High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.     

The cell envelope of Mycobacterium smegmatis: cytochemistry and architectural implications

In sections stained for localizing both carbohydrates (Thiery's method) and lipids (the O.T.O. method), the cell envelope of Mycobacterium smegmatis appeared to consists of an asymmetric cytoplasmic membrane surrounded by a three-layered cell wall. The outer layer of the cytoplasmic membrane was found to contain more glycoconjugate molecules (probably phosphatidyl inositol mannosides) than the inner one. The cell wall consists of the peptidoglycan (the innermost layer) surrounded by a layer containing both arabinogalactan and mycolates (the electron-dense layer), whereas the outermost layer was unstainable. There is clearly a difficulty in reconciling such a cell wall organization with the models so far proposed.     

Malaria transmission in a central area of Futa Djalon (Guinea): results of a parasitological survey during the 1989 rainy season.

A malaria survey based on household surveys and dispensary visits without notice was carried out during the rainy season of 1989 in three selected areas of central Futa Djalon (Republic of Guinea). Preliminary entomological evidence showed that Anopheles gambiae was the main vector in the area with a CS positive rate of 7.6% and a human blood index of 78% in August 1989. Indoor resting densities were highest in the rural village, lowest in Labé and intermediate in the town of Timbi Madina (median density per room of 18, 2 and 4.5, respectively). The household survey showed different parasite rates in the three areas: 78.2% of children had parasitaemia in Sombili, 45.8% in Timbi Madina and 16.7% in the urban sector of Labé. Prevalence and levels of P. falciparum antisporozoite antibodies were lowest in the urban area (seroprevalence of 51.9% and median titre of 2.4 arbitrary units), intermediate in the town (70.1% and 5.2) and highest in the rural village (78.9% and 5.8). Serological findings produced by dispensary visits were similar to those obtained with the household surveys except in the rural area, while parasitological data obtained with the two sampling methods were different both in rural and in urban areas. Routine malaria diagnosis made on a presumptive basis in the health services in the survey period was able to detect 30.1% of cases with parasitaemia and 53.2% of cases with hyperparasitaemia (sensitivity) and malaria was correctly not suspected in 82.8% of non parasitized people (specificity).     

An Intact U5-Leader Stem Is Important for Efficient Replication of Simian Immunodeficiency Virus

Previous work has shown that four deletions in simian immunodeficiency virus (SIV), termed SD1a, SD1b, SD1c, and SD6, which eliminated sequences at nucleotide positions 322 to 362, 322 to 370, 322 to 379, and 371 to 379, respectively, located downstream of the primer binding site, impaired viral replication capacity to different extents. Long-term culturing of viruses containing the SD1a, SD1b, and SD6 deletions led to revertants that possessed wild-type replication kinetics. We now show that these revertants retained the original deletions in each case but that novel additional mutations were also present. These included a large deletion termed D1 (nt +216 to +237) within the U5 region that was shown to be biologically relevant to reversion of both the SD1a and SD1b constructs. In the case of SD6, two compensatory point mutations, i.e., A+369G, termed M1, located immediately upstream of the SD6 deletion, and C+201T, termed M2, within U5, were identified and could act either singly or in combination to restore viral replication. Secondary structure suggests that an intact U5-leader stem is important in SIV for infectiousness and that the additional mutants described played important roles in restoration of this motif.     

