bluuebunny/crossref_metadata_2025 huggingface dataset:
Carnosic acid activates AMPK, inhibits Akt and inhibits H1299 human lung cancer cell survival
Authors: Nyforovskyy Dmytro , Moore Jessy , Tsiani Evangelina
Published: June 2021

Non‐small cell lung cancer (NSCLC) represents 80% of all lung cancers and is characterized by low survival rates due to chemotherapy and radiation resistance. Thus, finding alternative treatments to reduce NSCLC proliferation and survival is of high importance. The survival pathways of Akt, the energy sensor AMP‐activated protein kinase (AMPK), and the apoptotic protein poly (ADP‐ribose) polymerase (PARP) are key modulators of cancer cell growth and survival. In previous studies, we found inhibition of NSCLC proliferation and survival by rosemary extract but the exact components responsible for the anticancer effects and the cellular mechanisms involved are not known. Rosemary extract contains many polyphenols including carnosic acid (CA). The objectives of the present study were to examine the effects of CA on NSCLC cell survival and apoptosis, and to investigate its effects on Akt, AMPK and PARP. The human NSCLC cell line H1299 was used. Clonogenic cell survival assays were performed to examine the effects of CA on cell survival, and immunoblotting with phospho‐specific antibodies was performed to examine signaling events. CA dose‐dependently inhibited H1299 cell survival. A significant inhibition of Akt phosphorylation/activation and enhanced AMPK phosphorylation/activation was seen with 25 and 50 mM CA while the total levels of each protein were not affected. PARP cleavage, an indicator of apoptosis, was enhanced by carnosic acid treatment. Our findings indicate that CA may have robust anticancer properties in NSCLC, and strongly support the need for further studies to investigate its role against lung cancer.

Support or Funding Information

Supported by a Brock University Advancement Fund (BUAF) grant to ET

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of A Novel RON Targeted Antibody‐Drug Conjugates using Cysteine Bridging Technology for Potential Treatment of Pancreatic Cancer
Authors: suthe sreedhar reddy , Yao Hang‐Ping , trippier paul c. , wang ming‐hai
Published: June 2021

Therapeutics targeting known oncoproteins have been applied for pancreatic cancer treatment but clinical outcomes are not promising. Hence, there is an urgent need to identify novel targets and develop effective drugs to improve pancreatic cancer therapeutic index. Antibody‐drug conjugates (ADC) represent a promising class of drugs for targeted cancer therapy. Here we developed a novel ADC targeting RON receptor tyrosine kinase for potential pancreatic cancer treatment. To this end, we have synthesized a bis‐alkylating linker (BL), attached to a lysosomal protease‐cleavable dipeptide with payload Monomethyl auristatin E (MMAE). The BL‐MMAE was then conjugated to Zt/g4 (anti‐RON mAb) through cysteine bridging technology to produce Zt/g4‐BL‐MMAE with a relatively homogeneous conjugation profile and an antibody to drug ratio of 1:4. Zt/g4‐BL‐MMAE showed significant improvement in drug conjugation homogeneity and serum stability over conventional ADCs prepared by maleimide‐based linkers. In pancreatic cancer cell lines overexpressing RON, Zt/g4‐BL‐MMAE specifically targeted RON‐expressing tumor cells and was effective in rapid induction of cell surface RON endocytosis. Functional analysis revealed that Zt/g4‐BL‐MMAE caused cell cycle arrest at G2/M phase, reduction of cell viability and subsequently resulted in massive cell death. The calculated IC50 is in the range of 1 to 2 μg/ml. Therapeutic studies using mouse xenograft models are currently underway to determine the efficacy of Zt/g4‐BL‐MMAE for pancreatic and other epithelial cancers. We conclude that Zt/g4‐BL‐MMAE is a novel anti‐RON ADC with excellent conjugation profile, serum stability, and selective cytotoxicity for pancreatic cancer cells. This work provides a pharmaceutical opportunity for evaluating potentials of RON‐targeted ADCs in pancreatic cancer treatment in the future.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
How cAMP Homeostasis is Controlled in Leishmania by Receptor Adenylate Cyclases and Acidocalcisomal Pyrophosphatases Aiding its Survival in Phagolysosomal Conditions?
Authors: BISWAS ARUNIMA , BHATTACHARYA ARIJIT , Vij Amit , Das Pijush K.
Published: June 2021

