To date, more than 200 microRNAs have been described in humans; however, the precise functions of these regulatory, non-coding RNAs remains largely obscure. One cluster of microRNAs, the mir-17-92 polycistron, is located in a region of DNA that is amplified in human B-cell lymphomas. Here we compared B-cell lymphoma samples and cell lines to normal tissues, and found that the levels of the primary or mature microRNAs derived from the mir-17-92 locus are often substantially increased in these cancers. Enforced expression of the mir-17-92 cluster acted with c-myc expression to accelerate tumour development in a mouse B-cell lymphoma model. Tumours derived from haematopoietic stem cells expressing a subset of the mir-17-92 cluster and c-myc could be distinguished by an absence of apoptosis that was otherwise prevalent in c-myc-induced lymphomas. Together, these studies indicate that non-coding RNAs, specifically microRNAs, can modulate tumour formation, and implicate the mir-17-92 cluster as a potential human oncogene.

DNA sequencing continues to decrease in cost with the Illumina HiSeq2000 generating up to 600 Gb of paired-end 100 base reads in a ten-day run. Here we present a protocol for community amplicon sequencing on the HiSeq2000 and MiSeq Illumina platforms, and apply that protocol to sequence 24 microbial communities from host-associated and free-living environments. A critical question as more sequencing platforms become available is whether biological conclusions derived on one platform are consistent with what would be derived on a different platform. We show that the protocol developed for these instruments successfully recaptures known biological results, and additionally that biological conclusions are consistent across sequencing platforms (the HiSeq2000 versus the MiSeq) and across the sequenced regions of amplicons.

Emerging infectious diseases, such as SARS and Zika, present a major threat to public health^1-3. Despite intense research efforts, how, when and where new diseases appear are still the source of considerable uncertainly. A severe respiratory disease was recently reported in the city Wuhan, Hubei province, China. Up to 25th of January 2020, at least 1,975 cases have been reported since the first patient was hospitalized on the 12th of December 2019. Epidemiological investigation suggested that the outbreak was associated with a seafood market in Wuhan. We studied one patient who was a worker at the market, and who was admitted to Wuhan Central Hospital on 26th of December 2019 experiencing a severe respiratory syndrome including fever, dizziness and cough. Metagenomic RNA sequencing⁴ of a bronchoalveolar lavage fluid sample identified a novel RNA virus from the family Coronaviridae, designed here as WH-Human-1 coronavirus. Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) previously sampled from bats in China. This outbreak highlights the ongoing capacity of viral spill-over from animals to cause severe disease in humans.

The outbreak of a novel betacoronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development, we determined a 3.5 Å-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.

Colon cancer is one of the best-understood neoplasms from a genetic perspective, yet it remains the second most common cause of cancer-related death, indicating that some of its cancer cells are not eradicated by current therapies. What has yet to be established is whether every colon cancer cell possesses the potential to initiate and sustain tumour growth, or whether the tumour is hierarchically organized so that only a subset of cells--cancer stem cells--possess such potential. Here we use renal capsule transplantation in immunodeficient NOD/SCID mice to identify a human colon cancer-initiating cell (CC-IC). Purification experiments established that all CC-ICs were CD133+; the CD133- cells that comprised the majority of the tumour were unable to initiate tumour growth. We calculated by limiting dilution analysis that there was one CC-IC in 5.7 x 10(4) unfractionated tumour cells, whereas there was one CC-IC in 262 CD133+ cells, representing >200-fold enrichment. CC-ICs within the CD133+ population were able to maintain themselves as well as differentiate and re-establish tumour heterogeneity upon serial transplantation. The identification of colon cancer stem cells that are distinct from the bulk tumour cells provides strong support for the hierarchical organization of human colon cancer, and their existence suggests that for therapeutic strategies to be effective, they must target the cancer stem cells.

