Temperature and water requirements for germination and effects of discontinuous hydration on germinated seed survival in Tillandsia recurvata L.

Tillandsia recurvata is an epiphytic bromeliad with a wide distribution in the Americas; however, little is known about the development of its post-seminal adaptations for survival in epiphytic environments. The purpose of this study was to define the temperature and water requirements for the germination of T. recurvata seeds. The absence of radicle emergence in T. recurvata seeds resulted in 2?stages of germination: swollen with broken seed coat (stage-1) and chlorophyllic embryos (stage-2). The effects of partial or discontinuous hydration on germinated seed survival were also assessed. The seeds were collected in a semi-arid shrubland of Mexico City. We explored: (1) whether water vapour can provide a sufficient water source for germination; (2) the temperature required for germination stage-1 and the optimal and critical osmotic potentials for germination in both germination stages; (3) the effect of seed incubation at different osmotic potentials that undergo subsequent dehydration on their survival in stage-2; and (4) the loss of dehydration tolerance during early post-seminal development. In addition, an image of T. recurvata seed anatomy was obtained to illustrate its structures. Germination stage-1 of T. recurvata seeds is rather similar across the tested temperature range. The seeds required to be in contact with liquid water to germinate. The interval of osmotic potential facilitating both germination stages was from 0 to ?0.6?MPa. Although germinated seeds displayed dehydration tolerance, this tolerance decreased in germination stage-2. The osmotic potential during germination affected the tolerance of the chlorophyllic embryos (stage-2) to subsequent dehydration.     

Germination of <Emphasis Type="Italic">Sedum oxypetalum</Emphasis> (Crassulaceae) in a primary lava-field shrubland

Sedum oxypetalum is one of the dominant species in the xerophilous shrublands in the lava fields of the Basin of Mexico. Germination of this species was evaluated to understand its ecological response. We tested the effects of different pre-germination treatments (cold, and dry and wet heat) and storage time, as well as those of natural priming in two microhabitats with different disturbance levels. Experiments were performed in laboratory conditions under constant (25 °C) and fluctuating (20/30 °C) temperatures. Seeds did not germinate during burial and proved to be positively photoblastic. Under pre-germination treatments, final germination percentage was higher at 20/30 °C in seeds after four or more months of storage. None of the pre-germination treatments favored germination. Seeds can survive for more than 1 year and form a seed bank. Thus, seeds underwent natural priming that favored final germination percentage; however, germination rate and lag time were not affected by this process. In natural conditions, germination is delayed until the rainy season, improving the success of seedling establishment and growth. We discuss the role of fluctuating temperature in germination processes and the adaptations of seeds to their seasonal environment.     

DNA methylation-independent reversion of gemcitabine resistance by hydralazine in cervical cancer cells

Background

Down regulation of genes coding for nucleoside transporters and drug metabolism responsible for uptake and metabolic activation of the nucleoside gemcitabine is related with acquired tumor resistance against this agent. Hydralazine has been shown to reverse doxorubicin resistance in a model of breast cancer. Here we wanted to investigate whether epigenetic mechanisms are responsible for acquiring resistance to gemcitabine and if hydralazine could restore gemcitabine sensitivity in cervical cancer cells.

Methodology/Principal Findings

The cervical cancer cell line CaLo cell line was cultured in the presence of increasing concentrations of gemcitabine. Down-regulation of hENT1 & dCK genes was observed in the resistant cells (CaLoGR) which was not associated with promoter methylation. Treatment with hydralazine reversed gemcitabine resistance and led to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner. No changes in HDAC total activity nor in H3 and H4 acetylation at these promoters were observed. ChIP analysis showed H3K9m2 at hENT1 and dCK gene promoters which correlated with hyper-expression of G9A histone methyltransferase at RNA and protein level in the resistant cells. Hydralazine inhibited G9A methyltransferase activity in vitro and depletion of the G9A gene by iRNA restored gemcitabine sensitivity.

Conclusions/Significance

Our results demonstrate that acquired gemcitabine resistance is associated with DNA promoter methylation-independent hENT1 and dCK gene down-regulation and hyper-expression of G9A methyltransferase. Hydralazine reverts gemcitabine resistance in cervical cancer cells via inhibition of G9A histone methyltransferase.     

