“Obtaining and selecting microalgal genotypes suitable for the bioremediation of matrices containing hydrocarbons, starting from Haematococcus pluvialis (Flotow,1844)”

The world economy, even today, is based on the use of fossil fuels to obtain energy and specifically coal and oil. Oil consumption in 2020 increased by 0.9 million barrels per day while the demand for liquid fuels reached historic highs reaching 100 million barrels per day. The use of oil governs stock exchanges and world markets few and specific cartels determine the selling price while maintaining a sort of economic monopoly. Precisely for this reason, the extraction and refining of crude oil remain an extremely intense activity. Basilicata, a region of southern Italy, in particular, represents the largest onshore site on the European continent, contributing alone to 63% of Italian crude oil production. The process of extraction, refining and transport, however, are crucial points for the environmental pollution of soil, air and water. During this thesis paper, we focused on water pollution, caused both by transport (ships and/or underwater pipelines) subject to numerous accidents, and by extraction, during which processing waters come into contact with toxic hydrocarbons and harmful. Petroleum consists of aliphatic and non-aliphatic hydrocarbons, as well as other elements, main heteroatoms such as sulfur and nitrogen. Furthermore, hydrocarbons can be highly toxic: volatile hydrocarbons (VOCs) such as benzene, toluene, ethylbenzene and xylene (BTEX), or polycyclic aromatic hydrocarbons (PAHs) are the cause of the onset of various neurological, respiratory and tumour pathologies. It is therefore evident that the entire industrial process must be regulated to avoid contact with these compounds and that, if accidental spills occur, they must be immediately confined and treated. The methodologies used for this purpose include physicochemical processes that break down the molecules of the crude oil, making it more sensitive to attacks by atmospheric and natural agents. Among these, the evaporation of low molecular weight molecules, the emulsion of the oily fraction with water, solubilization and sedimentation contribute in part to the removal of the crude oil, but there is also a great contribution from the microbiological world, especially prokaryotic, which through bioremediation processes degrades and removes many toxic compounds. The limitations of these technologies lie in the use of chemical compounds that are more harmful to the ecosystem than the oil itself and the saturation of microorganisms that can act only on certain compounds. Precisely by the need to find a methodology that can constitute an innovative solution to the aforementioned problems, microalgae were thought of. Microalgae are unicellular, microscopic, photosynthetic and photoautotrophic organisms capable of removing carbon dioxide from the atmosphere, using it in the photosynthesis process to produce energy and release oxygen. There are different classes of microalgae that can also be placed in different domains (prokaryotes and eukaryotes), with different characteristics and physiologies, but the object of our study was green, eukaryotic and photoautotrophic microalgae. Their ability to use organic carbon, modifying their metabolism, thus becoming mixotrophic, has made them excellent candidates for their use in the world of bioremediation. Several studies have focused on their use for bioremediation, for example, some microalgae have been able to remove sulphate, and nitrates in significant percentages. The most investigated microalgae in this area was Chlorella spp. which removed after 5 days of treatment, 80% of oil emulsified in the middle in a 2020 study, and efficiently reduced the PAHs present in a 2013 study. In this thesis, the analyzed microalgae was Haematococcus pluvialis, known to the scientific world for its ability to produce one of the most powerful antioxidants in nature, astaxanthin (ASX) but never considered for the bioremediation of hydrocarbons of petroleum origin. This PhD project aimed to research a new and innovative technology that, thanks to the use of microalgae, can effectively remove petroleum hydrocarbons in aqueous matrices, creating different genotypes capable of acting more efficiently than the unchanged genotype. (WT). Thanks to a selective pressure process, H. pluvialis was induced to mutate and adapt to an extremely toxic condition. Once several mutant strains were selected, they were followed in their growth and their ability to produce astaxanthin, always comparing them with WT. Since our knowledge did not allow us to determine whether the mutations that occurred were genetic or epigenetic, a genetic analysis was also carried out through RAPD-PCR on the transcript to highlight the diversity of expression of the different genotypes between them and with the non-mutated species. under conditions of non-stress and in subsequent generations. It was thus possible to obtain a dendrogram through “cluster analysis” which highlighted the genetic expression distances between them.This result indicates that the difference in expression under standard conditions is attributable to underlying genetic modifications. Subsequently, after trying different stress and bioremediation conditions, they were grown in the presence of 1% of pure crude in anaerobic conditions, to evaluate the removal and/or degradation activity. The chemical analyses carried out only after 10 days of treatment immediately showed that the amount of carbon present in the culture medium had almost doubled while other parameters such as fluorides, chlorides and sulphates decreased. After 40 days of treatment, preliminary analyzes by GS-MS and GC-FID showed that both in the aqueous medium and in the algal biomass, there were hydrocarbons and oily substances, results confirmed, then, by a quantitative analysis at 20 and 160 days in which, with different kinetics, a decrease in hydrocarbons is highlighted, which for the mutant strain PA1004 had been removed both from the medium and from the biomass. Furthermore, after 120 days the genetic analysis was repeated to evaluate any adaptive mechanisms that occurred following the stress induced by oil. By maintaining the identical amplification conditions (primers, temperatures and concentrations), the result of the "cluster analysis" showed that, indeed, there had been changes in genetic expression, leading to a reorganization of the distances previously obtained in optimal conditions. These distances reflect the behaviour of the genotypes in the presence of the exogenous agent. Some have completely removed the oil, others have acted only on the oily fraction, helping to create a bituminous particulate by grouping as per the generated dendrogram. The absence of hydrocarbons within the aqueous medium and algal biomass is in itself an extremely interesting result, confirming the objectives set at the beginning of this research path. Not only was H. pluvialis able to survive in the presence of crude oil, but also the genotypes generated, proved to be a promising alternative in hydrocarbon bioremediation. In the future, we prepare ourselves to analyze not only the pathways involved in the mutations, to understand the mechanisms underlying this process, but also to analyze the by-products generated to reuse them in a circular economy perspective suitable for the removal and reuse of a potentially harmful product. for man and the environment.L’economia mondiale, ancora oggi, si basa sull’utilizzo di combustibili fossili per ricavare energia e nello specifico carbone e petrolio. Il consumo di petrolio nel 2020 è aumentato di 0.9 milioni di barili al giorno mentre la domanda di combustibili liquidi ha raggiunto massimi storici arrivando a 100 milioni di barili al giorno. L’utilizzo del petrolio governa borse e mercati mondiali, infatti pochi e specifici cartelli ne determinano il prezzo di vendita mantenendo una sorta di monopolio economico. Proprio per questo, l’estrazione e la raffinazione del greggio rimane ancora un’attività estremamente intensa. La Basilicata, regione del sud Italia, in particolare, rappresenta il sito onshore più grande del continente europeo, contribuendo da solo al 63% della produzione di greggio italiana. Il processo di estrazione, di raffinazione e di trasporto, tuttavia, costituiscono dei punti nevralgici per l’inquinamento ambientale di suolo, aria e acqua. Durante questo elaborato di tesi ci si è soffermati sull’inquinamento delle acque, causato sia dal trasporto (navi e/o condutture sottomarine) soggetto a numerosi incidenti, sia dall’estrazione, durante la quale acque di lavorazione vengono in contatto con idrocarburi tossici e nocivi. Il petrolio è costituito principalmente da idrocarburi alifatici e non, oltre che da altri elementi, principalmente eteroatomi come zolfo e azoto. Gli idrocarburi, inoltre, possono essere altamente tossici: gli idrocarburi volatili (VOCs) come il benzene, il toluene, l’etil-benzene e lo xilene (BTEX), o gli idrocarburi policiclici aromatici (PAHs) rappresentano la causa dell’insorgenza di diverse patologie neurologiche, respiratorie e tumorali. Risulta evidente, quindi, come tutto il processo industriale vada regolamentato per evitare contatti con questi composti e che, qualora avvengano, fuoriuscite accidentali, queste debbano essere immediatamente confinate e trattate. Le metodologie utilizzate a tal scopo comprendono processi fisico-chimici che rompono le molecole del greggio, rendendolo più sensibile agli attacchi degli agenti atmosferici e naturali. Tra queste l’evaporazione delle molecole a basso peso molecolare, l’emulsione della frazione oleosa con l’acqua, la solubilizzazione e la sedimentazione contribuiscono in parte, alla rimozione del greggio, ma vi è un grande contributo anche da parte del mondo microbiologico, soprattutto procariotico, il quale tramite processi di bioremediation degrada e rimuove molti composti tossici. I limiti di queste tecnologie risiedono nell’utilizzo di composti chimici che risultano essere più dannosi del petrolio stesso per l’ecosistema e la saturazione dei microrganismi che riescono ad agire solo su determinati composti. Proprio in virtù della necessità di trovare una metodologia che possa costituire una