Physical properties of two types of calcium stores and SERCAs in human platelets

The aim of this work was to obtain experimental data depending on the properties of calcium stores and SERCAs, to analyse these data in terms of the models based on simulation of the cellular compartments as communicating vessels, and to relate this way the data to the above properties. The main characteristics of the stores and corresponding SERCAs have been estimated. Calcium content in the DTS is ∼1.5 × 10⁶ ions per cell, that in the acidic stores, ∼0.64 × 10⁶ ions per cell. The rate constant of background calcium leakage from the DTS is ∼0.0033 s⁻¹, that from the acidic stores, ∼0.1 s⁻¹. The background activity of SERCA2b is ∼0.22 × 10⁶ s⁻¹ ions per cell, that of SERCA3, ∼2.5 × 10⁶ s⁻¹ ions per cell. The properties of both calcium stores and the SERCAs and the characteristics found might be related to physiological or pathological state of the cells.     

Genotyping faeces of red pandas (<Emphasis Type="Italic">Ailurus fulgens</Emphasis>): implications for population estimation

The red panda (Ailurus fulgens) is an endangered species distributed in the Himalaya and Hengduan Mountains and extremely difficult to monitor because it is elusive, wary and nocturnal. However, recent advances in noninvasive genetics are allowing conservationists to indirectly estimate population size of this animal. Here, we present a pilot study of individual identification of wild red pandas using DNA extracted from faeces. A chain of optimal steps in noninvasive studies were used to maximize genotyping success and minimize error rate across sampling, selection of microsatellite loci, DNA extraction and amplification and data checking. As a result, 18 individual red pandas were identified successfully from 33 faecal samples collected in the field using nine red panda-specific microsatellite loci with a low probability of identity of 1.249 × 10⁻³ for full siblings. Multiple methods of tracking genotyping error showed that the faecal genetic profiles possessed very few genotyping errors, with an overall error rate of 1.12 × 10⁻⁵. Our findings demonstrate the feasibility and reliability of using faeces as an effective source of DNA for estimating and monitoring wild red panda populations.     

How old can we go? Evaluating the age limit for effective DNA recovery from historical insect specimens

Historical museum specimens are valuable for exploring population genetics and evolutionary questions because they can provide snapshots of morphological and genetic characteristics from populations over space and time. Unfortunately, DNA found in older museum specimens is frequently degraded, so obtaining genotypes from many individual samples necessary for rigorous molecular population genetic studies is challenging. Previous studies have varied greatly in their success at obtaining genotypes from older preserved insect material. Many well-intentioned collection curators have used research results showing poor preservation of DNA preserved in museum specimens to inform curatorial best practices, in some cases choosing not to allow DNA extraction by destructive sampling because, in their estimation, the likelihood of success would be low. Recent methodological advances in DNA extraction, amplification, and genotyping have allowed some researchers to include mid-19th century samples in molecular genetic analyses. Here we present a robust, high-throughput, and low-cost DNA extraction and genotyping protocol for historical insect specimens employing restriction digests of PCR products followed by high sensitivity electrophoresis. Using this technique, we obtained mitochondrial haplotypes for 100% of 48 New World Junonia butterfly specimens (Nymphalidae) ranging in age from pre-1813 to 1909 and show that the haplotype frequencies obtained are statistically indistinguishable from 20th-century and contemporary reference populations of Junonia (1632 specimens) matched by geographic region. As most extant insect specimens were collected after 1813, based on our findings we would expect that many or even most pinned specimens preserved in museum collections contain usable DNA for mitochondrial haplotyping.     

Molecular aspects on the interaction of isatin-3-isonicotinylhydrazone to deoxyribonucleic acid: model for intercalative drug-DNA binding

Isatin-3-isonicotinylhydrazone was synthesized and characterized. The interaction of native calf thymus DNA with isatin-3-isonicotinylhydrazone (IINH) in 10 mM Tris–HCl aqueous solutions at neutral pH 7.4 has been investigated by spectrophotometric, circular dichroism (CD), melting temperature (T _m ), spectrofluorimetric, and viscometric techniques. It is found that IINH molecules could intercalate between base pairs of DNA as are evidenced by: hypochromism in UV absorption band of IINH, induced CD spectral changes, sharp increase in specific viscosity of DNA, and increase in the fluorescence of methylene blue (MB)-DNA solutions in the presence of increasing amounts of IINH, which indicates that it is able to release the intercalated MB completely. The binding constants of IINH–DNA complex at four different temperatures (277, 288, 298, and 310 K) were calculated to be 4.7 × 10⁴, 2.2 × 10⁴, 1.75 × 10⁴ and 1.1 × 10⁴ M⁻¹, respectively. Furthermore, the enthalpy and entropy of the reaction between IINH and CT-DNA showed that the reaction is enthalpy-favored and entropy- disfavored (∆H = −30.187 kJ mol⁻¹; ∆S = −20.46 J mol⁻¹K⁻¹) which are other evidences to indicate the IINH is able to be intercalated in the DNA base pairs.     

Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN₈+pN₄+pN′₈ Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN₈ is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′₈ Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10⁻¹⁴ mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.     

DNA content and distribution in ancient feathers and potential to reconstruct the plumage of extinct avian taxa

Feathers are known to contain amplifiable DNA at their base (calamus) and have provided an important genetic source from museum specimens. However, feathers in subfossil deposits generally only preserve the upper shaft and feather ‘vane’ which are thought to be unsuitable for DNA analysis. We analyse subfossil moa feathers from Holocene New Zealand rockshelter sites and demonstrate that both ancient DNA and plumage information can be recovered from their upper portion, allowing species identification and a means to reconstruct the appearance of extinct taxa. These ancient DNA sequences indicate that the distal portions of feathers are an untapped resource for studies of museum, palaeontological and modern specimens. We investigate the potential to reconstruct the plumage of pre-historically extinct avian taxa using subfossil remains, rather than assuming morphological uniformity with closely related extant taxa. To test the notion of colour persistence in subfossil feathers, we perform digital comparisons of feathers of the red-crowned parakeet (Cyanoramphus novaezelandiae novaezelandiae) excavated from the same horizons as the moa feathers, with modern samples. The results suggest that the coloration of the moa feathers is authentic, and computer software is used to perform plumage reconstructions of moa based on subfossil remains.     

Seasonal changes of Antarctic marine bacterioplankton and sea ice bacterial assemblages

The distributions of bacterial populations in sea ice and underlying seawater were investigated on the continental shelf of the “Terre Adélie” area. A reference station was sampled weekly from January 1991 to January 1992. In winter, the survey included a minimum of six sampling layers: surface and bottom ice, brine, seawater from the interface, and at 0.5 and 2 m depth. In seawater, the total bacterial abundance ranged from 0.5 × 10⁵ cells ml⁻¹ in July to 6.0 × 10⁵ cells ml⁻¹ after ice break. Values reaching 2.5 × 10⁶ cells ml⁻¹ were recorded in the overlying ice cover. Mean cell volumes were twice as high in brine as in seawater. The saprophytic bacterial abundance ranged from 5.0 × 10⁴ CFU (colony-forming units) ml⁻¹ in some winter interface samples to less than 1.0 × 10³ CFU ml⁻¹ in most of the summer seawater samples. In sea ice a clear decreasing gradient for most of the studied bacterial parameters from the surface layers towards the bottom layer was found. The ice cover had a discernible impact on underlying seawater, but its influence was restricted to a limited interface layer.     

Development and application of real-time PCR for quantification of specific ammonia-oxidizing bacteria in activated sludge of sewage treatment systems

In this study, four real-time polymerase chain reaction (PCR) primer sets were developed for the 16S rRNA genes of specific ammonia-oxidizing bacteria (AOB) found in activated sludge of sewage treatment systems. The primer sets target two of several sequence types of the Nitrosomonas oligotropha cluster, members within the Nitrosomonas communis cluster, and all members of the Nitrosomonas europaea–Nitrosococcus mobilis cluster. The detection limit of each primer set was in the range of 3×10¹–6×10² genes reaction⁻¹. Reliable quantification of the target AOB DNA was obtained when the target AOB DNA comprised more than 0.1% of total AOB DNA in the sample. The application of the primer sets to samples taken from five sewage treatment systems showed that, in all systems, the majority of the AOB population was comprised of one sequence type of the N. oligotropha cluster (3.9±1.5×10⁹–1.7±0.5×10¹⁰ cell l⁻¹) and, in most systems, followed by members within the N. communis cluster (2.8±0.3×10⁹–1.0±0.1×10¹⁰ cell l⁻¹) or/and another sequence type of the N. oligotropha cluster (1.5±0.6×10⁸–5.5±0.5×10⁸ cell l⁻¹). N. europaea–N. mobilis cluster arose solely in small numbers (4.9±0.8×10⁸ cell l⁻¹) in one system. Real-time PCR-amplified products obtained from genomic DNA extracted from samples were verified using clone library, and it revealed that only the target AOB DNA were PCR amplified, without amplification of the nontarget sequences.     

