Integration of hybridization-based markers (overgos) into physical maps for comparative and evolutionary explorations in the genus Oryza and in Sorghum

BACKGROUND: With the completion of the genome sequence for rice (Oryza sativa L.), the focus of rice genomics research has shifted to the comparison of the rice genome with genomes of other species for gene cloning, breeding, and evolutionary studies. The genus Oryza includes 23 species that shared a common ancestor 8–10 million years ago making this an ideal model for investigations into the processes underlying domestication, as many of the Oryza species are still undergoing domestication. This study integrates high-throughput, hybridization-based markers with BAC end sequence and fingerprint data to construct physical maps of rice chromosome 1 orthologues in two wild Oryza species. Similar studies were undertaken in Sorghum bicolor, a species which diverged from cultivated rice 40–50 million years ago. RESULTS: Overgo markers, in conjunction with fingerprint and BAC end sequence data, were used to build sequence-ready BAC contigs for two wild Oryza species. The markers drove contig merges to construct physical maps syntenic to rice chromosome 1 in the wild species and provided evidence for at least one rearrangement on chromosome 1 of the O. sativa versus Oryza officinalis comparative map. When rice overgos were aligned to available S. bicolor sequence, 29% of the overgos aligned with three or fewer mismatches; of these, 41% gave positive hybridization signals. Overgo hybridization patterns supported colinearity of loci in regions of sorghum chromosome 3 and rice chromosome 1 and suggested that a possible genomic inversion occurred in this syntenic region in one of the two genomes after the divergence of S. bicolor and O. sativa. CONCLUSION: The results of this study emphasize the importance of identifying conserved sequences in the reference sequence when designing overgo probes in order for those probes to hybridize successfully in distantly related species. As interspecific markers, overgos can be used successfully to construct physical maps in species which diverged less than 8 million years ago, and can be used in a more limited fashion to examine colinearity among species which diverged as much as 40 million years ago. Additionally, overgos are able to provide evidence of genomic rearrangements in comparative physical mapping studies

Quantitative miRNA Expression Analysis Using Fluidigm Microfluidics Dynamic Arrays

MicroRNA (miRNA) is a small non-coding RNA that can regulate gene expression in both plants and animals. Studies showed that miRNAs play a critical role in human cancer by targeting messenger RNAs that are positive or negative regulators of cell proliferation and apoptosis. Here, we evaluated miRNA expression in formalin fixed, paraffin embedded (FFPE) samples and fresh frozen (FF) samples using a high throughput qPCR-based microfluidic dynamic array technology (Fluidigm). We compared the results to hybridization-based microarray platforms using the same samples. We obtained a highly correlated Ct values between multiplex and single-plex RT reactions using standard qPCR assays for miRNA expression. For the same samples, the microfluidic technology (Fluidigm 48.48 dynamic array systems) resulted in a left shift towards lower Ct values compared to those observed by standard TaqMan (ABI 7900HT, mean difference, 3.79). In addition, as little as 10ng total RNA was sufficient to reproducibly detect up to 96 miRNAs at a wide range of expression values using a single 96-multiplexing RT reaction in either FFPE or FF samples. Comparison of miRNAs expression values measured by microfluidic technology with those obtained by other array and Next Generation sequencing platforms showed positive concordance using the same samples but revealed significant differences for a large fraction of miRNA targets. The qPCRarray based microfluidic technology can be used in conjunction with multiplexed RT reactions for miRNA gene expression profiling. This approach is highly reproducible and the results correlate closely with the existing singleplex qPCR platform while achieving much higher throughput at lower sample input and reagent usage. It is a rapid, cost effective, customizable array platform for miRNA expression profiling and validation. However, comparison of miRNA expression using different platforms requires caution and the use of multiple platforms

Prevalence and pattern of hypodontia in the permanent dentition of 3374 Iranian orthodontic patients

