Among the most promising approaches to activating therapeutic antitumour immunity is the blockade of immune checkpoints. Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. It is now clear that tumours co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumour antigens. Because many of the immune checkpoints are initiated by ligand-receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies were the first of this class of immunotherapeutics to achieve US Food and Drug Administration (FDA) approval. Preliminary clinical findings with blockers of additional immune-checkpoint proteins, such as programmed cell death protein 1 (PD1), indicate broad and diverse opportunities to enhance antitumour immunity with the potential to produce durable clinical responses.

Overlap extension represents a new approach to genetic engineering. Complementary oligodeoxyribonucleotide (oligo) primers and the polymerase chain reaction are used to generate two DNA fragments having overlapping ends. These fragments are combined in a subsequent 'fusion' reaction in which the overlapping ends anneal, allowing the 3' overlap of each strand to serve as a primer for the 3' extension of the complementary strand. The resulting fusion product is amplified further by PCR. Specific alterations in the nucleotide (nt) sequence can be introduced by incorporating nucleotide changes into the overlapping oligo primers. Using this technique of site-directed mutagenesis, three variants of a mouse major histocompatibility complex class-I gene have been generated, cloned and analyzed. Screening of mutant clones revealed at least a 98% efficiency of mutagenesis. All clones sequenced contained the desired mutations, and a low frequency of random substitution estimated to occur at approx. 1 in 4000 nt was detected. This method represents a significant improvement over standard methods of site-directed mutagenesis because it is much faster, simpler and approaches 100% efficiency in the generation of mutant product.

Traditional approaches to mechanical ventilation use tidal volumes of 10 to 15 ml per kilogram of body weight and may cause stretch-induced lung injury in patients with acute lung injury and the acute respiratory distress syndrome. We therefore conducted a trial to determine whether ventilation with lower tidal volumes would improve the clinical outcomes in these patients.

Patients with acute lung injury and the acute respiratory distress syndrome were enrolled in a multicenter, randomized trial. The trial compared traditional ventilation treatment, which involved an initial tidal volume of 12 ml per kilogram of predicted body weight and an airway pressure measured after a 0.5-second pause at the end of inspiration (plateau pressure) of 50 cm of water or less, with ventilation with a lower tidal volume, which involved an initial tidal volume of 6 ml per kilogram of predicted body weight and a plateau pressure of 30 cm of water or less. The primary outcomes were death before a patient was discharged home and was breathing without assistance and the number of days without ventilator use from day 1 to day 28.

The trial was stopped after the enrollment of 861 patients because mortality was lower in the group treated with lower tidal volumes than in the group treated with traditional tidal volumes (31.0 percent vs. 39.8 percent, P=0.007), and the number of days without ventilator use during the first 28 days after randomization was greater in this group (mean [+/-SD], 12+/-11 vs. 10+/-11; P=0.007). The mean tidal volumes on days 1 to 3 were 6.2+/-0.8 and 11.8+/-0.8 ml per kilogram of predicted body weight (P<0.001), respectively, and the mean plateau pressures were 25+/-6 and 33+/-8 cm of water (P<0.001), respectively.

In patients with acute lung injury and the acute respiratory distress syndrome, mechanical ventilation with a lower tidal volume than is traditionally used results in decreased mortality and increases the number of days without ventilator use.

