bluuebunny/crossref_metadata_2025 huggingface dataset:
Studying the Effects of MDS Mutations in HSH155 and PRP21 on the Splicing of Introns
Authors: Luo George , Carrocci Tucker , Zoerner Douglas , Xu Jiacui , Hoskins Aaron
Published: November 2023

The spliceosome is responsible for the excision of introns from pre‐mRNA in eukaryotes and alterations of the spliceosome can cause serious consequences. Recently, studies have shown mutations in certain genes associated with the spliceosome are linked with certain types of Myelodysplastic Syndromes (MDS). Specifically, mutations in SF3B1, an U2snRNP associated splicing factor, have been found in approximately 20% of MDS cases. SF3B1 is thought to bind pre‐mRNA and to help link U2snRNP to pre‐mRNA. In our research, we investigated the corresponding mutations in the S. cerevisiae homolog, HSH155, which is well‐conserved between humans and yeast. We have introduced MDS‐related mutations into S. cerevisiae HSH155 and showed the cells have no significant growth differences or temperature sensitive defects compared to the wild type. We expected splicing will be affected by the mutations, resulting in an increased ratio of pre‐mRNA to mRNA. To check this hypothesis, we carried out Reverse‐Transcription Polymerase Chain Reaction (RT‐PCR) but found no differences in splicing efficiency of tested pre‐mRNAs (SUS1, DYN2, GLC7, YFR045w, PMI40, MATa1) in S. cerevisiae. Our results suggest that changes in splicing due to SF3B1 mutations may not be apparent when studying endogenous yeast pre‐mRNAs. Simultaneously we are investigating the effects of mutations in a second spliceosomal protein associated with MDS cases, PRP21p. PRP21p is homologous to the human SF3A1 protein and is also associated with the U2 snRNP. For this project, we have produced the necessary yeast strains and have used growth assays to check for growth differences and temperature sensitivity as well as RT‐PCR experiments to test for splicing of endogenous genes. Additionally, we will use an ACT1‐CUP1 copper reporter gene that will help us determine splicing efficiency in cells. Through this assay, we will determine if splicing of wild type and mutated intron sequences are affected by mutations in PRP21.
Modifying Trypsin to Weaken Inhibitor Interaction
Authors: Nzessi Anthony , Huynh Hanh , Baird Teaster
Published: November 2023

Serine proteases have been used as therapeutics to help with health maladies such as Christmas Disease, a blood coagulation disorder. They may potentially be administered as drugs in order to aid many other conditions, but their activity is limited by natural regulatory inhibitors which serve to regulate the action of proteolytic enzymes and prevent diseases such as pancreatitis. A number of these inhibitors bind to the hydroxyl group of Y39 in the S1′ pocket of trypsin‐fold serine proteases. We expect that substituting this tyrosine with a leucine will weaken inhibitor interaction, thus increasing the half‐life of serine proteases in the body. Using trypsin as a model serine protease, the variant Y39L was designed via PCR mutagenesis, expressed in P. Pastoris, and purified through hydrophobic interaction chromatography. Before determining if inhibitor interaction with Y39L was weaker than with wild‐type trypsin, we compared their kinetics. In this study, we show that Y39L has comparable activity (kcat = 2.20 × 103 min‐1) and binding capability to Z‐GPR‐pNA (KM = 6.89 ± 1.28 μM) as wild‐type trypsin. Using the commercial competitive inhibitor benzamidine, we also show that the active site of Y39L has not been compromised by this amino acid replacement (KI = 5.28 μM). This implies that if the inhibition of this variant with macromolecular inhibitors is not consistent with that of WT, any difference must be due to the change made in the S1’ site. These studies will be instrumental in the development of trypsin‐fold serine proteases that may potentially be used as therapeutic agents to aid in several biological processes.

Support or Funding Information

National Institute of Health ‐ MARC

National Science Foundation ‐ CSU‐LSAMP
Trypsin as a model for investigating serine protease‐inhibitor interaction
Authors: Diaz Parris , Zhuo Weichao , Sharma Abriti , Baird Teaster
Published: November 2023

For years proteases have been drug targets, but recently, several have been used as therapeutic agents themselves. However, macromolecular inhibitors present in the human body that inactivate proteases have been a hindrance in their further development as therapeutic agents. In this study, we use trypsin as a model protease to understand protease‐inhibitor interaction. We have substituted amino acids in S1’ pocket and determined if these variants are resistant to inhibitors while also retaining catalytic activity. The S1’ pocket contains residues Tyr‐39, Phe‐41, and Lys‐60 and has been chosen as a target site due to it being a common location at which multiple inhibitors bind to trypsin. The large phenyl group of Phe‐41 likely locks its backbone carbonyl into a conformation where it participates in a conserved hydrogen bond with inhibitors. If this interaction were disrupted, the interaction between enzyme and inhibitor may be weakened. To test this hypothesis, we created the F41L variant of trypsin (F41L‐Tn). In our initial experiments, we have observed that F41L‐Tn has a high binding affinity for the substrate Z‐GPR‐pNA (Km = 14.1 ± 2.8 μM) along with catalytic capability comparable to wild‐type (kcat = 2.30 × 102 s‐1). In addition, F41L‐Tn has similar affinity for the inhibitor p‐aminobenzamidine (KI = 5.8 μM) when compared to WT (KI = 6 μM). These results suggest that the substitution has not affected the active site is still intact and can still interact with substrates such as Z‐GPR‐pNA as well as competitive inhibitors such as p‐aminobenzamidine. Consequently, any differences observed between trypsin and macromolecular inhibitors may be attributed to changes at the S1’ subsite.

Support or Funding Information

NIH MARC: T34‐GM008574LSAMP: HRD‐0802628
Characterization of Ecm14, a Fungal Pseudopeptidase
Authors: Idowu Temitope A , Schott Matthew J , Lyons Peter J
Published: November 2023

Many peptidase families contain inactive enzyme homologs due to the alteration of critical active site amino acids. The function of these inactive homologs is in many cases an open question, and their study made more difficult by their lack of activity. For example, the M14 family of metallocarboxypeptidases is a large family of peptidases made up of 23 members in humans; three of these are inactive enzyme homologs for which we have little functional information. Interestingly, the yeast Saccharomyces cerevisiae has only one M14 metallocarboxypeptidase homolog, Ecm14, and this sole homolog is predicted to be inactive. Our goal was to investigate the molecular and biological function of this pseudopeptidase. An analysis of available fungal amino acid sequences showed that the Ecm14 gene was present and conserved in ascomycete fungi only. In order to characterize the function of Ecm14, we expressed a C‐terminally histidine tagged version of Ecm14 using the Sf9/baculovirus system. The protein was secreted and subsequently purified by metal affinity chromatography. Incubation of Ecm14 with trypsin or chymotrypsin resulted in the specific removal of the Ecm14 prodomain. The mature form of Ecm14 lacked any detectable enzymatic activity, but was able to prevent the cleavage of standard substrates by an active metallocarboxypeptidase. Preliminary results from a screen of knockout fitness in response to chemical stressors suggest a biological function for Ecm14. We propose that Ecm14 may perform a regulatory function through binding to specific C‐terminal amino acids, thus blocking their cleavage by active carboxypeptidases.

Support or Funding Information

Supported by Andrews University Faculty Research Grants to PJL
Strategies for Getting Promoted in a Difficult Granting Environment
Authors: Guttman Julian Andrew
Published: November 2023

Research, teaching and service. These are the areas that we all know we need to excel in to get promoted. We can easily control our teaching quality and service levels, but it's the research component where many of us are currently facing our greatest challenges. We're all too aware that research costs are escalating, national research funds are dwindling and those funds are becoming more targeted than ever before. So how can we get enough money to do our research and get the quality publications needed for promotion? In this session I will discuss some of the unconventional strategies I have used to supplement funds for my labs, international collaborations that are funneling money to my research program and how these tactics have helped me develop a strong internationally recognized research component and (thus far) unimpeded promotion as a faculty member. I will also point out the key steps that I took throughout my career that have helped in my career progression and how I've overcome obstacles that we all deal with as faculty members.
The Role of Subunit Interfaces of Malate Dehydrogenase in Protein Folding & Unfolding
Authors: Abano John , Bell Ellis , Bell Jessica
Published: November 2023

