The production of ROS (reactive oxygen species) by mammalian mitochondria is important because it underlies oxidative damage in many pathologies and contributes to retrograde redox signalling from the organelle to the cytosol and nucleus. Superoxide (O2(*-)) is the proximal mitochondrial ROS, and in the present review I outline the principles that govern O2(*-) production within the matrix of mammalian mitochondria. The flux of O2(*-) is related to the concentration of potential electron donors, the local concentration of O2 and the second-order rate constants for the reactions between them. Two modes of operation by isolated mitochondria result in significant O2(*-) production, predominantly from complex I: (i) when the mitochondria are not making ATP and consequently have a high Deltap (protonmotive force) and a reduced CoQ (coenzyme Q) pool; and (ii) when there is a high NADH/NAD+ ratio in the mitochondrial matrix. For mitochondria that are actively making ATP, and consequently have a lower Deltap and NADH/NAD+ ratio, the extent of O2(*-) production is far lower. The generation of O2(*-) within the mitochondrial matrix depends critically on Deltap, the NADH/NAD+ and CoQH2/CoQ ratios and the local O2 concentration, which are all highly variable and difficult to measure in vivo. Consequently, it is not possible to estimate O2(*-) generation by mitochondria in vivo from O2(*-)-production rates by isolated mitochondria, and such extrapolations in the literature are misleading. Even so, the description outlined here facilitates the understanding of factors that favour mitochondrial ROS production. There is a clear need to develop better methods to measure mitochondrial O2(*-) and H2O2 formation in vivo, as uncertainty about these values hampers studies on the role of mitochondrial ROS in pathological oxidative damage and redox signalling.

In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21-nucleotide (nt) RNAs, respectively, which function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as small temporal RNAs (stRNAs). We show that many 21- and 22-nt expressed RNAs, termed microRNAs, exist in invertebrates and vertebrates and that some of these novel RNAs, similar to let-7 stRNA, are highly conserved. This suggests that sequence-specific, posttranscriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.

OBJECTIVE

Most patients with advanced pancreas cancer experience pain and must limit their daily activities because of tumor-related symptoms. To date, no treatment has had a significant impact on the disease. In early studies with gemcitabine, patients with pancreas cancer experienced an improvement in disease-related symptoms. Based on those findings, a definitive trial was performed to assess the effectiveness of gemcitabine in patients with newly diagnosed advanced pancreas cancer.

METHODS

One hundred twenty-six patients with advanced symptomatic pancreas cancer completed a lead-in period to characterize and stabilize pain and were randomized to receive either gemcitabine 1,000 mg/m2 weekly x 7 followed by 1 week of rest, then weekly x 3 every 4 weeks thereafter (63 patients), or to fluorouracil (5-FU) 600 mg/m2 once weekly (63 patients). The primary efficacy measure was clinical benefit response, which was a composite of measurements of pain (analgesic consumption and pain intensity), Karnofsky performance status, and weight. Clinical benefit required a sustained >> or = 4 weeks) improvement in at least one parameter without worsening in any others. Other measures of efficacy included response rate, time to progressive disease, and survival.

RESULTS

Clinical benefit response was experienced by 23.8% of gemcitabine-treated patients compared with 4.8% of 5-FU-treated patients (P = .0022). The median survival durations were 5.65 and 4.41 months for gemcitabine-treated and 5-FU-treated patients, respectively (P = .0025). The survival rate at 12 months was 18% for gemcitabine patients and 2% for 5-FU patients. Treatment was well tolerated.

CONCLUSIONS

This study demonstrates that gemcitabine is more effective than 5-FU in alleviation of some disease-related symptoms in patients with advanced, symptomatic pancreas cancer. Gemcitabine also confers a modest survival advantage over treatment with 5-FU.

The prime objective for every life form is to deliver its genetic material, intact and unchanged, to the next generation. This must be achieved despite constant assaults by endogenous and environmental agents on the DNA. To counter this threat, life has evolved several systems to detect DNA damage, signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management.

