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.

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.

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.

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.

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.

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%.

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.

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.

HBV Vaccines: Advances and Development.

Hepatitis B virus (HBV) infection is a global public health problem that is closely related to liver cirrhosis and hepatocellular carcinoma (HCC). The prevalence of acute and chronic HBV infection, liver cirrhosis, and HCC has significantly decreased as a result of the introduction of universal HBV vaccination programs. The first hepatitis B vaccine approved was developed by purifying the hepatitis B surface antigen (HBsAg) from the plasma of asymptomatic HBsAg carriers. Subsequently, recombinant DNA technology led to the development of the recombinant hepatitis B vaccine. Although there are already several licensed vaccines available for HBV infection, continuous research is essential to develop even more effective vaccines. Prophylactic hepatitis B vaccination has been important in the prevention of hepatitis B because it has effectively produced protective immunity against hepatitis B viral infection. Prophylactic vaccines only need to provoke neutralizing antibodies directed against the HBV envelop proteins, whereas therapeutic vaccines are most likely needed to induce a comprehensive T cell response and thus, should include other HBV antigens, such as HBV core and polymerase. The existing vaccines have proven to be highly effective in preventing HBV infection, but ongoing research aims to improve their efficacy, duration of protection, and accessibility. The routine administration of the HBV vaccine is safe and well-tolerated worldwide. The purpose of this type of immunization is to trigger an immunological response in the host, which will halt HBV replication. The clinical efficacy and safety of the HBV vaccine are affected by a number of immunological and clinical factors. However, this success is now in jeopardy due to the breakthrough infections caused by HBV variants with mutations in the S gene, high viral loads, and virus-induced immunosuppression. In this review, we describe various types of available HBV vaccines, along with the recent progress in the ongoing battle to develop new vaccines against HBV.

Transcriptomics based identification of S100A3 as the key anti-hepatitis B virus factor of 16F16.

More than 250 million people worldwide have chronic hepatitis B virus (HBV) infections, resulting in over 1 million annual fatalities because HBV cannot be adequately treated with current antivirals. Hepatocellular carcinoma (HCC) risk is elevated in the presence of the HBV. Novel and powerful medications that specifically target the persistent viral components are needed to remove infection. This study aimed to use HepG2.2.15 cells and the rAAV-HBV1.3 C57BL/6 mouse model established in our laboratory to examine the effects of 16F16 on HBV. The transcriptome analysis of the samples was performed to examine the impact of 16F16 therapy on host factors. We found that the HBsAg and HBeAg levels significantly decreased in a dose-dependent manner following the 16F16 treatment. 16F16 also showed significant anti-hepatitis B effects in vivo. The transcriptome analysis showed that 16F16 regulated the expression of several proteins in HBV-producing HepG2.2.15 cells. As one of the differentially expressed genes, the role of S100A3 in the anti-hepatitis B process of 16F16 was further investigated. The expression of the S100A3 protein significantly decreased following the 16F16 therapy. And upregulation of S100A3 caused an upregulation of HBV DNA, HBsAg, and HBeAg in HepG2.2.15 cells. Similarly, knockdown of S100A3 significantly reduced the levels of HBsAg, HBeAg, and HBV DNA. Our findings proved that S100A3 might be a new target for combating HBV pathogenesis. 16F16 can target several proteins involved in HBV pathogenesis, and may be a promising drug precursor molecule for the treatment of HBV.

An efficient CRISPR-Cas12a promoter editing system for crop improvement.

