Exosome as Nanoscale Vesicles | Pancreatic Cancer Research

Exosome as Nanoscale Vesicles Pancreatic Cancer ResearchAbstractExosome ar nanoscale vesicles that argon released from flowerpotcer carrels, playing an important role in the microenvironment of crab louseous tumour cellular telephones.6,8 The exosomal vesicles (EV) contribute to the progression and growth of the tumour, and can be tushed exploitation gold nanoparticles (GNP).1,6,7 The exosomes are isolated from the strain plasma, and with their stability in bodily fluids, they can be targeted and inhibited using GNP. The GNP exit inhibit the cancer cell, and will backtrack tumour growth and production. Using proteomic and quantitative methods, the exosome EphA2, generateed the highest signal detection of specificity and sensitivity in pancreatic cancer patients.1,2,4,8 Further turn ups confirmed that EphA2-EV has electric electric potential in previous(predicate) detection for pancreatic cancer, due to the levels specificity and sensitivity being high in comparison to pancreatitis patients and the control patients.1KeywordsBiomarkers, Exosome, Pancreatic cancer, Nanoparticle, Proteomics, Vesicles, Plasma, AntibodyIntroductionPancreatic cancer, is vastly terminal, with a survival rate of less than 5%. Pancreatic ductal adenocarcinoma (PDAC), is the roughly common form of exocrine pancreatic cancer, accounting for around 95% of pancreatic cancer cases.10 PDAC is a silent cancer, and with the lack of mental testinging, the need for novel biomarkers to embolden in early detection is imperative. With no valid early detection methods, and no symptoms of early stage PDAC, the cancer will progress rapidly passim the body before it is detected. There is need to find a biomarker, aiding in the early detection PDAC, so that a treatment can be provided to stop the cancer from progressing. The current tumour biomarker, CA19-9, is non reliable in early detection due to falsely elevated results of diseases other than PDAC.10Recent studies suggest that exosomes, a nanovesicle, has a high potential as the future biomarker of PDAC, due to the stability and detection sensitivity in human gunstock plasma.1,2,4,8 Malicious exosomes, are veil by cancer cells, screening the importance in tumour matu balancen and progression.7 Studies show how to optimize a method, to isolate exosomes from the blood plasma, to further assist in biomarker discovery. In regards to analysing exosomes as a potential biomarker, exosomes need to purified and isolated with variedial centrifugation paired with ultracentrifugation (UC). Another technique utilize, is affinity purification of the exosomal membrane antigens using minginess gradient (DG) centrifugation, separating the vesicles based on their density.8 A practise needs to be conducted, to purify exosomes, from only a sm either volume of blood plasma.With studies being conducted on the stability in fluids of circulating exosomes, it can be confirmed that these nanovesicles have the ability of target ing to uptake to hinder or delay tumour development. With the size of the exosomes ranging from 40 100 nm, they are distinguished for tumour microenvironment. Exosomes, a potential biomarker, for the early detection of PDAC, are also being studied as potential nanocarriers to target cancer cells and delaying tumour growth.1,7,8 The most common nanocarrier being studied is the gold nanoparticle (GNP), due to its imaging, diagnostics, and therapy abilities. The GNP can be easily synthesized via the citrate reduction, which is why is has potential in medical theranostics.7Extracellular vesicles (EV), are secreted into extracellular space, they are involved in tumour initiation, progression as salubrious as metastasis. EVs can be utilize as non-invasive biomarkers, but the current studied methods are time consuming in regards to EV closing off.1,7,8 The EV membrane markers which are part of the tetraspanin family, are CD9, CD63, and CD81, respectively, and an assay demonstrates simil ar features.6 A nanoparticle EV assay, will be captured by an EV-specific antibody with the dual binding of EV, using 2 nanoparticle probes. The 2 nanoparticle probes, will upraise a plasmon, promoting an summation in sensitivity and specificity for the discovery of an exosomal biomarker. Ephrin type A receptor 2 (EphA2), has recently been identified as a biomarker, of the tumour derived pancreatic cell line, and enriched on EV.1 EphA2, shows over expection, increases in vitro invasiveness and anoikis resistance in pancreatic cancer cell lines.1 A recent use up has been conducted with healthy control patients, pancreatitis patients as well as pancreatic cancer patients, with the use of nanoplasmon-enhanced scattering (nPES) assay a fast, sensitive, and specific method in biomarker detection.Convention Tumour profanekers in Pancreatic CancerCarcinoembryonic Antigen CEACEA, is a glycoprotein, that is measured in a common blood test apply for testing patients with cancer, includi ng pancreatic cancer. This will measure the amount of the CEA protein that is in the blood of a patient who may have cancer, and the CEA levels can be used to determine whether treatment is working or if the cancer is sp chartering. A CEA level of 5 ng/mL, is considered a normal level of this protein, but there are several conditions that can alter the levels of the CEA in your blood, which is why this is not a valid biomarker in the detection of pancreatic cancer.5 CEA testing can be useful in regards to recurrent colon cancer as well seeing if treatment is successful. Levels of CEA can be elevated due to smoking, as well as in other diseases such as Crohns disease. Due to the unreliability of CEA levels in cancer, this blood test confirms that CEA is not a consistent biomarker for the early detection of pancreatic cancer.CEA is expected to be paired with other biomarkers, for early detection reasons. When paired with CA19-9, there is an increase in detection for sensitivity and sp ecificity, showing an betterment in diseases including pancreatitis as well as pancreatic pseudocyst. Despite the improved results for pancreatic diseases, CEA is still not valid for the detection of pancreatic cancer, even when it is paired with another marker such as CA19-9.5Carbohydrate Antigen CA19-9Carbohydrate antigen (CA19-9) was discovered in 1981, and is considered a sialyl lewis a (sLea).9 CA19-9 is found on the prove cancer cell, expressed as a glycolipid and an O linked glycoprotein, and is related to the Lewis blood group antigens.3 Patients with Le (alpha beta +) or Le (alpha + beta -) blood group, express levels of CA19-9 in their blood, whereas approximately 5 10% of patients with Le (alpha beta -) blood group do not express CA19-9, limiting the use as a valid biomarker.9 Due to the low, and uncertain sensitivity of CA19-9, it is a poor interpreter of PDAC, therefore it is not a precious biomarker.CA19-9 is unable to differentiate between benign, precursor lesi ons and malignant conditions in PDAC patients, and it gives elevated results in many other gastrointestinal cancers.3 This blood test can show elevated CA19-9 levels in patients with other non-cancer diseases including pancreatitis and cirrhosis.3,9 The CA19-9 blood test can be beneficial in regards to knowing if a pancreatic tumour is secreting it, and to judge the qualification of treatment, and look for pancreatic cancer recurrence. A healthy patient will have a CA19-9 level of 0 37 U/mL, therefore with change magnitude levels of CA19-9, this could indicate tumour growth.3For more accurate results, a PDAC marker needs to be discovered and paired with CA19-9, to increase the sensitivity and specificity in early detection. With CA19-9 as the only marker, studies show it was only elevated in 50 75% of patients having PDAC, confirming that is not consistent as a biomarker and should not be used in diagnostic testing.3,9 Expressing elevated levels in other diseases such as benign jaundice, pancreatitis, and ovarian cancer, confirms the lack of consistency using the CA19-9 marker and that it cannot be used as an accurate indication of early pancreatic cancer detection.3Emerging BiomarkersWith the absence of reliability using the current PDAC biomarker, C19-9, it is a necessity to discover a biomarker with improved sensitivity as well specificity for the early detection of PDAC. Recent studies suggest, that exosomes can be detected in body fluid such as blood, and they have potential as disease biomarkers. Exosomes, found in blood plasma, need to be collected from healthy patients to obtain individual and pooled proves. The collected blood plasma, will need to be separated, by centrifugation, to isolate the exosomes for further proteomic and quantitative studies.8Isolation MethodsIsolation of exosomes using the UC method, involves normal human plasma, and diluting it with phosphate buffer solution. The sample will be differentially centrifuged, to eradicate c ell debris, which is followed by UC. The subsequent pellet, is washed in PBS, and filtered, and the filtrate was ultracentrifuged. The resulting exosomal pellet, used for the study, will be resuspended in PBS.8Using the EI isolation method, the plasma, is weaken in PBS and centrifuged. The supernant is filtered, and the filtrate will be incubated using a blocking agent. A microcolumn was placed in magnetic separators, where the column was rinsed with rinsing solution. string of beads were bound to the exosome, and were applied to the magnetic column. The column will be washed with rinsing solution, and the immune captured exosomes were recovered by removing them from the column and placing them in a exhibition tube. The exosome bound microbeads are washed to elute the exosomes, and centrifuged to obtain the exosomal pellet. The exosomal pellet will be resuspended in PBS.8Lastly, isolation using DG method, involved the exosomal pellet that was obtained from UC as well as normal bl ood plasma that was layered on iodixanol solution and centrifuged. To the top of the tube, there were 12 fractions, with increasing densities. The fractions are diluted with PBS and centrifuged, the resulting pellet was washed with PBS, centrifuged and resuspended in PBS.8Western Blot and Microscopic AnalysesThe western blot method, shows the enrichment of the exosomal marker proteins. Gel electrophoresis is used to separate and identify the different proteins. The thickness of the band, indicates the amount of the protein that is present. There is a labelled antibody, that is bound to the protein of arouse. AFM is used, to get a 3D image of the exosomal vesicle.Recent studies confirma that the exosomal markers CD9 and CD63 are enriched in exosomes purified using UC and EI methods.8 The study indicates that the UC method, had four exosomal markers whereas the EI method had only two exosomal markers, CD9 and CD63. Transmission electron microscopy (TEM) and atomic force microscopy (A FM), were used on the isolated exomes, from the three exosomal isolation techniques. In the DG sample, the TEM reported homogeneous vesicle, with diameter ranging from 40 100 nm, confirming the characteristics of exosomes. The AFM produced a 3D image of the exosome, and after further analysis it was revealed that the exosomes had round membranous vesicle characteristics.LC-MS/MSLiquid chromatography mass spectrometry (LC-MS/MS), is a quantitative method used for the identification of proteins at the peptide level. The first quadrupole is for the selection of the precursor and the second quadrupole is super specific for detection. In comparison to gas chromatography mass spectrometry (GC-MS), LC-MS/MS is not limited to volatile substances, it is better for the detection of molecules. LC-MS/MS can produce many quantitative results, and has a high specificity and sensitivity.The study was carried out, using an LTQ Orbitrap Velos with a nanoelectrospray interface coupled to an Ultim ate 3000 RSLC nanosystem and the LTQ Orbitrap Velos mass spectrometer operates using a nano -ESI spray. The LC-MS/MS spectra are searched against the human protein database using MASCOT. Equal amounts of protein from the three exosomal samples were separated, reduced, alkylated and digested with trypsin. The DG sample had the highest number of protein identifications, followed by the UC isolation method. Therefore, the western blotting, microscopy and MS results confirm that the DG isolation method is the most effective, in regards to isolating exosomes from blood plasma.8Targeting with halcyon NanoparticlesMalicious ExosomesThe exosomes are formed from endosomal pathways, after they are fused from multivesicular bodies (MVBs) with plasma membrane. The formation of leering exosomes, also starts in the endosomal pathway. The early exosome is formed from the migration from the cell periphery to the nucleus, by the formation of intraluminal vesicles (ILV). The process interceded by e xosomal complexes required for transport (ESCRT) and other proteins. Late exosomes/ MVB, reincarnate to the periphery and fuse with the membrane, releasing the ILV, which are called exosomes. The proteins, Rab GTPases, mediate the endosome migration.7 The malicious exosomes, are released from cancer cells found in the tumour microenvironment. Exosomes play a role in variation and shaping of that tumour microenvironment.1,2,4,6,8Malicious exosomes have potential as biomarkers, due to their stability in biological fluids including blood plasma. There have been change magnitude levels of circulating exosomes seen in several cancers including pancreatic cancer.1,6,7,8 Nanovesicles can be used to carry therapeutics, and have potential to limit cancer progression.1,7 The method consists of inhibiting the malicious exosomes biogenesis.Gold Nanoparticles The GNPs can be easily synthesised, as well they consist of a variety of shapes and sizes. These nanoparticles exhibit intense light abs orption and scattering, and they are deemed to be extremely stable.1,7 They have potential in targeting, therapeutics as well as diagnostic capabilities. Regarding rapid tumour growth, a compressed lymphatic vessel will hand causing lymph drainage, which will thusly allow for the nanosized molecule to be taken at the tumour site.7 This process will allow for passive targeting with nanosized molecules. The cellular interest will be dependent upon the size and shape of the GNP.1,7 The tumour cells will overexpress their cell number receptors, which can be used for potential biomarkers.1,2,4,6,7,8 These cell surface receptors, will aid in the direction of the GNP to the tumour cells.Gold Nanoparticle TargetingThe GNP will target malicious exosomes, by undertaking the malicious exosomes biogenesis with GNP specific targeting moieties as well as silencing moieties.7 Using antibodies to aim at the exosome for capture and selective retention. Lateral flow immunoassay, will aid in exosom e detection with CD9 and CD81 as antibodies, and CD63 with GNP.1,7 Therefore, GNP are being studied as a potential candidate for cancer therapy as well as for malicious exosome targeting. The use of nanotheranostics to help quantify and inhibit the malicious exosomes.Sample Collection and ProcessingThis recent study, developed a method for the purification of exosomes in blood plasma, as well as finding the EV concentrations in the plasma samples. A three-probe EV capture was used, with a capture antibody that recognizes an EV membrane protein (anti-CD81), with antibody immix AuS and AuR to serve as two EV probes. This EV capture was designed to form a plasmon, with the different GNP binding on an EV to improve sensitivity and specificity of EV detection.1 The study examined 59 pancreatic cancer patients, 48 pancreatitis patients, and 48 control patients, to see if early pancreatic cancer stage could be distinguished from pancreatitis patients and the control patients.1MethodThe E V isolation consisted of cells grown in culture media, and washed with PBS. The culture supernatants were collected and centrifuged to pellet cells, and centrifuged again to remove cell debris. Concentrated with centrifugal filtering units, and centrifuged, the precipitates were collected and resuspended in PBS and centrifuged. The resulting precipitates were collected and dissolved in PBS. The ELISA assay, consisted of ninety-six well plates, which were incubated with antibody CD81. The ELISA assay was analysed for absorbance, and the standard hack plotted the light absorbance versus the log10 EV standard concentration in pg/uL.1The peptides were separated using Ultimate 3000 nano-LC, with an enrichment column as well as an analytical column. The peptide fractions were analysed with Velos Dual-Pressure unidimensional Ion Trap mass spectrometer, and one MS scan, was followed by eight MS/MS scans.The nPES platform was constructed by filling sample wells with plasma sample or cell c ulture EV samples, followed by incubation and being washed three times with PBST and three times with PBS. The sample wells were then filled with AuS and AuR PBST solution, and were incubated and washed three times each with PBST and PBS, respectively. The sample wells were fitted with a cover slip and dark-field microscopy (DFM) was used for imaging. The DFM images, that had image empyreans with brightness equal to 225 were selected, and the ratio of the image area to the whole image gave area ratios that were indicative to the nPES EV signal.1 A standard concentration curve was generated with a linear regression of nPES area ratio with log10 concentrations.1SEM images analysed the images of GNP binding to EV, from EVs that were purified from human plasma. The purified EVs were hybridized with anti-CD63-AuS and anti-CD9-AuR. The SEM fields were analysed to calculate the total EVs, as well as the number of GNP-bound EVs per um2 of each assay.Proteomics and the Early Detection of PC An nPES was previously designed, for EV detection using GNP, that can scatter light at different wavelengths indicative to their shape and size. Using both gold nanospheres (AuS) as well as gold nanorods (AuR), a plasmon is formed, increasing the scattering intensity. With the plasmon, antibodies against CD9, CD81, and CD63 can capture and detect EV in a sample.1,7 AuS and AuR are detectable using dark field microscopy (DFM), and will form the complexes AuS-EV-AuR, AuS-EV and AuR-EV. These complexes can be analysed using scanning electronic microscopy (SEM), examining the binding and distribution. Following the pure preparation of EV samples, EV plasma was added to give the EV plasma standard. The anti CD81 was incubated with the standard and two antibodies conjugated GNPs, AuS-Anti-CD63 and AuR-Anti-CD9, which exhibited ratios 0.35%. A comparison was done with nPES and enzyme linked immunosorbent assay (ELISA), of the sensitivity and linearity of their EV values. The nPES assays showed to be highly sensitive, requiring less plasma as well has exhibiting more advantages over ELISA in regards to measuring EV concentrations.1Since CA19-9 is the only received pancreatic cancer marker that is not valid, pancreatic cancer derived EV marker is a more feasible biomarker due to the multiple factors that the pancreatic cancer cells express. The nPES assay will quantitate tumour derived EV from blood samples, and one of the two EV specific GNP were replaced with one specific for the membrane protein. LC-MS/MS proteomics, bioinformatics is used to identify trans-membrane proteins on EV PC (PANC-1 and MIAPaCa-2) and PDAC (BxPC-3).1 There were 128 membrane proteins identified, and 26 were expressed on EV. The EphA2 showed the highest expression and is associated with cancer progression, metastasis, and prognosis. The EphA2, was also not expressed by EV in HPNE. EphA2 was chosen as the potential marker, and CD81 and CD9 were chosen for EV capture. The nPES was modified , using one capture antibody (anti-CD81) and two antibody-conjugated GNP probes (anti-EphA2-AuS and anti-CD9-AuR).1The plasma EphA2-EV levels were higher in pancreatic cancer patients, in comparison to pancreatitis patients and the normal control (NC). With the strong association between the circulating EphA2-EV and early stage pancreatic cancer, there is potential for EphA2-EV to be used as an early detection marker.1 The CA19-9 levels were increased in the pancreatic cancer patients in comparison to the pancreatitis patients and the NC, but the levels were not increased in the early stages of PC. The receiver operating characteristic (ROC) curves, showed that the plasma EphA2-EV levels are promising in the classification of pancreatic cancer stages.The current EV analysis methods are tedious and lengthy for the isolation procedures, as will having volume requirements. The nPES platform that has been studied, assimilates EV capture and detection with the use of the plasmon couplin g effect, to have an increase in both detection sensitivity and specificity in small volume samples and fast sensitive biomarker quantification. This EV nPES platform, can be generalizable for any disease state that has a specific EV marker.1 The nPES EphA2-EV blood assay shows substantial value regarding pancreatic cancer screening tests, due to being a rapid, accurate and non-invasive blood test for the early diagnosis of pancreatic cancer.ConclusionsThis round off article explains the need to find a valid biomarker in the early detection of pancreatic cancer, as well as discussing how exosomes have potential to be that marker in the early detection process.1,2,4,6,7,8 The existing biomarkers for pancreatic cancer, are not valid markers in the early detection due to the lack of sensitivity and specificity that they exhibit when differentiating between benign and malignant stages. The use of exosomes for the early detection of pancreatic cancer, shows potential as a biomarker, wit h the use of nPES platform.1 The platform allows for EV capture using plasmon coupling, which increases in detection sensitivity and specificity, which allows for the discovery of an ultrasensitive biomarker. The nPES EphA2-EV assay could differentiate between pancreatic cancer patients (stage I and II) and pancreatitis and NC patients.1 The role of EphA2-EV, could help to improve early detection rates as well as improving patient outcome, and this blood test is inexpensive, accurate and non-invasive. This review involved proteomic and quantitative methods, to find a novel biomarker for the early detection of pancreatic cancer, and non-invasive nPES EphA2-EV analysis can aid in improving early pancreatic cancer detection and treatment.ReferencesLiang, K. Liu, F. Fan, J. Sun, D. Liu, C. Lyon, C. J. Bernard, D. W. Li, Y. Yokoi, K. Katz, M. H. Koay, E. J. Zhao, Z. Hu, Y. 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Simpson, R. J. Hulett, M. D. Mathivanan, S. Proteomics 2013, 13 (22), 3354-3364.Ballehaninna, U. K. Chamberlain, R. S. In dian Journal of Surgical Oncology 2011, 2 (2), 88-100.Pancreatic Cancer https//www.cancer.org/cancer/pancreatic-cancer.html (accessed Mar 20, 2017).Eldorado by Edgar Allan Poe AnalysisEldorado by Edgar Allan Poe AnalysisIn the poem Eldorado, poet Edgar Allan Poe delivers a fundamental message that can be understood if cautiously evaluated. Poe gives the account of a knight in search of a land called Eldorado, which holds wealthiness and fortune. After much futile searching, the knights enthusiastic quest for treasure ends in death. The foremost theme of this poem is the desire for wealth and treasure. This theme is an influence from Poes life and the relevant Gold Rush of 1849 (Coad 60). The literary devices, symbols, relevancy, and personal experiences offer a deeper meaning to the poem than what lies on the surface. Poes skillful use of these elements helps to stress the ignorant desire humans have for wealth and fortune.The poem delivers a reflective moral issue many readers c an, in some way or another, relate to. Poe uses the word poop in each of the four stanzas of the poem, each stanza consisting of six lines. The 3rd line in each stanza is where the use of the word prat is introduced. Though the word occurs multiple times, it has a different meaning each time. The first shadow represents a literal shadow, a casting shadow of the sun. It could also be interpreted as rejoicing and sadness. The second shadow represents the shadow that has overcome the knights rawness after much unsuccessful searching. The third shadow represents a live figure, possibly his or maybe an angel. And the fourth shadow figuratively refers to Valley of the phantasma (21). The fact the knight has grown old and weak, and must cross Over the Mountains Of the Moon, Down the Valley of the Shadow is seen as a symbol of the knights death, relating to the Biblical vale of death (19-21). Through Poes use of the word shadow and the period in which the poem was scripted, readers can understand Poes message.Poe uses the shadow in each stanza to convey his message. As the meaning of the shadow changes, so do readers emotional state. As he begins the first stanza, readers see a happy, gaily, bedighted knight who is enthusiastic about going on his search for gold. This start gives readers a sense of happiness and jolt of energy. His shadow could also be a foreshadowing of future events. However, Poe begins the second stanza with the word But. This irrelevant word signals a shift between the first and second stanza and also a shift in emotion. The knight has become old, disheartened, and dismayed as the shadow is used in context to signal the emotional state of the knight. This signal causes readers to suddenly have a change in emotion readers become forgiving towards the knight. Poe continues to elaborate on the disap hitchment of the knight in stanza three. The knight encounters a live or possibly imaginative figure and asks the shadow where is Eldorado, reflec ting on his black journey in which he wasted his life. This figure could possibly be an angel providing guidance, an angel of death, or even himself. As the shadow replies to the question in stanza 4, readers are left with the idea that he has come to the end of his life and has died. With the closing of the poem, the interview can relate to the pain the knight feels. In all, the clamant shadow becomes engraved in the readers mind, helping to sway the emotions. The life of the knight also provides a moral for people to learn.Poes moral in Eldorado is not to seek for riches on earth. The only true riches are the riches one receives after death. The knight in the poem seeks for physical riches for many years without any hope, leaving him disheartened and at the end of his life. When asked where Eldorado could be, the knight was told Down the Valley of the Shadow (21). This insinuation emphasizes the main point that true riches are found in Heaven, not earth, and any riches sought on earth leads to despair and death.As suggested by The pith of Poes Eldorado by the John Hopkins University Press, it can be argued Poe portrayed himself as the knight (Coad 60). Poe published his poem in 1849, the same year as his death. alike the knight, Poe had sought after an accomplished life, which he failed to do during his life. He was also unstable in the last years of his life. However, the knight most probably was a reference to the many prospectors of the California Gold Rush, which took place during the time the poem was written. The poem may have been Poes warning to the many prospectors that would experience the same hardships of the knight.Poes repeat of shadow and Eldorado and use of other symbols play an important part in his poem. It helps to further stress his main point. Other such devices Poe uses to communicate to his audience is through auditory and imagery senses. Poe uses aabccb rhyme scheme in the first three stanzas and xxabba rhyme scheme in the fourth stanza of his poem . Poes creatively written stressed and unstressed poem is one way Poe remarkably appeals to the readers auditory and imagery senses. The use of this rhyme scheme creates a thumpity, thump sound when read aloud, bringing the clattering of the horses trot to life. The symbols and rhyme scheme helps to immerse the reader into the scene of the poem and drives them to continue reading until the end.Eldorado is a poem by Edgar Allan Poe that has a stressed message to readers. It tells the story of a knight who traveled for a period of his life searching for a city of gold, Eldorado. It provides a message to all readers that true riches and happiness are only acquired through Heaven after death. If one attempts to search for wealth, in hopes happiness will follow, that person will come to the end of their life saddened and in despair. Poes use of symbols, rhyme scheme, and repetition brings life to his poem, which keeps the readers entertained and helps to convey his mes sage. The poem brings light to the life of everyone and anyone searching for happiness and wealth on earth. Thus, Eldorado is Over the Mountains Of the Moon, Down the Valley of the Shadow, Ride, boldly ride . . . If you seek for Eldorado (19-24).