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.
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.
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.
The development of new sequencing platforms, technologies, and bioinformatics tools in the past decade fostered key discoveries in human genomics. Among the most recent sequencing technologies, ...nanopore sequencing (NS) has caught the interest of researchers for its intriguing potential and flexibility. This up-to-date review highlights the recent application of NS in the hematology field, focusing on progress and challenges of the technological approaches employed for the identification of pathologic alterations. The molecular and analytic pipelines developed for the analysis of the whole-genome, target regions, and transcriptomics provide a proof of evidence of the unparalleled amount of information that could be retrieved by an innovative approach based on long-read sequencing.
Since the longer a read is, the more overlap it has with other reads, third generation sequencing technology facilitates the assembly of genomic regions and could resolve complex and wide structural variants or regions enriched for repetitive elements.Nanopore sequencing (NS) provides significant advantages for whole-genome studies and, thanks to the ability to span huge regions, it could be exploited to screen specific genes of pathologic relevance affected by unknown rearrangements (i.e., F8, BCR-ABL) or composed of repetitive elements and high GC content (i.e., CEBPA, FLT3).Owing to the nature of NS technology, the ability to direct sequence-untouched nucleic acids minimizes the bias associated with PCR amplification and enables the simultaneous detection of nucleotide sequence and the epigenetic changes of the template.The rapid workflow required to prepare NS libraries, along with the possibility to set real-time data analysis, allows retrieval of information of clinical utility at whole-genome and target region level in a unique reaction in 24/48 h.
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‐β1 (TGF‐β1) is a key player in PMF pathogenesis, we further investigated the effect of TGF‐β1 on ROS and miR‐382‐5p levels. Our data showed that TGF‐β1 treatment enhances miR‐382‐5p expression and reduces SOD2 activity leading to ROS accumulation. Finally, inhibition of TGF‐β1 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‐β1/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‐β1 in PMF patients.
Database linking
GEO: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE103464.
TGF‐β1/miR‐382‐5p/SOD2 axis is involved in the pathogenesis of primary myelofibrosis (PMF). TGF‐β1/miR‐382‐5p/SOD2 axis deregulation induces reactive oxygen species (ROS) overproduction in PMF stem/progenitor cells. Increased levels of TGF‐β1 in plasma enhance miR‐382‐5p expression, which in turn leads to ROS accumulation by targeting SOD2. The TGF‐β‐receptor I kinase inhibitor galunisertib decreases ROS production by reducing the expression level of miR‐382‐5p and restoring SOD2 activity.
Pyridine- and phosphine-based ligands modified with ethacrynic acid (a broad acting glutathione transferase inhibitor) were prepared and coordinated to ruthenium(II)–arene complexes and to a ...ruthenium(III) NAMI-A type complex. All the compounds (ligands and complexes) were fully characterized by analytical and spectroscopic methods and, in one case, by single-crystal X-ray diffraction. The in vitro anticancer activity of the compounds was studied, with the compounds displaying moderate cytotoxicity toward the human ovarian cancer cell lines. All the complexes led to similar levels of residual GST activity in the different cell lines, irrespective of the stability of the Ru–ligand bond.
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The JAK1/JAK2 inhibitor Ruxolitinib (ruxo) produced rapid and sustained responses in splenomegaly and symptomatic improvement in ...patients (pts) with myelofibrosis (MF), supporting the central role of dysregulated JAK2 signaling in myeloproliferative neoplasms (MPN). Splenomegaly and constitutional symptoms improved also after treatment with Everolimus (RAD001), a rapamycin-derivative inhibitor of the serine/threonine kinase mTOR, in a phase I/II study, pointing to mTOR pathway as a novel target for MPN therapy. BEZ235 is a dual phosphatidylinositol-3-kinase (PI3K) and mTOR inhibitor currently in clinical trials in solid cancer. Aim of the study was to investigate the efficacy of BEZ235, alone and in combination with ruxo, against MPN cells in vitro and in preclinical mouse models.
We used mouse (Ba/F3 and Ba/F3-EPOR expressing wild-type (WT) or V617F(VF) mutated JAK2) and human (VF HEL and SET2 or WT K562) cell lines and primary MPN CD34+ cells from pts with MF or polycythemia vera (PV); cell proliferation, colony formation, apoptosis, cell cycle and protein phosphorylation status were evaluated. Effects of drug combination were analyzed according to Chou and Talalay to calculate the combination index (CI); a CI <1.0 indicates synergistic activity. For in vivo studies, two mouse models were used. (1) SCID mice receiving iv Ba/F3-EPOR VF-luciferase (luc) cells (gift of T. Radimerski) were randomized on d6 to treatment groups (either drugs alone and in combination or vehicle (VE)) based on baseline luminescence. Bioluminescence measurement was done at week intervals until death. (2) A C57Bl6/J JAK2 V617F Knock-in mouse model was generated by the flex switch strategy with insertion of the reversed JAK2V617F exon 13 sequence; mating with Vav-Cre transgenic mice activates the VF allele producing a MPN phenotype in progenies from VF heterozygous expression. Mice were treated for 5d, then blood, spleen and bone marrow cells were analyzed.