Each year, the American Cancer Society estimates the numbers of new cancer cases and deaths that will occur in the United States and compiles the most recent data on population-based cancer occurrence. Incidence data (through 2016) were collected by the Surveillance, Epidemiology, and End Results Program; the National Program of Cancer Registries; and the North American Association of Central Cancer Registries. Mortality data (through 2017) were collected by the National Center for Health Statistics. In 2020, 1,806,590 new cancer cases and 606,520 cancer deaths are projected to occur in the United States. The cancer death rate rose until 1991, then fell continuously through 2017, resulting in an overall decline of 29% that translates into an estimated 2.9 million fewer cancer deaths than would have occurred if peak rates had persisted. This progress is driven by long-term declines in death rates for the 4 leading cancers (lung, colorectal, breast, prostate); however, over the past decade (2008-2017), reductions slowed for female breast and colorectal cancers, and halted for prostate cancer. In contrast, declines accelerated for lung cancer, from 3% annually during 2008 through 2013 to 5% during 2013 through 2017 in men and from 2% to almost 4% in women, spurring the largest ever single-year drop in overall cancer mortality of 2.2% from 2016 to 2017. Yet lung cancer still caused more deaths in 2017 than breast, prostate, colorectal, and brain cancers combined. Recent mortality declines were also dramatic for melanoma of the skin in the wake of US Food and Drug Administration approval of new therapies for metastatic disease, escalating to 7% annually during 2013 through 2017 from 1% during 2006 through 2010 in men and women aged 50 to 64 years and from 2% to 3% in those aged 20 to 49 years; annual declines of 5% to 6% in individuals aged 65 years and older are particularly striking because rates in this age group were increasing prior to 2013. It is also notable that long-term rapid increases in liver cancer mortality have attenuated in women and stabilized in men. In summary, slowing momentum for some cancers amenable to early detection is juxtaposed with notable gains for other common cancers.

The American College of Medical Genetics and Genomics (ACMG) previously developed guidance for the interpretation of sequence variants.(1) In the past decade, sequencing technology has evolved rapidly with the advent of high-throughput next-generation sequencing. By adopting and leveraging next-generation sequencing, clinical laboratories are now performing an ever-increasing catalogue of genetic testing spanning genotyping, single genes, gene panels, exomes, genomes, transcriptomes, and epigenetic assays for genetic disorders. By virtue of increased complexity, this shift in genetic testing has been accompanied by new challenges in sequence interpretation. In this context the ACMG convened a workgroup in 2013 comprising representatives from the ACMG, the Association for Molecular Pathology (AMP), and the College of American Pathologists to revisit and revise the standards and guidelines for the interpretation of sequence variants. The group consisted of clinical laboratory directors and clinicians. This report represents expert opinion of the workgroup with input from ACMG, AMP, and College of American Pathologists stakeholders. These recommendations primarily apply to the breadth of genetic tests used in clinical laboratories, including genotyping, single genes, panels, exomes, and genomes. This report recommends the use of specific standard terminology-"pathogenic," "likely pathogenic," "uncertain significance," "likely benign," and "benign"-to describe variants identified in genes that cause Mendelian disorders. Moreover, this recommendation describes a process for classifying variants into these five categories based on criteria using typical types of variant evidence (e.g., population data, computational data, functional data, segregation data). Because of the increased complexity of analysis and interpretation of clinical genetic testing described in this report, the ACMG strongly recommends that clinical molecular genetic testing should be performed in a Clinical Laboratory Improvement Amendments-approved laboratory, with results interpreted by a board-certified clinical molecular geneticist or molecular genetic pathologist or the equivalent.

Experimental and computational approaches to estimate solubility and permeability in discovery and development settings are described. In the discovery setting 'the rule of 5' predicts that poor absorption or permeation is more likely when there are more than 5 H-bond donors, 10 H-bond acceptors, the molecular weight (MWT) is greater than 500 and the calculated Log P (CLogP) is greater than 5 (or MlogP>> 4.15). Computational methodology for the rule-based Moriguchi Log P (MLogP) calculation is described. Turbidimetric solubility measurement is described and applied to known drugs. High throughput screening (HTS) leads tend to have higher MWT and Log P and lower turbidimetric solubility than leads in the pre-HTS era. In the development setting, solubility calculations focus on exact value prediction and are difficult because of polymorphism. Recent work on linear free energy relationships and Log P approaches are critically reviewed. Useful predictions are possible in closely related analog series when coupled with experimental thermodynamic solubility measurements.

BACKGROUND

Phase 1 and 2 clinical trials of the BRAF kinase inhibitor vemurafenib (PLX4032) have shown response rates of more than 50% in patients with metastatic melanoma with the BRAF V600E mutation.

METHODS

We conducted a phase 3 randomized clinical trial comparing vemurafenib with dacarbazine in 675 patients with previously untreated, metastatic melanoma with the BRAF V600E mutation. Patients were randomly assigned to receive either vemurafenib (960 mg orally twice daily) or dacarbazine (1000 mg per square meter of body-surface area intravenously every 3 weeks). Coprimary end points were rates of overall and progression-free survival. Secondary end points included the response rate, response duration, and safety. A final analysis was planned after 196 deaths and an interim analysis after 98 deaths.

