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Toll-like receptor 1, when dimerized with Toll-like receptor 2, is a cell surface receptor that, upon recognition of bacterial lipoproteins, activates the innate immune system. Variants in TLR1 associate with the risk of a variety of medical conditions and diseases, including sepsis, leprosy, tuberculosis, and others. The foremost of these is rs5743618 c.2079T>G(p.(Ile602Ser)), the derived allele of which is associated with reduced risk of sepsis, leprosy, and other diseases. Interestingly, 602Ser, which shows signatures of selection, inhibits TLR1 surface trafficking and subsequent activation of NFκB upon recognition of a ligand. This suggests that reduced TLR1 activity may be beneficial for human health. To better understand TLR1 variation and its link to human health, we have typed all 7 high-frequency missense variants (>5% in at least one population) along with 17 other variants in and around TLR1 in 2548 individuals from 56 populations from around the globe. We have also found additional signatures of selection on missense variants not associated with rs5743618, suggesting that there may be multiple functional alleles under positive selection in this gene.

Enteropathogenic Escherichia coli (EPEC) continues to be a leading cause of mortality and morbidity in children around the world. Two EPEC genomes have been fully sequenced: those of EPEC O127:H6 strain E2348/69 (United Kingdom, 1969) and EPEC O55:H7 strain CB9615 (Germany, 2003). The O55:H7 serotype is a recent precursor to the virulent enterohemorrhagic E. coli O157:H7. To explore the diversity of O55:H7 and better understand the clonal evolution of O157:H7, we fully sequenced EPEC O55:H7 strain RM12579 (California, 1974), which was collected 1 year before the first U.S. isolate of O157:H7 was identified in California. Phage-related sequences accounted for nearly all differences between the two O55:H7 strains. Additionally, O55:H7 and O157:H7 strains were tested for the presence and insertion sites of Shiga toxin gene ( stx )-containing bacteriophages. Analysis of non-phage-associated genes supported core elements of previous O157:H7 stepwise evolutionary models, whereas phage composition and insertion analyses suggested a key refinement. Specifically, the placement and presence of lambda-like bacteriophages (including those containing stx ) should not be considered stable evolutionary markers or be required in placing O55:H7 and O157:H7 strains within the stepwise evolutionary models. Additionally, we suggest that a 10.9-kb region (block 172) previously believed unique to O55:H7 strains can be used to identify early O157:H7 strains. Finally, we defined two subsets of O55:H7 strains that share an as-yet-unobserved or extinct common ancestor with O157:H7 strains. Exploration of O55:H7 diversity improved our understanding of the evolution of E. coli O157:H7 and suggested a key revision to accommodate existing and future configurations of stx -containing bacteriophages into current models.

In this study, we report a whole-genome single nucleotide polymorphism (SNP)-based evolutionary approach to study the epidemiology of a multistate outbreak of Salmonella enterica subsp. enterica serovar Montevideo. This outbreak included 272 cases that occurred in 44 states between July 2009 and April 2010. A case-control study linked the consumption of salami made with contaminated black and red pepper to the outbreak. We sequenced, on the SOLiD System, 47 isolates with XbaI PFGE pattern JIXX01.0011, a common pulsed-field gel electrophoresis (PFGE) pattern associated with isolates from the outbreak. These isolates represented 20 isolates collected from human sources during the period of the outbreak and 27 control isolates collected from human, food, animal, and environmental sources before the outbreak. Based on 253 high-confidence SNPs, we were able to reconstruct a tip-dated molecular clock phylogeny of the isolates and to assign four human isolates to the actual outbreak. We developed an SNP typing assay to rapidly discriminate between outbreak-related cases and non-outbreak-related cases and tested this assay on an extended panel of 112 isolates. These results suggest that only a very small percentage of the human isolates with the outbreak PFGE pattern and obtained during the outbreak period could be attributed to the actual pepper-related outbreak (20%), while the majority (80%) of the putative cases represented background cases. This study demonstrates that next-generation-based SNP typing provides the resolution and accuracy needed for outbreak investigations of food-borne pathogens that cannot be distinguished by currently used subtyping methods.

Autosomal DNA polymorphisms can pro- vide new information and understanding of both the ori- gins of and relationships among modern Native American populations. At the same time that autosomal markers can be highly informative, they are also susceptible to ascer- tainment biases in the selection of the markers to use. Identifying markers that can be used for ancestry inference among Native American populations can be considered sep- arate from identifying markers to further the quest for his- tory. In the current study, we are using data on nine Native American populations to compare the results based on a large haplotype-based dataset with relatively small inde- pendent sets of single nucleotide polymorphisms. We are interested in what types of limited datasets an individual laboratory might be able to collect are best for addressing two different questions of interest. First, how well can we differentiate the Native American populations and/or infer ancestry by assigning an individual to her population(s) of origin? Second, how well can we infer the historical/ evolutionary relationships among Native American popula- tions and their Eurasian origins? We conclude that only a large comprehensive dataset involving multiple autosomal markers on multiple populations will be able to answer both questions; different small sets of markers are able to answer only one or the other of these questions. Using our largest dataset, we see a general increasing distance from Old World populations from North to South in the New World except for an unexplained close relationship between our Maya and Quechua samples. Am J Phys Anthropol 146:495-502, 2011. V

The purpose of this study was to evaluate the system capabilities of a new single-molecule detection platform capable of both genomic and proteomic analysis of cellular pathways using very small amounts of tumor material. The system has 4-color optics, single-fluorophore detection capability, localization of molecules to within a 20nm area, a flow cell with an area of 940 mm2 and the ability to detect > 109 molecules on the surface. NSCLC cell lines were cultured and untreated or treated with the EGFR tyrosine kinase inhibitor erlotinib. Cells were harvested and used to generate protein lysates, isolate mRNA and gDNA. Cellular molecules were covalently attached to an epoxysilane-coated surface on the flow cell and then repeatedly probed 10-15 times with labeled antibodies and oligonucleotides to detect, count and quantitate proteins, protein phosphorylation levels, mRNAs, fusion transcripts, and DNA mutations. We detected protein phosphorylation changes for EGFR, ERK, MET and MEK comparable to that observed by traditional western blots; however, the system required only 0.5 to 2 cell equivalents of protein lysate, containing sub pico-Molar levels of protein. We demonstrated robust and statistically significant detection of 2-fold changes in protein levels across 3 slides, in triplicate lanes and performed over 3 non-consecutive days by 3 operators (Tukey-Kramer tests). We also detected 8 mRNAs, with no PCR amplification required, in a multiplexed format with relative levels similar to that observed with TaqMan® qPCR tests. We demonstrated protein detection at ~ 100% recovery compared to an ELISA test and nucleic acid recovery at ~ 30% with no amplification. We also detected exon 19 deletions in EGFR, point mutations at L858R, T790M in EGFR and V600E in BRAF, at 0.5% minor allele levels, by using an oligonucleotide ligation assay off-chip and then attaching the ligated product to the surface. mRNA levels were detected using 10-20 cell equivalents of RNA. We believe the system will enable comprehensive analysis of cancer-related pathways, requiring only a few cells, to help decipher cellular pathway activity induced by driver mutations and consequently help with selection of efficacious drugs tailored for specific individuals based on comprehensive molecular analysis. Citation Format: Manohar R. Furtado, Rixun Fang, Norman Burns, Rebekah Burich, Catherine Le, Soumya Ivaturi, Rob Hartlage, Ed Zizminskas, Jim Jahncke, Philip C. Mack, Bryan P. Staker. A single molecule detection system for comprehensive analysis of cancer related cellular pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 415.

The HID EVOlution—qPCR/STR Setup System enables automation of DNA quantitative real-time polymerase chain reaction (PCR) setup, normalization of DNA sample, and PCR setup for short tandem repeat (STR) analysis. The HID EVOlution System tracks sample and reagent information and facilitates data transfer of DNA quantification, normalization, and PCR setup for STR analysis steps, eliminating the need for manual processing and repetitive data entry. Instruments for the automated system include a Tecan Freedom EVO 150 robot for liquid handling, the 7500 Real-Time PCR System for DNA quantification, the GeneAmp PCR System 9700 for STR amplification, and the 3130 xl Genetic Analyzer for the detection of amplified STR fragments. Validation studies including reproducibility, accuracy, correlation, and contamination studies were performed. Results demonstrated clean liquid-handling capabilities and maintenance of sample integrity. Variation in average allele peak height obtained using automated protocol was similar to that obtained using the manual protocol.

Nonrecombining Y-chromosomal microsatellites (Y-STRs) are widely used to infer population histories, discover genealogical relationships, and identify males for criminal justice purposes. Although a key requirement for their application is reliable mutability knowledge, empirical data are only available for a small number of Y-STRs thus far. To rectify this, we analyzed a large number of 186 Y-STR markers in nearly 2000 DNA-confirmed father-son pairs, covering an overall number of 352,999 meiotic transfers. Following confirmation by DNA sequence analysis, the retrieved mutation data were modeled via a Bayesian approach, resulting in mutation rates from 3.78 x 10(-4) (95% credible interval [CI], 1.38 x 10(-5) - 2.02 x 10(-3)) to 7.44 x 10(-2) (95% Cl, 6.51 x 10(-2) - 9.09 x 10(-2)) per marker per generation. With the 924 mutations at 120 Y-STR markers, a nonsignificant excess of repeat losses versus gains (1.16:1), as well as a strong and significant excess of single-repeat versus multirepeat changes (25.23:1), was observed. Although the total repeat number influenced Y-STR locus mutability most strongly, repeat complexity, the length in base pairs of the repeated motif, and the father's age also contributed to Y-STR mutability. To exemplify how to practically utilize this knowledge, we analyzed the 13 most mutable Y-STRs in an independent sample set and empirically proved their suitability for distinguishing close and distantly related males. This finding is expected to revolutionize Y-chromosomal applications in forensic biology, from previous male lineage differentiation toward future male individual identification.