DNA polymerases and carcinogenesis

There are many various chromosomal and gene mutations in human cancer cells. The total mutation rate in normal human cells is 2·10⁻⁷ mutations/gene/division. From 6 to 12 carcinogenic mutations can arise by the end of the life, and these can affect the structure of ∼150 protooncogenes and genes encoding suppressors of tumor growth. However, this does not explain the tens and hundreds of thousands of mutations detectable in cancer cells. Mutation is any change of nucleotide sequence in cellular DNA. Gene mutations are mainly consequences of errors of DNA polymerases, especially of their specialized fraction (inaccurate DNA polymerases β, ζ, η, θ, ι, κ, λ, μ, σ, ν, Rev1, and terminal deoxynucleotidyl transferase, and only polymerases θ and σ manifest a slight 3′-exonuclease activity) and also consequences of a decrease in the rate of repair of these errors. Inaccurate specialized human polymerases are able to synthesize DNA opposite lesions in the DNA template, but their accuracy is especially low during synthesis on undamaged DNA. In the present review fundamental features of such polymerases are considered. DNA synthesis stops in the area of its lesion, but this block is overcome due to activities of inaccurate specialized DNA polymerases. After the lesion is bypassed, DNA synthesis is switched to accurate polymerases α, δ, ɛ, or γ. Mechanisms of direct and reverse switches of DNA polymerases as well as their modifications during carcinogenesis are discussed.     

Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics

Ancient and archival DNA samples are valuable resources for the study of diverse historical processes. In particular, museum specimens provide access to biotas distant in time and space, and can provide insights into ecological and evolutionary changes over time. However, archival specimens are difficult to handle; they are often fragile and irreplaceable, and typically contain only short segments of denatured DNA. Here we present a set of tools for processing such samples for state-of-the-art genetic analysis. First, we report a protocol for minimally destructive DNA extraction of insect museum specimens, which produced sequenceable DNA from all of the samples assayed. The 11 specimens analyzed had fragmented DNA, rarely exceeding 100 bp in length, and could not be amplified by conventional PCR targeting the mitochondrial cytochrome oxidase I gene. Our approach made these samples amenable to analysis with commonly used next-generation sequencing-based molecular analytic tools, including RAD-tagging and shotgun genome re-sequencing. First, we used museum ant specimens from three species, each with its own reference genome, for RAD-tag mapping. Were able to use the degraded DNA sequences, which were sequenced in full, to identify duplicate reads and filter them prior to base calling. Second, we re-sequenced six Hawaiian Drosophila species, with millions of years of divergence, but with only a single available reference genome. Despite a shallow coverage of 0.37±0.42 per base, we could recover a sufficient number of overlapping SNPs to fully resolve the species tree, which was consistent with earlier karyotypic studies, and previous molecular studies, at least in the regions of the tree that these studies could resolve. Although developed for use with degraded DNA, all of these techniques are readily applicable to more recent tissue, and are suitable for liquid handling automation.     

“False” thymine—1H-enol guanine base pair. Low misinsertion rate by DNA polymerase explained by computational chemistry consideration

Formation of correct TA and GC and “false” thymine—1H-enol guanine (TGenol) base pairs is here considered to control nucleotide insertion into DNA via low substrate concentration Michaelis-Menten controlled kinetics. Contributions of base pairing to formation of Gibbs free energies in water solution, ΔΔG, are calculated for the correct and false base pairs with the semi-empiric MNDO/PM3 method for base pairing energies in vacuum and the BEM method for hydration effects. The results for ΔΔG indicate equal insertion rates for correct base pairing and a 10⁻³−10⁻⁴ error probability for false insertion controlled by the TGenol false pair. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 402–406.     

Historical and non‐invasive samples: a study case of genotyping errors in newly isolated microsatellites for the lesser anteater (Tamandua tetradactyla L., Pilosa)

Tamandua tetradactyla (Pilosa), the lesser anteater, is a medium‐size mammal from South America. Its wide distribution through different landscapes, solitary and nocturnal habits, and the difficulty to capture and contain specimens limit the amount of individuals and populations sampled during fieldworks. These features along with the lack of specific molecular markers for the lesser anteater might be the causes for paucity in population genetic studies for the species. Historical samples from museum specimens, such as skins, and non‐invasive samples, such as plucked hair, can be supplementary sources of DNA samples. However, the DNA quantity and quality of these samples may be limiting factors in molecular studies. In this study, we describe nine microsatellite loci for T. tetradactyla and test the amplification success, data reliability and estimate errors on both historical and non‐invasive sample sets. We tested nine polymorphic microsatellites and applied the quality index approach to evaluate the relative performance in genotype analysis of 138 historical samples (study skin) and 19 non‐invasive samples (plucked hair). The observed results show a much superior DNA quality of non‐invasive over historical samples and support the quality index analysis as a practical tool to exclude samples with doubtful performance in genetic studies. We also found a relationship between the age of non‐invasive samples and DNA quality, but lack of evidence of this pattern for historical samples.     

