Structure-function analyses of {\it PAX6\/} and the molecular bases of aniridia

PAX6 is a transcription activator that regulates eye development in animals ranging from Drosophila to human. The C-terminal region of PAX6 is proline/serine/threonine-rich (PST) and functions as a potent transactivation domain when attached to a heterologous DNA-binding domain of the yeast transcription factor, GAL4. The PST region comprises 152 amino acids encoded by four exons. The transactivation function of the PST region has not been defined and characterized in detail by in vitro mutagenesis. I dissected the PST domain in two independent systems, a heterologous system using a GAL4 DNA-binding site and the native system of PAX6. In both systems, the results show consistently that all four constituent exons of the PST domain are responsible for the transactivation function. The four exon fragments act cooperatively to stimulate transcription, although none of them can function individually as an independent transactivation domain. Combinations of two or more exon fragments can reconstitute substantial transactivation activity when fused to the DNA-binding domain of GAL4, but they surprisingly do not produce much activity in the context of native PAX6 even though the mutant PAX6 proteins are stable and their DNA-binding function remains unaffected. I conclude that the PAX6 protein contains an unusually large transactivation domain that is evolutionarily conserved to a high degree, and that its full transactivation activity relies on the cooperative action of the four exon fragments.^ Most PAX6 mutations detected in patients with aniridia result in truncations of the protein. Some of the truncation mutations occur in the PST region of PAX6, resulting in mutant proteins that retain their DNA-binding ability but have no significant transactivation activity. It is not clear whether such mutants are true loss-of-function or dominant-negative mutants. I show that these mutants are dominant-negative if they are coexpressed with wild-type PAX6 in cultured cells and that the dominant-negative effects result from enhanced DNA-binding ability of these mutants due to removal of the PST domain. These mutants are able to repress the wild-type PAX6 activity not only at target genes with paired domain binding sites but also at target genes with homeodomain binding sites.^ Mutations in the human PAX6 gene produce various phenotypes, including aniridia, Peters' anomaly, autosomal dominant keratitis, and familial foveal dysplasia. The various phenotypes may arise from different mutations in the same gene. To test this theory, I performed a functional analysis of two missense mutations in the paired domain: the R26G mutation reported in a case of Peters' anomaly, and the I87R mutation identified in a patient with aniridia. While both the R26 and the I87 positions are conserved in the paired boxes of all known PAX genes, X-ray crystallography has shown that only R26 makes contact with DNA. I found that the R26G mutant failed to bind a subset of paired domain binding sites but, surprisingly, bound other sites and successfully transactivated promoters containing those sites. In contrast, the I87R mutant had lost the ability to bind DNA at all tested sites and failed to transactivate promoters. My data support the haploinsufficiency hypothesis of aniridia, and the hypothesis that R26G is a hypomorphic allele. ^

Hyalotekite, (Ba,Pb,K)(4)(Ca,Y)(2)Si-8(B,Be)(2)(Si,B)(2)O28F, a Tectosilicate Related to Scapolite: New Structure Refinement, Phase Transitions and a Short-Range Ordered 3B Superstructure

Hyalotekite, a framework silicate of composition (Ba,Pb,K)(4)(Ca,Y)(2)Si-8(B,Be)(2) (Si,B)(2)O28F, is found in relatively high-temperature(greater than or equal to 500 degrees C) Mn skarns at Langban, Sweden, and peralkaline pegmatites at Dara-i-Pioz, Tajikistan. A new paragenesis at Dara-i-Pioz is pegmatite consisting of the Ba borosilicates leucosphenite and tienshanite, as well as caesium kupletskite, aegirine, pyrochlore, microcline and quartz. Hyalotekite has been partially replaced by barylite and danburite. This hyalotekite contains 1.29-1.78 wt.% Y2O3, equivalent to 0.172-0.238 Y pfu or 8-11% Y on the Ca site; its Pb/(Pb+Ba) ratio ranges 0.36-0.44. Electron microprobe F contents of Langban and Dara-i-Pioz hyalotekite range 1.04-1.45 wt.%, consistent with full occupancy of the F site. A new refinement of the structure factor data used in the original structural determination of a Langban hyalotekite resulted in a structural formula, (Pb1.96Ba1.86K0.18)Ca-2(B1.76Be0.24)(Si1.56B0.44)Si8O28F, consistent with chemical data and all cations with positive-definite thermal parameters, although with a slight excess of positive charge (+57.14 as opposed to the ideal +57.00). An unusual feature of the hyalotekite framework is that 4 of 28 oxygens are non-bridging; by merging these 4 oxygens into two, the framework topology of scapolite is obtained. The triclinic symmetry of hyalotekite observed at room temperature is obtained from a hypothetical tetragonal parent structure via a sequence of displacive phase transitions. Some of these transitions are associated with cation ordering, either Pb-Ba ordering in the large cation sites, or B-Be and Si-B ordering on tetrahedral sites. Others are largely displacive but affect the coordination of the large cations (Pb, Ba, K, Ca). High-resolution electron microscopy suggests that the undulatory extinction characteristic of hyalotekite is due to a fine mosaic microstructure. This suggests that at least one of these transitions occurs in nature during cooling, and that it is first order with a large volume change. A diffuse superstructure observed by electron diffraction implies the existence of a further stage of short-range cation ordering which probably involves both (Pb,K)-Ba and (BeSi,BB)-BSi.

