Green Fluorescent Signal Research Articles

Recording and Interpretation of Active Calcium Transients in Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Calcium plays a pivotal role in the excitation-contraction coupling process in cardiomyocytes, a critical multi-parametric event leading to rhythmic contraction. Over the past few decades, calcium signaling in cardiomyocytes has been extensively studied in cardiovascular sciences. However, a standard methodology is needed not only to trace the calcium within cells but also to remove signal processing biases and to accurately interpret the features of calcium transient signals in relation to cardiomyocyte electrophysiology. This article outlines the use of genetically encoded calcium indicator (GCaMP) human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to record calcium transients. These cells express a green fluorescent signal when calcium binds to intracellular calmodulin, a key regulator of calcium signaling. The extraction and processing of calcium transient waveforms are performed using ImageJ and MATLAB software. Key features of these waveforms are then identified and categorized based on their physiological relevance to cardiomyocyte function. Additionally, this work includes a Support Protocol for the successful replating of cardiomyocytes onto non-traditional culture platforms, such as metallic sensors and polymer-based substrates, to facilitate data multiplexing. The three Basic Protocols outlined here provide a comprehensive approach for maintaining, expanding, and differentiating the GCaMP hiPSCs, video recording of calcium transients, and the subsequent signal extraction, preprocessing, analysis, and data visualization. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Maintenance, expansion, and differentiation of genetically encoded calcium indicator hiPSCs Support Protocol: Replating GCaMP hiPSC-CMs for stimulation and multielectrode array studies Basic Protocol 2: Video recording from calcium transients of GCaMP hiPSC-CMs Basic Protocol 3: Signal extraction, preprocessing, analysis, and data visualization.

Effects of anti-rainbow trout germ-cell monoclonal antibody on germ cells and gonadal tissues in rainbow trout (Oncorhynchus mykiss)

The development of sterilization technology is important to improve the production efficacy of aquaculture and prevent genetically modified fish from escaping. Immunosterilization is a novel sterilization method that has already been used in mammals. In this study, anti-rainbow trout germ-cell monoclonal antibodies (anti-GC mAbs) were injected into immature rainbow trout Oncorhynchus mykiss, and their effects were analyzed. First, anti-GC mAbs labeled with Alexa Fluor 488 were injected into the body cavity of larvae 20 days (group 1) and 102 days (group 2) after fertilization. Second, anti-GC mAbs were injected into the body cavity of yearling trout (group 3), and their impacts on gonadal tissue were analyzed in terms of histology and gene expression levels. As a result, anti-GC mAbs were observed to bind to some primordial GCs in group 1, whereas there were no obvious positive cells in male testes of group 2. In contrast, green fluorescent signals were observed in an arrangement parallel to the ovarian lamina in female ovaries of group 2, which was similar to the arrangement of positive signals on ovarian tissue sections immunostained with anti-GC mAbs. GCs in group 1 were observed again after 76 days, and double-positive cells remained. In group 3, there were changes in the expression levels of GC-specific genes, but the changes were not seen as biologically meaningful. Moreover, there was no histological abnormality in gonadal tissues after immunization. In conclusion, these results suggested that anti-GC mAbs could only reach GCs in early developmental stages or in the ovary, and injection of anti-GCs mAbs did not have harmful effects on GCs after binding.

Parathyroid gland identification and angiography classification using simple machine learning methods.

