The MSH1 and MSH2 genes of Saccharomyces cerevisiae are predicted to encode proteins that are homologous to the Escherichia coli MutS and Streptococcus pneumoniae HexA proteins and their homologs. Disruption of the MSH1 gene caused a petite phenotype which was established rapidly. A functional MSH1 gene present on a single-copy centromere plasmid was incapable of rescuing the established msh1 petite phenotype. Analysis of msh1 strains demonstrated that mutagenesis and large-scale rearrangement of mitochondrial DNA had occurred. 4',6-Diamidino-2-phenylindole (DAPI) staining of msh1 yeast revealed an aberrant distribution of mtDNA. Haploid msh2 mutants displayed an increase of 85-fold in the rate of spontaneous mutation to canavanine resistance. Sporulation of homozygous msh2/msh2 diploids gave rise to a high level of lethality which was compounded during increased vegetative growth prior to sporulation. msh2 mutations also affected gene conversion of two HIS4 alleles. The his4x mutation, lying near the 5' end of the gene, was converted with equal frequency in both wild-type and msh2 strains. However, many of the events in the msh2 background were post-meiotic segregation (PMS) events (46.4%) while none (< 0.25%) of the aberrant segregations in wild type were PMS events. The his4b allele, lying 1.6 kb downstream of his4x, was converted at a 10-fold higher frequency in the msh2 background than in the corresponding wild-type strain. Like the his4x allele, his4b showed a high level of PMS (30%) in the msh2 background compared to the corresponding wild-type strain where no (< 0.26%) PMS events were observed. These results indicate that MSH1 plays a role in repair or stability of mtDNA and MSH2 plays a role in repair of 4-bp insertion/deletion mispairs in the nucleus.

OBJECTIVE

To examine body size and fat measurements of babies born in rural India and compare them with white Caucasian babies born in an industrialised country.

METHODS

Community-based observational study in rural India, and comparison with data from an earlier study in the UK, measured using similar methods.

METHODS

A total of 631 term babies born in six rural villages, near the city of Pune, Maharashtra, India, and 338 term babies born in the Princess Anne Hospital, Southampton, UK.

METHODS

Maternal weight and height, and neonatal weight, length, head, mid-upper-arm and abdominal circumferences, subscapular and triceps skinfold thicknesses, and placental weight.

RESULTS

The Indian mothers were younger, lighter, shorter and had a lower mean body mass index (BMI) (mean age, weight, height and BMI: 21.4 y, 44.6 kg, 1.52 m, and 18.2 kg/m(2)) than Southampton mothers (26.8 y, 63.6 kg, 1.63 m and 23.4 kg/m(2)). They gave birth to lighter babies (mean birthweight: 2.7 kg compared with 3.5 kg). Compared to Southampton babies, the Indian babies were small in all body measurements, the smallest being abdominal circumference (s.d. score: -2.38; 95% CI: -2.48 to -2.29) and mid-arm circumference (s.d. score: -1.82; 95% CI: -1.89 to -1.75), while the most preserved measurement was the subscapular skinfold thickness (s.d. score: -0.53; 95% CI: -0.61 to -0.46). Skinfolds were relatively preserved in the lightest babies (below the 10th percentile of birthweight) in both populations.

CONCLUSIONS

Small Indian babies have small abdominal viscera and low muscle mass, but preserve body fat during their intrauterine development. This body composition may persist postnatally and predispose to an insulin-resistant state.

Sparling, Philip F. (Communicable Disease Center, Atlanta, Ga.). Genetic transformation of Neisseria gonorrhoeae to streptomycin resistance. J. Bacteriol. 92:1364-1371. 1966.-Eight strains of Neisseria gonorrhoeae were transformed to streptomycin resistance by deoxyribonucleic acid (DNA) extracted from a streptomycin-resistant strain of N. gonorrhoeae. In all strains, competence was greatest in the naturally occurring, virulent clonal types 1 and 2, which gave transformation frequencies up to 1%. Clonal types 3 and 4, which arise on laboratory transfer and are avirulent, gave maximal transformation frequencies of 0.00005%. Competence was maximal in lag and early log phases of growth, but was maintained throughout the growth cycle. A complex broth was required for the physiological expression of competence. The kinetics of DNA uptake, dose-response curve of DNA versus transformants, time required for phenotypic expression, and other features were similar to those in other bacterial transformation systems.

