Gene Quantitative Analysis Research Articles

5-HMF affects cardiovascular development in zebrafish larvae via reactive oxygen species and Wnt signaling pathways

5-Hydroxymethylfurfural (5-HMF) is a small molecule aldehyde compound produced by the Maillard reaction. As 5-HMF exists in a variety of foods and drugs and is easily ingested by humans, it has attracted extensive toxicological attention in recent years. Relevant research showed that 5-HMF has cytotoxicity, genotoxicity, and tumor effects. However, the cardiovascular effects of 5-HMF are unknown. To investigate the cardiovascular effects of 5-HMF in zebrafish, wild-type and transgenic embryos were treated with 10, 25, and 50 μg/mL of 5-HMF, followed by toxicological evaluation, histological observation, fluorescence observation, cell apoptosis staining, and gene quantitative analysis. High 5-HMF concentrations led to a significant increase in the heart rate and pericardial edema ratio, larger venous sinus–arterial bulb distance, more apoptosis of cardiac cells, cardiac linearization, defects in angiogenesis and cardiovascular development, and apoptosis-related gene expression disorders in zebrafish larvae. The abnormal phenotype caused by 5-HMF can be rescued by antioxidant N-acetyl-L-cysteine (NAC) and Wnt signaling pathway activator BML-284. It is inferred that high 5-HMF concentrations increased the level of reactive oxygen species, inhibited the transduction of the Wnt signaling pathway, and resulted in abnormal cardiovascular development in zebrafish larvae. This study provides a reference for understanding the mechanism of 5-HMF effects on cardiac development.

Zinc pyrithione (ZPT) -induced embryonic toxicogenomic responses reveal involvement of oxidative damage, apoptosis, endoplasmic reticulum (ER) stress and autophagy.

Zinc pyrithione (ZPT) is a frequently used organometallic biocide, carrying potentially adverse consequences to multiple species in the environment. Previously we have demonstrated its embryonic, organ developmental and liver metabolic toxicity of zebrafish. However, details of ZPT toxicity during embryogenesis are still limited. The present study was designed to evaluate the effects and possible mechanisms of ZPT-induced embryonic toxicogenomic responses by morphological investigations, transcriptome and gene quantitative analysis, as well as biochemical assays. The results revealed that treatment with ZPT caused embryogenesis toxicity, specifically in irregular cell division and rearrangement, delayed differentiations of eyes and notochords, the epiboly and germ ring formation and somite segmentation defects. In addition, ZPT exposure altered gene expression during early embryonic development, especially related with morphological abnormities and metabolic dysfunctions including reduction of oxidoreductase activity. Activities of antioxidants and caspases examinations showed inductions of oxidative stress and apoptosis by ZPT and quantitative analysis of marker genes further indicated that ZPT also triggered endoplasmic reticulum (ER) stress and autophagy. Thus, we deduce here that ZPT-induced embryonic toxicogenomic responses reveal involvement of oxidative damage, apoptosis, endoplasmic reticulum (ER) stress and autophagy.

Assessment of the Appearance and Toxin Production Potential of Invasive Nostocalean Cyanobacteria Using Quantitative Gene Analysis in Nakdong River, Korea.

Invasive nostocalean cyanobacteria (INC) were first reported in tropical regions and are now globally spreading rapidly due to climate change, appearing in temperate regions. INC require continuous monitoring for water resource management because of their high toxin production potential. However, it is difficult to analyze INC under a microscope because of their morphological similarity to nostocalean cyanobacteria such as the genus Aphanizomenon. This study calculates the gene copy number per cell for each target gene through quantitative gene analysis on the basis of genus-specific primers of genera Cylindrospermopsis, Sphaerospermopsis, and Cuspidothrix, and the toxin primers of anatoxin-a, saxitoxin, and cylindrospermopsin. In addition, quantitative gene analysis was performed at eight sites in the Nakdong River to assess the appearance of INC and their toxin production potential. Genera Cylindrospermopsis and Sphaerospermopsis did not exceed 100 cells mL−1 at the maximum, with a low likelihood of related toxin occurrence. The genus Cuspidothrix showed the highest cell density (1759 cells mL−1) among the INC. Nakdong River has potential for the occurrence of anatoxin-a through biosynthesis by genus Cuspidothrix because the appearance of this genus coincided with that of the anatoxin-a synthesis gene (anaF) and the detection of the toxin by ELISA.

