Light-directed 5'-->3' synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3'-->5' direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5'-->3' direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5'-->3' direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150,000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R(2) = 0.998). We have also shown that the 3' ends of array probes are available for sequence-specific primer extension and ligation reactions.     

Evaluating machine learning approaches for aiding probe selection for gene-expression arrays

MOTIVATION: Microarrays are a fast and cost-effective method of performing thousands of DNA hybridization experiments simultaneously. DNA probes are typically used to measure the expression level of specific genes. Because probes greatly vary in the quality of their hybridizations, choosing good probes is a difficult task. If one could accurately choose probes that are likely to hybridize well, then fewer probes would be needed to represent each gene in a gene-expression microarray, and, hence, more genes could be placed on an array of a given physical size. Our goal is to empirically evaluate how successfully three standard machine-learning algorithms-na?ve Bayes, decision trees, and artificial neural networks-can be applied to the task of predicting good probes. Fortunately it is relatively easy to get training examples for such a learning task: place various probes on a gene chip, add a sample where the corresponding genes are highly expressed, and then record how well each probe measures the presence of its corresponding gene. With such training examples, it is possible that an accurate predictor of probe quality can be learned. RESULTS: Two of the learning algorithms we investigate-na?ve Bayes and neural networks-learn to predict probe quality surprisingly well. For example, in the top ten predicted probes for a given gene not used for training, on average about five rank in the top 2.5% of that gene's hundreds of possible probes. Decision-tree induction and the simple approach of using predicted melting temperature to rank probes perform significantly worse than these two algorithms. The features we use to represent probes are very easily computed and the time taken to score each candidate probe after training is minor. Training the na?ve Bayes algorithm takes very little time, and while it takes over 10 times as long to train a neural network, that time is still not very substantial (on the order of a few hours on a desktop workstation). We also report the information contained in the features we use to describe the probes. We find the fraction of cytosine in the probe to be the most informative feature. We also find, not surprisingly, that the nucleotides in the middle of the probes sequence are more informative than those at the ends of the sequence.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

Factors that drive the increasing use of FFPE tissue in basic and translational cancer research

The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available.     

Nonrandom location of IS1 elements in the genomes of natural isolates of Escherichia coli

We have studied the spatial distribution of IS1 elements in the genomes of natural isolates comprising the ECOR reference collection of Escherichia coli. We find evidence for nonrandomness at three levels. Many pairs of IS1 elements are in much closer proximity (< 10 kb) than can be accounted for by chance. IS1 elements in close proximity were identified by long-range PCR amplification of the genomic sequence between them. Each amplified region was sequenced and its map location determined by database screening of DNA hybridization. Among the ECOR strains with at least two IS1 elements, 54% had one or more pairs of elements separated by < 10 kb. We propose that this type of clustering is a result of "local hopping," in which we assume that a significant proportion of tranposition events leads to the insertion of a daughter IS element in the vicinity of the parental element. A second level of nonrandomness is found in strains with a modest number of IS1 elements that are mapped through the use of inverse PCR to amplify flanking genomic sequences: in these strains, the insertion sites tend to be clustered over a smaller region of chromosome than would be expected by chance. A third level of nonrandomness is observed in the composite distribution of IS elements across strains: among 20 mapped IS1 elements, none were found in the region of 48-77 minutes, a significant gap. One region of the E. coli chromosome, at 98 min, had a cluster of IS1 elements in seven ECOR strains of diverse phylogenetic origin. We deduce from sequence analysis that this pattern of distribution is a result of initial insertion in the most recent common ancestor of these strains and therefore not a hot spot of insertion. Analysis using long- range PCR with primers for IS2 and IS3 also yielded pairs of elements in close proximity, suggesting that these elements may also occasionally transpose by local hopping.      

Quantitative protein determination for CYP induction via LC-MS/MS

The Cytochrome P450 (CYP) proteins are a family of membrane bound proteins that function as a major metabolizing enzyme in the human body. Quantification of CYP induction is critical in determining the disposition, safety and efficacy of drugs in humans. Described is a gel-free, high-throughput LC-MS approach to quantitate the CYP isoforms 1A2, 2B6, 3A4 and 3A5 by measuring isoform specific peptides released by enzymatic digestion of the hepatocyte incubations. The method uses synthetic stable isotope-labeled peptides as internal standards and allows both relative and absolute quantification to be performed from hepatic microsomal preparations. CYP protein determined by this LC-MS method correlated well with the mRNA and activity for induced levels of CYP1A2, CYP2B6 and CYP3A4. Interestingly, a small fold change was observed for the induction of 3A5 with phenobarbital. The results were reproducible with an average CV less then 10% for repeat analysis of the sample. This LC-MS method offers a robust assay for CYP protein quantitation for use in CYP induction assays.