Unusual activation pathways of amines in the reactions with molybdenum pentachloride

The 1-:-1 molar reactions at room temperature of MoCl5 with aliphatic amines were investigated in dichloromethane. Pyrrolidine, diethylamine and dibenzylamine underwent dehydrogenative oxidation when allowed to react with MoCl5; the compounds [MoCl5NCH(CH2)3], 1, and [CH3CHNHEt][MoOCl4], 2, were isolated in moderate to low yields from MoCl5/pyrrolidine and MoCl5/NHEt2, respectively. The chloride-amide complex [MoCl4(NEt2)], 3, was afforded in 65% yield from MoCl5 and Et2NSiMe3. The interaction of MoCl5 with Me2NSiMe3 was accompanied by activation of the solvent, and the complexes [MoCl3(NMe2)(κ2-Me2NCH2NMe2)], 4a, and [MoCl3(NMe)(κ2-Me2NCH2NMe2)], 4b, co-crystallized from the reaction mixture. The reactions of MoCl5 with a series of primary amines afforded mixtures of products, and the Mo(vi) chloride imido complexes [MoCl4(NR)]2 (R = Cy, 5a; tBu, 5b) were isolated in ca. 40% yield from MoCl5/NH2R (R = Cy, tBu). C-H bond activation may be viable in the reactions of MoCl5 with tertiary amines: the compounds [(CH2Ph)2NCHPh]2[MoCl6]·CH2Cl2, 6, and [NHEt3]2[Mo2Cl10], 7, were obtained from MoCl5/tribenzylamine and MoCl5/triethylamine, respectively. Pyrrolidine and tribenzylamine underwent analogous activation pathways when allowed to react with [MoCl3OCH(CF3)2]2 in the place of MoCl5. The isolated metal products were characterized by analytical and spectroscopic techniques, in addition the structures of 1, 2, 4, 5a, 6·CH2Cl2 and 7 were ascertained by single crystal X-ray diffraction studies. The organic products were identified by NMR and GC-MS after hydrolysis of the reaction mixtures. DFT calculations were carried out in order to assist the IR assignments, and clarify structural and mechanistic aspects

Resolving complex structural variants via nanopore sequencing

The recent development of high-throughput sequencing platforms provided impressive insights into the field of human genetics and contributed to considering structural variants (SVs) as the hallmark of genome instability, leading to the establishment of several pathologic conditions, including neoplasia and neurodegenerative and cognitive disorders. While SV detection is addressed by next-generation sequencing (NGS) technologies, the introduction of more recent long-read sequencing technologies have already been proven to be invaluable in overcoming the inaccuracy and limitations of NGS technologies when applied to resolve wide and structurally complex SVs due to the short length (100–500 bp) of the sequencing read utilized. Among the long-read sequencing technologies, Oxford Nanopore Technologies developed a sequencing platform based on a protein nanopore that allows the sequencing of “native” long DNA molecules of virtually unlimited length (typical range 1–100 Kb). In this review, we focus on the bioinformatics methods that improve the identification and genotyping of known and novel SVs to investigate human pathological conditions, discussing the possibility of introducing nanopore sequencing technology into routine diagnostics

Role of TGF-\u3b21/miR-382-5p/SOD2 axis in the induction of oxidative stress in CD34+ cells from primary myelofibrosis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm characterized by an excessive production of pro-inflammatory cytokines resulting in chronic inflammation and genomic instability. Besides the driver mutations in JAK2, MPL, and CALR genes, the deregulation of miRNA expression may also contribute to the pathogenesis of PMF. To this end, we recently reported the upregulation of miR-382-5p in PMF CD34+ cells. In order to unveil the mechanistic details of the role of miR-382-5p in pathogenesis of PMF, we performed gene expression profiling of CD34+ cells overexpressing miR-382-5p. Among the downregulated genes, we identified superoxide dismutase 2 (SOD2), which is a predicted target of miR-382-5p. Subsequently, we confirmed miR-382-5p/SOD2 interaction by luciferase assay and we showed that miR-382-5p overexpression in CD34+ cells causes the decrease in SOD2 activity leading to reactive oxygen species (ROS) accumulation and oxidative DNA damage. In addition, our data indicate that inhibition of miR-382-5p in PMF CD34+ cells restores SOD2 function, induces ROS disposal, and reduces DNA oxidation. Since the pro-inflammatory cytokine transforming growth factor-\u3b21 (TGF-\u3b21) is a key player in PMF pathogenesis, we further investigated the effect of TGF-\u3b21 on ROS and miR-382-5p levels. Our data showed that TGF-\u3b21 treatment enhances miR-382-5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF-\u3b21 signaling in PMF CD34+ cells by galunisertib significantly reduced miR-382-5p expression and ROS accumulation and restored SOD2 activity. As a whole, this study reports that TGF-\u3b21/miR-382-5p/SOD2 axis deregulation in PMF cells is linked to ROS overproduction that may contribute to enhanced oxidative stress and inflammation. Our results suggest that galunisertib may represent an effective drug reducing abnormal oxidative stress induced by TGF-\u3b21 in PMF patients. Database linking: GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464

Inhibition of ERK1/2 signaling prevents bone marrow fibrosis by reducing osteopontin plasma levels in a myelofibrosis mouse model

Clonal myeloproliferation and development of bone marrow (BM) fibrosis are the major pathogenetic events in myelofibrosis (MF). The identification of novel antifibrotic strategies is of utmost importance since the effectiveness of current therapies in reverting BM fibrosis is debated. We previously demonstrated that osteopontin (OPN) has a profibrotic role in MF by promoting mesenchymal stromal cells proliferation and collagen production. Moreover, increased plasma OPN correlated with higher BM fibrosis grade and inferior overall survival in MF patients. To understand whether OPN is a druggable target in MF, we assessed putative inhibitors of OPN expression in vitro and identified ERK1/2 as a major regulator of OPN production. Increased OPN plasma levels were associated with BM fibrosis development in the Romiplostim-induced MF mouse model. Moreover, ERK1/2 inhibition led to a remarkable reduction of OPN production and BM fibrosis in Romiplostim-treated mice. Strikingly, the antifibrotic effect of ERK1/2 inhibition can be mainly ascribed to the reduced OPN production since it could be recapitulated through the administration of anti-OPN neutralizing antibody. Our results demonstrate that OPN is a novel druggable target in MF and pave the way to antifibrotic therapies based on the inhibition of ERK1/2-driven OPN production or the neutralization of OPN activity

Activation of CN bonds by high-valent group 6 metal chlorides, including the conversion of an α-diimine into a functionalized imidazolium

The α-diimine (Xyl)NCHCHN(Xyl) (DADMe; Xyl = 2,6-C6H3Me2) was converted by the reaction with WCl6 into the iminomethyl-imidazolium compound [(Xyl)NCHCHN(Xyl)CCHN(Xyl)][WCl6], 1, in 47% yield. Mono (N-protonated) α-diimine salts were isolated from the reactions of WCl6 with (2,6-C6H3Et2)NC(Me)C(Me)N(2,6-C6H3Et2) (Me-DADEt) and of MoCl5 with (2,6-C6H3Et2)NCHCHN(2,6-C6H3Et2) (DADEt) and (2,6-C6H3tBu2)NCHCHN(2,6-C6H3tBu2) (DADtBu), giving respectively [(2,6-C6H3Et2)NHCHCHN(C6H2Et2Me)][WCl6], 2 (minor product), [DADEt(H)]2[Mo2Cl10], 3a, and [DADtBu(H)]2[Mo2Cl10], 3b. MoCl5 reacted with the carbodiimide (4-C6H4Me)NCN(4-C6H4Me) affording 70% yield of MoCl4[(4-C6H4Me)NC(Cl)N(4-C6H4Me)], 4. All the products were characterized by analytical and spectroscopic methods, and the X-ray structures of 1, 2 and 4 were elucidated by X-ray diffraction. DFT calculations were performed to shed light on mechanistic and structural aspects

NanoR: A user-friendly R package to analyze and compare nanopore sequencing data.

MinION and GridION X5 from Oxford Nanopore Technologies are devices for real-time DNA and RNA sequencing. On the one hand, MinION is the only real-time, low cost and portable sequencing device and, thanks to its unique properties, is becoming more and more popular among biologists; on the other, GridION X5, mainly for its costs, is less widespread but highly suitable for researchers with large sequencing projects. Despite the fact that Oxford Nanopore Technologies' devices have been increasingly used in the last few years, there is a lack of high-performing and user-friendly tools to handle the data outputted by both MinION and GridION X5 platforms. Here we present NanoR, a cross-platform R package designed with the purpose to simplify and improve nanopore data visualization. Indeed, NanoR is built on few functions but overcomes the capabilities of existing tools to extract meaningful informations from MinION sequencing data; in addition, as exclusive features, NanoR can deal with GridION X5 sequencing outputs and allows comparison of both MinION and GridION X5 sequencing data in one command. NanoR is released as free package for R at https://github.com/davidebolo1993/NanoR

Ubiquity of cis-Halide → Isocyanide Direct Interligand Interaction in Organometallic Complexes

We recently reported a density functional theory (DFT) analysis of the Nb(V)-C bond in various NbCl5(L) complexes, discovering that the carbon ligand L receives electronic density from the metal (classical back-donation) and from the chlorides in the cis position (direct interligand interaction). Here we report the synthesis and the structural characterization of two new coordination compounds of niobium pentahalides, i.e., NbX5(CNXyl) (X = Cl, Br; Xyl = 2,6-C6H3Me2), and the corresponding DFT analyses of the Nb(V)-C bond using the Natural Orbitals for Chemical Valence-Charge Displacement (NOCV-CD) approach, confirming the presence of a cis-halide → isocyanide direct interligand interaction. To verify whether the latter is limited to Nb complexes or not, we performed a NOCV-CD analysis on a series of several organometallic complexes based on Ti(IV), Nb(V), Ta(V), Rh(III), Pd(II), and Au(III), all of which bear one halide ligand and m-xylyl-isocyanide in a mutual cis position, revealing that the cis-halide → isocyanide interaction is always present

mTOR Inhibitors Alone and in Combination with JAK2 Inhibitors Effectively Inhibit Cells of Myeloproliferative Neoplasms

<div><h3>Background</h3><p>Dysregulated signaling of the JAK/STAT pathway is a common feature of chronic myeloproliferative neoplasms (MPN), usually associated with <em>JAK2</em>V617F mutation. Recent clinical trials with JAK2 inhibitors showed significant improvements in splenomegaly and constitutional symptoms in patients with myelofibrosis but meaningful molecular responses were not documented. Accordingly, there remains a need for exploring new treatment strategies of MPN. A potential additional target for treatment is represented by the PI3K/AKT/mammalian target of rapamycin (mTOR) pathway that has been found constitutively activated in MPN cells; proof-of-evidence of efficacy of the mTOR inhibitor RAD001 has been obtained recently in a Phase I/II trial in patients with myelofibrosis. The aim of the study was to characterize the effects <em>in vitro</em> of mTOR inhibitors, used alone and in combination with JAK2 inhibitors, against MPN cells.</p> <h3>Findings</h3><p>Mouse and human <em>JAK2</em>V617F mutated cell lines and primary hematopoietic progenitors from MPN patients were challenged with an allosteric (RAD001) and an ATP-competitive (PP242) mTOR inhibitor and two JAK2 inhibitors (AZD1480 and ruxolitinib). mTOR inhibitors effectively reduced proliferation and colony formation of cell lines through a slowed cell division mediated by changes in cell cycle transition to the S-phase. mTOR inhibitors also impaired the proliferation and prevented colony formation from MPN hematopoietic progenitors at doses significantly lower than healthy controls. JAK2 inhibitors produced similar antiproliferative effects in MPN cell lines and primary cells but were more potent inducers of apoptosis, as also supported by differential effects on cyclinD1, PIM1 and BcLxL expression levels. Co-treatment of mTOR inhibitor with JAK2 inhibitor resulted in synergistic activity against the proliferation of <em>JAK2</em>V617F mutated cell lines and significantly reduced erythropoietin-independent colony growth in patients with polycythemia vera.</p> <h3>Conclusions/Significance</h3><p>These findings support mTOR inhibitors as novel potential drugs for the treatment of MPN and advocate for clinical trials exploiting the combination of mTOR and JAK2 inhibitor.</p> </div

Inhibition of proliferation of CD34⁺ cells from PMF patients by mTOR and JAK2 inhibitors.

<p>CD34⁺ cells from patients with PMF (n = 6) or control subjects (Ctr; n = 5) were plated at 2×10⁵/mL in 96-well culture tissue plates with increasing concentrations of the drugs, in triplicate; viable cells were assessed after 48 hrs using the WST-1 assay and normalized to wells containing an equivalent volume of vehicle (DMSO) only. Values shown are the mean±SD The concentration at which 50% inhibition of proliferation occurred (IC₅₀) was calculated (see Results section for details).</p