bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression, purification, and characterization of halophilic Pph_Pro1 protease isolated from Pseudoalteromonas phenolica
Authors: Choi Kwon‐Young , Kim Yun‐Gon , Yang Yung‐Hun
Published: June 2021

In this study protease Pph_Pro1 from Pseudoalteromonas phenolica which was reported to have extracellular proteolytic activity and salt resistance properties was investigated for cloning, expression and purification. Through the optimization it was determined that when Pph_Pro1 was co‐expressed with pTf16 vector chaperone and grown with LB medium supplemented with CaCl3, optimum soluble recombinant expression was achieved. The Pph_Pro1 was purified using osmotic shock and immobilized metal‐affinity chromatography (IMAC) methods. The activity of the isolated Pph_Pro1 was determined as 0.44 U/mg against casein. Interestingly the Pph_Pro1 displayed halophilic, alkaliphilic, and unexpected thermostability properties. Furthermore, it was also resistant to a number of hydrophilic and hydrophobic organic solvents. The substrate specificity and kinetic values such as Km and Vmax were determined against casein, Bovine Serum Albumin (BSA) and algal waste protein, which indicated that the wild type proteases had a greater affinity for casein. Based on the outstanding characteristics of the wild type protease, obtained through experiments, it can find application in many biotechnological industries.

Support or Funding Information

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (NRF‐2015R1A2A2A04006014).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Radical SAM mechanism mediated by the Interferon‐Inducible Protein Viperin
Authors: Dumbrepatil Arti Baban , Malec Paige , Ghosh Soumi , Patel Ayesha , Kennedy Robert T. , Marsh E. Neil G.
Published: June 2021

To survive infection the immune system deploys an arsenal of defensive measures to combat invading pathogens. The type I interferon (IFN) response presents an early host defense by generating an intracellular environment that restricts virus replication and signals the presence of a viral pathogen to the adaptive arm of the immune response. Viperin (virus inhibitory protein, endoplasmic reticulum‐associated, interferon‐inducible) is an interferon stimulated gene (ISG), that is induced by type I, II, and III IFNs, or after infection by a broad range of DNA and RNA viruses. Viperin contains an S‐adenosyl‐L‐methionine (SAM) domain and an N‐terminal amphipathic helix that contributes to its antiviral activity by localizing viperin to ER membranes or lipid droplets. Viperin has been found to require ER localization for full antiviral activity and mutation of the cysteine residues (CX3CX2C motif) ligating the Fe‐S cluster in the central radical SAM domain entirely abolished antiviral activity. Viperin induces a cascade of signaling events to mount an immune response against the viral replication; however, the exact molecular basis for viperin's cellular activity and the role of SAM‐dependent reactions in inhibiting viral synthesis remains unknown. Our studies suggest that the radical SAM activity of viperin is critical for activation of innate immune signaling. The enzyme localizes the E3 ubiquitin ligase TRAF6 and the interleukin 1 receptor‐associated kinase (IRAK1) to the endoplasmic reticulum and activates TRAF6 to catalyze polyubiquitylation of IRAK1, key step in the Toll‐like receptor‐7 (TLR) and TLR‐9 signaling pathways. Our data implicates the radical SAM activity of viperin is essential in this activation step. We found that in presence of IRAK1 and TRAF6 the reductive cleavage of SAM to form 5′‐deoxyadenosine is increased ~ 4‐fold. Thus, our data provides the first evidence that viperin's radical SAM activity is linked to activation of a ubiquitin ligase. This observation has implications for how the enzyme may exert its multifaceted roles in the cellular antiviral response.

Support or Funding Information

This work was supported in part by NIH grants GM 093088 to E.N.G.M. and DK 046960 to R.T.K.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis of 1,4‐diaminoanthraquinones for the purification of lactate dehydrogenase
Authors: Campbell Meagan Elizabeth , Mueller Jaime
Published: June 2021

Cibacron blue 3G‐A is a dye commonly used for the purification of enzymes using affinity chromatography, often utilized in undergraduate biochemistry laboratory studies. Unfortunately, companies only have this product available intermittently, and when available it is sold at a fairly high price. Due to these issues, an alternative molecule that could be easily synthesized and would be similarly efficient at enzyme purification is desired. A distinct feature of Cibacron blue is its anthraquinone core. Anthraquinones are naturally‐occurring aromatic compounds commonly used in the manufacture of dyes. This synthesis utilizes a method of converting 1,4‐diaminoanthraquinones into diazonium salts to then allow the addition of various other functional groups, including anilines and phenols. After synthesis has been conducted, this small library of aminoanthraquinone derivatives will be tested for biological activity by examining spectrophotometric activity in the presence of lactate dehydrogenase (LDH). This will determine whether the products synthesized are efficient at binding to LDH and whether they can be successfully utilized for purification methods. This experiment details a simple, two‐step synthesis of various 1,4‐disubstituted aminoanthraquinone derivatives, which may be found to be useful alternatives to Cibacron blue. In addition, these products could potentially have other biological activity, due to the highly‐activated anthraquinone core.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
High Throughput Proteomic Applications Using Anti‐DYKDDDDK Magnetic Agarose
Authors: Benton Betsy , Geddes Joanna , Vattem Krishna , Patel Bhavin , Kaboord Barbara
Published: June 2021

