Competitive Hybridization Research Articles

The increasing spread of the European barbel in the Italian large lowland rivers is threatening the native species

Freshwater ecosystems are heavily altered by human activities, with anthropic introductions of non-native species substantially contributing to their biotic degradation. The invasion by alien species can alter ecosystem balances with direct and indirect impacts across different levels of biotic organization. The number of invasive alien species is particularly high in Mediterranean fresh waters including rivers draining in the northern Adriatic basins. Here, the Padanian barbel Barbus plebejus endemic to the Padano-Venetian district, is threatened by the introduction of the European barbel Barbus barbus via competition and introgressive hybridization. In this study, we genetically characterised using the partial mitochondrial DNA Cytochrome b gene, barbels present in two main tributaries of the Po River, the lower Ticino and Oglio rivers where the two species are suspected to co-occur. Since the two river sections are highly regulated and present severe hydrological alterations caused by dams and other hydraulic structures, the aim of the study was to provide information on the barbel populations composition in these systems to foster the implementation of more suitable management plans aimed at the conservation of the native species. A total of 86 barbel have been analysed (50 in the Ticino River and 36 in the Oglio River) corresponding to 13 haplotypes of which 8 were phylogenetically attributed to B. barbus and five to B. plebejus. The high haplotypic diversity and the unimodal trend evidenced by the mismatch distribution analysis for B. barbus haplotypes support a potential demographic expansion. Altogether, these results highlighted that B. barbus is progressively expanding in northern Italy while posing a serious risk for the conservation of the native B. plebejus, stressing the need to actively focus ecosystem and fisheries management regulations to stop B. barbus from spreading further throughout northern Italy.

Competitive aptamer switch for modulating ligand binding affinity

Aptamers are short, single-stranded DNA or RNA molecules that selectively bind to a target molecule. Aptamer-complement duplex (ACD) is often used to design molecular switches capable of producing a detectable signal or triggering a structural change upon aptamer binding to a target. However, such aptamer switch generally faces an increased dissociation constant (Kd) due to the energy barrier of the complementary duplex. We reported a competitive hybridization mechanism to modulate the binding affinity of an ACD to a target adenosine. Using the computation-guided design, we calculated the aptamer folding energy for the duplex length from 11-nt to 15-nt, and experimentally measured increased apparent Kd values resulted from these extended duplexes. Using a set of strands to compete with the ACD hybridization, it reduced the aptamer folding energy to facilitate aptamer switches with decreased apparent Kd values ranging from over 400 μM without a competing strand to ∼30 μM with a competing strand. This competitive aptamer switch was also found sensitive to single-nucleotide mutations of a competing strand. Our work provides an approach to modulate the binding affinity and the sensitivity of aptamer-complement duplexes, which could be useful in the nucleic acids-based sensing and nanomedicine.

Rapid detection of SARS-CoV-2 genetic targets using nanoporous waveguide based competitive displacement assay

Rapid detection of unlabeled SARS-CoV-2 genetic target was demonstrated using a competitive displacement hybridization assay made by a nanostructured anodized alumina oxide (AAO) membrane. The assay applied the toehold-mediated strand displacement reaction. The nanoporous surface of the membrane was functionalized with a complementary pair consisting of Cy3-labeled probe and quencher-labeled nucleic acids through a chemical immobilization process. In the presence of the unlabeled SARS-CoV-2 target, the quencher-tagged strand of the immobilized probe-quencher duplex was separated from the Cy3-modifed strand. A stable probe-target duplex formed and regained a strong fluorescence signal, thus enabling real-time and label-free SARS-CoV-2 detection. Assay designs with different numbers of base pair (bp) matches were synthesized to compare their affinities. Because of the large surface of a free-standing nanoporous membrane, two orders enhancement of the fluorescence was observed, where the detection limit of the unlabeled concentration can be improved to 1 nM. The assay was miniaturized by integrating a nanoporous AAO layer onto an optical waveguide device. The detection mechanism and the sensitivity improvement of the AAO-waveguide device were illustrated from the finite difference method (FDM) simulation and the experimental results. Light-analyte interaction was further improved due to the presence of the AAO layer, which created an intermediate refractive index and enhanced the waveguide's evanescent field. Our competitive hybridization sensor is an accurate and label-free testing platform applicable to the deployment of compact and sensitive virus detection strategies.

