bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the Potential for Na+‐H+ Exchanger Isoform 1 (NHE1) Inhibitors as Adjuvant Therapies in the Treatment of Ovarian Cancer
Authors: Corradi Anna R. , Provost Joseph J. , Wallert Mark
Published: June 2021

Each year in the United States over 200,000 women are diagnosed with ovarian cancer leading to more than 130,000 deaths. A major challenge in ovarian cancer treatment is the development of drug resistance to the therapeutic agents. Increases in the expression and activity Na+‐H+ Exchanger Isoform 1 (NHE1) was recently demonstrated to regulate cell proliferation in ovarian cancer patients. NHE1 is an 815 amino acid transmembrane transport protein that regulates intracellular pH. We hypothesize that combining common ovarian cancer therapeutic agents along with compounds that directly or indirectly inhibit NHE1 will enhance the ability of the therapeutic agents to slow ovarian cancer cell proliferation and migration. Three ovarian adenocarcinoma cell lines; CAOV‐3, SKOV‐3, and OVCAR‐3 are used in this research. Using a proliferation assay, we assessed the impact of different ovarian cancer therapeutics (Rucaparib, Olaparib, Paclitaxel and Cisplatin) in these three cell lines in the presence and absence of inhibitors that decrease NHE1 activity directly or indirectly. Our previous work demonstrated that the NHE1 inhibitor cariporide and a number of protein kinase inhibitors decreased cell proliferation rates in all three cell types. Treatment with Cisplatin reduced cell proliferation most significantly in the CAOV‐3 line. Paclitaxel and Cisplatin each had significant and comparable impacts on the SKOV‐3 and OVCAR‐3 cell lines. Cell migration was evaluated using electric cell‐substrate impedance sensing (ECIS). In the SKOV‐3 and CAOV‐3 cells, inhibition with cariporide and the Rsk inhibitor BID1870 each dramatically reduced the rate of cell migration. The research presented here supports the role of NHE1 in cell proliferation, and for the first time implicates NHE1 in the regulation of cell migration in ovarian cancer cells. Data will be presented that evaluated the potential for the inhibition of NHE1 as an adjuvant therapy in ovarian cancer.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Proof‐of‐Concept in a 3D Culture Model of Glioma Invasion: Towards Personalized Therapeutics in Brain Cancer
Authors: van Pel Derek M. , Naus Christian C. , Sin Wun Chey
Published: June 2021

Glioma is a highly aggressive form of brain cancer, with some subtypes having 5‐year survival rates of less than 5%. Tumor cell invasion into the surrounding parenchyma seems to be the primary driver of these poor outcomes, as most gliomas recur within 2 cm of the original surgically‐resected tumor. Many current approaches to the development of anticancer therapy attempt to target genetic weaknesses in a particular cancer, but may not take into account the microenvironment experienced by a tumor and patient‐specific genetic differences in susceptibility to treatment.

The primary aim of our research program is to overcome these limitations by ultimately developing 3D cerebral organoids generated from patient‐derived induced pluripotent stem cells (iPSCs), in which we will screen to identify an optimal treatment regimen by combining the organoids with patient‐derived tumor cells. As a proof‐of‐concept of this approach, we developed a system in which we could model the invasion of human glioma cells into mouse neural progenitor cell‐derived spheroids. Here we demonstrate the utility of such an approach, and find that we can follow invasion of human tumor cells using cell tracking dyes and 3D laser scanning confocal microscopy, both in real time and in fixed samples. These results were validated using conventional cryosectioning. We go on to show broad applicability of this system by using two different tumor cell lines, and two different mouse genetic backgrounds, attempting to mimic differences that might be observed in tumor and patient brain tissue, respectively. Having obtained this proof‐of‐concept, we will next recapitulate these results in a fully humanized system using spheroids and then cerebral organoids from human iPSCs, before moving on to patient‐derived cultures. This stepwise approach could ultimately lead to truly personalized treatments for brain cancer.

Support or Funding Information

DvP is supported by the UBC Faculty of Medicine, the UBC Summer Student Research Program, and the Mach‐Gaensslen Foundation. CCN is supported by the Canada Research Chair program and a grant from the Canadian Cancer Society. WCS is supported by the Canadian Cancer Society.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of N‐type Calcium Channels in inflammatory pain and associated sensory nerve growth
Authors: Pitake Saumitra , Mishra Santosh
Published: June 2021

