ALBERT

Description: Strata movement of drilling grouting is distinctly different from that of traditional caving mining, especially for overlying thin rock stratum. Considering the horizontal shear transfer of foundation, a mechanical model of overlying strata movement was established using the theory of Pasternak double-parameter foundation beam. The deflection and internal force equations of rock beam were derived, and the influence mechanisms of grouting material parameter were discussed. The results showed that the thickness of layer grouting was the key influential factor than the foundation modulus coefficient and grout diffusion radius. The comparison with the theory of Winkler elastic foundation beam also showed that double-parameter foundation beam method is more applicable to reinforced strata.

Description: IJERPH, Vol. 15, Pages 425: The Impact of Ambient Air Pollution on Daily Hospital Visits for Various Respiratory Diseases and the Relevant Medical Expenditures in Shanghai, China International Journal of Environmental Research and Public Health doi: 10.3390/ijerph15030425 Authors: Hao Zhang Yue Niu Yili Yao Renjie Chen Xianghong Zhou Haidong Kan The evidence concerning the acute effects of ambient air pollution on various respiratory diseases was limited in China, and the attributable medical expenditures were largely unknown. From 2013 to 2015, we collected data on the daily visits to the emergency- and outpatient-department for five main respiratory diseases and their medical expenditures in Shanghai, China. We used the overdispersed generalized additive model together with distributed lag models to fit the associations of criteria air pollutants with hospital visits, and used the linear models to fit the associations with medical expenditures. Generally, we observed significant increments in emergency visits (8.81–17.26%) and corresponding expenditures (0.33–25.81%) for pediatric respiratory diseases, upper respiratory infection (URI), and chronic obstructive pulmonary disease (COPD) for an interquartile range increase of air pollutant concentrations over four lag days. As a comparison, there were significant but smaller increments in outpatient visits (1.36–4.52%) and expenditures (1.38–3.18%) for pediatric respiratory diseases and upper respiratory infection (URI). No meaningful changes were observed for asthma and lower respiratory infection. Our study suggested that short-term exposure to outdoor air pollution may induce the occurrences or exacerbation of pediatric respiratory diseases, URI, and COPD, leading to considerable medical expenditures upon the patients.

Description: Abstract 292 Background: Allelic deficiency for the RPS14 gene impairs differentiation and survival of erythroid progenitors in del(5q) MDS (Nature 2008; 451:335). Nucleolar stress arising from disruption of ribosome assembly fosters MDM2 sequestration by free ribosome components resulting in p53 stabilization and erythroid hypoplasia (Nat Cell Biol 2009; 11:501). We recently reported that reduced gene dosage of the lenalidomide (LEN) inhibitable, haplodeficient phosphatases CDC25C and PP2Acα is a key determinant of drug sensitivity in del(5q) MDS (PNAS 2009; 106: 12974). We now show that shRNA suppression of these genes to levels commensurate with haplodeficiency reinforces p53 accumulation, and that treatment with LEN promotes MDM2-mediated p53 degradation to transition del(5q) clones to G2/M arrest. We hypothesized that emergence of resistance to LEN in del(5q) MDS arises from two possible mechanisms: (1) up-regulation of haplodeficient drug targets or compensatory isotypes, or (2) inactivating mutations of the TP53 or CDC25C genes. Methods: To investigate mechanisms of LEN resistance, we studied sequential bone marrow (BM) specimens obtained at baseline (BL), response to treatment (TR) and treatment failure (TF) from 12 LEN treated patients with Low/INT-1 risk, transfusion-dependent del(5q) MDS. Eleven patients achieved clonal suppression and transfusion independence; 7 patients developed clinical drug resistance with primary clonal recovery. Immunohistochemical (IHC) staining for cdc25-C, -A and -B; PP2A–Ca and p53 were performed using a biotin-streptavidin-horseradish peroxidase method and compared to 6 age-matched controls; intensity of cytoplasmic or nuclear staining in hematopoietic elements was recorded after blinded review. DNA and RNA were extracted from cryopreserved BM mononuclear cells (BM-MNC) or fixed paraffin blocks from BM clot and biopsy sections. Expression of CDC25C splice variants was assessed by RT-PCR and total gene expression by real time (QT)-PCR. Exonic DNA encoding the catalytic [exons 8–14] and nuclear export domains [exon 11] of CDC25C and the DNA-binding domain of TP53 [exons 4–9] was sequenced for gene mutation analysis. Differences in mean values were compared by paired t-test. Results: P53 immunostaining was significantly higher in del(5q) BL specimens compared to controls ( relative expression [RE] 9.6 vs. 0.25; P =0.007). An admixture of nuclear and cytoplasmic staining for p53 and each cdc25 isotype was observed at BL that was largely restricted to erythroid precursors, whereas at TR cdc25-C and -A expression was primarily cytoplasmic, consistent with drug-induced nuclear exclusion. At TR, RE of only cdc25C (BL, 75 vs. TR, 49; P=0.05) and PP2A (29.2 vs. 12.3; P=0.025) was significantly reduced; whereas at TF cdc25C (TR, 43 vs. TF, 166; P=0.003), cdc25A (42.4 vs. 150; P=0.006), PP2A (7.3 vs. 65.6; P=0.028) and p53 (0.92 vs. 25.4; P=0.024) RE significantly increased. Nuclear localization of cdc25C and p53 but not cdc25A predominated at TF, consistent with escape from cdc25C inhibition. QT-PCR confirmed transcriptional up-regulation of CDC25C at TF with a mean 8.8-fold increase in gene expression vs. BL. DNA sequencing revealed no acquisition of somatic mutations within the CDC25C and TP53 exons studied [n=5]. Conclusions: Secondary resistance to LEN in del(5q) MDS is associated with over-expression and activation of the haplodeficient drug-inhibitable phosphatases, cdc25C and PP2A, with consequent restoration of wt-p53 activation. Absence of gene mutations within the coding exons analyzed suggests that transcriptional compensation alone is responsible for drug resistance. Novel agents targeting transcriptional repression of CDC25C may restore LEN sensitivity and merit investigation in drug resistant del(5q) MDS. Disclosures: List: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Komrokji:Celgene: Research Funding, Speakers Bureau. Lancet:Celgene: Research Funding. Maciejewski:Esai: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Description: We previously reported that S100A9 promotes ineffective hematopoiesis and the development of MDS in a feed forward age-dependent fashion. Nonetheless, the precise mechanism by which S100A9 may foster DNA damage in MDS remains unclear. We recently showed that S100A9 directs overexpression of the fat-mass and obesity-associated gene (FTO) encoding an m6A RNA demethylase, which leads to nuclear exclusion of SRSF2. Removal of SRSF2 from its functional domain in the nucleosome leads to stalling of RNA polymerase II and formation of the nucleic acid R-loops, comprising DNA:RNA hybrids with the associated non-template single-stranded DNA. S100A9/FTO axis activation leads to SRSF2 deregulation through suppression of its main nuclear transport protein RanBP2, thereby stalling transcription machinery with resulting accumulation of nuclear R-loops and cytosolic/extracellular RNA:DNA hybrids. Persistent R-loops induce DNA damage while also compromising DNA repair. Here we identify an S100A9/FTO-regulated pathway responsible for induction of genomic instability through the accumulation of cytoplasmic RNA:DNA hybrids and modification of the spliceosomal patterns of aged S100A9Tg mice matching MDS hematopoietic stem and progenitor cells (HSPC). We first investigated which components of the S100A9/FTO axis are critical to hematopoiesis and those that are important for both the development of RNA:DNA hybrids and γH2AX activation. We analyzed the contribution of RanBP2 and the effects of elimination of R-loop formation via overexpression of RNAse H1, an enzyme that removes stalled R-loops in the nucleus by degrading DNA-hybridized RNA, thereby reducing the accumulation of cytoplasmic RNA:DNA hybrids. CRISPR knock-down of RanBP2 showed that the protein is critical for accumulation of yH2AX defined by double stranded breaks (DSB). Importantly, overexpression of RNAse H1 degraded R-loops and restored colony-forming capacity, indicating that RNA:DNA hybrids induced by the S100A9/FTO have profound effects on hematopoietic potential. However, while the FTO exclusion of SRSF2 from the nucleus explains the accumulation of γH2AX, it should potentially impact global RNA splicing. To investigate this, we performed a comparative RNAseq analysis on WT and S100A9Tg mice (young and old) to understand both changes induced through the normal aging process as well as those compounded by S100A9. We found that genes linked to splicing, RNA development, nucleotide excision repair and genomic instability and ribosome function were downregulated in aged S100A9Tg mice. Further analysis comparing splicing patterns of S100A9Tg and WT mice with human MDS BM HSPC led to ~200 common genes that were analyzed further. These genes showed that there are splicing changes enriched in spliceosomal assembly and mRNA splice selection site genes. We also found that the enriched genes affect the nucleolus and ribosome formation matching what is seen phenotypically with MDS. Dysregulation of these pathways are highly consistent with our observations of the pathways affected by the S100A9/FTO-induced inflammaging process, validating our hypothesis of S100A9 as a common initiator of dysfunction that can give rise to MDS. Importantly, our data demonstrates the potential for spliceosomal dysfunction regardless of the presence of