bluuebunny/crossref_metadata_2025_split huggingface dataset:
Functional Characterization of a Novel Role for Rfa2 N‐terminal Hyper‐phosphorylation During Checkpoint Adaptation
Authors: Wilson Timothy M , Baumgartner Trevor A , Senger Barbara L , Miles Nolan M , Haring Stuart J 
Published: November 2023

Replication Protein A (RPA) is an essential heterotrimeric complex found in all eukaryotic organisms and has critical roles in DNA metabolism and cell cycle progression. In human cells, RPA is known to be hyper‐phosphorylated, primarily on the N‐terminus of the second subunit (Rpa2), in response to genotoxic stress. The significance and function of Rpa2 N‐terminal (NT) phosphorylation has been the subject of over 25 years of research; however, the physiological function of this modification has yet to be clearly determined. In budding yeast, Rpa2 N‐terminal phosphorylation is detectable upon prolonged exposure to commonly used clastogens or upon generation of an irreparable break at the MAT locus using HO endonuclease. Upon cleavage of the MAT locus, the master checkpoint kinase Rad53 becomes hyper‐phosphorylated, leading to signaling events that arrest yeast cells at the G2/M checkpoint to allow adequate time for DNA repair.During checkpoint adaptation, cell cycle progression restarts, despite the presence of unrepaired DNA. Checkpoint adaptation affords cells the ability to bypass an established checkpoint, with the potential negative consequence of cellular division with broken chromosomes, leading to mutations and/or chromosome loss. Through the use of functional and phenotypic analysis of yeast containing Rpa2 NT mutations, we have determined that the phospho‐state of the Rpa2 NT may play a key role in mediating checkpoint adaptation. We demonstrate here that the Rpa2 NT is necessary for, and readily hyper‐phosphorylated during, checkpoint adaptation. We have determined the subregions of the Rpa2 NT that promote checkpoint adaptation when phosphorylated, as well as some of the conserved kinases that appear to phosphorylate the Rpa2 NT during checkpoint adaptation. Finally, we propose a model by which Rfa2 NT hyper‐phosphorylation directly leads to dephosphorylation/deactivation of the master checkpoint signaling kinase Rad53 in order to coordinate checkpoint adaptation in yeast. Understanding the mechanistic role the Rpa2 NT plays in coordinated checkpoint exit in the face of persistent damage may shed light on events that lead to deregulation of checkpoint signaling, and subsequently, mechanisms that lead to aberrant cell growth.Support or Funding InformationThis research was supported by NSF‐CAREER 1253723 to SJH
Dissecting the TORC1 Gene Interaction Network by Perturbing Different Subunits
Authors: Gutierrez Cesar Julian Torres , Santos Sean M. , Hartman John L. 
Published: November 2023

Target of Rapamycin Complex 1 (TORC1) is a kinase that controls cell growth. Regulation of TORC1 activity contributes to aging and the human orthologous complex mTORC1 often becomes dysregulated in cancer cells. Rapamycin (a TORC1 inhibitor) is an immunosuppressive agent used for patients with organ transplantation or cancer. In Saccharomyces cerevisiae, high Rapamycin concentrations create a nutrient starvation physiology resulting in decreased protein synthesis and entrance into a G0 state. TORC1 is composed of multiple subunits (Kog1/Lst8/Tco89/Tor1/Tor2) that have been extensively studied previously, however their function and interactions with other genes await further characterization. We aim to determine which S. cerevisiae genes have an undiscovered role in the TORC1 complex in relation to decreased expression of Kog1 and to compare these results with previous data from decreased expression of Tor1/Tor2. Tet‐Off‐Kog1 S. cerevisiae (Tetracycline‐Controlled Transcriptional Activation) were grown in different concentrations of Doxycycline (0.5μg/ml‐10μg/ml) and a non‐growth‐inhibitory Rapamycin concentration (5nM) in order to find a concentration that would inhibit cell growth. Tet‐Off‐Kog1 S. cerevisiae were mated with the yeast knockout and knockdown library (YKO/KD) of 5,851 genes using the Synthetic Genetic Array method. The Tet‐Off‐Kog1 YKO/KD was then plated with growth‐inhibiting concentrations of Rapamycin (5nM) and Doxycycline (0.5–1.0–2.0–5.0μg/ml). Cell growth was measured until carrying capacity was reached and analyzed using a quantitative high throughput cell array phenotyping (Q‐HTCP) method. By analyzing the growth of the Tet‐Off‐Kog1 YKO/KD and comparing them to a distribution of references and previous Tor1/Tor2 data, we identified gene deletions buffering cell proliferation against loss of TORC1.Support or Funding InformationThis research was partially supported by the UAB Preparation for Graduate and Medical Education (PARAdiGM) Program through a grant from the National Institutes of Health (NIH/NHLBI R25 HL120883) and the RISE Program (NIH Grant #R25GM096955).
Anticancer activity of novel benzimidazole derivatives against MCF‐7 cancer cells
Authors: Özdemir Aysun , Uzunoğlu Sümeyye , Çalışkan Burcu , Banoğlu Erden , Ark Mustafa 
Published: November 2023