Eosinophil accumulation is a prominent feature of allergic inflammatory reactions, such as those occurring in the lung of the allergic asthmatic, but the endogenous chemoattractants involved have not been identified. We have investigated this in an established model of allergic inflammation, using in vivo systems both to generate and assay relevant activity. Bronchoalveolar lavage (BAL) fluid was taken from sensitized guinea pigs at intervals after aerosol challenge with ovalbumin. BAL fluid was injected intradermally in unsensitized assay guinea pigs and the accumulation of intravenously injected 111In-eosinophils was measured. Activity was detected at 30 min after allergen challenge, peaking from 3 to 6 h and declining to low levels by 24 h. 3-h BAL fluid was purified using high performance liquid chromatography techniques in conjunction with the skin assay. Microsequencing revealed a novel protein from the C-C branch of the platelet factor 4 superfamily of chemotactic cytokines. The protein, "eotaxin," exhibits homology of 53% with human MCP-1, 44% with guinea pig MCP-1, 31% with human MIP-1 alpha, and 26% with human RANTES. Laser desorption time of flight mass analysis gave four different signals (8.15, 8.38, 8.81, and 9.03 kD), probably reflecting differential O-glycosylation. Eotaxin was highly potent, inducing substantial 111In-eosinophil accumulation at a 1-2 pmol dose in the skin, but did not induce significant 111In-neutrophil accumulation. Eotaxin was a potent stimulator of both guinea pig and human eosinophils in vitro. Human recombinant RANTES, MIP-1 alpha, and MCP-1 were all inactive in inducing 111In-eosinophil accumulation in guinea pig skin; however, evidence was obtained that eotaxin shares a binding site with RANTES on guinea pig eosinophils. This is the first description of a potent eosinophil chemoattractant cytokine generated in vivo and suggests the possibility that similar molecules may be important in the human asthmatic lung.

BACKGROUND

Currently, there is no effective intervention for a primary cytomegalovirus (CMV) infection during pregnancy.

METHODS

We studied pregnant women with a primary CMV infection. The therapy group comprised women whose amniotic fluid contained either CMV or CMV DNA and who were offered intravenous CMV hyperimmune globulin at a dose of 200 U per kilogram of maternal weight. A prevention group, consisting of women with a recent primary infection before 21 weeks' gestation or who declined amniocentesis, was offered monthly hyperimmune globulin (100 U per kilogram intravenously).

RESULTS

In the therapy group, 31 women received hyperimmune globulin, only 1 (3 percent) of whom gave birth to an infant with CMV disease (symptomatic at birth and handicapped at two or more years of age), as compared with 7 of 14 women who did not receive hyperimmune globulin (50 percent). Thus, hyperimmune globulin therapy was associated with a significantly lower risk of congenital CMV disease (adjusted odds ratio, 0.02; 95 percent confidence interval, -infinity to 0.15; P<0.001). In the prevention group, 37 women received hyperimmune globulin, 6 (16 percent) of whom had infants with congenital CMV infection, as compared with 19 of 47 women (40 percent) who did not receive hyperimmune globulin. Thus, hyperimmune globulin therapy was associated with a significantly lower risk of congenital CMV infection (adjusted odds ratio, 0.32; 95 percent confidence interval, 0.10 to 0.94; P=0.04). Hyperimmune globulin therapy significantly (P<0.001) increased CMV-specific IgG concentrations and avidity and decreased natural killer cells and HLA-DR+ cells and had no adverse effects.

CONCLUSIONS

Treatment of pregnant women with CMV-specific hyperimmune globulin is safe, and the findings of this nonrandomized study suggest that it may be effective in the treatment and prevention of congenital CMV infection. A controlled trial of this agent may now be appropriate.

Short peptides that contain the basic region of the HIV-1 Tat protein bind specifically to a bulged region in TAR RNA. A peptide that contained nine arginines (R9) also bound specifically to TAR, and a mutant Tat protein that contained R9 was fully active for transactivation. In contrast, a peptide that contained nine lysines (K9) bound TAR poorly and the corresponding protein gave only marginal activity. By starting with the K9 mutant and replacing lysine residues with arginines, a single arginine was identified that is required for specific binding and transactivation. Ethylation interference experiments suggest that this arginine contacts two adjacent phosphates at the RNA bulge. Model building suggests that the arginine eta nitrogens and the epsilon nitrogen can form specific networks of hydrogen bonds with adjacent pairs of phosphates and that these arrangements are likely to occur near RNA loops and bulges and not within double-stranded A-form RNA. Thus, arginine side chains may be commonly used to recognize specific RNA structures.

Cell-based therapies are attractive approaches to promote myelin repair. Recent studies demonstrated a reduction in disease burden in mice with experimental allergic encephalomyelitis (EAE) treated with mouse mesenchymal stem cells (MSCs). Here, we demonstrated human bone marrow-derived MSCs (BM-hMSCs) promote functional recovery in both chronic and relapsing-remitting models of mouse EAE, traced their migration into the injured CNS and assayed their ability to modulate disease progression and the host immune response. Injected BM-hMSCs accumulated in the CNS, reduced the extent of damage and increased oligodendrocyte lineage cells in lesion areas. The increase in oligodendrocytes in lesions may reflect BM-hMSC-induced changes in neural fate determination, since neurospheres from treated animals gave rise to more oligodendrocytes and less astrocytes than nontreated neurospheres. Host immune responses were also influenced by BM-hMSCs. Inflammatory T-cells including interferon gamma producing Th1 cells and IL-17 producing Th17 inflammatory cells and their associated cytokines were reduced along with concomitant increases in IL-4 producing Th2 cells and anti-inflammatory cytokines. Together, these data suggest that the BM-hMSCs represent a viable option for therapeutic approaches.

A model system for studying Candida biofilms growing on the surface of small discs of catheter material is described. Biofilm formation was determined quantitatively by a colorimetric assay involving reduction of a tetrazolium salt or by [3H]leucine incorporation; both methods gave excellent correlation with biofilm dry weight (r = 0.997 and 0.945, respectively). Growth of Candida albicans biofilms in medium containing 500 mM galactose or 50 mM glucose reached a maximum after 48 h and then declined; however, the cell yield was lower in low-glucose medium. Comparison of biofilm formation by 15 different isolates of C. albicans failed to reveal any correlation with pathogenicity within this group, but there was some correlation with pathogenicity when different Candida species were tested. Isolates of C. parapsilosis (Glasgow), C. pseudotropicalis, and C. glabrata all gave significantly less biofilm growth (P < 0.001) than the more pathogenic C. albicans. Evaluation of various catheter materials showed that biofilm formation by C. albicans was slightly increased on latex or silicone elastomer (P < 0.05), compared with polyvinyl chloride, but substantially decreased on polyurethane or 100% silicone (P < 0.001). Scanning electron microscopy demonstrated that after 48 h, C. albicans biofilms consisted of a dense network of yeasts, germ tubes, pseudohyphae, and hyphae; extracellular polymeric material was visible on the surfaces of some of these morphological forms. Our model system is a simple and convenient method for studying Candida biofilms and could be used for testing the efficacy of antifungal agents against biofilm cells.

Recently, we identified neuregulin 1 (NRG1) as a susceptibility gene for schizophrenia in the Icelandic population, by a combined linkage and association approach. Here, we report the first study evaluating the relevance of NRG1 to schizophrenia in a population outside Iceland. Markers representing a core at-risk haplotype found in Icelanders at the 5' end of the NRG1 gene were genotyped in 609 unrelated Scottish patients and 618 unrelated Scottish control individuals. This haplotype consisted of five SNP markers and two microsatellites, which all appear to be in strong linkage disequilibrium. For the Scottish patients and control subjects, haplotype frequencies were estimated by maximum likelihood, using the expectation-maximization algorithm. The frequency of the seven-marker haplotype among the Scottish patients was significantly greater than that among the control subjects (10.2% vs. 5.9%, P=.00031). The estimated risk ratio was 1.8, which is in keeping with our report of unrelated Icelandic patients (2.1). Three of the seven markers in the haplotype gave single-point P values ranging from .000064 to .0021 for the allele contributing to the at-risk haplotype. This direct replication of haplotype association in a second population further implicates NRG1 as a factor that contributes to the etiology of schizophrenia.

BACKGROUND

The beneficial effects of interferon beta have only been shown for patients in the relapsing-remitting phase of multiple sclerosis (MS). The role of interferon beta in the treatment of patients who are in the secondary progressive phase of the disease (SP-MS), and for whom no effective drug treatment is available, has not been assessed.

METHODS

In this multicentre, double-masked, randomised, placebo-controlled trial, outpatients with SP-MS having scores of 3.0-6.5 on the Expanded Disability Status Scale (EDSS) received either 8 million IU interferon beta-1b every other day subcutaneously, or placebo, for up to 3 years. The primary outcome was the time to confirmed progression in disability as measured by a 1.0 point increase on the EDSS, sustained for at least 3 months, or a 0.5 point increase if the baseline EDSS was 6.0 or 6.5. A prospectively planned interim analysis of safety and efficacy of the intention-to-treat population was done after all patients had been in the study for at least 2 years.

