bluuebunny/crossref_metadata_2025 huggingface dataset:
Structure of a Key Intermediate in the Reaction Catalyzed by the Dual Specificity Methylase RlmN
Authors: Schwalm Erica Lynne , Grove Tyler , Boal Amie , Booker Squire
Published: November 2023

Both rRNA and tRNA are highly modified, contributing to their structural and functional integrity. RlmN is a dual‐specificity RNA methylase that modifies C2 of adenosine 2503 (A2053) in 23S ribosomal RNA and C2 of adenosine 37 (A37) is several Escherichia coli tRNAs, representing one of only two enzymes known to act on both rRNA and tRNA. Cfr, a related methylase, modifies C8 of A2503, conferring resistance to more than five classes of antibiotics. These two methylases employ a unique radical mechanism to catalyze these difficult transformations. While a structure of RlmN was previously solved, it lacked the RNA substrate. As shown previously, a Cys118Ala substitution in RlmN results in the capture of a covalent intermediate in which the protein is cross‐linked to the RNA substrate at the C2 carbon of the target adenosine. The 2.4 Å resolution structure of this key intermediate formed using a tRNAGlu substrate was solved, providing key insight into the mode of substrate recognition and unresolved mechanistic details. RlmN contacts the entire length of the tRNA, similar to tRNA synthetases, but forms few nucleotide sequence specific interactions. Access to A37 is achieved through an induced‐fit strategy that completely unfolds the tRNA anticodon stem loop. This binding strategy likely facilitates recognition of the diverse sequences found in the tRNA and rRNA substrates. Mutagenesis of interacting residues suggests key interactions that are necessary for the modification of the tRNA substrates. The observation of reaction products in the active site suggests the order of substrate binding and product release, providing previously unknown mechanistic details. This structure represents the first structure of a dual specificity methylase in complex with the entire tRNA substrate.

Support or Funding Information

This work has been supported by NIH grants GM100011 (A.K.B.) and GM101957 (S.J.B.), the Searle Scholars Program (A.K.B.), and Tobacco Settlement Funds (TSF13/14 SAP_4100062216) to S.J.B.
A Spatiotemporal‐Controlled Targeting Tool for Understanding the Roles of Redox Signals on Bacterial Growth
Authors: Pham Vanha Nhat
Published: November 2023

Redox‐mediated modifications are becoming increasingly appreciated as finely‐tuned signaling events that affect organism fitness. Greater understanding of these redox signals and cellular communication is imperative to being able to influence specific cellular functions and growth. However, precise spatiotemporal control to investigate these redox‐mediated inter‐ and intracellular communications has thus far been impossible. A recently developed method, targetable reactive electrophiles and oxidants (T‐REX), has achieved the precision necessary to explore the impacts of discrete redox signals in live mammalian cells. T‐REX employs an inert photo‐caged precursor of a bioactive redox signal and is targeted to a protein of interest (POI) through a HaloTag. Upon UV light, the redox signal is released onto the POI. This project adapts the T‐REX approach to bacteria to better resolve the roles of redox signals in bacteria stress responses and growth pathways. We have shown that T‐REX in bacteria—using E. coli as the model organism—yields comparable specificity as in mammalian cells, highlighting the generality of the methodology. Using this platform, we also demonstrate the capability of delivering redox signals to specific subpopulations of cells expressing different fluorescent proteins. With this approach, we are making headway toward identification of novel bacterial stress response pathways. This project highlights a novel chemical biology tool used to address the longstanding challenges in precise targeting of small‐molecule redox signals to specific protein targets in living cells.

Support or Funding Information

We acknowledge Frank L. and Lynnet Douglas Fellowship for Undergraduate Summer Research for Vanha N. Pham and NIH Director's New Innovator Award (1DP2GM114850‐01) for Yimon Aye.
Sensitive Serologic Detection of Pathogenic Mycoplasma via Electrochemical Biosensor
Authors: Jacobs Jena , Bonham Andrew J
Published: November 2023

Mycoplasma pneumonia infects 2 million people every year and is responsible for upper respiratory infections and “walking pneumonia.” Here, we describe the creation of a novel electrochemical biosensor capable of detecting these pathogenic Mycoplasma for use in academic, research, and clinical applications. Current diagnostics of Mycoplasma, such as molecular‐based assays, PCR and serological analysis, are time consuming, expensive, and not particularly accurate. Serological analysis is our main focus, not only because it is not available in the United States, but due to the fact that current “serological kits” do not measure the presence of the microorganism, but instead measure the host immune response, creating the possibility of a false negative result for most infected individuals. In response, our biosensor is designed for rapid, reliable, and reagentless serological detection of several common Mycoplasma strains. We are able to detect this subgroup of Mollicute bacteria by detecting a protein that is commonly secreted by many pathogenic mycoplasmas, P48 protein. A modified aptamer against P48 was incorporated into a custom oligonucleotide scaffold and is used in a gold‐electrode‐bound fashion to give a robust, dose‐dependent electrochemical signal change upon binding the secreted P48 target. Ultimately, this biosensor should bring improvements to diagnosis and thus treatment of Mycoplasma in patients who present a proposed infection.
Mechanisms and Physiology of Fat Synthesis and Storage
Authors: Farese Robert V.
Published: November 2023