Leishmania donovani while infecting macrophages is exposed to phagolysosome conditions (PC) (37 °C and pH 5.5) from the sandfly gut environment of 22 °C and pH 7.4. This shift in temperature and pH led to increased intracellular cAMP and cAMP‐mediated PKA activity in the parasites, which help in intra‐macrophage survival, a pre‐requisite for infectivity and transformation to amastigotes. Though cAMP and cAMP‐triggered signaling pathways seemed to be one of the important factors for parasite survival and infectivity, the mechanism of cAMP production and its stringent control is obscure. In the absence of typical orthologs for G‐proteins and G‐protein coupled receptors, the precise mechanisms for positive modulation of cAMP production during exposure to PC seemed an important aspect to be studied. Amongst various membrane bound isoforms of receptor adenylate cyclase, two promastigote‐stage specific membrane bound receptor adenylate cyclases (LdRAC‐A and LdRAC‐B), LdRAC‐A appeared to function as a major cAMP generator following PC exposure. Following PC exposure, pyrophosphate (PPi), an energy storage compound as well as a by‐product of cAMP biosynthesis by adenylate cyclase, was found to be decreased. This may be due to microtubule and microfilament‐driven translocation of acidocalcisomes near plasma membrane vicinity with concomitant increase of acidocalcisome membrane pyrophosphatase (LdV‐H+PPase) and acidocalcisomal soluble pyrophosphatase (LdVSP1). Episomal over‐expression and conditional silencing demonstrated regulatory role of V‐H+PPase on cAMP trigger and consequent induction of resistance to macrophage‐derived pro‐oxidants and parasite killing. Moreover, immunofluorescence analysis indicated towards a possible colocalization of LdV‐H+PPase and LdRAC‐A during PC exposure. The results suggested that translocation of acidocalcisome in membrane vicinity functions as a trigger for LdRAC‐A‐driven cAMP generation through depletion of PPi pool by LdV‐H+PPase. For the first time, one of the molecular mechanisms behind the positive modulation of cAMP by leishmanial adenylate cyclases in absence of Gproteins was deciphered by us.

Support or Funding Information

DST‐INSPIRE, Department of Science and Technology (DST), Govt of India, DST‐PURSE, Department of Science and Technology (DST), Govt of India, UGC‐SAP, UGC, Govt of India

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Polyamines for Proliferation, Survival, and Infectivity of the Protozoan Parasite Leishmania donovani
Authors: Roberts Sigrid , Perdeh Jasmine , Abuan Irene , Le Linh , Love Quintin , LoGiudice Nicole , Turcu Kristen , Harrelson John
Published: June 2021

A better understanding of parasite biology and host‐parasite interactions is critical for the development of much needed new therapeutic strategies against the neglected tropical disease leishmaniasis, which affects an estimated 12 million people annually. Polyamines are metabolites that play central roles in the biology of all eukaryotes, and recent studies have highlighted their critical nature in Leishmania parasites. In Leishmania, the polyamine biosynthetic pathway consists of four enzymes: arginase (ARG), ornithine decarboxylase (ODC), spermidine synthase (SPD), and S‐adenosylmethionine decarboxylase (ADOMETDC). These enzymes sequentially generate ornithine, putrescine and spermidine. We have generated a complete set of gene deletion mutants in Leishmania donovani (Δarg, Δodc, LdΔspd, and Δadometdc), in order to evaluate the polyamine biosynthetic enzymes as therapeutic targets and to study the functions of polyamines. In vivo studies revealed surprising differences in infectivity phenotypes, with the Δodc deletion exhibiting the most profound reduction in infectivity. In vitro studies showed that the gene deletion mutants depend on the provision of downstream metabolites to survive. An evaluation of cell growth between the different cell lines uncovered that the only vital role of ornithine is as precursor for polyamine synthesis. However, putrescine, which has previously been postulated to be merely a substrate for spermidine formation, has additional essential functions. Our studies revealed that putrescine is especially important for replication and proliferation. Both putrescine and spermidine were essential for cell survival, but the provision of either polyamine alone allowed parasites to enter a quiescent‐like state for several weeks. Future studies utilizing the gene deletion mutants will continue to elucidate the functions of polyamines for cellular proliferation, survival, and persistence.

Support or Funding Information

National Institute of Allergy and Infectious Disease Grant AI041622

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Chai Tea Promotes Ampicillin Susceptibility in MRSA
Authors: Hinsdale Shannon , Pace Joel , Anderson Emeli , Favoreto Silvio
Published: June 2021

Staphylococcus aureus (S. aureus) is a pathogenic organism that asymptomatically resides on the skin and in nasal mucosal membranes of 11–40% of the worldwide population. S. aureus has mutated in response to long‐term antibiotic exposure, generating strains known as methicillin‐resistant Staphylococcus aureus (MRSA). MRSA is involved in approximately 80,000 infections, coupled with 11,000 deaths per year in the United States alone; costing the U. S. healthcare system up to $9.7billion, or approximately $60,000 per patient, annually. Consequently, MRSA infections require stronger, more expensive drugs, accompanied by extended hospitalization. Treatment options are dwindling as costs are rising, both in mortality/morbidity rates and financially. Antimicrobial resistance is an ongoing process that will require alternative and creative interventions.

Masala chai tea (chai tea) is a traditional tea beverage originating from India. This tea beverage is composed of a strong black tea base infused with various spices; typically cinnamon, cloves, cardamom, ginger, and black peppercorn. Each constituent listed above is composed of at least one bioactive plant compound with proven antimicrobial activity. We have taken these antimicrobial properties of each component into account, thus opting to use chai tea as a means to modify the resistance of MRSA to beta‐lactam antibiotics.