In recent years, many new cortical areas have been identified in the macaque monkey. The number of identified connections between areas has increased even more dramatically. We report here on (1) a summary of the layout of cortical areas associated with vision and with other modalities, (2) a computerized database for storing and representing large amounts of information on connectivity patterns, and (3) the application of these data to the analysis of hierarchical organization of the cerebral cortex. Our analysis concentrates on the visual system, which includes 25 neocortical areas that are predominantly or exclusively visual in function, plus an additional 7 areas that we regard as visual-association areas on the basis of their extensive visual inputs. A total of 305 connections among these 32 visual and visual-association areas have been reported. This represents 31% of the possible number of pathways if each area were connected with all others. The actual degree of connectivity is likely to be closer to 40%. The great majority of pathways involve reciprocal connections between areas. There are also extensive connections with cortical areas outside the visual system proper, including the somatosensory cortex, as well as neocortical, transitional, and archicortical regions in the temporal and frontal lobes. In the somatosensory/motor system, there are 62 identified pathways linking 13 cortical areas, suggesting an overall connectivity of about 40%. Based on the laminar patterns of connections between areas, we propose a hierarchy of visual areas and of somatosensory/motor areas that is more comprehensive than those suggested in other recent studies. The current version of the visual hierarchy includes 10 levels of cortical processing. Altogether, it contains 14 levels if one includes the retina and lateral geniculate nucleus at the bottom as well as the entorhinal cortex and hippocampus at the top. Within this hierarchy, there are multiple, intertwined processing streams, which, at a low level, are related to the compartmental organization of areas V1 and V2 and, at a high level, are related to the distinction between processing centers in the temporal and parietal lobes. However, there are some pathways and relationships (about 10% of the total) whose descriptions do not fit cleanly into this hierarchical scheme for one reason or another. In most instances, though, it is unclear whether these represent genuine exceptions to a strict hierarchy rather than inaccuracies or uncertainities in the reported assignment.

BACKGROUND

A sustained virological response (SVR) rate of 41% has been achieved with interferon alfa-2b plus ribavirin therapy of chronic hepatitis C. In this randomised trial, peginterferon alfa-2b plus ribavirin was compared with interferon alfa-2b plus ribavirin.

METHODS

1530 patients with chronic hepatitis C were assigned interferon alfa-2b (3 MU subcutaneously three times per week) plus ribavirin 1000-1200 mg/day orally, peginterferon alfa-2b 1.5 microg/kg each week plus 800 mg/day ribavirin, or peginterferon alfa-2b 1.5 microg/kg per week for 4 weeks then 0.5 microg/kg per week plus ribavirin 1000-1200 mg/day for 48 weeks. The primary endpoint was the SVR rate (undetectable hepatitis C virus [HCV] RNA in serum at 24-week follow-up). Analyses were based on patients who received at least one dose of study medication.

RESULTS

The SVR rate was significantly higher (p=0.01 for both comparisons) in the higher-dose peginterferon group (274/511 [54%]) than in the lower-dose peginterferon (244/514 [47%]) or interferon (235/505 [47%]) groups. Among patients with HCV genotype 1 infection, the corresponding SVR rates were 42% (145/348), 34% (118/349), and 33% (114/343). The rate for patients with genotype 2 and 3 infections was about 80% for all treatment groups. Secondary analyses identified bodyweight as an important predictor of SVR, prompting comparison of the interferon regimens after adjusting ribavirin for bodyweight (mg/kg). Side-effect profiles were similar between the treatment groups.

CONCLUSIONS

In patients with chronic hepatitis C, the most effective therapy is the combination of peginterferon alfa-2b 1.5 microg/kg per week plus ribavirin. The benefit is mostly achieved in patients with HCV genotype 1 infections.

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.

Based upon observations on 48 cases of retinoblastoma and published reports, the hypothesis is developed that retinoblastoma is a cancer caused by two mutational events. In the dominantly inherited form, one mutation is inherited via the germinal cells and the second occurs in somatic cells. In the nonhereditary form, both mutations occur in somatic cells. The second mutation produces an average of three retinoblastomas per individual inheriting the first mutation. Using Poisson statistics, one can calculate that this number (three) can explain the occasional gene carrier who gets no tumor, those who develop only unilateral tumors, and those who develop bilateral tumors, as well as explaining instances of multiple tumors in one eye. This value for the mean number of tumors occurring in genetic carriers may be used to estimate the mutation rate for each mutation. The germinal and somatic rates for the first, and the somatic rate for the second, mutation, are approximately equal. The germinal mutation may arise in some instances from a delayed mutation.

