Visible light-promoted C(sp3)-H α-carbamoylation of cyclic ethers with isocyanides.

A protocol exploiting isocyanides as carbamoylating agents for the α-C(sp3)-H functionalization of cyclic ethers has been optimized via a combined visible light-driven hydrogen atom transfer/Lewis acid-catalyzed approach. The isocyanide substrate scope revealed an exquisite functional group compatibility (18 examples, with yields up to 99%). Both radical and polar trapping, kinetic isotopic effect and real-time NMR studies support the mechanistic hypothesis and provide insightful details for the design of new chemical processes involving the generation of oxocarbenium ions.

Glucopeptides derived from myelin-relevant proteins and hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin cross-react with multiple sclerosis specific antibodies: A step forward in the identification of native autoantigens in multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory and autoimmune disorder, in which an antibody-mediated demyelination mechanism plays a critical role. We prepared two glucosylated peptides derived from the human myelin proteins, that is, oligodendrocyte-myelin glycoprotein (OMGp) and reticulon-4 receptor (RTN4R), selected by a bioinformatic approach for their conformational homology with CSF114(Glc), a designed ß-turn antigenic probe derived from myelin oligodendrocyte glycoprotein (MOG), a glycoprotein present in the CNS. This synthetic antigen is specifically recognized by antibodies in sera of MS patients. We report herein the antigenic properties of these peptides, showing, on the one hand, that MS patient antibodies recognize the two glucosylated peptides and, on the other hand, that these antibodies cross-react with CSF114(Glc) and with the previously described hyperglucosylated nontypeable Haemophilus influenzae bacterial adhesin protein HMW1ct(Glc). These observations point to an immunological association between human and bacterial protein antigens, underpinning the hypothesis that molecular mimicry triggers the breakdown of self-tolerance in MS and suggesting that RTN4R and OMGp can be considered as autoantigens.

Introduction of constrained Trp analogs in RW9 modulates structure and partition in membrane models.

Over the past decades, many cell-penetrating peptides (CPP) have been studied for their capacity to cross cellular membranes, mostly in order to improve cellular uptake of therapeutic agents. Even though hydrophobic and anionic CPPs have been described, many of them are polycationic, due to the presence of several arginine (Arg) residues. Noteworthy, however, the presence of aromatic amino acids such as tryptophan (Trp) within CPPs seems to play an important role to reach high membranotropic activity. RW9 (RRWWRRWRR) is a designed CPP derived from the polyarginine R9 presenting both features. In general, when interacting with membranes, CPPs adopt an optimal conformation for membrane interactions - an amphipathic helical secondary structure in the case of RW9. Herein, we assumed that the incorporation of a locally constrained amino acid in the peptide sequence could improve the membranotropic activity of RW9, by facilitating its structuration upon contact with a membrane, while leaving a certain plasticity. Therefore, two cyclized Trp derivatives (Tcc and Aia) were synthesized to be incorporated in RW9 as surrogates of Trp residues. Thus, a series of peptides containing these building blocks has been synthesized by varying the type, position, and number of modifications. The membranotropic activity of the RW9 analogs was studied by spectrofluorescence titration of the peptides in presence of liposomes (DMPG), allowing to calculate partition coefficients (Kp). Our results indicate that the partitioning of the modified peptides depends on the type, the number and the position of the modification, with the best sequence being [Aia4]RW9. Interestingly, both NMR analysis and molecular dynamic (MD) simulations indicate that this analog presents an extended conformation similar to the native RW9, but with a much-reduced structural flexibility. Finally, cell internalization properties were also confirmed by confocal microscopy.

Lateralized overgrowth with vascular malformation caused by a somatic PTPN11 pathogenic variant: Another piece added to the puzzle of mosaic RASopathies.

