Molecular Marker Studies Research Articles

Amplification ofPlasmodium falciparumMultidrug Resistance Gene 1 in Isolates from Gabon

The study of molecular markers of drug resistance is particularly important in surveillance studies of drugs, such as mefloquine, that still retain efficacy in sub-Saharan Africa yet have encountered resistance elsewhere. In a recent study in Thailand, we identified amplification of the Plasmodium falciparum multidrug resistance gene 1 (pfmdr1) as being the most important predictor of in vitro drug resistance and in vivo failure of mefloquine monotherapy. Here we report amplification of pfmdr1 in >5% of patient samples from Lambarene, Gabon, collected in 1995. None of the samples collected 7 years later showed pfmdr1 amplification, suggesting that parasites with increased numbers of pfmdr1 copies have not substantially spread through the population. Nevertheless, the detection of multicopy pfmdr1 in African parasites suggests a high potential for rapid selection for resistance, implying that mefloquine use in Africa should be considered only as part of combination therapy.

Comparative Proteomic Analysis of Intra- and Interindividual Variation in Human Cerebrospinal Fluid

Cerebrospinal fluid (CSF) is a potential source of biomarkers for many disorders of the central nervous system, including Alzheimer disease (AD). Prior to comparing CSF samples between individuals to identify patterns of disease-associated proteins, it is important to examine variation within individuals over a short period of time so that one can better interpret potential changes in CSF between individuals as well as changes within a given individual over a longer time span. In this study, we analyzed 12 CSF samples, composed of pairs of samples from six individuals, obtained 2 weeks apart. Multiaffinity depletion, two-dimensional DIGE, and tandem mass spectrometry were used. A number of proteins whose abundance varied between the two time points was identified for each individual. Some of these proteins were commonly identified in multiple individuals. More importantly, despite the intraindividual variations, hierarchical clustering and multidimensional scaling analysis of the proteomic profiles revealed that two CSF samples from the same individual cluster the closest together and that the between-subject variability is much larger than the within-subject variability. Among the six subjects, comparison between the four cognitively normal and the two very mildly demented subjects also yielded some proteins that have been identified in previous AD biomarker studies. These results validate our method of identifying differences in proteomic profiles of CSF samples and have important implications for the design of CSF biomarker studies for AD and other central nervous system disorders.

Molecular evidence for natural interspecific hybridization in Prosopis

Interspecific hybridization can occur under natural conditions among species of the Prosopis genus, potentially giving rise to hybrid individuals with increased adaptation values. A study was conducted to assess the occurrence of interspecific hybridization among Prosopis in the Argentinian Chaco region and the species involved. The survey included seven hybrid individuals and their potential parents. Principal component analysis (PCA) was carried out based on 16 morphological characters. The molecular marker study was carried out by the comparison of RAPD patterns of amplification using six primers. A total of 91 RAPD fragments were analysed, yielding 33 species-specific markers, 24 bands that were common to two or three parental lines, and 22 bands of unknown origin. The dendrograms arising from Nei’s genetic distances derived from the molecular data showed a clustering pattern in agreement with the grouping obtained by the PCA of the morphological traits. The results demonstrate the occurrence of interspecific hybridization involving Prosopis alba, P. nigra and P. hassleri in the Argentinian Chaco.

Adjuvant treatment of colon cancer

Colon cancer is the second leading cause of cancer death in Western countries. Although most colon cancer patients will undergo curative surgery, survival, depending on the pathologic stage, varies from 30-60% for Dukes C to 60-80% for Dukes B. Until very recently, the absolute benefit for survival obtained with adjuvant therapy compared with control was about 6%. The present review analyzes the results of the new combination of 5-fluorouracil/leucovorin/oxaliplatin (MOSAIC study), the oral fluoropyrimidines, and the studies of genetic and molecular markers, attempting to identify the patients at risk of recurrence. Both 5-fluorouracil/leucovorin/oxaliplatin and the oral fluoropyrimidines show a statistically significant benefit of experiments treatment compared with 5-fluorouracil/leucovorin and no treatment, respectively. A reduction of 23% in the risk of relapse (disease-free survival rates of 78.2% in the 5-fluorouracil/leucovorin/oxaliplatine arm compared with 72.9% for 5-fluorouracil/leucovorin; P = 0.002) for the former and an overall hazard ratio of 0.89 for survival (95% CI, 0.80-0.99; P = 0.04) and 0.85 for disease-free survival (95% CI, 0.77-0.93; P < 0.001) in favor of oral therapy compared with control. A review of the literature on translational research indicates that patients at risk of recurrence can be identified by a 23-gene signature as well as by the combination of p27, p53, and vascular endothelial growth factor molecular alterations. The present review shows that adjuvant therapy of colon cancer is still improving and that the use of genetic/molecular markers together with the new targeted therapies has the potential to improve survival further.

