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Stem cells can self-renew and also produce daughter cells destined for differentiation. The precise control of the balance between these two outcomes is essential to ensure tissue homeostasis and to prevent uncontrolled proliferation resulting in tumor formation. As self-renewal and differentiation are likely to be controlled by different gene expression programs, unraveling the underlying gene regulatory networks is crucial for understanding the molecular logic of this system. In this study, we have characterized by next generation RNA sequencing (RNA-seq) the transcriptome of germline stem cell (GSC)-like cells isolated from bag of marbles (bam) mutant Drosophila ovaries and compared it to the transcriptome of germ line cells isolated from wild type ovaries. We have complemented this dataset by utilizing an RNA-immunoprecipitation strategy to identify transcripts bound to the master differentiation factor Bam. Protein complex enrichment analysis on these combined datasets allows us to delineate known and novel networks essential for GSC maintenance and differentiation. Further comparative transcriptomics illustrates similarities between GSCs and primordial germ cells and provides a molecular footprint of the stem cell state. Our study represents a useful resource for functional studies on stem cell maintenance and differentiation.

The PICO Collaboration wishes to thank SNOLAB and its staff for support through underground space, logistical and technical services. SNOLAB operations are supported by the Canada Foundation for Innovation and the Province of Ontario Ministry of Research and Innovation, with underground access provided by Vale at the Creighton mine site. We wish to acknowledge the support of the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Foundation for Innovation (CFI) for funding. We acknowledge the support from National Science Foundation (NSF) (Grants No. 0919526, No. 1506337, No. 1242637, No. 1205987, and No. 1806722). We acknowledge that this work is supported by the U.S. Department of Energy (DOE) Office of Science, Office of High Energy Physics (under Award No. DE-SC-0012161), by DGAPA-UNAM (PAPIIT No. IA100118) and Consejo Nacional de Ciencia y Tecnologia (CONACyT, M?exico, Grants No. 252167 and No. A1-S-8960), by the Department of Atomic Energy (DAE), Government of India, under the Centre for AstroParticle Physics II project (CAPP-II) at the Saha Institute of Nuclear Physics (SINP), European Regional Development Fund?Project ?Engineering Applications of Microworld Physics? (Project No. CZ.02.1.01/0.0/0.0/ 16_019/0000766), and the Spanish Ministerio de Ciencia, Innovacion y Universidades (Red Consolider MultiDark, Grant No. FPA2017-90566-REDC). This work is partially supported by the Kavli Institute for Cosmological Physics at the University of Chicago through NSF Grant No. 1125897, and an endowment from the Kavli Foundation and its founder Fred Kavli. We also wish to acknowledge the support from Fermi National Accelerator Laboratory under Contract No. DE-AC02-07CH11359, and Pacific Northwest National Laboratory, which is operated by Battelle for the U.S. Department of Energy under Contract No. DE-AC05- 76RL01830. We also thank Compute Canada [75] and the Center for Advanced Computing, ACENET, Calcul Qu?ebec, Compute Ontario, and WestGrid for computational support.

Stem cells can self-renew and produce daughter cells destined for differentiation. The precise control of the balance between these two outcomes is essential to ensure tissue homeostasis and to prevent uncontrolled proliferation resulting in tumor formation. As self-renewal and differentiation are likely to be controlled by different gene expression programs, unraveling the underlying gene regulatory networks is crucial for understanding the molecular logic of this system. In this study, we have characterized by next generation RNA sequencing (RNA-seq) the transcriptome of germline stem cell (GSC)-like cells isolated from bag of marbles (bam) mutant Drosophila ovaries and compared it to the transcriptome of germ line cells isolated from wild-type ovaries. We have complemented this dataset by utilizing an RNA-immunoprecipitation strategy to identify transcripts bound to the master differentiation factor Bam. Protein complex enrichment analysis on these combined datasets allows us to delineate known and novel networks essential for GSC maintenance and differentiation. Further comparative transcriptomics illustrates similarities between GSCs and primordial germ cells and provides a molecular footprint of the stem cell state. Our study represents a useful resource for functional studies on stem cell maintenance and differentiation., Summary: Fruit fly germline stem cell differentiation is accompanied by major changes of the transcriptome that may be regulated at the post-transcriptional level.

The TET enzyme family of Ten-Eleven Translocation methylcytosine (5mC) dioxygenases comprising 3 members, TET1–3, play key roles in DNA demethylation. These processes regulate transcription programs that determine cell lineage, survival, proliferation, and differentiation. The impetus for our investigations described here is derived from the need to develop illuminating small molecule probes for TET enzymes with cellular activity and specificity. The studies were done so in the context of the importance of TET2 in the hematopoietic system and the preponderance of loss of function somatic TET2 mutations in myeloid diseases. We have identified that 2-hydroxy-4-methylene-pentanedicarboxylic acid 2a reversibly competes with the co-substrate α-KG in the TET2 catalytic domain and inhibits the dioxygenase activity with an IC(50) = 11.0±0.9 μM at 10 μM KG in a cell free system and binds in the TET2 catalytic domain with K(d) = 0.3± 0.12 μM.

Myelin pathology is present in many neurological conditions such as multiple sclerosis (MS) and traumatic spinal cord injury (SCI). To facilitate development of novel therapies aimed at myelin repair, we set out to develop imaging agents that permit direct quantification of myelination in vivo. In this work, we designed and synthesized a series of fluorescent fluorinated myelin imaging agents that can be used for in vivo positron emission tomography (PET) imaging combined with subsequent post-mortem fluorescent cryoimaging. Structure-activity relationship (SAR) studies of the newly developed myelin imaging agents led us to identify a lead compound (TAFDAS, 21) that readily enters the brain and spinal cord and selectively binds to myelin. By conducting sequential PET and 3D cryoimaging in an SCI rat model, we demonstrated for the first time that PET and cryoimaging can be combined as a novel technique to image the spinal cord with high sensitivity and spatial resolution.

