bluuebunny/crossref_metadata_2025_split huggingface dataset:
YKL40: a key modulator of the CF inflammatory response?
Authors: Bouvet Guillaume F , Bulka Olivia , Coriati Adèle , Massé Chantal , Berthiaume Yves 
Published: November 2023

YKL‐40 is a human circulating chitinase‐like protein and inflammatory biomarker whose levels are elevated in the blood serum of cystic fibrosis (CF) patients and CF patients with dysglycemia. We explored the effects of treatment with YKL‐40 on peripheral blood mononuclear cells (PBMC) isolated from the whole blood of 70 CF patients, in the context of the inflammatory gene response. The relative expressions of genes for both proinflammatory and immunosuppressive molecules in response to YKL‐40 treatment were quantified and analyzed in conjugation with biological characteristics obtained from each patient, including forced expiratory volume (FEV1%), a functional measure of pulmonary ability or disease progression, as well as sex and glycemic status. We found significant positive correlations between the expression of IL‐10 and TGF‐b in response to YKL‐40. Additionally, the FEV1% was found to be negatively correlated with IL‐10 expression. After analysis against sex and glycemic status, we found that a significant proportion of the FEV1%low/IL‐10high group consisted of dysglycemic women. These correlations suggest that as the degree of pulmonary function decreases across patients, CF‐PBMC express IL‐10/TGF‐b gene response to YKL‐40, and that dysglycemic women with CF are more predisposed to developing this phenotype in response to elevated YKL‐40 in the peripheral blood. In addition to being a proinflammatory protein and a biological marker of disease evolution, YKL‐40 looks to act as a complex inflammatory modulator in CF.Support or Funding InformationCystic Fibrosis Canada
Elucidating the Effects of Antibiotics on the Release of Pal from Escherichia coli and P6 from Nontypeable Haemophilus influenzae
Authors: Fernandez Nicole , McNamara Carlie , Pichichero Michael , Hellman Judith , Michel Lea 
Published: November 2023

Outer membrane protein P6 is a lipoprotein from nontypeable Haemophilus influenzae (NTHi), a Gram‐negative bacterium and the cause of acute otitis media (ear infections) across the world and sepsis in developing countries. P6's homologue in Escherichia coli (E. coli), Pal, has been shown to be implicated in the pathogenesis of sepsis through its release from E. coli. We hypothesized that P6 might also be released from NTHi during sepsis. To elucidate the effects of antibiotics on the release of Pal from E. coli and P6 from NTHi, we performed in vitro release experiments in human serum. Bacterial cells grown to mid‐log phase were incubated with healthy human serum and/or various antibiotics. Released Pal and P6 were detected using immunoblotting. Preliminary data suggest that antibiotics greatly enhance Pal's release from E. coli, and P6 is minimally released from NTHi in the presence of certain antibiotics.Support or Funding InformationThe Camille and Henry Dreyfus Foundation: Special Grant Program in the Chemical Sciences (L. Michel)
The Effects of Thapsigargin and Concanavalin A on Jurkat T‐Cell Interleukin‐2 (IL‐2) Production: A Preliminary Study
Authors: Lee Ruth M , Jones Ward 
Published: November 2023

Interleukin‐2 production is a key indicator of T cell activation. Specifically, IL‐2 leads to T cell expansion and signal amplification in vivo. Thus, understanding the control of IL‐2 production is important to T cell differentiation. Previous studies suggest a combination of phorbol myristate acetate (PMA) and Thapsigargin (TG) increase IL‐2 production (Sei and Reich et al. 1995). TG works by mimicking Inositol Tri‐Phosphate (IP3) and PMA works by mimicking Diacylglycerol (DAG). Together, both TG and PMA work to bypass the original pathway and activate Protein Kinase C (PKC). However, PKC will not activate when TG or PMA are used alone. Concanavalin A (ConA) activates T‐Cells by binding to cell‐surface glycoproteins. In addition, ConA and PMA are known to activate IL‐2 production. However, it is unknown whether TG, when paired with ConA, will stimulate production of IL‐2. We proposed that TG and ConA, when used simultaneously, would increase IL‐2 levels. Preliminary ELISA data suggests elevated ConA increases IL‐2 production while high concentrations of TG inhibit ConA induced IL‐2 production.Support or Funding InformationAll funding came from Viterbo University's Summer Research Fellowship.
HIV‐1 gp120 JRFL Mediated Human α7 nAChR's Up‐regulation and its Implications in the Cholinergic Anti‐inflammatory Response (CAP)
Authors: Cotto Sonnieliz , Colon Jose Orlando , Quesada Orestes , Lasalde Jose Antonio 
Published: November 2023

In 2014, there were approximately 36.9 million of people living with HIV‐1 (UNAIDS, WHO). Of those only 15 million people had access to antiretroviral therapy. However, despite the successful reduction of the development of HIV/AIDS in patients receiving therapy, this population is at a higher risk to develop non‐AIDS related diseases, which are often associated with inflammatory process. It is known that HIV‐1 gp120 proteins can cause increased cytokines release that generates sustained immune activation in macrophages, which are the principal target for HIV‐1 derived R5‐ tropism strains. In our laboratory, we recently demonstrate that in human macrophages, gp120 (X4‐ derived strains) protein induces the up‐regulation of the α7 nicotinic acetylcholine receptor (α7‐nAChR), a key regulator of systemic inflammation that inhibits the production of pro‐inflammatory cytokines. Moreover, recent findings suggest that inflammation processes in HIV positive patients are characterized by high levels of α7‐nAChR in macrophages, up regulation that resulted in cholinergic anti‐inflammatory pathway (CAP) disruption. Our project therefore, examines the functional relationship between the findings with the principal objective of determining whether HIV‐1 gp120JRFL (R5‐strain) pre‐incubation, that up‐regulate α7‐nAChR in macrophages, may also affects the CAP and the mechanism that mediates this up‐regulation.We previously demonstrated that gp120IIIB disrupt this signal transduction pathway through α7 nAChRs up‐regulation however the mechanism has not been reported. Here we show that M‐tropic gp120JRFL, induces a higher level of α7 nAChRs protein in human derived macrophages leading to CAP response disruption by a previous unknown mechanism. Our results demonstrated that HIV gp120 /CD4‐CCR5 receptor complex promotes α7 nAChRs up‐regulation via a mechanism that is associated with the activation of p38‐MAPK signaling that has been shown to up‐regulates early gene (Egr1), a transcription factor known to drive the expression of α7 nAChRs. Using α7 antagonist, bupropion attenuation of pro‐inflammatory cytokines production was prevented. In contrast, treatment with Maraviroc (CCR5, receptor antagonist), results in a reduction of IL‐8 and I‐309 pro‐inflammatory cytokines levels in up‐regulated MDMs but did not affect CAP functioning in terms of ILs. Taking together, these findings suggest that HIV‐1 glycoproteins can conferred different macrophages phenotypes, in terms of the α7 nAChRs up‐regulation mediated by gp120JRFL, normal functioning of the cholinergic anti‐inflammatory response control the excessive immune responses. Hence, our data demonstrate the possible molecular mechanism that may be responsible for α7 nAChRs up‐regulation that failed in inhibiting the release of pro‐inflammatory cytokines.Support or Funding InformationNHI Grant Number 2R25GM061151‐16
Lentivirus‐mediated RNA Interference Knockdown of Inositol Requiring 1 Enhances Sorafenib Lethality in Hepatoma Hep3B Cells Involving Autophagy Inhibition
Authors: Hu Ye , Ye Hui‐Lan , Zhang Guo , He Li‐Jun , Liu Ze‐Feng , Chen Li 
Published: November 2023

Inositol requiring 1 (IRE1), a component of the unfolded protein response signaling pathway, has been found to be correlated with sorafenib resistance in hepatocellular carcinoma (HCC). The present study aimed to investigate the effects of lentivirus vector mediated short hairpin RNA interference knockdown of IRE1 on hepatoma Hep3B cells and explore the possibility of IRE1 silencing as a therapeutic target for HCC. Firstly, three pairs of human IRE1 targeted specific interference sequences tagged with green fluorescent protein were designed, synthesized and subcloned into lentiviral vectors(pL‐shRNA‐IRE1‐GFP) successfully, which were further identified by PCR and sequencing. Then, recombinant and lentiviral packaging plasmids were co‐transfected into 293T cell lines effectively in order to produce lentiviral particles using fluorescence determined the titer. After stable infected Hep3B cell lines established with pL‐shRNA‐IRE1‐GFP and pL‐GFP‐Negative control at the optimum multiplicity of infection, flow cytometry was used to sort green fluorescent protein‐positive cells which showed a very high degree of positive rate. QPCR and western blot analysis were employed to identify the interference silencing sequence with the most efficient silencing profile. Results showed that the expression profiles of IRE1 mRNA and protein were significantly lower (P<0.05) in the specific interference group (LV142‐pL‐shRNA‐IRE1‐GFP) than that of the control group with a maximum of 90% inhibition. Although there was no significant difference of proliferation, growth as well as apoptosis in basal condition, sorafenib (5μM) was found to cause a substantial increment about 50% on cell death in IRE1 silencing LV142 cells vs control. Interestingly, mechanistic investigations showed that such a stronger sorafenib lethality was coupled with decreasing expression of autophagic molecule light chain 3 (LC3)‐II as well as accumulation of p62 in these cells. Collectively, these data suggests that RNA interference mediated IRE1 knockdown might enhance sorafenib lethality in hepatoma Hep3B cells involving autophagy inhibition, which brings insight into a novel and potential HCC therapeutic strategy in the future.Support or Funding InformationSupported by NSFC Grants 881172260, 30630145, 81360077 and GXNSFC Grants 2015GXNSFDA139022 to Guo Zhang
TRPM7 kinase domain rather than the channel regulates breast cancer cell migration and tumor metastasis
Authors: Kaoud Tamer S , Xie Xuemei , Mangieri Regina A. , Park Jihyun , Tavares Clint D.J. , Ebelt Nancy D , Cano Micael , Van Ravenstein Sabrina , Mitra Shreya , Radwan Mohamed F. , Morrisett Richard A , Bartholomeusz Chandra , Dalby Kevin N 
Published: November 2023

The channel‐kinase TRPM7 (transient receptor potential melastatin 7) is a bifunctional protein consisting of a cation channel that is permeable to Mg2+, Ca2+ and Mn2+ ions fused to a C‐terminal kinase domain. A growing number of studies with clinical significance suggest that TRPM7 is linked to adhesion and migration of breast cancer cells and promotes breast tumor metastasis. While the channel properties of TRPM7 have been studied extensively, little is known about its kinase activity and how it is linked to the control of actomyosin contractility. To understand the functions of the kinase domain we identified the first cell‐permeable inhibitor of the kinase domain (TRPM7‐IN‐1) and developed MDA‐MB‐231 cell lines in which TRPM7 is knocked out by CRISPR/Cas9 (KO), and in which various forms of TRPM7 were stably re‐expressed. These were wild type TRPM7 (WT), a kinase‐inactive mutant of TRPM7 (KD), and TRPM7 containing a truncated kinase domain (KT). TRPM7 KO significantly inhibited MDA‐MB‐231 cells migration. Only expression of the wild type TRPM7 (WT) rescued the migration phenotype, supporting a role for the kinase domain and not the channel in the regulation of cell migration. TRPM7‐IN‐1 decreased the binding of Myosin IIB to TRPM7 in HEK293 and MDA‐MB‐231 cells. And when MDA‐MB‐231 cells were treated with increasing doses of TRPM7‐IN‐1, TRPM7 phosphorylation at Ser‐1569 and its downstream Myosin IIa phosphorylation at Ser1943 were completely abrogated at a concentration of 5 μM. TRPM7‐IN‐1 inhibited MDA‐MB‐231 and BT 549 cell migration and invasion, while treatment of the KO cells with TRPM7‐IN‐1 showed no further inhibition of migration. Electrophysiological assessment of the TRPM7 channel revealed that the inhibitor did not affect the channel function in MD‐MB‐231 cells, supporting the notion that the inhibitor affects the migration exclusively through the inhibition of the TRPM7 kinase domain. Finally, in an experimental metastasis model, TRPM7‐IN‐1 significantly impeded metastasis to the lung. In conclusion, inhibition of TRPM7 kinase activity may reduce or block breast tumor progression and/or metastasis.Support or Funding InformationFinancial support by grants from the National Institute of General Medical Sciences (R01GM059802), the Welch Foundation (F‐1390) and Cancer Prevention and Research Institute of Texas to KND.
The Tyrosine Phosphatase PRL3 Drives T‐Cell Acute Lymphoblastic Leukemia Progression
Authors: Sieg Rachel Elizabeth , Lammers Sydney , Hausman Shea , Blackburn Jessica 
Published: November 2023

