Although adult mouse hematopoietic stem cells (HSCs) have been purified to near homogeneity, it remains impossible to achieve this with fetal HSCs. Adult HSC purity recently has been enhanced using the SLAM family receptors CD150, CD244, and CD48. These markers are expressed at different stages of the hematopoiesis hierarchy, making it possible to highly purify adult HSCs as CD150(+)CD48(-)CD244(-) cells. We found that SLAM family receptors exhibited a similar expression pattern in fetal liver. Fetal liver HSCs were CD150(+)CD48(-)CD244(-), and the vast majority of colony-forming progenitors were CD48(+)CD244(-)CD150(-) or CD48(+)CD244(+)CD150(-), just as in adult bone marrow. SLAM family markers enhanced the purification of fetal liver HSCs. Whereas 1 (11%) of every 8.9 Thy(low)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution in irradiated mice, 1 (18%) of every 5.7 CD150(+)CD48(-)CD41(-) cells and 1 (37%) of every 2.7 CD150(+)CD48(-)Sca-1(+)lineage(-)Mac-1(+) fetal liver cells gave long-term multilineage reconstitution. These data emphasize the robustness with which SLAM family markers distinguish progenitors at different stages of the hematopoiesis hierarchy and enhance the purification of definitive HSCs from diverse contexts. Nonetheless, CD150, CD244, and CD48 are not pan-stem cell markers, as they were not detectably expressed by stem cells in the fetal or adult nervous system.

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.

Subtractive expressed sequence tag analysis and screening of cDNA libraries derived from Brassica napus leaves subjected to mechanical wounding, flea beetle feeding or cold temperatures revealed eight genes encoding NAC-domain transcription factors. The genes were found to be differentially regulated in response to biotic and abiotic stresses including wounding, insect feeding, Sclerotinia sclerotiorum infection, cold shock and dehydration. Five BnNAC proteins were orthologous to Arabidopsis thaliana ATAF1 or ATAF2 and gave rise to developmental abnormalities similar to the A. thaliana nam and cuc mutants when expressed ectopically in A. thaliana. Transgenic lines expressing BnNAC14, exhibited large leaves, thickened stems and hyper-developed lateral root systems similar to that observed with A. thaliana NAC1, but also were delayed in bolting and lacked an apical dominant tap root. Several of the BnNAC proteins were capable of activating gene expression in yeast and recognized an element within the CaMV35S promoter. A yeast two-hybrid screen revealed that BnNAC14 interacted with other select BnNAC proteins in vitro and identified an additional BnNAC gene, BnNAC485. The protein interaction and transcriptional activation domains were mapped by deletion analysis.

Both of the major immediate-early (IE) proteins IE1 and IE2 of human cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE transcription colocalize with or adjacent to punctate PML domains (PML oncogenic domains [PODs] or nuclear domain 10) in the nucleus within the first few hours after infection of permissive human fibroblasts. However, colocalization of IE1 and PML in PODs is only transient, with both proteins subsequently redistributing into a nuclear diffuse form. These processes are believed to promote efficient viral IE transcription and initiation of DNA synthesis especially at low multiplicities of infection. To examine the mechanism of PML displacement by IE1, we carried out indirect immunofluorescence assay experiments with plasmids expressing intact or deleted forms of PML and IE1 in DNA-transfected cells. The results demonstrated that deletion of the C-terminal acidic region of IE1 uncouples the requirements for displacement of both endogenous and coexpressed PML from those needed to target to the PODs. Mutant PML proteins containing either a Cys point mutation within the N-terminal RING finger domain or a small deletion (of positions 281 to 304) within the coiled-coil region did not localize to the PODs but instead gave a nuclear diffuse distribution, similar to that produced by intact PML in the presence of IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis, implying that there may be a direct physical interaction between IE1 and PML. Indeed, a specific interaction between IE1 and PML was observed in a yeast two-hybrid assay, and the strength of this interaction was comparable to that of IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a threefold-lower interaction with IE1 in the yeast system, and deletion of the N-terminal RING finger domain of PML abolished the interaction. Consistent with the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region was sufficient for interaction with PML in the yeast system. The two-hybrid interaction assay also showed that both the N-terminal RING finger domain and the intact coiled-coil region of PML are required cooperatively for efficient self-interactions involving dimerization or oligomerization. Furthermore, truncated or deleted GAL4/PML fusion proteins that retained the RING finger domain but lacked the intact coiled-coil region displayed an unmasked cryptic transactivator function in both yeast and mammalian cells, and the RING finger mutation abolished this transactivation property of PML. Therefore, we suggest that a direct interaction between IE1 and the N-terminal RING finger domain of PML may inhibit oligomerization and protein-protein complex formation by PML, leading to displacement of PML and IE1 from the PODs, and that this interaction may also modulate a putative conditional transactivator function of PML.

