bluuebunny/crossref_metadata_2025 huggingface dataset:
Examining FOXO1 and DNA Interactions through Computational and Direct Methods
Authors: McDaniel Brianyell , Cherlin Tess , Haynes Rashad , Mottamal Madhusoodanan , Anderson David , Johanson Kelly
Published: November 2023

FOXO transcription factors are a subgroup of the FOX (forkhead) family and are important for regulation of cellular processes, gene expression, apoptosis, cell‐ cycle progression, and oxidative stress resistance. Several FOXO translocations have also been implicated in cancer development, with the most common being the PAX3‐FOXO1 translocation linked to the development of Alveolar Rhabdomyosarcoma (ARMS). All FOXO proteins share sequence similarity and a highly conserved DNA‐ binding domain (DBD). This DNA‐binding domain consists of three alpha helices accompanied by two winged domains; this domain binds the Daf‐16 binding element (DBE) and the insulin response element (IRE), with the crystal structure indicating that the majority of the DNA contacts are located within Helix 3. There have been reports in the literature of sequences flanking the IRE causing an increase in binding affinity; however, no mechanism has been reported to explain this effect. Using Molecular Dynamics Simulation (MDS) we observed that the number of direct interactions between protein and DNA increases over time. Interestingly, MDS showed that wing 1 has no contact with DNA at 0 ns but eventually makes contact with a region flanking the IRE consensus sequence at 10 ns. In order to verify the new interactions observed in the 10 ns model, we mutated several bases within the IRE and observed the effect of these mutations on the interaction between protein and DNA through Electrophoretic Mobility Shift Assays (EMSA) and intrinsic fluorescence. Mutation of several bases in the IRE consensus sequence as well as in the immediate 5′ and 3′ flanking regions decreased the interaction between FOXO1 and DNA. In support of our model, mutation of the new base contact identified with Wing 1 also decreased FOXO1 DNA‐binding. The truncated FOXO1 DNA‐binding domain found in the PAX3‐FOXO1 fusion was also examined by MDS, and was predicted to retain several contacts within Helix 3 as well as the newly identified contact in Wing 1. Using this information, we can further examine the DNA‐binding of PAX3‐FOXO1 to determine if the presence of flanking sequences surrounding the IRE consensus sequence also affect the affinity of the fusion protein for DNA. Knowledge of the contribution of the FOXO1 domain to PAX3‐FOXO1 DNA targeting is important for understanding the activity of this fusion protein and its role in ARMS development.

Support or Funding Information

NIH SC2GM095430

RCMI‐Molecular Structure and Modeling core facility (NIH‐RCMI 2G12MD007595‐06 )

NIH 5RL5GM118966‐03

RISE (R25GM060926)
Gene Expression Profiles of Various Rat Brain Regions Following Exposure to Soman
Authors: Gautam Aarti , Sowe Bintu , Donohue Duncan , Miller Stacy‐Ann , Hammamieh Rasha , Meyerhoff James , Pham Michael , Lumley Lucille , Jett Marti
Published: November 2023

Soman (GD) is a highly toxic man‐made substance and a potential chemical weapon. It belongs to a class of chemicals known as organophosphorus (OP) nerve agents, which may cause seizures, respiratory failure, and death. Soman undergoes aging and is one of the more difficult nerve agent exposures to treat since field available oxime therapy is not effective against soman. Understanding the effects of soman is vital to developing a method of detection and treatment for exposure. Exposure to GD can consequently result in neuropathological abnormalities in various regions of the brain, including the hippocampus, piriform cortex, amygdala, thalamus, and hypothalamus. In this study, rats were exposed to either 0.8 LD50 GD or 1.0 LD50 GD versus a saline control group and evaluated for seizure activity. Rats were sacrificed and brain regions were harvested at 72 hours post‐exposure. Brain tissues were homogenized and RNA was isolated using TRIzol reagent. In order to explore the pathways and biological processes associated with GD exposure, transcripts in the aforementioned brain regions were assayed using Agilent's rat gene expression arrays. Detailed analysis is currently underway to compare the expression profile generated from different regions of the brain and to understand the differences in response to the variation in the concentration of GD exposure.

DISCLAIMER “Research was conducted in compliance with the Animal Welfare Act and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.”
Signaling regulators of TGF cleavage predict mechanisms for RTK re‐activation and therapeutic resistance in gastric cancer
Authors: Harrison Christina , Wilson Jennifer L. , Fraenkel Ernest , Herrlich Andreas , Lauffenburger Doug
Published: November 2023

Alternative growth factor pathway activation causes resistance to targeted receptor tyrosine kinase (RTK) therapies, yet there is little understanding of the intracellular control of this process and how ligand shedding may affect therapeutic response. Already, the EGFR family and multiple matrix metalloproteinases are associated with altered growth and metastasis in gastric cancers, though, we do not know how signaling in these growth pathways may affect resistance. We use a network‐based approach to study the intracellular events affecting alternative pathway activation, specifically investigating the role of TGFα, an EGFR ligand, in gastric cancer. From the network‐based optimization, we identify a list of candidate intracellular targets. We indentified potential targets using randomization and robustness testing and targets were selected as significant if they were consistent in these metrics. While the approach predicts new targets as relevant to ligand shedding and growth factor activation, it does not assign directionality to their potential effects. As such, it is necessary to validate their effects directly. We performed shRNA knockdowns for top targets: GOLGA2, PAK1, and RAB1A and measured their effects on ligand shedding. PAK1 and RAB1 increased ligand shedding in vitro. This shows that PAK1 and RAB1 might be involved in intracellular signaling processes that induce TGFα ligand shedding. To further validate the model, we searched public databases for these targets in patient samples with gastric cancer and in other cancer types. This comparison allows us to better understand mechanisms that may be unique to gastric cancer and what mechanisms might be consistent across cancer types. Through the use of further network analysis and predictive modeling, we have picked additional targets to validate in vitro; CASP9, RAF1, XIAP, and CFTR. As these additional targets were also not in the original screen, we plan to measure ligand shedding after shRNA knockdown of these genes.
Effect of over the counter teeth bleaching products on the proteins of human teeth
Authors: Swanson Derek , Keenan Kelly
Published: November 2023