soluzione innovativa alle problematiche sopracitate, si è pensato alle microalghe. Le microalghe sono organismi unicellulari, microscopici, fotosintetici e fotoautotrofi in grado di rimuovere anidride carbonica dall’atmosfera, utilizzandola nel processo di fotosintesi per produrre energia e rilasciare ossigeno. Esistono diverse classi di microalghe collocabili anche in diversi domini (procarioti ed eucarioti), con caratteristiche e fisiologie diverse, ma l’oggetto del nostro studio sono state le microalghe verdi, eucariotiche e fotoautotrofe. La loro capacità di utilizzare il carbonio organico, modificando il loro metabolismo, divenendo così mixotrofici, le ha rese ottime candidate per il loro impiego nel mondo della bioremediation. Diversi studi si sono concentrati sul loro utilizzo per la bioremediation, ad esempio alcune microalghe sono state in grado di rimuovere solfato, e nitrati in percentuali significative. La microalga più investigata in questo ambito è stata Cholrella spp. che ha rimosso dopo 5 giorni di trattamento, l’80% di petrolio emulsionato nel mezzo in uno studio del 2020, e ha ridotto in maniera efficiente i PAHs presenti in uno studio del 2013. In questo elaborato di tesi la microalga analizzata è stata Haematococcus pluvialis, nota all’ambiente scientifico per la sua capacità di produrre uno degli antiossidanti più potenti in natura, l’astaxantina (ASX) ma mai considerata per la bioremediation di idrocarburi di origine petrolifera. Lo scopo di questo progetto di dottorato è stato quello di ricercare una nuova e innovativa tecnologia che grazie all’impiego di microalghe possa rimuovere efficacemente gli idrocarburi petroliferi in matrici acquose, creando diversi genotipi in grado di agire in maniera più efficiente rispetto al genotipo non mutato (WT). Grazie a un processo di pressione selettiva si è indotto H. pluvialis a mutare e ad adattarsi ad una condizione estremamente tossica. Una volta selezionati diversi ceppi mutanti, questi sono stati seguiti nella loro crescita e nella loro capacità di produrre astaxantina, comparandoli sempre con il WT. Poiché le nostre conoscenze non permettevano di determinare se le mutazioni avvenute fossero genetiche o epigenetiche, è stata effettuata anche un’analisi genetica tramite RAPD-PCR su trascritto per evidenziare le diversità di espressione dei diversi genotipi tra di loro e con la specie non mutata, in condizioni di non stress e in generazioni successive. È stato così possibile ottenere tramite “cluster analysis” un dendrogramma che evidenziasse le distanze di espressione genetica tra essi. Questo risultato indica che la differenza di espressione in condizioni standard è ricollegabile a modificazioni genetiche alla base. Successivamente dopo aver provato diverse condizioni di stress e bioremediation, sono stati fatti crescere in presenza dell’1% di greggio puro in condizioni anaerobiche, per valutare l’attività di rimozione e/o degradazione. Le analisi chimiche effettuate solo dopo 10 giorni di trattamento hanno subito evidenziato come la quantità di carbonio presente nel mezzo di coltura era quasi raddoppiata mentre altri parametri come fluoruri, cloruri e solfati sono diminuiti. Dopo 40 giorni di trattamento, analisi preliminari tramite GS-MS e GC-FID hanno evidenziato come sia nel mezzo acquoso, che nella biomassa algale, vi erano idrocarburi e sostanze oleose, risultati confermati, poi, da un’analisi quantitativa a 20 e 160 giorni in cui si evidenzia, con diverse cinetiche, una diminuzione degli idrocarburi, che per il ceppo mutante PA1004 erano stati totalmente rimossi sia dal mezzo che dalla biomassa. Inoltre, dopo 120 giorni è stata ripetuta l’analisi genetica per valutare eventuali meccanismi adattativi avvenuti in seguito allo stress indotto dal petrolio. Mantenendo le stesse e identiche condizioni di amplificazione (primer, temperature e concentrazioni) il risultato della “cluster analysis” ha dimostrato come, effettivamente, ci fossero stati dei cambiamenti a livello di espressione genetica, portando a una riorganizzazione delle distanze ottenute precedentemente in condizioni ottimali. Queste distanze rispecchiano il comportamento dei genotipi in presenza dell’agente esogeno. Alcuni hanno rimosso totalmente il petrolio, altri hanno agito solo sulla frazione oleosa contribuendo a creare un particolato bituminoso raggruppandosi come da dendrogramma generato. L’assenza di idrocarburi all’interno del mezzo acquoso e della biomassa algale costituisce di per sé un risultato estremamente interessante andando a confermare gli obiettivi preposti all’inizio di questo percorso di ricerca. Non solo H. pluvialis è stato in grado di sopravvivere in presenza di petrolio greggio, ma anche i genotipi generati, sono risultati essere una promettente alternativa nella bioremediation degli idrocarburi. In futuro ci si predispone ad analizzare non solo le pathways coinvolte dalle mutazioni, per comprendere i meccanismi sottesi a tale processo, ma anche analizzare i sottoprodotti generati per riutilizzarli in un’ottica di economia circolare atta alla rimozione e al riutilizzo di un prodotto potenzialmente dannoso per l’uomo e l’ambiente