Null mutant mice lacking the transcription factor pancreatic and duodenal homeobox 1 (Pdx1) are apancreatic and survive only a few days after birth. The role of Pdx1 in regulating intestinal gene expression has therefore yet to be determined in viable mice with normal pancreatic development. We hypothesized that conditional inactivation of Pdx1 restricted to the intestinal epithelium would alter intestinal gene expression and cell differentiation. Pdx1flox/flox; VilCre mice with intestine-specific Pdx1 inactivation were generated by crossing a transgenic mouse strain expressing Cre recombinase, driven by a mouse villin 1 gene promoter fragment, with a mutant mouse strain homozygous for loxP site-flanked Pdx1. Pdx1 protein is undetectable in all epithelial cells in the intestinal epithelium of Pdx1flox/flox; VilCre mice. Goblet cell number and mRNA abundance for mucin 3 and mucin 13 genes in the proximal small intestine are comparable between Pdx1flox/flox; VilCre and control mice. Similarly, Paneth cell number and expression of Paneth cell-related genes Defa1, Defcr-rs1, and Mmp7 in the proximal small intestine remain statistically unchanged by Pdx1 inactivation. Although the number of enteroendocrine cells expressing chromogranin A/B, gastric inhibitory polypeptide (Gip), or somatostatin (Sst) is unaffected in the Pdx1flox/flox; VilCre mice, mRNA abundance for Gip and Sst is significantly reduced in the proximal small intestine. Conditional Pdx1 inactivation attenuates intestinal alkaline phosphatase (IAP) activity in the duodenal epithelium, consistent with an average 91% decrease in expression of the mouse enterocyte IAP gene, alkaline phosphatase 3 (a novel Pdx1 target candidate), in the proximal small intestine following Pdx1 inactivation. We conclude that Pdx1 is necessary for patterning appropriate gene expression in enterocytes and enteroendocrine cells of the proximal small intestine.

An efficient method to uniquely identify every individual would have value in quality control and sample tracking of large collections of cell lines or DNA as is now often the case with whole genome association studies. Such a method would also be useful in forensics. SNPs represent the best markers for such purposes. We have developed a globally applicable resource of 92 SNPs for individual identification (IISNPs) with extremely low probabilities of any two unrelated individuals from anywhere in the world having identical genotypes. The SNPs were identified by screening over 500 likely/candidate SNPs on samples of 44 populations representing the major regions of the world. All 92 IISNPs have an average heterozygosity >0.4 and the F st values are all

We present data from a multi-laboratory validation study of the Genplex typing system [C. Phillips, et al., Evaluation of the Genplex SNP typing system and a 49plex forensic marker panel, Forensic Sci. Int.: Genet. 1 (2) (2007) 180–185.] (Applied Biosystems), which interrogates a subset of 48 SNPs selected from the panel of 52 previously developed for forensic analysis by the SNP for ID consortium [J.J. Sanchez, et al., A multiplex assay with 52 single nucleotide polymorphisms for human identification, Electrophoresis 27 (9) (2006) 1713–1724.], plus amelogenin. The Genplex technology was developed through modification of the SNPlex™ system (also Applied Biosystems) and utilises oligo-ligation of PCR products followed by probe hybridisation to generate dye-labeled, allele-specific oligonucleotides that are detected with capillary electrophoresis. We compare the success rate of Genplex in typing 55 samples in three laboratories with results obtained from STR typing of the same samples using Powerplex ® 16 (Promega) and SGMPlus ® (Applied Biosystems). The sample set was chosen to mimic extracts encountered in forensic situations and includes low concentration and degraded material as well as mixtures. We demonstrate that the Genplex technique provides a significantly higher success rate than STR-based methods when typing degraded DNA and is also capable of mixture detection up to a level of 1 in 10.

A highly automated RT-PCR-based approach has been established to validate novel human gene predictions with no prior experimental evidence of mRNA splicing (ab initio predictions). Ab initio gene predictions were selected for high-throughput validation using predicted protein classification, sequence similarity to other genomes, colocalization with an MPSS tag, or microarray expression. Initial microarray prioritization followed by RT-PCR validation was the most efficient combination, resulting in approximately 35% of the ab initio predictions being validated by RT-PCR. Of the 7252 novel genes that were prioritized and processed, 796 constituted real transcripts. In addition, high-throughput RACE successfully extended the 5′ and/or 3′ ends of >60% of RT-PCR-validated genes. Reevaluation of these transcripts produced 574 novel transcripts using RefSeq as a reference. RT-PCR sequencing in combination with RACE on ab initio gene predictions could be used to define the transcriptome across all species.

The small intestine matures from a primitive tube into morphologically and functionally distinct regions during gut development. Maximal expression of the genes encoding the digestive enzymes lactase-phlorizin hydrolase and sucrase-isomaltase is spatially restricted to distinct segments along the anterior-posterior axis of the small intestine and is temporally regulated during postnatal maturation. Transcription factors capable of interacting with the intestinal lactase and sucrase gene promoters are candidate regulators of spatio-temporal patterning during gut development and maturation. We aimed to quantitatively examine and compare the relative expression levels of a set of intestine-specific transcription factors along the anterior-posterior gut axis during postnatal maturation. Our analysis was focused on the transcription factors capable of regulating the intestinal lactase and sucrase-isomaltase genes. A real-time PCR protocol was used to quantitatively examine and compare spatially and temporally the relative transcript abundance levels for intestine-specific factors during postnatal intestinal maturation. Distinct spatial expressions patterns were detected along the length of the small intestine for PDX-1, Cdx-2, GATA-4, GATA-5, GATA-6, HNF-1alpha, HNF-1beta and CDP transcription factor genes. There is a general decline in transcript abundance for the factor genes during postnatal maturation. Defining the spatio-temporal expression patterns for intestine-specific transcription factor genes contributes to investigation of the roles that factor gradients play in mediating gut development and differentiation.

