Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Nanomolar detection of hydrogen peroxide at a new polynuclear cluster of tin pentacyanonitrosylferrate nanoparticle-modified carbon ceramic electrode

This work describes a new electrochemical sensor for hydrogen peroxide based on tin pentacyanonitrosylferrate (SnPCNF)-modified carbon ceramic electrode (CCE). The modified electrode was constructed by using a sol-gel technique involving two steps: construction of CCE containing metallic tin (Sn) powder and then electrochemical creation of SnPCNF film on the surface of CCE. The modified electrode was characterized by energy-dispersive X-ray, Fourier transform infrared, scanning electron microscopy, and cyclic voltammetry (CV) techniques. The charge transfer coefficient (α) and charge transfer rate constant (k_s) for the modifying film were calculated. The electrocatalytic activity of the modified electrode toward the reduction of hydrogen peroxide was studied by CV and chronoamperometry. A linear calibration curve was obtained over the hydrogen peroxide concentration range of 0.5 to 69.4 μM using a hydrodynamic amperometric technique. The limit of detection (for a signal-to-noise ratio of 3) and sensitivity were found to be 92 nM and 0.89 μA/μM, respectively. Furthermore, the diffusion coefficient of hydrogen peroxide (D) and catalytic rate constant (k_cat) were calculated.     

The colonization of palynomorphs by chytrids and thraustochytrids during pre–depositional taphonomic processes in tropical mangrove ecosystems

Degrees of colonization of palynomorphs from six mangrove plants by chytrids and thraustochytrids in four mangrove plant communities at each stage of the pre–depositional taphonomic process were investigated using a pollen–baiting method. Chytrids and thraustochytrids were the dominant group colonizing palynomorphs in mangroves, gaining access to the cytoplasm through the wall or aperture. There were no significant differences in the degree of colonization between the different plant communities. Pollen with a larger size and/or extensive apertural region appeared to be the most colonized, while the absence of apertures and the presence of a thick wall seemed to reduce the chance of colonization by these microorganisms. Taphonomic experiments showed that the longer palynomorphs take to settle into the mangrove sediments, the lower the possibility of survival from the destructive colonization by chytrids and thraustochytrids and consequently the less carbon that will be sequestered.     

Reply to the first systematic response by the Global Footprint Network to criticism: A real debate finally?

We respond to a reaction of the Global Footprint Network/GFN on our 8-point criticism of the ecological footprint. We also refer to, and comment on, an associated debate in this journal between Giampietro and Saltelli, 2014a, Giampietro and Saltelli, 2014b, on the one hand, and Goldfinger et al. (2014), on the other. We conclude that criticism on the footprint is accumulating and coherent across the various studies and disciplines and among the different authors. This was the first time that Wackernagel/GFN systematically responded to our criticisms. Hence, our response contains several original elements and the resulting exchange can be seen to add value to the existing literature. It ultimately allows readers to better make up their mind about the different viewpoints on the ecological footprint.     

An automated technique for monitoring carbon dioxide respired by insects*

ABSTRACT. A non-dispersive infrared gas analyser equipped with a Luft-type sonic detector and flow-through reference cell was automated to monitor the total volume of carbon dioxide (CO₂) respired by single insects or groups of insects. The infrared analyser was interfaced with an integrator for quantification, a microprocessor to control intermittent air flow through the insect respiration chambers, and a microcomputer for data storage and reduction. This technique has been used to monitor the CO₂ Output of diapausing and non-diapausing mature fifth instar larvae and of developing pupae of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). The resulting data were accurate, quantitative and reproducible.     

