Advances in the molecular design of potential anticancer agents via targeting of human telomeric DNA

Telomerases are an attractive drug target to develop new generation drugs against cancer. A telomere appears from the chromosomal termini and protects it from double-stranded DNA degradation. A short telomere promotes genomic instability, like end-to-end fusion and regulates the over-expression of the telomere repairing enzyme, telomerase. The telomerase maintains the telomere length, which may lead to genetically abnormal situations, leading to cancer. Thus, the design and synthesis of an efficient telomerase inhibitor is a viable strategy toward anticancer drugs development. Accordingly, small molecule induced stabilization of the G-quadruplex structure, formed by the human telomeric DNA, is an area of contemporary scientific art. Several such compounds efficiently stabilize the G-quadruplex forms of nucleic acids, which often leads to telomerase inhibition. This Feature article presents the discovery and development of the telomere structure, function and evolution in telomere targeted anticancer drug design and incorporates the recent advances in this area, in addition to discussing the advantages and disadvantages in the methods, and prospects for the future.

Distribution, relative abundance, and conservation status of Asian elephants in Karnataka, southern India

Karnataka state in southern India supports a globally significant and the country's largest population of the Asian elephant Elephas maximus. A reliable map of Asian elephant distribution and measures of spatial variation in their abundance, both vital needs for conservation and management action, are unavailable not only in Karnataka, but across its global range. Here, we use various data gathered between 2000 and 2015 to map the distribution of elephants in Karnataka at the scale of the smallest forest management unit, the `beat', while also presenting data on elephant dung density for a subset of `elephant beats.' Elephants occurred in 972 out of 2855 forest beats of Karnataka. Sixty percent of these 972 beats and 55% of the forest habitat lay outside notified protected areas (PM), and included lands designated for agricultural production and human dwelling. While median elephant dung density inside protected areas was nearly thrice as much as outside, elephants routinely occurred in or used habitats outside PM where human density, land fraction under cultivation, and the interface between human-dominated areas and forests were greater. Based on our data, it is clear that India's framework for elephant conservation which legally protects the species wherever it occurs, but protects only some of its habitats while being appropriate in furthering their conservation within PM, seriously falters in situations where elephants reside in and/or seasonally use areas outside PAs. Attempts to further elephant conservation in production and dwelling areas have extracted high costs in human, elephant, material and monetary terms in Karnataka. In such settings, conservation planning exercises are necessary to determine where the needs of elephants or humans must take priority over the other, and to achieve that in a manner that is based not only on reliable scientific data but also on a process of public reasoning. (C) 2015 Elsevier Ltd. All rights reserved.

Insights into the catalytic mechanism of synthetic glutathione peroxidase mimetics

Glutathione Peroxidase (GPx) is a key selenoenzyme that protects biomolecules from oxidative damage. Extensive research has been carried out to design and synthesize small organoselenium compounds as functional mimics of GPx. While the catalytic mechanism of the native enzyme itself is poorly understood, the synthetic mimics follow different catalytic pathways depending upon the structures and reactivities of various intermediates formed in the catalytic cycle. The steric as well as electronic environments around the selenium atom not only modulate the reactivity of these synthetic mimics towards peroxides and thiols, but also the catalytic mechanisms. The catalytic cycle of small GPx mimics is also dependent on the nature of peroxides and thiols used in the study. In this review, we discuss how the catalytic mechanism varies with the substituents attached to the selenium atom.

Pricing and Power allocation in Femtocells using Stackelberg Game Theory

We consider a system with multiple Femtocells operating in a Macrocell. The transmissions in one Femtocell interfere with its neighboring Femtocells as well as with the Macrocell Base Station. We model Femtocells as selfish nodes and the Macrocell Base Station protects itself by pricing subchannels for each usage. We use Stackelberg game model to study this scenario and obtain equilibrium policies that satisfy certain quality of service.

How much Dillenia indica seed predation occurs from Asian elephant dung?