Neomorphic mutations in IDH1/2 producing R-2-Hydroxyglutrate (R-2HG), are common in myeloid malignancies and in various solid cancers. A diffuse hypermethylated status is the biological consequence of the R-2HG-mediated inhibition of several α-ketoglutarate (αKG)-dependent enzymes including DNA dioxygenases TET1, TET2 and TET3.1,2 Specifically, the inhibition of TET2, either induced by the interaction with R-2HG or by direct genomic silencing (as in case of TET2 loss of function mutations) is responsible for the block of the DNA cytosine demethylation pathway, inducing changes in expression patterns, (e.g. decreasing expression of tumor suppressor genes) and impairing execution of differentiation programs. Analysis of genomic data from a Cleveland Clinic (CCF) cohort of AML/MDS patients combined in a meta-analytic fashion with BeatAML3 and Tumor Cancer Genome Atlas (TCGA) cohorts (1119 profiled patients) showed that IDH1/2 mutations are mutually exclusive (only 3% [N=4/106] of AML IDH1/2 mutated cases had TET2 mutations, expected to be at a frequency of 18% [N=110/585] in IDH1/2 wild type cases, p=.000125). In this scenario we suggest that the loss of TET2 activity due to mutations prevents the expansion of IDH1/2 mutant myeloid neoplasms (MNs) because of phenotypic redundancies inducing synthetic lethality. With this premise we stipulated that a critical level of DNA dioxygenase activity exists and thus cells with low TET2 activity will not tolerate further inhibition by R-2HG. Here we propose to apply pharmacologic inhibition of TET2 to produce an additive effect on DNA dioxygenases to investigate whether this will result in a synthetic lethality of IDH1/2 mutant cells. Specifically we hypothesize that TET-dioxygenase inhibition may be implemented as a possible therapeutic strategy in neomorphic IDH1/2 mutant MNs. To explore this hypothesis we conducted a series of in vitro experiments in different isogenic cell lines expressing either mutant or wild type IDH1 or IDH2, that were simultaneously mutant, wild type (WT) or knock down (KD) for TET2 (TF1-IDH2R140Q, K562-IDH1R132C both WT for TET2 gene, and K18-IDH1R132CTET2KD and SIGM5-IDH1R132C TET2MT, both with a doxycycline inducible promoter for mutant IDH1). First we found that the doxycycline induction of ectopic IDH1R132C expression led to R-2HG increase (~10,000-fold over the baseline) and induced cell death in TET2-deficient cells (experiments conducted in SIGM5-IDH1R132C cells showing 70% of decrease in cell growth after five days of IDH induction with doxycycline), confirming the cytotoxic effect of cellular R-2HG. We then tested in IDH1/2MT cells sensitivity towards TETi76, a specific TET inhibitor designed on R-2HG scaffold (with more than 200 fold potency compared to R-2HG in cell-free assays of 5-hydroxy-methyl cytosine [5hMC] production).4 This compound showed particular selectivity towards inhibition of DNA dioxygenases when a set of 23 other dioxygenase inhibitors were screened. Most importantly, consistent with our hypothesis, TETi76 preferentially inhibited the proliferation of IDH1/2MT cells either following doxycycline-induction both in TET2WTand TET2 deficient models (K562 TET2WT, K18 TET2kD, SIGM-5 TET2MT cell lines), or in models not carrying the inducible promoter (TF1 TETWT) (Growth inhibition: 20-25% in IDHWT vs 70-80% in IDHMT cell lines after 72h of co-culture with TETi76 treatment for concentrations ranging between 1 and 5 µM. P-value range: 0.04-0.001 in pairwise comparisons with untreated controls). Overall, our findings are consistent with the idea that neomorphic IDH1/2MT phenocopies loss of function TET2MT, through R-2HG, down-modulating pathways fundamental for cell homeostasis, division and differentiation. If a residual TET-activity is needed for the function of IDH1/2MT cells, the complete block of the residual activity appears to inevitably disrupt this phenotype impairing cell growth and proliferation. This is also in agreement with the paucity of TET3 and TET1 mutation in the context of TET2MT carriers. In summary, results shown here represent an important proof of concept that the increased inhibition of DNA dioxygenase activity, instead of being more leukemogenic, can be synthetically lethal. Our observations may have implications with regard to the therapy of IDH1/2 mutated neoplasms including AML and MDS Disclosures Saunthararajah: EpiDestiny: Consultancy, Current equity holder in private company, Patents & Royalties: University of Illinois at Chicago. Maciejewski:Alexion, BMS: Speakers Bureau; Novartis, Roche: Consultancy, Honoraria.

Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. We addressed this question in Drosophila neural stem cells called neuroblasts. We identified the Tip60 chromatin remodeling complex and its interaction partner Myc as regulators of genes required for neuroblast maintenance. Knockdown of Tip60 complex members results in loss of cortical polarity, symmetric neuroblast division, and premature differentiation through nuclear entry of the transcription factor Prospero. We found that aPKC is the key target gene of Myc and the Tip60 complex subunit Domino in regulating neuroblast polarity. Our transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes previously identified as direct Myc targets. Our findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex, and the molecular network controlling cell polarity and asymmetric cell division.

We report magnetic field dependence of magnetization on nickel oxide nanoparticles, ferrihydrite nanoparticles, and a nanocomposite of both nanoparticle systems. The process of magnetization in these samples is studied. Possibility for estimating concentration of an individual nanoparticle system in a nanocomposite consisting of two or more magnetic phases is discussed in detail.

Magnetization as a function of applied magnetic field at different temperatures for antiferromagnetic nanoparticles of ferrihydrite is measured and analyzed considering a distribution in particle magnetic moment. We find that the magnetization of this nanoparticle system is affected by the presence of particle magnetic moment distribution. This particle magnetic moment distribution is estimated at different temperatures.

Apico-basal polarity is the defining characteristic of epithelial cells. In Drosophila, apical membrane identity is established and regulated through interactions between the highly conserved Par complex (Bazooka/Par3, atypical protein kinase C and Par6), and the Crumbs complex (Crumbs, Stardust and PATJ). It has been proposed that Bazooka operates at the top of a genetic hierarchy in the establishment and maintenance of apico-basal polarity. However, there is still ambiguity over the correct sequence of events and cross-talk with other pathways during this process. In this study, we reassess this issue by comparing the phenotypes of the commonly used baz(4) and baz(815-8) alleles with those of the so far uncharacterized baz(XR11) and baz(EH747) null alleles in different Drosophila epithelia. While all these baz alleles display identical phenotypes during embryonic epithelial development, we observe strong discrepancies in the severity and penetrance of polarity defects in the follicular epithelium: polarity is mostly normal in baz(EH747) and baz(XR11) while baz(4) and baz(815) (-8) show loss of polarity, severe multilayering and loss of epithelial integrity throughout the clones. Further analysis reveals that the chromosomes carrying the baz(4) and baz(815-8) alleles may contain additional mutations that enhance the true baz loss-of-function phenotype in the follicular epithelium. This study clearly shows that Baz is dispensable for the regulation of polarity in the follicular epithelium, and that the requirement for key regulators of cell polarity is highly dependent on developmental context and cell type. Open-Access Publikationsfonds 2015 peerReviewed

Stem cells establish cortical polarity and divide asymmetrically to simultaneously maintain themselves and generate differentiating offspring cells. Several chromatin modifiers have been identified as stemness factors in mammalian pluripotent stem cells, but whether these factors control stem cell polarity and asymmetric division has not been investigated so far. We addressed this question in Drosophila neural stem cells called neuroblasts. We identified the Tip60 chromatin remodeling complex and its interaction partner Myc as regulators of genes required for neuroblast maintenance. Knockdown of Tip60 complex members results in loss of cortical polarity, symmetric neuroblast division, and premature differentiation through nuclear entry of the transcription factor Prospero. We found that aPKC is the key target gene of Myc and the Tip60 complex subunit Domino in regulating neuroblast polarity. Our transcriptome analysis further showed that Domino regulates the expression of mitotic spindle genes previously identified as direct Myc targets. Our findings reveal an evolutionarily conserved functional link between Myc, the Tip60 complex, and the molecular network controlling cell polarity and asymmetric cell division.

Two-line ferrihydrite nanoparticles are synthesized by a chemical method. The sample is characterized with X-ray diffractometer, thermogravimetric analyzer, differential scanning calorimeter, and vibrating sample magnetometer. The system is found to undergo two-phase transitions on heating. Nature of these transitions and magnetic behavior of original and heated samples are reported.

As there is growing concern about the best quality of power to be delivered, multilevel inverters had a great performance in raising the power quality by lessen the amount of switches thus access the level. This exert includes CMLI based analysis suitable for changing input from solar PV. This analysis endeavor to trace twain governing boundary and power circuitry to supply distinct amount of voltage levels by changing proper sequences. Therefore to achieve the requisite stage, the conservative methods need more switches and filter circuits whereas in our analysis only 10 switches are selected to have the prescribed levels. The approach is to minimise the rate of entire harmonics deformation by maximising the levels and hence improving the capacity of power. simulation for multistage inverter is presented according to IEEE standards and comparisons are made.

