Fungal and Bacterial Biodeterioration of Outdoor Canvas Paintings: The Case of the Cloisters of Quito, Ecuador

The historic center of Quito, Ecuador, was one of the first World Cultural Heritage Sites declared by UNE-ISCO in 1978. here are numerous religious buildings built during the Spanish colonial period reflecting the cultural her- itage in this area. Between them, the cloisters of San Francisco, Santo Domingo, and Santa Clara should be highlighted. The specific problems of conservation of the outdoor canvas paintings are not well known at the moment. The objective of this paper is to achieve a conservation study of the canvas paintings exhibited in these three cloisters of the historic center of Quito in order to identify the microbial agents and the main bioclimatic parameters of deterioration. For this, a study of the state of conservation of five canvas paintings has been carried out, as well as a sampling and identification of the main microorganisms present on the obverse and reverse of the works, employing diverse techniques, traditional and biomolecular ones. An analysis of climatic conditions has also been achieved in the cloister of San Francisco. The results of the study indicate that the exhibition conditions in the cloisters are really problematic for the conservation of paintings. Important biodeteriorating agents have been isolated, including fungi and bacteria species belonging, among others, to the genera Bacillus, Penicillium, Alternaria, Mucor, and Aspergillus. We have also researched its relationship with the deterioration state of the artworks and the exhibi ion conditions in each case, proposing guidelines for the proper conservation of this important World Cultural Heritage

Artificial Protein Coronas Enable Controlled Interaction with Corneal Epithelial Cells: New Opportunities for Ocular Drug Delivery

Topical administration is the most convenient route for ocular drug delivery, but only a minor fraction is retained in the precorneal pocket. To overcome this limitation, numerous drug delivery systems (DDS) have been developed. The protein corona (PC) is the layer of biomolecules (e.g., proteins, sugars, lipids, etc.) that forms around DDS in physiological environments by non-covalent interaction. The PC changes the DDS physical–chemical properties, providing them with a completely novel biological identity. The specific involvement of PC in ocular drug delivery has not been addressed so far. To fulfill this gap, here we explored the interaction between a library of four cationic liposome-DNA complexes (lipoplexes) and mucin (MUC), one of the main components of the tear film. We demonstrate that MUC binds to the lipoplex surface shifting both their size and surface charge and reducing their absorption by primary corneal epithelial cells. To surpass such restrictions, we coated lipoplexes with two different artificial PCs made of Fibronectin (FBN) and Val-Gly-Asp (VGA) tripeptide that are recognized by receptors expressed on the ocular surface. Both these functionalizations remarkedly boosted internalization in corneal epithelial cells with respect to pristine (i.e., uncoated) lipoplexes. This opens the gateway for the exploitation of artificial protein corona in targeted ocular delivery, which will significantly influence the development of novel nanomaterials

Combined effects of PI3K and SRC kinase inhibitors with imatinib on intracellular calcium levels, autophagy, and apoptosis in CML-PBL cells

Imatinib induces a complete cytogenetic regression in a large percentage of patients affected by chronic myeloid leukemia (CML) until mutations in the kinase domain of BCR-ABL appear. Alternative strategies for CML patients include the inhibition of phosphatidylinositol 3-kinase (PI3K)-Akt-mammalian target of rapamycin (mTOR ) pathway, which is constitutively activated in leukemia cells and seems important for the regulation of cell proliferation, viability, and autophagy. In this study, we verified the effect of imatinib mesylate (IM), alone or in association with LY294002 (LY) (a specific PI3K protein tyrosine kinase inhibitor) or 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]- pyrimidine (PP1) (a Src tyrosine kinase inhibitor), on viability, intracellular calcium mobilization, apoptosis, and autophagy, in order to verify possible mechanisms of interaction. Our data demonstrated that PP1 and LY interact synergistically with IM by inducing apoptosis and autophagy in Bcr/Abl+ leukemia cells and this mechanism is related to the stress of the endoplasmic reticulum (ER ). Our findings suggest a reasonable relationship between apoptotic and autophagic activity of tyrosine kinase inhibitors (TKIs) and the functionality of smooth ER Ca2+-AT Pase and inositol triphosphate receptors, independently of intracellular calcium levels. Therapeutic strategies combining imatinib with PI3K and/or Src kinase inhibitors warrant further investigations in Bcr/Abl+ malignancies, particularly in the cases of imatinib mesylate-resistant diseas