RESULTS

358 patients with SP-MS were allocated placebo and 360 were allocated interferon beta-1b; 57 patients (31 placebo, 26 interferon beta-1b) were lost to follow-up. There was a highly significant difference in time to confirmed progression of disability in favour of interferon beta-1b (p=0.0008). Interferon beta-1b delayed progression for 9-12 months in a study period of 2-3 years. The odds ratio for confirmed progression was 0.65 (95% CI 0.52-0.83). This beneficial effect was seen in patients with superimposed relapses and in patients who had only progressive deterioration without relapses. Positive results were also obtained regarding time to becoming wheelchair-bound, relapse rate and severity, number of steroid treatments and hospital admissions, as well as on magnetic resonance imaging variables. The drug was safe and side effects were in line with previous experience with interferon beta-1b. The study was stopped after the interim results gave clear evidence of efficacy.

CONCLUSIONS

Treatment with interferon beta-1b delays sustained neurological deterioration in patients with SP-MS. Interferon beta-1b is the first treatment to show a therapeutic effect in patients with SP-MS.

BACKGROUND

Enumeration of extracellular vesicles has clinical potential as a biomarker for disease. In biological samples, the smallest and largest vesicles typically differ 25-fold in size, 300,000-fold in concentration, 20,000-fold in volume, and 10,000,000-fold in scattered light. Because of this heterogeneity, the currently employed techniques detect concentrations ranging from 10(4) to 10(12) vesicles mL(-1) .

OBJECTIVE

To investigate whether the large variation in the detected concentration of vesicles is caused by the minimum detectable vesicle size of five widely used techniques.

METHODS

The size and concentration of vesicles and reference beads were measured with transmission electron microscopy (TEM), a conventional flow cytometer, a flow cytometer dedicated to detecting submicrometer particles, nanoparticle tracking analysis (NTA), and resistive pulse sensing (RPS).

RESULTS

Each technique gave a different size distribution and a different concentration for the same vesicle sample.

CONCLUSIONS

Differences between the detected vesicle concentrations are primarily caused by differences between the minimum detectable vesicle sizes. The minimum detectable vesicle sizes were 70-90 nm for NTA, 70-100 nm for RPS, 150-190 nm for dedicated flow cytometry, and 270-600 nm for conventional flow cytometry. TEM could detect the smallest vesicles present, albeit after adhesion on a surface. Dedicated flow cytometry was most accurate in determining the size of reference beads, but is expected to be less accurate on vesicles, owing to heterogeneity of the refractive index of vesicles. Nevertheless, dedicated flow cytometry is relatively fast and allows multiplex fluorescence detection, making it most applicable to clinical research.

OBJECTIVE

A transgenic mouse model has revealed parameters of the angiogenic switch during multistep tumorigenesis of pancreatic islets, and demonstrated efficacy of antiangiogenic therapies. Pericytes have been revealed as functionally important for tumor neovasculature, using kinase inhibitors targeting their platelet-derived growth factor receptors (PDGFRs). Additionally, vascular endothelial growth factor receptor (VEGFR) inhibitors and metronomic chemotherapy show modest benefit against early- but not late-stage disease.

METHODS

Seeking to improve efficacy against otherwise intractable end-stage pancreatic islet tumors, two receptor tyrosine kinase inhibitors, imatinib and SU11248, were used to disrupt PDGFR-mediated pericyte support of tumor endothelial cells in concert with maximum-tolerated dose (MTD) or metronomic chemotherapy and/or VEGFR inhibition.

RESULTS

Imatinib, despite equivocal efficacy as monotherapy, reduced pericyte coverage of tumor vessels and enhanced efficacy in combination with metronomic chemotherapy or VEGFR inhibition. A regimen involving all three was even better. MTD using cyclophosphamide caused transitory regression, but then rapid regrowth, in contrast to metronomic cyclophosphamide plus imatinib, which produced stable disease. The MTD regimen elicited apoptosis of tumor cells but not endothelial cells, whereas the other regimens increased endothelial cell apoptosis concordant with efficacy. A "chemo-switch" protocol, involving sequential MTD and then metronomic chemotherapy, overlaid with multitargeted inhibition of PDGFR and VEGFR, gave complete responses and unprecedented survival advantage in this model.

CONCLUSIONS

This study demonstrates a potentially tractable clinical strategy in a stringent preclinical model, wherein standard-of-care chemotherapy is followed by a novel maintenance regimen: PDFGR is targeted to disrupt pericyte support, while metronomic chemotherapy and/or VEGFR inhibitors target consequently sensitized endothelial cells, collectively destabilizing pre-existing tumor vasculature and inhibiting ongoing angiogenesis.