Affinity purification, immunoprecipitation and mass spectrometry analysis are essential tools used in proteomic studies. A successful outcome is facilitated by high capacity supports, efficient sample preparation, and reproducibility provided by automation. We demonstrate that an anti‐DYKDDDDK magnetic agarose fulfills these criteria. The DYKDDDDK epitope tag is commonly used in protein expression systems as a short purification handle. Using an antibody against DYKDDDDK tag covalently attached to high binding capacity magnetic agarose, we demonstrate effective purification of recombinant proteins expressed in bacteria, the Invitrogen™ ExpiCHO Expression System and by a HeLa in vitro translation (cell‐free) system. Biological activity was demonstrated for ExpiCHO‐expressed DYKDDDDK‐Tumor necrosis factor alpha (TNFa) which was used to successfully stimulate cells and activate Nuclear Factor kappa B (NFkB). In addition, successful co‐immunoprecipitation of DYKDDDDK‐tagged BAD protein with 14‐3‐3 proteins was confirmed using an optimized sample preparation protocol in conjunction with the Thermo Scientific KingFisher Flex magnetic particle processor and mass spectrometry. In summary, anti‐DYKDDDDDK magnetic agarose can be used to effectively isolate, purify, and enrich functional protein targets in a variety of proteomic applications.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mass spectrometry of single mammalian cells quantifies proteome heterogeneity during cell differentiation
Authors: Levy Ezra , Budnik Bogdan , Slavov Nikolai
Published: June 2021

Cellular heterogeneity is important to biological processes, including cancer and development. However, proteome heterogeneity is largely unexplored because of the limitations of existing methods for quantifying protein levels in single cells. To alleviate these limitations, we developed Single Cell ProtEomics by Mass Spectrometry (SCoPE‐MS), and validated its ability to identify distinct human cancer cell types based on their proteomes. We used SCoPE‐MS to quantify over a thousand proteins in differentiating mouse embryonic stem (ES) cells. The single‐cell proteomes enabled us to deconstruct cell populations and infer protein abundance relationships. Comparison between single‐cell proteomes and transcriptomes indicated coordinated mRNA and protein covariation. Yet many genes exhibited functionally concerted and distinct regulatory patterns at the mRNA and the protein levels, suggesting that post‐transcriptional regulatory mechanisms contribute to proteome remodeling during lineage specification, especially for developmental genes. SCoPE‐MS is broadly applicable to measuring proteome configurations of single cells and linking them to functional phenotypes, such as cell type and differentiation potentials.

Support or Funding Information

Northeastern University

NIH New Innovator Award DP2GM123497

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cell surface GRP78 activation by anti‐GRP78 autoantibodies in relation to prostate tumour growth via tissue factor activation.
Authors: Al‐Hashimi Ali , Shayegan Bobby , Austin Richard
Published: June 2021

Background

Prostate cancer (PC) presentation is associated with the production of anti‐GRP78 autoantibodies that enhance tissue factor (TF) expression and procoagulant activity (PCA). We and others observed that GRP78 is expressed on the surface of PC cells where it functions as a signaling receptor to promote cell proliferation and survival. Normally, GRP78 is an endoplasmic reticulum resident chaperon that ensure the proper folding of newly synthesized polypeptides. We report that binding of these autoantibodies to cell surface GRP78 PC progression in mice, mediated by TF. Further, this observed increase in tumour growth can be reversed with the use of heparin or low molecular weight heparin molecules. We hypothesize that the disruption of the autoantibody/cell surface GRP78 complex represent a viable surrogate treatment of PC.

Methods

Wild type, TF knockdown DU145 cells, and NOD/SCID mouse model system was used to investigate the effect of anti‐GRP78 autoantibodies on tumor growth. Protein expression was determined using western blotting and qRT‐PCR. TF activity was determined using the TF PCA continuous assay. Blood samples from patients diagnosed with PC were obtained from the Ontario Tumour Bank and St. Joseph's Hamilton.

Results

‐Anti‐GRP78 autoantibodies correlate with PC stage: PC patients demonstrated high levels of anti‐GRP78 autoantibodies (60–100 μg/ml, pre‐prostatectomy stage) vs. healthy individuals (5–10 μg/ml). These titers were significantly reduced 24‐weeks post‐prostatectomy.

‐Anti‐GRP78 autoantibodies drive tumour progression: We show here that anti‐GRP78 autoantibodies activate the unfolded protein response, a pro‐survival cellular pathway in cancer cells. Furthermore, these autoantibodies were shown to accelerate tumor growth in a NOD/SCID mouse model.

‐Heparin and low molecular weight heparin abolished the effect of anti‐GRP78 autoantiboides on PC progression. In vitro: We show that heparin and low molecular weight heparin inhibit the binding of anti‐GRP78 autoantibodies. In vivo: a low molecular weight heparin was used as an intervention treatment for anti‐GRP78 autoantibodies and was shown to reverse the effect of anti‐GRP78 autoantibodies on increased tumour growth.