Cancer takes the third place in the cause of death worldwide after cardiovascular and infectious disease. Benzimidazole derivatives have demonstrated as potential new therapeutics for the treatment of cancer in vitro and in vivo. In this study in order to develop more potent anticancer agents, 27 novel benzimidazole derivatives have been designed and synthesized to evaluate for their in vitro cytotoxic activity in MCF‐7 cells.Cytotoxic effects of synthesized compounds were evaluated using MTT assay and the most active ones were further studied to determine the mechanism of cytotoxic activity. Four active benzimidazole derivatives, compounds A, 13, 22 and 23, were chosen with the IC50 values of 19.93±1.84, 16.06±7.47, 3.98±0.12, 10.59±1.61 μM in MCF‐7 cells, respectively. Treatment of these compounds at 18 μM for 48 h resulted in rounded and shrunken‐cell shape in MCF‐7 cells. Additionally, change in nuclear morphology after the treatments was determined by DAPI staining of nucleus. We found that selected benzimidazole derivatives significantly increased the nuclear fragmentation comparing to control group in MCF‐7 cells. Morever compounds A, 13, 22 and 23 significantly increased the cleavage of PARP‐1 and led to form an inactive fragment of 89 kDa demonstating that caspases are activated after the treatment of compunds. Taken all together our findings indicate that cytotoxic activities of these benzimidazole‐derived compounds are mediated by apoptotic cell death mechanism. These selected benzimidazole derivatives have the potential in the development of chemotherapeutic agents.Support or Funding InformationThis work was supported by TUBITAK in 115S016 project.
Exploiting Immunogenic Modulation in Chordoma: Sublethal Radiation Increases EGFR Expression and Sensitizes Tumor Cells to Cetuximab
Authors: Griner Jake , Padget Michelle , Hodge James 
Published: November 2023

Chordoma is a rare primary spinal neoplasm for which standard therapy consists of surgery and radiation. However, the high dose of radiation required to kill chordoma is not always possible given their proximity to the brain and spinal cord. Cancer cells surviving radiation often have phenotypic changes making them more susceptible to immunotherapy, a process termed immunogenic modulation. Among these phenotypic changes is the upregulation of surface markers for which FDA approved monoclonal antibodies exist, such as trastuzumab (Anti‐HER2), rituximab (anti‐CD20), and cetuximab (anti‐EGFR). Upregulation of monoclonal antibody targets have previously been shown to increase natural killer (NK) cell lysis by antibody dependent cell‐mediated cytotoxicity (ADCC) in both chordoma and other carcinoma types. It is known that approximately 80% of chordomas express Epidermal Growth Factor Receptor (EGFR), though to varying degrees of positivity. We hypothesized that sublethal radiation of chordoma cells would increase EGFR expression and enhance the therapeutic effects of cetuximab. Three distinct chordoma cell lines were exposed to sublethal doses of radiation and changes in surface EGFR expression were monitored via flow cytometry. ADCC assays were then performed using NK cells from normal donors and cetuximab. We show here that chordoma cells surviving low dose ionizing radiation express increased EGFR levels. The functional consequence of this upregulation is significantly decreased proliferation compared to either radiation or cetuximab alone. ADCC assays also show increased killing in cells that have been irradiated compared to those which have not. While both radiotherapy and cetuximab have had mixed results as monotherapies for chordoma, our results suggest that cetuximab in combination with radiation will produce a more complete response than either therapy alone.Support or Funding InformationNational Cancer Institute
Connections between NF‐κB Misregulation and Carcinogenesis
Authors: Link Tina , Chyong Donian , Naderi Aurian , Lee Chase , Arpornsuksant Ty , Lu Allison , Dilip Megha , Zhang Margaret 
Published: November 2023

