bluuebunny/crossref_metadata_2025_split huggingface dataset:
Inhibition of osteotropic breast cancer cell motility by zoledronic acid in a 3D spheroid culture
Authors: Whalen William A , Bush Jason A 
Published: November 2023

Conventional cell culture techniques grow cells in a two‐dimensional (2D) format on compatible surfaces, but cells do not grow like this in vivo. A fast‐emerging alternative is three‐dimensional (3D) cell culture, which encourages cells to work together while suspended in a matrix rather than relying on adhesion to a treated surface. Most studies utilize a commercially available, industry standard recombinant basement membrane substitute—Matrigel®; however, this is proprietary and expensive. For these studies, the objective was to refine an alternative to commercial matrices for use as a suspension media. Our efforts focused on economical Methocel™ (methylcellulose dissolved into basal growth media) in combination with rat tail collagen Type I. Methocel promoted the formation of spheroids among several cell lines including three isogenic variants of a triple‐negative metastatic breast cancer cell line, MDA‐MB‐231, with differing organotropic propensity and difficult to grow in spheroid culture. While not required for all cell lines, the addition of Type I collagen greatly increased the efficacy of spheroid formation among all cell lines tested. Once harvested, spheroids were suspended within a Methocel‐collagen gel. Cultures were treated with various chemotherapy drugs including bisphosphonates (ibandronic and zoledronic acids) and docetaxol. The effectiveness of these drugs to inhibit migration through a surrogate extracellular matrix was determined by the amount of invasion as measured by distance migrated from the spheroid periphery. Osteotropic cells (MDA‐MB‐231Bo) showed a high degree of translocation through the matrix compared to MDA‐MB‐231 control cells but were dramatically more affected by the cytostatic action of zoledronic acid, a drug know to suppress skeletal‐related events. Furthermore, using RT‐PCR to evaluate canonical cell adhesion molecules including the a2b1 integrin, Cadherins, Twist, and Snail, we see a time‐dependent change in gene expression among the three MDA‐MB‐231 variants when grown in the presence of chemotherapeutic drugs. Taken together, our results demonstrate a simple, cost‐effective system that provides an in vivo‐like microenvironment to study gene expression alterations in tumor progression and metastasis.Support or Funding InformationFresno State faculty‐student scholarly research award.
Inhibitory Effects of Substituted Pyrazoline Derivatives on Entamoeba histolytica
Authors: Tight Alexander , Rossi Lauren , Espinosa Avelina 
Published: November 2023

Entamoeba histolytica is an intestinal parasite that causes disease in humans. Amoebiasis is a major public health risk, with about 50 million cases, and 100,000 deaths per year worldwide. The disease is primarily treated with metronidazole, which is effective but has adverse side effects such as neurological complications. Previous work in our laboratory generated 25 compounds within three series of pyrazoline derivatives that block the bifunctional enzyme alcohol dehydrogenase 2 (EhADH2). We tested these compounds in their ability to inhibit growth and survival of Entamoeba histolytica trophozoites. The inhibitors were tested at 60 and 120 μM concentrations and compared to the drug of choice metronidazole. Our results demonstrate that series 1a and 1b are the most effective. Compared to series 2, these series are significantly less bulky which could give the compound a greater affinity to bind to the enzyme. Although compounds from series 1b show greater growth inhibition, more data is needed to establish the relevance of each structural moiety. Future studies will test the efficiency in blocking the essential ADHE metabolic enzyme in Entamoeba and toxicity in human cells in comparison to metronidazole.Support or Funding InformationSupported by Rhode Island National Institutes of Health INBRE Grant
A Surprising Shift in the Biosynthesis of Marine Meroterpenoids
Authors: Miles Zachary , Deithelm Stefan , George Jonathan , Moore Bradley 
Published: November 2023

A disproportionate number of halogenated natural products are derived from marine sources primarily due to the stark difference in elemental availability of halides in oceanic environments in comparison to their terrestrial cohorts. Vanadium‐dependent haloperoxidases (VHPOs), one class of enzymes that install these atoms, have historically been associated with non‐specific halogenation of compounds in various fungal and seaweed species. We previously characterized VHPOs from marine bacteria that control regiospecificity of halogenation to facilitate complex cyclizations in the biosynthesis of antimicrobial natural products. Recently, we observed a surprising chlorine‐mediated rearrangement reaction that accounts for the structures of two compounds from the merochlorin series of antibiotics that had yet to be explained. Ultimately, this observation appears to unexpectedly unify the biosyntheses of many related meroterpenoid natural products and expands the biochemical function of VHPOs.Support or Funding InformationThis work was supported by NIH grant AI047818 awarded to B.M.
Investigating Mycobacterium tuberculosis L,D‐transpeptidases as novel drug targets
Authors: Basta Leighanne , Pan Ying , Kaushik Amit , Lloyd Evan , Townsend Craig , Staker Bart , Lukacs Christine , Bianchet Mario , Lamichhane Gyanu 
Published: November 2023

The final step of peptidoglycan (PG) biosynthesis in bacteria involves cross‐linking of peptide side chains. This step in Mycobacterium tuberculosis is catalyzed by L,D‐ and D,D‐transpeptidases that generate 3→3 and 4→3 transpeptide linkages, respectively. M. tuberculosis PG is predominantly 3→3 cross‐linked, and LdtMt2 is the dominant L,D‐transpeptidase. There are four additional sequence paralogs of LdtMt2 encoded by the genome of this pathogen, and the reason for this apparent redundancy is unknown. Here, we present microbiological, biochemical, and structural data suggesting paralogs LdtMt2, LdtMt3, and LdtMt5 are not functionally redundant. Further, we demonstrate that at least two of these paralogs are acylated by carbapenem antibiotics. Importantly, the work presented here provides structural evidence of acylation of LdtMt2 by tebipenem, an orally available carbapenem, and biapenem.We have generated strains of M. tuberculosis that lack functional copies of LdtMt2, LdtMt3, or LdtMt5, and these strains are more susceptible to killing by select carbapenems, osmotic shock, and/or chemical probes. Additionally, the different strains display unique phenotypes when cultured under different conditions, further suggesting these enzymes serve non‐redundant roles in maintaining the physiology of the M. tuberculosis cell wall.Taken together, the results from our studies highlight the essentiality of L,D‐transpeptidases in proper cell wall maintenance. Further investigation into the physiological roles of these enzymes is warranted, as these enzymes represent unexploited drug targets.Support or Funding InformationNIH Grants R21AI111739 and DP2OD008459 to GL.
Camel VHH for the Prevention of Listeria monocytogenes Cellular Invasion
Authors: Toride Moeko , Huh Ian , Mendoza Matthew , Brooks Cory L. 
Published: November 2023

Listeriosis is a central nervous system infection caused by the Gram‐positive bacteria Listeria monocytogenes (Listeria). Listeria is a serious threat in the food industry, because it is commonly transmitted by ingesting the contaminated food products. Pregnant women are one of the most vulnerable populations to listeriosis as the bacteria can cross the placental barrier leading to miscarriage or stillborn. The prophylactic therapy or detection technologies of Listeria in pre‐natal care would greatly reduce the risk of listeriosis.Listeria accomplishes invasion of epithelial cells in the intestinal tract by binding to the receptors on the target cell surface using its surface protein Internalin A (InlA) and Internalin B (InlB). InlB is one of the major virulence factors of Listeria and binds to the cMet receptor on the host epithelial cells to mediate cell internalization. The high resolution structure of InlB shows that it binds to cMet receptor at its Leucine‐Rich Region (LRR) domain where it has concave structure made by 310‐helixes and β‐strands.Single‐domain antibodies (VHH) are the smallest known antibody fragments and are derived from heavy‐chain antibodies found in Camalidae. Due to their small size, they can target enzyme active sites or protein‐protein interaction sites. Moreover, VHH possess Complementarity‐Determining Region (CDR) loops where it can bind to antigens with concave structure. Therefore, we hypothesized that VHH can bind to the LRR domain of InlB and inhibit Listeria entry into host cells. The objective of this research is to determine the effectiveness of VHH to inhibit Listeria invasion and determine the structural basis for their action.Using gentamicin protection assays and fluorescent microscopy, we demonstrated that several InlB‐specifics VHH are capable of inhibiting Listeria invasion of HeLa cell. We also plan to co‐crystallize InlB and VHH to determine the specific binding site of the two proteins. Our research demonstrates the potential of VHH as a new class of therapeutics to protect women from Listeria during pregnancy.Support or Funding Information‐ MT was supported by rGrant from the Associated Students, Inc. at California State University, Fresno.‐ This research was supported by NIH Score Grant (1SC3GM11253‐01).
Dynamic Interactions of TATA‐Box Binding Protein with Promoters is Regulated by O‐GlcNAcylation
Authors: Hardivillé Stéphan , Han Guanghui , Ma Junfeng , Hu Ping , Banerjee Partha S. , Hart Gerald W. 
Published: November 2023