Drug therapy for hypercholesterolaemia has remained controversial mainly because of insufficient clinical trial evidence for improved survival. The present trial was designed to evaluate the effect of cholesterol lowering with simvastatin on mortality and morbidity in patients with coronary heart disease (CHD). 4444 patients with angina pectoris or previous myocardial infarction and serum cholesterol 5.5-8.0 mmol/L on a lipid-lowering diet were randomised to double-blind treatment with simvastatin or placebo. Over the 5.4 years median follow-up period, simvastatin produced mean changes in total cholesterol, low-density-lipoprotein cholesterol, and high-density-lipoprotein cholesterol of -25%, -35%, and +8%, respectively, with few adverse effects. 256 patients (12%) in the placebo group died, compared with 182 (8%) in the simvastatin group. The relative risk of death in the simvastatin group was 0.70 (95% CI 0.58-0.85, p = 0.0003). The 6-year probabilities of survival in the placebo and simvastatin groups were 87.6% and 91.3%, respectively. There were 189 coronary deaths in the placebo group and 111 in the simvastatin group (relative risk 0.58, 95% CI 0.46-0.73), while noncardiovascular causes accounted for 49 and 46 deaths, respectively. 622 patients (28%) in the placebo group and 431 (19%) in the simvastatin group had one or more major coronary events. The relative risk was 0.66 (95% CI 0.59-0.75, p < 0.00001), and the respective probabilities of escaping such events were 70.5% and 79.6%. This risk was also significantly reduced in subgroups consisting of women and patients of both sexes aged 60 or more. Other benefits of treatment included a 37% reduction (p < 0.00001) in the risk of undergoing myocardial revascularisation procedures. This study shows that long-term treatment with simvastatin is safe and improves survival in CHD patients.

pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColE1. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2067) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColE1 plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.

Methods for the quantification of beta-cell sensitivity to glucose (hyperglycemic clamp technique) and of tissue sensitivity to insulin (euglycemic insulin clamp technique) are described. Hyperglycemic clamp technique. The plasma glucose concentration is acutely raised to 125 mg/dl above basal levels by a priming infusion of glucose. The desired hyperglycemic plateau is subsequently maintained by adjustment of a variable glucose infusion, based on the negative feedback principle. Because the plasma glucose concentration is held constant, the glucose infusion rate is an index of glucose metabolism. Under these conditions of constant hyperglycemia, the plasma insulin response is biphasic with an early burst of insulin release during the first 6 min followed by a gradually progressive increase in plasma insulin concentration. Euglycemic insulin clamp technique. The plasma insulin concentration is acutely raised and maintained at approximately 100 muU/ml by a prime-continuous infusion of insulin. The plasma glucose concentration is held constant at basal levels by a variable glucose infusion using the negative feedback principle. Under these steady-state conditions of euglycemia, the glucose infusion rate equals glucose uptake by all the tissues in the body and is therefore a measure of tissue sensitivity to exogenous insulin.

Eukaryotic cells make many types of primary and processed RNAs that are found either in specific subcellular compartments or throughout the cells. A complete catalogue of these RNAs is not yet available and their characteristic subcellular localizations are also poorly understood. Because RNA represents the direct output of the genetic information encoded by genomes and a significant proportion of a cell's regulatory capabilities are focused on its synthesis, processing, transport, modification and translation, the generation of such a catalogue is crucial for understanding genome function. Here we report evidence that three-quarters of the human genome is capable of being transcribed, as well as observations about the range and levels of expression, localization, processing fates, regulatory regions and modifications of almost all currently annotated and thousands of previously unannotated RNAs. These observations, taken together, prompt a redefinition of the concept of a gene.

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.

Methods which use the scintillator PPO to record film images of 3H in chromatograms and polyacrylamide gels (fluorography) have been described elsewhere. This paper demonstrates that pre-exposure of the film to a brief flash of light greatly increases the sensitivity of fluorography. Pre-exposure also permits quantitative interpretation of the film image, because it corrects the non-linear relationship between radioactivity of the sample and absorbance of the film image. Therefore the distribution of radioactivity in the sample is accurately represented by microdensitometry of the image obtained on pre-exposed film. Using pre-exposed film 300 dis. 3H/min or 30 dis. 14C/min can be detected in a band in a gel in a 24-h exposure. The Appendix describes revisions and extensions of existing fluorographic procedures, including application to agarose gels and a rapid procedure for recovering PPO for re-use.