Cyclin D1 protein and CCND1 gene expression in colorectal cancer

AimsTo report the expression of cyclin D1 protein and its gene in a series of colorectal adenocarcinoma. MethodsOne hundred and eleven specimens of colorectal carcinomas and adjacent normal colorectal mucosa were investigated by staining with a monoclonal antibody against cyclin D1 and by RT-PCR. ResultsExpression of CCND1 gene was found in 54 out of 111 cases of colorectal cancers, while in normal mucosa the expression of this gene was not observed. Cyclin D1 protein expression was checked in the same group of adenocarcinoma cases. Presence of this protein was observed in 69 cases and for 43 of them also expression of its gene was found. Dependence between the presence of protein and the gene expression was statistically significant (p=0.0002). In the group of cases where CCND1 gene expression was detected, high level of its protein expression was found in 20 cases. The CCND1 gene expression was associated with metastases to lymph nodes (p=0.0181) and also with distant metastasis (p=0.0204). ConclusionsThe combined measurement of both the gene and its protein product, is an important contribution to the study of molecular markers in histological material.

Two-photon absorption and self-phase modulation measurements with shaped femtosecond laser pulses

We show that phase-sensitive detection of spectral hole refilling can yield information about self-phase modulation and two-photon absorption coefficients. We expect that, when applied to tissue microscopy, this technique will allow the study of endogenous molecular markers beneath the surface, even if those markers are nonfluorescent.

Sirenomelia inBmp7andTsgcompound mutant mice:requirement for Bmp signaling in the development of ventral posterior mesoderm

Sirenomelia or mermaid-like phenotype is one of the principal human congenital malformations that can be traced back to the stage of gastrulation. Sirenomelia is characterized by the fusion of the two hindlimbs into a single one. In the mouse, sirens have been observed in crosses between specific strains and as the consequence of mutations that increase retinoic acid levels. We report that the loss of bone morphogenetic protein 7 (Bmp7) in combination with a half dose or complete loss of twisted gastrulation (Tsg) causes sirenomelia in the mouse. Tsg is a Bmp- and chordin-binding protein that has multiple effects on Bmp metabolism in the extracellular space; Bmp7 is one of many Bmps and is shown here to bind to Tsg. In Xenopus, co-injection of Tsg and Bmp7 morpholino oligonucleotides (MO) has a synergistic effect, greatly inhibiting formation of ventral mesoderm and ventral fin tissue. In the mouse, molecular marker studies indicate that the sirenomelia phenotype is associated with a defect in the formation of ventroposterior mesoderm. These experiments demonstrate that dorsoventral patterning of the mouse posterior mesoderm is regulated by Bmp signaling, as is the case in other vertebrates. Sirens result from a fusion of the hindlimb buds caused by a defect in the formation of ventral mesoderm.

Development and characterization of microsatellite markers inCynara cardunculusL.

Cynara cardunculus L. is a species native to the Mediterranean basin that comprises 2 crops, globe artichoke (var. scolymus L.) and cultivated cardoon (var. altilis DC), as well as wild cardoon (var. sylvestris (Lamk) Fiori). Globe artichoke represents an important component of the South European agricultural economy but is also cultivated in North Africa, the Near East, South America, the United States, and China. Breeding activities and molecular marker studies have been, to date, extremely limited. Better knowledge of the genome of the species might be gained by developing a range of molecular markers. Here, we report on the development of 14 microsatellites (simple sequence repeats (SSRs)) through a novel approach that we have defined as the microsatellite amplified library (MAL). The approach represents a combination of amplified fragment length polymorphism and a primer extension based enriched library, is rapid, and requires no hybridization enrichment steps. The technique provided a approximately 40-fold increase in the efficiency of SSR identification compared with conventional library procedures. The developed SSRs were applied for genotyping 36 accessions of C. cardunculus, including a core of 27 varietal types of globe artichoke, 3 accessions of cultivated cardoon, and 6 Sicilian accessions of wild cardoon. Principal coordinates analysis made it possible to differentiate both cultivated and wild forms from each other.