Voltage‐gated calcium channels (VGCC) play an important role in pain neurotransmission by releasing neuropeptides substance P and calcitonin gene‐related peptide from the sensory neurons. Here, we identified a new role of VGCC in pain signaling by sensory nerve growth at the periphery in the skin. In the present study, we first assessed the role of N‐type calcium channels (Cav2.2) in inflammatory pain. Complete Freund's Adjuvant (CFA), a known inflammogen was injected into the hind‐paws of C57Bl6 mice to induce inflammation and dorsal root ganglia from the lumbar region (L4–L6) were isolated and cultured 24‐hour post injection. Voltage clamp recordings revealed a significant increase in Cav2.2 currents between contralateral and ipsilateral DRGs. Current clamp analysis of these DRGs showed a significant increase in action potential frequency upon CFA injection. A significant increase in Cav2.2 currents was also observed when CFA (1:1000) was directly added to DRG culture. The increase in Cav2.2 currents was significantly decreased upon addition of ω‐conotoxin (1 μM) into DRG culture. Immunocytochemical analysis of cultured DRGs with CFA (1:1000) revealed a significant increase in TRPV1, Cav2.2 and neuronal filament markers such as Synaptophysin and neuronal marker β‐III tubulin (TUJ1), indicate increased neuronal synthesis and neurite formation at the periphery. This was further corroborated by Western Blot analysis. Altogether, our study suggests a possible role of VGCC‐dependent sensory nerve growth that might be responsible for increased sensitivity to minimal stimuli in chronic pain.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Circadian Deadenylase Nocturnin in the Mitochondria
Authors: Ojo Anne , Laothamatas Isara , Green Carla
Published: June 2021

The circadian rhythm is an evolutionarily conserved process that drives rhythmic behavior and physiology with a period of approximately 24 hours. Circadian rhythms are generated by the heterodimeric transcription factors CLOCK/BMAL1. CLOCK/BMAL1 binds to E‐boxes across the genome and drives the transcription of many genes, including their own repressors Per and Cry. Transcription activation by CLOCK/BMAL1 and its inhibitory feedback loop generated by PER/CRY result in the rhythmic expression of thousands of clock‐controlled genes. Nocturnin (mNOC) is among those genes regulated by CLOCK/BMAL1. mNOC is highly expressed in the liver, where its expression is at maximum in the evening and minimum in the morning. Compared to WT mice, mNOC KO mice exhibit a resistance to high‐fat diet (HFD) induced obesity. Protein sequence analysis reveals that mNOC contains an EEP domain that is conserved in the CCR4 family, whose members function as deadenylases. Interestingly, mNOC also contains a putative mitochondrial targeting signal (MTS) in between two translation initiation sites. To investigate if mNOC does localize to the mitochondria, we performed immunocytochemistry in HEK293 cells overexpressing mNOC that does or does not code for the MTS; and performed a mitochondrial fractionation followed by proteinase k protection assay. To examine mNOC's function in the mitochondria, we overexpressed mNOC in HEK293 cells and performed RT‐qPCR to examine mitochondrial RNA stability. We found that mNOC is able to localize to the mitochondria, but we did not see changes in mitochondrial RNA levels. Further experiments will help conclude if mNOC is targeting mitochondrial translation.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Immunoregulatory role of melatonin in Helicobacter pylori‐induced gastric diseases
Authors: Jianhua Luo , Hui Zhang , Mi Zhang , Jun Song , Li Li , Dancen Lin , Meifang Lin , Feng Zhang , Hui Liu , Ruixiang Zhou
Published: June 2021

Melatonin has shown important immunoregulatory activities in inflammatory disorders; however, the immunoregulatory role of melatonin in Helicobacter pylori‐induced gastric diseases remains unclear until now. This study aimed to examine the in‐vivo activity of melatonin against H. pylori‐induced gastric diseases and explore the immunoregulatory mechanisms. HE staining showed obvious inflammatory cell infiltration in the gastric mucosa and submucosa, and the culture of the mouse stomach homogenate was positive for the rapid urease test. Immunohistochemistry revealed extensive MT1 and MT2 expression in the gastric mucosa of H. pylori‐infected mice. ELISA measured lower TGF‐β1 levels in the liver of H. pylori‐infected mice than in uninfected mice 2 weeks post‐treatment with 25, 50 or 100 mg/kg melatonin, while higher TGF‐β1 levels were detected 4 and 6 weeks post‐treatment. Western blotting determined lower Foxp3 expression in the spleen of infected mice than in uninfected mice 2 weeks post‐treatment with 25, 50 or 100 mg/kg melatonin, and higher Foxp3 levels were detected 4 weeks post‐treatment with 50 and 100 mg/kg melatonin; however, the Foxp3 level was comparative in infected and uninfected mice 6 weeks post‐treatment. In addition, higher RORβ expression was found in the gastric specimens of infected mice than in uninfected mice 2 weeks post‐treatment with 25, 50 or 100 mg/kg melatonin, and higher RORβ expression was detected 6 weeks post‐treatment with 25 mg/kg melatonin. At 2, 4 and 6 weeks post‐treatment with 25, 50 and 100 mg/kg melatonin, higher TGF‐β1 expression was detected in the liver of infected mice than in uninfected mice. The results demonstrate that melatonin may alleviate H. pylori‐induced gastric diseases through mediating TGF‐β1 and Foxp3 expression via membrane and nuclear receptors.