There are numerous bleaching products available to whiten the appearance of teeth: some are done only in the office of a dentist and some can be done by the consumers. There is evidence to show that those products (usually containing hydrogen peroxide) used in dentist's office does result in a decrease in the level of protein as well as the strength of the enamel of teeth. The goal of this project was to evaluate the effect of over the counter whitening agents on teeth by measuring both total protein as well as looking at individual proteins of the teeth. A method to extract protein from teeth was used and assessed and total protein was measured using the Lowry assay. The individual proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and the molecular weights of the proteins were compared to those proteins known to be in the enamel or dentin of teeth. These proteins were further characterized based on amino acid composition.
Electrochemical‐Aptamer Biosensor Against ENOX2 for Cancer Diagnostics
Authors: Fetter Lisa C , Bonham Andrew J
Published: November 2023

Human Ecto‐Nicotinamide Dinucleotide Oxidase Disulfide Thiol Exchanger 2 (ENOX2), is common isoform of a parent protein ubiquitously expressed in humans, ENOX1. The ENOX2 isoform is only highly expressed on the surface of tumors, and as such has been heavily researched as a potential diagnostic biomarker for cancer. Since ENOX2 is shed into the sera and its presence is cancer‐specific, ENOX2 is a highly attractive target for cancer‐specific diagnostic blood testing. To enable a robust sensing platform that can detect ENOX2 in a rapid, reagentless manner, we have created an aptamer‐scaffold electrochemical bionsensor directed against ENOX2. This project involved identification of an ENOX2‐selective aptamer sequence through SELEX procedures, followed by modifications of the aptamer to support a conformation‐switching response to target binding. This oligonucleotide was then incorporated into an electrode‐based electrochemical biosensor platform, creating a dose‐responsive readout for the presence of ENOX2 based on electrical current changes. Ultimately, this ENOX2 biosensor will function in whole blood and yield a novel, rapid, and clinically‐relevant diagnostic tool for early cancer detection.
Analysis of the expression of the Basigin subset of the immunoglobulin superfamily in mouse brain
Authors: Hall Tavia N , Ochrietor Judith D
Published: November 2023

Basigin gene products are members of the immunoglobulin (Ig) superfamily and are structurally related to immunoglobulin molecules. Basigin protein possesses two extracellular Ig domains, a single transmembrane domain, and a cytoplasmic tail, whereas the splice variant Basigin‐2 protein possesses three extracellular Ig domains, a single transmembrane domain, and a cytoplasmic tail. There are two other members of the Basigin subset of the Ig superfamily – the Neuroplastins and Embigin. Neuroplastin gp55 and Embigin are similar in structure to Basigin and Neuroplastin gp65 is similar in structure to Basigin‐2. All five of these proteins are cell adhesion molecules within neural tissues and play various roles in development, structure, and function of those tissues. The purpose of the present study was to analyze the expression of the Basigins, the Neuroplastins, and Embigin during the development of the mouse brain. Mouse brains were obtained using an accepted protocol and RNA was isolated for use in quantitative reverse transcription polymerase chain reaction (q‐RT‐PCR) using primer sets specific for Basigin gene products, Neuroplastin gene products, and Embigin. The values obtained were normalized to 18s rRNA. It was determined that Neuroplastin gene product expression is ten times greater than that of Basigin or Embigin. Their relative expression levels remain the same throughout the life of the mouse. These data suggest that Neuroplastin gene products have a more prominent role in the structure and function of the mouse brain than the other two members of the subset of the Ig superfamily.

Support or Funding Information

University of North Florida Office of Undergraduate Studies and the Office of Academic Affairs
Accurate Quantification of Choline and Ethanolamine Plasmalogen Molecular Species by Liquid Chromatography coupled with Tandem Mass Spectrometry
Authors: Otoki Yurika , Kato Shunji , Kimura Fumiko , Furukawa Katshutoshi , Arai Hiroyuki , Nakagawa Kiyotaka , Miyazawa Teruo
Published: November 2023

Tandem mass spectrometry (MS/MS) has been used for the analysis of plasmalogens (Pls), a physiologically important class of vinyl ether‐linked phospholipid. However, MS/MS generally causes little fragmentation of Pls, especially choline Pls (PC‐Pls). Previous MS/MS studies reported increased formation of product ions of PC‐Pls (and ethanolamine Pls (PE‐Pls)) in the presence of ‘alkali metals.’ Therefore, the use of alkali metals may considerably improve the analytical method for the analysis of both PC‐ and PE‐Pls. This was evaluated in the present study using quadrupole‐time‐of‐flight MS/MS and liquid chromatography (LC) coupled with MS/MS. Results from MS/MS confirmed that alkali metals (e.g., sodium) produced significant fragmentation of PC‐Pls and PE‐Pls. A number of structure‐diagnostic product ions exhibiting high intensities were observed under optimized MS/MS conditions using alkali metals. Moreover the ability to selectively and sensitively quantify both PC‐Pls and PE‐Pls at the molecular species level in biological samples (e.g., blood plasma from healthy and Alzheimer's disease subjects) was demonstrated using LC‐MS/MS. The herein developed method appears to be a powerful tool for analyzing Pls and may provide a better understanding of their physiological roles in vivo.
Identification and purification of a serine protease involved in cell‐mediated immunity in fish
Authors: Matsuura Yuta , Yabu Takeshi , Shiba Hajime , Nakanishi Teruyuki
Published: November 2023

