BACKGROUND

Genotype imputation can help reduce genotyping costs particularly for implementation of genomic selection. In applications entailing large populations, recovering the genotypes of untyped loci using information from reference individuals that were genotyped with a higher density panel is computationally challenging. Popular imputation methods are based upon the Hidden Markov model and have computational constraints due to an intensive sampling process. A fast, deterministic approach, which makes use of both family and population information, is presented here. All individuals are related and, therefore, share haplotypes which may differ in length and frequency based on their relationships. The method starts with family imputation if pedigree information is available, and then exploits close relationships by searching for long haplotype matches in the reference group using overlapping sliding windows. The search continues as the window size is shrunk in each chromosome sweep in order to capture more distant relationships.

RESULTS

The proposed method gave higher or similar imputation accuracy than Beagle and Impute2 in cattle data sets when all available information was used. When close relatives of target individuals were present in the reference group, the method resulted in higher accuracy compared to the other two methods even when the pedigree was not used. Rare variants were also imputed with higher accuracy. Finally, computing requirements were considerably lower than those of Beagle and Impute2. The presented method took 28 minutes to impute from 6 k to 50 k genotypes for 2,000 individuals with a reference size of 64,429 individuals.

CONCLUSIONS

The proposed method efficiently makes use of information from close and distant relatives for accurate genotype imputation. In addition to its high imputation accuracy, the method is fast, owing to its deterministic nature and, therefore, it can easily be used in large data sets where the use of other methods is impractical.

Several factors may alter apparent resting metabolic rate (RMR) during measurement with indirect calorimetry. Likewise, numerous indirect calorimetry measurement protocols have been developed over the years, and the methodology employed could influence test results. As part of a larger project to determine the role of indirect calorimetry in clinical practice, a systematic review of the literature was undertaken to determine the ideal subject condition and test methodology for obtaining reliable measurement of RMR with indirect calorimetry. Food, ethanol, caffeine, and nicotine affect RMR for a variable number of hours after consumption; therefore, intake of these items must be controlled before measurement. Activities of daily living increase metabolic rate, but a short rest (< or =20 minutes) before testing is sufficient for the effect to dissipate. Moderate or vigorous physical activity has a longer carryover effect and therefore must be controlled in the hours before a measurement of RMR is attempted. Limited data were found regarding ideal ambient conditions for RMR testing. Measurement duration of 10 minutes with the first 5 minutes deleted and the remaining 5 minutes having a coefficient of variation <10% gave accurate readings of RMR. Individuals preparing for RMR measurement via indirect calorimetry should refrain from eating, consuming ethanol and nicotine, smoking, and engaging in physical activity for varying times before measurement. The test site should be physically comfortable and the individual should have 10 to 20 minutes to rest before measurement commences. A 10-minute test duration with the first 5 minutes discarded and the remaining 5 minutes having a coefficient of variation of <10% will give an accurate measure of RMR.

OBJECTIVE

To determine whether clinical trials originating in certain countries always have positive results.

METHODS

Abstracts of trials from Medline (January 1966-June 1995).

METHODS

Two separate studies were conducted. The first included trials in which the clinical outcome of a group of subjects receiving acupuncture was compared to that of a group receiving placebo, no treatment, or a nonacupuncture intervention. In the second study, randomized or controlled trials of interventions other than acupuncture that were published in China, Japan, Russia/USSR, or Taiwan were compared to those published in England.

METHODS

Blinded reviewers determined inclusion and outcome and separately classified each trial by country of origin.

RESULTS

In the study of acupuncture trials, 252 of 1085 abstracts met the inclusion criteria. Research conducted in certain countries was uniformly favorable to acupuncture; all trials originating in China, Japan, Hong Kong, and Taiwan were positive, as were 10 out of 11 of those published in Russia/USSR. In studies that examined interventions other than acupuncture, 405 of 1100 abstracts met the inclusion criteria. Of trials published in England, 75% gave the test treatment as superior to control. The results for China, Japan, Russia/USSR, and Taiwan were 99%, 89%, 97%, and 95%, respectively. No trial published in China or Russia/USSR found a test treatment to be ineffective.

CONCLUSIONS

Some countries publish unusually high proportions of positive results. Publication bias is a possible explanation. Researchers undertaking systematic reviews should consider carefully how to manage data from these countries.

OBJECTIVE

This study described the natural history of depression in mothers who recently gave birth in a low-income country and to investigate the effect of risk factors, particularly related to infant gender bias, on the occurrence and outcome of depression.

METHODS

The authors studied a group of pregnant mothers recruited during their third trimester of pregnancy from a district hospital in Goa, India. The mothers were interviewed at recruitment, 6-8 weeks, and 6 months after childbirth. Interview data included presence of antenatal and postnatal depression, obstetric history, economic and demographic characteristics, and gender-based variables (preference for male infant, presence of marital violence).

RESULTS

Depressive disorder was detected in 59 (23%) of the mothers at 6-8 weeks after childbirth; 78% of these patients had had clinically substantial psychological morbidity during the antenatal period. More than one-half of the patients remained ill at 6 months after delivery. Economic deprivation and poor marital relationships were important risk factors for the occurrence and chronicity of depression. The gender of the infant was a determinant of postnatal depression; it modified the effect of other risk factors, such as marital violence and hunger. Depressed mothers were more disabled and were more likely to use health services than nondepressed mothers.

CONCLUSIONS

Maternal and infant health policies, a priority in low-income countries, must integrate maternal depression as a disorder of public health significance. Interventions should target mothers in the antenatal period and incorporate a strong gender-based component.

Humoral immune responses were characterized in mouse strains lacking either or both B7 molecules. Mice deficient in both B7-1 and B7-2 failed to generate antigen-specific IgG1 and IgG2a responses and lacked germinal centers when immunized by a number of routes and even in the presence of complete Freund's adjuvant. These results demonstrate that B7-mediated signaling plays a critical role in germinal center formation and immunoglobulin class switching in vivo. Mice lacking only B7-1 or B7-2 mounted high-titer antigen-specific IgG responses when immunized in complete Freund's adjuvant, indicating that B7-1 and B7-2 can have overlapping, compensatory functions for IgG responses. When immunized intravenously without adjuvant, B7-2-deficient mice failed to switch antibody isotypes or form germinal centers, whereas B7-1-deficient mice gave antibody responses comparable with wild-type mice. Thus, B7-2 has an important role in initiating antibody responses in the absence of adjuvant, but the induction of B7-1 by adjuvant in B7-2-deficient mice can compensate for the absence of B7-2.

Changes in genes encoding transcriptional regulators can alter development and are important components of the molecular mechanisms of morphological evolution. MADS-box genes encode transcriptional regulators of diverse and important biological functions. In plants, MADS-box genes regulate flower, fruit, leaf, and root development. Recent sequencing efforts in Arabidopsis have allowed a nearly complete sampling of the MADS-box gene family from a single plant, something that was lacking in previous phylogenetic studies. To test the long-suspected parallel between the evolution of the MADS-box gene family and the evolution of plant form, a polarized gene phylogeny is necessary. Here we suggest that a gene duplication ancestral to the divergence of plants and animals gave rise to two main lineages of MADS-box genes: TypeI and TypeII. We locate the root of the eukaryotic MADS-box gene family between these two lineages. A novel monophyletic group of plant MADS domains (AGL34 like) seems to be more closely related to previously identified animal SRF-like MADS domains to form TypeI lineage. Most other plant sequences form a clear monophyletic group with animal MEF2-like domains to form TypeII lineage. Only plant TypeII members have a K domain that is downstream of the MADS domain in most plant members previously identified. This suggests that the K domain evolved after the duplication that gave rise to the two lineages. Finally, a group of intermediate plant sequences could be the result of recombination events. These analyses may guide the search for MADS-box sequences in basal eukaryotes and the phylogenetic placement of new genes from other plant species.

OBJECTIVE

In the light of a report suggesting that insulin glargine may increase cancer occurrence, the EASD asked us to perform this study.

METHODS

We followed 114,841 individuals who had a prescription dispensed for insulin between 1 July and 31 December 2005. From 1 January 2006 to 31 December 2007, we noted the occurrence of malignancies. Seven different nationwide registers were used to obtain information on insulin exposure, outcome and possible confounders; these were linked using the unique personal identity number assigned to every Swedish resident.

RESULTS

After adjustment for age and, when appropriate, sex, users of insulin glargine alone (no other types of insulin), compared with users of types of insulin other than insulin glargine, had an RR of 1.99 (95% CI 1.31-3.03) for breast cancer, 0.93 (95% CI 0.61-1.40) for gastrointestinal cancer, 1.27 (95% CI 0.89-1.82) for prostate cancer and 1.07 (95% CI 0.91-1.27) for any type of malignancy. Adjustment for age, smoking, BMI, age at onset of diabetes, age at birth of first child, cardiovascular disease and oestrogen use gave an RR for breast cancer of 1.97 (95% CI 1.29-3.00). The 95% CIs crossed 1.0 for the RR calculated in all analyses of users of insulin glargine in combination with other types of insulin.

CONCLUSIONS

In Sweden, during 2006 and 2007, women using insulin glargine alone (no other types of insulin) had an increased incidence rate of breast cancer as compared with women using types of insulin other than insulin glargine. This result may be due to a random fluctuation; the possibilities for examining validity are limited, and no statistically significant results were obtained for any other individual cancer site or for the outcome 'all malignancies'. No definitive conclusions regarding a possible causal relationship between insulin glargine use and the occurrence of malignancies can be drawn from the results of this study.

A soluble nitric oxide (NO) synthase activity was purified 426-fold from a mouse macrophage cell line activated with interferon gamma and bacterial lipopolysaccharide by sequential anion-exchange, affinity, and gel filtration chromatography. SDS/PAGE of the purified NO synthase gave three closely spaced silver-staining protein bands between 125 and 135 kDa. When assayed in the presence of L-arginine, NADPH, tetrahydrobiopterin, FAD, and reduced thiol, purified NO synthase had a specific activity of 1313 nmol of NO2- plus NO3- per min per mg. The apparent Km of the enzyme for L-arginine and NADPH was 2.8 and 0.3 microM, respectively. Addition of calcium ions with or without calmodulin did not increase the activity of the purified enzyme, and NO synthesis was not altered by calmodulin inhibitors. Gel filtration chromatography indicated that the induced NO synthase was catalytically competent as a dimer of approximately 250 kDa but could be dissociated into inactive monomers of approximately 130 kDa in the absence of L-arginine, FAD, and tetrahydrobiopterin. Upon heat denaturation, NO synthase released 1.1 mol of FAD and 0.55 mol of FMN per mol of 130-kDa subunit. Thus, inducible macrophage NO synthase differs in several respects from constitutive NO synthases and is one of very few eukaryotic enzymes containing both FAD and FMN.

Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrP(Sc)). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-beta-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrP(Sc) plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrP(Sc) in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases.

The classification of cat retinal ganglion cells as X or Y on the basis of linearity or nonlinearity of spatial summation has been confirmed and extended. Recordings were taken from optic tract fibres of anaesthetized, paralysed cats. 2. When an alternating phase sine wave grating was used as a stimulus, X cells had null positions and Y cells responded at all positions of the grating. 3. These results did not depend on the temporal wave form or the temporal frequency of pattern alternation over a wide range. 4. At high spatial frequencies for the particular cell, a Y cell gave abig 'on-off' response, or frequency doubling, at all positions of the grating, while an X cell did not. 5. The use of contrast sensitivity versus spatial phase also served to differentiate the two cell types. With an alternating sine grating stimulus X cells had a sinusoidal dependence on spatial phase, while each Y cell's sensitivity depended in a complicated manner on spatial phase. 6. Sensitivity versus spatial phase for different Fourier components of the neural response also separated the two classes of cells. Significant second harmonic distortion was present in Y cells. The second harmonic component was spatial phase insensitive, and became dominant at high spatial frequencies. 7. The maximum of the 2nd/1st harmonic ratio was taken as an index of nonlinearity. X cells always had a nonlinearity index less than 1 while in Y cells this index always exceeded 1. 8. Response to spots, diffuse light and drifting gratings were compared to the nonlinearity index as a basis for classifying cells. The nonlinearity index was most reliable because it was least dependent on retinal eccentricity.

BACKGROUND

Infection with Clostridium difficile is the primary infective cause of antibiotic-associated diarrhoea. We aimed to compare efficacy and safety of fidaxomicin and vancomycin to treat patients with C difficile infection in Europe, Canada, and the USA.

METHODS

In this multicentre, double-blind, randomised, non-inferiority trial, we enrolled patients from 45 sites in Europe and 41 sites in the USA and Canada between April 19, 2007, and Dec 11, 2009. Eligible patients were aged 16 years or older with acute, toxin-positive C difficile infection. Patients were randomly allocated (1:1) to receive oral fidaxomicin (200 mg every 12 h) or oral vancomycin (125 mg every 6 h) for 10 days. The primary endpoint was clinical cure, defined as resolution of diarrhoea and no further need for treatment. An interactive voice-response system and computer-generated randomisation schedule gave a randomisation number and medication kit number for each patient. Participants and investigators were masked to treatment allocation. Non-inferiority was prespecified with a margin of 10%. Modified intention-to-treat and per-protocol populations were analysed. This study is registered with ClinicalTrials.gov, number NCT00468728.

RESULTS

Of 535 patients enrolled, 270 were assigned fidaxomicin and 265 vancomycin. After 26 patients were excluded, 509 were included in the modified intention-to-treat (mITT) population. 198 (91·7%) of 216 patients in the per-protocol population given fidaxomicin achieved clinical cure, compared with 213 (90·6%) of 235 given vancomycin, meeting the criterion for non-inferiority (one-sided 97·5% CI -4·3%). Non-inferiority was also shown for clinical cure in the mITT population, with 221 (87·7%) of 252 patients given fidaxomicin and 223 (86·8%) of 257 given vancomycin cured (one-sided 97·5% CI -4·9%). In most subgroup analyses of the primary endpoint in the mITT population, outcomes in the two treatment groups did not differ significantly; although patients receiving concomitant antibiotics for other infections had a higher cure rate with fidaxomicin (46 [90·2%] of 51) than with vancomycin (33 [73·3%] of 45; p=0·031). Occurrence of treatment-emergent adverse events did not differ between groups. 20 (7·6%) of 264 patients given at least one dose of fidaxomicin and 17 (6·5%) of 260 given vancomycin died.

CONCLUSIONS

Fidaxomicin could be an alternative treatment for infection with C difficile, with similar efficacy and safety to vancomycin.

BACKGROUND

Optimer Pharmaceuticals.

RNase MRP is a site-specific ribonucleoprotein endoribonuclease that cleaves RNA from the mitochondrial origin of replication in a manner consistent with a role in priming leading-strand DNA synthesis. Despite the fact that the only known RNA substrate for this enzyme is complementary to mitochondrial DNA, the majority of the RNase MRP activity in a cell is found in the nucleus. The recent characterization of this activity in Saccharomyces cerevisiae and subsequent cloning of the gene coding for the RNA subunit of the yeast enzyme have enabled a genetic approach to the identification of a nuclear role for this ribonuclease. Since the gene for the RNA component of RNase MRP, NME1, is essential in yeast cells and RNase MRP in mammalian cells appears to be localized to nucleoli within the nucleus, we utilized both regulated expression and temperature-conditional mutations of NME1 to assay for a possible effect on rRNA processing. Depletion of the RNA component of the enzyme was accomplished by using the glucose-repressed GAL1 promoter. Shortly after the shift to glucose, the RNA component of the enzyme was found to be depleted severely, and rRNA processing was found to be normal at all sites except the B1 processing site. The B1 site, at the 5' end of the mature 5.8S rRNA, is actually composed of two cleavage sites 7 nucleotides apart. This cleavage normally generates two species of 5.8S rRNA at a ratio of 10:1 (small to large) in most eukaryotes. After RNase MRP depletion, yeast cells were found to have almost exclusively the larger species of 5.8S rRNA. In addition, an aberrant 309-nucleotide precursor that stretched from the A2 to E processing sites of rRNA accumulated in these cells. Temperature-conditional mutations in the RNase MRP RNA gene gave an identical phenotype. Translation in yeast cells depleted of the smaller 5.8S rRNA was found to remain robust, suggesting a possible function for two 5.8S rRNAs in the regulated translation of select messages. These results are consistent with RNase MRP playing a role in a late step of rRNA processing. The data also indicate a requirement for having the smaller form of 5.8S rRNA, and they argue for processing at the B1 position being composed of two separate cleavage events catalyzed by two different activities.

Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.

1. Physiological noise in the visual transduction mechanism was studied by recording membrane current from single rod outer segments in pieces of isolated toad retina. 2. The inward current in darkness showed spontaneous fluctuations which disappeared during the response to bright light. 3. The dark noise consisted of two components, a continuous fluctuation of rms amplitude about 0.2 pA and occasional discrete events about 1 pA in size. 4. Intervals between discrete events followed the exponential distribution expected of a Poisson process with a mean rate of about one event per 50 sec (20 degrees C). 5. The amplitude and power spectrum of the discrete events resembled those of single photon effects in the same rod, suggesting that discrete events may arise from spontaneous activation of single rhodopsin molecules. 6. The temperature dependence of the mean frequency of occurrence of discrete events gave an activation energy of 22 kcal mole-1, probably characteristic of thermal isomerization of rhodopsin. 7. The variance of the continuous component of the dark noise rose linearly with the length of the outer segment drawn into the suction electrode, indicating that this component is generated in the outer segment. 8. The power spectrum of a rod's continuous noise was usually fitted by the square of a Lorentzian with the same time constant as that of the four first-order delays in the cell's single photon response. The shot effects composing the continuous component thus appear to be shaped by two of four sequential processes in transduction. 9. The variance and spectrum of the continuous noise are interpreted to reflect shot effects about 1/400 the size of a single photon effect occurring at a frequency of 6 x 10(3) sec-1. 10. The rod's flash sensitivity was halved by a steady light to giving about 8 photoisomerizations sec-1. The much lower mean rate of discrete events indicates that Io in increment sensitivity experiments on individual receptors is not set by thermal activation of rhodopsin. 11. Values of sensitivity and time-to-peak flash response collected from many cells in darkness were correlated by the same power law relation obtaining in the presence of backgrounds. The correlation observed would be explained if a single variable controlled both the gain and time scale of several stages of the transduction mechanism in background light and in darkness.

BACKGROUND

Breastfeeding is essential for child health and development in low-resource settings but carries a significant risk of transmission of HIV-1, especially in late stages of maternal disease. We aimed to assess the efficacy and safety of triple antiretroviral compared with zidovudine and single-dose nevirapine prophylaxis in pregnant women infected with HIV.

METHODS

Pregnant women with WHO stage 1, 2, or 3 HIV-1 infection who had CD4 cell counts of 200-500 cells per μL were enrolled at five study sites in Burkina Faso, Kenya, and South Africa to start study treatment at 28-36 weeks' gestation. Women were randomly assigned (1:1) by a computer generated random sequence to either triple antiretroviral prophylaxis (a combination of 300 mg zidovudine, 150 mg lamivudine, and 400 mg lopinavir plus 100 mg ritonavir twice daily until cessation of breastfeeding to a maximum of 6·5 months post partum) or zidovudine and single-dose nevirapine (300 mg zidovudine twice daily until delivery and a dose of 600 mg zidovudine plus 200 mg nevirapine at the onset of labour and, after a protocol amendment in December, 2006, 1 week post-partum zidovudine 300 mg twice daily and lamivudine 150 mg twice daily). All infants received a 0·6 mL dose of nevirapine at birth and, from December, 2006, 4 mg/kg twice daily of zidovudine for 1 week after birth. Patients and investigators were not masked to treatment. The primary endpoints were HIV-free infant survival at 6 weeks and 12 months; HIV-free survival at 12 months in infants who were ever breastfed; AIDS-free survival in mothers at 18 months; and serious adverse events in mothers and babies. Analysis was by intention to treat. This trial is registered with Current Controlled Trials, ISRCTN71468401.

RESULTS

From June, 2005, to August, 2008, 882 women were enrolled, 824 of whom were randomised and gave birth to 805 singleton or first, liveborn infants. The cumulative rate of HIV transmission at 6 weeks was 3·3% (95% CI 1·9-5·6%) in the triple antiretroviral group compared with 5·0% (3·3-7·7%) in the zidovudine and single-dose nevirapine group, and at 12 months was 5·4% (3·6-8·1%) in the triple antiretroviral group compared with 9·5% (7·0-12·9%) in the zidovudine and single-dose nevirapine group (p=0·029). The cumulative rate of HIV transmission or death at 12 months was 10·2% (95% CI 7·6-13·6%) in the triple antiretroviral group compared with 16·0% (12·7-20·0%) in the zidovudine and single-dose nevirapine group (p=0·017). In infants whose mothers declared they intended to breastfeed, the cumulative rate of HIV transmission at 12 months was 5·6% (95% CI 3·4-8·9%) in the triple antiretroviral group compared with 10·7% (7·6-14·8%) in the zidovudine and single-dose nevirapine group (p=0·02). AIDS-free survival in mothers at 18 months will be reported in a different publication. The incidence of laboratory and clinical serious adverse events in both mothers and their babies was similar between groups.

CONCLUSIONS

Triple antiretroviral prophylaxis during pregnancy and breastfeeding is safe and reduces the risk of HIV transmission to infants. Revised WHO guidelines now recommend antiretroviral prophylaxis (either to the mother or to the baby) during breastfeeding if the mother is not already receiving antiretroviral treatment for her own health.

BACKGROUND

Agence nationale de recherches sur le sida et les hépatites virales, Department for International Development, European and Developing Countries Clinical Trials Partnership, Thrasher Research Fund, Belgian Directorate General for International Cooperation, Centers for Disease Control and Prevention, Eunice Kennedy Shriver National Institute of Child Health and Human Development, and UNDP/UNFPA/World Bank/WHO Special Programme of Research, Development and Research Training in Human Reproduction.

The full-length genome of human cytomegalovirus strain AD169 was cloned as an infectious bacterial artificial chromosome (BAC) plasmid, pAD/Cre. The BAC vector, flanked by LoxP sites, was inserted immediately after the Us28 open reading frame without deletion of any viral sequences. The BAC vector contained the Cre recombinase-encoding gene disrupted by an intron under control of the simian virus 40 early promoter. When pAD/Cre was transfected into primary human foreskin fibroblast cells, Cre was expressed and mediated site-specific recombination between the two LoxP sites, excising the BAC DNA backbone. This gave rise to progeny virus that was wild type with the exception of an inserted 34-bp LoxP site. We performed site-directed mutagenesis on pAD/Cre to generate a series of viruses in which the TRL/IRL13 diploid genes were disrupted and subsequently repaired. The mutants reach the same titer as the wild-type virus, indicating that the TRL/IRL13 open reading frames are not required for virus growth in cell culture. The sequence of the TRL13 open reading frame in the low-passage Toledo strain of human cytomegalovirus is quite different from the corresponding region in the AD169 strain. One of multiple changes is a frameshift mutation. As a consequence, strain Toledo encodes a putative TRL13 protein whose C-terminal domain is larger (extending through the TRL14 coding region) and encodes in a reading frame different from that of strain AD169. We speculate that the strain AD169 coding region has drifted during passage in the laboratory. We propose that TRL13 has been truncated in strain AD169 and that the partially overlapping TRL14 open reading frame is not functional. This view is consistent with the presence of both TRL13 and -14 on all mRNAs that we have mapped from this region, an organization that would include the much longer strain Toledo TRL13 open reading frame on the mRNAs.

This paper summarizes twenty studies, published since 1989, that have measured experimentally the relationship between speech recognition in noise and some aspect of cognition, using statistical techniques such as correlation or factor analysis. The results demonstrate that there is a link, but it is secondary to the predictive effects of hearing loss, and it is somewhat mixed across study. No one cognitive test always gave a significant result, but measures of working memory (especially reading span) were mostly effective, whereas measures of general ability, such as IQ, were mostly ineffective. Some of the studies included aided listening, and two reported the benefits from aided listening: again mixed results were found, and in some circumstances cognition was a useful predictor of hearing-aid benefit.

Complementary DNAs derived from a mouse hybridoma messenger RNA were used to transform tobacco leaf segments followed by regeneration of mature plants. Plants expressing single gamma or kappa immunoglobulin chains were crossed to yield progeny in which both chains were expressed simultaneously. A functional antibody accumulated to 1.3% of total leaf protein in plants expressing full-length cDNAs containing leader sequences. Specific binding of the antigen recognized by these antibodies was similar to the hybridoma-derived antibody. Transformants having gamma- or kappa-chain cDNAs without leader sequences gave poor expression of the proteins. The increased abundance of both gamma- and kappa-chains in transformants expressing assembled gamma-kappa complexes was not reflected in increased mRNA levels. The results demonstrate that production of immunoglobulins and assembly of functional antibodies occurs very efficiently in tobacco. Assembly of subunits by sexual cross might be a generally applicable method for expression of heterologous multimers in plants.

Neurons are continuously generated from stem cells in discrete regions in the adult mammalian brain. We found that ependymal cells lining the lateral ventricles were quiescent and did not contribute to adult neurogenesis under normal conditions in mice but instead gave rise to neuroblasts and astrocytes in response to stroke. Ependymal cell quiescence was actively maintained by canonical Notch signaling. Inhibition of this pathway in uninjured animals allowed ependymal cells to enter the cell cycle and produce olfactory bulb neurons, whereas forced Notch signaling was sufficient to block the ependymal cell response to stroke. Ependymal cells were depleted by stroke and failed to self-renew sufficiently to maintain their own population. Thus, although ependymal cells act as primary cells in the neural lineage to produce neurons and glial cells after stroke, they do not fulfill defining criteria for stem cells under these conditions and instead serve as a reservoir that is recruited by injury.

In this study, psychometric principles were used to develop an outcomes questionnaire capable of measuring health state domains important to patients with hand disorders. These domains were hypothesized to include (1) overall hand function, (2) activities of daily living (ADL), (3) pain, (4) work performance, (5) aesthetics, and (6) patient satisfaction with hand function. An initial pool of 100 questions was pilot-tested for clarity in 20 patients; following factor analysis, the number of questions was reduced to a 37-item Michigan Hand Outcomes Questionnaire (MHQ). The MHQ, along with the Short Form-12, a generic health status outcomes questionnaire, was then administered to 200 consecutive patients at a university-based hand surgery clinic and was subjected to reliability and validity testing. The mean time required to complete the questionnaire was 10 minutes (range, 7-20 minutes). Factor analysis supported the 6 hypothesized scales. Test-retest reliability using Spearman's correlation demonstrated substantial agreement, ranging from 0.81 for the aesthetics scale to 0.97 for the ADL scale. In testing for internal consistency, Cronbach's alphas ranged from 0.86 for the pain scale to 0.97 for the ADL scale (values >0.7 for Cronbach's alpha are considered a good internal consistency). Correlation between scales gave evidence of construct validity. In comparing similar scales in the MHQ and the Short Form-12, a moderate correlation (range, 0.54-0.79) for the ADL, work performance, and pain scales was found. In evaluating the discriminate validity of the aesthetics scale, a significant difference (p = .0012) was found between the aesthetics scores for patients with carpal tunnel syndrome and patients with rheumatoid arthritis. The MHQ is a reliable and valid instrument for measuring hand outcomes. It can be used in a clinic setting with minimal burden to patients. The questions in the MHQ have undergone rigorous psychometric testing, and the MHQ is a promising instrument for evaluation of outcomes following hand surgery.