Support or Funding Information

National Natural Science Foundation Youth Fund:81302601 NSFC(National Natural Science Foundation of China):30971541

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the Effects of Chronic Interferon Stimulation on Heme Oxygenase‐1 in Human Monocyte Derived Macrophages
Authors: Cobo Bryan , Garza Rolando , Kolson Dennis L.
Published: June 2021

HIV‐associated neurocognitive disorders (HAND) affect ~40% of the HIV infected population even with the use of combined antiretroviral therapy (cART). We previously reported that the critical cytoprotective enzyme, Heme oxygenase‐1 (HO‐1) is reduced in the brains of HIV infected individuals and that this reduction correlates with increased expression of type 1 interferon response genes and markers of monocyte activation. Furthermore, reduction of HO‐1 protein expression is accompanied by increased HO‐1 RNA expression. We also showed that chronic type 2 interferon (IFN) stimulation in astrocytes leads to HO‐1 protein degradation via an immunoproteasome mediated mechanism. Here, we aim to determine the effects of chronic type 1 and type 2 IFN stimulation on human monocyte derived macrophage (MDM) HO‐1 protein expression and elucidate the mechanisms by which IFNs cause dysregulation of HO‐1 protein. To address this, we treated primary human MDMs with type 1 or type 2 IFNs for 24 hours or 10 days. We collected protein lysates for western blot analysis and total RNA for quantitative PCR. Supernatants were collected to measure glutamate production and supernatant neurotoxicity. We observed that chronic type 1 and type 2 IFN stimulation (0.1 ng/ml) did not alter HO‐1 protein expression, however, we observed increased HO‐1 RNA expression with chronic type 1 IFN stimulation. Furthermore, we observed increased immunoproteosome subunit expression and loss of constitutive proteasome expression. No differences were observed in glutamate production or supernant neurotoxicity. These results suggest that chronic type 1 and type 2 IFN stimulation induce immunoproteasome expression and can subsequently lead to accelerated HO‐1 protein degradation, which may contribute to neurodegeneration.

Support or Funding Information

I would like to thank Mr. Rolando Garza and Dr. Dennis L. Kolson for their insightful comments and for allowing me to aid with their research project this summer. I would also like to express my gratitude towards my program advisors, Dr. Arnaldo Diaz Vazquez and Dr. Raquel Castellanos, the Office of Research and Diversity Training at Penn, and the following organizations that made this opportunity possible: MARC program @ UPR‐RP (NIH Grant 5T34GM007821‐37) and the Perelman School of Medicine @ UPenn.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mechanotransduction via HA LFA‐1 promotes Kindlin‐3/RACK1/Orai1 engagement to mediate Calcium in PMN
Authors: Morikis Vasilios Aris , Simon Scott
Published: June 2021

Absence of Kindlin‐3 severely impairs β2‐integrin function on neutrophils, results in severe bleeding and resistance to arterial thrombosis and immunodeficiency. Kindlin‐3 binds LFA‐1 β‐integrin tail independent of Talin‐1 and is associated with integrin conversion to a high‐affinity state and the facilitation of micro‐cluster formation that allow for long‐lived shear resistance bonds with ICAM‐1. However, the molecular and mechanical events that link LFA‐1 to Kindlin‐3 at spatially distinct sites to allow for subsequent engagement with scaffolding protein RACK1 and calcium release activated channel Orai1 has not been rigorously shown. We show that an allosteric shift of LFA‐1 due to force generation on the β2‐integrin cytodomain promotes interaction with Kindlin‐3 F3 subdomain and when sufficient force is transduced through this mechanical linkage induces the onset of calcium flux. We utilize real time two‐line total internal reflection fluorescent and epifluorescence microscopy on neutrophil‐like PLB‐985 cells arrested to ICAM‐1 within our microfluidic vascular mimetic. We have been able to identify the stoichiometric binding rate of Kindlin‐3 and subsequent RACK‐1 recruitment to high‐affinity LFA‐1 sites in response to increasing shear profiles prior to calcium flux. In addition to outside‐in signaling we have shown that force acting on LFA‐1 amplifies calcium influx during CXCR1/2 ligation in a Kindlin‐3 dependent manner. To assess the spatial proximity and association between Kindlin‐3/RACK1 with focal adhesion clusters we are developing a FRET pair between the Kindlin‐3 PH domain and RACK1 propellers 5 to 7 to further elucidate the spatiotemporal events and requirements for outside‐in signaling and neutrophil progression to a migratory phenotype. This has allowed for us to identify key forces and proteins required for outside‐in signaling during neutrophil transition from rolling to arrest which will help further glyocomimetic development to treat various disease states.

Support or Funding Information

The work was supported by grants AI047294 (SIS).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Human milk exosomes dampen induced inflammatory response in human intestinal epithelial cells
Authors: Kraft Jamie Dale , Ferretti Emanuela , Tremblay Eric , Beaulieu Jean‐François , Altosaar Illimar
Published: June 2021

After birth, upon exposure to the ex utero environment, the gastrointestinal tract of a newborn transitions from near sterile conditions to being exposed to a plethora of bacteria. In normal, term births the gastrointestinal tract matures through enteral feeds and commensal bacteria colonization. However, premature birth perturbs in utero organ maturation, thus depriving preterm infants of immune tolerance and increasing their susceptibility to gastrointestinal disorders. Human milk is the gold standard of infant nutrition, but in some cases, it is unavailable or insufficient. It is necessary to understand the components of human milk which facilitate development and promote immune tolerance to optimize feeding methods, ensuring best possible outcomes. Human milk exosomes have been shown to have immunomodulatory properties. Previous reports have demonstrated that milk exosomes survive in vivo digestion in animals, in vitro digestion in humans and can be taken up by human intestinal epithelial cells. We hypothesized that exosomes may promote immune tolerance in human intestinal epithelial cells.

