Prognostic significance of the sequential detection of circulating melanoma cells in high-risk melanoma patients receiving adjuvant interferon

During the last decades the incidence of malignant melanoma steadily increases and the need for markers in detection of early metastasis and guidance of therapy has become urgent. Presence of melanoma cells in peripheral blood could indicate dissemination in the circulation, and thus a high risk of metastasis. For detecting tumor cells in melanoma patients RT-PCR has been used to identify expression of tyrosinase a key enzyme in melanin biosynthesis, in the circulation of melanoma patients. To address the prognostic implications of the presence of circulating melanoma cells we investigated the presence of tyrosinase mRNA by RT-PCR in the peripheral blood of stage IIB and III melanoma patients on high dose adjuvant interferon alpha-2β at multiple sequential time points from initiation of treatment. Total RNA was isolated from the mononuclear cell fraction of peripheral blood, and after reverse transcription, cDNA samples were amplified by nested PCR. Results were confirmed by Southern blotting and hybridization with radiolabeled probe. The sensitivity of the assay was determined by serial dilution of SK-mel28 melanoma cells and was high enough to detect 1 SK-mel28 cell in 10⁷ mononuclear cells, after Southern blotting and autoradiography. Four hundred eighty-eight blood samples from sixty melanoma patients were tested for tyrosinase expression. The correlation between relapse and RT-PCR results was evaluated by the x² test. Relapse-free and overall survival were analysed by the Kaplan-Meir method. Positive RT-PCR result during follow-up was the only prognostic factor that significantly correlated with higher risk of relapse. Our data suggest that circulating melanoma cells are markers of high relapse risk and shorter disease-free survival. Tyrosinase mRNA amplification by RT-PCR may be a useful tool for monitoring the efficacy of adjuvant treatment in stage IIB and III melanoma patients.Κατά τις τελευταίες δεκαετίες οι περιπτώσεις κακοήθους μελανώματος αυξάνονται σταθερά και υπάρχει άμεση ανάγκη να βρεθούν βιολογικοί παράγοντας ικανοί να δώσουν προγνωστικά στοιχεία για την κλινική συμπεριφορά της νόσου και τη βελτίωση της θεραπευτικής αντιμετώπισης της. Παρουσία κυττάρων μελανώματος σε δείγματα περιφερικού αίματος, θα μπορούσε να υποδηλώνει αυξημένο κίνδυνο υποτροπής. Προκειμένου να εξεταστεί η προγνωστική σημασία της παρουσίας κυκλοφορούντων στο περιφερικό αίμα μελανοκυττάρων, κατά την παρούσα εργασία μελετήθηκε η παρουσία του μεταγραφήματος του γονιδίου της τυροσινάσης, ενός σημαντικού ενζύμου της βιοσυνθετικής οδού της μελανίνης, με την τεχνική της Αλυσιδωτής Αντίδρασης Πολυμεράσης Αντίστροφης μεταγραφής (RT-PCR) στο περιφερικό αίμα ασθενών κλινικού σταδίου IIB και III, οι οποίοι βρίσκονταν υπό θεραπεία με υψηλή δόση ενισχυτικής ιντερφερόνης άλφα-2β, σε διαδοχικάχρονικά στάδια από την έναρξη της θεραπείας. Ολικό RNA απομονώθηκε από το κλάσμα των λευκοκυττάρων του περιφερικού αίματος, και ακολούθησε αντίστροφη μεταγραφή και ενίσχυση του cDNA με nested PCR. Τα αποτελέσματα επιβεβαιώθηκαν με μεταφορά κατά Southern και υβριδισμό με ραδιενεργό ανιχνευτή. Η ευαισθησία της μεθόδου ελέγχθηκε με διαδοχικές αραιώσεις μελανοκυττάρων της κυτταρικής σειράς SK-mel28 και βρέθηκε ικανή να ανιχνεύει 1 SK-mel28 κύτταρο σε 10⁷ λευκοκύτταρα, μετά από μεταφορά κατά Southern και αυτοραδιογραφία. Τετρακόσια δέκα οκτώ δείγματα από εξήντα ασθενείς ελέγχθηκαν για έκφραση του γονιδίου της τυροσινάσης. Η δοκιμασία x² χρησιμοποιήθηκε για την εκτίμηση της συσχέτισης υποτροπής και αποτελεσμάτων της RT-PCR. Το ελεύθερο νόσου διάστημα καθώς και το ολικό διάστημα επιβίωσης αναλύθηκαν με τη μέθοδο Kaplan-Meir. Μία ή περισσότερες θετικές RT-PCRs κατά τη διάρκεια παρακολούθησης ήταν ο μόνος παράγοντας που βρέθηκε πως σχετίζεται στατιστικώς σημαντικά με μικρότερο διάστημα άνευ νόσουεπιβίωσης. Τα ευρήματα μας συνηγορούν πως η παρουσία κυκλοφορούντων μελανοκυττάρων είναι δείκτης υψηλού κινδύνου υποτροπής και βραχύτερης άνευ νόσου επιβίωσης. Η ενίσχυση του μεταγραφήματος της τυροσινάσης με τη μέθοδο της RT-PCR μπορεί να είναι ένα χρήσιμο εργαλείο για την εκτίμηση της αποτελεσματικότητας της συμπληρωματικής θεραπείας σε ασθενείς με μελάνωμα κλινικού σταδίου IIB και III

