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Background Few studies have investigated the role of the phosphodiesterase type 5A (PDE5A) isoenzyme in female genital tissue disorders, exclusively taken from cadavers, as well as the epigenetic mechanisms responsible for the regulation of PDE5A levels. Aim The aim was to study the in vivo association between microRNA (miRNA) expression and the expression levels of PDE5A in women with female genital arousal disorder (FGAD) compared with healthy women. Methods Premenopausal women affected by FGAD (cases) and sexually healthy women (control group) underwent microbiopsy of the periclitoral anterior vaginal wall for the collection of tissue samples. Computational analyses were preliminarily performed in order to identify miRNAs involved in the modulation of PDE5A by using miRNA–messenger RNA interaction prediction tools. Differences in the expression levels of miRNAs and PDE5A were finally investigated in cases and control subjects by using the droplet digital polymerase chain reaction amplification system and stratifying women considering their age, number of pregnancies, and body mass index. Outcomes Expression levels of miRNAs were able to target PDE5A and the tissue expression in women with FGAD compared with healthy women. Results The experimental analyses were performed on 22 (43.1%) cases and 29 (56.9%) control subjects. Two miRNAs with the highest interaction levels with PDE5A, hsa-miR-19a-3p (miR-19a) and hsa-miR-19b-3p (miR-19b), were identified and selected for validation analyses. A reduction of the expression levels of both miRNAs was observed in women with FGAD compared with the control subjects (P < .05). Moreover, PDE5A expression levels were higher in women with FGAD and lower in women without sexual dysfunctions (P < .05). Finally, a correlation between body mass index and the expression levels of miR-19a was found (P < .01). Clinical Implications Women with FGAD had higher levels of PDE5 compared with control subjects; therefore, the administration of PDE5 inhibitors (PDE5 inhibitors) could be useful in women with FGAD. Strengths and Limitations The strength of the current study was to analyze genital tissue obtained in vivo from premenopausal women. A limitation was to not investigate other factors, including endothelial nitric oxide synthetases, nitric oxide, and cyclic guanosine monophosphate. Conclusion The results of the present study indicate that the modulation of selected miRNAs could influence PDE5A expression in genital tissues in healthy women or in those with FGAD. Such findings further suggest that treatment with PDE5 inhibitors, as a modulator of PDE5A expression, could be indicated for women with FGAD.

Ammonium ion/carboxylate ion pairing is a key interaction ubiquitous in biological systems, but amine recognition by ionizable molecular receptors, mediated by host-to-guest proton transfer, has too often been overlooked as a design element for molecular recognition. This survey will show that proton transfer mediated recognition is a powerful and versatile tool that can be made to work with different amines and diverse macrocyclic scaffolds, such as crown ethers, calixarenes or pillararenes. We will trace the history of this recognition motif since Cram's first report half a century ago down to the latest applications in supramolecular sensing, drug-delivery and materials science, highlighting along the way the impact of host-to-guest proton transfer on self-assembly and molecular recognition.

The synthesis of some bolaamphiphiles is described. It is a convergent approach that allows the linkage of a glucosyl derivative to a bis-functionalized platform

Lung cancer (LC) represents the second most diagnosed tumor and the malignancy with the highest mortality rate. In recent years, tremendous progress has been made in the treatment of this tumor thanks to the discovery, testing, and clinical approval of novel therapeutic approaches. Firstly, targeted therapies aimed at inhibiting specific mutated tyrosine kinases or downstream factors were approved in clinical practice. Secondly, immunotherapy inducing the reactivation of the immune system to efficiently eliminate LC cells has been approved. This review describes in depth both current and ongoing clinical studies, which allowed the approval of targeted therapies and immune-checkpoint inhibitors as standard of care for LC. Moreover, the present advantages and pitfalls of new therapeutic approaches will be discussed. Finally, the acquired importance of human microbiota as a novel source of LC biomarkers, as well as therapeutic targets to improve the efficacy of available therapies, was analyzed. Therapy against LC is increasingly becoming holistic, taking into consideration not only the genetic landscape of the tumor, but also the immune background and other individual variables, such as patient-specific gut microbial composition. On these bases, in the future, the research milestones reached will allow clinicians to treat LC patients with tailored approaches.

Several features of cancer cells such as proliferation, invasion, metastatic spreading, and drug resistance are affected by their interaction with several tumor microenvironment (TME) components, including neutrophil gelatinase-associated lipocalin (NGAL), solute carrier family 22 member 17 (SLC22A17), and matrix metallopeptidase 9 (MMP9). These molecules play a key role in tumor growth, invasion, and iron-dependent metabolism of cancer cells. However, the precise epigenetic mechanisms underlying the gene regulation of

The increasing use of pesticides in intensive agriculture has had a negative impact on human health. It was widely demonstrated how pesticides can induce different genetic and epigenetic alterations associated with the development of different diseases, including tumors and neurological disorders. Therefore, the identification of effective indicators for the prediction of harmful pesticide exposure is mandatory. In this context, the aim of the study was to evaluate the modification of hsa-miR-199a-5p expression levels in liquid biopsy samples obtained from healthy donors and farm workers with chronic exposure to pesticides. For this purpose, the high-sensitive droplet digital PCR assay (ddPCR) was used to detect variation in the expression levels of the selected microRNA (miRNA). The ddPCR analyses revealed a significant down-regulation of hsa-miR-199a-5p observed in individuals exposed to pesticides compared to control samples highlighting the good predictive value of this miRNA as demonstrated by statistical analyses. Overall, the obtained results encourage the analysis of miRNAs as predictive biomarkers of chronic pesticide exposure thus improving the current strategies for the monitoring of harmful pesticide exposure.