Sodium nitroprusside, nitroglycerin, sodium azide and hydroxylamine increased guanylate cyclase activity in particulate and/or soluble preparations from various tissues. While sodium nitroprusside increased guanylate cyclase activity in most of the preparations examined, the effects of sodium azide, hydroxylamine and nitroglycerin were tissue specific. Nitroglycerin and hydroxylamine were also less potent. Neither the protein activator factor nor catalase which is required for sodium azide effects altered the stimulatory effect of sodium nitroprusside. In the presence of sodium azide, sodium nitroprusside or hydroxylamine, magnesium ion was as effective as manganese ion as a sole cation cofactor for guanylate cyclase. With soluble guanylate cyclase from rat liver and bovine tracheal smooth muscle the concentrations of sodium nitroprusside that gave half-maximal stimulation with Mn2+ were 0.1 mM and 0.01 mM, respectively. Effective concentrations were slightly less with Mg2+ as a sole cation cofactor. The ability of these agents to increase cyclic GMP levels in intact tissues is probably due to their effects on guanylate cyclase activity. While the precise mechanism of guanylate cyclase activation by these agents is not known, activation may be due to the formation of nitric oxide or another reactive material since nitric oxide also increased guanylate cyclase activity.

BACKGROUND

Participation rates in large cohort studies have decreased during the last 2 decades. The consequences of this trend for relative risk estimation are unknown.

METHODS

The impact of a low participation rate (30%) on the Danish National Birth Cohort was examined among 49,751 women from the source population, including 15,373 participants in the cohort study. On the basis of independent data collection, we estimated odds ratios (ORs) in the source population and among participants for 3 exposure-risk associations: (a) in vitro fertilization and preterm birth, (b) smoking during pregnancy and birth of a small-for-gestational-age infant, and (c) prepregnancy body mass index and antepartum stillbirth. The effect of nonparticipation was described by a relative odds ratio (ROR), calculated as the OR(participants)/OR(source population). Two methods for calculation of confidence intervals for the relative odds ratio also were assessed.

RESULTS

The effect of nonparticipation on the selected ORs was small. The relative ORs were close to one and the bias was never larger than 16%, although some of the confidence intervals were wide. The 2 methods for calculation of confidence intervals gave very similar results and a small simulation study showed that the coverage probabilities were close to the 95% nominal level.

CONCLUSIONS

For the 3 chosen associations, the ORs were not biased by nonparticipation. The results are reassuring for studies based on the Danish cohort and similar cohorts of pregnant women. The methodology used to compute confidence intervals for the relative odds ratios performed well in the scenarios considered.

OBJECTIVE

The Bonferroni correction adjusts probability (p) values because of the increased risk of a type I error when making multiple statistical tests. The routine use of this test has been criticised as deleterious to sound statistical judgment, testing the wrong hypothesis, and reducing the chance of a type I error but at the expense of a type II error; yet it remains popular in ophthalmic research. The purpose of this article was to survey the use of the Bonferroni correction in research articles published in three optometric journals, viz. Ophthalmic & Physiological Optics, Optometry & Vision Science, and Clinical & Experimental Optometry, and to provide advice to authors contemplating multiple testing.

RESULTS

Some authors ignored the problem of multiple testing while others used the method uncritically with no rationale or discussion. A variety of methods of correcting p values were employed, the Bonferroni method being the single most popular. Bonferroni was used in a variety of circumstances, most commonly to correct the experiment-wise error rate when using multiple 't' tests or as a post-hoc procedure to correct the family-wise error rate following analysis of variance (anova). Some studies quoted adjusted p values incorrectly or gave an erroneous rationale.

CONCLUSIONS

Whether or not to use the Bonferroni correction depends on the circumstances of the study. It should not be used routinely and should be considered if: (1) a single test of the 'universal null hypothesis' (Ho ) that all tests are not significant is required, (2) it is imperative to avoid a type I error, and (3) a large number of tests are carried out without preplanned hypotheses.

BACKGROUND

Induced pluripotent stem (iPS) cells are a novel stem cell population induced from mouse and human adult somatic cells through reprogramming by transduction of defined transcription factors. However, detailed differentiation properties and the directional differentiation system of iPS cells have not been demonstrated.