Interest in the topic of tumour metabolism has waxed and waned over the past century of cancer research. The early observations of Warburg and his contemporaries established that there are fundamental differences in the central metabolic pathways operating in malignant tissue. However, the initial hypotheses that were based on these observations proved inadequate to explain tumorigenesis, and the oncogene revolution pushed tumour metabolism to the margins of cancer research. In recent years, interest has been renewed as it has become clear that many of the signalling pathways that are affected by genetic mutations and the tumour microenvironment have a profound effect on core metabolism, making this topic once again one of the most intense areas of research in cancer biology.

Two large-scale yeast two-hybrid screens were undertaken to identify protein-protein interactions between full-length open reading frames predicted from the Saccharomyces cerevisiae genome sequence. In one approach, we constructed a protein array of about 6,000 yeast transformants, with each transformant expressing one of the open reading frames as a fusion to an activation domain. This array was screened by a simple and automated procedure for 192 yeast proteins, with positive responses identified by their positions in the array. In a second approach, we pooled cells expressing one of about 6,000 activation domain fusions to generate a library. We used a high-throughput screening procedure to screen nearly all of the 6,000 predicted yeast proteins, expressed as Gal4 DNA-binding domain fusion proteins, against the library, and characterized positives by sequence analysis. These approaches resulted in the detection of 957 putative interactions involving 1,004 S. cerevisiae proteins. These data reveal interactions that place functionally unclassified proteins in a biological context, interactions between proteins involved in the same biological function, and interactions that link biological functions together into larger cellular processes. The results of these screens are shown here.

The parasite Plasmodium falciparum is responsible for hundreds of millions of cases of malaria, and kills more than one million African children annually. Here we report an analysis of the genome sequence of P. falciparum clone 3D7. The 23-megabase nuclear genome consists of 14 chromosomes, encodes about 5,300 genes, and is the most (A + T)-rich genome sequenced to date. Genes involved in antigenic variation are concentrated in the subtelomeric regions of the chromosomes. Compared to the genomes of free-living eukaryotic microbes, the genome of this intracellular parasite encodes fewer enzymes and transporters, but a large proportion of genes are devoted to immune evasion and host-parasite interactions. Many nuclear-encoded proteins are targeted to the apicoplast, an organelle involved in fatty-acid and isoprenoid metabolism. The genome sequence provides the foundation for future studies of this organism, and is being exploited in the search for new drugs and vaccines to fight malaria.

BACKGROUND

Despite wide promulgation, clinical practice guidelines have had limited effect on changing physician behavior. Little is known about the process and factors involved in changing physician practices in response to guidelines.

OBJECTIVE

To review barriers to physician adherence to clinical practice guidelines.

METHODS

We searched the MEDLINE, Educational Resources Information Center (ERIC), and HealthSTAR databases (January 1966 to January 1998); bibliographies; textbooks on health behavior or public health; and references supplied by experts to find English-language article titles that describe barriers to guideline adherence.

METHODS

Of 5658 articles initially identified, we selected 76 published studies describing at least 1 barrier to adherence to clinical practice guidelines, practice parameters, clinical policies, or national consensus statements. One investigator screened titles to identify candidate articles, then 2 investigators independently reviewed the texts to exclude articles that did not match the criteria. Differences were resolved by consensus with a third investigator.

METHODS

Two investigators organized barriers to adherence into a framework according to their effect on physician knowledge, attitudes, or behavior. This organization was validated by 3 additional investigators.

RESULTS

The 76 articles included 120 different surveys investigating 293 potential barriers to physician guideline adherence, including awareness (n = 46), familiarity(n = 31), agreement (n = 33), self-efficacy (n = 19), outcome expectancy (n = 8), ability to overcome the inertia of previous practice (n = 14), and absence of external barriers to perform recommendations (n = 34). The majority of surveys (70 [58%] of 120) examined only 1 type of barrier.

CONCLUSIONS

Studies on improving physician guideline adherence may not be generalizable, since barriers in one setting may not be present in another. Our review offers a differential diagnosis for why physicians do not follow practice guidelines, as well as a rational approach toward improving guideline adherence and a framework for future research.

