ALBERT

Description: Natural fiber-filled polymers offer good mechanical properties and economic competitiveness compared to traditional materials. Wood flour is one of the most widely used fillers, and the resulting material, known as wood plastic composite (WPC), has already found a wide applicability in many industrial sectors including automotive and building construction. This paper, as a followup of a previous study on a numerical-based approach to optimize the sound transmission loss of WPC panels, presents an extensive numerical and experimental vibro-acoustic analysis of an orthotropic panel made out of WPC boards. Both structural and acoustical excitations were considered. The panel radiation efficiency and its transmission loss were modeled using analytic and semi-analytic approaches. The mechanical properties of the structure, required as input data in the prediction models, were numerically determined in terms of wavenumbers by means of finite element simulations, and experimentally verified. The accuracy of the predicted acoustic performances was assessed by comparing the numerical results with the measured data. The comparisons highlighted a significant influence of the junctions between the WPC boards, especially on the panel’s transmission loss. The radiation efficiency results were mostly influenced by the boundary conditions of the plate-like structure. This latter aspect was further investigated through a finite element analysis.

Description: DNA damage and the attendant cellular responses of apoptosis, senescence, and altered differentiation are major drivers of hematopoietic stem cell (HSC) aging. A reservoir of persistent DNA damage signaling can derive from progressive telomere erosion, which occurs over the lifespan of humans. However, the molecular mechanisms by which telomere damage compromises HSC functions are largely unknown. Here, though combined single-cell RNA-seq and functional studies of highly-purified c-Kit+Sca+Lin-CD34-flk2-CD150+CD48-CD41-HSCs, we show that persistent telomeric damage does not activate programs of apoptosis or senescence but maintains HSCs in an activated metabolic state, which directly compromises their self-renewal capability. To dissect the biological and molecular mechanisms by which persistent DNA damage affects HSC function we analyzed the HSC compartment of mice with short telomeres (G5/G6 TERTER/ER), which developed age-related defects. Immunophenotypic analysis of the HSC compartment showed that, compared with G0 TERTER/+ (G0) mice with intact telomeres (n=12), 2 month-old G5/G6 mice (n=17), had a significantly decreased number of HSCs (p

Description: The use of wood fibers is a deeply investigated topic in current scientific research and one of their most common applications is as filler for thermoplastic polymers. The resulting material is a biocomposite, known as a Wood Polymer Composite (WPC). For increasing the sustainability and reducing the cost, it is convenient to increase the wood fiber content as much as possible, so that the polymeric fraction within the composite is thereby reduced. On the other hand, this is often thwarted by a sharp decrease in toughness and processability—a disadvantage that could be overcome by compounding the material with a toughening agent. This work deals with the mechanical properties in tension and impact of polypropylene filled with 50 wt.% wood flour, toughened with different amounts (0%, 10%, and 20%) of a polypropylene-based thermoplastic vulcanizate (TPV). Such properties are also investigated as a function of extrusion processing variables, such as the feeding mode (i.e., starve vs. flood feeding) and screw speed. It is found that the mechanical properties do depend on the processing conditions: the best properties are obtained either in starve feeding conditions, or in flood feeding conditions, but at a low screw speed. The toughening effect of TPV is significant when its content reaches 20 wt.%. For this percentage, the processing conditions are less relevant in governing the final properties of the composites in terms of the stiffness and strength.

Description: The mechanisms of HMA failure in MDS remain unclear, as recent advances in sequencing approaches did not enable the molecular characterization of the cells that survive therapy and drive resistance and disease progression. Here, through combined functional and transcriptomic analyses of highly-purified hematopoietic populations isolated from the BM of 132 MDS patients enrolled in clinical trials of single drug HMA therapy, we show that 2 immunophenotypically and molecularly distinct cell types maintain the disease and expand at progression, and we propose therapeutic approaches to overcome MDS evolution. Unsupervised hierarchical clustering followed by principal component analysis of 101 untreated MDS samples based on the frequency of immunophenotypically defined stem and myeloid progenitor cell populations identified the frequencies of the lymphoid-primed multipotent progenitors (LMPPs) and granulo-monocytic progenitors (GMPs) as the main sources of variation across the samples. Further logistic regression analysis enabled the systematic stratification of the samples in 2 main groups. "CMP pattern" MDS was characterized by the prevalence of common myeloid progenitors (CMPs) in the progenitor compartment while "GMP pattern" MDS was characterized by the increased frequency of GMPs in the progenitors and by increased LMPPs and decreased long-term (LT)-HSC frequencies in the BM (Fig A, B). Functional analysis of the CMPs by immunophenotypic characterization of lineage-primed fractions and colony assays revealed that this population was myeloid-biased only in "CMP pattern" MDS but not in "GMP pattern" MDS, suggesting that 2 distinct hierarchical differentiation routes underlie disease manifestation (Fig C). Further logistic regression analysis of 31 MDS samples from patients with progressive disease showed that whereas "CMP pattern" MDS patients with blast expansion were characterized by a significant increase in the BM frequency of LT-HSCs, "GMP pattern" MDS patients were characterized by the expansion of the LMPPs (Fig D). To investigate the molecular pathways underlying the 2 different hierarchical patterns of progression, we analyzed the transcriptional profiling of the HSCs that selectively expanded in the 2 groups of MDS patients. RNA-Seq analysis revealed that, compared with those isolated from patients at baseline, LT-HSCs isolated from "CMP pattern" MDS patients with blast expansion had significantly upregulated genes involved in promoting cell proliferation and survival, including the anti-apoptotic regulator BCL2. In contrast, genes involved in the response to TNF-a were significantly upregulated in the LMPPs from "GMP pattern" MDS patients with progressive disease as compared with the LMPPs isolated at baseline (Fig E). Then, we hypothesized that, despite genetic dissimilarities, the HSCs that expanded at progression were addicted to the molecular pathways that were upregulated and that targeting these pathways represented a potential therapeutic strategy to overcome HMA resistance. As proof-of-principle, we treated CD34+ cells from "CMP pattern" MDS isolated after HMA failure, co-cultured over a layer of stromal cells, with the BCL2 inhibitor ABT-199 for 72 hours. ABT-199, in combination with 5-azacytidine (AZA), significantly decreased the number of LT-HSCs from MDS patients with progressive disease (Fig F), but did not affect those from MDS patients in whom HMA therapy failed because of persistent MDS. These data suggest that ABT-199 selectively targets the LT-HSCs from "CMP pattern" MDS with progressive disease, which depend on increased levels of BCL2 to support their expansion. Treatment of WT/vav-cre-TET2L/L BM chimeras with ABT-199 therapy showed selective apoptosis and depletion of the MDS-like LT-HSCs and the concomitant proliferation of the WT LT-HSCs. This suggests that ABT-199 allows selective therapeutic targeting that eliminates abnormal HSCs while restoring normal hematopoiesis (Fig G). Furthermore, ABT-199 treatment of xenografts generated by transplanting the MDS-L cell line in NSGS mice reduced tumoral burden by depleting the human blast population (Fig H). Taken together, these findings demonstrate that targeting commonly deregulated pathways in MDS HSCs is a feasible approach to tackling disease progression and provide a rationale for the selective inhibition of BCL2 in "CMP pattern" MDS with progressive disease. Figure. Figure. Disclosures Giuliani: Celgene Italy: Other: Avisory Board, Research Funding; Takeda Pharmaceutical Co: Research Funding; Janssen Pharmaceutica: Other: Avisory Board, Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Colla:Abbvie: Research Funding.

Description: In previous studies of recurrently amplified 1q21 genes in multiple myeloma (MM), we identified ILF2 (Interleukin Enhancer Binding Factor 2) as a key modulator of the DNA repair pathway, which promotes adaptive responses to genotoxic stress in a dose-dependent manner, explaining why 1q21 patients benefit less from high-dose chemotherapy than non-1q21 patients do (Marchesini, Cancer Cell 2017). These findings prompted us to develop strategies for blocking ILF2 signaling to enhance the effectiveness of available DNA-damaging agent-based treatments. Given that ILF2 is selectively overexpressed in 1q21 MM cells and is not easily amenable to small-molecule or antibody depletion, we collaborated with IONIS Pharmaceuticals to develop antisense nucleotides targeting ILF2 (ILF2 ASOs). To exclude on-target toxicities that could arise from ILF2 inhibition, we injected 14 different ASOs targeting mouse ILF2 into male Balb/c mice. Of the 14 ILF2 ASOs we tested, 6 did not induce either notable histopathological findings or hematological and biochemical alterations, which suggests that ILF2 inhibition is well-tolerated in normal tissues. Thus, ILF2 ASOs were used for functional validation studies in myeloma. Consistent with our previous work using ILF2-targeting shRNAs, we observed that ILF2 ASO-induced ILF2 depletion resulted in significantly inhibited cell proliferation, increased ATM/Chk2 pathway activation, γH2AX accumulation, and caspase 3-mediated apoptosis in KMS11 and JJN3 cells and sensitized these cells to melphalan, bortezomib and olaparib treatment (Fig 1). However, whereas KMS11 cells had a high level of DNA damage activation and a significantly higher apoptosis rate after more than 2 weeks of ILF2 ASO treatment, JJN3 cells overcame ILF2 ASO-induced DNA damage activation and apoptosis and became resistant to ILF2 ASO treatment. To gain insights into the molecular mechanisms by which MM cells can overcome ILF2 ASO-induced DNA damage activation, we subjected ILF2 ASO-treated KMS11 and JJN3 cells to RNA sequencing analysis at early and late treatment times. We found that the genes that were significantly downregulated in JJN3 but not KMS11 cells treated with ILF2 ASOs for more than 2 weeks as compared with those treated for 1 week were mostly involved in the regulation of the DNA damage response (Fig 2). These findings suggest that MM cells can activate compensatory mechanisms to overcome the deleterious effects of DNA damage and survive. To identify DNA repair effectors whose loss of function suppresses 1q21 MM cells' capability to overcome ILF2 ASO-induced DNA damage, we performed a CRISPR/Cas9 screening using a pool of single-guide RNAs (sgRNAs) targeting 196 genes involved in the DNA damage response. Using the drugZ algorithm to assess differences in the representation of all sgRNAs between cells treated with NT or ILF2 ASOs for 3 weeks (Fig 3), we found that sgRNAs targeting the DNA replication helicase/nuclease 2 (DNA2) were among the most significantly depleted sgRNAs in ILF2 ASO-treated JJN3 cells, whereas sgRNAs targeting the Fanconi anemia core complex-associated protein 24 (FAAP24) were significantly depleted in ILF2 ASO-treated KSM11 cells. Using the DNA2 inhibitor C5, we further validated that targeting DNA2 significantly enhances ILF2 ASO-induced apoptosis in JJN3 cells (Fig 4). Functional validation experiments using inducible sgRNAs are ongoing to evaluate whether the inhibition of DNA2 or FAAP24 is a synthetic lethal approach to targeting 1q21 MM cells in the setting of therapies with DNA-damaging agents. Collectively, our study demonstrates that ILF2 ASO therapy may be exploited to optimize the use of DNA-damaging agents in patients with 1q21 MM. Disclosures Garcia-Manero: Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; Novartis: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Colla:Abbvie: Research Funding; Amgen: Research Funding; IONIS: Other: Intellectual property and research material IONIS).

Description: The biological mechanisms of abnormal terminal erythroid differentiation (TED) in MDS-RS and SF3B1 mutations (SF3B1MT) are largely unknown. This gap in understanding, which has dramatically delayed the design of second-line approaches for SF3B1MT patients whose disease has failed hypomethylating agent (HMA) therapy, is primarily due to a lack of studies molecularly characterizing how SF3B1MTaffect distinct stages of erythropoiesis. Here, we dissected at the single-cell level the cellular and transcriptomic changes induced by SF3B1MT in cells undergoing erythroid differentiation and elucidated how HMA therapy can overcome SF3B1MT-defective erythropoiesis. We first analyzed the expression profile of the lineage-negative CD34+ stem and progenitor (HSPC) compartment. Single-cell RNA sequencing (scRNA-seq) analysis of HSPCs isolated from 2 healthy donors (HDs) and 5 untreated MDS-RS patients with SF3B1MT revealed cell clusters driven by the cells' differentiation potential that we defined based on the differential expression of validated lineage-specific transcriptional factors and cellular markers. Whereas the HD HSPCs had equally distributed erythroid/megakaryocyte and myeloid/lymphoid differentiation trajectories, the SF3B1MT HSPCs had a predominant erythroid differentiation route (Fig. 1a). Differential expression analysis revealed that the SF3B1MT HSPCs undergoing erythroid differentiation were characterized by the expression of genes involved in translation, oxidative phosphorylation, and cell cycle progression, which underlines these cells' metabolic and proliferative activation. Consistent with these findings, scRNA-seq of bone marrow (BM) mononuclear cells (MNCs) from the same samples showed that SF3B1MT MNCs had a predominant population of erythroid cells at the expense of B lymphocytes and myeloid cells (Fig. 1b). An analysis of the erythroid cluster distribution inside the erythroid compartment showed that the frequency of early-stage maturation (BFU-E [cluster #18], CFU-E [#9]) was lower in the SF3B1MT erythroblasts than in the HD erythroblasts, owing to a significantly increased frequency of SF3B1MT cells in the latest stages of TED (proerythroblasts [#4]), basophilic normoblasts [#12], polychromatophilic normoblasts [#7], orthochromatic normoblasts [ONs; #1/11], pre-reticulocytes [#13]). These data suggest that SF3B1MT enhance HSPC differentiation towards the erythroid lineage but arrest erythroblasts at the last step of their maturation by inhibiting the transition of ONs to pre-reticulocytes, resulting in the accumulation of TED cells in the BM. Transcriptomic analysis of SF3B1MT ONs revealed a significant upregulation of genes regulating heme metabolism, including those involved in EIF2AK1's response to impaired heme production, and of major effectors of cell cycle checkpoint activation (e.g., CHK1, CDKN1A, GADD45). These data are consistent with previous studies showing that the SF3B1MT-induced defective accumulation of iron in erythroblasts' mitochondria leads to activated cell stress response, cell cycle arrest, and apoptosis. Of note, the growth differentiation factor 11 receptors ACVR1B, TGFBR1, and ACVR1C were not expressed at any step of erythroid differentiation, which challenges this factor's role as a target of luspatercept-induced differentiation of late-stage erythroblasts. To evaluate how HMA therapy can overcome inefficient erythropoiesis, we performed scRNA-seq of HSPCs and BM MNCs isolated from SF3B1MT MDS-RS patients before any therapy and at the time of hematological response to HMA therapy. HMA therapy did not reduce the aberrant differentiation of SF3B1MT HSPCs towards the erythroid lineage but temporarily induced the TED of SF3B1MT ONs into reticulocytes and these cells' release into peripheral blood. Accordingly, blast progression following the initial HMA therapy-induced hematological response coincided with the further expansion of the erythroid-primed cells arising from the earliest stem cell population (Fig. 1c), which suggests that failure to eradicate or genetically correct SF3B1MT stem cells leads to disease relapse. In conclusion, our results elucidate how SF3B1MT molecularly affect distinct stages of erythropoiesis and have implications for developing approaches that achieve lasting hematological remission in patients with MDS-RS. Disclosures Garcia-Manero: Helsinn Therapeutics: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Jazz Pharmaceuticals: Consultancy; Merck: Research Funding; AbbVie: Honoraria, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Novartis: Research Funding; Astex Pharmaceuticals: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Amphivena Therapeutics: Research Funding; Acceleron Pharmaceuticals: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Research Funding. Colla:Amgen: Other: Unspecified.