The synthesis and storage of neutral lipids, such as triglycerides, are crucial for both cellular and physiological energy homeostasis. When fuels are abundant, triglycerides are synthesized in the ER by DGAT enzymes and subsequently stored in cytosolic organelles called lipid droplets. We study the molecular processes that govern the synthesis of energy storage lipids as well as their storage in and mobilization from lipid droplets. Studies on genetic factors controlling lipid droplet growth and utilization will be presented, as will relevant studies of the physiology of lipid storage.
Anti‐Microbial Properties of Plants Native to the Philippines and to Minnesota
Authors: Pethke Bridget Marie
Published: November 2023

A growing area of research is the identification of plant chemicals that exhibit anti‐microbial properties. This study involved analyzing the anti‐microbial activity of chemical extracts isolated from two plants that are members of the plant family Araceae, Epipremnum pinnatum and Arisaema triphyllum, plants native to the Philippines and Minnesota, respectively. To do this, plant chemicals were isolated by ethanol extraction, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays were completed to determine whether the extracts inhibited the growth of (MIC assay) and killed (MBC assay) the microorganisms. The microorganisms used were Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Results suggest that extracts from both plants are able to inhibit the growth of and kill the microorganisms tested. Plant extract isolated from the leaves of E. pinnatum exhibited the greatest anti‐microbial activity while extract isolated from the rhizomes of A. triphyllum was most effective at inhibiting and killing the microorganisms. Out of all three microorganisms, E. coli appeared to be least sensitive to the anti‐microbial activity of the plant extracts. Results from these studies suggest that extracts from the plants E. pinnatum and A. triphyllum may contain chemicals that would be useful in treating diseases caused by these microorganisms.
Influence of NSAID Flurbiprofen on Protein Helix‐helix Interactions
Authors: Gugala Natalie , Turner Raymond J.
Published: November 2023

The mechanism of action of non‐steroidal anti‐inflammatory drugs (NSAIDs) is not fully understood, however, there is now some reports signifying that they may inter chelate into the membrane consequently influencing integral membrane protein folding. Given the recent appreciation of the microbiome, we considered investigating the influence of NSAIDS on the folding of bacterial multidrug resistance proteins. In this work, the Bacterial Adenylate Cyclase Two‐hybrid System (BACTH) was used to study the protein‐protein interactions of EmrE, from Escherichia coli, which is an inner integral membrane protein belonging to the Small Multi‐drug Resistant family. The in vivo BACTH assay was used as a technique to develop an enhanced understanding of the helix interactions of this protein as it resides within the membrane. In particular, a glycine motif found on helix 4 has been observed to be critical for the oligomerization and functionality of EmrE, in agreement to the known importance of these motifs in membrane proteins. In recent years it has been revealed that certain NSAIDs have the ability to bind to glycine motifs and enhance or disturb transmembrane segment interactions. Flurbiporfen, a common NSAID, was used in this work to explore if this drug would affect EmrE. Upon increasing concentrations of flurbiprofen it was found that the interactions between certain integral helix pairs were strengthened, whereas the interaction between the leucine zipper peptides, which was used as a positive for this work, demonstrated a decrease in helix interaction. Our work suggests that these drugs could influence bacterial protein structure and function.
The C. albicans adenylyl cyclase, CYR1p, binds bacterial cell wall fragments via its LRR domain
Authors: Burch Jason , Schaefer Amy , Lauro Mackezie , Melnyk James , Glowala Jakub , Wykoff Dennis , Grimes Catherine Leimkuhler
Published: November 2023

Every organism, from the complex to the single cellular, is surrounded by trillions of microorganisms. In order to survive an organism must be able to recognize and adapt to the microbiome that surrounds it. The ability to recognize these microorganisms occurs when the appropriate pathogen associated molecular pattern (PAMPs) binds to the proper pattern recognition receptor (PRR). PAMPs are molecules associate with microorganisms such as lipopolysaccharide, viral RNA, and peptidoglycan. Interestingly, yeast respond to small molecules generated by bacteria. Recognition of the bacterial cell wall fragment muramyl dipeptide (MDP) by the adenylyl cyclase, CYR1p, triggers C. albicans to enter pathogenic hyphal growth. While not characterized as a PRR, CYR1p contains a Leucine Rich Repeat (LRR) domain, which is a common protein motif found in many PRRs, including toll‐like receptors and nod‐like receptors in human, and is often the site of PAMP binding. Using a surface plasmon resonance (SPR) assay, we were able to determine the binding affinity for MDP to the LRR domain of CYR1p. Similar SPR assays were used to determine which portions of MDP is necessary for binding CYR1p. The molecules found to bind CYR1p will be tested in vivo, to determine which, if any, are able to trigger hyphal growth in C. albicans.
Introducing Glycans to Molecular Visualization Software
Authors: Thieker David F. , Hadden Jodi , Stone John , Woods Robert
Published: November 2023

