Genome sequences of two Bacillus anthracis strains utilized as veterinary vaccines in China.

In this report, we present the complete genome sequences of two Bacillus anthracis strains utilized as veterinary vaccines in China. The sequencing was conducted using a hybrid assembly methodology that combined Illumina short reads and PacBio long reads. This approach provides a high-quality representative sequence for the strains mentioned above.

Rapid Identification of Brucella Genus and Species In Silico and On-Site Using Novel Probes with CRISPR/Cas12a.

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

A low bleeding risk thrombolytic agent: citPA5.

AIMS: Alteplase is a cornerstone thrombolytic agent in clinical practice, but presents a potential bleeding risk. Stroke patients need pre-screening to exclude hemorrhagic stroke before using Alteplase. In this study, we develop a new thrombolytic agent citPA5, characterized by an enhanced safety profile and minimal bleeding tendency. METHODS AND RESULTS: A clot lysis agent, named citPA5, is developed based on rtPA with point mutations to completely suppress its proteolytic activity in the absence of fibrin. In the presence of fibrin, citPA5 exhibited significantly higher fibrinolytic activity (a 15.8-fold increase of kcat/Km). Furthermore, citPA5 showed resistance to endogenous fibrinolysis inhibitor, PAI-1, resulting in enhanced potency. In a series of safety evaluation experiments, including thrombelastography (TEG) assay, mice tail bleeding assay, and a murine intracerebral hemorrhage (ICH) model, citPA5 did not cause systemic bleeding or worsen intracerebral hemorrhage compared to Alteplase. This highlights the low risk of bleeding associated with citPA5. Finally, we found that citPA5 effectively improved cerebral blood flow and reduced infarct volume in a carotid embolism-induced stroke (CES) model. CONCLUSIONS: This clot lysis agent, citPA5, not only exhibits a low risk of bleeding but also demonstrates highly effective thrombolysis capabilities. As a result, citPA5 shows great potential for administration prior to the classification of stroke types, making it possible for use in ambulances at the onset of stroke when symptoms are identified. The findings presented in this study also suggest that this strategy could be applied to develop a new generation of fibrinolytic drugs that offer greater safety and specificity in targeting fibrin.

One-step preparation of a self-assembled bioconjugate nanovaccine against Brucella.

Brucellosis, caused by Brucella, is a severe zoonosis, and the current Brucella live attenuated vaccine cannot be used in humans due to major safety risks. Although polysaccharide antigens can be used to prepare the Brucella vaccine, their lower immunogenicity limits them from producing efficient and broad protection. In this study, we produced a high-performance bioconjugate nanovaccine against different species of Brucella by introducing a self-assembly nanoparticle platform and an O-linked glycosylation system into Yersinia enterocolitica serotype O:9, which has an O-polysaccharide composed of the same unit as Brucella. After successfully preparing the vaccine and confirming its stability, we subsequently demonstrated the safety of the vaccine in mice by high-dose immunization. Then, by a series of mouse experiments, we found that the nanovaccine greatly promoted antibody responses. In particular, the increase of IgG2a was more obvious than that of IgG1. Most importantly, this nanovaccine could provide cross-protection against B. abortus, B. melitensis, and B. suis strains by lethal dose challenged models, and could improve the clearance of B. melitensis, the most common pathogenic species in human brucellosis, by non-lethal dose infection. Overall, for the first time, we biocoupled polysaccharide antigens with nano carriers to prepare a Brucella vaccine, which showed pronounced and extensive protective effects in mice. Thus, we provided a potential candidate vaccine and a new direction for Brucella vaccine design.

Effect of localized electrochemical pH and temperature synergistic modification on the structural and antibacterial properties of pectin/polyvinyl alcohol/zinc oxide nanorod films.

Two low-methoxy pectins (LMPs) were obtained by local electrochemical pH modification using an H-type double-layer water bath sealed electrochemical cell at the voltage of 180 V for 3 h. The weight-average molecular weight (Mw) of citrus peel pectin (CPP) prepared in the anodic part at room temperature (CPP-A5/RT) and in the cathodic part at 5 °C (CPP-C5/RT) were 346 kDa and 328 kDa, respectively, and the degrees of methylation (DM) were 36.8 % and 11.9 %. Moreover, the second-order kinetic model was most appropriate for the degradation processes, as free radicals were generated in the anodic part and ß-elimination occurred in the cathodic part. Subsequently, CPP-A5/RT and CPP-C5/RT were utilized to fabricate food packaging film blending with polyvinyl alcohol (PVA), bcZnO (ZnO coupled with bentonite and colophony) nanorods, and Ca2+ ions by casting method. Then the prepared films were studied for their ability to maintain the freshness of strawberries. The addition of Ca2+ ions and bcZnO nanorods increased the thickness, water contact angle (WCA), and mechanical properties of the composite films, while decreased water vapor permeability (WVP). Therefore, the CPP-based films, supplemented with bcZnO nanorods and crosslinked with Ca2+ ions by "egg-box" model, can serve as an antibacterial food packaging material for food preservation.

