bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploring the Effectiveness of a Flipped Class using Game‐based Learning
Authors: Klein Barbie
Published: November 2023

Flipped classes are becoming ubiquitous in education. This research reviewed current literature regarding flipped classes and investigated student perceptions and exam performance after a flipped class was incorporated into a traditional medical curriculum. Students prepared for the flipped class by viewing a podcast, reading through material, and completing an assignment. The day of the flipped class began with a Classroom Assessment Technique followed by a group activity consisting of a web‐based, interactive narrative that is crafted from the learning objectives. A final debrief summarized major concepts from the lesson and answered questions. Students completed questionnaires after the flipped class composed of a variety of ranking and open‐ended items about preparation, preference, and previous experiences with other alternative instructional methods. Opinions were mixed, with a few students stating that they enjoyed the flipped class format, a few students disliked it, and many were indifferent. Exam performance was higher for material taught through the flipped class compared to material taught through traditional lecture, however the difference was not statistically significant. This flipped class design modeled application of the material and was easily integrated to complement traditional lecturing.
The (AAA+) ATPases PSMC5 and VCP/p97 control ERK1/2 signals transmitted through the Shoc2 scaffolding complex
Authors: Galperin Emilia , Jang Eun Ryoung , Anderson Daniel , Jang HyeIn
Published: November 2023

Signaling scaffolds guide the flow of information and the spatial organization of the enzymes within the ERK1/2 signaling pathway. However, mechanisms that control assembly and dynamics within scaffolding complexes as well as mechanisms regulating the cellular distribution of these complexes remain largely unknown. Our studies unravel a novel, multi‐level paradigm in which allosteric modifications alter the ability of the scaffold protein Shoc2 to actively accelerate transmission of ERK1/2 signals.

Shoc2 accelerates ERK1/2 signaling by means of integrating Ras and RAF‐1 into a multi‐protein complex. Germ‐line mutations in Shoc2, affecting spatial distribution of Shoc2 or its ability to assemble signaling complex, result in Noonan‐like RASopathy, a developmental disorder with a wide spectrum of symptoms. Recently we determined that the amplitude of the ERK1/2 signals transduced through the complex is fine‐tuned by the HUWE1‐mediated ubiquitination of Shoc2 and its signaling partner RAF‐1. This ubiquitination is a part of the negative feedback mechanism that actively controls ERK signals.

To provide the mechanistic basis of how ubiquitination of the Shoc2 scaffold and RAF‐1 is controlled, we identified previously unknown binding partners of Shoc2, the proteins of the ubiquitin machinery (AAA+) ATPases, PSMC5 and VCP/p97. We demonstrated that both ATPases modulate ubiquitination of Shoc2 and RAF‐1 through remodeling of the complex and sequestration of the E3 ligase HUWe1 from the complex. Fluorescence microscopy and biochemical analysis were utilized to demonstrate that PSMC5 and VCP/p97 are also involved in recruitment of the Shoc2 scaffolding complexes to endosomes. Importantly, the Noonan‐like RASopathy mutation that causes aberrant distribution of Shoc2 to the plasma membrane also affects mechanisms controlling Shoc2 ubiquitination as it becomes inaccessible to PSMC5 and VCP/p97.

In summary, our studies are the first to demonstrate that mechano‐enzymes PSMC5 and VCP/p97 are the novel players regulating Shoc2‐ERK1/2 signal transmission and to show that mislocalization of the Shoc2 complexes affects remodeling of the scaffolding module and may lead to the developmental pathology. Furthermore, this study makes a significant advance in our understanding of the mechanisms by which scaffolds regulate specificity and dynamics of the ERK1/2 signaling networks.

Support or Funding Information

R00CA126161 (NIH); R01GM113087 (NIH); RSG‐14‐172‐01‐CSM (ACS); 15PRE25090207 (AHA)
Galectin‐1 Modulates Focal Adhesion Turnover and Migration of Vascular Smooth Muscle Cells
Authors: Chau Lee‐Young , Chiang Ming‐Tsai , Tsai Dong‐Lin , Tsai Min‐Shao
Published: November 2023

Background

Vascular smooth muscle cell (VSMC) migration play a key role in the development of atherosclerosis and restenosis. Galectin‐1 (Gal‐1) is a galactose‐binding lectin highly expressed in VSMCs. Previous studies have shown that Gal‐1 binds to extracellular matrix and β1integrin. Whether Gal‐1 influences VSMC motility via regulating focal adhesion dynamics remains to be characterized.

Methods and Results

The motilities of primary VSMCs isolated from wild type (WT) and Gal‐1 null mice were assessed by wound healing and Boyden chamber assays. Gal‐1 null VSMCs were shown to exhibit greater motility than WT cells. Moreover, recombinant Gal‐1 (0.5~2 μg/ml) inhibited the migration of Gal‐1 null VSMCs in a dose‐dependent manner. On the other hand, adhesion assay revealed that WT VSMCs adhered better than Gal‐1 null cells on surfaces coated with matrix proteins, including fibronectin, laminin and collagen. Cell spreading and focal adhesion formation examined by phalloidin and vinculin staining following plating on fibronectin were significantly greater in WT SMCs. Concomitantly, FAK phosphorylation examined by Western blot analysis was induced to greater extent in WT cells. Nocodazole washout assay demonstrated that focal adhesion disassembly was facilitated in Gal‐1 null VSMCs, which was correlated with faster decrease in FAK phosphorylation in these cells. In line with the in vitro findings, the neointimal hyperplasia induced by carotid artery ligation was much greater in Gal‐1 null mice comparing to WT control.

Conclusion

These data demonstrate that Gal‐1 restricts neointimal formation post vascular injury at least in part through attenuating focal adhesion turnover in VSMCs.

Support or Funding Information

This work was supported by a grant from the Ministry of Science and Technology of Taiwan (MOST‐103‐2321‐B‐001‐071)
Serglycin Regulates Cytoskeletal‐related Proteins Associated with Cell Motility in Breast Cancer In Vitro
Authors: Bay Boon Huat , Chua Pei Jou , Yip George Wai‐Cheong , Gunaratne Jayantha
Published: November 2023

Despite advances in cancer treatment, triple‐negative (ER−/PR−/HER2−) breast cancer represents a complicated subtype with no specific treatment regime. Serglycin (SRGN) is an intracellular proteoglycan which has been shown to be highly expressed in the triple‐negative MDA‐MB‐231 breast cancer cell line. In this study, we aim to decipher the molecular networks and pathways regulated by SRGN in breast cancer cells using a global gene expression analysis approach. We first analyzed the expression of SRGN in a dataset containing 1,881 breast cancer tissues from the GOBO database (http://co.bmc.lu.se/gobo/gobo.pl). For the in vitro experiments, siRNA‐mediated down‐regulation of the SRGN gene in MDA‐MB‐231 cells was performed. Transcriptomic analysis of si‐SRGN breast cancer cells was then carried out. Subsequently real time PCR for specific gene targets and expression of selected proteins by Western Blot were performed. Cell motility was determined using commercially available cell migration chambers. The Gene Set analysis from the GOBO database verified that the SRGN gene is highly expressed in basal (triple negative) breast tumors. Knockdown of the SRGN gene in MDA‐MB‐231 cells induced differential expression of 225 genes. Gene ontology analysis revealed the presence of a cluster of genes that were highly enriched in cellular processes, including cell movement. Reduced expression of SRGN was accompanied by down‐regulation of several cytoskeletal‐related genes, namely ABLIM1, LIMA1, CFL1, RAC1, RAC2 and RHOA, concomitant with decreased cell motility. Decreased expression of RAC1 and RHOA proteins were also demonstrated. The results suggest that SRGN is important in regulating actin cytoskeletal organization associated with cell migration, a crucial event during cancer metastasis.

Support or Funding Information

This work is supported by the National Medical Research Council Singapore Grant NMRC/CIRG/1370/2013.
The ULK3 Kinase Regulates the ESCRT pathway in the Abscission Checkpoint
Authors: Wenzel Dawn Marie , McCullough John , Caballe Anna , Marin‐Serrano Juan , Sundquist Wesley I
Published: November 2023

The endosomal sorting complex required for transport (ESCRT) pathway mediates important membrane fission events, including the physical separation of daughter cells during the abscission step of cytokinesis. Late acting proteins in this pathway, called ESCRT‐III subunits, are recruited to the intercellular bridge that connects two daughter cells where they appear to polymerize into spiraling membrane‐bound filaments that draw membranes together and promote fission. Abscission is highly regulated; if a dividing cell cuts its membrane prematurely (e.g., in the presence of unsegregated DNA) or fails to divide entirely, the daughters may become aneuploid. Progression through abscission is regulated by the abscission checkpoint; a genome protection mechanism that functions through the Aurora B kinase and the ESCRT‐III subunit CHMP4C to delay abscission until upstream mitotic events have been resolved. Checkpoint inputs include lagging chromosomes within the intercellular bridge, aberrant nuclear pore assembly, and mechanical tension at the intercellular bridge. Our goal is to understand how ESCRT‐mediated abscission is coordinated with these other steps in the mitotic program.

We recently identified a second kinase, ULK3, that functions downstream of Aurora B in the abscission checkpoint. ULK3 contains tandem C‐terminal Microtubule Interacting and Trafficking (MIT) domains, which serve as docking sites for ESCRT‐III proteins. Notably, ULK3 binds and phosphorylates a subset of ESCRT‐III proteins, including CHMP4C and IST1. We have determined the crystal structure of the second ULK3 MIT domain in complex with its IST1 binding site, and characterized the unusually tight binding interaction. We further find that ULK3 phosphorylation provides a reversible inhibitory signal that stalls abscission until mitotic errors can be resolved; phospho‐mimetic IST1 fails to support cytokinesis, and non‐phosphorylateable IST1 cannot support a functional abscission checkpoint. We are now mapping ULK3 phosphorylation sites on other ESCRT‐III subunits, with the goal of identifying a consensus site for ULK3 substrates. We are also testing the mechanistic consequences of ULK3 phosphorylation on ESCRT‐III function using in vitro ESCRT‐III assembly assays and cytokinesis assays in cells. Preliminary studies suggest that ULK3 phosphorylation inhibits the ability of ESCRT‐III proteins to form the filaments that constrict the midbody. Thus, we are learning how ULK3 phosphorylation of ESCRT‐III proteins inhibits the abscission checkpoint and laying the groundwork for identifying additional ULK3 substrates.

Support or Funding Information

This work was supported in part by an American Cancer Society Postdoctoral Fellowship to DMW (“ULK3 regulation of the ESCRT pathway in abscission”, PF‐14‐102‐01‐CSM) and NIH GM112080 to WIS
Electrophilic Nitro‐oleic Acid Inhibits Triple Negative Breast Cancer Cell Migration via Suppression of NF‐kB activity
Authors: Woodcock Chen‐Shan , Woodcock Steven , Salvatore Sonia , Davidson Nancy , Huang Yi , Freeman Bruce
Published: November 2023