Asymptotic Dynamics of a Three-Species Hybrid Competition System

Asymptotic Dynamics of a Three-Species Hybrid Competition System

The complete mitochondrial genome of the Critically Endangered Saba Green Iguana, Iguana iguana (Squamata: Iguanidae)

The populations of native iguanas in the Caribbean Lesser Antilles are threatened by the wide occurrence and spread of non-native iguanas. Until recently, competitive hybridization was not believed to threaten the Saba Green Iguana, a subpopulation of Iguana iguana (Linnaeus, 1758) from the island of Saba. However, the arrival of non-native iguanas has put the native population at risk, leading to a change in the conservation status of the Saba Green Iguana to Critically Endangered, according to guidelines from the International Union for the Conservation of Nature. Here, we generated the complete mitogenome of the Saba Green Iguana using Oxford Nanopore long-read technology. The mitogenome is 16,626 bp long and has 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region (1194 bp). Noteworthy, this is only the second published mitogenome for the Iguana iguana species complex, despite the known high intraspecific genetic variation.

An integrative approach to assess non‐native iguana presence on Saba and Montserrat: Are we losing all native Iguana populations in the Lesser Antilles?

AbstractInvasive alien species are among the main drivers of the ongoing sixth mass extinction wave, especially affecting island populations. Although the Caribbean is well‐known for its high species richness and endemism, also for reptiles, equally important is the regional contribution of non‐native species to island biodiversity. The Lesser Antilles encompass high genetic diversity in Iguana, though most native populations either have gone extinct or are declining following competitive hybridization with invasive non‐native green iguanas. Here, we assessed non‐native presence in two poorly‐studied native melanistic Iguana iguana populations using available genetic tools and explored utilizing size‐dependent body measurements to discriminate between native and non‐native iguanas. Genetic samples from Saba and Montserrat were genotyped across 17 microsatellite loci with STRUCTURE, and multivariate analyses indicating non‐native iguana presence only on Saba. This was corroborated by mtDNA and nDNA sequences, highlighting a non‐native origin in Central America and the ABC islands. We identified preliminary evidence suggestive of hybridization. Morphological variation among size‐dependent characteristics showed that non‐native iguanas have significantly larger subtympanic plates than native iguanas. Non‐native individuals also differed in scalation and coloration patterns. Overall, our findings demonstrate the need for continuous monitoring of non‐native iguanas within remaining native Iguana populations in the Lesser Antilles, as those not directly threatened by non‐native green iguanas are restricted to only 8.7% of the historic range. Although genetic data allow for the identification of non‐native or hybrid iguana presence, this field‐to‐lab workflow is time‐consuming. Rapid in‐situ identification of non‐native individuals is crucial for conservation management. In addition to patterns of scalation and coloration, we have highlighted the utility of size‐dependent variables for rapid diagnosis. We urge regional partners to build morphometric databases for native Iguana populations allowing the quick detection of future incursions of non‐native green iguanas and the rapid implementation of effective countermeasures during the early phase of invasion.

An efficient recurrent neural network for defensive Stackelberg game

This study presents a robust and efficient recurrent neural network (RNN) to solve the continuous defensive location problem (CDLP). CDLP is a bilevel mathematical model, where a planner locates various types of defense facilities for stopping a offender from reaching a critical vertex called core in the network. Due to the NP-hardness of the CDLP, RNN is implemented to find satisfactory solutions on four small-sized, two medium-sized and two larger-sized examples of problem. The numerical experiments show that RNN is able to produce promising results with good execution time and precision when compared with the best previous methods including tabu search (TS), imperialist competitive algorithm (ICA) and hybridization of TS with Levenberg–Marquardt algorithm (HTS) and hybridization of ICA with BFGS algorithm (HICA). The objective values of RNN show that this method stops the attacker at a further distance from the core in comparison with other methods. As well, to validate the efficiency of the proposed RNN, statistical criteria are considered and results are analyzed. For small-sized examples, the computational experiments show that the RNN can obtain solutions as the same as exact approaches but in a shorter runtime.