Raw Data and Normalized Data ofhsa-miR-199a-5p expression levels obtained through droplet digital PCR. These data will be published in an original article entitled "Chronic pesticide exposure in farm workers is associated with the epigenetic modulation of hsa-miR-199a-5p"

Breast and ovarian cancer represent two of the most common tumor types in females worldwide. Over the years, several non‑modifiable and modifiable risk factors have been associated with the onset and progression of these tumors, including age, reproductive factors, ethnicity, socioeconomic status and lifestyle factors, as well as family history and genetic factors. Of note, BRCA1 and BRCA2 are two tumor suppressor genes with a key role in DNA repair processes, whose mutations may induce genomic instability and increase the risk of cancer development. Specifically, females with a family history of breast or ovarian cancer harboring BRCA1/2 germline mutations have a 60‑70% increased risk of developing breast cancer and a 15‑40% increased risk for ovarian cancer. Different databases have collected the most frequent germline mutations affecting BRCA1/2. Through the analysis of such databases, it is possible to identify frequent hotspot mutations that may be analyzed with next‑generation sequencing (NGS) and novel innovative strategies. In this context, NGS remains the gold standard method for the assessment of BRCA1/2 mutations, while novel techniques, including droplet digital PCR (ddPCR), may improve the sensitivity to identify such mutations in the hereditary forms of breast and ovarian cancer. On these bases, the present study aimed to provide an update of the current knowledge on the frequency of BRCA1/2 mutations and cancer susceptibility, focusing on the diagnostic potential of the most recent methods, such as ddPCR.

Inflammation is a protective response of the body to various injuries, which is strictly regulated by a variety of factors, including immune cells and soluble mediators. However, dysfunction of this defensive mechanism often results in inflammation‑driven diseases, such as deep vein thrombosis (DVT). The complex relationship between inflammatory cell activity and DVT has not been fully elucidated. The present study aimed to investigate the role of interleukin‑6 (IL6) signaling transduction in DVT. To this aim, the expression levels of transmembrane isoforms of the IL6 receptor (IL6R) and the glycoprotein 130 responsible for the IL6 cis‑signaling were evaluated in the peripheral blood mononuclear cells of patients with DVT and of healthy controls. The results indicated that leukocytes from patients with DVT exhibited overexpression of both IL6R and gp130 membrane isoforms and that these were strongly associated with the occurrence of DVT. Overall, the present findings indicated that IL6 cis‑signaling may have a direct involvement in the leukocyte activation in DVT and may serve as a predictive biomarker of DVT development.

Legionella pneumophila (L. pneumophila) is one of the most threatening nosocomial pathogens. The implementation of novel and more effective surveillance and diagnostic strategies is mandatory to prevent the occurrence of legionellosis outbreaks in hospital environments. On these bases, the present review is aimed to describe the main clinical and molecular features of L. pneumophila focusing attention on the latest findings on drug resistance mechanisms. In addition, a detailed description of the current guidelines for the disinfection and surveillance of the water systems is also provided. Finally, the diagnostic strategies available for the detection of Legionella spp. were critically reviewed, paying the attention to the description of the culture, serological and molecular methods as well as on the novel high-sensitive nucleic acid amplification systems, such as droplet digital PCR.

High-grade gliomas are the most fatal brain tumors. Grade 4 gliomas are called glioblastoma multiforme (GBM), which are associated with the poorest survival and a 5-year survival rate of less than 4%. Many patients with GBM developed concomitant cognitive dysfunctions and epilepsy. Although the cognitive decline is well defined in glioblastomas, the neurotoxic factors underlying this pathology are not well understood in GBM patients. In this study, we aimed to investigate whether GBM-derived exosomes play a role in neuronal toxicity. For this purpose, exosomes obtained from T98G and U373 GBM cells were applied to primary neuron culture at different concentrations. Subsequently, MTT, LDH, GSH, TAS, and TOS tests were performed. Both GBM-derived exosomes induced a dose-dependent and statistically significant increase of LDH release in cerebellar neurons. MTT assay revealed as both T98G and U373 GBM-derived exosomes induced dose-dependent neurotoxic effects in cerebellar neurons. To the best of our knowledge, this study is the first study demonstrating the toxic potential of GBM-derived exosomes to primary neurons, which may explain the peritumoral edema and cognitive decline in GBM patients.