Lateralized/segmental overgrowth disorders (LOs) encompass a heterogeneous group of congenital conditions with excessive body tissue growth. Documented molecular alterations in LOs mostly consist of somatic variants in genes of the PI3KCA/AKT/mTOR pathway or of chromosome band 11p15.5 imprinted region anomalies. In some cases, somatic pathogenic variants in genes of the RAS/MAPK pathway have been reported. We present the first case of a somatic pathogenic variant (T507K) in PTPN11 causing a LO phenotype characterized by severe lateralized overgrowth, vascular proliferation, and cerebral astrocytoma. The T507K variant was detected in DNA from overgrown tissue in a leg with capillary malformation. The astrocytoma tissue showed a higher PTPN11 variant allele frequency. A pathogenic variant in FGFR1 was also found in tumor tissue, representing a second hit on the RAS/MAPK pathway. These findings indicate that RAS/MAPK cascade overactivation can cause mosaic overgrowth phenotypes resembling PIK3CA-related overgrowth disorders (PROS) with cancer predisposition and are consistent with the hypothesis that RAS/MAPK hyperactivation can be involved in the pathogenesis of astrocytoma. This observation raises the issue of cancer predisposition in patients with RAS/MAPK pathway gene variants and expands genotype spectrum of LOs and the treatment options for similar cases through inhibition of the RAS/MAPK oversignaling.

COVID-19 Pandemic and How It Affected Sino-Italian Relations.

Italy was the most affected European country at the beginning of the COVID-19 pandemic. While the European Union was struggling to get its act together and provide assistance to an ally in need, Russia and China used the opportunity to promote their own agendas. The focus of this article is on the economic and social impact of the COVID-19 pandemic on Italy, China's disinformation campaign, and the uncertain future of the relations between the two countries.

Kaposiform hemangioendothelioma further broadens the phenotype of PIK3CA-related overgrowth spectrum.

Kaposiform hemangioendothelioma (KHE) is a rare locally aggressive mixed vascular tumor, with typical onset in early childhood and characterized by progressive angio- and lymphangiogenesis. Its etiopathogenesis and molecular bases are still unclear. Here, we report the first case of congenital KHE harboring a PIK3CA mosaic pathogenic variant (c.323G > A, p.Arg108His) in a boy with very subtle PIK3CA-related overgrowth spectrum (PROS) features. This finding provides insights into the pathophysiology of KHE, offering targeted therapeutic options by inhibition of the PI3K/Akt/mTOR pathway. We propose the inclusion of this mixed lymphatic and vascular anomaly within the PROS.

A novel ß-hairpin peptide derived from the ARC repressor selectively interacts with the major groove of B-DNA.

Transcription factors (TFs) have a remarkable role in the homeostasis of the organisms and there is a growing interest in how they recognize and interact with specific DNA sequences. TFs recognize DNA using a variety of structural motifs. Among those, the ribbon-helix-helix (RHH) proteins, exemplified by the MetJ and ARC repressors, form dimers that insert antiparallel ß-sheets into the major groove of DNA. A great chemical challenge consists of using the principles of DNA recognition by TFs to design minimized peptides that maintain the DNA affinity and specificity characteristics of the natural counterparts. In this context, a peptide mimic of an antiparallel ß-sheet is very attractive since it can be obtained by a single peptide chain folding in a ß-hairpin structure and can be as short as 14 amino acids or less. Herein, we designed eight linear and two cyclic dodeca-peptides endowed with ß-hairpins. Their DNA binding properties have been investigated using fluorescence spectroscopy together with the conformational analysis through circular dichroism and solution NMR. We found that one of our peptides, peptide 6, is able to bind DNA, albeit without sequence selectivity. Notably, it shows a topological selectivity for the major groove of the DNA which is the interaction site of ARC and many other DNA-binding proteins. Moreover, we found that a type I' ß-hairpin folding pattern is a favorite peptide structure for interaction with the B-DNA major groove. Peptide 6 is a valuable lead compound for the development of novel analogs with sequence selectivity.

Synthesis and Characterization of Bis-Triazolyl-Pyridine Derivatives as Noncanonical DNA-Interacting Compounds.

Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.

Disulfide Bond Replacement with 1,4- and 1,5-Disubstituted [1,2,3]-Triazole on C-X-C Chemokine Receptor Type 4 (CXCR4) Peptide Ligands: Small Changes that Make Big Differences.

Here we investigated the structural and biological effects ensuing from the disulfide bond replacement of a potent and selective C-X-C chemokine receptor type 4 (CXCR4) peptide antagonist, with 1,4- and 1,5- disubstituted 1,2,3-triazole moieties. Both strategies produced candidates that showed high affinity and selectivity against CXCR4. Notably, when assessed for their ability to modulate the CXCL12-mediated cell migration, the 1,4-triazole variant conserved the antagonistic effect in the low-mid nanomolar range, while the 1,5-triazole one displayed the ability to activate the migration, becoming the first in class low-molecular-weight CXCR4 peptide agonist. By combining NMR and computational studies, we provided a valuable model that highlighted differences in the interactions of the two peptidomimetics with the receptor that could account for their different functional profile. Finally, we envisage that our findings could be translated to different GPCR-interacting peptides for the pursuit of novel chemical probes that could assist in dissecting the complex puzzle of this fundamental class of transmembrane receptors.

Programming and reprogramming a human heart cell.

The latest discoveries and advanced knowledge in the fields of stem cell biology and developmental cardiology hold great promise for cardiac regenerative medicine, enabling researchers to design novel therapeutic tools and approaches to regenerate cardiac muscle for diseased hearts. However, progress in this arena has been hampered by a lack of reproducible and convincing evidence, which at best has yielded modest outcomes and is still far from clinical practice. To address current controversies and move cardiac regenerative therapeutics forward, it is crucial to gain a deeper understanding of the key cellular and molecular programs involved in human cardiogenesis and cardiac regeneration. In this review, we consider the fundamental principles that govern the "programming" and "reprogramming" of a human heart cell and discuss updated therapeutic strategies to regenerate a damaged heart.

Megabase-scale deletion using CRISPR/Cas9 to generate a fully haploid human cell line.

Near-haploid human cell lines are instrumental for genetic screens and genome engineering as gene inactivation is greatly facilitated by the absence of a second gene copy. However, no completely haploid human cell line has been described, hampering the genetic accessibility of a subset of genes. The near-haploid human cell line HAP1 contains a single copy of all chromosomes except for a heterozygous 30-megabase fragment of Chromosome 15. This large fragment encompasses 330 genes and is integrated on the long arm of Chromosome 19. Here, we employ a CRISPR/Cas9-based genome engineering strategy to excise this sizeable chromosomal fragment and to efficiently and reproducibly derive clones that retain their haploid state. Importantly, spectral karyotyping and single-nucleotide polymorphism (SNP) genotyping revealed that engineered-HAPloid (eHAP) cells are fully haploid with no gross chromosomal aberrations induced by Cas9. Furthermore, whole-genome sequence and transcriptome analysis of the parental HAP1 and an eHAP cell line showed that transcriptional changes are limited to the excised Chromosome 15 fragment. Together, we demonstrate the feasibility of efficiently engineering megabase deletions with the CRISPR/Cas9 technology and report the first fully haploid human cell line.

Imprinted Igf2r silencing depends on continuous Airn lncRNA expression and is not restricted to a developmental window.

The imprinted Airn macro long non-coding (lnc) RNA is an established example of a cis-silencing lncRNA. Airn expression is necessary to initiate paternal-specific silencing of the Igf2r gene, which is followed by gain of a somatic DNA methylation imprint on the silent Igf2r promoter. However, the developmental requirements for Airn initiation of Igf2r silencing and the role of Airn or DNA methylation in maintaining stable Igf2r repression have not been investigated. Here, we use inducible systems to control Airn expression during mouse embryonic stem cell (ESC) differentiation. By turning Airn expression off during ESC differentiation, we show that continuous Airn expression is needed to maintain Igf2r silencing, but only until the paternal Igf2r promoter is methylated. By conditionally turning Airn expression on, we show that Airn initiation of Igf2r silencing is not limited to one developmental 'window of opportunity' and can be maintained in the absence of DNA methylation. Together, this study shows that Airn expression is both necessary and sufficient to silence Igf2r throughout ESC differentiation and that the somatic methylation imprint, although not required to initiate or maintain silencing, adds a secondary layer of repressive epigenetic information.

Selective synthesis of thioethers in the presence of a transition-metal-free solid Lewis acid.

The synthesis of thioethers starting from alcohols and thiols in the presence of amorphous solid acid catalysts is reported. A silica alumina catalyst with a very low content in alumina gave excellent results in terms of both activity and selectivity also under solvent-free conditions. The reaction rate follows the electron density of the carbinol atom in the substrate alcohol and yields up to 99% and can be obtained for a wide range of substrates under mild reaction conditions.

A downstream CpG island controls transcript initiation and elongation and the methylation state of the imprinted Airn macro ncRNA promoter.

A CpG island (CGI) lies at the 5' end of the Airn macro non-protein-coding (nc) RNA that represses the flanking Igf2r promoter in cis on paternally inherited chromosomes. In addition to being modified on maternally inherited chromosomes by a DNA methylation imprint, the Airn CGI shows two unusual organization features: its position immediately downstream of the Airn promoter and transcription start site and a series of tandem direct repeats (TDRs) occupying its second half. The physical separation of the Airn promoter from the CGI provides a model to investigate if the CGI plays distinct transcriptional and epigenetic roles. We used homologous recombination to generate embryonic stem cells carrying deletions at the endogenous locus of the entire CGI or just the TDRs. The deleted Airn alleles were analyzed by using an ES cell imprinting model that recapitulates the onset of Igf2r imprinted expression in embryonic development or by using knock-out mice. The results show that the CGI is required for efficient Airn initiation and to maintain the unmethylated state of the Airn promoter, which are both necessary for Igf2r repression on the paternal chromosome. The TDRs occupying the second half of the CGI play a minor role in Airn transcriptional elongation or processivity, but are essential for methylation on the maternal Airn promoter that is necessary for Igf2r to be expressed from this chromosome. Together the data indicate the existence of a class of regulatory CGIs in the mammalian genome that act downstream of the promoter and transcription start.

Discovery of 2,3-Diaminoindole Derivatives as a Novel Class of NOD Antagonists.

NOD1 and NOD2 are members of the pattern recognition receptors involved in the innate immune response. Overactivation of NOD1 is implicated in inflammatory disorders, multiple sclerosis, and cancer cell metastases. NOD1 antagonists would represent valuable pharmacological tools to gain further insight into protein roles, potentially leading to new therapeutic strategies. We herein report the expansion of the chemical space of NOD1 antagonists via a multicomponent synthetic approach affording a novel chemotype, namely, 2,3-diaminoindoles. These efforts resulted in compound 37, endowed with low micromolar affinity toward NOD1. Importantly, a proof-of-evidence of direct binding to NOD1 of Noditinib-1 and derivative 37 is provided here for the first time. Additionally, the combination of computational studies and NMR-based displacement assays enabled the characterization of the binding modality of 37 to NOD1, thus providing key unprecedented knowledge for the design of potent and selective NOD1 antagonists.

Developmental control of imprinted expression by macro non-coding RNAs.

Genomic imprinting is a developmentally regulated epigenetic phenomenon. The majority of imprinted genes only show parent-of-origin specific expression in a subset of tissues or at defined developmental stages. In some cases, imprinted expression is controlled by an imprinted macro non-coding RNA (ncRNA) whose expression pattern and repressive activity does not necessarily correlate with that of the genes whose imprinted expression it controls. This suggests that developmentally regulated factors other than the macro ncRNA are involved in establishing or maintaining imprinted expression. Here, we review how macro ncRNAs control imprinted expression during development and differentiation and consider how this impacts on target choice in epigenetic therapy.