In this review poster, we researched the function, regulation, and structure of NF‐κB as it relates to carcinogenesis. Found in almost all animal cells, nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) is a transcription factor that plays various roles in cellular proliferation, cell survival, inflammation, and T cell activation. There are two different NF‐κB signaling pathways: the canonical pathway and the non‐canonical pathway. NF‐κB forms a p50/Rel A heterodimer in the canonical pathway and a p52/Rel B heterodimer in the non‐canonical pathway. NF‐κB protein is normally sequestered in the cytoplasm as an inactive complex with a κB inhibitor (IκB) protein. Outside stimuli such as reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF‐α) activate cell surface receptors such as Toll‐Like Receptors (TLRs) and Receptor Activators of NF‐κB (RANK). These surface receptors, in turn, activate IκB kinase (IKK). IKK phosphorylates IκB, which causes ubiquitination of IκB, signaling proteasomes to degrade it. NF‐κB then enters the nucleus through a nuclear pore and forms a transcription complex with coactivators and RNA polymerase. NF‐κB binds to specific DNA‐binding sites to induce transcription. One of the target genes of NF‐κB encodes IκBs, so NF‐κB regulates its own activity through a negative feedback loop. Because the cell maintains storage of the signaling protein NF‐κB in the cytoplasm, NF‐κB allows the cell to respond to sudden stimuli by activating quick changes in gene expression. Because proteins that promote cellular proliferation and cell survival are mainly transcribed through NF‐κB activation, uncontrolled overactivation of this transcription factor has major implications in carcinogenesis. Many types of cancer cells have high levels of constitutively active NF‐κB. Mutations in the genes for NF‐κB itself or its inhibitors such as IκBs often found in cancer cells can cause cells to accelerate their proliferation or confer resistance to apoptosis. Some types of cancer cells secrete signaling molecules, inducing improper NF‐κB activity. Because of the tumor promoting characteristics of NF‐κB activation, a focus of current research is to find methods of suppressing NF‐κB to kill cancer cells or slow their growth. We designed a 3D‐printed model of the p50/RelA NF‐κB protein using Jmol to highlight the relationship between its structure and function.Support or Funding InformationDiane Munzenmaier, Ph.D., Center for Biomolecular Modeling, Milwaukee School ofEngineering
Salicylates Promote ROS‐induced Endoplasmic Reticulum Stress Triggering Apoptosis on B16F10 Melanoma
Authors: Ausina Priscila , Albanese Jamille Mansur , Leandro João Gabriel Bernardo , Mendonça Ana Paula Miranda , Palhano Fernando , Foguel Débora , Oliveira Marcus Fernandes , Zancan Patricia , Sola‐Penna Mauro 
Published: November 2023

Endoplasmic reticulum stress (ERS) and activation response to this stress have been documented in several types of cancer, suggesting that these cells activate the stress response as a survival strategy. Thus, drugs that cause an imbalance in this response may impair cancer cell viability. Salicylates have proven preventive and anti‐tumor effects in various types of cancer. On regard of ERS, it has been reported to act as potent inhibitor of protein synthesis in rat islets and in the gastric mucosa of guinea pigs. Salicylates also activate ERS through PERK activation in human promonocytic cells. In addition, the treatment with salicylates is correlated to increased reactive oxygen species (ROS) production, probably due to the direct inhibition of catalase activity. Nevertheless, there is still no correlation among salicylates, ERS and cancer. The aim of the present work is to investigate a new mechanism for salicylic acid (SA) and acetylsalicylic acid (ASA) related to their antitumoral effects, which involves the induction of ERS by ROS, and corroborate the new perspectives for the use of these drugs. Melanoma B16F10 cell line was used in all experiments and treated with 10mM SA or ASA. We tested cell viability and apoptosis using Anexin V assay, and ROS production using DCFDA, both by cell sorting. Unfolded protein response (UPR) markers were evaluated by qPCR and Western blot analysis. The treatment of the cells with SA or ASA for 1 or 2 hours promoted an increase in ROS production, as well as in the mRNA levels of the UPR markers ATF4 and CHOP. Increased protein levels of other UPR markers, such as PERK, IRE1, ATF6, cleaved ATF6, BiP and CHOP, are detected within 24 hours of treatment with both drugs. Finally, the treatment triggers apoptosis, which was detected on its early stage after 24 hours. Taken together, our data indicate that treatment of melanoma B16F10 cells with SA or ASA reduces cell viability thought a ROS‐induced ERS mechanism.Support or Funding InformationFinancial support: FAPERJ, CAPES, CNPq
TG‐interacting Factor Can Elicit Hedgehog Pathway to Increase Resistance to Gemcitabine in Urothelial Carcinoma
Authors: Huang Huei‐Sheng , Huang Yue‐Jhu 
Published: November 2023