Cell‐mediated immunity is crucial immune response against non‐self antigen such as allograft, tumor and virus infected cells. Non‐self antigens could be eliminated by effector cells (e.g. cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells) with secretory and/or non‐secretory cell‐death pathways in mammal. Also in fish, cytotoxic effector cells have been characterized in lymphocyte subsets level; however, biochemical mechanisms of the killing remain unknown. Here we tried to identify the protease involved in the killing adopting ginbuna crucian carp which has been used as a nice model for the study on cell‐mediated immunity in fish.

In order to identify the protease involved in the killing of allogeneic target cells, we performed cytotoxicity assays in the presence of serine or cysteine protease inhibitors using leukocytes of ginbuna crucian carp. Cytotoxicity was significantly inhibited by serine protease inhibitor, 3,4‐Dichloroisocoumarin: “DCI”. By contrast the cytotoxicity was partially inhibited by cysteine protease inhibitor. Then we tried to explore a substrate of the serine protease involved in killing activity, and we found that a protease activity against a peptide substrate “z‐Gly‐Pro‐Arg‐4‐Methylcoumaryl‐7‐amide (MCA) was inhibited by “DCI”. In addition, z‐Gly‐Pro‐Arg‐MCA cleaving activity was significantly enhanced by allo‐antigen stimulation. These results suggest that the serine protease which cleaved z‐Gly‐Pro‐Arg‐MCA substrate was involved in killing against allogeneic cells.

In order to analyze the enzymatic function of the serine protease, we tried to purify the protease. Purification was sequentially performed on two steps of benzamidine‐sepharose and SP‐sepharose chromatography. The purified protease was visualized as a single protein band and a molecular mass was estimated to be approximately 27,000 Da by using SDS‐PAGE. The protease was certainly serine protease because it was not inhibited by cysteine, metallo‐ and aspartic protease inhibitors but serine protease inhibitor.

In conclusion, we succeeded to purify the serine protease involved in the killing against allogeneic cells. Now we initiate proceeding to examine a function and to find endogenous substrate of the protease with the purified protease.

Support or Funding Information

Sasakawa Scientific Research Grant from the Japan Science Society
Investigating the GCN5 Histone Acetyltransferase Chemical Mechanism
Authors: Young Byron H. , Berndsen Christopher E.
Published: November 2023

Enzyme catalyzed protein acetylation involves the transfer of an acetyl group to a lysine side chain within the substrate. This process serves many vital cellular purposes including the regulation of gene expression and controlling of the circadian rhythm. Defects in enzymatic acetylation have been linked to many common diseases such as cancer, insomnia, and anemia. Despite decades of research into the biological function of protein acetylation, the enzymatic mechanism of acetyl‐transfer is unknown. We have worked to characterize the mechanism of acetyl‐transfer utilized by the yeast protein acetyltransferase General Control Nonderepressible 5 (GCN5) using deuterium solvent isotope effects, Brønsted‐beta analysis, and competitive inhibition studies. These analyses have allowed us to characterize the occurrence of proton transfer during catalysis as well as gain a general knowledge of the transition state structure. These results will be coupled with QM/MM computational modeling of the GCN5 acetyl‐transfer reaction in order to gain an explicit understanding of the mechanism of catalysis. The collection of these results set the groundwork for further investigation into the catalysis mechanisms of other acetyltransferases and may aid in development of treatment for acetyl‐transfer associated illness.

Support or Funding Information

‐Jeffress Trust Awards Program in Interdisciplinary Research

‐NSF REU: CHE‐1461175
Substrate‐induced Structural Changes in the Mevalonate Diphosphate Decarboxylase from Enterococcus faecalis
Authors: Chen Chun‐Liang
Published: November 2023

Mevalonate diphosphate decarboxylases (MDD) (EC 4.1.1.33) convert mevalonate‐5‐diphosphate (MVAPP) to isopentenyl‐5‐diphosphate (IPP) through the ATP‐dependent decarboxylation in the last step of the mevalonate pathway, which is essential for the production of diverse isoprenoids in different organisms. In our research, MDD from Enterococcus faecalis (MDDEF) has been tested and analyzed functionally, thermodynamically and structurally. The enzyme mechanism of MDDEF belongs to an ordered bi‐substrate mechanism with MVAPP binding first based on the kinetic results. A substrate‐induced substrate binding model of MDDEF has been suggested according to the thermodynamic results using the isothermal titration calorimetry (ITC). The induced conformational changes of the b‐10‐a‐4 loop, the phosphate binding loop, and helix α1, α2 and α4 of MDDEF upon substrate binding have also been structurally observed using X‐ray crystallography, along with two metal binding sites created by the enzyme and substrates. These new findings provide insight into the details of the MDDEF enzyme mechanism. The unbound and bound forms of MDDEF representing different states in the enzyme reaction will certainly provide platforms for rational drug development.
The synthesis and characterization of potential covalent probes for chronic lymphocytic leukemic B‐cell receptors using sulfonyl fluoride
Authors: Rohweder Peter , Kodadek Thomas , Sarkar Mohosin , Dreyer Chris , Park Hajeung
Published: November 2023