It is well accepted that pain is a multidimensional experience, but little is known of how the brain represents these dimensions. We used positron emission tomography (PET) to indirectly measure pain-evoked cerebral activity before and after hypnotic suggestions were given to modulate the perceived intensity of a painful stimulus. These techniques were similar to those of a previous study in which we gave suggestions to modulate the perceived unpleasantness of a noxious stimulus. Ten volunteers were scanned while tonic warm and noxious heat stimuli were presented to the hand during four experimental conditions: alert control, hypnosis control, hypnotic suggestions for increased-pain intensity and hypnotic suggestions for decreased-pain intensity. As shown in previous brain imaging studies, noxious thermal stimuli presented during the alert and hypnosis-control conditions reliably activated contralateral structures, including primary somatosensory cortex (S1), secondary somatosensory cortex (S2), anterior cingulate cortex, and insular cortex. Hypnotic modulation of the intensity of the pain sensation led to significant changes in pain-evoked activity within S1 in contrast to our previous study in which specific modulation of pain unpleasantness (affect), independent of pain intensity, produced specific changes within the ACC. This double dissociation of cortical modulation indicates a relative specialization of the sensory and the classical limbic cortical areas in the processing of the sensory and affective dimensions of pain.

BACKGROUND

Access to mobile phones continues to increase exponentially globally, outstripping access to fixed telephone lines, fixed computers and the Internet. Mobile phones are an appropriate and effective option for the delivery of smoking cessation support in some contexts. This review updates the evidence on the effectiveness of mobile phone-based smoking cessation interventions.

OBJECTIVE

To determine whether mobile phone-based smoking cessation interventions increase smoking cessation in people who smoke and want to quit.

METHODS

For the most recent update, we searched the Cochrane Tobacco Addiction Group Specialised Register in April 2015. We also searched the UK Clinical Research Network Portfolio for current projects in the UK, and the ClinicalTrials.gov register for ongoing or recently completed studies. We searched through the reference lists of identified studies and attempted to contact the authors of ongoing studies. We applied no restrictions on language or publication date.

METHODS

We included randomised or quasi-randomised trials. Participants were smokers of any age who wanted to quit. Studies were those examining any type of mobile phone-based intervention for smoking cessation. This included any intervention aimed at mobile phone users, based around delivery via mobile phone, and using any functions or applications that can be used or sent via a mobile phone.

METHODS

Review authors extracted information on risk of bias and methodological details using a standardised form. We considered participants who dropped out of the trials or were lost to follow-up to be smoking. We calculated risk ratios (RR) and 95% confidence intervals (CI) for each included study. Meta-analysis of the included studies used the Mantel-Haenszel fixed-effect method. Where meta-analysis was not possible, we presented a narrative summary and descriptive statistics.

RESULTS

This updated search identified 12 studies with six-month smoking cessation outcomes, including seven studies completed since the previous review. The interventions were predominantly text messaging-based, although several paired text messaging with in-person visits or initial assessments. Two studies gave pre-paid mobile phones to low-income human immunodeficiency virus (HIV)-positive populations - one solely for phone counselling, the other also included text messaging. One study used text messages to link to video messages. Control programmes varied widely. Studies were pooled according to outcomes - some providing measures of continuous abstinence or repeated measures of point prevalence; others only providing 7-day point prevalence abstinence. All 12 studies pooled using their most rigorous 26-week measures of abstinence provided an RR of 1.67 (95% CI 1.46 to 1.90; I(2) = 59%). Six studies verified quitting biochemically at six months (RR 1.83; 95% CI 1.54 to 2.19).

CONCLUSIONS

The current evidence supports a beneficial impact of mobile phone-based smoking cessation interventions on six-month cessation outcomes. While all studies were good quality, the fact that those studies with biochemical verification of quitting status demonstrated an even higher chance of quitting further supports the positive findings. However, it should be noted that most included studies were of text message interventions in high-income countries with good tobacco control policies. Therefore, caution should be taken in generalising these results outside of this type of intervention and context.

Polyclonal antibodies were generated against the major polypeptide (73,000 mol. wt) present in a highly purified preparation of the [Na+ + K+]coupled L-glutamate transporter from rat brain. These antibodies were able to selectively immunoprecipitate the 73,000 mol. wt polypeptide as well as most of the L-glutamate transport activity--as assayed upon reconstitution--from crude detergent extracts of rat brain membranes. The immunoreactivity in the various fractions obtained during the purification procedure [Danbolt et al. (1990) Biochemistry 29, 6734-6740] closely correlated with the L-glutamate transport activity. Immunoblotting of a crude sodium dodecyl sulphate brain extract, separated by two-dimensional isoelectric focusing-sodium dodecyl sulphate-polyacrylamide gel electrophoresis, showed that the antibodies recognized one 73,000 mol. wt protein species only. Deglycosylation of the protein gave a 10,000 reduction in molecular mass, but no reduction in immunoreactivity. These findings establish that the 73,000 mol. wt polypeptide represents the L-glutamate transporter or a subunit thereof. The antibodies also recognize a 73,000 mol. wt polypeptide and immunoprecipitate L-glutamate transport activity in extracts of brain plasma membranes from rabbit, pig, cow, cat and man. Using the antibodies, the immunocytochemical localization of the transporter was studied at the light and electron microscopic levels in rat central nervous system. In all regions examined (including cerebral cortex, caudatoputamen, corpus callosum, hippocampus, cerebellum, spinal cord) it was found to be located in glial cells rather than in neurons. In particular, fine astrocytic processes were strongly stained. Putative glutamatergic axon terminals appeared non-immunoreactive. The uptake of glutamate by such terminals (for which there is strong previous evidence) therefore may be due to a subtype of glutamate transporter different from the glial transporter demonstrated by us.

Although missing outcome data are an important problem in randomized trials and observational studies, methods to address this issue can be difficult to apply. Using simulated data, the authors compared 3 methods to handle missing outcome data: 1) complete case analysis; 2) single imputation; and 3) multiple imputation (all 3 with and without covariate adjustment). Simulated scenarios focused on continuous or dichotomous missing outcome data from randomized trials or observational studies. When outcomes were missing at random, single and multiple imputations yielded unbiased estimates after covariate adjustment. Estimates obtained by complete case analysis with covariate adjustment were unbiased as well, with coverage close to 95%. When outcome data were missing not at random, all methods gave biased estimates, but handling missing outcome data by means of 1 of the 3 methods reduced bias compared with a complete case analysis without covariate adjustment. Complete case analysis with covariate adjustment and multiple imputation yield similar estimates in the event of missing outcome data, as long as the same predictors of missingness are included. Hence, complete case analysis with covariate adjustment can and should be used as the analysis of choice more often. Multiple imputation, in addition, can accommodate the missing-not-at-random scenario more flexibly, making it especially suited for sensitivity analyses.

BACKGROUND

RNA quality and integrity are critical for many studies in plant molecular biology. High-quality RNA extraction from grapevine and other woody plants is problematic due to the presence of polysaccharides, polyphenolics and other compounds that bind or co-precipitate with the RNA.

OBJECTIVE

To develop an optimised cetyltrimethylammonium bromide (CTAB)-based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from polysaccharide-rich tissues of several plants.

METHODS

Several changes were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4 degrees C, the sample weight was decreased and the concentrations of PVP-40 and LiCl were increased reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with six different RNA extraction methods from three grapevine tissues, namely, in vitro plantlets, and leaves and mature canes from actively growing field vines.

RESULTS

The rapid CTAB method gave high-quality RNA in only 3 h at low cost with efficiency equal to or higher than that obtained with other time-consuming and expensive protocols. The procedure was applied to RNA extraction from other grapevine tissues and other woody species including olive, lemon, poplar, chestnut, apple, pear, peach, cherry, apricot, plum and kiwi fruit. RNA of high quality could be isolated from all tissues and from all species.

CONCLUSIONS

The study has shown that the improvement of a CTAB-based protocol allows the rapid isolation of high-quality RNA from grapevine and many woody species.

One hundred and three gastroscopic biopsies from 80 patients were cultured for Campylobacter pyloridis and studied histologically. Active chronic gastritis, as shown by the presence of polymorphonuclear leucocytes, was diagnosed in 51 biopsies and C pyloridis was found in 47. Sixteen gastric biopsies showed normal histology (no inflammation); C pyloridis was detected in only one of these, and a second biopsy taken from this patient at the same time showed active gastritis. Biopsies could be kept at 4 degrees C for five hours without loss of viability of C pyloridis. An inoculum made by grinding the biopsy in a ground glass grinder consistently gave a much heavier growth of C pyloridis than one made by mincing the specimen. The campylobacter supplement ferrous sulphate, sodium metabisulphite, sodium pyruvate (FBP) (Oxoid) was inhibitory for some isolates; the inhibitory component was found to be sodium metabisulphite. Contaminants, but not C pyloridis, were inhibited by the incorporation of vancomycin 6 mg/l, nalidixic acid 20 mg/l, and amphotericin 2 mg/l, but higher concentrations inhibited C pyloridis. Undried plates kept in a plastic container at room temperature for up to two weeks were as satisfactory as freshly poured plates for the isolation of C pyloridis.

Transient transfection studies have shown that the probasin (PB) promoter confers androgen selectivity over other steroid hormones, and transgenic animal studies have demonstrated that the PB promoter will target androgen, but not glucocorticoid, regulation in a prostate-specific manner. Previous PB promoters either targeted low levels of transgene expression or became too large to be conveniently used. The goal was to design a PB promoter that would be small, yet target high levels of prostate-specific transgene expression. Thus, a composite probasin promoter (ARR2PB) coupled to the bacterial chloramphenicol acetyltransferase reporter (ARR2PBCAT) was generated and tested in prostatic and nonprostatic cell lines and in a transgenic mouse model. In PC-3, LNCaP, and DU145 prostate cancer cell lines, the ARR2PB promoter gave basal expression and was induced in response to androgen and glucocorticoid treatment after cotransfection with the respective steroid receptor. Basal expression of ARR2PBCAT in the nonprostatic COS-1, MCF-7, ZR-75-1, and PANC-1 cell lines was very low; however, CAT activity could be induced in response to androgens and glucocorticoids when cells were cotransfected with either the AR or GR. In contrast to the transfection studies, ARR2PBCAT transgene expression remained highly specific for prostatic epithelium in transgenic mice. CAT activity decreased after castration, and could be induced by androgens and, in addition, glucocorticoids. This demonstrates that the necessary sequences required to target prostate-specific epithelial expression are contained within the composite ARR2PB minimal promoter, and that high transgene expression can now be regulated by both androgens and glucocorticoids. The ARR2PB promoter represents a novel glucocorticoid inducible promoter that can be used for the generation of transgenic mouse models and in viral gene therapy vectors for the treatment of prostate cancer in humans.

An inhibitory rat mAb, SER-4, has been raised to the mouse macrophage (M phi)-restricted hemagglutinin, sheep erythrocyte receptor (SER), which binds unopsonized sheep erythrocytes through recognition of sialylated glycoconjugates. This receptor was originally defined on mouse resident bone marrow M phi where it was implicated in adhesive interactions of these cells with proliferating hematopoietic cells. In the present study using mouse serum-induced thioglycollate-elicited peritoneal M phi (TPM) as a model system for SER expression, mAb SER-4 IgG2a completely blocked rosette formation at 1 microgram/ml. The inhibition was likely to be via steric hindrance rather than through a direct interaction with the putative sialic acid binding site of SER because F(ab')2 and Fab fragments of mAb SER-4 gave a maximum inhibition of 50-60% and 0% respectively, despite binding effectively to the SER-4 antigen (Ag). Immunoprecipitation and Western blotting experiments with cultured M phi or tissue extracts demonstrated that the Ag recognized by SER-4 mAb is a single chain molecule with an apparent Mr by SDS-PAGE of 185 x 10(3) (reduced) or 170 x 10(3) (non-reduced) and is distinct from members of the leukocyte common Ag family. Expression of SER and SER-4 Ag in culture were closely correlated and depended on the presence of mouse serum for optimal induction. Further evidence that the SER-4 Ag is functionally equivalent to SER was provided by immunocytochemistry in which the overall pattern of staining in tissues was consistent with previous rosetting experiments. In the bone marrow, expression of the SER-4 Ag was restricted to the resident bone marrow M phi population with no expression on monocytes. High expression was also observed on stromal M phi within the subcapsular sinus and medullary cords in lymph nodes and on marginal metallophils in the spleen. These results therefore confirm that SER is a novel M phi-restricted receptor whose distribution and properties indicate a role in cellular interactions in hematopoietic and lymphoid tissues.

Micellar complexes of human apolipoprotein A-I and phosphatidylcholine, with or without cholesterol, were prepared by adding apolipoprotein A-I (apo A-I) to sodium cholate-lipid mixtures. Cholate was removed by dialysis and the apo A-I.lipid complexes were isolated by gel filtration chromatography or by density gradient ultracentrifugation. The lipid mixtures consisted of dipalmitoylphosphatidylcholine or egg yolk phosphatidylcholine in the presence of various molar ratios of cholesterol. The formation of complexes was examined at different phosphatidylcholine (PC)-to-apo A-I ratios, PC-to-cholate ratios, and cholate concentrations. Yields of complexes were maximal when incubation and dialysis were performed near the transition temperature of the PC. Upon lipid binding and complex formation, apo A-I experienced a significant increase in alpha-helix content, and a blue shift in the intrinsic tryptophan fluorescence. In all lipid-protein incubation mixtures, from 600:1 to 75:1, PC/apo A-I (molar ratios), relatively small, stable complexes were present which gave maximum yields at incubation ratios similar to their isolated stoichiometries of 75:1 to 140:1, PC/apo A-I (molar ratios). For the isolated complexes, molecular weights were determined by sedimentation equilibrium to be in the range from 220,000 to 260,000; fluorescence polarization using the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene showed a broadened and shifted gel to liquid-crystalline phase transition, characteristic of micellar complexes of apo A-I with PC. Complexes prepared using apo A-I, covalently labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride, had an overall particle rotational relaxation time of 530 ns. On electron micrographs, the complexes, negatively stained with phosphotungstate, appeared as lamellar, discoidal particles.

BACKGROUND

Many injection drug users (IDUs) seek care at emergency departments and some require hospital admission because of late presentation in the course of their illness. We determined the predictors of frequent emergency department visits and hospital admissions among community-based IDUs and estimated the incremental hospital utilization costs incurred by IDUs with early HIV infection relative to costs incurred by HIV-negative IDUs.

METHODS

The Vancouver Injection Drug User Study (VIDUS) is a prospective cohort study involving IDUs that began in 1996. Our analyses were restricted to the 598 participants who gave informed consent for our study. We used the participants' responses to the baseline VIDUS questionnaire and, from medical records at St. Paul's Hospital, Vancouver, we collected detailed information about the frequency of emergency department visits, hospital admissions and the primary diagnosis for all visits or hospital stays between May 1, 1996, and Aug. 31, 1999. The incremental difference in hospital utilization costs by HIV status was estimated, based on 105 admissions in a subgroup of 64 participants.

RESULTS

A total of 440 (73.6%) of the 598 IDUs made 2763 visits to the emergency department at St. Paul's Hospital during the study period. Of these 440, 265 (160.2%) made frequent visits (3 or more). The following factors were associated with frequent use: HIV-positive status (seroprevalent: adjusted odds ratio [OR] 1.7, 95% confidence interval [CI] 1.2-2.6; seroconverted during study period: adjusted OR 3.0, 95% CI 1.6-5.7); more than 4 injections daily (adjusted OR 1.5, 95% CI 1.1-2.1); cocaine use more frequent than use of other drugs (adjusted OR 2.0, 95% CI 1.2-3.6); and unstable housing (adjusted OR 1.5, 95% CI 1.1-2.2). During the study period 210 of the participants were admitted to hospital 495 times; 118 (56.2%) of them were admitted frequently (2 or more admissions). The 2 most common reasons for admission were pneumonia (132 admissions among 79 patients) and soft-tissue infections (cellulitis and skin abscess) (90 admissions among 59 patients). The following factors were independently associated with frequent hospital admissions: HIV-positive status (seroprevalent: adjusted OR 5.4, 95% CI 3.4-8.6; seroconverted during study period: adjusted OR 2.9, 95% CI 1.4-6.0); and female sex (adjusted OR 1.8, 95% CI 1.1-3.1). The incremental hospital utilization costs incurred by HIV-positive IDUs relative to the costs incurred by HIV-negative IDUs were $1752 per year.

CONCLUSIONS

Hospital utilization was significantly higher among community-based IDUs with early HIV disease than among those who were HIV negative. Much of the hospital use was related to complications of injection drug use and may be reduced with the establishment of programs that integrate harm reduction strategies with primary care and addiction treatment.

Soon after infection, retroviruses synthesize a DNA copy of the genomic RNA and insert that DNA into the cellular genome by recombination at inverted repeat sequences at the termini of the viral genome. We have generated mutations that alter one terminus of the genome of Moloney murine leukemia virus (M-MuLV). Some mutations did not prevent integration of the viral DNA even though the very terminal bases were disrupted. Other mutations had dramatic effects on the efficiency of infection; in these cases the formation of preintegrative DNA was normal but the establishment of the productive provirus was prevented. One of these defective mutants gave rise to a pseudorevertant which differed from the wild type but displayed normal infectivity. An unusual number of bases of viral DNA were removed during the integration reaction carried out by this virus.

The fast evolving human KIR gene family encodes variable lymphocyte receptors specific for polymorphic HLA class I determinants. Nucleotide sequences for 24 representative human KIR haplotypes were determined. With three previously defined haplotypes, this gave a set of 12 group A and 15 group B haplotypes for assessment of KIR variation. The seven gene-content haplotypes are all combinations of four centromeric and two telomeric motifs. 2DL5, 2DS5 and 2DS3 can be present in centromeric and telomeric locations. With one exception, haplotypes having identical gene content differed in their combinations of KIR alleles. Sequence diversity varied between haplotype groups and between centromeric and telomeric halves of the KIR locus. The most variable A haplotype genes are in the telomeric half, whereas the most variable genes characterizing B haplotypes are in the centromeric half. Of the highly polymorphic genes, only the 3DL3 framework gene exhibits a similar diversity when carried by A and B haplotypes. Phylogenetic analysis and divergence time estimates, point to the centromeric gene-content motifs that distinguish A and B haplotypes having emerged ~6 million years ago, contemporaneously with the separation of human and chimpanzee ancestors. In contrast, the telomeric motifs that distinguish A and B haplotypes emerged more recently, ~1.7 million years ago, before the emergence of Homo sapiens. Thus the centromeric and telomeric motifs that typify A and B haplotypes have likely been present throughout human evolution. The results suggest the common ancestor of A and B haplotypes combined a B-like centromeric region with an A-like telomeric region.