Urothelial carcinoma (UC) of the bladder is the 4th most commonly diagnosed cancer and the 8th most common cancer‐related death in male in the United States in 2016. Using gemcitabine/cisplatin as a chemotherapy after surgery of patients with UC is a common treatment. However, the drug‐resistance to the therapy and high recurrence of UC patients usually occurred. Previously, we have demonstrated that TG‐interacting factor (TGIF) is involved in the migration/invasion, poor prognosis, and gemcitabine‐resistance of UC via PI3K/AKT pathway. It also has been proven that the aberrant hedgehog pathway is associated with many cancers and drug‐resistance. Therefore, whether the hedgehog pathway was involved in the gemcitabine‐resistance of UC was addressed. In the present study, we observed that expression of TGIF, pAKTser473, pGSK3βser9 and hedgehog components (Gli2, Smo, and Ptch) was increased in gemcitabine‐resistant NTUB1 (NGR) cells. Overexpression of TGIF or AKT1 in NTUB1 cells increased activity of GLI‐luciferase reporter and Gli2 expression. Furthermore, overexpression of AKT1 could phosphorylate pGSK3βser9 to inactivate its kinase activity and induce activation of hedgehog pathway. In addition, overexpression of GSKS9D could inactivate GSK3β kinase and increase activity of hedgehog pathway. However, overexpression of GSKS9A could activate GSK3β kinase to suppress hedgehog pathway activation. Finally, overexpression of Gli2 increased resistance to gemcitabine in NTUB1 cells, whereas, knockdown of Gli2 reduced resistance to gemcitabine in NGR cells. Accordingly, we suggest that gemcitabine‐induced TGIF expression can activate AKT/GSK3β signaling to elicit hedgehog pathway, which results in the gemcitabine resistance of UC.Support or Funding InformationThis work was supported by grants from the National Science Council (Taipei, Taiwan; NSC102‐2321‐B‐006‐019‐MY2; MOST104‐2314‐B‐006‐055‐MY3)
The chemosensory bitter taste receptors (T2Rs) are involved in proliferation and migration of breast cancer
Authors: Singh Nisha , Shaik Feroz Ahmed , Bhullar Rajinder Pal , Chelikani Prashen 
Published: November 2023

Background and ObjectiveBreast cancer is a very complex disease and involves interactions between many proteins. The human bitter taste receptors (T2Rs) are a group of 25 chemosensory proteins that belong to the G protein‐coupled receptor family. T2Rs mediate signal transduction in response to stimulation by a wide variety of bitter compounds. Earlier studies showed that bitter agonists such as quinidine and chloroquine triggered apoptosis in MCF‐7 breast cancer cells, and bitter melon extract inhibited breast cancer cell proliferation by modulating cell cycle regulatory genes and promoting apoptosis. However, the cell surface receptor (T2R) targets for these bitter compounds, and their expression and characterization in the cancer cells is not yet elucidated.MethodsTo delineate the expression profile of T2Rs in breast cancer MDA‐MB‐231 and MCF‐7 cells and non‐cancerous MCF‐10A cell line, quantitative PCR (qPCR) and flow cytometry analysis was done. Next, analysis of T2R4 and T2R49 in human breast cancer tissue panel was performed using qPCR array (Origene Inc.). Calcium mobilization experiment was pursued to analyze the functional response after stimulation of these cells using bitter compounds. Cell proliferation was pursued using MTT assay and migration analysis by RTCA xCELLigence cell system after stimulating the cells with T2R ligands.ResultsOur results suggest differential expression of T2Rs is breast cancer cells. Stimulation of endogenous T2Rs with bitter agonists leads to increase in intracellular calcium release. Breast cancer cells treated with T2R4 agonist show decrease in cell proliferation and migration compared to non‐cancerous cells. This effect was reversed upon T2R4 antagonist treatment. Furthermore, knockdown and/or overexpression studies of T2R4 in these cells suggest its role in inhibition of cell proliferation and migration of breast cancer cells.ConclusionsOur results suggest that T2Rs are involved in decrease of breast cancer cell proliferation and migration. Investigating the mechanistic role of these chemosensory T2Rs in breast cancer would aid in developing strategies to prevent and/or treat breast cancer.Support or Funding InformationThe Natural Sciences and Engineering Research Council of Canada (NSERC)
Analysis of Telomere Length in Aging and Age ‐Related Illness
Authors: Connon Scott M , Einstein George P , Tulp Orien L 
Published: November 2023

