Multiple testing in fetal gender determination from maternal blood by polymerase chain reaction

Fetal male DNA can be identified in maternal blood by polymerase chain reaction (PCR) amplification of Y-specific sequences. This technology has not reached a satisfactory accuracy and reproducibility in fetal gender determination because of the very low concentration of fetal cells. Our purpose was to evaluate the possibility of improving the reliability of this test by setting up a repeated amplification system. We amplified, by nested PCR of the Y-specific sequence DYS14, 137 DNA samples extracted from maternal peripheral blood (93 from male-bearing and 44 from female-bearing pregnancies ranging from the 6th to the 36th gestational week). Each maternal DNA sample was tested doubly, in two different PCR sessions, with a total of four amplifications. We obtained discordant results in the four amplifications in 82/137 (60%) samples. The best interpretation of these discordant results was obtained by applying a positivity cutoff of at least two positive amplifications for considering a DNA sample as belonging to a male-bearing pregnancy. We obtained a sensitivity of 83%, a specificity of 93%, a positive predictive value of 96% and a negative predictive value of 72% in fetal male gender diagnosis. By applying this quadruple testing system, we significantly improved PCR accuracy and predictive values compared with single and double testing of the same samples. We conclude that, for future investigations of fetal DNA retrieved from maternal blood, the application of a quadruple testing system is better than the single PCR test. Received: 18 August 1997 / Accepted: 12 January 1998     

Molecular Evolution of the Pathogenicity Island of Enterotoxigenic Bacteroides fragilis Strains

Enterotoxigenic Bacteroides fragilis (ETBF) strains, which produce a 20-kDa zinc metalloprotease toxin (BFT), have been associated with diarrheal disease in animals and young children. Studying a collection of ETBF and nontoxigenic B. fragilis (NTBF) strains, we found that bft and a second metalloprotease gene (mpII) are contained in an approximately 6-kb pathogenicity island (termed B. fragilis pathogenicity island or BfPAI) which is present exclusively in all 113 ETBF strains tested (pattern I). Of 191 NTBF strains, 100 (52%) lack both the BfPAI and at least a 12-kb region flanking BfPAI (pattern II), and 82 of 191 NTBF strains (43%) lack the BfPAI but contain the flanking region (pattern III). The nucleotide sequence flanking the left end of the BfPAI revealed a region with the same organization as the mobilization region of the 5-nitroimidazole resistance plasmid pIP417 and the clindamycin resistance plasmid pBFTM10, that is, two mobilization genes (bfmA and bfmB) organized in one operon and a putative origin of transfer (oriT) located in a small, compact region. The region flanking the right end of the BfPAI contains a gene (bfmC) whose predicted protein shares significant identity to the TraD mobilization proteins encoded by plasmids F and R100 from Escherichia coli. Nucleotide sequence analysis of one NTBF pattern III strain (strain I-1345) revealed that bfmB and bfmC are adjacent to each other and separated by a 16-bp GC-rich sequence. Comparison of this sequence with the appropriate sequence of ETBF strain 86-5443-2-2 showed that in this ETBF strain the 16-bp sequence is replaced by the BfPAI. This result defined the BfPAI as being 6,036 bp in length and its precise integration site as being between the bfmB and bfmC stop codons. The G+C content of the BfPAI (35%) and the flanking DNA (47 to 50%) differ greatly from that reported for the B. fragilis chromosome (42%), suggesting that the BfPAI and its flanking region are two distinct genetic elements originating from very different organisms. ETBF strains may have evolved by horizontal transfer of these two genetic elements into a pattern II NTBF strain.     

Biophysical properties and functional significance of stem water storage tissues in Neotropical savanna trees

Biophysical characteristics of sapwood and outer parenchyma water storage compartments were studied in stems of eight dominant Brazilian Cerrado tree species to assess the impact of differences in tissue capacitance on whole-plant water relations. The rate of decline in tissue water potential with relative water content (RWC) was greater in the outer parenchyma than in the sapwood for most of the species, resulting in tissue-and species-specific differences in capacitance. Sapwood capacitance on a tissue volume basis ranged from 40 to 160 kg m-3 MPa-1, whereas outer parenchyma capacitance ranged from 25 to only 60 kg m-3 MPa-1. In addition, osmotic potentials at full turgor and at the turgor loss point were more negative for the outer parenchyma compared with the sapwood, and the maximum bulk elastic modulus was higher for the outer parenchyma than for the sapwood. Sapwood capacitance decreased linearly with increasing sapwood density across species, but there was no significant correlation between outer parenchyma capacitance and tissue density. Midday leaf water potential, the total hydraulic conductance of the soil/leaf pathway and stomatal conductance to water vapour (gs) all increased with stem volumetric capacitance, or with the relative contribution of stored water to total daily transpiration. However, the difference between the pre-dawn water potential of non-transpiring leaves and the weighted average soil water potential, a measure of the water potential disequilibrium between the plant and soil, increased asymptotically with total stem capacitance across species, implying that overnight recharge of water storage compartments was incomplete in species with greater capacitance. Overall, stem capacitance contributes to homeostasis in the diurnal and seasonal water balance of Cerrado trees.     