Chronic lymphocytic leukemia is a cancer of the B‐cells. Patients afflicted can become monoclonal for a single B‐cell population and become immuno‐compromised. Current treatments focus on the ablation of all B‐cells without discrimination between healthy and cancerous cells. Following destruction of the immune cells, the patient's immune system is expected to begin producing healthy, non‐cancerous B‐cells again. This is often not the case though and cancerous B‐cell lines often reemerge. A more specific treatment protocol is needed for chronic treatment of patients. The discovery of small molecule, non‐covalent ligands for cancerous B‐cell receptors has been previously reported by the Kodadek lab, but fast off‐rates contributed to challenges in reproducing protein binding success in whole cell systems. The introduction of a sulfonyl fluoride warhead to established non‐covalent probes was performed with the intention of producing selective, covalent probes for cancerous B‐cell receptors. Non‐covalent binding assays by biolayer interferometry confirmed binding to cancerous B‐cell targets. Additionally, incubation of probes with soluble cancerous B‐cell receptor proteins and subsequent trypsin digest and MALDI mass spectrometry analysis seemed to confirm covalent bond formation. Further experiments are ongoing to confirm and characterize this covalent bond formation. Should these probes prove selective for cancerous B‐cell receptors, subsequent attachment to a toxic payload or incorporation into antibody‐drug conjugates could be carried out and tested for efficacy for the selective killing of cancerous B‐cell lines over their healthy counterparts.
In Vivo Activity of Ethanolic Extracts of Leaves and Stems Bark of Alstonia Boonei against Plasmodium Berghei in Female Albino Mice
Authors: Onifade Olayinka Fisayo , Atasie Nkechi V , Fakankun Irene U , Maganda Vera
Published: November 2023

Malaria is commonly associated with poverty and also a cause of poverty. It is a major hindrance to economic development in tropical and subtropical regions, including parts of America, Asia and Africa. A total of 109 countries were endemic for malaria in 2008, with 45 in the African region (WHO, 2008; Batista et al., 2009). The vast majority of the existing antimalarial chemotherapeutic agents are based on natural products and this fact anticipates that new drugs may certainly emerge from the tropical plant sources. The ethanolic extracts of Alstonia boonei leaves and stem were used for this study to investigate the qualitative phytochemical component of the extracts alongside acute toxicity and suppressive effect. The mice were administered 200mg/kg, 400mg/kg and 800mg/kg body weight of leave extract and stem extract separately, positive control were given standard drug chloroquine (5mg/kg) and the negative control were given distilled water. From the results, phytochemical analysis showed that the stem and leaves of Alstonia boonei had some compounds in common and also had some differences. After 24 hours of acute toxicity, the test mice did not die nor showed any sign of toxicity or any strange behavior. The result showed that the Alstonia boonei was safe and the lethal dose was >5000mg/kg. The suppressive treatment showed that the leaves and stem extracts of Alstonia boonei were dose independent. The stem extract of Alstonia boonei was more effective at the dose of 200mg/kg body weight and the leaves extracts of Alstonia boonei was more effective at the dose of 400mg/kg body weight. The standard drug (chloroquine) used for both test extracts gave 100% suppression. This study demonstrated that crude extract of Alstonia boonei possess strong activity against P.berghei indicating presence of chemical compositions that may be useful in preparing antimalarial drugs.
Synthesis of Effective Water‐Soluble Fluorescein Dibenzyl Ether Cytochrome P450 Substrates
Authors: Stepler Cameron M , Gallivan Maxwell A , Wysocki Laura M
Published: November 2023

Cytochrome P450 is a key family of enzymes in the breakdown of drugs and toxins in the body. We are interested in probing the activity of these enzymes with different organic ethers through a library of fluorescein diether derivatives. These compounds are hydrolytically stable and exhibit very low background fluorescence before unmasking with high contrast, making them ideal targets for sensitive assays. The first‐generation library revealed challenges related to solubility of the fluorogenic compounds in the desired assay. Through the design and synthesis of a variety of substrates, progress was made toward water‐soluble probes that display reasonable kinetics for evaluation and investigation of enzymatic activity.

Support or Funding Information

This work was supported by the Henry and Nellie Pence Trust.
Experimentally‐Induced Melanoma has an Heterogeneous Oxidative Metabolism and Induces Peri‐Tumoral Innervation Remodeling
Authors: Noriega‐Cisneros Ruth , Rosas‐Plaza F. Ximena , Ramírez‐Santos Jesús , Gutierrez‐Ospina Gabriel
Published: November 2023

Conventional wisdom assumes that carcinogenesis is caused by mutations in genes involved in the control of cell proliferation and survival, replicative senescence, oxidative metabolism, cell adhesion, immune recognition and angiogenesis. Over the last two decades, however, mounting evidence supports that “healthy” leukocytes, endothelial and fibroblasts “invade” the extracellular matrix of tumors and provide soluble factors that promote malignancy. Somatic and autonomic innervation, however, are factors that have remained out of the equation in spite of the evidence that shows that malignant tumors may attract or repel them. In addition, various neuro‐messengers are now known to modulate cancer progression. This is why in this work we begin studying the pattern of autonomic innervation in melanoma, putting emphasis on the cytological relationship between innervation and tumor metabolic compartmentalization; it is known that innervation regulates oxidative metabolism in normal tissues.

Methodology

Non‐metastatic, B16F0 melanoma cell line were inoculated subcutaneously (5×105 cells) in the back of C57BL/6 mice (n=5). Twenty days after inoculation, the mice were euthanized (pentobarbital 45mg/Kg of body weight) and perfused with buffered 4% paraformaldehyde. The excised tumors and the surrounding skin were then dissected and processed through histological procedures that allowed the tumor mapping of cytochrome oxidase and of cholinergic and catecholaminergic innervation.

Results

1) Oxidative metabolism was heterogeneous throughout the tumor. A trend for the tumor's core to display greater activity of cytochrome oxidase was observed. 2) Under normal conditions, cholinergic innervation was observed surrounding hair follicles. This innervation was absent in hair follicles adjacent to the tumor in melanoma‐inoculated mice. 3) Non‐inoculated mice showed numerous catecholaminergic fibers running throughout the dermis in association or not with blood vessels. This innervation qualitatively increased in melanoma‐inoculated mice. 4) No autonomic innervation was observed within the tumor. Only catecholaminergic fibers surrounded it.

Conclusions

Autonomic innervation remodels significantly around the tumor. Melanoma likely prevents the autonomic innervation to “invade” it. No obvious associations between tumor metabolic activity and autonomic innervation distribution were observed.

Support or Funding Information

The work was supported by PAPIIT Project, UNAM IN203912‐3. NCR is a postdoctoral fellow supported by CONACyT (No. 247328).
Expression of Folate Transport Proteins are Decreased in Head and Neck Squamous Cell Carcinoma
Authors: Green Jacalyn M. , Cuadros Kayla , Goral Joanna , Jham Bruno , Guimaraes Eduardo Pereira , Hanneman João Adolfo Costa
Published: November 2023

Introduction

Head and neck squamous cell carcinoma (HNSCC) is currently ranked tenth and eleventh in incidence and mortality, respectively, in the US. These cancers are often diagnosed at an advanced stage, making the prognosis poor for these patients. Cancers often have an increased need for folate, an essential vitamin that is involved in cell division and growth. Folate is transported into cells through three proteins: folate receptors (FR), reduced folate carrier (RFC), and proton coupled folate transporter (PCFT). Several other cancers have been found to have increased expression of at least one of these transporters. Changes in expression of these transporters have not been studied systematically in HNSCC.

Hypothesis

Compared to normal cells, HNSCC cells will show different patterns of expression in at least one of the three folate transporters.

Methods

Pathological samples with head and neck cancer were obtained from fifteen patients; eleven (80%) were male and 4 (20%) were female. The average age was 54 years; the range was from 38 to 76 years old. Cadaveric samples of tongue were used as controls. Expression of FR, RFC, and PCFT in formalin‐fixed tissues was detected by immunohistochemistry. Patient records on age, sex, lesion location, symptoms, treatment and prognosis were collected. Statistical analysis was performed to determine the correlation of expression of the protein of interest and clinical data.

Results

Normal tongue samples demonstrated maximal expression of all three folate transport proteins in the oral epithelium with wide distribution of expression in all cell layers of the stratified squamous epithelium. In contrast, HNSCC samples generally demonstrated low intensity of expression of FR, RFC, and PCFT, with wide distribution of expression in the invading cancer cells. There were no observed differences related to gender or age in patterns of FR, RFC, and PCFT expression in HNSCC and control tissues.

Conclusion

The majority of HNSCC showed decreased expression in folate transport proteins compared to normal tongue epithelial cells. This would be consistent with the idea that decreased folate availability might influence carcinogenesis through alterations in DNA methylation, and concomitant alterations in gene expression.

Support or Funding Information

Acknowledgements: Kayla Cuadros was supported by the Midwestern University Biomedical Sciences Program and Joanna Goral, Jacalyn Green and Bruno Jham were supported by Midwestern University. We thank Alice Meyer and Sabiha Raziuddin for their technical assistance.
CD44+/CD25−/CD100+ Cells Contribute to the Development of Basal Epithelial‐like Metaplastic Breast Cancer in Adiponectin‐deficient MMTV‐PyVT Mice
Authors: Chen Jie , Cao Handi , Liu Jing , Tse Wai Choi , Lee In‐Kye , Vanhoutte Paul M. , Xu Aimin , Yu Wang
Published: November 2023

Cancer development depends on the interactions between epithelial carcinoma cells and the stromal microenvironment. The present study investigated the role of adiponectin, an adipokine with potent tumor‐suppressive activities, in modulating the stromal microenvironment during breast cancer development in mice. Trans‐implantation of the stromal vascular fractions (SVF) of mammary adipose tissue from adiponectin‐deficient MMTV‐PyVT mice promotes the development of MDA‐MB‐231 human breast cancers in NOD/SCID mice, which exhibit the aggressive metaplastic basal epithelial‐like molecular features. In MMTV‐PyVT mammary tumors, adiponectin deficiency significantly decreases the density of CD3+ tumor‐infiltrating lymphocytes and enhances the SVF composition of CD44+/CD25−/CD100+ cells that are originated from thymus. Adiponectin plays an important role in T‐cell development and maturation partly by modulating the epithelial stromal microenvironment of thymus. Adiponectin deficiency prevents the positive selection of CD3+ T‐cells and the negative selection of CD44+CD25−CD100+ T‐cells by thymic nurse cells (TNCs). Thus, reduced production of CD3+ T‐cells as well as enhanced release of CD44+/CD25−/CD100+ cells from thymus into the circulation give rise to a tumor‐permissive microenvironment in mammary tissues of MMTV‐PyVT mice lacking adiponectin. Moreover, CD44+/CD25−/CD100+ cells represent a major metaplastic component contributing to the development of more aggressive basal epithelial‐like mammary tumors in MMTV‐PyVT or NOD/SCID mice implanted with human MDA‐MB‐231 breast cancer cells. Treatment with adiponectin restores thymic T‐cell maturation/selection process and prevents the development of metaplastic basal epithelial‐like breast cancer in MMTV‐PyVT mice. In summary, the present study reveals that adiponectin acts as an immunomodulatory stromal factor in both the thymus and mammary tumor microenvironment to elicit the multifaceted anti‐tumorigenic functions.