Exosomes were isolated from human milk through a series of centrifugations and ultracentrifugations. Post‐confluent Caco‐2/15 cells were incubated with either culture medium containing milk exosomes or PBS (control) for 24 hours. Caco‐2/15 cells, once maintained at post‐confluency, recapitulate mid‐gestation small intestine absorptive cells morphologically, functionally and at the gene level. An inflammatory response was induced by incubating the Caco‐2/15 cells with heat‐killed bacteria (E. coli + S. typhimurium). Expression of pro‐inflammatory markers, CXCL8, IL‐6 and CXCL10, was significantly decreased in cells pretreated with human milk exosomes compared to the control. Milk exosomes and their cargo caused a dampened immune response in human intestinal epithelial cells, suggesting that they may be promoting immune tolerance in the gastrointestinal tract of newborns. Infants born prematurely would benefit from such effects as their intestine is underdeveloped and more permeable, therefore, more susceptible to react to bacteria, causing an inflammatory cascade that could lead to sepsis. This is a relevant finding in identifying future directions to optimize feeding strategies for preterm infants.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Specificity of Liposomes Coated with Isolated Membranes from Cancer and Macrophage Cell Lines
Authors: Mason Thomas , Tamulonis Erin , Miller Keith R.
Published: June 2021

Chemotherapy is currently one of the mainstream treatments for cancers and is often unsuccessful in the treatment role due to a lack of target specificity. This lack of specificity leads to an abundance of off‐target effects and inadequate therapeutic concentrations due to the harsh side effects of the chemotherapeutics which commonly include alopecia, fatigue, and a compromised immune system. More specific drug delivery mechanisms are needed to address these issues and increase chemotherapeutic efficacy. One promising method was recently developed when investigators successfully synthesized and tested a macrophage membrane encapsulated liposome (MEL) in murine models. The chemotherapeutic loaded MEL demonstrated increased targeting, biomimetic capability, and was more effective at eliminating metastatic lung lesions of 4T1 murine breast cancer when compared to chemotherapeutic loaded liposomes and free chemotherapeutics alone. Based on that information, this study proposed that THP‐1 human macrophage membrane could be used to synthesize human compatible MELs and successfully target MDA MB 231 human breast adenocarcinoma cells while demonstrating decreased uptake in THP‐1 cells. Human MELs were successfully synthesized as confirmed by SDS‐PAGE, but preliminary uptake studies of fluorescent cargo disproved the initial hypothesis as MEL uptake was significantly increased in THP‐1 cells and statistically static in MDA MB 231 cells compared to control liposomes. However, these findings presented a new interest in investigating MDA MB 231 membrane encapsulated liposomes and uptake testing is currently ongoing. Investigation into membrane encapsulated liposome stability and cytotoxicity in MDA MB 231 and THP‐1 cultures will be performed, as well as testing to compare macrophage activation in the presence of the encapsulated liposome variants. This work will beneficial in understanding the specificity to MDA MB 231 and THP‐1 cells conferred by various cell membranes and can ultimately aid in the development of more specific drug delivery systems to improve the efficacy of chemotherapy while decreasing off‐target effects.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allosteric p97 inhibitors to overcome ATP‐competitive inhibitors resistance in anticancer therapy
Authors: WANG FENG , Gan Taiping , Chou Tsui‐Fen
Published: June 2021

Resistance mutations occurring on desired targets inside cancer cells, leading to loss of treatment effectiveness is a major challenge of targeted therapy; therefore, developing related small molecules with different mechanisms of action at different binding sites could be an effective strategy to overcome the resistant mutations. The ATPase enzyme, p97, is a promising target for cancer therapy because it is vital to cancer cell growth and survival. Development of resistance to existing ATP‐competitive p97 inhibitors such as CB‐5083 inspired identification of p97 inhibitors with different mechanisms of action. One approach to overcome CB‐5083 resistance is to use different classes of p97 inhibitors and therefore, we determined the potency of four ATP competitive p97 inhibitors (DBeQ, ML240, ML241, and CB‐5083) and two allosteric p97 inhibitors (NMS‐873 and UPCDC‐30245) using recombinantly purified CB‐5083 resistant p97 mutants and CB‐5083 resistant HCT116 colon cancer cells. Our results demonstrated that two known CB‐5083 resistant mutants (N660K and T688A p97) were consistently resistant to ATP‐competitive p97 inhibitors whereas allosteric p97 inhibitors NMS‐873 and UPCDC‐30245 were still active toward these mutants. The binding affinity of CB‐5083 to p97 mutants N660K and T688A is lower than to WT p97, which may be the mechanism of CB‐5083 drug resistance. To evaluate the effect of a single site mutation at N660 or T688 on p97 itself, we conducted steady state kinetic analysis. The results suggested that these mutations change the conformation of the p97 ATPase. N660K mutation decreased the Km of ATP to p97 whereas T688A decreased kcat of ATP hydrolysis. While CB‐5083, NMS‐873 and UPCDC‐30245 all effectively inhibited parent HCT116 cancer cell proliferation, allosteric p97 inhibitors (NMS‐873 and UPCDC‐30245) exhibited similar potency in inhibiting CB‐5083 resistant HCT116 cancer cell proliferation, which was more than 50‐fold resistance to CB‐5083.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
STAT6 Promoter Polymorphism is Essential for Malaria Infection and Suppression of Parasitemia Among Infected Children
Authors: Seamans Brooke , Liou Gialeigh , Ojurongbe Olusola , Thomas Bolaji
Published: June 2021

