Synthesis and Exon-Skipping Properties of a 3'-Ursodeoxycholic Acid-Conjugated Oligonucleotide Targeting DMD Pre-mRNA: Pre-Synthetic versus Post-Synthetic Approach.

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.

Can Asymptomatic or Non-Severe SARS-CoV-2 Infection Cause Medium-Term Pulmonary Sequelae in Children?

Pulmonary complications in adults who recovered from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported even in minimally symptomatic patients. In this study, lung ultrasound (LUS) findings and pulmonary function of children who recovered from an asymptomatic or mildly symptomatic SARS-CoV-2 infection were evaluated. We prospectively followed up for at least 30 days patients younger than 18 years who recovered from SARS-CoV-2 infection at the Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan (Italy). All enrolled patients underwent LUS. Airway resistance measured by the interrupter technique test was assessed in subjects aged 4-6 years, whereas forced spirometry and measurement of diffusing capacity of the lungs for carbon monoxide were performed in subjects older than 6 years. To evaluate a possible correlation between pulmonary alterations and immune response to SARS-CoV-2, two semiquantitative enzyme immune assays were used. We enrolled 16 out of 23 eligible children. The median age of enrolled subjects was 7.5 (0.5-10.5) years, with a male to female ratio of 1.7. No subject presented any abnormality on LUS, airway resistance test, forced spirometry, and diffusing capacity of the lungs for carbon monoxide. On the other hand, all subjects presented Ig G against SARS-CoV-2. In contrast in adults, we did not detect any pulmonary complications in our cohort. These preliminary observations suggest that children with an asymptomatic or mildly symptomatic SARS-CoV-2 infection might be less prone to develop pulmonary complications than adults.

Evaluation of the Anticancer Activity of a Bile Acid-Dihydroartemisinin Hybrid Ursodeoxycholic-Dihydroartemisinin in Hepatocellular Carcinoma Cells.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and accounts for 85-90% of all primary liver cancer. Based on the estimation by the International Agency for Research on Cancer in 2018, liver cancer is the fourth leading cause of cancer death globally. Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is a well-known drug for the treatment of malaria. Previous studies have demonstrated that DHA exhibits antitumor effects toward a variety of human cancers and has a potential for repurposing as an anticancer drug. However, its short half-life is a concern and may limit the application in cancer therapy. We have reported that UDC-DHA, a hybrid of bile acid ursodeoxycholic acid (UDCA) and DHA, is ∼12 times more potent than DHA against a HCC cell line HepG2. In this study, we found that UDC-DHA was also effective against another HCC cell line Huh-7 with an IC50 of 2.16 µM, which was 18.5-fold better than DHA with an IC50 of 39.96 µM. UDC-DHA was much more potent than the combination of DHA and UDCA at 1:1 molar ratio, suggesting that the covalent linkage rather than a synergism between UDCA and DHA is critical for enhancing DHA potency in HepG2 cells. Importantly, UDC-DHA was much less toxic to normal cells than DHA. UDC-DHA induced G0/G1 arrest and apoptosis. Both DHA and UDC-DHA significantly elevated cellular reactive oxygen species generation but with different magnitude and timing in HepG2 cells; whereas only DHA but not UDC-DHA induced reactive oxygen species in Huh-7 cells. Depolarization of mitochondrial membrane potential was detected in both HepG2 and Huh-7 cells and may contribute to the anticancer effect of DHA and UDC-DHA. Furthermore, UDC-DHA was much more stable than DHA based on activity assays and high performance liquid chromatography-MS/MS analysis. In conclusion, UDC-DHA and DHA may exert anticancer actions via similar mechanisms but a much lower concentration of UDC-DHA was required, which could be attributed to a better stability of UDC-DHA. Thus, UDC-DHA could be a better drug candidate than DHA against HCC and further investigation is warranted.

Polydopamine Nanoparticle-Coated Polysulfone Porous Granules as Adsorbents for Water Remediation.