Support or Funding Information

This work was supported in part by grants from Seeding Funds for basic research of the University of Hong Kong, Research Grant Council Grants (17121714, HKU779712M, HKU780613M) of Hong Kong; And the National Basic Research Program of China (973 program 2015CB553603).
Calcium Sulfide (CaS) Nanostructure Treatment on Non‐Small Cell Lung Cancer
Authors: Forti Kevin Muñoz , Figueroa Maria , Torres Bryan , Bernard Faviola , Rivera Daniel , Castro Miguel E , Suarez Edu B
Published: November 2023

Lung cancer is the number one cause of death among deaths due to cancer in the United States independent of gender. Current treatments focus on surgical removal of tumors and surround tissue, chemotherapy and radiation therapy. Side effects of the aforementioned treatments have been shown to be detrimental to a patient's health and wellbeing and are capable of causing secondary neoplasms. Recently, nanotechnology has shown promise in providing safer and more efficient therapy options for cancer patients. Our colleagues from the University of Puerto Rico at Mayagüez synthesized nanostructures composed of calcium sulfide (CaS). Due to both calcium and sulfur being incorporated in numerous biological pathways with the former being a mediator in apoptosis, we expect these nanostructures to be biocompatible and with low side effects. In this study, we evaluated the effect of CaS nanostructures on pulmonary adenocarcinoma cells by incubating ATCC CRL‐2124 cell line in the presence of CaS nanostructures [3.8 μM], Etoposide [10 μM], and DMSO (vehicle) for 24‐, 48‐, and 72‐hours after a single dose at time 0. We hypothesized that CaS nanostructures would suppress the cells’ proliferation by interfering with their cell cycle phases and increasing apoptosis. We performed cell‐based assays to study the cell cycle and apoptosis‐mediated cell death. There were no statistically significant observations after 24 hours in non‐cancerous cells (MRC5). Our results showed that at 48‐hours post treatment the progress of malignant cells from Sub‐G1 into G0/G1 was suppressed along with an increase in apoptosis (p≤0.05). After initial treatment with CaS nanoclusters we observed a built up of malignant cells in S phase at 24‐hours (p≤0.05). These results provide a platform for elucidating the mechanism of action of CaS interfering with proliferation, which may lead to new alternative therapies for lung cancer. Currently we are studying apoptosis mediators and calcium‐dependent pathways believed to be influenced by CaS in cancerous cells.

Support or Funding Information

This work was supported by: UPR‐PRISE Program NIH Grant #R25GM096955
A Model for the Function of COQ9: the Isoprenoid‐Precursor Presenter
Authors: Stefely Matthew S , Lohman Danielle C , Pagliarini David J
Published: November 2023

Coenzyme Q (CoQ) is a vital part of the electron transport chain that is synthesized de novo in mitochondria by the combined action of at least 10 proteins (COQ1–10). Defects in CoQ biosynthesis lead to primary CoQ deficiency, which has serious human health implications ranging from encephalomyopathy to cerebellar ataxia. Coenzyme Q biosynthesis protein 9 (COQ9) is a crucial part of this pathway, but its exact role remains unknown. A recently‐published manuscript from our lab revealed that COQ9 contains a small‐molecule binding domain, which co‐crystallized with a phospholipid, and demonstrated that COQ9 associates functionally and physically with COQ7. COQ7 catalyzes the hydroxylation of the CoQ intermediate, demethoxy‐CoQ (DMQ), in one of the last steps of CoQ biosynthesis. CoQ and its isoprenylated intermediates are highly hydrophobic, with mature CoQ residing predominantly within the inner mitochondrial membrane, while the CoQ biosynthetic enzymes are peripheral. This apparent lack of accessibility produces a biosynthetic barrier that we envision may be overcome by a protein capable of chaperoning and presenting lipid intermediates to the CoQ biosynthetic enzymes. Due to its ability to bind lipids, its physical association with COQ7, and the accumulation of DMQ in both yeast and mice harboring mutant versions of COQ9, we hypothesize that COQ9 fills this role. To test this hypothesis, I have enriched DMQ from bacteria using organic lipid extraction and column chromatography, and have optimized assays using liposomes to test the isoprenoid transport abilities of COQ9. In this presentation, I discuss the results of these experiments and the insights they have provided toward elucidating the biochemical role of COQ9 in CoQ biosynthesis, understanding CoQ biosynthesis as a whole, and ultimately treating CoQ deficiency.
Differential Effects of Diets Rich in Fats, Carbohydrates and/or Fructose on the Development of Hepatic Steatosis and Mitochondrial Dysfunction
Authors: GARCIA‐BERUMEN CLAUDIA I , ALEJANDRE‐BUITRON ALAN , MONTOYA‐PEREZ ROCIO , RODRIGUEZ‐OROZCO ALAIN R , CALDERON‐CORTES ELIZABETH , PIÑA‐ZENTELLA ROSA MARIA , RAMOS‐GOMEZ MINERVA , SAAVEDRA‐MOLINA ALFREDO , CORTES‐ROJO CHRISTIAN
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) comprises the progressive development of steatosis, steatohepatitis and cirrhosis. The progression of the disease is a multifactorial process involving risk factors such as obesity, diabetes mellitus, and dyslipidemia. Besides, NAFLD has been closely related with increased mitochondrial ROS production because excessive fat and carbohydrates dietary intake, with fructose playing a prominent role in this disease. The main goal of this work was to elucidate the contribution of these dietary components to the development of mitochondrial dysfunction and oxidative stress during NAFLD. We feed rats during 1 month with different diets containing standard diet, standard diet plus fructose (C+FR), high saturated fat plus high carbohydrates (HFHC), high saturated fat plus high carbohydrates plus fructose (HFHC+FR), only high fat (HF) and high fat plus fructose (HF+FR). A notable inhibition of mitochondrial respiration was observed with HFHC and HFHC+FR diets. This was associated to strong decline in the activity of complex I, particularly with the HFHC+FR diet. Rats feed with HFHC and HFHC+FR exhibited prominent ROS production. Moreover, the liver weight in the latter group increased significantly with respect to the other diets, indicating that this diet is the most harmful to liver health, which was further confirmed by an excessive fat accumulation seen in histopathological analyses. Therefore, it is concluded that the combination of saturated fat and carbohydrates in excess favors the development of mitochondrial dysfunction and ROS production, with the addition of fructose to this diet accelerating the early development of liver damage.

