These findings identify the presence of the rd8 mutation in the C57BL/6N mouse substrain used widely to produce transgenic and knockout mice. The results have grave implications for the vision research community who develop mouse lines to study eye disease, as presence of rd8 can produce significant disease phenotypes unrelated to the gene or genes of interest. It is suggested that researchers screen for rd8 if their mouse lines were generated on the C57BL/6N background, bear resemblance to the rd8 phenotype, or are of indeterminate origin.

The transcription factor Meis1 is expressed preferentially in hematopoietic stem cells (HSCs) and overexpressed in certain leukemias. However, the functions of Meis1 in hematopoiesis remain largely unknown. In the present study, we found that Meis1 is required for the maintenance of hematopoiesis under stress and over the long term, whereas steadystate hematopoiesis was sustained in the absence of Meis1 in inducible knock-out mice. BM cells of Meis1-deficient mice showed reduced colony formation and contained significantly fewer numbers of long-term HSCs, which exhibited loss of quiescence. Further, we found that Meis1 deletion led to the accumulation of reactive oxygen species in HSCs and decreased expression of genes implicated in hypoxia response. Finally, reac- IntroductionThe transcription factor Meis1 belongs to the 3-amino acid loop extension (TALE) family of homeodomain proteins, which also includes Pbx1. Meis1 was originally discovered as a target of activation by retroviral integration in the leukemic mouse strain BXH-2. In these mice, Meis1 was always found coactivated with Hoxa7 or Hoxa9. 1 Experimental overexpression of Meis1 and Hoxa9 in hematopoietic cells leads to an aggressive leukemia in mice. 2 In normal hematopoiesis, Meis1 expression among the various cell compartments is correlated with the degree of selfrenewal, with the levels being highest in hematopoietic stem cells (HSCs) and declining with differentiation. 3,4 These results, combined with those from studies in leukemia, suggest a role for Meis1 in HSC self-renewal. However, the functions of Meis1 in established hematopoiesis remain unknown. Meis1 Ϫ/Ϫ mice die by embryonic day 14.5 and the embryos display extensive hemorrhaging, particularly in the CNS. In addition, fetal liver cells obtained from Meis1 Ϫ/Ϫ embryos show reduced myeloid colony formation in vitro and perform poorly in competitive transplantation assays in vivo. 5,6 These results show that Meis1 is critical in the development of hematopoiesis. Further, Meis1 Ϫ/Ϫ embryos display defects in capillary formation, suggesting additional roles for Meis1 in mechanisms that regulate the process of angiogenesis, at least during development.Previous studies showed that Meis1 and Pbx1 can form heterodimeric and heterotrimeric complexes with HOX proteins, augmenting the binding and activation of target genes by HOX proteins such as HOXA9. 7,8 Deletion of Pbx1 in the hematopoietic system led to loss of quiescence in HSCs, resulting in hematopoietic failure. 9 Given that both Pbx1 and Meis1 are expressed in HSCs, we hypothesized that Meis1 might also be required for the maintenance of the self-renewing HSCs. In the present study, we investigated the role of Meis1 in established hematopoiesis using inducible knock-out mice. We found that Meis1 is required for the maintenance of HSCs by preserving quiescence in these mice. Maintenance of quiescence is critical for preserving self-renewal of long-term stem cells, and various signaling pathways have been shown to modulate quiescence in HSC...

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Purpose Classical pharmacology allows the use and development of conventional phytomedicine faster and more economically than conventional drugs. This approach should be tested for their efficacy in terms of complementarity and disease control. The purpose of this study was to determine the molecular mechanisms by which nimbolide, a triterpenoid found in the well-known medicinal plant Azadirachta indica controls glioblastoma (GBM) growth. Experimental Design Using in vitro signaling, anchorage-independent growth, kinase assays, and xenograft models, we investigated the mechanisms of its growth inhibition in glioblastoma. Results We show that nimbolide or an ethanol soluble fraction of A. indica leaves (Azt) that contains nimbolide as the principal cytotoxic agent is highly cytotoxic against GBM in vitro and in vivo. Azt caused cell cycle arrest, most prominently at the G1-S stage in GBM cells expressing EGFRvIII, an oncogene present in about 20-25% of GBMs. Azt/nimbolide directly inhibited CDK4/CDK6 kinase activity leading to hypophosphorylation of the retinoblastoma (RB) protein, cell cycle arrest at G1-S and cell death. Independent of RB hypophosphorylation, Azt also significantly reduced proliferative and survival advantage of GBM cells in vitro and in tumor xenografts by downregulating Bcl2 and blocking growth factor induced phosphorylation of Akt, Erk1/2 and STAT3. These effects were specific since Azt did not affect mTOR or other cell cycle regulators. In vivo, Azt completely prevented initiation and inhibited progression of GBM growth. Conclusions Our preclinical findings demonstrate Nimbolide as a potent anti-glioma agent that blocks cell cycle and inhibits glioma growth in vitro and in vivo.

Age modulates FasL function where increased MMP cleavage leads to a loss of function in the eye. The released form of FasL (sFasL) preferentially induces the migration of proangiogenic M2 macrophages into the laser lesions and increases proangiogenic cytokines promoting CNV. FasL may be a viable target for therapeutic intervention in aged-related neovascular disease.