Because most colorectal carcinomas appear to arise from adenomas, studies of different stages of colorectal neoplasia may shed light on the genetic alterations involved in tumor progression. We looked for four genetic alterations (ras-gene mutations and allelic deletions of chromosomes 5, 17, and 18) in 172 colorectal-tumor specimens representing various stages of neoplastic development. The specimens consisted of 40 predominantly early-stage adenomas from 7 patients with familial adenomatous polyposis, 40 adenomas (19 without associated foci of carcinoma and 21 with such foci) from 33 patients without familial polyposis, and 92 carcinomas resected from 89 patients. We found that ras-gene mutations occurred in 58 percent of adenomas larger than 1 cm and in 47 percent of carcinomas. However, ras mutations were found in only 9 percent of adenomas under 1 cm in size. Sequences on chromosome 5 that are linked to the gene for familial adenomatous polyposis were not lost in adenomas from the patients with polyposis but were lost in 29 to 35 percent of adenomas and carcinomas, respectively, from other patients. A specific region of chromosome 18 was deleted frequently in carcinomas (73 percent) and in advanced adenomas (47 percent) but only occasionally in earlier-stage adenomas (11 to 13 percent). Chromosome 17p sequences were usually lost only in carcinomas (75 percent). The four molecular alterations accumulated in a fashion that paralleled the clinical progression of tumors. These results are consistent with a model of colorectal tumorigenesis in which the steps required for the development of cancer often involve the mutational activation of an oncogene coupled with the loss of several genes that normally suppress tumorigenesis.

BACKGROUND

We conducted a randomized, placebo-controlled, double-blind trial to determine whether the epidermal growth factor receptor inhibitor erlotinib prolongs survival in non-small-cell lung cancer after the failure of first-line or second-line chemotherapy.

METHODS

Patients with stage IIIB or IV non-small-cell lung cancer, with performance status from 0 to 3, were eligible if they had received one or two prior chemotherapy regimens. The patients were stratified according to center, performance status, response to prior chemotherapy, number of prior regimens, and prior platinum-based therapy and were randomly assigned in a 2:1 ratio to receive oral erlotinib, at a dose of 150 mg daily, or placebo.

RESULTS

The median age of the 731 patients who underwent randomization was 61.4 years; 49 percent had received two prior chemotherapy regimens, and 93 percent had received platinum-based chemotherapy. The response rate was 8.9 percent in the erlotinib group and less than 1 percent in the placebo group (P<0.001); the median duration of the response was 7.9 months and 3.7 months, respectively. Progression-free survival was 2.2 months and 1.8 months, respectively (hazard ratio, 0.61, adjusted for stratification categories; P<0.001). Overall survival was 6.7 months and 4.7 months, respectively (hazard ratio, 0.70; P<0.001), in favor of erlotinib. Five percent of patients discontinued erlotinib because of toxic effects.

CONCLUSIONS

Erlotinib can prolong survival in patients with non-small-cell lung cancer after first-line or second-line chemotherapy.

Morphological assessment of the degree of differentiation has been shown in numerous studies to provide useful prognostic information in breast cancer, but until recently histological grading has not been accepted as a routine procedure, mainly because of perceived problems with reproducibility and consistency. In the Nottingham/Tenovus Primary Breast Cancer Study the most commonly used method, described by Bloom & Richardson, has been modified in order to make the criteria more objective. The revised technique involves semiquantitative evaluation of three morphological features--the percentage of tubule formation, the degree of nuclear pleomorphism and an accurate mitotic count using a defined field area. A numerical scoring system is used and the overall grade is derived from a summation of individual scores for the three variables: three grades of differentiation are used. Since 1973, over 2200 patients with primary operable breast cancer have been entered into a study of multiple prognostic factors. Histological grade, assessed in 1831 patients, shows a very strong correlation with prognosis; patients with grade I tumours have a significantly better survival than those with grade II and III tumours (P less than 0.0001). These results demonstrate that this method for histological grading provides important prognostic information and, if the grading protocol is followed consistently, reproducible results can be obtained. Histological grade forms part of the multifactorial Nottingham prognostic index, together with tumour size and lymph node stage, which is used to stratify individual patients for appropriate therapy.

