Alternative Model Organism Research Articles

Use of alternative assays to identify and prioritize organophosphorus flame retardants for potential developmental and neurotoxicity

Due to their toxicity and persistence in the environment, brominated flame retardants (BFRs) are being phased out of commercial use, leading to the increased use of alternative chemicals such as the organophosphorus flame retardants (OPFRs). There is, however, limited information on the potential health effects of OPFRs. Due to the structural similarity of the OPFRs to organophosphorus insecticides, there is concern regarding developmental toxicity and neurotoxicity. In response, we evaluated a set of OPFRs (triphenyl phosphate [TPHP]), isopropylated phenyl phosphate [IPP], 2-ethylhexyl diphenyl phosphate [EHDP], tert-butylated phenyl diphenyl phosphate [BPDP], trimethyl phenyl phosphate [TMPP], isodecyl diphenyl phosphate [IDDP], (tris(1,3-dichloroisopropyl) phosphate [TDCIPP], and tris(2-chloroethyl)phosphate [TCEP]) in a battery of cell-based in vitro assays and alternative model organisms and compared the results to those obtained for two classical BFRs (3,3′,5,5′-tetrabromobisphenol A [TBBPA] and 2,2′4,4′-brominated diphenyl ether [BDE-47]). The assays used evaluated the effects of chemicals on the differentiation of mouse embryonic stem cells, the proliferation and growth of human neural stem cells, rat neuronal growth and network activity, and development of nematode (Caenorhabditis elegans) and zebrafish (Danio rerio). All assays were performed in a concentration-response format, allowing for the determination of the point of departure (POD: the lowest concentration where a chemically-induced response exceeds background noise). The majority of OPFRs (8/9) were active in multiple assays in the range of 1–10μM, most of which had comparable activity to the BFRs TBBPA and BDE-47. TCEP was negative in all assays. The results indicate that the replacement OPFRs, with the exception of TCEP, showed comparable activity to the two BFRs in the assays tested. Based on these results, more comprehensive studies are warranted to further characterize the potential hazard of some of these OPFR compounds.

Performance of pairwise shape dissimilarity morphometrics on nonmammalian taxa (Insecta: Neuroptera: Mantispidae).

Morphometric dissimilarity metrics aim to quantify the variation between compared specimens such that inferences about their relatedness and alpha taxonomy can be made. Recently, the technique has developed metrics that purport to quantify shape dissimilarity between specimens-employing the use of least squares regression analysis. These metrics have been well applied by studies in the hominin fossil record with an arguably unsubstantiated backing for the technique. Originally postulated was the log10 sem metric which subsequently led to the standard error test of the null hypothesis metric. Following this, the standard deviation of logged ratios (SLR ) metric arose as a pairwise dissimilarity metric that constrains the regression to a zero-intercept, that is, a significant development in the robustness of the technique. This metric was tested on extant primates in order to evaluate its effectiveness alongside the two other metrics. It was shown to be the most reliable for comparisons between specimens of primates, but was unable to discriminate between heterospecific and conspecific comparisons. Arguably, an alternative model organism with which to compare the technique is lacking. This study considers shape dissimilarity metrics with respect to a group of nonmammalian organisms (mantidflies) and tests the metrics against three lines of evidence (morphology, CO1-DNA, and geographic distribution) that can delimit the species-level taxonomy for the group. It is shown that the metrics are unable to discriminate between pairwise comparisons of closely related species, resulting in biologically erroneous groupings, and contradicting the groupings derived from morphological, CO1-DNA, and distributional comparisons. It is thus asserted that the technique is unsuitable for use in alpha taxonomy as an additional line of evidence in mantidflies. It is further supposed that morphometrics in general should be employed with caution in studies of evolutionary history as phylogeny is not the only information contained within morphometric data.

The Evolutionary History of MAPL (Mitochondria-Associated Protein Ligase) and Other Eukaryotic BAM/GIDE Domain Proteins.

