Investigation of copper-induced intestinal damage and proteome alterations in Takifugu rubripes: Potential health risks and environmental toxicology detection.

Copper is one of the predominant water pollutants. Excessive exposure to copper can cause harm to animal health, affecting the central nervous system and causing blood abnormalities. Cuproptosis is a novel form of cell death that differs from previous programmed cell death methods. However, the impact of copper on the intestines remains unclear. Therefore, we investigated the effects of different concentrations of copper exposure on the intestinal proteome of Takifugu rubripes (T. rubripes). Relevant biomarkers were used to detect cuproptosis. We revealed the crosstalk relationship between cuproptosis and self-rescue at different concentrations, and discussed the feasibility of using potential cuproptosis indicators as anti-infection factors. We observed intestinal damage in the three copper exposure groups, especially in T. rubripes treated with 100 and 500 µg/L copper, with shedding and breakage of intestinal villus and fuzzy and loose structure of intestinal mucosa. The presence of copper stress not only causes cuproptosis but also oxidative damage caused by reactive oxygen species (ROS). The results of quantitative proteomics by TMT showed that compared to the 50 and 100 µg/L copper exposure groups, the expression of glutaminase, pyruvate kinase, and skin mucus lectin in the 500 µg/L group was significantly increased. The positive mediators COX5A and CTNNB1, as well as the negative mediators CD4 and FDXR, were found to be differentially expressed. Using the protein expression trends of cuproptosis indicator factors FDX1 and DLAT to indicate the concentration of copper ions in the environment. In addition, we found a new effect of promoting ferroptosis: providing additional copper ions can activate the phenomenon of ferroptosis. Our results expand our understanding of the potential health risks of copper in T. rubripes. At the same time, it is of great significance for the process of copper poisoning and the development of new environmental toxicology detection reagents.

Copper exposure causes alteration in the intestinal microbiota and metabolites in Takifugu rubripes.

Copper is an environmental pollutant, and copper in aquatic environments mainly comes from soil and water. It enters the environment through atmospheric deposition, sewage discharge, and industrial production, and enters aquatic organisms, causing toxicity. Takifugu rubripes (T. rubripes) is a marine fish with high economic value. Due to the toxic effects of heavy metals on aquatic organisms such as fish, it can affect the gut community and metabolites of fish. The gut is an important channel for fish to communicate with the outside world and a necessary pathway for the metabolism of nutrients and toxic substances in the fish body. Studies have shown that due to changes in global water emissions and the high sensitivity of aquatic organisms to the environment, copper may pose greater potential hazards to aquatic organisms. Copper poses a greater risk to aquatic species than other heavy metals and metal/metal like pollutants (such as cadmium, lead, mercury, arsenic, etc.) . In order to elucidate the effects of copper exposure on the gut of T. rubripes. In this study, we exposed T. rubripes to 0, 50, 100, or 500 µg/L of copper for three days, the effects of copper exposure on the gut microbiota structure and metabolites of the T. rubripes were investigated using 16 S rRNA gene and metabolomics techniques. The research results indicate that with the increase copper concentration, the intestinal tissue of T. rubripes undergoes significant damage. 16 S rRNA sequencing results show that copper exposure alters the structure and metabolites of intestinal microbiota. Copper exposure of 100 and 500 µg/L inhibited the colonization of the bacterial gut, disrupted the intestinal barrier, and made the fish susceptible to the pathogens. Liquid chromatography-mass spectrometry analysis showed that copper regulated the production of metabolites such as L-histidine, arachidonic acid, and L-glutamic acid, which are related to energy and immunity. Microbiome-metabolome correlation analysis showed that Subdoligranulum, Family_XIII_AD3011_group, and Clostridium_sensu_stricto_1 were the key bacteria for copper ion intervention, and they might up-regulate the levels of metabolites such as indole-3-acetic acid, 3-indoleacrylic acid, and 5-hydroxyindole in the tryptophan metabolism pathway. In summary, our research has demonstrated that copper exposure can cause pathological changes in the intestinal tissue of the T. rubripes. High concentrations of copper ions can affect the colonization of the T. rubripes microbiota in the intestine, damage the fish's immune system, and alter the structure and metabolites of the intestinal microbiota, this can lead to intestinal metabolic dysfunction. providing a reference for the evaluation of the biological toxicity effects of heavy metal elements in the marine environment. This study provides a reference for evaluating the biological toxicity effects of heavy metal elements in marine environments.

One-Base-Gap Circular Probe-Mediated Dual Amplification for Isothermal Detection of N6-Methyladenosine Modifications.

N6-Methyladenosine (m6A) stands out as the predominant internal modification in mammalian RNA, exerting crucial regulatory functions in the metabolism of mRNA. Currently available methods have been limited by an inability to quantify m6A modification at precise sites. In this work, we screened a Bst 2.0 warm start DNA polymerase with the capability of discriminating m6A from adenosine (A) and developed a robust m6A RNA detection method that enables isothermal and ultrasensitive quantification of m6A RNA at single-base resolution. The detection limit of the assay could reach about 0.02 amol, and the quantitative accuracy of the assay was verified in real cell samples. Furthermore, we applied this assay to single-cell analysis and found that the coefficients of variation of the MALAT1 m6A 2611 site in glioblastoma U251 cells showed over 20% higher than in oligodendrocytes MO3.13 cells. This method provides a highly sensitive analytical tool for site-specific m6A detection and quantification, which is expected to provide a basis for precise disease diagnosis and epigenetic transcriptional regulation.

Genome-wide integrated analysis reveals functions of lncRNA-miRNA-mRNA interactions in Atlantic salmon challenged by Aeromonas salmonicida.

Aeromonas salmonicida (A. salmonicida) is a pathogenic bacterium that causes serious problems in the global Atlantic salmon aquaculture industry. In this study, we comprehensively analyzed the profiles of lncRNAs, miRNAs and mRNAs in gills of Atlantic salmon at high-dose A. salmonicida infection (3.06 × 108 CFU/mL), low-dose A. salmonicida infection (3.06 × 105 CFU/mL), and a PBS (100 µL) control. We identified 65 differentially expressed lncRNAs, 41 miRNAs, and 512 mRNAs between the control group and infection groups. Functional analysis showed that these genes were significantly enriched in the p53 signaling pathway, Wnt signaling pathway, mTOR signaling pathway, JAK-STAT signaling pathway, and Toll-like receptor signaling pathway. In addition, we predicted key genes in immune-related pathways and constructed a lncRNA-miRNA-mRNA network based on whole transcriptomic analysis. We further predicted three lncRNA-miRNA-mRNA axes as potential novel biomarkers in regulating the immune response of Atlantic salmon against A. salmonicida infection.

Combination of RCA and DNAzyme for Dual-Signal Isothermal Amplification of Exosome RNA.

The RNA contained in exosomes plays a crucial role in information transfer between cells in various life activities. The accurate detection of low-abundance exosome RNA (exRNA) is of great significance for cell function studies and the early diagnosis of diseases. However, their intrinsic properties, such as their short length and high sequence homology, represent great challenges for exRNA detection. In this paper, we developed a dual-signal isothermal amplification method based on rolling circle amplification (RCA) coupled with DNAzyme (RCA-DNAzyme). The sensitive detection of low-abundance exRNA, the specific recognition of their targets and the amplification of the detection signal were studied and explored. By designing padlock probes to specifically bind to the target exRNA, while relying on the ligation reaction to enhance recognition, the precise targeting of exosome RNA was realized. The combination of RCA and DNAzyme could achieve a twice-as-large isothermal amplification of the signal compared to RCA alone. This RCA-DNAzyme assay could sensitively detect a target exRNA at a concentration as low as 527 fM and could effectively distinguish the target from other miRNA sequences. In addition, this technology was successfully proven to be effective for the quantitative detection of miR-21 by spike recovery, providing a new research approach for the accurate detection of low-abundance exRNA and the exploration of unknown exRNA functions.

Dual-Functional Nanocluster Probe-Based Single-Cell Analysis of RNA Splice Variants.