The Mulberry SPL Gene Family and the Response of MnSPL7 to Silkworm Herbivory through Activating the Transcription of MnTT2L2 in the Catechin Biosynthesis Pathway.

SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, as unique plant transcription factors, play important roles in plant developmental regulation and stress response adaptation. Although mulberry is a commercially valuable tree species, there have been few systematic studies on SPL genes. In this work, we identified 15 full-length SPL genes in the mulberry genome, which were distributed on 4 Morus notabilis chromosomes. Phylogenetic analysis clustered the SPL genes from five plants (Malus × domestica Borkh, Populus trichocarpa, M. notabilis, Arabidopsis thaliana, and Oryza sativa) into five groups. Two zinc fingers (Zn1 and Zn2) were found in the conserved SBP domain in all of the MnSPLs. Comparative analyses of gene structures and conserved motifs revealed the conservation of MnSPLs within a group, whereas there were significant structure differences among groups. Gene quantitative analysis showed that the expression of MnSPLs had tissue specificity, and MnSPLs had much higher expression levels in older mulberry leaves. Furthermore, transcriptome data showed that the expression levels of MnSPL7 and MnSPL14 were significantly increased under silkworm herbivory. Molecular experiments revealed that MnSPL7 responded to herbivory treatment through promoting the transcription of MnTT2L2 and further upregulating the expression levels of catechin synthesis genes (F3′H, DFR, and LAR).

Evaluation method for asymmetric uncertainty of quantitative polymerase chain reaction measurements of deoxyribonucleic acids with low copy number

Recently, in food safety and various other fields, qualitative and quantitative gene analysis using real-time polymerase chain reaction (PCR) method has become increasingly popular. The limit of detection (LOD) and quantifiable range for these measurements depends on the range and precision of DNA calibrators’ concentrations. Low-copy-number nucleic acid reference materials with low uncertainty produced by an inkjet system have been developed to allow for precise measurements in a low-copy-number region. However, when using a calibrator with a low copy number near one, the copy number distribution is asymmetric. Consequently, the confidence intervals of estimated copy numbers can include negative values when conventional methods of uncertainty estimation are used. A negative confidence interval is irrelevant in the context of copy number, which is always positive value or zero. Here, we propose a method to evaluate the uncertainty of real-time PCR measurements with representative values and an asymmetric 95% confidence interval. Moreover, we use the proposed method for the actual calculation of uncertainty of real-time PCR measurement results for low-copy-number DNA samples and demonstrate that the proposed method can evaluate the precision of real-time PCR measurements more appropriately in a low-copy-number region.

Reference gene selection for gene expression study in tissues of long-tailed chickens

Quantitative real-time polymerase chain reaction (RT-qPCR) is a rapid and precise method of analysis to quantify the level of gene expression and is widely used in the diagnosis of diseases and quantitative analysis of genes. In RT-qPCR analysis, a reference gene (or housekeeping gene) is used for normalization of experimental results. Since this method of analysis detects a small quantity of the product, it is highly sensitive and it is important for the accuracy and reproducibility of the experiment to select a reference gene suitable for gene expression studies. As the expression levels of the reference gene are affected under different conditions, in order to determine the suitability of the housekeeping gene used as the reference gene, it is necessary to verify the expression stability. In the current study, the stability of the expression of 11 housekeeping genes (B2M, SDHA, GAPDH, RPL13, VIM, EEF1A1, HPRT1, GUSB, RPL19, ACTB, and ABL1) was investigated in the tissues of long-tailed chickens (heart, thigh, and breast). Expression stability evaluation was analyzed with four software: BestKeeper, NormFinder, geNorm, and RefFinder. In our study, GAPDH in heart tissue, HPRT1 in thigh tissue, and RPL13 in breast tissue were selected as the most stable reference genes. Evaluation of the expression stability of housekeeping genes can provide important data in gene expression studies by selecting an appropriate reference gene according to various conditions.

Different effects of exposure to penconazole and its enantiomers on hepatic glycolipid metabolism of male mice

(±) - PEN is a chiral fungicide widely used to control powdery mildew in agriculture. Currently, only a few studies have investigated the toxic effects of (±) – penconazole ((±) – PEN) on non-target organisms, and whether (±) - PEN from the enantiomeric level have toxic effects remains unclear. In this study, we systematically evaluated the effects of exposure to (±) – PEN, (+) – PEN and (−) – PEN on liver function in mice. Biochemical and histopathological analyses showed that exposure to (±) – PEN and (−) – PEN led to significant liver damage and inflammation. However, exposure to (+) – PEN treatment did not cause no adverse effects on liver function and inflammation. 1H-NMR-based metabolomics revealed that exposure to (±) – PEN, (+) – PEN and (−) – PEN led to the animals developing liver metabolic disorder that was caused by changes in glycolipid metabolism. Quantitative analysis of genes regulating glycolipid metabolism revealed that expression of gluconeogenesis and glycolytic pathway genes were altered in individuals exposed to (±) – PEN, (+) – PEN and (−) – PEN. We also found that (±) – PEN, (+) – PEN and (−) – PEN have different effects on lipid metabolism of the liver. Exposure to (±) – PEN and (−) – PEN resulted in significant accumulation of lipids by regulating fatty acid synthesis, triglyceride synthesis, and fatty acid β oxidation pathways. In summary, we found different toxicological effects in individuals exposed to (±) – PEN, (+) – PEN and (−) – PEN. The results of this study are important for assessing the potential health risks of (±) – PEN.

High throughput quantification of the functional genes associated with RDX biodegradation using the SmartChip real-time PCR system.

The explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a contaminant at many military sites. RDX bioremediation as a clean-up approach has been gaining popularity because of cost benefits compared to other methods. RDX biodegradation has primarily been linked to six functional genes (diaA, nfsI, pnrB, xenA, xenB, xplA). However, current methods for gene quantification have the risk of false negative results because of low theoretical primer coverage. To address this, the current study designed new primer sets using the EcoFunPrimer tool based on sequences collected by the Functional Gene Pipeline and Repository and these were verified based on residues and motifs. The primers were also designed to be compatible with the SmartChip Real-Time PCR system, a massively parallel singleplex PCR platform (high throughput qPCR), that enables quantitative gene analysis using 5,184 simultaneous reactions on a single chip with low volumes of reagents. This allows multiple genes and/or multiple primer sets for a single gene to be used with multiple samples. Following primer design, the six genes were quantified in RDX-contaminated groundwater (before and after biostimulation), RDX-contaminated sediment, and uncontaminated samples. The final 49 newly designed primer sets improved upon the theoretical coverage of published primer sets, and this corresponded to more detections in the environmental samples. All genes, except diaA, were detected in the environmental samples, with xenA and xenB being the most predominant. In the sediment samples, nfsI was the only gene detected. The new approach provides a more comprehensive tool for understanding RDX biodegradation potential at contaminated sites.

Inhibition of Notch signaling pathway using γ-secretase inhibitor delivered by a low dose of Triton-X100 in cultured oral cancer cells

How to effectively delivering therapeutic agents, including γ-secretase inhibitors (GSIs), into live cells, remains a significant challenge. This study assessed the effect of Notch signaling inhibition by examining levels of the Notch1 intracellular domain (N1ICD) in cultured oral cancer cells analyzed with random stitched images (2D) and 3D visualizations using confocal microscopy and quantitative gene analysis. Substantially, we have developed a novel method to assist the delivery of γ-secretase inhibitor, DAPT, into live cells in the presence of an effective minimum concentration of Triton-X100 (0.001%) without damaging cell activity and membrane integrity assessed with cell proliferation assays. The images obtained in this study showed that DAPT alone could not block the γ-secretase inhibitor despite inhibiting cell growth. Further analysis of quantitative gene expressions of Notch signaling canonical pathway to verify the effectiveness of the novel method for delivering inhibitor into live cells, displayed deregulation of Notch1, Delta-like ligand 1 (DLL1) and hairy and enhancer of split 1 (Hes1). Our data suggest that Notch1/Hes1 signaling pathway is deactivated using DAPT with a low dose of Triton-X100 in this cancer cells. And the finding also suggests that Notch1 could be engaged by DLL1 to promote differentiation in oral cancer cells. Using this approach, we demonstrate that Triton-X100 is a promising and effective permeabilization agent to deliver γ-secretase inhibitor DAPT into live oral epithelial cells. This strategy has the potential to implicate in the treatment of cancer diseases.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase deficiency: urinary organic acid profiles and expanded spectrum of mutations.