Promoter editing represents an innovative approach to introduce quantitative trait variation (QTV) in crops. However, an efficient promoter editing system for QTV needs to be established. Here we develop a CRISPR-Cas12a promoter editing (CAPE) system that combines a promoter key-region estimating model and an efficient CRISPR-Cas12a-based multiplexed or singular editing system. CAPE is benchmarked in rice to produce QTV continuums for grain starch content and size by targeting OsGBSS1 and OsGS3, respectively. We then apply CAPE for promoter editing of OsD18, a gene encoding GA3ox in the gibberellin biosynthesis pathway. The resulting lines carry a QTV continuum of semidwarfism without significantly compromising grain measures. Field trials demonstrated that the OsD18 promoter editing lines have the same yield performance and antilodging phenotype as the Green Revolution OsSD1 mutants in different genetic backgrounds. Hence, promoter editing of OsD18 generates a quantitative Green Revolution trait. Together, we demonstrate a CAPE-based promoter editing and tuning pipeline for efficient production of useful QTV continuum in crops.

A multi-enzyme-like activity exhibiting mussel-inspired nanozyme hydrogel for bacteria-infected wound healing.

Bacterial infection, tissue hypoxia, and inflammatory and oxidative stress are several key problems in wound healing of chronic infections. Herein, a multi-enzyme-like activity exhibiting multifunctional hydrogel made up of mussel-inspired carbon dot reduced-Ag (CDs/AgNPs) and Cu/Fe-nitrogen-doped carbon (Cu,Fe-NC) was designed. Due to the loss of glutathione (GSH) and oxidase (OXD)-like activity of the nanozyme (decomposes O2 to generate a superoxide anion radical (O2˙-) and hydroxyl radical production (˙OH)), the multifunctional hydrogel exhibited excellent antibacterial performance. More importantly, during the bacterial elimination within the inflammatory phase of wound healing, the hydrogel could act as a catalase (CAT)-like agent to supply adequate O2 by catalyzing intracellular H2O2 for hypoxia abatement. The catechol groups on the CDs/AgNPs endowed them with the dynamic redox equilibrium properties of phenol-quinones, thus providing the hydrogel with mussel-like adhesion properties. The multifunctional hydrogel was shown to excellently promote bacterial infection wound healing and maximize the efficiency of nanozymes.

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.

Development of alkaline phosphatase-scFv and its use for one-step enzyme-linked immunosorbent assay for His-tagged protein detection.

A histidine (His)-tag is composed of six His residues and typically exerts little influence on the structure and solubility of expressed recombinant fusion proteins. Purification methods for recombinant proteins containing His-tags are relatively well-established, thus His-tags are widely used in protein recombination technology. We established a one-step enzyme-linked immunosorbent assay (ELISA) for His-tagged recombinant proteins. We analyzed variable heavy and light chains of the anti-His-tag monoclonal antibody 4C9 and used BLAST analyses to determine variable zones in light (VL) and heavy chains (VH). VH, VL, and alkaline phosphatase (ALP) regions were connected via a linker sequence and ligated into the pGEX-4T-1 expression vector. Different recombinant proteins with His tags were used to evaluate and detect ALP-scFv activity. Antigen and anti-His-scFv-ALP concentrations for direct ELISA were optimized using the checkerboard method. ZIKV-NS1, CHIKV-E2, SCRV-N, and other His-tag fusion proteins demonstrated specific reactions with anti-His-scFv-ALP, which were accurate and reproducible when the antigen concentration was 50 µg mL-1 and the antibody concentration was 6.25 µg mL-1. For competitive ELISA, we observed a good linear relationship when coating concentrations of recombinant human anti-Müllerian hormone (hAMH) were between 0.78 and 12.5 µg mL-1. Our direct ELISA method is simple, rapid, and accurate. The scFv antibody can be purified using a prokaryotic expression system, which provides uniform product quality and reduces variations between batches.

Wound healing mechanism of antimicrobial peptide cathelicidin-DM.