Despite the essential role of glycoconjugates in biological processes, molecular visualization software fail to recognize carbohydrates as a unified class of macromolecules. Although proteins and DNA are detected by default, carbohydrate residues are treated as ‘non‐standard’ (HETATM) and grouped together with solvent and ions. As a result, carbohydrates currently lack a simplistic representation for 3D structure, and are predominantly visualized with all atoms displayed. Discerning the glycan sequence from an atomic view is challenging for short fragments and prohibitive for oligosaccharides. The following work describes the implementation of algorithms for the automatic identification and representation of carbohydrate moieties within the highly‐cited Visual Molecular Dynamics (VMD) program. Carbohydrate residues are now detected with a robust algorithm that distinguishes carbohydrate moieties from other ring‐based structures based upon atomic coordinates, rather than residue names. A 3D adaptation of the symbolic nomenclature developed by the Consortium for Functional Glycomics (CFG) has been implemented as a cartoon representation of carbohydrate residues, analogous to the canonical depiction of protein backbones with ribbons. Incorporation of a standard representation scheme familiar to the glycobiology community should improve adoption and allow researchers to more efficiently convey complex structural concepts pertaining to carbohydrate‐containing biomolecules (i.e. glycoforms of proteins, ligand sequences, etc.). Alternatives to the 3D‐CFG representation have also been included to enhance the atomic view of carbohydrate moieties. These additions to VMD finally create a program capable of treating carbohydrates as a unified class of molecules, complete with custom representations.

Support or Funding Information

The authors thank the NIH for support (R01 GM100058).
Glycosylation Alters Oligomerization Status of the Human CEACAM1‐IgV Domain
Authors: Prestegard James H , Moremen Kelley , Zhuo You , Yang Jeong‐Yeh
Published: November 2023

Human carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is a cell‐surface signaling molecule involved in cell adhesion, proliferation and immune response. It is also implicated in cancer angiogenesis, progression and metastasis. This diverse set of roles likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other members of the CEACAM superfamily. Its N‐terminal Ig‐like variable (IgV) domain has been suggested to be a principal player in these interactions, facilitating cis and trans homo‐dimer formation, as well as complex formation with other molecules. The IgV domain crystal structure, which used E. coli expressed material that lacks glycosylation, shows a dimer interface that obscures a glycosylation site, while other studies using mutagenesis and electron microscopy suggest dimer formation may also be mediated by a non‐glycosylated surface on the opposite side of the molecule. Here, we use NMR cross correlation measurements and residual dipolar coupling measurements to examine the effect of glycosylation on CEACAM1‐IgV domain oligomerization status. Both a non‐glycosylated form expressed in E. coli and a glycosylated form expressed in HEK293 GnT1‐cells and trimmed to a single GlcNAc residue, were examined. Our findings demonstrate that IgV with a single N‐linked GlcNAc at all glycosylation sites inhibits dimer formation of any type, whereas formation of a dimer similar in structure to that found in the crystal structure occurs in solution when glycosylation is absent. Future studies will be directed at the role additional domains and more complex glycosylation may play in native dimer formation.

Support or Funding Information

Financial support provided by the National Institutes of Health, R01GM033225
Common Jamaican Herbal Medicine, Bizzy Nut, Inhibits Cell Cycle in Prostate Cancer Cells
Authors: Gray Wesley , Chumbow Anne , Phillips Orville
Published: November 2023

Prostate cancer development is dependent on androgens, and the majority of patients respond to androgen ablation. However, virtually every patient will develop hormone‐resistant prostate cancer and no longer respond to androgen deprivation therapy (ADT). Biz‐2 (the common Jamaican herbal medicine) Kola acuminate elicited anti‐proliferative effects in both hormone‐responsive (LNCaP cells) and hormone‐resistant (DU145 cells) prostate cancer cells. In this study we determined the cytotoxic efficacy of Biz‐2 in hormone‐responsive (LNCaP cells), the hormone‐resistant (DU145 cells) and a normal transformed (RWPE‐1) prostate cells. We determined growth inhibitory parameters (GI50, TGI and LC50) over 24hrs to 96 hrs using the MTT assay and cell count. Biz‐2 was found to inhibit the proliferation of all tested cell lines in a concentration‐dependent and time‐dependent manner. Both cancer cells showed significantly higher cytostatic and cytocidal (TGI 230ppm in DU145 verses 860ppm in RWPE‐1 and 156ppm in LNCaP verses 860ppm in WRPE‐1) as compared to the normal non‐transformed RWPE‐1. To begin to understand the mechanism of Biz‐2 anti‐proliferative activity, we used flow cytometry to determine where in the cell‐cycle Biz‐2 induced proliferation blockage. Analysis of untreated synchronized LNCaP cells by flow cytometry shows that 63% of the cells were in the Gi/Go, and 36% in the G2/M phases of the cell cycle. Upon treatment with Biz‐2, over 95% of the cells were in the Gi/Go phases. When these experiments were performed in DU‐145 we observed an accumulation of cells in the G2/M phases of the cell cycle. Both western blot and RT‐PCR showed a down regulation in the levels of the cell cycle protein. Our results suggest that the mechanism of action of Biz‐2 in androgen‐dependent and androgen‐independent prostate cancer cells is different.