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase (HMCS2) deficiency results in episodes of hypoglycemia and increases in fatty acid metabolites. Metabolite abnormalities described to date in HMCS2 deficiency are nonspecific and overlap with other inborn errors of metabolism, making the biochemical diagnosis of HMCS2 deficiency difficult. Urinary organic acid profiles from periods of metabolic decompensation were studied in detail in HMCS2-deficient patients from four families. An additional six unrelated patients were identified from clinical presentation and/or qualitative identification of abnormal organic acids. The diagnosis was confirmed by sequencing and deletion/duplication analysis of the HMGCS2 gene. Seven related novel organic acids were identified in urine profiles. Five of them (3,5-dihydroxyhexanoic 1,5 lactone; trans-5-hydroxyhex-2-enoate; 4-hydroxy-6-methyl-2-pyrone; 5-hydroxy-3-ketohexanoate; 3,5-dihydroxyhexanoate) were identified by comparison with synthesized or commercial authentic compounds. We provisionally identified trans-3-hydroxyhex-4-enoate and 3-hydroxy-5-ketohexanoate by their mass spectral characteristics. These metabolites were found in samples taken during periods of decompensation and normalized when patients recovered. When cutoffs of adipic >200 and 4-hydroxy-6-methyl-2-pyrone >20 μmol/mmol creatinine were applied, all eight samples taken from five HMCS2-deficient patients during episodes of decompensation were flagged with a positive predictive value of 80% (95% confidence interval 35-100%). Some ketotic patients had increased 4-hydroxy-6-methyl-2-pyrone. Molecular studies identified a total of 12 novel mutations, including a large deletion of HMGCS2 exon 1 in two families, highlighting the need to perform quantitative gene analyses. There are now 26 known HMGCS2 mutations, which are reviewed in the text. 4-Hydroxy-6-methyl-2-pyrone and related metabolites are markers for HMCS2 deficiency. Detection of these metabolites will streamline the biochemical diagnosis of this disorder.

Linking alkaline phosphatase activity with bacterial phoD gene abundance in soil from a long-term management trial

Changes in land management practices may have significant implications for soil microbial communities important in organic P turnover. Soil bacteria can increase plant P availability by excreting phosphatase enzymes which catalyze the hydrolysis of ester-phosphate bonds. Examining the diversity and abundance of alkaline phosphatase gene harboring bacteria may provide valuable insight into alkaline phosphatase production in soils. This study examined the effect of 20years of no input organic (ORG), organic with composted manure (ORG+M), conventional (CONV) and restored prairie (PRA) management on soil P bioavailability, alkaline phosphatase activity (ALP), and abundance and diversity of ALP gene (phoD) harboring bacteria in soils from the northern Great Plains of Canada. Management system influenced bioavailable P (P<0.001), but not total P, with the lowest concentrations in the ORG systems and the highest in PRA. Higher rates of ALP were observed in the ORG and ORG+M treatments with a significant negative correlation between bioavailable P and ALP in 2011 (r2=0.71; P=0.03) and 2012 (r2=0.51; P=0.02), suggesting that ALP activity increased under P limiting conditions. The phoD gene abundance was also highest in ORG and ORG+M resulting in a significant positive relationship between bacterial phoD abundance and ALP activity (r2=0.71; P=0.009). Analysis of phoD bacterial community fingerprints showed a higher number of species in CONV compared to ORG and ORG+M, contrary to what was expected considering greater ALP activity under ORG management. In 2012, banding profiles of ORG+M showed fewer phoD bacterial species following the second manure application, although ALP activity is higher than in 2011. This indicates that a few species may be producing more ALP and that quantitative gene analysis was a better indicator of activity than the number of species present.

Deep conservation of genes required for both Drosphila melanogaster and Caenorhabditis elegans sleep includes a role for dopaminergic signaling.

