Molecular and phylogenetic analyses of human Parvovirus B19 isolated from Brazilian patients with sickle cell disease and beta-thalassemia major and healthy blood donors

Human Parvovirus B19 (B19V) is a recognized cause of life-threatening conditions among patients with hemoglobinopathies. This study investigates B19V infection in patients with sickle cell disease and beta-thalassemia using different experimental approaches. A total of 183 individuals (144 with sickle cell disease and 39 with beta-thalassemia major) and 100 healthy blood donors were examined for B19V using anti-B19V IgG enzyme immunoassay, quantitative PCR, DNA sequencing, and phylogenetic analysis. Viremia was documented in 18.6% of patients and 1% of donors, and was generally characterized by low viral load (VL); however, acute infections were also observed. Anti-B19V IgG was detected in 65.9% of patients with sickle cell disease and in 60% of donors, whereas the patients with thalassemia exhibited relatively low seroreactivity. The seroprevalence varied among the different age groups. In patients, it progressively increased with age, whereas in donors it reached a plateau. Based on partial NS1 fragments, all isolates detected were classified as subgenotype 1A with a tendency to elicit genetically complex infections. Interestingly, quasispecies occurred in the plasma of not only patients but also donors with even higher heterogeneity. The partial NS1 sequence examined did not exhibit positive selection. Quantitation of B19V with a conservative probe is a technically and practically useful approach. The extensive spread of B19V subgenotype 1A in patients and donors and its recent introduction into the countryside of the Sao Paulo State, Brazil were demonstrated; however, it is difficult to establish a relationship between viral sequences and the clinical outcomes of the infection. J. Med. Virol. 84:16521665, 2012. (c) 2012 Wiley Periodicals, Inc.

Fumarate hydratase isoforms of Leishmania major: Subcellular localization, structural and kinetic properties

Fumarate hydratases (FHs; EC 4.2.1.2) are enzymes that catalyze the reversible hydration of fumarate to S-malate. Parasitic protists that belong to the genus Leishmania and are responsible for a complex of vector-borne diseases named leishmaniases possess two genes that encode distinct putative FH enzymes. Genome sequence analysis of Leishmania major Friedlin reveals the existence of genes LmjF24.0320 and LmjF29.1960 encoding the putative enzymes LmFH-1 and LmFH-2, respectively. In the present work, the FH activity of both L. major enzymes has been confirmed. Circular dichroism studies suggest important differences in terms of secondary structure content when comparing LmFH isoforms and even larger differences when comparing them to the homologous human enzyme. CD melting experiments revealed that both LmFH isoforms are thermolabile enzymes. The catalytic efficiency under aerobic and anaerobic environments suggests that they are both highly sensitive to oxidation and damaged by oxygen. Intracellular localization studies located LmFH-1 in the mitochondrion, whereas LmFH-2 was found predominantly in the cytosol with possibly also some in glycosomes. The high degree of sequence conservation in different Leishmania species, together with the relevance of FH activity for the energy metabolism in these parasites suggest that FHs might be exploited as targets for broad-spectrum antileishmanial drugs. (c) 2012 Elsevier B.V. All rights reserved.

Using complex networks concepts to assess approaches for citations in scientific papers

The number of citations received by authors in scientific journals has become a major parameter to assess individual researchers and the journals themselves through the impact factor. A fair assessment therefore requires that the criteria for selecting references in a given manuscript should be unbiased with regard to the authors or journals cited. In this paper, we assess approaches for citations considering two recommendations for authors to follow while preparing a manuscript: (i) consider similarity of contents with the topics investigated, lest related work should be reproduced or ignored; (ii) perform a systematic search over the network of citations including seminal or very related papers. We use formalisms of complex networks for two datasets of papers from the arXiv and the Web of Science repositories to show that neither of these two criteria is fulfilled in practice. By representing the texts as complex networks we estimated a similarity index between pieces of texts and found that the list of references did not contain the most similar papers in the dataset. This was quantified by calculating a consistency index, whose maximum value is one if the references in a given paper are the most similar in the dataset. For the areas of "complex networks" and "graphenes", the consistency index was only 0.11-0.23 and 0.10-0.25, respectively. To simulate a systematic search in the citation network, we employed a traditional random walk search (i.e. diffusion) and a random walk whose probabilities of transition are proportional to the number of the ingoing edges of the neighbours. The frequency of visits to the nodes (papers) in the network had a very small correlation with either the actual list of references in the papers or with the number of downloads from the arXiv repository. Therefore, apparently the authors and users of the repository did not follow the criterion related to a systematic search over the network of citations. Based on these results, we propose an approach that we believe is fairer for evaluating and complementing citations of a given author, effectively leading to a virtual scientometry.

Space-time relation of post-collisional granitic magmatism in Santa Catarina, southern Brazil: U-Pb LA-MC-ICP-MS zircon geochronology of coeval mafic-felsic magmatism related to the Major Gercino Shear Zone

LA-MC-ICP-MS U-Pb zircon dating was performed on syntectonic, early post-collisional granitic and associated mafic rocks that are intrusive in the Brusque Metamorphic Complex and in the Florianopolis Batholith, major tectonic domains separated by the Neoproterozoic Major Gercino Shear Zone (MGSZ) in south Brazil. The inferred ages of magmatic crystallization are consistent with field relationships, and show that the syntectonic granites from both domains are similar, with ages around 630-620 Ma for high-K calc-alkaline metaluminous granites and ca. 610 Ma for slightly peraluminous granites. Although ca. 650 Ma inherited zircon components are identified in granites from both domains, important contrasts on the crustal architecture in each domain are revealed by the patterns of zircon inheritance, indicating different crustal sources for the granites in each domain. The granites from the southern domain (Floriandpolis Batholith) have essentially Neoproterozoic (650-700 Ma and 900-950 Ma) inheritance; with a single 2.0-2.2 Ga inherited age obtained in the peraluminous Mariscal Granite. In the northern Brusque Metamorphic Complex, the metaluminous Rio Pequeno Granite and associated mafic rocks have scarce inherited cores with ages around 1.65 Ga, whereas the slightly peraluminous Serra dos Macacos Granite has abundant Paleoproterozoic (1.8-2.2 Ga) and Archean (2.9-3.4 Ga) inherited zircons. Our results are consistent with the hypothesis that the MGSZ separates domains with distinct geologic evolution; however, the contemporaneity of 630-610 Ma granitic magmatism with similar structural and geochemical patterns on both sides of this major shear zone indicates that these domains were already part of a single continental mass at 630 Ma, reinforcing the post-collisional character of these granites. (C) 2012 Elsevier B.V. All rights reserved.

Cryptic Species of the Anastrepha fraterculus Complex (Diptera: Tephritidae): A Multivariate Approach for the Recognition of South American Morphotypes

Although a large amount of data have been published in past years on the taxonomic status of the Anastrepha fraterculus (Wiedemann) species complex, there is still a need to know how many species this complex comprises, the distribution of each one, and their distinguishing features. In this study, we assessed the morphometric variability of 32 populations from the A. fraterculus complex, located in major biogeographical areas from the Neotropics. Multivariate techniques for analysis were applied to the measurements of 21 variables referring to the mesonotum, aculeus, and wing. For the first time, our results identified the presence of seven distinct morphotypes within this species complex. According to the biogeographical areas, populations occurring in the Mesoamerican dominion (Mexico, Guatemala, and Panama) were clustered within a single natural entity labeled as the "Mexican" morphotype; whereas in the northwestern South American dominion, samples fell into three distinct groups: the "Venezuelan" morphotype with a single population from the Caribbean lowlands of Venezuela, the "Andean" morphotype from the highlands of Venezuela and Colombia, and the third group or "Peruvian" morphotype comprised the samples from the Pacific coastal lowlands of Ecuador and Peru. Three additional groups were identified from the Chacoan and Paranaense sub-regions: the morphotype "Brazilian-1" was recognized as including the Argentinean samples with most pertaining to Brazil, and widely distributed in these biogeographical areas; the morphotype "Brazilian-2" was recognized as including two samples from the state of Sao Paulo (Ilha-Bela and Sao Sebastiao); whereas the morphotype "Brazilian-3" included a single population from Botucatu (state of Sao Paulo). Based on data published by previous authors showing genetic and karyotypic differentiation, as well as reproductive isolation, we have concluded that such morphotypes indeed represent natural groups and distinct taxonomic entities.

The Banhado Alkaline Complex, Southeastern Brazil: source and evolution of potassic SiO2-undersaturated high-Ca and low-Ca magmatic series

The Cretaceous Banhado alkaline complex in southeastern Brazil presents two potassic SiO2-undersaturated series. The high-Ca magmatic series consist of initially fractionated olivine (Fo(92-91)) + diopside (Wo(48-43)En(49-35)Ae(0-7)), as evidenced by the presence of xenocrysts and xenoliths. In that sequence, diopside (Wo(47-38)En(46-37)Ae(0-8)) + phlogopite + apatite + perovskite (Prv(> 92)) crystallized to form the phlogopite melteigite and led to the Ca enrichment of the magma. Diopside (Wo(47-41)En(32-24) Ae(3-14)) continued to crystallize as an early mafic mineral, followed by nepheline (Ne(74.8-70.1)Ks(26.3-21.2)Qz(7.6-0.9)) and leucite (Lc(65-56)) and subsequently by melanite and potassic feldspar (Or(85-99)Ab(1-7)) to form melanite ijolites, wollastonite-melanite urtites and melanite-nepheline syenites. Melanite-pseudoleucite-nepheline syenites are interpreted to be a leucite accumulation. Melanite nephelinite dykes are believed to represent some of the magmatic differentiation steps. The low-Ca magmatic series is representative of a typical fractionation of aegirine-augite (Wo(36-29)En(25-4)Ae(39-18)) + alkali feldspar (Or(57-96)Ab(3-43)) + nepheline (Ne(76.5-69.0)Ks(19.9-14.4)Qz(15.1-7.7)) + titanite from phonolite magma. The evolution of this series from potassic nepheline syenites to sodic sodalite syenites and sodalitolites is attributed to an extensive fractionation of potassic feldspar, which led to an increase of the NaCl activity in the melt during the final stages forming sodalite-rich rocks. Phonolite dykes followed a similar evolutionary process and also registered some crustal assimilation. The mesocratic nepheline syenites showed interactions with phlogopite melteigites, such as compatible trace element enrichments and the presence of diopside xenocrysts, which were interpreted to be due to a mixing/mingling process of phonolite and nephelinite magmas. The geochemical data show higher TiO2 and P2O5 contents and lower SiO2 contents for the high-Ca series and different LILE evolution trends and REE chondrite-normalized patterns as compared to the low-Ca series. The Sr-87/Sr-86, Nd-143/Nd-144, Pb-206/Pb-204 and Pb-208/Pb-204 initial ratios for the high-Ca series (0.70407-0.70526, 0.51242-0.51251, 17.782-19.266 and 38.051-39.521, respectively) were slightly different from those of the low-Ca series (0.70542-0.70583, 0.51232-0.51240, 17.758-17.772 and 38.021-38.061, respectively). For both series, a CO2-rich potassic metasomatized lithospheric mantle enriched the source with rutile-bearing phlogopite clinopyroxenite veins. Kamafugite-like parental magma is attributed to the high-Ca series with major contributions from the melting of the veins. Potassic nephelinite-like parental magma is assigned to the low-Ca series, where the metasomatized wall-rock played a more significant role in the melting process.

Crystal structure of dihydroorotate dehydrogenase from Leishmania major

Dihydroorotate dehydrogenase (DHODH) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and has been exploited as the target for therapy against proliferative and parasitic diseases. In this study, we report the crystal structures of DHODH from Leishmania major, the species of Leishmania associated with zoonotic cutaneous leishmaniasis, in its apo form and in complex with orotate and fumarate molecules. Both orotate and fumarate were found to bind to the same active site and exploit similar interactions, consistent with a ping-pong mechanism described for class 1A DHODHs. Analysis of LmDHODH structures reveals that rearrangements in the conformation of the catalytic loop have direct influence on the dimeric interface. This is the first structural evidence of a relationship between the dimeric form and the catalytic mechanism. According to our analysis, the high sequence and structural similarity observed among trypanosomatid DHODH suggest that a single strategy of structure-based inhibitor design can be used to validate DHODH as a druggable target against multiple neglected tropical diseases such as Leishmaniasis, Sleeping sickness and Chagas' diseases. (C) 2012 Elsevier Masson SAS. All rights reserved.

Phylogeography of the neotropical Anopheles triannulatus complex (Diptera: Culicidae) supports deep structure and complex patterns

Abstract Background The molecular phylogenetic relationships and population structure of the species of the Anopheles triannulatus complex: Anopheles triannulatus s.s., Anopheles halophylus and the putative species Anopheles triannulatus C were investigated. Methods The mitochondrial COI gene, the nuclear white gene and rDNA ITS2 of samples that include the known geographic distribution of these taxa were analyzed. Phylogenetic analyses were performed using Bayesian inference, Maximum parsimony and Maximum likelihood approaches. Results Each data set analyzed septely yielded a different topology but none provided evidence for the seption of An. halophylus and An. triannulatus C, consistent with the hypothesis that the two are undergoing incipient speciation. The phylogenetic analyses of the white gene found three main clades, whereas the statistical parsimony network detected only a single metapopulation of Anopheles triannulatus s.l. Seven COI lineages were detected by phylogenetic and network analysis. In contrast, the network, but not the phylogenetic analyses, strongly supported three ITS2 groups. Combined data analyses provided the best resolution of the trees, with two major clades, Amazonian (clade I) and trans-Andean + Amazon Delta (clade II). Clade I consists of multiple subclades: An. halophylus + An. triannulatus C; trans-Andean Venezuela; central Amazonia + central Bolivia; Atlantic coastal lowland; and Amazon delta. Clade II includes three subclades: Panama; cis-Andean Colombia; and cis-Venezuela. The Amazon delta specimens are in both clades, likely indicating local sympatry. Spatial and molecular variance analyses detected nine groups, corroborating some of subclades obtained in the combined data analysis. Conclusion Combination of the three molecular markers provided the best resolution for differentiation within An. triannulatus s.s. and An. halophylus and C. The latest two species seem to be very closely related and the analyses performed were not conclusive regarding species differentiation. Further studies including new molecular markers would be desirable to solve this species status question. Besides, results of the study indicate a trans-Andean origin for An. triannulatus s.l. The potential implications for malaria epidemiology remain to be investigated.

Interspecific variation of the bacterial community structure in the phyllosphere of the three major plant components of mangrove forests

Mangrove forests encompass a group of trees species that inhabit the intertidal zones, where soil is characterized by the high salinity and low availability of oxygen. The phyllosphere of these trees represent the habitat provided on the aboveground parts of plants, supporting in a global scale, a large and complex microbial community. The structure of phyllosphere communities reflects immigration, survival and growth of microbial colonizers, which is influenced by numerous environmental factors in addition to leaf physical and chemical properties. Here, a combination of culture-base methods with PCR-DGGE was applied to test whether local or plant specific factors shape the bacterial community of the phyllosphere from three plant species (Avicenia shaueriana, Laguncularia racemosa and Rhizophora mangle), found in two mangroves. The number of bacteria in the phyllosphere of these plants varied between 3.62 x 10(4) in A. schaeriana and 6.26 x 10³ in R. mangle. The results obtained by PCR-DGGE and isolation approaches were congruent and demonstrated that each plant species harbor specific bacterial communities in their leaves surfaces. Moreover, the ordination of environmental factors (mangrove and plant species), by redundancy analysis (RDA), also indicated that the selection exerted by plant species is higher than mangrove location on bacterial communities at phyllosphere.

Thermochronologic and geodynamic evolution of the Pontides: Sakarya terrane (Karakaya Complex) and Istanbul terrane, Turkey.