Support or Funding Information

NIH grant, Grant No. NIH R15AT006821
Novel Role for Lysocardiolipin Acyltransferase (LYCAT) in Non‐Small Cell Lung Cancer
Authors: Kotha Sainath R , Huang Longshuang , Van Scoyk Michelle Mai , Avasarala Sreedevi , Winn Robert A , Bikkavilli Rama Kamesh , Natarajan Viswanathan
Published: November 2023

Lung cancer is the leading cause of deaths in United States and non‐small cell lung cancer (NSCLC) accounts for ~85% of all lung cancers with a 5‐year survival rate of approximately ~16%. Therefore there is an immediate need to develop new strategies for early detection and more effective treatments options. Mitochondrial dysfunction including but not limited to defects in mitochondrial genomics and dynamics has long been implicated to play a role in human health and disease particularly in cancer initiation, progression and treatment options since it plays a pivotal role in cell death and survival. Lysocardiolipin acyltransferase (LYCAT), a Cardiolipin remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, plays a crucial role in maintaining normal mitochondrial function and vascular development. LYCAT was shown to be up‐regulated in cancers; however, the role of LYCAT in lung cancer is yet unclear. Probing the protein expression of LYCAT in lung cancer specimens, non‐transformed bronchial epithelial cell lines and 5 lung cancer cell lines revealed increased LYCAT expression and activity in all the lung cancer samples and cell lines tested in comparison to the control lung tissues and non‐transformed epithelial cell lines. To determine the role of LYCAT in lung cancer, NSCLC cell lines H2122 and H23 were transfected with either scrambled or LYCAT shRNA and differences in serum‐induced cell proliferation, migration, clonogenecity and mitochondrial dynamics were determined. Our results demonstrated that down‐regulation of LYCAT by shRNA significantly attenuated cell migration, proliferation, and invasion in NSCLC cell lines compared to control cell lines. Furthermore knockdown of LYCAT expression in NSCLC cell lines inhibited mitochondrial fragmentation and enhanced mitochondrial fusion. Taken together, these data demonstrate a strong association between increased LYCAT expression and cell proliferation, motility, invasion and mitochondrial dynamics in NSCLC cells. Thus, development of targeted therapies to reduce LYCAT expression in NSCLC should be beneficial.

Support or Funding Information

This work in part was supported by funds from the College of Medicine, UIC and NIH HL98050 to VN.
Enzymes Running in Reverse: Catalytic Studies on Isocitrate Dehydrogenase Mutations Implicated in Cancer
Authors: Sohl Christal , Matteo Diego Avellaneda , Grunseth Adam , Anselmo Stacy , Gonzalez Eric , Kennedy Madison
Published: November 2023

While it has been nearly a century since metabolic alterations in tumors were first described, only relatively recently have mutations in metabolic enzymes been implicated in cancer. Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), the enzymes responsible for the NADP+– dependent oxidation of isocitrate (ICT) to α‐ketoglutarate (αKG), are mutated in over 80% of adult grade II and grade III gliomas and secondary glioblastomas, and up to 20% of acute myeloid leukemias. These mutations have tumor suppressive properties in that they are significantly deficient in the normal oxidation reaction. Surprisingly, a class of these mutations can also serve as oncoproteins in that they can catalyze a new reduction reaction, generating the proposed oncometabolite 2‐hydroxyglutarate (2HG). This suggests intriguing catalytic and structural mechanisms are at work in both types of mutations. By using kinetic and structural methods, we are exploring the molecular mechanisms of dysfunction of mutations identified in tumors that are proposed to serve as either oncoproteins or tumor suppressors depending on their ability to catalyze 2HG formation. Through these structure‐function relationships, we can understand the catalytic attributes that result in these dysfunctional activities that can promote tumorigenesis.

Support or Funding Information

This work is supported by NIH R00CA187594.
New Insights into the Anatomy of the Bulbospongiosus Muscle and Its Role in the Composition of the External Anal Sphincter in Humans
Authors: Hall Margaret , Plochocki Jeffrey H , Adrian Brent , Rodriguez‐Sosa Jose R.
Published: November 2023

Traditional descriptions of the bulbospongiosus muscle in males and females describe a striated muscle overlying the most proximal portion of the corpus spongiosus, the bulb of the penis in males, and the bulbs of the vestibule in females. Likewise, in both sexes the bulbospongiosus muscle and the superficial transverse perineal muscles are described as inserting at the midline on the perineal body. In dissections of forty (40) human cadavers we found both of these descriptions do not accurately characterize the anatomy. In all forty cadavers examined, as suggested by the embryology literature, the adult bulbospongiosus continues from the urogenital triangle to the anal triangle as an uninterrupted ring of muscle that forms the superficial portion of the external anal sphincter and passes deep and lateral to the perineal body without insertion. Additionally, the superficial transverse perineal muscle passes anteriorly and superiorly to insert onto the bulbospongiosus muscle, and does not follow traditional descriptions asserting that this muscle continues medially from the ischial tuberosities to the perineal body. The perineal body is inferior to the rest of the perineum and in the great majority of cadavers we did not observe any muscle fibers inserting on the perineal body, again contradicting the published literature. Dissections of the external anal sphincter demonstrate that the subcutaneous external anal sphincter has a different fiber direction and is not continuous with the superficial external anal sphincter. Also, the deep external anal sphincter is continuous with the puborectalis muscle and is located deep to and separate from the superficial external anal sphincter. Therefore, we suggest that the traditionally conceived external anal sphincter, with three parts of a single muscle, is more usefully described as three individual muscles.