Most human acute myeloid leukaemia (AML) cells have limited proliferative capacity, suggesting that the leukaemic clone may be maintained by a rare population of stem cells. This putative leukaemic stem cell has not been characterized because the available in vitro assays can only detect progenitors with limited proliferative and replating potential. We have now identified an AML-initiating cell by transplantation into severe combined immune-deficient (SCID) mice. These cells homed to the bone marrow and proliferated extensively in response to in vivo cytokine treatment, resulting in a pattern of dissemination and leukaemic cell morphology similar to that seen in the original patients. Limiting dilution analysis showed that the frequency of these leukaemia-initiating cells in the peripheral blood of AML patients was one engraftment unit in 250,000 cells. We fractionated AML cells on the basis of cell-surface-marker expression and found that the leukaemia-initiating cells that could engraft SCID mice to produce large numbers of colony-forming progenitors were CD34+ CD38-; however, the CD34+ CD38+ and CD34- fractions contained no cells with these properties. This in vivo model replicates many aspects of human AML and defines a new leukaemia-initiating cell which is less mature than colony-forming cells.

Geometrical validation around the Calpha is described, with a new Cbeta measure and updated Ramachandran plot. Deviation of the observed Cbeta atom from ideal position provides a single measure encapsulating the major structure-validation information contained in bond angle distortions. Cbeta deviation is sensitive to incompatibilities between sidechain and backbone caused by misfit conformations or inappropriate refinement restraints. A new phi,psi plot using density-dependent smoothing for 81,234 non-Gly, non-Pro, and non-prePro residues with B < 30 from 500 high-resolution proteins shows sharp boundaries at critical edges and clear delineation between large empty areas and regions that are allowed but disfavored. One such region is the gamma-turn conformation near +75 degrees,-60 degrees, counted as forbidden by common structure-validation programs; however, it occurs in well-ordered parts of good structures, it is overrepresented near functional sites, and strain is partly compensated by the gamma-turn H-bond. Favored and allowed phi,psi regions are also defined for Pro, pre-Pro, and Gly (important because Gly phi,psi angles are more permissive but less accurately determined). Details of these accurate empirical distributions are poorly predicted by previous theoretical calculations, including a region left of alpha-helix, which rates as favorable in energy yet rarely occurs. A proposed factor explaining this discrepancy is that crowding of the two-peptide NHs permits donating only a single H-bond. New calculations by Hu et al. [Proteins 2002 (this issue)] for Ala and Gly dipeptides, using mixed quantum mechanics and molecular mechanics, fit our nonrepetitive data in excellent detail. To run our geometrical evaluations on a user-uploaded file, see MOLPROBITY (http://kinemage.biochem.duke.edu) or RAMPAGE (http://www-cryst.bioc.cam.ac.uk/rampage).

METHODS

Aldosterone is important in the pathophysiology of heart failure. In a doubleblind study, we enrolled 1663 patients who had severe heart failure and a left ventricular ejection fraction of no more than 35 percent and who were being treated with an angiotensin-converting-enzyme inhibitor, a loop diuretic, and in most cases digoxin. A total of 822 patients were randomly assigned to receive 25 mg of spironolactone daily, and 841 to receive placebo. The primary end point was death from all causes.

RESULTS

The trial was discontinued early, after a mean follow-up period of 24 months, because an interim analysis determined that spironolactone was efficacious. There were 386 deaths in the placebo group (46 percent) and 284 in the spironolactone group (35 percent; relative risk of death, 0.70; 95 percent confidence interval, 0.60 to 0.82; P<0.001). This 30 percent reduction in the risk of death among patients in the spironolactone group was attributed to a lower risk of both death from progressive heart failure and sudden death from cardiac causes. The frequency of hospitalization for worsening heart failure was 35 percent lower in the spironolactone group than in the placebo group (relative risk of hospitalization, 0.65; 95 percent confidence interval, 0.54 to 0.77; P<0.001). In addition, patients who received spironolactone had a significant improvement in the symptoms of heart failure, as assessed on the basis of the New York Heart Association functional class (P<0.001). Gynecomastia or breast pain was reported in 10 percent of men who were treated with spironolactone, as compared with 1 percent of men in the placebo group (P<0.001). The incidence of serious hyperkalemia was minimal in both groups of patients.

