Microevolutionary analysis of the nematode genus Pristionchus suggests a recent evolution of redundant developmental mechanisms during vulva formation

SUMMARY To identify the mechanisms by which molecular variation is introduced into developmental systems, microevolutionary approaches to evolutionary developmental biology have to be taken. Here, we describe the molecular and developmental characterization of laboratory strains of the nematode genus Pristionchus , which lays a foundation for a microevolutionary analysis of vulva development. We describe 13 laboratory strains of the Pristionchus genus that are derived from natural isolates from around the world. Mating experiments and ITS sequence analysis indicated that these 13 strains represent four different species: the gonochoristic species P. lheritieri and three hermaphroditic species, P. pacificus , P. maupasi , and an as yet undescribed species Pristionchus sp., respectively. P. pacificus is represented by five different strains isolated from California, Washington, Hawaii, Ontario, and Poland. Developmental differences during vulva formation are observed between strains from different species but also between strains of P. pacificus , like the strains from California and Poland. In particular, redundant developmental mechanisms present during vulva formation in P. pacificus var. California are absent in other strains. Amplified restriction fragment length polymorphism (AFLP) analyses of the P. pacificus strains revealed that the American strains are highly polymorphic. In contrast, the developmentally distinct strain from Poland is identical to the Californian strain, suggesting that the developmental differences rely on a small number of changes in developmental control genes rather than the accumulation of changes at multiple loci.     

Membrane-bound neuregulin1 type III actively promotes Schwann cell differentiation of multipotent Progenitor cells

Many steps of peripheral glia development appear to be regulated by neuregulin1 (NRG1) signaling but the exact roles of the different NRG1 isoforms in these processes remain to be determined. While glial growth factor 2 (GGF2), a NRG1 type II isoform, is able to induce a satellite glial fate in neural crest stem cells, targeted mutations in mice have revealed a prominent role of NRG1 type III isoforms in supporting survival of Schwann cells at early developmental stages. Here, we investigated the role of NRG1 isoforms in the differentiation of Schwann cells from neural crest-derived progenitor cells. In multipotent cells isolated from dorsal root ganglia, soluble NRG1 isoforms do not promote Schwann cell features, whereas signaling by membrane-associated NRG1 type III induces the expression of the Schwann cell markers Oct-6/SCIP and S100 in neighboring cells, independent of survival. Thus, axon-bound NRG1 might actively promote both Schwann cell survival and differentiation.     

Confidence measures for protein fold recognition

MOTIVATION: We present an extensive evaluation of different methods and criteria to detect remote homologs of a given protein sequence. We investigate two associated problems: first, to develop a sensitive searching method to identify possible candidates and, second, to assign a confidence to the putative candidates in order to select the best one. For searching methods where the score distributions are known, p-values are used as confidence measure with great success. For the cases where such theoretical backing is absent, we propose empirical approximations to p-values for searching procedures. RESULTS: As a baseline, we review the performances of different methods for detecting remote protein folds (sequence alignment and threading, with and without sequence profiles, global and local). The analysis is performed on a large representative set of protein structures. For fold recognition, we find that methods using sequence profiles generally perform better than methods using plain sequences, and that threading methods perform better than sequence alignment methods. In order to assess the quality of the predictions made, we establish and compare several confidence measures, including raw scores, z-scores, raw score gaps, z-score gaps, and different methods of p-value estimation. We work our way from the theoretically well backed local scores towards more explorative global and threading scores. The methods for assessing the statistical significance of predictions are compared using specificity--sensitivity plots. For local alignment techniques we find that p-value methods work best, albeit computationally cheaper methods such as those based on score gaps achieve similar performance. For global methods where no theory is available methods based on score gaps work best. By using the score gap functions as the measure of confidence we improve the more powerful fold recognition methods for which p-values are unavailable. AVAILABILITY: The benchmark set is available upon request.     

Robust dosage-PCR for detection of heterozygous chromosomal deletions

Robust dosage-PCR (RD-PCR) was developed to detect heterozygous large deletions, an important class of mutations missed by conventional PCR strategies. PCR-based methods are available for distinguishing between the dosage of one or two template copies, but general application is limited by the laborious nature of the method and/or the optimization required for each new set of gene exons to be analyzed. RD-PCR depends on a combination of (i) co-amplification of an autosomal and an X-chromosomal segment so that internal dosage controls are available for any segment to be analyzed and (ii) a robust primer design that includes a 5'tail and a 3'sequence-specific region in the PCR protocol. The ratio of yields (ROY) of the target to the internal control segment is directly proportional to the ratio of the two input templates over a wide range (at least 1:1 to 1:258 with a correlation coefficient of 0.99). The ROY is not dependent on the amount of genomic DNA or the number of cycles of amplification under typical conditions. RD-PCR eliminates errors in the preparation and manipulation steps by using an internal dosage control. A blinded analysis of gene dosage was performed to detect deletions of the human factor IX gene with 100% accuracy. Prospective analyses demonstrate that exons and flanking splice junctions can be analyzed for gene dosage with minimal optimization.     

Chromosomal aberrations and micronuclei in lymphocytes of breast cancer patients after an accident during radiotherapy with 8 MeV electrons

In February 2001 a radiation accident occurred in a radiotherapy unit of an oncology hospital in Poland. Five breast cancer patients undergoing radiotherapy received a single high dose of 8 MeV electrons. The exact doses are not known, but they were heterogeneous and may have reached about 100 Gy. To assess whether such exposure would be detectable in peripheral blood lymphocytes, chromosomal aberrations and micronuclei were analyzed in lymphocytes from the accident patients and compared to values for lymphocytes from 10 control patients who were not involved in the accident but who received similar radiotherapy treatments. Lymphocytes were harvested for analysis of chromosomal aberrations at three different culture times to determine whether heavily damaged cells reached mitosis with a delay. There was no effect of harvest time on the frequencies of chromosomal aberrations, indicating that there was no delay of heavily damaged cells in entering mitosis. A good correlation was observed between micronuclei and chromosomal aberrations. In lymphocytes from three of the accident patients, significantly enhanced frequencies of both aberrations and micronuclei were found. The great individual variability observed in the frequency of cytogenetic damage in lymphocytes from both control and accident patients precluded the unambiguous identification of all accident patients.     

Peripheral neuropathy and light-preliminary report indicating prevalence of nanobacteria in HIV

Peripheral neuropathy is a common condition in HIV-positive patients and is often experienced in diabetes mellitus. The primary mechanism of the disease, which can considerably aggravate the patient's state, is unknown. The perineurium of patients with peripheral neuropathy is frequently enveloped by apatite. Nanobacteria (NB) are protectd by a mineral shell consisting of apatite. Light has been shown to elevate the vitality level of cells, and was predicted to inhibit deposition of stressed NB in the cardiovascular system. Results indicate that light can durably restore the condition of patients with severe peripheral neuropathy.     

Sealing porous nanovesicles--solutions inspired by primordial biosystems

Microcapsules designed for slow drug release have preferably some porosity. There are, however, applications in which a hermetical sealing of the microcapsules is desired. Sealing is not a trivial problem and could be necessary to durably encapsulate toxic compounds which cannot be eliminated from the body, or to encapsulate harmful substances stored in the atmosphere. Nature may have one solution: Nanobacteria have developed surprisingly simple mechanisms to access and use primal energies, and to survive arid periods by sealing their surface.     

Changes in the physiological and agricultural characteristics of peat-based Bradyrhizobium japonicum inoculants after long-term storage

Commercial soybean inoculants processed with sterilised peat and stored at 20 °C for 1–8 years were used as experimental materials to assess the changes in the physiological activity of Bradyrhizobium japonicum after storage. Viable counts decreased and physiological characteristics of the bacterium changed during storage, with an increase in the time taken for colony appearance on a medium without yeast extract, an increase in the lag time for nodule appearance on soybean grown in glass tubes and a decrease in survival on seeds. All the inoculants produced a significant increase in grain yield in a field experiment. The percentage of efficient cells in the field (relative to the plate counts) decreased as the length of storage increased. These results suggest that the physiological activity of B. japonicum cells changes after storage. Practical implications for inoculant quality control are discussed. Received: 20 September 1999 / Received revision: 3 March 2000 / Accepted: 6 March 2000