Support or Funding Information

Supported by grants from Programa de Investigación 2016 de la Coordinación de la Investigación Científica de la UMSNH (CIC‐UMSNH) (CCR).
EXCITATION‐METABOLISM COUPLING: ROLE OF MEMBRANE DEPOLARIZATION ON THE MITOCHONDRIAL FUNCTION
Authors: Díaz‐Vegas Alexis , Cordova Alex , Utreras Yildy , Rosales Giovanni , Llanos Paola , Hidalgo Cecilia , Arias Manuel , Jaimovich Enrique , Contreras‐Ferrat Ariel Eduardo
Published: November 2023

Introduction

Depolarizing stimuli induce signaling pathways in muscle that have pleiotropic effects. Two new voltage‐dependent phenomena, excitation‐transcription and excitation‐metabolism coupling (EMC), add to the canonical excitation‐contraction coupling process. The mechanisms involved in EMC, however, remain poorly understood.

Material and Methods

Mice were used at 6–8 weeks. Changes in mitochondrial and cytoplasmic Ca2+ levels were evaluated in living fibers from Flexor digitorum brevis muscle via imaging of fibers transfected with suitable plasmids. The mitochondrial potential and oxygen consumption were evaluated with Seahorse system and TMRE+ respectively.

Results

Depolarization of skeletal muscle fibers increased the cytoplasmic and mitochondrial Ca2+ levels, as detected using R‐CaM and CEPIA‐mt3, respectively. The depolarization‐dependent mitochondrial Ca2+ increase was partly prevented by dantrolene (50 μM), xestospongin B (10 μM) or IP3R1 knockdown, and was completely suppressed when using both inhibitors. Fibers pre‐incubated with apyrase (2 U/mL) displayed partially inhibited stimulation‐induced mitochondrial Ca2+ increase. Muscle cells were pre‐incubated with caged‐IP3 (5 μM); photo‐release of IP3 induced a transient increase in mitochondria Ca2+ levels that was inhibited by xestospongin B infibers. Finally, depolarization incrased mitocrondrial membrane potencial and oxygen consumption and these effects were partially reduced by IP3R knockdown.

Discussion

All these results suggest that activation of IP3R1 and RyR1 mediated Ca2+ release contribute to the mitochondrial Ca2+ increase and mitochondrial function produced by muscle activation.

Support or Funding Information

FONDECYT (11130267, 1151293), PIA‐ACT1111, CONICYT 21150604
Determining Kinetics of Reversible and Irreversible Biofilm Formation by Staphylococcus aureus and Pseudomonas aeruginosa
Authors: Tran Laura , Heuertz Rita
Published: November 2023

Biofilms have become increasingly problematic in efficacious treatment of patient infections due to their tenacious survival, association with chronic and medical device‐associated infections, and resistance to antimicrobial treatments. According to the National Institutes of Health, biofilms play a role in approximately 80% of all chronic infections. This study focused on assessment of biofilm formation by bacterial models with P. aeruginosa, being the Gram negative model, and S. aureus as the Gram positive model. Inherent with the Gram positive model selected was the ability to discern biofilm characteristics of methicillin‐resistant S. aureus (MRSA) and methicillin‐susceptible strains (MSSA). The research design was use of a crystal violet‐based quantitative biofilm microplate assay performed at different time points (2–24 hours) on different strains of the above‐mentioned bacteria. To discern reversible attachment from irreversible adherence, wash conditions at two points in the procedure (one after bacteria were incubated and microwells were coated with bacterially‐produced biofilm; and the other after crystal violet staining of the biofilm) were changed as to number and rigor. Fewer number and less rigorous wash steps allowed for reversible attachment whereas greater number and more rigorous washes allowed for firmly adherent biofilm to be quantitated. Results indicated that MRSA produced higher amounts of biofilm than MSSA at most time points but both followed similar kinetic patterns (e.g., peaks at 8 h). Assessing MRSA and MSSA together, S. aureus showed peak biofilm at 6–8 hours and P. aeruginosa produced a similar magnitude of biofilm as S. aureus. Regarding number and rigor of wash steps, overall magnitude of biofilm was much lower when greater number and more rigorous washes were employed rather than fewer, less rigorous washes. These results indicate that number and rigor of wash steps was critical in quantitation of irreversibly attached biofilm.