Conclusions

We have identified the function of an agent in patients' blood, anti‐GRP78 autoantibodies, that correlate with PC stage in patients and increase TF PCA and promote PC progression in mice. The effect of this autoantibody can be reversed using heparin, thus, this acts as a new potential therapeutic target for PC.

Support or Funding Information

This work was supported in part by Prostate Cancer Canada (PCC) Discovery Grant (Discovery Grants # 2010‐590, and D2017‐1949), and McMaster Surgical Associate Grant 176725.
27‐hydroxycholesterol decreases cell proliferation in colon cancer cells
Authors: Warns Jessica A. , Freking Natalie , Ghribi Othman
Published: June 2021

In the United States, Colorectal Cancer is the third most diagnosed cancer, affecting 1 out of approximately 22 people in their lifetime. A major molecule involved in cancer progression is the AKT signaling pathway, which regulates cell cycle progression, protein synthesis, and cellular survival. Several cholesterol metabolites, including 27‐hydroxycholesterol (27‐OHC) have been shown to be involved in breast and prostate cancers. However, the role of 27‐OHC in colon cancer has yet to be determined. The goal of our study is to investigate the potential role of 27‐OHC in colon cancer progression. To study the effects of 27‐OHC, SW620 and Caco2 colon cancer cells were treated with 0.5 μM to 300 μM of 27‐OHC for 24 hours and MTT proliferation assay, LDH cytotoxicity, TUNEL assay, and western blotting was performed. Additionally, cellular migration was measured with a scratch assay. We found a significant decrease in cellular proliferation at supraphysiological concentrations of 27‐OHC (10 μM to 300 μM) with no significant cellular cytotoxicity or apoptotic cell death. Western blotting showed a significant decrease in p‐AKT expression with 10 μM of 27‐OHC in Caco2 cells. In both cell lines there was a significant increase in cellular migration. This data demonstrates that 27‐OHC increases cellular migration through a loss of cellular proliferation. The decrease in cellular proliferation is dependent on p‐AKT in Caco2 cells and an AKT‐independent pathway in SW620 cells, suggesting that 27‐OHC has differential effects on different colon cancer cells.

Support or Funding Information

R01AG0145264 awarded to OG

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanosensitivity Analysis of Breast Cancer Tumor Cells from Needle Biopsy
Authors: Bedoya Santiago Acero , Ghosh Deepraj , Dawson Michelle
Published: June 2021

Tumor stiffness has been associated with malignancy and increased risk for metastasis. Extensive research has been done investigating breast cancer cell lines' responsiveness to surfaces of varying rigidities as well as examining the biophysical properties of breast cancer tumor samples. However, there is a critical gap regarding the relationship between cells' mechanosensitivity in conjunction to biophysical properties of their extracellular matrix environment. To explore this relationship, we will analyze single‐cell mechanosensitivity in comparison to tumor rigidity via shearwave ultrasound elastogrophy (SWE). Given the putative affiliation, we hypothesize that cells expressing invasive mechanosensitivity profiles will correlate with stiffer tumor regions. Using collagen gels containing different cell types, we derived biopsy‐sized samples allowing us to optimize single‐cell mechanosensitivity analysis. Cells were stained using different dyes corresponding to invasiveness. Subsequently, we analyzed their morphology. Morphological identification within organoid environments would allow for single‐cell analysis without the aggression of tissue digestion, though preliminary results suggest high heterogeneity may not allow for confident cell identification solely on morphology. Thus, inquisition into cell viability and integrity was explored by analyzing the effects of tissue digestion with HyQtase on single‐cells. Cell count and live‐dead stain via flow cytometry allowed for analysis of single‐cell viability. Lastly, cell integrity was evaluated by a 2D adhesion assay of isolated cells. The live/dead stain revealed that digestion resulted in isolation of approximately 10% of the original 500,000 cell population with 90–97% of the isolated population being live‐cells (invasive and non‐invasive respectively). Furthermore, the adhesion assay showed that these isolated single cells retained the ability to adhere to new surfaces, with no difference between the invasive and non‐invasive cell types. These results show that cells are able to retain mechanosensitive properties following enzymatic digestion. However, they also suggest our digestion procedure is not aggressive enough to isolate invasive subpopulations that are more strongly imbedded in the original tissues. Development of these novel techniques will allow for accurate and confident analysis of precious human biopsy samples. Insight into the relationship between single‐cell mechanosensitivity and tumor biophysical properties could elucidate pathways for metastasis inhibition and prevention.

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 Combined Effect of Canagliflozin and Metformin in Human Prostate Cancer Cells
Authors: Ware Kierra , Stewart LaMonica
Published: June 2021

Background

There is still a need to identify effective treatments for castration‐resistant prostate cancers. Recent studies have shown that two drugs used to treat type 2 diabetes, metformin and canagliflozin, may be effective treatments for castration‐resistant prostate cancer. Both drugs have individually been shown to inhibit proliferation of 22Rv1 and PC3 prostate cancer cell lines. However, it is not known if combined treatment with metformin and canagliflozin suppresses the growth of prostate cancer cells better than each drug alone. The goal of this study was to determine the effectiveness of the combination of canagliflozin and metformin as an alternative treatment for prostate cancer.

