Cambi di conformazione dell'RNA indotti da ioni metallici e intercalanti

Riassunto contenuto della Tesi Luisa Menichetti Recentemente è sorto un grande interesse nei confronti di strutture non classiche degli acidi nucleici, in particolare verso le eliche triple [A. Rich, Gene, 1993, vol. 135, 99-109]. Tale interesse è giustificato tra l’altro dai successi delle terapie geniche in cui giocano un ruolo di primaria importanza le strategie “antigene” e “antisenso”, che consistono nell’indurre la formazione di triple eliche in tratti di DNA o RNA [D. Prosenth, A.L. Guieysse, C. Hélène, Biochim. Biophy. Acta 1999, 1489(1), 181-206]. E’ noto che le triple eliche si formano preferenzialmente lungo sequenze del tipo A-T nel DNA oppure A-U nell’RNA e che la loro formazione è indotta da ioni metallici e da molecole intercalanti. E’ stato recentemente osservato, in uno studio condotto nel laboratorio dove è stata svolta questa tesi, che la formazione della tripla elica nell’RNA in struttura di doppia elica (poly(A)∙poly(U)) è indotta dalla Coralina, un intercalante che si pone a ponte tra due doppie eliche formando un’elica quadrupla, che evolve verso una tripla elica e un’elica singola [Biver et al. Nucleic Acids Research 2010]. D’altra parte, in questo studio, condotto con concentrazione di cloruro di sodio costante, non è stato esplorato il ruolo dei cationi metallici nell’indurre la formazione del triplex poli(A)∙2poli(U). Scopo della tesi è quello di studiare il meccanismo della formazione di triple eliche, indotti da cationi di diversa carica (Na+,Mg2+ e Ni2+) a partire dal poly(A)∙poly(U) in doppia elica. In seguito è stato analizzato l’effetto sulla struttura del poly(A)∙poly(U) prodotto dalla Coralina in presenza dei cationi sopra menzionati. Lo studio ha previsto un’analisi sia termodinamica sia cinetica dei sistemi. Nel primo caso le tecniche utilizzte nello studio sono la fluorimetria (titolazioni spettrofluorimetriche), la spettrofotometria (per l’ottenimento delle curve di melting), il dicroismo circolare e la calorimetria a scansione differenziale. Lo studio cinetico è stato condotto utilizzando il metodo spettrofotometrico. I risultati ottenuti forniscono informazioni circa la capacità relativa dei cationi analizzati (Ni2+ > Mg2+ >> Na+) di stabilizzare strutture multiple nell’RNA, nonché sul meccanismo della loro formazione, in presenza ed in assenza dell’intercalante Coralina. Un importante risultato è quello di aver osservato che, in opportune condizioni saline, aumenta significativamente la stabilità di una quadrupla elica di RNA, tanto che questa non evolve più verso una tripla elica e un’elica singola ma sembra rimanere invece come specie predominante in soluzione

Mg(II) and Ni(II) induce aggregation of poly(rA)poly(rU) to either tetra-aggregate or triplex depending on the metal ion concentration

The ability of magnesium(II) and nickel(II) to induce dramatic conformational changes in the synthetic RNA poly(rA)poly(rU) has been investigated. Kinetic experiments, spectrofluorometric titrations, melting experiments and DSC measurements contribute in shedding light on a complex behaviour where the action of metal ions (Na+, Mg2+, Ni2+), in synergism with other operators as the intercalating dye coralyne and temperature, all concur in stabilising a peculiar RNA form. Mg2+ and Ni2+ (M) bind rapidly and almost quantitatively to the duplex (AU) to give a RNA/metal ion complex (AUM). Then, by the union of two AUM units, an unstable tetraaggregate (UAUA(M2)*) is formed which, in the presence of a relatively modest excess of metal, evolves to the UAUM triplex by releasing a single AM strand. On the other hand, under conditions of high metal content, the UAUA(M2)* intermediate rearranges to give a more stable tetra-aggregate (UAUA(M2)). As concerns the role of coralyne (D), it is found that D strongly interacts with UAUA(M2). Also, in the presence of coralyne, the ability of divalent ions to promote the transition of AUD into UAUD is enhanced, according to the efficiency sequence [Ni2+]≫[Mg2+]≫[Na+]

Tumor-selective peptide-carrier delivery of Paclitaxel increases in vivo activity of the drug

Taxanes are highly effective chemotherapeutic drugs against proliferating cancer and an established option in the standard treatment of ovarian and breast cancer. However, treatment with paclitaxel is associated with severe side effects, including sensory axonal neuropathy, and its poor solubility in water complicates its formulation. In this paper we report the in vitro and in vivo activity of a new form of paclitaxel, modified for conjugation with a tumor-selective tetrabranched peptide carrier (NT4). NT4 selectively targets tumor cells by binding to membrane sulfated glycosaminoglycans (GAG) and to endocytic receptors, like LRP1 and LRP6, which are established tumor markers. Biological activity of NT4-paclitaxel was tested in vitro on MDA-MB 231 and SKOV-3 cell lines, representing breast and ovarian cancer, respectively, and in vivo in an orthotopic mouse model of human breast cancer. Using in vivo bioluminescence imaging, we found that conjugation of paclitaxel with the NT4 peptide led to increased therapeutic activity of the drug in vivo. NT4-paclitaxel induced tumor regression, whereas treatment with unconjugated paclitaxel only produced a reduction in tumor growth. Moreover, unlike paclitaxel, NT4-paclitaxel is very hydrophilic, which may improve its pharmacokinetic profile and allow the use of less toxic dilution buffers, further decreasing its general chemotherapic toxicity

