Since the decoding of the human genome, the quest to obtain more and more molecular information from smaller and smaller samples is intensifying. Today the burden of this challenge is being borne by ...planar arrays, but the quality of the data provided by this approach is limited by variations in performance between different arrays. Suspension arrays of encoded microspheres provide higher quality data, but the amount of molecular information that can be acquired with them is limited by the number of codes that can be distinguished in the same sample. New methods of preparing encoded particles promise to alleviate this problem, but in the face of a growing number of new technologies it is sometimes difficult to decide which, if any, will succeed. Herein we appraise these new forms of encoded particle critically, and ask if they can deliver the necessary multiplexing power and whether they will perform well in multiplexed assays.
Faster, smaller, more sensitive: Since the decoding of the human genome a defining trait has been the quest to obtain more and more molecular information from smaller and smaller samples. Latex microspheres encoded with fluorescent dyes allow dozens of molecules to be detected in the same sample at the same time, but the race is now on to develop new types of encoded particle that can extract even more information.
Extended breath-hold endurance enables the exploitation of the aquatic niche by numerous mammalian lineages and is accomplished by elevated body oxygen stores and adaptations that promote their ...economical use. However, little is known regarding the molecular and evolutionary underpinnings of the high muscle myoglobin concentration phenotype of divers. We used ancestral sequence reconstruction to trace the evolution of this oxygen-storing protein across a 130-species mammalian phylogeny and reveal an adaptive molecular signature of elevated myoglobin net surface charge in diving species that is mechanistically linked with maximal myoglobin concentration. This observation provides insights into the tempo and routes to enhanced dive capacity evolution within the ancestors of each major mammalian aquatic lineage and infers amphibious ancestries of echidnas, moles, hyraxes, and elephants, offering a fresh perspective on the evolution of this iconic respiratory pigment.
Sex differences in dioecious animals are pervasive and result from gene expression differences. Elevated sexual selection has been predicted to increase the number and expression of male-biased ...genes, and experimentally imposing monogamy on Drosophila melanogaster has led to a relative feminisation of the transcriptome. Here, we test this hypothesis further by subjecting another polyandrous species, D. pseudoobscura, to 150 generations of experimental monogamy or elevated polyandry. We find that sex-biased genes do change in expression but, contrary to predictions, there is usually masculinisation of the transcriptome under monogamy, although this depends on tissue and sex. We also identify and describe gene expression changes following courtship experience. Courtship often influences gene expression, including patterns in sex-biased gene expression. Our results confirm that mating system manipulation disproportionately influences sex-biased gene expression but show that the direction of change is dynamic and unpredictable.
How do organisms respond adaptively to environmental stress? Although some gene-specific responses have been explored, others remain to be identified, and there is a very poor understanding of the ...system-wide integration of response, particularly in complex, multitissue animals. Here, we adopt a transcript screening approach to explore the mechanisms underpinning a major, whole-body phenotypic transition in a vertebrate animal that naturally experiences extreme environmental stress. Carp were exposed to increasing levels of cold, and responses across seven tissues were assessed by using a microarray composed of 13,440 cDNA probes. A large set of unique cDNAs (≈3,400) were affected by cold. These cDNAs included an expression signature common to all tissues of 252 up-regulated genes involved in RNA processing, translation initiation, mitochondrial metabolism, proteasomal function, and modification of higher-order structures of lipid membranes and chromosomes. Also identified were large numbers of transcripts with highly tissue-specific patterns of regulation. By unbiased profiling of gene ontologies, we have identified the distinctive functional features of each tissue's response and integrate them into a comprehensive view of the whole-body transition from one strongly adaptive phenotype to another. This approach revealed an expression signature suggestive of atrophy in cooled skeletal muscle. This environmental genomics approach by using a well studied but nongenomic species has identified a range of candidate genes endowing thermotolerance and reveals a previously unrecognized scale and complexity of responses that impacts at the level of cellular and tissue function.
We report a microarray format for the detection of proteins and protein functionality (kinase activity) based on marking either specific antibody−protein binding or peptide phosphorylation events by ...attachment of gold nanoparticles followed by silver deposition for signal enhancement. The attachment of the gold nanoparticles is achieved by standard avidin−biotin chemistry. The detection principle is resonance light scattering. Highly selective recognition of standard proteins (proteins A and G) down to 1 pg/mL for proteins in solution and 10 fg for proteins on the microarray spots is demonstrated. Enzyme activity of the kinase (PKA) is detected with high specificity down to a limit of 1 fg for an established peptide substrate (kemptide) on the microarray spots. Kinase inhibition by the inhibitor (H89) is shown, demonstrating the potential for high-throughput screening for inhibitors.
Hypoxic vasodilation is a physiological response to low oxygen tension that increases blood supply to match metabolic demands. Although this response has been characterized for >100 years, the ...underlying hypoxic sensing and effector signaling mechanisms remain uncertain. We have shown that deoxygenated myoglobin in the heart can reduce nitrite to nitric oxide (NO·) and thereby contribute to cardiomyocyte NO· signaling during ischemia. On the basis of recent observations that myoglobin is expressed in the vasculature of hypoxia-tolerant fish, we hypothesized that endogenous nitrite may contribute to physiological hypoxic vasodilation via reactions with vascular myoglobin to form NO·.
