bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effect of Phytochemicals on a Chemoresistant, Epithelial‐Mesenchymal Transitioned, Colorectal Cancer Cell Line
Authors: Murden Kathryn , San KayKay , Martino Alice , Ezekiel Uthayashanker
Published: November 2023

In 2012, colorectal cancer was the second most common cancer diagnosed in women and the third most common in men. In 2015, an estimated 132,700 new individuals will be diagnosed and 49,700 people will die from the disease. Colorectal cancer, though one of the most common cancers worldwide, is not distributed evenly among the world's population. It is concentrated mainly in countries with a Western culture, specifically the Western diet, such as the United States and Western Europe. A considerably smaller rate of incidence of disease is found in Africa and parts of Asia. This difference in colorectal cancer distribution may be attributed to the increased presence of certain phytochemicals in non‐Western diets. Our hypothesis is that phytochemicals inhibit proliferation of both metastatic and non‐metastatic cancer cell lines. Studies have shown that transdifferentiation into epithelial‐mesenchymal transition (EMT) by chemotherapy leads to the formation of resistant cells. EMT is a process in which epithelial cells acquire a mesenchymal phenotype, characterized by increased migratory and invasive properties. We developed a chemoresistance‐induced epithelial to mesenchymal transitioned colon cancer cell line (DLD‐1 OxR) by exposing the parental cell line (DLD‐1) to increasing concentrations of oxaliplatin (a drug used in the treatment of metastatic colon cancer). The DLD‐1 OxR cell line has undergone EMT and lost its epithelial properties to become a more motile mesenchymal cell type. We used a wound healing assay and transwell assay to assess the migration and invasion of the EMT‐chemoresistant cell line respectively. DLD‐1 and DLD‐1 OxR cells were exposed to curcumin and kaempferol. Curcumin is a phytochemical derived from turmeric, which has been shown to suppress inflammation and inhibit cell proliferation. Kaempferol, a phytochemical found in plants such as broccoli, kale, beans, and leeks, has also shown anti‐inflammatory and anti‐cancerous activity. Both phytochemicals inhibited proliferation of DLD‐1 and DLD‐1 OxR cell lines.

Support or Funding Information

KM, KS, and AM are supported by the DeNardo Education and Research Foundation Fellowship.
Anonna squamosa leaves ethanolic extract mediates apoptosis through heat shock protein mitigation and G2/M cell cycle arrest in Human Hepatocellular carcinoma (SK‐Hep1) cells
Authors: Lumbera Wenchie Marie Lara , Vergara Emil Joseph Sanvictores , Kim Yeon Soo , dela Cruz Joseph Flores , Hwang Seong Gu
Published: November 2023

The current study aimed to evaluate the chemotherapeutic property of Anonna squamosa (AS) leaves ethanolic extract in human hepatocellular carcinoma (HHC) SK‐Hep1 cells and identify its specific mechanism of action on the molecular level. Powdered AS leaves were soaked in 80% ethanol, subjected to rotary evaporator for extraction then used as treatment for SK‐Hep1 cells in the cell viability assay, cell cycle analysis, RT‐PCR and Western blot analysis. The cell viability assay demonstrated an increase in the cell proliferation among normal liver cell (CCL‐13) and cytotoxicity among SK‐Hep1 cells incubated with different AS concentration (0, 12.5, 25, 50, 100 and 200μg/ml) as treatment for both 24 and 48 hours. These results were further evaluated in the flow cytometry analysis which exhibited cell accumulation in the Sub/G1 and G2/M phase thereby cell cycle arrest identified at these points. Apoptotic and G2/M cell cycle arrest related genes and proteins expression were examined with RT‐PCR and Western blot to determine the specific molecular mechanism by which AS ethanolic extract causes HHC cell death. Both revealed a downstream expression of heat shock protein 70 (HSP70) and 27 (HSP27) unblocking apoptosis exhibited by an upstream expression of CASPASE‐3. G2/M cell cycle arrest was further verified by the down regulation of cell division cycle 25c (CDC25C) inhibiting the Cyclin B‐cyclin dependent kinase 1 (CDK1) complex exhibited by their decreased mRNA and protein expressions. Therefore, AS leaves ethanolic extract have a great chemotherapeutic potential against HHC (SK‐Hep1 cells) through HSP70 and HSP27 inhibition allowing programmed cell death through CASPASE‐3enhancement together with G2/M phase cell cycle arrest through Cyclin B‐CDK1 complex interference by CDC25C inactivation.
Characterization of the Role of Phenolic Compounds in the Inhibition of α‐Amylase by Kombucha Tea
Authors: Armando Samantha Renee , Schneider Rebecca Anne , Mann Francis M.
Published: November 2023