Differential expression of RNA splice variants among individual cells accounts for cell heterogeneity of gene expression, which plays a key role in the regulation of the immune system. However, currently available techniques face difficulties in achieving single-cell analysis of RNA splice variants with high base resolution, high spatial resolution and accurate quantification. Herein, we constructed DNA-templated dual-functional nanocluster probes to achieve in situ imaging and accurate quantification of RNA splice variants at the single-cell level. By designing ultrasmall nanocluster labeled probes to directly target the splicing junction sequence of RNA splice variants, the base recognition resolution is significantly improved. Benefit from the controllable fluorescence of nanoclusters, in situ imaging and genotyping of RNA splice variants are achieved. Due to the atom-precise nanocluster, RNA splice variants can be accurately quantified by laser ablation inductively coupled plasma mass spectrometry at the single-cell level. We further applied the probes to explore the function of MyD88 splice variants in mononuclear macrophages under immune activation. This strategy provides a novel single-cell analysis tool for studying the functional diversity of the immune system and splicing-related immune diseases.

Coupled CFD-DEM modeling to predict how EPS affects bacterial biofilm deformation, recovery and detachment under flow conditions.

The deformation and detachment of bacterial biofilm are related to the structural and mechanical properties of the biofilm itself. Extracellular polymeric substances (EPS) play an important role on keeping the mechanical stability of biofilms. The understanding of biofilm mechanics and detachment can help to reveal biofilm survival mechanisms under fluid shear and provide insight about what flows might be needed to remove biofilm in a cleaning cycle or for a ship to remove biofilms. However, how the EPS may affect biofilm mechanics and its deformation in flow conditions remains elusive. To address this, a coupled computational fluid dynamic- discrete element method (CFD-DEM) model was developed. The mechanisms of biofilm detachment, such as erosion and sloughing have been revealed by imposing hydrodynamic fluid flow at different velocities and loading rates. The model, which also allows adjustment of the proportion of different functional groups of microorganisms in the biofilm, enables the study of the contribution of EPS toward biofilm resistance to fluid shear stress. Furthermore, the stress-strain curves during biofilm deformation have been captured by loading and unloading fluid shear stress to study the viscoelastic properties of the biofilm. Our predicted emergent viscoelastic properties of biofilms were consistent with relevant experimental measurements.

A NIR-emissive probe with a remarkable Stokes shift for CO-releasing molecule-3 detection in cells and in vivo.

Carbon monoxide (CO) is regarded as one of the most important gaseous transmitters, playing a vital role in biological systems; meanwhile, abnormal levels of CO can be correlated with conditions such as lung disease, Alzheimer's disease, and cardiovascular disease. CO-releasing molecules (CORMs) are chemical agents used to release CO as an endogenous, biologically active molecule in order to treat diseases. CO-releasing molecule-3 (CORM-3), as a convenient and safe CO donor and therapeutic drug molecule, has been widely used to release exogenous CO in living cells to study the physiological and pathological roles of CO in living systems. Herein, we designed a NIR-emitting probe (NIR-CORM-3) with a large Stokes shift based on a 4-(dimethylamino)cinnamaldehyde lepidine derived fluorophore. A 4-nitrobenzyl group was selected as the CORM-3 recognizing moiety, and the probe is able to selectively and sensitively respond to CORM-3 (within only 15 min). Upon encountering CORM-3, NIR-CORM-3 releases a fluorophore with a response at 670 nm, and it shows a remarkable Stokes shift (up to 250 nm). In addition, NIR-CORM-3 has low cytotoxicity and exhibits outstanding NIR imaging abilities in living cells and mice.

The Optimization of Chlorella vulgaris Flocculation Harvesting by Chitosan and Calcium Hydroxide.

The high cost for microalgae harvesting still is the bottleneck of microalgae commercial production. In the present study, the effect of adjusting pH to alkaline conditions with sodium hydroxide/calcium hydroxide and the addition of chitosan together with pH adjustments on the flocculation of Chlorella vulgaris (C. vulgaris) was studied, respectively. A single-factor experiment showed a maximum flocculation efficiency of 96.7% when adjusting the pH to 12 with calcium hydroxide. There was synergistic action between chitosan and calcium hydroxide. Flocculation conditions of C. vulgaris for the combined use of calcium hydroxide and chitosan was optimized by response surface methodology (RSM) with a Box-Behnken design (BBD). Flocculation efficiency reached 97.08% under optimal flocculation conditions when adjustion of pH to 8.97 with 2 g/L calcium hydroxide, a chitosan dosage of 20 mg/L, and a flocculation time of 60 min. The current study presents one method for efficient flocculation harvesting of C. vulgaris at weak alkaline conditions and low chitosan dosage. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01004-1.

CFD-DEM modelling of biofilm streamer oscillations and their cohesive failure in fluid flow.

Biofilm streamer motion under different flow conditions is important for a wide range of industries. The existing work has largely focused on experimental characterisations of these streamers, which is often time-consuming and expensive. To better understand the physics of biofilm streamer oscillation and their interactions in fluid flow, a computational fluid dynamics-discrete element method model has been developed. The model was used to study the flow-induced oscillations and cohesive failure of single and multiple biofilm streamers. We have studied the effect of streamer length on the oscillation at varied flow rates. The predicted single biofilm streamer oscillations in various flow rates agreed well with experimental measurements. We have also investigated the effect of the spatial arrangement of streamers on interactions between two oscillating streamers in parallel and tandem arrangements. Furthermore, cohesive failure of streamers was studied in an accelerating fluid flow, which is important for slowing down biofilm-induced clogging.

Tumor promoter TPA activates Wnt/ß-catenin signaling in a casein kinase 1-dependent manner.

The tumor promoter 12-O-tetra-decanoylphorbol-13-acetate (TPA) has been defined by its ability to promote tumorigenesis on carcinogen-initiated mouse skin. Activation of Wnt/ß-catenin signaling has a decisive role in mouse skin carcinogenesis, but it remains unclear how TPA activates Wnt/ß-catenin signaling in mouse skin carcinogenesis. Here, we found that TPA could enhance Wnt/ß-catenin signaling in a casein kinase 1 (CK1) ε/δ-dependent manner. TPA stabilized CK1ε and enhanced its kinase activity. TPA further induced the phosphorylation of LRP6 at Thr1479 and Ser1490 and the formation of a CK1ε-LRP6-axin1 complex, leading to an increase in cytosolic ß-catenin. Moreover, TPA increased the association of ß-catenin with TCF4E in a CK1ε/δ-dependent way, resulting in the activation of Wnt target genes. Consistently, treatment with a selective CK1ε/δ inhibitor SR3029 suppressed TPA-induced skin tumor formation in vivo, probably through blocking Wnt/ß-catenin signaling. Taken together, our study has identified a pathway by which TPA activates Wnt/ß-catenin signaling.

Transcriptome analysis in Takifugu rubripes and Dicentrarchus labrax gills during Cryptocaryon irritans infection.

Takifugu rubripes and Dicentrarchus labrax are important commercial fish in China that are under serious threat from Cryptocaryon irritans. C. irritans is a ciliated obligate parasite that causes marine white spot disease and leads to heavy economic losses. We analysed the transcriptome in the gills of T. rubripes and D. labrax to compare differentially expressed genes (DEGs) and pathways during infection with C. irritans. In total, we identified 6,901 and 35,736 DEGs from T. rubripes and D. labrax, respectively. All DEGs were annotated into GO terms; 6,901 DEGs from T. rubripes were assigned into 991 sub-categories, and 35,736 DEGs from D. labrax were assigned into 8,517 sub-categories. We mapped DEGs to the KEGG database and obtained 153 and 350 KEGG signalling pathways from T. rubripes and D. labrax, respectively. Immune-related categories included Toll-like receptors, MAPK, lysosome, C-type lectin receptor and NOD-like receptor signalling pathways were significantly enriched pathways. In immune-related signalling pathways, we found that AP-1, P38, IL-1ß, HSP90 and PLA were significantly up-regulated DEGs in T. rubripes, but P38 and PLA were significantly down-regulated in D. labrax. In this study, transcriptome was used to analyse the difference between scaly and non-scaly fish infection by C. irritans, which not only provided a theoretical basis for the infection mechanism of C. irritans, but also laid a foundation for effectively inhibiting the occurrence of this disease. Our work provides further insight into the immune response of host resistance to C. irritans.