Fifty cases of allergic contact dermatitis from Parthenium hysterophorus have been studied in detail. Clinico-epidemiological features have been described. Patients present with any of the four observed patterns of dermatitis viz. atopic, photosensitivity, seborrhoeic and miscellaneous. Patients were patch tested with 12 extracts of the plants in six solvents producing differing reaction. Limited chemical analysis of the plant extracts yielded various fractions of which three were found to be immunologically active. Chemical nature of parthenin which is a known allergen of this plant has been studied. Possibility of cross sensitivity with other plants is briefed.

The clinical cand lipid abnormalities in 45 cases of xanthelasma are reported. Their findings suggest that xanthelasma is seen in some patients as an isolated clinical symptom with normal serum lipid level% representing a local derangement of cellular lipid metabolism. Nevertheless it is noted in many patients with moderate elevation of different serum lipid fractions without any other clinical manifestations, occurring probably as a resultof benign derangement of systemic lipid metabolism. Less frequently, it is observed with significant elevation of serum lipid levels in association with certain grave abnormalities like hypertension, ischaemic heart disease,' familial hypercholesterolemia, familial xanthomatosis, diabetes etc. Thus it is inferred that the symptom xanthelasma signifies disturbed lipid metabolism of several and the, patient needs thorough investigation.

Six hundred and fifty seven cases suspected to have contact dermatitis reporting at 14 dermatological centres of armed forces hospitals during a 12-month period were investigated. One hundred sixty one cases Showed positive patch tests. Sixty-five cases showed positive patch tests with footwear materials including rubber, leather and canvas. Clothing, topical medicaments, airborne allergens and marking ink were responsible in 5.75%, 25%, 3.82% and 0.85% patients respectively.

Fifteen male soldiers having dermatitis cruris pustulosa et atrophicans were studied clinically, bacteriologically, histopathologicafly and immunologically. Average age at detection was 26 years. Skin lesions were classically distributed on the legs. Staphylococcus aure was grown from the lesions. Histopathological findings were of eczematous pattern with polymorphic cellular infiltrate in the dermis. IgG and IgA levels in blood were increased.

This investigation explores the macrofungi with their identification and documentation of indigenous knowledge. The study area occupies 633 hector and lies within a narrow limit of altitude between 225 and 265msl. The collected samples represented 31 species of Basidiomycetes belonging to 7 orders, 17 families and 22 genera. The dried specimens are deposited in the Tribhuvan University Central Department of Botany, Pathology Unit, Kathmandu, Nepal. The area embraces many mycophagous ethnic groups. The mycoelements prevailing in this area need sustainable development.

Benzotriazole surrogates showing higher stabilities than the corresponding chlorophosphates, allow phosphonylation of a variety of N-, O-, and S-nucleophiles in good yields.

N-Acyl threonine isopeptides undergo acyl transfer in chemical ligations via 5-, 8-, 9and 10membered cyclic transition states to yield natural peptides, representing the first examples of successful isopeptide ligations from N-acyl threonine units.

Benzotriazole-mediated synthesis provides novel fluorescent-labeled amino acid–quinolone antibiotic conjugates in good yields with retention of chirality. The photophysical properties of all the fluorescent-labeled antibiotic conjugates were determined.

Knowledge management helps organizations to exploit and develop resources, improve their competitiveness and develop sustainable competitive advantage. Main focus of the research in KM is on theoretical concepts, approaches, and sustaining tools for human knowledge management. KM plans to manage knowledge by organizing formal and direct method to manage organizational knowledge in the workplace. There are many literatures that have shown a number of KM approaches developed with the purpose of managing organizational knowledge. However, there is a need to focus on study of ontology based collaborative filtering in the existing business environment. This paper reviews ontology-based KM approaches. The primary goal of this work is to understand and evaluate each approach by analyzing and using.

Myelodysplastic syndromes (MDS) ultimately will progress to a higher-risk form or acute myeloid leukemia. This evolution is accompanied by acquisition of genetic hits following ancestral mutations, with further subclonal diversification of the clonal architecture. Conceptually, therapeutic approaches targeting ancestral hits have the potential to eradicate MDS at early stages of ontogenesis. Founder SF3B1 mutations are frequent in MDS and therefore represent rational targets for drug development. During our drug discovery efforts we identified an existing drug that selectively inhibits the growth of SF3B1 mutant (SF3B1MT) cells. We consequently examined the effects of known compounds possibly arresting clonal expansion of SF3B1MT MDS cells. For our studies, we generated CRISPR/Cas9 knock-in human cells stably expressing the recurrent SF3B1 K700E mutation and a chromophore-tagged reporter (mRFP). Because of the high frequency of SF3B1 mutations and their effects on the splicing of a multitude of genes, we hypothesized that some of the corresponding downstream effects could be effectively targeted. Using luminescent cell viability assays and flow cytometry analysis, we screened a 3,000 compound library for selective sensitivity against isogenic SF3B1MT cells. A subset of this library contained 23 calcium2+ channel blockers (CCBs) of which one specific dihydropirimidine (DHP) showed the highest inhibitory activity against SF3B1MT cells. Several studies point towards a role of calcium signaling in MDS. Proteins active in calcium metabolism (GPR68, calpain, calpastatin) are involved in modulating sensitivity to drugs used in MDS (e.g., lenalidomide). Divalent substrates including Fe2+ may use CCs and it's plausible that our DHP can inhibit iron overload by blocking Fe2+ trafficking through L-type CCs. In this regard, CCBs have been shown to stimulate the activity of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters. Furthermore, it is known that the SF3B1 mutations reduce the expression of the iron transporter ABCB7, leading to increased iron accumulation. All of this led us to further evaluate the effects of DHP in vitro and in vivo. DHP had higher growth inhibitory activity against SF3B1MT cells when tested in vitro using 8 serial concentrations in half-log dilutions for a period of 3 days (20% and 60% growth inhibition at 1 and 3μM). Mixed competitive experiment of K700EmRFP and WTGFP cells treated with increased doses of DHP (1, 3, 5, 10, 20, 50 μM) reduced the competitiveness of K700EmRFP over the time inducing a 3-fold reduction at 50μM. K700EmRFP cells expressed half the amount of ABCB7 mRNA compared to WTGFP cells by RT-PCR. Therapeutic index provided by DHP was determined in vivo. Bone marrow cells of B6. Gt(ROSA)26Sortm1Sor/J (CD45.2) donors were transplanted in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients. Two weeks after transplantation, average engraftment (measured as percentage of CD45.2(+) donor cells) was 96% ± 0.02. DHP (10 mg/Kg) was orally administered. No decrease in the proportion of CD45.2(+) donor cells was seen post-treatment compared to pre-treatment (96±0.01 % vs. 96%±0.009). Similarly no change in the proportion of CD45.2(+) donor cells was observed in competitive repopulation experiments when B6. Gt(ROSA)26Sortm1Sor/J cells were mixed 1:1 with transgenic Sf3b1+/K700E cells (40%±0.13 vs. 44%±0.02). In contrast, preliminary experiments showed that DHP had an effect on reducing Sf3b1+/K700E allele burden in two chimeric mice (to 21% and 24%) compared to pre-treatment. DHP is structurally unique in comparison to other DHP-based CCBs; in that it possess a -CH20-CH2CH2NH2 moiety linked directly to the DHP scaffold that may in itself provide opportunities or modes for Fe2+ chelation as well as its L-type CCB activity. Our observations and structural analyses therefore provide impetus to explore this feature to possibly improve the drug's efficacy. In sum, we have demonstrated that the clonal growth of cells carrying SF3B1 mutations might be suppressed using the known L-type CCB DHP. This might represent a novel modulator of ATPase activity of ABC transporters in SF3B1MT expressing low ABCB7 levels. Disclosures Sekeres: Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.

SF3B1 mutations disrupt normal pre-mRNA splicing to cause disease. Drugs inhibiting the interaction between the SF3b complex and RNA or agents degrading auxiliary splicing factors are being tested as new avenues for targeted therapy in myeloid neoplasia (MN) with SF3B1 mutations. Here we describe the ability of small molecules to restore altered RNA processes in SF3B1MT MN. We previously reported (Visconte, ASH 2018) the identification of the small molecule 4-pyridyl-2-anilinothiazole (PAT) which showed growth inhibition of CRISPR/Cas9 SF3B1+/K700E cells and primary SF3B1MT cells. PAT did not influence the growth of other cell models without (THP1, MOLM13FLT3, OCIAML3DNMT3A, SIGM5TET2/DNMT3A, K562PHF6) and with other splicing factor mutations (K562U2AF1, K562LUC7L2). We now describe data from medicinal chemistry, transcriptome, and in vivo studies to advance drug development for SF3B1MT MN. SAR studies focused on logical and systematic modifications of PAT, e.g., i) replacement of the 2,4-disubstituted thiazole spacer ring with other heteroatom containing rings (5,6,7 membered aromatic or aliphatic ring structures); ii) alternative linking groups for the NH linker of the aniline of the tail region (sulfonamide, amide, substituted amine linkers); iii) alternative substituted aromatic and aliphatic ring structures for the phenyl head region substituent, led us to identify permissive sites for further chemical optimization. For example, a 4-chlorophenyl analog demonstrated activity [IC50, 3μM] similar to PAT. Competitive repopulation assays of bone marrow (BM) cells from dual reporters (ACTBtdTomato; EGFP) B6.GtROSA26 mixed with BM cells from conditional knock-in Sf3b1+/K700E mice injected in pre-lethally irradiated B6.SJL-PtprcaPepcb/BoyJ (CD45.1) recipients (n=18) were used as a preclinical murine model. This model then allowed i) demonstration of drug efficacy in reducing the competitiveness of SF3B1MT cells and ii) evaluation of therapeutic index in normal hematopoiesis. Post-transplant recovery, recipients of B6.GtROSA26 cells underwent PAT treatment (10 mg/Kg/IP/5 days weekly) for a period of 6 weeks without showing any signs of distress or drug intolerance (drop in blood count, weight loss, abdominal swelling, liver or kidney toxicity). Two weeks after transplantation, donor Sf3b1+/K700E cells had an engraftment capability similar to that of donor B6.GtROSA26 cells (83.6 ± 4 vs. 86.4 ± 2.4) when transplanted as a sole graft in CD45.1 recipients. PAT reduced almost half the percentage of Sf3b1+/K700E donor cells at 6 weeks of treatment (47.4%) vs. pre-treatment (83.6%). In mixed (1:1) BM transplants, Sf3b1+/K700E cellshad a repopulative disadvantage against competitors B6.GtROSA26 contributing for 16% of the marrow reconstitution. Similar to single graft transplants, PAT decreased the percentage of Sf3b1+/K700E cells at 6 weeks vs. pre-treatment (average, 6% vs. 16%) in chimeras. Consistent with the lack of toxicity of PAT treatment B6.GtROSA26 cells in chimeras were not affected by PAT and gradually repopulated the host (post-treatment, 80% vs. pre-treatment, 64%). Subsequently, we focused our efforts identifying important genes known to be dysregulated in MDS that were mostly influenced by drug treatment and minimally affected in normal cells. Our approach was based on the analyses of genes linked to erythropoiesis (a key hallmark of low-risk MDS). In normal hematopoiesis TGF-β signaling inhibits terminal erythroid maturation. Out of 13,775 genes, 5% (664/13,775) were found differentially expressed between CRISPR/Cas9 SF3B1+/K700E and parental cells of which 60% of these genes were significantly up-regulated and 40% down-regulated. Pathway analysis showed that the expression levels of SMAD family of genes and GDF factors changed significantly upon drug treatment. SMAD7 mRNA levels are 3-fold lower in MDS CD34+ cells (n=159) compared to the ones of healthy subjects (n=17) (GEO accession GSE58831) leading to TGF-β over activation. PAT treatment normalized SMAD7 expression levels in CRISPR/Cas9 SF3B1+/K700E cells by 3-fold while reducing the levels of GDF11. In summary, we have identified new drug entities that are modulators of transcriptomic changes which decrease the competitiveness of SF3B1MT cells. These results suggest combination therapies with current TGF-β pathway inhibitors. Disclosures Advani: Glycomimetics: Consultancy, Research Funding; Kite Pharmaceuticals: Consultancy; Amgen: Research Funding; Pfizer: Honoraria, Research Funding; Macrogenics: Research Funding; Abbvie: Research Funding. Kelly:Novartis, Bayer, Janssen, Pharmacyclics, Celgene, Astrazeneca, Seattle Genetics: Honoraria, Speakers Bureau; Takeda: Research Funding; Genentech, Verastem: Consultancy. Sekeres:Syros: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