Malate dehydrogenase (MDH) is an essential enzyme for several metabolic pathways including the production of NADH as a precursor for ATP synthesis in the mitochondrion and glyoxysome. In the cytosol MDH is involved in the reduction of oxaloacetate as part of the aspartate‐malate shuttle.. The enzyme is a dimer of chemically identical subunits and while much is known about the enzymatic steps, little is known about the folding mechanism of MDH and the link between folding and regulation or catalysis. The goal of the current project is to understand the folding process and explore the role of subunit interactions to test the hypothesis that the native dimer conformation of MDH governs the correct folding of the enzyme subunits in order to impose the observed spatial asymmetry of the subunits and facilitate active site communication across the interface thought to be involved in both overall catalysis and regulation. Clustal analysis identified a number of interface conserved residues and 4 mutants, H90Q, E256Q, S266A and L269A were constructed. The wild type protein and various mutants were expressed in E.Coli and purified using NiNTA Chromotography using the engineered His‐Tag. Purity was established by SDS‐Page or MALDI‐tof. Quaternary structure was established by size exclusion chromatography. Reversible denaturation of wild type and mutants was studied by fluorescence spectroscopy and CD to determine tertiary and secondary structures respectively, with activity loss and regain followed by enzymatic assay. The protein unfolding/folding dynamics were also probed using limited proteolysis in conjunction with mass spectrometry.
A Novel Immunoassay, AGILE Research, Enables Detection of Analyte in Quantity Limited Samples
Authors: Barron Francie Elizabeth , Scannura Ryan , Tavares Andre LP , Walker Amy , Pan Deng , Lerner Mitch , Clouthier David E , Goldsmith Brett
Published: November 2023

Proteomics work can be sample‐limited, making some experiments difficult to impossible to complete. One example is proteome analysis in mouse embryos, in which limited protein yield from embryonic tissue results in the requirement of sample pooling to obtain sufficient material for analysis. Due to this sample limitation and expense, RNA studies are often utilized to assess changes in gene expression, with the assumption that protein levels should concomitantly change with RNA levels. To overcome these obstacles, we have developed a novel direct detection approach that leverages advances in nanoelectronic sensors to quantify protein concentrations. AGILE Research is a new protein binding analysis tool using graphene biological field effect transistors that eliminates the need for secondary labeled antibodies to obtain results, and it can detect proteins in the fM range in as little as 15 minutes via a simple USB connection. We show data from use of AGILE Research demonstrating protein concentration from in vivo and in vitro quantity limited samples.
Whole‐Blood Optimization of Electrochemical Biosensors for Biomedical Diagnostics
Authors: Daniel Jessica , McCoy Michael , Bonham Andrew J
Published: November 2023

Electrochemical aptamer‐scaffold biosensors are able to detect target proteins, such as botulism and ricin toxins, in small concentrations with reliable results. Although these rationally designed biosensors perform well in buffered media under lab‐bench conditions, this is insufficient for real world diagnostic performance. Thus, we have developed methods utilizing polyacrylamide hydrogels and hydroxyl‐polyethylene glycol‐thiol passivants to optimize these biosensors for function with both small sample volumes and in whole blood, delivering rapid and accurate analysis. Through these surface optimizations and the creation of rapid software analysis front‐ends, we have created a platform for usage of this class of biosensors that successfully functions in complex media and delivers rapid, clear results for toxins in their diagnostic range. This work will enable greater adoption of electrochemical biosensors in industrial fields and as point‐of‐care diagnostics.
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 Information

Fresno 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 Information

Supported by Rhode Island National Institutes of Health INBRE Grant
Examination of Turnip Mosaic Virus Genome‐Linked Protein (VPg) as an Effective Novel Inhibitor of Ricin Toxin A Chain (RTA)
Authors: De Los Santos Sabrina , Domashevskiy Artem V
Published: November 2023

Ricin, a toxin originating from the seeds of Ricinus communis, is a type II ribosome inactivating protein (RIP) that is potent against eukaryotic ribosomes. The A chain of ricin, RTA, with functionality as an RNA N‐glycosidase, is capable of terminating protein synthesis via cleavage of the N‐glycosidic bond of a particular adenine residue in the highly conserved sarcin/ricin (S/R) loop of 28S ribosomal RNA. Pokeweed antiviral protein (PAP), stemming from the leaves of Phytolacca americana, is a type I RIP with both depurinating and antiviral properties. A previous study established that turnip mosaic virus genome‐linked protein (VPg), required for viral functions such as infectivity, inhibits the aforementioned characteristics of PAP. Based on the aforementioned study, we hypothesize that VPg will interact with RTA, relying on similarities in PAP and RTA active sites, and will inhibit RTA activity against ribosomal RNA. Following purification of VPg and RTA, steady state fluorescence will aid in the determination of the binding affinity pertaining to the protein‐protein complex. Quantification of the cleaved adenines, with and without VPg present, will be conducted through a bioluminescent assay that incorporates the conversion of adenine into adenosine triphosphate to produce luminescence via an enzymatic reaction involving firefly luciferase.
Development of an efficient structure‐based drug discovery platform for BACE1 Inhibitors for the treatment of Alzheimer's Disease
Authors: Yen Yu‐Chen , Kammeyer Annalissa , Jensen Katherine C. , Ghosh Arun K. , Mesecar Andrew D.
Published: November 2023