RESULTS

At 6 months, overall survival was 84% (95% confidence interval [CI], 78 to 89) in the vemurafenib group and 64% (95% CI, 56 to 73) in the dacarbazine group. In the interim analysis for overall survival and final analysis for progression-free survival, vemurafenib was associated with a relative reduction of 63% in the risk of death and of 74% in the risk of either death or disease progression, as compared with dacarbazine (P<0.001 for both comparisons). After review of the interim analysis by an independent data and safety monitoring board, crossover from dacarbazine to vemurafenib was recommended. Response rates were 48% for vemurafenib and 5% for dacarbazine. Common adverse events associated with vemurafenib were arthralgia, rash, fatigue, alopecia, keratoacanthoma or squamous-cell carcinoma, photosensitivity, nausea, and diarrhea; 38% of patients required dose modification because of toxic effects.

CONCLUSIONS

Vemurafenib produced improved rates of overall and progression-free survival in patients with previously untreated melanoma with the BRAF V600E mutation. (Funded by Hoffmann-La Roche; BRIM-3 ClinicalTrials.gov number, NCT01006980.).

We describe the Phase II HapMap, which characterizes over 3.1 million human single nucleotide polymorphisms (SNPs) genotyped in 270 individuals from four geographically diverse populations and includes 25-35% of common SNP variation in the populations surveyed. The map is estimated to capture untyped common variation with an average maximum r2 of between 0.9 and 0.96 depending on population. We demonstrate that the current generation of commercial genome-wide genotyping products captures common Phase II SNPs with an average maximum r2 of up to 0.8 in African and up to 0.95 in non-African populations, and that potential gains in power in association studies can be obtained through imputation. These data also reveal novel aspects of the structure of linkage disequilibrium. We show that 10-30% of pairs of individuals within a population share at least one region of extended genetic identity arising from recent ancestry and that up to 1% of all common variants are untaggable, primarily because they lie within recombination hotspots. We show that recombination rates vary systematically around genes and between genes of different function. Finally, we demonstrate increased differentiation at non-synonymous, compared to synonymous, SNPs, resulting from systematic differences in the strength or efficacy of natural selection between populations.

Ionizing radiation represents the most effective therapy for glioblastoma (World Health Organization grade IV glioma), one of the most lethal human malignancies, but radiotherapy remains only palliative because of radioresistance. The mechanisms underlying tumour radioresistance have remained elusive. Here we show that cancer stem cells contribute to glioma radioresistance through preferential activation of the DNA damage checkpoint response and an increase in DNA repair capacity. The fraction of tumour cells expressing CD133 (Prominin-1), a marker for both neural stem cells and brain cancer stem cells, is enriched after radiation in gliomas. In both cell culture and the brains of immunocompromised mice, CD133-expressing glioma cells survive ionizing radiation in increased proportions relative to most tumour cells, which lack CD133. CD133-expressing tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activate the DNA damage checkpoint in response to radiation, and repair radiation-induced DNA damage more effectively than CD133-negative tumour cells. In addition, the radioresistance of CD133-positive glioma stem cells can be reversed with a specific inhibitor of the Chk1 and Chk2 checkpoint kinases. Our results suggest that CD133-positive tumour cells represent the cellular population that confers glioma radioresistance and could be the source of tumour recurrence after radiation. Targeting DNA damage checkpoint response in cancer stem cells may overcome this radioresistance and provide a therapeutic model for malignant brain cancers.

BACKGROUND

Measuring disease and injury burden in populations requires a composite metric that captures both premature mortality and the prevalence and severity of ill-health. The 1990 Global Burden of Disease study proposed disability-adjusted life years (DALYs) to measure disease burden. No comprehensive update of disease burden worldwide incorporating a systematic reassessment of disease and injury-specific epidemiology has been done since the 1990 study. We aimed to calculate disease burden worldwide and for 21 regions for 1990, 2005, and 2010 with methods to enable meaningful comparisons over time.