TET2 is one of the most commonly mutated genes in myeloid neoplasia. Somatic TET2 mutations (TET2MT) cause complete or partial loss of enzymatic activity. TET2 (along with TET1/3) are Fe2+ and αKG-dependent DNA-dioxygenases that catalyze the oxidation of 5mC→5hmC→5fC→5caC. Ultimately, 5hmC generated by TET2-dioxygenase passively prevents maintenance methylation due to DNA methyltransferase's inability to recognize 5hmC. Alternatively, demethylation may also be a result of base excision repair of fC and caC. TET2MT can serve as therapeutic targets because they are often initiating lesions and present in a large fraction of patients. In this study, a comprehensive analysis of the configurations of TET2MT in myeloid neoplasia including MDS (n=1809) and AML (n=808), showed a remarkable exclusivity with 2-HG producing neomorphic IDH1/2MT (Fig.A). TET2 expression in 97 healthy and 909 MDS/MPN or AML patients from two independent studies showed that IDH1/2MT cases have significantly higher TET2 expression and were also mutually exclusive in cases with lower TET2 expression. Doxycycline inducible expression of IDH1MT led to profound growth inhibition of both a natural TET2MT cell line SIG-M5 and engineered TET2-/- K562, while the effect to parental K562 was mild (Fig.B-E). These observations suggest that mutual exclusivity of TET2MT and IDH1/2MT is due to synthetic lethality of TET2MT cell caused by 2-HG production, rather than redundancy of the consequences of IDH1/2MT and TET2MT. In TET2MT cell 2-HG further inhibit the residual TET-activity (TET1/3) and may cause synthetic lethality to cells with affected TET2 function. SIG-M5 cells expresses significant amount of TET3 while negligible levels of TET1. The reliance on relative compensation through residual TET3 activity has been confirmed in cells by inducible TET3 knockdown. We hypothesized that transient suppression of the residual DNA dioxygenase activity with inhibitors may selectively eliminate TET2-deficient clones. The known TET inhibitors 2-HG, N-oxalylglycine (NOG) and dimethyl methyl fumarate (DMF) lack specificity, pharmacologic properties and potency. Based on the results of in silico docking simulations, we designed and synthesized 16 aKG derivatives. Among them, TETi76 showed best inhibition effect in both TET activity and cell growth of TET2 low expressing cell. TETi76 binds to the α-KG co-factor site of TET2 that principally involves H1801, H1381 and S1898. These amino acids are conserved in all three TET enzymes. To test the in vitro efficacy and specificity of TETi, we used several human myeloid cell lines that harbor loss of function TET2 mutations or constitutively express low TET2 levels as well as bone marrow derived from Tet2+/+, Tet2+/- and Tet2-/- mice (Fig.F-G). Results showed that cells with low 5hmC level were more sensitive to TETi76 treatment. Specificity of TETi76 was further confirmed by RNAseq analyses of TETi76 treated K562, TET2-/- K562 and parental control cells. Moreover, TETi treatment did not appear to affect the function of α-KG-dependent histone dioxygenases. Mechanistically, treatment of SIG-M5 cells with TETi76 induced early and late stages of apoptotic cell death, a finding further confirmed by PARP1 and caspase-3 cleavage. RNAseq analyses of SIGM5 cells after treatment with TETi demonstrated a significant down-regulation of genes involved in transcription and peptide elongation, consistent with the consequences of TET inhibition. Interestingly, we also observed significant up-modulation of oxidative stress response pathway genes consistent with the inhibition of dioxygenases. In particular, TETi76 treatment induces 8-fold increase of oxidative stress sensor NQO1 a NRF2 target gene. To further probe the effects of TETi76 on TET2 deficient cells, Tet2MT/Tet2WT BM cells were co-cultured at fixed ratios to mimic the evolving Tet2MT clones. TETi76 effectively eliminated otherwise dominating Tet2MT cells (Fig.H). To determine the in vivo effects of TETi e.g., on elimination of Tet2MT clones, we performed bone marrow competitive reconstitution assays in PEP mice. TETi treatment selectively restricted the proliferative advantage of Tet2MT HSC compared to vehicle control where, as expected, TET2 mutant clones took over the WT cells. In clinical applications, TET inhibitors may constitute a new class of agents to be used in a targeted fashion in TET2 mutant neoplasia. Figure. Disclosures Meggendorfer: MLL Munich Leukemia Laboratory: Employment. Abazeed:Bayer AG: Honoraria, Other: Travel Support, Research Funding; Siemens: Research Funding. Sekeres:Millenium: Membership on an entity's Board of Directors or advisory committees; Syros: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Novartis: Consultancy; Alexion: Consultancy.

Aims:Succinylcholine is a depolarizing type of neuromuscular blocking drug. Aim of this study is to evaluate and compare serum potassium level with upper dose limit of succinylcholine (1.5mg/kg), which is used normally in anesthetic practice with low dose of succinylcholine (0.5mg/kg). Materials and methods:A total of 100 patients attending for elective surgery in College of Medical Sciences -Teaching Hospital( CMSTH)), Bharatpur, Chitwan, Nepal were studied. Two blood samples were collected, one before surgery and another at 4 min after injection of succinylcholine and send to central lab for estimation of serum potassium level. Result:The serum potassium levels recorded during the study showed that there were neither significant differences observed within the groups nor was there any difference between the groups. Conclusion:In absence of pathological conditions related to raised serum potassium level, both high and low doses of succinylcholine can be used safely as there is no statistically significant differences in serum potassium level between high and low dose of succinylcholine. Journal of College of Medical Sciences-Nepal, 2013, Vol-9, No-1, 19-22 DOI: http://dx.doi.org/10.3126/jcmsn.v9i1.9669

Background:Suxamethonium having its rapid onset and short duration of action makes this drug unique amongst the neuromuscular blocking drugs described so far. However, use of suxamethonium is associated with a large number of undesirable side effects. Objective:To evaluate clinical effects of high and low dose of suxamethonium and to determine whether lower dose of suxamethonium can be used for any beneficial effects in terms of its various adverse effects e.g. cardiovascular responses, post-operative muscle pains and intraocular pressure. Methods:A total of 100 patients were included in this prospective study. All these patients on preoperative clinical evaluation were assessed to have adequate airway. All the patients were divided in two groups, low dose group (group I) and High dose group (group II) with 50 patients in each at random. A standard anesthetic technique was adhered to all the patients and following parameters were observed on comparative basis: a. Fasciculation and post operative myalgia. b. Cardiovascular effects, c. Intraocular pressure. Observation:The incidence of post Suxamethonium pain was significantly greater in group II. Increase in heart rate from baseline was significant in both groups. There was no significant difference between the two groups in the diastolic pressure but rise in systolic blood pressure was significant at all assessment times in both groups. This rise from control was statistically significant. Conclusion:Suxamethonium can be used in lower doses (0.5 mg/kg) in elective cases without airway compromise. It gives benefits of reduced muscle pains, cardiovascular responses and intraocular hypertension. Journal of College of Medical Sciences-Nepal, 2013, Vol-9, No-2, 1-8 DOI: http://dx.doi.org/10.3126/jcmsn.v9i2.9677

The study area is very diverse due to its protectness on one hand and thus there is a vast scope of scientific study on the other hand. The parent material of the study area represents crystalline rocks and comprises of garnetiferous mica, schists, garnet mica and mica quartzite. The soil bulk density varies from 1.14 to 1.91 gm cm-3 under various soil depths ranging from 0 to 15, 15 to 30 and 30 to 45 cm. Whereas, an average water holding capacity ranged from 36.50 ± 0.52 to 67.39 ± 1.79% for 0 to 45 cm soil depth. The soil was found acidic in nature, which ranged from 5.09 ± 0.06 to 6.46 ± 0.05 for 0 to 45 cm depth. With increasing soil depths, the percentage of gravel particles (>4.75 mm) was found in ascending proportion under Abies pindrow, Betula utilis and Quercus semecarpifoliadominated forests. Whereas, this sequence was observed entirely different under other forests. However, fine particles showed the marked variation among other forests. While they were found to be in higher percentage in Pinus wallichiana and A. pindrow dominated forests under 30 to 45 and 0 to 15 cm depths, respectively. The least percentage of fine particles was found in Q. semecarpifolia dominated forest under 30 to 45 cm soil depth. The organic carbon was highest (6.10 ± 0.39%) and lowest (0.52 ± 0.13%) as observed underQ. semecarpifolia and A. pindrow dominated forests for 0to 15 and 30 to 45 cm depths during March to June, 1998 and December to March, 1999 respectively. Whereas, the seasonal variation of C : N : P ratio was highest (118.571:4.286:1) and lowest (8.000:2.682:1) when observed under A. pindrow dominated forest for 0 to 15 and 15 to 30 cm soil depths during June to September and September to December, 1998 respectively. Key words: Forest, soil, nutrients, N, P, K.

This work describes the stabilisation of silver and copper nanoparticles in chemically modified chitosan colloidal solution. Chitosan-N-2-methylidene-hydroxy-pyridine-6-methylidene hydroxy thiocarbohydrazide (CSPTH) was used as a stabilising and reducing agent for silver and copper nanoparticles. The modified chitosan derivatives and the synthesised nanoparticles were characterised by Fourier transform infrared (FT-IR) spectroscopy, Ultraviolet-visible (UV-Vis) spectroscopy and X-ray diffraction (XRD). Particle size, morphology and segregation of the nanoparticles were determined by transmission electron microscopy (TEM). The size of the nanoparticles was found to be less than 20 nm and 50 nm for silver and copper nanoparticles, respectively. These nanoparticles were stabilised in a chemically modified chitosan solution and their properties were studied using fluorescence spectroscopy, photoluminescence spectroscopy and surface-enhanced Raman scattering (SERS). The optical properties of silver nanoparticles in surface plasmon band (SPB) were enhanced at 407 nm compared to those of copper nanoparticles. Fluorescence (400 nm and 756 nm), photoluminescence (450 and 504 nm) and Raman scattering (1382 and 1581 cm(-1)) properties for the copper nanoparticles were superior to those of the silver nanoparticles.

A truly nonintrusive optical sensing method suitable for accurate and precise measurement of liquid level is proposed and demonstrated in this paper. Sensing principle of the proposed method is based on the phenomenon of lateral displacement effect, which is observed when oblique light rays propagate through material media with different thicknesses and refractive indices. Experimental results of this method demonstrate its ability to measure liquid level very precisely up to a resolution of about 40-50 μm. The proposed method has the potential for use in in situ measurement of liquid level and volume. It can also be exploited for measurement of refractive index of liquid.

Adenoid cystic carcinoma commonly arises from the major and minor salivary glands. Primary cutaneous adenoid cystic carcinoma is extremely rare. The tumour was hard, non-tender, and fixed to the underlying stucture with visible congested veins. Histopathologically, large cell masses with adenoid or cribriform pattern, forming cystic spaces at places were seen. Solid epithelial nests and a few ductal structures were also seen. Plumonary metastases were another unusual feature in our patient perhaps not reported before.

Myelination is one of the fundamental processes in vertebrates. A major challenge is to quantitatively image myelin distribution in the central nervous system. For this reason, we designed and synthesized a series of fluorinated radioligands that can be radiolabeled as radiotracers for positron emission tomography (PET) imaging of myelin. These newly developed radioligands readily penetrate the blood-brain barrier and selectively bind to myelin membranes in the white matter region. Structure-activity relationship studies of such ligands suggested that optimal permeability could be achieved with calculated lipophilicty in the range of 3-4. After radiolabeling with fluorine-18, the brain uptake and retention of each radioligand were determined by microPET/CT imaging studies. These pharmacokinetic studies led us to identify a lead compound ([(18)F]FMeDAS, 32) with promising in vivo binding properties, which was subsequently validated by ex vivo autoradiography.

An understanding of the epidemiological trend in hospital admissions, including morbidity and mortality patterns and the economic impact, is critical for healthcare planning and appropriate resource allocation. As we find very few literature on the pattern of paediatric admissions outside Kathmandu Valley, it is essential to conduct studies in the various parts of the country to determine the paediatric inpatient burden as well resource allocations.A retrospective study was carried out at Department of Pediatrics, College of Medical Sciences-Teaching Hospital, Bharatpur, Chitwan, Nepal over a period of one year. All the admissions were analyzed for age, sex, address, seasonal variation, frequency of diseases according to the organ system involved, duration of hospital stay, and the outcome.Out of the total 814 cases admitted 68.8% of the patients were younger than five years. There was male preponderance with male to female ratio of 1.9:1. Majority (43.6%) admissions were from Chitwan itself. Respiratory, gastrointestinal and neurologic conditions were the main cause for admission. The mean duration of hospital stay was 5.28 days and the mortality was only 0.1%.In this study under five-year patients contributed to 2/3rd of the hospital admissions. Respiratory, gastrointestinal and neurologic conditions were the main cause for admission with pneumonia, URTI and acute gastroenteritis being the most common diseases.

Magnetization of nanoparticles of NiO are measured and analyzed taking into account a distribution in particle magnetic moment. We find that disregarding this distribution in the analysis is the reason for the many anomalous observations reported on this system in the literature., Comment: 4 pages, 2 figures

Ionosonde measurements from Trivandrum (77°E, 8.5°N, dip 0.5°N) are used to investigate the seasonal and solar flux dependence of peak height, and vertical drift of F layer in post sunset hours. The study reveals that pre- and post-reversal peak vertical drift of F layer increases with solar flux in all seasons. However, the rate of increase is found to be season dependent. Clear difference is observed in the solar flux dependence of peak height and vertical drift for two equinoxes. The post reversal peak westward electric field shows weak (strong) dependence on solar flux during November–February solstice (March–April, September–October equinox).

SummaryDegenerative-calcific aortic stenosis is mainly a disease of old age. Patients with mild to moderate stenosis without symptoms and those with aortic valve sclerosis do not require mechanical intervention. There is no firm evidence that the rate of progression can be modified by medical therapies, though statins might have some effect. Patients who develop severe stenosis with symptoms have a very poor prognosis if managed medically. Surgical aortic valve replacement greatly improves symptoms and mortality rates and remains the treatment of choice for those fit for major surgery, even above the age of 80 years. For those not suitable for surgery, or who are unwilling to have an operation, outcomes can be significantly improved by trans-catheter aortic valve replacement, which has become an important option for frail elderly patients. Balloon aortic valvuloplasty improves symptoms and short-term survival, but has a less clear impact on mortality beyond 1 year. It is useful as a palliative treatment and as a bridging procedure for patients who are not ready for valve replacement. There is less agreement on the best approach to patients with asymptomatic severe aortic stenosis. Trials indicate that early valve replacement results in outcomes that are comparable to those seen in symptomatic patients, though guidelines advocate delaying surgery until symptoms occur or left ventricular function begins to decline rapidly. All elderly patients with severe aortic stenosis should be considered for a mechanical intervention unless there is a properly considered reason for not doing so, or they are not willing to receive such treatment.

Reaction of tin dichloride and tin tetrachloride with cyclohexylamine-N-thiohydrazide (ChaThz) [L1] and 1,3-propanediamine-N-thiohydrazide (PdaThz) [L2] results in [Sn(ChaThz)2] (1), Sn(ChaThz)2Cl2] (2), [Sn(PdaThz)2] (3), and [Sn(PdaThz)2Cl2] (4), in which the thiohydrazide coordinates to tin through imine nitrogen and thioamide sulfur. The ratio metal : ligand was 1 : 2 for all complexes. The tin(IV) thiohydrazide complexes were characterized by elemental analysis, IR, UV-Vis, 1H-NMR, 119Sn NMR, and mass spectral studies. Using the disc diffusion method, the ligands and metal complexes were screened for in vitro antibacterial activities against four pathogenic bacteria, Escherichia coli, Staphylococcus aureus, P. aeruginosa, and Bacillus cereus and for antifungal activities against Aspergillus flavus, A. carbonarius, A. niger, and A. fumigatus. While the tin(IV) complexes exhibited moderate antifungal activities, their parent ligands showed much higher and long-lasting broad spectrum of bioactivity agai...

Chitosan-N-2-methylhydroxypyridine-6-methylcorboxylate (Ch-PDC) and chitosan-N-2-methylhydroxypyridine-6-methylhydroxy thiocarbohydrazide (Ch-PDC-Th) were synthesized for the first time using chitosan as precursor. Chitosan, Ch-PDC, Ch-PDC-Th were used in the synthesis of gold nanoparticles (AuNP) in aqueous medium. Chitosan and Ch-PDC-Th possess reducing properties which enabled the 'green' synthesis of AuNPs. The stabilization of the AuNPs was as a result of the thiocarbide (SC) and amine (NH(2)) groups in the chitosan matrix. The modified chitosan, its derivatives and the resulting AuNPs were characterized by Fourier transform infrared (FTIR) spectroscopy, Ultraviolet-visible (UV-vis) spectroscopy, Raman scattering measurements, powder X-ray diffraction (PXRD) and thermo gravimetric analysis (TGA). Particle size, morphology, segregation and individuality of the AuNPs were examined by transmission electron microscope (TEM) and energy dispersion spectroscopy (EDS). An average AuNPs size of 20 nm was observed for chitosan and Ch-PDC-Th while Ch-PDC was 50 nm. In comparison, AuNPs resulting from Ch-PDC-Th precursor has the most enhanced Raman and fluorescent intensities and was stable for over 2 months.

is emerging with trade practices in the global scenario moving mainly towards the brands both for the products and service equally. Most of the Companies are trying to establish the brand name in the market progressively for the global recognition. As a result, Trademarks play today a very critical and important role. Every Company or Financial organization wants a distinguished trademark for brand name and market viability. Trademark registration and its evaluation for distinctiveness is thus becoming very tedious job for registration offices. Millions of trademarks already registered and millions of applications filed for trademarks registration are aggravating the problem of issuing the trademark certificates. There are different techniques and approaches currently in use for distinctness check for trademarks. The present paper gives the overview of the most popular and appreciated image processing techniques and approaches for the trademark distinctness check. Content Based Image retrieval techniques are widely used for that purpose and some other approaches like shape and texture based similarity finding techniques are also used. In the present paper, our approach is to summarize the most widely used techniques for the trademark distinctness check.

Human hematopoietic system produces various types of differentiated and short-lived cells with specialized functions, which require continuous replenishment through the function of hematopoietic stem cells (HSC). HSC failure is a common distal endpoint of various pathogenic mechanisms in almost all bone marrow failure (BMF) syndromes and associated diseases. Hematopoietic growth factor cocktails (HGF) used in expanding bone marrow cells e.g., to increase cellularity of the HSC grafts, lead to differentiation and decreased HSC count. Theoretically, when used in vivo, they may act on progenitors rather than HSC and lead to stimulation of clonal outgrowth. Our current ability to stimulate HSC self-renewal to provide reconstitution of long-term hematopoiesis is limited. Nicotinamide adenine dinucleotide (NAD+) serves as an essential cofactor and substrate for a number of critical cellular processes. NAD+ depletion may occur in response to DNA damage due to free radical/ionizing radiation attack, resulting in significant activation of NAD+ consuming PARPs. Because of their long lifespan, maintenance of the genomic integrity of HSCs by efficient and accurate DNA repair to reduce the risk of BMF and cellular transformation is essential. NAD+ is also required for the maintenance of sirtuins activity, important class III HDAC essential for the prevention of senescence. Aging or chronic immune activation and inflammatory cytokine production result in upmodulation of NAD+ degrading enzyme CD38 that rapidly depletes cellular and extracellular levels of NAD+. Various lines of evidence suggest that regulation of CD38 NADase activity is essential for maintenance of physiologic NAD+ levels. Enhancing NAD+ level can profoundly reduce oxidative cell damage in catabolic tissue, including blood. Consequently, promotion of intracellular NAD+ by preventing NAD+ catabolism represents a promising therapeutic strategy for degenerative diseases in general, and BMF and associated diseases in particular. Therefore; CD38, a major NAD+ degrading enzyme, can be an excellent therapeutic target to increase the cellular levels of NAD+ and consequently improve the function of HSC. Here we report the development of inhibitors of CD38 NADase activity that extends the self-renewal and proliferative life span of HSC. We used structure-guided virtual screening followed by docking simulation to develop CD38 inhibitors. The compounds were synthesized using rational chemical synthesis and characterized by high-resolution mass spectroscopy and C13 & H1NMR. HPLC based assays were performed to assess the ability of compounds to inhibit NAD+ degradation by recombinant CD38. Using an iterative approach of synthesis characterization and activity, we selected the most potent compound, designated as ccf1172, for further studies. Docking simulations, surface plasmon resonance, and HPLC based assays demonstrate that ccf1172 binds (KD=12 nM) and inhibits CD38 (IC50=10 nM) (Fig.1B, C&D). To further characterize the ability of ccf1172, colony forming assays (CFU-A) and long-term culture-initiating cell assays (LTCIC-A) were performed with cord blood, human and murine bone marrows. No GF-like activity was observed, but in combination with GF mix ccf1172 increased the number of erythroid and myeloid colonies (n=9) in dose-dependent manner with a maximal effect seen at 100 nM in a serial replating assay. Significant extension of proliferative life span of hematopoietic progenitors (n=5) were observed (Fig 1E). When we studied the ability of CD38 inhibitor to expand LTCICs in stromal cultures (n=3) as best in vitro surrogates of HSC, ccf1172 increased LTCIC numbers 2.6-fold at 10 nM. The effect did not require the presence of accessory cells as ccf1172 treatment resulted in ~2-fold increase in CD34+Lin-/CD45+ cells in stem cell culture media supplemented with growth factors over a period of 25 days (Fig 1F). The CD38 inhibitor demonstrated cytotoxic effects on nine different leukemic cell lines with IC 50 ranging from 1 to 5 µM while no effect was observed on normal bone marrow. Here, we demonstrate that CD38 inhibition may be a potential therapeutic principle for ex vivo and in vivo expansion of HSC. Decreasing levels of NAD+ have been linked to aging and stem cell dysfunction, as a key aspect of various BMF syndromes. The strategy of CD38 inhibition to preserve NAD+ is innovative and relevant therapeutic strategy. Disclosures Saunthararajah: Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy.