SS18-SSX Antibody: A Useful Tool to Save Time and Reduce Costs in Synovial Sarcoma Diagnosis. Proposal of a Novel Diagnostic Algorithm.

Synovial sarcoma is a rare malignant mesenchymal neoplasm mostly affecting young adults, characterized by a specific translocation which results in the fusion of the SS18 gene on chromosome 18 with one of the three highly homologous SSX genes on chromosome X. Its morphological diagnosis, especially in monophasic or poorly differentiated variants, can be challenging because histological features often overlap with other malignant mesenchymal tumors. Until recently, the differential diagnosis mostly relied on the use of cytogenetic or molecular analyses to detect the specific t(X;18)(p11;q11) translocation, thus virtually restricting its correct identification to referral centers with a high histological and molecular pathology workflow. The recently commercialized highly sensitive and fusion-specific SS18-SSX antibody has significantly improved the approach to these tumors, representing a relatively cheap and easy to access tool for synovial sarcoma diagnosis. Through a retrospective analysis of 79 synovial sarcomas and histological mimickers, this study confirms the usefulness of the SS18-SSX antibody in the diagnosis of synovial sarcoma, particularly focusing on its application in the pathological response evaluation after neoadjuvant treatment as well as its time- and cost-saving advantages. Finally, we here propose a new diagnostic algorithm to apply into the routine practice.

Solitary fibrous tumor of the thyroid: report of three cases with a focus on cytological features and histological clues for malignancy.

Solitary fibrous tumor is a mesenchymal tumor of intermediate malignant potential characterized by a recurrent NAB2::STAT6 fusion and STAT6 nuclear expression. Primary thyroid solitary fibrous tumor is relatively uncommon, with 45 cases described in the English literature to date. Although its histologic features are characteristic, its diagnosis in the thyroid can be problematic, especially in small biopsies or cytology specimens. We herein present three new cases of thyroid solitary fibrous tumor, one of which is malignant, with new insights on the morphological spectrum and malignant potential of this tumor. We additionally provide a review of the literature with a focus on the clues and challenges of a preoperative cytological diagnosis of this tumor, which can nowadays be supported by STAT6 nuclear expression, when appropriately suspected.

Silencing and transcriptional properties of the imprinted Airn ncRNA are independent of the endogenous promoter.

The Airn macro ncRNA is the master regulator of imprinted expression in the Igf2r imprinted gene cluster where it silences three flanking genes in cis. Airn transcription shows unusual features normally viewed as promoter specific, such as impaired post-transcriptional processing and a macro size. The Airn transcript is 108 kb long, predominantly unspliced and nuclear localized, with only a minority being variably spliced and exported. Here, we show by deletion of the Airn ncRNA promoter and replacement with a constitutive strong or weak promoter that splicing suppression and termination, as well as silencing activity, are maintained by strong Airn expression from an exogenous promoter. This indicates that all functional regions are located within the Airn transcript. DNA methylation of the maternal imprint control element (ICE) restricts Airn expression to the paternal allele and we also show that a strong active promoter is required to maintain the unmethylated state of the paternal ICE. Thus, Airn expression not only induces silencing of flanking mRNA genes but also protects the paternal copy of the ICE from de novo methylation.

Photomicellar Catalyzed Synthesis of Amides from Isocyanides: Optimization, Scope, and NMR Studies of Photocatalyst/Surfactant Interactions.

The merging of micellar and photoredox catalysis represents a key issue to promote "in water" photochemical transformations. A photomicellar catalyzed synthesis of amides from N-methyl-N-alkyl aromatic amines and both aliphatic and aromatic isocyanides is herein presented. The mild reaction conditions enabled a wide substrate scope and a good functional groups tolerance, as further shown in the late-stage functionalization of complex bioactive scaffolds. Furthermore, solution 1D and 2D NMR experiments performed, for the first time, in the presence of paramagnetic probes enabled the study of the reaction environment at the atomic level along with the localization of the photocatalyst with respect to the micelles, thus providing experimental data to drive the identification of optimum photocatalyst/surfactant pairing.