Standard reporting requirements for biological samples in metabolomics experiments: microbial and in vitro biology experiments

With the increasing use of metabolomics as a means to study a large number of different biological research questions, there is a need for a minimal set of reporting standards that allow the scientific community to evaluate, understand, repeat, compare and re-investigate metabolomics studies. Here we propose, a first draft of minimal requirements to effectively describe the biological context of metabolomics studies that involve microbial or in vitro biological subjects. This recommendation has been produced by the microbiology and in vitro biology working subgroup of the Metabolomics Standards Initiative in collaboration with the yeast systems biology network as part of a wider standardization initiative led by the Metabolomics Society. Microbial and in vitro biology metabolomics is defined by this sub-working group as studies with any cell or organism that require a defined external medium to facilitate growth and propagation. Both a minimal set and a best practice set of reporting standards for metabolomics experiments have been defined. The minimal set of reporting standards for microbial or in vitro biology metabolomics experiments includes those factors that are specific for metabolomics experiments and that critically determine the outcome of the experiments. The best practice set of reporting standards contains both the factors that are specific for metabolomics experiments and general aspects that critically determine the outcome of any microbial or in vitro biological experiment.     

Current halo structures in high-current plasma experiments: θ-pinch

Experimental data elucidating mechanisms for halo formation in θ-pinch discharges are presented and discussed. The experiments were performed with different gases (H₂, D₂, He, and Ar) in a theta-pinch device with a porcelain vacuum chamber and an excitation coil 15 cm in diameter and 30 cm in length. The stored energy, the current in the excitation coil, and the current half-period were W = 10 kJ, I = 400 kA, and T/2 = 14 μs, respectively. It is found that the plasma rings (halos) surrounding the pinch core arise as a result of coaxial pinch stratification due to both the excitation of closed currents (inductons) inside the pinch and the radial convergence of the plasma current sheaths produced after the explosion of T-layers formed near the wall in the initial stage of the discharge. It is concluded that halo structures observed in pinches, tokamaks, and other high-current devices used in controlled fusion research have the same nature.     

Are there allelopathic interactions in zooplankton? Laboratory experiments with Daphnia

Summary Daphnia hyalina and Daphnia cucullata were kept in flow-through chambers in lake water (I), water from mass cultures of D. hyalina (II) and water from mass cultures of D. cucullata (III). A good correlation between body length and clutch size of the Daphnia was found in all cases. The regression lines of the different treatments were compared with respect to different slopes and different intercepts. D. hyalina showed the same reaction to medium II and III, but a lower regression coefficient and higher intercept in medium I. In contrast D. cucullata showed the same reaction to medium I and II, but a higher regression coefficient in medium III.     

Can video images imitate real stimuli in animal behaviour experiments?

The use of video images in place of natural stimuli in animal behaviour experiments is reviewed. Unlike most other artificial means of stimulus presentation, video stimuli can depict complex moving objects such as other animals, preserving the temporal and spatial patterns of movement precisely as well as colour and sounds for repeated playback. Computer editing can give flexibility and control over all elements of the stimulus. A variety of limitations of video image presentation are also considered. Televisions and video monitors are designed with human vision in mind, and some non-human animals that differ in aspects of visual processing such as their colour vision, critical flicker-fusion threshold, perception of depth and visual acuity, may perceive video images differently to ourselves. The failure of video stimuli to interact with subjects can be a drawback for some studies. For video to be useful, it is important to confirm that the subject animal responds to the image in a comparable way to the real stimulus, and the criteria used to assess this are discussed. Finally, the contribution made by video studies to date in the understanding of animal visual responses is considered, and recommendations as to the future uses of video are made.     

Drugs and parasites: global experiments in life history evolution?

Recent work has shown that the evolution of Drosophila melanogaster resistance to attack by the parasitoid Asobara tabida is constrained by a trade-off with larval competitive ability. However, there are two very important questions that need to be answered. First, is this a general cost, or is it parasitoid specific? Second, does a selected increase in immune response against one parasitoid species result in a correlated change in resistance to other parasitoid species? The answers to both questions will influence the coevolutionary dynamics of these species, and also may have a previously unconsidered, yet important, influence on community structure.     

Logic of experiments in ecology: is pseudoreplication a pseudoissue?

Hurlbert divides experimental ecologist into ‘those who do not see any need for dispersion (of replicated treatments and controls), and those who do recognize its importance and take whatever measures are necessary to achieve a good dose of it’. Experimental ecologists could also be divided into those who do not see any problems with sacrificing spatial and temporal scales in order to obtain replication, and those who understand that appropriate scale must always have priority over replication. If an experiment is conducted in a spatial or temporal scale, where the predictions of contesting hypotheses are convergent or ambiguous, no amount of technical impeccability can make the work instructive. Conversely, replication can always be obtained afterwards, by conducting more experiments with basically similar design in different areas and by using meta‐analysis. This approach even reduces the sampling bias obtained if resources are allocated to a small number of well‐replicated experiments. For a strict advocate of the hypothetico‐deductive method, replication is unnecessary even as a matter of principle, unless the predicted response is so weak that random background noise is a plausible excuse for a discrepancy between predictions and results. By definition, a prediction is an ‘all‐statement’, referring to all systems within a well‐defined category. What applies to all must apply to any. Hence, choosing two systems and assigning them randomly to a treatment and a control is normally an adequate design for a deductive experiment. The strength of such experiments depends on the firmness of the predictions and their a priori probability of corroboration. Replication is but one of many ways of reducing this probability. Whether the experiment is replicated or not, inferential statistics should always be used, to enable the reader to judge how well the apparent patterns in samples reflect real patterns in statistical populations. The concept ‘pseudoreplication’ amounts to entirely unwarranted stigmatization of a reasonable way to test predictions referring to large‐scale systems.     

The composting of tree bark in small reactors— self-heating experiments

The effects of aeration, moisture content and temperature on the rate of decomposition of tree bark were studied in small reactors (26 litres) in the laboratory. Aeration was found to be essential in the rapid self-heating phase; thereafter vigorous aeration only served to cool the reactor. Decomposition was optimal at a moisture content of 60% with constant rates of aeration. When aeration was reduced to prevent cooling, decomposition was not appreciably different in bark with moisture contents of 60 or 70%, although higher temperatures were achieved at 60%. It was postulated that the lower thermal capacity of bark at a moisture content of 60% was partly responsible for this latter difference.     

Can results from limnocorral experiments be transferred to in situ conditions?

In 1982–1984 eight limnocorral (LC) experiments, each lasting for two weeks, were performed in mesotrophic Lake Lucerne, Switzerland, to study the effects of biomanipulation (removal of crustaceans by 95 µm filter nets) on zooplankton — phytoplankton relationships and epilimnetic carbon and phosphorus fluxes. Seston concentrations and to a lesser extent primary production rates were reduced in control LCs through zooplankton grazing, and settling flux increased through fecal pellet production. But C and P regeneration were not significantly affected.We found several indications that the LCs, despite of their large size ( 70 m³, 3 m in diameter and 11 m long) were artificial systems when compared to the surrounding lake: The eddy diffusion was diminished by about one order of magnitude, nutrients depleted (but phosphorus was supplied to the LCs), the phyto- and zooplankton showed lower standing crops in the control LCs than in the lake, the phytoplankton showed a shift from nannoplankton to netplankton, the crustacean zooplankton was mostly limited in their vertical migration, and the POC and PP sedimentation rates were increased. Moreover, in our set of experiments we always found outliers, which may have been caused by the different in situ conditions at the beginning of the experiments, and further enhanced by the complexity of the enclosed system.The problems of the LC-technique, such as replicability, scaling (size and time) and data extrapolation are discussed. The impact of crustacean zooplankton on particulate matter, i.e. seston reduction and sedimentation enhancement, can be extrapolated on a qualitative rather than on a quantitative basis for the Lake Lucerne ecosystem.     

Diseases of protein conformation: what do in vitro experiments tell us about in vivo diseases?

Certain human diseases are associated with proteins that misfold and exhibit decreased solubility under physiological conditions. They result either from mutations that change the amino acid sequence of a protein, or from misfolded wild-type proteins, such as in Parkinson's disease and Alzheimer's disease. One subset--the amyloidoses--cause extracellular deposits that stain with the dye Congo red. Another subset is associated with intracellular deposits with non-Congophilic nuclear or cytoplasmic inclusions. Purified, recombinantly produced versions of some of the proteins that form intracellular aggregates can also display Congophilia, as well as other properties associated with the in vivo amyloidoses when examined under non-physiological conditions in vitro. Some of these purified proteins or protein fragments have never been identified as pathogenic in humans or animals. Despite potentially shared thermodynamic and kinetic processes involving the target molecules, the biology of these two subsets differs significantly.     

Can oxysterols work in anti-glioblastoma therapy? Model studies complemented with biological experiments

Despite the progress made in recent years in the field of oncology, the results of glioblastoma treatment remain unsatisfactory. In this paper, cholesterol derivatives - oxysterols - have been investigated in the context of their anti-cancer activity. First, the influence of three oxysterols (7-K, 7β-OH and 25-OH), differing in their chemical structure, on the properties of a model membrane imitating glioblastoma multiforme (GBM) cells was investigated. For this purpose, the Langmuir monolayer technique was applied. The obtained results clearly show that oxysterols modify the structure of the membrane by its stiffening, with the 7-K effect being the most pronounced. Next, the influence of 7-K on the nanomechanical properties of glioblastoma cells (U-251 line) was verified with AFM. It has been shown that 7-K has a dose-dependent cytotoxic effect on glioblastoma cells leading to the induction of apoptosis as confirmed by viability tests. Interestingly, significant changes in membrane structure, characteristic for phospholipidosis, has also been observed. Based on our results we believe that oxysterol-induced apoptosis and phospholipidosis are related and may share common signaling pathways. Dysregulation of lipids in phospholipidosis inhibit cell proliferation and may play key roles in the induction of apoptosis by oxysterols. Moreover, anticancer activity of these compounds may be related to the immobilization of cancer cells as a result of stiffening effect caused by oxysterols. Therefore, we believe that oxysterols are good candidates as new therapeutic molecules as an alternative to the aggressive treatment of GBM currently in use.     

Do western boxelder bugs sunbathe for sanitation? Inferences from in vitro experiments

When western boxelder bugs, Boisea rubrolineata (Barber) (Hemiptera: Rhopalidae), form aggregations in warm sunlight, they release from their posterior dorsal abdominal gland an odorous blend of monoterpenes with heretofore unknown biological function. In laboratory analyses and experiments, we show that bugs in warm sunlight, but not in shade, exude and spread copious amounts of monoterpenes onto their cuticle. These monoterpenes do not serve as a pheromone, but rather as a means of sanitation. They inhibit germination of conidia of the fungal pathogen Beauveria bassiana (Bals.‐Criv.) Vuill. (Hypocreales) as well as halt the growth of germinated spores. This prophylactic defense against pathogens appears adaptive for phytophagous insects, like B. rubrolineata, that are prone to infections by microbes thriving on leaf surfaces and in the insects' overwintering microhabitat.     

Correcting for base rates in multidimensional “Who said what?” experiments

The “Who said what?” protocol is a popular experimental paradigm and has been used for 40?years to study spontaneous mental categorization. This paper offers a crucial methodological improvement to calculate unbiased estimates in multidimensional “Who said what?” studies. Previous studies predominantly corrected for base rates by first correcting the base rates and consequently aggregating errors for the two dimensions separately. The paper demonstrates that this procedure's estimates are biased. A large simulation of over 175,000 experiments and the re-analysis of a pivotal study show that this may increase both false-positive and false-negative error rates in treatment effects and might therefore, respectively, strengthen or weaken evidence for past hypotheses. The paper offers a simple remedy: researchers should first aggregate errors for each dimension and then correct for base rates relying on the method known from single-dimensional studies.     

Is a compromise possible in Russia between animal advocates and researchers who use animals in harmful experiments?

The current situation relating to the use of laboratory animals in Russia, which is primarily characterised by the complete absence of legislation for their protection, is examined and discussed. This lack of regulation causes well-founded protests by animal protection organisations and a number of reputable politicians. It also has a negative influence on the quality of medical and biological research results that are obtained through the use of experimental animals in Russia. The opinion is expressed that the Russian scientific community should be able to build upon the experience of other countries - in particular, members of the European Union, where there is an effective system of self-control over the ethical and legislative regulation of animal-based research. It is suggested that, in Russia, the basic animal protection principles of the Three Rs should be introduced, when the decision on whether to finance scientific projects involving the use of animals is being made.     

Does age matter in song bird vocal interactions? Results from interactive playback experiments

The song of oscines provides an extensively studied model of age-dependent behaviour changes. Male and female receivers might use song characteristics to obtain information about the age of a signaller, which is often related to its quality. Whereas most of the age-dependent song changes have been studied in solo singing, the role of age in vocal interactions is less well understood. We addressed this issue in a playback study with common nightingales (Luscinia megarhynchos). Previous studies showed that male nightingales had smaller repertoires in their first year than older males and males adjusted their repertoire towards the most common songs in the breeding population. We now compared vocal interaction patterns in a playback study in 12 one year old and 12 older nightingales (cross-sectional approach). Five of these males were tested both in their first and second breeding season (longitudinal approach). Song duration and latency to respond did not differ between males of different ages in either approach. In the cross-sectional approach, one year old nightingales matched song types twice as often as did older birds. Similarly, in the longitudinal approach all except one bird reduced the number of song type matches in their second season. Individuals tended to overlap songs at higher rates in their second breeding season than in their first. The higher levels of song type matches in the first year and song overlapping by birds in their second year suggest that these are communicative strategies to establish relationships with competing males and/or choosy females.     

Natural fishing experiments in marine reserves 1983–1993: community and trophic responses

 This study examined the effect of fishing on the density, biomass, species richness and overall structure of the reef fish community at two islands (Sumilon and Apo) in the Philippines from 1983 to 1993. A series of natural fishing experiments over this period involving marine reserves were monitored at each island, where estimates of fishing intensity and selectivity were available. Fishing intensity (15% and 25% of biomass removed per year at Sumilon and Apo, respectively) was high enough to affect total community biomass, but not density, significantly. Species richness was not affected significantly by fishing, except at Sumilon reserve. The fishery was relatively non-selective with most families/trophic groups caught roughly in proportion to their contribution to community biomass. Thus fishing did not alter the relative abundance of the major families/trophic groups significantly, except during a period of use of explosives and drive nets in the Sumilon reserve. At the level of family/trophic group the community displayed strong resilience of structure. There was little evidence of secondary effects e.g. declines in abundance of large predators resulting in measurable increases in abundance of their prey. This resilience of the community to the effects of fishing most likely results from three important community attributes (open nature of the component populations, likely maintenance of upstream recruitment supply and apparent lack of any obvious “keystone” species or families) and one important characteristic of the fishery (relatively non-selective with respect to the components of the community). Accepted: 30 June 1998     

Active promoters give rise to false positive ‘Phantom Peaks’ in ChIP-seq experiments

Chromatin immunoprecipitation (ChIP) is widely used to identify chromosomal binding sites. Chromatin proteins are cross-linked to their target sequences in living cells. The purified chromatin is sheared and the relevant protein is enriched by immunoprecipitation with specific antibodies. The co-purifying genomic DNA is then determined by massive parallel sequencing (ChIP-seq).We applied ChIP-seq to map the chromosomal binding sites for two ISWI-containing nucleosome remodeling factors, ACF and RSF, in Drosophila embryos. Employing several polyclonal and monoclonal antibodies directed against their signature subunits, ACF1 and RSF-1, robust profiles were obtained indicating that both remodelers co-occupied a large set of active promoters.Further validation included controls using chromatin of mutant embryos that do not express ACF1 or RSF-1. Surprisingly, the ChIP-seq profiles were unchanged, suggesting that they were not due to specific immunoprecipitation. Conservative analysis lists about 3000 chromosomal loci, mostly active promoters that are prone to non-specific enrichment in ChIP and appear as ‘Phantom Peaks’. These peaks are not obtained with pre-immune serum and are not prominent in input chromatin.Mining the modENCODE ChIP-seq profiles identifies potential Phantom Peaks in many profiles of epigenetic regulators. These profiles and other ChIP-seq data featuring prominent Phantom Peaks must be validated with chromatin from cells in which the protein of interest has been depleted.     

Dissociation-reconstitution experiments support the presence of two catalytic β-subunits in mitochondrial F1

Mitochondrial F₁, inactivated to various extents with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), was dissociated with LiCl and reconstituted after removal of the salt. This procedure resulted in a reactivation that corresponded with a reactivation theoretically expected on the basis of the assumption that the reassociation of β-subunits into native F₁ molecules is random and that two out of the three β-subunits are directly involved in catalysis. Repeated inactivation of such reactivated F₁, followed by the same dissociation-association procedure, resulted in similar data. After inactivation of F₁ by covalent binding of 2-N-AT(D)P to one catalytic site, no reactivation upon dissociation-reassociation was obtained due to the fact that such modified F₁ did not dissociate under the experimental conditions used.     

What's wrong with correlative experiments?

Here, we make a case for multivariate measurements in cell biology with minimal perturbation. We discuss how correlative data can identify cause-effect relationships in cellular pathways with potentially greater accuracy than conventional perturbation studies.