spliceosomal mutations in MDS. We are currently in the process of performing both DRIPseq and m6A-seq of primary human MDS specimens and S100A9Tg mice to further assess the role of the S100A9/FTO pathway in the selection of sites for RNA/DNA hybrid formation and rise of genomic dysfunction that gives rise to MDS. We conclude that S100A9/FTO-induced nuclear exclusion of SRSF2 aids in the formation of RNA:DNA-hybrids that lead to genomic instability and the disruption of normal spliceosomal patterns in both human HSPC and the S100A9Tg MDS murine model, representing a previously uncharacterized mechanism contributing to MDS pathogenesis. Our studies provide evidence that targeting this cascade offers significant potential for development of novel, biologically rational therapeutics for MDS. Disclosures List: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Abstract 597 The acquisition of genetic abnormalities that lead to ineffective hematopoiesis is a characteristic of MDS. This event is mediated in part by an interaction of inflammatory intermediates with the bone marrow (BM) microenvironment; however, the mechanism by which chromosomal damage occurs to the stem/progenitor compartment (HSC/HPC) is unknown. We have identified a unique population of myeloid derived suppressor cells (MDSC) bearing a LIN-HLA-DR-CD33+ surface phenotype. These MDSC elicit hematopoietic suppression through the elaboration of nitric oxide, arginase, and inflammatory cytokines. This study found that MDSCs accumulated in excess in the BM of patients with lower risk MDS (mean, 32.32% ± 13.3; n=12) compared to BM from healthy age matched controls (mean 2.1%±0.9; n=8) and non-MDS cancer patients (n=8 mean 2.06%±1.5 p

Description: Abstract 788 Understanding the pathophysiology of myelodysplastic syndrome (MDS) is limited by a complex molecular mechanism and lack of an adequate animal model that recapitulates the role of inflammation in the abnormal hematopoiesis. We recently showed that patients with MDS have expansion of inflammation-related hematopoietic suppressive cells called immature myeloid-derived suppressor cells (MDSC) that display direct cytotoxic and suppressive effects on autologous hematopoietic progenitor cells (HPCs). Expansion of bone marrow (BM) MDSCs contributed to the production of inflammatory cytokines and reduced HPC survival underlying BM failure in lower risk patients. Here we provide evidence that MDSC activation, expansion and development is driven by overexpression of inflammatory-related signaling molecules, myeloid-related protein 8 (MRP8, encoded by S100A8) and MRP14 (encoded by S100A9). Both MRP proteins serve as the native endogenous ligands for Toll-like receptor 4 (TLR4), which is an important damage-associated molecular pattern (DAMP) mediating inflammatory response. We found higher expression of MRP8 and MRP14 in BM mononuclear cells from MDS patients compared to healthy donors, in whom these proteins were not detectable. High surface expression of both TLR2 and TLR4 in MDS MDSCs compared to healthy donor MDSCs confirmed that this signaling pathway is activated in MDS. Inhibition of MRP8/MRP14 proteins in MDSCs using specific shRNAs dramatically attenuated IL-10 and TGF-β production and rescued BFU-E and CFU-GM colony formation of autologous bone marrow progenitors. These data show that inflammation-associated MRP8/MRP14 expression plays a critical role in the suppressive activities of MDS MDSCs. We therefore generated S100A9 transgenic mice (S100A9Tg) overexpressing the murine MRP14 homologue and investigated the role of this protein in bone marrow failure. Significant MDSC accumulation was evident in the BM of S100A9Tg mice by 6 weeks, but not in S100 knockout (KO) or wild type (WT) mice. Similar to human MDS, MDSCs from S100A9Tg mice, but not S100KO or WT mice, significantly inhibited BFU-E colony formation. Depletion of MDSCs in vitro rescued BM colony formation in the S100A9Tg mice indicating that the BM suppression is mediated by MDSC cells. TGF-β and IL-10 secretion was significantly increased in S100A9Tg mice, substantiating the role of S100A9 as an essential inflammatory factor that regulating MDSC suppressive activity. Analogous to human MDS, 6-month old S100A9Tg mice developed ineffective hematopoiesis with severe anemia, leukopenia, and thrombocytopenia accompanied by MDS-like morphological features. BM aspirates and core biopsies from S100A9Tg mice were hypercellular with trilineage cytological dysplasia characteristic of MDS. Treatment with ATRA, which induced the differentiation of MDSCs rescued hematopoiesis in S100A9Tg mice. Our findings indicate that primary BM expansion of MDSC is sufficient to perturb hematopoiesis and result in the development of MDS, supporting the notion of microenvironment-conducive oncogenesis. S100A9Tg transgenic mice provide a novel in vivo model of human MDS for target discovery and testing of novel therapeutics. Disclosures: No relevant conflicts of interest to declare.

Description: Recent studies suggest that aging-associated inflammation, or "inflammaging", contributes to genetic instability and MDS predisposition. Although innumerable somatic genetic events have been annotated in recent years, including many that are not unique to MDS, they are not sufficient for disease initiation. The precise underlying mechanisms conducive to the emergence of these genetic events also remain to be delineated. We reported that bone marrow (BM) myeloid derived suppressor cells (MDSC) activated by the damage associated molecular pattern (DAMP) protein S100A9, promote ineffective hematopoiesis and the development of MDS. Inflammaging associated alterations in metabolism have been implicated in predisposition to cancer development with age. Here we report that S100A9 and ROS-induced inflammaging are associated with insulin resistance and hyperglycemia in the BM microenvironment that triggers activation of adaptive oncogenic pathways and genomic instability in HSPC. Glucose concentrations were markedly elevated in MDS BM plasma vs. age-matched controls, and directly correlated with S100A9 concentration (r=0.513, P=0.003, n=41). The magnitude of BM-glucose elevation significantly exceeded that in the peripheral blood and negatively correlated with the proportion of HSPC while directly correlating with BM MDSC percentage. S100A9 transgenic (Tg) mice displayed age-dependent elevation of glucose in peripheral blood and BM when compared to wild type mice accompanied by accumulation of somatic mutations (SM) by sequencing in aged S100A9-Tg compared to younger counterparts. NGS of 38 primary MDS BM specimens showed that SM common to MDS, such as those involving ASXL1, U2AF1 and DNMT3A, we re present only in high glucose stratified MDS-BM specimens (glucose 〉 110 ug/ml). Furthermore, there was a strong correlation between the cellular ROS/nuclear-β-catenin to DNA damage (γH2AX+ cells) linking S100A9-induced ROS accumulation to genetic instability. Elevation in BM plasma glucose was specifically associated with upregulation of the fat mass and obesity associated (FTO) transcript and protein in both MDS BM and S100A9Tg mice. FTO is a risk factor for type 2 diabetes, and encodes an α-ketoglutarate-dependent dioxygenase that functions as an RNA demethylase specific for N 6-methyladenosine (m6A) residues, targeted by splicing factors. Both human and murine MDS specimens displayed decreased m6A mRNA methylation compared to controls. FTO knockdown with CRISPR increased mRNA m6A methylation in MDS primary specimens, whereas overexpression of FTO led to a corresponding decrease that was enhanced by S100A9 stimulation. Moreover, S100A9 treatment induced FTO and demethylation of m6A mRNA in human and murine BM cells. These effects were accompanied by disruption of spliceosomes in the nucleus, as demonstrated by delocalization of SRSF2 from nuclear speckles into the cytoplasm where they colocalize with FTO in MDS patients. Interestingly, from the S100A9Tg mouse sequencing studies, we discovered that the mutation hotspots were in locations specific for histone H3K27 acetylation which have been previously linked to genomic instability, as well as splicing regulation, and regulated by histone deacetylase 1 (HDAC1). Reduced HDAC1 levels are known to make cells hypersensitive to DNA-damaging insults, such as those inducing ROS, similar to what we observed with treatment of S100A9 in healthy human bone marrow, MDS patient samples and S100A9Tg mice. Furthermore, this correlated with increased levels of DNA damage in our patient samples and in our murine S100A9Tg model as measured by phosphorylated γH2AX histones. Our studies provide a biological rationale for the initiation of DNA-genetic damage under inflammatory senescence conditions in MDS. These findings demonstrate that S100A9-induced inflammation activates a signaling cascade that enhances development of splicing variants and somatic gene mutations critical to MDS pathogenesis. Disclosures List: Celgene Corporation: Honoraria, Research Funding.