Support or Funding Information

Midwestern University Intramural Funds
Mitochondrial Calcium Entry Induces Mitochondrial Reactive Oxygen Species Production by ATP Synthase‐uncoupled, But Proton Leak‐coupled Mechanism – A New System of How Atherogenic Stimuli Activate Aortic Endothelial Cells without Compromising Endothelial Cell Survival
Authors: Li Xinyuan , Fu Hangfei , Fang Pu , Koch Walter J , Wang Hong , Yang Xiao‐Feng
Published: November 2023

Previously considered as toxic byproducts of cellular metabolism, it was recently discovered that mitochondrial reactive oxygen species (mtROS) are in fact signaling molecules, which drive inflammatory cytokine production and T cell activation. In addition, pathological conditions as diverse as cancer, autoimmune diseases, and cardiovascular diseases all share common feature of increased mtROS production above basal levels. MtROS are generated from the partial oxygen reduction in the electron transport chain and a number of factors have been linked to mtROS production, including mitochondrial calcium (Ca2+), mitochondrial membrane potential, and proton leak. However, since mtROS and ATP production are both coupled to electron transport chain activity, it is unclear whether mitochondrial ROS could be induced independently from ATP synthesis in the cell. By using Seahorse XF96 mitochondrial function analyzer and other molecular biology approaches, we found that during endothelial cell (EC) activation process induced by proatherogenic stimuli lysophosphatidylcholine (lysoPC), mitochondrial calcium entry mediates proton leak‐coupled, but ATP‐synthesis‐uncoupled mitochondrial respiratory chain acceleration and increased mtROS production. By doing this, EC could upregulate mtROS production without compromising mitochondrial membrane potential and ATP generation, and consequently without causing mitochondrial damage and endothelial cell death. Thus, we uncovered a novel pathophysiological role of proton leak in fine‐tuning mtROS production for endothelial activation purpose without compromising endothelial cell survival. This new working model explains how mtROS could be increasingly generated independently from ATP synthesis. Our finding is significant towards the development of novel mitochondrial ROS‐modulating therapies to combat cardiovascular inflammatory diseases, atherosclerosis, autoimmune diseases and cancers.

Support or Funding Information

NIH RO1 HL 108910‐01
Hypoglycaemic Activity of the aqueous extract of Costus afer stems alone and in combination with metformin
Authors: Monago Comfort , Anacletus Francis Chukwuma , Nwauche ThankGod Kelechi
Published: November 2023

Medicinal plants are reservoirs of natural products with anti‐diabetic potentials. In the present study we screened Costus afer for the presence of plant products and to study the effects of the aqueous stem extract of the plant on fasting blood glucose of streptozotocin induced diabetic male rats. Adult male wistar strain albino rats were given graded doses (500, 1000 and 1500mg/kg) of the aqueous stem extract of Costus afer, and a standard hypoglycaemic drug, metformin was co‐administered with 500mg/kg of the plant extract for a period of 3, 6 and 9 weeks. The statistical analysis was carried out using one way ANOVA followed by post hoc LSD multiple comparison on SPSS 19. Costus afer showed significant hypoglycaemic effect (p<0.05) compared to glucose level of the normal control rats. Also, a combination of plant extract and metformin caused a significant reduction in fasting blood sugar of the streptozotocin induced diabetic rats. In histological evaluation of the pancreas, the islet cells of the Langerhan of the pancreas were repopulated and preserved in all the groups treated with the plant extract and its combination with metformin against the diabetic control group whose islet cells were reduced. The results validate the use of Costus afer as a hypoglycaemic plant in native medicine. Costus afer could be a promising plant for the development of anti‐diabetic drugs.

Support or Funding Information

Research was funded by the authors.
Sub‐Acute Administration of Ethanol Extracts of Erythrophloem suavolens Induced Hepatotoxicity and Oxidative Stress in Wistar Strain Albino Rats
Authors: ADISA RAHMAT ADETUTU , NWALIOBA COSMOS U , AKUJOBI SYLVIA , ORIBABOR JUDE
Published: November 2023

Erythrophloem suavolens is a known poisonous plant to livestock, insects and humans. However, there is no known scientific basis for the toxic effects of this specie. The present study was designed to investigate the hepatotoxic and antioxidative effects of 28 days sub‐acute administration of ethanol extracts of E. suavolens stem bark (EESB) to albino rats. Graded doses of EESB (50, 100, 150, 200 mg/kg) were administered to each of groups II – V of 5 animals each for 28 days, respectively. Group I served as control and received distilled water. Hepatotoxicity, hepatic antioxidant status and levels of plasma lipid profile were determined. Sub‐acute administration of EESB decreased the activities of superoxide dismutase and catalase as well as levels of glutathione in liver homogenate, post‐mitochondrial and mitochondrial fractions, respectively. The amount of malondialdehyde produced increased significantly (p<0.05, p<0.01) and dose‐dependently in all the hepatic fractions, respectively compared to control. The activities of alkaline phosphatase, alanine and aspartate amino transferases were decreased non‐significantly (p>0.05, p>0.01), respectively compared to control animals. Low density lipoprotein, total cholesterol and triglyceride levels decreased non‐significantly but markedly at 200 mg/kg of EESB. Non‐significant increase in level of high density lipoprotein was observed in the EESB‐treated animals. Plasma albumin and total protein concentrations of EESB‐treated group were not significantly different from control. Photomicrographs of liver sections showed necrotic lesions at 200 mg/kg dose.