Kombucha is a tea blend that is fermented by a symbiotic culture of bacteria and yeast (SCOBY), and is of high interest in the United States for its health benefits. Research indicates that Kombucha can improve resistance against cancer, prevent cardiovascular disease, promote digestive functions, reduce inflammatory problems, and lower blood glucose levels. The hypoglycemic activity is the primary focus of this study. Kombucha brewed in black tea contains phenolics and this class of molecules has previously demonstrated inhibitory effects on α‐amylase. Previous research indicated that unfermented black tea is not appreciably inhibitory and thus it was hypothesized that the black tea phenolics undergo biotransformation during fermentation. In order to discriminate the specific inhibitory molecules found in Kombucha, Mock Tea (MT) replicates were made containing no tea polyphenols and compared to fermented Black Tea (BT) replicates. After fermentation, the replicates were treated to remove glycosides and separated over C18 resin. Three fractions were collected and measured for α‐amylase inhibition and polyphenolic content. Prior to evaluation of the data, the pH of the tea was neutralized to negate the effect of inhibition due to acidity. The results demonstrate an acid‐independent inhibition of α‐amylase by BT but not MT (−30% activity at 2.5% (v/v) analyte in most inhibitory fractions). This does not correlate to black tea phenolic content, as the phenolic content of the BT was or lower than that of the MT in most inhibitory fractions (BT=4.7ug/mL vs. MT= 37.6 ug/mL). However, this may be correlated to oxidative biotransformation of the black tea phenolics as polar fractions resulted in BT dependent inhibition and increased phenolic content (−50% activity at 2.5% (v/v) analyte with phenolic concentrations of BT= 106.3 ug/mL vs. MT= 62.8 ug/mL). While this indicates that the mechanism of α‐amylase inhibition is indeed dependent on black tea components, it is likely there are multiple inhibitory molecules with diverse chemical properties. Furthermore, it appears that the SCOBY synthesizes hydrophobic phenolic compounds, but the molecules are not inhibitors of α‐amylase.
Mechanism of SAMT Analog Inactivation of HIV‐1 Gag Polyprotein
Authors: Paine Elliott L. , George Rosenker Kara M. , Scerba Michael T. , Deshmukh Lalit , Appella Daniel H. , Miller Jenkins Lisa M.
Published: November 2023

Although antiretroviral therapy has advanced, there remains a critical need for new therapeutics, especially those targeted at resistant strains of HIV. The Gag polyprotein, and in particular its constituent nucleocapsid protein, NCp7, represents a prime target for antiretroviral inhibition. NCp7 is composed of two highly conserved zinc‐binding domains and plays multiple roles throughout the virus replication cycle. We have developed a class of small molecule inhibitors of NCp7, based upon an S‐acyl‐2‐mercaptobenzamide thioester (SAMT) scaffold, that covalently modify the zinc‐binding domains of NCp7 in vitro and in cells. These compounds display potent antiviral activity without evidence of cytotoxicity in cell models, ex vivo cervical explants, and in animal models. We investigated the mechanism of viral inactivation of the Gag polyprotein both in vitro and in virus‐like particles (VLPs) released from cells. We utilized mass spectrometry and NMR spectroscopy to characterize the covalent SAMT reaction sites within the Gag polyprotein. Our results indicate that reaction occurs first within the NCp7 domain, but that sites within both matrix and capsid are also modified. These results represent an increased understanding of the mechanism of SAMT activity and will help to develop better inhibitors.

Support or Funding Information

This research was supported by the Intramural Research Programs of the NCI and the NIDDK, NIH.
New Tricks for an Old Drug: Inhibition of Glyceraldehyde‐3‐Phosphate Dehydrogenase by Dithiolethiones to Suppress Cancer Cell Proliferation
Authors: White Michael R , Miller Thomas W , Switzer Christopher , Chavis John , Xu Pauline , Roberts David , Wink David , Garcin Elsa D
Published: November 2023

Dithiolethione compounds, such as the well‐characterized chemotherapeutic oltipraz, possess chemopreventive, cytoprotective, anti‐angiogenic, and chemotherapeutic properties. The chemopreventive effects of these compounds are mediated in part by the activation of antioxidant response elements and the increased expression of the tumor repressor PP2a and other cancer‐preventive proteins. Much of the transcriptional control of this response occurs via modification of thiol residues in the Keap1‐Nrf2 transcription factor complex. The 1,2‐dithiole‐3‐thione compound, ACS‐1, has also been shown to inhibit NF‐kB and downstream inflammatory molecules that promote tumor progression. As a result, these compounds can potentially be considered as promising multi‐targeting anticancer drugs.

In addition to these established roles for dithiolethione compounds, we show here that ACS‐1 plays a novel role in inhibiting glycolysis, important for survival and proliferation of cancer cells. Here we show that ACS‐1 inhibits glycolysis in human tumor cell lines by targeting the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). To understand the mechanism of action of ACS‐1, we have combined cell‐based assays, mutational analysis, kinetic assays, mass spectrometry, and x‐ray crystallography. We propose a mechanism whereby ACS‐1 inhibits glycolysis via covalent modification of GAPDH. Our results suggest that inhibition of GAPDH glycolytic function can be exploited in synergy with other cytotoxic chemotherapies to treat cancer. Cancer cells usually exhibit increased glycolysis (Warburg effect) in order to meet cellular energy requirements. Increasing evidence suggests that inhibition of glycolysis may be an effective strategy in chemoprevention. As such, these studies may provide new avenues for the treatment of cancer with the dithiolethione class of molecules.

Support or Funding Information

National Institutes of Health Grant T32 GM066706 (MRW)
Chemoenzymatic Synthesis of a Novel Sugar Scaffold for Targeting Ligand Development
Authors: Erickson Katelyn M , Hazel Joseph M , Troutman Jerry M
Published: November 2023

The capsular polysaccharides (CPSs) that encapsulate bacteria aids in overall virulence of the microbe. The bacterial capsules differ across species of bacteria from benign, pathogenic, or symbiotic bacteria. Specific targeting of the surface of the differing organisms would be advantageous in order to select certain types of bacteria over others, for either targeting or detection purposes.

