Sphingosine,a Modulator of Human Translesion DNA Polymerase Activity

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼3000 small molecules, including one comprising ∼600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.     

Dendrobium tamenglongense sp. nov. (Orchidaceae) from Manipur,India

Dendrobium tamenglongense R. Kishor, Y. N. Devi, H. B. Sharma, J. Tongbram & S. P. Vij sp. nov. from Manipur, India is described and illustrated as a new species. The new species differs from D. jaintianum in having larger and glabrous leaves with acute apices, green sepal apices, a 1–3‐flowered inflorescence, smaller and non‐fragrant flowers and a 3‐lobed labellum with fimbriate midlobe.     

Clinically Approved Iron Chelators Influence Zebrafish Mortality,Hatching Morphology and Cardiac Function

Iron chelation therapy using iron (III) specific chelators such as desferrioxamine (DFO, Desferal), deferasirox (Exjade or ICL-670), and deferiprone (Ferriprox or L1) are the current standard of care for the treatment of iron overload. Although each chelator is capable of promoting some degree of iron excretion, these chelators are also associated with a wide range of well documented toxicities. However, there is currently very limited data available on their effects in developing embryos. In this study, we took advantage of the rapid development and transparency of the zebrafish embryo, Danio rerio to assess and compare the toxicity of iron chelators. All three iron chelators described above were delivered to zebrafish embryos by direct soaking and their effects on mortality, hatching and developmental morphology were monitored for 96 hpf. To determine whether toxicity was specific to embryos, we examined the effects of chelator exposure via intra peritoneal injection on the cardiac function and gene expression in adult zebrafish. Chelators varied significantly in their effects on embryo mortality, hatching and morphology. While none of the embryos or adults exposed to DFO were negatively affected, ICL -treated embryos and adults differed significantly from controls, and L1 exerted toxic effects in embryos alone. ICL-670 significantly increased the mortality of embryos treated with doses of 0.25 mM or higher and also affected embryo morphology, causing curvature of larvae treated with concentrations above 0.5 mM. ICL-670 exposure (10 µL of 0.1 mM injection) also significantly increased the heart rate and cardiac output of adult zebrafish. While L1 exposure did not cause toxicity in adults, it did cause morphological defects in embryos at 0.5 mM. This study provides first evidence on iron chelator toxicity in early development and will help to guide our approach on better understanding the mechanism of iron chelator toxicity.     

Co-Encapsulation of Doxorubicin With Galactoxyloglucan Nanoparticles for Intracellular Tumor-Targeted Delivery in Murine Ascites and Solid Tumors

Doxorubicin (Dox) treatment is limited by severe toxicity and frequent episodes of treatment failure. To minimize adverse events and improve drug delivery efficiently and specifically in cancer cells, encapsulation of Dox with naturally obtained galactoxyloglucan polysaccharide (PST001), isolated from Tamarindus indica was attempted. Thus formed PST-Dox nanoparticles induced apoptosis and exhibited significant cytotoxicity in murine ascites cell lines, Dalton’s lymphoma ascites and Ehrlich’s ascites carcinoma. The mechanism contributing to the augmented cytotoxicity of nanoconjugates at lower doses was validated by measuring the Dox intracellular uptake in human colon, leukemic and breast cancer cell lines. PST-Dox nanoparticles showed rapid internalization of Dox into cancer cells within a short period of incubation. Further, in vivo efficacy was tested in comparison to the parent counterparts - PST001 and Dox, in ascites and solid tumor syngraft mice models. Treatment of ascites tumors with PST-Dox nanoparticles significantly reduced the tumor volume, viable tumor cell count, and increased survival and percentage life span in the early, established and prophylactic phases of the disease. Administration of nanoparticles through intratumoral route delivered more robust antitumor response than the intraperitoneal route in solid malignancies. Thus, the results indicate that PST-Dox nanoparticles have greater potential compared to the Dox as targeted drug delivery nanocarriers for loco regional cancer chemotherapy applications.     

Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy

Chloroquine (CQ) is a 4-aminoquinoline drug used for the treatment of diverse diseases. It inhibits lysosomal acidification and therefore prevents autophagy by blocking autophagosome fusion and degradation. In cancer treatment, CQ is often used in combination with chemotherapeutic drugs and radiation because it has been shown to enhance the efficacy of tumor cell killing. Since CQ and its derivatives are the only inhibitors of autophagy that are available for use in the clinic, multiple ongoing clinical trials are currently using CQ or hydroxychloroquine (HCQ) for this purpose, either alone, or in combination with other anticancer drugs. Here we show that in the mouse breast cancer cell lines, 67NR and 4T1, autophagy is induced by the DNA damaging agent cisplatin or by drugs that selectively target autophagy regulation, the PtdIns3K inhibitor LY294002, and the mTOR inhibitor rapamycin. In combination with these drugs, CQ sensitized to these treatments, though this effect was more evident with LY294002 and rapamycin treatment. Surprisingly, however, in these experiments CQ sensitization occurred independent of autophagy inhibition, since sensitization was not mimicked by Atg12, Beclin 1 knockdown or bafilomycin treatment, and occurred even in the absence of Atg12. We therefore propose that although CQ might be helpful in combination with cancer therapeutic drugs, its sensitizing effects can occur independently of autophagy inhibition. Consequently, this possibility should be considered in the ongoing clinical trials where CQ or HCQ are used in the treatment of cancer, and caution is warranted when CQ treatment is used in cytotoxic assays in autophagy research.     