Lactase-phlorizin hydrolase gene expression is spatially restricted along the anterior-posterior gut axis. Lactase gene transcription is maximal in the distal duodenum and jejunum in adult mammals and is barely detectable in the proximal duodenum. By contrast, pancreatic duodenal homeobox-1 (PDX-1) protein is expressed maximally in the proximal duodenum. This study aimed to determine the role of PDX-1 in regulating lactase gene promoter activity in intestinal epithelial cells. Caco-2 cells were cotransfected with lactase promoter-reporter constructs in the presence of a PDX-1 expression vector and assayed for luciferase activity. PDX-1 cotransfection results in repression of lactase promoter activity. Sequence analysis of the lactase promoter revealed a putative PDX-1 DNA binding site in the proximal 100-bp lactase gene promoter. EMSAs demonstrated that PDX-1 can interact with the lactase promoter binding site but not with a site in which the core PDX-1 binding sequence TAAT is mutated. Site-directed mutagenesis of the PDX-1 core binding site in the lactase promoter-reporter construct suggests that PDX-1 can function independently of DNA binding to its consensus binding site. Stable overexpression of PDX-1 results in repression of the endogenous human lactase gene in differentiated Caco-2 cells. Given the contrasting spatial expression pattern, PDX-1 may function to specify the anterior boundary of lactase expression in the small intestine and is thus a candidate regulator of anterior spatial restriction in the gut.

The residual small bowel undergoes profound adaptive alterations after surgical resection. GH is considered to have a role in regulation of these adaptive changes, but its precise role is unknown. We investigated the role of GH by studying the response to intestinal resection in rats with isolated GH deficiency. Spontaneous dwarf rats, a strain of rats with congenital isolated GH deficiency, underwent 60% resection of the small intestine and parameters of the response of the intestinal remnant were compared with age-matched GH-deficient rats undergoing transection, GH-normal rats undergoing 60% resection, and nonmanipulated GH-normal rats. Deficiency of GH did not inhibit hyperplasia of the mucosal mass of the intestinal remnant, indicating that GH is not required for regulation of this aspect of the adaptive response. However, GH deficiency resulted in lack of accumulation of mucosal protein, including lack of accumulation of digestive hydrolases. In addition, GH deficiency resulted in alterations in processing of digestive hydrolases of the distal intestine, indicating that GH may have region-specific effects on small intestinal function. We conclude that GH is required for the normal expression of specific components of the adaptive response to massive small intestinal resection, but not for all aspects. The aspects that require GH appear to involve protein synthesis and processing.

Forensic analysts routinely encounter samples containing DNA mixtures from male and female contributors. To obtain interpretable Short Tandem Repeat (STR) profiles and select the appropriate STR analysis methodology, it is desirable to determine relative quantities of male and female DNA, and detect PCR inhibitors. We describe a multiplex assay for simultaneous quantification of human and human male DNA using the ribonuclease P RNA component H1 (RPPH1) human target and the sex determining region Y (SRY) male-specific target. A synthetic oligonucleotide sequence was co-amplified as an internal PCR control. Standard curves were generated using human male genomic DNA. The SRY and RPPH1 assays demonstrated human specificity with minimal cross-reactivity to DNA from other species. Reproducible DNA concentrations were obtained within a range of 0.023–50ng/μl. The assay was highly sensitive, detecting as little as 25pg/μl of human male DNA in the presence of a thousand-fold excess of human female DNA. The ability of the assay to predict PCR inhibition was demonstrated by shifted IPC Ct values in the presence of increasing quantities of hematin and humic acid. We also demonstrate the correlation between the multiplex assay quantification results and the strength of STR profiles generated using the AmpFlSTR ® PCR Amplification kits.

Identification of tissue parts and body fluids is frequently required in crime scene investigations. We analysed expression data from public databases and internal studies to identify 110 highly expressed mRNAs as potential tissue specific markers. Further, we identified specific assays targeting mRNA in body fluids like saliva, semen, vaginal secretions and blood. The capability to pre-amplify small amounts of RNA enabling testing for the presence of multiple mRNA species was demonstrated. D 2005 Elsevier B.V. All rights reserved.