In this study, we utilized seven MRSA strains. The microbes were isolated from carriers at the Cuesta College Allied Health program and were characterized by culture and PCR properties. Aqueous based chai infusion (CW) or ethanol‐based chai extract (CA) was incorporated into the media used for antibiogram diffusion assays, according to standard protocols. Compared to the control conditions, the aqueous‐based chai infusion and ethanol extract promoted a significant increase in MRSA susceptibility to Ampicillin, as demonstrated by the diameter of the inhibition halo (ANOVA, F=4.92, p<0.05).

The results indicate that aqueous based chai infusion or ethanol‐based chai extract potentiated the bacteriocidal effect of Ampicillin against all seven isolates of MRSA in vitro. Interestingly, only Ampicillin, from all tested antibiotics, showed synergistic activity with chai. The data suggest the potential benefits from the association of established antimicrobial drugs with natural plant compounds as a means of modifying drug resistance in MRSA. The synergism shown between chai tea and ampicillin holds the possibility of assisting in deconstructing mechanisms of resistance, helping stage for future studies to progress towards reducing the MRSA global crisis.

Support or Funding Information

Work supported by Cuesta College FoundationShannon Hill ‐ Advancement Foundation Executive Directorshannon_hill@cuesta.edu

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Benzimidazole Carboxamide Derivative, DDB‐506 Inhibits Bacterial Proliferation by Targeting FtsZ
Authors: TIWARI RISHU , DHAMELIYA TEJAS M. , CHAKRABORTY ASIT K. , PANDA DULAL
Published: June 2021

FtsZ, a conserved cell division protein in prokaryotes has emerged as an attractive target for antibacterial drugs. FtsZ plays a key role in bacterial cell division; therefore targeting FtsZ assembly may be an efficient approach to inhibit bacterial cell division. In this work, we have checked the effect of several benzimidazole carboxamide derivatives on the bacterial cell proliferation. A benzimidazole carboxamide derivative, DDB‐506 was found to inhibit the proliferation of Bacillus subtilis (B. subtilis) with an MIC of 6 μM. DDB‐506 increased the length of B. subtilis cells by six times indicating that it inhibits bacterial cytokinesis. Light scattering, sedimentation and electron microscopic analysis of the effects of DDB‐506 on the assembly of Bacillus subtilis FtsZ (BsFtsZ) indicated that DDB‐506 inhibits the polymerization of BsFtsZ in vitro. DDB‐506 also inhibited the GTPase activity of BsFtsZ. Molecular docking analysis showed that DDB‐506 possibly binds to the nucleotide binding site of BsFtsZ. DDB‐506 also inhibited the binding of MANT‐GTP, a fluorescent analogue of GTP to FtsZ suggesting that the compound may bind to the GTP binding site of FtsZ.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Computational Studies of the NUDIX Hydrolase Superfamily
Authors: Richman Spencer Chase , O'Donovan Kevin , Craig Paul , Mills Jeffrey , O'Handley Suzanne
Published: June 2021

The Protein Structure Initiative (PSI) was an effort by consortiums to solve as many unique protein structures as possible; the Protein Data Bank contains a number of enzymes whose structures have been solved, but for which no enzymatic activity has been determined. The Enzyme Function Initiative (EFI) is an effort to determine as many unique enzyme functions as possible. There are a number of putative NUDIX Hydrolase superfamily members for which structures have been solved, but for which no enzymatic activity has been determined. We have catalogued the structurally‐determined enzymes within the NUDIX Hydrolase superfamily using the PDB, BLAST, Dali, and SCOP. We are now in the process of characterizing these enzymes in the biochemistry teaching laboratory.

Computational programs, such as ProMOL and UCSF Chimera, aid in annotating uncharacterized enzymes. ProMOL works by aligning the relative spatial arrangement of the catalytic residues of a reference enzyme with query searches. Chimera is a molecular visualization program that provides various tools for the structural analysis of proteins, including comparative modeling and conformational analysis. We are using these programs to design catalytic motifs for the NUDIX hydrolase superfamily and subfamilies within, which then can be used to uncover family members whose structures have been solved. Likewise, we have used the docking program PyRx, which utilizes AutoDock Vina, to predict substrate specificity, and have discovered that due to its “static” nature, it does not satisfactorily predict substrate specificity. Thus, next we will use a “dynamic” docking program to reevaluate substrate specificity prediction. Through this process, we have discovered the strengths and limitations of these programs.

Bioinformatics tools, including databases and computational programs, are good starting points for determining protein function; however, the only way to definitively determine protein function is through experimentally determining activity. Our ultimate goal is to discover and characterize new enzymes from human pathogens as potential novel antibiotic targets.

Support or Funding Information

NSF IUSE grant (PAC, JLM, and SOH). RIT College of Science FEAD grant (SOH and JLM).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discovery of Novel Small Molecule Inhibitors of Bacterial Pyruvate Carboxylase
Authors: Wyatt Brittney , Burkett Daniel , Mews Mallory , Donaldson William , Dockendorff Christopher , St. Maurice Martin
Published: June 2021

Staphylococcus aureus and Listeria monocytogenes are pathogenic bacteria that can cause serious infections for immunocompromised individuals1, 2. Bacterial metabolism has been linked to pathogenicity and, in particular, the metabolic enzyme pyruvate carboxylase (PC) has been shown to be an important contributor to L. monocytogenes virulence1, 2. Small molecules that could inhibit bacterial PC activity would better reveal the role of this enzyme in a wide range of bacterial infections and could have the potential to be developed into antimicrobial agents. However, there are currently no known specific or potent small molecule inhibitors of PC activity3. We aim to find, develop, and characterize novel small molecule inhibitors of PC that will aid in understanding the structure, mechanism, and regulation of this enzyme. PC catalyzes the conversion of pyruvate to oxaloacetate (OAA) and a novel fixed‐time assay has been developed to detect OAA with a diazonium salt, Fast Violet B (FVB). The validated assay has been used to screen specific subsets of rationally designed inhibitors as well as compounds in an unbiased high‐throughput screen. Using both approaches, we have discovered novel small molecule inhibitors of bacterial PC that have the potential to be developed into valuable research tools and future antimicrobial agents.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
THE GENE EXPRESSION PROFILE AND TETRASPANIN PROTEIN CO029 ON THE HUMAN COLORRETAL CANCER LATERALITY
Authors: Braga Lucélia Antunes , Assis Jéssica Vieira , Moraes Vanessa Silva , Silva Istéfani Dayse , Grenfell Rafaella Fortini
Published: June 2021

Colorectal cancer (CRC) is one of the most prevalent neoplasms and presents a multifactorial and complex etiopathogenesis, not yet fully elucidated. Studies have shown that tumors of the right colon differ significantly from neoplasms of the left colon, because they present greater risks of metastasis, are less responsive and exhibit different predictive and prognostic markers. Some of these markers have been studied, such as the tetraspanin CO029 protein, which favors cell motility, and epithelial‐mesenchymal (EMT) and neoplastic stem cell (CSC) markers, which are associated with loss of cell differentiation. According to studies, the three markers cited are closely associated with tumor progression. It has already been observed that in RCC tumor cells these markers present a high expression. Therefore, the analysis of the expression profile of tetraspanin CO029 and other markers in the RCC may become potential predictive tools for this neoplasia. Thus, the objective of the study is to evaluate the gene and protein expression of tetraspanin CO029 and correlate with laterality of RCC and with EMT and CSC markers. Therefore, it is proposed the use of human samples affected by the neoplasia proposed in different histopathological stages, for the evaluation of the immunohistochemical expression of tetraspanin CO029 protein. In addition, we will evaluate the gene expression of the three mentioned markers through the extraction of messenger RNA (mRNA) from these samples, for quantitative PCR (qRT‐PCR). The clinical data of the patients will be used for correlation with the expression of the proposed markers. Currently our efforts are concentrated in the evaluation by qRT‐PCR of the samples affected by CCR, where we observed that the samples located in the right colon presents a greater expression of tetraspanin CO029, corroborating with works that associate this protein with intensification of metastasis, due to the function of give motility to the cell. We are jointly conducting the production of anti‐tetraspanin CO029 monoclonal antibodies by in vitro methodology, which will be used for immunohistochemical labeling and in future projects, tested as monoclonal therapy. The production is in the phase of selection of positive hybridomas. Considering that the number of RCC cases is increasing in Brazil and in the world, it is important to carry out studies to elucidate this neoplasia. This project is relevant when we refer to possible predictive markers and future therapeutic targets, as it may make treatment personalized and possibly more efficient. Financial support: Fiocruz, CNPq, FIOCÂNCER IRR.

Support or Funding Information

Financial support: Fiocruz, CNPq, FIOCÂNCER IRR.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding the Impact of IDH2 Mutations on the Redox Balance of Cancer Cells
Authors: Gelman Susan J. , McKenzie Lisa , Chheda Milan G. , Patti Gary J.
Published: June 2021

Study Objective

We seek to answer the following question: how does consumption of NADPH by mutant IDH2 for 2‐HG synthesis affect other NADPH‐dependent pathways in the mitochondria?

Methods

HCT116 IDH2 mutants were incubated in the presence of H2O2 or irradiated with 3,6, or 9 Gy ionizing radiation (IR) and cell viability was assessed with a trypan blue exclusion assay. Stable isotope tracing was performed by incubating HCT116 cells (n=3) with the specified isotope for 12–24 hours, then harvested in ice‐cold methanol. After extracting metabolites from cells in a mixture of methanol, acetonitrile, and water, samples were run on a Thermo QExactive Plus using liquid chromatography/mass spectrometry (LC/MS) with a HILIC column in negative mode.

