Utilizing geometric morphometrics to investigate gene function during organ growth: Insights through the study of beetle horn shape allometry.

Static allometry is a major component of morphological variation. Much of the literature on the development of allometry investigates how functional perturbations of diverse pathways affect the relationship between trait size and body size. Often, this is done with the explicit objective to identify developmental mechanisms that enable the sensing of organ size and the regulation of relative growth. However, changes in relative trait size can also be brought about by a range of other distinctly different developmental processes, such as changes in patterning or tissue folding, yet standard univariate biometric approaches are usually unable to distinguish among alternative explanations. Here, we utilize geometric morphometrics to investigate the degree to which functional genetic manipulations known to affect the size of dung beetle horns also recapitulate the effect of horn shape allometry. We reasoned that the knockdown phenotypes of pathways governing relative growth should closely resemble shape variation induced by natural allometric variation. In contrast, we predicted that if genes primarily affect alternative developmental processes, knockdown effects should align poorly with shape allometry. We find that the knockdown effects of several genes (e.g., doublesex, Foxo) indeed closely aligned with shape allometry, indicating that their corresponding pathways may indeed function primarily in the regulation of relative trait growth. In contrast, other knockdown effects (e.g., Distal-less, dachs) failed to align with allometry, implicating these pathways in potentially scaling-independent processes. Our findings moderate the interpretation of studies focusing on trait length and highlight the usefulness of multivariate approaches to study allometry and phenotypic plasticity.

Multiple source direction-of-arrival estimation applicable under near-, far-, and mixed-field scenarios.

Traditionally, direction-of-arrival (DOA) estimations under near- and far-field scenarios are treated as independent tasks based on the corresponding acoustic model, hence necessitating a proper soundfield detector as an upstream processing tool, whereas there may not be a distinct boundary between different soundfield types, especially the mixed-field scenarios where both near- and far-field sources coexist simultaneously. To handle this issue, this article investigates a multisource DOA estimator that equally localizes multiple near-, far-, and mixed-field sources, not requiring any specialized adjustments. We (i) define a signal-invariant multichannel feature denoted generalized relative harmonic coefficients in the spherical harmonics domain; (ii) derive the analytical expression of this feature and summarize its unique properties, exhibiting consistence for both near- and far-field sources; (iii) estimate source elevation and azimuth using the magnitude and phase parts of this feature, respectively; (iv) detect single-source dominated periods from the mixed measurements based on an investigated distance measure; and (v) count the number of sources and localize their DOAs by clustering the single-source dominated estimates. Extensive experimental results, in both simulated and real-life environments, finally confirm the effectiveness of the proposed algorithm under diverse acoustic scenarios, and a superiority over baseline approaches in localizing mixed-field sources.

PfAgo-based dual signal amplification biosensor for rapid and highly sensitive detection of alkaline phosphatase activity.

A Pyrococcus furiosus Argonaute (PfAgo)-based biosensor is presented for alkaline phosphatase (ALP) activity detection in which the ALP-catalyzed hydrolysis of 3'-phosphate-modified functional DNA activates the strand displacement amplification, and the amplicon mediates the fluorescent reporter cleavage as a guide sequence of PfAgo. Under the dual amplification mode of PfAgo-catalyzed multiple-turnover cleavage activity and pre-amplification technology, the developed method was successfully applied to ALP activity determination with a detection limit (LOD) of 0.0013 U L-1 (3σ) and a detection range of 0.0025 to 1 U L-1 within 90 min. The PfAgo-based method exhibits satisfactory analytic performance in the presence of potential interferents and in complex human serum samples. The proposed method shows several advantages, such as rapid analysis, high sensitivity, low-cost, and easy operation, and has great potential in disease evolution fundamental studies and clinical diagnosis applications.

Unique Fluorescent Protein-Encoded Microbeads with Supramaximal Encoding Capacity and High Sensitivity for Multiplex Bioassays.

Fluorescence-encoded microbeads (FEBs) have been widely used as a critical component in multiplexed biomolecular assays. Here, we propose a simple, sustainable, low-cost, and safe strategy for preparing FEBs by assembling fluorescent proteins (FPs) onto magnetic microbeads (MBs) via chemical coupling. Combining the type of FP, the concentration of FP, and the size of the magnetic microbeads as encoding elements, an ultralarge encoding capacity with 506 barcodes was obtained. We demonstrate that the FP-based FEBs have good stability during long-term storage and tolerate the use of an organic solution. Multiplex detection of femtomolar ssDNA molecules was achieved via flow cytometry, and the detection procedure is simple and fast because it does not require amplification and washing strategies. The advantages of this advanced method for multiplex detections including high sensitivity, specificity, accuracy, repeatability, rapidity, and cost-effectiveness show a broad application prospect in basic and applied research fields such as disease diagnosis, food safety, environmental protection, proteomics, genomics, and drug screening.