T‐cell acute lymphoblastic leukemia (T‐ALL) is a major clinical challenge, representing 15–25% of all acute lymphoblastic leukemias in children and adults. The survival rate for pediatric patients with relapsed T‐ALL is a disheartening <30% in pediatric patients and <10% in adults. Current drugs against T‐ALL include highly cytotoxic chemotherapies, which are generally ineffective against relapse and are particularly hard for young children to receive. These harsh therapies can result in a spectrum of developmental issues and potent side‐effects. Therefore, it is imperative to develop molecularly targeted therapies that can effectively inhibit T‐ALL progression without contributing to the adverse side‐effects of general chemotherapy.We have found that zebrafish T‐ALL cells acquired amplification and high expression of the Protein Tyrosine Phosphatase 4A3 (PRL3) concurrent with evolution of enhanced relapse potential in a transplantation screen involving over 6,000 animals. Further, approximately 50% of human T‐ALL patients have high PRL3 expression. PRL3 has not been associated with leukemia onset or progression, but here we show overexpression of PRL3 leads to increased T‐ALL onset and aggressiveness in an in vivo transgenic zebrafish T‐ALL model. Small molecule inhibitors of PRL3 significantly reduced viability of established cultures of human T‐ALL cells in vitro. Based on these data, we hypothesize that PRL3 plays an important role in T‐ALL cell survival at all stages of leukemia progression. We are currently using fluorescent and confocal microscopy in zebrafish over‐expressing PRL3 to determine the cellular mechanisms by which PRL3 enhances leukemia onset. We are also generating PRL3 knockout lines using a CRISPR‐Cas9 for similar studies. Finally, we are treating zebrafish bearing T‐ALL with two specific, research‐grade PRL inhibitors to determine if blocking PRL3 phosphatase activity prevents leukemia onset and relapse. We have also identified several FDA‐approved, general phosphatase inhibitors that have potent anti‐PRL activity and are capable of killing ALL cells in vitro. Current work is focused on screening these inhibitors in vivo in our zebrafish models.We conclude that PRL3 is a novel gene that may be important for T‐ALL onset and progression. As a phosphatase, it is a unique druggable target. Future directions will focus on identifying the molecular mechanisms by which PRL3 drives leukemia progression, and moving top drug hits from our zebrafish screens into pre‐clinical testing in patient derived xenografts models in mice. We hope that our findings may lead to eventual clinical trials as alternatives to the traditional cytotoxic chemotherapy offered to pediatric T‐ALL patients now.Support or Funding InformationNCI 4R00CA181500NIGMS P30GM110787The V Foundation for Cancer Research
Characterization of Asialoglycoprotein Receptor (ASGPR) Directed Hepatocellular Delivery Using a Pfizer Developed Targeting Ligand PF‐06853291
Authors: Roy Marc , Finley James , Coskran Timothy , Shen Amy , Xia Shuhua , Thuma Benjamin , Mascitti Vincent 
Published: November 2023

Asialoglycoprotein receptor (ASGPR) is a transmembrane protein that plays a critical role in serum glycoprotein homeostasis by mediating the endocytosis and lysosomal degradation of glycoproteins with exposed terminal galactose or N‐acetylgalactosamine. This receptor is highly expressed on hepatocytes while extra‐hepatic expression is minimal making the receptor an ideal candidate for targeted delivery of therapeutics to the liver (D'Souza, 2015).PF‐06853291 is an ASGPR ligand, discovered at Pfizer, conjugated to a fluorophore (Alexa Fluor® 647) and PF‐06868566 is a trivalent version of the natural ASGPR ligand N‐acetylgalactosamine (GalNAc) conjugated to the same fluorophore. This trivalent GalNac ligand is presently the standard for ASGPR mediated hepatic delivery both in preclinical studies and clinical trials (Chan, 2015; Rajeev, 2015; Simon, 2015). Demonstrating a more desirable hepatocellular delivery profile of the Pfizer ligand over current ligands in use would bolster confidence in using our internal ligand for hepatic delivery of therapeutics, including macromolecules. To this end, we have evaluated the hepatic bio‐distribution and pharmacokinetics of PF‐06853291 and PF‐06868566 in vivo following administration of a single intravenous (IV) dose in male C57BL/6N mice.Data from this study confirms each compound achieves similar serum exposure at early time points, and is rapidly cleared by a combination of liver accumulation and renal clearance. Quantification of PF‐06853291 and PF‐06969566 liver exposure by multiple endpoints demonstrates that the Pfizer trivalent ASGPR ligand facilitates increased ASGPR‐directed hepatocellular uptake and prolonged retention compared to the trivalent GalNAc. These results confirm the superiority of the Pfizer ASGPR ligand in hepatocellular delivery.
The Development of Novel Small Molecule Inhibitors of Listeria monocytogenes and Staphylococcus aureus Pyruvate Carboxylase
Authors: Wyatt Brittney N , Matloub Sara , St. Maurice Martin 
Published: November 2023