Support or Funding Information

Financial support for LAT was received from the DeNardo Education and Research Foundation.
Ascorbate anti‐cancer activity is increased by Mn(III)N‐alkylpyridylporphyrins through production of reactive oxygen species
Authors: Bader Bader Hassan , Tovmayasan Artak , Craik James , Benov Ludmil , Batinic Haberle Ines
Published: November 2023

Ascorbate (Asc) has shown potential for cancer therapy, and cancer cells typically accumulate porphyrins and display aberrant redox homeostasis. Mn(III)N‐alkylpyridylporphyrins (MnPs) are redox‐active superoxide dismutase mimetics with many prospective therapeutic applications. This study examined how structural modifications of MnPs might affect their anticancer activity through selective cellular targeting of production of reactive oxygen species (ROS) produced by redox‐cycling with ascorbate.

Methods

PII/MDA ER‐negative human breast cancer and non‐tumorigenic HBL100 human breast epithelial cell lines were used as model systems. Uptake of MnPs and ascorbate oxidation rates were determined spectrophotometrically. MTT/SRB assays were used to investigate cytotoxic and anti‐proliferative actions of MnPs and Asc, and combinations thereof. Membrane damage and cell death mechanisms were investigated by flow cytometry.

Results

Ascorbate and MnPs redox‐cycle to generate cytotoxic ROS, mainly H2O2. Anticancer efficiency of MnPs correlated with ability to oxidize ascorbate; differences in cellular uptake and subcellular distribution had only minor effect.

Conclusions

The major factor determining MnPs’ anticancer activity in cell culture models is redox potential

Support or Funding Information

Support: College of Graduate Studies, Kuwait University and Research Sector, Kuwait University; Research Grant YM04/14 and University Grant SRUL02/13
Effects of in vivo knockdown of a Cold‐Inducible RNA Binding Protein (CIRP) on Breast Tumorigenesis
Authors: Ochoa Joey L , Lujan Daniel A , Peterson Melanie N , Hathaway Helen J , Hartley Rebecca S
Published: November 2023

RNA binding proteins (RBPs) are essential to processes that involve RNAs, beginning from their transcription through to their degradation. In this study, we utilize the polyomavirus middle T antigen (PyMT) mouse model to evaluate the role of cold‐inducible RBP (CIRP) in breast tumorigenesis. When PyMT is expressed in the mammary epithelium of mice, it causes aggressive tumorigenesis. CIRP has been shown to incite the translation of mRNAs encoding stress‐induced proteins, and is observed to have increased expression in breast cancer. We have shown that human CIRP overexpression in the PyMT mouse appears to inhibit breast tumorigenesis during early and late stages. Based on these results, the current study set out to evaluate the effects of CIRP knockdown on tumor development. We hypothesize that CIRP knockdown will allow greater progression of tumor growth, as well as a higher rate of metastasis. To address this hypothesis, CIRP was knocked down using siRNA in a PyMT ‐mammary tumor‐derived cell line. PyMT cells transiently transfected with siCIRP and siControl were injected into the mammary fat pad of a wild type mouse, and tumor growth assessed. CIRP knockdown in PyMT cells is being analyzed via RT‐qPCR and western blotting to determine the effective timing tor mammary fat pad injections. Tumor growth kinetics, final tumor burden and metastasis will be determined, as will expression of CIRP targets, proliferation, apoptosis and histopathological analysis. The results of this study will tell us if CIRP overexpression has the potential to protect against breast cancer.

Support or Funding Information

Initiatives to Maximize Student Development (IMSD GM‐0602010)

Grant from the UNM‐HSC allocation Committee (RAC)
Structural Impact of Phosphorylation on the Dynamic Properties of SIKE
Authors: Minzer Ian , Machek Megan , Standen Heather , Kim Seung‐Hwan , Latzer Joachim , Bell Ellis , Bell Jessica
Published: November 2023

Suppressor of IKKe (SIKE)inhibits TBK1 mediated phosphorylation of interferon regulatory factor 3 (IRF3) and misregulation of TBK1 is involved in both autoimmune disorders and cancer. Our work (Marion et al, J.Biol.Chem 288, 18612–23, 2013) demonstrated that TBK1 can phosphorylate SIKE in up to 6 positions, altering its ability to inhibit TBK1. Little however is known concerning the three dimensional structure of SIKE. Clustal analysis of available SIKE sequences suggests that some phosphorylation sites are highly conserved, and in comparison with WIT3 may be involved more specifically with SIKE function. Molecular dynamics studies using models created with Phyre2 are exploring the impact of differently phosphorylated forms of SIKE on the structure and dynamics of the molecule. C‐terminal Histidine tagged SIKE has been expressed and purified using Nickel‐NTA chromatography and limited proteolysis in conjunction with tandem mass spectrometry is being used to explore both the validity of the models and potential differences in dynamic structure of the molecule induced by phosphorylation.

Support or Funding Information

This work was supported by NIH grant R21A1 107447 to JKB