An integrated array of analytical methods -including clay mineralogy, vitrinite reflectance, Raman spectroscopy on carbonaceous material, and apatite fission-track analysis- was employed to constrain the thermal and thermochronological evolution of selected portions of the Pontides of northern Turkey. (1) A multimethod investigation was applied for the first time to characterise the thermal history of the Karakaya Complex, a Permo-Triassic subduction-accretion complex cropping out throughout the Sakarya Zone. The results indicate two different thermal regimes: the Lower Karakaya Complex (Nilüfer Unit) -mostly made of metabasite and marble- suffered peak temperatures of 300-500°C (greenschist facies); the Upper Karakaya Complex (Hodul and the Orhanlar Units) –mostly made of greywacke and arkose- yielded heterogeneous peak temperatures (125-376°C), possibly the result of different degree of involvement of the units in the complex dynamic processes of the accretionary wedge. Contrary to common belief, the results of this study indicate that the entire Karakaya Complex suffered metamorphic conditions. Moreover, a good degree of correlation among the results of these methods demonstrate that Raman spectroscopy on carbonaceous material can be applied successfully to temperature ranges of 200-330°C, thus extending the application of this method from higher grade metamorphic contexts to lower grade metamorphic conditions. (2) Apatite fission-track analysis was applied to the Sakarya and the İstanbul Zones in order to constrain the exhumation history and timing of amalgamation of these two exotic terranes. AFT ages from the İstanbul and Sakarya terranes recorded three distinct episodes of exhumation related to the complex tectonic evolution of the Pontides. (i) Paleocene - early Eocene ages (62.3-50.3 Ma) reflect the closure of the İzmir-Ankara ocean and the ensuing collision between the Sakarya terrane and the Anatolide-Tauride Block. (ii) Late Eocene - earliest Oligocene (43.5-32.3 Ma) ages reflect renewed tectonic activity along the İzmir-Ankara. (iii) Late Oligocene- Early Miocene ages reflect the onset and development of the northern Aegean extension. The consistency of AFT ages, both north and south of the tectonic contact between the İstanbul and Sakarya terranes, suggest that such terranes were amalgamated in pre-Cenozoic times. (3) Fission-track analysis was also applied to rock samples from the Marmara region, in an attempt to constrain the inception and development of the North Anatolian Fault system in the region. The results agree with those from the central Pontides. The youngest AFT ages (Late Oligocene - early Miocene) were recorded in the western portion of the Marmara Sea region and reflect the onset and development of northern Aegean extension. Fission-track data from the eastern Marmara Sea region indicate rapid Early Eocene exhumation induced by the development of the İzmir-Ankara orogenic wedge. Thermochronological data along the trace of the Ganos Fault –a segment of the North Anatolian Fault system- indicate the presence of a tectonic discontinuity active by Late Oligocene time, i.e. well before the arrival of the North Anatolian Fault system in the area. The integration of thermochronologic data with preexisting structural data point to the existence of a system of major E-W-trending structural discontinuities active at least from the Late Oligocene. In the Early Pliocene, inception of the present-day North Anatolian Fault system in the Marmara region occurred by reactivation of these older tectonic structures. 


EPR spectroscopic investigation of membrane protein structure and folding on light harvesting complex LHCIIb

Structure and folding of membrane proteins are important issues in molecular and cell biology. In this work new approaches are developed to characterize the structure of folded, unfolded and partially folded membrane proteins. These approaches combine site-directed spin labeling and pulse EPR techniques. The major plant light harvesting complex LHCIIb was used as a model system. Measurements of longitudinal and transversal relaxation times of electron spins and of hyperfine couplings to neighboring nuclei by electron spin echo envelope modulation(ESEEM) provide complementary information about the local environment of a single spin label. By double electron electron resonance (DEER) distances in the nanometer range between two spin labels can be determined. The results are analyzed in terms of relative water accessibilities of different sites in LHCIIb and its geometry. They reveal conformational changes as a function of micelle composition. This arsenal of methods is used to study protein folding during the LHCIIb self assembly and a spatially and temporally resolved folding model is proposed. The approaches developed here are potentially applicable for studying structure and folding of any protein or other self-assembling structure if site-directed spin labeling is feasible and the time scale of folding is accessible to freeze-quench techniques.