In our lab, we have developed a new strategy to specifically target surfaces of certain types of bacteria. The developed targeting strategy is based on the surface coating of Vibrio vulnificus, and specifically the rare N‐acetyl‐quinovosamine (QuiNAc) sugar contained in the sugar polymer, as well as other sugars from the V. vulnificus polysaccharide biosynthesis pathway. The targeting strategy involves naturally occurring enzyme sugar products, as well as an isoprenoid anchor analogue that was developed for use with elucidation of polysaccharide biosynthesis pathways in our lab.

Determination of the sugar linkage to the isoprenoid anchor has been analyzed by RP‐HPLC with the use of a modified fluorescent isoprenoid analogue. A further developed analogue has been synthesized in our lab, which contains a different reactive group. This reactive group is used in the development of the targeting ligand for the rare QuiNAc sugar. Following the development of the targeting ligand associated with the rare sugars on specific types of bacteria, the use of the targeting agent is useful in applications of bacterial detection or in antibacterial schemes.

Support or Funding Information

National Institutes of Health AREA Grants R15GM100402 (J.M.T.), R15GM114773 (J.M.T.) and National Science Foundation Instrumentation Grant 1337873 (UNC‐Charlotte Chemistry)
ABO Blood Type Correlates with Survival on Prostate Cancer Vaccine Therapy
Authors: Gildersleeve Jeff
Published: November 2023

Cancer vaccines and other immunotherapies have significant potential for treating cancer patients, but clinical responses vary considerably from patient to patient. Simple, inexpensive strategies to target treatment to likely responders could substantially improve efficacy while simultaneously reducing health care costs, but identification of reliable biomarkers has proven challenging. Previously, we found that pre‐treatment serum IgM to blood group A (BG‐A) correlated with survival for patients treated with PROSTVAC‐VF, a therapeutic cancer vaccine in phase III clinical trials for the treatment of prostate cancer. These results suggested that ABO blood type might influence efficacy. Unfortunately, blood types were not available in the clinical records for all but 8 patients and insufficient amounts of sera were left for standard blood typing methods. To test the hypothesis, therefore, we developed a new glycan microarray‐based method for determining ABO blood type. The method requires only 4 μL of serum, provides 97% accuracy, and allows simultaneous profiling of many other serum anti‐glycan antibodies. After validation with 220 healthy subjects of known blood type, the method was then applied to 74 PROSTVAC‐VF patients and 37 control patients from a phase II trial. In this retrospective study, we found that type B and O PROSTVAC‐VF patients demonstrated markedly improved clinical outcomes relative to A and AB patients, including longer median survival, longer median survival relative to Halabi predicted survival, and improved overall survival via Kaplan‐Meier survival analysis (p=0.006). Consequently, blood type may provide an inexpensive screen to pre‐select patients likely to benefit from PROSTVAC‐VF therapy.

Support or Funding Information

This research was supported by the intramural research program of the NCI/NIH.
High Affinity Binding of a MUC1 Specific Antibody to a Glycopeptide Mediated by Conformational Selection
Authors: Brooks Cory L , Movahedin Reza , Brooks Teresa M , Supekar Nitin , Boons Geert‐Jan
Published: November 2023

MUC1 is a transmembrane glycoprotein and is a target for antibody‐mediated immunotherapy. In cancer cells, MUC1 is upregulated, and undergoes abnormal glycosylation resulting in truncated carbohydrate chains being added to the protein. The truncated glycosylation exposes cryptic peptide epitopes that can be recognized by antibodies. Since these immunogenic regions are cancer specific, they represent ideal targets for therapeutic antibodies. The peptide sequence, and the cancer specific truncated glycans are both available for antibody recognition. There is significant interest in determining which is the more suitable target for antibody immunotherapy. We investigated the role of tumor specific glycosylation in antigen recognition by the therapeutic antibody AR20.5. Using microscale thermophoresis we explored the affinity of AR20.5 to cancer specific MUC1 glycopeptide and peptide. The antibody bound to the glycopeptide with an order of magnitude stronger affinity than against the naked peptide. Given these results we postulated that A20.5 must specifically bind the carbohydrate as well as the peptide. Using X‐ray crystallography we examined this hypothesis by determining the structure of AR20.5 in complex with both peptide and glycopeptide. Surprisingly, the structure revealed that the carbohydrate did not form any specific contacts with the antibody. The high affinity of AR20.5 for the glycopeptide and the lack of specific binding contacts supports a hypothesis that glycosylation of MUC1 stabilizes an extended bioactive conformation of the peptide that is recognized by the antibody. High affinity binding of AR20.5 to the MUC1 glycopeptide is not driven by specific antibody‐antigen contacts, but rather glycosylation alters the conformational equilibrium of the antigen which allows the antibody to select the correct coformation. This study demonstrates a novel mechanism of antibody‐antigen interaction and also suggests that glycosylation of MUC1 is important for the generation of high affinity therapeutic antibodies.

Support or Funding Information

This work was supported through start‐up funds from the College of Science and Math at CSUF, and by a research grant from Quest Pharmatech Inc. to CLB. MR was supported by rGrants from ASI at CSUF and FSSRA awards from the College of Science and Math at CSUF.
Longitudinal Monitoring of Nanoparticle Accumulation in PC‐3 Tumors
Authors: Nneji Joseph Chidi , Gharagouzloo Codi Amir
Published: November 2023

Currently there isn't any definitive and non‐destructive test for predicting the accumulation of nanoparticles within tumors, and longitudinal monitoring of delivery requires a baseline using current methodologies. Diagnostic tools that make quantitative, independent measurements are needed to move product to the market. We utilized a subcutaneous PC‐3 tumor modal in 5 mice to predict and quantify accumulation of ferumoxytol. Diffusion tensor imaging (DTI) was utilized pre‐contrast to map out the diffusion anisotropy on a per‐voxel basis throughout the tumor to generate a predictive map for passive accumulation. QUTE‐CE MRI, a novel imaging modality developed in our lab, along with T1 and T2 measurements was used to follow accumulation.

We are developing a methodology for both predicting and unambiguously characterizing the heterogeneous accumulation of nanoparticles within tumors from the enhanced permeability and retention (EPR) effect. This impacts the market of cancer pharmaceuticals, currently limited by its predictive ability of exclusive drug delivery to the tumor.

We accurately predicted the heterogeneous accumulation of ferumoxytol with DTI. QUTE‐CE MRI proved to be more efficacious than T1 or T2 measurements, producing a better or comparable CNR, as well as clear, unambiguous definition of uptake without the need for comparison to pre‐contrast images. We have developed a novel strategy for predicting and following the accumulation of nanoparticles within the tumor. Ferumoxytol may be used as surrogate, combined with our methodology, to predict the EPR effect of other nanoparticles for image guided drug delivery.
Level of Polycyclic Aromatic Hydrocarbons and Heavy Metals in Selected Facial Makeup Cosmetics Commonly Used in Lagos, Nigeria
Authors: IGWO‐EZIKPE MIRIAM N , Taiwo Funmilayo O. , Sotinala Olayiwola T.
Published: November 2023

The use of cosmetics makeup has continually found growing market in Nigeria with different types and brands being introduced to consumers. This has raised concern regarding the adherence to standard limit of chemical composition in these cosmetic products. In this study, we investigated the level of polycyclic aromatic hydrocarbons and heavy metals in some cosmetics makeup products (powder, lipstick, lip gloss, eye shadow and brow pencils) commonly used in Lagos, Nigeria. Result obtain indicated that all samples analyzed for PAHs had minimum of two PAHs in significant concentration. Varying significant (p<0.05) concentration of Lead, Nickel and chromium were detected in samples analyzed, however cadmium was only detected in one brand of powder as its content in other samples were below detection limit. It is obvious from the present study that the use of facial cosmetics exposes users to PAHs and heavy metals which are persistent pollutants that are implicated in the etiology of several forms of oxidative stress related disorders including cancers, mutational deformities and reproduction abnormalities.
Activation of LXR inhibits the Development of Pulmonary Carcinomas Induced by 3‐Methylcholanthrene and Butylated Hydroxytoluene
Authors: Liu Ying , Wang Qixue , Ma Xingzhe , Chen Yuanli , Sun Lei , Duan Yajun , Han Jihong
Published: November 2023

We previously demonstrated that activation of liver X receptor (LXR), a transcription factor regulating cholesterol metabolism, by T0901317 inhibited the growth of inoculated Lewis lung carcinoma in C57BL/6 wild type mice by activating IFN‐gamma production (Biochemical Journal 2014;459:345–354). However, it remains unknown if LXR activation can function on carcinogens‐induced pulmonary carcinomas. In this study, we initially conducted a statistical analysis on the lung cancer samples extracted from the TCGA database, and found that both survival rate and time of lung cancer patients were correlated, while the grade of lung adenocarcinoma was inversely related to IFN‐gamma levels in lung samples of the patients. We then determined the effect of LXR activation on the development of pulmonary carcinomas induced by 3‐methylcholanthrene (MCA) and butylated hydroxytoluene (BHT) in mouse models. MCA/BHT induced pulmonary carcinomas to different degrees depending on the mouse strain. T0901317 reduced morbidity and mortality in the MCA/BHT‐injected BALB/c wild type mice. It also protected C57BL/6 wild type mice, but not IFN‐gamma deficient (C57BL/6 background, IFN‐gamma−/−) mice, against MCA/BHT‐induced death and tumor formation. IFN‐gamma levels in the lung and serum, which were associated with inhibition of tumor growth in wild type mice, were substantially increased by T0901317 administration. In addition, we determined that LXR activation inhibited the growth and metastasis of 4T1 human breast cancer tumors. Taken together, our study demonstrates that LXR activation is also able to inhibit MCA/BHT‐induced pulmonary carcinomas which is mainly attributed to induction of IFN‐gamma production.
Claudin‐7 regulates survival and glucose metabolism of human lung cancer cells
Authors: Kim Do Hyung , Jackson Spencer M , Chen Yan‐Hua , Lu Qun
Published: November 2023

Claudins are a family of tight junction membrane proteins engaged in a variety of human diseases including cancer. It has been reported that lung cancer patients with high claudin‐7 expression showed better survival than those with low claudin‐7 expression. Our study has shown that suppressing claudin‐7 expression (KD) in a human lung adenocarcinoma HCC827 cell line significantly increased cell proliferation in culture and tumor growth in mice. To investigate how tumor microenvironment plays a role in claudin‐7 regulation of cell survival, we treated the control and claudin‐7 KD cells under the hypoxia (1% O2) condition for 3 days. Light microscopic study revealed KD cells with fairly‐normal looking morphology whereas control cells displayed shrunken cell morphology. Immunofluorescence staining confirmed substantially decreased expression of cleaved PARP in KD cells than that of control cells (p < 0.01). To examine how claudin‐7 modulates glucose metabolism for cell survival, the cells were pre‐treated with normal (2g/L) glucose‐containing medium in normal oxygen condition for 2 days, followed by being sub‐cultured with normal or low (0.2g/L) glucose‐containing medium in either hypoxia or normal oxygen condition up to additional 24 hours. Western blot revealed an elevation in phosphorylation (or inhibition) of a negative regulator of glycogen synthesis GSK3β, phospho‐glycogen synthase kinase 3 beta (p‐GSK3β), and a substantial decrease in a cellular energy sensor, phospho‐AMP kinase (p‐AMPK), in KD cells when compared to control cells after the cells were treated in low glucose‐containing media for 24 hours, indicating the enhanced glycogen synthesis in KD cells for cell survival. This observation was not evident when the cells were cultured in the normal oxygen condition. We propose that claudin‐7 participates in regulating glucose metabolism and cell survival in human lung cancer cells through modulating GSK3β and AMPK signaling pathways.