Flow through a Collapsible Tube: Experimental Analysis and Mathematical Model

Flow through thin-wall axisymmetric tubes has long been of interest to physiologists. Analysis is complicated by the fact that such tubes will collapse when the transmural pressure (internal minus external pressure) is near zero. Because of the absence of any body of related knowledge in other sciences or engineering, previous workers have directed their efforts towards experimental studies of flow in collapsible tubes. More recently, some attention has been given towards analytical studies. Results of an extensive series of experiments show that the significant system parameter is transmural pressure. The cross-sectional area of the tube depends upon the transmural pressure, and changes in cross-section in turn affect the flow geometry. Based on experimental studies, a lumped parameter system model is proposed for the collapsible tube. The mathematical model is simulated on a hybrid computer. Experimental data were used to define the functional relationship between cross-sectional area and transmural pressure as well as the relation between the energy loss coefficient and cross-sectional area. Computer results confirm the validity of the model for both steady and transient flow conditions.     

Larvicidal potential of cell wall degrading enzymes from Trichoderma asperellum against Aedes aegypti (Diptera: Culicidae)

Aedes aegypti is a mosquito vector of arboviruses such as dengue, chikungunya, zika and yellow fever that cause important public health diseases. The incidence and gravity of these diseases justifies the search for effective measures to reduce the presence of this vector in the environment. Bioinsecticides are an effective alternative method for insect control, with added ecological benefits such as biodegradability. The current study demonstrates that a chitinolytic enzyme complex produced by the fungus Trichoderma asperellum can disrupt cuticle formation in the L3 larvae phase of A. aegypti, suggesting such biolarvicidal action could be used for mosquito control. T. asperellum was exposed to chitin from different sources. This induction of cell wall degrading enzymes, including chitinase, N-acetylglucosaminidase and β-1,3-glucanase. Groups of 20 L3 larvae of A. aegypti were exposed to varying concentrations of chitinolytic enzymes induced with commercial chitin (CWDE) and larvae cell wall degrading enzymes (L-CWDE). After 72 h of exposure to the CWDE, 100% of larvae were killed. The same percent mortality was observed after 48 h of exposure to L-CWDE at half the CWDE enzyme mixture concentration. Exoskeleton deterioration was further observed by scanning and electron microscopy. Our findings indicate that L-CWDE produced by T. asperellum reflect chitinolytic enzymes with greater specificity for L3 larval biomolecules. This specificity is characterized by the high percentage of mortality compared with CWDE treatments and also by abrupt changes in patterns of the cellular structures visualized by scanning and transmission electron microscopy. These mixtures of chitinolytic enzymes could be candidates, as adjuvant or synergistic molecules, to replace conventional chemical insecticides currently in use.     

Culture of mouse peritoneal macrophages with mouse serum induces lipid bodies that associate with the parasitophorous vacuole and decrease their microbicidal capacity against Toxoplasma gondii

Lipid bodies [lipid droplets (LBs)] are lipid-rich organelles involved in lipid metabolism, signalling and inflammation. Recent findings suggest a role for LBs in host response to infection; however, the potential functions of this organelle in Toxoplasma gondii infection and how it alters macrophage microbicidal capacity during infection are not well understood. Here, we investigated the role of host LBs in T. gondii infection in mouse peritoneal macrophages in vitro. Macrophages cultured with mouse serum (MS) had higher numbers of LBs than those cultured in foetal bovine serum and can function as a model to study the role of LBs during intracellular pathogen infection. LBs were found in association with the parasitophorous vacuole, suggesting that T. gondii may benefit from this lipid source. Moreover, increased numbers of macrophage LBs correlated with high prostaglandin E2 (PGE2) production and decreased nitric oxide (NO) synthesis. Accordingly, LB-enriched macrophages cultured with MS were less efficient at controlling T. gondii growth. Treatment of macrophages cultured with MS with indomethacin, an inhibitor of PGE2 production, increased the microbicidal capacity against T. gondii. Collectively, these results suggest that culture with MS caused a decrease in microbicidal activity of macrophages against T. gondii by increasing PGE2 while lowering NO production.     

Hydroxylated metabolites of the antimalarial drug primaquine. Oxidation and redox cycling.

Oxidation and redox cycling of the hydroxylated metabolites of the antimalarial drug primaquine (i.e. 5-hydroxyprimaquine, 5-hydroxydemethylprimaquine, and 5,6-dihydroxy-8-aminoquinoline) were studied. The three metabolites readily oxidized under physiological conditions, forming hydrogen peroxide and the corresponding quinone-imine derivatives as the main products. The latter compounds were characterized by visible, NMR, and infrared spectroscopy. Concomitant formation of drug-derived radicals and hydroxyl radicals was attested by direct and spin-trapping EPR experiments, respectively. The use of the spin stabilization method indicated that the radicals derived from 5-hydroxydemethylprimaquine and 5,6-dihydroxy-8-aminoquinoline are of the o-semiquinone type. Tentative structures are proposed for the radicals based on product identification and computer simulation of the experimental EPR spectra. The quinone-imines obtained from the reduced metabolites did not react at appreciable rates with NADPH but underwent redox cycling upon addition of ferredoxin:NADP+ oxidoreductase, forming hydrogen peroxide and hydroxyl radicals. The effect of antioxidant enzymes on hydroxyl radical yield obtained during oxidation and redox cycling indicates that the main route for hydroxyl radical formation is the metal ion-catalyzed reaction between the drug-derived radicals and hydrogen peroxide. Taken together, the results indicate that hydrogen peroxide is the potential toxic product formed from the primaquine metabolites.     

Quantitative analysis of race-specific resistance to Colletotrichum lindemuthianum in common bean

Molecular genetic maps continue to play a major role in breeding of crop species. The common bean genetic map of the recombinant inbred line population IAC-UNA × CAL 143 (UC) has been used to detect loci controlling important agronomic traits in common bean. In the current study, new microsatellite markers were added to the UC map and the linkage analysis was refined using current genomic resources of common bean, in order to identify quantitative resistance loci (QRL) associated with different races of the anthracnose pathogen. A single race inoculation was conducted in greenhouse using four plants per plot. Both race-specific and joint-adjusted disease severity means, obtained from linear-mixed model, were used to perform multiple interval mapping (MIM) and multi-trait MIM (MTMIM). In total, 13 and 11 QRL were identified by MIM and MTMIM analyses, respectively; with nine being observed in both analyses. ANT02.1^UC and ANT07.1^UC showed major effects on resistance both for MIM and MTMIM. Common major QRL for resistance to the three anthracnose races were expected, since high genetic pairwise-correlation was observed between the race-specific and joint-adjusted disease severity means. Therewith, both ANT02.1 and ANT07.1 can be regarded as valuable targets for marker-assisted selection; and so, putative genes potentially involved in the resistance response were identified in these QRL regions. Minor effect QRL were also observed, showing differential affects either on race-specific or multi-trait analyses and may play a role on durable horizontal resistance. These results contribute to a better understanding of the host-pathogen interaction and to breeding for enhancing resistance to Colletotrichum lindemuthianum in common bean.     

Genetic diversity and structure of natural populations of Gossypium mustelinum,a wild relative of cotton,in the basin of the De Contas River in Bahia,Brazil

Gossypium mustelinum is a wild cotton relative found only in the semiarid region of Bahia state in Brazil, and changes caused by humans in the natural habitat of this species have endangered the existence of several natural populations. Information about the occurrence and genetic composition of these populations is necessary to design effective conservation measures. The aim of this study was to characterize the in situ maintenance mode and assess the genetic diversity of G. mustelinum populations in the basin of the De Contas River. A sample of 205 G. mustelinum specimens was collected from the margins of the Jacaré, Riacho Quixaba, Riacho Serra Azul, and Riacho Riachão rivers and genotyped using 13 SSR primer pairs. In general, all G. mustelinum populations exhibit inadequate in situ maintenance, predominantly due to the deforestation of riparian vegetation and herbivory. The observed total genetic diversity of G. mustelinum was significant (H _E = 0.489), highly structured (F _ST = 0.534), and organized in homozygous genotypes (F _IS = 0.873). The high observed inbreeding level is consistent with the predominance of self-fertilization and geitonogamy (t _m = 0.234). In addition, the pattern of genetic structure tended to form groups that coincided with the collection sites, i.e., first clustering within subpopulations, then within populations, and finally within the closest populations. Thus, the observed genetic diversity is likely to be rapidly lost, and conservation measures should therefore be undertaken.