Results and Conclusion

Isocitrate dehydrogenase 2 (IDH2) mutants are found in cases of acute myeloid leukemia (AML), glioblastoma, chondrosarcoma, and lymphoma cancers. IDH2 is one of two nicotinamide adenine dinucleotide (NADPH)‐dependent isoforms of IDH. While IDH1 is found exclusively in the cytosol, IDH2 is localized to the mitochondria. Both enzymes are mutated in cancer to produce 2‐hydroxyglutarate (2‐HG) in millimolar amounts. The neomorphic reaction consumes the essential redox factor NADPH. Previous research indicates that IDH1 mutants are dependent upon upregulation of the pentose phosphate pathway (PPP) to supply more NADPH for 2‐HG synthesis. However, this pathway is localized to the cytosol and NADPH is not capable of crossing the mitochondrial membrane to supply the IDH2 reaction. To study the effects of NADPH consumption/production with respect to compartmentalization, we used stable isotope labeling and LC/MS to assess central carbon pathway flux in comparison to IDH1 and WT cells. Our preliminary data suggests that IDH2 mutants may be dependent on compensatory mechanisms to generate essential metabolic factors (such as ME1 and the folate cycle). We conclude that IDH2 mutations lead to metabolic rewiring of the TCA cycle to support the demand of 2‐HG synthesis in the mitochondria.

Support or Funding Information

GJP received financial support for this work from the National Institutes of Health Grants R35ES028365 and R21CA191097 as well as the Alfred P. Sloan Foundation, the Pew Scholars Program in the Biomedical Sciences, and the Edward Mallinckrodt, Jr 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.
Profiling Bis(monoacylglycero)phosphate Lipids in Cancer Cell Lysosomes as Therapeutic Targets
Authors: Showalter Megan , Sa Michael , Tsugawa Hiroshi , Berg Anastasia , VanderVorst Kacey , Kind Tobias , Carraway Kermit L. , Fiehn Oliver
Published: June 2021

Background

Tumor resistance to apoptosis underlies the clinical failure of conventional chemotherapeutics and commonly employed molecularly targeted drugs. Cancer cells frequently activate survival pathways to evade destruction by apoptosis‐inducing therapies and persist following treatment to seed tumor recurrence and metastatic outgrowth. Lysosomal cell death is an alternative death pathway that triggers non‐apoptotic mechanisms. The lysosome, a major degradative component of eukaryotic cells, is altered by oncogenic transformation. Key to the function and stability of lysosome‐associated enzymes are bis(monoacylglycero)‐phosphate lipids (BMP). BMPs are negatively charged and highly enriched in the internal lysosome membrane. Untargeted analytical methods for the measurement of BMPs and in silico libraries to support the discovery of new BMP species are currently lacking.

Method

We present here an expanded BMP library to include over 1800 BMP species in both positive and negative ESI modes. We have chromatographic separation of BMP from isobaric species, and have used this method to profile cancerous and non‐cancerous immortalized cell lines. Lipidomic analysis was performed using Waters Acquity UPLC CSH C18 (100 mm length × 2.1 mm id; 1.7 μm) column coupled to a Thermo Scientific Q‐Exactive HF mass spectrometer. Data processing was done using MS‐DIAL software and in‐silico structure predictions were done using MS‐FINDER.

Results

In breast cancer cells (Met‐1), we found that all BMP species measured were significantly less abundant relative to non‐transformed mammary epithelial cells (nMUMG), (n = 10). As BMPs are key to lysosome stability, we hypothesize the dramatic loss of BMPs observed in transformed cells renders cancer‐associated lysosomes unstable. Therefore, targeting BMPs might represent an effective therapeutic strategy for selective cancer cell destruction.

Support or Funding Information

This work was funded by NIH U24 DK097154.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Herbal Mixture Extracts on Degenerative Arthritis In Vivo Models
Authors: Moon Yun Jeong , Dasom Lee , Dakyung Kim , Minhee Lee , Sujeung Park , Yongwook Lee , Junkee Hong , Jeongmin Lee
Published: June 2021

This study was to investigate the effects of herbal mixture extract complex (S1, S2) on the monosodium iodoacetate (MIA)‐induced osteoarthritis in rats. Arthritis was induced by injection of MIA (6 mg/m L) into knee joints of rats. Arthritic rats were divided into control and treated (S1L, S1H, S2L and S2H) group. Control group was taken AIN93G diet and treated group was taken samples (S1L, S1H, S2L and S2H) by orally for 21 days. Body weights were measured at 0, 7, 14, 21 days after MIA injection. At the end of experiment (21day after MIA injection), gross and histopathological examination on the articular structures of knee joints were performed. Prostaglandin E2 (PGE2) contents in serum were measured by ELISA method. TNF‐α, IL‐1β, COX2 and, NF‐kB contents in chondrocyte were measured by Real‐time PCR method. And also, proteoglycan aggregate (Aggrecan), collagen type 1,2,10, TIMP‐1, MMP‐3 and MMP‐7 content in chondrocyte were measured by Real‐time PCR method.

PGE2 contents in serum level of the treated group were decreased compared with control group. TNF‐α, COX2 and IL‐1β contents in chondrocyte of the treated group were significantly decreased compared with control group. NF‐kB contents in chondrocyte of the S2H treated group were significantly decreased compared with control group. Collagen type 1 and 2 contents in chondrocyte of the treated group were significantly increased compared with control group. MMP‐3 and MMP‐7 contents in chondrocyte of the S2H treated group were significantly decreased compared with control group. The results of the histopathology showed that the severity of the arthritis was slightly reduced compared to the control group.