Near-infrared indocyanine green angiography allows experienced surgeons to reliably evaluate parathyroid gland vitality during thyroid and parathyroid operations in order to predict postoperative function. To facilitate equal performance between surgeons, we developed an automatic computational quantification method using computer vision that portrays expert interpretation of visualized parathyroid gland near-infrared indocyanine green angiographic fluorescence signals. Near-infrared indocyanine green-parathyroid gland angiography video recordings (Fluobeam® LX, Fluoptics, Grenoble-part of Getinge-Göteborg) from patients undergoing endocrine cervical surgery in a high-volume unit were used for model development. Computation (MATLAB, Mathworks, Ireland) included segmentation-identification of the parathyroid gland (by autofluorescence), image stabilization (by linear translation) and adjusted time-fluorescence intensity profile generation. Relative upslope and maximum intensity ratios then trained a simple logistic regression model based on expert interpretation and outcome (including hypoparathyroidism), with subsequent unseen testing for validation. The model was trained on 37 patient videos (45 glands, 29 judged well perfused by parathyroid gland angiography experts), achieving feature data separation with 100% accuracy, and tested on 22 unseen videos (27 glands, 15 judged well perfused), including four in real time. Segmentation-guided parathyroid gland detection correctly identified all parathyroid glands during unseen testing along with three additional non-parathyroid gland regions (90% positive predictive value). Subsequent time-fluorescence intensity profile extraction with vitality prediction was shown feasible in all cases within 5 min, with a 96.3% model accuracy (sensitivity and specificity were 93.3 and 100% respectively) when compared with expert judgement. Automatic parathyroid gland perfusion quantification using simple machine learning computational methods discriminates parathyroid gland perfusion in concordance with expert surgeon interpretation, providing a means for near-infrared indocyanine green-parathyroid gland signal evaluation.

Constructing Mechanochromic Fluorescent Interphase via Core-Shell Microcapsules: A Route for Interface Reinforcing and Damage Self-Reporting of CFRP Composites.

Perylenediimide-chitosan/γ-poly (glutamic acid) microcapsules sizing (PDI-CS/γ-PGA) core-shell microcapsule is designed and used to establish a novel interphase in carbon fiber/epoxy (CF/EP) composite, and the interfacial property, as well as the damage self-reporting of the composite, is compared with desized carbon fiber (CF-desized)/EP and commercial carbon fiber (CF-COM)/EP composite. The ruptured PDI-CS/γ-PGA microcapsule exhibits strong "turn-on" green fluorescence from the released PDI upon mechanical stimuli. The anchoring of PDI-CS/γ-PGA microcapsule on carbon fiber with PDI-CS/γ-PGA microcapsules sizing (CF@PDI-CS/γ-PGA) surface results in increased chemical activity and roughness, exhibiting a weak green fluorescence signal instead of non-fluorescence on CF-desized and CF-COM surface. The transverse fiber bundle tensile (TFBT) strength of CF@PDI-CS/γ-PGA composite is 80.97% and 31.09% higher than those of CF-desized/EP and CF-COM/EP composite, which is attributed to the mechanical interlocking and chemical bonding interaction between carbon fiber and epoxy matrix by introducing PDI-CS/γ-PGA microcapsule with spherular structure and active groups. After microdroplet testing, the strong "turn-on" green fluorescence signal of the released PDI from the microcapsules is detected in the interfacial debonding regions, realizing the microscopic damage self-reporting of CF@PDI-CS/γ-PGA composite.

Dual response signal CdTe QDs@ZIF-8 with butterfly spectrum for dual-mode fluorescence/colorimetric detection of tetracycline in animal feeds.

In this study, a ratiometric fluorescent sensor CdTe QDs@ZIF-8 with butterfly spectra is successfully constructed by in situ encapsulating mercaptopropionic acid-modified CdTe quantum dots in zeolitic imidazolate framework-8 (ZIF-8) with a simple strategy, and used for the detection of tetracycline in fluorescence/smartphone colorimetry dual-mode. ZIF-8 not only reduces the agglomeration of the quantum dots but also surprisingly generates a new green fluorescence signal at 524nm while the red fluorescence of the CdTe quantum dots at 650nm quenches when tetracycline is added. The two opposing fluorescence signals create a butterfly-shaped fluorescence spectrum, allowing the sensor to detect tetracycline over a linear range of 0-70μM with the detection limit (LOD) of 0.0155μM by using a ratiometric fluorescence technique. What is more, based on the obvious color change of the fluorescent sensor gradually from red to green under UV light, a highly stable point-of-care testing sensor has been developed for on-site detection of tetracycline through color recognition by smartphones, which can be used for real-time detection of this antibiotic in the range of 0-1000μM with the LOD of 0.0249μM. This work provides a simple and efficient method for the on-site detection of tetracycline.

Visual detection of anti‐icing fluids freezing by a low‐temperature viscosity‐sensitive aggregation‐induced emission probe

AbstractIcing detection is critically important for preventing safety accidents and economic losses, especially concerning ice formation from invalidated anti‐icing fluids (water and ethylene glycol) under extreme conditions. Traditional technologies like ultrasonics and capacitor‐antenna face challenges with limited detection areas, lower accuracy, and susceptibility to electromagnetic interference. Here, we introduce a novel viscosity‐ultrasensitive fluorescent probe 4′,4‴‐(2,2‐diphenylethene‐1,1‐diyl) bis‐(3,5‐dicarboxylate) (TPE‐2B4C) based on AIEgens for monitoring ice formation of anti‐icing fluids in low‐temperature environments. TPE‐2B4C, consisting of four sodium carboxylate groups and multiple freely rotating benzene rings, demonstrates outstanding solubility in anti‐icing fluids and exhibits no fluorescent background signal even at low temperatures (<−20°C). Upon freezing, TPE‐2B4C relocates from the water phase to higher viscosity ethylene glycol, causing restriction of benzene rings and a significantly increased green fluorescence signal. TPE‐2B4C can successfully determine whether the anti‐icing fluids are icing from −5 to −20°C with a high contrast ratio. Due to its simple setup, fast operation, and broad applicability, our new method is anticipated to be employed for rapid, real‐time, and large‐scale icing detection.

A Rapid Method for Screening Pathogen-Associated Molecular Pattern-Triggered Immunity-Intensifying Microbes.

PAMP-triggered immunity (PTI) is the first layer of plant defense response that occurs on the plant plasma membrane. Recently, the application of a rhizobacterium, Bacillus amyloliquefaciens strain PMB05, has been demonstrated to enhance flg22Pst- or harpin-triggered PTI response such as callose deposition. This PTI intensification by PMB05 further contributes to plant disease resistance to different bacterial diseases. Under the demand for rapid and large-scale screening, it has become critical to establish a non-staining technology to identify microbial strains that can enhance PTI responses. Firstly, we confirmed that the expression of the GSL5 gene, which is required for callose synthesis, can be enhanced by PMB05 during PTI activation triggered by flg22 or PopW (a harpin from Ralstonia solanacearum). The promoter region of the GSL5 gene was further cloned and fused to the coding sequence of gfp. The constructed fragments were used to generate transgenic Arabidopsis plants through a plant transformation vector. The transgenic lines of AtGSL5-GFP were obtained. The analysis was performed by infiltrating flg22Pst or PopW in one homozygous line, and the results exhibited that the green fluorescent signals were observed until after 8 h. In addition, the PopW-induced fluorescent signal was significantly enhanced in the co-treatment with PMB05 at 4 h after inoculation. Furthermore, by using AtGSL5-GFP to analyze 13 Bacillus spp. strains, the regulation of PopW-induced fluorescent signal was observed. And, the regulation of these fluorescent signals was similar to that performed by callose staining. More importantly, the Bacillus strains that enhance PopW-induced fluorescent signals would be more effective in reducing the occurrence of bacterial wilt. Taken together, the technique by using AtGSL5-GFP would be a promising platform to screen plant immunity-intensifying microbes to control bacterial wilt.

One-pot Synthesis of FITC-Doped Mesoporous Silica Nanoparticles: Synthesis, Characterization and Cellular Imaging

This study aimed to develop, evaluate and analyse the potential of mesoporous silica nanoparticles doped with fluorescein isothiocyanate (MSNFITC) as an agent for bioimaging. The synthesis of MSNFITC nanoparticles was achieved by using a CTAB formaldehyde method. Transmission electron microscopy (TEM), Scanning electron microscopy (SEM) and Atomic force microscopy (AFM) were employed to conduct a detailed characterization of the size and morphology of MSNFITC. The efficacy of dye doping was validated via fluorescence and UV absorbance spectroscopy. The produced MSNFITC had a uniform size distribution (65.5 ± 0.825nm) and a spherical shape. Spectroscopic investigations showed distinct peaks at 495 nm (absorbance) and 520 nm (fluorescence emission), indicative of FITC. Biocompatibility experiments on HeLa cells, even exposed to high concentrations (100 μg/mL) of MSNFITC, showed minimal cytotoxicity effects. Furthermore, the effective uptake of nanoparticles by HeLa cells was demonstrated through a strong green fluorescence signal that is distinctive for FITC. Given their advantageous features and biocompatibility with living organisms, the MSNFITC nanoparticles can potentially be highly effective agents for studying cells through bioimaging.

Stimulus-Responsive Four-Stranded DNA Nanoring Assembly to Host Multiple Nanosilver Clusters for Cooperatively Enhanced Fluorescence Biosensing.

Exploring the ability of four-stranded DNA nanorings (fsDNRs) to host multiple nanosilver clusters (NAgCs) for cooperatively amplifiable fluorescence biosensing to a specific initiator (tI*) is fascinating. By designing three DNA single strands and three analogous stem-loop hairpins, we developed a functional fsDNR through sequential cross-opening and overlapped hybridization. Note that a substrate strand (SS) was programmed with six modules: two severed splits (sT and sT') of NAgCs template, two sequestered segments by a middle unpaired spacer, and a partition for tI*-recognizable displacement, while sT and sT' were also tethered in two ends of three hairpins. At first, a triple dsDNA complex with stimulus-responsiveness was formed to guide the specific binding to tI*, while the exposed toehold of the SS activated the forward cascade hybridization of three hairpins, until the ring closure in the tailored self-assembly pathway for forming the fsDNR. The resulting four duplexes forced each pair of sT/sT' to be merged as the parent template in four nicks, guiding the preferential synthesis of four clusters in the shared fsDNR, thereby cooperatively amplifying the green fluorescence signal for sensitive assay of tI*. Meanwhile, the topological conformation of fsDNR can be stabilized by the as-formed cluster adducts to rivet the pair of two splits in the nicks. Benefitting from the self-enhanced effect of multiple emitters, this label-free fluorescent sensing strategy features simplicity, rapidity, and high on-off contrast, without involving complicated nucleic acid amplifiers.

A novel surface marker CD49d promotes TNF expression in oyster agranulocytes by mediating the MAPK pathway

CD49d, encoded by the gene Integrin α4, is a significant member of cell adhesion receptors, which is widely expressed in various immune cells to trigger immune responses against invading pathogens. In the present study, the expression of CgCD49d and its regulatory role in TNF expression were investigated in the Pacific oyster Crassostrea gigas. There were five Int-alpha domains, an Integrin_alpha2 region and a unique FG-GAP repeat region inserted identified in CgCD49d. CgCD49d transcript was specifically expressed in haemocytes, and its mRNA expression level in haemocytes increased after LPS and Vibrio splendidus stimulation. After CgCD49d was blocked by using its antibody, the phosphorylation level of CgJNK in the MAPK signaling pathway and CgTNF transcripts decreased significantly post V. splendidus stimulation. After phosphorylation level of CgJNK was inhibited by using its inhibitor, the nuclear translocation of CgRel was restrained and CgTNF transcripts also decreased significantly post V. splendidus stimulation. Furthermore, CgCD49d was found to be mainly expressed in the agranulocyte subpopulation, and Alexa Fluor 488-conjugated CgCD49d antibody labeled agranulocytes with a circle of green fluorescence signals on CgCD49d+ agranulocyte surface under Confocal microscopy, which accounted for 24.9 ± 4.53% of total haemocytes. Collectively, these results suggested that CgCD49d promoted TNF expression in oyster haemocytes against bacterial invasion by mediating MAPK pathway, and it could be used as a surface marker to type and sort a subset of agranulocyte subpopulation among haemocytes.

A high-throughput lysosome trafficking assay guides ligand selection and elucidates differences in CD22-targeted nanodelivery

ABSTRACT Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.

Bioinformatics, expression analysis, and functional verification of allene oxide synthase gene HvnAOS1 and HvnAOS2 in qingke.

Allene oxide synthase (AOS) is a key enzyme involved in the jasmonic acid (JA) synthesis pathway in plants. To explore its function on the regulatory mechanism of JA synthesis, we screened and identified two AOS genes HvnAOS1 and HvnAOS2 in qingke. Both HvnAOS1 and HvnAOS2 contained conserved heme-binding motif, which is most closely related to AtsAOS2, indicating controlled dehydration of fatty acid hydroperoxides to allene oxides. Molecular docking simulations identified the key amino acid sites that were important for heme binding and interaction with 13(S)-HPOT, respectively. The expression pattern also indicated that HvnAOS1 and HvnAOS2 were highly induced by JA, abscisic acid, and salicylic acid. Subcellular localization of HvnAOS1 and HvnAOS2 using transient expression of Agrobacterium tumefaciens showed the green fluorescent protein signal in the cell cytoplasm of the N. benthamiana leaves. Overexpression of HvnAOS1 and HvnAOS2 in Arabidopsis aos mutant restored male fertility and plant resistance to Botrytis cinerea, indicating that HvnAOS1 and HvnAOS2 can restore the functions of AOS in Arabidopsis aos mutant.

Investigating the Effect of RNA Scaffolds on the Multicolor Fluorogenic Aptamer Pepper in Different Bacterial Species.

RNA synthetic biology tools have primarily been applied in E. coli; however, many other bacteria are of industrial and clinical significance. Thus, the multicolor fluorogenic aptamer Pepper was evaluated in both Gram-positive and Gram-negative bacteria. Suitable HBC-Pepper dye pairs were identified that give blue, green, or red fluorescence signals in the E. coli, Bacillus subtilis, and Salmonella enterica serovar Typhimurium (S. Typhimurium). Furthermore, we found that different RNA scaffolds have a drastic effect on in vivo fluorescence, which did not correlate with the in vitro folding efficiency. One such scaffold termed DF30-tRNA displays 199-fold greater fluorescence than the Pepper aptamer alone and permits simultaneous dual color imaging in live cells.

Self-calibrating probes constructed on a unique dual-emissive fluorescence platform for the precise tracking of cellular senescence

Human β-galactosidase (β-gal) is recognized as a crucial biomarker for evaluating senescence at the cellular and tissue levels in humans. However, tools to precisely track the endogenous β-gal are still limited. Herein, we present two novel self-calibrating β-gal probes 7a and 7b which were constructed on a unique green/red dual-emissive fluorescence platform. The two probes inherently exhibited a stable green fluorescence signal impervious to β-gal activity, serving as a reliable internal reference. They also displayed a progressively diminishing red fluorescence signal with the increasing of β-gal expression levels. The dual behavior endows them with self-calibration capacity and then renders excellently selective and sensitive for precisely monitoring β-gal activity. Notably, compared with E. coli β-gal, the two probes are more effectively response to A. oryzae β-gal homologous to human β-gal, indicating their unique species-selectivity. Furthermore, 7a was validated for its effectiveness in determining senescence-associated β-galactosidase (SA-β-gal) expression in senescent NRK-52E and HepG2 cells, underscoring its practical applicability in senescence research.

The Molecular Characterization of the Cyprinid herpesvirus 3 (CyHV-3) ORF24 Protein and its effect on the expression of immune genes (in vitro)

Cyprinid herpesvirus 3 (CyHV-3), known as koi herpesvirus (KHV), is highly contagious and lethal. In this study, we aimed to characterize the ORF24-encoding protein of CyHV-3, investigate its sub-cellular localization, and determine its impact on the expression of immune factors through in vitro experiments. The results showed that the CyHV-3 ORF24 protein comprises 579 amino acids. Interestingly, multiple comparisons with homologous proteins from three carp herpesvirus origins showed no significant similarity. The fluorescence localization experiment showed that the green fluorescence signal, representing the protein pEGFP-ORF24, was primarily diffused in the cytoplasm. Notably, the overexpression of ORF24 effectively suppressed the expression of immune factors in both CCO (Channel catfish ovary) and FHM (Fathead minnow muscle cell line) cells. Bioinformatic analysis indicated that the CyHV-3 ORF24 gene exhibited significant differences from the corresponding genes in the other two carp herpesviruses. This suggests its unique functional role in the evolutionary context. Moreover, our findings demonstrated that overexpression of CyHV-3 ORF24 can effectively inhibit the expression of immune factors, underscoring its crucial role as a viral immune escape factor. These results provide further insights into the immune function of the CyHV-3 ORF24 protein and offer a theoretical foundation for developing new vaccines against CyHV-3 virus infections.

Ratiometric Fluorescent Probe for Real-Time Detection of β-Galactosidase Activity in Lysosomes and Its Application in Drug-Induced Senescence Imaging.

Senescence is an important biological process, which leads to the gradual degradation of its physiological function and increases morbidity and mortality. Herein, a novel ratiometric fluorescent probe (P1) was constructed by using benzothiazolyl acetonitrile dye as fluorophore, exhibiting significantly enhanced blue-shifted emission to indicate the activity of β-galactosidase (β-gal), a commonly used biomarker for the detection of senescent cells. After incubation with β-gal, the excimer emission of P1 at 620 nm was weakened, while the emission at 533 nm was significantly enhanced, forming an obvious ratiometric probe with high sensitivity and low detection limit (2.7 mU·mL-1). More importantly, probe P1 can locate lysosomes accurately, allowing us to monitor the emergence of living cell senescence in real time. P1 was successfully used to detect β-gal activity in PC-12 cells, Hep G2 cells, and RAW 264.7 cells. It showed strong green fluorescence signal in senescent cells and red fluorescence signal in normal cells, indicating that it can detect endogenous senescence-related β-gal content in living cells. For in vivo drug-induced senescence imaging, after 5 weeks of injection of D-galactose or hydroxyurea, the mice showed significant fluorescence enhancement in specific channels to indicate the activity of β-gal in vivo. At the same time, the senescence of cell-specific organs and skin tissues at the organ level were also detected, which proved that the drug-induced senescence of brain, skin, and muscle tissues was the most serious. These results supported the important application value of P1 in senescence biomedical research.

Spatiotemporally Precise Optical Manipulation of Intracellular Molecular Activities.

Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, a closed-loop optoelectronic control system is developed that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical-selective fluorescence signals are utilized to command lasers that trigger specific chemical processes or control the activation of photoswitchable inhibitors at desired targets. This technology is fully compatible with laser scanning confocal fluorescence microscopes. The authors demonstrate selective interactions of a 405nm laser with targeted organelles and simultaneous monitoring of cell responses by fluorescent protein signals. Notably, blue laser interaction with the endoplasmic reticulum leads to a more pronounced reduction in cytosolic green fluorescent protein signals in comparison to that with nuclei and lipid droplets. Moreover, when combined with a photoswitchable inhibitor, microtubule polymerization is selectively inhibited within the subcellular compartments. This technology enables subcellular spatiotemporal optical manipulation over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non-targeted locations.

Cellular localization and potential ligands of a novel scavenger receptor class B/CD36 protein homolog (Pt-SRB2) identified in the marine crab, Portunustrituberculatus

The scavenger receptor class B family proteins (SRB) are multiligand membrane receptor proteins. Herein, a novel SRB homolog (Pt-SRB2) was identified in Portunus trituberculatus. The open reading frame of Pt-SRB2 was predicted to encode 520 amino acid residues comprising a typical CD36 domain. Phylogenetic analysis showed that Pt-SRB2 distinctly clustered with the SRB homologs of most crustaceans and Drosophila but was separate from all vertebrate CD36/SRB. Semi-quantitative and Real-time quantitative PCR revealed that the abundance of Pt-SRB2 transcripts was the highest in hepatopancreas than in other tested tissues. Overexpressed Pt-SRB2 was distributed primarily in the cell membrane and cytoplasm of HEK293T or Drosophila Schneider 2 cells. In crab hemocytes, Pt-SRB2 was distributed primarily in the cell membrane by immunofluorescence staining. In addition, the immunofluorescence staining showed that green fluorescence signals were mainly located in the inner lumen membrane of the hepatopancreatic tubules. Moreover, solid-phase enzyme-linked immunosorbent assay revealed that rPt-SRB2-L exhibited relative high affinity with lipopolysaccharides, and relative moderate binding affinity with lipoteichoic acid or peptidoglycan. Of note, rPt-SRB2-L showed high binding affinity with eicosapentaenoic acid among a series of long-chain polyunsaturated fatty acids. Taken together, this study provided valuable data for understanding the functions of the crab CD36/SRB.

Endometrial organoids: a reservoir of functional mitochondria for uterine repair.

Background: Asherman's syndrome (AS) is a dreadful gynecological disorder of the uterus characterized by intrauterine adhesion with severe fibrotic lesions, resulting in a damaged basalis layer with infertility. Despite extensive research on overcoming AS, evidence-based effective and reproducible treatments to improve the structural and functional morphology of the AS endometrium have not been established. Methods: Endometrial organoids generated from human or mouse endometrial tissues were transplanted into the uterine cavity of a murine model of AS to evaluate their transplantable feasibility to improve the AS uterine environment. The successful engraftment of organoid was confirmed by detection of human mitochondria and cytosol (for human endometrial organoid) or enhanced green fluorescent protein signals (for mouse endometrial organoid) in the recipient endometrium. The therapeutic effects mediated by organoid transplantation were examined by the measurements of fibrotic lesions, endometrial receptivity and angiogenesis, and fertility assessment by recording the number of implantation sites and weighing the fetuses and placenta. To explore the cellular and molecular mechanisms underlying the recovery of AS endometrium, we evaluated the status of mitochondrial movement and biogenetics in organoid transplanted endometrium. Results: Successfully engrafted endometrial organoids with similar morphological and molecular features to the parental tissues dramatically repaired the AS-induced damaged endometrium, significantly reducing fibrotic lesions and increasing fertility outcomes in mice. Moreover, dysfunctional mitochondria in damaged tissues, which we propose might be a key cellular feature of the AS endometrium, was fully recovered by functional mitochondria transferred from engrafted endometrial organoids. Endometrial organoid-originating mitochondria restored excessive collagen accumulation in fibrotic lesions and shifted uterine metabolic environment to levels observed in the normal endometrium. Conclusions: Our findings suggest that endometrial organoid-originating mitochondria might be key players to mediate uterine repair resulting in fertility enhancement by recovering abrogated metabolic circumstance of the endometrium with AS. Further studies addressing the clinical applicability of endometrial organoids may aid in identifying new therapeutic strategies for infertility in patients with AS.

Dual-channel fluorescence dye: Fluorescent color-dependent visual detection of microplastics and selective polyurethane

In this study, we developed a dual-channel fluorescent dye ((E)-N′-(4-(diphenylamino)benzylidene)pyrazine-2-carbohydrazide) DPC for visual detection of 8 types of microplastics (MPs; HDPE, MDPE, LDPE, PET, PU, PVC, PS, and PP) and selective PU. The intramolecular charge transfer (ICT) and aggregation-induced emission (AIE) properties of DPC were demonstrated by the spectroscopic analysis, DFT calculations, and Tyndall effect. MPs and nonplastics (cellulose, chitin, sand, shell, and wood) were stained with DPC in water and their respective fluorescence signals in the blue and green channels were analyzed. The staining procedure using DPC was optimized with the concentration of DPC and staining time as parameters. DPC was able to effectively stain 8 types of MPs and only PU in blue and green fluorescence signals, respectively. Furthermore, false positive detections of DPC were minimized through additional ethanol treatment after staining. Moreover, the effects of temperature, pH, and salinity on the staining ability of DPC were investigated. Surprisingly, DPC was able to selectively detect PU through the green fluorescence signal even in a single environment where various MPs existed. Most importantly, DPC is the first fluorescent dye capable of selectively monitoring PU in the green channel as well as staining 8 types of MPs in the blue channel. DPC showed promising potential to be used for MP monitoring on real environmental samples.