Asymmetric Nanopore Sensor for Logic Detection of Dam and M.SssI Methyltransferases in Combination of DNA Walker and Autocatalytic Hybridization Reaction.

The detection of DNA methyltransferase (MTase) was crucial for understanding gene expression regulation, cancer mechanisms, and various biological processes, contributing significantly to disease diagnosis and drug development. Herein, a nanopore sensor based on cascaded signal amplification of DNA walker and autocatalytic hybridization reaction (AHR) was developed for the ultrasensitive determination of various MTases. In the presence of Dam MTase, the hairpin structure HD underwent methylation and cleavage by DpnI endonuclease, forming T-DNA fragments. These T-DNA fragments were used to activate the DNA walker, which moved across the surface of magnetic beads step by step, generating a large quantity of initiator I by cleaving the substrate. The initiator I subsequently activated the AHR. The AHR included a hybridization chain reaction (HCR) amplifier and a catalytic hairpin assembly (CHA) convertor. The HCR amplifier generated multiple novel CHA triggers, which activated the CHA convertor. This, in turn, stimulated the HCR amplifier, creating an AHR circuit that resulted in the formation of numerous DNA nanowires. These DNA nanowires were adsorbed onto the G4-PAMAM-modified nanopore surface under the influence of an electric field, thereby altering the surface charge of the nanopore and changing the ionic rectification curve. The detection limit of the Dam MTase nanopore sensor reached 0.0002 U/mL. By modification of the recognition sites of the probes, this nanopore system could also be used for the detection of M.SssI MTase. Moreover, a four-input parallel concatenated logic circuit (AND//INHIBIT-OR) had been constructed and applied for the multivariate detection of Dam MTase and M.SssI MTase, presenting a novel conceptual model for advancing the construction of nanopore logic gate systems and their applications in biosensing.

Effects of essential amino acids supplementation in a low-protein diet on growth performance, intestinal health and microbiota of juvenile blotched snakehead (Channa maculata).

Developing a low-protein feed is important for the sustainable advancement of aquaculture. The aim of this study was to investigate the effects of essential amino acid (EAA) supplementation in a low-protein diet on the growth, intestinal health, and microbiota of the juvenile blotched snakehead, Channa maculata in an 8-week trial conducted in a recirculating aquaculture system. Three isoenergetic diets were formulated to include a control group (48.66 % crude protein (CP), HP), a low protein group (42.54 % CP, LP), and a low protein supplementation EAA group (44.44 % CP, LP-AA). The results showed that significantly lower weight gain (WG), specific growth rate (SGR), protein efficiency ratio (PER), and feed efficiency ratio (FER) were observed in fish that were fed LP than in the HP and LP-AA groups (P < 0.05). The HP and LP-AA groups exhibited a significant increase in intestinal villus length, villus width, and muscular thickness compared to the LP group (P < 0.05). Additionally, the HP and LP-AA groups demonstrated significantly higher levels of intestinal total antioxidant capacity (T-AOC), catalase (CAT), and superoxide dismutase (SOD) and lower levels of malondialdehyde (MDA) compared to the LP group (P < 0.05). The apoptosis rate of intestinal cells in the LP group was significantly higher than those in the LP and HP groups (P < 0.05). The mRNA expression levels of superoxide dismutase (sod), nuclear factor kappa B p65 subunit (nfκb-p65), heat shock protein 70 (hsp70), and inhibitor of NF-κBα (iκba) in the intestine were significantly higher in the LP group than those in the HP and LP-AA groups (P < 0.05). The 16s RNA analysis indicated that EAA supplementation significantly increased the growth of Desulfovibrio and altered the intestinal microflora. The relative abundances of Firmicutes and Cyanobacteria were positively correlated with antioxidant parameters (CAT and T-AOC), whereas Desulfobacterota was negatively correlated with sod and T-AOC. The genera Bacillus, Bacteroides, and Rothia were associated with the favorable maintenance of gut health. In conclusion, dietary supplementation with EAAs to achieve a balanced amino acid profile could potentially reduce the dietary protein levels from 48.66 % to 44.44 % without adversely affecting the growth and intestinal health of juvenile blotched snakeheads.

Production of all-male non-transgenic zebrafish by conditional primordial germ cell ablation.

Many fish species exhibit remarkable sexual dimorphism, with males possessing numerous advantageous traits for commercial production by aquaculture such as faster growth rate, more efficient food energy utilization for muscle development, and better breeding performance. Several studies have shown that a decrease in the number of primordial germ cells (PGCs) during early development leads predominantly to male progeny. In this study, we developed a method to obtain all-male zebrafish (Danio rerio) by targeted PGC ablation using the nitroreductase/metronidazole (NTR/Mtz) system. Embryos generated by female heterozygous Tg(nanos3:nfsB-mCherry-nanos3 3'UTR) and male wild-types (WTs) were treated with vehicle or Mtz. Compared to vehicle-treated controls, 5.0 and 10.0 mM Mtz treatment for 24 h significantly reduced the number of PGCs and yielded an exclusively male phenotype in adulthood. The gonads of offspring treated with 5.0 mM Mtz exhibited relatively normal morphology and histological characteristics. Furthermore, these males were able to chase females, spawn, and produce viable offspring, while about 20.0% of males treated with 10.0 mM Mtz were unable to produce viable offspring. The 5.0 mM Mtz treatment protocol may thus be suitable for large-scale production of fertile male offspring. Moreover, about half of these males were WT as evidenced by the absence of nfsB gene expression. It may thus be possible to breed an all-male WT fish population by Mtz-mediated PGC ablation.

Ultrasensitive and Label-Free Detection of Multiple DNA Methyltransferases by Asymmetric Nanopore Biosensor.

DNA methylation is catalyzed by a family of DNA methyltransferases that play crucial roles in various biological processes. Therefore, an ultrasensitive methyltransferase assay is highly desirable in biomedical research and clinical diagnosis. However, conventional assays for the detection of DNA methyltransferase activity often involve radioactive labeling, costly equipment, and laborious operation. In this study, an ultrasensitive and label-free method for detecting DNA adenine methyltransferase (Dam) and CpG methyltransferase (M.SssI) was developed using the nanopore technique coupled with DNA cascade signal amplification reactions. A hairpin DNA (HD) comprising of the methylation-responsive sequences was skillfully designed. In the presence of Dam methyltransferase, the corresponding recognition site of hairpin HD was methylated and specifically cleaved by DpnI endonuclease, thus forming a DNA fragment that induces the catalytic hairpin assembly and hybridization chain reaction (CHA-HCR). The generated products could be absorbed onto the Zr4+-coated nanopore, resulting in an ion current rectification signal change. Considering the high sensitivity of the nanopore and excellent specificity toward the recognition of methyltransferase/endonuclease, our developed method could detect both Dam and M.SssI methyltransferases in the same sensing platform. Furthermore, the designed nanopore sensor could realize the multiplex detection of Dam and M.SssI methyltransferases after integration with the cascaded INHIBIT-AND logic gate. This ultrasensitive methyltransferase assay holds great promise in the field of cancer diagnosis.

Fabrication of a Biomimetic Nanochannel Logic Platform and Its Applications in the Intelligent Detection of miRNA Related to Liver Cancer.

Nanochannel-based analytical techniques have great potential applications for nucleic acid sequencing and high sensitivity detection of biological molecules. However, the sensitivity of conventional solid-state nanochannel sensors is hampered by a lack of effective signal amplification strategies, which has limited its utility in the field of analytical chemistry. Here we selected a solid-state nanochannnel modified with polyethylenimine and Zr4+ in combination with graphene oxide as the sensing platform. The high-performance sensor is based upon the change of the surface charge of the nanochannel, which is resulted from DNA cascade signal amplification in solution. The target miRNA (miR-122) can be indirectly quantitated with a detection limit of 97.2 aM with an excellent selectivity. Depending on the nucleic acid's hybridization and configuration transform, the designed nanochannel sensing systems can realize the intelligent detection of multiple liver cancer-related miRNA (miR-122 and miR Let-7a) integrating with cascaded INHIBIT-OR logic gate to provide theoretical guidance and technical support for clinical diagnosis and therapeutic evaluation of liver cancer.

Effects of norfloxacin nicotinate on the early life stage of zebrafish (Danio rerio): Developmental toxicity, oxidative stress and immunotoxicity.

Norfloxacin nicotinate (NOR-N), an adduct of norfloxacin (NOR) and nicotinic acid, has been widely used for replacing NOR in animal husbandry and fishery industry. Nowadays, increasing evidences showed that NOR could pose toxic effects on fish and other aquatic organisms, but as its adduct, whether NOR-N could cause adverse effects on aquatic organisms is still unclear. To evaluate the toxic effects of NOR-N on the early life stage of zebrafish, we determined the changes in embryonic development (hatching rate, body length, malformation rate and mortality), antioxidant enzyme (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (Gpx)) activities, malondialdehyde (MDA) content and gene expression levels related to antioxidant enzymes (Cu/Zn-sod, Mn-sod, CAT and Gpx) and innate immune system (tumor necrosis factor α (TNFα), interferon (IFN), Interleukin-1 beta (IL-1ß), IL-8, CXCL-clc, CC-chemokine, lysozyme (Lzy) and complement factors (C3)) after embryonic exposure to NOR-N till 96 hpf. The results showed that NOR-N exposure could decreased the hatching rate and body length, and increased abnormality and mortality as concentration-dependent during embryonic development process. NOR-N induced oxidative stress in zebrafish larvae through increasing the contents of MDA and the activities of SOD, CAT and Gpx, as well as the mRNA levels of genes related to these antioxidant enzymes. Moreover, the expression of TNFα, IFN, IL-1ß, IL-8, CXCL-clc, CC-chemokine, Lzy and C3 genes were significantly up-regulated after exposure to high concentration (5 and/or 25 mg/L) of NOR-N till 96 hpf, indicating that the innate immune system in zebrafish larvae was disturbed by NOR-N. Overall, our results suggested that NOR-N caused development toxicity, oxidative stress and immunotoxicity on the early life stage of zebrafish. Thus, widespread application of NOR-N might pose potential ecotoxicological risk on aquatic ecosystems.

Detection and quantification of Aeromonas schubertii in Channa maculata by TaqMan MGB probe fluorescence real-time quantitative PCR.

Aeromonas schubertii is a major epidemiological agent that threatens cultured snakeheads (Channidae) and has caused great economic losses in fish-farming industries in China in recent years. In present study, a specific TaqMan minor groove binder (MGB) probe fluorescence real-time quantitative PCR (qPCR) assay was developed to rapidly detect and quantify A. schubertii. A pair of qPCR primers and a TaqMan MGB probe were selected from the rpoD gene, which were shown to be specific for A. schubertii. A high correlation coefficient (R2  = 0.9998) in a standard curve with a 103% efficiency was obtained. Moreover, the qPCR method's detection limit was as low as 18 copies/µl, which was 100 times more sensitive than that of conventional PCR. The detection results for the A. schubertii in pond water and fish tissue were consistent with those of the viable counts. Bacterial load changes detected by qPCR in different tissues of snakeheads infected with A. schubertii showed that the gills and intestines may be the entry for A. schubertii, and the spleen and kidney are major sites for A. schubertii replication. The established method in present study should be a useful tool for the early surveillance and quantitation of A. schubertii.

A tri-site fluorescent probe for simultaneous sensing of hydrogen sulfide and glutathione and its bioimaging applications.

Hydrogen sulfide (H2S) and biothiol molecules, such as glutathione (GSH), cysteine (Cys), and homocysteine (Hcy), play an important role in biology. However, understanding the complicated relationship between H2S and biothiols remains an enormous challenge owing to the difficulty in sensing H2S and biothiols simultaneously. Therefore, the development of probes for detecting H2S and biothiols is of great importance in biological science. In this work, we reported a novel fluorescent probe for the sensitive and selective detection of H2S and glutathione (GSH) simultaneously in different buffer solutions. The key design principle is based on a coumarin as the fluorophore structuring a fluorescent probe with three potential sites which could react with H2S and biothiols. This probe displays a rapid response with highly sensitive and selective detection of H2S and GSH (the detection limit of 75 nM and 280 nM, respectively). Moreover, with the assistance of a confocal fluorescence microscope, we demonstrated that the probe can be successfully applied for imaging H2S and GSH in MCF-7 cells.

Joint Effects of Multiple UV Filters on Zebrafish Embryo Development.

The widespread use of UV filters has resulted in significant amounts of these chemicals appearing not only in the environment but also in organisms. This study first assessed the levels of nine UV filters in waters along the coast of Shenzhen, China, in tapwater, and in a nearby reservoir. UV filters were found to be high, in both winter and summer at most locations. Then, using zebrafish as a model, the influence of a UV filter mixture after dietary and aqueous exposure was assessed. After exposing artemia to three dominant UV filters at two levels and then feeding these artemia to zebrafish adults, concentrations in both were up to 4 times higher when exposed to the mixtures than when exposed to only a single UV filter. A short-term 25-day dietary exposure to the zebrafish adults did not appear to significantly influence early life stage development of the second generation; however, relatively long exposure over 47 days had significant adverse effects on embryo development. Aqueous exposure of fish embryos to mixtures of the three UV filters demonstrated a general trend of decreased heart/hatching rate as doses increased, coupled with significant changes in activities of catalase and malate dehydrogenase.

Förster Resonance Energy Transfer Switchable Self-Assembled Micellar Nanoprobe: Ratiometric Fluorescent Trapping of Endogenous H2S Generation via Fluvastatin-Stimulated Upregulation.

H2S produced in small amounts by mammalian cells has been identified in mediating biological signaling functions. However, the in situ trapping of endogenous H2S generation is still handicapped by a lack of straightforward methods with high selectivity and fast response. Here, we encapsulate a semi-cyanine-BODIPY hybrid dye (BODInD-Cl) and its complementary energy donor (BODIPY1) into the hydrophobic interior of an amphiphilic copolymer (mPEG-DSPE), especially for building up a ratiometric fluorescent H2S nanoprobe with extraordinarily fast response. A remarkable red-shift in the absorption band with a gap of 200 nm in the H2S response can efficiently switch off the Förster resonance energy transfer (FRET) from BODIPY1 to BODInD-Cl, subsequently recovering the donor fluorescence. Impressively, both the interior hydrophobicity of supramolecular micelles and electron-withdrawing nature of indolium unit in BODInD-Cl can sharply increase aromatic nucleophilic substitution with H2S. The ratiometric strategy based on the unique self-assembled micellar aggregate NanoBODIPY achieves an extremely fast response, enabling in situ imaging of endogenous H2S production and mapping its physiological and pathological consequences. Moreover, the amphiphilic copolymer renders the micellar assembly biocompatible and soluble in aqueous solution. The established FRET-switchable macromolecular envelope around BODInD-Cl and BODIPY1 enables cellular uptake, and makes a breakthrough in the trapping of endogenous H2S generation within raw264.7 macrophages upon stimulation with fluvastatin. This study manifests that cystathione γ-lyase (CSE) upregulation contributes to endogenous H2S generation in fluvastatin-stimulated macrophages, along with a correlation between CSE/H2S and activating Akt signaling pathway.

Effects of norfloxacin on hepatic genes expression of P450 isoforms (CYP1A and CYP3A), GST and P-glycoprotein (P-gp) in Swordtail fish (Xiphophorus Helleri).

The presence of antibiotics including norfloxacin in the aquatic environment may cause adverse effects in non-target organisms. But the toxic mechanisms of fluoroquinolone to fish species are still not completely elucidated. Thus, it is essential to investigate the response of fish to the exposure of fluoroquinolone at molecular or cellular level for better and earlier prediction of these environmental pollutants toxicity. The sub-chronic toxic effects of norfloxacin (NOR) on swordtail fish (Xiphophoru s helleri) were investigated by measuring mRNA expression of cytochrome P450 1A (CYP1A), cytochrome P450 3A (CYP3A), glutathione S-transferase (GST) and P-glycoprotein (P-gp) and their corresponding enzyme activities (including ethoxyresorufin O-deethylase, erythromycin N-demethylase and GST. Results showed that NOR significantly affected the expression of CYP1A, CYP3A, GST and P-gp genes in swordtails. The gene expressions were more responsive to NOR exposure than their corresponding enzyme activities. Moreover, sexual differences were found in gene expression and enzyme activities of swordtails exposed to NOR. Females displayed more dramatic changes than males. The study further demonstrated that the combined biochemical and molecular parameters were considered as useful biomarkers to improve our understanding of potential ecotoxicological risks of NOR exposure to aquatic organisms.

[Construction and application of bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer].

As a key component of life science, bioinformatics has been widely applied in genomics, transcriptomics, and proteomics. However, the requirement of high-performance computers rather than common personal computers for constructing a bioinformatics platform significantly limited the application of bioinformatics in aquatic science. In this study, we constructed a bioinformatic analysis platform for aquatic pathogen based on the MilkyWay-2 supercomputer. The platform consisted of three functional modules, including genomic and transcriptomic sequencing data analysis, protein structure prediction, and molecular dynamics simulations. To validate the practicability of the platform, we performed bioinformatic analysis on aquatic pathogenic organisms. For example, genes of Flavobacterium johnsoniae M168 were identified and annotated via Blast searches, GO and InterPro annotations. Protein structural models for five small segments of grass carp reovirus HZ-08 were constructed by homology modeling. Molecular dynamics simulations were performed on out membrane protein A of Aeromonas hydrophila, and the changes of system temperature, total energy, root mean square deviation and conformation of the loops during equilibration were also observed. These results showed that the bioinformatic analysis platform for aquatic pathogen has been successfully built on the MilkyWay-2 supercomputer. This study will provide insights into the construction of bioinformatic analysis platform for other subjects.

Transforming waste into valuables: Preparation and evaluation of dual-ligand hydrophobic charge-induction chromatography using two poor performing ligands.

In conventional chromatographic ligand screening, underperforming ligands are often dismissed. However, this practice may inadvertently overlook potential opportunities. This study aims to investigate whether these underperforming ligands can be repurposed as valuable assets. Hydrophobic charge-induction chromatography (HCIC) is chosen as the validation target for its potential as an innovative chromatographic mode. A novel dual-ligand approach is employed, combining two suboptimal ligands (5-Aminobenzimidazole and Tryptamine) to explore enhanced performance and optimization prospects. Various dual-ligand HCIC resins with different ligand densities were synthesized by adjusting the ligand ratio and concentration. The resins were characterized to assess appearance, functional groups, and pore features using SEM, FTIR, and ISEC techniques. Performance assessments were conducted using single-ligand mode resins as controls, evaluating the selectivity against human immunoglobulin G and human serum albumin. Static adsorption experiments were performed to understand pH and salt influence on adsorption. Breakthrough experiments were conducted to assess dynamic adsorption capacity of the novel resin. Finally, chromatographic separation using human serum was performed to evaluate the purity and yield of the resin. Results indicated that the dual-ligand HCIC resin designed for human antibodies demonstrates exceptional selectivity, surpassing not only single ligand states but also outperforming certain high-performing ligand types, particularly under specific salt and pH conditions. Ultimately, a high yield of 83.9 % and purity of 96.7 % were achieved in the separation of hIgG from human serum with the dual-ligand HCIC, significantly superior to the single-ligand resins. In conclusion, through rational design and proper operational conditions, the dual-ligand mode can revitalize underutilized ligands, potentially introducing novel and promising chromatographic modes.

Hydrogen-bond tuned conjugated architectures for nitric oxide sensing in single-benzene framework: Advances and mechanistic insights.

In contrast to the conventional fluorescence enhancement resulting from the cessation of the photoinduced electron transfer effect upon capturing nitric oxide (NO) by o-phenylenediamine, we found an interesting fluorescence quench within small molecule fluorophores characterized by intramolecular hydrogen bonding. Herein, the integration of a push-pull electron system with intramolecular hydrogen bonding onto an ultra-small fluorophore was employed to fabricate a hydrogen bond-tuned single benzene core fluorescent probe with an exceptional fluorescence quantum yield of 26 %, denoted as HSC-1. By virtue of its small size and low molecular weight (mere 192 g/mol), it demonstrated superior solubility and biocompatibility. Given the optimized conditions, HSC-1 manifested extraordinary linearity in detecting NO concentrations ranging from 0.5 to 60 µM, with an outstanding detection limit of 23.8 nM. Theoretical calculations unraveled the photophysical properties of hydrogen bonding-related probe molecules and highlighted the NO sensing mechanism. This pioneering work offers an important platform for the design of small fluorescence probes only with a single benzene core applied to NO sensing, which will potentially emerge as a new frontier in the area.

The New Roles of traf6 Gene Involved in the Development of Zebrafish Liver and Gonads.

Traf6, an adaptor protein, exhibits non-conventional E3 ubiquitin ligase activity and was well studied as an important factor in immune systems and cancerogenesis. In mice, the traf6-null caused a perinatal death, so that the underlying pathophysiology of traf6-defeciency is still largely unclear in animals. Here, in the present study, a traf6 knockout zebrafish line (traf6-/-) was generated and could survive until adulthood, providing a unique opportunity to demonstrate the functions of traf6 gene in animals' organogenesis beyond the mouse model. The body of traf6-/- fish was found to be significantly shorter than that of the wildtype (WT). Likewise, a comparative transcriptome analysis showed that 866 transcripts were significantly altered in the traf6-/- liver, mainly involved in the immune system, metabolic pathways, and progesterone-mediated oocyte maturation. Especially, the mRNA expression of the pancreas duodenum homeobox protein 1 (pdx1), glucose-6-phosphatase (g6pcb), and the vitellogenesis genes (vtgs) were significantly decreased in the traf6-/- liver. Subsequently, the glucose was found to be accumulated in the traf6-/- liver tissues, and the meiotic germ cell was barely detected in traf6-/- testis or ovary. The findings of this study firstly implied the pivotal functions of traf6 gene in the liver and gonads' development in fish species.

Revisit of a dipropargyl rhodamine probe reveals its alternative ion sensitivity in both a solution and live cells.

This study reveals that a dipropargyl rhodamine B derivative previously described as a reaction-based irreversible palladium probe responds, however, more sensitively to mercury with a reversible "turn-on" fluorescence. The probe also shows a much better imaging ability for mercury than for palladium in live cells.

Nanofluidic sensing platform for PNK assay using nonlinear hybridization chain reaction and its application in DNA logic circuit.

In this study, a polyethyleneimine (PEI)/Zr4+-functionalized nanofluidic sensing platform based on nonlinear hybridization chain reaction (NHCR) was developed for PNK activity assay. With the existence of PNK, the hairpin HPNK was cleaved by λ exonuclease, liberating the initiator T-DNA. Then T-DNA triggered the nonlinear HCR in solution and the reaction products were absorbed onto the nanopore, which changed the surface charge of nanofluidic device and could be detected by current-voltage characteristic curves. Compared to traditional linear HCR, the nonlinear HCR exhibits a higher sensitivity and order of growth kinetics, making it a powerful signal amplifier in bioanalysis. Due to the powerful amplification efficiency of nonlinear HCR, high sensitivity of the nanopore and specific recognition site of PNK/λ-Exo, an ultrasensitive and selective PNK sensing approach had been developed and applied to precisely quantitate the PNK activity with a LOD of 0.0001 U/mL. Moreover, utilizing this nanofluidic system as a foundation, we constructed a logic circuit that utilized PNK, adenosine diphosphate (ADP), and (NH4)2SO4 as input elements. ADP and (NH4)2SO4 had a crucial function in facilitating the PNK to regulate the DNA logic gate. By modifying the target and inhibitors, the nanofluidic device could detect a variety of stimuli and execute more advanced logical operations.

Ultrasensitive fluorescence detection of multiple DNA methyltransferases based on DNA walkers and hyperbranched rolling circle amplification.

The accurate and ultrasensitive detection of multiple methyltransferases was in great request for clinical diagnosis and epigenetic therapy. Here, a novel fluorescence assay was proposed for ultrasensitive CpG methyltransferase (M.SssI) and DNA adenine methyltransferase (Dam) activity detection based on hyperbranched rolling circle amplification (HRCA) and DNA walkers. The biosensor showed an extremely high sensitivity due to the dual-amplification strategy of HRCA and DNA walker. The LOD of the biosensor for M.SssI and Dam methyltransferase was estimated at 0.0004 U/mL and 0.001 U/mL, respectively. Without the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was cleaved by HpaII endonuclease, generating a DNA fragment (T-DNA) and inducing the DNA walker-HRCA reaction. Since the HRCA products contained numerous double-strand DNA (dsDNA), SYBR Green I could be embedded in the dsDNA, leading to a high fluorescent signal. In the presence of M.SssI methyltransferase, the corresponding recognition site of hairpin HM was methylated and the HpaII endonuclease-catalyzed stem of hairpin HM dissociation was hindered, leading to no DNA fragment (T-DNA) present. Hence, the DNA walker-HRCA reaction was not initiated and the fluorescent signal of SYBR Green I remained at a low level. Similarly, DNA adenine methyltransferase (Dam) and its inhibitors could also be detected by redesigning hairpin HD with the Dam recognition sequences. Furthermore, the sensing system was applied to analyze the endogenic Dam methyltransferase in the real samples such as E. coli cell lysate.

Effects of Myostatin b Knockout on Offspring Body Length and Skeleton in Yellow Catfish (Pelteobagrus fulvidraco).

Based on obtaining mstnb gene knockout in Pelteobagrus fulvidraco, a study on the effect of the mstn gene on skeletal morphology and growth was performed by comparing the number and length of the vertebrae of mutant and wild-type fish in a sibling group of P. fulvidraco, combined with the differences in cells at the level of vertebral skeletal tissue. It was found that mstnb gene knockdown resulted in a reduction in the number of vertebrae, the length, and the intervertebral distance in P. fulvidraco, and these changes may be the underlying cause of the shorter body length in mutant P. fulvidraco. Further, histological comparison of the same sites in the mstn mutant and wild groups of P. fulvidraco also revealed that the number and density of osteocytes were greater in mstnb knockout P. fulvidraco than in wild-type P. fulvidraco. Our results demonstrated that when using genome editing technology to breed new lines, the effects of knockout need to be analyzed comprehensively and may have some unexpected effects due to insufficient study of the function of certain genes.

Ultrastructure, development, and molecular phylogeny of Pleistophora hyphessobryconis, a broad host microsporidian parasite of Puntius tetrazona.

A potentially fatal microsporidial infection targeting the skeletal muscles of the tiger barb Puntius tetrazona was described. Ultrastructural and molecular analyses of infected tissues confirmed that the causative parasite was Pleistophora hyphessobryconis. Compared to P. hyphessobryconis observed in other hosts, those infecting tiger barb demonstrated differences in ultrastructure that may be related to host adaptation. Phylogenetic analysis revealed that classifications based on different methods of analysis (molecular, morphologic, or developmental) do not always coincide, and suggesting that the genetic relationships between Pleistophora and Ovipleistophora may need to be redefined. Transparent mutants of tiger barb can be artificially infected by P. hyphessobryconis, and the dynamic process and spatial distribution of P. hyphessobryconis infection can be observed in real time. These transparent fish mutants are a valuable model to study microsporidial infection in vivo.