Structure and inferred dynamics of a large grove of Microberlinia bisulcata trees in central African rain forest: the possible role of periods of multiple disturbance events

A 272-ha grove of dominant Microberlinia bisulcata (Caesalpinioideae) adult trees greater than or equal to 50 cm stem diameter was mapped in its entirety in the southern part of Korup National Park, Cameroon. The approach used an earlier-established 82.5-ha permanent plot with a new surrounding 50-m grid of transect lines. Tree diameters were available from the plot but trees on the grid were recorded as being greater than or equal to 50 cm. The grove consisted of 1028 trees in 2000. Other species occurred within the grove. including the associated subdominants Tetraberlinia bifoliolata and T. korupensis. Microberlinia bisulcata becomes adult at a stein diameter of c. 50 cm and at an estimated age of 50 y. Three oval-shaped subgroves with dimensions c. 8 50 in x 13 50 in (90 ha) were defined. For two of them (within the plot) tree diameters were available. Subgroves differed in their scales and intensities of spatial tree patterns, and in their size frequency distributions, these suggesting differing past dynamics. The modal scale of clumping was 40-50 m. Seed dispersal by pod ejection (to c. 50 in) was evident from the semi-circles of trees at the grove's edge and from the many internal circles (100-200 m diameter). The grove has the capacity. therefore, to increase at c. 100 m per century. To form its present extent and structure. it is inferred that it expanded and infilled from a possibly smaller area of lower adult-tree density. This possibly happened in three waves of recruitment, each one determined by a period of several intense disturbances. Climate records for Africa show that 1740-50 and 1820-30 were periods of drought, and that 1870-1895 was also regionally very dry. Canopy openings allow the light-demanding and fast-growing ectomycorrhizal M. bisulcata to establish, but successive releases are thought to be required to achieve effective recruitment. Nevertheless, in the last 50 y there were no major events and recruitment in the grove was very poor. This present study leads to a new hypothesis of the role of periods of multiple extreme events being the driving factor for the population dynamics of many large African tree species such as M. bisulcata.

Evaluating perspectives for PRRS virus elimination from pig dense areas with a risk factor based herd index

Porcine reproductive and respiratory syndrome virus (PRRSV) is wide-spread in pig populations globally. In many regions of Europe with intensive pig production and high herd densities, the virus is endemic and can cause disease and production losses. This fuels discussion about the feasibility and sustainability of virus elimination from larger geographic regions. The implementation of a program aiming at virus elimination for areas with high pig density is unprecedented and its potential success is unknown. The objective of this work was to approach pig population data with a simple method that could support assessing the feasibility of a sustainable regional PRRSV elimination. Based on known risk factors such as pig herd structure and neighborhood conditions, an index characterizing individual herds' potential for endemic virus circulation and reinfection was designed. This index was subsequently used to compare data of all pig herds in two regions with different pig- and herd-densities in Lower Saxony (North-West Germany) where PRRSV is endemic. Distribution of the indexed herds was displayed using GIS. Clusters of high herd index densities forming potential risk hot spots were identified which could represent key target areas for surveillance and biosecurity measures under a control program aimed at virus elimination. In an additional step, for the study region with the higher pig density (2463 pigs/km(2) farmland), the potential distribution of PRRSV-free and non-free herds during the implementation of a national control program aiming at national virus elimination was modeled. Complex herd and trade network structures suggest that PRRSV elimination in regions with intensive pig farming like that of middle Europe would have to involve legal regulation and be accompanied by important trade and animal movement restrictions. The proposed methodology of risk index mapping could be adapted to areas varying in size, herd structure and density. Interpreted in the regional context, this could help to classify the density of risk and to accordingly target resources and measures for elimination.

