Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.

Cognitive and neurobiologic markers of early Alzheimer disease

Recent studies indicate that impairments in two cognitive domains characterize the cognitive abnormalities that appear earliest in the course of Alzheimer disease (AD). These cognitive domains pertain to memory and executive function ability; in particular, memory test scores reflecting the difference between immediate and delayed recall and tasks that assess cognitive flexibility (e.g., set-shifting). Preliminary data indicate that tasks of this nature, along with specific genetic information (i.e., APOE-4 status), are important in identifying which individuals with recent cognitive changes (considered to have “questionable” disease) will progress to the point where they meet criteria for AD over time. When this cognitive and genetic information is combined with neuroimaging measures targeted at the brain regions demonstrating pathology early in AD, it may serve as specific and accurate prognostic markers of AD.

Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers.

A maximum likelihood approach of half tetrad analysis (HTA) based on multiple restriction fragment length polymorphism (RFLP) markers was developed. This procedure estimates the relative frequencies of 2n gametes produced by mechanisms genetically equivalent to first division restitution (FDR) or second division restitution and simultaneously locates the centromere within a linkage group of RFLP marker loci. The method was applied to the diploid alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production. HTA was based on four RFLP loci for which PG-F9 was heterozygous with codominant alleles that were absent in the tetraploid tester. Models including three linked and one unlinked RFLP loci were developed and tested. Results of the HTA showed that PG-F9 produced 6% FDR and 94% second division restitution 2n eggs. Information from a marker locus belonging to one linkage group was used to more precisely locate the centromere on a different linkage group. HTA, together with previous cytological analysis, indicated that in PG-F9, FDR 2n eggs are likely produced by diplospory, a mechanism common among apomictic species. The occurrence of FDR 2n eggs in plant species and their importance for crop evolution and breeding is discussed together with the potential applicability of multilocus HTA in the study of reproductive mutants.

A rat genetic map constructed by representational difference analysis markers with suitability for large-scale typing.

Representational difference analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot analysis of amplicons, hybridizing only with tester amplicons. Dot blot analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.

Molecular markers reveal cryptic sex in the human pathogen Coccidioides immitis.

Coccidioides immitis, cause of a recent epidemic of "Valley fever" in California, is typical of many eukaryotic microbes in that mating and meiosis have yet to be reported, but it is not clear whether sex is truly absent or just cryptic. To find out, we have undertaken a population genetic study using PCR amplification, screening for single-strand conformation polymorphisms, and direct DNA sequencing to find molecular markers with nucleotide-level resolution. Both population genetic and phylogenetic analyses indicate that C. immitis is almost completely recombining. To our knowledge, this study is the first to find molecular evidence for recombination in a fungus for which no sexual stage has yet been described. These results motivate a directed search for mating and meiosis and illustrate the utility of single-strand conformation polymorphism and sequencing with arbitrary primer pairs in molecular population genetics.

Cyclins as markers of tumor proliferation: immunocytochemical studies in breast cancer.

We have developed methods to use anticyclin A, B, and E antibodies as reagents to specifically detect proliferating cells in specific phases of the cell cycle in formalin-fixed, paraffin-embedded sections of tissues and cells. Staining of 48 archival cases of breast cancer showed that these antibodies estimate the tumor proliferation fraction and therefore are potentially useful for the prediction of prognosis. A subset of cancers had a high frequency of tumor cells expressing cyclins A and E, out of proportion to other proliferation markers, suggesting that these tumors may have deregulated cyclin expression. In addition to recognizing authentic cyclin E in the nucleus of proliferating cells, anticyclin E antibody cross-reacted with a cytoplasmic protein in nonproliferating endothelial cells. This cross-reaction allows the simultaneous visualization and quantitation of microvessels in the tumors, measuring a second potential predictor of breast cancer prognosis, tumor angiogenesis.

Direct detection and isolation of restriction landmark genomic scanning (RLGS) spot DNA markers tightly linked to a specific trait by using the RLGS spot-bombing method.