Target-mediated competitive hybridization of hairpin probes for kanamycin detection based on exonuclease III cleavage and DNAzyme catalysis.

Based on aptamer recognition and target-mediated competitive hybridization of hairpin probes, we developed a fluorescence sensor for kanamycin (KAN) detection. The aptamer and KAN binding will open hairpin H1 to release the trigger DNA fragment, which can initiate the competitive hybridization between hairpins H2 and H3. Then, exonuclease III (Exo III) can cleave H2 and H3 to produce numerous DNA3 and DNA4. Through the synergetic hybridization among DNA1, DNA2, DNA3, and DNA4, an active Mg2+-DNAzyme can be formed. The cleavage reaction toward FAM-BHQ-modified DNA2 will produce a high fluorescence signal for KAN assay. Through Exo III-guided cleavage and Mg2+-DNAzyme-based catalysis, the sensor exhibits high sensitivity, with a detection limit of 3.1 fM. This method is robust and has been applied to the detection of KAN in milk and water samples with good accuracy and reliability. Our developed fluorescence sensor exhibits the advantages of simple operation, high sensitivity, and good robustness, which are beneficial for KAN detection in food samples.

Asymptotic behavior of solutions of an ODE-PDE hybrid competition system

We study the large-time behavior of a two-species competition model in a spatially heterogeneous environment and investigate the influence of dispersal strategy on the competition. In particular, we allow one species to exhibit a random dispersal movement while the second species is constrained to a non-spatial movement dynamic. We show that there is a nonnegative threshold number (possibly equal to infinity and determined by both species' local intrinsic growth rates) such that the species adopting the random dispersal strategy persists uniformly in space if its diffusion rate is kept below this number while it goes extinct if its diffusion rate is greater than this number. Furthermore, in the critical case that the diffusing species' movement rate equals this number, exactly two scenarios are possible: the non-diffusing species wins the competition if it has a sink area; otherwise, both species coexist.

Depletion of wild-type target enhances the hybridization-based sensitivity of low-abundant mutation detection by reference capture probes

Nucleic acids duplex formation via hybridization is a crucial reaction in many processes and application across different disciplines. In life sciences the detection of mutations is an important application for which hybridization is used, e.g. in diagnostics via single-nucleotide variants (SNVs). This paper deals with the physico-chemical aspects of hybridization-based detection of low-abundance mutations, which is challenging due to unavoidable competitive hybridization of high-abundant wild type sequence with the low-abundant variants. We apply two experimental methods based on theoretical hybridization models to show how sensing of DNA mutation can be significantly improved. This is implemented on two SNV biomarkers for which we first select a reference capture probe. This is a probe designed to match neither the wild type nor the SNV sequence, but to have an equal affinity to the wild-type as the SNV-matching probe. This allows the mutation-specific signal to be expressed as a ratiometric quantity, leading to increased assay robustness. Secondly, we selectively deplete the wild-type species by introducing an excess of wild-type-specific capture probes, and account for these depletion effects in the theoretical model. We demonstrate the detection of 0.05% mutant species in a wild-type background, which is an improvement of an order of magnitude in the limit of detection in comparison with the no-depletion case. This sensitivity is comparable with digital PCR results, showing performance suitable for e.g. clinical applications in liquid biopsy context. The principles of this work apply to a wide range of hybridization-based DNA biosensing technologies, irrespective of the underlying transducer principle.

Turning coordination environment of 2D nickel-based metal-organic frameworks by π-conjugated molecule for enhancing glucose electrochemical sensor performance

The design and synthesis of the high-efficiency electrochemical sensor is the key to electrochemical conversion technology. This work synthesized Ni-based metal-organic framework (Ni-MOF) nanosheets by a competitive double ligands hybridization strategy. They were used as precursors to construct Ni-MOF@Ni-2,3,6,7,10,11-hexahydroxytriphenylene (HHTP) core@shell structures by introducing π - conjugated molecule, HHTP. EXAFS further confirmed the interaction between Ni atoms and hexahydroxytriphenylene molecules. This process helps to promote more exposure of its active sites and enhance its electrochemical performance to become a high-performance enzyme glucose sensor. After coating, glucose detection sensitivity increased from 139.82 to 2124.90 μA/(mM cm2). These findings lay the foundation for preparing two-dimension hybrid functional MOF materials.

Competitive Hybridization of a Microarray Identifies CMKLR1 as an Up-Regulated Gene in Human Bone Marrow-Derived Mesenchymal Stem Cells Compared to Human Embryonic Fibroblasts.

Mesenchymal stem cells (MSCs) have been widely applied to the regeneration of damaged tissue and the modulation of immune response. The purity of MSC preparation and the delivery of MSCs to a target region are critical factors for success in therapeutic application. In order to define the molecular identity of an MSC, the gene expression pattern of a human bone marrow-derived mesenchymal stem cell (hBMSC) was compared with that of a human embryonic fibroblast (hEF) by competitive hybridization of a microarray. A total of 270 and 173 genes were two-fold up- and down-regulated with FDR < 0.05 in the hBMSC compared to the hEF, respectively. The overexpressed genes in the hBMSC over the hEF, including transcription factors, were enriched for biological processes such as axial pattern formation, face morphogenesis and skeletal system development, which could be expected from the differentiation potential of MSCs. CD70 and CD339 were identified as additional CD markers that were up-regulated in the hBMSC over the hEF. The differential expression of CD70 and CD339 might be exploited to distinguish hEF and hBMSC. CMKLR1, a chemokine receptor, was up-regulated in the hBMSC compared to the hEF. RARRES2, a CMKLR1 ligand, stimulated specific migration of the hBMSC, but not of the hEF. RARRES2 manifested as ~two-fold less effective than SDF-1α in the directional migration of the hBMSC. The expression of CMKLR1 was decreased upon the osteoblastic differentiation of the hBMSC. However, the RARRES2-loaded 10% HA-silk scaffold did not recruit endogenous cells to the scaffold in vivo. The RARRES2–CMKLR1 axis could be employed in recruiting systemically delivered or endogenous MSCs to a specific target lesion.

Characterizing Aptamers with Reconfigurable Chiral Plasmonic Assemblies.

Aptamers have emerged as versatile affinity ligands and as promising alternatives to protein antibodies. However, the inconsistency in the reported affinities and specificities of aptamers has greatly hindered the development of aptamer-based applications. Herein, we present a strategy to characterize aptamers by using DNA origami-based chiral plasmonic assemblies as reporters and establishing a competitive hybridization reaction-based thermodynamic model. We demonstrate the characterization of several DNA aptamers, including aptamers for small molecules and macromolecules, as well as aptamers with high and low affinities. The presented characterization scheme can be readily adapted to a wide selection of aptamers. We anticipate that our approach will advance the development of aptamer-based applications by enabling reliable and reproducible characterization of aptamers.

Lessons of the Cold War

Lessons of the Cold War

Two-Step Competitive Hybridization Assay: A Method for Analyzing Cancer-Related microRNA Embedded in Extracellular Vesicles.

With an increased understanding of the role of microRNAs (miRNAs) in cancer evolution, there is a growing interest in the use of these non-coding nucleic acids in cancer diagnosis, prognosis, and treatment monitoring. miRNAs embedded in extracellular vesicles (EVs) are of particular interest given that circulating EVs carry cargo that are strongly correlated to their cells of origin such as tumor cells while protecting them from degradation. As such, there is a tremendous interest in new simple-to-operate vesicular microRNA analysis tools for widespread use in performing liquid biopsies. Herein, we present a two-step competitive hybridization assay that is rationally designed to translate low microRNA concentrations to large electrochemical signals as the measured signal is inversely proportional to the microRNA concentration. Using this assay, with a limit-of-detection of 122 aM, we successfully analyzed vesicular miRNA 200b from prostate cancer cell lines and human urine samples, demonstrating the expected lower expression levels of miRNA 200b in the EVs from prostate cancer cells and in the prostate cancer patient's urine samples compared to healthy patients and non-tumorigenic cell lines, validating the suitability of our approach for clinical analysis.