Cross-species conservation of sleep-like behaviors predicts the presence of conserved molecular mechanisms underlying sleep. However, limited experimental evidence of conservation exists. Here, this prediction is tested directly. During lethargus, Caenorhabditis elegans spontaneously sleep in short bouts that are interspersed with bouts of spontaneous locomotion. We identified 26 genes required for Drosophila melanogaster sleep. Twenty orthologous C. elegans genes were selected based on similarity. Their effect on C. elegans sleep and arousal during the last larval lethargus was assessed. The 20 most similar genes altered both the quantity of sleep and arousal thresholds. In 18 cases, the direction of change was concordant with Drosophila studies published previously. Additionally, we delineated a conserved genetic pathway by which dopamine regulates sleep and arousal. In C. elegans neurons, G-alpha S, adenylyl cyclase, and protein kinase A act downstream of D1 dopamine receptors to regulate these behaviors. Finally, a quantitative analysis of genes examined herein revealed that C. elegans arousal thresholds were directly correlated with amount of sleep during lethargus. However, bout duration varies little and was not correlated with arousal thresholds. The comprehensive analysis presented here suggests that conserved genes and pathways are required for sleep in invertebrates and, likely, across the entire animal kingdom. The genetic pathway delineated in this study implicates G-alpha S and previously known genes downstream of dopamine signaling in sleep. Quantitative analysis of various components of quiescence suggests that interdependent or identical cellular and molecular mechanisms are likely to regulate both arousal and sleep entry.

Quantitative gene analysis of methylation and expression (Q‐GAME) in fresh or fixed cells and tissues

Epigenetic regulation of gene expression by DNA methylation is a central mechanism governing the silencing of tumor suppressor genes in many forms of cancer. Current methods have not proven optimal for the quantitative analysis of DNA methylation and corresponding in situ protein expression within cells in small specimens like skin biopsies. We have overcome this limitation by combining and modifying several techniques: target cell enrichment, DNA micro-isolation, one-step denaturation/bisulphite conversion/in-column desulphonation, specially designed PCR amplification, pyrosequencing and multispectral image analysis. Using this approach optimized for small samples, we can quantify minor alterations in gene methylation and protein expression using minimal amounts of tissue. Comparative studies of fresh and processed cells showed that our method is valid for DNA in both fresh and formalin-fixed, paraffin-embedded specimens. We can measure the effects of DNA methylation inhibitors, administered in vitro or in vivo, on the promoter methylation and protein expression of selected genes in specific cells. This novel approach should prove useful for a wide variety of investigative and clinical applications in dermatology and other specialties where the collection of small, routinely processed biopsy specimens is common. We refer to this method as Q-GAME (quantitative gene analysis of methylation and expression).

Lymphocytes of Patients with Alzheimer’s Disease Display Different DNA Damage Repair Kinetics and Expression Profiles of DNA Repair and Stress Response Genes

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, characterized by loss of memory and cognitive capacity. Given the limitations to analyze brain cells, it is important to study whether peripheral lymphocytes can provide biological markers for AD, an interesting approach, once they represent the overall condition of the organism. To that extent, we sought to find whether lymphocytes of AD patients present DNA damage and repair kinetics different from those found in elderly matched controls (EC group) under in vitro treatment with hydrogen peroxide. We found that AD patient cells indeed showed an altered DNA repair kinetics (comet assay). Real-time quantitative analysis of genes associated with DNA stress response also showed that FANCG and CDKN1A are upregulated in AD, while MTH1 is downregulated, compared with the control group. In contrast, the expression of ATM, ATR and FEN1 genes does not seem to differ between these groups. Interestingly, TP53 protein expression was increased in AD patients. Therefore, we found that kinetics of the stress response in the DNA were significantly different in AD patients, supporting the hypothesis that repair pathways may be compromised in AD and that peripheral lymphocytes can reveal this condition.

Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling

Much is known regarding the role of Indian hedgehog (Ihh) in endochondral ossification, where Ihh regulates multiple steps of chondrocyte differentiation. The Ihh-/- phenotype is most notable for severely foreshortened limbs and a complete absence of mature osteoblasts. A far less explored phenotype in the Ihh-/- mutant is found in the calvaria, where bones form predominately through intramembranous ossification. We investigated the role of Ihh in calvarial bone ossification, finding that proliferation was largely unaffected. Instead, our results indicate that Ihh is a pro-osteogenic factor that positively regulates intramembranous ossification. We confirmed through histologic and quantitative gene analysis that loss of Ihh results in reduction of cranial bone size and all markers of osteodifferentiation. Moreover, in vitro studies suggest that Ihh loss reduces Bmp expression within the calvaria, an observation that may underlie the Ihh-/- calvarial phenotype. In conjunction with the newly recognized roles of Hedgehog deregulation in craniosynostosis, our study defines Ihh as an important positive regulator of cranial bone ossification.