Methods

Presto Blue Assays were used to assess the effect of metformin and canagliflozin on proliferation of the 22Rv1 and PC3 prostate cancer cell lines. Western blot analysis was also performed to determine the effect of metformin and canagliflozin (both individually and combined) on androgen receptor (AR) protein levels. Western blots were stripped and re‐probed for the protein actin to confirm that gels were evenly loaded.

Results

Canagliflozin at a concentration of 30 uM produced a significant decrease in the proliferation of PC3 and 22Rv1 cells. While metformin showed no significant decrease in proliferation of PC3 cells, metformin (5 mM) did significantly decrease 22Rv1 cell proliferation. The combination of canagliflozin and metformin showed a significant decrease in both PC3 and 22Rv1 cells. However, the effect of the combination was no greater than canagliflozin alone. AR protein levels were reduced by canagliflozin, metformin, and the combination of both drugs. Moreover, the combination of metformin and canagliflozin was the most effective in reducing AR protein levels.

Conclusion

The combination of canagliflozin and metformin was equally effective as canagliflozin alone at reducing prostate cancer cell proliferation. However, this drug combination was more effective at suppressing AR protein levels within castration‐resistant prostate cancer cells. Taken together, these data suggest that combination treatments involving metformin and canagliflozin could be used to decrease tumor growth and AR expression in prostate cancer patients who have developed castration‐resistant prostate cancer.

Support or Funding Information

Supported by NIH grants MD007593 and T34GM007663

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 vitamin B12 receptor serves a role in membrane stability of Caulobacter crescentus
Authors: Barraza Daniela , Strebe Sierra , Menikpurage Inoka , Melendez Ady , Mera Paola E.
Published: June 2021

This project focuses on studying the function of a highly expressed receptor in bacteria. In E. coli, the BtuB transmembrane receptor is involved in the active transport of vitamin B12. The role of BtuB in our model organism C. crescentus is currently unknown. Recently, this protein was shown to be highly expressed (levels comparable to ribosomal proteins) throughout the cell cycle of C. crescentus (Schrader et al 2016). This is intriguing since C. crescentus is a fresh water bacterium that is not likely to be exposed to vitamin B12. We hypothesize that this membrane receptor serves to stabilize the outer membrane of C. crescentus. We began by constructing a btuB deletion mutant and collecting data regarding morphology, growth rate, motility, and viability characteristics as compared to wild type. We showed that BtuB in C. crescentus plays a role in vitamin B12 transport when nutrient availability is limited. Using Transmission Electron Microscopy we observed the mutant to have a potential defect in membrane stability when the cells are growing at high doubling rates. In conclusion, our data suggests that this membrane receptor is playing a role in membrane integrity aside from vitamin B12 transport. Currently, we are in the process of testing membrane stability characteristics of the ΔbtuB mutant. In addition, we are employing a combination of genetics, biochemistry, and microscopy techniques to see if BtuB's role in membrane stability is dependent on vitamin B12 transport.

Support or Funding Information

Funding by Maximizing Access to Research Careers (MARC), grant number: GM07667‐39

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Aggregation of Insulin on Langmuir Monolayers
Authors: Saulcy Kate , Crans Debbie C. , Sostarecz Audra Goach
Published: June 2021

The polypeptide hormone, insulin, is known to aggregate into a hexamer in the presence of zinc ions. The Langmuir monolayer technique can be used to examine the transition between active monomeric insulin molecules and the hexameric form which is more difficult for the body to use. Distinctive isotherm characteristics for both monomeric insulin and hexameric insulin were observed. Zinc (II) chloride is added to the model membrane system to create hexamers of insulin. Conditions of the system are altered to learn about the dependence of insulin's molecular conformation on factors such as pH and concentration. Chelating agents, such as (ethylenediaminetetraacetic acid) EDTA and citrate are added to effectively remove the zinc ion from the solution and observe the behavior of the insulin molecules. With the addition of EDTA to the monolayer of zinc exposed insulin, monomeric isotherm characteristics were noted, indicating the removal of zinc from the insulin hexamer and the creation of monomeric insulin. These molecular conformational changes affect the function of the insulin molecule in the human body.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Age‐Related Loss of Mitochondrial Glutathione Exacerbates Vulnerability to Redox‐Cycling Challenge
Authors: Thomas Nicholas , Shay Kate , Butler Judy , Kelley Amanda , Hagen Tory
Published: June 2021

