PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping

Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.     

Ecological genomics of local adaptation in Cornus florida L. by genotyping by sequencing

Discovering local adaptation, its genetic underpinnings, and environmental drivers is important for conserving forest species. Ecological genomic approaches coupled with next‐generation sequencing are useful means to detect local adaptation and uncover its underlying genetic basis in nonmodel species. We report results from a study on flowering dogwood trees (Cornus florida L.) using genotyping by sequencing (GBS). This species is ecologically important to eastern US forests but is severely threatened by fungal diseases. We analyzed subpopulations in divergent ecological habitats within North Carolina to uncover loci under local selection and associated with environmental–functional traits or disease infection. At this scale, we tested the effect of incorporating additional sequencing before scaling for a broader examination of the entire range. To test for biases of GBS, we sequenced two similarly sampled libraries independently from six populations of three ecological habitats. We obtained environmental–functional traits for each subpopulation to identify associations with genotypes via latent factor mixed modeling (LFMM) and gradient forests analysis. To test whether heterogeneity of abiotic pressures resulted in genetic differentiation indicative of local adaptation, we evaluated F_st per locus while accounting for genetic differentiation between coastal subpopulations and Piedmont‐Mountain subpopulations. Of the 54 candidate loci with sufficient evidence of being under selection among both libraries, 28–39 were Arlequin–BayeScan F_st outliers. For LFMM, 45 candidates were associated with climate (of 54), 30 were associated with soil properties, and four were associated with plant health. Reanalysis of combined libraries showed that 42 candidate loci still showed evidence of being under selection. We conclude environment‐driven selection on specific loci has resulted in local adaptation in response to potassium deficiencies, temperature, precipitation, and (to a marginal extent) disease. High allele turnover along ecological gradients further supports the adaptive significance of loci speculated to be under selection.     

New insights into adaptation and population structure of cork oak using genotyping by sequencing

Species respond to global climatic changes in a local context. Understanding this process, including its speed and intensity, is paramount due to the pace at which such changes are currently occurring. Tree species are particularly interesting to study in this regard due to their long generation times, sedentarism, and ecological and economic importance. Quercus suber L. is an evergreen forest tree species of the Fagaceae family with an essentially Western Mediterranean distribution. Despite frequent assessments of the species’ evolutionary history, large‐scale genetic studies have mostly relied on plastidial markers, whereas nuclear markers have been used on studies with locally focused sampling strategies. In this work, “Genotyping by sequencing” is used to derive 1,996 single nucleotide polymorphism markers to assess the species’ evolutionary history from a nuclear DNA perspective, gain insights into how local adaptation is shaping the species’ genetic background, and to forecast how Q. suber may respond to global climatic changes from a genetic perspective. Results reveal (a) an essentially unstructured species, where (b) a balance between gene flow and local adaptation keeps the species’ gene pool somewhat homogeneous across its distribution, but still allowing (c) variation clines for the individuals to cope with local conditions. “Risk of Non‐Adaptedness” (RONA) analyses suggest that for the considered variables and most sampled locations, (d) the cork oak should not require large shifts in allele frequencies to survive the predicted climatic changes. Future directions include integrating these results with ecological niche modeling perspectives, improving the RONA methodology, and expanding its use to other species. With the implementation presented in this work, the RONA can now also be easily assessed for other organisms.     

Checkpoint adaptation precedes spontaneous and damage-induced genomic instability in yeast

Despite the fact that eukaryotic cells enlist checkpoints to block cell cycle progression when their DNA is damaged, cells still undergo frequent genetic rearrangements, both spontaneously and in response to genotoxic agents. We and others have previously characterized a phenomenon (adaptation) in which yeast cells that are arrested at a DNA damage checkpoint eventually override this arrest and reenter the cell cycle, despite the fact that they have not repaired the DNA damage that elicited the arrest. Here, we use mutants that are defective in checkpoint adaptation to show that adaptation is important for achieving the highest possible viability after exposure to DNA-damaging agents, but it also acts as an entrée into some forms of genomic instability. Specifically, the spontaneous and X-ray-induced frequencies of chromosome loss, translocations, and a repair process called break-induced replication occur at significantly reduced rates in adaptation-defective mutants. This indicates that these events occur after a cell has first arrested at the checkpoint and then adapted to that arrest. Because malignant progression frequently involves loss of genes that function in DNA repair, adaptation may promote tumorigenesis by allowing genomic instability to occur in the absence of repair.     

Simulation of spontaneous discharge and short-term adaptation in acoustic nerve fibers

A model for firing of the auditory nerve fibres was carried out on a general purpose digital computer. In the model a noise with small correlation time and with assigned standard deviation (when modeling a spontaneous discharge) or a sum of a noise and a determinated signal (when modeling an elicited discharge) is compared with incremental threshold. When the threshold is exceeded a spike occurs and the threshold is increased. The threshold qualitative properties and quantitative values were chosen in a way to provide the most reliable patterns of spontaneous discharge, according to the literature data obtained from the cat's auditory nerve. When stimulated by tone-bursts the model reveals intrinsic ability of mimicking the phenomena of discharge rate short-term adaptation. Thus according to our model the short-term adaptation is entirely due to the properties of the incremental threshold.     