Water purification technologies possibly based on eco-sustainable, low cost, and multifunctional materials are being intensively pursued to resolve the current water scarcity and pollution. In this scenario, polysulfone hollow porous granules (PS-HPGs) prepared from scraps of the industrial production of polysulfone hollow fiber membranes were recently introduced as adsorbents and filtration materials for water and air treatment. Here, we report the functionalization of PS-HPGs with polydopamine (PD) nanoparticles for the preparation of a new versatile and efficient adsorbent material, namely, PSPD-HPGs. The in situ growth of PD under mild alkaline oxidative polymerization allowed us to stably graft PD on polysulfone granules. Enhanced removal efficiency of ofloxacin, an antibiotic drug, with an improvement up to 70% with respect to the pristine PS-HPGs, and removal of Zn(II) and Ni(II) were also observed after PD modification. Remarkably, removal of Cu(II) ions with an efficiency up to 80% was observed for PSPD-HPGs, whereas no adsorption was found for the PD-free precursor. Collectively, these data show that modification with a biocompatible polymer such as PD provides a simple and valuable tool to enlarge the field of application of polysulfone hollow granules for water remediation from both organic and metal cation contaminants.

Dihydroartemisinin-Bile Acid Hybridization as an Effective Approach to Enhance Dihydroartemisinin Anticancer Activity.

A series of hybrid compounds based on natural products-bile acids and dihydroartemisinin-were prepared by different synthetic methodologies and investigated for their in vitro biological activity against HL-60 leukemia and HepG2 hepatocellular carcinoma cell lines. Most of these hybrids presented significantly improved antiproliferative activities with respect to dihydroartemisinin and the parent bile acid. The two most potent hybrids of the series exhibited a 10.5- and 15.4-fold increase in cytotoxic activity respect to dihydroartemisinin alone in HL-60 and HepG2 cells, respectively. Strong evidence that an ursodeoxycholic acid hybrid induced apoptosis was obtained by flow cytometric analysis and western blot analysis.

Anti-cancer activity of di- and tri-organotin(IV) compounds with D-(+)-Galacturonic acid on human tumor cells.

We have compared the anti-proliferative activity in vitro, of R2SnGala (1-3) [R = Me, n-Bu, Ph] and novel R3SnGala (4, 5) [R = Me, n-Bu] with D-(+)-Galacturonic acid [HGala; Galaq-, q = (2) and (1) for R2SnGala and R3SnGala, respectively] compounds, towards human tumor cell lines of intestinal carcinoma (HCT-116) and breast adenocarcinoma (MCF-7). The new synthesized 4 and 5 compounds were characterized, in solution, by 1H, 13C and 119Sn NMR, that showed that HGala acts as monoanionic moiety and evidenced the dynamic behavior of the compounds, due to inter-conversions involving the anomeric carbon atom of the ligand. Cell viability, apoptosis induction and cell cycle distribution were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay and flow cytometry, respectively. The cytotoxicity of the compounds, in the micro-submicromolar range, changed in the order of the organotin(IV) moieties, according to 5 > 3 > 2, while 1 and 4, containing MenSn(IV) (n = 2,3) moieties, were ineffective. Compound 5 showed peculiar cytotoxic effects. It did not cause time dependent inhibition of cell growth nor accumulated into the cells. Cell death induced by the active 2, 3, and 5, was shown to be apoptotic by measuring the exposure of phosphatidylserine to the outer membrane and the loss of mitochondrial potential. All the cytotoxic compounds induced an accumulation of cells in the subG0/G1phase, while only 2 and 3 perturbed the cell cycle confining viable cells in G0/G1phase. Finally, none of the compounds investigated affected the viability of normal intestinal or liver cells, indicating selectivity towards tumor cells.

Omni-resonant optical micro-cavity.