In order to determine the role of mitochondrial GSH (mGSH) loss in increased susceptibility to xenobiotic insult with age, markers of mitochondrial function were measured in intact and digitonin‐permeabilized hepatocytes from young and old F344 rats under a redox cycling (300 μM menadione) challenge. While the rate of mGSH loss under menadione challenge was similar in both age groups, a 47% decline in initial mGSH levels with age resulted in a 50% loss of mGSH in old rat hepatoctyes versus 28% in young within 10 minutes of exposure. Examination of mitochondrial membrane potential (Δψm), which is acutely regulated by mGSH content, showed a 21% basal loss with age. Additionally, within five minutes of menadione challenge, Δψm in young was not markedly reduced but had collapsed in old rat hepatocytes. Further characterization demonstrated that basal respiration and respiratory reserve capacity, indicators of cellular bioenergetic capacity, were both significantly reduced upon menadione treatment in old rat hepatocytes but not in young. Examination of proton leak, complex I, and complex II contributions to mitochondrial oxygen consumption rates under menadione challenge revealed no marked effect in young rat hepatocytes, while old rat hepatocytes demonstrated a significant loss in complex I activity as well as increases in proton leak and a compensatory increase in complex II activity. These data clearly demonstrate an age‐related increase in mitochondrial susceptibility to a redox‐cycling challenge, particularly in complex I, and provide a plausible mechanism that links this vulnerability to mGSH perturbations.

Support or Funding Information

N.T. is supported by an NIH grant. Title: CAM RESEARCH TRAINING IN NEUROSCIENCE & STRESS; number: 2T32AT002688.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Osteopenia in a Spontaneous Mouse Model of Systemic Lupus Erythematosus
Authors: Lotinun Sutada , Atjanasuppat Korakot , Visitchanakun Peerapat , Saiworn Worasit , Pisitkun Prapaporn , Leelahavanichkul Asada
Published: June 2021

Systemic lupus erythematosus (SLE), a chronic inflammatory autoimmune disease, is characterized by loss of B‐cell tolerance to autoantigens. SLE patients are at increased risk for osteoporosis. Mice lacking Fc gamma receptor IIb (Fc©RIIB) exhibit SLE and its restoration rescues the disease. To determine whether deletion of Fc©RIIB induces osteopenia, we analyzed a skeletal phenotype of Fc©RIIB knockouts at 3, 6 and 10 months old. The analysis of anti‐dsDNA antibodies indicated that mice deficient in Fc©RIIB developed spontaneous SLE at 6 months of age. μCT analysis showed that deletion of Fc©RIIB had no effect on cortical and cancellous bone at 3 months of age. In contrast, 6‐ and 10‐month‐old Fc©RIIB −/− males were osteopenic and a severity of bone loss increased with disease duration. Deletion of Fc©RIIB decreased cortical bone area and cancellous bone volume with increased structural model index. Histomorphometry indicated a decrease in cancellous bone volume, trabecular thickness and trabecular number with concomitant increase in trabecular separation in Fc©RIIB−/− males. Ablation of Fc©RIIB increased osteoclast number without any change in osteoblast number. A skeletal phenotype was milder in females. Deletion of Fc©RIIB increased osteoclast differentiation but did not alter alkaline phosphatase activity and mineralization. Breaking strength parameters, including J, Imax, Imin, Imin/Cmin, and Imax/Cmax were decreased in Fc©RIIB deficient mice compared to WT controls. Our data indicate that Fc©RIIB contributes to the regulation of bone turnover and homeostasis subsequent to SLE and that absence of Fc©RIIB induces osteopenia and decreases breaking strength.

Support or Funding Information

The Ratchadapisek Sompoch Endowment Fund (2014, CU‐57‐091‐IC) and the Faculty of Dentistry, Chulalongkorn University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Group A Streptococcus Prevents Acidification of the Phagolysosome: Role of Lysosomal Leakage
Authors: Zhang Robert , Snipper Joshua , Zheng Kelvin , No Hyewon , Okumura Cheryl
Published: June 2021

Group A Streptococcus (GAS) is a Gram‐positive bacterium that is among the top 10 causes of infection‐related mortality in humans, with infections ranging from minor illnesses such as pharyngitis, to life threatening diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. As the first response to infection, the innate immune system recruits leukocytes such as macrophages to the site of GAS infection, which are able to phagocytose GAS. Once GAS is inside the phagosome, the lysosome fuses with the phagosome to form the phagolysosome. To degrade pathogens such as GAS, the phagolysosome reduces its internal pH to activate proteolytic enzymes. To monitor this process, we infected THP‐1 macrophages with M1 serotype GAS transformed with a plasmid (pWASABI) that expresses a pH‐sensitive green fluorescent protein that is degraded at low pH. Our results showed that GAS expressing pWASABI has a persistent fluorescent signal when bacteria were present in phagolysosomes. In comparison, the non‐pathogenic Gram‐positive species Lactococcus lactis expressing pWASABI had an appropriate decreased fluorescence signal. These results suggest that GAS inhibits the acidification of the phagolysosome. To further confirm the pH of the phagolysosome, we stained infected macrophages with Lysotracker, an acidotropic fluorescent dye. The preliminary results of this experiment confirmed that phagolysosomes containing GAS are not acidified. Previous research has suggested that pore‐forming toxins such as streptolysin O (SLO) and streptolysin S (SLS) induce phagolysosomal leakage of protons. To examine this, we transformed a mutant strain lacking SLO (ΔSLO) with pWASABI. Our preliminary results showed no difference in fluorescence signal when the ΔSLO strain was present in phagolysosomes, suggesting that SLO may not be inducing lysosomal leakage. We are currently exploring whether pore formation by SLS plays a role in lysosomal leakage. The results of these experiments will help provide a clearer image of the molecular mechanism of innate immune resistance of GAS, and may aid in the development of therapeutics aimed to enhance the innate immune system's ability to degrade GAS.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Discriminative Stimulus Effects of Gabapentin
Authors: Zuidema Michael Ryan , Galbo Lindsey Kay , Lekander Alex Duncan , Esterline Lea , Prus Adam Joseph
Published: June 2021