Developmentally regulated sphingolipid degradation in Leishmania major

Leishmania parasites alternate between extracellular promastigotes in sandflies and intracellular amastigotes in mammals. These protozoans acquire sphingolipids (SLs) through de novo synthesis (to produce inositol phosphorylceramide) and salvage (to obtain sphingomyelin from the host). A single ISCL (Inositol phosphoSphingolipid phospholipase C-Like) enzyme is responsible for the degradation of both inositol phosphorylceramide (the IPC hydrolase or IPCase activity) and sphingomyelin (the SMase activity). Recent studies of a L. major ISCL-null mutant (iscl(-)) indicate that SL degradation is required for promastigote survival in stationary phase, especially under acidic pH. ISCL is also essential for L. major proliferation in mammals. To further understand the role of ISCL in Leishmania growth and virulence, we introduced a sole IPCase or a sole SMase into the iscl(-) mutant. Results showed that restoration of IPCase only complemented the acid resistance defect in iscl(-) promastigotes and improved their survival in macrophages, but failed to recover virulence in mice. In contrast, a sole SMase fully restored parasite infectivity in mice but was unable to reverse the promastigote defects in iscl(-). These findings suggest that SL degradation in Leishmania possesses separate roles in different stages: while the IPCase activity is important for promastigote survival and acid tolerance, the SMase activity is required for amastigote proliferation in mammals. Consistent with these findings, ISCL was preferentially expressed in stationary phase promastigotes and amastigotes. Together, our results indicate that SL degradation by Leishmania is critical for parasites to establish and sustain infection in the mammalian host.     

Synthesis and thermochemical study of ligand substitution reactions of aminobis(phosphines), Ph2P(R)NPPh2, with [Me2Pt(COD)]

The reactions of aminobis(phosphines), Ph₂PN(R)PPh₂ (R = H, Me, Et, ⁿPr, ⁿBu, Ph), with Me₂Pt(COD) led to the formation of four-membered metallacycles in good yield. The enthalpies of the ligand substitution reactions are measured by the anaerobic solution calorimetry in THF at 30 °C.     

Identification of critical residues of subunit H in its interaction with subunit E of the A-ATP synthase from Methanocaldococcus jannaschii

The boomerang-like H subunit of A(1)A(0) ATP synthase forms one of the peripheral stalks connecting the A(1) and A(0) sections. Structural analyses of the N-terminal part (H1-47) of subunit H of the A(1)A(0) ATP synthase from Methanocaldococcus jannaschii have been performed by NMR spectroscopy. Our initial NMR structural calculations for H1-47 indicate that amino acid residues 7-44 fold into a single alpha-helical structure. Using the purified N- (E1-100) and C-terminal domains (E101-206) of subunit E, NMR titration experiments revealed that the N-terminal residues Met1-6, Lys10, Glu11, Ala15, Val20 and Glu24 of H1-47 interact specifically with the N-terminal domain E1-100 of subunit E. A more detailed picture regarding the residues of E1-100 involved in this association was obtained by titration studies using the N-terminal peptides E1-20, E21-40 and E41-60. These data indicate that the N-terminal tail E41-60 interacts with the N-terminal amino acids of H1-47, and this has been confirmed by fluorescence correlation spectroscopy results. Analysis of (1)H-(15)N heteronuclear single quantum coherence (HSQC) spectra of the central stalk subunit F in the presence and absence of E101-206 show no obvious interaction between the C-terminal domain of E and subunit F. The data presented provide, for the first time, structural insights into the interaction of subunits E and H, and their arrangement within A(1)A(0) ATP synthase.     

Yale school of public health symposium on lifetime exposures and human health: the exposome; summary and future reflections

The exposome is defined as “the totality of environmental exposures encountered from birth to death” and was developed to address the need for comprehensive environmental exposure assessment to better understand disease etiology. Due to the complexity of the exposome, significant efforts have been made to develop technologies for longitudinal, internal and external exposure monitoring, and bioinformatics to integrate and analyze datasets generated. Our objectives were to bring together leaders in the field of exposomics, at a recent Symposium on “Lifetime Exposures and Human Health: The Exposome,” held at Yale School of Public Health. Our aim was to highlight the most recent technological advancements for measurement of the exposome, bioinformatics development, current limitations, and future needs in environmental health. In the discussions, an emphasis was placed on moving away from a one-chemical one-health outcome model toward a new paradigm of monitoring the totality of exposures that individuals may experience over their lifetime. This is critical to better understand the underlying biological impact on human health, particularly during windows of susceptibility. Recent advancements in metabolomics and bioinformatics are driving the field forward in biomonitoring and understanding the biological impact, and the technological and logistical challenges involved in the analyses were highlighted. In conclusion, further developments and support are needed for large-scale biomonitoring and management of big data, standardization for exposure and data analyses, bioinformatics tools for co-exposure or mixture analyses, and methods for data sharing.