RESULTS

Previously, we established a novel mouse embryonic stem (ES) cell differentiation system that can reproduce the early differentiation processes of cardiovascular cells. We applied our ES cell system to iPS cells and examined directional differentiation of mouse iPS cells to cardiovascular cells. Flk1 (also designated as vascular endothelial growth factor receptor-2)-expressing mesoderm cells were induced from iPS cells after approximately 4-day culture for differentiation. Purified Flk1(+) cells gave rise to endothelial cells and mural cells by addition of vascular endothelial growth factor and serum. Arterial, venous, and lymphatic endothelial cells were also successfully induced. Self-beating cardiomyocytes could be induced from Flk1(+) cells by culture on OP9 stroma cells. Time course and efficiency of the differentiation were comparable to those of mouse ES cells. Occasionally, reexpression of transgene mRNAs, including c-myc, was observed in long-term differentiation cultures.

CONCLUSIONS

Various cardiovascular cells can be systematically induced from iPS cells. The differentiation properties of iPS cells are almost completely identical to those of ES cells. This system would greatly contribute to a novel understanding of iPS cell biology and the development of novel cardiovascular regenerative medicine.

1. The low-molecular-weight components of myosin freshly prepared by the standard procedure from adult rabbit skeletal muscle migrated as four main bands Ml(1), Ml(2), Ml(3) and Ml(4) on polyacrylamide-gel electrophoresis in 8m-urea. 2. The number of bands increased on storage. This change was accelerated by increasing the temperature and pH. 3. None of the bands had electrophoretic mobilities identical with those of the well-characterized proteins of the myofibril or with the sarcoplasmic proteins. 4. By varying the ionic conditions and concentration of muscle mince used for the initial extraction it was possible to change the relative proportions of the two electrophoretic bands of intermediate mobility, Ml(2) and Ml(3). 5. The four-band picture similar to that obtained with rabbit was observed with myosin isolated from skeletal muscle of the rat, mouse, hamster, pigeon and chicken. 6. Rabbit cardiac myosin gave only two bands on electrophoresis. Myosin from rabbit red muscle gave a pattern intermediate between cardiac and white-skeletal-muscle myosin, i.e. the two fastest bands were present in decreased relative amounts. 7. It is suggested that the differences in the low-molecular-weight components of myosin from different types of muscle are a consequence of differences in the isoenzyme composition of the myosins.

Between 1 and 1.5 billion years ago, eukaryotic organisms acquired the ability to convert light into chemical energy through endosymbiosis with a Cyanobacterium (e.g.,). This event gave rise to "primary" plastids, which are present in green plants, red algae, and glaucophytes ("Plantae" sensu Cavalier-Smith). The widely accepted view that primary plastids arose only once implies two predictions: (1) all plastids form a monophyletic group, as do (2) primary photosynthetic eukaryotes. Nonetheless, unequivocal support for both predictions is lacking (e.g.,). In this report, we present two phylogenomic analyses, with 50 genes from 16 plastid and 15 cyanobacterial genomes and with 143 nuclear genes from 34 eukaryotic species, respectively. The nuclear dataset includes new sequences from glaucophytes, the less-studied group of primary photosynthetic eukaryotes. We find significant support for both predictions. Taken together, our analyses provide the first strong support for a single endosymbiotic event that gave rise to primary photosynthetic eukaryotes, the Plantae. Because our dataset does not cover the entire eukaryotic diversity (but only four of six major groups in), further testing of the monophyly of Plantae should include representatives from eukaryotic lineages for which currently insufficient sequence information is available.

CHROMagar Candida is a novel, differential culture medium that is claimed to facilitate the isolation and presumptive identification of some clinically important yeast species. We evaluated the use of this medium with 726 yeast isolates, including 82 isolated directly on the medium from clinical material. After 2 days of incubation at 37 degrees C, 285 C. albicans isolates gave distinctive green colonies that were not seen with any of 441 other yeast isolates representing 21 different species. A total of 54 C. tropicalis isolates also developed distinctive dark blue-gray colonies with a halo of dark brownish purple in the surrounding agar. C. krusei isolates (n = 43) also formed highly characteristic rough, spreading colonies with pale pink centers and a white edge that was otherwise encountered only rarely with isolates of C. norvegensis. Trichosporon spp. (n = 34) formed small, pale colonies that became larger and characteristically rough with prolonged incubation. Most of the other 310 yeasts studied formed colonies with a color that ranged from white to pink to purple with a brownish tint. The only exceptions were found among isolates identified as Geotrichum sp. or Pichia sp., some of which formed colonies with a gray to blue color and which in two instances formed a green pigment or a dark halo in the agar. The specificity and sensitivity of the new medium for the presumptive identification of C. albicans, C. krusei, and C. tropicalis exceeded 99% for all three species. A blinded reading test involving four personnel and 57 yeast isolates representing nine clinically important species confirmed that colonial appearance after 48 h of incubation on CHROMagar Candida afforded the correct presumptive recognition of C. albicans, C. tropicalis, C, krusei, and Trichosporon spp. None of nine bacterial isolates grew on CHROMagar Candida within 72 h, and bacteria (Escherichia coli) grew from only 4 of 104 vaginal, 100 oral, and 99 anorectal swabs. The new medium supported the growth of 19 of 23 dermatophyte fungi tested and 41 of 43 other molds representing a broad range of fungal pathogens and contaminants. In parallel cultures of 348 clinical specimens set up on Sabourand agar and CHROMagar Candida, both media grew yeasts in the same 78 instances. CHROMagar Candida is recommended as a useful isolation medium capable of the presumptive identification of the yeast species most commonly isolated from clinical material and facilitating recognition of mixed yeast cultures.