Gabapentin (Neurontin) is synthetic analog of GABA that functions as a voltage‐gated calcium channel (VDCC) blocker and is FDA approved for the management of neuropathic pain and seizure disorders. While not a controlled substance, gabapentin misuse is associated with illicit opioid use, suggesting, along with anecdotal reports, that gabapentin may elicit positive subjective effects. The present study was conducted to examine the subjective effects of gabapentin using rats (N=10) trained to discriminate a 30.0 mg/kg dose of gabapentin (60 min pretreatment time) versus saline in a two‐choice drug discrimination task. The discrimination was acquired in all 10 animals (M = 67.4 sessions, +/− 7.2 SEM). A time course assessment using the gabapentin training dose revealed full substitution (> 80% Gabapentin‐appropriate responding) at 30 and 60 minutes post‐injection. In addition to the training dose, full substitution also occurred for a 60 and 120 mg/kg dose of Gabapentin. Full substitution was shown for the barbiturate and GABAA receptor positive modulator pentobarbital and for the gabapentin analog and VDCC blocker pregabalin. Partial substitution was shown with the psychostimulant d‐amphetamine and the anxiolytic drug and 5‐HT1A partial agonist buspirone. The present findings demonstrate gabapentin's discriminative stimulus effects appear to be primarily mediated by GABAA receptors and VDCC blockade and partially mediated by dopamine and 5‐HT receptors. Full substitution by pentobarbital and partial substitution by amphetamine suggests the potential for positive subjective effects produced by gabapentin.

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 nanotechnology system for augmenting the intranasal delivery of oxytocin
Authors: Murnane Kevin Sean , Oppong‐Damoah Aboagyewaah , Zaman Rokon Uz , D'Souza Martin
Published: June 2021

Neuropeptides such as oxytocin (OT) have shown promise as treatments for many central nervous system (CNS) disorders, such as Dravet Syndrome and Autism Spectrum Disorder (ASD). The primary barriers to the use of OT as a treatment for ASD are that, due to its size and hydrophicity, it does not cross the blood brain barrier (BBB) and penetrate the CNS, and that it is rapidly metabolized in the blood stream, with a half‐life of 1–6 minutes following IV administration. Ongoing studies are attempting to overcome its poor brain penetrance using intranasal delivery of OT. Although the development of intranasal delivery of OT has been a seminal advance in ASD research, this administration method is an inefficient means of transferring drug to the brain and requires frequent dosing (because of the short half‐life of OT). As such, there is legitimate concern regarding the long‐term effects of high‐dose and frequent daily administration of intranasal OT. We have collected compelling data showing that a nanotechnology is a promising means of augments the brain penetrance of OT and protecting OT from metabolism and sustaining its delivery. We have found that our nanoscale drug carriers do not exhibit in vitro or in vivo toxicity, have sizes on the order of 100–200nm (which is highly amenable to brain delivery), increase the brain penetrance of an IR‐marker (of similar molecular size to OT) using bioimaging, engender stronger prosocial effects than OT alone given intranasally, and exhibit sustained release effects both in vitro and in vivo. Although we have focused on the development of intranasal OT, we believe that this new technology will be applicable to many other neuropeptides (such as neuropeptide Y) that may have promise for ASD and other disorders because of their anti‐inflammatory, neuroprotective, and/or anti‐seizure effects.

Support or Funding Information

This work was supported by R21NS100512.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synthesis and Evaluation of Bioactive Estrogen Conjugated Naphthalimides
Authors: Skubic Kristin , Arnatt Christopher K.
Published: June 2021

Small molecule DNA intercalators as antineoplastic agents, for breast cancers, have gained a significant amount of success within recent years. Therefore, previously tested naphthalimide compounds were coupled to an estrogen receptor antagonist in offering a useful method in delivering cytotoxic drugs selectively to sites expressing high levels of estrogen receptors. A series of novel conjugate compounds incorporating a linker, with an anhydride component and a diamine component, joining estrogen and known potent naphthalimides were synthesized to demonstrate apoptotic effects with antiproliferative activity in breast cancer cell lines. These conjugates are promising compounds for further studies in treatment of estrogen receptor positive breast cancers. Calculated LD50 values for the antiproliferation assays illustrate a clear structure‐activity relationship for this conjugated naphthalimide‐estrogen series.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altered mitochondrial dynamics contributes to endothelial dysfunction in hypertension
Authors: Li Guohua , Wu Jie , Hou Zuoxu , Zhang Xing , Gao Feng
Published: June 2021

Aims

Impaired mitochondrial dynamics is implicated in cardiovascular diseases. Alterations in mitochondrial dynamics and reactive oxygen species production have been shown to be associated with the development of hypertension. However, the role of mitochondrial dynamics in endothelial dysfunction during the development of hypertension remains unknown.