Primer relevamiento de marcadores de resistencia a antibióticos en Enterobacteriaceae en Cochabamba, Bolivia

Se llevó a cabo un relevamiento molecular de la resistencia a antibióticos de importancia clínica en aislamientos recuperados en Cochabamba, Bolivia. Se estudiaron los genes codificantes de β-lactamasas de espectro extendido y de resistencia a quinolonas de localización plasmídica (PMQR) en un total de 101 aislamientos de enterobacterias resistentes a oximinocefalosporinas recuperados en distintos centros de salud, durante 4 meses (2012-2013). En todos ellos se detectó la presencia de cefotaximasas, las CTX-M grupo 1 fueron las más prevalentes (88,1%). La presencia de blaOXA-1 se detectó en el 76,4% de estos aislamientos. Se observó una elevada proporción de aislamientos resistentes a quinolonas. El gen aac(6′)-Ib-cr fue el determinante PMQR más frecuentemente identificado (83%). Además, 6 aislamientos resultaron ser portadores de qnrB. Por otro lado, cabe remarcar que 7 Escherichia coli presentaron qepA1 (6) y oqxAB (1); se documenta así por primera vez la presencia de oqxAB en Bolivia. Este estudio constituye el primer relevamiento de marcadores de resistencia en aislamientos clínicos de enterobacterias en Cochabamba, Bolivia; de este modo se contribuye al conocimiento regional de la situación epidemiológica, la cual presenta un escenario similar al observado en el resto de Latinoamérica.A molecular survey was conducted in Cochabamba, Bolivia, to characterize the mechanism involved in the resistance to clinically relevant antibiotics. Extended Spectrum β-lactamase encoding genes and plasmid-mediated quinolone resistance (PMQR) markers were investigated in a total of 101 oxyimino-cephalosporin-resistant enterobacteria recovered from different health centers during four months (2012?2013). CTX-M enzymes were detected in all isolates, being the CTX-M-1 group the most prevalent (88.1%). The presence of blaOXA-1 was detected in 76.4% of these isolates. A high quinolone resistance rate was observed among the included isolates. The aac(6′)-Ib-cr gene was the most frequent PMQR identified (83.0%). Furthermore, 6 isolates harbored the qnrB gene. Interestingly, qepA1 (6) and oqxAB (1), were detected in 7 Escherichia coli, being the latter the first to be reported in Bolivia. This study constitutes the first molecular survey on resistance markers in clinical enterobacterial isolates in Cochabamba, Bolivia, contributing to the regional knowledge of the epidemiological situation. The molecular epidemiology observed herein resembles the scene reported in South America.Fil: Saba Villarroel, Paola M.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Gutkind, Gabriel Osvaldo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Di Conza, José Alejandro. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Radice, Marcela Alejandra. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

MDA5-positive dermatomyositis: An uncommon entity in Europe with variable clinical presentations