Optical cavities transmit light only at discrete resonant frequencies, which are well-separated in micro-structures. Despite attempts at the construction of planar 'white-light cavities', the benefits accrued upon optically interacting with a cavity - such as resonant field buildup - have remained confined to narrow linewidths. Here, we demonstrate achromatic optical transmission through a planar Fabry-Pérot micro-cavity via angularly multiplexed phase-matching that exploits a bio-inspired grating configuration. By correlating each wavelength with an appropriate angle of incidence, a continuous spectrum resonates and the micro-cavity is rendered transparent. The locus of a single-order 0.7-nm-wide resonance is de-slanted in spectral-angular space to become a 60-nm-wide achromatic resonance spanning multiple cavity free-spectral-ranges. The result is an 'omni-resonant' planar micro-cavity in which light resonates continuously over a broad spectral span. This approach severs the link between the resonance bandwidth and the cavity-photon lifetime, thereby promising resonant enhancement of linear and nonlinear optical effects over broad bandwidths in ultrathin devices.

Octave-spanning coherent perfect absorption in a thin silicon film.

Although optical absorption is an intrinsic materials property, it can be manipulated through structural modification. Coherent perfect absorption increases absorption to 100% interferometrically but is typically realized only over narrow bandwidths using two laser beams with fixed phase relationship. We show that engineering a thin film's photonic environment severs the link between the effective absorption of the film and its intrinsic absorption while eliminating, in principle, bandwidth restrictions. Employing thin aperiodic dielectric mirrors, we demonstrate coherent perfect absorption in a 2 µm thick film of polycrystalline silicon using a single incoherent beam of light at all the resonances across a spectrally flat, octave-spanning near-infrared spectrum, ≈800-1600 nm. Critically, these mirrors have wavelength-dependent reflectivity devised to counterbalance the decline in silicon's intrinsic absorption at long wavelengths.

NO Photoreleaser-Deoxyadenosine and -Bile Acid Derivative Bioconjugates as Novel Potential Photochemotherapeutics.

This contribution reports the synthesis of some novel bioconjugates with anticancer activity and able to release nitric oxide (NO) under visible light excitation. The 4-nitro-2-(trifluoromethyl)aniline derivative, a suitable NO photodonor, was conjugated with 2'-deoxyadenosine and urso- and cheno-deoxycholic acid derivatives, through a thioalkylic chain or the 4-alkyl-1,2,3-triazole moiety. Photochemical experiments demonstrated the effective release of NO from 2'-deoxyadenosine and ursodeoxycholic acid conjugates under the exclusive control of visible light inputs. Studies for the in vitro antiproliferative activity against leukemic K562 and colon carcinoma HCT116 cell lines are reported for all the compounds as well as a case study of photocytotoxicity against HCT116.

Analytical model for coherent perfect absorption in one-dimensional photonic structures.

Coherent perfect absorption (CPA) is the phenomenon where a linear system with low intrinsic loss strongly absorbs two incident beams but only weakly absorbs either beam when incident separately. We present an analytical model that captures the relevant physics of CPA in one-dimensional photonic structures. This model elucidates an absorption-mediated interference effect that underlies CPA-an effect that is normally forbidden in Hermitian systems but is allowed when conservation of energy is violated due to the inclusion of loss. By studying a planar cavity model, we identify the optimal mirror reflectivity to guarantee CPA in the cavity at resonances extending in principle over any desired bandwidth. As a concrete example, we design a resonator that produces CPA in a 1-µm-thick layer of silicon over a 200-nm bandwidth in the near-infrared.

Gold-nanoparticle extraction and reversed-electrode-polarity stacking mode combined to enhance capillary electrophoresis sensitivity for conjugated nucleosides and oligonucleotides containing thioether linkers.

We present a capillary electrophoresis method for determining two different C8-conjugated deoxyadenosines, and for oligonucleotides containing them, in which a psoralen or an acridine molecule is bonded to the base via a short alkyl chain containing sulfur ethers at both ends. The sensitivity of the micellar electrokinetic chromatography (MEKC) method was increased by using two preconcentration techniques, micro solid-phase extraction (µSPE) followed by reversed-electrode-polarity stacking mode (REPSM). Variables that affect the efficiency of the extraction in µSPE and preconcentration by REPSM, including the type and volume of extraction nanoparticle, concentration, and injection time, were investigated. Under the optimum conditions, enrichment factors obtained were in the range 360-400. The limits of detection (LODs) at a signal-to-noise ratio of 3 ranged from 2 to 5 nmol L(-1). The relative recoveries of labelled adenosines from water samples were 95-103%. The proposed method provided high enrichment factors and good precision and accuracy with a short analysis time. On the basis of the advantages of simplicity, high selectivity, high sensitivity, and good reproducibility, the proposed method may have great potential for biochemical applications.

Labeling deoxyadenosine for the preparation of functional conjugated oligonucleotides.

Herein we present a versatile synthetic method for the 8-thioalkylation of (deoxy)adenosine with a short carbon linker having on the other side a variety of molecules (psoralen, acridine) and functional groups (alkyne). After conventional protections, the modified adenosine can be phosphytylated and inserted into an oligonucleotide without affecting the standard protocols for supported oligonucleotide synthesis. The hybridization properties of a generic oligonucleotide containing the above conjugated moieties toward both DNA and RNA are evaluated both in the case of a perfectly complementary strand and in the case of a single mismatch. This methodology is suitable for the preparation of several types of derivatives and­through the alkynyl moiety­provides fast access to click-chemistry transformations.

Severe side effects of health migration: stress and anger.

A great deal of immigrants of very sick children treated in our country are unable to elaborate an effective coping strategy and a methodology of resilience. Their culture and communication difficulties do not allow them to build a strong self help system. Herein, we report five stories of mothers and children who were cared for in our Children's Hospital. Anger; this is quite a common emotion among many immigrant parents of sick children with high risk diseases who have been treated in our hospital for long periods of time. They live together in community housing with families from other countries, and of different religions and habits. Anger affects their personal and social well-being. Self-blame is a common expression of their condition, and they are unable to make helpful self sacrifices. They feel anger as a result of what has happened to them, and they do not have the abilities they need to activate a good defence mechanism. Resilience is completely unknown to them. In most cases, their relatives do not intend to help them. These mothers are far from their families and habits, they are alone with their child, they suffer but their relatives push them to go back home and to renounce to the hope of cure given by the physicians. They experience a loss in their self-monitoring ability to build a coping strategy and resilience. In these cases their display of anger is often exaggerated. The anger of the immigrants is an additional problem for physicians and other caregivers working in hospitals which treat immigrant children.

Oligothiophenes as fluorescent markers for biological applications.

This paper summarizes some of our results on the application of oligothiophenes as fluorescent markers for biological studies. The oligomers of thiophene, widely known for their semiconductor properties in organic electronics, are also fluorescent compounds characterized by chemical and optical stability, high absorbance and quantum yield. Their fluorescent emission can be easily modulated via organic synthesis by changing the number of thiophene rings and the nature of side-chains. This review shows how oligothiophenes can be derivatized with active groups such as phosphoramidite, N-hydroxysuccinimidyl and 4-sulfotetrafluorophenyl esters, isothiocyanate and azide by which the (bio)molecules of interest can be covalently bound. This paper also describes how molecules such as oligonucleotides, proteins and even nanoparticles, tagged with oligothiophenes, can be used in experiments ranging from hybridization studies to imaging of fixed and living cells. Finally, a few multilabeling experiments are described.

Adolescents and cancer: discrepancy between untroubled appearance and insight disturbance.

The care of adolescents suffering from high risk disease is a demanding challenge. At times, adolescents may well be too old for the pediatrician and Pediatric Units, and yet too young for the adult practitioner. A dedicated approach that builds on the insight deriving from special attention to, and reflection on the adolescent patient's true desire to share feelings is pivotal to the efforts that must be made to improve the perceived quality of life. The specific age class of adolescence is the focus of few papers, and we feel that a broader discussion of the subject would be useful. In our short article we report the pieces written by three very positive-minded girls suffering from cancer for the journal of our Department. They show deep and severe insight disturbance.

Fluoroquinolones as potential photochemotherapeutic agents: covalent addition to guanosine monophosphate.

The triplet aryl cation photochemically generated from fluoroquinolones bearing a fluoro atom at position 8 attacks guanosine monophosphate (k(r) > 10(9) M(-1)s(-1)) and forms covalent adducts. The reaction is a model for the implementation of oxygen-independent photochemotherapy.