On the basis of these results, we concluded that S1 and S2 treatment had anti‐arthritic effects on the monosodium iodoacetate‐induced osteoarthritis in rats.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improvement of joint health by anti‐inflammatory activity of natural substances in chondrocyte of osteoarthritis
Authors: Kim Dakyung , Lee Minhee , Park Soo‐Jeung , Yun Jeong‐Moon , Lee Dasom , Sohn Johann , Yun Min‐Kyu , Lee Jeongmin
Published: June 2021

Osteoarthritis (OA) is a chronic degenerative disease that involves inflammation and pain due to damage or degenerative changes in the cartilage that protects the joints. OA has a high incidence in the elderly. Treatment methods include relieving symptoms and preventing cartilage damage. Drugs used to treat osteoarthritis have side effects and it is important to reduce them. Therefore, it is necessary to develop a therapeutic agent for osteoarthritis using natural substances. The purpose of this experiment was the anti‐inflammation effect of natural substances on osteoarthritis in primary cultured rat cartilage chondrocytes. Aggrecan, collagen type I, □, □, MMP‐3, 7, NK‐kB and COX‐2 expression were measured by real‐time PCR. TNF‐α and IL‐1β secretion was measured by ELISA kit. H2O2 and LPS‐induced chondrocytes were used to measure concentrations of 100, 200, and 400 (μg/ml). As a result, S400 significantly increased the gene expression level of aggrecan, collagen type I, II, III and decreased gene expression of pro‐inflammatory factors NF‐kB, COX‐2 and MMPs (3 and 7). The cytokine productions of TNF‐α and IL‐1β also decreased in S400. Therefore, S400 had an anti‐inflammatory effect and alleviated the inflammation occurs in OA.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary Restriction modulates sleep in Drosophila Melanogaster
Authors: Varamini Behzad , Joel Hayley , Lee Jin , Newman Emma , Robinson Krystal , Smith Zachary
Published: June 2021

Sleep deprivation has catastrophic metabolic and physiological consequences in numerous organisms, from fruit flies to humans. Dietary restriction (DR), a limitation of calories in the absence of malnutrition, has been shown to dramatically improve the health and longevity of a number of species, and evidence shows that this intervention may have the potential to overcome some of the molecular and neurological deficits seen in aging and sleep decline. Using a 17‐beam infrared‐light paradigm as well as gene expression analysis, this study demonstrates that restriction of amino acids has significant effects on sleep/wake patterns in Drosophila Melanogaster, as well as alters the expression of genes essential to maintaining homeostatic sleep.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Myeloperoxidase‐Derived 2‐Chlorofatty Acids Make Neutrophils Go NETs
Authors: Palladino Elisa N. D. , Katunga Lalage A. , Ford David A.
Published: June 2021

Neutrophils represent the major effector cells of innate immunity. One mechanism for neutrophil‐mediated microbe killing is through the release of neutrophil extracellular traps (NETs). NET formation (NETosis) involves the fusion of granule and nuclear contents, which are then released in the extracellular space to capture and kill microbes. Chlorinated fatty acids (2‐ClFAs) are a product of neutrophil activation as a result of the myeloperoxidase (MPO) product, HOCl, targeting plasmalogen phospholipids. In this study, we examined the role of 2‐ClFA as an important lipid mediator of NETosis. Treating human peripheral blood neutrophils with physiological levels of 2‐ClFAs led to the formation of NETs, characterized by MPO association with DNA and neutrophil elastase (NE) redistribution to the perinuclear area. NETs induced by 2‐ClFA treatment were as efficient in killing bacteria as those produced by stimulating neutrophils with phorbol myristate acetate (PMA), a potent activator of NETosis. Like PMA, 2‐ClFA‐induced NETosis is calcium‐ and protein arginine deiminase 4 (PAD4)‐dependent. Interestingly, while PMA‐induced NETosis requires neutrophil activation, 2‐ClFAs initiate the NETosis process without neutrophil activation and degranulation. Furthermore, unlike PMA, 2‐ClFA elicits NETosis in bone‐marrow derived neutrophils from MPO deficient mice. Overall, 2‐ClFA may have an important role in directing neutrophil fate following activation, mediating the production on NETs and providing a new pharmacological target to either enhance bacterial killing or reduce host injury during inflammation.

Support or Funding Information

This study was supported by research funding from the National Institute of Health R01 GM‐115553 to D.A.F.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
2‐Chlorofatty Acid: A Functional Connection between Neutrophils and Endothelial Weibel‐Palade Body Mobilization
Authors: Hartman Celine L. , Duerr Mark A. , Albert Carolyn J. , Neumann William L. , McHowat Jane , Ford David A.
Published: June 2021

Endothelial dysfunction has been characterized as a hallmark of multiple inflammatory diseases, including sepsis and atherosclerosis. Neutrophil interactions with the endothelium may have profound effects on vascular wall biology and pathophysiology. Myeloperoxidase (MPO)‐derived oxidant products released from neutrophils can mediate inflammation and endothelial dysfunction. 2‐Chlorofatty acid (2‐ClFA) is a product of MPO‐derived HOCl targeting plasmalogen phospholipids, found abundantly throughout the body. Chlorinated lipids, produced and released by neutrophils, have recently been associated with multiple inflammatory diseases, but their impact on nearby endothelial cells has not been examined. This study tested the biological properties of the 16‐carbon 2‐ClFA molecular species, termed 2‐chlorohexadecanoic acid (2‐ClHA), on endothelial cells. An alkyne analog of 2‐ClHA, 2‐chlorohexadec‐15‐ynoic acid (2‐ClHyA), was synthesized and utilized to determine the subcellular localization of 2‐ClFA in human coronary artery endothelial cells. Experiments using a novel Click chemistry approach revealed 2‐ClHyA localizes to Weibel‐Palade bodies, which are endothelial‐specific storage granules containing multiple key bioactive molecules. 2‐ClHA and 2‐ClHyA significantly increase the surface expression of P‐selectin, as well as the release of von Willebrand factor and angiopoietin‐2 from endothelial cells. Functionally, 2‐ClHA and 2‐ClHyA increase neutrophil adherence to and platelets aggregation on the endothelium, as well as increase permeability of the endothelial barrier which has been tied to the release of angiopoietin‐2. Ongoing studies utilize Click chemistry to pulldown proteins which bind to 2‐ClHyA through biotinylated azide. Initial studies indicate multiple, unique proteins interact with 2‐ClHyA when compared to the non‐chlorinated fatty acid, and identification of these proteins may provide further mechanistic insight into 2‐ClHyA‐induced Weibel‐Palade body mobilization. These findings suggest that 2‐chlorofatty acid, a novel lipid produced by neutrophils under inflammatory conditions, promotes endothelial cell dysfunction, which may have broad implications in inflammation, thrombosis, and blood vessel stability.

Support or Funding Information

This work was supported in part by National Institutes of Health Grant R01 GM‐115552 (DAF) and American Heart Association Grant‐in‐Aid 15GRNT25750022 (DAF).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SAC1 degrades its lipid substrate PtdIns4P in the ER to maintain a steep electrochemical gradient on donor membranes
Authors: Hammond Gerry , Zewe James , Sangappa Sahana , Wills Rachel , Goulden Brady
Published: June 2021

Phosphatidylinositol 4‐phosphate (PtdIns4P) is one of the most functionally diverse molecules utilized by eukaryotic cells. It is both a metabolic hub for other crucial signaling lipids such as PtdIns(4,5)P2 and PtdInsP3, and a key molecule for recruitment of membrane and lipid transport proteins in its own right. Most recently, it has been proposed to form a “phosphoinositide‐motive force”, whereby transport of PtdIns4P molecules down their concentration gradient from the plasma membrane (PM) or Golgi to the ER drives counter transport of other lipids up their own concentration gradients. A critical requirement for this model is that the main PtdIns4P degrading enzyme, the integral ER phosphatase SAC1, hydrolyzes PtdIns4P in the ER in a “cis” configuration. Alternatively, it has been suggested that other functions of PtdIns4P are regulated by SAC1 hydrolyzing PtdIns4P directly in the PM in a “trans” configuration at membrane contact sites (MCS). However, such activity would surely disrupt lipid counter transport. Therefore, we sought to determine whether SAC1 acts in “cis”, “trans”, or both in mammalian cells. Acute chemical ablation of SAC1 activity drives ectopic accumulation of PtdIns4P in the ER, revealing “cis” activity. Furthermore, endogenous or ectopically expressed SAC1 localizes to the ER and Golgi, but does not constitutively or dynamically enrich at ER‐PM MCS. Forced recruitment of SAC1 to experimentally induced MCS does not produce robust “trans” activity on PM PtdIns4P. However, “trans” activity can be induced by adding an approximately 6 nm long helical linker between the ER anchor and the catalytic domain. Together, our results reveal that SAC1 operates in a “cis” configuration. This ensures a “phosphoinositide‐motive force” for lipid transport is effective, and also implies regulation of PM phosphoinositide signaling is tightly linked to non‐vesicular traffic of PtdIns4P at ER‐PM MCS.

Support or Funding Information

This work was supported by National Institutes of Health grant 1R35GM119412‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Anthrax Toxin: A Molecular Trojan Horse
Authors: Patel Heeral , Edmondson Riley Grace , Baugh Jessica Renee , Shabbir Sanah Zehra , Kenana Sireen
Published: June 2021