Natural Aldehyde-Chitosan Schiff Base: Fabrication, pH-Responsive Properties, and Vegetable Preservation.

The aim of the present work was to fabricate Schiff base compounds between chitosan and aldehydes and use the resultant aldehyde-chitosan Schiff bases for broccoli preservation. Using an element analyzer, the degree of substitution was calculated as 68.27-94.65%. The aldehyde-chitosan Schiff bases showed acidic sensitivity to rapid hydrolysis for releasing aldehyde at a buffer solution of pH 4-6, in which more than 39% of the aldehydes were released within 10 h. The release of aldehydes endows the aldehyde-chitosan Schiff bases with a better antibacterial activity at pH 5 than at pH 7. In a simulated CO2 (5-15%) atmosphere with high humidity (92%), the hydrolysis of imine bonds (C=N) was triggered and continuously released aldehyde, even without direct contact with the aqueous phase. The application of aldehyde-chitosan Schiff bases significantly extended the shelf life of broccoli from 4 d to 5-7 d and decreased the weight loss of broccoli during storage. In summary, the fabrication of aldehyde-chitosan Schiff bases and the strategy of using pH-response imine bond (C=N) hydrolysis (thus releasing aldehyde to kill microorganisms) were feasible for use in developing EO-incorporated intelligent food packages for vegetable preservation.

Reusable flexible poly(vinyl alcohol)/chitosan-based polymer carbon dots composite film for acid blue 93 dye adsorption.

The design and synthesis of water-insoluble chitosan-based polymer carbon dots [P(CS-g-CA)CDs] are described. A polyvinyl alcohol/chitosan-based polymer carbon dot [PVA/P(CS-g-CA)CDs] composite film was prepared using a simple casting method to be used in dye adsorption. The composite film was characterized using FT-IR, XPS, transparency, contact angle, and mechanical properties tests, which showed the successful incorporation of P(CS-g-CA)CDs into the film and also revealed that hydrogen bonding improved the mechanical properties of the PVA film. Furthermore, the composite film displayed substantially enhanced hydrophobicity, making it suitable for use in aqueous environments. In addition, the composite film exhibited stable adsorption of acid blue 93 (AB93) at pH 2-9, with an enhanced adsorption capacity of 433.24 mg/g. The adsorption obeyed Langmuir law with an efficiency of more than 89% even after five cycles. Therefore, the PVA/P(CS-g-CA)CDs film is a promising material for the treatment of organic dye-polluted wastewater.

Effect of different NaCl concentrations on the structure-function relationship of citrus peel pectins modified by electrochemistry.

In this study, the modified citrus peel pectins (CPPs) were successfully produced by electrochemistry with varying NaCl concentrations of 0, 0.01 % and 0.1 % w/v using an H-type cell at 40 mA current. After 4 h, the pH and oxidation-reduction potential (ORP) of oxidized CPP solution in the anodic region were 2.00- 2.52 and 371.17- 564.45 mV, respectively, due to the electrolysis of water, whereas those of reduced CPP solution in cathodic region were 9.46- 10.84 and - 202.77 âˆ¼ -230.57 mV. The modified CPPs in the anodic region (A-0, A-0.01, and A-0.1) exhibited significantly higher weight-average molecular weights and methyl esterification degrees than those in the cathodic region (C-0, C-0.01 and C-0.1). In contrast, the K+, Mg2+, and Ca2+ contents of A-0, A-0.01, and A-0.1 were lower than those of C-0, C-0.01 and C-0.1 due to the electrophoretic migration. Furthermore, the antioxidant activities of A-0 and A-0.01 solutions were stronger than those of the C-0, C-0.01, and C-0.1, while rheological and texture properties of their hydrogels showed contradictory results. Finally, the potential structure-function relationships of CPPs were explored by combining PCA and correlation analysis. Overall, this study introduced a potential approach for pectin purification and functional low-methoxyl pectin manufacturing.

Structural Dynamics-Driven Discovery of Anticancer and Antimetastatic Effects of Diltiazem and Glibenclamide Targeting Urokinase Receptor.

Diltiazem and glibenclamide are commonly used hypotensive and antidiabetic drugs. This study reports the discovery of the potential antitumor and antimetastatic effects of these two drugs using a structural dynamics-driven virtual screening targeting urokinase receptor (uPAR). Owing to uPAR's high flexibility, currently resolved crystal structures of uPAR, all in ligand-bound states, provide limited representations of its physiological conformation. To improve the accuracy of screening, we performed a long-timescale molecular dynamics simulation and obtained the representative conformations of apo-uPAR as the targets for our screening. Experimentally, we demonstrated that diltiazem and glibenclamide bound uPAR with KD values in the micromolar range. In addition, both compounds effectively suppressed tumor growth and metastasis in a uPAR-dependent manner in vitro and in vivo. This work not only provides two potent uPAR inhibitors but also reports a proof-of-concept study on the potential off-label antitumor and antimetastatic uses of diltiazem and glibenclamide.

Albumin-based drug carrier targeting urokinase receptor for cancer therapy.

Urokinase plasminogen activator receptor (uPAR) is a key participant in extracellular proteolysis, tissue remodeling and cell motility. uPAR overexpresses in most solid tumors and several hematologic malignancies, but has low levels on normal tissues, thus is advocated as a molecular target for cancer therapy. One of the obstacles for the evaluation of uPAR targeting agents in preclinical study is the species specificity, where targeting agents for human uPAR  usually not bind to murine uPAR. Here, we develop a targeting agent that binds to both murine and human uPAR. This targeting agent is genetically fused to human serum albumin, a commonly used drug carrier, and the final construct is named as uPAR targeting carrier (uPARTC). uPARTC binds specifically to uPAR-overexpressing 293T/huPAR and 293T/muPAR as demonstrated by flow cytometry. A cytotoxic compound, celastrol, is embedded into uPARTC non-covalently. The resulting macromolecular complex show effective proliferation inhibition on both murine and human uPAR overexpressing cells, and exhibit potent antitumor efficacy on hepatoma H22-bearing mice. This work demonstrates that uPARTC is a promising tumor targeting drug carrier, which address the species-specificity challenge of uPAR targeting agents and can be used to load other cytotoxic compounds.

Sensory evaluation, chemical structures, and threshold concentrations of bitter-tasting compounds in common foodstuffs derived from plants and maillard reaction: A review.

The bitterness of foodstuffs is often associated with toxicity, which negatively influences product acceptability. However, bitter compounds have many benefits, and a slight bitter taste is sometimes favored. In this review, we summarize the methods used to isolate and evaluate the taste of bitter compounds in different foods. The chemical structures and threshold concentrations of these compounds are also recapped. Although the structures and thresholds of many bitter compounds have been confirmed, further studies are needed to develop detailed bitter-masking strategies and establish the relation between functional groups (hetero-cyclic substituents and bonding types) and taste quality. Furthermore, a comprehensive bitterness database and chemometric data must be provided in order to quickly assess the bitterness of unfamiliar products.

Nafamostat Mesylate in Combination with the Mouse Amino-Terminal Fragment of Urokinase-Human Serum Albumin Improves the Treatment Outcome of Triple-Negative Breast Cancer Therapy.

Triple-negative breast cancer (TNBC) is highly aggressive and causes a higher proportion of metastatic cases. However, therapies directed to specific molecular targets have rarely achieved clinically meaningful improvements in the outcome of TNBC therapy. A urokinase-type plasminogen activator (uPA), one of the best-validated biomarkers of breast cancer, is an extracellular proteolytic serine protease involved in many pathological and physiological processes, including tumor cell invasion and metastasis. Nafamostat mesylate (NM) is a synthetic compound that inhibits various serine proteases and has been used as a therapeutic agent for the treatment of TNBC. Nevertheless, NM has poor specificity for serine proteases and is easy be hydrolyzed; moreover, the inhibitory mechanism of TNBC therapy is unclear. In this study, we combine NM with a macromolecular drug delivery vehicle, mouse amino-terminal fragment of urokinase-human serum albumin (mATF-HSA), to form a complex (mATF-HSA:NM) using the dilution-incubation-purification method. mATF specifically targets uPAR overexpressed on the surface of TNBC cells; moreover, HSA prevents NM from being hydrolyzed by numerous serine proteases. mATF-HSA:NM showed stronger inhibitory effects on the proliferation and metastasis of TNBC in vitro and in vivo without significant cytotoxicity on normal cells and tissues. In addition, we demonstrated that NM mediates metastasis of TNBC cells through inhibition of uPA using a stable uPA knockdown cell line (MDA-MB231 shuPA). Overall, we have developed a macromolecular complex targeted to treat high uPAR-expressing tumor types, and mATF-HSA can potentially be used to load other types of drugs with tumor-targeting specificity for mouse tumor models and is a promising tool to study tumor biology in mouse tumor models.

The clinicopathological features and treatments of odontogenic keratocysts.

An odontogenic keratocyst (OKC) is a benign but aggressive intraosseous tumor derived from the remains of the original tooth germ or dental lamina. It has a marked ability to recur and become cancerous. However, patients with early-stage OKC often have no symptoms and manifestations. The common clinical manifestation is swelling. Hence, it is critical to precisely diagnose the disease, to use differential diagnosis in combination with auxiliary examination methods, and to select the most appropriate treatment option to reduce the loss of bone tissue and the related damage to patients. In recent years, with the advancement in understanding the molecular basis of this disease and the development of early detection and targeted therapy, the diagnosis and the prognosis of OKC have been improved. The aim of this study was to provide an overview on the clinical features, diagnosis, and treatment of OKC. The molecular and genetic basis of this disease and the characteristics of malignant transformation of OKC were also discussed. Finally, we presented patient cases from our clinical practice to provide some advice on the diagnosis and treatment of OKC.

Enhancing the Emulsification and Photostability Properties of Pectin from Different Sources Using Genipin Crosslinking Technique.

Pectin is a potential polysaccharide-based emulsifier, but the stabilized emulsions suffer from insufficient emulsion stability. Therefore, modification is needed to enhance its emulsification performance to cater to practical applications. The genipin-crosslinking strategy was used in this work to modify pectin with different sources and extraction conditions. Chemical composition analysis, molecular weight (Mw), and radius of gyration (Rg) measurement revealed that sugar beet pectin (SBP) has a more compact and flexible conformation than commercial citrus pectin (CP) and apple pectin (AP), indicated by the significantly (p < 0.05) larger Mw/Rg of SBP (18.1−11.3 kg/mol/nm) than CP (8.3 kg/mol/nm) and AP (8.0 kg/mol/nm). Crosslinking modification significantly increased the Mw, radius of gyration, and viscosity. This significantly (p < 0.05) improved the emulsifying stability (a smaller increase in droplets size) by the contribution of both thicker adsorbed hydrated layers at the oil-water interface with a stronger steric-hindrance effect and larger viscosity effect to slow down droplet collision. The formation of a blue-black substance from crosslinking reaction was able to block the UV radiation, which significantly improved the photostability of ß-carotene-loaded emulsions. Altogether, genipin-crosslinking is feasible to modify pectin of different sources to enhance the emulsion stability and for use as a vehicle for delivering bioactive compounds.

Tannic Acid-Aminated Sugar Beet Pectin Nanoparticles as a Stabilizer of High-Internal-Phase Pickering Emulsions.

Pickering stabilizers with additional antioxidant, photostabilizing, and metal-chelating properties are suitable for structuring multifunctional Pickering emulsion systems. Tannic acid (TA) is a potential material which when adsorbed onto the interface may impart antioxidant, UV-light-shielding, and chelating properties to Pickering stabilizers. Herein, we report a type of TA polyelectrolyte nanoparticles (NPs) fabricated following a complexation between TA and aminated sugar beet pectin (SBP-NH2). This study is geared toward investigating the performance of TA/SBP-NH2 NPs in stabilizing Pickering emulsions and protecting ß-carotene from degradation. TA/SBP-NH2 NPs formed under optimum conditions had a mean diameter of 82 nm with a sphere-like shape. Because of their favorable surface wettability (91.2°), TA/SBP-NH2 NPs promoted formation of the low-, medium-, and high-internal-phase Pickering emulsions (HIPEs) in an oil volume fraction (φ)-dependent manner; the TA/SBP-NH2 NP-stabilized HIPE demonstrated viscoelastic properties increasing with the increasing concentration (c) of nanoparticles. Due to the excellent storage stability and UV light-absorbing capacity, the photostability of ß-carotene was significantly improved by a TA/SBP-NH2 NP-stabilized HIPE (φ = 0.75; c = 3 mg/mL). Altogether, this study highlights that TA/SBP-NH2 NPs have potential applications in structuring Pickering emulsions with improved protective effects on loaded lipophilic compounds.

Physicochemical properties of whey protein isolate and alkaline soluble polysaccharide from sugar beet pulp conjugates formed by Maillard reaction and genipin crosslinking reaction: A comparison study.

This study aim to investigate the effect of alkaline soluble polysaccharide from sugar beet pulp (ASP2) grafted with whey protein isolate (WPI) by two linking models (grafting on amino group or carbonyl group) on its emulsifying properties. Results demonstrated that the d 4,3 value of WPI, M-AW, M-AA, G-AW and G-AA stabilized emulsions was 0.18 µm, 0.28 µm, 0.72 µm, 0.56 µm and 0.83 µm, respectively, suggesting the higher emulsifying activity of the products prepared by Maillard reaction compared with the products obtained from genipin crosslinking reaction. After storage, the d 4,3 increment was 1.05 µm, 0.21 µm, 0.31 µm, 0.2 µm and 0.15 µm for WPI, M-AW, M-AA, G-AW and G-AA stabilized emulsions, respectively, indicating that the new generated polymers held stronger emulsifying stability compared with WPI. However, the aggregates emerged in high calcium emulsions system indicated that grafting with WPI could not efficiently reduce the sensitivity of ASP2 to calcium.

Effects of moisture content on the enolization products formation in glucose-proline Maillard reaction models.

BACKGROUND: 2,3-Dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one (DDMP) and 5-hydroxymethylfurfural (HMF) are two main enolization products in the Maillard reaction and found in some foodstuffs. For many years, whether they are functional or noxious to human health has been a matter of debate. Thus, insight into their formation pathways is important to manage Maillard reaction products. In this study, DDMP and HMF were quantified and compared with regard to their formation and degradation in the d-glucose and l-proline Maillard reaction models using different moisture contents (0, 0.1, 0.5, 1.0, and 4.0 mL) at 150 °C for various heating times. RESULTS: DDMP was predominantly generated in dry or low water-content heating models along with n increased 1-deoxyglucosone (1-DG) generation via 2,3-enolization. However, increasing moisture content resulted in a decay of reaction intensity, 1-DG, and DDMP due to a change in the reaction mechanism from 2,3-enolization to 1,2-enolization, which facilitated 3-deoxyglucosone (3-DG) and HMF formation. CONCLUSION: Increased moisture content in glucose-proline models reduced reaction intensity and also inhibited DDMP and facilitated HMF formation by promoting the pathway change from 2,3-enolization to 1,2-enolization to generate more 3-DG. A water content of 1.0 mL was identified as a critical value, from which the 1,2-enolization became a primary pathway occurring in the Maillard reaction. © 2022 Society of Chemical Industry.

Crystal structure and cellular functions of uPAR dimer.

Receptor dimerization of urokinase-type plasminogen activator receptor (uPAR) was previously identified at protein level and on cell surface. Recently, a dimeric form of mouse uPAR isoform 2 was proposed to induce kidney disease. Here, we report the crystal structure of human uPAR dimer at 2.96 Å. The structure reveals enormous conformational changes of the dimer compared to the monomeric structure: D1 of uPAR opens up into a large expanded ring that captures a ß-hairpin loop of a neighboring uPAR to form an expanded ß-sheet, leading to an elongated, highly intertwined dimeric uPAR. Based on the structure, we identify E49P as a mutation promoting dimer formation. The mutation increases receptor binding to the amino terminal fragment of its primary ligand uPA, induces the receptor to distribute to the basal membrane, promotes cell proliferation, and alters cell morphology via ß1 integrin signaling. These results reveal the structural basis for uPAR dimerization, its effect on cellular functions, and provide a basis to further study this multifunctional receptor.

A versatile insertion point on albumin to accommodate peptides and maintain their activities.

Genetic fusion of human serum albumin to peptides is an important strategy to enhance the plasma half-life of the peptide. An inherent challenge of such method is the reduction of specific activity of the cargo peptides upon connecting at N- or C-termini of albumin. Here, we report a finding that residue 363-364 of albumin can be inserted with a peptide while maintaining the peptide activities. We insert a peptide inhibitor into this site, and at the N-terminus of albumin, for comparison. The chimeric protein displays potent inhibition (IC50 value of 30 nM) to its target (uPAR), but not the N-terminally fused construct. We also study the chimera of HSA with a cyclic peptide inhibitor of murine urokinase-type plasminogen activator grafted at either the internal site or the N-terminus. The internally peptide-grafted protein possesses a much more potent inhibition compared to the N-terminally located fusion (IC50 value of 32 nM vs 19 µM). We further demonstrate that such internal fusion does not affect albumin expression, secondary structure, and inherent drug binding activity. Thus, this work identifies a versatile insertion point inside albumin for maintaining fusion peptide activity, and opens a new avenue to expand the applications of albumin fusion technology.