Phylogenetic analysis informed by geological history supports multiple, sequential invasions of the Mediterranean Basin by the angiosperm family Araceae

Despite the remarkable species richness of the Mediterranean flora and its well-known geological history, few studies have investigated its temporal and spatial origins. Most importantly, the relative contribution of geological processes and long-distance dispersal to the composition of contemporary Mediterranean biotas remains largely unknown. We used phylogenetic analyses of sequences from six chloroplast DNA markers, Bayesian dating methods, and ancestral area reconstructions, in combination with paleogeographic, paleoclimatic, and ecological evidence, to elucidate the time frame and biogeographic events associated with the diversification of Araceae in the Mediterranean Basin. We focused on the origin of four species, Ambrosina bassii, Biarum dispar, Helicodiceros muscivorus, Arum pictum, subendemic or endemic to Corsica, Sardinia, and the Balearic Archipelago. The results support two main invasions of the Mediterranean Basin by the Araceae, one from an area connecting North America and Eurasia in the Late Cretaceous and one from the Anatolian microplate in western Asia during the Late Eocene, thus confirming the proposed heterogeneous origins of the Mediterranean flora. The subendemic Ambrosina bassii and Biarum dispar likely diverged sympatrically from their widespread Mediterranean sister clades in the Early-Middle Eocene and Early-Middle Miocene, respectively. Combined evidence corroborates a relictual origin for the endemic Helicodiceros muscivorus and Arum pictum, the former apparently representing the first documented case of vicariance driven by the initial splitting of the Hercynian belt in the Early Oligocene. A recurrent theme emerging from our analyses is that land connections and interruptions, caused by repeated cycles of marine transgressions-regressions between the Tethys and Paratethys, favored geodispersalist expansion of biotic ranges from western Asia into the western Mediterranean Basin and subsequent allopatric speciation at different points in time from the Late Eocene to the Late Oligocene.     

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.     

Tomato transcriptional changes in response to Clavibacter michiganensis subsp. michiganensis reveal a role for ethylene in disease development

Clavibacter michiganensis subsp. michiganensis (Cmm) is a gram-positive actinomycete, causing bacterial wilt and canker disease in tomato (Solanum lycopersicum). Host responses to gram-positive bacteria and molecular mechanisms associated with the development of disease symptoms caused by Cmm in tomato are largely unexplored. To investigate plant responses activated during this compatible interaction, we used microarray analysis to monitor changes in host gene expression during disease development. This analysis was performed at 4 d postinoculation, when bacteria were actively multiplying and no wilt symptoms were yet visible; and at 8 d postinoculation, when bacterial growth approached saturation and typical wilt symptoms were observed. Of the 9,254 tomato genes represented on the array, 122 were differentially expressed in Cmm-infected plants, compared with mock-inoculated plants. Functional classification of Cmm-responsive genes revealed that Cmm activated typical basal defense responses in the host, including induction of defense-related genes, production and scavenging of free oxygen radicals, enhanced protein turnover, and hormone synthesis. Cmm infection also induced a subset of host genes involved in ethylene biosynthesis and response. After inoculation with Cmm, Never ripe (Nr) mutant plants, impaired in ethylene perception, and transgenic plants with reduced ethylene synthesis showed significant delay in the appearance of wilt symptoms, compared with wild-type plants. The retarded wilting in Nr plants was a specific effect of ethylene insensitivity, and was not due to altered expression of defense-related genes, reduced bacterial populations, or decreased ethylene synthesis. Taken together, our results indicate that host-derived ethylene plays an important role in regulation of the tomato susceptible response to Cmm.     

Vulnerability, livelihood assets and institutional dynamics in the management of wetlands in Lake Victoria watershed basin

This paper uses data from 600 households in the Lake Victoria watershed in Tanzania, Kenya, and Uganda to analyze the effects of vulnerabilities and shocks on the management and exploitation of wetlands within the context of agricultural activities and high poverty levels. A multinomial logit model is used to determine variables that influence the perception of wetlands degradation, while a tobit model is used to establish the determinants of willingness to pay for wetland conservation and the imputed value of wetland product extracts. The model results show that although the perception of wetland degradation is modest, it is influenced by attributes of social capital. Variables such as floods, diseases and droughts significantly influence the households’ willingness to pay for wetland conservation. Land size and ownership, education level and household size all influence households’ likelihood to actively engage in wetland resource exploitation and willingness to pay for its conservation. The implications of these results hinge on measures that would moderate the effects of shocks, mobilize collective action, and improve physical infrastructure within the context of sustainable wetland resource use.     