Estimating Diagnostic Test Accuracies for Brachyspira hyodysenteriae Accounting for the Complexities of Population Structure in Food Animals

For swine dysentery, which is caused by Brachyspira hyodysenteriae infection and is an economically important disease in intensive pig production systems worldwide, a perfect or error-free diagnostic test ("gold standard") is not available. In the absence of a gold standard, Bayesian latent class modelling is a well-established methodology for robust diagnostic test evaluation. In contrast to risk factor studies in food animals, where adjustment for within group correlations is both usual and required for good statistical practice, diagnostic test evaluation studies rarely take such clustering aspects into account, which can result in misleading results. The aim of the present study was to estimate test accuracies of a PCR originally designed for use as a confirmatory test, displaying a high diagnostic specificity, and cultural examination for B. hyodysenteriae. This estimation was conducted based on results of 239 samples from 103 herds originating from routine diagnostic sampling. Using Bayesian latent class modelling comprising of a hierarchical beta-binomial approach (which allowed prevalence across individual herds to vary as herd level random effect), robust estimates for the sensitivities of PCR and culture, as well as for the specificity of PCR, were obtained. The estimated diagnostic sensitivity of PCR (95% CI) and culture were 73.2% (62.3; 82.9) and 88.6% (74.9; 99.3), respectively. The estimated specificity of the PCR was 96.2% (90.9; 99.8). For test evaluation studies, a Bayesian latent class approach is well suited for addressing the considerable complexities of population structure in food animals.

Trypanosoma brucei RRM1 is a nuclear RNA-binding protein and modulator of chromatin structure

TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 by RNA interference caused widespread changes in mRNA abundance, but these changes did not correlate with the binding of the protein to transcripts, and most splice sites were unchanged, negating a general role for TbRRM1 in splice site selection. When changes in mRNA abundance were mapped across the genome, regions with many downregulated mRNAs were identified. Two regions were analyzed by chromatin immunoprecipitation, both of which exhibited increases in nucleosome occupancy upon TbRRM1 depletion. In addition, subjecting cells to heat shock resulted in translocation of TbRRM1 to the cytoplasm and compaction of chromatin, consistent with a second role for TbRRM1 in modulating chromatin structure. IMPORTANCE: Trypanosoma brucei, the parasite that causes human sleeping sickness, is transmitted by tsetse flies. The parasite progresses through different life cycle stages in its two hosts, altering its pattern of gene expression in the process. In trypanosomes, protein-coding genes are organized as polycistronic units that are processed into monocistronic mRNAs. Since genes in the same unit can be regulated independently of each other, it is believed that gene regulation is essentially posttranscriptional. In this study, we investigated the role of a nuclear RNA-binding protein, TbRRM1, in the insect stage of the parasite. We found that TbRRM1 binds nuclear mRNAs and also affects chromatin status. Reduction of nuclear TbRRM1 by RNA interference or heat shock resulted in chromatin compaction. We propose that TbRRM1 regulates RNA polymerase II-driven gene expression both cotranscriptionally, by facilitating transcription and efficient splicing, and posttranscriptionally, via its interaction with nuclear mRNAs.

Unique Sm core structure of U7 snRNPs: assembly by a specialized SMN complex and the role of a new component, Lsm11, in histone RNA processing.

A set of seven Sm proteins assemble on the Sm-binding site of spliceosomal U snRNAs to form the ring-shaped Sm core. The U7 snRNP involved in histone RNA 3' processing contains a structurally similar but biochemically unique Sm core in which two of these proteins, Sm D1 and D2, are replaced by Lsm10 and by another as yet unknown component. Here we characterize this factor, termed Lsm11, as a novel Sm-like protein with apparently two distinct functions. In vitro studies suggest that its long N-terminal part mediates an important step in histone mRNA 3'-end cleavage, most likely by recruiting a zinc finger protein previously identified as a processing factor. In contrast, the C-terminal part, which comprises two Sm motifs interrupted by an unusually long spacer, is sufficient to assemble with U7, but not U1, snRNA. Assembly of this U7-specific Sm core depends on the noncanonical Sm-binding site of U7 snRNA. Moreover, it is facilitated by a specialized SMN complex that contains Lsm10 and Lsm11 but lacks Sm D1/D2. Thus, the U7-specific Lsm11 protein not only specifies the assembly of the U7 Sm core but also fulfills an important role in U7 snRNP-mediated histone mRNA processing.

A Synthetic Factor XIIa Inhibitor Blocks Selectively Intrinsic Coagulation Initiation.