An explainable model of host genetic interactions linked to COVID-19 severity

We employed a multifaceted computational strategy to identify the genetic factors contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing (WES) dataset of a cohort of 2000 Italian patients. We coupled a stratified k-fold screening, to rank variants more associated with severity, with the training of multiple supervised classifiers, to predict severity based on screened features. Feature importance analysis from tree-based models allowed us to identify 16 variants with the highest support which, together with age and gender covariates, were found to be most predictive of COVID-19 severity. When tested on a follow-up cohort, our ensemble of models predicted severity with high accuracy (ACC = 81.88%; AUCROC = 96%; MCC = 61.55%). Our model recapitulated a vast literature of emerging molecular mechanisms and genetic factors linked to COVID-19 response and extends previous landmark Genome-Wide Association Studies (GWAS). It revealed a network of interplaying genetic signatures converging on established immune system and inflammatory processes linked to viral infection response. It also identified additional processes cross-talking with immune pathways, such as GPCR signaling, which might offer additional opportunities for therapeutic intervention and patient stratification. Publicly available PheWAS datasets revealed that several variants were significantly associated with phenotypic traits such as "Respiratory or thoracic disease", supporting their link with COVID-19 severity outcome.A multifaceted computational strategy identifies 16 genetic variants contributing to increased risk of severe COVID-19 infection from a Whole Exome Sequencing dataset of a cohort of Italian patients

The polymorphism L412F in TLR3 inhibits autophagy and is a marker of severe COVID-19 in males

The polymorphism L412F in TLR3 has been associated with several infectious diseases. However, the mechanism underlying this association is still unexplored. Here, we show that the L412F polymorphism in TLR3 is a marker of severity in COVID-19. This association increases in the sub-cohort of males. Impaired macroautophagy/autophagy and reduced TNF/TNFα production was demonstrated in HEK293 cells transfected with TLR3L412F-encoding plasmid and stimulated with specific agonist poly(I:C). A statistically significant reduced survival at 28 days was shown in L412F COVID-19 patients treated with the autophagy-inhibitor hydroxychloroquine (p = 0.038). An increased frequency of autoimmune disorders such as co-morbidity was found in L412F COVID-19 males with specific class II HLA haplotypes prone to autoantigen presentation. Our analyses indicate that L412F polymorphism makes males at risk of severe COVID-19 and provides a rationale for reinterpreting clinical trials considering autophagy pathways. Abbreviations: AP: autophagosome; AUC: area under the curve; BafA1: bafilomycin A1; COVID-19: coronavirus disease-2019; HCQ: hydroxychloroquine; RAP: rapamycin; ROC: receiver operating characteristic; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; TLR: toll like receptor; TNF/TNF-α: tumor necrosis factor

Neurotensin branched peptide as a tumor-targeting agent for human bladder cancer

Despite recent advances in multimodal therapy, bladder cancer still ranks ninth in worldwide cancer incidence. New molecules which might improve early diagnosis and therapeutic efficiency for tumors of such high epidemiological impact therefore have very high priority. In the present study, the tetrabranched neurotensin peptide NT4 was conjugated with functional units for cancer-cell imaging or therapy and was tested on bladder cancer cell lines and specimens from bladder cancer surgical resections, in order to evaluate its potential for targeted personalized therapy of bladder cancer. Fluorophore-conjugated NT4 distinguished healthy and cancer tissues with good statistical significance ( < 0.05).NT4 conjugated to methotrexate or gemcitabinewas cytotoxic for human bladder cancer cell lines at micromolar concentrations.Their selectivity for bladder cancer tissue and capacity to carry tracers or drugsmake NT4 peptides candidate tumor targeting agents for tracing cancer cells and for personalized therapy of human bladder cancer

Protective role of benzoselenophene derivatives of resveratrol on the induced oxidative stress in intestinal myofibroblasts and osteocytes

Resveratrol (RE), a polyphenolic compound present in some food and plants, is characterized by anti-inflammatory and antioxidant properties. However, it is quickly metabolized with consequent loss of its efficacy. In this study, the antioxidant effect of 2-phenyl-benzoselenophene derivatives (VD0, VD1 and VD2) was detected in intestinal myofibroblast and osteocyte cell lines in which the oxidative stress was induced by GSH depletion or starvation, respectively. In fact, the oxidative stress is involved in pathogenesis of inflammatory bowel diseases (IBD) and in increased osteoclastogenesis in osteoporosis. Our results show that these derivatives have major antioxidant power in reducing and/or restoring radical oxygen species to control values than RE itself in both cell types. Moreover, derivatives have different antioxidant capacity in myofibroblasts and in osteocytes and this can be due to different degree of oxidative stress and structural characteristics of these compounds. Some of the synthesized RE analogs have shown anti-bacterial role in IBD and anti-resorptive activity in bone pathologies related to inflammatory and osteoporotic processes. Thus, we suggest benzoselenophene derivatives as good candidates for alternative therapy and/or therapeutic support in these pathologie