BACKGROUND

Erlotinib has been shown to improve progression-free survival compared with chemotherapy when given as first-line treatment for Asian patients with non-small-cell lung cancer (NSCLC) with activating EGFR mutations. We aimed to assess the safety and efficacy of erlotinib compared with standard chemotherapy for first-line treatment of European patients with advanced EGFR-mutation positive NSCLC.

METHODS

We undertook the open-label, randomised phase 3 EURTAC trial at 42 hospitals in France, Italy, and Spain. Eligible participants were adults >> 18 years) with NSCLC and EGFR mutations (exon 19 deletion or L858R mutation in exon 21) with no history of chemotherapy for metastatic disease (neoadjuvant or adjuvant chemotherapy ending ≥ 6 months before study entry was allowed). We randomly allocated participants (1:1) according to a computer-generated allocation schedule to receive oral erlotinib 150 mg per day or 3 week cycles of standard intravenous chemotherapy of cisplatin 75 mg/m(2) on day 1 plus docetaxel (75 mg/m(2) on day 1) or gemcitabine (1250 mg/m(2) on days 1 and 8). Carboplatin (AUC 6 with docetaxel 75 mg/m(2) or AUC 5 with gemcitabine 1000 mg/m(2)) was allowed in patients unable to have cisplatin. Patients were stratified by EGFR mutation type and Eastern Cooperative Oncology Group performance status (0 vs 1 vs 2). The primary endpoint was progression-free survival (PFS) in the intention-to-treat population. We assessed safety in all patients who received study drug (≥ 1 dose). This study is registered with ClinicalTrials.gov, number NCT00446225.

RESULTS

Between Feb 15, 2007, and Jan 4, 2011, 174 patients with EGFR mutations were enrolled. One patient received treatment before randomisation and was thus withdrawn from the study; of the remaining patients, 86 were randomly assigned to receive erlotinib and 87 to receive standard chemotherapy. The preplanned interim analysis showed that the study met its primary endpoint; enrolment was halted, and full evaluation of the results was recommended. At data cutoff (Jan 26, 2011), median PFS was 9·7 months (95% CI 8·4-12·3) in the erlotinib group, compared with 5·2 months (4·5-5·8) in the standard chemotherapy group (hazard ratio 0·37, 95% CI 0·25-0·54; p < 0·0001). Main grade 3 or 4 toxicities were rash (11 [13%] of 84 patients given erlotinib vs none of 82 patients in the chemotherapy group), neutropenia (none vs 18 [22%]), anaemia (one [1%] vs three [4%]), and increased amino-transferase concentrations (two [2%] vs 0). Five (6%) patients on erlotinib had treatment-related severe adverse events compared with 16 patients (20%) on chemotherapy. One patient in the erlotinib group and two in the standard chemotherapy group died from treatment-related causes.

CONCLUSIONS

Our findings strengthen the rationale for routine baseline tissue-based assessment of EGFR mutations in patients with NSCLC and for treatment of mutation-positive patients with EGFR tyrosine-kinase inhibitors.

BACKGROUND

Spanish Lung Cancer Group, Roche Farma, Hoffmann-La Roche, and Red Temática de Investigacion Cooperativa en Cancer.

Quantitative study of protein-protein and protein-ligand interactions in solution requires accurate determination of protein concentration. Often, for proteins available only in "molecular biological" amounts, it is difficult or impossible to make an accurate experimental measurement of the molar extinction coefficient of the protein. Yet without a reliable value of this parameter, one cannot determine protein concentrations by the usual uv spectroscopic means. Fortunately, knowledge of amino acid residue sequence and promoter molecular weight (and thus also of amino acid composition) is generally available through the DNA sequence, which is usually accurately known for most such proteins. In this paper we present a method for calculating accurate (to +/- 5% in most cases) molar extinction coefficients for proteins at 280 nm, simply from knowledge of the amino acid composition. The method is calibrated against 18 "normal" globular proteins whose molar extinction coefficients are accurately known, and the assumptions underlying the method, as well as its limitations, are discussed.