MIC distribution data were obtained from a variety of international sources, and pooled after selection by a defined criterion. Sixty-seven of these datasets were subjected to a range of statistical goodness-of-fit tests. The log-normal distribution was selected for subsequent modelling. Cumulative counts of MIC distribution data were fitted to the cumulative log-normal distribution using non-linear least squares regression for a range of data subsets from each antibiotic-bacterium combination. Estimated parameters in the regression were the number of isolates in the subset, and (the log(2) values of) the mean and standard deviation. Optimum fits for the cumulative log-normal curve were then used to determine the wild-type MIC range, determined by calculating the MICs associated with the lower and upper 0.1% of the distribution, rounding to the nearest two-fold dilution, and calculating the probabilities of values higher and lower than these values. When plotted logarithmically, histograms of MIC frequencies appeared normal (Gaussian), but standard goodness-of-fit tests showed that the two-fold dilution grouping of MICs fits poorly to a log-normal distribution, whereas non-linear regression gave good fits to population (histogram) log-normal distributions of log(2) MIC frequencies, and even better fits to log-normal cumulative distributions. Optimum fits were found when the difference between the estimated and true number of isolates in the fitted subset was minimal. Sixteen antibiotic-bacterium datasets were fitted using this technique, and the log(2) values of the means and standard deviations were used to determine the 0.1% and 99.9% wild-type cut-off values. When rounded to the nearest two-fold dilution,>> or = 98.5% of MIC values fall within the cut-off value range. Non-linear regression fitting to a cumulative log-normal distribution is a novel and effective method for modelling MIC distributions and quantifying wild-type MIC ranges.

BACKGROUND

Shear wave elastography is a new method of obtaining quantitative tissue elasticity data during breast ultrasound examinations. The aims of this study were (1) to determine the reproducibility of shear wave elastography (2) to correlate the elasticity values of a series of solid breast masses with histological findings and (3) to compare shear wave elastography with greyscale ultrasound for benign/malignant classification.

METHODS

Using the Aixplorer® ultrasound system (SuperSonic Imagine, Aix en Provence, France), 53 solid breast lesions were identified in 52 consecutive patients. Two orthogonal elastography images were obtained of each lesion. Observers noted the mean elasticity values in regions of interest (ROI) placed over the stiffest areas on the two elastography images and a mean value was calculated for each lesion. A sub-set of 15 patients had two elastography images obtained by an additional operator. Reproducibility of observations was assessed between (1) two observers analysing the same pair of images and (2) findings from two pairs of images of the same lesion taken by two different operators. All lesions were subjected to percutaneous biopsy. Elastography measurements were correlated with histology results. After preliminary experience with 10 patients a mean elasticity cut off value of 50 kilopascals (kPa) was selected for benign/malignant differentiation. Greyscale images were classified according to the American College of Radiology (ACR) Breast Imaging Reporting and Data System (BI-RADS). BI-RADS categories 1-3 were taken as benign while BI-RADS categories 4 and 5 were classified as malignant.

RESULTS

Twenty-three benign lesions and 30 cancers were diagnosed on histology. Measurement of mean elasticity yielded an intraclass correlation coefficient of 0.99 for two observers assessing the same pairs of elastography images. Analysis of images taken by two independent operators gave an intraclass correlation coefficient of 0.80. Shear wave elastography versus greyscale BI-RADS performance figures were sensitivity: 97% vs 87%, specificity: 83% vs 78%, positive predictive value (PPV): 88% vs 84%, negative predictive value (NPV): 95% vs 82% and accuracy: 91% vs 83% respectively. These differences were not statistically significant.

CONCLUSIONS

Shear wave elastography gives quantitative and reproducible information on solid breast lesions with diagnostic accuracy at least as good as greyscale ultrasound with BI-RADS classification.

OBJECTIVE

To assess whether the breath carbon monoxide (CO) concentration can be used to determine a patient's smoking habits in a respiratory outpatient clinic.

METHODS

To provide a normal range for smokers and nonsmokers, 41 outpatients and 24 healthy subjects were questioned on their smoking habits and asked to provide two breaths into a CO monitor (EC50 Smokerlyser; Bedfont Instruments; Kent, UK). In a subsequent single-blind study, 51 different outpatients were not told of the purpose of the study and were assessed by extensive questionnaire, spirometry, and Smokerlyser estimation.

METHODS

The Chest Clinic and Pulmonary Medicine Department at the Northern General Hospital, Sheffield, UK.