These findings suggest that sub‐acute administration of EESB is hepatotoxic which seems mediated by induction of oxidative stress.
The Role of Semen‐Derived Exosomes on Acute HIV Infection in T Lymphocytes
Authors: Moricz Bridget , Madison M. Nia , Welch Jennifer , Okeoma Chioma
Published: November 2023

About 35 million people are infected with HIV and even with antiretroviral therapy, the infected individuals are still not living a normal life expectancy. Exosomes, nanovesicles with host proteins and small RNAs, have been shown to possess antiviral properties including suppression of HIV infection. However, these studies were performed in a short‐term setting within 24–48 hours post infection. Here we sought to determine the role of semen exosomes on HIV infection in a longer setting, up to ten days post infection. Semen exosomes were added to acutely infected Jurkat cells and aliquots of the cells and media were taken every two days. The samples were then analyzed for viral integration, replication and fitness either by qPCR, gel electrophoresis or reverse transcriptase assays. Based on the results, exosomes decrease HIV integration, replication and progeny fitness.

Support or Funding Information

NSF REU in Microbiology at The University of Iowa through grant number DBI‐1262222
Uncovering the mechanism of trogocytosis in Entamoeba histolytica
Authors: Feeney Shea E. , Ralston Katherine S.
Published: November 2023

Entamoeba histolytica (histolytica: tissue dissolving) is a eukaryotic pathogen that is the causative agent of amoebiasis. Amoebiasis is a global concern that is responsible for 50 million cases of diarrheal infections, and 100,000 deaths per year primarily in developing countries. This infection causes diarrhea and can also lead to invasive colitis and extraintestinal disease. In some cases, amoebae can spread to the liver and cause abscesses. Killing of human cells by the amoebae is likely to drive this tissue destruction. Our lab recently showed that the amoebae kill human cells by nibbling off bites via trogocytosis (trogo‐: nibble) and discard dead cell corpses, whereas dead cells are ingested whole via phagocytosis. While trogocytosis also occurs in a variety of other microbes and multicellular organisms, the mechanism is not known. In immune cell trogocytosis, two small GTPases have been identified that are involved in its regulation, and one has been shown to be specific to trogocytosis. Thus although trogocytosis and phagocytosis may share some mechanistic components, there are likely to be unique proteins specifically involved in trogocytosis that have not yet been identified. Therefore, I propose that amoebic trogocytosis in E. histolytica requires specific signaling machinery that is distinct from phagocytosis. The discovery of genes involved in amoebic trogocytosis in E. histolytica is not only relevant to the pathogenesis of amoebiasis, but also to the many other organisms that nibble. Two complementary approaches are being utilized to address this hypothesis, a forward genetic screen and transcriptomics analysis, to identify genes uniquely involved in trogocytosis that are not involved in phagocytosis. I am taking advantage of the endogenous RNAi pathway in E. histolytica in order to perform two different forward screens to isolate trogocytosis or phagocytosis mutants. Preliminary studies provide proof of principle that ingestion mutants can be isolated by selection. Since there is evidence that both phagocytosis and trogocytosis are transcriptionally regulated in E. histolytica, I am also performing RNA‐seq analysis to examine differential gene expression in amoebae undergoing trogocytosis versus phagocytosis. My preliminary results with imaging flow cytometry have shown a method to quantify and optimize the number of amoebae performing either trogocytosis or phagocytosis respectively. These results will help to define the mechanism for trogocytosis, an important cellular function in a variety of unicellular and multicellular organisms.

Support or Funding Information

NIH MCB T32 GM007377; NIH NIAID K22 Career Transition Award Al108814
Culture Of Urine Sediment Reveals Differences Between Patients With Overactive Bladder (OAB) And Controls: Are Changes In The Bladder Microbiome Responsible For Symptoms Of OAB?
Authors: Krska Louise , Balachandran Aswini , Caballero Nadine , Curtiss Natasha , Peppiatt‐Wildman Claire , Strutt Matthew , Duckett Jonathan , Wildman Scott S.P.
Published: November 2023

Overactive bladder (OAB) is a common syndrome characterized by increased frequency, urinary urgency, urinary incontinence and nocturia. The cause of idiopathic OAB is unknown, however, a recent investigation has shown that antibiotics may reduce the symptoms of OAB in some patients (1). There is also an increasing body of evidence to suggest that the human bladder has a microbiome, missed by conventional urinalysis and culture (2). Consequently, infection may be a possible cause for the symptoms of OAB in some patients affected by this syndrome.

We hypothesise that changes in the urinary tract microbiome are responsible for the symptoms of OAB. The aim of this investigation is to compare the species of bacteria in the urine of patients with OAB and age‐matched controls.

Urine samples (5 ml), taken from females aged 35–87 via MSU sampling techniques, were centrifuged, re‐suspended and plated on chocolate agar plates in aerobic and anaerobic conditions for 7 days. Purity plating was performed and isolates identified by sequencing of the 16s rRNA gene.

Sediment culture of MSU samples reveals a variety of species of bacteria in urine of both patients with OAB and controls. The average number of species, as identified by our methodology, is 5 per patient in both groups. In controls, the most notable species are Staphylococcus, Streptococcus and Lactobacillus. With increased age, these notable species decrease and other species such as Escherichia coli (E.coli), Campylobacter and Bifidobacterium increase in prevalence. Comparing the microbiome of age‐matched controls with that of patients with OAB, shows that the incidence of E.coli, Campylobacter, Enterococcus and Proteus are further increased.