BACKGROUND

Postoperative chemoradiotherapy is the recommended standard therapy for patients with locally advanced rectal cancer. In recent years, encouraging results with preoperative radiotherapy have been reported. We compared preoperative chemoradiotherapy with postoperative chemoradiotherapy for locally advanced rectal cancer.

METHODS

We randomly assigned patients with clinical stage T3 or T4 or node-positive disease to receive either preoperative or postoperative chemoradiotherapy. The preoperative treatment consisted of 5040 cGy delivered in fractions of 180 cGy per day, five days per week, and fluorouracil, given in a 120-hour continuous intravenous infusion at a dose of 1000 mg per square meter of body-surface area per day during the first and fifth weeks of radiotherapy. Surgery was performed six weeks after the completion of chemoradiotherapy. One month after surgery, four five-day cycles of fluorouracil (500 mg per square meter per day) were given. Chemoradiotherapy was identical in the postoperative-treatment group, except for the delivery of a boost of 540 cGy. The primary end point was overall survival.

RESULTS

Four hundred twenty-one patients were randomly assigned to receive preoperative chemoradiotherapy and 402 patients to receive postoperative chemoradiotherapy. The overall five-year survival rates were 76 percent and 74 percent, respectively (P=0.80). The five-year cumulative incidence of local relapse was 6 percent for patients assigned to preoperative chemoradiotherapy and 13 percent in the postoperative-treatment group (P=0.006). Grade 3 or 4 acute toxic effects occurred in 27 percent of the patients in the preoperative-treatment group, as compared with 40 percent of the patients in the postoperative-treatment group (P=0.001); the corresponding rates of long-term toxic effects were 14 percent and 24 percent, respectively (P=0.01).

CONCLUSIONS

Preoperative chemoradiotherapy, as compared with postoperative chemoradiotherapy, improved local control and was associated with reduced toxicity but did not improve overall survival.

BACKGROUND

Results of previous randomised trials have shown that interventions that lower LDL cholesterol concentrations can significantly reduce the incidence of coronary heart disease (CHD) and other major vascular events in a wide range of individuals. But each separate trial has limited power to assess particular outcomes or particular categories of participant.

METHODS

A prospective meta-analysis of data from 90,056 individuals in 14 randomised trials of statins was done. Weighted estimates were obtained of effects on different clinical outcomes per 1.0 mmol/L reduction in LDL cholesterol.

RESULTS

During a mean of 5 years, there were 8186 deaths, 14,348 individuals had major vascular events, and 5103 developed cancer. Mean LDL cholesterol differences at 1 year ranged from 0.35 mmol/L to 1.77 mmol/L (mean 1.09) in these trials. There was a 12% proportional reduction in all-cause mortality per mmol/L reduction in LDL cholesterol (rate ratio [RR] 0.88, 95% CI 0.84-0.91; p<0.0001). This reflected a 19% reduction in coronary mortality (0.81, 0.76-0.85; p<0.0001), and non-significant reductions in non-coronary vascular mortality (0.93, 0.83-1.03; p=0.2) and non-vascular mortality (0.95, 0.90-1.01; p=0.1). There were corresponding reductions in myocardial infarction or coronary death (0.77, 0.74-0.80; p<0.0001), in the need for coronary revascularisation (0.76, 0.73-0.80; p<0.0001), in fatal or non-fatal stroke (0.83, 0.78-0.88; p<0.0001), and, combining these, of 21% in any such major vascular event (0.79, 0.77-0.81; p<0.0001). The proportional reduction in major vascular events differed significantly (p<0.0001) according to the absolute reduction in LDL cholesterol achieved, but not otherwise. These benefits were significant within the first year, but were greater in subsequent years. Taking all years together, the overall reduction of about one fifth per mmol/L LDL cholesterol reduction translated into 48 (95% CI 39-57) fewer participants having major vascular events per 1000 among those with pre-existing CHD at baseline, compared with 25 (19-31) per 1000 among participants with no such history. There was no evidence that statins increased the incidence of cancer overall (1.00, 0.95-1.06; p=0.9) or at any particular site.