METHODS

Phase 1 involved 41 outpatients attending the Chest Clinic and 24 nonoutpatient colleagues. In phase 2, an additional 51 different outpatients were studied.

RESULTS

The mean (SD) breath CO levels were 17.4 (11.6) parts per million (ppm) for smokers and 1.8 (1.3) ppm for nonsmokers (p < 0.001). A level of 6 ppm was taken as the cutoff, as this gave a selectivity of 96% and a sensitivity of 94% for outpatients. Of the 51 study patients, 5 admitted to smoking in the administered questionnaire. Eight denied smoking but had a mean breath CO>> 6 ppm (7.5 to 42 ppm). Of these, three admitted to smoking after being explained the implication of the reading.

CONCLUSIONS

Breath CO concentration provides an easy, noninvasive, and immediate way of assessing a patient's smoking status. A reading>> 6 ppm strongly suggests that an outpatient is a smoker.

Defining language lateralization is important to minimize morbidity in patients treated surgically for temporal lobe epilepsy (TLE). Functional magnetic resonance imaging (fMRI) offers a promising, noninvasive, alternative strategy to the Wada test. Here we have used fMRI to study healthy controls and patients with TLE in order to (i) define language-related activation patterns and their reproducibility; (ii) compare lateralization determined by fMRI with those from of the Wada test; and (iii) contrast different methods of assessing fMRI lateralization. Twelve healthy right-handed controls and 19 right-handed preoperative patients with TLE (12 left- and seven right-TLE) were studied at 3T using fMRI and a verbal fluency paradigm. A Wada test also was performed on each of the patients. Greater activation was found in several areas in the right hemisphere for the left-TLE group relative to controls or right-TLE patients. Relative hemispheric activations calculated based on either the extent or the mean signal change gave consistent results showing a more bihemispheric language representation in the left-TLE patients. There was good agreement between the Wada and fMRI results, although the latter were more sensitive to involvement of the nondominant right hemisphere. The reproducibility of the fMRI values was lowest for the more bihemispherically represented left-TLE patients. Overall, our results further demonstrate that noninvasive fMRI measures of language-related lateralization may provide a practical and reliable alternative to invasive testing for presurgical language lateralization in patients with TLE. The high proportion (33%) of left-TLE patients showing bilateral or right hemispheric language-related lateralization suggests that there is considerable plasticity of language representation in the brains of patients with intractable TLE.

A novel Mr 28,000 erythrocyte transmembrane protein was recently purified and found to exist in two forms, "28kDa" and "gly28kDa," the latter containing N-linked carbohydrate (Denker, B. M., Smith, B. L., Kuhajda, F. P., and Agre, P. (1988) J. Biol. Chem. 263, 15634-15642). Although 28kDa protein resembles the Rh polypeptides biochemically, structural homologies were not identified by immunoblot or two-dimensional iodopeptide maps. The NH2-terminal amino acid sequence for the first 35 residues of purified 28kDa protein is 37% identical to the 26-kDa major intrinsic protein of lens (Gorin, M. B., Yancey, S. B., Cline, J., Revel, J.-P., and Horwitz, J. Cell 39, 49-59). Antisera to a synthetic peptide corresponding to the NH2-terminus of 28kDa protein gave a single reaction of molecular mass 28kDa on immunoblots of erythrocyte membranes. Selective digestions of intact erythrocytes and inside-out membrane vesicles with carboxypeptidase Y indicated the existence of a 5-kDa COOH-terminal cytoplasmic domain. Multiple studies indicated that 28kDa and gly28kDa proteins exist together as a multisubunit oligomer: 1) similar partial solubilizations in Triton X-100; 2) co-purification during ion exchange and lectin affinity chromatography; 3) cross-linking in low concentrations of glutaraldehyde; and 4) physical analyses of purified proteins and solubilized membranes in 1% (v/v) Triton X-100 showed 28kDa and gly28kDa proteins behave as a large single unit with Stokes radius of 61 A and sedimentation coefficient of 5.7 S. These studies indicate that the 28kDa and gly28kDa proteins are distinct from the Rh polypeptides and exist as a multisubunit oligomer. The 28kDa protein has NH2-terminal amino acid sequence homology and membrane organization similar to major intrinsic protein and other members of a newly recognized family of transmembrane channel proteins.