Clinically amyopathic dermatomyositis (CADM), described almost 50 years ago, is defined on the basis of still not validated criteria and characterized by skin findings almost without muscle weakness. Autoantibodies directed against the cytosolic pathogen sensor MDA5 (CADM 140) can mark this subtype of dermatomyositis which has been reported to associate, in particular ethnic groups, with severe progressive interstitial lung disease, poor prognosis and an hyperferritinemic status resembling hemophagocytic-like syndromes. MDA5 may be relevant in that Interferon-signature claimed to characterize inflammatory myopathies and dermatomyosits itself, but its role is not clear. However, the titre of anti-MDA5 autoantibodies seems to correlate with the outcome. In Caucasian populations the association between anti-MDA5 positive CADM and rapidly progressive interstitial lung disease seems to be weaker, but the limited numbers of patients described so far could explain the lack of statistical significance. As a fact, European patients with circulating anti-MDA5 autoantibodies may be clinically inhomogeneous and exhibit different rates of severity. The two patients affected by anti-MDA5 positive dermatomyositis described hereafter provide a clear example of the extreme variability of the disease in terms of laboratory findings and clinical features

Bioremediation of Crude Oil by Haematococcus Pluvialis: A Preliminary Study

Nowadays, oil pollution is one of the main environmental problems. The current methods for recovering spills mainly involve chemical agents, but scientific research has focused on more natural and less harmful techniques for the environment, including a consortium of bacteria and microalgae to clean up water contaminated by hydrocarbons. The purpose of this preliminary study was to evaluate the ability of a microalga belonging to Chlorophyceae to grow in the presence of crude oil and remove the principal contaminants. H. pluvialis, which is usually used for nutraceutical purposes, thanks to the production of astaxanthin, was able to grow in anaerobic conditions, varying its metabolism from autotrophic to heterotrophic, exploiting the carbon present in the solution deriving from the presence of 1% of crude oil. Furthermore, the results of bioremediation showed a relevant reduction in chemical pollutants such as nitrate, fluoride, sulfate, and phosphate. The most important aspect of the study was the reduction after 160 days in the hydrocarbon concentration inside not only the culture medium (−32%) but also the algal biomass (−80.25%), demonstrating an optimized degradation rather than a simple absorption inside the alga

Crude Oil Bioremediation: From Bacteria to Microalgae

Crude oil is one of the major pollutants present. Its extraction and processing generate processing waters contaminated by hydrocarbons which are harmful to both human health and the flora and fauna that come into contact with it. Hydrocarbon contamination can involve soil and water, and several technologies are used for recovery. The most used techniques for the recovery of spilt oil involve chemical-physical methods that can remove most of the pollutants. Among these, must consider the bioremediation by microorganisms, mostly bacterial capable of degrading many of the toxic compounds contained within the petroleum. Microalgae participate in bioremediation indirectly, supporting the growth of degrading bacteria, and directly acting on contaminants. Their direct contribution is based on the activation of various mechanisms ranging from the production of enzymes capable of degrading hydrocarbons, such as lipoxygenases, to the attack through the liberation of free radicals. The following review analyzed all the works published in the last ten years concerning the ability of microalgae to remove hydrocarbons, intending to identify in these microorganisms an alternative technology to the use of bacteria. The advantages of using microalgae concern not only their ability to remove toxic compounds and release oxygen into the atmosphere but their biomass could then be used in a circular economy process to produce biofuels

Genetic Improvement to Obtain Specialized Haematococcus pluvialis Genotypes for the Production of Carotenoids, with Particular Reference to Astaxanthin

Nowadays, the search for natural substances with a high nutraceutical effect positively impact the world market. Among the most attractive macromolecules are antioxidants, capable of preventing the development of various pathologies. Astaxanthin (ASX) is antioxidant molecule produced by the microalga H. pluvialis as a response to different types of stress. Usually, astaxanthin production involves the first phase of accumulation of the biomass of H. pluvialis (green phase), which is then stressed to stimulate the biosynthesis and accumulation of ASX (red phase). In this study, the H. pluvialis wild-type strain was subjected to random mutagenesis by UV. Among the different mutant strains obtained, only two showed interesting bio-functional characteristics, such as a good growth rate. The results demonstrated that the HM1010 mutant not only has a higher growth trend than the WT mutant but accumulates and produces ASX even in the green phase. This innovative genotype would guarantee the continuous production of ASX, not linked to the two-step process and the uniqueness of the product obtained

