti‐inflammatory, anti‐diabetic and anti‐tumor effects. To control the quality of the components in diverse ginseng products, we developed a new quantitative method using LC‐MS/MS for 13 ginsenosides: Rb1, Rb2, Rc, Rd, Re, Rf, 20(S)‐Rh1, 20(S)‐Rh2, Rg1, 20(S)‐Rg3, F1, F2, and compound K. The method was successfully validated and applied in four representative ginseng products. This quantification method was applied to several processed ginseng products including fresh ginseng powder (FGP), white ginseng powder (WGP), red ginseng powder (WGP), and red ginseng concentrates (RGC). The amounts of the 13 ginsenosides in the various type of ginseng samples could be analyzed simultaneously. This method was expected to be suitable for quality control of ginseng products

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MICROINJECTION OF UROTENSIN II INTO THE PEDUNCULOPONTINE TEGMENTNUM LEADS TO AN INCREASED PERCEPTION OF SENSORY STIMULI
Authors: Ettaro Robert , Daniels Derek , MacLaren Duncan , Markovic Tamara , Clark Stewart
Published: June 2021

The pedunculopontine tegmentum (PPTg) plays a role in processing multiple sensory inputs and innervates brain regions associated with reward‐related behaviors. The urotensin II receptor, activated by the urotensin II peptide (UII), is selectively expressed by the cholinergic neurons of the PPTg. Although the exact function of cholinergic neurons of the PPTg are unknown, they are thought to modify the perception of reward magnitude or salience detection. We hypothesized that the activation of PPTg cholinergic neurons would alter sensory processing across multiple modalities (ex. taste and hearing). Here we had three aims: first, determine if cholinergic activation is involved in consumption behavior. Second, if so, distinguish the impact of the caloric value by using saccharin, a zero calorie sweetener. Lastly, we tested the perception of acoustic stimuli by measuring the acoustic startle reflex (ASR).

Male Sprague‐Dawley rats were bilaterally cannulated into the PPTg, then housed in a contact lickometer and placed under food restriction lasting the entire consumption experiment (water ad lib.). The lickometer recorded each individual lick to give insight into the microstructure of the consumption behavior. Treatment consisted of a microinjection of either 1 μL of aCSF or 1 μL of 10 μM UII into the PPTg, and the rats were immediately given access to either sucrose or saccharin. For the remaining five days rats were allowed one hour access per day to the same sweet solution without any further treatments. ASR testing consisted of a baseline (no treatment), treatment day, and two additional days (no treatment). Immediately following the microinjection of UII, consumption of both saccharin and sucrose increased compared to controls. The significant difference in sucrose consumption lasted two days post‐treatment while saccharin consumption was significantly different for three days after treatment. The ASR was significantly increased the day after treatment in the rats treated with UII.

Activating cholinergic PPTg neurons may lead to a miscalculation of the salience of external stimuli, implicating the importance of cholinergic input in modulating a variety of behaviors. This potential plastic change can give rise to further studies in the role of sensory processing on reward related‐behaviors at the level of the PPTg cholinergic neurons.

Support or Funding Information

The Howard T. Blane Director's Award in the Addictions (BDAA) from Research Institute on Addiction

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Adult Neural Stem Cells Show Regional and Sex‐Dependent Responses to Chronic Poly‐Drug Administration
Authors: Schlagal Caitlin , McGrath Erica , Gao Junling , Dunn Tiffany , Fox Robert , Stutz Sonja , Dineley Kelly , Kaphalia Bhupendra , Cunningham Kathryn , Wu Ping
Published: June 2021

Cocaine use disorder (CUD) and alcohol use disorder (AUD) are highly prevalent substance use disorders in the US. Cocaine and alcohol co‐abuse is the third most lethal substance combination and affects 12 million Americans annually. Interestingly, it has been reported that females experience greater euphoria when cocaine and alcohol are combined. Individual use of cocaine and alcohol cause neurodegeneration; however little is known about the combined effects of these substances on the neural stem cell (NSC) population. NSCs are a population of cells in the brain maintained throughout life that are characterized by their ability to self‐renew and give rise to new neurons and astrocytes. Efforts in drug addiction research primarily focus on preventing or stopping abuse, however little work is being done to reverse brain damage incurred by chronic drug abuse. Therefore, the NSC population represents a potential therapeutic target due to their regenerative capabilities. The aim of our study is to evaluate regional and sex differences of endogenous adult neural stem cells to chronic treatment with alcohol and/or cocaine. We sought to elucidate the response of adult endogenous NSCs to chronic alcohol and cocaine treatment using an inducible lineage tracing mouse model. Adult mice were randomly divided into 1 of 4 groups: control, cocaine, ethanol, or combination treatment. Daily i.p. injections of cocaine were administered and ethanol was provided in a complete nutrient liquid diet to appropriate groups. Brain tissue was analyzed for markers of NSC survival and differentiation in two distinct brain regions, the subventricular zone of lateral ventricle (SVZ), and subgranular zone of dentate gyrus (SGZ). The SGZ was also evaluated for changes in metabolic enzymes associated with ethanol and cocaine metabolism. We found that NSCs have a unique response to drug depending on the regional location and sex of the animal. Females had more robust decreases in neural stem cell (NSC) survival in the SVZ compared to males. SGZ neurogenesis was reduced drastically in the combination group. There were significant increases in metabolic enzyme expression in response to drug treatment, with notable differences between sexes. Overall, we show drastic changes to the NSC population following exposure to cocaine and ethanol, as well as alterations in metabolic enzyme expression.

Support or Funding Information

John S. Dunn Foundation NIDA T32 Award (Grant# 5T32DA007287‐17 and 3T32DA007287‐18S1)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Desensitization of PGE2‐stimulated cAMP Signaling Due to Upregulated Phosphodiesterase Activity in Human Lung Fibroblasts
Authors: Nuñez Francisco , Corpuz Maia , Kazarian Austin , Johnstone Timothy , Ostrom Rennolds
Published: June 2021

Pulmonary fibroblasts play an essential role in maintaining the structure and function of the lung. In idiopathic pulmonary fibrosis, fibroblast cells persist in an activated form and produce excessive fibrous material, leading to loss of alveolar structure. PGE2, which specifically activates EP2 and EP4 receptor subtypes, is a cAMP elevating agent and thus has anti‐fibrotic potential, since cAMP has been shown to have anti‐fibrotic properties. Although PGE2 levels are increased in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis (and in animal models of pulmonary fibrosis), it does not prevent pulmonary fibrosis. One hypothesis is that PGE2 effects are self‐limiting because of desensitization caused by prolonged exposure. To test this hypothesis, we pretreated HFL‐1 fibroblasts with PGE2 and other cAMP‐elevating agents then measured PGE2‐stimulated cAMP levels using the downward cAMP difference detector in situ (cADDis) cAMP sensor (Montana Molecular, Bozeman MT). After 24‐hour pretreatment with 100 nM PGE2, we changed the media and measured cAMP kinetics in response to various concentrations of PGE2. Pretreatment with PGE2 shifted the concentration‐response curve of PGE2 rightward approximately 50‐fold. Neither 100 nM isoproterenol or 1μM forskolin pretreatment for 24 hours altered PGE2 response, implying that other cAMP elevating agents do not induce desensitization. We sought to uncover the underlying mechanism responsible for PGE2 desensitization. Desensitization could result from either receptor internalization through the GRK/β‐arrestin mediated pathway or via increase PDE activity. To determine if PGE2 pretreatment increases PDE activity, we used the PDE3 inhibitor, cilostazol, and the PDE4 inhibitor, rolipram after PGE2 pretreatment. Rolipram, increased both the EC50 and Emax of PGE2 in vehicle pretreated HFL‐1 fibroblasts. In cells pretreated with PGE2, rolipram partially reversed the desensitization, indicating that PDE4 may be induced by long term PGE2 exposure. Cilostazol had minimal effects on either control or desensitized PGE2 responses, implying that PDE3 is not involved. Examination of PDE isoform mRNA levels via quantitative RT‐PCR, show that PGE2 treatment upregulates PDE3A, PDE4C and PDE4D, while pretreatment with isoproterenol or forskolin only upregulated PDE4D. Taken together, these results show that long‐term exposure to PGE2 causes desensitization of EP receptor‐stimulated cAMP signaling, partly through the increased expression of a PDE4 isoform. Defining the precise PDE isoform involved in the desensitization of PGE2 responses may lead to new therapeutic strategies for treating pulmonary fibrosis.

Support or Funding Information

This work was supported by NIH grant GM107094 (RO)

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