Single isocenter SRS using CAVMAT offers improved robustness to commissioning and treatment delivery uncertainty compared to VMAT.

PURPOSE: In this study, we evaluate and compare single isocenter multiple target VMAT (SIMT) and Conformal Arc Informed VMAT (CAVMAT) radiosurgery's sensitivity to uncertainties in dosimetric leaf gap (DLG) and treatment delivery. CAVMAT is a novel planning technique that uses multiple target conformal arcs as the starting point for limited inverse VMAT optimization. METHODS: All VMAT and CAVMAT plans were recalculated with DLG values of 0.4, 0.8, and 1.2 mm. DLG effect on V6Gy [cc], V12Gy [cc], and V16Gy [cc], and target dose was evaluated. Plans were delivered to a Delta4 (ScandiDos, Madison, WI) phantom and gamma analysis performed with varying criteria. Log file analysis was performed to evaluate MLC positional error. Sixteen targets were delivered to a SRS MapCHECK (Sun Nuclear Corp., Melbourne, FL) to evaluate VMAT and CAVMAT's dose difference (DD) as a function of DLG. RESULTS: VMAT's average maximum and minimum target dose sensitivity to DLG was 9.08 ±3.50%/mm and 9.50 ± 3.30%/mm, compared to 3.20 ± 1.60%/mm and 4.72 ± 1.60%/mm for CAVMAT. For VMAT, V6Gy [cc], V12Gy [cc], and V16Gy [cc] sensitivity was 35.83 ± 9.50%/mm, 34.12 ± 6.60%/mm, and 39.23 ± 8.40%/mm. In comparison, CAVMAT's sensitivity was 23.19 ± 4.50%/mm, 22.45 ± 4.40%/mm, and 24.88 ± 4.90%/mm, respectively. Upon delivery to the Delta4 , CAVMAT offered superior dose agreement compared to VMAT. For a 1%/1 mm gamma analysis, VMAT and CAVMAT had a passing rate of 94.53 ± 4.40% and 99.28 ± 1.70%, respectively. CAVMAT was more robust to DLG variation, with the SRS MapCHECK plans yielding an absolute average DD sensitivity of 2.99 ± 1.30%/mm compared to 5.07 ± 1.10%/mm for VMAT. Log files demonstrated minimal differences in MLC positional error for both techniques. CONCLUSIONS: CAVMAT remains robust to delivery uncertainties while offering a target dose sensitivity to DLG less than half that of VMAT, and 65% of that of VMAT for V6Gy [cc], V12Gy [cc], and V16Gy [cc]. The superior dose agreement and reduced sensitivity of CAVMAT to DLG uncertainties indicate promise as a robust alternative to VMAT for SIMT SRS.

CCDC102B functions in centrosome linker assembly and centrosome cohesion.

The proteinaceous centrosome linker is an important structure that allows the centrosome to function as a single microtubule-organizing center (MTOC) in interphase cells. However, the assembly mechanism of the centrosome linker components remains largely unknown. In this study, we identify CCDC102B as a new centrosome linker protein that is required for maintaining centrosome cohesion. CCDC102B is recruited to the centrosome by C-Nap1 (also known as CEP250) and interacts with the centrosome linker components rootletin and LRRC45. CCDC102B decorates and facilitates the formation of rootletin filaments. Furthermore, CCDC102B is phosphorylated by Nek2A (an isoform encoded by NEK2) and is disassociated from the centrosome at the onset of mitosis. Together, our findings reveal a molecular role for CCDC102B in centrosome cohesion and centrosome linker assembly.This article has an associated First Person interview with the first authors of the paper.

Quantitative proteomic analysis in serum of Takifugu rubripes infected with Cryptocaryon irritans.

Cryptocaryon irritans can cause cryptocaryonosis (white spot disease) in marine fish but the pathogenesis of the disease is unclear. In this work, we used high-throughput proteomics to identify differentially expressed proteins in the serum of Takifugu rubripes challenged with C. irritans. By using quantitative proteomic assays combined with Tandem Mass Tag-labeled quantitative proteomic analysis, we identified a total of 2088 differentially abundant proteins (1706 proteins were quantified, p < 0.05, fold-change threshold ≥ 2), including 21 up-regulated and 44 down-regulated. Combined with STRING-based functional analysis, we ultimately obtained eight proteins including glucokinase-like, integrin beta-1-like isoform X2, H4, H2A.V, histone H1-like, histone H2AX-like, histone H2B 1/2-like and myosin-9 isoform X1, which could be considered as potential biomarkers for T. rubripes immune responses. Eight proteins that were selected to validate significant differentially expressed genes at the proteomic level were consistent with qPCR at the transcriptomic level. The proteins identified in our work may serve as candidates for elucidating the molecular mechanism of cryptocaryonosis in T. rubripes. Our collective findings could provide new insights into searching for disease-specific targets and biomarkers, which may be effective indicators of C. irritans infection in T. rubripes.

miR-290 contributes to the low abundance of cyclin D1 protein in mouse embryonic stem cells.

Mouse miR-290 cluster miRNAs are expressed specifically in early embryos and embryonic germ cells. These miRNAs play critical roles in the maintenance of pluripotency and self-renewal. Here, we showed that Cyclin D1 is a direct target gene of miR-290 cluster miRNAs. Negative relationships between the expression of Cyclin D1 protein and miR-290 cluster miRNAs in pluripotent and non-pluripotent cells, as well as in differentiating CGR8 cells were observed. Inhibition of miR-290 cluster miRNAs could arrest cells at the G1 phase and slow down the cell proliferation in CGR8 mouse stem cells. Since miR-290 cluster miRNAs are the most dominant stem-cell-specific miRNAs, our results revealed an important cause for the absence of Cyclin D1 in mouse embryonic stem cells.

An Adaptive S-Method to Analyze Micro-Doppler Signals for Human Activity Classification.

In this paper, we propose the multiwindow Adaptive S-method (AS-method) distribution approach used in the time-frequency analysis for radar signals. Based on the results of orthogonal Hermite functions that have good time-frequency resolution, we vary the length of window to suppress the oscillating component caused by cross-terms. This method can bring a better compromise in the auto-terms concentration and cross-terms suppressing, which contributes to the multi-component signal separation. Finally, the effective micro signal is extracted by threshold segmentation and envelope extraction. To verify the proposed method, six states of motion are separated by a classifier of a support vector machine (SVM) trained to the extracted features. The trained SVM can detect a human subject with an accuracy of 95.4% for two cases without interference.

Mollow sidebands in high order harmonic spectra of molecules.

Novel feature of high order harmonic generation process for molecules is presented for several molecules at their equilibrium geometries. The high order harmonic spectra reveal additional sidebands for each odd harmonic, which are a consequence of the resonant coupling of two valence orbitals, a mechanism analogous to Mollow triplets known from quantum optics. Strong modification of the high order harmonic generation process is illustrated with time frequency analysis in which there appear additional minima dependent on the Rabi frequency for the corresponding transition. The orbital coupling further leads to the modification of the electron dynamics which is presented using total electron density difference maps.

Multielectron contributions in elliptically polarized high-order harmonic emission from nitrogen molecules.

We have studied the polarization and ellipticity of high-order harmonics from nitrogen molecules using the time-dependent density functional theory. The results of our numerical calculations are in excellent agreement with the data of recent experiments. The theoretical analysis of our results reveals that at least three contributions, namely those from the HOMO, the HOMO-1, and the HOMO-2 orbitals, contribute to the observed high harmonic spectra. Furthermore, we confirm that a proper account of the distribution of the alignment in the molecular ensemble is necessary to obtain agreement with the experimental data.