Cancer Progression Mediated by Horizontal Gene Transfer in an In Vivo Model

It is known that cancer progresses by vertical gene transfer, but this paradigm ignores that DNA circulates in higher organisms and that it is biologically active upon its uptake by recipient cells. Here we confirm previous observations on the ability of cell-free DNA to induce in vitro cell transformation and tumorigenesis by treating NIH3T3 recipient murine cells with serum of colon cancer patients and supernatant of SW480 human cancer cells. Cell transformation and tumorigenesis of recipient cells did not occur if serum and supernatants were depleted of DNA. It is also demonstrated that horizontal cancer progression mediated by circulating DNA occurs via its uptake by recipient cells in an in vivo model where immunocompetent rats subjected to colon carcinogenesis with 1,2-dimethylhydrazine had increased rate of colonic tumors when injected in the dorsum with human SW480 colon carcinoma cells as a source of circulating oncogenic DNA, which could be offset by treating these animals with DNAse I and proteases. Though the contribution of biologically active molecules other than DNA for this phenomenon to occur cannot be ruled out, our results support the fact that cancer cells emit into the circulation biologically active DNA to foster tumor progression. Further exploration of the horizontal tumor progression phenomenon mediated by circulating DNA is clearly needed to determine whether its manipulation could have a role in cancer therapy.     

Crystal structure of a ring-cleaving cyclohexane-1,2-dione hydrolase, a novel member of the thiamine diphosphate enzyme family

The thiamine diphosphate (ThDP) dependent flavoenzyme cyclohexane-1,2-dione hydrolase (CDH) (EC 3.7.1.11) catalyses a key step of a novel anaerobic degradation pathway for alicyclic alcohols by converting cyclohexane-1,2-dione (CDO) to 6-oxohexanoate and further to adipate using NAD(+) as electron acceptor. To gain insights into the molecular basis of these reactions CDH from denitrifying anaerobe Azoarcus sp. strain 22Lin was structurally characterized at 1.26 ? resolution. Notably, the active site funnel is rearranged in an unprecedented manner providing the structural basis for the specific binding and cleavage of an alicyclic compound. Crucial features include a decreased and displaced funnel entrance, a semi-circularly shaped loop segment preceding the C-terminal arm and the attachment of the C-terminal arm to other subunits of the CDH tetramer. Its structural scaffold and the ThDP activation is related to that observed for other members of the ThDP enzyme family. The selective binding of the competitive inhibitor 2-methyl-2,4-pentane-diol (MPD) to the open funnel of CDH reveals an asymmetry of the two active sites found also in the dimer of several other ThDP dependent enzymes. The substrate binding site is characterized by polar and non-polar moieties reflected in the structures of MPD and CDO and by three prominent histidine residues (His28, His31 and His76) that most probably play a crucial role in substrate activation. The NAD(+) dependent oxidation of 6-oxohexanoate remains enigmatic as the redox-active cofactor FAD seems not to participate in catalysis, and no obvious NAD(+) binding site is found. Based on the structural data both reactions are discussed.     

Vaccination against GIP for the treatment of obesity

Background

According to the WHO, more than 1 billion people worldwide are overweight and at risk of developing chronic illnesses, including cardiovascular disease, type 2 diabetes, hypertension and stroke. Current therapies show limited efficacy and are often associated with unpleasant side-effect profiles, hence there is a medical need for new therapeutic interventions in the field of obesity. Gastric inhibitory peptide (GIP, also known as glucose-dependent insulinotropic polypeptide) has recently been postulated to link over-nutrition with obesity. In fact GIP receptor-deficient mice (GIPR^−/−) were shown to be completely protected from diet-induced obesity. Thus, disrupting GIP signaling represents a promising novel therapeutic strategy for the treatment of obesity.

Methodology/Principal Findings

In order to block GIP signaling we chose an active vaccination approach using GIP peptides covalently attached to virus-like particles (VLP-GIP). Vaccination of mice with VLP-GIP induced high titers of specific antibodies and efficiently reduced body weight gain in animals fed a high fat diet. The reduction in body weight gain could be attributed to reduced accumulation of fat. Moreover, increased weight loss was observed in obese mice vaccinated with VLP-GIP. Importantly, despite the incretin action of GIP, VLP-GIP-treated mice did not show signs of glucose intolerance.

Conclusions/Significance

This study shows that vaccination against GIP was safe and effective. Thus active vaccination may represent a novel, long-lasting treatment for obesity. However further preclinical safety/toxicology studies will be required before the therapeutic concept can be addressed in humans.     