CONCLUSIONS

Blockade of aldosterone receptors by spironolactone, in addition to standard therapy, substantially reduces the risk of both morbidity and death among patients with severe heart failure.

BACKGROUND

In systematic reviews and meta-analyses, time-to-event outcomes are most appropriately analysed using hazard ratios (HRs). In the absence of individual patient data (IPD), methods are available to obtain HRs and/or associated statistics by carefully manipulating published or other summary data. Awareness and adoption of these methods is somewhat limited, perhaps because they are published in the statistical literature using statistical notation.

METHODS

This paper aims to 'translate' the methods for estimating a HR and associated statistics from published time-to-event-analyses into less statistical and more practical guidance and provide a corresponding, easy-to-use calculations spreadsheet, to facilitate the computational aspects.

RESULTS

A wider audience should be able to understand published time-to-event data in individual trial reports and use it more appropriately in meta-analysis. When faced with particular circumstances, readers can refer to the relevant sections of the paper. The spreadsheet can be used to assist them in carrying out the calculations.

CONCLUSIONS

The methods cannot circumvent the potential biases associated with relying on published data for systematic reviews and meta-analysis. However, this practical guide should improve the quality of the analysis and subsequent interpretation of systematic reviews and meta-analyses that include time-to-event outcomes.

Poly(ADP-ribose) polymerase (PARP1) facilitates DNA repair by binding to DNA breaks and attracting DNA repair proteins to the site of damage. Nevertheless, PARP1-/- mice are viable, fertile and do not develop early onset tumours. Here, we show that PARP inhibitors trigger gamma-H2AX and RAD51 foci formation. We propose that, in the absence of PARP1, spontaneous single-strand breaks collapse replication forks and trigger homologous recombination for repair. Furthermore, we show that BRCA2-deficient cells, as a result of their deficiency in homologous recombination, are acutely sensitive to PARP inhibitors, presumably because resultant collapsed replication forks are no longer repaired. Thus, PARP1 activity is essential in homologous recombination-deficient BRCA2 mutant cells. We exploit this requirement in order to kill BRCA2-deficient tumours by PARP inhibition alone. Treatment with PARP inhibitors is likely to be highly tumour specific, because only the tumours (which are BRCA2-/-) in BRCA2+/- patients are defective in homologous recombination. The use of an inhibitor of a DNA repair enzyme alone to selectively kill a tumour, in the absence of an exogenous DNA-damaging agent, represents a new concept in cancer treatment.

BACKGROUND

We present the combined results of two trials that compared adjuvant chemotherapy with or without concurrent trastuzumab in women with surgically removed HER2-positive breast cancer.

METHODS

The National Surgical Adjuvant Breast and Bowel Project trial B-31 compared doxorubicin and cyclophosphamide followed by paclitaxel every 3 weeks (group 1) with the same regimen plus 52 weeks of trastuzumab beginning with the first dose of paclitaxel (group 2). The North Central Cancer Treatment Group trial N9831 compared three regimens: doxorubicin and cyclophosphamide followed by weekly paclitaxel (group A), the same regimen followed by 52 weeks of trastuzumab after paclitaxel (group B), and the same regimen plus 52 weeks of trastuzumab initiated concomitantly with paclitaxel (group C). The studies were amended to include a joint analysis comparing groups 1 and A (the control group) with groups 2 and C (the trastuzumab group). Group B was excluded because trastuzumab was not given concurrently with paclitaxel.

RESULTS

By March 15, 2005, 394 events (recurrent, second primary cancer, or death before recurrence) had been reported, triggering the first scheduled interim analysis. Of these, 133 were in the trastuzumab group and 261 in the control group (hazard ratio, 0.48; P<0.0001). This result crossed the early stopping boundary. The absolute difference in disease-free survival between the trastuzumab group and the control group was 12 percent at three years. Trastuzumab therapy was associated with a 33 percent reduction in the risk of death (P=0.015). The three-year cumulative incidence of class III or IV congestive heart failure or death from cardiac causes in the trastuzumab group was 4.1 percent in trial B-31 and 2.9 percent in trial N9831.

