bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationLaboratory Support and Faculty Development Award, College of Health Sciences and Technology, Rochester Institute of TechnologyThis 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 InformationNIH MCB T32 GM007377; NIH NIAID K22 Career Transition Award Al108814This 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 InformationNIH R01GM111926This 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 InformationTexas Tech University Health Sciences Center start‐up fund of Dr. Nadezhda GermanThis 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 InformationSupported 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.