BACE1 (Beta‐site Amyloid precursor protein (APP) Cleaving Enzyme 1) is a promising therapeutic target for Alzheimer's disease (AD). However, efficient expression, purification and crystallization systems are not well described or detailed in the literature nor are approaches for treatment of enzyme kinetic data for tight‐binding inhibitors well described. Therefore, we have developed a platform for expression and purification of BACE1, including protein refolding from inclusion bodies, from E. coli in addition to a reproducible crystallization procedure of BACE1 bound with inhibitors. We also report a detailed approach to the proper analysis of enzyme kinetic data for compounds that exhibit either rapid‐equilibrium or tight‐binding inhibitor behavior. Our methods allow for the purification of 16 mg of BACE1 enzyme from 1 L of culture scale, which is higher than reported yields in the current literature. For treatment of inhibitory data, we have identified binding affinity ranges where fitting kinetic data using the Morrison equation and plot and running a standard inhibitor to accurately determine the active enzyme concentration, is favored over classical models. To evaluate our platform from purification to X‐ray crystallography, we have tested known potent inhibitors previously reported by the Ghosh lab. The inhibitory constants and X‐ray structures of these three compounds in complex with BACE1 were readily obtained and provide important insight into the structure and thermodynamics of the BACE1‐inhibitor interactions.
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 Information

This work was supported by NIH grant AI047818 awarded to B.M.
Synthesis and biological evaluation of 10‐chloro‐9h‐5‐oxa‐6b,7,9,11,12‐pentaaza‐benzo[5,6]pentaleno[2,1‐b]naphthalen‐6‐one
Authors: Govori Sevdije , Haziri Arben , Neziraj Nexhat , Ibrahimi Hamide
Published: November 2023

10‐chloro‐9h‐5‐oxa‐6b,7,9,11,12‐pentaaza‐benzo[5,6]pentaleno[2,1‐b]naphthalen‐6‐one 2 was synthesized by reaction of 4‐(6‐Chloro‐7H‐purin‐8‐ylamino)‐3‐nitro‐chromen‐2‐one 1 with P(OEt)3 under reflux conditions. Structure of synthesized compound was characterized using IR, NMR spectra and elemental analyses The purified synthesized compound 2 was screened for its antibacterial activity. Compound was screened against Escherichia coli and Bacillus cereus. The antibacterial activity of synthesized compound 2 is compared to antibacterial activity of the standard antibiotics Cephalexine and Streptomycine.
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 Information

NIH 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

Introduction

Pseudomonas 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.

Methods

P. 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.

Results

There 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.

Conclusion

Proteins in lysates of P. aeruginosa are glycosylated and may contribute to the regulation of QS by an undetermined mechanism.

Support or Funding Information

Supported 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 Information

This 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

Objectives

To determine if indices of acyl‐CoA desaturase can differentiate stages of dementia.

Methods

We 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.

Results

The 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.

Conclusions

The 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 Information

L.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 Information

2013 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 Information

University of North Florida Office of Undergraduate Studies and the Office of Academic Affairs
Administration of a novel β‐hydroxybutyrate ester after radiation exposure suppresses in vitro lethality and chromosome damage, attenuates bone marrow suppression in vivo
Authors: Kemper Martin F , Miller Alexandra , Pawlosky Robert J , Veech Richard
Published: November 2023

Cellular mechanisms to protect against and repair radiation damage depend on reducing equivalents from oxidative metabolism. Two recent studies explored compounds (ex. Ex‐Rad and TP‐508) with efficacy in preventing injury or reducing its severity but neither takes advantage of cellular metabolism to this end. We report for the first time that a novel ketone body ester (KE) protects against injury and death in cells and animals exposed to several types of radiation.

The unique metabolism of ketone bodies shifts biochemical set points, particularly of the NADP/H system which is essential to cellular antioxidant function and oxidative damage repair. Additionally, the demonstrated activity of the ketone bodies as histone deacetylase inhibitors and enhancers of DNA repair may make these compounds uniquely suited to combat radiation injury.