METHODS

We calculated DALYs as the sum of years of life lost (YLLs) and years lived with disability (YLDs). DALYs were calculated for 291 causes, 20 age groups, both sexes, and for 187 countries, and aggregated to regional and global estimates of disease burden for three points in time with strictly comparable definitions and methods. YLLs were calculated from age-sex-country-time-specific estimates of mortality by cause, with death by standardised lost life expectancy at each age. YLDs were calculated as prevalence of 1160 disabling sequelae, by age, sex, and cause, and weighted by new disability weights for each health state. Neither YLLs nor YLDs were age-weighted or discounted. Uncertainty around cause-specific DALYs was calculated incorporating uncertainty in levels of all-cause mortality, cause-specific mortality, prevalence, and disability weights.

RESULTS

Global DALYs remained stable from 1990 (2·503 billion) to 2010 (2·490 billion). Crude DALYs per 1000 decreased by 23% (472 per 1000 to 361 per 1000). An important shift has occurred in DALY composition with the contribution of deaths and disability among children (younger than 5 years of age) declining from 41% of global DALYs in 1990 to 25% in 2010. YLLs typically account for about half of disease burden in more developed regions (high-income Asia Pacific, western Europe, high-income North America, and Australasia), rising to over 80% of DALYs in sub-Saharan Africa. In 1990, 47% of DALYs worldwide were from communicable, maternal, neonatal, and nutritional disorders, 43% from non-communicable diseases, and 10% from injuries. By 2010, this had shifted to 35%, 54%, and 11%, respectively. Ischaemic heart disease was the leading cause of DALYs worldwide in 2010 (up from fourth rank in 1990, increasing by 29%), followed by lower respiratory infections (top rank in 1990; 44% decline in DALYs), stroke (fifth in 1990; 19% increase), diarrhoeal diseases (second in 1990; 51% decrease), and HIV/AIDS (33rd in 1990; 351% increase). Major depressive disorder increased from 15th to 11th rank (37% increase) and road injury from 12th to 10th rank (34% increase). Substantial heterogeneity exists in rankings of leading causes of disease burden among regions.

CONCLUSIONS

Global disease burden has continued to shift away from communicable to non-communicable diseases and from premature death to years lived with disability. In sub-Saharan Africa, however, many communicable, maternal, neonatal, and nutritional disorders remain the dominant causes of disease burden. The rising burden from mental and behavioural disorders, musculoskeletal disorders, and diabetes will impose new challenges on health systems. Regional heterogeneity highlights the importance of understanding local burden of disease and setting goals and targets for the post-2015 agenda taking such patterns into account. Because of improved definitions, methods, and data, these results for 1990 and 2010 supersede all previously published Global Burden of Disease results.

BACKGROUND

Bill & Melinda Gates Foundation.

A gene expression system based on bacteriophage T7 RNA polymerase has been developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amount of T7 RNA polymerase provided from a cloned copy of T7 gene 1 is sufficient to direct high-level transcription from a T7 promoter in a multicopy plasmid. Such transcription can proceed several times around the plasmid without terminating, and can be so active that transcription by E. coli RNA polymerase is greatly decreased. When a cleavage site for RNase III is introduced, discrete RNAs of plasmid length can accumulate. The natural transcription terminator from T7 DNA also works effectively in the plasmid. Both the rate of synthesis and the accumulation of RNA directed by T7 RNA polymerase can reach levels comparable with those for ribosomal RNAs in a normal cell. These high levels of accumulation suggest that the RNAs are relatively stable, perhaps in part because their great length and/or stem-and-loop structures at their 3' ends help to protect them against exonucleolytic degradation. It seems likely that a specific mRNA produced by T7 RNA polymerase can rapidly saturate the translational machinery of E. coli, so that the rate of protein synthesis from such an mRNA will depend primarily on the efficiency of its translation. When the mRNA is efficiently translated, a target protein can accumulate to greater than 50% of the total cell protein in three hours or less. We have used two ways to deliver active T7 RNA polymerase to the cell; infection by a lambda derivative that carries gene 1, or induction of a chromosomal copy of gene 1 under control of the lacUV5 promoter. When gene 1 is delivered by infection, very toxic target genes can be maintained silent in the cell until T7 RNA polymerase is introduced, when they rapidly become expressed at high levels. When gene 1 is resident in the chromosome, even the very low basal levels of T7 RNA polymerase present in the uninduced cell can prevent the establishment of plasmids carrying toxic target genes, or make the plasmid unstable.(ABSTRACT TRUNCATED AT 400 WORDS)

BACKGROUND

Type 2 diabetes mellitus is increasingly common, primarily because of increases in the prevalence of a sedentary lifestyle and obesity. Whether type 2 diabetes can be prevented by interventions that affect the lifestyles of subjects at high risk for the disease is not known.