Triple negative breast cancer (TNBC) is an aggressive and therapy‐resistant metastatic cancer. Patients with metastatic TNBC are difficult to treat due to the absence of effective target therapies, thus leading to poor clinical outcome. Constitutive activation of the nuclear factor kB (NF‐kB) pathway is a main driver to promote TNBC tumor aggressive phenotypes including cell survival, migration, and invasion. This study investigates the anticancer effects of electrophilic anti‐inflammatory mediators nitroalkene fatty acids in TNBC in vitro and in vivo. Decreased inflammatory metastatic potential of TNBC cells by an exemplary electrophilic nitrolipid, nitro‐oleic acid (NO2‐OA), was shown through inhibition of cell motility using transwell cell migration and invasion assays. NO2‐OA treatment inhibited NF‐kB transcriptional activity and expression of two TNFa‐induced NF‐kB‐regulated genes involved in tumor metastasis, intercellular adhesion molecule‐1 (ICAM‐1) and urokinase‐type plasminogen activator (uPA) in TNBC cells. The mechanism of NF‐kB signaling inhibition by NO2‐OA was due to suppression of IKKb phosphorylation and IkBa degradation. Notably, NO2‐OA also caused polyubiquitination and degradation of NF‐kB RelA protein via protein nitroalkylation in TNBC cells. In an MDA‐MB‐231 xenograft model, NO2‐OA inhibited tumor burden resulted in reduced tumor growth and incidence of metastasis in mice. Taken together, electrophilic NO2‐OA shows potential as a therapeutic agent that may display high selectivity for inhibition of TNBC progression and metastasis.
3,4′,7‐O‐Trimethylquercetin as a Novel Agent to Inhibit in Vitro Ovarian Cancer Cell Migration and Invasion
Authors: Yamauchi Kosei , Afroze Syeda Humayra , Dean DeShaundra M , Mitsunaga Tohru , Zawieja David C , Uddin Mohammad Nasir
Published: November 2023

Objective

Quercetin is a representative flavonoid that presents widely in variety of foods and medicinal plants. So far quercetin has been reported to inhibit migration and invasion of cancer via decreasing the matrix metalloproteinase (MMP)‐2. 3,4′,7‐O‐trimethylquercetin (34′7TMQ) was synthesized from rutin as a starting material in order to evaluate the anti‐migration and anti‐invasion activity of ovarian cancer cells.

Study Design

We evaluated the effect of 34′7TMQ on three ovarian cancer cells SK‐OV‐3, CRL‐1978, and CRL‐11731 function in vitro. Each Cell lines were treated with different concentrations of 34′7TMQ (0.0, 3.125, 6.25 and 12.5 μM). For each cell line cell proliferation, migration and invasion were measured by using a CellTiter Assay (Promega), Cytoselect Assay (Cell Biolabs) and by using a FluoroBlock Assay (BD) respectively. Proliferating Cell Nuclear Antigen (PCNA) was also evaluated in cell lysates of 34′7TMQ‐treated these 3 ovarian cancer cells by western blot analysis. Expressions of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI‐1) and matrix metalloproteinase‐2 (MMP‐2) were also evaluated in 34′7TMQ‐treated ovarian cancer cells by western blot analysis. Statistical comparisons were performed using analysis of variance with Duncan's post hoc test.

Results

34′7TMQ significantly inhibited migration and invasion of three ovarian cancer cells. 34′7TMQ had no effect on proliferation and PCNA expression in ovarian cancer cells. Moreover, 34′7TMQ significantly inhibited the expressions of uPA and MMP‐2 for all ovarian cancer cells.

Conclusion

These data indicate that 34′7TMQ inhibited migration and invasion of ovarian cancer cell without inhibiting the proliferation. 34′7TMQ may inhibit the metastasis activity of ovarian cancer cell by decreasing uPA and MMP‐2 expression. 34′7TMQ might be further investigated as a novel anti‐ovarian cancer agent.

Support or Funding Information

The Discovery Foundation (MNU), Grimes Foundation Endowment Award (MNU), Japan Society for Promotion of Science (JSPS) (KY)
Hypoxia and Serum‐Deprivation Impacts Calcineurin B Homologous Protein Isoform 2 Expression and Activity in Non‐Small Cell Lung Cancer
Authors: Wallert Clarice H , Provost Joseph J , Wallert Mark A.
Published: November 2023

The Na+‐ H+ exchanger isoform 1 (NHE1) is a key regulator of cell proliferation, migration and invasion in cells from a variety of solid tumors. The calcineurin B homologous proteins (CHP1 and CHP2) appear to be essential cofactors to support NHE1 function. CHP1 appears to be expressed ubiquitously in healthy tissue, while CHP2 is predominantly expressed in tumor cells. While both CHP isoforms are highly homologous and bind in nearly identical regions on NHE1, each has been identified to have a distinct impact on NHE1 function. Our research has demonstrated an elevation in CHP2 expression in tumor samples from patients with either adenocarcinoma or squamous cell carcinoma of the lung as well as in a broad range of NSCLC cell lines. To evaluate the role of CHP2 in NSCLC cells we used NCI‐H1299, a carcinoma cell line, as the parent cell line and developed two new cell lines. One of these cell lines lacked CHP2 expression (H1299 CHP2KD) and the other lacked NHE1 expression (H1299 NHE1KD). We hypothesized that CHP2 expression enhances cell survival and the tumorigenic potential of these cells. We evaluated the impact of a low serum and hypoxic microenvironment on CHP2 expression and determined CHP2 levels increased when H1299 cells are grown in 0.5% serum and 1% O2 compared to cells grown in 10% serum and 21% O2 (normal conditions). This increased CHP2 expression drives changes that support the cells' ability to survive these low serum and hypoxic microenvironments. Proliferation and migration rates in normal conditions for H1299 CHP2KD cells were approximately 50% of H1299 cells. When these experiments were performed in low serum and hypoxic conditions, the proliferation and migration rates of the H1299 cells was reduced to about 50% of that seen in normal conditions, while the rates of proliferation and migration in H1299 CHP2KD was reduced to a rate equivalent to the H1299 NHE1KD cells. Finally, in a soft agar assay, a greater rate of tumor formation occurred for H1299 cells in both 10% and 0.5% serum than either the H1299 CHP2KD or H1299 NHE1KD cells. These data suggest a major role for CHP2 expression in the development and progression of non‐small cell lung cancer.
Role of Bcl‐2 in Hematological Cancer Formation
Authors: Link Tina , Fortier Abigail , Jain Supriya , Kepner Alisa , Koh Ally , Stanovski Lisa , Ul‐Islam Misha , Walter Ellie
Published: November 2023

B cell leukemia/lymphoma 2 (BCL‐2) is a 26 kilodalton, amphipathic, anti‐apoptotic protein located on the outer mitochondrial membrane. BCL‐2 interacts with other anti‐apoptotic and pro‐apoptotic factors in order to regulate apoptosis. If a structural change occurs on the BCL‐2 gene, overexpression of the BCL‐2 protein can result. This causes a change in the ratio of BCL‐2 to its BCL‐2 Homologous 3 (BH3) ligands, preventing the release of the apoptotic signal. The primary change associated with BCL‐2 overexpression in lymphomas is a t(14;18) chromosomal translocation, which causes an increase in BCL‐2 transcription. The upregulation of the oncogene allows for the survival of the mutated B cells by raising the apoptotic threshold, which then dislodges regular lymphocytes in the bone marrow or lymph nodes. Lack of cell death due to overexpression of BCL‐2 has been linked to hematological malignancies, specifically lymphomas and myelomas. Both B‐lymphocytic cancers can result from alterations of the immunoglobulin heavy chain locus on chromosome 14. While BCL2 translocation can cause Follicular Lymphoma, BCL2 over‐expression is observed in many other tumor types and likely contributes to cancer progression and drug resistance in these tumors. Treatments to inhibit BCL‐2 include attempts at activation and overexpression of the pro apoptotic BH3 proteins, BCL‐2 Associated X Protein (BAX) and BCL‐2 Associated K protein (BAK), and creation of BH3 mimetics. Indeed, the FDA approved the BCL2 inhibitor Venetoclax (ABT‐199) earlier this year for the treatment of Chronic Lymphocytic Leukemia. These treatments could help cause cancer cell death by binding to targeted anti‐apoptotic proteins and causing the signal for apoptosis to be released. This could neutralize the cancerous effects of the BCL‐2 oncogene upregulation. Walton High School SMART (Students Modeling A Research Topic) Team used the computer program Jmol to create a 3D printed model of BCL‐2 and composed a review poster of our secondary research to investigate and demonstrate the relationship between the structure and function of this molecule.

Support or Funding Information

Diane Munzenmaier, Ph.D., Center for Biomolecular Modeling, Milwaukee School of Engineering
CINOBUFOTALIN INHIBITS OVARIAN CANCER CELL METASTASIS VIA APOPTOTIC SIGNALING AND TARGETING THE mTOR PATHWAY
Authors: Afroze Syeda Humayra , Peddaboina Chander , Dean DeShaundra M , McDowell Anthony , McCormick Timothy C , Newell‐Rogers Karen , Zawieja David C , Kuehl Thomas J , Uddin Mohammad N
Published: November 2023

Objective

Cinobufotalin (CINO), a cardiotonic steroid (CTS) or bufadienolide, is extracted from the skin secretions of the traditional Chinese medicine giant toads (Chan su). CINO has been used as a cardiotonic, diuretic and a hemostatic agent. Previously we have shown that CINO inhibits the cytotrophoblast cell function. Recently other study has shown that CINO inhibits A549, a lung cancer cell function. We demonstrated that CINO inhibited the proliferation, migration and invasion of three ovarian cancer cell lines; SK‐OV‐3, CRL‐1978 and CRL‐11731. In this study we evaluated the cellular mechanisms by which CINO inhibits ovarian cancer cell function.

Study Design

We evaluated the effect of CINO on three ovarian cancer cells SK‐OV‐3, CRL‐1978, and CRL‐11731 function in vitro. Each Cell lines were treated with different concentrations of CINO (0.1, 1, 5 and 10 μM). For each cell line cell proliferation, migration and invasion were measured by using a CellTiter Assay (Promega), Cytoselect Assay (Cell Biolabs) and by using a FluoroBlock Assay (BD) respectively. Proliferating Cell Nuclear Antigen (PCNA), mTOR and pAKT were also evaluated in cell lysates of CINO treated these 3 ovarian cancer cells by western blot analysis. Cell Cycle arrest and Cell viability were determined by fluorescence‐activated cell sorting (FACS) analysis. We also performed Annexin V staining on CINO treated these 3 ovarian cancer cell lines by immunofluorescence to evaluate the pro‐apoptotic protein expression. In addition mitochondrial membrane potential (MMP) has also been measured for all these 3 ovarian cell lines after CINO treatment using MMP kit, by FACS analysis.

Results

Concentration of CINO at 0.5μM inhibit SK‐OV‐3, CRL‐1978, and CRL‐11731 ovarian cancer cells proliferation, migration and invasion without cell death and loss of cell viability but cell viability differs for each cell line. Each cell lines differ in response to CINO doses for PCNA expression as well as Annexin V pro‐apoptotic protein expression. CINO decreases mitochondrial membrane potential for SK‐OV‐3 but not for CRL‐1978 and CRL‐11731 increases in response to CINO treatment. CINO causes the decrease in mTOR and pAKT expression in SK‐OV‐3 and CRL‐1978 but not in CRL‐11731.

Conclusion

CINO is cell specific, as each cancer cell line responds differently. These data demonstrate that the mode of action of CINO is different on these 3 types of ovarian cancer cells. Moreover, it also can be suggested that CINO inhibits the ovarian cancer cell metastasis via apoptotic signaling and targeting the mTOR pathway.

