Comparative analyses of complex formation and binding sites between human tumor necrosis factor-alpha and its three antagonists elucidate their different neutralizing mechanisms

Tumor necrosis factor-alpha (TNFα)-blocking therapy, using biologic TNFα antagonists, has been approved for the treatment of several diseases including rheumatoid arthritis, psoriasis and Crohn's disease. There have been few detailed studies of binding characterizations for the complex formation by TNFα and clinically relevant antagonists, particularly Infliximab (Remicade®) and Etanercept (Enbrel®). Here we characterized the binding stoichiometry and size of soluble TNFα–antagonist complexes and identified energetically important binding sites on TNFα for the three antagonists, Etanercept, Infliximab, and the recently developed humanized TNFα neutralizing monoclonal antibody, YHB1411-2. Size-exclusion chromatography and dynamic light scattering analyses revealed that the three antagonists formed distinct thermodynamically stable TNFα–antagonist complexes that exhibited differences in their size and composition. Energetically important binding residues on TNFα were identified for each antagonist by a sequence of experiments that consisted of competition binding assays, fragmentations, loop mutations, and single-point mutations using yeast surface-displayed TNFα, which was further confirmed for solubly purified TNFα mutants by surface plasmon resonance technique. Analyses of the binding geometry based on binding site location, spatial constraints, and valency satisfaction allowed us to interpret the thermodynamically stable complexes as follows: one molecule of Etanercept and one molecule of trimeric TNFα (Etanercept₁––TNFα₁), Infliximab₆–TNFα₃, and YHB1411–2₄-TNFα₂. The distinct features of the soluble antagonist–TNFα complex formation among the antagonists may give further insights into their different neutralizing mechanisms and pharmacokinetic profiles.     

Morphological and Molecular Characterization of Fusarium Isolated From Maize in Syria

Maize is the third most important cereal after wheat and barley in Syria. Maize plants are attacked by several Fusarium species causing mainly stalk and ear rot of maize which poses a major impact worldwide. Identification of Fusarium species is important for disease control and for assessment of exposure risk to mycotoxines. To identify Fusarium species attacking maize in Syria, a total of 32 Fusarium isolates were recovered from maize ears collected from four different geographical regions, mainly from Ghouta surrounding Damascus. Fusarium isolates were identified based on morphology and on partial DNA sequencing of the TEF1‐α and rDNA/ITS genes. The majority (26 of 32) of these isolates was identified as F. verticillioides (subdivided into four groups), whereas three isolates turned out to be F. thapsinum, F. equiseti and F. andiyazi. The remaining three isolates were close to F. andiyazi, although further investigation is needed to confirm whether they represent a yet undescribed species. Furthermore, our results showed that sequencing the TEF1‐α gene is much more informative than sequencing of the rDNA/ITS region for Fusarium identification at the species level. PCR analysis showed that only F. verticillioides isolates were potentially fumonisin producers and that only the F. equiseti isolate was potentially trichotecene producer. This is the first report on Fusarium thapsinum, F. equiseti and F. andiyazi attacking maize in Syria.     

Roads to isolation: Similar genomic history patterns in two species of freshwater crabs with contrasting environmental tolerances and range sizes

Freshwater species often show high levels of endemism and risk of extinction owing to their limited dispersal abilities. This is exemplified by the stenotopic freshwater crab, Johora singaporensis which is one of the world's 100 most threatened species, and currently inhabits less than 0.01 km² of five low order hill streams within the highly urbanized island city‐state of Singapore. We compared populations of J. singaporensis with that of the non‐threatened, widespread, abundant, and eurytopic freshwater crab, Parathelphusa maculata, and found surprisingly high congruence between their population genomic histories. Based on 2,617 and 2,470 genome‐wide SNPs mined via the double‐digest restriction‐associated DNA sequencing method for ~90 individuals of J. singaporensis and P. maculata, respectively, the populations are strongly isolated (F_ST = 0.146–0.371), have low genetic diversity for both species (also for COI), and show signatures of recent genetic bottlenecks. The most genetically isolated populations for both species are separated from other populations by one of the oldest roads in Singapore. These results suggest that anthropogenic developments may have impacted stream‐dependent species in a uniform manner, regardless of ubiquity, habitat preference, or dispersal modes of the species. While signs of inbreeding were not detected for the critically endangered species, the genetic distinctiveness and low diversity of the populations call for genetic rescue and connecting corridors between the remaining fragments of the natural habitat.     

Molecular Identification,Mycotoxin Production and Comparative Pathogenicity of Fusarium temperatum Isolated from Maize in China

A recently isolated Fusarium population from maize in Belgium was identified as a new species, Fusarium temperatum. From a survey of Fusarium species associated with maize ear rot in nineteen provinces in 2009 in China, ten strains isolated from Guizhou and Hubei provinces were identified as F. temperatum. Morphological and molecular phylogenetic analyses based on the DNA sequences of individual translation elongation factor 1‐alpha and β‐tubulin genes revealed that the recovered isolates produced macroconidia typical of four‐septate with a foot‐shaped basal cell and belonged to F. temperatum that is distinctly different from its most closely related species F. subglutinans and others within Gibberella fujikuroi complex species from maize. All the strains from this newly isolated species were able to infect maize and wheat in field, with higher pathogenicity on maize. Mycotoxin determination of maize grains infected by the strains under natural field condition by ultra‐high‐performance liquid chromatography–tandem mass spectrometry and gas chromatography–mass spectrometry analyses showed that among fifteen mycotoxins assayed, two mycotoxins fumonisin B₁ and B₂ ranging from 9.26 to 166.89 μg/g were detected, with massively more FB₂ mycotoxin (2.8‐ to 108.8‐fold) than FB₁. This mycotoxin production profile is different from that of the Belgian population in which only fumonisin B₁ was barely detected in one of eleven strains assayed. Comparative analyses of the F. temperatum and F. subglutinans strains showed that the highest fumonisin producers were present among the F. temperatum population, which were also the most pathogenic to maize. These results suggested a need for proper monitoring and controlling this species in the relevant maize‐growing regions.     

Population genetic analyses provide insights on the introduction pathway and spread patterns of the North American forest pathogen Heterobasidion irregulare in Italy

A population genetics approach is used to identify the most likely introduction site and introduction pathway for the North American forest pathogen Heterobasidion irregulare using 101 isolates from six sites in Italy and 34 isolates from five sites in North America. Diversity indices based on sequences from ten loci indicate the highest diversity in Italy is found in Castelfusano/Castelporziano and that diversity progressively decreases with increasing distance from that site. AMOVA, Bayesian clustering and principal coordinates analyses based on 12 SSR loci indicate high levels of gene flow among sites, high frequency of admixing, and fail to identify groups of genotypes exclusive to single locations. Cumulatively, these analyses suggest the current infestation is the result of multiple genotypes expanding their range from a single site. Based on two sequenced loci, a single source site in North America could provide enough variability to explain the variability observed in Italy. These results support the notion that H. irregulare was introduced originally in Castelporziano: because Castelporziano has been sealed off from the rest of the world for centuries except for a camp set up by the US military in 1944, we conclude the fungus may have been transported in infected wood used by the military. Finally, spatial autocorrelation analyses using SSR data indicate a significant under‐dispersion of alleles up to 0.5–10 km, while a significant overdispersion of alleles was detected at distances over 80 km: these ranges can be used to make predictions on the likely dispersal potential of the invasive pathogen.     

Foliar δ13C response patterns along a moisture gradient arising from genetic variation and phenotypic plasticity in grassland species of Inner Mongolia

Plants depend upon both genetic differences and phenotypic plasticity to cope with environmental variation over different timescales. The spatial variation in foliar δ¹³C levels along a moisture gradient represents an overlay of genetic and plastic responses. We hypothesized that such a spatial variation would be more obvious than the variation arising purely from a plastic response to moisture change. Leymus chinensis and Stipa spp. were sampled from Inner Mongolia along a dry‐wet transect, and some of these species were transplanted to an area with a moisture gradient. For Stipa spp., the slope of foliar δ¹³C and mean annual precipitation along the transect was significantly steeper than that of foliar δ¹³C and mean annual precipitation after the watering treatment. For L. chinensis, there was a general decreasing trend in foliar δ¹³C under the different (increasing) watering levels; however, its populations showed an irregular relationship between foliar δ¹³C and moisture origin. Therefore, support for our hypothesis was obtained from Stipa spp., but not from L. chinensis.     

Phylogeography of var gene repertoires reveals fine‐scale geospatial clustering of Plasmodium falciparum populations in a highly endemic area

Plasmodium falciparum malaria is a major global health problem that is being targeted for progressive elimination. Knowledge of local disease transmission patterns in endemic countries is critical to these elimination efforts. To investigate fine‐scale patterns of malaria transmission, we have compared repertoires of rapidly evolving var genes in a highly endemic area. A total of 3680 high‐quality DBLα‐sequences were obtained from 68 P. falciparum isolates from ten villages spread over two distinct catchment areas on the north coast of Papua New Guinea (PNG). Modelling of the extent of var gene diversity in the two parasite populations predicts more than twice as many var gene alleles circulating within each catchment (Mugil = 906; Wosera = 1094) than previously recognized in PNG (Amele = 369). In addition, there were limited levels of var gene sharing between populations, consistent with local parasite population structure. Phylogeographic analyses demonstrate that while neutrally evolving microsatellite markers identified population structure only at the catchment level, var gene repertoires reveal further fine‐scale geospatial clustering of parasite isolates. The clustering of parasite isolates by village in Mugil, but not in Wosera was consistent with the physical and cultural isolation of the human populations in the two catchments. The study highlights the microheterogeneity of P. falciparum transmission in highly endemic areas and demonstrates the potential of var genes as markers of local patterns of parasite population structure.     

Genetic drift or natural selection? Hybridization and asymmetric mitochondrial introgression in two Caribbean lizards (Anolis pulchellus and Anolis krugi)

Hybridization and gene introgression can occur frequently between closely related taxa, but appear to be rare phenomena among members of the species‐rich West Indian radiation of Anolis lizards. We investigated the pattern and possible mechanism of introgression between two sister species from Puerto Rico, Anolis pulchellus and Anolis krugi, using mitochondrial (ND2) and nuclear (DNAH3, NKTR) DNA sequences. Our findings demonstrated extensive introgression of A. krugi mtDNA (k‐mtDNA) into the genome of A. pulchellus in western Puerto Rico, to the extent that k‐mtDNA has mostly or completely replaced the native mtDNA of A. pulchellus on this part of the island. We proposed two not mutually exclusive scenarios to account for the interspecific matings between A. pulchellus and A. krugi. We inferred that hybridization events occurred independently in several populations, and determined that k‐mtDNA haplotypes harboured in individuals of A. pulchellus can be assigned to four of the five major mtDNA clades of A. krugi. Further, the spatial distribution of k‐mtDNA clades in the two species is largely congruent. Based on this evidence, we concluded that natural selection was the probable driving mechanism for the extensive k‐mtDNA introgression into A. pulchellus. Our two nuclear data sets yielded different results. DNAH3 showed reciprocal monophyly of A. pulchellus and A. krugi, indicating no effect of hybridization on this marker. In contrast, the two species shared nine NKTR alleles, probably due to incomplete lineage sorting. Our study system will provide an excellent opportunity to experimentally assess the behavioural and ecological mechanisms that can lead to hybridization in closely related taxa.     

Gender specific patterns of carbon uptake and water use in a dominant riparian tree species exposed to a warming climate

Air temperatures in the arid western United States are predicted to increase over the next century. These increases will likely impact the distribution of plant species, particularly dioecious species that show a spatial segregation of the sexes across broad resource gradients. On the basis of spatial segregation patterns, we hypothesized that temperature increases will have a greater negative impact on female plants compared with co‐occurring male plants of dioecious species. This hypothesis was tested by examining the whole‐plant carbon and water relations of 10‐year‐old female (n = 18) and male (n = 13) Acer negundo Sarg. trees grown in a common garden in Salt Lake City, UT. The trees were established from cuttings collected where the growing season temperature averaged about 6.5 °C cooler than at the common garden. During May and June, stem sap flux (J_s) was similar between genders, but averaged 25% higher in males during the warmer months of July and August. Daytime canopy stomatal conductance (g_s) per unit leaf area was 12% higher in females in May : June, but was 11% higher in males in July : August. We combined measurements of sap flux–scaled transpiration with measurements of tree allometry and δ¹³C of leaf soluble sugars to estimate whole‐tree carbon assimilation (A_tree) and water use efficiency (WUE) (A_tree : E_tree). A_tree was similar between genders until late August when A_tree was 32% higher in male trees. A_tree : E_tree was on average 7% higher in females than in males during the growing season. Patterns of J_s, g_s, A_tree and A_tree : E_tree in the present study were in contrast to those previously reported for A. negundo genders under native growing season temperatures. Results suggest that the spatial segregation of the sexes could shift under global warming such that female plants lose their dominance in high‐resource habitats, and males increase their dominance in relatively lower‐resource habitats.     

Phylogeography and molecular phylogeny of Macaronesian island Tarphius (Coleoptera: Zopheridae): why are there so few species in the Azores?

Aim We used a phylogenetic framework to examine island colonization and predictions pertaining to differentiation within Macaronesian Tarphius (Insecta, Coleoptera, Zopheridae), and explain the paucity of endemics in the Azores compared with other Macaronesian archipelagos. Specifically, we test whether low diversity in the Azores could be due to recent colonization (phylogenetic lineage youth), cryptic speciation (distinct phylogenetic entities within species) or the young geological age of the archipelago. Location Macaronesian archipelagos (Azores, Madeira and the Canary Islands), northern Portugal and Morocco. Methods Phylogenetic analyses of mitochondrial and nuclear genes of Tarphius beetles of the Azores, other Macaronesian islands and neighbouring continental areas were used to investigate the origin of island biodiversity and to compare patterns of colonization and differentiation. A comparative nucleotide substitution rate test was used to select the appropriate substitution rate to infer clade divergence times. Results Madeiran and Canarian Tarphius species were found to be more closely related to each other, while Azorean taxa grouped separately. Azorean taxa showed concordance between species and phylogenetic clades, except for species that occur on multiple islands, which segregated by island of origin. Divergence time estimates revealed that Azorean Tarphius are an old group and that the most recent intra‐island speciation event on Santa Maria, the oldest island, occurred between 3.7 and 6.1 Ma. Main conclusions Our phylogenetic approach provides new evidence to understand the impoverishment of Azorean endemics: (1) Tarphius have had a long evolutionary history within the Azores, which does not support the hypothesis of fewer radiation events due to recent colonization; (2) the current taxonomy of Azorean Tarphius does not reflect common ancestry and cryptic speciation is responsible for the underestimation of endemics; (3) intra‐island differentiation in the Azores was found only in the oldest island, supporting the idea that young geological age of the archipelago limits the number of endemics; and (4) the lack of evidence for recent intra‐island diversification in Santa Maria could also explain the paucity of Azorean endemics. Phylogenetic reconstructions of other species‐rich taxa that occur on multiple Macaronesian archipelagos will reveal whether our conclusions are taxon specific, or of a more general nature.     

Complete mitochondrial genome sequences of three bats species and whole genome mitochondrial analyses reveal patterns of codon bias and lend support to a basal split in Chiroptera

Order Chiroptera is a unique group of mammals whose members have attained self-powered flight as their main mode of locomotion. Much speculation persists regarding bat evolution; however, lack of sufficient molecular data hampers evolutionary and conservation studies. Of ~ 1200 species, complete mitochondrial genome sequences are available for only eleven. Additional sequences should be generated if we are to resolve many questions concerning these fascinating mammals. Herein, we describe the complete mitochondrial genomes of three bats: Corynorhinus rafinesquii, Lasiurus borealis and Artibeus lituratus. We also compare the currently available mitochondrial genomes and analyze codon usage in Chiroptera. C. rafinesquii, L. borealis and A. lituratus mitochondrial genomes are 16438 bp, 17048 bp and 16709 bp, respectively. Genome organization and gene arrangements are similar to other bats. Phylogenetic analyses using complete mitochondrial genome sequences support previously established phylogenetic relationships and suggest utility in future studies focusing on the evolutionary aspects of these species. Comprehensive analyses of available bat mitochondrial genomes reveal distinct nucleotide patterns and synonymous codon preferences corresponding to different chiropteran families. These patterns suggest that mutational and selection forces are acting to different extents within Chiroptera and shape their mitochondrial genomes.