Modulation of apoptosis by caprine herpesvirus 1 infection in a neuronal cell line

Caprine herpesvirus type 1 (CpHV-1), like other members of the alpha subfamily of herpesviruses, establishes latent infections in trigeminal ganglion neurons. Our groups previously demonstrated that CpHV-1 induces apoptosis in goat peripheral blood mononuclear cells and in an epithelial bovine cell line, but the ability of CpHV-1 to induce apoptosis in neuronal cells remains unexplored. In this report, the susceptibility of Neuro 2A cells to infection by CpHV-1 was examined. Following infection of cultured cells with CpHV-1, expression of cell death genes was evaluated using real-time PCR and Western blot assays. Analysis of virus-infected cells revealed activation of caspase-8, a marker for the extrinsic pathway of apoptosis, and caspase-9, a marker for the intrinsic pathway of apoptosis at 12 and 24 h post-infection. Significant increase in the levels of cleaved caspase-3 was also observed at the acme of cytopathic effect at 24 h post-infection. In particular, at 3 and 6 h post-infection, several proapototic genes were under-expressed. At 12 h post-infection several proapototic genes such as caspases, TNF, Cd70, and Traf1 were over expressed while Bcl2a1a, Fadd, and TNF genes were underexpressed. In conclusion, the simultaneous activation of caspase-8 and caspase-9 suggests that CpHV-1 can trigger the death-receptor pathway and the mitochondrial pathway separately and in parallel. Our findings are significant because this is the first published study showing the effect of CpHV-1 infection in neuronal cells in terms of gene expression and apoptosis modulation

Effect of rMnSOD on sodium reabsorption in renal proximal tubule in Ochratoxin A - treated rats

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium that represent toxic real threat for human beings and animal health. In this study we evaluated the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) on oxidative stress and on the alterations of fluid reabsorption in renal proximal tubule (PT) as possible causes of OTA nephrotoxicity. Finally, we have measured the concentration of O2 - in the kidney through dihydroethidium assay (DHE) and nitric oxide (NO) concentration through nitrites and nitrates assay. Male Sprague Dawley rats weighing 120-150 g were treated for 14 days by gavage, as follows: Control group, 12 rats received a corresponding amount of saline solution (including 10% DMSO); rMnSOD group, 12 rats treated with rMnSOD (10 μg/kg bw); OTA group, 12 rats treated with OTA (0,5 mg/Kg bw) dissolved in 10% DMSO and then scaled to required volume with corn oil; rMnSOD + OTA, 12 rats treated with rMnSOD (10 μg /kg bw) plus OTA (0,5 mg/Kg bw). Our results have shown that rMnSOD restores the alteration of reabsorption in PT in rats treated with OTA plus rMnSOD, probably through the response to pressure natriuresis, where nitric oxide plays a key role. Moreover, rMnSOD prevents the nephrotoxicity induced by OTA probably restoring the balance between superoxide and NO that is most probably the cause of hypertension and renal functional alterations through the inhibition of NO synthase. In conclusion these data provide important information for understanding of mechanism of toxic action of OTA

The Role of Curcumin in Prostate Cancer Cells and Derived Spheroids

A major challenge in the clinical management of prostate cancer (PC) is to inhibit tumor growth and prevent metastatic spreading. In recent years, considerable efforts have been made to discover new compounds useful for PC therapy, and promising advances in this field were reached. Drugs currently used in PC therapy frequently induce resistance and PC progresses toward metastatic castration-resistant forms (mCRPC), making it virtually incurable. Curcumin, a commercially avail- able nutritional supplement, represents an attractive therapeutic agent for mCRPC patients. In the present study, we compared the effects of chemotherapeutic drugs such as docetaxel, paclitaxel, and cisplatin, to curcumin, on two PC cell lines displaying a different metastatic potential: DU145 (moder- ate metastatic potential) and PC-3 (high metastatic potential). Our results revealed a dose-dependent reduction of DU145 and PC-3 cell viability upon treatment with curcumin similar to chemotherapeutic agents (paclitaxel, cisplatin, and docetaxel). Furthermore, we explored the EGFR-mediated signaling effects on ERK activation in DU145 and PC-3 cells. Our results showed that DU145 and PC-3 cells overexpress EGFR, and the treatment with chemotherapeutic agents or curcumin reduced EGFR expression levels and ERK activation. Finally, chemotherapeutic agents and curcumin reduced the size of DU145 and PC-3 spheroids and have the potential to induce apoptosis and also in Matrigel. In conclusion, despite different studies being carried out to identify the potential synergistic curcumin combinations with chemopreventive/therapeutic efficacy for inhibiting PC growth, the results show the ability of curcumin used alone, or in combinatorial approaches, to impair the size and the viability of PC-derived spheroids

Pathological Role of Peptidyl-Prolyl Isomerase Pin1 in the Disruption of Synaptic Plasticity in Alzheimer’s Disease

Synaptic loss is the structural basis for memory impairment in Alzheimer’s disease (AD). While the underlying pathological mechanism remains elusive, it is known that misfolded proteins accumulate as β-amyloid (Aβ) plaques and hyperphosphorylated Tau tangles decades before the onset of clinical disease. The loss of Pin1 facilitates the formation of these misfolded proteins in AD. Pin1 protein controls cell-cycle progression and determines the fate of proteins by the ubiquitin proteasome system. The activity of the ubiquitin proteasome system directly affects the functional and structural plasticity of the synapse. We localized Pin1 to dendritic rafts and postsynaptic density (PSD) and found the pathological loss of Pin1 within the synapses of AD brain cortical tissues. The loss of Pin1 activity may alter the ubiquitin-regulated modification of PSD proteins and decrease levels of Shank protein, resulting in aberrant synaptic structure. The loss of Pin1 activity, induced by oxidative stress, may also render neurons more susceptible to the toxicity of oligomers of Aβ and to excitation, thereby inhibiting NMDA receptor-mediated synaptic plasticity and exacerbating NMDA receptor-mediated synaptic degeneration. These results suggest that loss of Pin1 activity could lead to the loss of synaptic plasticity in the development of AD

Definition of the pharmacological profile of conventional chemotherapeutic drugs based on pharmacogenetics determinant in CRC patients