Can the Geranium Bronze, <Emphasis Type="Italic">Cacyreus marshalli</Emphasis>, become a threat for European biodiversity?

Cacyreus marshalli Butler is an invasive species in many parts of Europe and Mediterranean area. In Europe, its larvae normally feed on pelargoniums. We investigated its potential to spread to native Geranium spp. and evaluated the conservation risks that such a shift would pose for both native geraniums and cohabitant butterflies. The host plant preferences of the Geranium Bronze were investigated under controlled conditions. Studies included both no-choice and multi-choice tests, respectively using 9 and 6 Italian native Geranium spp. Host plant preferences were evaluated by counting the number of eggs laid on individual plants and following butterfly development until adult emergence. Under no-choice conditions, at least one egg was recorded on each tested plant, except for G. phaeum L. All the plants on which oviposition occurred were fully suitable for larval development. The butterfly, however, clearly preferred three species, i.e. G. pratense L., G. sanguineum L. and G. sylvaticum L. for oviposition. In multi-choice trials, females laid at least one egg on all the tested plants, with a preference for G. pratense and G. sylvaticum. In presence of Pelargonium spp. plants, however, no oviposition was observed on any Geranium spp. We assessed offspring fitness measuring their wingspan. No statistical differences were detected in the wingspan between adults emerged from Geranium and Pelargonium. Cacyreus marshalli represents a potential threat for both native geraniums and for Geranium-consuming lycaenids, such as Aricia nicias Meigen and Eumedonia eumedon Esper.     

Sugar feeding protects against arboviral infection by enhancing gut immunity in the mosquito vector Aedes aegypti

As mosquito females require a blood meal to reproduce, they can act as vectors of numerous pathogens, such as arboviruses (e.g. Zika, dengue and chikungunya viruses), which constitute a substantial worldwide public health burden. In addition to blood meals, mosquito females can also take sugar meals to get carbohydrates for their energy reserves. It is now recognised that diet is a key regulator of health and disease outcome through interactions with the immune system. However, this has been mostly studied in humans and model organisms. So far, the impact of sugar feeding on mosquito immunity and in turn, how this could affect vector competence for arboviruses has not been explored. Here, we show that sugar feeding increases and maintains antiviral immunity in the digestive tract of the main arbovirus vector Aedes aegypti. Our data demonstrate that the gut microbiota does not mediate the sugar-induced immunity but partly inhibits it. Importantly, sugar intake prior to an arbovirus-infected blood meal further protects females against infection with arboviruses from different families. Sugar feeding blocks arbovirus initial infection and dissemination from the gut and lowers infection prevalence and intensity, thereby decreasing the transmission potential of female mosquitoes. Finally, we show that the antiviral role of sugar is mediated by sugar-induced immunity. Overall, our findings uncover a crucial role of sugar feeding in mosquito antiviral immunity which in turn decreases vector competence for arboviruses. Since Ae. aegypti almost exclusively feed on blood in some natural settings, our findings suggest that this lack of sugar intake could increase the spread of mosquito-borne arboviral diseases.     

Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation

Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases.     

Transformation of white poplar (Populus alba L.) with a novel Arabidopsis thaliana cysteine proteinase inhibitor and analysis of insect pest resistance

Transgenic white poplar (Populus alba L.) plants expressing a novel Arabidopsis thaliana cysteine proteinase inhibitor (Atcys) gene have been produced using Agrobacterium tumefaciens-mediated gene transfer. Internodal stem segments of cv. Villafranca were co-cultivated with the EHA105 pBI-Atcys A. tumefaciens strain. Sixteen putative transgenic plant lines were regenerated from different calli with a transformation efficiency of 11%. The integration and expression of the cysteine proteinase inhibitor (Atcys) gene into the plant genome was confirmed by Southern and northern blot analyses. Papain inhibitory activity was detected in poplar transgenic tissues by means of a specific in vitro assay. Such activity was sufficient to inhibit most of the digestive proteinase activity of chrysomelid beetle (Chrysomela populi L.) and confer resistance to C. populi larvae on selected transgenic plants. A close correspondence between the inhibition of papain and resistance to poplar leaf beetle was observed in all tested transgenic lines. Our results indicate that Atcys could be succesfully employed in breeding programmes aimed at the selection of new poplar genotypes resistant to major insect pests.