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RLGS-SB). RLGS-SB allows us to scan the genome of higher organisms quickly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyzing a C57BL/6-GusS mouse congenic strain. We identified 33 variant markers out of 10,565 total loci in a 4.2-centimorgan (cM) interval surrounding the Gus locus in 4 days of laboratory work. The validity of RLGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny by analyzing the spot intensity of already mapped RLGS loci. Finally, we used RLGS-SB to identify DNA markers closely linked to the mouse reeler (rl) locus on chromosome 5 by phenotypic pooling. A total of 31 RLGS loci were identified and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl. The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were closely linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a trait or gene of interest.

Trans-Pacific migrations of the loggerhead turtle (Caretta caretta) demonstrated with mitochondrial DNA markers.

Juvenile loggerhead turtles (Caretta caretta) have recently been documented in the vicinity of Baja California and thousands of these animals have been captured in oceanic fisheries of the North Pacific. The presence of loggerhead turtles in the central and eastern North Pacific is a prominent enigma in marine turtle distribution because the nearest documented nesting concentrations for this species are in Australia and Japan, over 10,000 km from Baja California. To determine the origin of the Baja California feeding aggregate and North Pacific fishery mortalities, samples from nesting areas and pelagic feeding aggregates were compared with genetic markers derived from mtDNA control region sequences. Overall, 57 of 60 pelagic samples (95%) match haplotypes seen only in Japanese nesting areas, implicating Japan as the primary source of turtles in the North Pacific Current and around Baja California. Australian nesting colonies may contribute the remaining 5% of these pelagic feeding aggregates. Juvenile loggerhead turtles apparently traverse the entire Pacific Ocean, approximately one-third of the planet, in the course of developmental migrations, but mortality in high-seas fisheries raises concern over the future of this migratory population.

Blood markers and recurrence of stroke in patients with transient ischemic attack

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Development of new analytical tools for monitoring of cardiovascular disease markers – towards the detection of homocysteine-thiolactone

Poster presented at the 4th International Conference on Bio-Sensing Technology, 10-13 May 2015, Lisbon, Portugal

Markers for silent atrial fibrillation in esophageal long-term electrocardiography.

PURPOSE Paroxysmal atrial fibrillation (PAF) often remains undiagnosed. Long-term surface ECG is used for screening, but has limitations. Esophageal ECG (eECG) allows recording high quality atrial signals, which were used to identify markers for PAF. METHODS In 50 patients (25 patients with PAF; 25 controls) an eECG and surface ECG was recorded simultaneously. Partially A-V blocked atrial runs (PBARs) were quantified, atrial signal duration in eECG was measured. RESULTS eECG revealed 1.8‰ of atrial premature beats in patients with known PAF to be PBARs with a median duration of 853ms (interquartile range (IQR) 813-1836ms) and a median atrial cycle length of 366ms (IQR 282-432ms). Even during a short recording duration of 2.1h (IQR 1.2-17.2h), PBARs occurred in 20% of PAF patients but not in controls (p=0.05). Left atrial signal duration was predictive for PAF (72% sensitivity, 80% specificity). CONCLUSIONS eECG reveals partially blocked atrial runs and prolonged left atrial signal duration - two novel surrogate markers for PAF.

Verbs as Linguistic Markers of Agency - The Social Side of Grammar

Basic grammatical categories may carry social meaning irrespective of their semantic content. In a set of four studies, we demonstrate that verbs – a basic linguistic category present and distinguishable in most languages – are related to the perception of agency, a fundamental dimension in social perception. In an archival analysis on actual language use in Polish and German, we found that targets stereotypically associated with high agency (men and young people) are presented in the immediate neighborhood of a verb more often than non-agentic social targets (women and old people). Moreover, in three experiments using a pseudo-word paradigm, verbs (but not adjectives and nouns) were consistently associated with agency (but not communion). These results provide consistent evidence that verbs, as grammatical vehicles of action, are linguistic markers of agency. In demonstrating meta-semantic effects of language, these studies corroborate the view of language as a social tool and of language as an integral part of social perception.