We found that BEZ235 inhibited proliferation of Ba/F3 VF and Ba/F3-EPOR VF cells at concentrations significantly lower than the wt counterparts (IC50 was 64±10nM vs 10,000±500nM and 87±50nM vs 676±200nM; P<0.01); similar preferential inhibition was observed in HEL and SET2 cells compared to K562 cells (IC50, 387±90nM and 334±40nM vs 5,000±1,000nM; P<0.01). BEZ235 dose-dependently increased the percentage of cells in G0/G1 and induced apoptosis. Western blot analysis showed marked reduction of the mTOR target p4EBP1 as well as appreciable downregulation of pSTAT5 and pSTAT3 at 6 to 24h of treatment. BEZ235 impaired the proliferation of CD34+ cells from MF pts with an IC50= 43±20nM vs 780±150nM in healthy donors (P<0.01), and reduced colony formation of MF and PV hematopoietic progenitors at doses statistically lower (2 to 15-fold) than normal cells. The growth of EPO-independent colonies (EEC) in PV pts was potently inhibited (IC50=20±10nM). Co-treatment of BEZ235+ruxo resulted in synergistic inhibition of proliferation in SET2 (median CI=0.37) and BaF3-EPOR VF (CI=0.77) cells and increased apoptosis rate in SET2 (CI=0.25); drug combination was highly effective also in the EEC assay (CI=0.17). In the Ba/F3VF luc model, the median bioluminescence index (No. of pixel) on day 7 of treatment was 4.7×106 in VE animals, 7.2×106 in ruxo (P=ns), 1.1×106 in BEZ235 (P=0.04) and 4.6×105 in animals receiving the BEZ+ruxo combination (P<0.01). The median survival in mice treated with the combination of BEZ+ruxo was 30d, significantly longer than either ruxo (18.5d; P<0.01) or BEZ (24d; P<0.04) alone (15.0d in VE animals). In JAK2V617F KI mice treated for 5d, we found that drug combination was significantly more effective in reducing enlarged spleen (median spleen index (spleen weight/body weightx100): 38, 35, 27 and 7 for VE, BEZ, Ruxo and BEZ+Ruxo) and reticulocyte count (median No. per HPF: 48, 50, 44, and 3 for VE, BEZ, Ruxo and BEZ+Ruxo) than either drugs alone. The phosho levels of STAT5 and 4EBP1 in the spleen were significantly reduced in mice receiving BEZ+Ruxo as compared to single treatment.
Combined inhibition of PI3K/mTOR and JAK2 signaling resulted in enhanced activity in preclinical models of MPN compared with either treatment alone, providing a rationale for the development of combination clinical trials.
Vannucchi:Novartis: Membership on an entity's Board of Directors or advisory committees.
Somatic mutations of calreticulin (CALR) have been described in approximately 60-80% of JAK2 and MPL unmutated Essential Thrombocythemia and Primary Myelofibrosis patients. CALR is an endoplasmic ...reticulum (ER) chaperone responsible for proper protein folding and calcium retention. Recent data demonstrated that the TPO receptor (MPL) is essential for the development of CALR mutant-driven Myeloproliferative Neoplasms (MPNs). However, the precise mechanism of action of CALR mutants haven't been fully unraveled. In this study, we showed that CALR mutants impair the ability to respond to the ER stress and reduce the activation of the pro-apoptotic pathway of the unfolded protein response (UPR). Moreover, our data demonstrated that CALR mutations induce increased sensitivity to oxidative stress, leading to increase oxidative DNA damage. We finally demonstrated that the downmodulation of OXR1 in CALR-mutated cells could be one of the molecular mechanisms responsible for the increased sensitivity to oxidative stress mediated by mutant CALR. Altogether, our data identify novel mechanisms collaborating with MPL activation in CALR-mediated cellular transformation. CALR mutants negatively impact on the capability of cells to respond to oxidative stress leading to genomic instability and on the ability to react to ER stress, causing resistance to UPR-induced apoptosis.
International collaborations over the years have produced a series of prognostic models for primary myelofibrosis (PMF), including the recently unveiled mutation-enhanced international prognostic ...scoring systems for transplant-age patients (MIPSS70 and MIPSS70-plus). In the current study, we considered the feasibility of a genetically inspired prognostic scoring system (GIPSS) that is exclusively based on genetic markers. Among 641 cytogenetically annotated patients with PMF and informative for previously recognized adverse mutations, multivariable analysis identified "VHR" karyotype, "unfavorable" karyotype, absence of type 1/like CALR mutation and presence of ASXL1, SRSF2, or U2AF1Q157 mutation, as inter-independent predictors of inferior survival; the respective HRs (95% CI) were 3.1 (2.1-4.3), 2.1 (1.6-2.7), 2.1 (1.6-2.9), 1.8 (1.5-2.3), 2.4 (1.9-3.2), and 2.4 (1.7-3.3). Based on HR-weighted risk points, a four-tiered GIPSS model was devised: low (zero points; n = 58), intermediate-1 (1 point; n = 260), intermediate-2 (2 points; n = 192), and high (≥3 points; n = 131); the respective median (5-year) survivals were 26.4 (94%), 8.0 (73%), 4.2 (40%), and 2 (14%) years; the model was internally validated by bootstrapping and its predictive accuracy was shown to be comparable to that of MIPSS70-plus. GIPPS offers a low-complexity prognostic tool for PMF that is solely dependent on genetic risk factors and, thus, forward-looking in its essence.
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