Support or Funding Information

The Discovery Foundation Grant (MNU), Grimes Foundation Endowment Award (MNU)
A Novel Endoglin Variant in Pancreatic Ductal Adenocarcinoma
Authors: Kumar Sanjay , Shah Nirav , Zaman Naveed , Lee Nam Y
Published: November 2023

Advanced pancreatic ductal adenocarcinoma (PDAC) is among the most aggressive and lethal malignancies with poor long‐term prognosis. Although early diagnosis for surgical resection is considered essential for improved survival outcome, most patients have delayed presentation of signs and symptoms until the disease has advanced or metastasized. Hence, significant improvements in treatment options will require a greater understanding of PDAC progression at the molecular level along with identification of new therapeutic targets and associated biomarkers. Endoglin is a TGF‐b co‐receptor essential for normal and tumor angiogenesis. Its expression is generally restricted to proliferating vascular endothelial cells (ECs) during neovascularization, although more recently endoglin expression has been found in a subset of breast, prostate and pancreatic tumors. While the functional roles of endoglin in tumors are still emerging, here we report a unique PDAC‐specific endoglin isoform (PDAC‐Eng) not expressed in normal human pancreatic ductal epithelia or ECs. We provide evidence that PDAC‐Eng is a novel splice variant and not a proteolytic byproduct. Additionally, PDAC‐Eng is secreted as an unexpected component of PDAC exosomes, which we found exerts strong autocrine and paracrine effects by enhancing endothelial and PDAC cell growth inhibition and deregulates TGF‐b signaling. Taken together, these findings support endoglin as a potentially new prognostic marker and a therapeutic target in pancreatic cancer.
Role of autophagy in temozolomide‐induced apoptosis in Rhabdomyosarcoma cells
Authors: REZAEIMOGHADAM ADEL , Alizadeh Javad , Field Jared , da Silva Rosa Simone , Gordon Joseph W. , Ghavami Saeid
Published: November 2023

Rhabdomyosarcoma (RMS) is a muscle‐derived tumor and is the most common pediatric soft tissue sarcoma representing 5% of all childhood cancers. RMS is a major clinical problem in pediatric oncology. Treatment of RMS with the oral alkylating agent temozolomide (TMZ), alone or in combination with other drugs, has recently received considerable interest. However, the mechanism of action of TMZ remains unclear. The aim of this investigation was to determine if autophagy modulates TMZ‐induced cell death in RMS cell lines. MTT assay and Nicoletti flow cytometry analysis were used to measure cell death and apoptosis in SJCRH30 (RH30) human RMS cells and the non‐transformed mouse myoblast cell line C2C12, following TMZ treatment (100 μM). We monitored autophagy using transmission electron microcopy (autophagosome and autophagolysosome formation) and immunoblotting (LC3 lipidation, Atg15‐12 conjugation, p62 degredation). The effect of bafilomycin A1 on TMZ‐induced cytotoxicity was also evaluated. We showed that TMZ decreased the viability of RMS cells in a dose/time‐dependent manner and induced accumulation of sub‐G1 cell population, representing apoptotic cells. Interestingly, TMZ induced apoptosis by 17‐fold in the RH30 cells, but only increased apoptosis by 2.7‐fold in C2C12 cells. In RH30 cells, TMZ decreased the expression of antiapoptotic proteins Bcl‐XL and Mcl‐1, and increased the expression of the death gene Nix. Moreover, we showed that TMZ altered biochemical markers of autophagy, such as LC3 lipidation, Atg5–12 conjugation, and p62 degradation, and induced morphological evidence autophagy, including accumulation of autophagosomes and autophagolysosome, determined by transmission electron. Finally, treatment of RMS cells with the autophagy flux inhibitor (Bafilomycin A1, 4 and 6 nM) had no significant effect TMZ‐induced cell death in either cell line. In conclusion, our investigation showed that TMZ induced simultaneous autophagy and apoptosis in both RH30 and C2C12 cells; however, cell death induction by TMZ does not appear to be dependent on Bafilomycin A1 inhibited processes, such as autophagosome‐lysosome fusion or autolysosome acidification.
Characterizing the specificity of E3 ubiquitin ligase MARCH1 for CD98
Authors: Najera Susana Sarai , Ablack Jailal , Ginsberg Mark
Published: November 2023

CD98 is a type II transmembrane protein that supports adhesive signaling and amino acid‐transport enabling rapid cell proliferation. Our genetic ablation studies have revealed that CD98 is essential for proliferation of multiple cell types including cancer cells. CD98 is found at the cell surface where it interacts with integrins and its light chains to amplify adhesive signaling and amino acid transport, respectively. Surface expression of CD98 is regulated post‐translationally by MARCH1 and MARCH8, members of a novel class of E3 ubiquitin‐ligases. MARCH1 limits CD98 dependent proliferation of lymphocytes, and potentially cancer cells, by selectively targeting CD98 for ubiquitination and subsequent lysosomal degradation. However, the mechanism by which MARCH1 targets and interacts with CD98 is still poorly understood. Our goal is to decipher how MARCH1 achieves specificity for its substrates using CD98 as a model substrate. Since MARCH1 isoforms have unique N‐termini, we initially hypothesized that the N‐terminus of MARCH1 mediated the interaction with CD98. Ectopic expression of either isoform of MARCH1 in HeLa cells resulted in down regulation of CD98 from the cell surface, while ectopic expression of a catalytically inactive mutant of MARCH1 had no effect. Thus, the N‐terminus alone does not determine MARCH1 specificity for CD98. We next expanded our hypothesis to include other domains of MARCH1 that could be responsible for imparting specificity for CD98. To test this hypothesis, we generated chimeras of MARCH1 and MARCH3 (MARCH3 has no specificity for CD98) to determine which regions of MARCH1 impart specificity for CD98. Our preliminary results confirmed that the N‐terminus of MARCH1 alone, is not sufficient for targeting of CD98. Likewise, the RING domain, the transmembrane domains and C‐terminus of MARCH1 on their own are not sufficient to impart CD98 specificity to MARCH3. Together these observations suggest that the domains of MARCH1 likely work in combination to selectively interact with CD98 and target it for degradation.