The purpose of this study was to create a detailed analysis of the factual concepts and theoretical principles of telomeres and their application in aging and disease. The analysis is based on the current genetic knowledge of the telomere and its main enzyme telomerase as well considerayion and evaluation of theoretical applications of telomere status. The assessment of telomere lengths were the main concepts being analyzed in this thesis. This area of genetics is being studied to explore its many benefits to understanding key functions of the human chromosome as it relates to wellness and disease during the lifespan. There are many challenges facing the advancement of telomere studies and its application to disease and wellness. For the purposes of this analysis the telomere length and its role in the immune system and aging were the focus of the meta analysis. A strong immune system and ability to aid in the prevention and proliferation of disease can theoretically be associated with the telomere. Free radical and oxidative stress of the cell may also be comprising factors. Cancer is one of the largest chalenges facing modern medicine today. There are many theoretical correlations between the cancer cell mortality and the length of the telomere. Additionally, the amount of telomerase can influence the life span of the cancer cell and making the cell immortal. A greater understanding of this aspect of the chromosome will encourage more research and testing of the telomere. Published data from meta‐analysis and research have shown interesting results in the correlation of decreasing telomere lengths with aging diseases and cancers. Further research may be extremely valuable to patients suffering from age associated disease and tumor related cancers.Support or Funding InformationSupported by Institutional Resources of USAT College of Graduate Studies and Einstein Medical Institute
Follicle Stimulating Hormone and Reproductive Aging
Authors: Pardue Avery , Dvoretz Illay , Wright Collette , Custard Alaura , Barbara Ella , Langat Diana 
Published: November 2023

Follicle‐stimulating hormone (FSH) is a member of the glycoprotein hormone (GPH) family. It is secreted by gonadotropes, which are found in the anterior pituitary gland. FSH is an important hormone for the production and development of gametes in both men and women. The hormone is a part of a cascade that begins with the release of gonadotropin‐releasing hormone that is released from the hypothalamus and stimulates gonadotropes in the pituitary gland. These stimulated gonadotropes release FSH and the luteinizing hormone (LH) into the bloodstream where they travel through the bloodstream to the gonads. In males, FSH stimulate the Sertoli cells to produce a ABP, or androgen‐binding protein, and inhibin. ABP keeps testicular testosterone concentrations elevated for spermatogenesis, while inhibin feeds back to the anterior pituitary to cease production of FSH. LH stimulates testicular Leydig cells to synthesize and secrete testosterone. In females, FSH stimulates ovarian follicular granulosa cells to continue to grow and divide, secrete estradiol, and become responsive to LH stimulation. LH stimulates ovarian theca cells to produce the androgen precursor converted to estradiol by granulosa cells. The LH surge triggers ovulation and LH stimulates progesterone synthesis and release by the corpus luteum during the luteal phase of the menstrual cycle. FSH binds to the FSH receptor (FSHR) to stimulate cellular responses. The FSHR is a G protein‐coupled receptor (GPCR) that possesses an exceptionally large N‐terminal ectodomain composed of leucine‐rich repeats. Glycoprotein hormones, which FSH is member, are identified by a common alpha‐ and hormone‐specific beta‐subunit heterodimeric structure. The alpha‐subunit peptide sequence and disulfide bond pattern are consistent throughout all glycoprotein hormones. The beta‐subunit is what dictates the function of the hormone and dictates binding to the cognate receptor. The alpha and beta subunits are folded into non‐globular, cystine knot structures with three loops extending from the core motif, which consists of three disulfide bridges. An additional peptide emerges from the beta subunit and wraps around the L2 loop of the alpha subunit in a manner similar to a seatbelt. The seatbelt loop possesses the hormone‐specific determinants of the FSH beta‐subunit. FSH binds to the FSHR. Current research is focused on the effect of FSH glycosylation variations on the activation of the receptor.Support or Funding InformationThis is a SMART Team project supported through the contributions of Dr. George R. Bousfield of Wichita State University, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy.