FAST, a method based on split-GFP for the detection in solution of proteins synthesized in cell-free expression systems.

Cell-free protein synthesis (CFPS) systems offer a versatile platform for a wide range of applications. However, the traditional methods for detecting proteins synthesized in CFPS, such as radioactive labeling, fluorescent tagging, or electrophoretic separation, may be impractical, due to environmental hazards, high costs, technical complexity, and time consuming procedures. These limitations underscore the need for new approaches that streamline the detection process, facilitating broader application of CFPS. By harnessing the reassembly capabilities of two GFP fragments-specifically, the GFP1-10 and GFP11 fragments-we have crafted a method that simplifies the detection of in vitro synthesized proteins called FAST (Fluorescent Assembly of Split-GFP for Translation Tests). FAST relies on the fusion of the small tag GFP11 to virtually any gene to be expressed in CFPS. The in vitro synthesized protein:GFP11 can be rapidly detected in solution upon interaction with an enhanced GFP1-10 fused to the Maltose Binding Protein (MBP:GFP1-10). This interaction produces a fluorescent signal detectable with standard fluorescence readers, thereby indicating successful protein synthesis. Furthermore, if required, detection can be coupled with the purification of the fluorescent complex using standardized MBP affinity chromatography. The method's versatility was demonstrated by fusing GFP11 to four distinct E. coli genes and analyzing the resulting protein synthesis in both a homemade and a commercial E. coli CFPS system. Our experiments confirmed that the FAST method offers a direct correlation between the fluorescent signal and the amount of synthesized protein:GFP11 fusion, achieving a sensitivity threshold of 8 ± 2 pmol of polypeptide, with fluorescence plateauing after 4 h. Additionally, FAST enables the investigation of translation inhibition by antibiotics in a dose-dependent manner. In conclusion, FAST is a new method that permits the rapid, efficient, and non-hazardous detection of protein synthesized within CFPS systems and, at the same time, the purification of the target protein.

A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

High-Plex ELISPOT: FOLISPOT Based on Fluorescence Detection and DNA Complementary Pairing.

Enzyme-linked immunospot (ELISPOT) is one of the most important methods to measure the number of specific cells by detecting protein secretion at a single-cell level. However, traditional ELISPOT based on enzyme-substrate color development can only detect one target. Therefore, scientists developed multiple-target ELISPOT based on enzyme-substrate coloring. Besides, FluoroSPOT that can detect 2-4 fluorescent signals are developed. Nevertheless, the maximum detection targets of multiple-target ELISPOT and FluoroSPOT are around 4, and the signal amplification system can be further optimized. Fluorescence-based oligo-linked immunospot (FOLISPOT), which utilized DNA-barcoded antibodies to provide a highly multiplexed method with signal amplification, was developed to detect multiple targets simultaneously. In this method, multiple targets can be detected in one round and multiple rounds of detection can be conducted, and thus a large number of targets can be detected. Besides, signal amplification is achieved by DNA complementary pairing and modular orthogonal DNA concatemers, and thus cells secreting limited amounts of proteins can be detected. According to the studies, FOLISPOT can detect more spots than ELISPOT and can detect targets that are undetectable by ELISPOT. Furthermore, FOLISPOT can be utilized to detect more than 6 targets, by allowing sequential detection of multiple targets in one round and sequential detection in multiple rounds.

Decolorization and detoxication of malachite green by engineered Saccharomyces cerevisiae expressing novel thermostable laccase from Trametes trogii.

Malachite Green (MG) is a widely used industrial dye that is hazardous to health. Herein, the decolourisation and detoxification of MG were achieved using the engineered Saccharomyces cerevisiae expressing novel thermostable laccase lcc1 from Trametes trogii. The engineered strain RCL produced a high laccase activity of 121.83 U L-1. Lcc1 was stable at temperatures ranging from 20 ℃ to 60 ℃ and showed a high tolerance to organic solvents. Moreover, Lcc1 could decolorize different kinds of dyes (azo, anthraquinone and triphenylmethane), among which, the decolorization ability of MG is the highest, reaching 95.10 %, and the decolorization rate of other triphenylmethane dyes also over 50 %. The RCL decolorized about 95 % of 50 mg L-1 of MG dye in 10 h at 30 ℃. The MG degradation products were analyzed. The industrial application potential of the RCL was evaluated by treating industrial wastewater and the decolourisation rates were over 90 %.

Promotion of anaerobic biodegradation of azo dye RR2 by different biowaste-derived biochars: Characteristics and mechanism study by machine learning.

The addition of biochar resulted in a 31.5 % to 44.6 % increase in decolorization efficiency and favorable decolorization stability. Biochar promoted extracellular polymeric substances (EPS) secretion, especially humic-like and fulvic-like substances. Additionally, biochar enhanced the electron transfer capacity of anaerobic sludge and facilitated surface attachment of microbial cells. 16S rRNA gene sequencing analysis indicated that biochar reduced microbial species diversity, enriching fermentative bacteria such as Trichococcus. Finally, a machine learning model was employed to establish a predictive model for biochar characteristics and decolorization efficiency. Biochar electrical conductivity, H/C ratio, and O/C ratio had the most significant impact on RR2 anaerobic decolorization efficiency. According to the results, the possible mechanism of RR2 anaerobic decolorization enhanced by different types of biochar was proposed.

Biotransformation of the azo dye reactive orange 16 by Aspergillus flavus A5P1: Performance, genetic background, pathway, and mechanism.

This study reports the strain Aspergillus flavus A5P1 (A5P1), which is with the capable of degrading the azo dye reactive orange 16 (RO16). The mechanism of RO16 degradation by A5P1 was elucidated through genomic analysis, enzymatic analysis, degradation pathway analysis and oxidative stress analysis. Strain A5P1 exhibited aerobic degradation of RO16, with optimal degradation at an initial pH of 3.0. Genomic analysis indicates that strain A5P1 possesses the potential for acid tolerance and degradation of azo dye. Enzymatic analysis, combined with degradation product analysis, demonstrated that extracellular laccase, intracellular lignin peroxidase, and intracellular quinone reductase were likely key enzymes in the RO16 degradation process. Oxidative stress analysis revealed that cell stress responses may participate in the RO16 biotransformation process. The results indicated that the biotransformation of RO16 may involves biological processes such as transmembrane transport of RO16, cometabolism of the strain with RO16, and cell stress responses. These findings shed light on the biodegradation of RO16 by A5P1, indicating A5P1's potential for environmental remediation.

Visualizing Low-Abundance Proteins and Post-Translational Modifications in Living Drosophila Embryos via Fluorescent Antibody Injection.

Visualization of proteins in living cells using GFP (Green Fluorescent Protein) and other fluorescent tags has greatly improved understanding of protein localization, dynamics, and function. Compared to immunofluorescence, live imaging more accurately reflects protein localization without potential artifacts arising from tissue fixation. Importantly, live imaging enables quantitative and temporal characterization of protein levels and localization, crucial for understanding dynamic biological processes such as cell movement or division. However, a major limitation of fluorescent tagging approaches is the need for sufficiently high protein expression levels to achieve successful visualization. Consequently, many endogenously tagged fluorescent proteins with relatively low expression levels cannot be detected. On the other hand, ectopic expression using viral promoters can sometimes lead to protein mislocalization or functional alterations in physiological contexts. To address these limitations, an approach is presented that utilizes highly sensitive antibody-mediated protein detection in living embryos, essentially performing immunofluorescence without the need for tissue fixation. As proof of principle, endogenously GFP-tagged Notch receptor that is barely detectable in living embryos can be successfully visualized after antibody injection. Furthermore, this approach was adapted to visualize post-translational modifications (PTMs) in living embryos, allowing the detection of temporal changes in tyrosine phosphorylation patterns during early embryogenesis and revealing a novel subpopulation of phosphotyrosine (p-Tyr) underneath apical membranes. This approach can be modified to accommodate other protein-specific, tag-specific, or PTM-specific antibodies and should be compatible with other injection-amenable model organisms or cell lines. This protocol opens new possibilities for live imaging of low-abundance proteins or PTMs that were previously challenging to detect using traditional fluorescent tagging methods.

Minicells as an Escherichia coli mechanism for the accumulation and disposal of fluorescent cadmium sulphide nanoparticles.

BACKGROUND: Bacterial biosynthesis of fluorescent nanoparticles or quantum dots (QDs) has emerged as a unique mechanism for heavy metal tolerance. However, the physiological pathways governing the removal of QDs from bacterial cells remains elusive. This study investigates the role of minicells, previously identified as a means of eliminating damaged proteins and enhancing bacterial resistance to stress. Building on our prior work, which unveiled the formation of minicells during cadmium QDs biosynthesis in Escherichia coli, we hypothesize that minicells serve as a mechanism for the accumulation and detoxification of QDs in bacterial cells. RESULTS: Intracellular biosynthesis of CdS QDs was performed in E. coli mutants ΔminC and ΔminCDE, known for their minicell-producing capabilities. Fluorescence microscopy analysis demonstrated that the generated minicells exhibited fluorescence emission, indicative of QD loading. Transmission electron microscopy (TEM) confirmed the presence of nanoparticles in minicells, while energy dispersive spectroscopy (EDS) revealed the coexistence of cadmium and sulfur. Cadmium quantification through flame atomic absorption spectrometry (FAAS) demonstrated that minicells accumulated a higher cadmium content compared to rod cells. Moreover, fluorescence intensity analysis suggested that minicells accumulated a greater quantity of fluorescent nanoparticles, underscoring their efficacy in QD removal. Biosynthesis dynamics in minicell-producing strains indicated that biosynthesized QDs maintained high fluorescence intensity even during prolonged biosynthesis times, suggesting continuous QD clearance in minicells. CONCLUSIONS: These findings support a model wherein E. coli utilizes minicells for the accumulation and removal of nanoparticles, highlighting their physiological role in eliminating harmful elements and maintaining cellular fitness. Additionally, this biosynthesis system presents an opportunity for generating minicell-coated nanoparticles with enhanced biocompatibility for diverse applications.

Bacteria as a source of biopigments and their potential applications.

From the prehistoric period, the utilization of pigments as colouring agents was an integral part of human life. Early people may have utilized paint for aesthetic motives, according to archaeologists. The pigments are either naturally derived or synthesized in the laboratory. Different studies reported that certain synthetic colouring compounds were toxic and had adverse health and environmental effects. Therefore, knowing the drawbacks of these synthetic colouring agents now scientists are attracted towards the harmless natural pigments. The main sources of natural pigments are plants, animals or microorganisms. Out of these natural pigments, microorganisms are the most important source for the production and application of bioactive secondary metabolites. Among all kinds of microorganisms, bacteria have specific benefits due to their short life cycle, low sensitivity to seasonal and climatic variations, ease of scaling, and ability to create pigments of various colours. Based on these physical characteristics, bacterial pigments appear to be a promising sector for novel biotechnological applications, ranging from functional food production to the development of new pharmaceuticals and biomedical therapies. This review summarizes the need for bacterial pigments, biosynthetic pathways of carotenoids and different applications of bacterial pigments.

Dye-decolorizing peroxidase of Thermobifida halotolerance displays complex kinetics with both substrate inhibition and apparent positive cooperativity.

Dye-decolorizing peroxidases (DyPs) have been intensively investigated for the purpose of industrial dye decolourization and lignin degradation. Unfortunately, the characterization of these peroxidases is hampered by their non-Michaelis-Menten kinetics, exemplified by substrate inhibition and/or positive cooperativity. Although often observed, the underlying mechanisms behind the unusual kinetics of DyPs are poorly understood. Here we studied the kinetics of the oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydroquinones, and anthraquinone dyes by DyP from the bacterium Thermobifida halotolerans (ThDyP) and solved its crystal structure. We also provide rate equations for different kinetic mechanisms explaining the complex kinetics of heme peroxidases. Kinetic studies along with the analysis of the structure of ThDyP suggest that the substrate inhibition is caused by the non-productive binding of ABTS to the enzyme resting state. Strong irreversible inactivation of ThDyP by H2O2 in the absence of ABTS suggests that the substrate inhibition by H2O2 may be caused by the non-productive binding of H2O2 to compound I. Positive cooperativity was observed only with the oxidation of ABTS but not with the two electron-donating substrates. Although the conventional mechanism of cooperativity cannot be excluded, we propose that the oxidation of ABTS assumes the simultaneous binding of two ABTS molecules to reduce compound I to the enzyme resting state, and this causes the apparent positive cooperativity.

An ER-targeted, Viscosity-sensitive Hemicyanine Dye for the Diagnosis of Nonalcoholic Fatty Liver and Photodynamic Cancer Therapy by Activating Pyroptosis Pathway.

The concept of molecular design, integrating diagnostic and therapeutic functions, aligns with the general trend of modern medical advancement. Herein, we rationally designed the smart molecule ER-ZS for endoplasmic reticulum (ER)-targeted diagnosis and treatment in cell and animal models by combining hemicyanine dyes with ER-targeted functional groups (p-toluenesulfonamide). Owing to its ability to target the ER with a highly specific response to viscosity, ER-ZS demonstrated substantial fluorescence turn-on only after binding to the ER, independent of other physiological environments. In addition, ER-ZS, being a small molecule, allows for the diagnosis of nonalcoholic fatty liver disease (NAFLD) via liver imaging based on high ER stress. Importantly, ER-ZS is a type I photosensitizer, producing O2 ⋅- and ⋅OH under light irradiation. Thus, after irradiating for a certain period, the photodynamic therapy inflicted severe oxidative damage to the ER of tumor cells in hypoxic (2 % O2 ) conditions and activated the unique pyroptosis pathway, demonstrating excellent antitumor capacity in xenograft tumor models. Hence, the proposed strategy will likely shed new light on integrating molecular optics for NAFLD diagnosis and cancer therapy.

A fluorescent splice-switching mouse model enables high-throughput, sensitive quantification of antisense oligonucleotide delivery and activity.

While antisense oligonucleotides (ASOs) are used in the clinic, therapeutic development is hindered by the inability to assay ASO delivery and activity in vivo. Accordingly, we developed a dual-fluorescence, knockin mouse model that constitutively expresses mKate2 and an engineered EGFP that is alternatively spliced in the presence of ASO to induce expression. We first examined free ASO activity in the brain following intracerebroventricular injection revealing EGFP splice-switching is both ASO concentration and time dependent in major central nervous system cell types. We then assayed the impact of lipid nanoparticle delivery on ASO activity after intravenous administration. Robust EGFP fluorescence was observed in the liver and EGFP+ cells were successfully isolated using fluorescence-activated cell sorting. Together, these results show the utility of this animal model in quantifying both cell-type- and organ-specific ASO delivery, which can be used to advance ASO therapeutics for many disease indications.

Cancer-cell-specific Self-Reporting Photosensitizer for Precise Identification and Ablation of Cancer Cells.

Cancer-cell-specific fluorescent photosensitizers (PSs) are highly desired molecular tools for cancer ablation with minimal damage to normal cells. However, such PSs that can achieve cancer specification and ablation and a self-reporting manner concurrently are rarely reported and still an extremely challenging task. Herein, we have proposed a feasible strategy and conceived a series of fluorescent PSs based on simple chemical structures for identifying and killing cancer cells as well as monitoring the photodynamic therapy (PDT) process by visualizing the change of subcellular localization. All of the constructed cationic molecules could stain mitochondria in cancer cells, identify cancer cells specifically, and monitor cancer cell viability. Among these, IVP-Br has the strongest ability to produce ROS, which serves as a potent PS for specific recognition and killing of cancer cells. IVP-Br could translocate from mitochondria to the nucleolus during PDT, self-reporting the entire therapeutic process. Mechanism study confirms that IVP-Br with light irradiation causes cancer cell ablation via inducing cell cycle arrest, cell apoptosis, and autophagy. The efficient ablation of tumor through PDT induced by IVP-Br has been confirmed in the 3D tumor spheroid chip. Particularly, IVP-Br could discriminate cancer cells from white blood cells (WBCs), exhibiting great potential to identify circulating tumor cells (CTCs).

Contamination of textile dyes in aquatic environment: Adverse impacts on aquatic ecosystem and human health, and its management using bioremediation.

Textile dyes are the burgeoning environmental contaminants across the world. They might be directly disposed of from textile industries into the aquatic bodies, which act as the direct source for the entire ecosystem, ultimately impacting the human beings. Hence, it is essential to dissect the potential adverse outcomes of textile dye exposure on aquatic plants, aquatic fauna, terrestrial entities, and humans. Analysis of appropriate literature has revealed that textile dye effluents could affect the aquatic biota by disrupting their growth and reproduction. Various aquatic organisms are targeted by textile dye effluents. In such organisms, these chemicals affect their development, behavior, and induce oxidative stress. General populations of humans are exposed to textile dyes via the food chain and drinking contaminated water. In humans, textile dyes are biotransformed into electrophilic intermediates and aromatic amines by the enzymes of the cytochrome family. Textile dyes and their biotransformed products form the DNA and protein adducts at sub-cellular moiety. Moreover, these compounds catalyze the production of free radicals and oxidative stress, and trigger the apoptotic cascades to produce lesions in multiple organs. In addition, textile dyes modulate epigenetic factors like DNA methyltransferase and histone deacetylase to promote carcinogenesis. Several bioremediation approaches involving algae, fungi, bacteria, biomembrane filtration techniques, etc., have been tested and some other hybrid systems are currently under investigation to treat textile dye effluents. However, many such approaches are at the trial stage and require further research to develop more efficient, cost-effective, and easy-to-handle techniques.

Revealing the degrading-possibility of methyl red by two azoreductases of Anoxybacillus sp. PDR2 based on molecular docking.

Azo dyes, as the most widely used synthetic dyes, are considered to be one of the culprits of water resources and environmental pollution. Anoxybacillus sp. PDR2 is a thermophilic bacterium with the ability to degrade azo dyes, whose genome contains two genes encoding azoreductases (named AzoPDR2-1 and AzoPDR2-2). In this study, through response surface methodology (RSM), when the initial pH, inoculation volume and Mg2+ addition amount were 7.18, 10.72% and 0.1 g/L respectively, the decolorization rate of methyl red (MR) (200 mg/L) could reach its maximum (98.8%). The metabolites after biodegradation were detected by UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and liquid chromatography mass spectrometry (LC-MS/MS), indicating that MR was successfully decomposed into 4-aminobenzoic acid and other small substrates. In homologous modeling, it was found that both azoreductases were flavin-dependent azoreductases, and belonged to the α/ß structure, using the Rossmann fold. In their docking results with the cofactor flavin mononucleotide (FMN), FMN bound to the surface of the protein dimer. Nicotinamide adenine dinucleotide (NADH) was superimposed on the plane of the pyrazine ring between FMN and the activity pocket of protein. Besides, both azoreductase complexes (azoreductase-FMN-NADH) exhibited a substrate preference for MR. Asn104 and Tyr74 played an important role in the combination of the azoreductase AzoPDR2-1 complex and the azoreductase AzoPDR2-2 complex with MR, respectively. This provided assistance for studying the mechanism of azoreductase biodegradation of azo dyes in thermophilic bacteria.

Biodegradation of commercial textile reactive dye mixtures by industrial effluent adapted bacterial consortium VITPBC6: a potential technique for treating textile effluents.

Textile industries release major fraction of dyestuffs in effluents leading to a major environmental concern. These effluents often contain more than one dyestuff, which complicates dye degradation. In this study ten reactive dyes (Reactive Yellow 145, Reactive Yellow 160, Reactive Orange 16, Reactive Orange 107, Reactive Red 195, Reactive Blue 21, Reactive Blue 198, Reactive Blue 221, Reactive Blue 250, and Reactive Black 5) that are used in textile industries were subjected to biodegradation by a bacterial consortium VITPBC6, formulated in our previous study. Consortium VITPBC6 caused single dye degradation of all the mentioned dyes except for Reactive Yellow 160. Further, VITPBC6 efficiently degraded a five-dye mixture (Reactive Red 195, Reactive Orange 16, Reactive Black 5, Reactive Blue 221, and Reactive Blue 250). Kinetic studies revealed that the five-dye mixture was decolorized by VITPBC6 following zero order reaction kinetic; Vmax and Km values of the enzyme catalyzed five-dye decolorization were 128.88 mg L-1 day-1 and 1003.226 mg L-1 respectively. VITPBC6 degraded the dye mixture into delta-3,4,5,6-Tetrachlorocyclohexene, sulfuric acid, 1,2-dichloroethane, and hydroxyphenoxyethylaminohydroxypropanol. Phytotoxicity, cytogenotoxicity, microtoxicity, and biotoxicity assays conducted with the biodegraded metabolites revealed that VITPBC6 lowered the toxicity of five-dye mixture significantly after biodegradation.

Synergistic molecular mechanism of degradation in dye wastewater by Rhodopseudomonas palustris intimately coupled carbon nanotube - Silver modified titanium dioxide photocatalytic composite with sodium alginate.

The intimately coupled photocatalysis and biodegradation (ICPB), which combined the advantages of high oxidation capacity of photocatalysis and high mineralization rate of biodegradation, has demonstrated excellent removal performance in the degradation of azo dyes with highly toxic, refractory, mutagenic and carcinogenic. In order to explore the metagenomics mechanism of the ICPB system, a novel ICPB was prepared by coupling Rhodopseudomonas palustris (R. Palustris), carbon nanotube - silver modified titanium dioxide photocatalytic composite (CNT-Ag -TiO2, CAT) and sodium alginate (SA) (R. palustris/CAT@SA, R-CAT). Metagenomics sequencing was used to investigate the molecular mechanism of adaptation and degradation of dyes by photosynthetic microorganisms and the adaptive and synergistic interaction between photosynthetic microorganisms and photocatalyst. Experiments on the adaptability and degradability of photosynthetic microorganisms have proved that low concentration azo dyes could be utilized as carbon sources for growth of photosynthetic microorganisms. Metagenomics sequencing revealed that R. palustris was the main degrading bacterium in photosynthetic microorganisms and the functional genes related to carbohydrate metabolism, biological regulation and catalytic activity were abundant. It was found that the addition of photocatalyst significantly up-regulated the functional genes related to the catabolic process, electron transport, oxidoreductase activity and superoxide metabolism of organic matter in the photosynthetic microorganisms. Moreover, many key gene such as alpha-amylase, 1-acyl-sn-glycerol-3-phosphate acyltransferase, aldehyde dehydrogenase enrichment in microbial basal metabolism, such as enoyl-CoA hydratase, malate dehydrogenase, glutathione S-transferase enrichment in degrading azo dyes and electron transport, and many key gene such as undecaprenyl-diphosphatase, carbon storage regulator, DNA ligase enrichment in response to dyes and photocatalysts were discovered. These findings would contribute to a comprehensive understanding of the mechanism of degradation of dye wastewater by ICPB system, a series of genes was produced to adapt to environmental changes, and played synergistic role in terms of intermediate product degradation and electron transfer for degrading azo dyes. The photosynthetic microorganisms might be a promising microorganism for constructing ICPB system.

Electricity generation and oxidoreductase potential during dye discoloration by laccase-producing Ganoderma gibbosum in fungal fuel cell.

Color chemicals contaminate pure water constantly discharged from different points and non-point sources. Physical and chemical techniques have certain limitations and complexities for bioenergy production, which motivated the search for a novel sustainable production approaches during dye wastewater treatment. The emerging environmental problem of dye decolorization has attracted scientist's attention to a new, cheap, and economical way to treat dye wastewater and power production via fungal fuel cells. Ganoderma gibbosum was fitted in the cathodic region with laccase secretion in the fuel cell. At the same time, dye water was placed in the anodic region to move electrons and produce power. This study treated wastewater using the oxidoreductase enzymes released extracellularly from Ganoderma gibbosum for dye Remazol Brilliant Blue R (RBBR) degradation via fungal-based fuel cell. The maximum power density of 14.18 mW/m2 and the maximum current density of 35 mA/m2 were shown by the concentration of 5 ppm during maximum laccase activity and decolorization of RBBR. The laccase catalysts have gained considerable attention because of eco-friendly and alternative easy handling approaches to chemical methods. Fungal Fuel Cells (FFCs) are efficiently used in dye treatment and electricity production. This article also highlighted the construction of fungal catalytic cells and the enzymatic performance of fungal species in energy production during dye water treatment.

Bone Formation in Zebrafish: The Significance of DAF-FM DA Staining for Nitric Oxide Detection.

DAF-FM DA is widely used as a live staining compound to show the presence of nitric oxide (NO) in cells. Applying this stain to live zebrafish embryos is known to indicate early centers of bone formation, but the precise (cellular) location of the signal has hitherto not been revealed. Using sections of zebrafish embryos live-stained with DAF-FM DA, we could confirm that the fluorescent signals were predominantly located in areas of ongoing bone formation. Signals were observed in the bone and tooth matrix, in the notochord sheath, as well as in the bulbus arteriosus. Surprisingly, however, they were exclusively extracellular, even after very short staining times. Von Kossa and Alizarin red S staining to reveal mineral deposits showed that DAF-FM DA stains both the mineralized and non-mineralized bone matrix (osteoid), excluding that DAF-FM DA binds non-specifically to calcified structures. The importance of NO in bone formation by osteoblasts is nevertheless undisputed, as shown by the absence of bone structures after the inhibition of NOS enzymes that catalyze the formation of NO. In conclusion, in zebrafish skeletal biology, DAF-FM DA is appropriate to reveal bone formation in vivo, independent of mineralization of the bone matrix, but it does not demonstrate intracellular NO.

Biomimetic nanoplatform with selectively positioned indocyanine green for accurate sentinel lymph node imaging.

The status of draining lymph nodes (LNs) is critical for determining the treatment and prognosis of cancer that spreads through the lymphatic system. Indocyanine green (ICG) fluorescence imaging has been widely used in sentinel LN (SLN) biopsy technology and has shown favorable effects. However, this too has its own limitations, such as fluorescence instability and diffusion imaging. In this study, we developed macrophage cell membrane-camouflaged ICG-loaded biomimetic nanoparticles (M@F127-ICG) for accurate SLN imaging. ICG selectively positioned at the hydrophobic-hydrophilic interfaces of pluronic F127 micelles protected itself from quenching in aqueous solution, thereby maintaining fluorescence stability and improving fluorescence intensity. In addition, to further improve the aggregation in SLN, the micellar surface was coated with a layer of biomimetic macrophage cell membrane to target LN-resident macrophages. In vivo fluorescence imaging demonstrated that M@F127-ICG significantly enhanced the fluorescence signal and improved the imaging efficiency of SLN. Thus, selectively positioning ICG in the biomimetic nanoplatform enhanced the fluorescence intensity and stability, providing a novel tracer for timely and accurate SLN imaging.