Coagulation factor XII (FXII) inhibitors are of interest for the study of the protease in the intrinsic coagulation pathway, for the suppression of contact activation in blood coagulation assays, and they have potential application in antithrombotic therapy. However, synthetic FXII inhibitors developed to date have weak binding affinity and/or poor selectivity. Herein, we developed a peptide macrocycle that inhibits activated FXII (FXIIa) with an inhibitory constant Ki of 22 nM and a selectivity of >2000-fold over other proteases. Sequence and structure analysis revealed that one of the two macrocyclic rings of the in vitro evolved peptide mimics the combining loop of corn trypsin inhibitor, a natural protein-based inhibitor of FXIIa. The synthetic inhibitor blocked intrinsic coagulation initiation without affecting extrinsic coagulation. Furthermore, the peptide macrocycle efficiently suppressed plasma coagulation triggered by contact of blood with sample tubes and allowed specific investigation of tissue factor initiated coagulation.

Mechanism of subunit interaction and DNA binding of the heterotrimeric CCAAT binding factor CBF

Many eukaryotic promoters contain a CCAAT element at a site close ($-$80 to $-$120) to the transcription initiation site. CBF (CCAAT Binding Factor), also called NF-Y and CP1, was initially identified as a transcription factor binding to such sites in the promoters of the Type I collagen, albumin and MHC class II genes. CBF is a heteromeric transcription factor and purification and cloning of two of the subunits, CBF-A and CBF-B revealed that it was evolutionarily conserved with striking sequence identities with the yeast polypeptides HAP3 and HAP2, which are components of a CCAAT binding factor in yeast. Recombinant CBF-A and CBF-B however failed to bind to DNA containing CCAAT sequences. Biochemical experiments led to the identification of a third subunit, CBF-C which co-purified with CBF-A and complemented the DNA binding of recombinant CBF-A and CBF-B. We have recently isolated CBF-C cDNAs and have shown that bacterially expressed purified CBF-C binds to CCAAT containing DNA in the presence of recombinant CBF-A and CBF-B. Our experiments also show that a single molecule each of all the three subunits are present in the protein-DNA complex. Interestingly, CBF-C is also evolutionarily conserved and the conserved domain between CBF-C and its yeast homolog HAP5 is sufficient for CBF-C activity. Using GST-pulldown experiments we have demonstrated the existence of protein-protein interaction between CBF-A and CBF-C in the absence of CBF-B and DNA. CBF-B on other hand, requires both CBF-A and CBF-C to form a ternary complex which then binds to DNA. Mutational studies of CBF-A have revealed different domains of the protein which are involved in CBF-C interaction and CBF-B interaction. In addition, CBF-A harbors a domain which is involved in DNA recognition along with CBF-B. Dominant negative analogs of CBF-A have also substantiated our initial observation of assembly of CBF subunits. Our studies define a novel DNA binding structure of heterotrimeric CBF, where the three subunits of CBF follow a particular pathway of assembly of subunits that leads to CBF binding to DNA and activating transcription. ^

Application of factor decomposition techniques to vertical specialisation measurements

The increasing importance of vertical specialisation (VS) trade has been a notable feature of rapid economic globalisation and regional integration. In an attempt to understand countries’ depth of participation in global production chains, many Input-Output based VS indicators have been developed. However, most of them focus on showing the overall magnitude of a country’s VS trade, rather than explaining the roles that specific sectors or products play in VS trade and what factors make the VS change over time. Changes in vertical specialisation indicators are, in fact, determined by mixed and complex factors such as import substitution ratios, types of exported goods and domestic production networks. In this paper, decomposition techniques are applied to VS measurement based on the OECD Input-Output database. The decomposition results not only help us understand the structure of VS at detailed sector and product levels, but also show us the contributions of trade dependency, industrial structures of foreign trade and domestic production system to a country’s vertical specialisation trade.

Fracture strength of drilled wafers: study of the stress concentration factor and determination of the weibull distribution of the fracture stress

In the photovoltaic field, the back contact solar cells technology has appeared as an alternative to the traditional silicon modules. This new type of cells places both positive and negative contacts on the back side of the cells maximizing the exposed surface to the light and making easier the interconnection of the cells in the module. The Emitter Wrap-Through solar cell structure presents thousands of tiny holes to wrap the emitter from the front surface to the rear surface. These holes are made in a first step over the silicon wafers by means of a laser drilling process. This step is quite harmful from a mechanical point of view since holes act as stress concentrators leading to a reduction in the strength of these wafers. This paper presents the results of the strength characterization of drilled wafers. The study is carried out testing the samples with the ring on ring device. Finite Element models are developed to simulate the tests. The stress concentration factor of the drilled wafers under this load conditions is determined from the FE analysis. Moreover, the material strength is characterized fitting the fracture stress of the samples to a three-parameter Weibull cumulative distribution function. The parameters obtained are compared with the ones obtained in the analysis of a set of samples without holes to validate the method employed for the study of the strength of silicon drilled wafers.