Methods

Effects of mitochondrial fission inhibitor mdivi‐1 on vascular contraction and relaxation were examined on mesenteric arteries isolated from WKY or SHR rats. Mitochondrial networks in aortic endothelia were assessed using transmission electron microscope, and further evaluated with laser confocal after labeling with 100 nmol/L MitoTracker Green FM. In vitro, effects of Angiotensin□(10−6 M, 24 h) on mitochondrial morphology in HUVECs were investigated.

Results

Pretreatment with mdivi‐1 inhibited PE‐induced mesenteric artery constriction in WKY rats, and the inhibitory effect of mdivi‐1 was attenuated on endothelium‐denuded mesenteric arteries. Then we observed mitochondrial fragmentation, increased mitochondrial reactive oxygen species production and increased expression of dynamin‐related protein 1 (Drp1) in aortic endothelial cells from SHR rats. Mdivi‐1 pretreatment ameliorated acetylcholine induced mesenteric artery relaxation in SHR rats. In cultured HUVECs exposed to Angiotensin□, we observed a similar loss of mitochondrial networks and increased expression of Drp1. Altered mitochondrial dynamics was associated with increased mitochondrial reactive oxygen species production and a marked impairment of insulin‐stimulated activation of endothelial nitric oxide synthase and endothelial autophagy. Silencing Drp1 with siRNA blunted Angiotensin□induced alterations in mitochondrial networks, reactive oxygen species production, endothelial nitric oxide synthase activation and endothelial autophagy.

Conclusions

These findings show that up‐regulation of Drp1 during hypertension exacerbates endothelial dysfunction by increasing mitochondrial reactive oxygen species and subsequently inhibiting endothelial nitric oxide synthase activation and autophagy.

Support or Funding Information

This study was supported by the grants from the National Natural Science Foundation of China (Grant Nos. AWS16J018, 81670253)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological inhibition of TRPV4 channels protects against early renal hypoperfusion induced by ischemia‐reperfusion in neonatal pigs
Authors: Soni Hitesh , Peixoto‐Neves Dieniffer , Olushoga Michael A. , Adebiyi Adebowale
Published: June 2021

Renal ischemia‐reperfusion (IR) remains a significant cause of acute kidney injury (AKI) in neonates. Early renal vasoconstriction in ischemic AKI induces hypoperfusion and a rapid decline in kidney function. Mechanisms that underlie post‐ischemic renal hypoperfusion are poorly understood, but possibilities include alterations in ion channel‐mediated renal vasoregulation. Here, we show that pharmacological activation of TRPV4 channels promotes renal vasoconstriction and hypoperfusion in neonatal pigs. Bilateral renal ischemia followed by short‐term reperfusion increased TRPV4 channel protein expression and activity in preglomerular microvessels and vascular smooth muscle cells (SMCs), respectively. Renal IR caused oliguria and elevated serum or urine levels of AKI biomarkers. Renal IR also increased renal vascular resistance (RVR) and attenuated renal blood flow and glomerular filtration rate in the pigs. However, TRPV4 channel blockers HC 067047 and RN 1734 mitigated renal IR‐induced oliguria, hypoperfusion, and alterations in AKI biomarkers. Furthermore, renal IR increased serum angiotensin II (ANG II) concentration, but not renal vascular expression of type 1 ANG II receptors in the pigs. TRPV4 channel blockade reduced ANG II‐induced renal vasoconstriction, RVR increase, and renal hypoperfusion in the pigs. Together, our data suggest that increased renal vascular SMC TRPV4 channel expression, ANG II‐induced TRPV4 activation, and subsequent increase in RVR contribute to hypoperfusion and kidney insufficiency elicited by renal IR in neonatal pigs. We propose that multimodal signaling by renal vascular SMC TRPV4 channels regulates neonatal renal hemodynamics in health and disease.

