NopB, a soybean cultivar-specificity protein from Sinorhizobium fredii USDA257, is a type III secreted protein

The type III secretion system (TTSS) of plant- and animal-pathogenic bacteria is involved in translocation of virulence factors into the host cell cytosol where they modulate cellular processes. Sinorhizobium fredii USDA257 is a gram-negative soil bacterium that forms nitrogen-fixing nodules on specific soybean cultivars (Glycine max (L.) Merr.). This microsymbiont is known to secrete at least five nodulation outer proteins (Nops) in response to flavonoid induction. Some of these Nops have been shown to be secreted by TTSS in this symbiotic bacterium. We have isolated and purified an 18-kDa extracellular protein from flavonoid-induced cultures of USDA257. The N-terminal amino acid sequence of this purified protein was identical to the published sequence of the soybean cultivar-specificity protein, NopB (formerly NoIB). Inactivation of rhcN, which encodes an ATPase, abolished secretion of NopB. Similarly, a nonpolar nopB deletion mutant was compromised in its ability to secrete several Nops. A construct containing the coding region of nopB under control of a T7 promoter was expressed successfully in Escherichia coli and, subsequently, the recombinant NopB was purified by nickel-affinity column chromatography. Polyclonal antibodies raised against purified recombinant NopB were used in Western blot analysis to demonstrate the association of NopB with pilus-like surface appendages. Deletion analysis indicated that the first 33 N-terminal residues of NopB were necessary and sufficient to mediate the secretion of a green fluorescent protein reporter. Introduction of plasmid-borne extra copies of nopB into USDA257 resulted in lower accumulation of native NopB. We also show that USDA257 and its nonpolar nopB deletion mutant exhibited discernible differences in their ability to nodulate legume hosts.     

A cloud-based enhanced differential evolution algorithm for parameter estimation problems in computational systems biology

Metaheuristics are gaining increasing recognition in many research areas, computational systems biology among them. Recent advances in metaheuristics can be helpful in locating the vicinity of the global solution in reasonable computation times, with Differential Evolution (DE) being one of the most popular methods. However, for most realistic applications, DE still requires excessive computation times. With the advent of Cloud Computing effortless access to large number of distributed resources has become more feasible, and new distributed frameworks, like Spark, have been developed to deal with large scale computations on commodity clusters and cloud resources. In this paper we propose a parallel implementation of an enhanced DE using Spark. The proposal drastically reduces the execution time, by means of including a selected local search and exploiting the available distributed resources. The performance of the proposal has been thoroughly assessed using challenging parameter estimation problems from the domain of computational systems biology. Two different platforms have been used for the evaluation, a local cluster and the Microsoft Azure public cloud. Additionally, it has been also compared with other parallel approaches, another cloud-based solution (a MapReduce implementation) and a traditional HPC solution (a MPI implementation)     

Timing of the evolutionary history of Corallinaceae (Corallinales,Rhodophyta)

The temporal dimension of the most recent Corallinaceae (order Corallinales) phylogeny was presented here, based on first occurrence time estimates from the fossil record. Calibration of the molecular clock of the genetic marker SSU entailed a separation of Corallinales from Hapalidiales in the Albian (Early Cretaceous ~105 mya). Neither the calibration nor the fossil record resolved the succession of appearance of the first three emerging subfamilies: Mastophoroideae, Corallinoideae, and Neogoniolithoideae. The development of the tetra/bisporangial conceptacle roofs by filaments surrounding and interspersed among the sporangial initials was an evolutionary novelty emerging at the Cretaceous–Paleogene boundary (~66 mya). This novelty was shared by the subfamilies Hydrolithoideae, Metagoniolithoideae, and Lithophylloideae, which diverged in the early Paleogene. Subclades within the Metagoniolithoideae and Lithophylloideae diversified in the late Oligocene–middle Miocene (~28–12 mya). The most common reef corallinaceans (Hydrolithon, Porolithon, Harveylithon, “Pneophyllum” conicum, and subclades within Lithophylloideae) appeared in this interval in the Indo‐Australian Archipelago.     

Inhibition of basal and calmodulin-activated Ca2+-pump ATPase by fractionated compound 48/80

