OsKANADI1 and OsYABBY5 regulate rice plant height by targeting GIBERELLIN 2-OXIDASE6.

Plant height is an important agronomic characteristic of rice (Oryza sativa L.). Map-based cloning analyses of a natural semi-dwarf rice mutant with inwardly curled leaves found in the field revealed that the defects were due to a mutation of a SHAQKYF-class MYB family transcription factor, OsKANADI1 (OsKAN1). OsKAN1 directly bound to the OsYABBY5 (OsYAB5) promoter to repress its expression and interacted with OsYAB5 to form a functional OsKAN1-OsYAB5 complex. GIBERELLIN 2-OXIDASE6 (OsGA2ox6), encoding an enzyme in the gibberellin (GA) catabolic pathway, was activated by OsYAB5. Furthermore, the OsKAN1-OsYAB5 complex suppressed the inhibitory effect of OsKAN1 toward OsYAB5 and inhibited OsYAB5-induced OsGA2ox6 expression. The proOsKAN1:OsYAB5 transgenic plants were taller than wild-type plants, whereas oskan1 proOsKAN1:OsYAB5 plants exhibited a severe dwarf phenotype due to the absence of the OsKAN1-OsYAB5 complex. The OsKAN1-OsYAB5 complex modulated OsGA2ox6 expression, thereby regulating the levels of bioactive gibberellins and, consequently, plant height. This study elucidated the mechanism underlying the effect of the OsKAN1-OsYAB5-OsGA2ox6 regulatory pathway on plant height at different positions in rice stems and provided insights on stem development and candidate genes for the aerial architecture improvement of crop plants.

A SYBR Green I-based aptasensor for the label-free, fluorometric, and anti-interference detection of MeHg.

Methylmercury (MeHg+) is a common form of organic mercury that is substantially more toxic than inorganic mercury and is more likely to accumulate in organisms through biological enrichment. Therefore, developing a method to enable the specific and rapid detection of MeHg+ in seafood is important and remains challenging to accomplish. Herein, a rapid, label-free fluorescence detection method for MeHg+ determination was developed based on SYBR Green I. The detection system implemented "add and measure" detection mode can be completed in 10 min. Under optimal assay conditions, the detection platform showed a linear relationship with the concentration of MeHg+ within 1-50 nM (Y = 8.573x + 42.89, R2 = 0.9928), with a detection limit of 0.3218 nM. The results obtained for competitive substances, such as inorganic mercury ions and anions, show a high specificity of the method. In addition, this method successfully detected MeHg+ in seawater and marine products, with an accompanying spike recovery rate of 96.45-105.1%.

Raman-enhanced sensor based on CRISPR-SERS technology for the rapid and hypersensitive detection of Mycobacterium tuberculosis.

Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis, and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)-based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10x + 1594, with R2 = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis-specific sequence was detected at 5 copy/µL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.

Exploration of the Antibacterial and Anti-Inflammatory Activity of a Novel Antimicrobial Peptide Brevinin-1BW.

Antibiotic resistance has emerged as a grave threat to global public health, leading to an increasing number of treatment failures. Antimicrobial peptides (AMPs) are widely regarded as potential substitutes for traditional antibiotics since they are less likely to induce resistance when used. A novel AMP named Brevinin-1BW (FLPLLAGLAASFLPTIFCKISRKC) was obtained by the Research Center of Molecular Medicine of Yunnan Province from the skin of the Pelophylax nigromaculatus. Brevinia-1BW had effective inhibitory effects on Gram-positive bacteria, with a minimum inhibitory concentration (MIC) of 3.125 µg/mL against Enterococcus faecalis (ATCC 29212) and 6.25 µg/mL against both Staphylococcus aureus (ATCC 25923) and multidrug-resistant Staphylococcus aureus (ATCC 29213) but had weaker inhibitory effects on Gram-negative bacteria, with a MIC of ≥100 µg/mL. Studies using scanning electron microscopy (SEM) and flow cytometry have revealed that it exerts its antibacterial activity by disrupting bacterial membranes. Additionally, it possesses strong biofilm inhibitory and eradication activities as well as significant lipopolysaccharide (LPS)-binding activity. Furthermore, Brevinin-1BW has shown a significant anti-inflammatory effect in LPS-treated RAW264.7 cells. In conclusion, Brevinin-1BW is anticipated to be a promising clinical agent with potent anti-Gram-positive bacterial and anti-inflammatory properties.

Outer membrane protein A (OmpA) may be used as a novel target to enrich and detect Escherichia coli in milk samples.

In recent years, food safety incidents caused by Escherichia coli have occurred and have endangered human health. Due to the complex matrix of milk samples and the long pretreatment time, the existing methods cannot quickly detect E. coli in milk samples. It is necessary to enrich the E. coli in the complex matrix to improve the detection sensitivity. The E. coli outer membrane protein A (OmpA) is widely present on the cell membrane of E. coli and may be used as a new target to enrich E. coli. In this study, the purified recombinant OmpA protein was used to immunize BALB/c mice to produce polyclonal antibody. Immunomagnetic beads were combined with the polyclonal antibody to enrich the E. coli in the artificially contaminated milk samples. The products of immunoprecipitation were further used for PCR assay. The bacteria in the PCR sample can be pre-enriched, and the limit of detection is 10 × 100 cfu/mL, which is about 100 times more sensitive than samples not processed by this method. Then, the artificially contaminated milk, coffee, juice, and soybean milk samples were tested separately, and it was found that the E. coli gene could be amplified. The whole analysis time was about 120 min, including the enrichment of bacteria and the detection of eluate. We found that OmpA combined with immunomagnetic beads was more efficient, fast, and convenient than the conventional method. Bacteria can be enriched more efficiently without extracting genomic DNA and culturing bacteria. Therefore, this method has potential value for improving the detection sensitivity and shortening the detection time of E. coli in food samples.

Establishment of an Improved ELONA Method for Detecting Fumonisin B1 Based on Aptamers and Hemin-CDs Conjugates.

Fumonisin B1 (FB1) is a strong mycotoxin that is ubiquitous in agricultural products. The establishment of rapid detection methods is an important means to prevent and control FB1 contamination. In this study, an improved enzyme-linked oligonucleotide assay (ELONA) method was designed and tested to detect the contents of FB1 in maize (corn) samples. F10 modified with biotin was bound to an enzyme label plate that was coated with streptavidin (SA) in advance, and carbon dots (CDs) were used to catalyze the color of tetramethylbenzidine (TMB). The complementary chain of F10 was modified with an amino group and coupled with CDs to obtain conjugates. The sample and conjugates were then added to the enzyme plate coated with F10 (an FB1 aptamer). Upon completion of the color reaction, the absorbance was measured at 450 nm. The LOD of this method was 4.30 ng/mL and the LOQ was 13.03 ng/mL. We observed a linear relationship in the FB1 concentration range of 0-100 ng/mL. The standard curve was y = -0.001482 × x + 0.3463, R2 = 0.9918, and the experimental results could be directly measured visually. The recovery of the maize sample was 97.5-99.23% and 94.54-99.25%, and the total detection time was 1 h.

Determination of Fumonisin B1 by Aptamer-Based Fluorescence Resonance Energy Transfer.

Fumonisin FB is produced by Fusarium moniliforme Sheld, of which FB1 is the most common and the most toxic. The establishment of a rapid detection method is an important means to prevent and control FB1 pollution. A highly sensitive fluorescent sensor based on an aptamer for the rapid detection of fumonisin B1 (FB1) in corn was established. In this study, 5-carboxyfluorescein (FAM) was labeled on the aptamer of FB1 (F10). F10 was adsorbed on the surface of graphene oxide (GO) by π-π stacking. The FAM fluorescence signal could be quenched by fluorescence resonance energy transfer between fluorescent molecules and graphene oxide (GO). In the presence of FB1, the binding efficiency of the aptamer to GO was reduced. Therefore, the content of FB1 in corn samples was determined by fluorescence measurements of mixed FAM-labeled F10, GO and corn samples. This method had a good linear relationship in an FB1 concentration range of 0-3000 ng/mL. The equation was y = 0.2576x + 10.98, R2 = 0.9936. The limit of detection was 14.42 ng/mL, and the limit of quantification was 43.70 ng/mL. The recovery of a spiked standard in the corn sample was 89.13-102.08%, and the time of detection was 30 min.

Utilizing the DNA Aptamer to Determine Lethal α-Amanitin in Mushroom Samples and Urine by Magnetic Bead-ELISA (MELISA).

Amanita poisoning is one of the most deadly types of mushroom poisoning. α-Amanitin is the main lethal toxin in amanita, and the human-lethal dose is about 0.1 mg/kg. Most of the commonly used detection techniques for α-amanitin require expensive instruments. In this study, the α-amanitin aptamer was selected as the research object, and the stem-loop structure of the original aptamer was not damaged by truncating the redundant bases, in order to improve the affinity and specificity of the aptamer. The specificity and affinity of the truncated aptamers were determined using isothermal titration calorimetry (ITC) and gold nanoparticles (AuNPs), and the affinity and specificity of the aptamers decreased after truncation. Therefore, the original aptamer was selected to establish a simple and specific magnetic bead-based enzyme linked immunoassay (MELISA) method for α-amanitin. The detection limit was 0.369 µg/mL, while, in mushroom it was 0.372 µg/mL and in urine 0.337 µg/mL. Recovery studies were performed by spiking urine and mushroom samples with α-amanitin, and these confirmed the desirable accuracy and practical applicability of our method. The α-amanitin and aptamer recognition sites and binding pockets were investigated in an in vitro molecular docking environment, and the main binding bases of both were T3, G4, C5, T6, T7, C67, and A68. This study truncated the α-amanitin aptamer and proposes a method of detecting α-amanitin.

Rapid and Simultaneous Detection of Five, Viable, Foodborne Pathogenic Bacteria by Photoinduced PMAxx-Coupled Multiplex PCR in Fresh Juice.

Escherichia coli, Staphylococcus aureus, Shigella, Pseudomonas aeruginosa, and Klebsiella pneumoniae are common foodborne pathogens. In this study, the light-induced PMAxx-coupled multiplex PCR (PMAxx-mPCR) was established to detect the aforementioned five foodborne pathogens in fresh juice at the same time. Moreover, PMAxx pretreatment could effectively distinguish live bacteria from dead bacteria. The optimized PMAxx pretreatment conditions were incubation with a final concentration of 10 µmol/L PMAxx for 10 min and then photolysis for 8 min. After PMAxx pretreatment, the difference in Ct values with or without PMAxx was determined by quantitative real-time PCR. The results showed a significant difference in Ct value before and after PMAxx treatment. Finally, the bacteria-contaminated fresh juice samples treated with PMAxx dye were detected by mPCR. The detection limit of PMAxx-mPCR was 102 colony-forming units (CFU)/mL for E. coli, Shigella, P. aeruginosa, and K. pneumoniae and 103 CFU/mL for S. aureus. Compared with mPCR detection of samples without PMAxx treatment, the proposed method solved the false-positive problem due to dead bacteria. Hence, an accurate and efficient method for the simultaneous detection of five types of pathogenic bacteria was established. This method could be applied to analytical procedures for ensuring food safety.

Instrument-Free and Visual Detection of Salmonella Based on Magnetic Nanoparticles and an Antibody Probe Immunosensor.

Salmonella, a common foodborne pathogen, causes many cases of foodborne illness and poses a threat to public health worldwide. Immunological detection systems can be combined with nanoparticles to develop sensitive and portable detection technologies for timely screening of Salmonella infections. Here, we developed an antibody-probe-based immuno-N-hydroxysuccinimide (NHS) bead (AIB) system to detect Salmonella. After adding the antibody probe, Salmonella accumulated in the samples on the surfaces of the immuno-NHS beads (INBs), forming a sandwich structure (INB-Salmonella-probes). We demonstrated the utility of our AIB diagnostic system for detecting Salmonella in water, milk, and eggs, with a sensitivity of 9 CFU mL-1 in less than 50 min. The AIB diagnostic system exhibits highly specific detection and no cross-reaction with other similar microbial strains. With no specialized equipment or technical requirements, the AIB diagnostic method can be used for visual, rapid, and point-of-care detection of Salmonella.

Development and characterization of sandwich-type enzyme-linked aptamer assay for the detection of rongalite in food.

Rongalite is an essentially strong carcinogen, which due to its properties as a bleaching and brightening, is illegally added to the food processing. In this study, a sandwich-type enzyme-linked aptamer assay (ELAA) is developed by using a rongalite-specific aptamer G02 modified fluorescein amidite (FAM) as a capture probe and aptamer C01 modified biotin as a signal element. In the presence of rongalite, the aptamer G02-rongalite-aptamer C01 complex is produced, and the absorbance value can be subsequently measured. The sandwich-type ELAA was shown to detect rongalite with high specificity and affinity, with a KD value of 19.91 ±â€¯1.321 nM. In addition, the standard curve was established, with the limit of quantification (LOQ) for rongalite at 10 ng mL-1. By calculating the slope of the standard curve and the standard deviation of the blank values, the method detection limit (MDL) was 0.572 ng mL-1. Additionally, the accuracy of the sandwich-type ELAA was demonstrated in real food samples. Compared with high-pressure liquid chromatography (HPLC) assay, the sandwich-type ELAA can detect rongalite directly, and it has great advantages in pre-treatment, operation technique and cost. In short, our data suggest that the sandwich-type ELAA may be applicable as a molecular detection technique for rongalite.

Auxin enhances aluminium-induced citrate exudation through upregulation of GmMATE and activation of the plasma membrane H+-ATPase in soybean roots.

Background and Aims Aluminium (Al) toxicity is a limiting factor for plant growth and crop production in acidic soils. Citrate exudation and activation of the plasma membrane H+-ATPase are involved in soybean responses to Al stress. Auxin has crucial functions in plant growth and stress responses. However, little is known about possible interactions between auxin and citrate exudation under Al stress. In this study, we elucidated the regulatory roles of IAA in Al-induced citrate exudation in soybean roots. Methods We measured IAA content, Al concentration, citrate exudation, plasma membrane H+-ATPase activity, expression of the relevant genes and phosphorylation of the plasma membrane H+-ATPase by integrating physiological characterization and molecular analysis using hydroponically grown soybean. Key Results The concentration of IAA was increased by 25 and 50 µm Al, but decreased to the control level at 200 µm Al. External addition of 50 µm IAA to the root medium containing 25, 50 or 200 µm Al decreased root Al concentration and stimulated Al-induced citrate exudation and the plasma membrane H+-ATPase activity. Reverse transcription-PCR analysis showed that exogenous IAA enhanced the expression of citrate exudation transporter (GmMATE) but not the plasma membrane H+-ATPase gene. The western blot results suggested that IAA enhanced phosphorylation of the plasma membrane H+-ATPase under Al stress. Conclusions Auxin enhanced Al-induced citrate exudation through upregulation of GmMATE and an increase in phosphorylation of the plasma membrane H+-ATPase in soybean roots.

Colorimetric assay for α-amanitin based on inhibition of carbon dots/AuNPs nanoenzyme activity.

Accidental ingestion of poisonous mushrooms leading to poisoning is a global issue. The most important and lethal toxin causing mushroom poisoning is α-amanitin, with a lethal dose of about 0.1 mg kg-1. Rapid detection of wild mushrooms before consumption or rapid identification of toxins after poisoning can effectively reduce the occurrence of fatalities. This study established a method for detecting α-amanitin using carbon dots/AuNPs nanoenzymes (D-Glu-CDs/AuNPs) with robust peroxidase-like activity. This nanoenzyme was prepared employing glucose carbon dots and sodium citrate as reducing and stabilizing agents, respectively. It could oxidize the substrate TMB (tetramethylbenzidine) to produce blue o-TMB. When α-amanitin specifically bound to the active site of the nanoenzyme, a resultant decrease was observed in catalytic activity and the absorbance value at 652 nm. The regression equation Y = -0.06083x + 0.9643, with an R2 value of 0.996, was obtained. The limit of detection was determined to be 48.03 ng mL-1, and the recoveries in urine ranged from 91.2% to 97.6%. This method enabled the visualization of α-amanitin, and the whole detection process was completed within 20 min. The approach holds promise for the quantitative and qualitative determination of α-amanitin in urine samples.

Evaluation of the activity of antimicrobial peptides against bacterial vaginosis.

New drugs for the treatment of bacterial vaginosis (BV) are yet to be developed due to concerns that they may contribute to the increase in antibiotic resistance in BV. Antimicrobial peptides (AMPs) are one of the most promising options for next-generation antibiotics. In this study, we investigated the bacteriostatic activity of the AMPs Pexiganan, plectasin, melittin, and cathelicidin-DM against Gram-negative and Gram-positive bacteria both in vitro and in a mouse model of BV infection. The results showed that Pexiganan, melittin, and cathelicidin-DM had significant antibacterial activity against both Gram-negative and Gram-positive bacteria. AMPs have great potential for clinical application in the treatment of vaginitis, and this study provides an experimental basis for their use in the active immunoprophylaxis of BV.

Enhancing diabetic wound healing with a pH-responsive nanozyme hydrogel featuring multi-enzyme-like activities and oxygen self-supply.

Diabetic wound treating remains a challenging due to bacterial infections, oxidative stress, tissue hypoxia, and high glucose levels. Herein, a multi-enzyme-like activities nanocomposite (Mo,Fe/Cu,I-Ag@GOx) was designed and anchored to a multifunctional fluorescence hydrogel. The nanozyme gel, loaded with glucose-oxidase (GOx), exhibits intrinsic GOx, peroxidase (POD)-, oxidase (OXD)-, catalase (CAT)- and superoxide dismutase (SOD)-like activities with pH-switchable glucose-initiated cascade reaction for diabetic wound healing. In the first cascade-reaction, initiated by GOx, the nanozyme gel catalyzes glucose and O2 into gluconic acid and H2O2 to further generate superoxide anion radical (O2·-) and hydroxyl radicals (·OH) to eradicate bacteria. In the second cascade-reaction, as the wound pH changes alkalescent microenvironment, the nanozyme gel simulates SOD to transform O2·- into O2 and H2O2, and then decomposes endogenous and exogenous H2O2 into O2 via CAT-like activity to reduce oxidative stress and alleviate hypoxia. The gel by calcium ion (Ca2+) cross-linked sodium alginate (SA) and chitosan (CS) containing nanozyme was constructed with injectability, adhesion and fluorescence properties, as well as beneficial biocompatible. Importantly, the water/alcohol solubility of the nanozyme gel allows it to be used as a dressing without causing secondary injury to the wound. The multifunctional fluorescence hydrogel exhibits efficiently promote pro-angiogenesis and bacteria-infected wound healing.

Cu,Zn,I-Doped Carbon Dots with Boosted Triple Antioxidant Nanozyme Activity for Treatment of DSS-Induced Colitis.

Nanozyme-mediated antioxidative therapy is a promising star for treating a myriad of important diseases through eliminating excessive reactive oxygen species (ROS) such as O2·- and H2O2, a critical mechanism for inflammatory bowel disease (IBD). This work provides a high biocompatibility iodine-copper-zinc covalent doped carbon dots (Cu,Zn,I-CDs) with the catalase (CAT)-, superoxide dismutase (SOD)- and glutathione peroxidase (GPx)-like catalytic activities for treating ulcerative colitis (UC) by scavenging overproduced ROS. We found that I dopant aids in counteracting the positive charge at Cu,Zn dopants brought on by low pH, enabling Cu,Zn,I-CDs to process strong triple antioxidant nanozyme activities rather than Cu,Zn-CDs. Vitro experiments displayed that the Cu,Zn,I-CDs could scavenge the excessive ROS to protect cellular against oxidative stress and reduce the expression of proinflammatory cytokines, such as TNF-α, IL-1ß, and IL-6. In sodium dextran sulfate (DSS)-induced colitis mice models, Cu,Zn,I-CDs with excellent biocompatibility could effectively relieve the inflammation of the colon, containing the reduction of the colon length, the damaged epithelium, the infiltration of inflammatory cells, and upregulation of antioxidant genes. Therefore, the therapy of Cu,Zn,I-CD antioxidant nanozymes is an effective approach and provides a novel strategy for UC treatment.

Boosting genome editing in plants with single transcript unit surrogate reporter systems.

CRISPR-Cas-based genome editing holds immense promise for advancing plant genomics and crop enhancement. However, the challenge of low editing activity complicates the identification of editing events. In this study, we introduce multiple single transcript unit surrogate reporter (STU-SR) systems to enhance the selection of genome-edited plants. These systems use the same single guide RNAs designed for endogenous genes to edit reporter genes, establishing a direct link between reporter gene editing activity and that of endogenous genes. Various strategies are used to restore functional reporter genes after genome editing, including efficient single-strand annealing (SSA) for homologous recombination in STU-SR-SSA systems. STU-SR-base editor systems leverage base editing to reinstate the start codon, enriching C-to-T and A-to-G base editing events. Our results showcase the effectiveness of these STU-SR systems in enhancing genome editing events in the monocot rice, encompassing Cas9 nuclease-based targeted mutagenesis, cytosine base editing, and adenine base editing. The systems exhibit compatibility with Cas9 variants, such as the PAM-less SpRY, and are shown to boost genome editing in Brassica oleracea, a dicot vegetable crop. In summary, we have developed highly efficient and versatile STU-SR systems for enrichment of genome-edited plants.

Development of a novel multi-epitope oral DNA vaccine for rabies based on a food-borne microbial vector.

Rabies has been with humans for a long time, and its special transmission route and almost 100 % lethality rate made it once a nightmare for humans. In this study, by predicting the rabies virus glycoprotein outer membrane region and nucleoprotein B-cell antigenic epitopes, the coding sequence of the predicted highly antigenic polypeptide region obtained was assembled using the eukaryotic expression vector pcDNA3.1(-), and then E. coli was used as the delivery vector. The immunogenicity and protective properties of the vaccine were verified by in vivo and in vitro experiments, which demonstrated that the vaccine could produce antibodies in mice and prolong the survival time of mice exposed to the strong virus without any side effects. This study demonstrated that the preparation of an oral rabies DNA vaccine using food-borne microorganisms as a transport vehicle is feasible and could be a new strategy to eradicate rabies starting with wild animals.

A fusion protein of vimentin with Fc fragment inhibits Japanese encephalitis virus replication.

Japanese encephalitis virus (JEV), a member of the Flaviviridae family and a flavivirus, is known to induce acute encephalitis. Vimentin protein has been identified as a potential receptor for JEV, engaging in interactions with the viral membrane protein. The Fc fragment, an integral constituent of immunoglobulins, plays a crucial role in antigen recognition by dendritic cells (DCs) or phagocytes, leading to subsequent antigen presentation, cytotoxicity, or phagocytosis. In this study, we fused the receptor of JEV vimentin with the Fc fragment of IgG and expressed the resulting vimentin-Fc fusion protein in Escherichia coli. Pull-down experiments demonstrated the binding ability of the vimentin-Fc fusion protein to JEV virion in vitro. Additionally, we conducted inhibition assays at the cellular level, revealing the ability of vimentin-Fc protein suppressing JEV replication, it may be a promising passive immunotherapy agent for JEV. These findings pave the way for potential therapeutic strategies against JEV.

Molecular cloning and characterization of cystatin, a cysteine protease inhibitor, from bufo melanostictus.

Cystatins are efficient inhibitors of papain-like cysteine proteinases, and they serve various important physiological functions. In this study, a novel cystatin, Cystatin-X, was cloned from a cDNA library of the skin of Bufo melanostictus. The single nonglycosylated polypeptide chain of Cystatin-X consisted of 102 amino acid residues, including seven cysteines. Evolutionary analysis indicated that Cystatin-X can be grouped with family 1 cystatins. It contains cystatin-conserved motifs known to interact with the active site of cysteine proteinases. Recombinant Cystatin-X expressed and purified from Escherichia coli exhibited obvious inhibitory activity against cathepsin B. rCystatin-X at a concentration of 8 µM inhibited nearly 80% of cathepsin B activity within 15 s, and about 90% of cathepsin B activity within 15 min. The Cystatin-X identified in this study can play an important role in host immunity and in the medical effect of B. melanostictus.