Several features of cancer cells such as proliferation, invasion, metastatic spreading, and drug resistance are affected by their interaction with several tumor microenvironment (TME) components, including neutrophil gelatinase-associated lipocalin (NGAL), solute carrier family 22 member 17 (SLC22A17), and matrix metallopeptidase 9 (MMP9). These molecules play a key role in tumor growth, invasion, and iron-dependent metabolism of cancer cells. However, the precise epigenetic mechanisms underlying the gene regulation of Lipocalin 2 (LCN2), SLC22A17, and MMP9 in cancer still remain unclear. To this purpose, computational analysis was performed on TCGA and GTEx datasets to evaluate the expression and DNA methylation status of LCN2, SLC22A17, and MMP9 genes in different tumor types. Correlation analysis between gene/isoforms expression and DNA methylation levels of LCN2, SLC22A17, and MMP9 was performed to investigate the role of DNA methylation in the modulation of these genes. Protein network analysis was carried out using reverse phase protein arrays (RPPA) data to identify protein–protein interactions of the LCN2–SLC22A17–MMP9 network. Furthermore, survival analysis was performed according to gene expression and DNA methylation levels. Our results demonstrated that LCN2 and MMP9 were mainly upregulated in most tumor types, whereas SLC22A17 was largely downregulated, representing a specific hallmark signature for all gastrointestinal tumors. Notably, the expression of LCN2, SLC22A17, and MMP9 genes was negatively affected by promoter methylation. Conversely, intragenic hypermethylation was associated with the overexpression of SLC22A17 and MMP9 genes. Protein network analysis highlighted the role of the LCN2–SLC22A17–MMP9 network in TME by the interaction with fibronectin 1 and claudin 7, especially in rectal tumors. Moreover, the impact of expression and methylation status of LCN2, SLC22A17, and MMP9 on overall survival and progression free interval was tumor type–dependent. Overall, our analyses provide a detailed overview of the expression and methylation status of LCN2, SLC22A17, and MMP9 in all TCGA tumors, indicating that the LCN2–SLC22A17–MMP9 network was strictly regulated by DNA methylation within TME. Our findings pave the way for the identification of novel DNA methylation hotspots with diagnostic and prognostic values and suitable for epi-drug targeting.

Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) is the gold standard method for the diagnosis of COVID‑19 infection. Due to pre‑analytical and technical limitations, samples with low viral load are often misdiagnosed as false‑negative samples. Therefore, it is important to evaluate other strategies able to overcome the limits of RT‑qPCR. Blinded swab samples from two individuals diagnosed positive and negative for COVID‑19 were analyzed by droplet digital PCR (ddPCR) and RT‑qPCR in order to assess the sensitivity of both methods. Intercalation chemistries and a World Health Organization (WHO)/Center for Disease Control and Prevention (CDC)‑approved probe for the SARS‑CoV‑2 N gene were used. SYBR‑Green RT‑qPCR is not able to diagnose as positive samples with low viral load, while, TaqMan Probe RT‑qPCR gave positive signals at very late Ct values. On the contrary, ddPCR showed higher sensitivity rate compared to RT‑qPCR and both EvaGreen and probe ddPCR were able to recognize the sample with low viral load as positive even at 10‑fold diluted concentration. In conclusion, ddPCR shows higher sensitivity and specificity compared to RT‑qPCR for the diagnosis of COVID‑19 infection in false‑negative samples with low viral load. Therefore, ddPCR is strongly recommended in clinical practice for the diagnosis of COVID‑19 and the follow‑up of positive patients until complete remission.

A four component system consisting of two water-soluble calix[5]arenes (1 and 2) with differently substituted upper-rims and two α,ω-alkanediyldiammonium ions of different lengths (NC8N and NC10N) was found to assemble in a high-fidelity fashion providing exclusively the two homocalixarene capsules 1⊃NC8N⊂1 and 2⊃NC10N⊂2, irrespective of the order of guest addition. When the longer linker (NC10N) was left out from the mixture altogether, however, 1⊃NC8N⊂2 heterocapsules were formed.

A calix[5]arene monomer bearing two different ammonium sites assembles into a supramolecular polymer that self-sorts into a polypseudorotaxane upon addition of dibenzo-24-crown-8, but only in the presence of superweak counterions.

A new bis-(3-hydroxy-4-pyridinone) ligand (NTA(PrHP)2), derived from nitrilotriacetic acid (NTA), was synthesized and evaluated for its selective solution metal complexation capacity as well as its in vivo sequestering power for hard metal cations. After the study of its acid-base properties and determination of the protonation constants, the binding ability of NTA(PrHP)2 towards divalent (Ca2+, Cu2+, Zn2+) and trivalent (Al3+, Fe3+) metal cations was investigated by means of UV–Vis spectrophotometric, potentiometric and 1H NMR measurements. The determined speciation models consist of MpLqHr species with different stoichiometry. The obtained stability constants for the ML species follow the trend: Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+. Furthermore, the sequestering ability of the ligand towards these metal cations was investigated by the determination of the pL0.5 and pM parameters calculated at different pHs and pH = 7.4, respectively. High pL0.5 values were obtained with significant variation of the sequestering ability with the investigated pH range. Analysis of the pM values at pH = 7.4 showed that the metal-ligand affinity follows the trend: Fe3+ > Al3+ > Cu2+ > Zn2+ > Ca2+. Finally, in vivo assays were performed to verify the efficacy of NTA(PrHP)2 as sequestering agent towards trivalent metal cations, when administered to mice preloaded with the radiotracer 67Ga–citrate.