Abstract Cancer patients do not always respond in the same way to the same drug treatment: the administration of the same dose of an antiblastic drug in a population of patients induces the manifestation of a wide range of toxicity, which sometimes can even be deadly. The intersubject variation, that is observed in efficacy and toxicity of anticancer drugs, can be determined by complex interactions among components of the physiological, environmental and genetic factors. The activities carried out in the framework of the PhD project have focused the attention on the influence of genetic factors on the outcome of drug treatment, identifying possible prognostic and predictive genetic biomarkers. To evaluate the correlation between genotype and phenotype of the patient, we considered a pharmacogenetics study which defined the relationship between a specific polymorphism (UGT1A1 * 28) and the effects of the drug. A phase Ib clinical trial was conducted at the CRO, Aviano (PN). The purpose of this study was to modulate the dosage of CPT-11 in the presence of the inhibitor angiogenetic BV for each individual patient, based on genotype of UGT1A1, in order to obtain an improvement of the therapeutic index . To do this, it was considered necessary to develop a specific method of pharmacokinetic analysis and we proceeded as follows : * Development of a quantitative method for the analysis of the drug of interest * Validation of the method in accordance with FDA guidelines * Measurement of plasma concentrations of the drug * Determination of the pharmacokinetics of the drug * Correlation of pharmacokinetics and pharmacogenetics We enrolled patients with a histological diagnosis of metastatic colorectal adenocarcinoma, naïve or treated with adjuvant chemotherapy (excluding irinotecan), and corresponding to the criteria for eligibility/exclusion described in the protocol. The patients were assigned to their treatment group based on genotype (*1/*1 or *1/*28) until the completion of recruitment for each dose level in each group of patients. Patients with genotype *28/*28 were excluded because of high risk of toxicity. The starting dose of irinotecan administered in patients carrying the *1 (wild-type and heterozygous) was 260mg/m2. The dose of BV was 5mg/kg and it was administered in infusion after two weeks. In the treatment group with lower dose, the irinotecan dose was increased to 310 and 370mg/m2 in case of lack of toxicity. The evaluation of pharmacokinetic and pharmacodynamic interactions between bevacizumab (BV) and irinotecan (CPT-11) was conducted describing the pharmacokinetic profile of the CPT -11 (and its metabolites) in the absence and in the presence of BV in the same patient. The obteined pharmacokinetic parameters exclude an effect of BV on the pharmacokinetics of CPT-11. However, the registered values ??of MTD were lower than those determined by previous works with FOLFIRI alone, and that might suggest that the addition of BV results in changes in the manifestation of toxicity in the high-dose regimens. The drugs that have been investigated in this protocol have been present in the clinical practice since several years, and the high variability of the response and toxicity makes it essential the definition of useful criteria for treatment personalization. Basic criteria for personalization of therapy are clinical parameters, like gender and age of the patients. Many of the elderly subjects, in fact, receive reduced treatments comparing standard clinical protocols, with a considerable reduction of the dosage of the drug or of the number of therapy cycles. Due to their physiological characteristics, the older cancer patients reported greater toxicity effects associated with the treatment. In clinical trials, elderly patients are under-represented because they don’t satisfy all the inclusion criteria, like comorbid conditions and baseline functional status of the patient. Similarly, women are often excluded from some protocols especially for the variability that characterizes the hormonal status. In a second part of the PhD project, we considered a very large series of patients with CRC treated with other medications associated with fluoropyrimidine (FOLFOX or FOLFIRI) at the CRO, Aviano (PN), and other centers participating in the program. We performed a pharmacogenetic study to see if there are genetic biomarkers useful to define a personalization of therapy for subjects considered elderly (age at diagnosis >70 years). In particular, the aim was to identify possible pharmacogenetic (PG) determinants which influence drug effects (toxicity and response) in a different way between young and elderly subjects, and between the two genders (male and female). We analyzed several polymorphisms of genes coding for proteins involved in the mechanisms of activation, metabolism and elimination of drugs mainly used in therapies against CRC and other polymorphisms in genes that regulate the cell cycle and involved in the process of carcinogenesis and tumor progression. Many allelic variations confirm an association with the risk of developing cancer risk, while others with the survival of patients. In particular, the variation in the copy number of GSTM1 and GSTT1 genes, showed a correlation with the age of diagnosis of the patients, as well as with the gender. The increase in copies of GSTM1 or GSTT1 reduction are associated with greater survival in male patients older than 70 years (PGSTM1=0.047, HR=3.937, 95% CI=0.89-0:31; PGSTT1=0.039, HR=4.246). Moreover, we further found a specific association of genotype GSTnull with an increased risk of tumor in young subjects, but not in the elderly. It can therefore be assumed that there is a deficiency in the enzymatic activity of glutathione S-transferase deficiency resulting in reduced cell detoxification mechanisms that involve an increase in damage to the DNA damnage. Additional data allowed us to conclude that genetic characteristics may be associated with both gender and age of the patients: there are polymorphisms predictive of risk related to one of the two parameters (MTHFR 1298A>C in young women); polymorphisms localized in repair genes are mainly associated with a greater risk and a lower survival in the elderly population. Translational research is a fundamental step for the application of experimental research to clinical practice. One of the most important aspects in this area is the customization of the therapy that should be considerate important to get treatment optimization especially in the field of oncology.Riassunto Pazienti oncologici, con lo stesso tipo di tumore, non sempre rispondono in modo uguale al medesimo trattamento farmacologico: la somministrazione della stessa dose di un dato farmaco antiblastico in una popolazione di pazienti implica spesso la manifestazione di un vasto range di tossicità, che in alcuni casi può risultare addirittura mortale. La variabilità intersoggettiva che si osserva nell’efficacia e nella tossicità dei farmaci antiblastici impiegati nella chemioterapia può pertanto essere determinata da interazioni complesse tra le componenti fisiologiche, ambientali e fattori genetici individuali. L’attività svolta in ambito del progetto di dottorato ha puntato l’attenzione su come i fattori genetici possano influenzare l’esito di un trattamento farmacologico, individuando dei possibili biomarcatori genetici prognostici e predittivi. Per valutare la correlazione tra genotipo e fenotipo del paziente siamo partiti da uno studio di farmacogenetica che ha definito la relazione esistente tra un determinato polimorfismo (UGT1A1*28) e l’alterazione dell’effetto del farmaco che ne consegue. Successivamente, ci si è indirizzati verso il trasferimento e l’applicazione nella pratica clinica attraverso uno studo clinico di fase Ib (“Studio di fase I guidato dal genotipo dell’irinotecano in combinazione con 5-fluorouracile/leucovorina (FOLFIRI) e bevacizumab in pazienti con carcinoma colonrettale”), condotto presso il Centro di Riferimento Oncologico di Aviano (PN). Lo scopo di tale studio è stato quello di modulare il dosaggio dell’irinotecano (CPT-11) in presenza dell’inibitore angiogenetico bevacizumab (BV), non essendo ancora nota l’interazione tra i due farmaci, in base al genotipo di UGT1A1, al fine di ottenere un miglioramento dell’indice terapeutico per ogni singolo paziente. Per fare ciò, si è ritenuto necessario sviluppare un metodo di analisi di farmacocinetica specifico e si è pensato di procedere nel seguente modo: * Sviluppo di un metodo quantitativo per l’analisi del farmaco d’interesse * Validazione del metodo in accordo con le linee guida dell’FDA * Misurazione delle concentrazioni plasmatiche del farmaco * Determinazione della farmacocinetica del farmaco * Correlazione dei dati di farmacocinetica e farmacogenetica Sono stati arruolati pazienti con diagnosi istologica di adenocarcinoma colorettale (CRC) metastatico, non pretrattati con chemioterapia o trattati con terapia adiuvante (escluso irinotecano), e rispondenti ai criteri di eleggibilità/esclusione previsti dal protocollo. I pazienti sono stati assegnati al loro gruppo di trattamento in base al genotipo (*1/*1 o *1/*28) fino al completamento del reclutamento per ogni livello di dose in ogni gruppo di pazienti. I pazienti con genotipo *28/*28 sono stati esclusi perché ad alto rischio di tossicità. La dose iniziale di irinotecano somministrata nei pazienti portatori dell’allele *1 (wild type ed eterozigoti) è di 260 mg/m2. La dose di BV è di 5 mg/kg, somministrata anch’essa in infusione ogni due settimane. Il dosaggio dell’irinotecano è stato incrementato a 310 e 370 mg/m2 qualora nel gruppo di trattamento con dosaggio più basso non vi sia stata tossicità. La valutazione delle interazioni farmacocinetiche e farmacodinamiche tra bevacizumab (BV) ed irinotecano (CPT-11) è stata condotta descrivendo il profilo farmacocinetico del CPT-11 (e dei suoi metaboliti) in assenza ed in presenza di BV nello stesso paziente. Da una prima analisi dei parametri farmacocinetici sembra che si possa escludere un effetto del BV sulla farmacocinetica dell’irinotecano. La sovrapponibilità del dato farmacocinetico con o senza BV, è valida per entrambi i dosaggi di irinotecano considerati. Tuttavia, sono stati riscontrati dei valori di Dose Massima Tollerata (MTD) inferiori rispetto a quelli determinati da lavori precedenti, e ciò potrebbe suggerire che l’aggiunta del BV comporti una variazione nella manifestazione di tossicità in regimi ad alto dosaggio. Una percentuale non trascurabile di pazienti ha ottenuto una riduzione del numero e delle dimensioni delle lesioni secondarie epatiche tanto da renderle aggredibili chirurgicamente o tramite termoablazione, facendo pertanto concludere che il regime terapeutico si configuri quindi come una conversion therapy. Poiché i farmaci che sono stati studiati in questo protocollo sono presenti nella pratica clinica da diversi anni, la notevole variabilità riscontrata nella risposta e nello sviluppo di tossicità rende indispensabile trovare dei criteri utili alla personalizzazione del trattamento. Criteri fondamentali per la personalizzazione della terapia sono i parametri clinici, principalmente il genere e l’età dei pazienti. Molti dei soggetti anziani, infatti, ricevono dei trattamenti ridotti poiché tollerano meno le terapie dei protocolli clinici standard, con una notevole riduzione del dosaggio di farmaco o del numero di cicli di trattamento. Proprio per le loro caratteristiche fisiologiche, i pazienti oncologici anziani riportano maggiori effetti di tossicità associati al trattamento rispetto ai soggetti definiti giovani. Analogamente, anche le donne sono spesso escluse da alcuni protocolli soprattutto per la variabilità ormonale che caratterizza l’età fertile e il periodo della menopausa. A tale proposito, in una seconda parte del progetto di dottorato, abbiamo considerato una casistica molto ampia di pazienti affetti da CRC trattati con fluoropirimidine associate ad altri farmaci (FOLFIRI o FOLFOX) presso il Centro di Riferimento Oncologico di Aviano (PN) e altri centri aderenti al programma. Lo scopo è stato quello di individuare dei possibili determinanti farmacogenetici (PG) che condizionano gli effetti del farmaco (tossicità e risposta) in maniera differente tra soggetti giovani e anziani, e tra i due generi (maschio e femmina), nonché dei possibili marcatori di rischio tumorale specifici. Sono stati analizzati diversi polimorfismi di geni codificanti per proteine coinvolte nei meccanismi di attivazione, metabolizzazione ed eliminazione dei farmaci principalmente usati nelle terapie contro il CRC e altri polimorfismi di geni che regolano il ciclo cellulare in quanto coinvolti nel processo di carcinogenesi e di progressione tumorale. Molte delle variazioni alleliche analizzate confermano un’associazione con il rischio d’insorgenza del tumore, altre con la sopravvivenza dei pazienti. In particolare, la variazione del copy number dei geni codificanti per l’enzima Glutatione S-trasferasi, GSTT1 e GSTM1, ha messo in evidenza una correlazione con l’età di diagnosi dei pazienti, nonché con il genere. L’aumento delle copie di GSTM1 o la riduzione del GSTT1 sono associati a una maggiore sopravvivenza nei soggetti con età superiore a 70 anni e di sesso maschile (PGSTM1=0.047, HR=3.937, 95% CI=0.31–0.89; PGSTT1=0.039, HR=4.246). Abbiamo ulteriormente riscontrato una specifica associazione del genotipo GSTnull con un aumentato rischio d’insorgenza tumorale nei soggetti giovani, ma non nei soggetti anziani. Si può quindi ipotizzare che vi sia una riduzione dell’attività enzimatica della Glutatione-S Transferasi con conseguente deficit ai meccanismi di detossificazione cellulare che comportano un incremento dei danni a carico del DNA. Gli ulteriori dati ottenuti, ci permettono di concludere che ci possono essere delle differenze associate alle caratteristiche genotipiche relative sia al genere che all’età dei pazienti: ci sono polimorfismi predittivi di rischio prevalentemente associati ad uno dei due generi (MTHFR 1298A>C nelle giovani donne); altri polimorfismi localizzati nei geni del riparo del DNA sono prevalentemente associati ad un rischio maggiore e ad una sopravvivenza minore nella popolazione anziana. È pertanto fondamentale che, grazie alle nuove conoscenze nell’ambito della medicina molecolare e alle nuove tecnologie, si arrivi alla personalizzazione della terapia, al fine di massimizzare la risposta e ridurre gli effetti tossici in ciascun paziente

CDK9 inhibitors in acute myeloid leukemia

Abstract Current treatment for acute myeloid leukemia (AML) is less than optimal, but increased understanding of disease pathobiology and genomics has led to clinical investigation of novel targeted therapies and rational combinations. Targeting the cyclin-dependent kinase 9 (CDK9) pathway, which is dysregulated in AML, is an attractive approach. Inhibition of CDK9 leads to downregulation of cell survival genes regulated by super enhancers such as MCL-1, MYC, and cyclin D1. As CDK9 inhibitors are nonselective, predictive biomarkers that may help identify patients most likely to respond to CDK9 inhibitors are now being utilized, with the goal of improving efficacy and safety