Email to a Friend

Posts tagged 'Cancer'

RSS Feed

Cancer Research (White Paper)

By Bioline 8 March 2016

Cancer Research

Cancer cells represent the manifestation of a breakdown in any number of normal cell states involving a wide range of cellular processes and cell types, thus the complexity of this disease cannot be understated. Mutations or changes anywhere from somatic DNA, through to active stem cells, as well as epigenetic changes, and environmental factors can all contribute to the resulting disease, and cancer research areas are just as diverse as these contributing factors. The range of reagents, kits and assays from Bioline support all aspects of cancer research, including oncogenes and genetic markers, epigenetic changes, as well as biomarkers and personalised medicine approaches.

Cancer - a mixed bag of disease states
Our bodies are composed of a broad range of cell types that grow, divide, and function in a controlled manner - interacting with other cells to form organised matrices, differentiating into specific types of cells where required, and breaking down when damaged or no longer needed. The processes that control normal cell activity are complex and multi-level, and mutations in any number of them can lead to cancer – the continual, unregulated proliferation of cells that grow, reproduce and eventually migrate throughout the body.

There are over 200 different types of known cancers, classified based on the type of cell from which they arise, falling into three main groups:

  • Carcinomas – malignancies of epithelial cells, representing over 90% of known cancers and manifesting as abnormal growth of tissue (tumours).

  • Leukemias/Lymphomas – produced from blood-forming cells and cells of the immune system, accounting for roughly 8% of human malignancies, supressing normal blood cell production and immune system function.

  • Sarcomas – solid tumours of connective tissues such as muscle or bone, these cancers are relatively rare in humans.

Further classification is based on the type of cell involved and the tissue of origin – for example erythroid leukemias are precursors of erythrocytes. The four most common cancers are breast, prostate, lung and colon, accounting for more than 50% of all cancer cases.

Cancer – mutations within basic cellular processes
The genes within a genome hold the blueprint for creation of the protein molecules that perform many of the important functions of a healthy cell. Mutations within this blueprint have the potential to affect protein production and resulting cellular function. Bioline offers an excellent range of end-point and Real Time PCR reagents and kits to support research into the genomics of cancer and cancer-related mutations. Usually a number of mutations are required before a cell exhibits cancerous properties, typically involving abnormalities in the mechanisms that control cell proliferation, differentiation and survival.

A primary hallmark of cancerous cells is their ability to sustain chronic cell proliferation – growing to high cell densities and larger cell sizes, with limited spatial regulation. Normal cells exhibit density-dependent growth and proliferation, controlled by complex pathways of growth-promotion and inhibitory signalling. There are a number of ways through which cancer cells can bypass normal proliferative mechanisms including producing their own growth-factor ligands, stimulating neighbour cells to produce various growth factors, or by altering cell surfaces to become hyper-responsive to growth factors. Research in this area looks at a number of different aspects of cell-growth mechanisms, such as mutations that lead to disruptions in the negative feedback mechanisms that reduce proliferative signalling, or those that aid in the evasion of growth suppressors, as well as somatic mutations that activate downstream pathways involved in cell proliferation.

All normal cells are subject to a process called apoptosis, the programmed cell death process that occurs in response to the absence of growth factors or other environmental stimulations, or as a result of DNA damage. The ability to evade apoptotic processes is another hallmark of cancerous cells, increasing their lifespans and significantly contributing to tumour growth. Upstream regulators and downstream effector components contribute to the apoptotic machinery of the cell environment and consist of both extra- and intra-cellular signalling pathways. There are a number of ways through which tumour cells circumvent or limit apoptosis, such as increasing expression of antiapoptotic regulators, downregulating proaptotic factors, or through the total loss of tumour suppressor genes.