Mitochondrial respiratory supercomplex association limits production of reactive oxygen species from Complex I

Evidence accumulated in the last ten years has demonstrated that a large proportion of the mitochondrial respiratory chain complexes in a variety of organisms is arranged in supramolecular assemblies called supercomplexes or respirasomes. Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. Following this line of thought we have decided to directly investigate ROS production by Complex I under conditions in which the complex is arranged as a component of the supercomplex I1III2 or it is dissociated as an individual enzyme. The study has been addressed both in bovine heart mitochondrial membranes and in reconstituted proteoliposomes composed of complexes I and III in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. The results of this investigation provide experimental evidence that the production of ROS is strongly increased in either model; supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I . This is the first demonstration that dissociation of the supercomplex I1III2 in the mitochondrial membrane is a cause of oxidative stress from Complex I. Previous work in our laboratory demonstrated that lipid peroxidation can dissociate the supramolecular assemblies; thus, here we confirm that preliminary conclusion that primary causes of oxidative stress may perpetuate reactive oxygen species (ROS) generation by a vicious circle involving supercomplex dissociation as a major determinant.

Engineering Complex Computational Ecosystems

Self-organising pervasive ecosystems of devices are set to become a major vehicle for delivering infrastructure and end-user services. The inherent complexity of such systems poses new challenges to those who want to dominate it by applying the principles of engineering. The recent growth in number and distribution of devices with decent computational and communicational abilities, that suddenly accelerated with the massive diffusion of smartphones and tablets, is delivering a world with a much higher density of devices in space. Also, communication technologies seem to be focussing on short-range device-to-device (P2P) interactions, with technologies such as Bluetooth and Near-Field Communication gaining greater adoption. Locality and situatedness become key to providing the best possible experience to users, and the classic model of a centralised, enormously powerful server gathering and processing data becomes less and less efficient with device density. Accomplishing complex global tasks without a centralised controller responsible of aggregating data, however, is a challenging task. In particular, there is a local-to-global issue that makes the application of engineering principles challenging at least: designing device-local programs that, through interaction, guarantee a certain global service level. In this thesis, we first analyse the state of the art in coordination systems, then motivate the work by describing the main issues of pre-existing tools and practices and identifying the improvements that would benefit the design of such complex software ecosystems. The contribution can be divided in three main branches. First, we introduce a novel simulation toolchain for pervasive ecosystems, designed for allowing good expressiveness still retaining high performance. Second, we leverage existing coordination models and patterns in order to create new spatial structures. Third, we introduce a novel language, based on the existing ``Field Calculus'' and integrated with the aforementioned toolchain, designed to be usable for practical aggregate programming.

BPAG1 isoform-b: Complex distribution pattern in striated and heart muscle and association with plectin and α-actinin

BPAG1-b is the major muscle-specific isoform encoded by the dystonin gene, which expresses various protein isoforms belonging to the plakin protein family with complex, tissue-specific expression profiles. Recent observations in mice with either engineered or spontaneous mutations in the dystonin gene indicate that BPAG1-b serves as a cytolinker important for the establishment and maintenance of the cytoarchitecture and integrity of striated muscle. Here, we studied in detail its distribution in skeletal and cardiac muscles and assessed potential binding partners. BPAG1-b was detectable in vitro and in vivo as a high molecular mass protein in striated and heart muscle cells, co-localizing with the sarcomeric Z-disc protein alpha-actinin-2 and partially with the cytolinker plectin as well as with the intermediate filament protein desmin. Ultrastructurally, like alpha-actinin-2, BPAG1-b was predominantly localized at the Z-discs, adjacent to desmin-containing structures. BPAG1-b was able to form complexes with both plectin and alpha-actinin-2, and its NH(2)-terminus, which contains an actin-binding domain, directly interacted with that of plectin and alpha-actinin. Moreover, the protein level of BPAG1-b was reduced in muscle tissues from plectin-null mutant mice versus wild-type mice. These studies provide new insights into the role of BPAG1-b in the cytoskeletal organization of striated muscle.

Crystal structure of a CAP-DNA complex: the DNA is bent by 90 degrees

The 3 angstrom resolution crystal structure of the Escherichia coli catabolite gene activator protein (CAP) complexed with a 30-base pair DNA sequence shows that the DNA is bent by 900. This bend results almost entirely from two 400 kinks that occur between TG/CA base pairs at positions 5 and 6 on each side of the dyad axis of the complex. DNA sequence discrimination by CAP derives both from sequence-dependent distortion of the DNA helix and from direct hydrogen-bonding interactions between three protein side chains and the exposed edges of three base pairs in the major groove of the DNA. The structure of this transcription factor-DNA complex provides insights into possible mechanisms of transcription activation