METHODS

We randomly assigned 522 middle-aged, overweight subjects (172 men and 350 women; mean age, 55 years; mean body-mass index [weight in kilograms divided by the square of the height in meters], 31) with impaired glucose tolerance to either the intervention group or the control group. Each subject in the intervention group received individualized counseling aimed at reducing weight, total intake of fat, and intake of saturated fat and increasing intake of fiber and physical activity. An oral glucose-tolerance test was performed annually; the diagnosis of diabetes was confirmed by a second test. The mean duration of follow-up was 3.2 years.

RESULTS

The mean (+/-SD) amount of weight lost between base line and the end of year 1 was 4.2+/-5.1 kg in the intervention group and 0.8+/-3.7 kg in the control group; the net loss by the end of year 2 was 3.5+/-5.5 kg in the intervention group and 0.8+/-4.4 kg in the control group (P<0.001 for both comparisons between the groups). The cumulative incidence of diabetes after four years was 11 percent (95 percent confidence interval, 6 to 15 percent) in the intervention group and 23 percent (95 percent confidence interval, 17 to 29 percent) in the control group. During the trial, the risk of diabetes was reduced by 58 percent (P<0.001) in the intervention group. The reduction in the incidence of diabetes was directly associated with changes in lifestyle.

CONCLUSIONS

Type 2 diabetes can be prevented by changes in the lifestyles of high-risk subjects.

BACKGROUND

In 2010, overweight and obesity were estimated to cause 3·4 million deaths, 3·9% of years of life lost, and 3·8% of disability-adjusted life-years (DALYs) worldwide. The rise in obesity has led to widespread calls for regular monitoring of changes in overweight and obesity prevalence in all populations. Comparable, up-to-date information about levels and trends is essential to quantify population health effects and to prompt decision makers to prioritise action. We estimate the global, regional, and national prevalence of overweight and obesity in children and adults during 1980-2013.

METHODS

We systematically identified surveys, reports, and published studies (n=1769) that included data for height and weight, both through physical measurements and self-reports. We used mixed effects linear regression to correct for bias in self-reports. We obtained data for prevalence of obesity and overweight by age, sex, country, and year (n=19,244) with a spatiotemporal Gaussian process regression model to estimate prevalence with 95% uncertainty intervals (UIs).

RESULTS

Worldwide, the proportion of adults with a body-mass index (BMI) of 25 kg/m(2) or greater increased between 1980 and 2013 from 28·8% (95% UI 28·4-29·3) to 36·9% (36·3-37·4) in men, and from 29·8% (29·3-30·2) to 38·0% (37·5-38·5) in women. Prevalence has increased substantially in children and adolescents in developed countries; 23·8% (22·9-24·7) of boys and 22·6% (21·7-23·6) of girls were overweight or obese in 2013. The prevalence of overweight and obesity has also increased in children and adolescents in developing countries, from 8·1% (7·7-8·6) to 12·9% (12·3-13·5) in 2013 for boys and from 8·4% (8·1-8·8) to 13·4% (13·0-13·9) in girls. In adults, estimated prevalence of obesity exceeded 50% in men in Tonga and in women in Kuwait, Kiribati, Federated States of Micronesia, Libya, Qatar, Tonga, and Samoa. Since 2006, the increase in adult obesity in developed countries has slowed down.

CONCLUSIONS

Because of the established health risks and substantial increases in prevalence, obesity has become a major global health challenge. Not only is obesity increasing, but no national success stories have been reported in the past 33 years. Urgent global action and leadership is needed to help countries to more effectively intervene.