We show in the present study that myoglobin is expressed in vascular smooth muscle and contributes significantly to nitrite-dependent hypoxic vasodilation in vivo and ex vivo. The generation of NO· from nitrite reduction by deoxygenated myoglobin activates canonical soluble guanylate cyclase/cGMP signaling pathways. In vivo and ex vivo vasodilation responses, the reduction of nitrite to NO·, and the subsequent signal transduction mechanisms were all significantly impaired in mice without myoglobin. Hypoxic vasodilation studies in myoglobin and endothelial and inducible NO synthase knockout models suggest that only myoglobin contributes to systemic hypoxic vasodilatory responses in mice.
Endogenous nitrite is a physiological effector of hypoxic vasodilation. Its reduction to NO· via the heme globin myoglobin enhances blood flow and matches O(2) supply to increased metabolic demands under hypoxic conditions.
Post copulatory interactions between the sexes in internally fertilizing species elicits both sexual conflict and sexual selection. Macroevolutionary and comparative studies have linked these ...processes to rapid transcriptomic evolution in sex‐specific tissues and substantial transcriptomic post mating responses in females, patterns of which are altered when mating between reproductively isolated species. Here, we tested multiple predictions arising from sexual selection and conflict theory about the evolution of sex‐specific and tissue‐specific gene expression and the post mating response at the microevolutionary level. Following over 150 generations of experimental evolution under either reduced (enforced monogamy) or elevated (polyandry) sexual selection in Drosophila pseudoobscura, we found a substantial effect of sexual selection treatment on transcriptomic divergence in virgin male and female reproductive tissues (testes, male accessory glands, the female reproductive tract and ovaries). Sexual selection treatment also had a dominant effect on the post mating response, particularly in the female reproductive tract – the main arena for sexual conflict – compared to ovaries. This effect was asymmetric with monandry females typically showing more post mating responses than polyandry females, with enriched gene functions varying across treatments. The evolutionary history of the male partner had a larger effect on the post mating response of monandry females, but females from both sexual selection treatments showed unique patterns of gene expression and gene function when mating with males from the alternate treatment. Our microevolutionary results mostly confirm comparative macroevolutionary predictions on the role of sexual selection on transcriptomic divergence and altered gene regulation arising from divergent coevolutionary trajectories between sexual selection treatments.
Fish offer important advantages for defining the organism-environment interface and responses to natural or anthropogenic stressors. Genomic approaches using fish promise increased investigative ...power, and have already provided insights into the mechanisms that underlie short-term and long-term environmental adaptations. The range of fish species for which genomic resources are available is increasing, but will require significant further expansion for the optimal application of fish environmental genomics.
We have reconstructed the events that led to the evolution of a key physiological innovation underpinning the large adaptive radiation of fishes, namely their unique ability to secrete molecular ...oxygen (O₂). We show that O₂ secretion into the swimbladder evolved some 100 million years after another O₂-secreting system in the eye. We unravel the likely sequence in which the functional components of both systems evolved. These components include ocular and swimbladder countercurrent exchangers, the Bohr and Root effects, the buffering power and surface histidine content of hemoglobins, and red blood cell Na⁺/H⁺ exchange activity. Our synthesis reveals the dynamics of gains and losses of these multiple traits over time, accounting for part of the huge diversity of form and function in living fishes.
The Hawaiian strain (CB4856) of Caenorhabditis elegans is one of the most divergent from the canonical laboratory strain N2 and has been widely used in developmental, population, and evolutionary ...studies. To enhance the utility of the strain, we have generated a draft sequence of the CB4856 genome, exploiting a variety of resources and strategies. When compared against the N2 reference, the CB4856 genome has 327,050 single nucleotide variants (SNVs) and 79,529 insertion-deletion events that result in a total of 3.3 Mb of N2 sequence missing from CB4856 and 1.4 Mb of sequence present in CB4856 but not present in N2. As previously reported, the density of SNVs varies along the chromosomes, with the arms of chromosomes showing greater average variation than the centers. In addition, we find 61 regions totaling 2.8 Mb, distributed across all six chromosomes, which have a greatly elevated SNV density, ranging from 2 to 16% SNVs. A survey of other wild isolates show that the two alternative haplotypes for each region are widely distributed, suggesting they have been maintained by balancing selection over long evolutionary times. These divergent regions contain an abundance of genes from large rapidly evolving families encoding F-box, MATH, BATH, seven-transmembrane G-coupled receptors, and nuclear hormone receptors, suggesting that they provide selective advantages in natural environments. The draft sequence makes available a comprehensive catalog of sequence differences between the CB4856 and N2 strains that will facilitate the molecular dissection of their phenotypic differences. Our work also emphasizes the importance of going beyond simple alignment of reads to a reference genome when assessing differences between genomes.
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