Significant morbidity and mortality among children remain the most consequential outcome of malaria infection in sub‐Saharan Africa, with host genetic variants reported to modulate infectivity and regulation of disease markers. Effector T cells are essential for a proper immune response, such responses ameliorated by regulatory T cells, which are encoded by genes implicated in cytokine cross regulation. One such gene is the signal transducer and activator of transcription 6 (STAT6), whose intronic (rs3024974) single nucleotide polymorphism (SNP), is suspected in malaria pathogenesis. To this end, regulatory SNP of the STAT6 gene was genotyped in a cohort of P. falciparum malaria infected (n=106) and uninfected, control children (n=225), ages 1–5 years, recruited from Nigeria. The genotypic distribution of rs3024974 was in Hardy‐Weinberg equilibrium, among all groups. Even though the wild type genotype (rs3024974CC) was commoner in both groups; 83.0% (infected) versus 70.7% (uninfected); the percentile frequency of rs3024974TT genotype of STAT6 was significantly higher among uninfected children compared to infected (4.9% versus 1.0% respectively). In addition, the minor allele rs3024974T was doubly prevalent among uninfected controls than infected children (17.1% versus 8.9% respectively; p=0.01). Significantly, children with the mutant genotype (rs3024974TT) were less likely to develop clinical malaria (OR 0.19; 95% CI=0.00–1.31), with the odds increased tenfold among children with wild‐type genotype (OR=2.03; 95% CI=1.10–3.86). This observation was further confirmed when examining markers of disease among infected children, with parasitemia significantly less (tenfold reduction) in those with the mutant genotype (mean parasite count: 2,986 parasites/ml of blood) versus children with wild‐type genotype (mean parasite count: 30,242 parasites/ml of blood). Despite the fact there were differences in other markers (age, weight, packed cell volume, temperature) based on rs3024974 variants, these differences were not significant. We conclude that STAT‐6 promoter polymorphism rs3024974 is a susceptibility factor for malaria infection and significant downregulation of P. falciparum parasitemia levels among infected children.

Support or Funding Information

Laboratory Support and Faculty Development Award, College of Health Sciences and Technology, Rochester Institute of Technology

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Genes Involved in Trogocytosis (cell‐nibbling) in Entamoeba histolytica
Authors: Feeney Shea E. , Ralston Katherine S.
Published: June 2021

Entamoeba histolytica (histo‐: tissue; lytic‐: dissolving) is a eukaryotic pathogen that causes amoebic dysentery in humans. Amoebic dysentery is a considerable global burden resulting in about 50 million diarrheal infections and 100,000 deaths per year. The ability of E. histolytica to cause profound tissue damage is likely to be driven by human cell killing. While many other cell‐killing organisms release cell‐killing effector toxins, this is not the case for E. histolytica, and the mechanism by which amoebae kill cells was previously unclear. We recently uncovered a new paradigm for cell killing by showing that amoebae kill human cells by performing trogocytosis (trogo‐: nibble), or taking ‘nibbles', of living human cells leading to cell death. In contrast, amoebae engulf dead (pre‐killed) human cells whole via phagocytosis (phago‐: devour). While there are some shared features between trogocytosis and phagocytosis, we hypothesize that amoebic trogocytosis requires signaling machinery distinct from phagocytosis. There is evidence that phagocytosis is under feed‐forward gene regulation in E. histolytica. We have shown that amoebae that are exposed to living human cells perform more trogocytosis after a second round of exposure to living human cells, suggesting that trogocytosis is likewise under feed‐forward gene regulation. To identify genes differentially involved in trogocytosis versus phagocytosis, we will feed E. histolytica live or dead human cells, respectively, to prompt genes that are involved in these processes to be up‐regulated. We will then perform RNA‐seq analysis to identify genes that have changed substantially in comparison to the reference RNA from amoebae that have not been fed human cells. From this, we expect to develop a list of candidate genes that are differentially important for either trogocytosis or phagocytosis, and genes that are important for both processes. To prepare for performing RNA‐seq analysis on amoebae that have undergone trogocytosis and phagocytosis, we have established amoebic ingestion conditions to maximize ingestion, quantified methods of digestion via imaging flow cytometry, and used qRT‐PCR to analyze gene expression before and after ingestion. After performing RNA‐seq analysis and generating a list of candidate genes that appear to be uniquely involved in trogocytosis, we will then perform functional studies in order to validate the roles of these genes in trogocytosis. Since trogocytosis appears to be a fundamental form of cell‐cell interaction that is present in many eukaryotes, and exaggerated and exploited for cell killing by eukaryotic microbes, this work will have broad impact.