A DNA walker triggered isothermal amplification method based on freezing construction of AuNP probes and its application in ricin detection.

DNA molecular machines are widely used in the fields of biosensors and biological detection. Among them, DNA walkers have attracted much attention due to their simple design and controllability. Herein, we attempt to develop a DNA walker triggered exponential amplification method and explore its application. The AuNP probes in the DNA walker are constructed by a freezing technology, instead of the time-consuming and complex synthesis process of the traditional method. Meanwhile, after the "recognition-cleavage-relative motion" cycle of this DNA walker reaction, the exponential amplification reaction is initiated, and leads to the fluorescence recovery of the molecular beacon. Taking ricin as a target, this new method shows a limit of detection of 2.25 pM by selecting aptamers with strong binding affinity, and exhibits a wide detection range, satisfactory specificity, and excellent stability in practical application. Therefore, our method provides a universal sensing platform and has great prospects in the fields of biosensors, food safety detection, and clinical diagnostics.

Closed-form multiple source direction-of-arrival estimator under reverberant environments.

Accurate direction-of-arrival (DOA) estimation of multiple sources, simultaneously active in a reverberant environment, remains a challenge, as the multi-path acoustic reflections and overlapped periods dramatically distort the direct-path wave propagation. This article proposes a prominent solution localizing multiple sources in a reverberant environment using closed-form estimates, circumventing any exhaustive search over the two-dimensional directional space. Apart from a low complexity cost, the algorithm has robustness to reverberant, inactive, and overlapped periods and an ease of operation in practice, achieving sufficient accuracy compared to state-of-the-art approaches. Specifically, this algorithm localizes an unknown number of sources through four steps: (i) decomposing the frequency domain signals on a spherical array to the spherical harmonics domain; (ii) extracting the first-order relative harmonic coefficients as the input features; (iii) achieving direct-path dominance detection and localization using closed-form estimation; and (iv) estimating the number of sources and their DOAs based on those pass the direct-path detection. Experimental results, using extensive simulated and real-life recordings, confirm the algorithm with a significantly reduced computational complexity, while preserving competitive localization accuracy as compared to the baseline approaches. Additional tests confirm this low-complexity algorithm even with a potential capacity for online DOA tracking of multiple moving sources.

Biomimetic Assembly of Spore@ZIF-8 Microspheres for Vaccination.

Vaccines have been one of the most powerful weapons to defend against infectious diseases for a long time now. Subunit vaccines are of increasing importance because of their safety and effectiveness. In this work, a Bacillus amyloliquefaciens spore@zeolitic imidazolate framework-8 (ZIF-8) vaccine platform is constructed. The ovalbumin (OVA) is encapsulated in the ZIF-8 shells as a model antigen to form a spore@OVA@ZIF-8 (SOZ) composite. The assembly of ZIF-8 improves the loading content of OVA on the spores and provides OVA with long-term protection. The SOZ composite enhances the immunization efficacy in multiple ways, such as facilitation of antigen uptake and lysosome escape, stimulation of dendritic cells to mature and secrete cytokines, boosting of antibody production and formation of an antigen depot. This platform shows several advantages including easy preparation, cost-effectiveness, long life, convenience of transportation and storage, and no need for the cold chain. These findings may have promising implications for the rational design of safe and effective spore-based composite vaccine platforms.

Developmental bias in the evolution and plasticity of beetle horn shape.

The degree to which developmental systems bias the phenotypic effects of environmental and genetic variation, and how these biases affect evolution, is subject to much debate. Here, we assess whether developmental variability in beetle horn shape aligns with the phenotypic effects of plasticity and evolutionary divergence, yielding three salient results. First, we find that most pathways previously shown to regulate horn length also affect shape. Second, we find that the phenotypic effects of manipulating divergent developmental pathways are correlated with each other as well as multivariate fluctuating asymmetry-a measure of developmental variability. Third, these effects further aligned with thermal plasticity, population differences and macroevolutionary divergence between sister taxa and more distantly related species. Collectively, our results support the hypothesis that changes in horn shape-whether brought about by environmentally plastic responses, functional manipulations or evolutionary divergences-converge along 'developmental lines of least resistance', i.e. are biased by the developmental system underpinning horn shape.