Support or Funding Information

R01DK101668

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evolution of Naturally High Plasma Glucose Concentrations in Birds
Authors: Basile Anthony J. , Jarrett Catherine L. , Witt Christopher C. , Sweazea Karen L.
Published: June 2021

Blood glucose is a circulating fuel source for animals. However, species vary in glucose requirements due to distinct fuel physiology. Birds naturally have higher plasma glucose concentrations (PGlu) by a factor of 1.5–2 compared to mammals when adjusted for body mass. Currently, the physiological and evolutionary cause of the high PGlu is unknown. The goal of this study was to begin to elucidate the evolution of high PGlu in birds by comparing PGlu between Aves and Mammalia as well as analyzing the phylogenetic distribution of PGlu among avian species. Data were collected for PGlu and body mass for 284 species (Aves = 163, Mammalia = 121) housed at The Phoenix Zoo in Arizona. We used these data to 1) examine the relationship between body mass and PGlu in Aves and Mammalia, 2) examine inter‐order differences in PGlu within Aves, and 3) examine the maximum‐likelihood ancestral state for glucose in Aves. Consistent with prior studies, birds maintain the highest PGlu among vertebrates (16.20 mM/L ± 0.26, p < 0.05). Further, PGlu was negatively correlated with body mass for Aves (Linear Regression, p<0.05). A similar negative correlation for mammals was not observed within this data set. The highest and lowest PGlu in birds were Coraciiformes (22.35 ± 1.17 mM/L) and Struthioniformes (10.58 ± 0.63 mM/L), respectively (one‐way ANOVA, p<0.05). As Coraciiformes are small (~0.21kg) and Struthioniformes are large (~64kg), these variations in PGlu may be explained by the relationship between body mass and PGlu presented above. This was confirmed by phylogenetic generalized linear model analyses, which showed that body mass alone explained PGlu within bird species. Collectively, these data allow us to better understand the evolution of PGlu within birds. Future research aims to determine the physiological mechanisms driving the differences in PGlu among avian species.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
TNFSF14 inhibits neutrophil extracellular trap formation in neutrophil‐like HL‐60 cells.
Authors: Petreaca Melissa , Kloczkowski Breanna
Published: June 2021

Tissue injury and infection induce a rapid inflammatory response, in which inflammatory cells, primarily neutrophils, leave the blood vessels and move into the affected tissue. Upon their arrival in this tissue, neutrophils use a variety of methods to kill potential pathogens, including phagocytosis, the generation of reactive oxygen species (ROS) that non‐specifically damage microbes, and formation of neutrophil extracellular traps (NETs) that trap microbes in an anti‐microbial environment. In addition to eliminating potential pathogens, ROS and NETs damage host tissues, promoting a positive feedback loop of tissue damage and inflammation that normally ends during the resolution of inflammation. The resolution of inflammation is associated with the inhibition of neutrophil recruitment and function, as well as induction of neutrophil apoptosis. Previous studies comparing wound healing in wild type and knockout mice found that the cytokine TNFSF14 is necessary for timely resolution of inflammation in mouse wounds, though the mechanism remains unclear. TNFSF14 could promote the resolution of inflammation by reducing cytokine production in the affected tissue, increasing neutrophil apoptosis, and/or reducing pro‐inflammatory neutrophil functions, such as ROS production and NET formation. In this study, we investigated whether TNFSF14 decreases HL‐60 neutrophil‐like cell production of ROS or NETs in response to multiple stimuli, using fluorimetry of DCF‐DA‐stained HL‐60 cells to quantify ROS production and fluorimetry of Sytox Green‐stained extracellular DNA to quantify NET formation. We found that, while TNFSF14 does not inhibit ROS production in these cells, it does decrease NET formation in response to PMA and LPS, suggesting that TNFSF14 may promote the resolution of inflammation through its inhibition of NET formation.

Support or Funding Information

Research start‐up and faculty development funding from DePauw University

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Preparation Techniques for Optimal Hepcidin Detection in Human Hepatocyte Culture
Authors: Morehouse Zachary Patrick , Easparro Brandon , Proctor Caleb , Nash Rodney J. , Atwood James
Published: June 2021

Approximately 40% of adults aged 65 and older are affected by Chronic Kidney Disease (CKD). Anemia is an associated symptom seen in many patients diagnosed with CKD, promoting elevated cardiovascular risk, decreased quality of life, and increased morbidity and mortality in affected populations. Hepcidin‐25 (hepcidin) is a key iron regulating hormone secreted by hepatocytes to regulate and maintain body iron homeostasis and dietary iron absorption. Hepcidin does this by stifling iron release from macrophages, preventing the normal recycling of iron in the body needed to support erythropoiesis. Elevated levels of hepcidin have been observed in both adults and children suffering from CKD, while its pathological mechanism of action is still relatively unstudied. There is a growing need to further the understanding of hepcidin's role in CDK and other iron related pathologies. Currently there is no standardized methodology for hepcidin evaluation studies on relevant human cell lines, and there is a growing need for studies to be done in this field to assist biomedical researchers in the translation of clinically relevant lab findings from the benchtop to the bedside. Hep G2 is a perpetual human hepatic cancer cell line confirmed to produce hepcidin and a commonly used lineage when studying hepatic function in vitro. While it is confirmed that Hep G2 cells produce hepcidin when cultured, there is currently no standardized procedure for detection of this peptide in vitro from these, or any other human derived cell lines. The expanding clinical and biomedical implications of hepcidin has prompted the need to determine an optimal method for detection of hepcidin in vitro. In this study we use of Hep G2 cells as a model for hepcidin peptide detection and various tissue processing techniques to prepare the cells hepcidin quantification via ELISA.

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