CONCLUSIONS

Statin therapy can safely reduce the 5-year incidence of major coronary events, coronary revascularisation, and stroke by about one fifth per mmol/L reduction in LDL cholesterol, largely irrespective of the initial lipid profile or other presenting characteristics. The absolute benefit relates chiefly to an individual's absolute risk of such events and to the absolute reduction in LDL cholesterol achieved. These findings reinforce the need to consider prolonged statin treatment with substantial LDL cholesterol reductions in all patients at high risk of any type of major vascular event.

In order to extract the maximum amount of information from the rapidly accumulating genome sequences, all conserved genes need to be classified according to their homologous relationships. Comparison of proteins encoded in seven complete genomes from five major phylogenetic lineages and elucidation of consistent patterns of sequence similarities allowed the delineation of 720 clusters of orthologous groups (COGs). Each COG consists of individual orthologous proteins or orthologous sets of paralogs from at least three lineages. Orthologs typically have the same function, allowing transfer of functional information from one member to an entire COG. This relation automatically yields a number of functional predictions for poorly characterized genomes. The COGs comprise a framework for functional and evolutionary genome analysis.

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.

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).

An improved dynamic programming algorithm is reported for RNA secondary structure prediction by free energy minimization. Thermodynamic parameters for the stabilities of secondary structure motifs are revised to include expanded sequence dependence as revealed by recent experiments. Additional algorithmic improvements include reduced search time and storage for multibranch loop free energies and improved imposition of folding constraints. An extended database of 151,503 nt in 955 structures? determined by comparative sequence analysis was assembled to allow optimization of parameters not based on experiments and to test the accuracy of the algorithm. On average, the predicted lowest free energy structure contains 73 % of known base-pairs when domains of fewer than 700 nt are folded; this compares with 64 % accuracy for previous versions of the algorithm and parameters. For a given sequence, a set of 750 generated structures contains one structure that, on average, has 86 % of known base-pairs. Experimental constraints, derived from enzymatic and flavin mononucleotide cleavage, improve the accuracy of structure predictions.

Phylogenetic analyses are central to many research areas in biology and typically involve the identification of homologous sequences, their multiple alignment, the phylogenetic reconstruction and the graphical representation of the inferred tree. The Phylogeny.fr platform transparently chains programs to automatically perform these tasks. It is primarily designed for biologists with no experience in phylogeny, but can also meet the needs of specialists; the first ones will find up-to-date tools chained in a phylogeny pipeline to analyze their data in a simple and robust way, while the specialists will be able to easily build and run sophisticated analyses. Phylogeny.fr offers three main modes. The 'One Click' mode targets non-specialists and provides a ready-to-use pipeline chaining programs with recognized accuracy and speed: MUSCLE for multiple alignment, PhyML for tree building, and TreeDyn for tree rendering. All parameters are set up to suit most studies, and users only have to provide their input sequences to obtain a ready-to-print tree. The 'Advanced' mode uses the same pipeline but allows the parameters of each program to be customized by users. The 'A la Carte' mode offers more flexibility and sophistication, as users can build their own pipeline by selecting and setting up the required steps from a large choice of tools to suit their specific needs. Prior to phylogenetic analysis, users can also collect neighbors of a query sequence by running BLAST on general or specialized databases. A guide tree then helps to select neighbor sequences to be used as input for the phylogeny pipeline. Phylogeny.fr is available at: http://www.phylogeny.fr/

We sought to create a comprehensive catalog of yeast genes whose transcript levels vary periodically within the cell cycle. To this end, we used DNA microarrays and samples from yeast cultures synchronized by three independent methods: alpha factor arrest, elutriation, and arrest of a cdc15 temperature-sensitive mutant. Using periodicity and correlation algorithms, we identified 800 genes that meet an objective minimum criterion for cell cycle regulation. In separate experiments, designed to examine the effects of inducing either the G1 cyclin Cln3p or the B-type cyclin Clb2p, we found that the mRNA levels of more than half of these 800 genes respond to one or both of these cyclins. Furthermore, we analyzed our set of cell cycle-regulated genes for known and new promoter elements and show that several known elements (or variations thereof) contain information predictive of cell cycle regulation. A full description and complete data sets are available at http://cellcycle-www.stanford.edu