Life Sciences Commons^™

Mir-770-5p-Induced Cellular Switch To Sensitize Trastuzumab Resistant Breast Cancer Cells Targeting Her2/Egfr/Igf1r Bidirectional Crosstalk, Senem Noyan, Bala Gür Dedeoğlu

Turkish Journal of Biology

Background/aim: Studies highlighted the bidirectional crosstalk between the HER family members in breast cancer as resistance mechanism to anti-HER agents. Cross-signaling between HER2/EGFR and ER/IGF1R could play role in the development of resistance to therapeutics hence stimulating cell growth. To overcome this resistance, combined therapies targeting both pathways simultaneously have been proposed as an effective strategy. The involvement of miRNAs in resistance of targeted therapies like trastuzumab was demonstrated in recent studies. Hence the regulation of miRNAs in resistance state could reverse the cell behaviour to drugs. Previously we found that overexpression of miR-770-5p downregulated AKT and ERK expression through …

Theranostic Potential Of A Novel Aptamer Specifically Targeting Her2 In Breast Cancer Cells, Fulya Küçükcankurt, Samet Uçak, Nedret Altiok

Turkish Journal of Biology

Background/aim: The overexpression of HER2 is correlated with poorer outcomes and therapeutic resistance in breast cancer patients. While HER2-targeted therapies have shown improvement, prognosis remains poor for HER2-positive breast cancer patients, and these treatments have limitations. Therefore, it is crucial to explore effective molecular strategies for early detection and treatment of HER2-positive breast cancers. Materials and methods: In this study, we employed the cell-SELEX method to generate a selective aptamer capable of recognizing HER2 in its native conformation within breast cancer cells, for theranostic applications. Utilizing an adherent cell-SELEX approach, we developed and explored a DNA aptamer, named HMAP7, which …

Targeting Strategies To Optimize The Therapeutic Potential Of Gold Compounds Against Her2-Positive Breast Cancers, Afruja Ahad

Dissertations, Theses, and Capstone Projects

The overexpression of HER2 accounts for 20-30% of breast cancer tumors and not only serves as a marker for poor predictive clinical outcomes but also as a target for treatment. Antibody-drug conjugates (ADCs) combine the selectivity of monoclonal antibodies (mAbs) with the efficacy of chemotherapeutic drugs to provide targeted treatment without toxicity to normal tissue. Most of the ADCs currently in the clinic for cancer chemotherapy are based on complex organic molecules. In contrast, the conjugation of metallodrugs to mAbs has been overlooked when there is enormous potential in this area with the resurgence of metal-based drugs as prospective cancer …

9s1r Nullomer Peptide Induces Mitochondrial Pathology, Metabolic Suppression, And Enhanced Immune Cell Infiltration, In Triple-Negative Breast Cancer Mouse Model, Nilufar Ali, Cody Wolf, Swarna Kanchan, Shivakumar R. Veerabhadraiah, Laura Bond, Matthew W. Turner, Cheryl L. Jorcyk, Greg Hampikian

Biology Faculty Publications and Presentations

Nullomers are the shortest strings of absent amino acid (aa) sequences in a species or group of species. Primes are those nullomers that have not been detected in the genome of any species. 9S1R is a 5-aa peptide prime sequence attached to 5-arginine aa, used to treat triple negative breast cancer (TNBC) in an in vivo mouse model. This unique peptide, administered with a trehalose carrier (9S1R-NulloPT), offers enhanced solubility and exhibits distinct anti-cancer effects against TNBC. In our study, we investigated the effect of 9S1R-NulloPT on tumor growth, metabolism, metastatic burden, tumor immune-microenvironment (TME), and transcriptome of aggressive mouse …

Pyrrolidine Derivative Targets Actin Cytoskeleton In Mcf-7 Breast Cancer Cells, An Pham, Jeff Hansen, Sarah Mordan-Mccombs

Annual Student Research Poster Session

Recent research has brought pyrrolidine derivatives into consideration for the development of anticancer drugs with high efficacy and low toxicity. Dr. Hansen’s lab at DePauw has synthesized a pyrrolidine derivative that demonstrated anticancer activity. However, there are many ways a compound can affect cancer cells. In this research, we decided to investigate the mechanism of action of this new compound, specifically on MCF-7 breast cancer cell line. Based on the results, we believe that there is a great likelihood that the pyrrolidine derivative can induce apoptosis (cell death) and disrupt cell movement in MCF-7 cells. In other words, these are …

Soloxolone Methyl Induces Apoptosis And Oxidative/Er Stress In Breast Cancer Cells And Target Cancer Stem Cell Population, Eli̇f Ertürk, Oğuzhan Akgün, Yaren Yildiz, Pinar Kalkan, Oksana V. Salomatina, Nariman F. Salakhutdinov, Engi̇n Ulukaya, Ferda Ari

Turkish Journal of Biology

One of the most prevalent malignancies in women and one of the leading causes of cancer-related death is breast cancer. There is a need for new treatment approaches and drugs for breast cancer. Many studies show the high potential of triterpene compounds and their semisynthetic derivatives as anticancer agents due to their ability to induce apoptosis and suppress tumorigenesis. The effects of soloxolone methyl (SM), a semisynthetic derivative of 18-H-glycyrrhetinic acid, on the cytotoxicity and apoptosis of human breast cancer cell line (T-47D) and cancer stem cell (CSCs) population (mammospheres; CD44+/CD24-antigen) derived from breast cancer cells, were examined in this …

Pre-Clinical Trials With Precision-Medicine Based Therapeutics In Basal-Like Patient-Derived Xenografts, David C. Boyd

Theses and Dissertations

Breast cancer treatments have improved over time, but the diseases seeing the most benefit from these improvements have the estrogen receptor, progesterone receptor, or are positive for HER2. Basal-like breast cancer tends to not have these biomarkers, which necessitates their treatment to be traditional, untargeted therapeutics which are less effective and tend to have harsh adverse effect profiles – this is an important unmet need. These studies utilize a variety of techniques, including tissue culture, viability assays, high-throughput screening, in vivo drug treatments and imaging, pathway analyses, molecular techniques such as Western blot, antibody arrays, RNA sequencing, sc RNA sequencing, …

Mir-216b-5p Promotes Late Apoptosis/Necroptosis In Trastuzumab-Resistant Sk-Br-3 Cells, İştar Burcu Dolapçi, Senem Noyan, Ayşegül Yücel Polat, Hakan Gürdal, Bala Gür Dedeoğlu

Turkish Journal of Biology

Breast cancer is the most common cancer in women. The human epidermal growth factor receptor 2 (HER2) overexpressing subtype is related to poor prognosis with an aggressive phenotype and is reported as one of the most commonly seen subtypes. Trastuzumab is prevalently used as a treatment method for HER2+ breast cancer however, resistance to the drug frequently occurs following the treatment. MicroRNAs (miRNAs) are 19-23 nucleotide long small RNAs, which regulate gene expression at posttranscriptional level and studies show that there are differentially expressed miRNAs between drug sensitive and resistant groups, indicating that they might have some key roles in …

The Influence Of Administrative Timing In Triple-Negative Breast Cancer Treatments, Emily Mailloux

Electronic Theses and Dissertations

Breast cancer accounts for 25% of all cancers in Canadian women, and 15-20% of these are triple-negative breast cancers (TNBC), which have a poorer prognosis than other breast cancer subtypes. TNBC lacks expression of the estrogen receptor, progesterone receptor, and the human epidermal growth factor receptor 2 (HER2), which are common therapeutic targets in breast cancer. Due to the lack of target therapy, generalized chemotherapy treatments are used instead. The standard of care for treatment of TNBC instead consists of doxorubicin (A), cyclophosphamide (C) paclitaxel (T), and carboplatin (Carbo), that target various aspects of the cell cycle to induce cell …

Characterization Of The Cell Cycle Dependent Kinase Inhibitor P27kip1 In The Stem-Like Properties Of Breast Cancer Stem Cells, Jaz-Mine Mccovins

Theses (2016-Present)

Breast cancer is the second leading cause of cancer death among American women. Traditional therapies, such as chemotherapy and radiation therapy, target proliferative cells within the tumor bulk. However, these treatments do not provide long-term disease survival because they fail to eliminate a small population of long-lived tumor initiating cells known as breast cancer stem cells (CSCs). Two features of CSCs are that they slowly divide and are more resistant to conventional anti-cancer treatments due to altered cellular machinery. It is the breast CSC subpopulation that develops new tumors which have the potential to lead to recurrence. However, the mechanism …

Potentially Cis-Acting Super-Enhancer Long Noncoding Rnas As Biomarkers In Triple Negative Breast Cancer Progression, Kavipriya Kovai Palanivel

Biological Sciences

Triple negative breast cancer (TNBC) is defined by the lack of estrogen receptors (ER), the lack of progesterone receptors (PR), and normal human epidermal growth factor 2 (HER2) protein levels in breast tumors. Therefore, TNBC is an aggressive type of breast cancer due to limited treatment options for patients. Determining genes that regulate the progression of tumors to TNBC may help develop new therapeutic techniques. Many super-enhancer long noncoding RNAs (SElncRNAs) are found to be differentially expressed in cancer cells. Super-enhancer activity heavily influences target gene transcription and is often augmented through cis-acting SE-lncRNA activity. Therefore, we hypothesize cis-acting SE-lncRNAs …

The Comparison Of Differentially Expressed Micrornas In Bag-1 Deficient And Wild Type Mcf-7 Breast Cancer Cells By Small Rna Sequencing, Peli̇n Özfi̇li̇z Kilbaş, Gi̇zem Alkurt, Pinar Obakan Yerli̇kaya, Ajda Çoker Gürkan, Gi̇zem Di̇nler Doğanay, Eli̇f Damla Arisan

Turkish Journal of Biology

The multifunctional BAG-1 (Bcl-2 athanogene-1) protein promotes breast cancer survival through direct or indirect interaction partners. The number of the interacting partners determines its cellular role in different conditions. As well as interaction partner variability, the amount of BAG-1 protein in the cells could cause dramatic alterations. According to previous studies, while the transient silencing of Bag-1 enhanced drug-induced apoptosis, deletion of BAG-1 could induce stemness properties and Akt-mediated actin remodeling in MCF-7 breast cancer cells. Considering the heterogeneity of breast cancer and the variability of BAG-1 -mediated cell response, it has become essential to determine microRNA (miRNA) functions in …

The Role Of The Nkr-P1b:Clr-B Inhibitory Axis In Nk Cell-Mediated Tumour Immunosurveillance, Abd El Aziz Hendy

Electronic Theses and Dissertations

Natural killer (NK) cells are lymphocytes of the innate immune system which do not require prior priming to target infected or altered-self cells, such as tumour cells. The basic functions of NK cells include cytotoxicity and cytokine production. These cells possess numerous activating and inhibitory receptors which regulate their function based on a signalling balance. In mice, NK cells possess NKR-P1B inhibitory receptors which recognize the C-type lectin-related protein-b (Clr-b) ligand. This ligand is present on healthy self cells and may be used by cancer cells to evade NK cell effector functions. The disruption of the NKR-P1B:Clr-b axis has been …

The Impact Of Nanopulse Treatment On The Tumor Microenvironment In Breast Cancer: Overturning The Treg Immunosuppressive Dominance, Anthony Nanajian

Biomedical Sciences Theses & Dissertations

Nanopulse treatment (NPT) is a high-power electric engineering modality that has been shown to be an effective local tumor treatment approach in multiple cancer models. Our previous studies on the orthotopic 4T1-luc breast cancer model demonstrated that NPT ablated local tumors. The treatment consequently conferred protection against a second live tumor challenge and minimized spontaneous metastasis. This study aims to understand how NPT mounts a potent immune response in a predominantly immunosuppressive tumor.

NPT changed the local and systemic dynamics of immunosuppressive cells by significantly reducing the numbers of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages …

Investigation Of The Efficacy Of Paclitaxel On Some Mirnas Profiles In Breast Cancer Stem Cells, Eli̇f Ertürk, Ferda Ari, Oğuzhan Akgün, Engi̇n Ulukaya, Cem İsmai̇l Küçükali̇, Şaki̇r Ümi̇t Zeybek

Turkish Journal of Biology

Understanding of the functions of microRNAs in breast cancer and breast cancer stem cells have been a hope for the development of new molecular targeted therapies. Here, it is aimed to investigate the differences in the expression levels of let-7a, miR-10b, miR-21, miR-125b, miR-145, miR-155, miR-200c, miR-221, miR-222 and miR-335, which associated with gene and proteins in MCF-7 (parental) and MCF-7s (Mammosphere/stem cell-enriched population/CD44+/CD24-cells) cells treated with paclitaxel. MCF-7s were obtained from parental MCF-7 cells. Cytotoxic activity of paclitaxel was determined by ATP assay. Total RNA isolation and cDNA conversion were performed from the samples. Changes in expression levels of …

Endoplasmic Reticulum Stress And Oncomir-Associated Chemotherapeutic Drug Resistance Mechanisms In Breast Cancer Tumors, Leila Mehdizadehtapeh, Pinar Obakan Yerli̇kaya

Turkish Journal of Biology

Breast cancer, as a heterogenous malign disease among the top five leading causes of cancer death worldwide, is defined as by far the most common malignancy in women. It contributes to 25% of all cancer-associated deaths after menopause. Breast cancer is categorized based on the expression levels of cell surface and intracellular steroid receptors [estrogen, progesterone receptors, and human epidermal growth factor receptor (HER2)], and the treatment approaches frequently include antiestrogen, aromatase inhibitors, and Herceptin. However, the management and prevention strategies due to adverse side effects stress the patients. The unsuccessful treatments cause to raise the drug levels, leading to …

Pvt1 Exon 9 A Regulator Of Claudin Expression In Triple Negative Breast Cancer, Fayola Levine

Theses and Dissertations

Claudin low triple negative breast cancer (TNBC) has the worst clinical outcomes in comparison to other breast cancer subtypes. The underlying molecular mechanisms associated with the aberrant expression of claudins are not well understood. The transcript from PVT1 exon 9 is a regulator of claudin expression, and migration, in TNBC.

Phytochemical Content And Anti-Breast Cancer Activity Of Kentucky Native Plants., Nicole A. Mcgrath

College of Arts & Sciences Senior Honors Theses

Plants are a proven and rich source of chemical diversity for the development of pharmaceuticals. Plants that are native to Kentucky are an underexplored resource that could lead to advances in treating human diseases such as cancer. Kentucky native plants, especially those with highly diverse or enriched chemical compositions are possible leads for therapeutic drug development. To explore the potential of native Kentucky plants for treatment of breast cancer, a list of prospective target plants was developed based on literature reports. This list was narrowed to 10 tissues for further in-depth chemical analysis including estimation of alkaloids, terpenes, and phenolics, …

The Role Of Apkcs And Apkc Inhibitors In Cell Proliferation And Invasion In Breast And Ovarian Cancer, Tracess B. Smalley

USF Tampa Graduate Theses and Dissertations

Research has demonstrated that the atypical protein kinase C-zeta (PKC-ζ) is a component of many dysregulated pathways in breast and ovarian cancer, including cellular proliferation, survival, and cell cycle upregulation. Breast and ovarian cancers affect women every day and are second and fifth leading cause of cancer death. Women who seek treatments are commonly met with invasive surgeries or chemotherapy. Protein kinase C (PKC) is a family of serine and threonine phosphorylating kinases that have been shown to modulate and transduce signaling cascades that play roles in the development and survival of cancers. Atypical PKC (aPKC), have been heavily suggested …

Statin Drugs Plus Th1 Cytokines Potentiate Apoptosis And Ras Delocalization In Human Breast Cancer Lines And Combine With Dendritic Cell-Based Immunotherapy To Suppress Tumor Growth In A Mouse Model Of Her-2 Disease, Crystal M. Oechsle, Loral E. Showalter, Colleen M. Novak, Brain J. Czerniecki, Gary K. Koski

Biological Sciences Faculty Publications

A dendritic cell-based, Type 1 Helper T cell (Th1)-polarizing anti-Human Epidermal Growth Factor Receptor-2 (HER-2) vaccine supplied in the neoadjuvant setting eliminates disease in up to 30% of recipients with HER-2-positive (HER-2) ductal carcinoma in situ (DCIS). We hypothesized that drugs with low toxicity profiles that target signaling pathways critical for oncogenesis may work in conjunction with vaccine-induced immune effector mechanisms to improve efficacy while minimizing side effects. In this study, a panel of four phenotypically diverse human breast cancer lines were exposed in vitro to the combination of Th1 cytokines Interferon-gamma (IFN-γ) and Tumor Necrosis Factor-alpha (TNF-α) and lipophilic …

Single Nucleotide Polymorphisms In Micrornas Action As Biomarkers For Breast Cancer, Thanh Thi Ngoc Nguyen, Minh Thi Hong Tran, Vy Thi Lan Nguyen, Uyen Doan Phuong Nguyen, Giang Dien Thanh Nguyen, Luan Huu Huynh, Hue Thi Nguyen

Turkish Journal of Biology

MicroRNAs (miRNAs) have been recently described as small noncoding RNAs that are involved in numerous crucial physiological processes, such as cell cycles, differentiation, development, and metabolism. Thus, dysregulation of these molecules could lead to several severe disorders, including breast cancer (BC). Ongoing investigations in malignant growth diagnostics have distinguished miRNAs as promising disease biomarkers. As with any other mRNAs, single nucleotide polymorphisms (SNPs) in DNA sequence encoding for miRNA (miR-SNPs) indeed lead to potential changes in the function of miRNA. In this study, miR-SNPs located in different miRNA sequence regions, which have been associated with BC in different ways, and …

Transcriptional Splice Variants Of Cd40 And Its Prognostic Value In Breast Cancer, Neşe Ünver, Di̇ğdem Yöyen Ermi̇ş, Bahar Zehra Weber, Güneş Esendağli

Turkish Journal of Biology

CD40 is an important tumor necrosis factor receptor (TNFR) family protein for the development of antitumor response against cancer cells, apart from its role in the regulation of the immune system as a costimulatory molecule. It is broadly expressed on the surface of immune cells and in diverse cancer types, including breast cancer. Here, we analyzed both CD40/CD40 ligand expression in breast cancer cells and tissues using public data sets and overall survival analysis in ungrouped breast cancer patients, as well as in the triple-negative breast cancer subtype. We detected CD40 gene expression along with its 3 different splice variants …

Ubiquitin-Specific Protease 7 Downregulation Suppresses Breast Cancer In Vitro, Taha Bartu Hayal, Ayşegül Doğan, Hati̇ce Burcu Şi̇şli̇, Bi̇nnur Kiratli, Fi̇kretti̇n Şahi̇n

Turkish Journal of Biology

Because breast cancer is complicated at the pathological, histological, clinical, and molecular levels, identification of new genetic targets against carcinogenic pathways is required to generate clinically relevant treatment options. In the current study, ubiquitinspecific protease 7 (USP7), which regulates various cellular pathways including Mdm2, p53, and NFκB, was selected as a potential gene editing strategy for breast cancer in vitro. Anticancer activity of USP7 gene suppression has been evaluated through cell proliferation, gene expression, cell cycle, sphere dissemination, and cell migration analysis. Here, siRNA and shRNA strategies and an allosteric small-molecule inhibitor of USP7 were used to define potential anticancer …

Akt-Mediated Phosphorylation Of Twist1 Is Essential For Breast Cancer Cell Metastasis, Mustafa Gökhan Ertosun, Suray Pehli̇vanoğlu, Sayra Di̇lmaç, Gamze Tanriöver, Osman Ni̇dai̇ Özeş

Turkish Journal of Biology

Previously, it was shown that human TWIST1 (basic helix-loop-helix (b-HLH) is phosphorylated by Akt kinase at S42, T121, and S123. To show in vivo effect of these phosphorylations, we created mouse TWIST1 expression vector and converted the codons of S42, T125, and S127 to unphosphorylatable alanine and phosphorylation mimicking Glutamic acid. We hypothesized that alanine mutants would inhibit the metastatic ability of 4T1 cells while glutamic acid mutants would convert nonmetastatic 67NR cells into metastatic phenotype. To confirm this hypothesis, we created metastatic 4T1 and nonmetastatic 67NR cells expressing alanine mutants and glutamic acid mutants mouse TWIST1, respectively. Then, we …

Relationships Of Protein Biomarkers Of The Urokinase Plasminogen Activator System With Expression Of Their Cognate Genes In Primary Breast Carcinomas., Seth B. Sereff

College of Arts & Sciences Senior Honors Theses

Background: Urokinase plasminogen activator (uPA), its receptor uPAR and serine protease inhibitors PAI-1 or PAI-2 play key roles in tissue membrane remodeling and invasion of basement membranes by induction of a fibrinolytic pathway. Earlier studies reported that uPA and PAI-1 protein levels in breast carcinomas assist in prediction of response to chemotherapy. Our goal is to develop molecular signatures of candidate genes and identify novel relationships with these four protein biomarkers that demonstrate clinical utility for assessment of breast carcinoma outcomes.

Methods: This retrospective study used de-identified biomarker results and clinical outcomes from primary breast cancers that were stored in …

Deciphering The Molecular Mechanism Through Which Rhus Coriaria Exerts Its Anti-Cancer Activity, Khawlah Najib Saeed Athamneh

Dissertations

Cancer remains a major health problem around the globe. Among various types of treatments, plants have been shown to have great capacity in cancer treatment, one of which is Rhus coriaria. Commonly known as sumac, Rhus coriaria is a culinary herb that is known to possess different therapeutic values including anti-oxidant and anti-microbial activities.

In this Ph.D. project, we tested the effect of Rhus coriaria extract (RCE) on the migration, invasion, and metastasis of MDA-MB-231 human breast cancer cells. We showed that non-cytotoxic concentrations of RCE inhibited migration and invasion, blocked adhesion to fibronectin, and downregulated MMP-9. Additionally, we found …

Utilizing Immunopet To Measure Tumor Response To Treatment In Breast Cancer, Brooke Mcknight

Wayne State University Dissertations

With a broad spectrum of therapies available for treating breast cancer, the need for personalized medicine tailoring the cure according to phenotype is evident. Such an approach may be fully realized with the development of quantitative imaging technologies for disease detection, staging and diagnosis, without increasing patient burden. Immuno-positron emission tomography (PET) combines the targeted specificity of antibodies with the sensitivity of PET for whole body imaging by targeting molecular features amplified in lesions. ImmunoPET probes targeting different antigens and their utility to measure response to treatment were explored. 89Zr-trastuzumab was employed as a surrogate readout of Src inhibition after …

Response Of Breast Cancer Cells To Ifnî±-2b In 2d And 3d Cell Cultures, Tetiana Herheliuk, Olena Perepelytsina, Andrij Ugnivenko, Lyudmila Ostapchenko, Mychailo Sydorenko

Turkish Journal of Biology

The effect of IFNα-2b on the migration, proliferation, and expression of epithelial and mesenchymal markers of MCF-7 tumor adenocarcinoma cells in 2D and 3D cell cultures was examined. A significant cytostatic effect of IFNα-2b on the tumor population was detected. It was found that changes in the expression of epithelial (CKs and EpCAM) and mesenchymal markers were caused by changing the growth type of the tumor population. IFNα-2b inhibited migration of tumor cells to the suspension fraction and promoted an increase in expression of CK and EpCAM in 2D and 3D cell cultures, but only in the 3D culture was …

Effects Of Notch Signalling On The Expression Of Sema3c, Hmga2, Cxcl14, Cxcr7, And Ccl20 In Breast Cancer, Cansu Küçükköse, Özden Yalçin Özuysal

Turkish Journal of Biology

Metastasis is the main reason for death in breast cancer. Understanding the molecular players in metastasis is crucial for diagnostic and therapeutic purposes. Notch signalling plays an oncogenic role in breast tumorigenesis and is involved in metastasis. Downstream mediators of Notch signalling in prometastatic processes are not yet fully discovered. Here we aimed to investigate whether Notch signalling regulates the expression of SEMA3C, HMGA2, CXCL14, CXCR7, and CCL20, which are involved in prometastatic processes, in breast cell lines. To this end, expression of the selected genes was analysed following Notch activation by overexpression of the Notch1 intracellular domain in the …

E2f6 Is Essential For Cell Viability In Breast Cancer Cells During Replication Stress, Inam Lafta

Turkish Journal of Biology

E2F6 is a member of the E2F family of transcription factors involved in regulation of a wide variety of genes through both activation and repression. E2F6 has been reported as overexpressed in breast cancers but whether or not this is important for tumor development is unclear. We first checked E2F6 expression in tumor cDNAs and the protein level in a range of breast cancer cell lines. RNA interference-mediated depletion was then used to assess the importance of E2F6 expression in cell lines with regard to cell cycle profile using fluorescence-activated cell sorting and a cell survival assay using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide …