Identification and functional characterization of annexin A2 in half-smooth tongue sole (Cynoglossus semilaevis).

Annexin A2 (AnxA2), belonging to the annexin family, plays a crucial role in immune responses. In this study, the cDNA of the AnxA2 gene was identified in half-smooth tongue sole, Cynoglossus semilaevis. The transcript of AnxA2 gene in C. semilaevis (CsAnxA2) showed broad tissue distribution, with the highest expression level observed in the gut. CsAnxA2 expression was significantly up-regulated in the intestine, spleen, and kidney tissues following exposure to Shewanella algae. Immunohistochemical staining revealed that CsAnxA2 was predominantly expressed in epithelial cells and significantly elevated after S. algae challenge. Subcellular localization showed that CsAnxA2 was primarily localized in the cytoplasmic compartment. Moreover, proinflammatory cytokines (IL-6, IL-8 and IL-1ß) exhibited significant upregulation after CsAnxA2 was overexpressed in vivo. One hundred and fifty-eight CsAnxA2-interacting proteins were captured in the intestinal tissue, showing the top two normalized abundance observed for actin beta (ACTB) and protein S100-A10 (p11). Fifty-four high abundance CsAnxA2-interacting proteins (HIPs) were primary enriched in ten pathways, with the top three significantly enriched pathways being Salmonella infection, glycolysis/gluconeogenesis, and peroxisome proliferator-activated receptor (PPAR) signaling pathway. These results provide valuable information for further investigation into the functional mechanism of AnxA2 in C. semilaevis.

Dysfunction of the intestinal physical barrier in the intestinal inflammation of tongue sole, Cynoglossus semilaevis, induced by Shewanella algae infection.

Bacterial intestinal inflammation occurs frequently in cultured fish. However, research on the dysfunction of the intestinal physical barrier in fish intestinal inflammation is scarce. In this study, intestinal inflammation in tongue sole Cynoglossus semilaevis was induced by Shewanella algae and the intestinal permeability was investigated. Gene expression patterns in inflammatory factors, tight junction molecules, and keratins 8 and 18 in the intestines were further explored. Histological examinations of the middle intestines showed that S. algae induced pathological lesions of intestinal inflammation and significantly increased the total number of mucous cells (p < 0.01). Ultrastructural observation in the middle intestines showed that intercellular spaces between epithelial cells were significantly wider in infected fish compared with the control (p < 0.01). The positive result of fluorescence in situ hybridization confirmed the presence of S. algae in the intestine. Enhanced Evans blue exudation and increased levels of serum d-lactate and intestinal fatty acid binding protein were suggestive of increased intestinal barrier permeability. The mRNA levels of four pro-inflammatory cytokines, namely IL-6, IL-8, IL-ß, and TNF-α, were significantly increased after S. algae infection at most tested time points (p < 0.01 or p < 0.05), while there was an alternating increasing and decreasing trend in the gene expression patterns of IL-10, TGF-ß, TLR-2, AP-1, and CASP-1. The mRNA expression of tight junction molecules (claudin-1, claudin-2, ZO-1, JAM-A, and MarvelD3) and keratins 8 and 18 in the intestines was significantly decreased at 6, 12, 24, 48, or 72 h post infection (p < 0.01 or p < 0.05). In conclusion, S. algae infection induced intestinal inflammation accompanied by increased intestinal permeability in tongue sole, and tight junction molecules and keratins were probably associated with the pathological process.

A deep ensemble learning-based automated detection of COVID-19 using lung CT images and Vision Transformer and ConvNeXt.

Since the outbreak of COVID-19, hundreds of millions of people have been infected, causing millions of deaths, and resulting in a heavy impact on the daily life of countless people. Accurately identifying patients and taking timely isolation measures are necessary ways to stop the spread of COVID-19. Besides the nucleic acid test, lung CT image detection is also a path to quickly identify COVID-19 patients. In this context, deep learning technology can help radiologists identify COVID-19 patients from CT images rapidly. In this paper, we propose a deep learning ensemble framework called VitCNX which combines Vision Transformer and ConvNeXt for COVID-19 CT image identification. We compared our proposed model VitCNX with EfficientNetV2, DenseNet, ResNet-50, and Swin-Transformer which are state-of-the-art deep learning models in the field of image classification, and two individual models which we used for the ensemble (Vision Transformer and ConvNeXt) in binary and three-classification experiments. In the binary classification experiment, VitCNX achieves the best recall of 0.9907, accuracy of 0.9821, F1-score of 0.9855, AUC of 0.9985, and AUPR of 0.9991, which outperforms the other six models. Equally, in the three-classification experiment, VitCNX computes the best precision of 0.9668, an accuracy of 0.9696, and an F1-score of 0.9631, further demonstrating its excellent image classification capability. We hope our proposed VitCNX model could contribute to the recognition of COVID-19 patients.

First record of isolation and characterization of Vibrio sinaloensis from diseased orange-spotted grouper Epinephelus coioides.

A dominant bacterium, ZYL-12, isolated from the liver of a diseased orange-spotted grouper Epinephelus coioides, was identified as Vibrio sinaloensis, based on phenotypic and molecular analysis. The median lethal dosage of ZYL-12 was calculated as 1.6 × 105 CFU g-1 fish weight. The infection experiment indicated that ZYL-12 caused noticeable histological lesions to the liver, kidney and spleen of the fish. Growth characteristics showed that ZYL-12 possessed strong environmental adaptability. This note is the first report about the pathogenicity of V. sinaloensis isolated from diseased fish.

The delimitation of intestine segments of koi carp (Cyprinus carpio var. koi) based on histological features.

In this study, the delimitation of intestine segments of koi carp (Cyprinus carpio var. koi) was conducted using a histological approach with the measurements of height of mucosa folds (HF), width of mucosa folds (WF), thickness of muscularis (TM) and cross-sectional area (CSA). According to the change trends for these four parameters, the intestine of the koi carp was divided into anterior intestine, middle intestine and posterior intestine. The locations of the three intestine segments were defined, and their ratios along the entire intestine were accounted for 23.84 ± 1.18%, 46.77 ± 2.29% and 29.39 ± 1.65%, respectively. The anterior intestine had a significantly higher HF, compared with the middle (p < .01) or posterior intestines (p < .01). The muscularis became thin gradually from the anterior intestine to posterior part. TM was significantly different among the anterior, middle and posterior intestines (p < .01). The anterior intestine had a significantly higher CSA, compared with the middle (p < .01) and posterior intestines (p < .01), and the latter two segments had similar CSA values (p > .05). The procedure of the delimitation of the koi carp intestine segments can offer useful information for future studies on other fish species. The presented results are meaningful for studies on differential functions of the different intestine segments in fish.

Differentially expressed proteins in the intestine of Cynoglossus semilaevis Günther following a Shewanella algae challenge.

Fish intestine is an important constituent of the mucosal immune system. The gut and gut-associated lymphoid tissue construct a local immune environment. A Shewanella algae strain was previously reported to be a pathogen causing ascitic disease accompanied with intestinal inflammation in Cynoglossus semilaevis. This study aimed to investigate the intestine immune response in C. semilaevis to S. algae infection at the protein level. Two-dimensional electrophoresis coupled with mass spectrometry proteomics was utilized to compare protein expression in the intestines from normal and S. algae-infected C. semilaevis. A total of 70 differentially expressed proteins (DEPs), consisting of 16 upregulated and 54 downregulated proteins, were identified in the intestine tissue of C. Semilaevis. These protein expression changes were further validated using western blot analysis and quantitative real-time PCR. Gene ontology enrichment analysis showed that these 70 DEPs could be assigned across three categories: "cellular components", "molecular function", and "biological process". Forty-one DEPs (six up-regulated and 35 down-regulated proteins) related to metabolic processes were identified. In addition, 20 DEPs (eight up-regulated and 12 down-regulated proteins) related to stress and immune responses were identified. A protein-protein interaction network generated by the STRING (Search Tool for the Retrieval of Interacting Genes/protein) revealed that 30 DEPs interacted with one another to form an integrated network. Among them, 29 DEPs were related to stress, immune, and metabolism processes. In the network, some of the immune related proteins (C9, FGB, KNG1, apolipoprotein A-IV-like, and PDIA3) were up-regulated and most DEPs involved in metabolism processes were down-regulated. These results indicate that the immune defense response of the intestine was activated and the intestinal function associated with metabolism processes was disturbed. This study provides valuable information for further research into the functions of these DEPs in fish.

Sensitivity of SWIFT spectroscopy.

SWIFT spectroscopy (Shifted Wave Interference Fourier Transform Spectroscopy) is a coherent beatnote technique that can be used to measure the temporal profiles of periodic optical signals. While it has been essential in understanding the physics of various mid-infrared and terahertz frequency combs, its ultimate limits have not been discussed. We show that the envelope of a SWIFTS interferogram is physically meaningful and is directly related to autocorrelation. We derive analytical expressions for the SWIFTS signals of two prototypical cases-chirped pulses from a mode-locked laser and a frequency-modulated comb-and derive scaling laws for the noise of these measurements, showing how it can be mitigated. Finally, we confirm this analysis by performing the first SWIFTS measurements of near-infrared pulses from femtosecond lasers, establishing the validity of the technique for highly-dispersed sub-picojoule pulses.

Isolation, Identification and Characteristics of Aeromonas veronii From Diseased Crucian Carp (Carassius auratus gibelio).

Aeromonas species often cause disease in farmed fish. In the present study, dominant bacteria were isolated from diseased crucian carp (Carassius auratus gibelio). Based on this, a bacterial isolate was tentatively named CFJY-623. This isolate was identified as Aeromonas veronii based on analysis of its morphological, physiological, and biochemical features, as well as 16S rRNA and gyrB gene sequences. Six virulence genes related to pathogenicity including aerolysin, cytotonic enterotoxins, elastase, glycerophospholipid: cholesterol acyltransferase, lipase, and serine protease were identified in this A. veronii isolate. The median lethal dosage (LD50) of the CFJY-623 isolate for crucian carp was determined as 1.31 × 107 CFU/mL. Artificial experimental infection showed that the CFJY-623 isolate caused considerable histological lesions in the fish, including tissue cell degeneration, necrosis, and inflammatory cell infiltrating. Drug sensitivity testing showed that the isolate was susceptible to aminoglycosides, carbapenemes, and nitrofurans. Exploring its growing features showed that this isolate exhibited a high level of environmental adaptability. These results provided a scientific basis for the identification of A. veronii and treatment for fish infected by this pathogen.

Biases from different DNA extraction methods in intestine microbiome research based on 16S rDNA sequencing: a case in the koi carp, Cyprinus carpio var. Koi.

This study examined the technical bias associated with different DNA extraction methods used in microbiome research. Three methods were used to extract genomic DNA from the same intestinal microbiota sample that was taken from the koi carp Cyprinus carpio var. koi, after which their microbial diversity and community structure were investigated on the basis of a 16S rDNA high-throughput sequencing analysis. Biased results were observed in relation to the number of reads, alpha diversity indexes and taxonomic composition among the three DNA extraction protocols. A total of 1,381 OTUs from the intestinal bacteria were obtained, with 852, 759, and 698 OTUs acquired, using the Lysozyme and Ultrasonic Lysis method, Zirmil-beating Cell Disruption method, and a QIAamp Fast DNA Stool Mini Kit, respectively. Additionally, 336 OTUs were commonly acquired, using the three methods. The results showed that the alpha diversity indexes (Rarefaction, Shannon, and Chao1) of the community that were determined using the Lysozyme and Ultrasonic Lysis method were higher than those obtained with the Zirmil-beating Cell Disruption method, while the Zirmil method results were higher than those measured, using the QIAamp Fast DNA Stool Mini Kit. Moreover, all the major phyla (ratio>1%) could be identified with all three DNA extraction methods, but the phyla present at a lower abundance (ratio <1%) could not. Similar findings were observed at the genus level. Taken together, these findings indicated that the bias observed in the results about the community structure occurred primarily in OTUs with a lower abundance. The results of this study demonstrate that possible bias exists in community analyses, and researchers should therefore be conservative when drawing conclusions about community structures based on the currently available DNA extraction methods.

Transcriptome profiling of immune-responsive genes in the intestine of Cynoglossus semilaevis Günther challenged with Shewanella algae.

To better understand gene expression in the intestine after Shewanella algae infection and provide insights into its immune roles in the tongue sole, Cynoglossus semilaevis, sequencing-based high-throughput RNA analysis (RNA-Seq) for the intestines between the control group and 12 h post-injection group was performed. After assembly, there was an average of 23,957,159 raw sequencing reads, and 23,943,491 clean reads were obtained after filtering out low-quality reads. Then, 383 differentially expressed genes (DEGs) in the intestines in response to S. algae infection were identified. Subsequently, gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the DEGs were conducted to further explore their functions. Among all of the pathways involved, sixteen pathways were related to the immune system, among which the complement and coagulation cascades pathway was the most prominent for immunity-related DEGs, followed by the leukocyte transendothelial migration pathway. Furthermore, the expression levels of twelve selected DEGs in the immune-related pathways were identified by quantitative real-time polymerase chain reaction, substantiating the reliability and reproducibility of the RNA-Seq results. In summary, this study represents an important genomic resource for understanding the potential immune role of the tongue sole intestine from the perspective of gene expression.

A modified method for genomic DNA extraction from the fish intestinal microflora.

A modified genomic DNA extraction method named the combination of lysozyme and ultrasonic lysis (CLU) method was used to analyze the fish intestinal microflora. In this method, the physical disruption and chemical lysis steps were combined, and some parameters in the key steps were adjusted. In addition, the results obtained by this method were compared with the results obtained by the Zirmil-beating cell disruption method and the QIAamp Fast DNA Stool Mini Kit. The OD260/OD280 ratio and concentration of the DNA extracted using the CLU method were 2.02 and 282.8 µg/µL, respectively; when the incubation temperatures for lysozyme and RNase were adjusted to 37 °C, those values were 2.08 and 309.8 µg/µL, respectively. On the agarose gel, a major high-intensity, discrete band of more than 10 kb was found for the CLU method. However, the smearing intensity of degraded DNA was lower when the incubation temperatures were 60 °C for lysozyme and 30 °C for RNase than when incubation temperatures of 37 °C for lysozyme and 37 °C for RNase were used. The V3 variable region of the prokaryotic 16S rDNA was amplified, and an approximately 600-bp fragment was observed when the DNA extracted using the CLU method was used as a template. The CLU method is simple and cost effective, and it yields high-quality, unsheared, high-molecular-weight DNA, which is comparable to that obtained with a commercially available kit. The extracted DNA has potential for applications in critical molecular biology techniques.