BackgroundResistance of Leishmania donovani to pentavalent antimonials, the first-line treatment of visceral leishmaniasis (VL), has become a critical issue worldwide. Second-line and new drugs are also not devoid of limitations. Suitable drug-delivery systems can improve the mode of administration and action of the existing antimonials, thus increasing their clinical life.Methodology/Principal FindingsWe investigated the efficacy of sodium stibogluconate (SSG) in phosphatidylcholine (PC)–stearylamine-bearing liposomes (PC-SA-SSG), PC-cholesterol liposomes (PC-Chol-SSG) and free amphotericin B (AmB) against SSG-resistant L. donovani strains in 8-wk infected BALB/c mice. Animals were sacrificed and parasites in liver, spleen and bone marrow were estimated 4-wk post-treatment by microscopic examination of stamp smears and limiting dilution assay. A set of PC-SA-SSG and AmB treated mice were further studied for protection against reinfection. Serum antibodies and cytokine profiles of ex-vivo cultured splenocytes were determined by ELISA. Uptake of free and liposomal SSG in intracellular amastigotes was determined by atomic absorption spectroscopy. Rhodamine 123 and 5-carboxyfluorescein, known substrates of Pgp and MRP transporter proteins, respectively, were used in free and liposomal forms for efflux studies to estimate intracellular drug retention. Unlike free and PC-Chol-SSG, PC-SA-SSG was effective in curing mice infected with two differentially originated SSG-unresponsive parasite strains at significantly higher levels than AmB. Successful therapy correlated with complete suppression of disease-promoting IL-10 and TGF-β, upregulation of Th1 cytokines and expression of macrophage microbicidal NO. Cure due to elevated accumulation of SSG in intracellular parasites, irrespective of SSG-resistance, occurs as a result of increased drug retention and improved therapy when administered as PC-SA-SSG versus free SSG.Conclusions/SignificanceThe design of this single-dose combination therapy with PC-SA-SSG for VL, having reduced toxicity and long-term efficacy, irrespective of SSG-sensitivity may prove promising, not only to overcome SSG-resistance in Leishmania, but also for drugs with similar resistance-related problems in other diseases.

• Meis1 is required for the maintenance of MLL-fusion gene leukemia; HLF is a key downstream mediator of Meis1.• Meis1 and HLF restrict oxidative stress; induction of oxidative phosphorylation may be therapeutic in leukemia.Leukemias with MLL translocations are often found in infants and are associated with poor outcomes. The pathogenesis of MLL-fusion leukemias has been linked to upregulation of HOX/MEIS1 genes. The functions of the Hox/Meis1 complex in leukemia, however, remain elusive. Here, we used inducible Meis1-knockout mice coupled with MLL-AF9 knockin mice to decipher the mechanistic role of Meis1 in established MLL leukemia. We demonstrate that Meis1 is essential for maintenance of established leukemia. In addition, in both the murine model and human leukemia cells, we found that Meis1 loss led to increased oxidative stress, oxygen flux, and apoptosis. Gene expression and chromatin immunoprecipitation studies revealed hepatic leukemia factor (HLF) as a target gene of Meis1. Hypoxia or HLF expression reversed the oxidative stress, rescuing leukemia development in Meis1-deficient cells. Thus, the leukemia-promoting properties of Meis1 are at least partly mediated by a lowoxidative state, aided by HLF. These results suggest that stimulants of oxidative metabolism could have therapeutic potential in leukemia treatment. (Blood. 2015;125(16):2544-2552 IntroductionReciprocal translocations of the 11q23 locus lead to acute leukemias of both myeloid and lymphoid lineages. These leukemias are usually resistant to conventional chemotherapies. The translocation generates an oncogenic fusion protein comprised of an amino terminus derived from MLL (now called KMT2A), the gene at 11q23, fused to a carboxy terminus derived from one of several different genes. Recent studies have revealed that these MLL-fusion proteins then join higher-order protein complexes containing various transcriptional activators and/or elongating factors such as DOT1L, PAFc, and p-TEFB. 1,2 Recruitment of these complexes to genomic loci was shown to be associated with chromatin modifications and subsequent changes in gene expression. 3 Regardless of the fusion partner involved in the translocation and of the consequential protein complex assembled, the downstream genes activated by the various MLL-fusion proteins are largely identical. Data from patientderived leukemia cells and from experimental models reveal that MLLfusion proteins upregulate the expression of posterior HOX-A genes and of the HOX cofactor MEIS1. [4][5][6] Further, retroviral overexpression of a Hox gene (with few exceptions) along with Meis1 in murine hematopoietic cells induces an aggressive leukemia in transplanted mice. 7,8 Thus, MLL-fusion protein induced upregulation of HOX and MEIS1 genes is considered central to the pathology of these leukemias.Studies to test the requirement of HOX-A and MEIS1 genes in MLL-fusion leukemias have yielded mixed results. Although shRNA knockdown of HOXA9 was shown to inhibit leukemia, mice lacking Hoxa9 protein developed leukemia induced...

SA-bearing liposomes specifically kill Leishmania, but are non-toxic to murine peritoneal macrophages and human erythrocytes.