Support or Funding Information

Research reported in this poster was supported by the National Cancer Institute of the National Institutes of Health under award numbers: U54CA132384 & U54CA132379
Kv3.1 and Kv3.4 as tumor hypoxia related voltage‐gated potassium channels
Authors: Song Min Seok
Published: November 2023

Hypoxia is one of the representative characteristics of cancer tissue that differs from normal tissue. Our data demonstrate a close relationship between several oxygen‐sensitive voltage‐gated K+ (Kv) channel subunits and tumor hypoxia, as well as an importance of these channels in cancer progression. Kv3.1, Kv3.3, and Kv3.4, which belong to the Kv3 subfamily, were examined as tumor hypoxia‐related Kv channels in A549, MDA‐MB‐231, and HT‐29 cells. All three cells lines highly expressed these channels. We found a cell‐density dependent increment of HIF‐1α, a tumor hypoxia marker, in A549 and MDA‐MB‐231 cells, whereas HIF‐1α showed no similar increment in HT‐29 cells, which grow in an aggregate form. Interestingly, expression of Kv3.1 and Kv3.4 showed the same pattern with HIF‐1α in all three cell lines, and we determined that Kv3.1 and Kv3.4 are tumor hypoxia‐related Kv channels. Furthermore, blood depressing substance (BDS) toxin, a Kv3 subfamily specific blocker, inhibited cancer cell migration and invasion. Our results demonstrate that tumor hypoxia related oxygen‐sensitive Kv channels, including Kv3.1 and Kv3.4, appear to be new therapeutic targets for tumor metastasis.
Sequence‐Dependent Sorafenib Therapy in Combination with Natural Phenolic Compounds for Hepatocellular Carcinoma and Possible Mechanism of Action
Authors: Abaza Mohamed Salah , Bahman Abdul‐Majed M , Khoushiash Sarah , Al‐Attiyah Rajaa
Published: November 2023

Sorafenib (NexavarH, BAY43‐9006, Sora) is the first molecular targeted‐therapy that has shown significantly therapeutic benefits in advanced hepatocellular carcinoma (HCC). However, not all HCC patients respond to Sora well and new therapeutic strategies to optimize the efficacy of Sora are urgently required. The efficacy of plant‐based drugs has received growing attention due to their excellent chemotherapeutic and chemopreventive activities beside they are well tolerated, nontoxic, easily available, and inexpensive. It is well known that natural active compounds may act in synergy with drugs used in clinical applications. Therefore, this study aimed to investigate whether a combination therapy with natural phenolic compounds including curcumin (Cur), quercetin (Que), kaempferol (Kmf) and resveratrol (Rsv) may allow dose reduction of Sora without loss of effectiveness at the same time. HCC cell lines (Hep3b, HepG2) were treated with Sora alone or in combination with natural phenolic compounds in concomitant, sequential and inverted sequential regimens. Cell proliferation, cell cycle, apoptosis and expression of proteins‐related to cell cycle and apoptosis were investigated. Phenolic compounds markedly potentiated the therapeutic efficacy of Sora in a schedule‐, type and dose of phenolic compound‐, cell line‐dependent manner. Concomitant treatment with Cur (Sensitization ratio, SR= 28), Kmf (SR = 18), Que (SR= 8), was found to be associated with the highest SR in Hep3b. Rsv markedly potentiated Sora effect (SR = 17) on Hep3b when administered in reverse sequential manner. In contrast, Rsv and Que did not improve the efficacy of Sora on HepG2, meanwhile simultaneous treatment with Cur (SR =10) and Kmf (SR = 4.01) potentiated Sora cytotoxicity. Concomitant treatment with Sora and Cur or Kmf growth arrested HCC cells in S‐phase and G2/M‐phase and markedly induced apoptosis compared to single treatments with Sora, Cur and Kmf. Concomitant combined treatment with Sora and Cur reduced the protein levels of cyclins A, B2 and D1, phosphorylated retinoblastoma and BcLxL. On the other hand, Sora/Cur co‐treatment increased the protein levels of Bax, cleaved caspase‐3, and cleaved caspase‐9 in dose‐dependent manner. In conclusion, concomitant treatment with Sora and Cur or Kmf seems to be a potent and promising therapeutic approach that can control HCC by triggering cell cycle arrest and apoptosis. Additional studies need to be performed in order to exam the in vivo potential of the combined treatment of Sora and natural phenolic compounds. This study was supported by Kuwait University, Research Grant No. YS01/15.
The potential role of NMDA receptor regulating TGF‐β/Smad pathway in radiation‐induced resistance in glioblastoma multiforme
Authors: Liu Chan‐Chuan , Wu Sheng‐Nan , Sze Chun‐I
Published: November 2023

Glioblastoma multiforme (GBM) is the most lethal primary brain cancer with poor prognosis despite the aggressive therapeutic treatment. Infiltrative growth, resistance to therapy, genetic heterogeneity, and the existence of cancer stem cells lead to tumor recurrence and poor outcome. The current standard of therapy is surgery followed by radiation and/or chemotherapy. Unfortunately, tumor cells establish resistance to the therapy. Glutamate is a neurotransmitter that induces post‐synaptic excitation of neurons for the cell communication. Glutamate activating ionotropic and metabotropic receptor is also essential in tissue development. Glutamate receptors are classified into NMDA, AMPA and kainate receptors. The activation of glutamate receptor involves in regulation of cell death and survival, proliferation, and migration. Previous studies revealed that activation of NMDA receptor via glutamate regulates proliferation and invasion in gliomas. In addition, cross talk between NMDA receptor and TGF‐β/Smad signaling leads to cell differentiation. We hypothesize that NMDA receptor may play role in the generation of radiation‐induced resistance in GBM.