Tissue Invasion and Metastasis
Cell-cell interactions and the phenomenon of contact inhibition control the orderly way in which normal cells grow, migrate and adhere to each other to form a healthy cell matrix. In contrast, cancerous cells continue migration regardless of cell contact, growing in multi-layered and disorderly patterns. In addition, many malignant cells secrete proteases to digest the components of the extracellular matrix and enable invasion of adjacent tissue. Another hallmark of cancer cells is the ability to promote the formation of new blood vessels – angiogenesis – required to supply much needed oxygen and nutrients to the proliferating tumour. The activation of this angiogenic switch can be related to factors that either induce or oppose angiogenesis, and most likely a number of countervailing factors. The ability to metastasise, that is to migrate or spread to another part of the body not directly connected with the original tumour, involves a complex invasion-metastasis cascade that is still being understood, and includes mechanisms that both allow for physical dissemination from the primary tumour, as well as adaptation to the foreign tissue environment at the secondary destination.

Cancer research – genetic predisposition through to personalized medicine
Bioline offers reagents that support all areas of cancer research, from individual reagents and kits for PCR and Real Time PCR, through to fully optimised custom designed assays. Real Time PCR Assay panels of a full range of miRNA targets associated with cancer-related processes are also available to assist researchers in identifying appropriate targets for further study, and ISO13485 manufactured reagents provide the required quality for developing assays for personalised medicine approaches.

Oncogene/tumour suppressor research
Oncogenes can be defined as any gene or gene cluster, typically involved in an important cellular function such as differentiation, proliferation or apoptosis, which can turn a normal cell cancerous under specific circumstances. Since the completion of the Human Genome Project and the continued progression of deep sequencing approaches for a range of tumours and cancer types, there has been much hope that identification of key oncogenes would become evident, and once identified, ways in which to counter their activation could be devised. What has instead come to light is an unpredicted level of diversity both within and between cancers that have been sequenced thus far. Mutations in several hundred different genes have been identified as drivers of cancer, and while data is still being gathered, no clear candidates for subsequent treatment development have emerged thus far, so research efforts continue.

Epigenetic cancer research
The presence of a gene, or indeed a gene mutation, does not necessarily result in subsequent translation of an affected protein. Physiological or phenotypic variations can be observed in genetically identical cells that contain, for example, differences in DNA methylation or histone modifications, properties that alter the transcriptional potential of a cell. Epigenetic changes can occur as a result of environmental influences, they are heritable as well as reversible, and epigenetic changes contribute to carcinogenesis, thus studies in cancer epigenetics are thriving and this area holds promise for the development of cancer treatment options.

Tumour microenvironment research
The tumour, or cancer microenvironment is the cellular environment within which a tumour exists, comprised of immune cells, fibroblasts, blood vessel cells, as well as the proteins produced by these non-cancerous cells that support the growth of cancer cells. It is difficult to dissociate the microenvironment from traditionally defined cancer cells, however data suggests that dysfunction within the microenvironment is linked to carcinogenesis, thus understanding the pathophysiology of the microenvironment is a path towards development of chemopreventive agents. Research in this area attempts to understand the dynamic and reciprocal interactions between tumour cells and the cells that orchestrate their growth, metastatic properties or drug resistance progression.

Cancer biomarkers and personalised medicine
Biomarkers are biological molecules, such as proteins or nucleic acids, which are found in body fluids or tissues and can be used to assess the state of a biological process, or differentiate normal from disease state. A wide range of biomarkers for cancer have been identified across the full spectrum of cellular processes involved in the disease, and these biomarkers can be used to estimate risk towards developing disease, screen for active disease, determine disease prognoses, and both predict response to therapy and monitor therapeutic responses. There are many researchers committed to unravelling the immense complexity of biological and cellular information that has been collected thus far across the 200-odd diseases that are collectively known as cancer. Insights into this diversity and individual correlations within disease state form the foundation of biomarker development and subsequent personalised medicine. Today almost half of the cancer medicines and treatments in development are linked to small-molecules or novel biologic agents that have been identified as biomarkers for disease, and this figure is only likely to increase as research continues in this area.


MicroRNAs and Their Role in Personalised Medicine

By Bioline 9 January 2016

MicroRNAs and Their Role in Personalised Medicine

Using mRNA qPCR panels as a tool to understand cancer

Circulating miRNAs are attracting interest in the burgeoning field of personalised medicine, with data supporting their diagnostic, prognostic and predictive biomarker potential. Effective miRNA profiling calls for reproducible, sensitive and specific tools with turn-around times fast enough to support investigations into what can be a rapidly changing disease progression and treatment environment. Introducing the latest miRNA RT-qPCR technology from Bioline, the EPIKTM Cancer miRNA Panel, offering sensitive SYBR® Green-based detection for 352 targets, including the most differentially expressed miRNAs and controls, for convenient, robust, and extremely specific miRNA analysis.

MicroRNAs in Cellular Processes and Disease

Since their discovery a little over 20 years ago1, miRNAs, previously overlooked within what was thought to be non-functional genome components, are now understood to be crucial regulators of important cellular functions2. The biogenesis of miRNA follows a complex path through a number of precursor forms resulting in the mature, single stranded miRNA which is about 20-25 nucleotides3. Mature miRNA interact with mRNA effecting post-translational gene regulation of cellular processes such as development, differentiation, proliferation, metabolism and apoptosis2, thus it is no surprise that aberrantly expressed miRNAs are a hallmark in many diseases, including cancer4. Recently, miRNAs were included within the traditional oncogene definition due to their vital role of controlling cell differentiation, proliferation and survival3, plus their role in the negative regulation of tumour suppressor genes is clearly evident4, and now over 12,600 publications are listed in the NCBI PubMed database relating miRNA with cancer5. Improvements in deep sequencing technology have allowed for genome-wide profiling of miRNA expression, revealing cancer-specific signatures that not only discriminate between cancer types with high accuracy, but identify tissues of origin in metastasised cancers4 – thus miRNA profiling as a cancer diagnostic became an attractive concept for development. Further, the highly regulated process of miRNA expression is sensitive to internal and external stimuli such as hormones, pharmacological molecules etc., leading to a unique miRNA profile within different tissue types, locations and time points, making them ideal candidates for prognostic and therapeutic oncology biomarkers5.

Liquid Biopsies and miRNA profiling

There is much excitement around the concept of ‘liquid biopsy’ – the ability to screen, monitor, and uniquely characterise tumours from, for example, a simple blood or plasma sample, foregoing the traditionally invasive, costly, and in many cases difficult to obtain tissue biopsy6. Although circulating cell-free tumour DNA (ctDNA) and circulating tumour cells (CTCs) are commonly the focus for these methods, circulating miRNAs are also attracting attention as viable candidates. Not only do they carry specific information about the patho-physiological state of an individual, miRNAs are remarkably stable in their protein-bound form and are present in cell-free body fluids such as plasma, serum, urine and saliva7. Correlations have been observed between specific circulating miRNAs and chemotherapy responses in a range of cancers5, and retrospective studies have also begun to identify miRNA signatures with strong predictive and prognostic potentials8. There are still many hurdles to overcome before miRNA profiling and liquid biopsy become mainstream diagnostic practice, not the least of which is establishing robust and reproducible protocols, in addition to identifying, among the growing list of candidates, which combination of targets can be linked to clinical relevance. Indeed, the very specificity with which cells express miRNA and the subsequent sensitivity to a range of stimuli, including age and gender, while attractive qualities for personalised medicine, make for a difficult moving target for applied research5.

Molecular Tools for miRNA profiling

Expression profile studies based on microarray platforms or large scale deep sequencing projects have been instrumental in the discovery and identification of miRNAs, serving to expand the current database to almost double the number of known human miRNAs in the past five years. Among the growing number of tools available for studying miRNAs, however, qPCR remains a routine favourite as it has potential to be extremely sensitive and accurate as well as being accessible to many in terms of access to instrumentation as well as overall cost. Notwithstanding the range of sample collection and extraction methods with all associated caveats, there are many challenges to overcome when applying RT-qPCR to miRNA. Targets are short, 18-22nt, as well as being highly homologous, often with as little as 1-2nt differences, in addition the mature miRNA target sequence is present in all the precursor forms9. EPIKTMmiRNA Panels are the latest release from Bioline that overcome these challenges and more, the unique assay design discriminates between even the most closely related miRNA targets, such as those within the let-7 family, with maximum sensitivity down to 10pg of total RNA, enabling low volume starting materials, such as required for blood and plasma samples and other liquid biopsy candidates10.

Advanced Design Concepts for miRNA qPCR Panels

There are many ways to overcome the difficulties of miRNA priming for RT-qPCR, many of which involve universal priming steps and additions of long tail nucleotides, resulting in a reduction of qPCR efficiency and target specificity9. EPIKTM miRNA Panel assays forego the universal priming approach and incorporate a unique 3-primer system, all of which are miRNA specific, including the initial RT stem-loop primer, allowing for clear discrimination of mature miRNAs as well as superior specificity to identify very closely related targets10. Despite the use of specific primer sets, the overall protocol is efficiently streamlined, less than 2 hours of instrument run-time leads to interpretation of profile changes in less than half a day, capitalising on the speed advantage that qPCR holds over sequencing or microarray approaches, and answering the turn-around time requirements of personalised medicine. To focus study efforts on the most differentially expressed miRNA candidates, panel design draws on a bioinformatics mega-study in collaboration with MiRXes, screening thousands of miRNA and cancer related articles and incorporating the top 340 targets. Additionally, assay performance is enhanced to ensure robust quantification over a 7-log linear dynamic range, covering low and high expressed targets within a single run10. In deference to the rising costs of laboratory technology, the RT step is linked to SYBR® Green detection, negating the use of sequence specific probes while maintaining specificity with the three specific primer approach.

As efforts continue towards the transition of miRNA profiling from bench to bedside, RT-qPCR panels offer an ideal tool to support current applied research and the resulting clinical processes. The latest technology in the field is represented by the strong assay design of the EPIKTM Cancer miRNA Panel, covering the most useful targets to monitor disease states, and offering the performance and sensitivity required for low level starting materials or liquid biopsy applications.


  1. Lee, R. C., Feinbaum, R. L., Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 75: 843-854 (1993).
  2. He, L. & Hannon, G. J. (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nature Reviews Genetics 5, 522–531
  3. MacFarlane, L-A., & Murphy, P. R. (2010) MicroRNA: Biogenesis, Function and Role in Cancer. Current Genomics 11: 537-561
  4. Lorio, M. V. & Croce, C. M. (2012) MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Molecular Medicine 4: 143–159
  5. Saumet, A., Mathelier, A. & Lecellier, C-H. (2014) The Potential of MicroRNAs in Personalized Medicine against Cancers. BioMed Research International 2014 (Article ID 642916): 10 pages
  6. Karachaliou, Niki et al. (2015) Real-Time Liquid Biopsies Become a Reality in Cancer Treatment. Annals of Translational Medicine 3(3):36.
  7. Madhavan, Dharanija et al. (2013) Cancer Diagnosis and Prognosis Decoded by Blood-Based Circulating microRNA Signatures. Frontiers in Genetics 4
  8. Boeri M, et al. (2011) MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proceedings of the National Academy of Sciences USA 108: 3713-3718
  9. Chen, C. et al. (2011) Quantitation of microRNAs by real-time RT-qPCR. Methods in Molecular Biology 687: 113-134.
  10. Wan, G., Qing ‘E. L, & Too, H-P. (2010) High-Performance Quantification of Mature microRNAs by Real-Time RT-PCR Using Deoxyuridine-Incorporated Oligonucleotides and Hemi-Nested Primers. RNA 16(7): 1436–1445.
  11. Saumet, A., Mathelier, A. & Lecellier, C-H. (2014) The Potential of MicroRNAs in Personalized Medicine against Cancers. BioMed Research International 2014 (Article ID 642916): 10 pages
  12. Karachaliou, Niki et al. (2015) Real-Time Liquid Biopsies Become a Reality in Cancer Treatment. Annals of Translational Medicine 3(3):36.

Madeline O’Donoghue (Application Scientist), Simon Baker (Senior Director of R&D), Bioline Reagents

MyTaq™ - How to Take Your PCR From 11 Hours to TWO

By Bioline: The PCR Company 14 June 2012 No comments

Fides LayWe caught Fides Lay in the lab on the day of a basketball game between UCLA and crosstown arch-rival USC. As a former UCLA undergrad now working on her PhD at USC, she never wants to miss a game between the two. Unfortunately, the long hours in the lab don’t always allow her to be home in time to watch. At least, that’s how it used to be for her. Thanks to Bioline’s MyTaq HS Mix, now she doesn’t miss a game.

In Peter Jones’ lab at USC, Fides is part of a team studying the epigenetic regulation of cancer. And as anyone who ever worked on epigenetics knows, you need to be a master of PCR to get any results at all. When you need to amplify bisulfite converted DNA to measure methylation status, you work with small samples that often have been digested with several enzymes, with DNA that is fragmented and damaged.

For the longest time the lab amplified bisulfite converted DNA with a tedious protocol; tedious because of the long set-up with using Taq from a previous supplier, DMSO, and other components added one at a time, and extremely slow cycling with a PCR protocol that was 7-11 hours long.

Anything could go wrong at any time, and the result was impossible to predict. Simply using a different thermocycler with a different ramping speed could cause the reaction to fail. And once you found out, it would be too late to fix because every new attempt takes a whole other day. Since the lab usually clones and sequences the PCR fragments, there was always still the risk that no clones would have inserts. Hard to predict, and that meant starting over.

All that changed when Fides first tried Bioline’s MyTaq HS Mix, an easy, all-in-one mix that contains the enzyme, dNTPs, buffer and all optimizers. There’s no need to add any DMSO, it works right away on almost all templates. And it's fast! Reactions are done in less than two hours, even on bisulfite converted DNA, with highly consistent results, and always with nice bands. The PCR products are much easier to clone, and on the rare occasion that something does go wrong, there’s still time to redo the experiment AND get the samples off to pyrosequencing the same day.

Now Fides has time to run multiple experiments and redo anything that goes wrong, all in time to get home, kick up her feet and watch the game.

Bioline Scholar Monthly: April 2012 Roundup – Breast Cancer

By Bioline: The PCR Company 8 May 2012 No comments

Focus on Breast Cancer

For years scientists have been treating breast cancer as a single disease. However, a new landmark study published in Nature has reclassified breast cancer into ten separate sub-diseases based on their genetic fingerprint. The culmination of decades of research, the study is the largest global study of breast cancer tissue ever performed.

The team, led by the British Columbia Cancer Center in Canada and the Cambridge Cancer Research Institute in the UK, used genome-wide microarrays to analyze the DNA and RNA of 2,000 tumor samples taken from women diagnosed with breast cancer. This huge pool of genetic information (copy number variants, SNPs and gene expression data), as well as survival data, allowed researchers to spot new and previously unacknowledged patterns for ten subtly different cancers that have, historically, been considered as one.

The challenge now is to understand the genetic drivers behind these newly discovered breast cancer variants and to develop new targeted therapies in the future. It could also lead to women with the best prognosis being spared side-effects of chemotherapy. The classification system will likely also form the basis for newer and better ways to diagnose and manage the disease.

Bioline offers a number of reagents that have helped further the study of cancers and, more specifically, breast cancer. So this edition of Bioline Scholar Monthly focuses on the use of Bioline reagents and kits in the field of breast cancer research.

SensiMix SYBR & Fluorescein Kit

In a diverse cohort of breast cancer patients with a 1–5 year tumor relapse versus those with up to 7 years relapse-free survival, RNA was extracted and subjected to microarray and real-time RT-PCR analysis. Among the 299 genes, five genes which included B cell response genes were found to predict with >85% accuracy relapse-free survival. Real-time RT-PCR confirmed the 5-gene prognostic signature that was distinct from an FDA-cleared 70-gene signature of MammaPrint panel and from the Oncotype DX recurrence score assay panel.

Ascierto, L. M., et al. Breast Can. Res. Treat. 131(3):871-880 (2012) - A signature of immune function genes associated with recurrence-free survival in breast cancer patients.

TRIsure reagent

MicroRNAs (miRNAs) are noncoding RNAs that function as key posttranscriptional regulators of gene expression. This paper found that BRCA1 recognizes the RNA secondary structure and directly binds with primary transcripts of miRNAs via a DNA-binding domain. The findings indicate novel functions of BRCA1 in miRNA biogenesis, which may be linked to its tumor suppressor mechanism and maintenance of genomic stability.

Kawai S. and Amano A. J. Cell Biol. 197 (2):201-208 (2012) - BRCA1 regulates microRNA biogenesis via the DROSHA microprocessor complex.


The bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P4) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. The magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P4 may have an important role in breast cancer progression.

Long J. S., et al. J. Biol. Chem. 285:35957-35966 (2010) - Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells.

SensiMix SYBR No-ROX Kit

CD44, the transmembrane receptor for hyaluronan, is implicated in tumor cell invasion and metastasis. The expression of CD44 and its variants is associated with poor prognosis in breast cancer. This paper investigated the effect of silibinin (a polyphenolic flavonolignan of the herbal plant of Silybum marianum, milk thistle) on the epidermal growth factor (EGF) ligand-induced CD44 expression in human breast cancer cells. The results suggest that silibinin prevents the EGFR signaling pathway and may be used as an effective drug for the inhibition of metastasis of human breast cancer.

Kim S., et al. Anticancer Res. 31(11): 3767-3773 (2011) - Silibinin Suppresses EGFR Ligand-induced CD44 Expression through Inhibition of EGFR Activity in Breast Cancer Cells.

Human papillomavirus (HPV) and Epstein Barr virus (EBV) have been found in breast carcinomas around the world. In this study, fifty-five BCs from Chile were analyzed for HPV and EBV presence. In addition, HPV- 16 viral load/physical status and E6/E7 expressions were determined. The results suggest that it is unlikely that HPV and/or EBV play a direct role in the etiology of breast carcinomas.

Aguayo F., et al. Infectious Agents and Cancer 6:7 (2011) - Human papillomavirus and Epstein-Barr virus infections in breast cancer from chile.

cDNA Synthesis Kit

This study suggests that melatonin may play a role in the desmoplastic reaction in breast cancer through a down regulatory action on the expression of antiadipogenic cytokines, which decrease the levels of these cytokines. Lower levels of cytokines stimulate the differentiation of fibroblasts and decrease both aromatase activity and expression, thereby reducing the number of estrogen-producing cells proximal to malignant cells.

Alonso-González C., et al. J. Pineal Res. 52(3): 282–290, (2012) - Melatonin interferes in the desmoplastic reaction in breast cancer by regulating cytokine production.

Melatonin reduces the development of breast cancer interfering with oestrogen-signalling pathways, and also inhibits aromatase activity and expression. This study shows that melatonin inhibits aromatase activity and expression by regulating the gene expression of specific aromatase promoter regions. A possible mechanism for these effects would be the regulation by melatonin of intracellular cAMP levels, mediated by an inhibition of cyclooxygenase activity and expression.

Martínez-Campa C., et al. British J. Can. 101: 1613–1619 (2009) - Melatonin inhibits aromatase promoter expression by regulating cyclooxygenases expression and activity in breast cancer cells.


Bisphenol A (BPA) has long been suspected to promote carcinogenesis, but the high doses of BPA used in many studies generated conflicting results. This paper shows that BPA at environmentally relevant doses reduces the efficacy of chemotherapeutic agents. These data provide considerable support to the accumulating evidence that BPA is hazardous to human health.

LaPensee E. W., et al. Environ Health Perspect. 117(2): 175–180 (2009) - Bisphenol A at Low Nanomolar Doses Confers Chemoresistance in Estrogen Receptor-a–Positive and –Negative Breast Cancer Cells.

BIOTAQ DNA Polymerase

This paper indicates that decreased CDH1, CDH13 and TIMP3 with increased CD44 gene expression levels can be used as an indicator for invasion in both ER-positive and ER-negative breast tumors. In double-negative tumor tissues, CD44 can be considered a marker for aggressive properties. However, additional assays in a larger series of patients with long follow up will be necessary to confirm these results of gene expressions in ER-positive and ER-negative tumors and their relationship with HER2 and ESR1.

Celebiler A., et al. Can. Sci. 100: 2341–2345. (2009) - Predicting invasive phenotype with CDH1, CDH13, CD44, and TIMP3 gene expression in primary breast cancer.

^ Back to Top