Aldehyde dehydrogenase-1A1 (ALDH1A1) expression characterizes a subpopulation of cells with tumor-initiating or cancer stem cell properties in several malignancies. Our goal was to characterize the phenotype of ALDH1A1-positive ovarian cancer cells and examine the biological effects of ALDH1A1 gene silencing. In our analysis of multiple ovarian cancer cell lines, we found that ALDH1A1 expression and activity was significantly higher in taxane- and platinum-resistant cell lines. In patient samples, 72.9% of ovarian cancers had ALDH1A1 expression in which the percentage of ALDH1A1-positive cells correlated negatively with progression-free survival (6.05 vs. 13.81 months; P < 0.035). Subpopulations of A2780cp20 cells with ALDH1A1 activity were isolated for orthotopic tumor-initiating studies, where tumorigenicity was approximately 50-fold higher with ALDH1A1-positive cells. Interestingly, tumors derived from ALDH1A1-positive cells gave rise to both ALDH1A1-positive and ALDH1A1-negative populations, but ALDH1A1-negative cells could not generate ALDH1A1-positive cells. In an in vivo orthotopic mouse model of ovarian cancer, ALDH1A1 silencing using nanoliposomal siRNA sensitized both taxane- and platinum-resistant cell lines to chemotherapy, significantly reducing tumor growth in mice compared with chemotherapy alone (a 74%-90% reduction; P < 0.015). These data show that the ALDH1A1 subpopulation is associated with chemoresistance and outcome in ovarian cancer patients, and targeting ALDH1A1 sensitizes resistant cells to chemotherapy. ALDH1A1-positive cells have enhanced, but not absolute, tumorigenicity but do have differentiation capacity lacking in ALDH1A1-negative cells. This enzyme may be important for identification and targeting of chemoresistant cell populations in ovarian cancer.

Plasmids pAD1 (37.8 megadaltons) and pAD2 (17.1 megadaltons) of Streptococcus faecalis strain DS16 have been mapped with restriction enzymes. The location of a hemolysin-bacteriocin determinant on the conjugative pAD1 plasmid was derived from analyses of transposon insertions. Electron microscope and hybridization analyses located Tn917(Em) and the streptomycin (Sm) and kanamycin (Km) resistance determinants on the nonconjugative pAD2 plasmid. It was shown previously that the erythromycin (Em) resistance associated with Tn917 is inducible and that transposition from pAD2 to pAD1 is also stimulated by exposure of cells to low concentrations of Em. Here we show that inducing concentrations of Em also increase the conjugative transfer potential of pAD1; this is possibly related to a mild and short-lived inhibitory stress placed on the cells before full induction of resistance. Selection of Em-resistant transconjugants arising from matings between DS16 and a plasmid-free recipient gave rise to transconjugants which primarily harbor stable pAD1::pAD2 cointegrates. A 30-min exposure of donors to Em (0.5 microgram/ml) before mating resulted in a severalfold increase in the number of such transconjugants. However, a small fraction (e.g., 3 of 40) of these Emr Smr Kmr transconjugants harbored pAD1::Tn917 and pAD2 molecules. Since we believe pAD2 is incapable of being mobilized by pAD1 without being covalently linked, it is likely that transfer in these cases involved cointegrates representing structural intermediates in the transposition of Tn917 from pAD2 to pAD1. It follows that such intermediates probably had two copies of Tn917 and readily resolved after transfer. (These cointegrates are different from the stable cointegrates which were shown to have only a single copy of Tn917; the latter are assumed not to be related to transposition.) Two variants with altered Tn917 transposition properties were derived. One of them transposed at an elevated frequency, whereas the other showed no detectabel transposition. In neither case was transposition influenced by Em exposure; however, both remained inducible for Em resistance.

