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.

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.

Ultrarobust stable ABTS radical cation prepared using Spore@Cu-TMA biocomposites for antioxidant capacity assay.

Herein, spore@Cu-trimesic acid (TMA) biocomposites were prepared by self-assembling Cu-based metal-organic framework on the surface of Bacillus velezensis spores. The laccase-like activity of spore@Cu-TMA biocomposites was enhanced by 14.9 times compared with that of pure spores due to the reaction of Cu2+ ions with laccase on the spore surface and the microporous structure of Cu-TMA shell promoting material transport and increasing substrate accessibility. Spore@Cu-TMA rapidly oxidized and transformed 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) into ABTS●+ without using H2O2. Under optimum conditions, the ABTS●+ could be stored for 21 days at 4 °C and 7 days at 37 °C without the addition of any stabilizers, allowing for the large-scale preparation and long-term storage of ABTS●+. The ultrarobust stable ABTS●+ obtained with the use of Cu-TMA could effectively reduce the "back reaction" by preventing the leaching of the metabolites released by the spores. On the basis of these findings, a rapid, low-cost, and eco-friendly colorimetric platform was successfully developed for the detection of antioxidant capacity. Determination of antioxidant capacity for several antioxidants such as caffeic acid, glutathione, and Trolox revealed their corresponding limits of detection at 4.83, 8.89, and 7.39 nM, respectively, with linear ranges of 0.01-130, 0.01-140, and 0.01-180 µM, respectively. This study provides a facile way to prepare ultrarobust stable ABTS●+ and presents a potential application of spore@Cu-TMA biocomposites in food detection and bioanalysis.

Histone deacetylases regulate organ-specific growth in a horned beetle.

BACKGROUND: Nutrient availability is among the most widespread means by which environmental variability affects developmental outcomes. Because almost all cells within an individual organism share the same genome, structure-specific growth responses must result from changes in gene regulation. Earlier work suggested that histone deacetylases (HDACs) may serve as epigenetic regulators linking nutritional conditions to trait-specific development. Here we expand on this work by assessing the function of diverse HDACs in the structure-specific growth of both sex-shared and sex-specific traits including evolutionarily novel structures in the horned dung beetle Onthophagus taurus. RESULTS: We identified five HDAC members whose downregulation yielded highly variable mortality depending on which HDAC member was targeted. We then show that HDAC1, 3, and 4 operate in both a gene- and trait-specific manner in the regulation of nutrition-responsiveness of appendage size and shape. Specifically, HDAC 1, 3, or 4 knockdown diminished wing size similarly while leg development was differentially affected by RNAi targeting HDAC3 and HDAC4. In addition, depletion of HDAC3 transcript resulted in a more rounded shape of genitalia at the pupal stage and decreased the length of adult aedeagus across all body sizes. Most importantly, we find that HDAC3 and HDAC4 pattern the morphology and regulate the scaling of evolutionarily novel head and thoracic horns as a function of nutritional variation. CONCLUSION: Collectively, our results suggest that both functional overlap and division of labor among HDAC members contribute to morphological diversification of both conventional and recently evolved appendages. More generally, our work raises the possibility that HDAC-mediated scaling relationships and their evolution may underpin morphological diversification within and across insect species broadly.

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.

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.

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.

Directed evolution of glyphosate oxidase and a chemiluminescence system for glyphosate detection: A comprehensive practical laboratory experiment on biotechnology.

This article describes a comprehensive practical laboratory method for developing an enzyme to more easily measure glyphosate levels in solution. Through this article, undergraduate students of biology majors can conduct research experiments in critical fields by utilizing various techniques, such as chemiluminescence (CL) biosensors with engineered enzymes and are guided in molecular biology laboratories. A glyphosate oxidase mutant library was constructed by DNA shuffling, and a glyphosate oxidase variant with increased glyphosate degradation activity was selected by using a high-throughput screening assay. Following protein overexpression in Escherichia coli (DE3) and purification by affinity chromatography, the glyphosate oxidase variant protein combined with luminol-H2 O2 reaction was constructed as a new CL biosensor for detecting glyphosate in soils.

Gas induced formation of inactive Li in rechargeable lithium metal batteries.

The formation of inactive lithium by side reactions with liquid electrolyte contributes to cell failure of lithium metal batteries. To inhibit the formation and growth of inactive lithium, further understanding of the formation mechanisms and composition of inactive lithium are needed. Here we study the impact of gas producing reactions on the formation of inactive lithium using ethylene carbonate as a case study. Ethylene carbonate is a common electrolyte component used with graphite-based anodes but is incompatible with Li metal anodes. Using mass spectrometry titrations combined with 13C and 2H isotopic labeling, we reveal that ethylene carbonate decomposition continuously releases ethylene gas, which further reacts with lithium metal to form the electrochemically inactive species LiH and Li2C2. In addition, phase-field simulations suggest the non-ionically conducting gaseous species could result in an uneven distribution of lithium ions, detrimentally enhancing the formation of dendrites and dead Li. By optimizing the electrolyte composition, we selectively suppress the formation of ethylene gas to limit the formation of LiH and Li2C2 for both Li metal and graphite-based anodes.

Microorganisms@aMIL-125 (Ti): An Amorphous Metal-Organic Framework Induced by Microorganisms and Their Applications.

Amorphous metal-organic framework (aMOF)-based materials have attracted considerable attention as an emerging class of nanomaterials. Herein, novel microorganisms@aMIL-125 (Ti) composites including yeast@aMIL-125 (Ti), PCC 6803@aMIL-125 (Ti), and Escherichia coli@aMIL-125 (Ti) composites were respectively synthesized by self-assembling aMOFs on the microorganisms' surface. The functional groups on the microorganisms' surface induced structural defects and participated in the formation of aMIL-125 (Ti) composites. Finally, the application of microorganisms@aMIL-125 (Ti) composites for the removal of glyphosate from aqueous solution was selected as a model reaction to illustrate their potential for environmental protection. The present method is not only economical but also has other advantages including ease of operation, environmentally friendly assay, and high adsorption. The maximum adsorption capacity of aMIL-125 (Ti) was 1096.25 mg g-1, which was 1.74 times that of crystalline MIL-125 (Ti). Therefore, the microorganisms@aMOFs composites will have broad application prospects in energy storage, drug delivery, catalysis, adsorbing toxic substances, sensing, encapsulating and delivering enzymes, and in other fields.

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.

A rapid and naked-eye on-site monitoring of biogenic amines in foods spoilage.

Due to growing food safety issues, developing economic, rapid, and sensitive strategies for food spoilage monitoring has attracted significant attention. Here, a Bacillus subtilis spore-based biosensor is presented for rapid, highly sensitive, visual biogenic amines detection. The biosensor is fabricated through biogenic amines-induced pH increase which inhibits the electron transfer between Cu ion sites within CotA-laccase on the spore surface, leading to decrease in catalytic oxidation activity towards the chromogenic substrates. The developed system integrated with smartphone analysis realized the on-site monitoring of histamine with a detection range of 0.17-120 mg L-1, and a detection limit of 0.17 mg L-1 (3σ). Moreover, the color change induced by histamine is observable by the naked eye. The smart biosensor was successfully applied for food freshness evaluation in raw meat samples, showing several advantages, including eco-friendliness, low cost, and high stability, meeting the demands of on-site monitoring in the food safety field.

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.

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.

DHA Ameliorates Cognitive Ability, Reduces Amyloid Deposition, and Nerve Fiber Production in Alzheimer's Disease.

Background: The etiology of Alzheimer's disease (AD) is very complex. Docosahexaenoic acid (DHA) is important in cognitive ability and nervous system development. A limited number of studies have evaluated the efficacy of DHA in the treatment of AD. Introduction: We detected neurofibrillary tangles (NFT) in the hippocampus and cortex of transgenic mice brain through silver glycine staining. We determined the activity of neurons by staining Nissl bodies, used liquid NMR to detect metabolites in the brain, and functional magnetic resonance imaging results to observe the connection signal value between brain regions. Materials and Methods: We fed 3-month-old APP/PS1 double transgenic mice with DHA mixed feeds for 4 months to assess the effects of DHA on cognitive ability in AD mice through the Morris water maze and open field tests. To evaluate its effects with AD pathology, continuous feeding was done until the mice reached 9 months of age. Results: Compared to AD mice, escape latency significantly decreased on the fifth day while swimming speed, target quadrant stay time, and the crossing number of platforms increased by varying degrees after DHA treatment. Brain tissue section staining revealed that DHA significantly reduced Aß and nerve fibers in the brain of AD mice. Conclusion: DHA significantly reduced the deposition of Aß in the brain and inhibited the production of nerve fibers, thereby increasing cognitive abilities in AD mice. In addition, DHA suppressed blood lipid levels, and restored uric acid and urea levels, implying that DHA is a potential therapeutic option for early AD.

Emerging Argonaute-based nucleic acid biosensors.

Recent advances in Argonaute (Ago)-mediated biotechnology have provided new insights into the development of programmable and highly sensitive nucleic acid detection platforms. This study provides an overview of recent research on Ago-based nucleic acid detection. The potential applications of these emerging nucleic acid biosensors and the challenges associated with their use have been discussed.

Advances in the Immunoassays for Detection of Bacillus thuringiensis Crystalline Toxins.

Insect-resistant genetically modified organisms have been globally commercialized for the last 2 decades. Among them, transgenic crops based on Bacillus thuringiensis crystalline (Cry) toxins are extensively used for commercial agricultural applications. However, less emphasis is laid on quantifying Cry toxins because there might be unforeseen health and environmental concerns. Immunoassays, being the preferred method for detection of Cry toxins, are reviewed in this study. Owing to limitations of traditional colorimetric enzyme-linked immunosorbent assay, the trend of detection strategies shifts to modified immunoassays based on nanomaterials, which provide ultrasensitive detection capacity. This review assessed and compared the properties of the recent advances in immunoassays, including colorimetric, fluorescence, chemiluminescence, surface-enhanced Raman scattering, surface plasmon resonance, and electrochemical approaches. Thus, the ultimate aim of this study is to identify research gaps and infer future prospects of current approaches for the development of novel immunosensors to monitor Cry toxins in food and the environment.

A spore-based portable kit for on-site detection of fluoride ions.

The excess residues of fluoride ions cause serious human health problems, making their detection highly valuable. In this work, a whole-cell-based biosensor was presented for the detection of fluoride ions, which can inhibit the color reaction of 3,3',5,5',-tetramethylbenzidine (TMB) catalyzed by the CotA-laccase of spore surface. This reaction for the detection of fluoride ions could be read out through UV-vis spectrophotometer, smartphone, or standard colorimetric card within 10 min. Under optimum conditions, a linear range of 1-600 µmol L-1 with a detection limit of 0.12 µmol L-1 (3σ/k) was achieved for fluoride ions detection by using UV-vis spectrophotometer. The biosensor coupling with smartphone had a good linear response to fluoride ions concentration in the range of 5-600 µmol L-1 with LOD of 0.90 µmol L-1 (3σ/k). The standard colorimetric card can be directly used for recognizing the fluoride ions level via naked-eyes. A portable kit based on a colorimetric card and smartphone was developed and has been successfully applied for fluoride ions monitoring in surface waters and groundwater. This developed method has several advantages such as rapid, outstanding selectivity and anti-interference, low-cost, ease of operation and storage, and eco-friendliness, meeting the demands of point-of-care testing of fluoride ions and disease prevention.

Light-Regulated Natural Fluorescence of the PCC 6803@ZIF-8 Composite as an Encoded Microsphere for the Detection of Multiple Biomarkers.

The use of color-encoded microspheres for a bead-based assay has attracted increasing attention for high-throughput multiplexed bioassays. A fluorescent PCC 6803@ZIF-8 composite was prepared as a bead-based assay platform by a self-assembled zeolitic imidazolate framework (ZIF-8) on the surface of inactivated PCC 6803 cells. The composite fluorescence owing to the presence of pigment proteins in PCC 6803 could be gradually bleached with the prolongation of the ultraviolet light irradiation time. The composites with different fluorescence intensities were therefore obtained as encoded microspheres for the multiplexed assay. ZIF-8 provides a stable, rigid shell and a large specific surface area for composites, which prevent the composites from breakage during use and storage, simplify the protein immobilization procedure, reduce non-specific adsorption, and enhance the detection sensitivity. The encoded composites were successfully used to detect multiple DNA insertion sequences of Mycobacterium tuberculosis. The presented strategy offers an innovative color-encoding method for high-throughput multiplexed bioassays without the need of using chemically synthesized fluorescent materials.