Elephants are thought to be effective seed dispersers, but research on whether elephant dung effectively protects seeds from seed predation is lacking. Quantifying rates of seed predation from elephant dung will facilitate comparisons between elephants and alternative dispersers, helping us understand the functional role of megaherbivores in ecosystems. We conducted an experiment to quantify the predation of Dillenia indica seeds from elephant dung in Buxa Reserve, India from December 2012 to April 2013. Using dung boluses from the same dung pile, we compared the number of seeds in boluses that are a) opened immediately upon detection (control boluses), b) made available only to small seed predators (<= 3 mm wide) for 1-4 months, and c) made available to all seed predators and secondary dispersers for 1-4 months. Using a model built on this experiment, we estimated that seed predation by small seed predators (most likely ants and termites) destroys between 82.9% and 96.4% of seeds in elephant dung between the time of defecation and the median germination date for D. indica. Exposure to larger seed predators and secondary dispersers did not lead to a significant additional reduction in the number of seeds per dung bolus. Our findings suggest that post-dispersal seed predation by small insects (<3 mm) substantially reduces but does not eliminate the success of elephants as dispersers of D. indica in a tropical moist forest habitat. (C) 2015 Elsevier Masson SAS. All rights reserved.

San Vicente de Paúl y la oración

Resumen: San Vicente de Paúl se revela no sólo como el santo defensor de los pobres, sino como un maestro de oración. Este artículo quiere detenerse en este último aspecto. Veremos cómo plantea San Vicente la vida de oración y de devoción. Como es un santo minucioso, propio del barroco del siglo XVII, nos señalará detalladamente como se puede mejorar la práctica de la oración. Al mismo tiempo, hará hincapié en que una vida de oración demuestra su coherencia en la medida que se abre a la caridad fraterna y al servicio a los hermanos que padecen. De tal modo, la oración nos capacita para cumplir cabalmente la Voluntad de Dios. Idea que se puede resumir en su frase: “Denme un hombre de oración y será capaz de todo”.

El caso Artavia Murillo y otros vs. Costa Rica (fecundación in vitro) : la redefinición del derecho a la vida desde la concepción, reconocido en la Convención Americana

Resumen: El Artículo 4(1) de la Convención Americana protege la vida de la persona por nacer desde el momento de la concepción. Esta nota analiza críticamente la interpretación restrictiva de este artículo realizada por la Corte Interamericana de Derechos Humanos en el caso Artavia Murillo y otros vs. Costa Rica, sobre la prohibición de la fecundación in vitro en ese país. La nota describe cómo la Corte revirtió parcialmente el reconocimiento del derecho a la vida del niño no nacido, qué hicieron los Estados latinoamericanos, al adoptar la Convención Americana, a través de una aplicación distorsionada de sus propias técnicas de interpretación y de las normas internacionales de interpretación de los tratados.

Novel mechanisms for regulation of cell survival and migration by ceramide 1-phosphate

201 p. : gráf.

Gewinnmaximierung der Fischerei schützt den Dorsch der westlichen Ostsee vor Überfischung – Nur ein theoretischer Ansatz ?

Profit maximization in fishery protects cod of western Baltic Sea against overfishing – Only a theoretical approach? The frame for the management of fish stocks politically given contains – apart from ecological and social goals – also an economic goal, which is considered here in particular. From the point of view of fishery enterprises the main management goal for the exploitation of fish stocks is the maximization of profit. There are models for the yield optimization since long time. They are mainly used so far to optimize fishing mortality. Here the Beverton and Holt yield model was used. Apart from the optimization of fishing effort the model was used to optimize age of first capture and thus mesh opening. Starting point of the considerations is a given age group of a fish stock. If this age group is completely fished the yield obtained from this age group is maximized. The investigations show that the term overfishing is not exclusively linked as frequently assumed with a too large fishing mortality, but likewise with a mismatch of the mesh opening. For the calculated example Baltic cod data are used. At present the cod is caught far from reaching its mass optimum. Therefore, the profit of fishery enterprises can in the long term be considerably increased by the optimization of the mesh opening. During the conversion from the state of the art to fishing with optimised mesh sizes, however, a loss of profit has to be expected. The title of the paper sounds provocative. However, the stock of the Baltic Sea cod is better protected by a long-term maximum-profit oriented exploitation than by the precautionary approach applied now.