Background : Hypodontia is the most common dental anomaly and might cause clinical complications. The aim of this study was to assess the prevalence and pattern of congenital missing in the permanent dentition dentition (excluding third molars), among Iranian orthodontic patients. Materials and Methods : In this descriptive cross-sectional study, all approved panoramic radiographs of 3374 orthodontic patients (aged 10 to 20 years old), who had visited the Orthodontic Departments of all Tehran Dentistry Universities and 10 private clinics during the years 1999 to 2009 were investigated, to establish the prevalence of hypodontia in the permanent dentition (excluding third molars). The data were analyzed using a chi-square test (α = 0.01). Results : Included were 2012 female and 1362 male patients. The prevalence of hypodontia was 5.21% (5.86% in females, 4.25% in males). The difference between the genders in terms of missing teeth was not significant (P = 0.202). A total of 298 teeth were missing (166 in females, 132 in males). The average of missing per individual was found to be 1.69 (1.40 missing for each girl, 2.32 for each boy). There was no significant difference between the number of missing teeth in males and females (P = 0.160). The most common missing teeth were maxillary lateral incisors (37.2%), mandibular second premolars (22.1%), and mandibular central incisors (10.7%). In both unilateral and bilateral hypodontia cases, the maxillary lateral had the highest prevalence of missing, followed by the mandibular second premolar. Missing was significantly more frequent (P = 0.001) in the maxilla (5.3%) compared to the mandible (3.5%). Conclusion : Out of every 20 Iranian orthodontic patients, one might have some missing permanent teeth, needing early attention. Hypodontia was more prevalent in females (though not significantly) and in the maxilla. Although more females were affected, the number of missing per individual was greater in males

Development of a larval-settlement assay protocol for the serpulid polychaete, Galeolaria caespitosa

Static settlement assays are considered the standard tool for determining the settlement preferences of marine invertebrates. Often used to assess and evaluate the properties of a given substrate or biofilm cues for coloni- sation, the static assay format is technically simple, rapid, and inexpensive. Galeolaria caespitosa is a sessile, filter-feeding polychaete worm that inhabits mid to low intertidal regions of exposed rocky shores. Mature adults of G. caespitosa are fertile throughout the year, easy to spawn and rear to settlement competency, and their gregarious settlement behaviour enables easy collection making them an ideal test species. Here we report an optimised protocol for larval settlement assays with G. caespitosa. Unlike other serpulid polychaetes, a bacterial biofilm alone was not sufficient to consistently induce settlement. Instead, a conspecific cue was required for reliable settlement under assay conditions. Yet empty tubes and a homogenate of crushed adult worms had no significant impact on settlement compared to a control, and both treatments showed high variance, indu- cing < 5% of larvae to settle. Only the presence of live conspecific worms consistently induced settlement to the extent necessary for useful assays. In this case, after 48 h the proportion of larvae to have successfully settled reached 44%. On average, live conspecific worms increased settlement by 87% compared to the presence of a biofilm alone. The static settlement assay protocol developed in this study provides a reproducible tool for assessing invertebrate settlement with G. caespitosa. Guidelines are provided for adapting this approach to other species

Transcriptomic and Immunohistochemical Profiling of SLC6A14 in Pancreatic Ductal Adenocarcinoma

We used a target-centric strategy to identify transporter proteins upregulated in pancreatic ductal adenocarcinoma (PDAC) as potential targets for a functional imaging probe to complement existing anatomical imaging approaches. We performed transcriptomic profiling (microarray and RNASeq) on histologically confirmed primary PDAC tumors and normal pancreas tissue from 33 patients, including five patients whose tumors were not visible on computed tomography. Target expression was confirmed with immunohistochemistry on tissue microarrays from 94 PDAC patients. The best imaging target identified was SLC6A14 (a neutral and basic amino acid transporter). SLC6A14 was overexpressed at the transcriptional level in all patients and expressed at the protein level in 95% of PDAC tumors. Very little is known about the role of SLC6A14 in PDAC and our results demonstrate that this target merits further investigation as a candidate transporter for functional imaging of PDAC

Additional file 1: Table S1. of The Hedgehog pathway as targetable vulnerability with 5-azacytidine in myelodysplastic syndrome and acute myeloid leukemia

A–D