Abundance and distribution of bacterioplankton in the Gambia River,West Africa

Four ecological zones of the Gambia River were sampled during four different hydrologic seasons for determination of microbial, nutrient, and physical parameters. A Greco-Latin Square experimental design was used to define the particular transect, station, depth, and tide/time-of-day of samples taken. Ranges of total bacterioplankton densities (10⁶ cells/ml) were similar to those of tropical and temperate environments. Numbers of free bacteria were similar temporally, whereas attached bacteria numbers were greater during periods of high stream flows when suspended solids concentrations were higher. Free bacteria were usually twice as numerous in the freshwater zones than in the estuarine zones. Attached bacterial densities were approximately four times greater in the estuarine zones than in the freshwater zones. Uptake of³H-glucose on both a sample volume and per-cell basis increased from the early stages of the flood (6.95±SE 1.37 ng/liter/hour and 3.8 pg/hour/10⁶ cells, respectively) and reached observed annual maximums during the dry season (21.01±SE 3.05 ng/ liter/hour and 13.0 pg/hour/10⁶ cells, respectively). Spatially,³H-glucose uptake per sample volume and per cell was highest in the upper river zone and lowest in the lower estuary zone. The lower estuary zone consistently acted out of concert with the other river zones in terms of³H-glucose and¹⁴C-bicarbonate uptake. Analysis of variance (ANOVA) indicated that free and attached bacterioplankton densities were not homogeneous among transects, stations, depths, and tide/time-of-day at the different zones during the four hydrologic seasons. The results suggested that heterotrophy overshadowed autotrophy in the river and that the bacterial abundance, distribution, and glucose uptake activity in this tropical floodplain river were greatly influenced by the annual flood and the presence of extensive mangrove forests in the estuary.     

Non-antibiotic selection systems for soybean somatic embryos: the lysine analog aminoethyl-cysteine as a selection agent

Background  

In soybean somatic embryo transformation, the standard selection agent currently used is hygromycin. It may be preferable to avoid use of antibiotic resistance genes in foods. The objective of these experiments was to develop a selection system for producing transgenic soybean somatic embryos without the use of antibiotics such as hygromycin.     

Retrospective study of maternal HIV-1 and HIV-2 infections and child survival in Abidjan,C?te d'Ivoire.

OBJECTIVES--To compare the effects of maternal HIV-1 and HIV-2 infections on outcome of pregnancy, infant mortality, and child survival, and to measure serological concordance between mothers and children. DESIGN--Retrospective cohort study with cross sectional study of concordance for HIV antibodies. SETTING--Hospital, tuberculosis clinic, and maternal and child health centre in Abidjan, Côte d''Ivoire, west Africa. SUBJECTS--986 women who had had a total of 2758 pregnancies since 1980. The last born children of 194 of these women. MAIN OUTCOME MEASURES--Pregnancy outcomes; mortality for all children born since 1980; and outcome for last born children. Serological concordance between mothers and last born children. RESULTS--Women with HIV-1 and HIV-2 infections had higher rates of spontaneous abortion and stillbirth than uninfected women (86/769 in HIV-1 positive women, 48/421 in HIV-2 positive, 31/234 in dually reactive, and 96/1131 in uninfected). Compared with children born to uninfected mothers (mortality 10.3%), greater proportions of children of HIV-1 positive (20.6%) and dually reactive (20.3%) mothers had died; mortality in children of HIV-2 infected women (13.1%) was not significantly increased. Infant mortalities for the last born children of HIV-1 positive, dually reactive, HIV-2 positive, and seronegative women were, respectively, 133, 82, 32, and 40 per 1000 live births. Nine of 77 last born children of HIV-1 positive mothers were concordantly seropositive compared with none of 21 children of HIV-2 infected mothers. CONCLUSIONS--Maternal HIV-2 infection has less influence on child survival than infection with HIV-1, probably because of a lower vertical transmission rate.     

Molecular Convergence of Neurodevelopmental Disorders

Neurodevelopmental disorders (NDDs) are caused by mutations in diverse genes involved in different cellular functions, although there can be crosstalk, or convergence, between molecular pathways affected by different NDDs. To assess molecular convergence, we generated human neural progenitor cell models of 9q34 deletion syndrome, caused by haploinsufficiency of EHMT1, and 18q21 deletion syndrome, caused by haploinsufficiency of TCF4. Using next-generation RNA sequencing, methylation sequencing, chromatin immunoprecipitation sequencing, and whole-genome miRNA analysis, we identified several levels of convergence. We found mRNA and miRNA expression patterns that were more characteristic of differentiating cells than of proliferating cells, and we identified CpG clusters that had similar methylation states in both models of reduced gene dosage. There was significant overlap of gene targets of TCF4 and EHMT1, whereby 8.3% of TCF4 gene targets and 4.2% of EHMT1 gene targets were identical. These data suggest that 18q21 and 9q34 deletion syndromes show significant molecular convergence but distinct expression and methylation profiles. Common intersection points might highlight the most salient features of disease and provide avenues for similar treatments for NDDs caused by different genetic mutations.