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP(3)Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP(3)Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by alpha subunit of the eukaryotic initiation factor 2 alpha phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. Cell Death and Disease (2010) 1, e54; doi:10.1038/cddis.2010.31; published online 15 July 2010

Oceans and human health: Sentinel habitats and sentinel species

The health of the oceans and people are inextricably linked. For many years we focused research and policy on anthropogenic impacts to oceans and coasts. Recently we have started to think about how the health of the oceans affects us. In response to the Oceans and Human Health Act of 2004, a NOAA initiative was created to explore the “One Health” of the oceans and coasts. The Center of Excellence in Oceans and Human Health at Hollings Marine Laboratory (HML) is one of three Centers dedicated to understanding the connections and forecasting changes in ocean and coastal health and human health. The Center at HML is developing new tools and approaches, including sentinel habitats and sentinel species, to evaluate linkages between ecological process and human health and wellbeing. The results provide environmental and public health managers, policy-makers and communities forecasts and assessments to improve ecosystem-based management that protects health and mitigates risks for the oceans, coasts and people.(PDF contains 4 pages)

Isolation and characterization of proteins that bind to yeast origins of DNA replication

Yeast chromosomes contain sequences called ARSs which function as origins of replication in vitro and in vivo. We have carried out a systematic deletion analysis of ARS1, allowing us to define three functionally distinct domains, designated A, B, and C. Domain A is a sequence of 11 to 19bp, containing the core consensus element that is required for replication. The core consensus sequence, A/TTTTATPuTTTA/T, is conserved at all ARSs sequenced to date. A fragment containing only element A and 8 flanking nucleotides enables autonomous replication of centromeric plasmids. These plasmids replicate very inefficiently, suggesting that flanking sequences must be important for ARS function. Domain B also provides important sequences needed for efficient replication. Deletion of domain B drastically increases the doubling times of transformants and reduces plasmid stability. Domain B contains a potential consensus sequence conserved at some ARSs which overlaps a region of bent DNA. Mutational analysis suggests this bent DNA may be important for ARS function. Deletion of domain C has only a slight effect on replication of plasmids carrying those deletions.

We have identified a protein called ARS binding factor I (ABF-I) that binds to the HMR-E ARS and ARS1. We have purified this protein to homogeneity using conventional and oligonucleotide affinity chromatography. The protein has an apparent molecular weight of 135kDa and is present at about 700 molecules per diploid cell, based on the yield of purified protein and in situ antibody staining. DNaseI footprinting reveals that ABF-I binds sequence-specifically to an approximately 24bp sequence that overlaps element Bat ARSl. This same protein binds to and protects a similar size region at the HMR-E ARS.

We also find evidence for another ARS binding protein, ABF-III, based on DN asei footprint analysis and gel retardation assays. The protein protects approximately 22bp adjacent to the ABF-I site. There appears to be no interaction between ABF-I and ABF-III despite the proximity of their binding sites.

To address the function of ABF-I in DNA replication, we have cloned the ABF-I gene using rabbit polyclonal anti-sera and murine monoclonal antibodies against ABF-I to screen a λgt11 expression library. Four EcoRI restriction fragments were isolated which encoded proteins that were recognized by both polyclonal and monoclonal antibodies. A gene disruption can now be constructed to determine the in vivo function of ABF-I.

A GIS-based model of soil erosion and transport

Soil erosion is a natural process that occurs when the force of wind, raindrops or running water on the soil surface exceeds the cohesive forces that bind the soil together. In general, vegetation cover protects the soil from the effects of these erosive forces. However, land management activities such as ploughing, burning or heavy grazing may disturb this protective layer, exposing the underlying soil. The decision making process in rural catchment management is often supported by the predictive modelling of soil erosion and sediment transport processes within the catchment, using established techniques such as the Universal Soil Loss Equation [USLE] and the Agricultural Nonpoint Source pollution model [AGNPS]. In this article, the authors examine the range of erosion models currently available and describe the application of one of these to the Burrishoole catchment on the north-west coast of Ireland, which has suffered heavy erosion of blanket peat in recent years.