Support or Funding Information

NIH MCB T32 GM007377; NIH NIAID K22 Career Transition Award Al108814

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structures of New Delhi Metallo‐Beta‐Lactamases in Pursuit of New Inhibitors
Authors: Morris Matthew , VanPelt Jamie , Page Richard
Published: June 2021

Antibiotic resistance is a cause of ever‐escalating concern throughout the global community. Antibiotics have been essential to the development of modern medicine and its techniques in the past two centuries; however, bacteria have developed mechanisms that have made many antibiotics almost entirely ineffective. Metallo‐Beta‐Lactamases (MBLs) confer resistance to carbapenems, cephalosporins, and penicillins in several clinically relevant Gram‐negative bacteria including Acinetobacter, Pseudomonas aeruginosa, and many Enterobacteriaceae. New Delhi Metallo‐Beta‐lactamases (NDMs) are amongst some of the most worrisome and prevalent MBLs. This family of MBLs is capable of hydrolyzing all generations of bicyclic beta‐lactams. Among this family, NDM‐1, NDM‐4, and NDM‐12 are key members. To combat these proteins, an inhibitor that targets and binds to the enzymes would be needed, preventing them from hydrolyzing antibiotics; however, not enough is known about the structure of the active site, which is crucial in order to develop specific inhibitors that could be used on patients. Herein we describe key differences between the residues in NDM‐1, NDM‐4, and NDM‐12 that alter biochemical activity of each enzyme, and biophysical stability. Together, our crystal structures of these NDM variants, including the first known structure of NDM‐12, biochemical analyses, and biophysical analyses provide a basis for understanding the roles of key residues surrounding the active site within NDM variants. Furthermore, we have determined the structure of NDM‐4 with a novel inhibitor that binds to the zinc ions at the active site. Our studies combine to provide a clearer picture of the biochemical and biophysical properties of New Delhi Metallo‐Beta‐lactamases and how we can exploit these properties to inform drug design efforts to develop new MBL inhibitors.

Support or Funding Information

NIH R01GM111926

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Class of PqsR Inhibitors to Reduce the Biofilm Formation of P. aeruginosa
Authors: Hossain Mohammad Anwar , Parikh Hardik I. , German Nadezhda
Published: June 2021

Microbial biofilms are significant contributors to the pathology of chronic infections. Chronic wounds, cystic fibrosis, diabetic ulcers, periodontitis, and endocarditis are relevant examples of chronic biofilm‐associated disorders. Pseudomonas aeruginosa is a gram‐negative bacterium that forms these biofilms in the wounds, causing increased resistance to antibiotics. It does so through the mechanisms associated with reduced permeability, decreased metabolic rate and altered genetic expression. Recent reports have shown that the use of combination therapy, where antibiotics are supplemented by compounds with the ability to disrupt biofilm integrity, increases the efficacy of the treatment and potentially reduces the rate of the resistance development. P. aeruginosa exerts its virulence program through three intertwined quorum sensing systems: LasR, RhlR and PqsR. Attenuation of the PqsR pathway was shown to affect the release of virulence factors, resulting in inhibited biofilm formation, and increasing the efficacy of antibiotics. In our research, we initiated a study that targeted identification of the structurally different compounds other than acyl homoserine lactone (AHL) moiety with antagonistic properties against PqsR. Using ligand‐based and structure‐based approaches, we performed virtual screening of various databases (ZINC, nine million drug‐like molecules) to identify new pharmacophore scaffolds with potential activity at PqsR. The top scored compounds were synthesized and evaluated against P. aeruginosa PAO1 as an inhibitor of biofilm formation. As a result, we have identified a novel series of PqsR inhibitors capable of reducing biofilm formation by 65% in a concentration range of 1 to 10 μM (in‐vitro static biofilm assay). At the same time, these compounds were shown not to interfere with the bacterial growth at the concentrations up to 200 μM. Altogether, our study identified a novel class of PqsR inhibitor with the ability to reduce the biofilm formation (in‐vitro) and to potentiate the efficacy of traditional antibiotics against P. aeruginosa associated infections.

Support or Funding Information

Texas Tech University Health Sciences Center start‐up fund of Dr. Nadezhda German

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insights into Glycogen Metabolic Inhibition‐Induced Death of Hepatocellular Carcinoma
Authors: Barot Shrikant , Abo‐Ali Ehab M. , Palaguachi Christian , Dukhande Vikas V.
Published: June 2021

Metabolic reprogramming is one of the prominent features of cancer. Cancer metabolic reprogramming offers various advantages for proliferation, invasion, metastasis, and chemoresistance. Cancer cells are highly glycolytic, a phenomenon termed as Warburg effect or aerobic glycolysis, with concomitant increase in the amount of glucose entering the cells. Glycogen is a storage form of glucose and the levels of glycogen have been found to change with biological processes in these reprogrammed cells. Glycogen Phosphorylase (Gp) is a rate‐limiting enzyme that catabolizes glycogen to glucose. The contribution of glycogen metabolism to carcinogenesis, cancer cell growth, metastasis, and chemoresistance is poorly understood. Thus, we aimed to mechanistically study the processes involved in GP inhibition‐induced hepatocellular carcinoma cell death. We pharmacologically inhibited glycogen catabolism in HepG2 cells and found that there was a decrease in cell viability in a dose‐dependent manner. Next, we measured the major markers for apoptosis, necrosis, and autophagy. Our results indicate that GP‐inhibition induces cancer cell death through apoptosis process. In addition, GP inhibition potentiates the effects of known anti‐cancer drugs. Our ongoing studies will identify the key molecular components involved in apoptotic and autophagic processes in glycogen inhibition‐induced cell death. Our study provides insights into cancer cell metabolism and identifies GP‐inhibition as a potential cancer pharmacology target.