Background and Purpose: Chronic wound infections and the development of antibiotic resistance are serious clinical problems that affect millions of people worldwide. Cathelicidin-DM, an antimicrobial peptide from Duttaphrynus melanostictus, has powerful antimicrobial activity and wound healing efficacy. So, it could be a potential candidate to address this problem. In this paper, we investigate the wound healing mechanism of cathelicidin-DM to establish a basis for preclinical studies of the drug. Experimental Approach: The effects of cathelicidin-DM on cell proliferation and migration, cytokines, and mitogen-activated protein kinase (MAPK) signaling pathways were examined. Then mice whole skin wound model was constructed to evaluate the wound healing activity of cathelicidin-DM, and further histological changes in the wounds were assessed by hematoxylin-eosin staining (H&E) and immunohistochemical assays. Key Results: Cathelicidin-DM promotes the proliferation of HaCaT, HSF, and HUVEC cells in a concentration-dependent manner and the migration of HSF, HUVEC, and RAW.264.7 cells. Moreover,cathelicidin-DM can involve in wound healing through activation of the MAPK signaling pathway by upregulating phosphorylation of ERK, JNK, and P38. However, cathelicidin-DM didn't affect the secretion of IL-6 and TNF-α. At the animal level, cathelicidin-DM accelerated skin wound healing and early debridement in mice as well as promoted re-epithelialization and granulation tissue formation, α-SMA expression, and collagen I deposition in mice. Conclusion and Implications: Our data suggest that cathelicidin-DM can be engaged in the healing of infected and non-infected wounds through multiple pathways, providing a new strategy for the treatment of infected chronic wounds.

Establishment of fumonisin B1 detection method for catalytic fluorescence detection of aptamer-regulated carbon dots.

Mycotoxin, common in agricultural products, is a small secondary metabolite with strong toxicity. Fumonisin B1 (FB1) is the most common and the most toxic. Establishing a rapid detection method is important for preventing and controlling FB1 pollution. This study prepared carbon dots (CDs) from 2,2'-dithiosalicylic acid (DTSA). Tetramethylbenzidine (TMB) can be catalyzed to produce fluorescence by CDs, while FB1 can adhere to the surface of CDs, decreasing fluorescence. Aptamer F10 of FB1 combines with FB1 attached to the surface of CDs to restore the catalytic ability of CDs and increase the fluorescence value. This method has good linearity in the FB1 concentration range from 0 to 1.0 µg mL-1. The standard curve was Y = -0.2512x + 661.4, R2 = 0.9903, the limit of detection (LOD) was 17.67 ng mL-1 and limit of quantitation (LOQ) was 53.55 ng mL-1. The recovery of the corn sample was 89.83-98.62%, and the detection time was 30 min.

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.

Polyaniline derived carbon membrane and its in-situ membrane fouling mitigation performance in MBR based on metal-free electro-Fenton.

An electro-enhanced membrane bioreactor (EMBR) was constructed with polyaniline-based carbon (PAC) separation membrane as the membrane-electrode, which could realize the in-situ electro-generation and activation of H2O2 to ·OH depending on the graphitic and pyridinic N as active sites without metal catalyst. After the continuous operation of the bioreactor for 74 days, approximately 77.41% irreversible membrane fouling occurred on the electrochemically enhanced membrane, which was less than that on the control membrane (85.96%). The ·OH oxidation combined with electrostatic barrier formed by -1.0 V enhanced PAC membrane suppressed the extracellular polymeric substances deposition on membrane. After operation, the strength of total cell, proteins, ß-polysaccharides and α-polysaccharides on the membrane without bias were 5.17, 4.32, 9.65 and 16.31, respectively. In EMBR, the corresponding strength were 2.03, 3.35, 2.15 and 6.73. After calculation, the unblocked pores accounted for 35.3% and 78.5% of the total membrane surface in MBR and EMBR, respectively, indicating the fouling was alleviated obviously. Meanwhile, the EMBR owned a satisfactory wastewater treatment effect with average effluent chemical oxygen demand and NH4+-N around 18.98 mg/L and 0.68 mg/L. The successful implementation of this strategy achieved a green and metal-free method for ·OH production with electrochemical effect for membrane fouling control in MBR.