Colorimetric Detection of Perfluorinated Compounds by All-Polymer Photonic Transducers

We report on the highly sensitive optical and colorimetric detection of perfluorinated compounds in the vapor phase achieved by all-polymer dielectric mirrors. High optical quality and uniformly distributed Bragg reflectors were fabricated by alternating thin films of poly(N-vinylcarbazole) and Hyflon AD polymers as high and low refractive index medium, respectively. A new processing procedure has been developed to compatibilize the deposition of poly(N-vinylcarbazole) with the highly solvophobic Hyflon AD polymer layers to achieve mutual processability between the two polymers and fabricate the devices. As a proof of principle, sensing measurements were performed using the Galden HT55 polymer as a prototype of the perfluorinated compound. The Bragg stacks show a strong chromatic response upon exposure to this compound, clearly detectable as both spectral and intensity variations. Conversely, Bragg mirrors fabricated without fluorinated polymers do not show any detectable response, demonstrating that the Hyflon AD polymer acts as the active and selective medium for sensing perfluorinated species. These results demonstrate that organic dielectric mirrors containing perfluorinated polymers can represent an innovative colorimetric monitoring system for fluorinated compounds, suitable to improve both environmental safety and quality of life

ACKR3 promotes CXCL12/CXCR4-mediated cell-to-cell-induced lymphoma migration through LTB4 production.

Chemotaxis is an essential physiological process, often harnessed by tumors for metastasis. CXCR4, its ligand CXCL12 and the atypical receptor ACKR3 are overexpressed in many human cancers. Interfering with this axis by ACKR3 deletion impairs lymphoma cell migration towards CXCL12. Here, we propose a model of how ACKR3 controls the migration of the diffused large B-cell lymphoma VAL cells in vitro and in vivo in response to CXCL12. VAL cells expressing full-length ACKR3, but not a truncated version missing the C-terminus, can support the migration of VAL cells lacking ACKR3 (VAL-ko) when allowed to migrate together. This migration of VAL-ko cells is pertussis toxin-sensitive suggesting the involvement of a Gi-protein coupled receptor. RNAseq analysis indicate the expression of chemotaxis-mediating LTB4 receptors in VAL cells. We found that LTB4 acts synergistically with CXCL12 in stimulating the migration of VAL cells. Pharmacologic or genetic inhibition of BLT1R markedly reduces chemotaxis towards CXCL12 suggesting that LTB4 enhances in a contact-independent manner the migration of lymphoma cells. The results unveil a novel mechanism of cell-to-cell-induced migration of lymphoma

New and old biomarkers in the differential diagnosis of lung cancer: Pro-gastrin-releasing peptide in comparison with neuron-specific enolase, carcinoembryonic antigen, and CYFRA 21-1.

Background: Testing for circulating biomarkers in lung cancer is hampered by the insufficient specificity. We aimed to assess the relative diagnostic accuracy of pro-gastrin-releasing peptide (ProGRP) for the differential diagnosis of small cell lung cancer and compare it with more conventional biomarkers. Methods: We enrolled a cohort of 390 patients with a clinical suspicion of lung cancer and for whom a histologic assessment was available. Serum or plasma samples were assessed for ProGRP, carcinoembryonic antigen, CYFRA 21-2, and neuron-specific enolase. The performance of each biomarker in discriminating the small cell lung cancer and squamous cell carcinoma/adenocarcinoma from non-malignant lung disease, and small cell lung cancer from squamous cell carcinoma/adenocarcinoma, was assayed by receiver operating characteristic curve analysis. Results: At the cut-off levels suggested by the manufacturers, ProGRP and neuron-specific enolase showed an almost identical sensitivity of 55.2% and 55.6%, respectively, in discriminating small cell lung cancer with respect to non-malignant lung disease. In order to quantify the added value of ProGRP to other conventional markers, we ran a multivariable logistic regression analysis, but the results showed that no markers improve the performance of ProGRP. Conclusions: ProGRP and neuron-specific enolase individually appear more accurate than other conventional biomarkers for small cell lung cancer, but the union of two markers does not increase the accuracy. The very small target group of patients with small cell lung cancer is a limitation of this study, which can explain why ProGRP alone does not show a sensitivity higher than neuron-specific enolase, as reported by other authors