Quantitative Determination of H2-Utilizing Acetogenic and Sulfate-Reducing Bacteria and Methanogenic Archaea from Digestive Tract of Different Mammals

Total number of bacteria, cellulolytic bacteria, and H₂-utilizing microbial populations (methanogenic archaea, acetogenic and sulfate-reducing bacteria) were enumerated in fresh rumen samples from sheep, cattle, buffaloes, deer, llamas, and caecal samples from horses. Methanogens and sulfate reducers were found in all samples, whereas acetogens were not detected in some samples of each animal. Archaea methanogens were the largest H₂-utilizing populations in all animals, and a correlation was observed between the numbers of methanogens and those of cellulolytic microorganisms. Higher counts of acetogens were found in horses and llamas (1 × 10⁴ and 4 × 10⁴ cells ml⁻¹ respectively).     

Spatial and temporal genetic dynamics of the grasshopper Oedaleus decorus revealed by museum genomics

Analyzing genetic variation through time and space is important to identify key evolutionary and ecological processes in populations. However, using contemporary genetic data to infer the dynamics of genetic diversity may be at risk of a bias, as inferences are performed from a set of extant populations, setting aside unavailable, rare, or now extinct lineages. Here, we took advantage of new developments in next‐generation sequencing to analyze the spatial and temporal genetic dynamics of the grasshopper Oedaleus decorus, a steppic Southwestern‐Palearctic species. We applied a recently developed hybridization capture (hyRAD) protocol that allows retrieving orthologous sequences even from degraded DNA characteristic of museum specimens. We identified single nucleotide polymorphisms in 68 historical and 51 modern samples in order to (i) unravel the spatial genetic structure across part of the species distribution and (ii) assess the loss of genetic diversity over the past century in Swiss populations. Our results revealed (i) the presence of three potential glacial refugia spread across the European continent and converging spatially in the Alpine area. In addition, and despite a limited population sample size, our results indicate (ii) a loss of allelic richness in contemporary Swiss populations compared to historical populations, whereas levels of expected heterozygosities were not significantly different. This observation is compatible with an increase in the bottleneck magnitude experienced by central European populations of O. decorus following human‐mediated land‐use change impacting steppic habitats. Our results confirm that application of hyRAD to museum samples produces valuable information to study genetic processes across time and space.     

Genomics and museum specimens

Nearly 25 years ago, Allan Wilson and colleagues isolated DNA sequences from museum specimens of kangaroo rats (Dipodomys panamintinus) and compared these sequences with those from freshly collected animals (Thomas et al. 1990 ). The museum specimens had been collected up to 78 years earlier, so the two samples provided a direct temporal comparison of patterns of genetic variation. This was not the first time DNA sequences had been isolated from preserved material, but it was the first time it had been carried out with a population sample. Population geneticists often try to make inferences about the influence of historical processes such as selection, drift, mutation and migration on patterns of genetic variation in the present. The work of Wilson and colleagues was important in part because it suggested a way in which population geneticists could actually study genetic change in natural populations through time, much the same way that experimentalists can do with artificial populations in the laboratory. Indeed, the work of Thomas et al. ( 1990 ) spawned dozens of studies in which museum specimens were used to compare historical and present‐day genetic diversity (reviewed in Wandeler et al. 2007 ). All of these studies, however, were limited by the same fundamental problem: old DNA is degraded into short fragments. As a consequence, these studies mostly involved PCR amplification of short templates, usually short stretches of mitochondrial DNA or microsatellites. In this issue, Bi et al. ( 2013 ) report a breakthrough that should open the door to studies of genomic variation in museum specimens. They used target enrichment (exon capture) and next‐generation (Illumina) sequencing to compare patterns of genetic variation in historic and present‐day population samples of alpine chipmunks (Tamias alpinus) (Fig. 1). The historic samples came from specimens collected in 1915, so the temporal span of this comparison is nearly 100 years.     

Historical DNA reveals the phylogenetic position of the extinct Alpine lynx

During the last two centuries, lynx populations have undergone severe declines and extinctions in Europe. The Alpine lynx, once distributed across the whole Alpine arc, became extinct due to direct human prosecution and deprivation of its main prey in the 1930s. Similar to the Iberian lynx Lynx pardinus , its taxonomy has been subject to several controversies. Moreover, knowing the taxonomic status of the Alpine lynx will help to define conservation units of extant lynx populations in Europe. In this study, we investigated two mitochondrial DNA regions in museum specimens ( n =15) representing the autochthonous Alpine population and in samples from extant Eurasian lynx Lynx lynx populations in Europe and Asia ( n =17). Phylogenetic analysis (cytochrome b , 345 bp) placed the Alpine lynx within the Eurasian lynx lineage. Among all individuals examined, seven different haplotypes (control region, 300 bp) were observed but no unique Alpine haplotype was discovered. Haplotypes of the extinct Alpine population were identical to previously described haplotypes in Scandinavian lynx signifying a recent genetic ancestry with current European populations. Moreover, our genetic data suggest two distinct glacial refugia for the Carpathian and Balkan population. Overall this study demonstrates that historical DNA from extinct populations can help to disentangle the phylogenetic relationships and historical biogeography of taxa with only a limited number of extant populations remaining.     

Insight into Danube sturgeon life history: trace element assessment in pectoral fin rays

Sturgeon populations in the Danube River have experienced severe decline during the last several decades, mostly due to the poorly regulated fishery, river fragmentation and water pollution. This study focuses on gaining better understanding of sturgeon life history primarily by addressing the assessment of microelement accumulation in sturgeon pectoral fin rays, especially of strontium and calcium, as a method that can reveal migration patterns of anadromous sturgeons. Analysis was performed on pectoral fin samples of three anadromous Danube sturgeon species (beluga, Russian sturgeon and stellate sturgeon) by the use of a Nuclear Microprobe technique. The most frequent pattern in analyzed samples was represented by a low Sr:Ca ratio in the innermost annuli, followed by an increased ratio in the middle annuli segment, and often with a decreased ratio in the outermost annuli. Probability density estimate has revealed three distinguished maxima of the Sr:Ca ratio, 7.08 × 10⁻³, 8.98 × 10⁻³ and 9.90 × 10⁻³, which might correspond, respectively, to fresh, brackish and saltwater. Although the analysis of the Sr:Ca ratio in sturgeon pectoral fin rays has revealed changes that might indicate probable migration between habitats with different water salinity, further studies are needed for improvement of this method. This study represents the first analysis of this kind that was conducted on sturgeon species from the Black Sea basin.     

DNA amounts in the nuclei ofParamecium aurelia andTetrahymena pyriformis

DNA amounts have been determined in the micronuclei and macronuclei of 8 strains ofParamecium aurelia and 6 strains ofTetrahymena pyriformis. In the case ofTetrahymena a distribution of values for the amount of DNA in the macronuclei of all the strains was observed but the lowest values were approximately the same, viz. 1.17×10⁻¹¹ g. There are two groups of strains in relation to micronuclear DNA values ofTetrahymena, one giving an average of 0.36×10⁻¹² g and the other 0.815×10⁻¹² g. The ratio of MIC/MAC DNA varies in the two groups.Paramecium again has a range of macronuclear values within each stock—lowest value 2.51×10⁻¹⁰ g—and the micronuclear values are similar in all stocks—approximately 0.613×10⁻¹² g. The ratio of MIC/MAC DNA is similar in each stock.—The haploid genome values calculated from these data show excellent agreement with the values obtained by DNA renaturation studies. Supported by a Research Grant B/SR/8276 from the Science Research Council. The Vickers densitometer was purchased with a grant from the Medical Research Council.     

Genetic variation in time and space: the use of herbarium specimens to reconstruct patterns of genetic variation in the endangered orchid Anacamptis palustris

Habitat alteration, fragmentation and destruction as a consequence of human impact are a global phenomenon. Here we document changes in genetic variation in the marsh orchid Anacamptis palustris as a consequence of such habitat changes. We examined historical specimens that were collected during the nineteenth and early twentieth centuries, prior to the most recent massive habitat changes affecting this species. Sequences of a hypervariable region in the plastid DNA, located in the tRNA^LEU intron, from herbarium vouchers were compared with those from a near-exhaustive survey of the extant A. palustris populations on the Italian peninsula. It was found that private haplotypes and alleles found in small, extant populations were once widespread and more common in historic populations and that alleles, once present in historic populations, have gone extinct. In addition, genetic differentiation among populations has increased over time and haplotype frequencies significantly differ among historic and extant populations. These results document that human induced habitat changes have reduced genetic diversity and increased the importance of random genetic drift in this species. It is concluded that the analysis of herbarium specimens may provide important insights into changes of genetic diversity over time and may be critical for correct inference of the evolutionary history of rare and endangered species.