Compound 48/80 (48/80), a mixture of polycationic compounds was fractionated using affinity chromatography on calmodulin-Sepharose. Unfractionated 48/80 and various fractions were tested for their potential inhibitory effects on ATPase activities of isolated human red blood cell membranes. ATPase activities tested included: Mg2+-ATPase, the Na+/K+-pump ATPase, and the Ca2+-pump ATPase in both its basal (calmodulin-independent) and calmodulin-activated state. Neither 48/80 nor its various fractions were very potent or efficacious inhibitors of the Mg2+-ATPase or the Na+/K+-pump ATPase. In agreement with previous reports, 48/80 was found to be an inhibitor of the calmodulin-activated Ca2+-pump ATPase. By contrast, we found that unfractionated, as well as some fractionated, material inhibited both the basal (calmodulin-independent) and calmodulin-activated Ca2+-pump ATPase activity. A fraction designated as Fraction III bound to calmodulin-Sepharose in the presence of Ca2+ and low salt and was eluted in the absence of Ca2+ and 0.15 M NaCl. By gel filtration, Fraction III had an apparent average molecular weight of 2064 (1320 for unfractionated material). Fraction III was the most potent inhibitor of the Ca2+-pump ATPase with IC50 values for the basal and calmodulin-activated forms of the enzyme of 0.6 and 1.2 micrograms/ml, respectively. Inhibition by Fraction III was cooperative with n apparent values of 2.4 and 5.7, respectively, for the basal and calmodulin-activated forms of the enzyme. Thus, binding of 48/80 constituents to calmodulin can not fully account for the observed data. Direct interaction of 48/80 constituent(s) with the enzyme and/or the lipid portion of the membrane is suggested.     

Babesia bigemina sexual stages are induced in vitro and are specifically recognized by antibodies in the midgut of infected Boophilus microplus ticks

Babesia bigemina, a causative agent of bovine babesiosis, is transmitted from one bovine to another only by infected ticks. The life cycle of B. bigemina includes a sexual phase in the tick host; however, molecules from sexual stages of any Babesia species have not been characterized. This is the first report of the induction of sexual stages of any Babesia species in vitro, free of tick antigens. Intraerythrocytic parasites were cultured in vitro for 20h using an induction medium. Extraerythrocytic parasites were first seen 3h post induction; elongated stages with long projections appeared at 6h post induction and by 9h they paired and fused to form larger stages. Round zygotes appeared 20h post induction. Moreover, by using Percoll gradients, sexual stages were purified free of contaminating intraerythrocytic stages. Purified parasites were used to generate polyclonal antibodies, which specifically bound to antigens expressed in sexual stages induced in vitro, but not to antigens expressed in intraerythrocytic stages. Importantly, these antibodies specifically identified sexual stages from midguts of female Boophilus microplus ticks fed on infected cattle.     

Temperature-dependent development,cold tolerance,and potential distribution of Gratiana boliviana (Coleoptera: Chrysomelidae), a biological control agent of tropical soda apple,Solanum viarum (Solanaceae)

The leaf beetle Gratiana boliviana Spaeth (Coleoptera: Chrysomelidae) was introduced from South America into the southeastern United States in 2003 as a classical biological control agent of tropical soda apple, Solanum viarum Dunal (Solanaceae). Temperature-dependent development and survival studies revealed that development was completed at temperatures >16°C and ≤34°C. The number of degree-days required to complete one generation was 341 and the estimated lower developmental threshold was 13.37°C. Using nonlinear regression, the upper lethal threshold was estimated to be 34–35°C. Cold tolerance studies revealed that the lethal time for 90% of adults (LT₉₀) was 12.6 days at 5°C and 8.68 days at 0°C. Based on the developmental and cold tolerance data, a map predicting the areas of establishment and number of generations per year was generated, which suggests that the northern extent of the G. boliviana range in the USA will be near 32–33° north latitude. Fewer generations per year in more northern areas of the southeastern USA may decrease the effectiveness of this biological control agent.     

Biochemical variation to determine phylogenetic relationships betweenHordeum chilense and other American species of the genusHordeum (Poaceae)

Slab gel electrophoresis techniques have been applied to the study of isozyme and kernel protein patterns in 20 accessions ofHordeum chilense and related species in order to elucidate their phylogenetic relationships. On the basis of our results we can conclude that: (1) Conventional classification based on morphological characters does not totally agree with biochemical data. (2) Sectt.Anisolepis andCritesion seem to be clearly differentiated. (3) The accessions classified asH. compressum present biochemical phenotypes quite different from the rest of the species. (4)H. stenostachys, H. muticum andH. chilense constitute a group of variable species with many biochemical similarities and close phylogenetic relationships. (5) The evolutionary pattern of these American species seems to follow a model of reticulate evolution.     

A new karyotype of an endangered primate species (Callicebus personatus) from the Brazilian Atlantic forests

The genus Callicebus has 28 species arranged in five groups: donacophilus, torquatus, moloch, cupreus and personatus. The personatus group occurs in the Brazilian Atlantic forests where it is isolated from the other Callicebus groups. The present paper is the first report on the karyotype of Callicebuspersonatus. We compared the karyotypes of C. personatus and C. nigrifrons utilizing the following classic banding techniques: G-, C-, NOR banding, G/C- and G/NOR sequential banding. The karyotype of C. personatus has 2n=44, while C. nigrifrons presented 2n=42. Both the species diverge in the diploid number in consequence of a rearrangement type centric fusion/fission. Our results support personatus as a valid group and the specific status for C. personatus and C. nigrifrons.