Red cell genotyping and the future of pretransfusion testing

The ABO blood group, based on molecular biological detection technology, has the advantages of simple operation, high sensitivity, and standardized result interpretation, and is not affected by sample immunological characteristics. However, clinically, performance verification, clinical application scope, quality management, abnormal result processing, and other issues associated with the ABO blood group molecular detection technology are relatively complex, and there is a lack of unified norms and standards. Therefore, from the perspective of the whole process of ABO molecular biology detection, this study aims to provide standardized opinions on important links affecting the detection results, common problems encountered in the detection process, and the assessment and treatment of abnormal results. Finally, a Chinese expert consensus on molecular biological technology based on genotyping and sequencing detection was put forward, which standardizes the detection process, improves the accuracy of results, and promotes the development of technology and broader clinical application.     

Single tube genotyping of sickle cell anaemia using PCR-based SNP analysis

Allele-specific amplification (ASA) is a generally applicable technique for the detection of known single nucleotide polymorphisms (SNPs), deletions, insertions and other sequence variations. Conventionally, two reactions are required to determine the zygosity of DNA in a two-allele system, along with significant upstream optimisation to define the specific test conditions. Here, we combine single tube bi-directional ASA with a ‘matrix-based’ optimisation strategy, speeding up the whole process in a reduced reaction set. We use sickle cell anaemia as our model SNP system, a genetic disease that is currently screened using ASA methods. Discriminatory conditions were rapidly optimised enabling the unambiguous identification of DNA from homozygous sickle cell patients (HbS/S), heterozygous carriers (HbA/S) or normal DNA in a single tube. Simple downstream mathematical analyses based on product yield across the optimisation set allow an insight into the important aspects of priming competition and component interactions in this competitive PCR. This strategy can be applied to any polymorphism, defining specific conditions using a multifactorial approach. The inherent simplicity and low cost of this PCR-based method validates bi-directional ASA as an effective tool in future clinical screening and pharmacogenomic research where more expensive fluorescence-based approaches may not be desirable.     

Molecular characterization of spontaneous mesenchymal stem cell transformation

Background

We previously reported the in vitro spontaneous transformation of human mesenchymal stem cells (MSC) generating a population with tumorigenic potential, that we termed transformed mesenchymal cells (TMC).

Methodology/Principal Findings

Here we have characterized the molecular changes associated with TMC generation. Using microarrays techniques we identified a set of altered pathways and a greater number of downregulated than upregulated genes during MSC transformation, in part due to the expression of many untranslated RNAs in MSC. Microarray results were validated by qRT-PCR and protein detection.

Conclusions/Significance

In our model, the transformation process takes place through two sequential steps; first MSC bypass senescence by upregulating c-myc and repressing p16 levels. The cells then bypass cell crisis with acquisition of telomerase activity, Ink4a/Arf locus deletion and Rb hyperphosphorylation. Other transformation-associated changes include modulation of mitochondrial metabolism, DNA damage-repair proteins and cell cycle regulators. In this work we have characterized the molecular mechanisms implicated in TMC generation and we propose a two-stage model by which a human MSC becomes a tumor cell.     

Classification of cell signalling in tissue development

The traditional classification of signalling in biological systems is insufficient and outdated and novel efforts must take into account advances in systems theory, information theory and linguistics. We present some of the classification systems currently used both within and outside of the biological field and discuss some specific aspects of the nature of signalling in tissue development. The analytical methods used in understanding non-biological networks provide a valuable vocabulary, which requires integration and a system of classification to further facilitate development.     

Ultrastructural characteristics of mitochondria during cell adaptation to rotenone

A study was made of respiration, heat production, K+ output and ultrastructure of wheat root cells treated for 6 h with rotenone (10 microM), an inhibitor of HADH-ubiquinone oxidoreductase (Complex I). Besides, the involvement of alternative pathways for adaptation to this inhibitor was studied. After 20 min of treatment, a brightened mitochondrial matrix and mitochondria with torus shapes were observed. We propose that the outer area of mitochondria increases due to their torus shapes, and this can point to the activating of extremal NAD(P)H-dehydrogenase, which uses enternal NAD(P)H. Further on the normal ultrastructure of mitochondria was observed, which may result from activation of succinate dehydrogenase and rotenone resistant NAD(P)H-dehydrogenase. After 1 h of treatment, a decrease in respiration, heat production, K+ output and pH increase of incubation medium were observed. Starting from 2 h of incubation and up to the end of the experiment, an increase of respiration and heat production was observed, pointing to the activation of oxidative phosphorilation. Besides, re-entry of K+ and pH decrease in the incubation medium were observed. We conclude that these findings may indicate to a possible adaptation of root cells to this inhibitor. We propose that the torus shape of mitochondria may be associated with function of external NAD(P)H-dehydrogenase.     

Mechanism of spontaneous inside-out vesiculation of red cell membranes

In certain conditions, human red cell membranes spontaneously form inside out vesicles within 20 min after hypotonic lysis. Study of the geometry of this process now reveals that, contrary to earlier views of vesiculation by endocytosis or by the mechanical shearing of cytoskeleton-depleted membrane, lysis generates a persistent membrane edge which spontaneously curls, cuts, and splices the membrane surface to form single or concentric vesicles. Analysis of the processes by which proteins may stabilize a free membrane edge led us to formulate a novel zip-type mechanism for membrane cutting-splicing and fusion even in the absence of free edges. Such protein-led membrane fusion represents an alternative to mechanisms of membrane fusion based on phospholipid interactions, and may prove relevant to processes of secretion, endocytosis, phagocytosis, and membrane recycling in many cell types.     

Enzymatic studies during spontaneous cell rupture of Saccharomyces cerevisiae

Summary Some of the extract and intracellular enzyme activities in K2nB strain of Saccharomyces cerevisiae that growing in the condition which induce spontaneous cell rupture, were measured. B-1-3-glucanase, invertase, acid phosphatase and active chitin synthetase zymogen showed a reduced activity in ruptured cell while alkaline phosphatase shows no differences in its activity.     

On spontaneous mutagenesis and cell cultivation conditions.

The leu2 revertant content of a Saccharomyces cerevisiae cell culture increases as the leucine concentration in the nutrient solid medium decreases. Reversions form in the S-phase of the cell cycle. If a cell culture from a medium with a low concentration of leucine containing the revertants which have just formed is transferred on a medium with a normal or higher than normal leucine content, these 'newborn' revertants disappear at the end of the G1-phase or at the beginning of the S-phase of the next cell cycle. These data can be explained either by a difference in the ability of revertants formed in the culture to compete with the cells of the initial strain on different media, or on the basis of the intermediate heteroduplex model proposed by F.W. Stahl (1988).     

Phylogeography and adaptation genetics of stickleback from the Haida Gwaii archipelago revealed using genome‐wide single nucleotide polymorphism genotyping

Threespine stickleback populations are model systems for studying adaptive evolution and the underlying genetics. In lakes on the Haida Gwaii archipelago (off western Canada), stickleback have undergone a remarkable local radiation and show phenotypic diversity matching that seen throughout the species distribution. To provide a historical context for this radiation, we surveyed genetic variation at >1000 single nucleotide polymorphism (SNP) loci in stickleback from over 100 populations. SNPs included markers evenly distributed throughout genome and candidate SNPs tagging adaptive genomic regions. Based on evenly distributed SNPs, the phylogeographic pattern differs substantially from the disjunct pattern previously observed between two highly divergent mtDNA lineages. The SNP tree instead shows extensive within watershed population clustering and different watersheds separated by short branches deep in the tree. These data are consistent with separate colonizations of most watersheds, despite underlying genetic connections between some independent drainages. This supports previous suppositions that morphological diversity observed between watersheds has been shaped independently, with populations exhibiting complete loss of lateral plates and giant size each occurring in several distinct clades. Throughout the archipelago, we see repeated selection of SNPs tagging candidate freshwater adaptive variants at several genomic regions differentiated between marine–freshwater populations on a global scale (e.g. EDA, Na/K ATPase). In estuarine sites, both marine and freshwater allelic variants were commonly detected. We also found typically marine alleles present in a few freshwater lakes, especially those with completely plated morphology. These results provide a general model for postglacial colonization of freshwater habitat by sticklebacks and illustrate the tremendous potential of genome‐wide SNP data sets hold for resolving patterns and processes underlying recent adaptive divergences.     

High cell density induces spontaneous bifurcations of dissolved oxygen controllers during CHO cell fermentations

High cell density cultures of CHO cells growing in a bioreactor under dissolved oxygen control were found to undergo spontaneous bifurcations and a subsequent loss of stability some time into the fermentation. This loss of stability was manifested by sustained and amplified oscillations in the bioreactor dissolved oxygen concentration and in the oxygen gas flow rate to the reactor. To identify potential biological and operational causes for the phenomenon, linear stability analysis was applied in a neighborhood of the experimentally observed bifurcation point. The analysis revealed that two steady state process gains, K(P1) and K(P2), regulated k(l)a and gas phase oxygen concentration inputs, respectively, and the magnitude of K(P1) was found to determine system stability about the bifurcation point. The magnitude of K(P1), and hence the corresponding open-loop steady state gain K(OL1), scaled linearly with the bioreactor cell density, increasing with increasing cell density. These results allowed the generation of a fermentation stability diagram, which partitioned K(C)-N operating space into stable and unstable regions separated by the loci of predicted critically stable controller constants, K(C,critical), as a function of bioreactor cell density. This consistency of this operating diagram with experimentally observed changes in system stability was demonstrated. We conclude that time-dependent increases in cell density are the cause of the observed instabilities and that cell density is the critical bifurcation parameter. The results of this study should be readily applicable to the design of a more robust controller.