STT3A, C1orf24, TFF3: Putative Markers for Characterization of Follicular Thyroid Neoplasms From Fine‐Needle Aspirates

The goals of this study were to characterize gene expression using fine-needle aspirates (FNAs) from follicular neoplasms to distinguish follicular adenomas (FAs) from follicular thyroid carcinomas (FTCs) and follicular variant of papillary thyroid carcinomas (FVPTCs); and to use FNA material to distinguish benign from malignant follicular neoplasms. Retrospective expression analysis of diagnosed follicular neoplasms (level of evidence 2b); prospective cohort of FNA from the operating room after thyroid lobectomy (level of evidence 1b). Gene expression analysis via reverse-transcription polymerase chain reaction (rt-PCR) of nine genes previously noted to be differentially expressed in follicular neoplasms was performed on formalin-fixed, paraffin-embedded archived normal thyroid tissue (n = 63) and follicular neoplasms as diagnosed on preoperative FNA: FA (n = 16), FTC (n = 13), FVPTC (n = 24), and papillary thyroid carcinomas (PTCs) (n = 10). All cases were originally read as follicular neoplasms on preoperative FNA. To determine if these results could be translated into fresh tissue, ex vivo FNA was performed on follicular neoplasms (n = 17) in the operating room after thyroidectomy. Quantitative gene analysis detected differential TFF3 expression in FA versus FTC, FVPTC, and PTC (P = .02). Rt-PCR of FNA samples demonstrated that malignant nodules overexpress STT3A as compared with benign disease (P = .046). The combination of STT3A overexpression/Clorf24 underexpression identified malignant disease (P = .03) on FNA samples. Gene-expression data suggest a difference in expression between STT3A, Clorf24, and TFF3 in FAs versus carcinomas that may be detected from an FNA sample. Findings must be validated from preoperative FNAs in larger numbers.

Ultrasensitive electrochemical detection of nucleic acid based on the isothermal strand-displacement polymerase reaction and enzyme dual amplification

We describe an ultrasensitive electrochemical detection of DNA protocol based on the isothermal strand-displacement polymerase reaction (ISDPR) and enzyme dual amplifications. Target DNA triggered an ISDPR to produce numerous bi-functionalized duplex DNA complexes. Following an immuno-magnetic collection via an immunoreaction between the attached digoxin on the duplex DNA and the anti-digoxin antibody on the magnetic bead, horseradish (HRP) tracers were bound to the duplex DNA through a biotin–streptavidin interaction. The quantification of DNA was realized by square wave voltammetric detection of the enzymatic products with a screen-printed gold electrode. The voltammetric response was proportional to the concentration of DNA in the range of 0.1 fM–0.5 pM, and the limit of detection was estimated to be 0.06 fM. The new protocol showed great promise for simple, cost-effective, and quantitative gene analysis.

Lactobacillus phylogenomics - towards a reclassification of the genus

The extremely diverse genus Lactobacillus is the largest among the lactic acid bacteria, with over 145 recognized species. In this work, which to our knowledge is the largest comparative phylogenomics study of a single genus to date, 12 genomes of Lactobacillus strains were subjected to an array of whole-genome and single-marker phylogenetic approaches, to investigate the case for extracting subgeneric groups and to determine whether a single congruent phylogeny could be identified. We conclude that GroEL is a more robust single-gene phylogenetic marker for the genus Lactobacillus than the 16S rRNA gene, when no whole-genome information is available. Significant incongruence was found, both within a set of trees based on 141 core proteins and within those phylogenies based on numbers of orthologues, concatenated RNA polymerase subunits and single gene/protein markers. This is possibly due to different evolutionary rates, hidden paralogies or horizontal gene transfer. Such phylogenetic ambiguities are efficiently visualized with cluster-networks. Although the genus contains some highly unstable taxa, four subgeneric groups were distinguished. Qualitative and quantitative gene analysis of these groups resulted in three findings: there is a relatively small number of group-specific proteins, the majority of which are poorly characterized; major groupings are functionally better distinguishable by absent genes rather than gained/retained genes; and, finally, a gene cluster possibly involved in purine metabolism is uniquely present in four lactobacilli associated with meat. In conclusion, because of either significantly different branching patterns or the availability of too few members, three of the four identified groups could not serve as the basis for identifying candidate novel genera within the current genus. We therefore suggest targeted sequencing of key taxonomic species identified here, which are likely to add sufficient depth for a future reclassification, followed by phylogenomic analysis involving the core proteins identified here. This will ideally be combined with phenotypic data using a polyphasic approach.

Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies

The leukemic stem cells in patients with chronic myeloid leukemia (CML) are well known to be clinically resistant to conventional chemotherapy and may also be relatively resistant to BCR-ABL-targeted drugs. Here we show that the lesser effect of imatinib mesylate (IM) on the 3-week output of cells produced in vitro from lin(-)CD34(+)CD38(-) CML (stem) cells compared with cultures initiated with the CD38(+) subset of lin(-)CD34(+) cells is markedly enhanced (>10-fold) when conditions of reduced growth factor stimulation are used. Quantitative analysis of genes expressed in these different CML subsets revealed a differentiation-associated decrease in IL-3 and G-CSF transcripts, a much more profound decrease in expression of BCR-ABL than predicted by changes in BCR expression, decreasing expression of ABCB1/MDR and ABCG2 and increasing expression of OCT1. p210(BCR-ABL) and kinase activity were also higher in the lin(-)CD34(+)CD38(-) cells and formal evidence that increasing BCR-ABL expression decreases IM sensitivity was obtained from experiments with a cell line model. Nevertheless, within the entire CD34(+) subset of CML cells, BCR-ABL expression was not strongly affected by changes in cell cycle status. Taken together, these results provide the first evidence of multiple mechanisms of innate IM resistance in primitive and quiescent CML cells.

Occurrence and persistence of Bacillus thuringiensis (Bt) and transgenic Bt corn cry1Ab gene from an aquatic environment

Genetically modified corn crops and suspensions of Bacillus thuringiensis (Bt) are currently used to control pest infestations of insects of the Lepidoptera family. For this purpose, the cry1Ab gene coding for protein δ-endotoxin derived from B. thuringiensis kurstaki (Btk), which is highly toxic to these insects, was inserted and expressed in corn. The aims of this study were to examine the occurrence and persistence of the cry1Ab gene from Btk and Bt corn in aquatic environments near fields where Bt corn was cultivated. First, an optimal DNA preparation and extraction methodology was developed to allow for quantitative gene analysis by real-time polymerase chain reaction (qPCR) in various environmental matrices. Second, surface water and sediment were spiked in vitro with genomic DNA from Bt or Bt corn to evaluate the persistence of cry1Ab genes. Third, soil, sediment, and water samples were collected before seeding, 2 weeks after pollen release, and after corn harvesting and mechanical root remixing in soils to assess cry1Ab gene content. DNA was extracted with sufficient purity (i.e., low absorbance at 230 nm and absence of PCR-inhibiting substances) from soil, sediment, and surface water. The cry1Ab gene persisted for more than 21 and 40 days in surface water and sediment, respectively. The removal of bacteria by filtration of surface water samples did not significantly increase the half-life of the transgene, but the levels were fivefold more abundant than those in unfiltered water at the end of the exposure period. In sediments, the cry1Ab gene from Bt corn was still detected after 40 days in clay- and sand-rich sediments. Field surveys revealed that the cry1Ab gene from transgenic corn and from naturally occurring Bt was more abundant in the sediment than in the surface water. The cry1Ab transgene was detected as far away as the Richelieu and St. Lawrence rivers (82 km downstream from the corn cultivation plot), suggesting that there were multiple sources of this gene and/or that it undergoes transport by the water column. Sediment-associated cry1Ab gene from Bt corn tended to decrease with distance from the Bt cornfield. Sediment concentrations of the cry1Ab gene were significantly correlated with those of the cry1Ab gene in surface water ( R = 0.83 ; P = 0.04 ) . The data indicate that DNA from Bt corn and Bt were persistent in aquatic environments and were detected in rivers draining farming areas.