We propose that haplotyped loci with high heterozygosity can be useful in human identification, especially within families, if recombination is very low among the sites. Three or more SNPs extending over small molecular intervals (

Forensic analysts routinely encounter samples containing mixtures of DNA from male and female contributors and PCR inhibitors due to exposure to environmental insults. In order to select the appropriate STR analysis methodology for such samples and obtain optimal results at first pass, it is desirable to determine the relative quantities of male and female DNA and to detect the presence of PCR inhibitors at an early stage in the sample processing workflow. Here we describe a multiplex real-time PCR assay that can provide the desired information in a single reaction. Briefly, the simultaneous quantification of human and human male DNA is achieved by measuring the RPPH1 human target and the SRY male-specific target. At the same time a synthetic sequence is co-amplified as an Internal PCR Control (IPC) to detect the presence of PCR inhibitors. The assay has a good dynamic range (0.023–50 ng/μL) and can detect 25 pg/μL of human male DNA in the presence of ten thousand-fold excess of human female DNA. In addition, the ability of the assay to predict PCR inhibition was demonstrated by shifted IPC C T values in the presence of increasing quantities of hematin. All the real-time PCR results showed a good correlation with the downstream STR profiles obtained from a large set of various sample types therefore demonstrating that this assay can be considered a guiding tool to predict the performance of the STR genotyping kits with forensic samples.

Genomic DNA preparations from 60 human cell lines from the National Cancer Institute (NCI), 48 human cell lines from American Type Culture Collection (ATCC) and 19 embryonic cell lines were profiled for autosomal short tandem repeat (STR) loci using the AmpFℓSTR ® Identifiler ® kit. Each DNA sample was profiled at least twice to ensure consistency and reproducibility of results. The resulting STR profiles were compared with the STR profiles in the database of ATCC. The allele calls for the common loci between the Identifiler ® kit and the database were identical except for one DNA sample, which we attribute to amplification artifacts. We have observed a high percentage of the STR loci exhibiting allelic imbalance. Certain STR loci for some cell lines exhibited 3 or more alleles. This type of observation can result from a unique profile for a given cell line or as the result of clonotypic heterozygosity and is not necessarily due to contamination. Our study demonstrates that STR based technologies are useful for cell line authentication applications. These data, combined with data from other researchers, will enable the development of a standard genotyping protocol for cell line authentication.

The potential value of SNPs for individual identification has been recognized by many researchers and different panels have been proposed. Here we present a new interface in the ALFRED database to access compendia of allele frequencies for several published panels of markers for forensic uses. One of those is our panel of individual identification SNPs (IISNPs) based on samples of 44 populations originating from many parts of the world. Here we also present additional data and additional statistical analyses that continue to support the value of our panel of IISNPs as a universal panel. We also describe initial developments of multiplex methods and various robustness analyses for our 45 marker IISNP panel.

The GenPlex (TM) HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex (TM) HID System using 250500 pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex (TM) HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex (TM) HID System with the most commonly used STR kits, 500 pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500 pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500 pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler (TM) kit (AB), GenPlex (TM) HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex (TM) HID showed a very low mean mach probability, while all STR kits except MiniFiler (TM) had very limited discriminatory power.

Background In the event of biocrimes or infectious disease outbreaks, high-resolution genetic characterization for identifying the agent and attributing it to a specific source can be crucial for an effective response. Until recently, in-depth genetic characterization required expensive and time-consuming Sanger sequencing of a few strains, followed by genotyping of a small number of marker loci in a panel of isolates at or by gel-based approaches such as pulsed field gel electrophoresis, which by necessity ignores most of the genome. Next-generation, massively parallel sequencing (MPS) technology (specifically the Applied Biosystems sequencing by oligonucleotide ligation and detection (SOLiD™) system) is a powerful investigative tool for rapid, cost-effective and parallel microbial whole-genome characterization. Results To demonstrate the utility of MPS for whole-genome typing of monomorphic pathogens, four Bacillus anthracis and four Yersinia pestis strains were sequenced in parallel. Reads were aligned to complete reference genomes, and genomic variations were identified. Resequencing of the B. anthracis Ames ancestor strain detected no false-positive single-nucleotide polymorphisms (SNPs), and mapping of reads to the Sterne strain correctly identified 98% of the 133 SNPs that are not clustered or associated with repeats. Three geographically distinct B. anthracis strains from the A branch lineage were found to have between 352 and 471 SNPs each, relative to the Ames genome, and one strain harbored a genomic amplification. Sequencing of four Y. pestis strains from the Orientalis lineage identified between 20 and 54 SNPs per strain relative to the CO92 genome, with the single Bolivian isolate having approximately twice as many SNPs as the three more closely related North American strains. Coverage plotting also revealed a common deletion in two strains and an amplification in the Bolivian strain that appear to be due to insertion element-mediated recombination events. Most private SNPs (that is, a, variant found in only one strain in this set) selected for validation by Sanger sequencing were confirmed, although rare false-positive SNPs were associated with variable nucleotide tandem repeats. Conclusions The high-throughput, multiplexing capability, and accuracy of this system make it suitable for rapid whole-genome typing of microbial pathogens during a forensic or epidemiological investigation. By interrogating nearly every base of the genome, rare polymorphisms can be reliably discovered, thus facilitating high-resolution strain tracking and strengthening forensic attribution.

