QPCR: Application for real-time PCR data management and analysis

Background  

Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline.     

Unstructured Hydrophilic Sequences in Prokaryotic Proteomes Correlate with Dehydration Tolerance and Host Association

Water loss or desiccation is among the most life-threatening stresses. It leads to DNA double-strand breakage, protein aggregation, cell shrinkage, and low water activity precluding all biological functions. Yet, in all kingdoms of life, rare organisms are resistant to desiccation through prevention or reversibility of such damage. Here, we explore possible hallmarks of prokaryotic desiccation tolerance in their proteomes. The content of unstructured, low complexity (LC) regions was analyzed in a total of 460 bacterial and archaeal proteomes.It appears that species endowed with proteomes abundant in unstructured hydrophilic LC regions are desiccation-tolerant or sporulating bacteria, halophilic archaea and bacteria, or host-associated species. In the desiccation- and radiation-resistant bacterium Deinococcus radiodurans, most proteins that contain large hydrophilic LC regions have unassigned function, but those with known function are mostly involved in diverse cellular recovery processes. Such LC regions are typically absent in orthologous proteins in desiccation-sensitive species. D. radiodurans encodes also special LC proteins, akin to those associated with desiccation resistance of plant seeds and some plants and animals. Therefore, we postulate that large unstructured hydrophilic LC regions and proteins provide for cellular resistance to dehydration and we discuss mechanisms of their protective activity.     

Demasculinization and feminization of male gonads by atrazine: consistent effects across vertebrate classes

Atrazine is the most commonly detected pesticide contaminant of ground water, surface water, and precipitation. Atrazine is also an endocrine disruptor that, among other effects, alters male reproductive tissues when animals are exposed during development. Here, we apply the nine so-called "Hill criteria" (Strength, Consistency, Specificity, Temporality, Biological Gradient, Plausibility, Coherence, Experiment, and Analogy) for establishing cause-effect relationships to examine the evidence for atrazine as an endocrine disruptor that demasculinizes and feminizes the gonads of male vertebrates. We present experimental evidence that the effects of atrazine on male development are consistent across all vertebrate classes examined and we present a state of the art summary of the mechanisms by which atrazine acts as an endocrine disruptor to produce these effects. Atrazine demasculinizes male gonads producing testicular lesions associated with reduced germ cell numbers in teleost fish, amphibians, reptiles, and mammals, and induces partial and/or complete feminization in fish, amphibians, and reptiles. These effects are strong (statistically significant), consistent across vertebrate classes, and specific. Reductions in androgen levels and the induction of estrogen synthesis - demonstrated in fish, amphibians, reptiles, and mammals - represent plausible and coherent mechanisms that explain these effects. Biological gradients are observed in several of the cited studies, although threshold doses and patterns vary among species. Given that the effects on the male gonads described in all of these experimental studies occurred only after atrazine exposure, temporality is also met here. Thus the case for atrazine as an endocrine disruptor that demasculinizes and feminizes male vertebrates meets all nine of the "Hill criteria".     

BHK 21 C13 cells for Aujeszky’s disease virus production using the multiple harvest process

Production of Aujeszky’s disease virus (ADV) from BHK 21 C13 suspension cells using a simple harvest and multiple harvest process mode was examined. We studied growth kinetics of BHK 21 C31 cells in 750 ml spinner flask containing 500 ml of culture medium. In the simple harvest process of ADV production, 425 ml of virus harvest was obtained with a virus titer of 10^6.4 TCID₅₀ ml⁻¹ which corresponds to 10,676 doses of vaccine. The multiple harvest process resulted in 850 ml of virus harvest with a virus titer of 10^6.5 TCID₅₀ ml⁻¹ corresponding to 26,877 AD vaccine doses. In conclusion, the multiple harvest process mode using BHK 21 C13 can be considered as a favorable process to produce ADV.     

Age-Dependent Effects of ELF-MF on Oxidative Stress in the Brain of Mongolian Gerbils

The aim of study was to investigate the effects of extremely low frequency magnetic field (ELF-MF; 50 Hz; 0.1, 0.25 and 0.5 mT) on oxidative stress in the brain of 3- (adult) and 10-month-old (middle-aged) gerbils. Nitric oxide (NO) level, superoxide (O₂ ^?) production, superoxide dismutase (SOD) activity, and index of lipid peroxidation (ILP) were measured in the forebrain cortex, striatum, hippocampus, and cerebellum immediately and 3 days after cessation of 7-day exposure. In all gerbils, ELF-MF significantly increased oxidative stress in all tested brain regions. This effect was correlated with the value of magnetic induction and was higher in middle-aged gerbils. Three days after cessation of exposure, the values of examined parameters were closer to control levels. In adult gerbils, the effect of ELF-MF of 0.1 mT on NO level, O₂ ^? production and SOD activity was almost fully disappeared, and ILP was at the control level regardless of the value of magnetic induction. In middle-aged gerbils, the effect of ELF-MF was still present but to a lesser degree than those observed immediately after cessation of exposure. These findings pointed out the ability of ELF-MF to induce age- and magnetic induction-dependent modification of oxidative stress in the brain.     

Species traits predict assembly of mayfly and stonefly communities along pH gradients

Much recent ecological research has centred on the interrelations between species diversity and ecological processes. In the present study, I show how species traits may aid in comprehending ecology by studying the link between an environmental variable and functional traits. I examined the composition of species traits with a theoretically underpinned relationship to ecological processes along a pH gradient. I focused on body size, reproductive output, life cycle length and feeding habit of mayflies and stoneflies. In mayfly assemblages, I found smaller body size, greater reproductive output, faster life cycles and a larger proportion of gathering collectors and scrapers with increasing pH. In stonefly assemblages, I found smaller body size, greater reproductive output and faster life cycles at sites with a history of long-term natural acidification, but no clear trends in feeding habits and in most traits where acidification is anthropogenic. The results suggest that mayflies and stoneflies exhibit different ecological functions following different ecological strategies. Mayflies follow an opportunistic strategy relative to stoneflies, likely facilitating high rates of ecological processes with respect to the autotrophic resource base at neutral sites. Relative to mayflies, stoneflies follow an equilibrium strategy contributing to ecological functioning in heterotrophic ecosystems and likely maintaining heterotrophic processes despite the erosion of species diversity in response to acidification. The rules governing an ecological community may be more readily revealed by studying the distribution of species traits instead of species diversity; by studying traits, we are likely to improve our understanding of the workings of ecological communities.     

Correction to: Pyrethrin from Dalmatian pyrethrum (Tanacetum cinerariifolium (Trevir.) Sch. Bip.): biosynthesis,biological activity,methods of extraction and determination

Phytochemistry Reviews -     

Influence of different ammonium,lactate and glutamine concentrations on CCO cell growth

In this study the effects of ammonium and lactate on a culture of channel catfish ovary (CCO) cells were examined. We also made investigation on the influence of glutamine, since our previous research revealed that this amino acid stimulated CCO cell growth more than glucose in a concentration-dependent manner. The effect of ammonium in cell culture included the considerable decrease in cell growth rate with eventual growth arrest as well as the retardation of glucose consumption. At ammonium concentrations above 2.5 mM, the cells displayed specific morphological changes. The effect of lactate was different to that of ammonium since the cell growth rate was progressively decreasing with the increase of lactate concentration, whereas the glucose consumption rate remained almost unchanged. Besides that, it was found that lactate was steadily eliminated from the culture medium when its initial concentration was relatively high. The influence of glutamine on CCO cell propagation showed that nutrient requirements of this cell line were mainly dependent on glutamine rather than glucose. The increase in glutamine concentration led to the increase in cell growth rate and consequent ammonia accumulation while the glucose utilization and lactate production were reduced. Without glutamine in culture medium cell growth was arrested. However, the lack of glucose reversed the stimulating effect of glutamine by decreasing cell growth rate and affecting amino acid utilization.     

Hypochlorite modification of sphingomyelin generates chlorinated lipid species that induce apoptosis and proteome alterations in dopaminergic PC12 neurons in vitro

Recent observations link myeloperoxidase (MPO) activation to neurodegeneration. In multiple sclerosis MPO is present in areas of active demyelination where the potent oxidant hypochlorous acid (HOCl), formed by MPO from H₂O₂ and chloride ions, could oxidatively damage myelin-associated lipids. The purpose of this study was (i) to characterize reaction products of sphingomyelin (SM) formed in response to modification by HOCl, (ii) to define the impact of exogenously added SM and HOCl-modified SM (HOCl-SM) on viability parameters of a neuronal cell line (PC12), and (iii) to study alterations in the PC12 cell proteome in response to SM and HOCl-SM. MALDI-TOF-MS analyses revealed that HOCl, added as reagent or generated enzymatically, transforms SM into chlorinated species. On the cellular level HOCl-SM but not SM induced the formation of reactive oxygen species. HOCl-SM induced severely impaired cell viability, dissipation of the mitochondrial membrane potential, and activation of caspase-3 and DNA damage. Proteome analyses identified differential expression of specific subsets of proteins in response to SM and HOCl-SM. Our results demonstrate that HOCl modification of SM results in the generation of chlorinated lipid species with potent neurotoxic properties. Given the emerging connections between the MPO–H₂O₂–chloride axis and neurodegeneration, this chlorinating pathway might be implicated in neuropathogenesis.     

Proteome sequence features carry signatures of the environmental niche of prokaryotes

Background  

Prokaryotic environmental adaptations occur at different levels within cells to ensure the preservation of genome integrity, proper protein folding and function as well as membrane fluidity. Although specific composition and structure of cellular components suitable for the variety of extreme conditions has already been postulated, a systematic study describing such adaptations has not yet been performed. We therefore explored whether the environmental niche of a prokaryote could be deduced from the sequence of its proteome. Finally, we aimed at finding the precise differences between proteome sequences of prokaryotes from different environments.