Social Interaction Deficits Caused by Chronic Phencyclidine Administration are Reversed by Oxytocin

Chronic administration of phencyclidine (PCP) has been advanced as a valid animal model of the social deficit symptoms of schizophrenia. In these studies, the cumulative time that male rats treated once a day for 14 days with PCP actively engaged in social behavior was decreased approximately 75% relative to saline-treated control animals. In addition, these socially impaired rats had an increase in the relative amount of noncontact interactions compared with saline-injected peers. Social behaviors were preferentially affected by PCP treatment because in two anxiety-related behavioral assays, the open field and light/dark emergence tests, there was a failure to differentiate between the PCP-treated rats and saline-injected control rats. Considering the general importance of the neuropeptides oxytocin and vasopressin in male social behaviors, studies of molecular markers related to these neuropeptides were performed. Hypothalamic oxytocin mRNA expression was significantly decreased while oxytocin receptor binding was increased in the central nucleus of the amygdala following chronic PCP treatment. Given the significance of central nucleus of the amygdala in social behavior, oxytocin was infused into the central nucleus of experimental and control male rats, and their postinfusion social interaction and open field behaviors were analyzed. A bilateral infusion of 1 mug of oxytocin into the central amygdala selectively restored the normal quantity and quality of social behavior in chronic PCP-treated male rats without altering open field behaviors. These findings suggest that deficits in the central oxytocinergic system may underlie the social impairment exhibited in this animal model of schizophrenia.

Discovery and utilization of QTLs for insect resistance in soybean

Insect resistance in soybean has been an objective in numerous breeding programs, but efforts to develop high yielding cultivars with insect resistance have been unsuccessful. Three Japanese plant introductions, PIs 171451, 227687 and 229358, have been the primary sources of insect resistance alleles, but a combination of quantitative inheritance of resistance and poor agronomic performance has hindered progress. Linkage drag caused by co-introgression of undesirable agronomic trait alleles linked to the resistance quantitative trait loci (QTLs) is a persistent problem. Molecular marker studies have helped to elucidate the numbers, effects and interactions of insect resistance QTLs in the Japanese PIs, and markers are now being used in breeding programs to facilitate transfer of resistance alleles while minimizing linkage drag. Molecular markers also make it possible to evaluate QTLs independently and together in different genetic backgrounds, and in combination with transgenes from Bacillus thuringiensis.

Similarity coefficients for molecular markers in studies of genetic relationships between individuals for haploid, diploid, and polyploid species

Determining true genetic dissimilarity between individuals is an important and decisive point for clustering and analysing diversity within and among populations, because different dissimilarity indices may yield conflicting outcomes. We show that there are no acceptable universal approaches to assessing the dissimilarity between individuals with molecular markers. Different measures are relevant to dominant and codominant DNA markers depending on the ploidy of organisms. The Dice coefficient is the suitable measure for haploids with codominant markers and it can be applied directly to (0,1)-vectors representing banding profiles of individuals. None of the common measures, Dice, Jaccard, simple mismatch coefficient (or the squared Euclidean distance), is appropriate for diploids with codominant markers. By transforming multiallelic banding patterns at each locus into the corresponding homozygous or heterozygous states, a new measure of dissimilarity within locus was developed and expanded to assess dissimilarity between multilocus states of two individuals by averaging across all codominant loci tested. There is no rigorous well-founded solution in the case of dominant markers. The simple mismatch coefficient is the most suitable measure of dissimilarity between banding patterns of closely related haploid forms. For distantly related haploid individuals, the Jaccard dissimilarity is recommended. In general, no suitable method for measuring genetic dissimilarity between diploids with dominant markers can be proposed. Banding patterns of diploids with dominant markers and polyploids with codominant markers represent individuals' phenotypes rather than genotypes. All dissimilarity measures proposed and developed herein are metrics.

Carcinoma micropapilar de vejiga: aportación de un caso y revisión de la bibliografía

Micropapillary carcinoma is an uncommon pathologic variant of bladder carcinoma with aggressive behavior. Its usual presentation is like a high grade and high stage carcinoma and associated with other histologic types in different proportion. It doesn´t differ clinically from normal transitional cell carcinoma of the bladder. Studies of molecular markers are still contradictories. Treatment should be early and aggresive, based on surgical therapy as radiotherapy and chemotherapy have shown limited results.We report a 72 years old man suffering from low urinary tract symptoms for years and recently presented gross hematuria. He was diagnosed as high stage micropapillary carcinoma. One year after radical cystectomy and subsequent chemotherapy based on carboplatin and gemcitabine , progression of the disease was shown on CT and the patient died 14 months after the diagnosis.

