Dysregulation of alternative splicing of coagulation factor V results in bleeding disorder, east Texas type and other disorders of hemostasis

The studies completed herein explore different phenotypes related to the genetic defects that predispose individuals to a disruption of normal hemostasis. In the first study, a novel autosomal dominant bleeding disorder, which is characterized by excessive bleeding with trauma or surgery and menorrhagia in affected women, was studied in a large family (16 affected individuals) from east Texas. Affected members had a prolongation of their PT and/or aPTT, but normal clinical coagulation studies. Previous linkage analysis by Kuang et. al. (2001) mapped the defective gene to 1g23-24 (LODmax 7.22), which contains the gene for coagulation factor V (FV). I identified an alteration (A2440G) in the FV gene in exon 13 that segregated with the disease and was not present in 62 controls. Interestingly, this alteration resulted in a 22-fold up-regulation of a novel alternative splicing variant in patients' RNA versus controls. This translated into a similar fold increase in a 250-kDa isoform of FV seen in patients' plasma versus controls. A recombinant of this splicing event exhibited an increased sensitivity to cleavage by activated protein C (APC) that was more striking in the presence of PS. In addition, this novel isoform had increased APC cofactor activity, thus increasing the degradation of FVIIIa. These data indicated that A2440G up-regulates an alternatively spliced transcript of FV, and increases a FV isoform that hinders coagulation as opposed to promoting it like its wild-type counterpart. ^ The second study reports the largest screening to date of African Americans in two independent cohorts for a rare prothrombin variant, C20209T, which is suspected to be associated with thrombotic disease. The Texas Medical Center Genetics Resource (TexGen) Stroke DNA repository revealed 1.67% (Fisher p=0.27) of African American stroke patients were heterozygous for the 20209*T allele. Screening of the Atherosclerosis Risk in Communities Study (ARIC) cohort (n=3470) for the 20209*T allele revealed a population prevalence of 0.58% in individuals of African American descent; however, all associations with thrombotic disease were negative. Analysis of these two independent cohorts revealed that, unlike its neighbor G20210A, the C20209T variant does not increase the risk of thrombotic events in the African American population. ^

Comparison of alternative methods of contact tracing in a syphilis control program

The purpose of this study was to compare the relative effectiveness of alternative methods of tracing named contacts of syphilis patients. A total of 236 contacts, identified by patients in two City of Houston Department of Health and Human Services clinics during the period April 1 through July 31, 1987, were studied. After contacts were grouped by sex and age, the proportion brought to examination by each of three methods, and by a combination of methods, was determined for each subgroup.^ The study found that 78.4% of all the 236 named sex contacts reported were located and brought to examination by the various methods of contact tracing and that 21.6% were missed. Of the 185 contacts examined, a combination of methods identified 47.7% of the cases, telephone contact, 28.6%, field contact, 16.9%, and patient referral, 11.8%.^ Of the 236 contacts reported, males made up 56.8% and females 43.2%. Contact tracing was more successful among females, with 81.4% of the 102 named female contacts, as compared to 76.1% of the 134 named male contacts being brought to examination. It is not known whether equal efforts were exerted in the follow-up of both male and female contacts. In both female and male subgroups, a combination of methods brought over 40% of sex contacts to examination. Telephone contact among females yielded 27.7% of the cases and field contact 18.1%, whereas in males, telephone contact identified 29.4% of the cases and field contact 15.7%. Patient referral was the least productive method in both sex groups, locating 12.8% in males as compared to 10.8% in females.^ On an age specific basis, a combination of methods was the most effective method in the 15-39 age group, whereas telephone contact was most effective in the 40-44 age group, and field contact in the 50-54 age group. Of all the methods of contact tracing, patient referral was the least productive in most age groups.^ Future studies of contact tracing should incorporate several important variables which were not examined in this study. ^