CONCLUSIONS

Trastuzumab combined with paclitaxel after doxorubicin and cyclophosphamide improves outcomes among women with surgically removed HER2-positive breast cancer. (ClinicalTrials.gov numbers, NCT00004067 and NCT00005970.)

SNPs discovered by genome-wide association studies (GWASs) account for only a small fraction of the genetic variation of complex traits in human populations. Where is the remaining heritability? We estimated the proportion of variance for human height explained by 294,831 SNPs genotyped on 3,925 unrelated individuals using a linear model analysis, and validated the estimation method with simulations based on the observed genotype data. We show that 45% of variance can be explained by considering all SNPs simultaneously. Thus, most of the heritability is not missing but has not previously been detected because the individual effects are too small to pass stringent significance tests. We provide evidence that the remaining heritability is due to incomplete linkage disequilibrium between causal variants and genotyped SNPs, exacerbated by causal variants having lower minor allele frequency than the SNPs explored to date.

Recent advances in cDNA and oligonucleotide DNA arrays have made it possible to measure the abundance of mRNA transcripts for many genes simultaneously. The analysis of such experiments is nontrivial because of large data size and many levels of variation introduced at different stages of the experiments. The analysis is further complicated by the large differences that may exist among different probes used to interrogate the same gene. However, an attractive feature of high-density oligonucleotide arrays such as those produced by photolithography and inkjet technology is the standardization of chip manufacturing and hybridization process. As a result, probe-specific biases, although significant, are highly reproducible and predictable, and their adverse effect can be reduced by proper modeling and analysis methods. Here, we propose a statistical model for the probe-level data, and develop model-based estimates for gene expression indexes. We also present model-based methods for identifying and handling cross-hybridizing probes and contaminating array regions. Applications of these results will be presented elsewhere.

A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.

The finite mixture (FM) model is the most commonly used model for statistical segmentation of brain magnetic resonance (MR) images because of its simple mathematical form and the piecewise constant nature of ideal brain MR images. However, being a histogram-based model, the FM has an intrinsic limitation--no spatial information is taken into account. This causes the FM model to work only on well-defined images with low levels of noise; unfortunately, this is often not the the case due to artifacts such as partial volume effect and bias field distortion. Under these conditions, FM model-based methods produce unreliable results. In this paper, we propose a novel hidden Markov random field (HMRF) model, which is a stochastic process generated by a MRF whose state sequence cannot be observed directly but which can be indirectly estimated through observations. Mathematically, it can be shown that the FM model is a degenerate version of the HMRF model. The advantage of the HMRF model derives from the way in which the spatial information is encoded through the mutual influences of neighboring sites. Although MRF modeling has been employed in MR image segmentation by other researchers, most reported methods are limited to using MRF as a general prior in an FM model-based approach. To fit the HMRF model, an EM algorithm is used. We show that by incorporating both the HMRF model and the EM algorithm into a HMRF-EM framework, an accurate and robust segmentation can be achieved. More importantly, the HMRF-EM framework can easily be combined with other techniques. As an example, we show how the bias field correction algorithm of Guillemaud and Brady (1997) can be incorporated into this framework to achieve a three-dimensional fully automated approach for brain MR image segmentation.

An algorithm was developed which facilitates the search for similarities between newly determined amino acid sequences and sequences already available in databases. Because of the algorithm's efficiency on many microcomputers, sensitive protein database searches may now become a routine procedure for molecular biologists. The method efficiently identifies regions of similar sequence and then scores the aligned identical and differing residues in those regions by means of an amino acid replacability matrix. This matrix increases sensitivity by giving high scores to those amino acid replacements which occur frequently in evolution. The algorithm has been implemented in a computer program designed to search protein databases very rapidly. For example, comparison of a 200-amino-acid sequence to the 500,000 residues in the National Biomedical Research Foundation library would take less than 2 minutes on a minicomputer, and less than 10 minutes on a microcomputer (IBM PC).