Polymer-coated bismuth film electrodes for the determination of trace metals by sequential-injection analysis/anodic stripping voltarnmetry

In this work. the utility of polymer-coated bismuth film electrodes (BiFEs) was assessed for the simultaneous on-line determination of Cd(II), Pb(II) and Zn(II) by square-wave anodic stripping voltammetry (SWASV) in the sequential-injection analysis (SIA) mode. The polymeric Nafion coating was initially plated on a glassy carbon electrode forming part of the flow-through electrochemical cell. The subsequent steps of the bismuth layer generation, analyte preconcentration, voltammetric measurement and electrode cleaning were conducted on-line. For a sample volume of 1.2 mL, the limits of detection (at S/N = 3) were 2 mu g L-1 for Cd(II) and Pb(H) and 6 mu g L-1 for Zn(II), the coefficients of variation at 20 mu g L-1 were 5.1% for Cd(II), 5.9% for Pb(H) and 6.2% for Zn(II) and the measurement frequency was between 10 and 20 h(-1) (depending on the sample volume aspirated). It is demonstrated that the polymer-coated BiFEs, combined with SIA, can provide an environmentally friendly, sensitive and robust tool to perform rapid and cost-efficient on-line monitoring of trace metals by ASV, even in the presence of surface-active compounds. (c) 2006 Elsevier B.V. All rights reserved

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapA (Rv2753c) from Mycobacterium tuberculosis

M. tuberculosis dihydrodipicolinate synthase, the enzyme that catalyzes the first unique reaction in the l-lysine biosynthesis pathway, has been cloned, expressed, purified and crystallized and the crystals have been characterized by X-ray diffraction

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DapC (Rv0858c) from Mycobacterium tuberculosis

M. tuberculosis N-succinyldiaminopimelate aminotransferase, the enzyme which catalyzes the sixth reaction in the lysine-biosynthesis pathway, has been cloned, expressed, purified and crystallized

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of LysA (Rv1293) from Mycobacterium tuberculosis

M. tuberculosis diaminopimelate decarboxylase, the enzyme that catalyzes the final step of lysine biosynthesis, has been cloned, expressed, purified and crystallized in the absence of cofactor or substrate

Changes in endothelial cell microrheology in medium modified by cancer cells

During the process of metastasis, cancer cells are detached from the primary tumour and may find their way into the blood system, from where they can migrate in a distant site. Endothelial cells line the vascular wall of all blood and lymphatic vessels and regulate its permeability for substances and migratory cells. Previous studies have focused on the properties of cancer cells (Abidine et al. 2018), as well as their interaction with endothelial cells (Rajan et al. 2016), as they attach to and transmigrate through the endothelium barrier. For many authors, the most widely accepted hypothesis was that transmigration is a characteristic of cancer cells, overlooking the possibility of endothelial cells actively participating in this process (Mierke et al. 2012). In this paper, endothelial monolayers were studied with a previously developed AFM microrheology technique (Abidine et al. 2018) when cultured in cancer cell conditioned medium, at different dilutions. This medium is enriched with molecules secreted by the cancer cells, which may play a role during the cancer cells’ migration and alter the endothelial cells’ behaviour (Ritchie et al. 2021). We show the first results of the viscoelastic properties of endothelial cells under these experimental conditions

Combined Operation of Wind-Pumped Hydro Storage Plant with a Concentrating Solar Power Plant for Insular Systems: A Case Study for the Island of Rhodes

Insular power systems are a special case of infrastructure for power production due to their particular land morphology with extensive hills and ridges. For a higher renewable energy share in the power production, a dedicated design according to local constraints is required. The high wind and solar resources of such cases can be utilized with offshore wind turbines and concentrating solar power, respectively. In addition, pumped-hydro storage is a mature and suitable technology for such terrain. A case study is presented in the island of Rhodes to obtain a renewable energy penetration higher than 70%. The technical and financial requirements for this implementation support the design of this system, while the introduction of concentrating solar power enables significant energy savings during the periods of peak demand of the island. An annual RES penetration close to 80% can be achieved with the combined operation of both plants. The economic viability of the required investment can be ensured with selling prices of the produced electricity in the range of 0.20 EUR/kWh