REGULATION OF ALTERNATIVE CARBON METABOLISM IN CANDIDA ALBICANS

Candida albicans is the most important fungal pathogen of humans. Transcript profiling studies show that upon phagocytosis by macrophages, C. albicans undergoes a massive metabolic reorganization activating genes involved in alternative carbon metabolism, including the glyoxylate cycle, β-oxidation and gluconeogenesis. Mutations in key enzymes such as ICL1 (glyoxylate cycle) and FOX2 (fatty acid β-oxidation) revealed that alternative carbon metabolic pathways are required for full virulence in C. albicans. These studies indicate C. albicans uses non-preferred carbon sources allowing its adaptation to microenvironments were nutrients are scarce. It has become apparent that the regulatory networks required for regulation of alternative carbon metabolism in C. albicans are considerably different from the Saccharomyces cerevisiae paradigm and appear more analogous to the Aspergillus nidulans systems. Well-characterized transcription factors in S. cerevisiae have no apparent phenotype or are missing in C. albicans. CTF1 was found to be a single functional homolog of the A. nidulans FarA/FarB proteins, which are transcription factors required for fatty acid utilization. Both FOX2 and ICL1 were found to be part of a large CTF1 regulon. To increase our understanding of how CTF1 regulates its target genes, including whether regulation is direct or indirect, the FOX2 and ICL1 promoter regions were analyzed using a combination of bioinformatics and promoter deletion analysis. To begin characterizing the FOX2 and ICL1 promoters, 5’ rapid amplification of cDNA ends (5’RACE) was used to identify two transcriptional initiation sites in FOX2 and one in ICL1. GFP reporter assays show FOX2 and ICL1 are rapidly expressed in the presence of alternative carbon sources. Both FOX2 and ICL1 harbor the CCTCGG sequence known to be bound by the Far proteins, hence rendering the motif as a putative CTF1 DNA binding element. In this study, the CCTCGG sequence was found to be essential for FOX2 regulation. However, this motif does not appear to be equally important for the regulation of ICL1. This study supports the notion that although C. albicans has diverged from the paradigms of model fungi, C. albicans has made specific adaptations to its transcription-based regulatory network that may contribute to its metabolic flexibility.

Use of alternative fuels obtained from renewable sources in brayton cycles

Analysis and simulation of the behaviour of gas turbines for power generation using different nonconventional fuels obtained from different renewable sources are presented. Three biomass-tobiofuel processes are considered: anaerobic digestion of biomass (biogas), biomass gasification (synthesis gas) and alcoholic fermentation of biomass and dehydration (bioethanol), each of them with two different biomass substrates (energy crops and municipal solid waste) as input. The gas turbine behaviour in a Brayton cycle is simulated both in an isolated operation and in combined cycle. The differences in gas turbine performance when fired with the considered biofuels compared to natural gas are studied from different points of view related with the current complex energetic context: energetic and exergetic efficiency of the simple/combined cycle and CO2 emissions. Two different tools have been used for the simulations, each one with a different approach: while PATITUG (own software) analyses the behaviour of a generic gas turbine allowing a total variability of parameters, GT-PRO (commercial software) is more rigid, albeit more precise in the prediction of real gas turbine behaviour. Different potentially interesting configurations and its thermodynamic parameters have been simulated in order to obtain the optimal range for all of them and its variation for each fuel.

Use of alternative fuels obtained from renowable sources in Brayton cycles

Analysis and simulation of the behaviour of gas turbines for power generation using different nonconventional fuels obtained from different renewable sources are presented. Three biomass-tobiofuel processes are considered: anaerobic digestion of biomass (biogas), biomass gasification (synthesis gas) and alcoholic fermentation of biomass and dehydration (bioethanol), each of them with two different biomass substrates (energy crops and municipal solid waste) as input. The gas turbine behaviour in a Brayton cycle is simulated both in an isolated operation and in combined cycle. The differences in gas turbine performance when fired with the considered biofuels compared to natural gas are studied from different points of view related with the current complex energetic context: energetic and exergetic efficiency of the simple/combined cycle and CO2 emissions. Two different tools have been used for the simulations, each one with a different approach: while PATITUG (own software) analyses the behaviour of a generic gas turbine allowing a total variability of parameters, GT-PRO (commercial software) is more rigid, albeit more precise in the prediction of real gas turbine behaviour. Different potentially interesting configurations and its thermodynamic parameters have been simulated in order to obtain the optimal range for all of them and its variation for each fuel.