Flavonols are a subclass of natural flavonoids characterized by a remarkable number of biotechnological applications and health-promoting properties. They attract researchers’ attention due to many epidemiological studies supporting their usage. They are phytochemicals commonly present in our diet, being ubiquitous in the plant kingdom and, in particular, relatively very abundant in fruits and vegetables. All these aspects make flavonols candidates of choice for the valorization of products, based on the presence of a remarkable number of different chemical structures, each one characterized by specific chemical features capable of influencing biological targets inside the living organisms in very different manners. In this review, we analyzed the biochemical and physiological characteristics of flavonols focalizing our attention on the most promising compounds to shed some light on their increasing utilization in biotechnological applications in processing industries, as well as their suitable employment to improve the overall wellness of the humankind.

The acid–base properties of two bifunctional 3-hydroxy-4-pyridinone ligands and their chelating capacity towards Zn2+, an essential bio-metal cation, were investigated in NaCl aqueous solutions by potentiometric, UV-Vis spectrophotometric, and 1H NMR spectroscopic titrations, carried out at 0.15 ≤ I/mol −1 ≤ 1.00 and 288.15 ≤ T/K ≤ 310.15. A study at I = 0.15 mol L−1 and T = 298.15 K was also performed for other three Zn2+/Lz− systems, with ligands belonging to the same family of compounds. The processing of experimental data allowed the determination of protonation and stability constants, which showed accordance with the data obtained from the different analytical techniques used, and with those reported in the literature for the same class of compounds. ESI-MS spectrometric measurements provided support for the formation of the different Zn2+/ligand species, while computational molecular simulations allowed information to be gained on the metal–ligand coordination. The dependence on ionic strength and the temperature of equilibrium constants were investigated by means of the extended Debye–Hückel model, the classical specific ion interaction theory, and the van’t Hoff equations, respectively.

Citrus fruit and juices represent one of the main sources of compounds with a high potential for health promoting properties. Among these compounds, flavanones (such as hesperetin, naringenin, eriodictyol, isosakuranetin, and their respective glycosides), which occur in quantities ranging from ∼180 to 740 mg/L (depending on the Citrus species and cultivar) are responsible for many biological activities. These compounds support and enhance the body's defenses against oxidative stress and help the organism in the prevention of cardiovascular diseases, atherosclerosis, and cancer. Moreover, among other properties, they also show anti-inflammatory, antiviral, and antimicrobial activities. This review analyzes the biochemistry, pharmacology, and biology of Citrus flavanones, emphasizing the occurrence in Citrus fruits and juices and their bioavailability, structure-function correlations and ability to modulate signal cascades both in vitro and in vivo. © 2017 BioFactors, 43(4):495-506, 2017.

A potentiometric study on the acid–base properties and formation of complexes with alkali metals (i.e., Na+ and K+) of some poly(methyl vinyl ether-co-maleic) acids, namely, Gantrez AN169 (2000 kDa), S95 (220 kDa), and S97 (1200 kDa), was carried out under different experimental conditions consisting of the ionic medium, ionic strength, and temperature. Owing to the possible formation of micelles, the critical micelle concentration of Gantrez AN169 was determined by means of different techniques in pure water and at T = 298.15 K. The diprotic-like model was used for the elaboration of the experimental protonation data, and the best model was obtained by assuming that a monomeric unit consists of two methyl vinyl ether-co-maleic acid residues. Moreover, the protonation data of two poly(acrylic comaleic acids), PCA 3 kDa and 70 kDa (published in a previous paper), under different experimental conditions were reanalyzed. The trend in the protonation constants of polyelectrolytes in different ionic media is (...

Through-the-annulus threading of calix[5]arene penta-O-ethers by dialkylammonium cations coupled to the loosely coordinating superweak TFPB– anion has been achieved. 1H NMR titration data show that preorganization of the calix[5]arene scaffold leads to great thermodynamic stability of the pseudorotaxane complexes as well as to a favorable threading kinetic. Thus, calix[5]arene 1c, bearing tert-butyl groups at the wide rim, was threaded by all of the cations under study (with the exception of the dibenzylammonium 2b+) more tightly than the other derivatives under investigation (Ka’s up to 2.02 ± 0.2 × 105 M–1) because of its preorganized cone conformation. According to DFT calculations, van der Waals interactions between the tert-butyl groups of 1c and the alkyl chain of the cationic axle are likely responsible for the remarkable stability observed. The threading of the calix[5]arene wheels with the asymmetric pentylbenzylammonium axle 2c+ led to the toposelective formation of the endo-pentylpseudorotaxane...

Adverse drug interactions due to grapefruit juice are well known prompting warnings even in drug labels. Similar issues have not been reported for clementines and available data is scarce, despite of genetic descent. We observed substantially increased tacrolimus trough concentrations in a renal transplant patient consuming high clementine amounts and, thus, scrutinised the effects of clementine juice on drug metabolism and drug transporters in vitro and compared it to the effects of mandarin and grapefruit juice. All citrus juices profoundly induced several drug transporters and drug metabolising enzymes, whereas the effects of grapefruit juice were most pronounced (e.g. 156-fold and 34-fold induction of cytochrome P450 (CYP) 3A4 mRNA by grapefruit juice and clementine juice, respectively). However, the juices also inhibited e.g. CYP3A4, raising the question which effect prevails in vivo. Using an enzymatic activity assay, we demonstrated that at least in vitro CYP3A4 inhibition prevails for both grapefruit and clementine juice, whereas for CYP1A2 induction appears to predominate. Thus, inhibition of CYP3A4 is presumably the underlying reason for the observed increase in the concentrations of the CYP3A4 substrate tacrolimus in the patient. Taken together, our data indicate that clementine juice as well as grapefruit juice and to a lesser extent also mandarin juice can induce several important drug metabolising enzymes and drug transporters, but also inhibit some of these proteins. Our data indicate that clementine juice similar to grapefruit juice bears the potential for profound interactions with drugs potentially leading to adverse drug effects e.g. through over-exposure to CYP3A4 substrates.

We have previously demonstrated that clementines have in vitro drug interaction potential. To assess the clinical relevance of clementine-drug interaction, two single case experiments with repetitive phenotyping of CYP3A activity were conducted. Although an increment of 43% in the estimated midazolam clearance (eCLmet) was observed during the first experiment in a renal transplant patient on tacrolimus after 4-d consumption of clementines (1 kg/d), and an increment of +89% of eCLmet was observed during chronic consumption of clementine juice in a healthy male volunteer, these changes lie within the range of intra-individual variability. Therefore one cannot assure a potential drug interaction due to the clementines, but prescribers should be cautious unless further data emerges. In contrast to the juice used for the in vitro assay comprising several flavonoids, this juice only contained hesperidin and narirutin indicating that the drug interactions potential of clementines might depend on the composition varying from batch to batch.

The pivotal role played by potassium ions in the noncovalent synthesis of discrete porphyrin-calixarene nanostructures has been examined. The flattened-cone conformation adopted by the two cavities of octa-cationic calix[4]tube C4T was found to prevent the formation of complexes with well-defined stoichiometry between this novel water-soluble calixarene and the tetra-anionic phenylsulfonate porphyrin CuTPPS. Conversely, preorganization of C4T into a C4v-symmetrical scaffold, triggered by potassium ion encapsulation (C4T@K+), allowed us to carry out an efficient hierarchical self-assembly process leading to 2D and 3D nanostructures. The stepwise formation of discrete CuTPPS/C4T@K+ noncovalent assemblies, containing up to 33 molecular elements, was conveniently monitored by UV/vis spectroscopy by following the absorbance of the porphyrin Soret band.

Simple Summary Despite the availability of screening programs, oral cancer is often diagnosed due to the lack of effective biomarkers. Therefore, the identification of new effective diagnostic and late prognostic biomarkers is of fundamental importance for the management of this tumor type. In our previous computational study, we have identified a set of microRNAs (miRNAs) significantly dysregulated in oral cancer and with a potential diagnostic and prognostic significance for oral cancer patients. Starting from our preliminary bioinformatics results, the aim of the present study was to validate the diagnostic potential of four selected miRNAs, hsa-miR-133a-3p, hsa-miR-375-3p, hsa-miR-503-5p and hsa-miR-196a-5p, in liquid biopsy samples obtained from oral cancer patients and healthy donors. For this purpose, the expression levels of the selected miRNAs were determined in plasma samples by using specific miRNA probes and droplet digital PCR (ddPCR). The ddPCR results showed that the hsa-miR-133a-3p and hsa-miR-375-3p were significantly down-regulated in oral cancer and their evaluation in liquid biopsy samples can predict the risk of oral cancer development with high sensitivity and specificity. Finally, the computational analysis of miRNA expression and clinical-pathological features of patients allowed us to establish the functional role and prognostic significance of the two validated miRNAs. Abstract Despite the availability of screening programs, oral cancer deaths are increasing due to the lack of diagnostic biomarkers leading to late diagnosis and a poor prognosis. Therefore, there is an urgent need to discover novel effective biomarkers for this tumor. On these bases, the aim of this study was to validate the diagnostic potential of microRNAs (miRNAs) through the analysis of liquid biopsy samples obtained from ten oral cancer patients and ten healthy controls. The expression of four selected miRNAs was evaluated by using droplet digital PCR (ddPCR) in a pilot cohort of ten oral cancer patients and ten healthy donors. Bioinformatics analyses were performed to assess the functional role of these miRNAs. The expression levels of the predicted down-regulated hsa-miR-133a-3p and hsa-miR-375-3p were significantly reduced in oral cancer patients compared to normal individuals while no significant results were obtained for the up-regulated hsa-miR-503-5p and hsa-miR-196a-5p. ROC analysis confirmed the high sensitivity and specificity of hsa-miR-375-3p and hsa-miR-133a-3p. Therefore, both miRNAs are significantly down-regulated in cancer patients and can be used as biomarkers for the early diagnosis of oral cancer. The analysis of circulating miRNAs in a larger series of patients is mandatory to confirm the results obtained in this pilot study.

A combined DOSY and XRD study indicates that a carboxylcalix[5]arene receptor is able to encapsulate alpha,omega-diamines of appropriate length by means of a proton-transfer-mediated recognition process followed by salt-bridge-assisted bis-endo-complexation.

Sanguinello and Tarocco are the blood orange ( Citrus sinensis (L.) Osbeck) cultivars most diffused worldwide. Reversed phase liquid chromatography coupled with MS-MS analysis showed that these two varieties have a similar chromatographic pattern, characterised by the presence of C - and O -glycosyl flavonoids. Of the two, Sanguinello was found to be far richer in flavonoids than Tarocco. In the juices, twelve individual components were identified for the first time, namely, four C -glycosyl flavones (lucenin-2, vicenin-2, stellarin-2, lucenin-2 4′-methyl ether and scoparin), three flavonol derivatives (quercetin-3- O -(2-rhamnosyl)-rutinoside, quercetin-3- O -hexoside, quercetin 3-hydroxy-3-methylglutaryl-glycoside), an O -triglycosyl flavanone (narirutin 4′- O -glucoside) and a flavone O -glycosides (chrysoeriol 7- O -neoesperidoside). Moreover, the influence of the identified C - and O -glycosyl flavonoids on the antioxidant and acetylcholinesterase activity of these juices has been evaluated.

For clementine juice, previous data indicate a possible food-drug interaction with substrates of key enzymes responsible for drug metabolism (i.e. cytochrome P450 [CYP] 3A4, CYP1A2). However, which compounds in clementine juice are responsible for these effects are unknown. Therefore, we aimed to identify the compounds in clementine juice provoking metabolic enzyme inhibition or induction. The results demonstrated that the flavonoid fraction of clementine juice provoked induction of several genes and inhibition of both CYP3A4 and CYP1A2, matching effects observed with whole clementine juice. CYP1A2 inhibition and induction can most likely be attributed to nobiletin, sinensetin, and tangeretin. Tangeretin was the only compound causing CYP3A4 induction while CYP3A4 inhibition was most likely the result of additive or synergistic effects caused by several compounds. Thus, whenever evaluating the clinical relevance of clementine interactions, flavonoid contents should be reported because these might explain differences between cultivars and harvests.

Amphiphilic PEGylated calix[5]arenes 1 and 2 were obtained in one step from the parent p-tert-butylcalix[5]arene and p-methylcalix[5]arene. Very low critical micellar concentrations (0.5 and 4.5 μM for 1 and 2 respectively) were determined by the Rose Bengal micellization method. Aggregation features were studied by means of 1H and DOSY NMR as well as by AFM. Release of Rose Bengal from the micellar environment was accomplished by increasing the temperature.

The stepwise synthesis of ionizable p-tert-butylcalix[5]arenes 1a·H and 1b·H, featuring a fixed cone cavity endowed with a carboxyl moiety at the narrow rim, is described. Single-crystal X-ray analyses have shown that in the solid state 1a·H and 1b·H adopt a cone-out conformation with the carboxylic OH group pointing in, toward the bottom of the aromatic cavity, as a result of a three- or two-center hydrogen-bonding pattern between the carboxyl group and the phenolic oxygen atom(s). The affinity of amines for calix[5]arene derivatives 1a·H and 1b·H was probed by (1)H NMR spectroscopy and single-crystal X-ray diffraction studies. These carboxylcalix[5]arenes are shown to selectively recognize linear primary amines--over branched, secondary, and tertiary amines--by a two-step process involving a proton transfer from the carboxyl to the amino group to provide the corresponding alkylammonium ion, followed by binding of the latter inside the cavity of the ionized calixarene. Proton transfer occurs only with linear primary amines, that is, when the best size and shape fit between host and substrate is achieved, while the other amines remain in their noncompeting unprotonated form. The role of the solvent in the ionization/complexation process is discussed. Structural studies on the n-BuNH(2) complexes with 1a·H and 1b·H provide evidence that binding of the in situ formed n-BuNH(3)(+) substrate to the cavity of the ionized macrocycle is ultimately secured, in the case of 1a·H, by the formation of an unprecedented salt-bridge interaction.

The self-assembly of internally ion-paired, neutral AA/BB-type supramolecular polymers composed of complementary di-ionizable homoditopic pairs of monomers is reported. Host-to-guest double-proton transfer mediates the recognition between bis-calix[5] arenedicarboxylic acids and a, w-diaminoalkanes to yield cyclic, doughnut-shaped assem-blies with morphologies (i.e., cyclic vs. linear) that can be controlled by means of external chemical stimuli. The behavior of these intriguing aggregates, both in solution and on surfaces, has been investigated by a combination of H-1 and DOSY NMR spectroscopy, light-scattering, and atomic force microscopy.

The remarkable affinity of deca-carboxylatopillar[5]arene WP5 towards the aminoglycoside antibiotic, amikacin, in aqueous media is reported; in vitro studies on Gram-positive bacteria (Staphylococcus aureus) show that drug entrapment inside WP5 also takes place in the presence of the microrganisms, thus pointing to WP5 as an appealing carrier for amikacin targeted delivery.

The interactions between hydrochloride salts of biologically relevant amines and micelles formed by a water-soluble p-tert-butylcalix[5]arene were investigated by means of 1H NMR spectroscopy, using a combination of NOESY and DOSY techniques. DOSY measurements were conveniently employed to assess the micelle affinity towards the target guests (phenylethylamine ≈ histamine ≈ serotonin > tyramine > dopamine > norepinephrine), whereas NOESY studies revealed the approximate position of these cationic guests within the different micellar regions. In general, all the guests were located in the palisade region of the micelle, with the exception of phenethylamine and histamine, the former being bound to the micellar core as well as the palisade, the latter preferring the outer shell of the micelle.

RP-LC-DAD-ESI-MS-MS separation/identification protocol has been employed for the identification and characterisation of nine C- and O-glycosyl flavonoids in Moro (Citrus sinensis (L.) Osbeck) juice grown in Southern Italy. For the first time we reported the presence of five C-glycosyl flavones (lucenin-2, vicenin-2, stellarin-2, lucenin-2 4'-methyl ether and scoparin), a 3-hydroxy-3-methylglutaryl glycosyl flavonol (3-hydroxy-3-methylglutaryl glycosyl quercetin) and a flavone O-glycosides (chrysoeriol 7-O-neoesperidoside). Moreover, the influence of the identified C- and O-glycosyl flavonoids on the total antioxidant activity of crude juice has been evaluated on the basis of its ability to scavenge DPPH•, OH• and ABTS•+ radicals and to reduce iron.

A separation/identification protocol based on RP-LC-DAD-ESI-MS-MS has been employed for the characterisation of the flavonoid fraction of the juice from tangelos (Citrus reticulata×Citrus paradisi) grown in Southern Italy. Eleven compounds were identified in a single chromatographic course. Of these, two C-glycosyl flavones (lucenin-2 and vicenin-2) and an O-triglycosyl flavanone (narirutin 4'-O-glucoside) were identified for the first time. Fruit juice antioxidant activity was evaluated on the basis of its ability to scavenge DPPH, O2(-), OH and ABTS(+) radicals, and to reduce iron (FRAP). Moreover, the influence of the identified polymethoxylated, C- and O-glycosyl flavonoids on the total antioxidant activity has been elucidated. We also checked the antimicrobial activity of a broad fraction, containing all the detected flavonoids obtained by preparative HPLC, in terms of MICs for Staphylococcus aureus.

A new water-soluble calix[5]arene 1, bearing 4-butylsulfonato pendant groups at the lower rim, has been synthesised. This derivative shows aggregation properties in water. Critical micellar concentration (cmc) for 1 has been calculated by means of DOSY NMR and fluorescence spectroscopy. When n-dodecylammonium chloride is added to 1 below its cmc, the resulting 1:1 endo-cavity inclusion complex behaves as a supramolecular amphiphile providing a new micellar aggregate.

The bioprotective action of the disaccharide trehalose has been studied against the well-known denaturating agent, guanidine hydrochloride. The results indicated a direct influence of trehalose on both enzymatic activity and conformational changes of lysozyme, as shown by the decrease of the inactivation rate constant of about 1.48-fold and the loss of α-helix structure of lysozyme. In addition, ESI-MS hydrogen-deuterium (H/D) exchange experiments allowed us to correlate the structural and dynamic features of the protein in the presence of the two additives, highlighting as trehalose remarkably influenced this exchange by decreasing local protein environment changes and solvent accessibility to the amide peptide backbone, as further evidenced by circular dichroism and (1)H NMR measurements.

This chapter focuses on calix[5]arene-based supramolecular oligo/polymers and, in doing so, examines the genesis and the evolution of this subgroup of the greater calixarene family from the viewpoint of the intermolecular host-guest interactions held to be responsible for first securing capsular complex self-assembly and subsequently polymer formation.

The first examples of photo-responsive azobenzene-containing oxacalixarenes have been synthesized via a 3+1 macrocyclization approach. Introduction of the photoresponsive unit was achieved by using 4-phenylazoresorcinol or ( E )-4-(4′-nitrophenylazo)resorcinol as the nucleophilic component in the macrocyclization reaction. These novel macrocycles have been characterized by means of 1D and 2D NMR spectroscopy and DFT calculations (B3LYP/6-31G(d)). According to thermal and photoisomerization studies, tetranitro-oxacalix[4]arenes are less prone to E → Z isomerization than oxacalix[2]arene[2]triazines and, within the two series, p -nitrophenylazo derivatives are more unwilling to isomerize than their phenylazo analogues.