The Quantifiler Duo DNA Quantification kit enables simultaneous quantification of human DNA and human male DNA as well as detection of inhibitors of PCR in a single real-time PCR well. Pooled human male genomic DNA is used to generate standard curves for both human (ribonuclease P RNA component H1) and human male (sex determining region Y) specific targets. A shift in the cycle threshold (C(T)) values for the internal positive control monitors the presence of PCR inhibitors in a sample. The assay is human specific and exhibits a high dynamic range from 0.023 to 50 ng/microL. In addition, the multiplex assay can detect as little as 25 pg/microL of human male DNA in the presence of a 1000-fold excess of human female DNA. The multiplex assay provides assessment of the DNA extract and guidance for the selection of the appropriate AmpFlSTR Amplification Kit to obtain interpretable short tandem repeat profiles.

Using a 52 SNP marker set previously developed for forensic analysis, a novel 49plex assay has been developed based on the Genplex typing system, a modification of SNPlex™ chemistry (both Applied Biosystems) using oligo-ligation of pre-amplified DNA and dye-labeled, mobility modified detection probes. This gives highly predictable electrophoretic mobility of the allelic products generated from the assay to allow detection with standard capillary electrophoresis analyzers. The loci chosen comprise the 48 most informative autosomal SNPs from the SNP for ID core discrimination set supplemented with the amelogenin gender marker. These SNPs are evenly distributed across all 22 autosomes, exhibit balanced polymorphisms in three major population groups and have been previously shown to be effective markers for forensic analysis. We tested the accuracy and reproducibility of the Genplex system in three SNP for ID laboratories, each using a different Applied Biosystems Genetic Analyzer. Genotyping concordance was measured using replicates of 44 standardized DNA controls and by comparing genotypes for the same samples generated by the TaqMan ® , SNaPshot ® and Sequenom iPLEX ® SNP typing systems. The degree of informativeness of the 48 SNPs for forensic analysis was measured using previously estimated allele frequencies to derive the cumulative match probability and in paternity analysis using 24 trios previously typed with 18 STRs together with three CEPH families with extensive sibships typed with the 15 STRs in the Identifiler ® kit.

Human embryonic stem (hES) cells hold promise for generating an unlimited supply of cells for replacement therapies. To characterize hES cells at the molecular level, we obtained 148,453 expressed sequence tags (ESTs) from undifferentiated hES cells and three differentiated derivative subpopulations. Over 32,000 different transcripts expressed in hES cells were identified, of which more than 16,000 do not match closely any gene in the UniGene public database. Queries to this EST database revealed 532 significantly upregulated and 140 significantly downregulated genes in undifferentiated hES cells. These data highlight changes in the transcriptional network that occur when hES cells differentiate. Among the differentially regulated genes are several components of signaling pathways and transcriptional regulators that likely play key roles in hES cell growth and differentiation. The genomic data presented here may facilitate the derivation of clinically useful cell types from hES cells.

Thyroid hormone (T3) is an important regulator of gut mucosal development and differentiation, inducing intestinal alkaline phosphatase (IAP) and repressing lactase gene transcription. In contrast, cyclin D1 (CD1) appears to be a growth promoter in the gut, functioning to maintain the undifferentiated state. The present studies were designed to examine the effects of CD1 on T3 action within intestinal epithelia. Caco-2 cells were maintained in hypothyroid medium and transiently transfected with either rat lactase (3.0 kb) or human IAP (2.4 kb) luciferase (Luc) reporter plasmids. Cotransfections were carried out using two T3 receptor (TR) isoforms, TR"-1 and TR$-1, as well as plasmids expressing CD1, CD3, CA, or CB1. Cells were then treated +/- 10 nmol/L T3 for 24 hours and luciferase activity was determined. With T3 treatment, IAP-Luc activity was induced (TR"-1 = eightfold, TR$-1 = ninefold), but these effects were dramatically inhibited (> 50%) by CD1 and CD3. In contrast, CA and CB1 did not alter T3-mediated IAP gene activation. The ability of CD1 and CD3 to inhibit T3 action was also tested in the context of the lactase gene, which is negatively regulated by T3. As expected, lactase reporter gene activity was repressed by T3 treatment in the case of both receptor isoforms, TR"-1 = 30% and TR$-1 = 40%. In contrast to its effects on the IAP gene, CD1 did not inhibit T3-mediated changes in lactase reporter gene activity. The D-type cyclins (CD1 and CD3), but not CA or CB1, specifically inhibit T3-mediated activation of the IAP gene. In contrast, the D-type cyclins do not inhibit T3-mediated repression of the lactase gene. These studies have identified a novel molecular interaction that exists between the pathways of growth and differentiation within intestinal epithelia.

The GATA family of transcription factors regulate tissue-specific patterns of gene expression during development. We have characterized the interaction between GATA proteins and the lactase gene promoter. Nuclear protein bound to the lactase gene GATA region cis element (−97 to −73) was analyzed by electrophoretic mobility shift assays (EMSA) and supershift assays with GATA antibodies. Lactase promoter activities were assayed in Caco-2 cells transfected with wild-type and mutated luciferase promoter-reporter constructs and GATA-4/5/6 expression constructs. EMSA with the GATA region probe yields a specific DNA-protein complex that requires the GATA factor binding site WGATAR. The complex is recognized by GATA-4- and GATA-6-specific antibodies. GATA-4/5/6 expression constructs are able to activate transcription driven by the wild-type promoter, but not by a promoter in which the GATA binding site is mutated, in Caco-2 and nonintestinal QT6 cells. GATA factor binding to the lactase cis element correlates with functional promoter activation. We conclude that each of the GATA family zinc finger proteins expressed in the intestine, GATA-4, -5, and -6, can interact with the lactase promoter GATA element and can function to activate the promoter in Caco-2 cells.

Forensic DNA analysis is a multi-step process involving extraction of DNA, quantification of human DNA in the extract, amplification using multiplex STR systems, separation of products, and data analysis. The backlog of forensic casework is increasing worldwide. Automation is one significant way to alleviate the bottleneck of sample processing in forensic labs. The HID EVOlution™ Combination System described here is a robust, reliable sample processing platform, easily adapted to forensic laboratory workflows. Using a variety of forensic sample types including: blood stained FTA paper, cotton fabric and denim, dried blood spiked with known PCR inhibitors, saliva on cotton swabs, and semen stains, we found that yields of human DNA and STR profiles obtained with AmpF l STR ® Idenitfiler ® kits were complete, highly reproducible, and equivalent to results obtained using the manual PrepFiler™ reagent extraction method. Automated operation was clean, and no cross-contamination was detected between extraction blanks and interspersed high DNA content samples.

SNP profiling is a very powerful tool for human identification. Two panels of 49 and 41 SNPs were selected based on high average heterozygosity (>0.4) and low global Fst values (

We report the results of an inter-laboratory exercise on typing autosomal single nucleotide polymorphisms (SNPs) with the GenPlex™ HID system (Applied Biosystems-AB). A total of 12 European and US forensic genetic laboratories typed 10 reference DNA samples and 3 samples with partly degraded DNA that only gave strong reactions with amplicons shorter than approximately 135 bp and weak reactions up to 185 bp. The samples and the critical reagents were sent to the participating laboratories from Copenhagen. In addition, the laboratories typed the three degraded DNA samples with one or more STR kits. When 500 pg degraded DNA was used, the success rates of SNP typing in the laboratories varied from 15.6% to 98.6% (median: 94.6%). Only 1.6% of the unsuccessful SNP types were due to wrong SNP calling, while the remaining 98.4% were due to lack of amplification and uncertain results. Two laboratories counted for more than two-thirds of the non-successful results while seven laboratories reported no wrong SNP types. Two laboratories typed the degraded DNA samples successfully with the MiniFiler kit, while other commonly used kits gave incomplete STR profiles in all laboratories. Titration of the degraded DNA showed that the SNP typing results became less reliable with 100 pg DNA and less. The median success rate of SNP typing of 10 reference samples with 50 pg reference DNA was 86% (range: 44.5-99.2% among laboratories). The success rate of SNP typing of the reference samples decreased with smaller amounts of DNA. SNP typing with the GenPlex™ HID system provided more information from degraded DNA samples than any of the commercially available kits used by the participating laboratories. © 2009 Elsevier Ireland Ltd.

Background & Aims: Lactase is the intestinal disaccharidase responsible for digestion of lactose, the predominant carbohydrate in milk. Transcription of the lactase gene is activated during enterocyte differentiation. We have characterized the interaction between the lactase promoter and Cdx2, a homeodomain protein involved in regulating intestinal development and differentiation. Methods: Nuclear protein bound to the lactase gene cis element, CE-LPH1, was analyzed by electrophoretic mobility shift assays and supershifts with Cdx2 antibody. Lactase promoter activities were assayed in cells transfected with luciferase reporter constructs and a Cdx2 expression construct. Results: Electrophoretic mobility shift assay with CE-LPH1 yields a specific DNA/protein complex that requires the caudal-related protein binding site, TTTAC. The complex is recognized by Cdx2 antibody and is more abundant in differentiated enterocytes. A Cdx2 expression construct is able to activate transcription driven by the wild-type, but not a mutated, promoter and results in increased endogenous lactase messenger RNA. Conclusions: The homeodomain protein Cdx2 interacts with the lactase promoter and is capable of activating transcription of the endogenous gene. In contrast to a previous report, Cdx2 interaction with the lactase promoter correlates with enterocyte differentiation. These conclusions are consistent with the role of Cdx2 in regulating intestinal cell differentiation. GASTROENTEROLOGY 2000;118:115-127

Rapid and accurate identification of SNPs and mutations are useful in ensuring that the correctly identified cell lines are being used in cancer research studies and for accurate identification of SNPs and indels in cancer cells. In this study we report on a method that uses nucleic acid isolation methods, multiplexed PCR to amplify up to 48 regions within the human genome followed by an oligonucleotide ligation assay that can detect SNPs, mutations and indels. The protocol takes about 4 hrs to complete and uses panels optimized for human cell identification and detection of mutations in EGFR and KRAS genes. We typed the NCI-60 panel and several cell lines from the ATCC using two panels. One is a panel of SNPs that has been tested with samples from over 46 human populations to identify a set of SNPs that would discriminate globally. This SNP set has 48 markers with high heterozygosity (>0.4) and low Fst values ( In conclusion, we have developed a sensitive, highly accurate, high throughput genotyping system. Studies have demonstrated its effectiveness for successful SNP/InDel detection and mutation screening. This system has been successfully used for oncogen mutation studies in cancer cell lines. Overall, the system can become a valuable tool for molecular and clinical diagnostics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3883. doi:10.1158/1538-7445.AM2011-3883

The S-type lectins have been shown to be components of mucosal scrapings, and in avian systems these lectins have been localized immunohistochemically to the mucosal surface and goblet cells of the intestine. The interaction of lectin specifically with purified mucin has not, however, been established. Quail intestinal mucin was purified by two subsequent isopycnic density-gradient centrifugations in CsCl and chromatography on Sepharose Cl-2B. Purified mucin, obtained from the void volume of the Sepharose column, was characterized by SDS/PAGE, amino acid and carbohydrate analyses, sensitivity to thiol reduction, and cross-reactivity with antibody preparations to rat and human intestinal mucins on Western blots. Antibody raised against purified quail mucin partially cross-reacts with purified rat, rabbit and human intestinal mucins, and specifically labels the mucosal surface and goblet cells of quail intestine by the immunoperoxidase technique. Protein eluted by lactose from an affinity matrix composed of quail intestinal mucin possessed the same molecular mass on SDS/PAGE as intestinal lectin and reacted on Western blots with a lectin-specific antibody. The data clearly demonstrate the co-localization of lectin and mucin in the quail intestine and also the ability of the lectin to specifically interact with the purified mucin, raising the question of the role of endogenous lectins in secretions.

Soluble extracts of quail intestine scrapings contain a lectin activity specific for chicken and rabbit trypsinized, glutaraldehyde-fixed erythrocytes. The lectin displayed a specificity for the simple sugar haptens lactose and galactose and for mucin. Quail lectin was purified by affinity chromatography on either asialofetuin- or mucin-Sepharose, followed by DEAE-Sepharose chromatography, and demonstrated an apparent molecular weight of 14 500 on sodium dodecyl sulfate – polyacrylamide gel electrophoresis and a pi of 6.2 upon isoelectric focusing. Immunohistochemical localization of this lectin in the intestine was carried out using polyclonal antibody raised in rabbits and tested for specificity in Western blots. Immunoperoxidase staining for quail lectin showed the lectin to be prominent in secretions at the mucosal surface and in goblet cells.Key words: endogenous lectin, intestinal, mucin, goblet cells.

Thyroid hormone (T3) is an important regulator of gut mucosal development and differentiation, inducing intestinal alkaline phosphatase (IAP) and repressing lactase gene transcription. In contrast, cyclin D1 (CD1) appears to be a growth promoter in the gut, functioning to maintain the undifferentiated state. The present studies were designed to examine the effects of CD1 on T3 action within intestinal epithelia. Caco-2 cells were maintained in hypothyroid medium and transiently transfected with either rat lactase (3.0 kb) or human IAP (2.4 kb) luciferase (Luc) reporter plasmids. Coaansfections were carried out using two T3 receptor (TR) isoforms, TRW1 and TRβ-1, as well as plasmids expressing CDl, CD3, CA, or CB1. Cells were then treated ±10 nmol/L T3 for 24 hours and luciferase activity was determined. with T3 treatment, IAP-Luc activity was induced (TRα-1 = eightfold, TRβ-1 = ninefold), but these effects were dramatically inhibited (>50%) by CD1 and CD3. In contrast, CA and CBl did not alter T3-mediated IAP gene activation. The ability of CD1 and CD3 to inhibit T3 action was also tested in the context of the lactase gene, which is negatively regulated by T3. As expected, lactase reporter gene activity was repressed by T3 treatment in the case of both receptor isoforms, TRW1 = 30% and TRP-1 = 40%. In contrast to its effects on the IAP gene, CD1 did not inhibit T3-mediated changes in lactase reporter gene activity. The D-type cyclins (CD1 and CD3), but not CA or CB1, specifically inhibit T3-mediated activation of the IAP gene. In contrast, the D-type cyclins do not inhibit T3-mediated repression of the lactase gene. These studies have identified a novel molecular interaction that exists between the pathways of growth and differentiation within intestinal epithelia.

Many panels of ancestry informative single nucleotide polymorphisms have been proposed in recent years for various purposes including detecting stratification in biomedical studies and determining an individual's ancestry in a forensic context. All of the panels have limitations in their generality and efficiency for routine forensic work. Some panels have used only a few populations to validate them. Some panels are based on very large numbers of SNPs thereby limiting the ability of others to test different populations. We have been working toward an efficient and globally useful panel of ancestry informative markers that is comprised of a small number of highly informative SNPs. We have developed a panel of 55 SNPs analyzed on 73 populations from around the world. We present the details of the panel and discuss its strengths and limitations.

The mechanism of phytohemagglutinin-induced bacterial overgrowth of the small bowel in the rat was studied. Interaction of the lectin with bacterial isolates selected at random from those that comprised the major population of the over-growth was determined. In both bacterial agglutination assays and glycocalyx stabilization, no specific association between lectin and bacteria was seen. In three independent binding assays phytohemagglutinin was not found to increase bacterial adherence to washed intestinal mucosa. Phytohemagglutinin would not appear to act, therefore, as a direct ligand to mediate bacterial adherence or to modify the mucosal surface to increase bacterial adherence.