BACKGROUND

Bill & Melinda Gates Foundation.

Reconstructing phylogenies from intraspecific data (such as human mitochondrial DNA variation) is often a challenging task because of large sample sizes and small genetic distances between individuals. The resulting multitude of plausible trees is best expressed by a network which displays alternative potential evolutionary paths in the form of cycles. We present a method ("median joining" [MJ]) for constructing networks from recombination-free population data that combines features of Kruskal's algorithm for finding minimum spanning trees by favoring short connections, and Farris's maximum-parsimony (MP) heuristic algorithm, which sequentially adds new vertices called "median vectors", except that our MJ method does not resolve ties. The MJ method is hence closely related to the earlier approach of Foulds, Hendy, and Penny for estimating MP trees but can be adjusted to the level of homoplasy by setting a parameter epsilon. Unlike our earlier reduced median (RM) network method, MJ is applicable to multistate characters (e.g., amino acid sequences). An additional feature is the speed of the implemented algorithm: a sample of 800 worldwide mtDNA hypervariable segment I sequences requires less than 3 h on a Pentium 120 PC. The MJ method is demonstrated on a Tibetan mitochondrial DNA RFLP data set.

Thrombolytic therapy for acute ischemic stroke has been approached cautiously because there were high rates of intracerebral hemorrhage in early clinical trials. We performed a randomized, double-blind trial of intravenous recombinant tissue plasminogen activator (t-PA) for ischemic stroke after recent pilot studies suggested that t-PA was beneficial when treatment was begun within three hours of the onset of stroke.

The trial had two parts. Part 1 (in which 291 patients were enrolled) tested whether t-PA had clinical activity, as indicated by an improvement of 4 points over base-line values in the score of the National Institutes of Health stroke scale (NIHSS) or the resolution of the neurologic deficit within 24 hours of the onset of stroke. Part 2 (in which 333 patients were enrolled) used a global test statistic to assess clinical outcome at three months, according to scores on the Barthel index, modified Rankin scale, Glasgow outcome scale, and NIHSS:

In part 1, there was no significant difference between the group given t-PA and that given placebo in the percentages of patients with neurologic improvement at 24 hours, although a benefit was observed for the t-PA group at three months for all four outcome measures. In part 2, the long-term clinical benefit of t-PA predicted by the results of part 1 was confirmed (global odds ratio for a favorable outcome, 1.7; 95 percent confidence interval, 1.2 to 2.6). As compared with patients given placebo, patients treated with t-PA were at least 30 percent more likely to have minimal or no disability at three months on the assessment scales. Symptomatic intracerebral hemorrhage within 36 hours after the onset of stroke occurred in 6.4 percent of patients given t-PA but only 0.6 percent of patients given placebo (P < 0.001). Mortality at three months was 17 percent in the t-PA group and 21 percent in the placebo group (P = 0.30).

Despite an increased incidence of symptomatic intracerebral hemorrhage, treatment with intravenous t-PA within three hours of the onset of ischemic stroke improved clinical outcome at three months.

Resistance to insulin-stimulated glucose uptake is present in the majority of patients with impaired glucose tolerance (IGT) or non-insulin-dependent diabetes mellitus (NIDDM) and in approximately 25% of nonobese individuals with normal oral glucose tolerance. In these conditions, deterioration of glucose tolerance can only be prevented if the beta-cell is able to increase its insulin secretory response and maintain a state of chronic hyperinsulinemia. When this goal cannot be achieved, gross decompensation of glucose homeostasis occurs. The relationship between insulin resistance, plasma insulin level, and glucose intolerance is mediated to a significant degree by changes in ambient plasma free-fatty acid (FFA) concentration. Patients with NIDDM are also resistant to insulin suppression of plasma FFA concentration, but plasma FFA concentrations can be reduced by relatively small increments in insulin concentration. Consequently, elevations of circulating plasma FFA concentration can be prevented if large amounts of insulin can be secreted. If hyperinsulinemia cannot be maintained, plasma FFA concentration will not be suppressed normally, and the resulting increase in plasma FFA concentration will lead to increased hepatic glucose production. Because these events take place in individuals who are quite resistant to insulin-stimulated glucose uptake, it is apparent that even small increases in hepatic glucose production are likely to lead to significant fasting hyperglycemia under these conditions. Although hyperinsulinemia may prevent frank decompensation of glucose homeostasis in insulin-resistant individuals, this compensatory response of the endocrine pancreas is not without its price. Patients with hypertension, treated or untreated, are insulin resistant, hyperglycemic, and hyperinsulinemic. In addition, a direct relationship between plasma insulin concentration and blood pressure has been noted. Hypertension can also be produced in normal rats when they are fed a fructose-enriched diet, an intervention that also leads to the development of insulin resistance and hyperinsulinemia. The development of hypertension in normal rats by an experimental manipulation known to induce insulin resistance and hyperinsulinemia provides further support for the view that the relationship between the three variables may be a causal one.(ABSTRACT TRUNCATED AT 400 WORDS)