Support or Funding Information

This study was supported by National Institute of Health grants HL085752.
A Novel Role for the Extracellular Matrix in Regulation of Hepcidin in Breast Cancer Spheroids
Authors: Blanchette Nicole , Tesfay Lia , Kita Daniel , Claffey Kevin P , Torti Frank M , Torti Suzy V
Published: November 2023

Iron is an essential nutrient for breast cancer cell growth and proliferation. Our laboratory has demonstrated that ferroportin (Fpn), an iron efflux pump, plays a critical role in maintenance of intracellular iron levels. Fpn is controlled by hepcidin, a secreted peptide hormone that binds to Fpn and triggers its degradation. We have observed that Fpn expression is down‐regulated and hepcidin expression is up‐regulated in breast cancer cells, promoting increased accumulation of iron in cancer cells to fuel their proliferative demands. Since expression of Fpn/hepcidin predicts outcome in breast cancer patients, we explored pathways that control these proteins. We used a 3D spheroid culture system to model in vivo breast tumor architecture, microenvironment and metabolism. We found that hepcidin expression is significantly up‐regulated from 2D to 3D culture of MCF‐7 and patient‐derived breast cancer cells, suggesting that hepcidin may be regulated by processes that exceed the realm of 2D culture. Culturing MCF‐7 cells as spheroids in the presence of Matrigel repressed the induction of hepcidin, suggesting that exogenous extracellular matrix (ECM) proteins present in Matrigel may negatively regulate hepcidin. Further analysis of the effect of exogenous ECM on hepcidin expression revealed that collagen IV, one of the most abundant basement membrane (BM) proteins in Matrigel, negatively regulates hepcidin expression in MCF‐7 spheroids. On the other hand, endogenous production of ECM components, such as collagen I and collagen II, matrix components frequently up‐regulated in breast cancer, was increased in MCF‐7 spheroids compared to monolayer culture. It is known that there is a modified microenvironement during breast tumorigenesis that includes breakdown of the structural BM proteins and deregulation of matrix components, including those that support tumor progression and promote metastatic spread. Together, our data suggest that components of the ECM play a dynamic role in both positive and negative regulation of hepcidin expression in breast cancer spheroids. This finding suggests that the ECM is an integral component in regulation of hepcidin within the tumor microenvironment. Understanding mechanisms of hepcidin regulation by ECM components will provide a greater understanding of the crosstalk between the ECM and iron metabolism in breast cancer.

Support or Funding Information

This research is supported by NIH R01 CA171101 and NIH R01 CA188025.
Upregulated Expression of GRP78 Supports ER Stress‐induced Apoptosis in Tunicamycin Treated Triple Negative Breast Tumor
Authors: Banerjee Dipak K , Banerjee Aditi , Zhang Zhenbo , Serrano Jesus E , Romero Eva C , Sanchez Neysharie , Baksi Krishna
Published: November 2023

Breast cancer affects nearly 1.5 million women (5–10%) worldwide per year. Triple negative breast cancer (TNBC) accounts for 15% of all breast cancer with a disproportionate share of mortality. The patients are younger and pre‐menopausal. The cancers are poorly differentiated, and most fall into the basal subgroup of breast cancers. The long‐term survival is extremely low because specific treatment guidelines are lacking. The patients are managed with standard treatment causing a high rate of local and systemic relapse. This make them resistant to existing targeted therapies (endocrine/biologics/adjuvant/neo‐adjuvant). Anthracycline/taxane combination therapy and PARP inhibitor are currently being explored but not known if they would achieve a pathological complete response (pCR). Protein glycosylation has been claimed as a universal feature of helping cancer cells escaping immune surveillance, facilitate tumor invasion, and increase malignancy with increased tumor burden and poor prognosis as well as enhanced angiogenesis. Based on the glycomics profile of human breast cancer cells as well as tumor specimen, we have hypothesized that targeting asparagine‐linked (N‐linked) protein glycosylation would evolve a new generation therapeutic preventing breast tumor progression and eliminating the disease. To test our hypothesis, we have used tunicamycin a natural product that competitively inhibits the N‐acetyl glucosaminyl 1‐phosphate transferase activity in the ER and consequently the N‐linked protein glycosylation. When tested in a triple negative breast tumor in nude mice, the tumor progression is inhibited by ~65% in one week at tunicamycin concentration of 0.25 mg/Kg administered orally twice a week. Human triple negative breast cancer cells (MDA‐MB‐231) are also equally susceptible to tunicamycin action. The cells are arrested in G1 and exhibit ER stress followed by induction of apoptosis mediated by unfolded protein response (upr) signaling. Western blotting and qRT‐PCR support increased expression of the ER stress master regulator GRP78. But, the immunofluorescence microscopy could not detect GRP78 on the surface of control or tunicamycin treated cells. The result is the same in cells cultured in the absence of serum. GRP78 fluorescence however, is detectable in cells after either they are fixed with ice‐cold methanol or permeabilized with digitonin. We, therefore, conclude that GRP78 is not expressed on the outer‐leaflet of the cell surface of the triple negative human breast cancer cells MDA‐MB‐231. But, its intracellular expression is anti‐tumorigenic.

Support or Funding Information

Supported in part by NIH U54‐CA096297 and Komen for the Cure BCTR06582 (DKB) and NIH/NIMHD 2G12MD007583 (KB).
Getting the Shaft?: Investigating Midshaft Location in Immature Femora
Authors: Eleazer Courtney D. , Kelso Rebecca Scopa , West Frankie L. , Williams Laura , Stradleigh Ryan , Shaeffer Ari
Published: November 2023

In skeletal samples, diaphyseal breadths of immature femora have traditionally been measured at 50% of diaphyseal length. However, research on the Denver growth sample suggests that 45.5% of diaphyseal length in younger individuals best represents the most analogous position to the 50% midshaft location in adult femora with fused epiphyses. Currently, direct comparisons have not been made between measures at 45.5% and 50% diaphyseal length. Lack of methodological consistency could affect comparisons across studies of immature skeletal remains.

Comparative analysis utilizing modern day radiographs of immature individuals (birth‐12 years) indicated that 50% of diaphyseal length is, on average, located 2.6 mm proximal to true midshaft. This discrepancy is caused by the distal epiphysis contributing more to total bone length than the proximal epiphysis and confirms that mid‐diaphyseal locations approximate, but do not exactly align with, midshaft locations.

Additionally, we analyzed immature (birth‐12 years)femora from several archaeological skeletal assemblages (n=170) to determine whether significant differences are present in anterioposterior (AP) and mediolateral (ML) breadths between the two diaphyseal locations. A repeated measures ANOVA indicates significantly wider AP and ML breadths at 45.5% when compared with 50% in all archaeological populations. Additionally, age is positively correlated with increases in AP breadth at 45.5% diaphyseal length. Morphological differences in the two locations support current understanding of shaft development and biomechanical changes with age. This finding, along with results from radiographs, suggests that studies utilizing one location may not be directly comparable to those utilizing the other. Future research will assess the impact of age on midshaft location to determine age‐specific methods for collecting diaphyseal breadths from immature femora.
Mitochondria as signaling organelles in aging
Authors: Titorenko Vladimir , Leonov Anna , Arlia‐Ciommo Anthony , Medkour Younes , Svistkova Veronika
Published: November 2023

Exogenously added lithocholic bile acid (LCA) is known to extend the chronological lifespan of the yeast Saccharomyces cerevisiae, accumulate in mitochondria and alter mitochondrial membrane lipidome. Using quantitative mass spectrometry, we demonstrated that LCA alters the age‐related dynamics of changes in levels of many mitochondrial proteins, as well as numerous proteins in cellular locations outside of mitochondria. These proteins belong to two regulons, each modulated by a different mitochondrial dysfunction; we call them a partial mitochondrial dysfunction regulon and an oxidative stress regulon. Proteins constituting these regulons (1) can be divided into several “clusters”, each of which denotes a distinct type of partial mitochondrial dysfunction that elicits a different signaling pathway mediated by a discrete set of transcription factors; (2) exhibit three different patterns of the age‐related dynamics of changes in their cellular levels; and (3) are encoded by genes whose expression is regulated by the transcription factors Rtg1p/Rtg2p/Rtg3p, Sfp1p, Aft1p, Yap1p, Msn2p/Msn4p, Skn7p and Hog1p, each of which is essential for longevity extension by LCA. Our findings suggest that LCA‐driven changes in mitochondrial lipidome alter mitochondrial proteome and functionality, thereby enabling mitochondria to operate as signaling organelles that orchestrate an establishment of an anti‐aging transcriptional program for many longevity‐defining nuclear genes. We propose a model for how such LCA‐driven changes early and late in life of chronologically aging yeast cause a stepwise development of an anti‐aging cellular pattern and its maintenance throughout lifespan.

Support or Funding Information

Supported by NSERC of Canada.
Methylene Blue Alleviates Nuclear and Mitochondrial Abnormalities in Hutchinson‐Gilford Progeria Syndrome
Authors: Xiong Zhengmei , Choi Ji Young , Wang Kun , Zhang Haoyue , Tariq Zeshan , Wu Di , Cao Kan
Published: November 2023

Hutchinson‐Gilford progeria syndrome (HGPS), a fatal premature aging disease, is caused by a single nucleotide mutation in the lamin A gene. Previous reports have focused on nuclear phenotypes in HGPS cells, and the potential contribution of the mitochondria, a key player in normal aging, remains unclear. Using high‐resolution microscopy analysis, we demonstrated a significantly increased fraction of swollen and fragmented mitochondria and a marked reduction in mitochondrial mobility in HGPS fibroblast cells. Notably, the expression of PGC‐1α, a central regulator of mitochondrial biogenesis, was inhibited by progerin. To rescue mitochondrial defects, we treated HGPS cells with a mitochondrial‐targeting antioxidant methylene blue (MB). Our analysis indicated that MB treatment not only alleviated the mitochondrial defects but also specifically rescued the hallmark nuclear abnormalities in HGPS cells. Additional analysis suggested that MB treatment released progerin from the nuclear membrane, rescued perinuclear heterochromatin loss and corrected misregulated gene expression in HGPS cells. Together, these results demonstrate a role of mitochondrial dysfunction in developing the premature aging phenotypes in HGPS cells and suggest MB as a promising therapeutic for HGPS.

Support or Funding Information

NIH/NHGRI Grants R01HG007104 (KC) and R21AG043801 (KC)
The Biosynthesis of the Indolic Acid Moiety from the Antibiotic Nosiheptide
Authors: Badding Edward , Booker Squire
Published: November 2023

Nosiheptide (NOS) is a highly modified thiopeptide natural product produced by Streptomyces actuosus that belongs to a unique class of thiazole‐containing peptide antibiotics. Recent studies have shown that NOS is as an effective antibacterial agent against a number of pathogenic bacteria, such as methicillin resistant Staphylococcus aureus, penicillin‐resistant Streptococcus pneumoniae, and vancomycin‐resistant enterococci. One key structural feature of NOS essential for its antibacterial properties is an indolic acid ring system characteristic of e‐series thiopeptides. The indolic acid moiety is derived from the amino acid tryptophan. In the first step of the proposed pathway, catalyzed by NosL, tryptophan is converted into 3‐methyl indolic acid (MIA). The remaining steps and enzymes involved in the formation and installation of MIA into the NOS core have not yet been definitively determined, but are believed to be catalyzed by the action of four enzymes: NosI, NosJ, NosK, and NosN. The research herein uses isothermal titration calorimetry (ITC), high‐pressure liquid chromatography (HPLC), mass spectrometry, and bioinformatics to delineate the exact roles of each of the enzymes in the biosynthesis of the indolic acid moiety. We show that NosJ is an acyl carrier protein that is post‐translationally modified with a 4′‐phosphopantetheine group, and that NosI catalyzes the ATP‐dependent adenylation of MIA. Upon incubation of NosI with ATP and MIA in the presence of NosJ, we observe transfer of MIA to NosJ. We believe that NosK then catalyzes the transfer of MIA from NosJ to the side‐chain of a cysteinyl residue of NOS. NosN, annotated as a radical S‐adenosylmethionine (SAM) methylase, subsequently attaches a C1 unit—suggested to be a methyl group—to C4 of the indolic acid moiety and then connects the C1 unit to Glu 43 of the macrocyclic core, completing the synthesis of NOS. We are particularly interested in understanding how NosN carries out this reaction. Preliminary ITC experiments show that two SAM molecules bind to the enzyme simultaneously. We suggest that one SAM molecule is converted into a 5′‐deoxyadenosyl 5′‐radical that initiates a novel methylation reaction by abstracting a hydrogen atom from the methyl moiety of the second SAM molecule. These studies shed new light on novel mechanisms for methylating unactivated carbon centers.

Support or Funding Information

This work was supported by NIH (GM103268), the President's Undergraduate Research Fund (Penn State), and the Rodney Erickson Discovery Grant (Penn State).
Investigating Mycobacteriohage‐Host Protein Interactions
Authors: Neevel Andrew , Chang Gloria , VandenBergh Wessel , McDonough Virginia , Stukey Joseph
Published: November 2023

Mycobacteriophages are viruses that infect bacterial cells of the genus Mycobacterium. With more than 400 mycobacteriophage genomes sequenced and available in GenBank, they represent the largest collection of sequenced phages that infect a single host (Mycobacterium smegmatis). They possess a multitude of unfamiliar or novel genes – genes encoding protein sequences that do not resemble any previously studied proteins – and thus encode products with functions not readily predicted. We hypothesized that some of those genes encode products that interfere with the normal metabolism of the host cell, possibly through specific phage‐host protein‐protein interactions, and thus have a role in enabling phage infection. Further, we predicted that those gene products when expressed alone in host cells would still be toxic and impair cell growth. We have investigated unfamiliar genes in two genetically distinct mycobacteriophages, Pumpkin and Vix, and have identified 4 single genes (Pumpkin_115, Pumpkin_119, Pumpkin_142, Vix_80) and several small genomic regions (Pumpkin gene segment 130–133 and Vix gene segments 65–66, 68–72, 87–88) that are cytotoxic to M. smegmatis. We are taking a multi‐prong approach to further identify the specific functions associated with these genes and their products and to determine their roles in the infection process: 1) we have identified mutants of M. smegmatis that are resistant to the expression of those genes, 2) we are using E. coli‐expressed phage genes to screen for interacting host proteins, 3) we are collecting microscopy data that could identity phage interruption of normal cellular function, and 4) we are in the process of deleting these genes from the phage genome to determine the effect on infection. 24‐hour expression of individual cytotoxic phage genes in M. smegmatis resulted in a significant increase in mean host cell length and some subtle effects on cell shape. Ongoing analysis of the mutants has identified a common mechanism of resistance to distinct phage gene expression, while protein‐protein interaction studies have not yet identified a potential host target involved in translation.

Support or Funding Information

Supported by a Jacob E. Nyenhuis Faculty Development Grant and an HHMI Faculty Research Award
Regulation of T cell Activation By Herpes Simplex Virus 1 Protein Us3
Authors: Pan Shuang , Wu Songfang , Liu Yaohui , Zhang Liming , Leng Qianli , Chen Da , Yang Mengmeng , Wang Jiyan , He Bin , Zhang Cuizhu , Cao Youjia
Published: November 2023

Herpes simplex virus 1 (HSV‐1) is the most prevalent human virus that infects a variety of cell types in latent and lytic states. Previous studies demonstrated that HSV‐1 productively infected human T cells and inhibited T cell receptor (TCR) mediated cell signaling. However, the precise way of virus‐host interactions remains to be solved. Here, we report that HSV‐1 encoded Us3 protein interfered TCR signaling and interleukine‐2 production by targeting the linker for activation of T cells (LAT). Us3 associated with LAT and sequestered the later from TNFR‐associated factor 6 (TRAF6), which plays important role in the activation of LAT. This study unveils a mechanism by which HSV‐1 intrudes early events of T cell activation, and may help to answer how HSV escapes from host immune surveillance.

Support or Funding Information

This work is supported by the National Natural Science Foundation of China 81171556, and 31370862.
Penetration of Staphylococcus aureus Biofilm by the Cinnamon‐Derived Compound, Cinnamaldehyde
Authors: Peach Aaron Preston , 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. Of grave concern is the observation that biofilm is frequently a contributory component of antimicrobic resistance and treatment failure in patient infections. Review of the literature reveals that many plant‐derived compounds have anti‐bacterial properties; however, biofilm effects have not been reported. Staphylococcus aureus is known for its multi‐drug resistant strains (MRSA) and plant‐based therapy may be appropriate for treatment of antimicrobic‐resistant bacteria. Our laboratory identified that the cinnamon‐derived compound, cinnamaldehyde, inhibited biofilm production by MRSA in a concentration‐dependent manner. To further this line of study, a penetration assay was developed to determine if cinnamaldehyde was able to penetrate biofilm produced by S. aureus. Briefly, a biofilm‐producing strain of S. aureus (ATCC 29213) was grown on disks of nitrocellulose (pore size 0.45 μm) or Whatman 1 filter paper (pore size 11 μm) and positioned onto Mueller Hinton agar plates streaked with a different S. aureus (ATCC 25923) so as to produce a lawn of growth after incubation. Cinnamaldehyde (at different concentrations) was placed onto the top of the biofilm‐coated disks and plates were incubated. If cinnamaldehyde was able to penetrate the bacterial biofilm on the disk, then it inhibited the lawn of S. aureus growth. Assay development results indicated that application of cinnamaldehyde directly to ATCC 25923 S. aureus inhibited its lawn of growth and that nitrocellulose and filter paper were both acceptable for use in the penetration assay. Early studies suggested that cinnamaldehyde penetrated the biofilm layer: these studies are on‐going. With many infections being associated with biofilms, it is timely to focus on plant‐derived compounds as antimicrobial agents.

Support or Funding Information

Financial support for AP was received from the DeNardo Education and Research Foundation.
Characterization of a Halotolerant Bacterium Isolated from Soil in Suburban Detroit
Authors: Sleda Melissa , Moricz Bridget , Zwiesler‐Vollick Julie
Published: November 2023

During cold weather, salt is sprayed on roads and sidewalks to prevent ice formation. We sought to determine if this excess salt affected the soil microbial community by increasing the presence of halotolerant or halophilic microbes. We collected a soil sample and isolated a single bacterial colony using nutrient agar plates supplemented with 5% NaCl. The microbe was able to grow indicating an ability to tolerate NaCl. To test if NaCl was required, we grew it on Nutrient Agar without additional NaCl. This microbe was able to grow at least as well without 5% NaCl, indicating that it is halotolerant but does not thrive in the presence of NaCl (halophilic). This bacterial isolate was characterized using multiple staining methods and differential/selective media. We also studied the metabolism of this microbe with respect to its ability to utilize various nutrient sources both in the presence and absence of oxygen. Genomic DNA was extracted and used to amplify the 16s ribosomal RNA gene. This was then sequenced and compared to the 16s RNA bacterial and archaeal database at the National Center for Biotechnology Information (NCBI) to further aid in identification. We were able to isolate a halotolerant microbe showing the presence of a microbe with salt tolerance in the community of salt‐treated soil. The characterization of this microbe will be discussed.

Support or Funding Information

Funded by the Department of Natural Science at Lawrence Technological University.
Delivery of paclitaxel and cetuximab by nanodiamond enhances mitotic catastrophe and tumor inhibition in colorectal cancer
Authors: Lin Yu‐Wei , Liao Wei‐Siang , Lin Johnson , Liu Kuang‐Kai , Cheng Hsiao‐Chun , Wang Chi‐Ching , Li Yi , Chen Chinpiao , Chao Jui‐I
Published: November 2023

Nanomaterials provide the opportunities to improve the efficacies for cancer therapy. Nanodiamond (ND) is a promising carbon nanomaterial developed for novel cancer treatment. Here, we show ND‐carried paclitaxel (PTX), a mitotic blocker, and cetuximab (Cet) (a specific EGFR antibody) that enhances mitotic catastrophe and tumor inhibition in human colorectal cancer (CRC). ND‐PTX induced the mitotic catastrophe and apoptosis in a variety of human CRC cell lines; in contrast, ND alone did not induce cell death. ND‐PTX blocked the mitotic progression, caused abnormal chromosomes separation and subsequently induced the caspase‐3 activation and PARP protein cleavage for apoptosis induction. ND‐PTX inhibited the xenografted human CRC tumors in nude mice. Furthermore, ND‐PTX‐Cet enhanced the cellular uptake ability and apoptosis induction and tumor growth inhibition in the EGFR‐expressed colorectal cancer cells. The mitotic marker of phospho‐histone H3 proteins was highly elicited by ND‐PTX‐Cet in CRC tissues. These results demonstrate that ND is a promising nanocarrier of PTX and Cet for developing novel CRC therapy.