Mechanisms of Transposable Element Repression by Piwi Proteins in the piRNA Pathway of Drosophila Germ Cells

The ability to reproduce is a defining characteristic of all living organisms. During reproduction, the integrity of genetic material transferred from one generation to the next is of utmost importance. Organisms have diverse strategies to ensure the fidelity of genomic information inherited between generations of individuals. In sexually reproducing animals, the piRNA pathway is an RNA-interference (RNAi) mechanism that protects the genomes of germ cells from the replication of ‘selfish’ genetic sequences called transposable elements (TE). When left unabated, the replication of TE sequences can cause gene disruption, double-stranded DNA breaks, and germ cell death that results in sterility of the organism. In Drosophila, the piRNA pathway is divided into a cytoplasmic and nuclear branch that involves the functions of three Piwi-clade Argonaute proteins—Piwi, Aubergine (Aub) and Argonaute-3 (Ago3)—which bind piwi-interacting RNA (piRNA) to form the effector complexes that represses deleterious TE sequences.

The work presented in this thesis examines the function and regulation of Piwi proteins in Drosophila germ cells. Chapter 1 presents an introduction to piRNA biogenesis and to the essential roles occupied by each Piwi protein in the repression of TE. We discuss the architecture and function of germ granules as the cellular compartments where much of the piRNA pathway operates. In Chapter 2, we present how Piwi in the nucleus co-transcriptionally targets genomic loci expressing TE sequences to direct the deposition of repressive chromatin marks. Chapter 3 examines the cytoplasmic function of the piRNA pathway, where we find that the protein Krimper coordinates Aub and Ago3 in the piRNA ping-pong pathway to adaptively target and destroy TE transcripts. Chapter 4 explores how interactions of Piwis with associated proteins are modulated by arginine methylation modifications. Lastly, in Chapter 5 I present evidence that the cytoplasmic branch of the piRNA pathway can potentially ‘cross-talk’ with the nuclear branch to transfer sequence information to better target and co-transcriptionally silence the genomic loci coding active TE sequences. Overall, the work presented in this thesis constitutes a part of the first steps in understanding the molecular mechanisms that protect germ cells from invasion by TE sequences.

Investigations of DNA-mediated protein oxidation

DNA charge transport (CT) involves the efficient transfer of electrons or electron holes through the DNA π-stack over long molecular distances of at least 100 base-pairs. Despite this shallow distance dependence, DNA CT is sensitive to mismatches or lesions that disrupt π-stacking and is critically dependent on proper electronic coupling of the donor and acceptor moieties into the base stack. Favorable DNA CT is very rapid, occurring on the picosecond timescale. Because of this speed, electron holes equilibrate along the DNA π-stack, forming a characteristic pattern of DNA damage at low oxidation potential guanine multiplets. Furthermore, DNA CT may be used in a biological context. DNA processing enzymes with 4Fe4S clusters can perform DNA-mediated electron transfer (ET) self-exchange reactions with other 4Fe4S cluster proteins, even if the proteins are quite dissimilar, as long as the DNA-bound [4Fe4S]^3+/2+ redox potentials are conserved. This mechanism would allow low copy number DNA repair proteins to find their lesions efficiently within the cell. DNA CT may also be used biologically for the long-range, selective activation of redox-active transcription factors. Within this work, we pursue other proteins that may utilize DNA CT within the cell and further elucidate aspects of the DNA-mediated ET self-exchange reaction of 4Fe4S cluster proteins.

Dps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. One aspect of their protection involves ferroxidase activity, whereby ferrous iron is bound and oxidized selectively by hydrogen peroxide, thereby preventing formation of damaging hydroxyl radicals via Fenton chemistry. Understanding the specific mechanism by which Dps proteins protect the bacterial genome could inform the development of new antibiotics. We investigate whether DNA-binding E. coli Dps can utilize DNA CT to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate oxidative DNA damage via the flash-quench technique, which localizes to a low potential guanine triplet. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps which lack available reducing equivalents, significantly attenuates the yield of oxidative DNA damage at the guanine triplet. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA CT may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.