OBJECTIVE

This study attempts to develop a comprehensive set of comorbidity measures for use with large administrative inpatient datasets.

METHODS

The study involved clinical and empirical review of comorbidity measures, development of a framework that attempts to segregate comorbidities from other aspects of the patient's condition, development of a comorbidity algorithm, and testing on heterogeneous and homogeneous patient groups. Data were drawn from all adult, nonmaternal inpatients from 438 acute care hospitals in California in 1992 (n = 1,779,167). Outcome measures were those commonly available in administrative data: length of stay, hospital charges, and in-hospital death.

RESULTS

A comprehensive set of 30 comorbidity measures was developed. The comorbidities were associated with substantial increases in length of stay, hospital charges, and mortality both for heterogeneous and homogeneous disease groups. Several comorbidities are described that are important predictors of outcomes, yet commonly are not measured. These include mental disorders, drug and alcohol abuse, obesity, coagulopathy, weight loss, and fluid and electrolyte disorders.

CONCLUSIONS

The comorbidities had independent effects on outcomes and probably should not be simplified as an index because they affect outcomes differently among different patient groups. The present method addresses some of the limitations of previous measures. It is based on a comprehensive approach to identifying comorbidities and separates them from the primary reason for hospitalization, resulting in an expanded set of comorbidities that easily is applied without further refinement to administrative data for a wide range of diseases.

Mammalian microRNAs (miRNAs) pair to 3'UTRs of mRNAs to direct their posttranscriptional repression. Important for target recognition are approximately 7 nt sites that match the seed region of the miRNA. However, these seed matches are not always sufficient for repression, indicating that other characteristics help specify targeting. By combining computational and experimental approaches, we uncovered five general features of site context that boost site efficacy: AU-rich nucleotide composition near the site, proximity to sites for coexpressed miRNAs (which leads to cooperative action), proximity to residues pairing to miRNA nucleotides 13-16, positioning within the 3'UTR at least 15 nt from the stop codon, and positioning away from the center of long UTRs. A model combining these context determinants quantitatively predicts site performance both for exogenously added miRNAs and for endogenous miRNA-message interactions. Because it predicts site efficacy without recourse to evolutionary conservation, the model also identifies effective nonconserved sites and siRNA off-targets.

BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.

Non-biological experimental variation or "batch effects" are commonly observed across multiple batches of microarray experiments, often rendering the task of combining data from these batches difficult. The ability to combine microarray data sets is advantageous to researchers to increase statistical power to detect biological phenomena from studies where logistical considerations restrict sample size or in studies that require the sequential hybridization of arrays. In general, it is inappropriate to combine data sets without adjusting for batch effects. Methods have been proposed to filter batch effects from data, but these are often complicated and require large batch sizes (>> 25) to implement. Because the majority of microarray studies are conducted using much smaller sample sizes, existing methods are not sufficient. We propose parametric and non-parametric empirical Bayes frameworks for adjusting data for batch effects that is robust to outliers in small sample sizes and performs comparable to existing methods for large samples. We illustrate our methods using two example data sets and show that our methods are justifiable, easy to apply, and useful in practice. Software for our method is freely available at: http://biosun1.harvard.edu/complab/batch/.

A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

BACKGROUND

In patients with type 2 diabetes, the effects of intensive glucose control on vascular outcomes remain uncertain.