Regional differences in the carbon isotopic compositions of teak from two monsoonal regimes of India

Inter and intra-annual carbon isotope compositions (δ¹³C) of several annual growth rings of teak trees from two monsoonal regimes from India were studied and compared with the corresponding oxygen isotopic (δ¹⁸O) variations. In teak from both the regimes, amplitudes of intra-annual δ¹³C were ∼2-3 times lower than that observed in δ¹⁸O. Seasonal cycle with lower δ¹³C values at the middle and higher at ring boundaries was observed for teak from central India, dominated by the southwest monsoon. Positive correlations of intra-annual δ¹³C values with the corresponding δ¹⁸O values of the same rings and with relative humidity (RH) of the concurrent period suggest a dominant role of RH in controlling δ¹³C values of teak from central India. Intra-annual δ¹³C variations of teak from southern India, receiving both the southwest and northeast monsoons, revealed an initial decreasing trend followed by an increasing trend before culminating in depleted ¹³C values at the end of the growing season. No correlation was observed between intra-annual δ¹³C and δ¹⁸O variations of teak trees from southern India. Regional differences in the climatology of δ¹³C of atmospheric CO₂ or the lengths of growing season could be likely reasons for differing intra-annual δ¹³C variations of teak from the two climatic regimes.     

The first crenarchaeon capable of growth by anaerobic carbon monoxide oxidation coupled with H2 production

The ability to grow by anaerobic CO oxidation with production of H₂ from water is known for some thermophilic bacteria, most of which belong to Firmicutes, as well as for a few hyperthermophilic Euryarchaeota isolated from deep-sea hydrothermal habitats. A hyperthermophilic, neutrophilic, anaerobic filamentous archaeon strain 1505 = VKM B-3180 = KCTC 15798 was isolated from a terrestrial hot spring in Kamchatka (Russia) in the presence of 30% CO in the gas phase. Strain 1505 could grow lithotrophically using carbon monoxide as the energy source with the production of hydrogen according to the equation CO + H₂O → CO₂ + H₂; mixotrophically on CO plus glucose; and organotrophically on peptone, yeast extract, glucose, sucrose, or Avicel. The genome of strain 1505 was sequenced and assembled into a single chromosome. Based on 16S rRNA gene sequence analysis and in silico genome-genome hybridization, this organism was shown to be closely related to the Thermofilum adornatum species. In the genome of Thermofilum sp. strain 1505, a gene cluster (TCARB_0867-TCARB_0879) was found that included genes of anaerobic (Ni,Fe-containing) carbon monoxide dehydrogenase and genes of energy-converting hydrogenase ([Ni,Fe]-CODH–ECH gene cluster). Compared to the [Ni,Fe]-CODH–ECH gene clusters occurring in the sequenced genomes of other H₂-producing carboxydotrophs, the [Ni,Fe]-CODH–ECH gene cluster of Thermofilum sp. strain 1505 presented a novel type of gene organization. The results of the study provided the first evidence of anaerobic CO oxidation coupled with H₂ production performed by a crenarchaeon, as well as the first documented case of lithotrophic growth of a Thermofilaceae representative.     

The glands of Tamarix aphylla: a system for salt recretion or for carbon concentration?

During periods of high atmospheric humidity, twigs of Tamarix aphylla (L.) Karst. become covered by an alkaline solution. The pH of that solution fluctuates between 8.0 – 8.5 in the dark and 10.5 during the light hours. Such a solution, produced by the glands, constitutes an efficient trap for atmospheric CO₂. Upon the periodic drop in pH, much of the preabsorbed carbon may gradually be released from the solution. This enriches the immediate surroundings of the twigs with CO₂ for prolonged periods of time. The expected concentrations of CO₂, at the boundary layer between the atmosphere and the surfaces of the twigs, are over 1 000 ppm. As net photosynthesis of T. aphylla reaches maximal rates only at CO₂ concentrations of above 500 ppm, the plants may benefit from this extra source of carbon and may exploit it for maximal assimilation during the early morning hours. Thus, the "salt glands'of Tamarix , which are liable for the production of the alkaline recretum, may serve a triple purpose: (a) removal of excess salts out of the twigs, (b) provision of a cover of hygroscopic solutes that moistens the twigs and shortens the duration of transpiration, and (c) providing the plants with an environment enriched in CO₂.