We show that addition of KE to culture media provides significant protection to cultured osteoblasts against γ‐radiation lethality when administered before or after radiation. KE significantly attenuated gamma radiation induced chromosomal injury in an escalating dose model (60–70% reduction). Ketone ester supplementation also significantly reduced proton radiation induced γH2Ax foci, a marker of double stranded DNA breaks, and overall proton radiation lethality. KE protection extends to other radiation types, with an average dose reducing factor of 30%.

In vivo confirmation of these findings is ongoing, but preliminary data indicates that many bone marrow chromosomal damage markers were attenuated by 50% when KE was administered by gavage 24 hours after γ‐radiation exposure. Importantly, the reticulocyte/erythrocyte ratio in bone marrow, a marker of erythrogenesis and an important indicator for survival at the radiation exposure level studied was significantly rescued by KE.

Support or Funding Information

Funding was provided by the NIH/NIAAA, Department of Defense and Defense Medical Research and Development Program
Novel Method for the Detection of Glucose Uptake: Direct Measurement of Glucose Levels in Cultured Cells
Authors: Yan Kevin , Li Shunan , Lin Yuan , Saddar Sonika , Tchaga Grigoriy , Yan Gordon
Published: November 2023

Glucose uptake is the transport process of glucose from the outside of cells into cells across the cell membrane. It is one of the key regulation steps of glucose metabolism. Study of glucose uptake provides important information for understanding glucose metabolism and its regulation in normal and disease development. Here we report the development of a novel glucose uptake detection method based on direct measurement of glucose levels in cultured cells. In this method, we utilized the inhibitor, 3‐bromopyruvate (3BP), to temporarily inhibit hexokinases, the first enzyme that metabolizes glucose in cells. Thus, glucose metabolism was blocked, which leads to glucose accumulation rather than its rapid metabolism inside the cells. Glucose uptake was then detected by direct measurement of the glucose levels inside of the cells using Glucose Oxidase/HRP/OxiRedTM enzyme based method. In comparison with current widely used methods, this direct glucose uptake detection method has the following advantages: i) No radioisotope materials are used; ii) It directly detects glucose without using structurally modified glucose derivatives; iii) Glucose from culture medium is transported inside of cells, and then detected with minimum medium changes and washing processes. This simple, sensitive and direct glucose uptake detection method provides a powerful tool for studying the process of glucose uptake and its regulation, as well as for screening and characterization of drugs that regulate glucose uptake during normal and disease development.

Support or Funding Information

This project was supported by the New Product Development Funds from R&D Division of BioVision Inc.
Characterizing the Structural Requirements of the Human Antiviral Protein Apobec3G
Authors: Casazza Rebecca , Sheehy Ann
Published: November 2023

Apobec3G (A3G) is an endogenous, human protein that restricts Human Immunodeficiency Virus Type 1 (HIV‐1) infection. A3G exhibits cytidine deaminase activity and when packaged into virus particles, has been shown to hypermutate the viral genome during the critical process of reverse transcription in the next round of target cells. HIV‐1 combats this cellular defense with Viral Infectivity Factor (Vif), a viral protein that binds to A3G resulting in its polyubiquitination and degradation in the 26S proteasome. Research has shown an uncoupling of cytidine deamination and antiviral activity, indicating that A3G may restrict infection via multiple mechanisms. It has been proposed that, in addition to its well‐characterized cytidine deamination activity, A3G may also sterically hinder the reverse transcription process by blocking reverse transcriptase from accessing the viral RNA. A3G has characterized RNA‐binding activity although the regions of the protein involved in this interaction have not been fully delineated.

To investigate the A3G structural requirements for this proposed alternate mechanism, an A3G mutant library was generated and examined for antiviral and deaminase activity. When tested for functional activity, several mutants identified in an N‐terminal domain appeared to demarcate a region that, when mutated, exhibited decreased antiviral activity, but retained wildtype levels of deaminase activity. This phenotype suggests that this putative domain may be implicated in A3G's alternate antiviral mechanism. Computational analysis of this region of A3G indicate that it is characterized by a highly‐positive surface charge. The region that the novel domain falls within has previously been implicated in nucleic acid binding. We hypothesize that this novel domain has a role in facilitating A3G binding to the negatively‐charged viral RNA backbone. This surface charge is primarily conferred by three specific residues within the domain of interest. Here, the importance of this charge in A3G's alternate mechanism is investigated through site‐directed mutagenesis of the three positively charged residues, and subsequent assessment for antiviral and deaminase activity.