Sample size under inverse negative binomial group testing for accuracy in parameter estimation

Background

The group testing method has been proposed for the detection and estimation of genetically modified plants (adventitious presence of unwanted transgenic plants, AP). For binary response variables (presence or absence), group testing is efficient when the prevalence is low, so that estimation, detection, and sample size methods have been developed under the binomial model. However, when the event is rare (low prevalence <0.1), and testing occurs sequentially, inverse (negative) binomial pooled sampling may be preferred.

Methodology/Principal Findings

This research proposes three sample size procedures (two computational and one analytic) for estimating prevalence using group testing under inverse (negative) binomial sampling. These methods provide the required number of positive pools (), given a pool size (k), for estimating the proportion of AP plants using the Dorfman model and inverse (negative) binomial sampling. We give real and simulated examples to show how to apply these methods and the proposed sample-size formula. The Monte Carlo method was used to study the coverage and level of assurance achieved by the proposed sample sizes. An R program to create other scenarios is given in Appendix S2.

Conclusions

The three methods ensure precision in the estimated proportion of AP because they guarantee that the width (W) of the confidence interval (CI) will be equal to, or narrower than, the desired width (), with a probability of . With the Monte Carlo study we found that the computational Wald procedure (method 2) produces the more precise sample size (with coverage and assurance levels very close to nominal values) and that the samples size based on the Clopper-Pearson CI (method 1) is conservative (overestimates the sample size); the analytic Wald sample size method we developed (method 3) sometimes underestimated the optimum number of pools.     

A transient,positive effect of habitat fragmentation on insect population densities

We conducted an experimental landscape study to test the hypotheses that: (1) habitat removal results in short-term increases in population density in the remaining habitat patches (the crowding effect); (2) following habitat removal, density is higher in landscapes with more, smaller patches and more habitat edge (i.e., a higher level of habitat fragmentation per se) than in less fragmented landscapes, for the same total amount of habitat on the landscapes; (3) this positive effect of fragmentation per se on density is larger in landscapes with smaller inter-patch distances; and (4) these last two effects should be reduced or disappear over time following habitat removal. Our results did not support the first hypothesis, but they provided some support for the other three hypotheses, for two of the four Coccinellid species studied. As in other empirical studies of fragmentation per se on population density, the effects of fragmentation per se were weak and positive (when they did occur). This is the first study to document a transient effect of fragmentation per se on population density, and to show that this effect depends on inter-patch distances. We suggest that fragmentation per se increased the rate of immigration to patches, resulting in higher population densities in more fragmented landscapes.     

Phylogeography and population genetics of the endangered Amazonian manatee, Trichechus inunguis Natterer, 1883 (Mammalia, Sirenia)

We used mitochondrial DNA control region sequences to examine phylogeography and population differentiation of the endangered Amazonian manatee Trichechus inunguis. We observe lack of molecular differentiation among localities and we find weak association between geographical and genetic distances. However, nested clade analysis supports restricted gene flow and/or dispersal with some long-distance dispersal. Although this species has a history of extensive hunting, genetic diversity and effective population sizes are relatively high when compared to the West Indian manatee Trichechus manatus. Patterns of mtDNA haplotype diversity in T. inunguis suggest a genetic disequilibrium most likely explained by demographic expansion resulting from secession of hunting and enforcement of conservation and protective measures. Phylogenetic analysis of T. manatus and T. inunguis haplotypes suggests that T. inunguis is nested within T. manatus, effectively making T. manatus a paraphyletic entity. Paraphyly of T. manatus and recent divergence times of T. inunguis and the three main T. manatus lineages suggest a possible need for a taxonomic re-evaluation of the western Atlantic Trichechus.     

The Pneumocystis carinii drug target S-adenosyl-L-methionine:sterol C-24 methyl transferase has a unique substrate preference

Pneumocystis is an opportunistic pathogen that can cause pneumonitis in immunodeficient people such as AIDS patients. Pneumocystis remains difficult to study in the absence of culture methods for luxuriant growth. Recombinant protein technology now makes it possible to avoid some major obstacles. The P. carinii expressed sequence tag (EST) database contains 11 entries of a sequence encoding a protein homologous to S-adenosyl-L-methionine (SAM):C-24 sterol methyl transferase (SMT), suggesting high activity of this enzyme in the organism. We sequenced the erg6 cDNA, identified the putative peptide motifs for the sterol and SAM binding sites in the deduced amino acid sequence and expressed the protein in Escherichia coli. Unlike SAM:SMT from other organisms, the P. carinii enzyme had higher affinities for lanosterol and 24-methylenelanosterol than for zymosterol, the preferred substrate in other fungi. Cycloartenol was not a productive substrate. With lanosterol and 24-methylenelanosterol as substrates, the major reaction products were 24-methylenelanosterol and pneumocysterol respectively. Thus, the P. carinii SAM:SMT catalysed the transfer of both the first and the second methyl groups to the sterol C-24 position, and the substrate preference was found to be a unique property of the P. carinii SAM:SMT. These observations, together with the absence of SAM:SMT among mammals, further support the identification of sterol C-24 alkylation reactions as excellent targets for the development of drugs specifically directed against this pathogen.     

Decline of neural tube defects cases after a folic acid campaign in Nuevo León,México

BACKGROUND: Nuevo León is a state in northeastern Mexico, near the border of Texas. Mean mortality rate from 1996-98 due to anencephaly cases was 0.6/1,000. In 1999 a surveillance program for the registry and prevention of neural tube defects (NTD) cases was initiated. METHODS: Cases were obtained from hospitals and OB-GYN clinics by immediate notification, death certificates, or fetal death registries. Only isolated cases of NTD were included. In August 1999 a folic acid campaign was initiated with the free distribution of the vitamin to low-income women with a recommendation to take a 5.0-mg pill once a week. Number of cases and rates from 1999 to 2001 were compared (chi(2) test). RESULTS: After 2 years there has been a significant reduction in the number of cases and rates. In 1999 there were 95 NTD cases and in the years 2000 and 2001 there were only 59 and 55 respectively (P < 0.001). NTD rate decreased from 1.04/1,000 in 1999 to 0.58/1,000 in 2001. Anencephaly and spina bifida rates decreased from 0.55/1,000 to 0.29/1,000 and from 0.47/1,000 to 0.22/1,000 respectively, from 1999-2001. Decrease of female cases was higher than male cases for both phenotypes. CONCLUSION: After 2 years there was a 50% decrease in the incidence of anencephaly and spina bifida cases with a significant reduction of infant mortality and disability. These results encourage us to propose the use of a single tablet of 5.0-mg of folic acid per week as an alternative to supplementation on a daily basis.     

Integrated approach to produce a recombinant, His-tagged human α-galactosidase A in mammalian cells

Successful production of recombinant proteins (r-proteins) by transient gene expression (TGE) depends on several parameters (including producer cells, culture conditions, transfection procedure, or expression vector) that should be optimized when producing any recombinant product. In this work, TGE-based production of human α-galactosidase A (GLA) is described. Producer cells, expression vectors, and parameters influencing cell metabolism after transfection have been tested. The enzyme is secreted, has the right molecular weight, and is enzymatically active. Productivities of up to 30-40 mg/L have been achieved, with a simple, fast procedure. A 6 × His tag allows enzyme purification in a single step, rendering a highly pure product. We propose a TGE-based protocol able to produce up to several milligrams per liter of highly pure, active GLA in a time as short as a few days. By this, enough amounts of engineered versions of the enzyme can be easily produced to be tested in vitro or in preclinical trials.     

In vitro rhizogenesis: histoanatomy of Cedrela odorata (Meliaceae) microcuttings

Cedrela odorata (Meliaceae) is considered as one of the most valuable forest tree in the tropics. Clonal propagation of this species provide an alternative method to propagate superior genotypes, being the production of good quality adventitious roots one of the most important steps in micropropagation techniques. The sequence of anatomical changes that takes place during the formation of adventitious roots in shoots of Cedrela odorata cultured in vitro is described in this study. Eigth-week-old shoots, from multiplication cultures, were rooted in Murashige and Skoog's medium (1962) with half-strength macronutrients and with 0 or 1 mg/l indole-3-butyric acid (IBA). Between 12 and 24h after the start of rooting, some cambium, phloem and interfascicular parenchyma cells became dense cytoplasm, nuclei with prominent nucleoli and the first cell divisions were observed, especially in shoots treated with auxin (dedifferentiation phase). After 3-4 days, the number of dedifferentiated cells and mitotic divisions increased considerably, and the formation of groups of some 30-40 meristematic cells (meristemoids) was observed (induction phase). The first primordial roots developed from the 4th-5th day. The vascular tissues of these primordia connected to those of the explant, and roots began to emerge from the base by day 6. Development of the primordial roots was similar in the control shoots and shoots treated with 1 mg/l IBA, although there were more roots per explant in the latter.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse