TALEN-mediated homologous-recombination-based fibroin light chain in-fusion expression system in Bombyx mori.

Silkworm was the first domesticated insect and has important economic value. It has also become an ideal model organism with applications in genetic and expression studies. In recent years, the use of transgenic strategies has made the silkworm silk gland an attractive bioreactor for the production of recombinant proteins, in particular, piggyBac-mediated transgenes. However, owing to differences in regulatory elements such as promoters, the expression levels of exogenous proteins have not reached expectations. Here, we used targeted gene editing to achieve site-specific integration of exogenous genes on genomic DNA and established the fibroin light chain (FibL) in-fusion expression system by TALEN-mediated homology-directed recombination. First, the histidine-rich cuticular protein (CP) was successfully site-directed inserted into the native FibL, and the FibL-CP fusion gene was correctly transcribed and expressed in the posterior silk gland under the control of the endogenous FibL promoter, with a protein expression level comparable with that of the native FibL protein. Moreover, we showed based on molecular docking that the fusion of FibL with cuticular protein may have a negative effect on disulfide bond formation between the C-terminal domain of fibroin heavy chain (FibH) and FibL-CP, resulting in abnormal spinning and cocoon in homozygotes, indicating a significant role of FibL in silk protein formation and secretion. Our results demonstrate the feasibility of using the FibL fusion system to express exogenous proteins in silkworm. We expect that this bioreactor system will be used to produce more proteins of interest, expanding the application value of the silk gland bioreactor.

Development of a Coumarin-derived Fluorescent Probe for Detection of HOCl and its Application in Cells and Zebrafish.

We have prepared a simple, universal and efficient coumarin-derived fluorescent probe (XDS1) to detecting HOCl. The experimental findings revealed that the introduction of HOCl produced an obvious quenching effect on the probe with high selectivity and sensitivity. The calculated limit of detection (LOD) was as low as 0.02 µM. Furthermore, an impressive response time of less than 10 s was observed when XDS1 detecting HOCl. Importantly, the probe XDS1 exhibited negligible cytotoxicity, thereby facilitating its application for imaging HOCl within biological environment. The probe XDS1 had been successfully used for specific detection in cells.

Bimetallic Organic Gel for Effective Methyl Orange Dye Adsorption.

A bimetallic organic gel (MOG-Fe/Al) was synthesized through the solvothermal method. The gel state of the product obtained under optimized gel formation conditions is sufficient to carry 2 g of weight for a long time. Scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared (FT-IR) spectroscopy, Brunauer-Emmett-Teller (BET) technique, and X-ray photoelectron spectroscopy (XPS) analysis confirmed the structures and morphologies of the synthesized materials. MOG-Fe/Al, with good stability, excellent durability, and wide applicability, exhibited efficient MO adsorption capacity as high as 335.88 mg/g at 25 °C. Adsorption-influencing factors including solution pH, contact time, and temperature were investigated. The adsorption performance of the bimetallic organic gel was better than that of the monometallic organic gels (MOG-Fe and MOG-Al), and its adsorption processes were in accordance with the pseudo-second-order kinetic and Langmuir isothermal models. The excellent adsorption capacity of the MOG-Fe/Al is due to its surface structure, pore volume, π-π interactions, hydrogen bonds, and electrostatic interactions.

Naphthalimide-based fluorescent probe for Hg2+ detection and imaging in living cells and zebrafish.

A simple naphthalimide-based fluorescent probe was designed and synthesized for the determination of mercury ion (Hg2+ ). The probe showed a noticeable fluorescence quenching response for Hg2+ . When added with Hg2+ , the fluorescence intensity of the probe at 560 nm was remarkably decreased with the color changed from yellow to colorless under ultraviolet (UV) light. The probe had a notable selectivity and sensitivity for Hg2+ and displayed an excellent sensing performance when detecting Hg2+ at low concentration (19.5 nM). The binding phenomenon between the probe and Hg2+ was identified by Job's method and high-resolution mass spectrometry (HRMS). Moreover, the probe was not only utilized to identify Hg2+ in real samples with satisfactory results (92.00%-110.00%) but also was successfully used for bioimaging in cells and zebrafish. The recognition mechanism has been verified by transmission electron microscopy (TEM) for the first time. All the results showed that the probe could be used as a potent useful tool for detection of Hg2+ .

High mechanical property silk produced by transgenic silkworms expressing the Drosophila Dumpy.

Extensive research has been conducted on utilizing transgenic silkworms and their natural spinning apparatus to produce high-performance spider silk fibers. However, research on using non-spider biological proteins to optimize the molecular structure of silk protein and improve the mechanical performance of silk fibers is still relatively scarce. Dumpy, a massive extracellular matrix polypeptide, is essential for preserving the shape and structural integrity of the insect cuticle due to its remarkable tension and elasticity. Here, we constructed two transgenic donor plasmids containing the fusion genes of FibH-Dumpy and FibL-Dumpy. The results indicated the successful integration of two exogenous gene expression cassettes, driven by endogenous promoters, into the silkworm genome using piggyBac-mediated transgenic technology. Secondary structure analysis revealed a 16.7% and 13.6% increase in the ß-sheet content of transgenic silks compared to wild-type (WT) silk fibers. Mechanical testing demonstrated that, compared to the WT, HDUY and LDUY transgenic silk fibers exhibited respective increases of 39.54% and 21.45% in maximum stress, 44.43% and 45.02% in toughness, and 24.91% and 28.51% in elastic recovery rate. These findings suggest that Drosophila Dumpy significantly enhanced the mechanical properties of silk, positioning it as an excellent candidate for the development of extraordinary-performance fibers. This study provides rich inspiration for using other biological proteins to construct high-performance silk fibers and expands the possibilities for designing and applying novel biomaterials.

Modeling the 3-dimensional structure of the silkworm's spinning apparatus in silk production.

The silk-spinning process of the silkworms transforms the liquid silk solution to a solid state under mild conditions, making it an attractive model for bioinspiration However, the precise mechanism behind silk expulsion remains largely unknown. Here we selected the silkworms as representative models to investigate the silk-spinning mechanism. We used serial block-face scanning electron microscopy (SBF-SEM) to reconstruct the three-dimensional structures of the spinnerets in silkworms at various stages and with different gene backgrounds. By comparing the musculature and duct deformation of these spinneret models during the spinning process, we were able to simulate the morphological changes of the spinneret. Based on the results, we proposed three essential factors for silkworm spinning: the pressure generated by the silk gland, the opening duct, and the pulling force generated by head movement. Understanding the silkworm spinning process provides insights into clarify the fluid-ejecting mechanism of a group of animals. Moreover, these findings are helpful to the development of biomimetic spinning device that mimics the push-and-pull dual-force system in silkworms. STATEMENT OF SIGNIFICANCE: The silkworms' spinning system produces fibers under mild conditions, making it an ideal candidate for bioinspiration. However, the mechanism of silk expulsion is unknown, and the three-dimensional structure of the spinneret is still uncertain. In this study, we reconstructed a detailed 3-dimensional model of the spinneret at near-nanometer resolution, and for the first time, we observed the changes that occur before and during the silk-spinning process. Our reconstructed models suggested that silkworms have the ability to control the spinning process by opening or closing the spinning duct. During the continuously spinning period, both the pressure generated by the silk gland and the pulling force resulting from head movement work in tandem to expel the silk solution. We believe that gaining a full understanding of the spinning process steps can advance our ability to spin synthetic fibers with properties comparable to those of native fibers by mimicking the natural spinning process.

In-situ preparation of lignin/Fe3O4 magnetic spheres as bifunctional material for the efficient removal of metal ions and methylene blue.

In this paper, magnetic composite of lignin/Fe3O4 spheres were synthesized via a straightforward one-step in-situ solvothermal method showing good capacity for adsorbing heavy metal ions and dyes. The physicochemical properties of lignin/Fe3O4 spheres are analyzed using a range of techniques such as SEM, XRD, FTIR, VSM, TG, and BET. Lignin/Fe3O4 spheres exhibited high adsorption capacities of 100.00, 353.36 and 223.71 and 180.18 mg/g for Cu (II), Ni (II) and Cr (VI) metal ions and methylene blue (MB) with equilibrium attained within 60 min. After the recycling experiments, lignin/Fe3O4 spheres still possesses excellent superparamagnetic properties and displays high adsorption capacity. The lignin/Fe3O4 spheres are an efficient and continuous adsorbent to remove heavy metal ions of Cu (II), Ni (II), Cr (VI) and cationic dyes of methylene blue in wastewater, which proves the great potential in practical pollutants treatment applications for water systems.

Rhodamine-based Fluorescent Probe With Quick Response and High Selectivity for Imaging Labile Ferrous Iron in Living Cells and Zebrafish.

The transition between its various oxidation states of Iron plays a crucial part in various chemical transformation of cells. Misregulation of iron can give rise to the iron-catalyzed reactive oxygen species disorder which have been linked to a variety of diseases, so it is crucial to monitor the labile iron pool in vivo for clinical diagnosis. According to iron autoxidation and hydrogen abstraction reaction, we reported a novel "off-on" fluorescent probe to response to ferrous (Fe2+) both in solutions and biological systems. The probe responds to Fe2+ with good selectivity toward competing metal ions. What's more, the probe presents significant fluorescent enhancement to Fe2+ in less than 1 min, making real-time sensing in biological system possible. The applications of the probe in bioimaging revealed the changes in labile iron pool by iron autoxidation or diverse stimuli.

The ratiometric fluorescent sensor based on the mixture of CdTe quantum dots and graphene quantum dots for quantitative analysis of silver in drinks.

Silver nanoparticles can be used in antibacterial packaging or disinfection. Research has shown that sugary fluid induces the leaching of silver nanoparticles into water, which may be harmful to humans. Single wavelength fluorescence analysis has been used for quantitative analysis of silver nanoparticles but suffers from low specificity and poor anti-interference ability. In this paper, a ratiometric fluorescence sensor system (GCS) was used for the detection of Ag+, which realized both visual detection and quantitative analysis of silver in drinks. The color changes of GCS with different concentrations of Ag+ are distinguishable and easy to analyze. There is also a good linear relationship between the concentrations of Ag+ and varieties of F424 nm/F570 nm, and the lowest detection limit reached 0.2266 nmol/L. This GCS shows good selectivity and recovery and could be used for the detection of Ag+ in drink samples.

An Ethyl-Thioglycolate-Functionalized Fe3O4@ZnS Magnetic Fluorescent Nanoprobe for the Detection of Ag+ and Its Applications in Real Water Solutions.

Ethyl-thioglycolate-modified Fe3O4@ZnS nanoparticles (Fe3O4@ZnS-SH) were successfully prepared using a simple chemical precipitation method. The introduction of ethyl thioglycolate better regulated the surface distribution of ZnS, which can act as a recognition group and can cause a considerable quenching of the fluorescence intensity of the magnetic fluorescent nanoprobe, Fe3O4@ZnS-SH. Benefiting from stable fluorescence emission, the magnetic fluorescent nanoprobe showed a highly selective fluorescent response to Ag+ in the range of 0-400 µM, with a low detection limit of 0.20 µM. The magnetic fluorescent nanoprobe was used to determine the content of Ag+ in real samples. A simple and environmentally friendly approach was proposed to simultaneously achieve the enrichment, detection, and separation of Ag+ and the magnetic fluorescent nanoprobe from an aqueous solution. These results may lead to a wider range of application prospects of Fe3O4 nanomaterials as base materials for fluorescence detection in the environment.

One-pot preparation of Mn3O4/GSH/CdTe quantum dots complex for T1-weighted MRI/fluorescence detection of H3PO4.

Magnetic resonance and fluorescence dual mode imaging with both advantages of high sensitivity and high spatial resolution, which could realize real-time, in situ, and dynamic imaging, is very suitable for detecting small molecules. And the synthesis of imaging materials with good imaging performance has become the core content. In this paper, based on biomimetic mineralization technology, GSH has been used as a material to design a one-pot method for the preparation of Mn3O4/GSH/CdTe quantum dots composites (MGQ in short). Furthermore, MGQ with good T1MR and fluorescence response to the variety concentration of H3PO4, has been used for quantitative analysis of H3PO4 in serum. And the lowest limit detection could reach 0.1769 nmol/L for fluorescent detection, 0.02207 mol/L for MRI detection. MGQ would be applied as a sensor in diagnostic and clinical applications.

Drosophila melanogaster resilin improves the mechanical properties of transgenic silk.

Resilin is a natural protein with high extensibility and resilience that plays a key role in the biological processes of insects, such as flight, bouncing, and vocalization. This study used piggyBac-mediated transgenic technology to stably insert the Drosophila melanogaster resilin gene into the silkworm genome to investigate whether exogenous protein structures improve the mechanical properties of silkworm silk. Molecular detection showed that recombinant resilin was expressed and secreted into silk. Secondary structure and mechanical property analysis showed that the ß-sheet content in silk from transgenic silkworms was higher than in wild-type silk. The fracture strength of silk fused with resilin protein was 7.2% higher than wild-type silk. The resilience of recombinant silk after one-time stretching and cyclic stretching was 20.5% and 18.7% higher than wild-type silk, respectively. In summary, Drosophila resilin can enhance the mechanical properties of silk, and this study is the first to improve the mechanical properties of silk using proteins other than spider silk, which broadens the possibilities for the design and application of biomimetic silk materials.

Synthesis and Application of Ion-Exchange Magnetic Microspheres for Deep Removal of Trace Acetic Acid from DMAC Waste Liquid.

In order to develop a deep method for removing trace acetic acid from industrial solvents, a type of quaternary ammonium-salt-modified magnetic microspheres was developed as a potential nanoadsorbent for low-concentration acetic-acid-enhanced removal from DMAC aqueous solution. The ion-exchange magnetic microspheres (Fe3O4@SiO2@N(CH3)3+) have been prepared by a two-step sol-gel method with N-trimethoxysilylpropyl-N, N, N-trimethylammonium chloride as functional monomer, tetraethyl orthosilicate as a cross-linking agent, Fe3O4@SiO2 as a matrix. The nanocomposite is characterized by SEM, FI-IR, XRD, VSM, and XPS. Moreover, the optimization of adsorption experiments shows that the maximum adsorption capacity of nanoadsorbent is 7.25 mg/g at a concentration = 30 mg/L, adsorbent dosage = 10 mg, V = 10 mL, and room temperature. Furthermore, the saturated Fe3O4@SiO2@N(CH3)3+ achieved an efficient regeneration using a simple desorption method and demonstrated a good regeneration performance after five adsorption/desorption cycles. In addition, Fe3O4@SiO2@N(CH3)3+ was used to remove acetic acid in DMAC waste liquid; the adsorption effect is consistent with that of a nanoadsorbent of acetic acid in an aqueous solution. These results indicate that Fe3O4@SiO2@N(CH3)3+ can efficiently treat acetic acid that is difficult to remove from DMAC waste liquid.

Sex separation by body color via a W-chromosome-linked transgene.

Sex separation processes are important for commercial insect production and sterile insect techniques. Here, we describe the transgenic insertion of a DsRed expression cassette driven by the enhancer HR3 and strong promoter IE1 into the silkworm W chromosome as a dominant visible marker of sex separation. The obtained transgenic lines showed female-specific body color visible to the naked eye at the second- to fifth-instar larval, pupal and adult stages, and their performance traits were comparable to those of a nontransgenic practical silkworm variety. This strategy can greatly facilitate the sex separation of silkworms for male-only rearing and to obtain hybrids while avoiding sibling mating, and it can also be applied to the sex separation of other light-colored insects.

Intravenous Administration of Hypertonic Glucose Solution to Prevent Dizziness in Patients Undergoing Gastrointestinal Endoscopy Under General Anesthesia: A Randomized Clinical Trial.

BACKGROUND AND OBJECTIVE: Dizziness is a common complication of gastrointestinal endoscopy under general anesthesia. Dizziness is primarily caused by a lack of energy and blood volume following fasting and water deprivation. Hypertonic glucose solution (HGS) is an intravenous energy replenishment, that increases blood volume due to its hyperosmotic characteristics and can be directly absorbed from blood circulation. This study aimed to HGS can prevent dizziness after gastrointestinal endoscopy. METHODS: This was a double-blind, randomized, controlled study. Eligible patients were randomly allocated into two groups based on the intravenous agent administered before gastrointestinal endoscopy: Group A, saline (0.9%; 20 mL); and group B, HGS (50%; 20 mL). Overall, 840 patients were included in the statistical analysis. The scores and incidence of dizziness were assessed. RESULTS: The dizziness score were higher in group A than in group B (1.92 ± 0.08 vs. 0.92 ± 0.06; p < 0.01). The incidence of mild dizziness and moderate-to-severe dizziness was significantly lower in group B than in group A (40.10% vs. 51.78% and 3.10% vs. 19.72%, respectively; p < 0.01). The incidence and score of dizziness were significantly lower in males than in females (30.81% vs. 51.82% and 0.64 ± 0.08 vs. 1.12 ± 0.08, respectively; p < 0.01) after pretreatment with HGS. CONCLUSION: Pretreatment with HGS effectively prevents dizziness after gastrointestinal endoscopy under general anesthesia. The mechanism of action is unclear but might be related to body energy replacement and an increase in blood volume following HGS administration.

Controllable Preparation of Superparamagnetic Fe3O4@La(OH)3 Inorganic Polymer for Rapid Adsorption and Separation of Phosphate.

Superparamagnetic Fe3O4 particles have been synthesized by solvothermal method, and a layer of dense silica sol polymer is coated on the surface prepared by sol-gel technique; then La(OH)3 covered the surface of silica sol polymer in an irregular shape by controlled in situ growth technology. These magnetic materials are characterized by TEM, FT-IR, XRD, SEM, EDS and VSM; the results show that La(OH)3 nanoparticles have successfully modified on Fe3O4 surface. The prepared Fe3O4@La(OH)3 inorganic polymer has been used as adsorbent to remove phosphate efficiently. The effects of solution pH, adsorbent dosage and co-existing ions on phosphate removal are investigated. Moreover, the adsorption kinetic equation and isothermal model are used to describe the adsorption performance of Fe3O4@La(OH)3. It was observed that Fe3O4@La(OH)3 exhibits a fast equilibrium time of 20 min, high phosphate removal rate (>95.7%), high sorption capacity of 63.72 mgP/g, excellent selectivity for phosphate in the presence of competing ions, under the conditions of phosphate concentration 30 mgP/L, pH = 7, adsorbent dose 0.6 g/L and room temperature. The phosphate adsorption process by Fe3O4@La(OH)3 is best described by the pseudo-second-order equation and Langmuir isotherm model. Furthermore, the real samples and reusability experiment indicate that Fe3O4@La(OH)3 could be regenerated after desorption, and 92.78% phosphate removing remained after five cycles. Therefore, La(OH)3 nanoparticles deposited on the surface of monodisperse Fe3O4 microspheres have been synthesized for the first time by a controlled in-situ growth method. Experiments have proved that Fe3O4@La(OH)3 particles with fast separability, large adsorption capacity and easy reusability can be used as a promising material in the treatment of phosphate wastewater or organic pollutants containing phosphoric acid functional group.

The preparation of MnO2/BSA/CdTe quantum dots complex for ratiometric fluorescence/ T1-weighted MRI detection of H2O2.

Hydrogen peroxide, as the second messenger in cells, plays an essential regulatory role in cell proliferation, differentiation and migration. How to effectively identify H2O2 signals, and detect H2O2 in circulating tumor cells, are essential for the early diagnosis of tumors. Herein a fluorescence and T1-weighted MR dual-mode imaging material, named MnBQ, has been successfully prepared and characterized. Based on the chemical redox reaction between MnBQ and H2O2, a fluorescence/T1-weighted MRI dual-mode sensor has been developed for quantitative analysis of H2O2, of which the lowest limit detection obtained from fluorescence and MRI could reach 2.841 µmol/L and 43.85 µmol/L, respectively. This imaging sensor has dual advantages of high sensitivity and high spatial resolution, which could be a good candidate for monitoring of H2O2 in vivo.

The detection of multiple analytes by using visual colorimetric and fluorometric multimodal chemosensor based on the azo dye.

In recent decades, researchers have conducted in-depth studies of the design and synthesis of colorimetric/fluorometric probes and the application of such probes to biological and practical samples. The multifunctional colorimetric and fluorescent azo benzene-based probe (4'-hydroxyl-2,4-diaminoazobenzene, MP) was designed to detect Al3+, Fe3+, Cu2+ and F¯. Based on the distinct redshift of the absorption band and a significant color change (yellow → purple), MP was utilized for both naked-eyed and quantitative detection of Al3+ and Fe3+ after formation of the 1:1 complex. Test paper coated with MP and used in conjunction with a cell phone was used for colorimetric detection of Al3+ and Fe3+ ions (20 µM-2.0 mM) in water samples through naked-eye and digital image colorimetry. The "MP-Fe3+" coordination shift that occurs in the presence of the competitive ligand F¯ was used in the colorimetric measurement of F¯ in toothpaste. In the presence of Cu2+ ion, the non-emissive MP has transformed into fluorescent benzotriazole product PMP (Φ = 0.53) through the bimolecular rate-limiting step, and the second-order rate constant k is calculated as 31 ± 2 M-1 s-1. MP exhibits a "turn-on" fluorescence response in the presence of Cu2+ that is greater than its response in the presence of competitive species such as Fe3+, Al3+, Co2+, Fe2+, Zn2+, Cd2+, Mg2+, Mn2+, Ni2+ and Ag+. MP was shown to have low toxicity to living HeLa cells and to present good imaging characteristics for tracking of Cu2+ in vivo.

A novel mitochondria-targetable NIR fluorescent probe for monitoring intracellular hypobromous acid levels.

The development of ultrasensitive in situ detection techniques for monitoring hypobromous acid (HBrO) levels in the biological systems is of great significance to reveal its complex pathological and physiological effects. A simple mitochondria-targetable hydrazine-based near-infrared (NIR) fluorescent probe (Mito-NIR) for detecting HBrO in the mitochondria of live cells is presented in this paper. Probe Mito-NIR displays the ultrafast (< 5 s) response for HBrO. It can detect HBrO with high sensitivity. Additionally, it shows high selectivity towards HBrO over other biologically important substances. Finally, it can monitor the changes of endogenous/exogenous HBrO levels in the mitochondria of live cells. A simple mitochondria-targetable NIR fluorescent probe with picomolar sensitivity for HBrO was developed to specifically track mitochondrial HBrO.

Mechanism of silk secretion revealed by proteomic analysis of silkworm cocoons with fibroin light chain mutations.

Silkworm is an economically important insect due to its efficient production of silk proteins. Silk itself and the silk trade have enriched human civilization through art and culture and contributed to early globalization in the Silk Road era for nearly two thousand years. Although a large number of studies on silk have been carried out, the mechanism of silk secretion in silkworms has not been thoroughly studied thus far. As the main component of fibroin, fibroin light chain (Fib-L) plays a key role in the secretion of silk. In this study, we constructed a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. Strikingly, fibrohexamerin (P25) was secreted almost normally in the homozygous mutant. Further analysis of cocoon proteins revealed that the mutant responded to greater environmental stress caused by a dramatic decrease in fibroin by significantly increasing the secretion of protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm. SIGNIFICANCE: This study generated a homozygous Fib-L gene mutant population of a nonpractical variety using the CRISPR/Cas9 system. The homozygous mutants displayed a thin cocoon layer, but their viability was not affected by the Fib-L mutation. Furthermore, a comparative proteomic analysis of homozygous mutant cocoons and wild-type cocoons was performed. The analysis of the abundance of silk proteins in the cocoons revealed that P25 could be secreted almost normally. The analysis of the abundance of cocoon proteins other than silk proteins showed that the homozygous mutants responded to greater environmental stress by increasing the secretion of defense-related proteins, such as protease inhibitors. These results will further help explain the silk secretion mechanism of silkworm.