Our laboratory had been using growth inhibition 50% and consecutive irradiation to establish radiation resistant GBM cell lines. Parts of radiation resistant cells showed morphological changes and we defined these cells as transformed cells. Immunocytochemistry staining showed the different expression patterns of NMDA receptor subunits, and its adaptor protein PSD95 between transformed cells and non‐transformed cells. In Western blotting results, we found that the expression of NMDA receptor and PSD95 are decreased in the development of radiation resistance. We also found that NMDA receptor related signaling proteins, such as nNOS, Akt, and ERK, are also decreased. In our previous discovery, radiation activates TGF‐β/Smad signaling which leads to mesenchymal differentiation. On the other hand, inhibition of TGF‐β/Smad pathway reverses mesenchymal differentiation in radiation resistant cell lines. In our current studies, TGF‐β treatment results in parental GBM cell lines reduced expression of the NMDA receptor and PSD95, however, the inhibition of TGF‐β receptor have no influence on these changes. We conclude that the decrement of NMDA receptor and its related pathways help GBM cells establish radiation resistance which suggests that NMDA receptor may regulate TGF‐β/Smad signaling.
An Ageless Wonder: Decreasing Cardiac CapZ Prevents Myocardial Decline in Aged Mice
Authors: Pyle W. Glen , Lorenzen‐Schmidt Ilka , Reitz Cristine J , Alibhai Faisal J , Martino Tami A.
Published: November 2023

Ageing populations are a worldwide trend: by 2030 the number of people over 65 years of age will double and this cohort will continue to increase by another 50% by 2050. Age is the primary risk factor for cardiovascular disease with a 22% increase in risk every 5 years after age 65. Mitigating the functional decline of the heart that comes with advanced age will reduce susceptibility to heart failure and prolong lifespans. The actin capping protein ‘CapZ’ is a key regulator of molecular remodeling in ageing skeletal muscle. Whether it plays a similar role in the heart has never been investigated. Our objective was to determine if cardiac CapZ is altered in the hearts of aged mice, and to ascertain the impact of these changes. Wildtype C57Bl6 mice of both sexes were aged to 21 months and cardiac function determined by echocardiography. Left ventricular ejection fraction decreased from 77.2 ± 0.3% to 69.7 ± 0.8% (p<0.05) in male mice at 4 and 21 months of age respectively, whereas female mice showed no significant changes with ageing. Left ventricular fractional shortening showed a similar reduction in aged male mice, declining from 40.2 ± 0.3% to 34.1 ± 0.6% (p<0.05) in male mice aged 4 and 21 months. Again, females maintained fractional shortening with ageing. Immunoblot analysis revealed no differences in cardiac CapZ expression in male myocardial samples between 4 and 21 month old mice. Samples from female hearts, however, showed a significant reduction in CapZ protein levels. To determine if the natural reduction in CapZ protein levels seen in female hearts mitigated the functional decline of ageing, we measured myocardial function of CapZ‐deficient transgenic mice by echocardiography. While female CapZ‐deficient transgenic mice again exhibited no differences in cardiac performance between 4 and 21 month old animals, the functional decline that characterized aged male wildtype mice was significantly alleviated in CapZ‐deficient transgenic mice. It has been proposed that one factor contributing to the age‐dependent decline in cardiac performance is a reduction in beta‐adrenergic reserve. We have previously shown that CapZ influences beta‐adrenergic signaling in the heart. We measured the myocardial response to beta‐adrenergic stimulation of Langendorff‐perfused murine hearts and discovered that aged CapZ‐deficient transgenic mice retain a robust positive inotropic response to myocardial beta‐adrenergic activation, unlike aged wildtype mice. These studies represent the first report showing that decreased cardiac CapZ is an endogenous mechanism by which the hearts of female mice protect themselves against ageing, and demonstrate that reduced cardiac CapZ is sufficient to mitigate the functional decline of advanced age.

Support or Funding Information

CIHR Catalyst Grant
The role of the sphingolipid metabolism pathway in healthy aging
Authors: Hrobuchak Hannah P , Ardasheva Anastasia P , Servello Dustin P , Nolan Abby P , Chan Jason P
Published: November 2023

Animals are exposed to stressors, such as temperature and chemical stress, which may contribute to their aging. Organisms respond to stress by mobilizing cell‐signaling pathways, which are critical to control cell function and stress response. One specific type of pathway is the sphingolipid‐signaling pathway, which interconverts between different sphingolipids that can function as critical cell survival and cell death factors. Two critical enzymes are sphingosine kinase and ceramide kinase, which converts a cell death signals to cell survival factors (ceramide‐1‐phosphate and sphingosine‐1‐phosphate). However, less is known regarding the role of these lipid signaling pathways in healthspan, or healthy aging in animals. To examine how these enzymes mediate healthspan, we use the genetic model organism C. elegans to examine sphingolipid metabolism. We identified that sphingosine kinase, in particular, is important for mediating lifespan and healthspan. Through neuromuscular and heat stress assays, we determined that locomotor behavior and thermoregulatory responses of animals declined at greater rates when they lacked sphingosine kinase. However, we found that there is no differences in locomotor response in ceramide kinase mutants. Further examination of how sphingosine kinase and ceramide kinase loss at developmental and post‐reproductive windows will help us better understand the role of lipids in aging.

Support or Funding Information

National Institute On Aging of the National Institutes of Health, Award Number R15AG052933
An artistic Approach for Purification of Adeno‐Associated Virus and Adenovirus
Authors: He Dao‐Yao , Xie May
Published: November 2023

Since “virus‐like” particles were first discovered in adenovirus (AdV) preparations, adeno‐associated virus (AAV) has been characterized and developed in the last 50 years as a potent viral vector to deliver genes in vitro in cultured cells and in vivo in animal models. Numerous investigations have revealed its attracting features including nonpathogenicity, efficient transduction and stable expression, thus laying foundations for recombinant AAV for use as one of the most successful gene delivery vehicles. Although there are a few of traditional techniques used for purification of the viruses, one rapid and facile procedure for AAV purification remains to be developed. Here we present a novel approach using combination of techniques that has been developed as products for purification of AAV viruses and adenoviruses as well. The whole procedure of the approach takes about 3 h to produce viral solutions of AAV or adenovirus with a titer up to 1013 genomic copies or 1010 transducing units per ml. Coomassie staining reveals that the purified AAV solution exhibits only three bands of capsid proteins. Besides, there are no any toxic compounds or reagents used in this procedure, so that the high‐titer, ultrapure viral solutions obtained by the novel technology can meet the quality requirements for most research purposes and may be also qualified for preclinical or clinical trials.
Immunohistofluorescence Localization of Biogenic Amine Receptors in Ganglia and Tissues of the Bivalve Mollusc, Mytilus edulis
Authors: Cummings Delilah , Jacobs Maxine , Carroll Margaret A , Catapane Edward J
Published: November 2023

Biogenic amines serve as neurotransmitters in both sensory and motor systems in a variety of invertebrates. In bivalves, particularly Mytilus edulis, the physiological roles of serotonin and dopamine have been well studied especially with respect to gill and heart. Recently, in the related bivalve Crassostrea virginica, other biogenic amines, including GABA and histamine along with their receptors, were demonstrated to be present, and these amines are being shown to have physiological roles. In light of this we hypothesize that M. edulis would have receptors for the biogenic amines, serotonin, dopamine, histamine and GABA. We tested this using immunohistofluorescence techniques. Primary antibodies to serotonin, dopamine, histamine and GABA receptors, and secondary FITC‐labeled antibodies were purchased from Abcam and Santa Cruz Biotechnology, and used to visualize the presence or absence of these biogenic amine receptors in cerebral ganglia, visceral ganglia, and the animals' innervated gill and mantle. Briefly, tissues were dissected, snap frozen, sectioned on a cryostat, fixed with EDAC (N‐Ethyl‐N′‐(3‐dimethylaminopropyl) carbodiimide hydrochloride), treated with blockers, and incubated with primary, then secondary antibodies. Controls were treated the same way except without primary antibodies. Treated cryostat sections were viewed with a Zeiss epilume fluorescence microscope fitted with a ProgRes C3 Peltier cooled camera, or with a Leica epilume fluorescence microscope fitted with a Leica DFC400 camera. Both microscopes had 100 watt mercury lamps and FITC excitation/emission filters (Ex 470 nm, Em 525 nm). The results show serotonin 2A‐like, dopamine D2‐like, histamine H2‐like and GABA R1‐6‐like receptors present in cerebral ganglia, visceral ganglia, gill and in the sensory tentacles of the mantle rim. This comparative study provides new knowledge of the neurobiology of M. edulis by confirming the presence of these biogenic amine receptors in the animals' nervous system and innervated organs, in a comparable manner to what has been reported in C. virginica. It also provides a foundation to further study the physiological roles for histamine and GABA in M. edulis.

Support or Funding Information

This work was supported in part by grant 2R25GM06003 of the Bridge Program of NIGMS, grant 690340047 of PSC‐CUNY and a Carnegie Foundation award.
High‐throughput Functional Annotation of the Caenorhabditis elegans Neural Network
Authors: Aoki Wataru , Yokoyama Haruki , Matsukura Hidenori , Ueda Mitsuyoshi
Published: November 2023

In order to investigate the relationship between behavior and neural networks, such a technology that can artificially control the activity of the particular neuron is required. Once we artificially manipulate the activity of neurons, we will be able to examine in detail how the neural networks function by evaluating how the artificial manipulation influences the behavior.

Optogenetics is a technology that is able to freely control the activity of neurons with opsin by light irradiation. Optogenetics provides a methodology that can precisely analyze the relationship between behavior and neural network, and has become a trigger of rapid progress of brain research in the circuit‐centric approaches.

However, there are some weaknesses in the optogenetics.

1. Optogenetics requires a hypothesis, because we have to consider in advance which group of neurons is related to particular behavior when choosing promoter regulating opsin expression. For this reason, although it is a powerful method for validation and development of existing hypothesis, a completely new discovery is hard to be achieved. 2. Throughput is low. We need to create different transgenic animals depending on the hypothesis to be tested. 3. Single cell level analysis is difficult. Cell‐specific promoter does not usually exist. In addition, it is difficult to converge light on the single neuron of moving animals.

In order to overcome these weaknesses, we propose a new method that does not need any hypothesis, can be high throughput and can analyze in single cell resolution (Aoki et al. submitted). A key technology for this methodology is Cre/lox system. By applying Cre/lox system, we have established a methodology to randomize single neuron that expresses opsin, among the all neurons that construct the neural network.

In this methodology, we conduct behavior experiment after opsin expressions are randomized, and identify the neuron that influences the behavior “later”. Therefore, it does not require the hypothesis in advance. In addition, since we can create a large number of individuals that have randomized opsin expression pattern, from single individual, our method is feasible for high throughput analysis. Since expression of opsin is determined in each cell at random, single cell level analysis is feasible.

Here we present stochastic expression of opsin and high‐throughput annotation of phenotype using C. elegans as an experimental model.