MAPL (mitochondria-associated protein ligase, also called MULAN/GIDE/MUL1) is a multifunctional mitochondrial outer membrane protein found in human cells that contains a unique BAM (beside a membrane) domain and a C-terminal RING-finger domain. MAPL has been implicated in several processes that occur in animal cells such as NF-kB activation, innate immunity and antiviral signaling, suppression of PINK1/parkin defects, mitophagy in skeletal muscle, and caspase-dependent apoptosis. Previous studies demonstrated that the BAM domain is present in diverse organisms in which most of these processes do not occur, including plants, archaea, and bacteria. Thus the conserved function of MAPL and its BAM domain remains an open question. In order to gain insight into its conserved function, we investigated the evolutionary origins of MAPL by searching for homologues in predicted proteomes of diverse eukaryotes. We show that MAPL proteins with a conserved BAM-RING architecture are present in most animals, protists closely related to animals, a single species of fungus, and several multicellular plants and related green algae. Phylogenetic analysis demonstrated that eukaryotic MAPL proteins originate from a common ancestor and not from independent horizontal gene transfers from bacteria. We also determined that two independent duplications of MAPL occurred, one at the base of multicellular plants and another at the base of vertebrates. Although no other eukaryote genome examined contained a verifiable MAPL orthologue, BAM domain-containing proteins were identified in the protists Bigelowiella natans and Ectocarpus siliculosis. Phylogenetic analyses demonstrated that these proteins are more closely related to prokaryotic BAM proteins and therefore likely arose from independent horizontal gene transfers from bacteria. We conclude that MAPL proteins with BAM-RING architectures have been present in the holozoan and viridiplantae lineages since their very beginnings. Our work paves the way for future studies into MAPL function in alternative model organisms like Capsaspora owczarzaki and Chlamydomonas reinhardtii that will help to answer the question of MAPL’s ancestral function in ways that cannot be answered by studying animal cells alone.

The molecular and structural characterization of two vitellogenins from the free-living nematode Oscheius tipulae.

This paper describes the purification of yolk proteins, which are important for the reproduction of egg-laying animals, and the structural characterization of two vitellogenins, VT1 and OTI-VIT-6, of the nematode Oscheius tipulae. O. tipulae is an alternative model organism to its relative, the widely used Caenorhabditis elegans, and is a good model to understand reproduction in insect parasitic nematodes of the genus Heterorhabditis. The native purified O. tipulae vitellogenin is composed of three polypeptides (VT1, VT2 and VT3), whereas in C. elegans, vitellogenin is composed of four polypeptides. The gene (Oti-vit-1) encoding yolk polypeptide VT1 has been recently identified in the genome of O. tipulae. Immunoblotting and N-terminal sequencing confirmed that VT1 is indeed coded by Oti-vit-1. Utilizing the same experimental approaches, we showed that the polypeptides VT2 and VT3 are derived from the proteolytic processing of the C- and N-terminal portions of the precursor OTI-VIT-6, respectively. We also showed that the recombinant polypeptide (P40), corresponding to the N-terminal sequence of OTI-VIT-6, preferentially interacts with a 100-kDa polypeptide found in adult worm extracts, as we have previously shown for the native vitellins of O. tipulae. Using the putative nematode vitellogenin amino acid sequences available in the UniProtKB database, we constructed a phylogenetic tree and showed that the O. tipulae vitellogenins characterized in this study are orthologous to those of the Caenorhabditis spp. Together, these results represent the first structural and functional comparative study of nematode yolk proteins outside the Caenorhabditis genus and provide insight into the evolution of these lipoproteins within the Nematode Phylum.

D469del-COMP Retention in Chondrocytes Stimulates Caspase-Independent Necroptosis

Mutations in the cartilage oligomeric matrix protein gene (COMP) cause pseudoachondroplasia (PSACH). This dysplasia results from the intracellular retention of mutant COMP protein and premature death of growth-plate chondrocytes. Toward better understanding of these underlying mechanisms, we examined D469del-COMP activation of the unfolded protein response and cell death pathways in rat chondrosarcoma cells. Using an inducible expression system, we examined the effects of D469del-COMP retention after 4 days of mRNA expression and then 5 days without inducing agent. Retention of D469del-COMP stimulated Chop (Ddit3) and Gadd34 (Ppp1r15a) and triggered reactivation of protein translation that exacerbated intracellular retention. High levels of Nox4 and endoplasmic reticulum receptor stress-inducible Ero1β generated reactive oxygen species, causing oxidative stress. Increased expression of Gadd genes and presence of γH2AX indicated that DNA damage was occurring. The presence of cleaved apoptosis inducing factor (tAIF) and the absence of activated caspases indicated that retention of D469del-COMP triggers cell death in chondrocytes by necroptosis, a caspase-independent programmed necrosis. Loss of growth-plate chondrocytes by necroptosis was also found in our pseudoachondroplasia mouse model. These results suggest a model in which D469del-COMP expression induces persistent endoplasmic reticulum stress, oxidative stress, and DNA damage, thus priming chondrocytes for necroptosis. We define for the first time the precise mechanisms underlying D469del-COMP pathology in pseudoachondroplasia and suggest that oxidative stress and AIF may be promising therapeutic targets.