Support or Funding Information

Supported by startup funds to Dr. Dukhande from St. John's University, College of Pharmacy and Health Sciences.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Unconventional Pathways of Nitrogen Metabolism in Lung Cancer
Authors: Kim Jiyeon
Published: June 2021

Lung cancer is the leading cause of cancer death worldwide. The critical barrier in lung cancer treatment is a lack of effective therapies at later stages of the disease. Lung cancer genome sequencing supports the idea that combinations of mutations act in concert to foster malignancy and disease progression. Among these, KRAS and LKB1 mutations represent “driver” changes in tumor development. Importantly, mutations in these genes also perturb signal transduction to promote a form of metabolism conducive to tumor growth. Metabolic reprogramming is considered to be fundamental to malignant transformation; thus, identifying molecular links between these mutations and metabolism, and targeting the resulting metabolic pathways, may produce better therapies.

By integrating metabolomics profiling with bioinformatics analysis, we discovered that cells and tumors harboring LKB1/KRAS co‐mutations share metabolomic signatures of perturbed nitrogen handling. In particular, KRAS/LKB1 co‐mutants are addicted to a urea cycle enzyme, carbamoyl phosphate synthetase 1 (CPS1). The selective dependence of KRAS/LKB1 co‐mutants on CPS1 is not due to the canonical activity of the enzyme. Instead, CPS1 stimulates an unconventional pathway of nitrogen flow from ammonia into pyrimidines. While further investigating why these cells need to recapture ammonia to initiate pyrimidine synthesis, we noted that co‐mutant human cells lines and mouse tumors up‐regulate the ‘hexosamine biosynthesis pathway (HBP)’ gene set. A focused RNAi screen and pharmacological inhibition of HBP showed that KRAS/LKB1 co‐mutants are selectively sensitive to suppression of Glutamine‐Fructose‐6‐Phosphate Transaminase 2 (GFPT2), a key enzyme in the HBP.

These experiments establish the functional significance of two nitrogen‐related metabolic pathways ‐ the urea cycle and the HBP ‐ in a select population of lung cancer cells. The results suggest a model where enhanced activity of the HBP limits the availability of glutamine to serve as a direct nitrogen for conventional pyrimidine biosynthesis, forcing the cells instead to recapture ammonia via CPS1. Altogether, the work identifies a novel metabolic co‐dependence in cancer and may point towards a new therapeutic strategy in KRAS/LKB1 co‐mutant tumors.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mogat1 is a fasting‐induced PPARα target gene that plays a role in coordinating the hepatic response to food deprivation
Authors: Lutkewitte Andrew John , McCommis Kyle S. , Chambers Kari T. , Graham Mark J. , Hall Angela M. , Finck Brian N.
Published: June 2021

During prolonged fasting, the liver plays a central role in maintaining systemic energy homeostasis by producing glucose and ketones in processes fueled by oxidation of fatty acids liberated from adipose tissue. In mice, this is also accompanied by transient hepatic accumulation of complex lipids in the form of triglycerides. In mice fasted for 24 hours, the hepatic expression of Mogat1 was significantly increased compared to mice given ad libitum access to food. Mogat1 encodes an enzyme with monoacylglycerol (MAG) acyltransferase (MGAT) activity, which is an understudied step in glycerolipid synthesis. Basal and fasting‐induced expression of Mogat1 was markedly diminished in liver of mice lacking the transcription factor peroxisome proliferator‐activated receptor α (PPARα). Suppressing Mogat1 expression with antisense‐oligonucleotides (ASO) reduced hepatic MGAT activity and hepatic triglyceride content compared to fasted ASO control treated mice. Surprisingly, the expression of many other known PPARα target genes (Dgat1, Mgll, Cpt1α, Acadm, and Acadl) was also decreased in mice treated with Mogat1 ASO. We postulated that inhibiting Mogat1 suppressed PPARα activity, potentially by loss of a ligand for PPARα. Fasted mice treated with control or Mogat1 ASO were gavaged with the synthetic PPARα ligand WY‐14643, which reversed the effects of Mogat1 ASO on liver triglyceride content as well as PPARα target gene expression. In conclusion, Mogat1 is a fasting‐induced PPARα target gene and MGAT and may feed forward to regulate liver PPARα activity during fasting.

Support or Funding Information

A.J.L. was supported by funds from NIH training grant T32 DK07120. K.S.M is supported by K99 HL136658 and A.M.H. is supported by 17‐IBS‐109 from the American Diabetes Association and R56 DK111735. This work was also funded by R01 DK078187 to B.N.F. The Core services of the Diabetes Research Center (P30 DK020579) and the Nutrition Obesity Research Center (P30 DK56341) at the Washington University School of Medicine also supported this work.

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 Elevated ω‐3/ω‐6 PUFA ratio on High‐Fat Diet and Acute Ethanol‐induced Obesity, Glucose Intolerance and Liver Injury in Mice
Authors: Dastidar Shubha Gosh , Warner Dennis , Song Ying , Warner Jeffrey , McClain Craig , Kirpich Irina
Published: June 2021

Rationale and Objectives

Nonalcoholic and alcoholic fatty liver disease (ALD and NAFLD) are both serious health and socioeconomic problems worldwide, with similar pathological spectra, ranging from simple hepatic steatosis to steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. ALD and NAFLD have also several similar risk factors, including certain dietary factors and obesity. Human and animal studies have demonstrated that a Western diet, rich in ω‐6 polyunsaturated fatty acids (PUFAs) and low in ω‐3 PUFAs, exacerbates NAFLD and ALD. In addition, obese alcohol drinkers might be at an increased risk of developing more severe liver injury. The objective of the present study was to investigate whether a decrease in ω6 PUFAs with subsequent increase in the ω‐3/ω‐6 PUFA ratio ameliorates metabolic derangements and liver injury in a two‐hit model of long‐term HFD feeding plus a single EtOH binge administration.

Methods

Female fat‐1+/− (mice with the ability to endogenously convert ω6 to ω3 PUFA) and WT littermates were fed low or high fat diets (LFD and HFD, 10% and 60%, respectively) for 12 weeks. On the last day of the experiment, mice received a single dose of EtOH (5 g/kg) by oral gavage. Animals were euthanized 9h‐post gavage. Metabolic parameters and liver injury were evaluated.

Results

Long‐term HFD feeding resulted in alterations of metabolic parameters in both genotypes. However, compared to WT, fat‐1+/− gained significantly less body weight (BW), and had less parametrial fat at the end of the study. This could be due to significantly lower food consumption in fat‐1+/− mice. Liver weight/BW ratio was equally reduced in HFD‐fed mice compared to LFD. While the type of the diet did not affect the pancreas weight/BW ratio, ethanol administration resulted in an opposite effect on this parameter in WT (increase) and fat1−/+ mice (decrease). Analysis of liver injury revealed that HFD led to similar induction of ALT levels (a marker of liver injury) in both genotypes, which was further equally elevated by ethanol administration. However, ethanol challenge resulted in significant increase in ALT levels in LFD fed mice in WT but not fat1−/+ littermates.

Conclusions

Increase in endogenous ω‐3/ω‐6 PUFA ratio was not beneficial against HFD and binge EtOH‐induced liver damage (two‐hit model), however, mice fed a LFD were protected against EtOH‐mediated liver injury. The molecular mechanisms underlying these effects needs to be further investigated.

Support or Funding Information

Supported by grants from NIH and Veterans Administration

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting molecular chaperone HSP90 to treat Niemann‐Pick type C1 disease
Authors: Pipalia Nina H. , Maxfield Frederick R.
Published: June 2021

NPC1 is a fatal, lysosomal neurovisceral storage disease caused by mutations in the npc1 gene. More than 200 different disease‐associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids and other lipids in late endosome/ lysosomes (LE/Ly) of many cell types. There is no FDA‐approved therapy to treat NPC1 disease. A limited initial human trial of hydroxyl propyl cyclodextrin (HPCD) is currently in progress, but HPCD does not cross the BBB. Based on our previous findings on NPC1I1061T skin fibroblast, a small clinical trial is also in progress on the treatment of histone deacetylase inhibitor (HDACi), Vorinostat. Our data suggested that HDACi treatment on skin fibroblasts results in increased expression of the Niemann‐Pick type C1 (NPC1) protein in mutant cells. Using an engineered human cell line, we have also observed that HDACis, can correct the NPC1 cholesterol storage defect in 60 of the 81 NPC1 mutants tested. Although significant progress has been made in understanding the pathology of NPC1 disease, the mechanisms governing the proteostatic control of NPC1 protein is not completely understood. However, being an epigenetic modifier, HDACi is known to alter the pattern of gene expression. As a result, off‐target effects are of a major concern in using HDACi's as a therapy. In order to understand the molecular mechanism of HDACi on NPC1, we considered potential cellular targets of HDACi other than histones. Disruption of molecular chaperone HSP90 (via hyperacetylation) is one of the many consequences of HDACi treatment.

We, therefore, evaluated the effect of direct inhibition of HSP90 family of proteins on NPC1 skin fibroblasts in terms of lowering lysosomal cholesterol accumulation. Our results indicated that out of the three HSP90 family proteins, selectively HSP90A and not HSP90B or TRAP inhibition resulted in clearance of cholesterol from lysosomes as well as aided in mobilizing unfolded mutant NPC1 protein from ER to lysosomes. Hence the data on the effect of several different HSP90 inhibitors on multiple human patient‐derived skin fibroblast in lowering lysosomal cholesterol burden using microscopy based filipin assay will be presented. Our previous published work using HDACi and recent preliminary results using heat shock protein HSP90 inhibitors suggests that cytosolic HSP90 and associated chaperones may be a target and indicates that protein folding quality control plays a critical role in NPC1 diseases. An attempt will be made to elucidate the role of cytosolic HSP90 and its associated co‐chaperones in the regulation of NPC1 mutant protein stability.

Support or Funding Information

Ara Parseghian Medical Research 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.