The Life Cycle of Converging Industries: The Evolution of the Tablet Sector and Its Impact on Competitive Dynamics

Background of the study. In recent years a series of academic research projects in the economic managerial field have investigated the relationship between innovation and industrial evolution, providing new interpretive keys to improve the understanding of one of the most important events in the industries&amp;rsquo; transformation in the current economy, that of digital convergence.&#x0D; &#x0D; Purpose of the paper and methodology. This research work provides an original contribution to the question of how market convergence affects industry evolution. The paper analyzes the convergence process that has influenced the evolution of the tablet sector and the dynamics of entry, exit, innovation and competition over the industry life cycle. From a methodological point of view, a historical-longitudinal&amp;nbsp;study was carried out, which was aimed at examining &amp;ndash; supported by a qualitative/quantitative analysis &amp;ndash; the sequence of events that, over 30 years, influenced the development of the tablet industry. &#x0D; &#x0D; Findings and implications. From the analysis some interesting findings emerged. Firstly, it was found that the life cycle curve of the converging tablet industry defined on the basis of sales data followed the &amp;lsquo;S-shaped pattern&amp;rsquo; empirically detected by product and industry life cycle studies. Secondly, we have verified that the evolution of firm population and the level of product innovation in the tablet industry are consistent with two temporal patterns that characterize the evolutionary model, identified by evolutionary economics and technology management studies. It has also been found that the sectoral convergence process has affected the dynamics of competition in the tablet industry. It emerged, in fact, that the leading companies in introductory stages of the development of the tablet market &amp;ndash; which came from the personal computer industry &amp;ndash; quickly lost their market position in favor to newcomer firms that came from different converging sectors. The analysis has also made it possible to highlight that tablet market leaders tend to compete with each other in multiple sectors within the scope of a broader convergent mobile digital device market. Therefore, a &amp;lsquo;hybrid competition&amp;rsquo; seems to have been affirmed between technological devices &amp;ndash; smartphones, notebooks, tablets, smartwatches, e-readers &amp;ndash; different in shape, but united by satisfying, in different ways, a need for simplified access on the move to a series of advanced digital functions and services.

Direct Amperometric Sensing of Fish Nodavirus RNA Using Gold Nanoparticle/DNA-Based Bioconjugates.

We describe the design of a simple and highly sensitive electrochemical bioanalytical method enabling the direct detection of a conserved RNA region within the capsid protein gene of a fish nodavirus, making use of nanostructured disposable electrodes. To achieve this goal, we select a conserved region within the nodavirus RNA2 segment to design a DNA probe that is tethered to the surface of nanostructured disposable screen-printed electrodes. In a proof-of-principle test, a synthetic RNA sequence is detected based on competitive hybridization between two oligonucleotides (biotinylated reporter DNA and target RNA) complimentary to a thiolated DNA capture probe. The method is further validated using extracted RNA samples obtained from healthy carrier Sparus aurata and clinically infected Dicentrarchus labrax fish specimens. In parallel, the sensitivity of the newly described biosensor is compared with a new real-time RT-PCR protocol. The current differences measured in the negative control and in presence of each concentration of target RNA are used to determine the dynamic range of the assay. We obtain a linear response (R2 = 0.995) over a range of RNA concentrations from 0.1 to 25 pM with a detection limit of 20 fM. The results are in good agreement with the results found by the RT-qPCR. This method provides a promising approach toward a more effective diagnosis and risk assessment of viral diseases in aquaculture.

Splitting hares: Current and future ecological niches predicted as distinctly different for two congeneric lagomorphs

The congeneric lagomorphs Lepus timidus and L. europaeus share allopatric distributions in many areas of Europe characterised by competitive exclusion and hybridisation. We investigated prospects for these species under climate change in northern England uplands. We created ensemble models predicting niche realisation for these species, influenced by abiotic and biotic factors, estimating niche overlap in geo-environmental space. The two species occupy distinctly different niches, influenced more by vegetation preferences than climatic differences. The current climate niche for L. timidus featured higher elevations with cooler temperatures and 168 km2 range extent. Its current habitat niche scale was larger at 269 km2, comprised entirely of upland dwarf shrubs: heather, cotton grass, moorland grasses. By contrast, the current climate niche predicted L. europaeus occupying lowland areas with a milder climate and range extent of 252 km2. Its current habitat niche was also greater, 401 km2, being mostly improved grassland. Competition was presently limited. The current niche predictions showed very little geographic overlap between the species. Niche overlap measured by Schoener Index was low: current climate niche 0.16; current habitat niche 0.07. The future climate niches for 2050 (IPCC RCP2.6), predicted L. timidus range contracting to 19 km2, on hilltops and L. europaeus range expanding to 765 km2. Consequently L. timidus range would be wholly within the L. europaeus range. In many contact zones throughout Europe, L. europaeus outcompetes L. timidus; however, in the Peak District their distributions are largely distinct. Future replacement of L. timidus by L. europaeus may be engendered by dietary convergence, should a warmer climate cause a transition of upland dwarf shrub vegetation to grasses.

Discrimination of a single nucleotide polymorphism in the haptoglobin promoter region, rs5472, using a competitive fluorophore-labeled probe hybridization assay following loop-mediated isothermal amplification.

Personalized peptide vaccination, which involves activation of the host immune system against cancer cells using personalized peptide vaccines (PPVs), can improve overall survival in multiple cancer types. However, the clinical efficacies of PPVs vary for unknown reasons. Recently, a single nucleotide polymorphism (NG_012651.1:g.4461_5460[4960A>G]) in the haptoglobin promoter region, rs5472, was significantly associated with clinical response of PPV. Therefore, rs5472 is expected to be a predictive biomarker for PPV therapy. Here, we described a single nucleotide discrimination method for rs5472 analysis by combining the loop-mediated isothermal amplification and quenching probe methods. In evaluation of saliva samples, this method showed high concordance with the results of Sanger sequencing (100%, n=36). Importantly, this method did not require calculation of melting temperature for single nucleotide discrimination and could therefore be carried out on a simple instrument. Accordingly, this method may be more robust and applicable to near-patient testing.

Conversion of full nitritation to partial nitritation/anammox in a continuous granular reactor for low-strength ammonium wastewater treatment at 20°C.

The feasibility of converting full nitritation to partial nitritation/anammox (PN/A) at ambient temperature (20°C) was investigated in a continuous granular reactor. The process was conducted without anammox bacteria inoculation for the treatment of 70mgL-1 of low-strength ammonium nitrogen wastewater. Following the stepwise increase of the nitrogen loading rate from 0.84 to 1.30kgNm-3d-1 in 320days of operation, the removal efficiency of total inorganic nitrogen (TIN) exceeded 80% under oxygen-limiting conditions. The mature PN/A granules, which had a compact structure and abundant biomass, exhibited a specific TIN removal rate of 0.11gNg-1 VSSd-1 and a settling velocity of 70.2mh-1. This was comparable with that obtained at above 30°C in previous reports. High-throughput pyrosequencing results revealed that the co-enrichment of aerobic and anaerobic ammonium-oxidizing bacteria identified as genera Nitrosomonas and Candidatus Kuenenia, which prompted a hybrid competition for oxygen and nitrite with nitrite-oxidizing bacteria (NOB). However, the overgrowth of novel NOB Candidatus Nitrotoga adapted to low temperatures and low nitrite concentration could potentially deteriorate the one-stage PN/A process by exhausting residual bulk ammonium under long-term excessive aeration.