Citrus myrtifolia Raf. (chinotto) flavedo, albedo and carpel membranes from green and ripe fruits, as well as seeds and leaves, were investigated for the first time for their flavonoid and furocoumarin composition. Twenty-three unique compounds distributed in the different fruit/plant parts were identified and quantified. All samples analysed contained flavanone O -glycosides, flavone C - and O -glycosides and furocoumarins; flavedos and albedos also contained significant amounts of polymethoxyflavones. Flavedo and albedo extracts were found to be richest in flavonoids and furocoumarins, containing up to 1.95 g/kg fresh weight. Flavedo, albedo and carpel membranes from ripe fruits were found to be richer than the corresponding tissues from unripe fruits. The remarkable radical-scavenging activity of all the extracts was tested by DPPH • , ABTS •+ and FRAP methods, revealing that (i) they were particularly efficient in quenching ABTS •+ radical cations (up to 9.8 mM Trolox equivalents), and (ii) flavedo and albedo extracts, on average, showed the highest antioxidant capacity.

A novel bis-calix[5]arene (1) with divergent cavities was prepared in four steps starting from p-methylcalix[5]arene. This molecule is composed of two calix[5]-bis-crown-3 units held together, via their narrow rims, by a N,N″-1,4-phenylene-bis[N′-(2-ethenyloxyethyl)]diureido bridging moiety. Owing to the anion-binding ability of the ureido moieties embedded in its framework, bis-calix[5]arene 1 acts as a heterotetratopic monomer and, in the presence of 1,8-octanediyldiammonium dichloride, self-assembles into AA/BB-type polycapsular oligomers. Extensive 1H NMR investigations have demonstrated that the nature and stoichiometry of these supramolecular oligomers are strongly dependent on the concentration and the ratio of the two monomers.

New AB-type supramolecular polymers have been prepared by acid-promoted self-assembly of an aminododecyloxy-calix[5]arene monomer precursor. The number-average degree of polymerization has been found to be dependent on the concentration of the salt monomer and on the nature of the counterion (i.e., chloride, picrate or hexafluorophosphate). Chain-length regulation experiments have been carried out, employing orthogonal chain stoppers capable of selectively interacting with a given moiety of the AB-type monomer/polymer. Competitive calix[5]arene ‘caps’ and n -butylammonium ion ‘plugs’ have been used to control the extent of self-assembly of the polymer, in turn interacting with the ammoniumdodecyloxy or with the cavity end-groups of the supramolecular calixarene assembly. These experiments, conveniently carried out at a 10 mM concentration, can be easily followed by 1 H NMR spectroscopy.

An investigation is hereby presented on the possible formation of a stacking (π–π) complex between adenosine-5′-triphosphate (ATP) and three xanthines (L), namely caffeine (Cf), theophylline (Tp) and theobromine (Tb), at different ionic strengths (0.05 ≤ I/mol L−1 ≤ 1) in NaCl(aq) and temperature (T = 283.15, 298.15 and 313.15 K), by using magnetic resonance spectroscopy 1H NMR. The values of log β of the [ATP(L)]4− species were: 1.32, 1.24 and ≤0.7 at I = 0.05 mol L−1 and T = 298.15 K for Cf, Tp and Tb, respectively. The stability constant of [ATP(Cf)]4− species shown a linear dependence on the temperature, and the values of log β obtained were: 1.18, 1.32 and 1.47 at T = 283.15, 298.15 and 313.15 K, respectively. By using the values of log β obtained at different temperatures a value of ΔH = −16 ± 3 kJ mol−1 was calculated for the formation of the stacking (π–π) complex species. The three systems were also studied potentiometrically at different ionic strengths (0.05 ≤ I/mol L−1 ≤ 1) in NaCl(aq) and T = 298.15 K. For all the investigated systems, the formation of [(ATP)(L)Hi](i−4) species (i = 1–3), with high yields (60–80%) was found; the stability trend obtained for the protonated complex species was (ATP–Tb) > (ATP–Tp) > (ATP–Cf). The stability constants of all the [(ATP)(L)Hi](i−4) species proved fairly dependent on the ionic strength that was studied by using the Debye–Huckel-type equation. Furthermore, by comparison with the stability of the species of analogous systems, such as ATP–aliphatic amines, ATP–aromatic amines and pyrophosphate–aliphatic amines, an empirical relationship between the stability of the complexes and the total basicity of the ligand was found.

Special Section Dendrons composed of three functionalized cone calix[4]arene substructures covalently bound to rigid C-3-symmetric tris-anilino cores through amide linkages have been obtained by a divergent synthetic protocol involving the coupling of p-nitrocalix[4]arene butanoic acid 9 with tris(4-aminophenyl)amine (TAPA), 1,3,5-tris(4-aminophenyl)benzene (TAPB), and 2,4,6-tris(4-aminophenyl)-s-triazine (TAPT). Two different amidation procedures were used to generate the dodecanitro intermediates (through activation of acid 9 with PyBOP catalyst or transformation into its acid chloride). The final reduction to the title dodecaamino dendrons was smoothly carried out with H-2 and Raney-Ni catalyst.