METHODS

We randomly assigned 11,140 patients with type 2 diabetes to undergo either standard glucose control or intensive glucose control, defined as the use of gliclazide (modified release) plus other drugs as required to achieve a glycated hemoglobin value of 6.5% or less. Primary end points were composites of major macrovascular events (death from cardiovascular causes, nonfatal myocardial infarction, or nonfatal stroke) and major microvascular events (new or worsening nephropathy or retinopathy), assessed both jointly and separately.

RESULTS

After a median of 5 years of follow-up, the mean glycated hemoglobin level was lower in the intensive-control group (6.5%) than in the standard-control group (7.3%). Intensive control reduced the incidence of combined major macrovascular and microvascular events (18.1%, vs. 20.0% with standard control; hazard ratio, 0.90; 95% confidence interval [CI], 0.82 to 0.98; P=0.01), as well as that of major microvascular events (9.4% vs. 10.9%; hazard ratio, 0.86; 95% CI, 0.77 to 0.97; P=0.01), primarily because of a reduction in the incidence of nephropathy (4.1% vs. 5.2%; hazard ratio, 0.79; 95% CI, 0.66 to 0.93; P=0.006), with no significant effect on retinopathy (P=0.50). There were no significant effects of the type of glucose control on major macrovascular events (hazard ratio with intensive control, 0.94; 95% CI, 0.84 to 1.06; P=0.32), death from cardiovascular causes (hazard ratio with intensive control, 0.88; 95% CI, 0.74 to 1.04; P=0.12), or death from any cause (hazard ratio with intensive control, 0.93; 95% CI, 0.83 to 1.06; P=0.28). Severe hypoglycemia, although uncommon, was more common in the intensive-control group (2.7%, vs. 1.5% in the standard-control group; hazard ratio, 1.86; 95% CI, 1.42 to 2.40; P<0.001).

CONCLUSIONS

A strategy of intensive glucose control, involving gliclazide (modified release) and other drugs as required, that lowered the glycated hemoglobin value to 6.5% yielded a 10% relative reduction in the combined outcome of major macrovascular and microvascular events, primarily as a consequence of a 21% relative reduction in nephropathy. (ClinicalTrials.gov number, NCT00145925.)

Cells of a multicellular organism are genetically homogeneous but structurally and functionally heterogeneous owing to the differential expression of genes. Many of these differences in gene expression arise during development and are subsequently retained through mitosis. Stable alterations of this kind are said to be 'epigenetic', because they are heritable in the short term but do not involve mutations of the DNA itself. Research over the past few years has focused on two molecular mechanisms that mediate epigenetic phenomena: DNA methylation and histone modifications. Here, we review advances in the understanding of the mechanism and role of DNA methylation in biological processes. Epigenetic effects by means of DNA methylation have an important role in development but can also arise stochastically as animals age. Identification of proteins that mediate these effects has provided insight into this complex process and diseases that occur when it is perturbed. External influences on epigenetic processes are seen in the effects of diet on long-term diseases such as cancer. Thus, epigenetic mechanisms seem to allow an organism to respond to the environment through changes in gene expression. The extent to which environmental effects can provoke epigenetic responses represents an exciting area of future research.

Toll-like receptors (TLRs) are a family of innate immune-recognition receptors that recognize molecular patterns associated with microbial pathogens, and induce antimicrobial immune responses. Double-stranded RNA (dsRNA) is a molecular pattern associated with viral infection, because it is produced by most viruses at some point during their replication. Here we show that mammalian TLR3 recognizes dsRNA, and that activation of the receptor induces the activation of NF-kappaB and the production of type I interferons (IFNs). TLR3-deficient (TLR3-/-) mice showed reduced responses to polyinosine-polycytidylic acid (poly(I:C)), resistance to the lethal effect of poly(I:C) when sensitized with d-galactosamine (d-GalN), and reduced production of inflammatory cytokines. MyD88 is an adaptor protein that is shared by all the known TLRs. When activated by poly(I:C), TLR3 induces cytokine production through a signalling pathway dependent on MyD88. Moreover, poly(I:C) can induce activation of NF-kappaB and mitogen-activated protein (MAP) kinases independently of MyD88, and cause dendritic cells to mature.