Further work focused on spectroscopic characterization of the DNA-mediated oxidation of ferrous iron-loaded Dps. X-band EPR was used to monitor the oxidation of DNA-bound Dps after DNA photooxidation via the flash-quench technique. Upon irradiation with poly(dGdC)₂, a signal arises with g = 4.3, consistent with the formation of mononuclear high-spin Fe(III) sites of low symmetry, the expected oxidation product of Dps with one iron bound at each ferroxidase site. When poly(dGdC)₂ is substituted with poly(dAdT)₂, the yield of Dps oxidation is decreased significantly, indicating that guanine radicals facilitate Dps oxidation. The more favorable oxidation of Dps by guanine radicals supports the feasibility of a long-distance protection mechanism via DNA CT where Dps is oxidized to fill guanine radical holes in the bacterial genome produced by reactive oxygen species.

We have also explored possible electron transfer intermediates in the DNA-mediated oxidation of ferrous iron-loaded Dps. Dps proteins contain a conserved tryptophan residue in close proximity to the ferroxidase site (W52 in E. coli Dps). In comparison to WT Dps, in EPR studies of the oxidation of ferrous iron-loaded Dps following DNA photooxidation, W52Y and W52A mutants were deficient in forming the characteristic EPR signal at g = 4.3, with a larger deficiency for W52A compared to W52Y. In addition to EPR, we also probed the role of W52 Dps in cells using a hydrogen peroxide survival assay. Bacteria containing W52Y Dps survived the hydrogen peroxide challenge more similarly to those containing WT Dps, whereas cells with W52A Dps died off as quickly as cells without Dps. Overall, these results suggest the possibility of W52 as a CT hopping intermediate.

DNA-modified electrodes have become an essential tool for the study of the redox chemistry of DNA processing enzymes with 4Fe4S clusters. In many cases, it is necessary to investigate different complex samples and substrates in parallel in order to elucidate this chemistry. Therefore, we optimized and characterized a multiplexed electrochemical platform with the 4Fe4S cluster base excision repair glycosylase Endonuclease III (EndoIII). Closely packed DNA films, where the protein has limited surface accessibility, produce EndoIII electrochemical signals sensitive to an intervening mismatch, indicating a DNA-mediated process. Multiplexed analysis allowed more robust characterization of the CT-deficient Y82A EndoIII mutant, as well as comparison of a new family of mutations altering the electrostatics surrounding the 4Fe4S cluster in an effort to shift the reduction potential of the cluster. While little change in the DNA-bound midpoint potential was found for this family of mutants, likely indicating the dominant effect of DNA-binding on establishing the protein redox potential, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we proposed that the electron transfer pathway in EndoIII can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.

While the 4Fe4S cluster of EndoIII is relatively insensitive to oxidation and reduction in solution, we have found that upon DNA binding, the reduction potential of the [4Fe4S]^3+/2+ couple shifts negatively by approximately 200 mV, bringing this couple into a physiologically relevant range. Demonstrated using electrochemistry experiments in the presence and absence of DNA, these studies do not provide direct molecular evidence for the species being observed. Sulfur K-edge X-ray absorbance spectroscopy (XAS) can be used to probe directly the covalency of iron-sulfur clusters, which is correlated to their reduction potential. We have shown that the Fe-S covalency of the 4Fe4S cluster of EndoIII increases upon DNA binding, stabilizing the oxidized [4Fe4S]³⁺ cluster, consistent with a negative shift in reduction potential. The 7% increase in Fe-S covalency corresponds to an approximately 150 mV shift, remarkably similar to DNA electrochemistry results. Therefore we have obtained direct molecular evidence for the shift in 4Fe4S reduction potential of EndoIII upon DNA binding, supporting the feasibility of our model whereby these proteins can utilize DNA CT to cooperate in order to efficiently find DNA lesions inside cells.

In conclusion, in this work we have explored the biological applications of DNA CT. We discovered that the DNA-binding bacterial ferritin Dps can protect the bacterial genome from a distance via DNA CT, perhaps contributing to pathogen survival and virulence. Furthermore, we optimized a multiplexed electrochemical platform for the study of the redox chemistry of DNA-bound 4Fe4S cluster proteins. Finally, we have used sulfur K-edge XAS to obtain direct molecular evidence for the negative shift in 4Fe4S cluster reduction potential of EndoIII upon DNA binding. These studies contribute to the understanding of DNA-mediated protein oxidation within cells.