O‐GlcNAcylation is a dynamic posttranslational modification tightly correlated to the concentration of UDP‐GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP), which varies according nutritional factors. Through HBP activity and production of UDP‐GlcNAc, OGT senses nutrient availability and forward the signal to downstream effectors by transferring a GlcNAc moiety to the targeted proteins. In return, the O‐GlcNAcase resets the signal. Interestingly, literature points out a regulation of the basal transcription machinery by O‐GlcNAc. TATA‐box binding protein (TBP) is a key player in regulating transcription since it is required for the activity of all three RNA polymerases. DNA‐bound TBP acts as a dock for recruitment of basal transcription factors and the RNA polymerase II to formation of a functional pre‐initiation complex (PIC). DNA‐bound TBP can also be recognized by NC2 and BTAF1. NC2 stabilizes the chromatin interaction of TBP, whereas the association of TBP with BTAF1 promotes its release.Here, we mapped tree O‐GlcNAc sites on the N‐terminus domain of TBP (T114, T126 and S158), and shown that O‐GlcNAc inhibitor/activator affect localization of TBP with specific chromatin marks. O‐GlcNAcylated TBP is bound to DNA and the sugar inhibits the interaction with BTAF1. TBP site‐specific O‐GlcNAc mutants have been used to point out the T114 as the main regulator of its interaction. Owing to the fact that BTAF1 dynamically regulates the chromatin interaction of TBP, we investigated the dynamic behavior of TBP and TBP O‐GlcNAc by Strip‐FRAP and ChIP assays. Collectively, our results indicate that O‐GlcNAcylation of TBP at T114 regulates TBP/BTAF1 dynamics on gene promoters.
Glycans in Pseudomonas aeruginosa
Authors: McClinton Anna Kathleen , Cioffi Donna L , Cioffi Eugene A 
Published: November 2023

IntroductionPseudomonas aeruginosa is an opportunistic pathogen known to encode a neuraminidase, PaNA. It is known that some strains of P. aeruginosa glycosylate pilin and flagellin and that pseudaminic acid is a component of the cell wall. PaNA has been associated with biofilm formation and has also been characterized to have more activity against pseudaminic acid than sialic acid. An allelic knockout of PaNA resulted in strain PAO1Δ2794. This strain was shown to generate less organized biofilms and also to have a somewhat decreased virulence in mice, depending on the route of inoculation. However, when PAO1Δ2794 is grown in culture, there are marked differences between wild type PAO1 and the mutant. Namely, the PAO1Δ2794 strain is likely to vigorously produce a green‐blue pigment as well as a layer of foam under certain media conditions, while PAO1 does not. These changes are indicators that quorum‐sensing (QS) is active in this strain in a population density independent manner compared to wild type. These observations led to the question of why the absence of PaNA might lead to this apparent mis‐regulation of QS. The objective of the study was to explore the role of PaNA in P. aeruginosa.MethodsP. aeruginosa strains PAO1 and PAO1Δ2794 were grown in LB media and harvested hourly for 12 hours. Bacteria were pelleted by centrifugation and lysed. Both lysates and culture supernatant were subjected to SDS‐PAGE and Lectin Blot analysis.ResultsThere are distinctive bands in the lectin blot of lysates with Limax flavus lectin, which recognizes generic sialic acids, including pseudaminic acid. The presence of these bands indicate that some internal protein(s) are in fact glycosylated. Interestingly, over the course of the growth curve and corresponding to the time point that the PAO1Δ2794 culture began to visibly produce pigments, the strongest set of bands is lost in the blot of the Δ2794 lysates, but maintained in PAO1 lysates.ConclusionProteins in lysates of P. aeruginosa are glycosylated and may contribute to the regulation of QS by an undetermined mechanism.Support or Funding InformationSupported by R01HL107778 and T32HL076125‐11.
Cellular O‐Glycome Reporter/Amplification (CORA) to Explore O‐Glycans of Living Cells
Authors: Kudelka Matthew R. , Antonopoulos Aristotelis , Wang Yingchun , Duong Duc M. , Song Xuezheng , Seyfried Nicholas T. , Dell Anne , Haslam Stuart M. , Cummings Richard D. , Ju Tongzhong 
Published: November 2023

O‐glycosylation is present on over 80% of proteins that traverse the secretory apparatus and plays key roles in many biological processes. However, the repertoire of O‐glycans synthesized by cells and thus their function are difficult to determine. Current strategies to evaluate O‐glycans from cells utilize chemical release, such as alkaline β‐elimination, prior to analysis by mass spectrometry or other technologies. However, β‐elimination is inefficient, potentially biased, and results in O‐glycan degradation. To address this challenge, we developed a technology to amplify and profile mucin‐type O‐glycans synthesized by living cells, termed Cellular O‐Glycome Reporter/Amplification (CORA). We developed a chemical O‐glycan precursor that when incubated with live cells crosses the plasma membrane, is taken up by the Golgi Apparatus, and is modified by glycosyltransferases in situ, before being secreted from cells as a variety of modified O‐glycan derivatives, allowing easy purification for analysis by HPLC and mass spectrometry (MS). CORA detected O‐glycans observed by β‐elimination as well as many additional complex structures with ~100–1000‐fold increase in sensitivity over conventional O‐glycan analyses. Furthermore, CORA coupled with computational modeling allowed us to predict the diversity of the human O‐glycome. To our knowledge, CORA is the first technology for glycome amplification and thus could offer new opportunities for understanding the role of glycans in health and disease.Support or Funding InformationThis work was supported by National Institutes of Health Grant U01CA168930 to TJ and RDC, P41GM103694 to RDC, Georgia Cancer Coalition (now Georgia Research Alliance, GRA) Award to TJ, and by Biotechnology and Biological Sciences Research Council grant BB/K016164/1 (AD and SMH for Core Support for Collaborative Research). AD is supported by a Wellcome Trust Senior Investigator Award. We also acknowledge support from the Emory Integrated Proteomics Core.
Indices of fatty acyl‐CoA desaturase activity are associated with cognitive function and preclinical changes in cerebrospinal fluid beta‐amyloid and tau levels in Alzheimer's disease
Authors: Fonteh Alfred N. , Cipolla Matthew , Chiang Jiarong , Arakaki Xianghong , Harrington Michael 
Published: November 2023

ObjectivesTo determine if indices of acyl‐CoA desaturase can differentiate stages of dementia.MethodsWe used gas chromatography‐mass spectrometry to quantify saturated (SAFA) and monounsaturated (MUFA) fatty acids in cerebrospinal fluid (CSF) fractions from cognitively normal study participants (CN, n=70), study participants with mild cognitive impairment (MCI, n=40), and Alzheimer's disease (AD, n=29). CSF amyloid and tau measures revealed two CN groups: CN with normal Aβ42/tau (CN‐nAT, n=36) and CN with pathological Aβ42/tau (CN‐pAT, n=34). We determined the ratio of each MUFA product to their corresponding SAFA precursor for even numbered (C14:1/C14:0, C16:1/C16:0, C18:1/C18:0) and odd numbered (C15:1/C15:0, C17:1/C17:0, C19:1/C19:0) chain fatty acids.ResultsThe product to substrate ratio (desaturase index) was different in the CSF nanoparticle fraction (NP) compared with the supernatant fluid (SF) or free fatty acid fractions. C18:1/C18:0 accounted for most of the desaturation index in CSF fractions. In the NP fraction, desaturation index progressively decreased in MCI and AD compared with CN. ROC curves show that free C18:1/C18:0 indices can differentiate CN from MCI and MCI from AD.ConclusionsThe rate‐limiting enzyme in the biosynthesis of MUFAs, stearoyl‐CoA desaturase (SCD‐1), is altered with cognitive changes. Desaturation indices for free fatty acids in CSF can differentiate CN from MCI and AD and are altered in pre‐symptomatic AD. These changes may reflect changes in energy balance, pro‐inflammatory parameters, or membrane properties linked to amyloid precursor protein processing.Support or Funding InformationL.K. Whittier and the Helen Posthuma Foundations
Do Sweeteners Affect the Expression of (Pro)renin Receptors in the Rat Kidney?
Authors: Young Natalie E , Salerno Theresa 
Published: November 2023

Excess amounts of sugars can cause diabetes and chronic kidney diseases. Recently stevia and saccharin have been used to lower calorie intake and reduce blood sugar. Aqueous extracts of stevia may reduce blood pressure and could be a useful hypotensive agent. The mechanism has not yet been determined but may relate to the renin angiotensin system (RAS). A key protein in the system is the (Pro)renin receptor (PRR) that binds both renin and prorenin. PRR may lead to upregulation of genes associated with hypertension and chronic kidney disease. Recent studies have shown that sweeteners affect RAS; however their effect on PRR is unknown. PRR is a transmembrane protein with a large extracellular domain. The extracellular domain can be cleaved in the Golgi by furin to make a soluble form of PRR. Additionally the mRNA of PRR has a long 3′UTR and may be targeted by microRNAs. We performed a preliminary study to test the effect of sweeteners on PRR expression in the rat kidney. Rats were fed standard Osmolite diets with supplements of sucrose, stevia or saccharin. Kidneys were obtained and small RNAs and mRNAs were purified from the tissues using the mirVana kit. Then the expression of PRR mRNA was measured. At the mRNA level, PRR mRNA expressions were not statistically different in the experimental samples versus the control sample. Then we tested the expression of miRNAs that can potentially target PRR protein expression. miRNA‐132 showed a decrease in expression in the stevia and saccharin samples; however, these samples were not statistically different from the control samples. miRNA‐152 was found to be expressed in the rat kidney and showed the same trend as miRNA‐132. Saccharin and stevia samples did not show any statistical differences from the control. This suggests that sweeteners do not affect PRR mRNA expression or the expression of miRNAs that might target PRR protein expression. In the third study, PRR protein expression was measured in the samples using Western blotting. The blot showed the presence of both intact and soluble PRR proteins. The blot suggested a significant decrease in the protein expression of the soluble PRR in the kidneys of rats fed stevia and sucrose. Increased levels of sPRR have been associated with increased hypotensive affects. However, more samples will be tested to further determine if sPRR is further downregulated or unregulated in the saccharin, stevia and sucrose diets. Our preliminary study indicates that sweeteners may play a role in the regulation of soluble PRR that may further affect kidney diseases.Support or Funding Information2013 and 2014 Minnesota State University, Mankato Foundation Grants
Characterization of Basigin and MCT1 expression in murine myocardium
Authors: Morse Grace E , Ochrietor Judith D 
Published: November 2023

Obesity is a condition that has systemic effects that include hypertension, diabetes, and atherosclerosis. The long‐term goal of this study is to examine the effects of obesity and inflammation in the mouse heart, specifically looking for potential biomarkers that can be used to predict cardiovascular disease long before its onset. Previous reports have established Basigin, a glycoprotein member of the immunoglobulin superfamily (IgSF), as a heterotypic cell‐adhesion molecule expressed in two splice‐variant forms varying in the expression of extracellular immunoglobulin (Ig) domains. Basigin variant‐2 and Basigin variant‐1 contain two and three C2 domain loops, respectively. The variant‐2 protein is known to be expressed in mouse heart. Monocarboxylate transporter 1 (MCT1), a proton‐linked lactate transporter, known to interact with Basigin gene products is expressed in murine myocardium during vascular remodeling following atherosclerotic conditions. Therefore, the purpose of this present study was to investigate the relative expression of Basigin and MCT1 gene‐products in murine myocardium during post‐natal development. Mouse hearts were obtained via an accepted protocol and protein lysates were generated. Enzyme‐linked immunosorbent assay (ELISA) was utilized to determine the relative expression of Basigin and MCT1 in murine myocardium samples at several ages. It was determined that expression of both molecules increases over the life of the animal. We concluded that expression of Basigin and MCT1 in murine myocardial tissue may be potential biomarkers for cardiovascular disease.Support or Funding InformationUniversity of North Florida Office of Undergraduate Studies and the Office of Academic Affairs