Central administration of the preproglucagon-derived peptide glucagon-like peptide-1 significantly inhibits ingestion of food and water, and glucagon-like peptide-1 binding sites are present in a multitude of central areas involved in the regulation of ingestional behaviour. To evaluate further the neuroanatomical organization of central glucagon-like peptide-1 containing neuronal circuits with potential implications on ingestional behaviour, we carried out a series of experiments in the rat demonstrating the topographical sites of synthesis and processing of the preproglucagon precursor followed by a chromatographic analysis of the processed fragments. In situ hybridization histochemistry revealed that preproglucagon encoding messenger RNA was expressed in a single population of neurons in the caudal portion of the nucleus of the solitary tract. Gel chromatographic analysis of hypothalamic and brainstem tissue extracts revealed that the preproglucagon precursor is processed in a fashion similar to that seen in the small intestine, preferentially giving rise to glicentin, glucagon-like peptide-1 and glucagon-like peptide-2. This single brain site of glucagon-like peptide-1 synthesis was subsequently confirmed by immunohistochemical demonstration of glucagon-like peptide-1-immunoreactive perikarya in the central and caudal parts of the nucleus of the solitary tract. Numerous sites containing glucagon-like peptide-1 immunoreactive fibres were, however, discovered in the forebrain including hypothalamic, thalamic and cortical areas. The densest innervation by glucagon-like peptide-1 immunoreactive nerve fibres was seen in the hypothalamic dorsomedial and paraventricular nuclei, but numerous glucagon-like peptide-1 immunoreactive fibres were also seen throughout the periventricular strata of the third ventricle. Dual-labelling immunohistochemistry for tyrosine hydroxylase and glucagon-like peptide-1 gave no evidence for co-localization of catecholamines and glucagon-like peptide-1 in neurons of the lower brainstem. To identify neurons of the nucleus of the solitary tract that project to the hypothalamic paraventricular nucleus, the retrograde tracer FluoroGold was injected into this hypothalamic target and dual immunocytochemical identification of glucagon-like peptide-1 and tyrosine hydroxylase-positive neurons was performed on brainstem sections containing retrogradely labelled perikarya. From this experiment it was seen that many of the retrogradely labelled neurons in the central portion of the nucleus of the solitary tract are catecholaminergic, while none is glucagon-like peptide-1 immunoreactive. In contrast, most of the retrogradely labelled neurons of the caudal portion of the nucleus of the solitary tract contain glucagon-like peptide-1. These observations further substantiate that glucagon-like peptide-1 neurons of the solitary tract constitute a distinct non-catecholaminergic cell group which projects to many targets, one of which is the hypothalamic paraventricular nucleus.

Both connective tissue mast cells and mast cells grown in vitro are derived from multipotential hematopoietic stem cells, but these two mast cell populations exhibit many differences in morphology, biochemistry, and function. We investigated whether the phenotype of cultured mast cells or their progeny was altered when the cells were transferred into different locations in vivo. Cultured mast cells were immature by ultrastructure, and stained with alcian blue but with neither safranin or berberine sulfate, a fluorescent dye that binds to the heparin of connective tissue mast cell granules. By contrast, mast cells recovered from the peritoneal cavity of congenitally mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice 10 wk after intraperitoneal injection of cultured WBB6F1-+/+ or C57BL/6-bgJ/bgJ mast cells stained with both safranin and berberine sulfate. Staining with berberine sulfate was prevented by treatment of the cells with heparinase but not chondroitinase ABC, suggesting that the adoptively transferred mast cell population had acquired the ability to synthesize and store heparin. Furthermore, the recovered mast cells were indistinguishable by ultrastructure from the normal mature peritoneal mast cells of WBB6F1-+/+ mice, and contained substantially more histamine than mast cells studied directly from culture. Intravenous injection of cultured mast cells resulted in the development of safranin-and berberine sulfate-positive mast cells in the peritoneal cavity, spleen, skin, and glandular stomach muscularis propria. Mast cells also developed on the glandular stomach mucosa, but these cells stained with alcian blue rather than safranin, and did not stain with berberine sulfate. This result suggests that cultured mast cells can give rise to mast cells of either the connective tissue type or mucosal phenotype, depending on anatomical location. Furthermore, transplantation of cultured mast cells into WBB6F1-W/Wv mice had no measurable effect on the anemia of the recipient mice, suggesting a possible strategy for repairing the mast cell deficiency of WBB6F1-W/Wv mice without affecting other bone marrow-derived populations such as erythrocytes. Intravenous injection of representative connective tissue type mast cells (30-50% pure peritoneal mast cells derived from WBB6F1-+/+ mice) gave results similar to those obtained with cultured mast cells: mast cells developing in the peritoneal cavity, skin, spleen, and glandular stomach muscularis propria of WBB6F1-W/Wv recipients stained with safranin and berberine sulfate, whereas mast cells developing in the mucosa of the glandular stomach stained only with alcian blue.(ABSTRACT TRUNCATED AT 400 WORDS)