Anthrax is a three protein component exotoxin released by the virulent bacterium Bacillus anthracis that causes cell lethality in its host. In humans and many animals, Bacillus anthracis can enter the body through the lungs, skin, intestines, or bloodstream. Symptoms of Anthrax poisoning are commonly displayed between one day to two months after initial contact. Once the bacterium spores enter the host organism, the spores germinate and the bacterium synthesizes and releases the anthrax endotoxin protein molecules in response to elevated CO2 levels. Anthrax toxin is composed of three protein molecules: protective antigen, edema factor, and the lethal factor. The protective antigen is released by the bacterium as an 83 kDa protein single chain, and then binds to the host cell receptors. It is then cleaved by proteases on the host cell into a smaller 63 kDa assembly competent monomer which assembles on the cell surface as a heptamer. This heptameric ring binds to edema factor and lethal factor. The pre pore three component complex is then endocytosed into the host cell. As the endosome acidifies to pH 5.0, the prepore protective antigen changes shape, and inserts into the endosomal lipid membrane. The bound edema factor and the lethal factor are gradually unfolded and threaded through the protective antigen pore translocon injection system. The translocated edema and lethal factors then refold in the cytoplasm. This threading is facilitated by a pH gradient. During the threading of the lethal and edema factors, the acidic residues of these proteins are first protonated, resulting in a positive charged state. As these enzymatic proteins are threaded through the pore, the proton is lost after crossing a critical phenylalanine clamp loop region and the protein continues to thread in a unidirectional manner into the cytoplasm. Once the translocated enzymes refold in the cytoplasm, the lethal factor disrupts cell signaling and the edema factor disrupts cell homeostasis. To study this translocation mechanism, one can use the 20 kDa N terminal portion of the lethal factor (called LFN) that binds to the pore and removes the enzymatic portion of the protein. Research is progressing where this translocation system is used to deliver drugs that are attached onto the LFN and be transported into cells in a very specific manner. Thus the understanding of this transport mechanism may be important for engineered drug delivery systems. The Olathe North MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function relationships of anthrax.

Support or Funding Information

The Olathe North SMART Team is joint venture between the Medical Professions Academy and the Milwaukee School of Engineering.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Membrane localization of HspA1A, a stress inducible 70‐kDa heat shock protein, is mediated by the lipid phosphatidylserine
Authors: Bilog Andrei Dominic , Nikolaidis Nikolas
Published: June 2021

Seventy‐kilodalton heat shock proteins (Hsp70s) are molecular chaperones essential for regulating cellular homeostasis. Apart from their indispensable roles in protein homeostasis, specific Hsp70s, including HspA1A, also localize at the plasma membrane (PM) of 90% of human and mouse tumors and its PM presence has been associated with bad patient prognosis. However, it is currently unknown how HspA1A, a protein that lacks membrane localization signals, is translocated and anchored to the PM. We have recently demonstrated that HspA1A interacts with several lipids, including phosphatidylserine (PS), a lipid found primarily in the inner leaflet of the PM during normal cell growth. Taking into account that lipids recruit proteins to the PM, we hypothesized that the interaction of HspA1A with PS allows the chaperone to localize at the PM. To test this hypothesis, we determined the translocation pattern of HspA1A in three human cell lines and also whether the C2 domain of lactadherin (Lact‐C2), a known PS‐biosensor, inhibits this translocation. Specifically, cells were subjected to mild heat‐shock, a condition that does not induce apoptosis, and the PM‐localized HspA1A was quantified using confocal microscopy and cell surface biotinylation. These experiments revealed that in the absence of Lact‐C2, HspA1A's membrane localization increases during recovery from non‐apoptotic heat‐shock and this phenomenon is unrelated to PM‐charge. In the presence of Lact‐C2, however, HspA1A's membrane localization is inhibited. Furthermore, we used TopFluor‐PS, a fluorescent PS analog, and established that it co‐localizes with HspA1A. Collectively, these results strongly suggest that HspA1A's membrane localization, anchorage, and embedding depends on its interaction with intracellular PS. This discovery institutes PS as a new and dynamic partner in the cellular stress response and establish the foundation to directly assess the biological implications of this newly described function of Hsp70s.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Induction of Osteogenesis by Means of Continuous Delivery of Testosterone and TQ Using in Bone Fracture Rat Model
Authors: Benghuzzi Hamed A. , Tucci Michelle A.
Published: June 2021

Potent anti‐oxidant extracts from natural products, as an alternative to conventional treatment of various diseases, have been utilized extensively in the recent years. For example, Nigella sativa, natural herbs have been used for many acute as well as chronic conditions. The specific objectives of this study were (1) to successfully deliver the active components of black seed, thymoquinone (TQ), and anabolic agent, testosterone (TE) at sustained levels using a tricalcium phosphate delivery system (TCPL); (2) to evaluate the effects TQ and TE on bone healing (induced femoral defect) in adult male rats. Sixty animals were randomly divided into five equal groups. Groups I and II animals served as controls, and shams, while groups III and IV served as TQ and TE experimental groups (40 mg of TQ or TE loaded TCPL). Group V animals served as TQ (40 mg)+TE (40 mg) treated group. Serum biomarkers, x‐rays, reproductive and vital organs were collected and analyzed. At the end of 30 days, animals were sacrificed and vital as well reproductive organs were collected and analyzed morphometrically. At the end of 60 days post treatment all animals were sacrificed and the results revealed the following: (i) gross anatomical observation indicated an increase in the healing pattern of animals in all experimental groups compared to the sham operated group, (ii) the progression of osteogenesis was in the following order: TQ+TE>TE>TQ>shams, and (iii) no significant differences where detected in the levels of total serum proteins, and maliondialdehyde; (iv) an increase in alkaline phosphatase (p<0.05) in all experimental animals compared to control and sham groups. Overall conclusion of this study demonstrated that TCPL sustained delivery of TQ plus TE can be used effectively to stimulate osteogenesis in rat model.

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