Staphylococcus aureus and Listeria monocytogenes are pathogenic bacteria that can cause serious infections for immunocompromised individuals1, 2. Bacterial metabolism has been linked to pathogenicity and, in particular, the metabolic enzyme pyruvate carboxylase (PC) has been shown to be an important contributor to L. monocytogenes virulence1, 2. PC is a metabolic enzyme that catalyzes the conversion of pyruvate to oxaloacetate (OAA) in a wide range of organisms3. Small molecules directed against PC would better reveal the cellular role of this enzyme in bacterial metabolism and have the potential to be developed into antimicrobial agents. However, there are currently no known specific or potent small molecule regulators of PC activity. We aim to find, develop, and characterize novel small molecule effectors of PC that will aid in understanding the structure, mechanism, and regulation of this enzyme and will ultimately lead to new chemical probes to enable studies on PC in its cellular context. A novel fixed‐time assay has been developed based on the reaction of OAA with the diazonium salt, Fast Violet B (FVB), which produces a colored complex with an absorbance maximum at 530 nm. The fixed time assay is reproducible, sensitive and responsive to known effectors of Rhizobium etli PC (RePC), Staphylococcus aureus PC (SaPC), and L. monocytogenes PC (LmPC). The assay has been used to assess specific subsets of rationally designed inhibitors as well as in an unbiased high‐throughput screen. The assay was validated in a plate uniformity test and in an initial pilot screen of 1,280 compounds. We subsequently screened over 13,000 compounds against RePC, LmPC and SaPC. Using a combination of approaches, we have found novel small molecule inhibitors of LmPC and SaPC that have the potential to be developed into valuable research tools and potential antimicrobial agents.Support or Funding InformationThis research has been funded by the GAAAN Fellowship through the United States Department of Education and the Marquette University Strategic Innovation Fund.
Synthesis of potential AAC(6′)‐Ib inhibitors to combat bacterial resistance to aminoglycoside antibiotics
Authors: Simes Miranda , Labby Kristin Jansen , Johnson Kathryn 
Published: November 2023

One of the greatest threats to human health is the rise of antibiotic resistance in pathogenic bacteria, and failure of antibiotic treatment is becoming far more common. Aminoglycosides, a family of antibiotics, are far too often being rendered ineffective by bacterial aminoglycoside modifying enzymes. Of the various types of aminoglycoside modifying enzymes, the most common in clinical isolates is the AAC(6′)‐Ib enzyme. In order to combat aminoglycoside modification by the AAC(6′)‐Ib enzyme, previous work has screened potential small molecule inhibitors and identified a lead molecule inhibitor. We are working to synthesize and test the potency of this lead inhibitor on the AAC(6′)‐Ib enzyme. Recently, the synthesis and purification of the lead inhibitor were optimized. Currently, work is underway to synthesize a library of derivatives of the lead inhibitor to also test on the AAC(6′)‐Ib enzyme, which will aid in determining structure‐activity relationships between the inhibitor and the AAC(6′)‐Ib resistance enzyme.Support or Funding InformationBeloit College Biomedical Research Scholars Program provided funding for this work.
MsbA as a Drug Discovery Target for Compounds with Antibacterial Activities
Authors: LaVigne Collette A , McCormick James W , Vogel Pia D , Wise John G 
Published: November 2023

There is an ever increasing need for novel antibiotics today as many bacteria adapt and become resistant to commonly used antibiotics. MsbA is a prokaryotic integral membrane protein, one of the ATP‐binding cassette (ABC) transporters, and transports lipid A from the outer to the inner leaflet of the bacterial plasma membrane. MsbA is essential in gram‐negative bacteria for proper membrane formation and might therefore be viewed as an antibiotic target. The fact that MsbA also transports a wide variety of drug‐like compounds across the membrane, however, complicates the search for effective inhibitors. In efforts similar to those described in (Brewer et al. Mol Pharmacol. 2014) for the search for inhibitors of the multidrug resistance protein, P‐glycoprotein, computational screens were performed to search for drug‐like compounds that effectively inhibit MsbA ATP hydrolysis without being themselves transported by it. Identified compounds were purchased and tested in both in vivo and in vitro models to determine efficacy. A His6‐tagged MsbA was overexpressed and purified from E. coli and reconstituted into nanodiscs, where MsbA has been shown in the past to have high catalytic activity. ATP hydrolysis rates of MsbA were measured in the presence and absence of potential inhibitors using a coupled enzyme assay. E. coli growth assays with in the presence of identified compounds were used as an in vivo model to evaluate potential growth inhibition. Synergistic effects were also studied using growth assays where potential inhibitor compounds were administered in addition to sub‐lethal concentrations of known antibiotics. Preliminary results suggest that several compounds identified by the computational screening inhibited MsbA ATP hydrolysis activity. Further experiments will aim to better characterize and optimize these compounds for potential use as novel antibiotics.Support or Funding InformationThis work is supported by the SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.