In an attempt to confirm that the bacteria observed was not a contamination artefact of using MSU samples, we performed similar analysis on CSU samples. Preliminary data indicates a trend for increases in the incidence of E.coli and Comamonadaceae and a decrease in Gardnerella and Staphylococcus in patients with OAB.

Evidence presented here supports recent observation that the bladder has a microbiome and urine is not sterile. Furthermore, there appears to be differences in the bladder microbiome between patients with OAB and controls, and these differences become more apparent in the aged. A more thorough analysis of the microbiome, including antibiotic protection assays on each sample, is now under way.

Support or Funding Information

Kidney Research UK
Irradiation of Red Sea Fennero penaeusindicus Shrimp Increased its Content of Vitamin A and Vitamin E
Authors: Mohammed Mohammed Elimam Ahamed , Abd Elhafise Eatimad A. , Elfaki Ahmed A.
Published: November 2023

This study was designed to investigate the effect of one and two Kilo Gray(KG) irradiation on red sea shrimp (Fennero penaeusindicus) with regard to its microbial content of E. coli, Salmonella SPP, Staphylococcus aureus, Vibrio spp, Listeria monocytegen and Proteus mirabilis and some biochemical parameters like vitamin A and E, proteins and iodine. The results showed that Vibrio spp was resistant to the one and two Kilo Gray doses. Surprisingly, vitamin A and vitamin E increased with the increase in the Vibrio spp count.
Novel cancer‐oocyte biomarker SAS1B as a potential target in head and neck squamous cell carcinoma (HNSCC)
Authors: Lee Christina Ju , Anderson‐Knapp Kiley , Pires Eusebio , Jameson Mark , Herr John
Published: November 2023

Head and neck squamous cell carcinoma (HNSCC) is the most common cancer of the head and neck region with a low overall 5‐year survival rate of 50%, highlighting the need for more effective therapeutics. A novel cancer‐oocyte biomarker, SAS1B (sperm acrosomal SLLP1 binding protein), presents substantial opportunities for development of novel diagnostics and therapeutics for patients with HNSCC. SAS1B has been recently discovered as a membrane‐associated zinc metalloprotease expressed in growing oocytes housed in the secondary follicle stage through ovulation, and is virtually absent in other tissues. SAS1B has been identified in approximately 77% uterine tumors including 74% endometrioid and 87% malignant mixed Mullerian tumors (MMMT). Given the range of cancers expressing SAS1B, probing for SAS1B gene and protein may increase the number of possible cancer patients in which a therapeutic method targeting SAS1B would potentially be beneficial. Twenty‐four primary human tumor specimens and eight HNSCC human‐derived cell lines were analyzed for ASTL message and SAS1B protein. A PCR assay unique to ASTL/SAS1B, excluding all of the other 134 human metalloproteases, has been developed with validated primers to amplify various domains of ASTL/SAS1B transcripts. SAS1B was identified in all samples of cell lines and primary tumors [100% incidence] using primers spanning a part of the c‐terminus of SAS1B. In addition, 46kDa and 65kDa SAS1B isoforms have been identified by Western blot using anti‐SAS1B polyclonal antibodies. To test the possibility of SAS1B as an immunotherapeutic target for HNSCC, live cell staining of an HNSCC cell line, OSC19 was carried out using indirect immunofluorescence with anti‐SAS1B polyclonal antibodies revealing cell surface localization of SAS1B. Future experiments will focus on in‐vitro cell killing assays using a duocarmycin‐conjugated antibody to test internalization of the antibody drug conjugate and subsequent growth arrest capabilities. The restricted localization of SAS1B presents opportunities for development of novel diagnostics and highly selective treatment approaches that spare both normal tissues and the ovarian reserve, thus potentially preventing unwanted negative side effects common in currently available therapeutics.

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Galangin‐induced cell arrest and apoptosis of nasopharyngeal carcinoma cells through suppression of PI3K/Akt signaling pathway
Authors: Lee Chuan‐Chun , Lin Meng‐Liang , Meng Meng‐Hsiao , Chen Shih‐Shun
Published: November 2023

Aberrant activation of phosphatidylinositol 3‐kinase (PI3K)–(protein kinase B) Akt signaling is associated with nasopharyngeal carcinoma (NPC) cells resistance to conventional chemo/radiotherapy. We show that natural active compound 3, 5, 7‐trihydroxyflavone (galangin), a type of bioflavonoid extracted from the galangal root (Alpinia galangal), can induce apoptosis and S‐phase arrest of NPC cells through p53‐medulated expression of Bax and p21. Using cell cycle synchronization techniques, we demonstrated that S‐phase synchronized NPC cells were more sensitive to galangin‐induced apoptosis. In ATP‐competitive inhibition assay and thin‐layer chromatography separation of the γ‐32P‐labeled phosphatidylinositol 3, 4, 5‐triphosphate product of kinase reaction with purified recombinant p85α and p110α proteins, we found that galangin might be an ATP‐competitive inhibitor by competing ATP binding site on PI3K catalytic subunit p110 alpha with ATP. Our results indicate that galangin induces p53‐mediated cell cycle arrest and apoptosis of NPC cells by interaction with ATP binding site on p110 alpha catalytic domain of PI3K.