Atomic force microscopy was used to measure the thickness of air-dried, collapsed murein sacculi from Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Air-dried sacculi from E. coli had a thickness of 3.0 nm, whereas those from P. aeruginosa were 1.5 nm thick. When rehydrated, the sacculi of both bacteria swelled to double their anhydrous thickness. Computer simulation of a section of a model single-layer peptidoglycan network in an aqueous solution with a Debye shielding length of 0.3 nm gave a mass distribution full width at half height of 2.4 nm, in essential agreement with these results. When E. coli sacculi were suspended over a narrow groove that had been etched into a silicon surface and the tip of the atomic force microscope used to depress and stretch the peptidoglycan, an elastic modulus of 2.5 x 10(7) N/m(2) was determined for hydrated sacculi; they were perfectly elastic, springing back to their original position when the tip was removed. Dried sacculi were more rigid with a modulus of 3 x 10(8) to 4 x 10(8) N/m(2) and at times could be broken by the atomic force microscope tip. Sacculi aligned over the groove with their long axis at right angles to the channel axis were more deformable than those with their long axis parallel to the groove axis, as would be expected if the peptidoglycan strands in the sacculus were oriented at right angles to the long cell axis of this gram-negative rod. Polar caps were not found to be more rigid structures but collapsed to the same thickness as the cylindrical portions of the sacculi. The elasticity of intact E. coli sacculi is such that, if the peptidoglycan strands are aligned in unison, the interstrand spacing should increase by 12% with every 1 atm increase in (turgor) pressure. Assuming an unstressed hydrated interstrand spacing of 1.3 nm (R. E. Burge, A. G. Fowler, and D. A. Reaveley, J. Mol. Biol. 117:927-953, 1977) and an internal turgor pressure of 3 to 5 atm (or 304 to 507 kPa) (A. L. Koch, Adv. Microbial Physiol. 24:301-366, 1983), the natural interstrand spacing in cells would be 1.6 to 2.0 nm. Clearly, if large macromolecules of a diameter greater than these spacings are secreted through this layer, the local ordering of the peptidoglycan must somehow be disrupted.

After whole-genome duplication (WGD), deletions return most loci to single copy. However, duplicate loci may survive through selection for increased dosage. Here, we show how the WGD increased copy number of some glycolytic genes could have conferred an almost immediate selective advantage to an ancestor of Saccharomyces cerevisiae, providing a rationale for the success of the WGD. We propose that the loss of other redundant genes throughout the genome resulted in incremental dosage increases for the surviving duplicated glycolytic genes. This increase gave post-WGD yeasts a growth advantage through rapid glucose fermentation; one of this lineage's many adaptations to glucose-rich environments. Our hypothesis is supported by data from enzyme kinetics and comparative genomics. Because changes in gene dosage follow directly from post-WGD deletions, dosage selection can confer an almost instantaneous benefit after WGD, unlike neofunctionalization or subfunctionalization, which require specific mutations. We also show theoretically that increased fermentative capacity is of greatest advantage when glucose resources are both large and dense, an observation potentially related to the appearance of angiosperms around the time of WGD.

1. EPSCs were recorded under whole-cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12- to 14-day-old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired-pulse depression and all-or-none appearance in response to a graded stimulus. 2. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non-uniform release probability. This 'multiple-probability fluctuation' (MPF) analysis gave an estimate of 510 +/- 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 +/- 0.03 nS (n = 6). 3. Control experiments, and simulations examining the effects of non-uniform release probability, indicate that MPF analysis provides a reliable estimate of quantal parameters. Direct measurement of quantal amplitudes in the presence of 5 mM Sr2+, which gave asynchronous release, yielded distributions with a mean quantal size of 0.55 +/- 0.01 nS and a CV of 0.37 +/- 0.01 (n = 4). Similar estimates of q were obtained in 2 mM Ca2+ when release probability was lowered with the calcium channel blocker Cd2+. The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 1 microM) reduced both the evoked current and the quantal size (estimated with MPF analysis) to a similar degree, but did not affect the estimate of N. 4. We used MPF analysis to identify those quantal parameters that change during frequency-dependent depression at climbing fibre-Purkinje cell synaptic connections. At low stimulation frequencies, the mean release probability (pr) was unusually high (0.90 +/- 0.03 at 0.033 Hz, n = 5), but as the frequency of stimulation was increased, pr fell dramatically (0.02 +/- 0.01 at 10 Hz, n = 4) with no apparent change in either q or N. This indicates that the observed 50-fold depression in EPSC amplitude is presynaptic in origin. 5. Presynaptic frequency-dependent depression was investigated with double-pulse and multiple-pulse protocols. EPSC recovery, following simultaneous release at practically all sites, was slow, being well fitted by the sum of two exponential functions (time constants of 0.35 +/- 0.09 and 3.2 +/- 0.4 s, n = 5). EPSC recovery following sustained stimulation was even slower. We propose that presynaptic depression at CF synapses reflects a slow recovery of release probability following release of each quantum of transmitter. 6. The large number of functional release sites, relatively large quantal size, and unusual dynamics of transmitter release at the CF synapse appear specialized to ensure highly reliable olivocerebellar transmission at low frequencies but to limit transmission at higher frequencies.