Physical Mapping and Chromosomal Location of the Photoperiod Response Gene Ppd2 in Common Wheat

Duration from sowing to flowering is of critical importance for crop adaptation. Breeding for the optimal flowering time is one of the major factors, which confer maximal yields in a given environment. The timing of flowering is modulated primarily by responsiveness to photoperiod and temperature, and there are large differences in sensitivity among genotypes. In most spring wheat cultivars the duration of the vegetative period is strongly influenced by genes controlling photoperiod response. In this paper we report the photoperiod responses of a set of ‘Chinese Spring’ substitution and partial deletion lines of homoeologous group 2 chromosomes studied under natural short days. We confirmed the presence of dominant genes Ppd1 and Ppd2 on chromosomes 2D and 2B, respectively, and compared the effects of Ppd1 and Ppd2 for daylength response. The photoperiodic responses of partial chromosome deletion lines of ‘Chinese Spring’ allowed us to map Ppd2 physically between breakpoints 0.27 and 0.53 of the short arm of chromosome 2B. This result is consistent with the finding of a possible location of Ppd2 based on molecular marker studies. A dosage effect of the dominant gene Ppd2 was shown.

Genetical and Mathematical Properties of Similarity and Dissimilarity Coefficients Applied in Plant Breeding and Seed Bank Management

A proper choice of a dissimilarity measure is important in surveys investigating genetic relationships among germplasm with molecular marker data. The objective of our study was to examine 10 dissimilarity coefficients widely used in germplasm surveys, with special focus on applications in plant breeding and seed banks. In particular, we (i) investigated the genetical and mathematical properties of these coefficients, (ii) examined consequences of these properties for different areas of application in plant breeding and seed banks, and (iii) determined relationships between these 10 coefficients. The genetical and mathematical concepts of the coefficients were described in detail. A Procrustes analysis of a published data set consisting of seven CIMMYT maize populations demonstrated close affinity between Euclidean, Rogers', modified Rogers', and Cavalli-Sforza and Edwards' distance on one hand, and Nei's standard and Reynolds dissimilarity on the other hand. Our investigations show that genetical and mathematical properties of dissimilarity measures are of crucial importance when choosing a genetic dissimilarity coefficient for analyzing molecular marker data. The presented results assist experimenters to extract the maximum amount of information from genetic data and, thus, facilitate the interpretation of findings from molecular marker studies on a theoretically sound basis.

Genome Relationship among Nine Species of Millettieae (Leguminosae: Papilionoideae) Based on Random Amplified Polymorphic DNA (RAPD)

Random amplified polymorphic DNA (RAPD) marker was used to establish intergeneric classification and phylogeny of the tribe Millettieae sensu Geesink (1984) (Leguminosae: Papilionoideae) and to assess genetic relationship between 9 constituent species belonging to 5 traditionally recognized genera under the tribe. DNA from pooled leaf samples was isolated and RAPD analysis performed using 25 decamer primers. The genetic similarities were derived from the dendrogram constructed by the pooled RAPD data using a similarity index, which supported clear grouping of species under their respective genera, inter- and intra-generic classification and phylogeny and also merger of Pongamia with Millettia. Elevation of Tephrosia purpurea var. pumila to the rank of a species (T. pumila) based on morphological characteristics is also supported through this study of molecular markers.

Tracking movement in small insect pests, with special reference to aphid populations

Insects are among the greatest pests of agriculture, horticulture and forestry world-wide, inflicting damage and economic costs both directly and by transmitting plant viruses. Many kinds of insects are now resistant or cross-resistant to pesticides. In order to combat these pests, including for purposes of immediate control or to follow movements in order to better understand pest biology, tracking studies are important. In turn, such studies become useful for modelling and forecasting outbreaks. This is especially so for flying insects, which constitute the greatest threat in terms of the dissemination of plant pathogenic viral diseases. Tracking the aerial displacement of small insects over large spatial scales is difficult mainly because of their size. Dilution effects upon take off, together with displacement by air currents, makes the recapture of individuals tagged e.g. by fluorescent or radio-labelling, highly unlikely and hence estimates of flight direction, speed, duration and distance are hampered, more especially over larger distances. This is generally true also for larger insects. Consequently, molecular markers have been employed in attempts to resolve population structure and dynamics by inference from estimates of gene flow. In this article, we describe some of the methodologies devised that have provided invaluable information relating to the movement of small insect pests, particularly aphids, and explain how these techniques can be added to by detail gained from molecular marker studies. We outline a national survey-system that could be managed by specialist centres and involving members of the Public, that would enable invasive pest insect populations to be tracked efficiently. The logistics of such a survey are discussed along with the benefits. Overall, an insect tracking mandate is described that will promote the standardisation of population movement measurements. Adopting this approach should allow more accurate intra- as well as interspecies comparisons.