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Kisspeptin inhibits cancer metastasis through PKR activation
Authors: Kim Tae‐Hun , Kim Jong‐Pil , Han Dong‐Hun , Cho Sung‐Gook
Published: November 2023

Kisspeptin encoded from KiSS1 gene has been known to suppress metastatic abilities of cancer cells via its endogenous G‐protein‐coupled receptor, GPR54. However, kisspeptin‐mediated signaling in the inhibition of cancer cell migration and invasion is yet clearly mapped. Our present study demonstrates that kisspeptin‐induced PKR activation inhibits metastatic ability of cancer cells. Kisspeptin inhibited both migratory and invasive abilities of highly metastatic cancer cells, which was blocked by silencing PKR. Consistently, kisspeptin failed to block the migration and invasion of mouse embryonic fibroblasts where Prkr gene was deleted. Moreover, kisspeptin activated RhoA‐ROCK‐PKR‐TCPTP signaling, resulting in the inhibition of STAT3 activity. Consequently, kisspeptin inhibition of STAT3 activity reduced IL‐6 level, which affected heterogeneous cancer cell population and repressed the distant metastasis in both in vitro and in vivo experimental sets. Therefore, our study conclude that kisspeptin activation of PKR is crucial for suppressing cancer metastasis, suggesting that this kisspeptin effect on PKR in cancer cells is not restricted on kisspeptin‐stimulated cancer cells, and affects IL‐6‐confluent tumor microenvironment.
Exosomes Secreted by Inflammatory Cytokine Stimulated Glioma Cells Carry a Repertoire of Proteins Which Influence Progression of Glioblastoma Multiforme
Authors: Kore Rajshekhar A , Abraham Edathara C , Griffin Robert J
Published: November 2023

Astrocytes constitutively secrete exosomes, a set of distinct nanovesicles (30–100 nm) of endosomal origin. In glioblastoma multiforme (GBM), exosomes are involved in mediating intercellular communication by their ability to deliver potentially oncogenic cargo leading to modulation of tissue microenvironment and progression of GBM. Inflammation and oxidative stress have been demonstrated to be connected in the development of GBM. Additionally, pro‐inflammatory cytokines like interleukin‐1 beta (IL‐1β) and tumor necrosis factor‐alpha (TNF‐α) are upregulated in the brain and elevated in serum following radiation therapy in patients with primary GBM tumors. Among others, these cytokines play an important role in progression of neuroinflammation and inflammation driven tumor re‐growth after treatment.

Methods

Media from cultured glial cell lines was subjected to sequential centrifugation to isolate exosomes. Exosomes were characterized by presence of exosomal markers and electron microscopy (EM). Exosomal proteins were analyzed by mass spectrometry (MS). Cell based assays were used to investigate the effect of exosomes secreted by cytokine stimulated cells.

Results

Here we present evidence showing that exosomes (~70–80nm in size) secreted by cytokine‐stimulated glioma cells rescue both glial and neuronal cells from oxidative stress induced cell death. Our MS analysis demonstrates profound changes in exosomal proteome following IL‐1β and TNF‐α stimulation of glioma cells which may explain how GBM tumors manipulate their microenvironment to grow and respond to treatment. Many of the proteins identified are involved in pathways promoting remodeling of extracellular matrix (ECM), biological adhesion, developmental processes and progression of inflammation leading to cancer growth and development.

Conclusions

Increases in cytokine levels in GBM patient brains, either due to disease or radiation, lead to qualitative and quantitative changes in the exosomal proteome. The role and function of proteins present in exosomes secreted by cytokine stimulated cells, in part, explains aggressiveness, resistance to anti‐cancer therapies and high incidence of recurrence of GBM. Our study demonstrates the role of the tissue microenvironment in dictating exosomal content secreted by glioma cells, in the aggressive progression of GBM and their ability to determine the fate of target or recipient cells.

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NIH/NEI EY011352‐14
Replisome Disassembly At The Termination Of Eukaryotic Replication Forks
Authors: Gambus Agnieszka
Published: November 2023

To ensure duplication of the whole genome DNA replication initiates from thousands of origins of replication. The pre‐loaded inactive helicase (Mcm2‐7 double hexameric complexes) becomes activated during replication initiation by loading of additional components: Cdc45 and GINS in a carefully regulated process. The progressing replication fork moves through the chromatin until it encounters fork from the neighboring origin. During the termination of replication forks the replisomes disassemble and topisomerase II resolves the daughter DNA molecules. If not resolved properly, the terminating forks are at risk of stalling and fork reversal, leading to DNA damage and genomic instability.

Using the Xenopus laevis egg extract system, we have shown (Priego Moreno et al. Science 2014) that blocking polyubiquitylation results in the prolonged association of the active helicase with replicating chromatin. It was accompanied by a defect in replication forks termination leading to conclusion that polyubiquitylation is essential for the disassembly of the active helicase at the termination of replication forks. The Mcm7 subunit of the Mcm2‐7 complex was the only component of the active helicase that we found polyubiquitylated during S‐phase and its ubiquitylation was blocked when forks could not terminate. Finally, disassembly of the helicase was dependent on p97/VCP/Cdc48 segregase activity.

This process of replisome dissolution is very well conserved through evolution (Maric et al. Science 2014), however, the ubiquitin ligase driving ubiquitylation of replicative helicase is not. I will present, therefore, our recent findings on the identity of the enzyme ubiquitylating Mcm7 in higher eukaryotes.

Support or Funding Information

This work was funded by MRC CDA Fellowship and Lister Institute Research Award for A.G.