BACKGROUND

As screening methods for colorectal cancer (CRC) are limited by uptake and adherence, further options are sought. A blood test might increase both, but none has yet been tested in a screening setting.

OBJECTIVE

We prospectively assessed the accuracy of circulating methylated SEPT9 DNA (mSEPT9) for detecting CRC in a screening population.

METHODS

Asymptomatic individuals ≥50 years old scheduled for screening colonoscopy at 32 US and German clinics voluntarily gave blood plasma samples before colon preparation. Using a commercially available assay, three independent blinded laboratories assayed plasma DNA of all CRC cases and a stratified random sample of other subjects in duplicate real time PCRs. The primary outcomes measures were standardised for overall sensitivity and specificity estimates.

RESULTS

7941 men (45%) and women (55%), mean age 60 years, enrolled. Results from 53 CRC cases and from 1457 subjects without CRC yielded a standardised sensitivity of 48.2% (95% CI 32.4% to 63.6%; crude rate 50.9%); for CRC stages I-IV, values were 35.0%, 63.0%, 46.0% and 77.4%, respectively. Specificity was 91.5% (95% CI 89.7% to 93.1%; crude rate 91.4%). Sensitivity for advanced adenomas was low (11.2%).

CONCLUSIONS

Our study using the blood based mSEPT9 test showed that CRC signal in blood can be detected in asymptomatic average risk individuals undergoing screening. However, the utility of the test for population screening for CRC will require improved sensitivity for detection of early cancers and advanced adenomas.

BACKGROUND

NCT00855348.

Two modifications to Western blots which enhance immunochemical recognition have been developed. The first is transfer in carbonate buffer at pH 9.9, rather than the more commonly used Tris-glycine buffer at pH 8.3. This alteration improved the recognition of four of the five subunits of Escherichia coli F1-ATPase by monoclonal antibodies, the smaller subunits showing the greatest effects. Recognition of dinitrophenyl groups attached to the subunits by polyclonal antibodies was improved by the carbonate buffer only for the smallest ATPase subunit, epsilon. The second modification was incubation of the gel in mild buffers, designed to promote the renaturation of proteins, before the electrophoretic transfer step. The most effective buffer was 20% glycerol in 50 mM Tris-HCl, pH 7.4. Improvements in the signal obtained with monoclonal antibodies to all the subunits of ATPase were obtained by this procedure. As the subunits vary markedly in size, isoelectric point, and other properties, this method should be useful for most proteins. The fate of the 15,000-Da epsilon subunit, labeled with 125I, was followed through a blotting experiment. As long as no sodium dodecyl sulfate was added to the transfer buffer, epsilon was bound to nitrocellulose efficiently in either Tris-glycine or carbonate buffer. However, the epsilon was retained much more strongly during the subsequent incubation steps if the transfer was done in the carbonate buffer. The binding of epsilon to the nitrocellulose was even more stable when the gel had been treated with the buffered glycerol solution before transfer. These results indicate that the conditions under which epsilon subunit first encounters the nitrocellulose markedly affect the stability of binding during subsequent steps. The F1-ATPase was partially fragmented by treatment with proteases and then run on a gel and either transferred immediately in Tris-glycine buffer or else treated with the buffered glycerol solution and transferred in the carbonate buffer. The second blot gave stronger recognition of residual alpha subunit and fragments by an anti-alpha monoclonal antibody, with the largest improvement for the smaller fragments. This result suggests that the modified procedure may be particularly useful in enhancing the detection of small proteins.

OBJECTIVE

The extensive neuroprotective literature describing the efficacy of candidate drugs in focal ischemia has yet to lead to the development of effective stroke treatments. Ideally, the choice of drugs taken forward to clinical trial should be based on an unbiased assessment of all available data. Such an assessment might include not only the efficacy of a drug but also the in vivo characteristics and limits--in terms of time window, dose, species, and model of ischemia used--to that efficacy. To our knowledge, such assessments have not been made. Nicotinamide is a candidate neuroprotective drug with efficacy in experimental stroke, but the limits to and characteristics of that efficacy have not been fully described.

METHODS

Systematic review and modified meta-analysis of studies of experimental stroke describing the efficacy of nicotinamide. The search strategy ensured ascertainment of studies published in full and those published in abstract only. DerSimonian and Laird random effects meta-analysis was used to account for heterogeneity between studies.

RESULTS

Nicotinamide improved outcome by 0.287 (95% confidence interval 0.227 to 0.347); it was more effective in temporary ischemia models, after intravenous administration, in animals without comorbidities, and in studies published in full rather than in abstract. Studies scoring highly on a quality measure gave more precise estimates of the global effect.

CONCLUSIONS

Meta-analysis provides an effective technique for the aggregation of data from experimental stroke studies. We propose new standards for reporting such studies and a systematic approach to aggregating data from the neuroprotective literature.

The product of the Philadelphia chromosome (Ph) translocation, the BCR/ABL oncogene, exists in three principal forms (P190, P210, and P230 BCR/ABL) that are found in distinct forms of Ph-positive leukemia, suggesting the three proteins have different leukemogenic activity. We have directly compared the tyrosine kinase activity, in vitro transformation properties, and in vivo leukemogenic activity of the P190, P210, and P230 forms of BCR/ABL. P230 exhibited lower intrinsic tyrosine kinase activity than P210 and P190. Although all three oncogenes transformed both myeloid (32D cl3) and lymphoid (Ba/F3) interleukin (IL)-3-dependent cell lines to become independent of IL-3 for survival and growth, their ability to stimulate proliferation of Ba/F3 lymphoid cells differed and correlated directly with tyrosine kinase activity. In a murine bone marrow transduction/transplantation model, the three forms of BCR/ABL were equally potent in the induction of a chronic myeloid leukemia (CML)-like myeloproliferative syndrome in recipient mice when 5-fluorouracil (5-FU)-treated donors were used. Analysis of proviral integration showed the CML-like disease to be polyclonal and to involve multiple myeloid and B lymphoid lineages, implicating a primitive multipotential target cell. Secondary transplantation revealed that only certain minor clones gave rise to day 12 spleen colonies and induced disease in secondary recipients, suggesting heterogeneity among the target cell population. In contrast, when marrow from non- 5-FU-treated donors was used, a mixture of CML-like disease, B lymphoid acute leukemia, and macrophage tumors was observed in recipients. P190 BCR/ABL induced lymphoid leukemia with shorter latency than P210 or P230. The lymphoid leukemias and macrophage tumors had provirus integration patterns that were oligo- or monoclonal and limited to the tumor cells, suggesting a lineage-restricted target cell with a requirement for additional events in addition to BCR/ABL transduction for full malignant transformation. These results do not support the hypothesis that P230 BCR/ABL induces a distinct and less aggressive form of CML in humans, and suggest that the rarity of P190 BCR/ABL in human CML may reflect infrequent BCR intron 1 breakpoints during the genesis of the Ph chromosome in stem cells, rather than intrinsic differences in myeloid leukemogenicity between P190 and P210.

All hospitalisations for pulmonary arterial hypertension (PAH) in the Scottish population were examined to determine the epidemiological features of PAH. These data were compared with expert data from the Scottish Pulmonary Vascular Unit (SPVU). Using the linked Scottish Morbidity Record scheme, data from all adults aged 16-65 yrs admitted with PAH (idiopathic PAH, pulmonary hypertension associated with congenital heart abnormalities and pulmonary hypertension associated with connective tissue disorders) during the period 1986-2001 were identified. These data were compared with the most recent data in the SPVU database (2005). Overall, 374 Scottish males and females aged 16-65 yrs were hospitalised with incident PAH during 1986-2001. The annual incidence of PAH was 7.1 cases per million population. On December 31, 2002, there were 165 surviving cases, giving a prevalence of PAH of 52 cases per million population. Data from the SPVU were available for 1997-2006. In 2005, the last year with a complete data set, the incidence of PAH was 7.6 cases per million population and the corresponding prevalence was 26 cases per million population. Hospitalisation data from the Scottish Morbidity Record scheme gave higher prevalences of pulmonary arterial hypertension than data from the expert centres (Scotland and France). The hospitalisation data may overestimate the true frequency of pulmonary arterial hypertension in the population, but it is also possible that the expert centres underestimate the true frequency.