The CARI guidelines. CMV disease and kidney transplant: treatment of cytomegalovirus disease in renal transplant recipients.

The CARI guidelines. CMV disease and kidney transplant: treatment of cytomegalovirus disease in renal transplant recipients.

Tissue microarrays (TMAS) in colorectal cancer: Study of clinical and molecular markers

Tissue microarrays (TMAS) in colorectal cancer: Study of clinical and molecular markers

Tissue microarrays (TMAS) in colorectal cancer: Study of clinical and molecular markers

9665 Background: TMAS are a powerful tool for testing a large number of molecular parameters in hundreds of tissue samples. This technique can help us to improve our knowledge of molecular markers used to predict disease evolution.To study clinical factors and the expression pattern of proteins including Rb, p53, ki67, p21, p16, p27, cyclins (A, B1, D1, D3 y E), cyclin dependent kinases (cdks) 1, 2 and 6, Survivin, bcl-2 and activated caspase 3 in 71 patients diagnosed of carcinoma colorectal, of which 31 had metastasic disease at diagnosis 22 relapsed and 18 were of free disease. Patients were included in multicentric clinical trials and had received chemotherapy. Methods: Two TMAS were constructed by acquiring 1 mm cylindrical biopsies from each of formalin-fixed paraffin-embedded tumors and controls and spaced 1.5 mm. As dependent variables we analysed overall survival (OS) and survival after relapsed (SV2). As independent variables we studied age, gender, tumor localization, TNM, lymph node involvement, differentiation grade, liver metastases, symptoms at metastatic disease, and single or multiple metastatic localizations. The univariate analysis was carried out for each one of the variables by means of the Kaplan-Meier method and the multivariate analysis was performed using the Cox regression model. Results: According to the univariate analysis the statistically significant variables were differentiation grade, liver metastasis, symptoms, number localization, cdk1,cdk 2,cdk6, cyclin A, D3, ki-67, p53 and survivin for OS, and differentiation grade, symtoms, number localization, activated caspase 3, cdk2, cdk6, cyclin D3, ki67, p21 and survivin for SV2. In the multivariate analysis those variables that reached a significant value p≤ 0.2 were included. Conclusions: In this study clinical factors such as differentiation grade, symptoms, number localization were independent prognostic factors for OS and symptoms for SV2; none of proteins analysed correlated with OS or SV2 in multivariate analysis. No significant financial relationships to disclose.

Genetic resources in maritime pine ( Pinus pinaster Aiton): molecular and quantitative measures of genetic variation and differentiation among maternal lineages

Pinus pinaster is a conifer native to western Europe and northern Africa. Following on-going breeding programmes, provenance and progeny trials were established in some of the countries of the species’ range (France, Portugal and Spain) and quantitative traits were measured: growth, stem form, survival and pest and disease resistance, amongst others. Populations from the wide range of P. pinaster were recently screened with molecular markers in order to assess their genetic diversity. Data were obtained using allozymes, chloroplast (cpSSRs) and nuclear (nuSSRs) microsatellites and amplified fragment length polymorphisms (AFLPs). Based on mtDNA-RFLP analysis, three maternal lineages (named “western”, “eastern” and “Moroccan”) were identified and no population showed a mixed composition. In this study, the imprint that differentiation in putatively isolated glacial refugia (identified by the different maternal lineages) might have left on the nuclear genome was analysed using a wide range of molecular markers and adaptive traits. Multivariate ordination of populations based on nuclear molecular markers (allozymes and nuSSRs) showed a clear clustering of provenances sharing a given mtDNA lineage. However, that clustering was found to be less tight when only quantitative traits were investigated. In P. pinaster, the within-population estimates of gene diversity using different traits were not correlated. Therefore, caution is advisable when designing conservation strategies based on molecular marker studies or a limited number of populations. After these results, we recommend a conservation strategy characterised by gene flow consistent with the current population structure, careful seed transfer among maternal lineages (if any), selection of populations for conservation based on the originality of their allelic composition and definition of Management Units (MUs) based on adaptive traits.