Wing serial homologues and the diversification of insect outgrowths: insights from the pupae of scarab beetles.

Modification of serially homologous structures is a common avenue towards functional innovation in developmental evolution, yet ancestral affinities among serial homologues may be obscured as structure-specific modifications accumulate over time. We sought to assess the degree of homology to wings of three types of body wall projections commonly observed in scarab beetles: (i) the dorsomedial support structures found on the second and third thoracic segments of pupae, (ii) the abdominal support structures found bilaterally in most abdominal segments of pupae, and (iii) the prothoracic horns which depending on species and sex may be restricted to pupae or also found in adults. We functionally investigated 14 genes within, as well as two genes outside, the canonical wing gene regulatory network to compare and contrast their role in the formation of each of the three presumed wing serial homologues. We found 11 of 14 wing genes to be functionally required for the proper formation of lateral and dorsal support structures, respectively, and nine for the formation of prothoracic horns. At the same time, we document multiple instances of divergence in gene function across our focal structures. Collectively, our results support the hypothesis that dorsal and lateral support structures as well as prothoracic horns share a developmental origin with insect wings. Our findings suggest that the morphological and underlying gene regulatory diversification of wing serial homologues across species, life stages and segments has contributed significantly to the extraordinary diversity of arthropod appendages and outgrowths.

Enhanced genome assembly and a new official gene set for Tribolium castaneum.

BACKGROUND: The red flour beetle Tribolium castaneum has emerged as an important model organism for the study of gene function in development and physiology, for ecological and evolutionary genomics, for pest control and a plethora of other topics. RNA interference (RNAi), transgenesis and genome editing are well established and the resources for genome-wide RNAi screening have become available in this model. All these techniques depend on a high quality genome assembly and precise gene models. However, the first version of the genome assembly was generated by Sanger sequencing, and with a small set of RNA sequence data limiting annotation quality. RESULTS: Here, we present an improved genome assembly (Tcas5.2) and an enhanced genome annotation resulting in a new official gene set (OGS3) for Tribolium castaneum, which significantly increase the quality of the genomic resources. By adding large-distance jumping library DNA sequencing to join scaffolds and fill small gaps, the gaps in the genome assembly were reduced and the N50 increased to 4753kbp. The precision of the gene models was enhanced by the use of a large body of RNA-Seq reads of different life history stages and tissue types, leading to the discovery of 1452 novel gene sequences. We also added new features such as alternative splicing, well defined UTRs and microRNA target predictions. For quality control, 399 gene models were evaluated by manual inspection. The current gene set was submitted to Genbank and accepted as a RefSeq genome by NCBI. CONCLUSIONS: The new genome assembly (Tcas5.2) and the official gene set (OGS3) provide enhanced genomic resources for genetic work in Tribolium castaneum. The much improved information on transcription start sites supports transgenic and gene editing approaches. Further, novel types of information such as splice variants and microRNA target genes open additional possibilities for analysis.

Developmental bias in horned dung beetles and its contributions to innovation, adaptation, and resilience.

Developmental processes transduce diverse influences during phenotype formation, thereby biasing and structuring amount and type of phenotypic variation available for evolutionary processes to act on. The causes, extent, and consequences of this bias are subject to significant debate. Here we explore the role of developmental bias in contributing to organisms' ability to innovate, to adapt to novel or stressful conditions, and to generate well integrated, resilient phenotypes in the face of perturbations. We focus our inquiry on one taxon, the horned dung beetle genus Onthophagus, and review the role developmental bias might play across several levels of biological organization: (a) gene regulatory networks that pattern specific body regions; (b) plastic developmental mechanisms that coordinate body wide responses to changing environments and; (c) developmental symbioses and niche construction that enable organisms to build teams and to actively modify their own selective environments. We posit that across all these levels developmental bias shapes the way living systems innovate, adapt, and withstand stress, in ways that can alternately limit, bias, or facilitate developmental evolution. We conclude that the structuring contribution of developmental bias in evolution deserves further study to better understand why and how developmental evolution unfolds the way it does.

Notch signaling patterns head horn shape in the bull-headed dung beetle Onthophagus taurus.

Size and shape constitute fundamental aspects in the description of morphology. Yet while the developmental-genetic underpinnings of trait size, in particular with regard to scaling relationships, are increasingly well understood, those of shape remain largely elusive. Here we investigate the potential function of the Notch signaling pathway in instructing the shape of beetle horns, a highly diversified and evolutionarily novel morphological structure. We focused on the bull-headed dung beetle Onthophagus taurus due to the wide range of horn sizes and shapes present among males in this species, in order to assess the potential function of Notch signaling in the specification of horn shape alongside the regulation of shape changes with allometry. Using RNA interference-mediated transcript depletion of Notch and its ligands, we document a highly conserved role of Notch signaling in general appendage formation. By integrating our functional genetic approach with a geometric morphometric analysis, we find that Notch signaling moderately but consistently affects horn shape, and does so differently for the horns of minor, intermediate-sized, and major males. Our results suggest that the function of Notch signaling during head horn formation may vary in a complex manner across male morphs, and highlights the power of integrating functional genetic and geometric morphometric approaches in analyzing subtle but nevertheless biologically important phenotypes in the face of significant allometric variation.

Microorganism@UiO-66-NH2 Composites for the Detection of Multiple Colorectal Cancer-Related microRNAs with Flow Cytometry.

High-throughput analyses of multitarget markers can facilitate rapid and accurate clinical diagnosis. Suspension array assays, a flow cytometry-based analysis technology, are among some of the most promising multicomponent analysis methods for clinical diagnostics and research purposes. These assays are appropriate for examining low-volume, complex samples having trace amounts of analytes due to superior elimination of background. Physical shape is an important and promising code system, which uses a set of visually distinct patterns to identify different assay particles. Here, we presented a morphology recognizable suspension arrays based on the microorganisms with different morphologies. In this study, UiO-66-NH2 (UiO stands for University of Oslo) metal-organic frameworks (MOFs), was wrapped on the microorganism surface to form an innovative class of microorganism@UiO-66-NH2 composites for suspension array assays. The use of microorganisms endowed composites barcoding ability with their different morphology and size. Meanwhile, the UiO-66-NH2 provided a stable rigid shell, large specific surface area, and metal(IV) ions with multiple binding sites, which could simplify the protein immobilization procedure and enhance detection sensitivity. With this method, simultaneous detection of three colorectal cancer-related microRNA (miRNA), including miRNA-21, miRNA-17, and miRNA-182, could be easily achieved with femtomolar sensitivity by using a commercial flow cytometer. The synergy between microorganisms and MOFs make the composites a prospective barcoding candidate with excellent characteristics for multicomponent analysis, offering great potential for the development of high throughput and accurate diagnostics.

CRISPR-Cas9 Triggered Two-Step Isothermal Amplification Method for E. coli O157:H7 Detection Based on a Metal-Organic Framework Platform.

Escherichia coli O157:H7 has been reported as an important pathogenic bacteria causing serious infection and economic loss. However, detection of Escherichia coli O157:H7 needs improvement, given its current complexity and sensitivity. Herein, we attempt to build a fluorescence sensing method to detect Escherichia coli O157:H7 with easy operation and high efficiency. The target virulence gene sequences are recognized and cleaved by the CRISPR-Cas9 system, and trigger strand displacement amplification and rolling circle amplification. After amplification reactions, massive products can hybridize with the probes, the fluorescence of which are quenched based on a metal-organic framework platform, leading to the fluorescence recovery at typical excitation/emission wavelengths of 480/518 nm. This method exhibits high sensitivity with the detection limit at 4.0 × 101 CFU mL-1 and a wide range from 1.3 × 102 CFU mL-1 to 6.5 × 104 CFU mL-1. Meanwhile, this assay also shows significant specificity and applies to practical samples with high accuracy. Therefore, our method would have great potential application in bacterial detection, food safety monitoring, or clinical diagnostics.

Bio-barcode triggered isothermal amplification in a fluorometric competitive immunoassay for the phytotoxin abrin.

Abrin is one of the most toxic phytotoxins to date, and is a potential biological warfare agent. A bio-barcode triggered isothermal amplification for fluorometric determination of abrin is described. Free abrin competes with abrin-coated magnetic microparticles (MMP) probes to bind to gold nanoparticle (AuNP) probes modified with abrin antibody and bio-barcoded DNA. Abundant barcodes are released from the MMP-AuNP complex via dithiothreitol treatment. This triggers an exponential amplification reaction (EXPAR) that is monitored by real-time fluorometry, at typical excitation/emission wavelengths of 495/520 nm. The EXPAR assay is easily operated, highly sensitive and specific. It was used to quantify abrin in spiked commercial samples. The detection limit (at S/N = 3; for n = 6) is 5.6 pg·mL-1 which is considerably lower than previous reports. This assay provides a universal sensing platform and has great potential for determination of various analytes, including small molecules, proteins, DNA, and cells. Graphical abstract Schematic representation of the bio-barcode triggered exponential amplification reaction (EXPAR) for a fluorometric competitive immunoassay for abrin. The limit of detection is 5.6 pg mL-1 with a large dynamic range from 10 pg mL-1 to 1 µg mL-1.

The origins of novelty from within the confines of homology: the developmental evolution of the digging tibia of dung beetles.

Understanding the origin of novel complex traits is among the most fundamental goals in evolutionary biology. The most widely used definition of novelty in evolution assumes the absence of homology, yet where homology ends and novelty begins is increasingly difficult to parse as evo devo continuously revises our understanding of what constitutes homology. Here, we executed a case study to explore the earliest stages of innovation by examining the tibial teeth of tunnelling dung beetles. Tibial teeth are a morphologically modest innovation, composed of relatively simple body wall projections and contained fully within the fore tibia, a leg segment whose own homology status is unambiguous. We first demonstrate that tibial teeth aid in multiple digging behaviours. We then show that the developmental evolution of tibial teeth was dominated by the redeployment of locally pre-existing gene networks. At the same time, we find that even at this very early stage of innovation, at least two genes that ancestrally function in embryonic patterning and thus entirely outside the spatial and temporal context of leg formation, have already become recruited to help shape the formation of tibial teeth. Our results suggest a testable model for how developmental evolution scaffolds innovation.

A morphological novelty evolved by co-option of a reduced gene regulatory network and gene recruitment in a beetle.

The mechanisms underlying the evolution of morphological novelties have remained enigmatic but co-option of existing gene regulatory networks (GRNs), recruitment of genes and the evolution of orphan genes have all been suggested to contribute. Here, we study a morphological novelty of beetle pupae called gin-trap. By combining the classical candidate gene approach with unbiased screening in the beetle Tribolium castaneum, we find that 70% of the tested components of the wing network were required for gin-trap development. However, many downstream and even upstream components were not included in the co-opted network. Only one gene was recruited from another biological context, but it was essential for the anteroposterior symmetry of the gin-traps, which represents a gin-trap-unique morphological innovation. Our data highlight the importance of co-option and modification of GRNs. The recruitment of single genes may not be frequent in the evolution of morphological novelties, but may be essential for subsequent diversification of the novelties. Finally, after having screened about 28% of annotated genes in the Tribolium genome to identify the genes required for gin-trap development, we found none of them are orphan genes, suggesting that orphan genes may have played only a minor, if any, role in the evolution of gin-traps.

A competitive chemiluminescence enzyme immunoassay method for ß-defensin-2 detection in transgenic mice.

A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine ß-defensin-2 (pBD-2) detection in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.05-80 ng/mL with a correlation coefficient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2.

Sustainable endospore-based microreactor system for antioxidant capacity assay.

A novel endospore-based microbial method for "post-additional" antioxidant capacity assay was developed. The technique was based on oxidation and catalysis of the 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) by Bacillus subtilis 168 endospores in the presence of dissolved oxygen. Coat protein A (CotA), which belongs to the endospore coat, was expressed, purified, and assessed for its ability to oxidize ABTS into the ABTS(•+) radical cation. The wild-type endospore necessary for oxidizing ABTS into ABTS(•+) radical cation was confirmed by knocking out the cotA gene from B. subtilis 168 by homologous double exchange. Findings revealed that the catalytic activity of the endospores may be attributed to the presence of the CotA protein. The use of endospores instead of purified enzymes to prepare ABTS(•+) greatly reduced the assay cost and eliminated the need to purify and store of enzymes. The self-life of the radical cation was kept stable for at least 12 days without addition of a stabilizer and laccase inhibitor. This behavior enables the large-scale preparation of ABTS(•+). The antioxidant capacities of the individual antioxidants and fruit samples were easily quantified and compared using the proposed method. The developed technique can be further developed as a high-throughput screening technique for antioxidants.