Analysis of alternative tendering mechanisms for transportation Public-Private Partnerships

This paper describes a theoretical model based primarily on transaction costs, for comparing the various tendering mechanisms used for transportation Public-Private Partnership (PPP) projects. In particular, the model contrasts negotiated procedures with the open procedure, as defined by the current European Union legislation on public tendering. The model includes both ex ante transaction costs (borne during the tendering stage) and ex post transaction costs (such as enforcement costs, re-negotiation costs, and costs arising from litigation between partners), explaining the trade-off between them. Generally speaking, it is assumed that the open procedure implies lower transaction costs ex ante, while the negotiated procedure reduces the probability of the appearance of new contingencies not foreseen in the contract, hence diminishing the expected value of transaction costs ex post. Therefore, the balance between ex ante and ex post transaction costs is the main criterion for deciding whether the open or negotiated procedure would be optimal. Notwithstanding, empirical evidence currently exists only on ex ante transaction costs in transportation infrastructure projects. This evidence has shown a relevant difference between the two procedures as far as ex ante costs are concerned, favouring the open procedure. The model developed in this paper also demonstrates that a larger degree of complexity in a contract does not unequivocally favour the use of a negotiated procedure. Only in those cases dealing with very innovative projects, where important dimensions of the quality of the asset or service are not verifiable, may we observe an advantage in favour of the negotiated procedure. The bottom line is that we find it difficult to justify the employment of negotiated procedures in most transportation PPP contracts, especially in the field of roads. Nevertheless, the field remains open for future empirical work and research on the levels of transaction costs borne ex post in PPP contracts, as well as on the probabilities of such costs appearing under any of the procurement procedures.

Effect of late weaning and use of alternative cages on performance of does, suckling and fattening rabbits under extensive reproductive management

The effects of the combined use of long lactation periods (46 days) with alternative cages on the reproductive and growth performance of 104 rabbit does and their litters during five consecutive reproductive cycles were studied. Half of does were housed in conventional polyvalent cages (39 cm×100 cm×30 cm) and the other half in alternative polyvalent cages (39 cm×100 cm×60 cm), with a raised platform. Half of the rabbit does in each type of cage were weaned at 32 and the other half at 46 days after parturition. Longer lactation negatively affected the body weight (P<0.001), fat and energy content (P<0.05) of rabbit does at the end of the lactation period, but this effect decreased with the number of parturitions. Fertility, prolificacy and doe mortality were not affected by lactation length. Late weaning led to higher litter size (by 8.9%) and litter weight (by 11.3%) at the end of growing period (P<0.001) and lower feed conversion ratio per cage during the experimental period (13.5%) than weaning at 32 day (P<0.001). These results were paralleled by lower mortality (12.6 vs. 17.6%; P<0.01) of young rabbits weaned later during the overall experimental period. Differences in performance as a result of different weaning ages were only observed during cycles with worst health status (third and fifth cycles) in which late weaning decreased mortality. Type of cage did not affect doe body weight and body condition, mortality, fertility, prolificacy and litter size during the five reproductive cycles. Nevertheless, at day 21 litter weight and feed conversion ratio between 3 and 21 day were 4.2% higher (P<0.01) and 5.0% lower (P<0.05), respectively, in animals housed in alternative rather than in conventional cages. Alternative cages also led to heavier litters at 59 days (P<0.01). It was concluded that the combined use of longer lactations and cages with higher available surface with a raised platform could be alternatives to improve animal welfare in farmed rabbit.

Effect of late weaning and use of alternative cages on performance of does, suckling and fattening rabbits under extensive reproductive management

The effects of the combined use of long lactation periods (46 days) with alternative cages on the reproductive and growth performance of 104 rabbit does and their litters during five consecutive reproductive cycles were studied. Half of does were housed in conven- tional polyvalent cages (39 cm x 100 cm x 30 cm) and the other half in alternative polyvalent cages (39 cm x 100 cm x 60 cm), with a raised platform. Half of the rabbit does in each type of cage were weaned at 32 and the other half at 46 days after parturition. Longer lactation negatively affected the body weight ( P o 0.001), fat and energy content ( P o 0.05) of rabbit does at the end of the lactation period, but this effect decreased with the number of parturitions. Fertility, prolificacy and doe mortality were not affected by lactation length. Late weaning led to higher litter size (by 8.9%) and litter weight (by 11.3%) at the end of growing period ( P o 0.001) and lower feed conversion ratio per cage during the experimental period (13.5%) than weaning at 32 day ( P o 0.001). These results were paralleled by lower mortality (12.6 vs. 17.6%; P o 0.01) of young rabbits weaned later during the overall experimental period. Differences in performance as a result of different weaning ages were only observed during cycles with worst health status (third and fifth cycles) in which late weaning decreased mortality. Type of cage did not affect doe body weight and body condition, mortality, fertility, prolificacy and litter size during the five reproductive cycles. Nevertheless, at day 21 litter weight and feed conversion ratio between 3 and 21 day were 4.2% higher ( P o 0.01) and 5.0% lower ( P o 0.05), respectively, in animals housed in alternative rather than in conventional cages. Alternative cages also led to heavier litters at 59 days ( P o 0.01). It was concluded that the combined use of longer lactations and cages with higher available surface with a raised platform could be alternatives to improve animal welfare in farmed rabbit.

Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries

Transport of peptides across the membrane of the endoplasmic reticulum for assembly with MHC class I molecules is an essential step in antigen presentation to cytotoxic T cells. This task is performed by the major histocompatibility complex-encoded transporter associated with antigen processing (TAP). Using a combinatorial approach we have analyzed the substrate specificity of human TAP at high resolution and in the absence of any given sequence context, revealing the contribution of each peptide residue in stabilizing binding to TAP. Human TAP was found to be highly selective with peptide affinities covering at least three orders of magnitude. Interestingly, the selectivity is not equally distributed over the substrate. Only the N-terminal three positions and the C-terminal residue are critical, whereas effects from other peptide positions are negligible. A major influence from the peptide backbone was uncovered by peptide scans and libraries containing d amino acids. Again, independent of peptide length, critical positions were clustered near the peptide termini. These approaches demonstrate that human TAP is selective, with residues determining the affinity located in distinct regions, and point to the role of the peptide backbone in binding to TAP. This binding mode of TAP has implications in an optimized repertoire selection and in a coevolution with the major histocompatibility complex/T cell receptor complex.

A test of alternative models of diversification in tropical rainforests: Ecological gradients vs. rainforest refugia

Comparison of mitochondrial and morphological divergence in eight populations of a widespread leaf-litter skink is used to determine the relative importance of geographic isolation and natural selection in generating phenotypic diversity in the Wet Tropics Rainforest region of Australia. The populations occur in two geographically isolated regions, and within each region, in two different habitats (closed rainforest and tall open forest) that span a well characterized ecological gradient. Morphological differences among ancient geographic isolates (separated for several million years, judging by their mitochondrial DNA sequence divergence) were slight, but morphological and life history differences among habitats were large and occurred despite moderate to high levels of mitochondrial gene flow. A field experiment identified avian predation as one potential agent of natural selection. These results indicate that natural selection operating across ecological gradients can be more important than geographic isolation in similar habitats in generating phenotypic diversity. In addition, our results indicate that selection is sufficiently strong to overcome the homogenizing effects of gene flow, a necessary first step toward speciation in continuously distributed populations. Because ecological gradients may be a source of evolutionary novelty, and perhaps new species, their conservation warrants greater attention. This is particularly true in tropical regions, where most reserves do not include ecological gradients and transitional habitats.

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1α and aggst1β) reported within are interrupted by introns. The gene aggst1α contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5′ exon encoding the N termini of the GST protein spliced to one of four distinct 3′ exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1α are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1β gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.

Control of alternative pre-mRNA splicing by distributed pentameric repeats

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3′ splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.