Docking of Antizyme to Ornithine Decarboxylase and Antizyme Inhibitor using Experimental Mutant and Double-Mutant Cycle Data

Antizyme (Az) is a highly conserved key regulatory protein bearing a major role in regulating polyamine levels in the cell. It has the ability to bind and inhibit ornithine decarboxylase (ODC), targeting it for degradation. Az inhibitor (AzI) impairs the activity of Az. In this study, we mapped the binding sites of ODC and AzI on Az using Ala scan mutagenesis and generated models of the two complexes by constrained computational docking. In order to scan a large number of mutants in a short time, we developed a workflow combining high-throughput mutagenesis, small-scale parallel partial purification of His-tagged proteins and their immobilization on a tris-nitrilotriacetic-acid-coated surface plasmon resonance chip. This combination of techniques resulted in a significant reduction in time for production and measurement of large numbers of mutant proteins. The data-driven docking results suggest that both proteins occupy the same binding site on Az, with Az binding within a large groove in AzI and ODC. However, single-mutant data provide information concerning the location of the binding sites only, not on their relative orientations. Therefore, we generated a large number of double-mutant cycles between residues on Az and ODC and used the resulting interaction energies to restrict docking. The model of the complex is well defined and accounts for the mutant data generated here, and previously determined biochemical data for this system. Insights on the structure and function of the complexes, as well as general aspects of the method, are discussed.     

On a Reproductive Stage-Structured Model for the Population Dynamics of the Malaria Vector

A reproductive stage-structured deterministic differential equation model for the population dynamics of the human malaria vector is derived and analysed. The model captures the gonotrophic and behavioural life characteristics of the female Anopheles sp. mosquito and takes into consideration the fact that for the purposes of reproduction, the female Anopheles sp. mosquito must visit and bite humans (or animals) to harvest necessary proteins from blood that it needs for the development of its eggs. Focusing on mosquitoes that feed exclusively on humans, our results indicate the existence of a threshold parameter, the vectorial reproduction number, whose size increases with increasing number of gonotrophic cycles, and is also affected by the female mosquito’s birth rate, its attraction and visitation rate to human residences, and its contact rate with humans. A stability analysis of the model indicates that the mosquito can establish itself in the environment if and only if the value of the vectorial reproduction number exceeds unity and that mosquito eradication is possible if the vectorial reproduction number is less than unity, since, then, the trivial steady state which always exist is unique and is globally and asymptotically stable. When a persistent vector population steady state exists, it is locally and asymptotically stable for a range of reproduction numbers, but can also be driven to instability via a Hopf bifurcation as the reproduction number increases further away from unity. The model derivation identifies and characterizes control parameters relating to activities such as human-mosquito contact and the mosquito’s survival chances between blood meals and egg laying. Our results show that the total mosquito population size increases with increasing number of gonotrophic cycles. Therefore understanding the fundamental aspects of the mosquito’s behaviour provides a pathway for the study of human-mosquito contact and mosquito population control. Control of the mosquito population densities would ultimately lead to malaria control.     

Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents

Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C–C bond cleavage in symmetrical β‐diketones by 6‐oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β‐diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily. Biotechnol. Bioeng. 2011;108: 2815–2822. © 2011 Wiley Periodicals, Inc.     

The effect of N-acetylcysteine on the antitumor activity of ifosfamide

Ifosfamide-induced nephrotoxicity is a serious adverse effect in children undergoing chemotherapy. Our previous cell and rodent models have shown that the antioxidant N-acetylcysteine (NAC), used extensively as an antidote for acetaminophen poisoning, protects renal tubular cells from ifosfamide-induced nephrotoxicity at a clinically relevant concentration. For the use of NAC to be clinically relevant in preventing ifosfamide nephrotoxicity, we must ensure there is no effect of NAC on the antitumor activity of ifosfamide. Common pediatric tumors that are sensitive to ifosfamide, human neuroblastoma SK-N-BE(2) and rhabdomyosarcoma RD114-B cells, received either no pretreatment or pretreatment with 400 μmol/L of NAC, followed by concurrent treatment with NAC and either ifosfamide or the active agent ifosfamide mustard. Ifosfamide mustard significantly decreased the growth of both cancer cell lines in a dose-dependent manner (p < 0.001). The different combined treatments of NAC alone, sodium 2-mercaptoethanesulfonate alone, or NAC plus sodium 2-mercaptoethanesulfonate did not significantly interfere with the tumor cytotoxic effect of ifosfamide mustard. These observations suggest that NAC may improve the risk/benefit ratio of ifosfamide by decreasing ifosfamide-induced nephrotoxicity without interfering with its antitumor effect in cancer cells clinically treated with ifosfamide.