Discovery of many somatic lesions in leukemia enables development of targeted therapies. TET2 is one of the most frequently mutated genes in MDS/related disorders and is also present in a significant proportion of CHIP (clonal hematopoiesis of indeterminate potential) carriers. TET2 mutations (TET2mt) are mostly loss of function and occur in biallelic, heterozygous and hemi/homozygous configurations. TET2 encodes for Fe2+-dependent DNA dioxygenase that utilizes 2-ketoglutarate (αKG) for oxidation of 5-methylcytosine (mC) that results in demethylation either actively, by base excision repair of further oxidation products (fC or caC), or passively, via replication, due to DNA methyltransferase's inability to recognize 5-hydroxymethylcytosine (hmC). TET2mt are good targets for drug discovery because they often initiate the clonal evolution and are present in a large fraction of patients. However, except for ascorbic acid (AA) applied to augment TET2 activity and hypomethylating agents to which TET2mtmay be more susceptible, no specific therapies have been conceptualized for TET2mt disease. Synthetic lethality can be applied to genetic loci affected by loss of function mutations never occurring in homo/hemi/homozygous configuration. However, many tumor suppressor genes (TSG) are similar to TET2, where biallelic inactivation promotes oncogenicity and thus synthetic lethality would not be directly applicable. However, TET2 has TET1/TET3 homologs and a transient inhibition of TET enzymes could still yield a synthetic lethality (particularly in TET2mt). The idea for the proposed therapeutic strategy of TET inhibition was conceptualized based on in vivo observations of mutual exclusivity of TET2mt and IDH1/2 mutations which produces 2HG as a bone-fide natural TET inhibitor. Indeed, in our study of 485 TET2mt cases only 9 carried IDH1/2mt (mostly tiny subclones). Conversely, among 157 IDH1/2mtcases, there were only 11TET2mt (p=5.5x10-9) of which 4 were non-deleterious missense alterations or had a small clonal burden. To further support our hypothesis, we knocked in IDH1mt controlled by the doxycycline-inducible promoter into TET2mt cells. Induction of IDH1R132C or IDH1R132H (or IDH2mt), expression resulted in rapid cytotoxicity in TET2mt, and no growth perturbation was observed for TET2wt cells. Similar results have been observed in mice, where knockdown of TET3 in TET2 background shortened the life span. These observations led us to the idea of developing an aKG antagonist TET specific inhibitors (TETi). Using a structure-guided targeted discovery approach we designed, synthesized and characterized TETi, which demonstrated dose dependent inhibition of dioxygenase activity in a cell-free system. Using an iterative approach of design synthesis and activity, we selected the most potent 'hit', designated as TETi76, for further evaluation. Esterified TETi76 decreased 5hmC production and selectively induced cell death in TET2mt leukemia cell lines, SIGM5 (TET2-/-) and OCI-AML5 (TET2+/-), while a minimal effect was observed in K562 and CMK which are TET2+/+. The LD50 of TETi was 250-fold lower than 2HG in TET2mt cells. In TET2-/-engineered K562, TETi76 also showed cytotoxicity leaving a therapeutic window as compared with wild type K562. Normal bone marrows were resistant to TETi76 in clonogenic assay. In vitro mixing experiments in which Tet2mt/Tet2wt were subjected to methocult cultures at fixed ratios to mimic evolving Tet2mt clones, CD45.2 (either Tet2+/-or Tet2-/-) outcompeted CD45.1 marrow in a control setting, while treatment with TETi76 led to a gradual elimination of mutant marrow. This result was further recapitulated in vivo. In a competitive repopulation transplantation model using graft consist of a mixture of CD45 isotypes mismatched Tet2+/- or Tet2-/-and Tet2+/+marrow cells, TETi76 treatment selectively eliminated Tet2 deficient marrow. Our data have several important implications. It is likely that compensatory function from other TETs or remaining allele is needed for survival and elimination of dioxygenase appears to be lethal. This explains the exclusivity of TET2/IDH1/2 mutations. Dioxygenase inhibitors may have therapeutic applicability for selective elimination of TET2mt cells in MDS or potentially as a preventive measure in CHIP. TETi may represent a novel class of antileukemic agents. Disclosures Nazha: MEI: Consultancy. Maciejewski:Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Acute myeloid leukemia (AML) cells express the PU.1/RUNX1/CEBPA master transcription factor circuit at levels similar to or exceeding that in normal granulocyte-monocyte progenitors (GMP), but somehow terminal granulo-monocytic fates do not occur. Gene expression analyses of AML cells compared to the normal hematopoietic hierarchy confirmed suppression of hundreds of monocyte and granulocyte terminal-differentiation genes that were PU.1 targets by chromatin-immunoprecipitation analyses, despite intact expression of proliferation and myeloid-commitment genes. To better understand this repression rather than activation by the PU.1/RUNX1/CEBPA circuit, we used liquid chromatography-tandem mass spectrometry (LCMS/MS) to analyze the protein composition of the PU.1/RUNX1/CEBPA master transcription factor hub in AML cells of various genotypes. A striking common finding was enrichment for corepressors, coregulators that repress gene transcription, over coactivators that activate genes (we have previously described how leukemia oncoproteins produce this corepressor/coactivator imbalance [e.g., Gu et al., JCI 2018]). Prominent amongst the enriched corepressors were ATP-dependent chromatin remodelers SMARCA5 and CHD4 that execute the critical and energetically expensive event in epigenetic repression of nucleosome positioning to obstruct gene transcription start sites. To translate these observations towards therapy, we used a high throughput screen to identify a first-in-class compound series (ED2-AD101) that inhibits SMARCA5/CHD4. In silico modeling, supported by structure-activity relationship (SAR) studies, surface plasmon resonance and ATP-ase assays, indicated that ED2-AD101 inhibits ISWI family ATP-ase activity allosterically by binding to the highly conserved HELICc-DExx domain. In cell-based assays, ED2-AD101 at doses of between 1 to 10 µM potently suppressed AML cell growth (THP1, KG-1, MV411, HL60, MOLM13) via terminal-differentiation (without early apoptosis) while simultaneously sparing exponential growth of normal CD34+ hematopoietic stem and progenitor cells (Fig.1 A, C, D). We also demonstrated terminal granulo-monocytic differentiation using CD11b/CD14 markers by flow cytometry in THP-1 cells treated with ED2-AD101 at 1 µM dose (Fig 1B). Importantly, these differentiation-based cell cycle exits occurred even in chemo-resistant p53-null AML and other cancer cells (MCF7, UC6, RPMI 8226, and HCT116 cancer cells treated with 10µM of ED2-AD101. In the first panel of Fig. 1E, MCF7 cells show morphologic changes indicative of epithelial differentiation after treatment. Giemsa-stained cytospin preparations of all the above cell lines displayed morphological features of cell differentiation, such as a lower nucleocytoplasmic ratio after treatment) (Fig.1E). The lead compound has pharmaceutical range efficacy in the cell-based assays with GI50 ~ 0.9 µM. The therapeutic index and p53-independent findings are consistent also with our previous pre-clinical and clinical observations using decitabine or 5-azacytidine to inhibit the corepressor DNMT1 in AML cells (reviewed in Velcheti et al., ASCO Ed. Book 2017). Drug pharmacology and pre-clinical in vivo proof-of-principle experiments using patient-derived xenotransplant models of leukemia are in progress. Thus, druggable corepressors are the barriers between AML (and other cancer) cells and the terminal lineage-fates intended by their master transcription factor content, opening the door to novel non-cytotoxic, normal hematopoiesis sparing, differentiation-based oncotherapy. Figure 1. Figure 1. Disclosures Saunthararajah: Novo Nordisk, A/S: Patents & Royalties; EpiDestiny, LLC: Patents & Royalties.

Strong encryption is an urgent need for e-commerce development, since it allows the privacy and secure transactions of the financial and other data. International regulations must support the spreading of e-commerce and the associated encryption techniques in order to establish a secure e-commerce environment that customers can trust and develop faith in international deployment of ecommerce solutions without restrictions. In the present paper, a pricing and license model for the fare use of the digital contents is proposed suitable for today’s digital environment. Protection by way of Digital Rights Management (DRM) systems is emerging as a natural solution because it remains in force wherever the content goes. In contrast, a file that sits on a server behind the server’s access control mechanism loses its protection once it is moved from the server. The meaning of the term “DRM” has been used so far in its most narrow sense and no longer reflects many of the recent developments in the technology used to manage copyrighted works in the digital age. The efforts made for a secure DRM approach for the secure content transactions over digital environment using latest technologies has been reviewed in the present paper to advocate finally for the most acceptable DRM architecture supporting audio, video, text, XML and other popular applications file formats to serve the dynamic needs of the market optimally.

The present study compares the efficacy of two different subspecies of Bacillus thuringiensis viz. aizawai and kurstaki, available as commercial preparations in market against different stages of Spodoptera litura. Leaf-dip bioassay was done to calculate the LC50 values of both the preparations, while microbiological analysis and total protein content estimation were also performed to co-relate the results. B. thuringiensis aizawai was found to be more effective (LC50 values being 0.505, 1.121, 5.009, 6.706 for 1st, 2nd, 3rd, and 4th instar, respectively) than kurstaki (LC50 values being 1.121, 3.974, 6.897, 9.471 for 1st, 2nd, 3rd, and 4th instar, respectively). The efficacy was found to depend upon the number of viable spores in a particular preparation as well as on the total protein content of that particular subspecies. The preparation containing B.t.a was more effective because its viable spore counts as well as total protein content values (at different time intervals) were higher as compa...