Implementation and enforcement of the 3Rs principle in the field of transgenic animals used for scientific purposes

In 2007, 2.7 million vertebrates were used for animal experiments and other scientific purposes in Germany alone. Since 1998 there has been an increase in the number of animals used for research purposes, which is partly attributable to the growing use of transgenic animals (Fig. 1). These animals are, for instance, used as in vivo models to mimic human diseases like diabetes, cancer or Alzheimer's disease. Here, transgenic model organisms serve as valuable tools, being instrumental in facilitating the analysis of the molecular mechanisms underlying human diseases, and might contribute to the development of novel therapeutic approaches. Due to variable and, sometimes low, efficiency (depending on the species used), however, the generation of such animals often requires a large number of embryo donors and recipients. The experts evaluated methods that could possibly be utilised to reduce, refine or even replace experiments with transgenic vertebrates in the mid-term future. Among the promising alternative model organisms available at the moment are the fruit fly Drosophila melanogaster and the roundworm Caenorhabditis elegans. Specific cell culture experiments or threedimensional (3D) tissue models also offer valuable opportunities to replace experiments with transgenic animals or reduce the number of laboratory animals required by assisting in decision-making processes. Furthermore, at the workshop an in vitro technique was presented which permits the production of complete human antibodies without using genetically modified (“humanised”) animals. Up to now, genetically modified mice are widely used for this purpose. Improved breeding protocols, enhanced efficiency of mutagenesis as well as training of laboratory personnel and animal keepers can also help to reduce the numbers of laboratory animals. Well-trained staff in particular can help to minimise the pain, suffering and discomfort of animals and, at the same time, improve the quality of data obtained from animal experiments. This, in turn, can lead to a reduction in the numbers of animals needed for each experiment. The experts also came to the conclusion that the numbers of laboratory animals can be reduced by open access to a central database that provides detailed documentation of completed experiments involving transgenic animals. This documentation should not be restricted to experiments with substantial scientific results that warrant publication, but should also include those with “negative” outcome, which are usually not published. Capturing all kinds of results within such a database provides added value to the respective scientists and the scientific community as a whole; it could also help to stimulate collaborations and to ensure funding for future research. An important aspect to be considered in the generation of this kind of database is the quality and standardisation of the information provided on existing in vitro models and the respective opportunities for their use.The experts felt that the greatest potential for reducing the numbers of laboratory animals in the near future realistically might not be offered by the complete replacement of transgenic animal models but by opportunities to examine specific questions to a greater degree using in vitro models, such as cell and tissue cultures including organotypic models. The use of these models would considerably reduce the number of in vivo experiments using transgenic animals. However, the overall number of experimental animals may still be increasing or remain unaffected, e.g. when transgenic animals continue to serve as the source of primary cells and organs/tissues for in vitro experiments.

Application of a Mathematical Model to Describe the Effects of Chlorpyrifos on Caenorhabditis elegans Development

BackgroundThe nematode Caenorhabditis elegans is being assessed as an alternative model organism as part of an interagency effort to develop better means to test potentially toxic substances. As part of this effort, assays that use the COPAS Biosort flow sorting technology to record optical measurements (time of flight (TOF) and extinction (EXT)) of individual nematodes under various chemical exposure conditions are being developed. A mathematical model has been created that uses Biosort data to quantitatively and qualitatively describe C. elegans growth, and link changes in growth rates to biological events. Chlorpyrifos, an organophosphate pesticide known to cause developmental delays and malformations in mammals, was used as a model toxicant to test the applicability of the growth model for in vivo toxicological testing.Methodology/Principal FindingsL1 larval nematodes were exposed to a range of sub-lethal chlorpyrifos concentrations (0–75 µM) and measured every 12 h. In the absence of toxicant, C. elegans matured from L1s to gravid adults by 60 h. A mathematical model was used to estimate nematode size distributions at various times. Mathematical modeling of the distributions allowed the number of measured nematodes and log(EXT) and log(TOF) growth rates to be estimated. The model revealed three distinct growth phases. The points at which estimated growth rates changed (change points) were constant across the ten chlorpyrifos concentrations. Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.ConclusionsEffects of chlorpyrifos on C. elegans growth and development were mathematically modeled. Statistical tests confirmed a significant concentration effect on several model endpoints. This confirmed that chlorpyrifos affects C. elegans development in a concentration dependent manner. The most noticeable effect on growth occurred during early larval stages: L2 and L3. This study supports the utility of the C. elegans growth assay and mathematical modeling in determining the effects of potentially toxic substances in an alternative model organism using high-throughput technologies.

Production of Germline Transgenic Prairie Voles (Microtus ochrogaster) Using Lentiviral Vectors1

The study of alternative model organisms has yielded tremendous insights into the regulation of behavioral and physiological traits not displayed by more widely used animal models, such as laboratory rats and mice. In particular, comparative approaches often exploit species ideally suited for investigating specific phenomenon. For instance, comparative studies of socially monogamous prairie voles and polygamous meadow voles have been instrumental toward gaining an understanding of the genetic and neurobiological basis of social bonding. However, laboratory studies of less commonly used organisms, such as prairie voles, have been limited by a lack of genetic tools, including the ability to manipulate the genome. Here, we show that lentiviral vector-mediated transgenesis is a rapid and efficient approach for creating germline transgenics in alternative laboratory rodents. Injection of a green fluorescent protein (GFP)-expressing lentiviral vector into the perivitelline space of 23 single-cell embryos yielded three live offspring (13 %), one of which (33%) contained germline integration of a GFP transgene driven by the human ubiquitin-C promoter. In comparison, transfer of 23 uninjected embryos yielded six live offspring (26%). Green fluorescent protein is present in all tissues examined and is expressed widely in the brain. The GFP transgene is heritable and stably expressed until at least the F(2) generation. This technology has the potential to allow investigation of specific gene candidates in prairie voles and provides a general protocol to pursue germline transgenic manipulation in many different rodent species.

Evolution: Biology's next top model?

From Antarctic icefish to Galapagos finches, there are some interesting characters at the fringes of developmental biology. Brendan Maher explores a world of alternative model organisms.

Oscheius tipulae as an Example of eEF1A Gene Diversity in Nematodes

We characterized four eEF1A genes in the alternative rhabditid nematode model organism Oscheius tipulae. This is twice the copy number of eEF1A genes in C. elegans, C. briggsae, and, probably, many other free-living and parasitic nematodes. The introns show features remarkably different from those of other metazoan eEF1A genes. Most of the introns in the eEF1A genes are specific to O. tipulae and are not shared with any of the other genes described in metazoans. Most of the introns are phase 0 (inserted between two codons), and few are inserted in protosplice sites (introns inserted between the nucleotide sequence A/CAG and G/A). Two of these phase 0 introns are conserved in sequence in two or more of the four eEF1A gene copies, and are inserted in the same position in the genes. Neither of these characteristics has been detected in any of the nematode eEF1A genes characterized to date. The coding sequences were also compared with other eEF1A cDNAs from 11 different nematodes to determine the variability of these genes within the phylum Nematoda. Parsimony and distance trees yielded similar topologies, which were similar to those created using other molecular markers. The presence of more than one copy of the eEF1A gene with nearly identical coding regions makes it difficult to define the orthologous cDNAs. As shown by our data on O. tipulae, careful and extensive examination of intron positions in the eEF1A gene across the phylum is necessary to define their potential for use as valid phylogenetic markers.

Ageing in a eusocial insect: molecular and physiological characteristics of life span plasticity in the honey bee

Commonly held views assume that ageing, or senescence, represents an inevitable, passive, and random decline in function that is strongly linked to chronological age. In recent years, genetic intervention of life span regulating pathways, for example, in Drosophila as well as case studies in non-classical animal models, have provided compelling evidence to challenge these views.Rather than comprehensively revisiting studies on the established genetic model systems of ageing, we here focus on an alternative model organism with a wild type (unselected genotype) characterized by a unique diversity in longevity - the honey bee.Honey bee (Apis mellifera) life span varies from a few weeks to more than 2 years. This plasticity is largely controlled by environmental factors. Thereby, although individuals are closely related genetically, distinct life histories can emerge as a function of social environmental change.Another remarkable feature of the honey bee is the occurrence of reverted behavioural ontogeny in the worker (female helper) caste. This behavioural peculiarity is associated with alterations in somatic maintenance functions that are indicative of reverted senescence. Thus, although intraspecific variation in organismal life span is not uncommon, the honey bee holds great promise for gaining insights into regulatory pathways that can shape the time-course of ageing by delaying, halting or even reversing processes of senescence. These aspects provide the setting of our review.We will highlight comparative findings from Drosophila melanogaster and Caenorhabditis elegans in particular, and focus on knowledge spanning from molecular- to behavioural-senescence to elucidate how the honey bee can contribute to novel insights into regulatory mechanisms that underlie plasticity and robustness or irreversibility in ageing.

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis

The soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a sigma(54) promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the alpha subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site (Prior & Dalton, 1985). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO(-) mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO(-) mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulation.

Inactivation of viruses, bacteria, spores and protozoa by ultraviolet irradiation in drinking water practice: a review

The inactivation rate constants (k-value) of pathogenic viruses, bacteria and protozoa and indicator or model microorganisms for monochromatic UV253.7 radiation have been calculated from literature studies describing experiments of seeded microorganisms in collimated beam apparatus with low-pressure lamps. Results show that data of most studies fitted well in the first-order Chick-Watson disinfection kinetics. Viruses require the highest UV fluence and the double-stranded DNA adenovirus is by far the most persistent species with an UV fluence requirement of 125 mJ/cm2 for 3 log inactivation. Translation of these k-values to full-scale UV systems requires accurate fluence assessment in these continuous flow systems with fluence distribution, variations in water quality (UV transmittance) and operational aspects like lamp age and fouling. Determination of the reduction equivalent fluence or REF with seeded model organisms is an accepted methodology to validate UV systems. Frequently used model organisms F-specific RNA phage MS2 and spores of Bacillus subtilis have low k-values. For validation of systems with low fluence requirement to inactivate UV sensitive organisms, alternative model organisms are suggested. Further research is needed to verify the potential of REF verification of full-scale systems by monitoring inactivation of indigenous spores (anaerobic or aerobic) with large volume sampling.

Developmental hematopoiesis from fly to human

Developmental hematopoiesis is an exciting multi-disciplinary field at the crossroads of developmental biology, hematopoiesis, stem cell biology and genetics. This field began nearly a century ago with detailed observations on the formation of the first blood cells, the primitive erythrocytes, in

Cell-death alternative model organisms: why and which?

Classical model organisms have helped greatly in our understanding of cell death but, at the same time, might have constrained it. The use of other, non-classical model organisms from all biological kingdoms could reveal undetected molecular pathways and better-defined morphological types of cell death. Here we discuss what is known and what might be learned from these alternative model systems.

Integrative Microbiology, 2003

OMICS: A Journal of Integrative BiologyVol. 6, No. 4 EditorialIntegrative Microbiology, 2003Eugene KolkerEugene KolkerSearch for more papers by this authorPublished Online:8 Jul 2004https://doi.org/10.1089/153623102321112728AboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail "Integrative Microbiology, 2003." , 6(4), p. 285FiguresReferencesRelatedDetailsCited byUnraveling the Complexities of Life Sciences Data Roger Higdon, Winston Haynes, Larissa Stanberry, Elizabeth Stewart, Gregory Yandl, Chris Howard, William Broomall, Natali Kolker, and Eugene Kolker13 February 2013 | Big Data, Vol. 1, No. 1 Volume 6Issue 4Oct 2002 To cite this article:Eugene Kolker.Integrative Microbiology, 2003.OMICS: A Journal of Integrative Biology.Oct 2002.285-285.http://doi.org/10.1089/153623102321112728Published in Volume: 6 Issue 4: July 8, 2004PDF download

Introductory remarks: Unconventional organisms as models in biological research

The use of unconventional organisms in experimental research is a timely subject that impacts upon current science policy in the areas of animal welfare, biodiversity, and biotechnology. Many recent reports have recommended a reduction in the use of mammals in biomedical research to promote animal welfare by utilizing alternative methods and unconventional model organisms. Currently we lack adequate models for all the biological phenomena worthy of study. Biologists must continue to take advantage of the functional biodiversity of organisms not only to identify new model organisms for studying more optimally those life processes we know something about, but also to discover entirely new biological principles, processes, and products. Increased emphasis on the need to preserve the diversity of life also requires more knowledge of the comparative biology of processes such as reproduction, growth, defense mechanisms, and nutrition. The functional biodiversity of life can be exploited not only for fundamentally new biological insights, but also for expanding the knowledge base needed for commercial biotechnology. There is a critical need of commercial biotechnology for basic functional information on more species, especially microorganisms and plants. The knowledge of biodiversity will mean little to most of mankind unless the motivation exists to use it for commercial benefit.