bluuebunny/crossref_metadata_2025 huggingface dataset:
Attenuation of Glia Maturation Factor Enhances Mitochondrial Biogenesis, Decreases Oxidative Stress and Apoptotic Cell Death in N27 Dopaminergic Neurons
Authors: Selvakumar Govindhasamy Pushpavathi , Kempuraj Duraisamy , Thangavel Ramasamy , Ahmed Mohammad Ejaz , Zaheer Smita , Iyer Shankar S , Zaheer Asgar
Published: November 2023

Parkinson's disease (PD) is a progressive loss of dopamine producing neurons in the brain, that leads to motor complications. But the exact molecular events underlying the neuronal death are still not known. Glia maturation factor (GMF), a brain specific protein, plays a crucial role in PD progression. Mitochondrial abnormalities and oxidative stress triggers apoptotic dopaminergic degeneration in PD. Peroxisome proliferator activated receptor‐gamma coactivator‐(PPARGC‐1α or PGC‐1 α) acts as a key regulating factor for the mitochondrial biogenesis and respiration in dopaminergic cells in PD. We examined if attenuation of GMF expression enhances the expression of PGC‐1 α and reduces oxidative stress, mitochondrial dysfunction and apoptotic cell death in dopaminergic neurons. N27 dopaminergic neuronal cells were treated with GMF (100 ng/ml) for 24 h and analyzed for PGC‐1α expression by immunocytochemistry. Cell lysates were also prepared from these cells to estimate oxidative stress markers (glutathione and superoxide dismutase) using microplate reader, and expression of apoptotic (Bax cytochrome‐c, caspases 3, 7, 8,9) and anti‐apoptotic (Bcl‐2) markers by immunoblotting. GMF treatment for 24 h reduces the expression of PGC‐1α and mitochondrial complexes in N27 cells as compared with control cells. GMF also increased oxidative stress and depolarized mitochondrial membrane potential (MMP) in these cells. Furthermore, GMF increases Bax and reduces Bcl‐2 expression, stimulates the release of cytochrome‐c leading to activation of initiator procaspase‐9 and effector procaspases in N27 cells. These results suggest a crucial role of GMF in mitochondrial abnormalities and oxidative stress‐mediated apoptosis through PGC‐1α in dopaminergic neurons. We conclude that GMF is a critical factor for PD pathology and may be a potential therapeutic target to inhibit PD progression.

Support or Funding Information

This work was supported by Veteran Affairs Merit Award I01BX002477 and National Institutes of Health Grants AG048205 and NS073670
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 Information

Cystic 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 Information

The 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 Information

All 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 Information

NHI 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 Information

Supported 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 Information

Financial 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 Information

NCI 4R00CA181500

NIGMS P30GM110787

The 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 Information

This 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 Information

Beloit College Biomedical Research Scholars Program provided funding for this work.
Phagosomal Copper Triggers a Peptidomimetic's Oxidative Activity and Enables Eradication of Intracellular Mycobacterium tuberculosis
Authors: Libardo Mark Daben Javate , Anand Kushi , Krishnamoorthy Gopinath , Kaufmann Stefan H.E. , Singh Amit , Angeles‐Boza Alfredo
Published: November 2023

Copper, due to its inherent antimicrobial properties is enriched at sites of infection, particularly in mycobacteria‐containing phagosomes. However, recent studies show that M tuberculosis (Mtb) has multiple copper detoxification pathways allowing it to persist inside macrophages. A thorough in vitro investigation also showed that Mtb is highly susceptible to Ascorbic acid‐induced Reactive Oxygen Species (ROS). Using these information, we designed an all‐D‐amino acid antimicrobial peptide (DL‐10, DVIHRAGLQFPVGRVHRLLRK‐NH2) containing the Amino Terminal Copper and Nickel (ATCUN) Binding Unit Val‐Ile‐His. The ATCUN Motif binds strongly to cupric ions to form a metallopeptide that directly generates ROS under physiological conditions. We find that DL‐10 displays high activity against Mtb both in vitro and inside THP‐1 macrophages. This antimycobacterial activity was found to be linked to levels of free copper and molecular oxygen. Using a genetically encoded fluorescent sensor, we found that DL‐10 generates oxidative stress in M smegmatis (Msm), leading to efficient DNA cleavage. Incidentally, the oxidative burst coincides with the onset of Msm killing, further implicating ROS in its bactericidal action. DL‐10 is internalized by RAW264.7 macrophages via COP1‐mediated endocytosis. Using strategically positioned fluorophores along the DL‐10 primary structure, we were able to confirm DL‐10 copper binding inside macrophages, while, Isothermal Titration Calorimetry revealed a 500 nM Kd of binding. Moreover, by confocal microscopy, we found that DL‐10 effectively co‐localizes with intracellular Msm. This suggests that DL‐10 likely utilizes the endogenous phagosomal copper pool to kill intracellular mycobacteria. Finally, DL‐10 exhibits low cytotoxicity against RAW264.7 macrophages up to 128 uM. The high potency and the fact that DL‐10 circumvents proteolytic cleavage suggests that it is a good drug candidate. Our study also reveals an efficient way of sterilizing mycobacterial infections using host‐derived metal ions.
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 Information

This 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.
Analysis of Lysin A from Two Novel Mycobacteriophages
Authors: Surendranathan Nandini
Published: November 2023

As antibiotic resistant bacteria increase in prevalence, bacteriophage have been studied as an alternative to traditional antimicrobials. Through the Phage Hunters project funded by the Howard Hughes Medical Institute (HHMI), students have discovered over fifteen hundred novel bacteriophages that infect Mycobacterium smegmatis (M.Smeg), a bacterium related to Mycobacterium tuberculosis. At Montclair State University, the program has resulted in the isolation and genomic sequencing of numerous phage variants over the past five years. However, while multiple phages have been identified and sequenced, the genes required for their infectivity have not been studied as extensively. In order to gain a better understanding of how these bacteriophage function, this project has focused on the bioinformatic analysis and protein characterization of one gene, Lysin A, in two isolated bacteriophage, ShiVal and TeardropMSU. Lysin A is a protein that enables the virus to break out of the host and has the potential to serve as an antibiotic in its own right. ShiVal is part of the mycobacteriophage cluster B, the second largest group, while TeardropMSU is member of the mycobacteriophage cluster E. Structurally, Lysin A requires a murimidase homology sequence as well as a Zn+ binding site and peptidoglycan binding site that enables the enzyme to hydrolyze peptidoglycan. The bioinformatics data indicates that, while some of these critical regions appear in both sequences, there is only a three percent identity between Lysin A of the two bacteriophage which suggests that both lysins could be structurally different from one other. In addition to analyzing sequence comparisons between the two genes, Lysin A was directly isolated from ShiVal and TeardropMSU using PCR. TeardropMSU Lysin A was inserted into a cloning vector to allow for insertion into an expression vector. Protein expression and analysis of these two forms of the protein will allow us to clarify the mechanism of Lysin A activity and determine how the structural differences impact enzyme function.

Support or Funding Information

I would like to thank the Science Honors Innovation Program (SHIP) and the department of Biology and Molecular Biology at Montclair State University.
Association of Acid Phosphatase‐1 (ACP1) Gene Polymorphism with MAI infection.
Authors: ANDAVOLU Radhika Gade , Al Sarabi Rubakovic Anastasia , Rodriguez Olivia Gade , Stafford Catherine
Published: November 2023

Human acid phosphatases are normally found at low concentrations. However, pronounced changes in their synthesis occur in particular diseases, where unusually high or low enzyme expression is seen as part of the pathophysiological process. Acid Phosphatase locus 1 (ACP1) is a polymorphic enzyme controlled by a locus on chromosome 2 with three common codominant alleles: *A, *B, and *C. ACP1 shows two major isoforms, F and S. Genetic polymorphisms could contribute to natural resistance or susceptibility to the disease. Genetic screens in humans link polymorphisms in or near the ACP1 locus to a variety of inflammatory diseases including allergy and asthma. ACP1 is involved in activation, adhesion, and differentiation of T cells. Though few studies have shown the presence of ACP in human tissues, no studies have been reported linking ACP1 gene polymorphism to susceptibility to infections. In this study we investigate the role of ACP1 gene polymorphism in patients infected with Mycobacterium avium and normal healthy subjects. Mycobacterium avium complex (MAC) consists of two species: M avium and M intracellulare; because these species are difficult to differentiate, they are also collectively referred to as Mycobacterium avium‐intracellulare (MAI). Mycobacterium avium is an environmental microorganism which also exists in multiple hosts, including humans. M. avium can invade and replicate in immunocompromised hosts and also enhances permissiveness for HIV‐1 infection. DNA was extracted from blood and PCR‐RFLP method was used to detect ACP‐1 gene polymorphism. Our study showed high frequency of ACP AB haplotype (44%) in patients with infection than in individuals with no infection (24.8%) showing a significant association with infection (p<0.006). This is the first study to show an association of ACP1 gene with MAI. Our study highlights the role of gene/s polymorphism and their possible association with infection.

Support or Funding Information

Eisenhower Medical Center Research funds for Infectious disease.,
Targeting sigma factor controlled signaling pathways to modulate biofilm growth and composition
Authors: Ryno Lisa M , Brezel Emma R , Loewus Sarel J , Zheng Erica J
Published: November 2023

Stress‐responsive transcription factors (TFs) and alternative sigma factor TFs control a multitude of downstream targets, some of which may have a direct impact on the formation of bacterial biofilm and the composition of the extracellular polymeric matrix (EPS) surrounding the biofilm. We explore the effect of moderate overexpression of several different alternative sigma factor TFs on biofilm growth using a variety of biochemical and analytical assays. Biofilm growth is monitored by a dye‐binding microtiter plate assay, and the EPS composition, specifically the concentration of proteins and polysaccharides, is monitored by colorimetric assays and FT‐IR spectroscopy. We monitor the known downstream targets of these TFs using quantitative real‐time PCR (qPCR) to confirm TF overexpression. Currently, we are exploring the impact of overexpressing sigma factors rpoH, normally upregulated upon heat shock, and rpoF, an alternative sigma factor responsible for the expression of chemotaxis receptors and flagellar assembly, on biofilm formation and composition.

Support or Funding Information

This work was supported by Oberlin College.
Suppression of biofilm formation in Staphylococcus aureus by targeting Staphylococcal accessory regulator X expression using siHybrids
Authors: Morrow Mikayla
Published: November 2023

Nosocomial infections are becoming increasingly prevalent due to the ability of bacteria to adhere to implanted medical devices. Over 600,000 prosthetic joint replacements are performed yearly in the United States, with approximately 2% of those cases developing infections. The implantation of cardiac valves has increased by 42%, but with a corresponding 210% increased infection rate of Staphylococcus gram‐positive bacteria. S. aureus accounts for up to 73% of these infections. The ability of bacteria to adhere to the hard surfaces of medical devices is due to production of a biofilm, which is initiated by the secretion of polysaccharide intercellular adhesion (PIA) molecules. In Staphylococcal species, PIA production and regulation is controlled by the icaADBC (ica) operon, a sequence of four genes that are all involved in the production of PIA molecules. The icaA gene produces an N‐acetyl‐glucosaminyltransferase like protein, with the icaD gene product enhancing its activity. The icaC gene encodes a membrane protein that initiates the production of longer chains of polysaccharides and their translocation to the cell surface, while the icaB product is responsible for deacetylating the poly‐N‐acetylglucosamine ica transcript.

The ica locus has several known activators and repressors. One of the best‐characterized factors is the staphylococcal accessory regulator A (sarA), which functions as an activator of the locus. Research has shown that when sarA transcripts are suppressed, biofilm growth is repressed. SarX is closely homologous to SarA and is believed to share similar, activating functions; however, the product of the sarX locus is not as well characterized. We investigated if sarX function can be suppressed through the use of sequence specific siHybrids, and if so, whether suppressing sarX has a repressing effect on biofilm development in Staphylococcus aureus. We hypothesized that suppression of sarX will cause repression of biofilm formation. S. aureus was passively transformed with siHybrids and the resulting biofilm growth was measured using crystal violet staining. RNA was isolated from transformed bacteria to determine if levels of sarX transcripts had changed. SiHybrid treatment groups were compared to three controls: an untreated S. aureus positive control, a sterile media negative control, and an E. coli negative control. The results indicated that repressing biofilm growth through the suppression of sarX transcripts with siHybrids led to a seventeen to forty percent decrease in biofilm formation. An ANOVA with a p‐value of 0.5 was used to determine significance. Inhibition of S. aureus at the molecular level, by targeting the RNA transcripts of the icaADBC activator, sarX, presents a novel treatment for nosocomial infections.
Understanding the mechanical stresses of sexually selected structures; is baculum remodelling influenced by the frequency of sex?
Authors: Dean Matthew , Schultz Nicholas G. , Huttenlocker Adam K
Published: November 2023

In about 80% of mammalian species, males have a bone in their penis called a baculum. The baculum displays astonishing morphological diversity, likely driven by a combination of adaptive processes that may relate to male‐male competition and male‐female interactions, although specific function(s) remain unknown. Bones continuously remodel themselves in response to the magnitude and direction of stresses they experience. Characterization of such remodelling in the baculum may help uncover its function during copulation. Here we compare patterns of baculum remodeling in mice, comparing two treatments (regular sex versus no sex) to understand whether copulation strains the baculum. These studies are important for illuminating the mechanical forces imparted by structures thought to be important for sexual selection.

Support or Funding Information

National Institutes of Health R01‐GM098536
Microbial Masterpieces: Bacterial community profiling in the Hastings College art buildings
Authors: Athy Sienna , Tidwell Elizabeth , Laederach Alain , Solem Amanda
Published: November 2023

Several large initiatives such as the Human Microbiome Project (NIH), the Earth Microbiome Project and Microbiology of the Built Environement (Alfred P. Sloan Foundation) as well as many smaller scale projects are exploring the composition and dynamics of bacterial communities to better understand how microbes impact our world from ecology to human health. The advent of high‐throughput sequencing techniques allows sequencing of the variable regions of the 16S rRNA gene from many samples. The 16S rRNA sequence is useful for differentiating between bacteria due to the fact that the gene is present in all bacteria, but contains variable regions. These 16S sequences can be used to identify the diversity and composition of Operational Taxonomic Units (OTUs) to characterize the bacterial community. Here we profile the bacterial communities found in the recently used Art building and the newly constructed Jackson Dinsdale Art Center on the Hastings College campus. Both Art buildings include different types of rooms such as faculty offices, a glass blowing studio and a painting studio. From the collected samples, DNA was extracted and PCR was used to amplify and isolate the 16S rRNA v4 sequence. These samples were then sent for high throughput sequencing for further analysis. This is an ongoing study; initial samples were collected shortly after construction finished and we plan to continue monitoring the bacterial community profiles in the Jackson Dinsdale Art Center periodically now that the facility is in use. The results will contribute to our overall understanding of the microbiology of the built environment in general and specifically at Hastings College.

Support or Funding Information

This work was supported by the Student Development Fund and the Excelsior Scholar program at Hastings College.
Association Between the Serum Metabolome and All‐Cause Mortality: A Prospective Analysis in the Alpha‐Tocopherol, Beta‐Carotene Cancer Prevention (ATBC) Study Cohort
Authors: Huang Jiaqi , Weinstein Stephanie J. , Moore Steven C. , Sampson Joshua N. , Albanes Demetrius
Published: November 2023

Tobacco smoking, hypertension, hyperglycemia, overweight, and inactivity are the leading causes of mortality worldwide. Beyond these factors, prospective metabolomic profiling may help identify individuals at higher risk of death and point to molecular factors and biological pathways that have preventive potential. We conducted a serum metabolomic analysis of 667 men included as controls in 6 nested case‐control studies in the Alpha‐Tocopherol, Beta‐Carotene Cancer Prevention (ATBC) Study. During up to 28 years of follow‐up, there were 457 deaths, including 199 deaths from cardiovascular disease (CVD) and 112 deaths from cancer. Ultra‐high performance LC/MS and GC/MS (Metabolon, Inc., NC) identified 1,173 known metabolites, and 375 metabolites were retained for analysis after excluding those missing in ≥2 of the study sets. We modeled the relationship between each metabolite and risk of death by cox regression and report the set of metabolites where the False Discovery Rate (FDR) q‐value<0.02. We next split the data into training and test sets. Using only the training set, we selected those metabolites (N=14) with an FDR q‐value of <0.15 and included all selected metabolites in a multivariable cox regression. In the test set, we constructed a metabolomic score, where each individual's metabolite levels were weighted by the coefficients from the regression model, and categorized individuals into tertiles according to their score. All models were adjusted for mortality risk factors. 7‐Methylguanine, N‐acetylvaline, taurocholate, N‐acetyltryptophan, 5,6‐dihydrothymine, homocitrulline, dimethylglycine, myristoleate (14:1n5), palmitoleate (16:1n7), C‐glycosyltryptophan, taurochenodeoxycholate, 5‐dodecenoate (12:1n7), erythronate, N1‐methylguanosine, N‐formylmethionine, and hexanoylcarnitine yielded the strongest metabolite risk signals at a threshold of FDR q‐value <0.02 (0.05≤β≤0.07, and 2.0×10−6≤P≤4.6×10−4, respectively). The validation analysis in the test‐set showed that individuals in the 2nd and 3rd tertiles of the metabolite risk score experienced twice the mortality of those in the lowest tertile (2nd tertile score: HR=2.0, 95% CI: 1.2–3.2; 3rd tertile highest score: HR=2.2, 95% CI: 1.3–3.6). Our analysis identified several serum metabolites independently associated with overall mortality, as was their composite risk score, which replicated in the independent test‐set. The metabolite signals are associated with pathways related to CVD, physical activity, ageing, inflammation and nucleoside tumor markers.

Research support: Intramural Research Program of the National Cancer Institute, NIH
The transmembrane sequences of amyloid precursor protein family members regulate their ectodomain shedding
Authors: Multhaup Gerhard , Schauenburg Linda , Mayer Magnus , Walter Corvin , Eravci Murat , Weise Chris , Liebsch Filip
Published: November 2023

It has been shown that APP, APLP1 and APLP2 are similarly cleaved by different secretases. The prerequisite for shedding of those proteins by γ‐secretase is the initial cleavage by α‐ or β‐secretase, which released the soluble ectodomains. Here, we analyzed the soluble fragments in the conditioned media of HEK 293T cells overexpressing either APP, APLP1 or APLP2 under the treatment of different secretase inhibitors.

Soluble fragments in the media of cells overexpressing APP, APLP1 and APLP2 were analyzed in the presence and absence of secretase inhibitors by Western blot using a variety of antibodies directed against individual domains of APP and APLPs. MS/MS analyses of APLP1 deletion mutants and of chimeric constructs allowed to differentiate between APLP1γ and APLP1α/β processing and to determine exact α‐, β‐ and γ‐cleavage sites.

Interestingly, in the medium of APLP1‐overexpressing cells a larger soluble fragment appears that is recognized by an antibody directed against the Aβ‐like region of APLP1. The secretion of sAPLP1α/β occurred independent from inhibitor treatment whereas there is an inhibitor dependent secretion of endogenous sAPLP1γ. To enrich the fragment, the conditioned medium of SH‐SY5Y cells with or without treatment of γ‐secretase inhibitor was subjected to immunoprecipitation. MS/MS analysis revealed the exact cleavage site within the C‐terminal sAPLP1γ fragment.

Our results reveal a novel secretion mechanism, which is specific for APLP1. Only this member of the APP protein family can be cleaved by γ‐secretase without the so far thought necessary proteolysis step of ectodomain shedding.

Support or Funding Information

DFG, CIHR, NSERC
Pyruvate Carboxylase is Essential for Breast Cancer Metastasis in Vivo
Authors: Wilmanski Tomasz M , Shinde Aparna , Donkin Shawn S , Burgess John , Wendt Michael , Teegarden Dorothy
Published: November 2023

Anaplerosis, or the replenishment of TCA cycle intermediates utilized for biosynthetic pathways, has recently emerged as an integral part of breast cancer cell metabolism. In particular, the enzyme pyruvate carboxylase (PC), responsible for facilitating pyruvate flux through oxaloacetate into the TCA cycle, has gained recognition as an essential component of breast tumor growth and metastatic capability. Therefore, the objective of the present studies was to investigate the role of PC in breast cancer utilizing the MCF10 model of breast cancer progression in vitro as well as in vivo utilizing metastatic murine 4T1 cells in immunocompetent Balb/C mice. PC mRNA expression was not significantly different between MCF10A untransformed and Harvey‐ras oncogene transfected cells (MCF10A‐ras), representing initiation. However metastatic MCF10CA1a cells, representing the latest stage in the model, had a 5.14(±0.21) fold higher PC mRNA abundance relative to both the MCF10A and MCF10A‐ras cells. In addition, PC overexpression achieved through transient transfection of a pCMV6‐PC plasmid increased cell migratory capability of both MCF10A‐ras (183%±46 relative to Neo control) and MCF10CA1a cells (191%±31 relative to Neo control), as measured by a Boyden Chamber migration assay. Interestingly, the same overexpression of PC in untransformed MCF10A cells resulted in their acquisition of a migratory phenotype in vitro (252%±34 relative to Neo vehicle). To test the role of PC in vivo, murine metastatic 4T1 breast epithelial cells expressing either PC silencing shRNA or a scramble control were engrafted onto the mammary fat pad of 6 week old Balb/C mice. Five weeks after engraftment, primary tumor and pulmonary metastasis were analyzed. Surprisingly, there was no difference in primary tumor size or volume between shPC and scramble control 4T1 breast epithelial cells. PC silencing, however, had a drastic impact on metastatic capability of cells with nearly complete inhibition of pulmonary metastasis (pulmonary mets per mouse of shPC#25=6±2 and shPC#28=8±4 scramble control=31±10). Together, these results suggest that PC plays an essential role in breast cancer progression, particularly in metastasis of cells from the primary tumor to distant secondary organs.

Support or Funding Information

This work was supported by a Project Development Team within the ICTSI NIH/NCRR (Grant Number UL1TR001108) and the National Institutes of Health, National Cancer Institute(R25CA128770) Cancer Prevention Internship Program. Additional support was received from the Indiana Elks Charities and the SIRG grant, both administered through the Purdue University Center for Cancer Research (Purdue University, West Lafayette, IN).