Cu-doped Fe3O4 nanoparticles for efficient detoxification of epsilon toxin: Toward substituting magnetically recyclable detoxifying agent for formaldehyde.

This research presents the synthesis and characterization of Cu-doped Fe3O4 (Cu-Fe3O4) nanoparticles as a magnetically recoverable and reusable detoxifying agent for the efficient and long-lasting neutralization of bacterial toxins. The nanoparticles were synthesized using the combustion synthesis method and characterized through SEM, XRD, BET, TGA, and VSM techniques. The detoxification potential of Cu-Fe3O4 was compared with traditional formaldehyde (FA) in detoxifying epsilon toxin (ETx) from Clostridium perfringens Type D, the causative agent of enterotoxemia in ruminants. In vivo residual toxicity tests revealed that Cu-Fe3O4 could detoxify ETx at a concentration of 2.0 mg mL-1 within 4 days at room temperature (RT) and 2 days at 37 °C, outperforming FA (12 and 6 days at RT and 37 °C, respectively). Characterization studies using dynamic light scattering (DLS) and circular dichroism (CD) highlighted lower conformational changes in Cu-Fe3O4-detoxified ETx compared to FA-detoxified ETx. Moreover, Cu-Fe3O4-detoxified ETx exhibited exceptional storage stability at 4 °C and RT for 6 months, maintaining an irreversible structure with no residual toxicity. The particles demonstrated remarkable reusability, with the ability to undergo five continuous detoxification batches. This study provides valuable insights into the development of an efficient and safe detoxifying agent, enabling the production of toxoids with a native-like structure. The magnetically recoverable and reusable nature of Cu-Fe3O4 nanoparticles offers practical advantages for easy recovery and reuse in detoxification reactions.

Optimization and scale-up of Clostridium perfringens type D culture and epsilon-toxin production: Effects of stirring, glucose and pH adjustment.

The effects of some main bacteria culture parameters including mixing rate, glucose (GC) concentration, steps of GC addition, and steps of pH adjustment on both C. perfringens bacteria growth and its epsilon toxin production in a bench-scale 20-L glass carboy were investigated. The optimized mixing rate of 300 rpm, GC concentration of 4 g L-1, and 3-step addition of GC resulted in the bacteria and toxin concentrations of 0.16 g L-1 and 330 ng mL-1, respectively. Also, the induction of a pH shock at the reaction time of 180 min led to the remarkable enhancement of toxin production (367 ng mL-1). Upon applying both optimized conditions for GC addition and pH adjustment, the high toxin concentration of 433 ng mL-1 was obtained. Using the constant mixing rate technique, the process was scaled up to a 1500-L industrial bioreactor, where its performance was close to the bench-scale bioreactor (i.e., toxin concentration of 419 ng mL-1). The results revealed the reliability of this method to economically improve and scale up the bacteria culture process, which can be further used for other microbial fermentations.

Effect of formalin percentage, incubation time and temperature on Clostridium chauvoei culture inactivation and immunogenicity.

OBJECTIVES: In order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied. METHODS: C. chauvoei was cultured with different formalin percentages (0.3, 0.5 or 0.7% V/V), inactivation temperatures (37 °C or room temperature) and incubation times (one or two weeks). Sterility tests were performed and residual formaldehyde and pH were measured. Rabbits were immunized twice with inactivated cultures and sera were used for detection of immune response. RESULTS: In the one-week experiment, 0.5 and 0.7% formalin inactivated the bacteria after one week, and the percentage of 0.3 inactivated after three weeks. The residual formaldehyde at weeks 1 and 8 was not significantly different. In the two-week experiment, cultures treated with 0.3 and 0.5% formalin were inactivated after four weeks, and those with 0.7% formalin were inactivated after three weeks. Residual formaldehyde at week 8 differed significantly from that of week 1. Residual formaldehyde was affected by incubation temperature since it was lower at 37 °C than in room temperature. Also, a significant effect was observed for formalin on pH, as higher formalin contents led to lower pH values of the cultures. ELISA showed the lowest antibody titer achieved by 0.7% formalin group. Antibody titer was not different between 0.3 and 0.5% formalin. CONCLUSIONS: The best condition for inactivation of C. chauvoei was considered as one-week incubation with 0.5% formalin at 37 °C, leading to a high antibody response.

Effect of the Structure of Magnetic Nanocomposite Adsorbents on the Lysozyme Separation Efficiency.

In this study, we prepared various anionic magnetic adsorbents through the carboxyl functionalization of core/shell-structured Fe3O4/SiO2 (FS) particles by either succinic anhydride (FSC), low-molecular-weight (MW 1800) polyacrylic acid (PAA) (FSP1), or high-molecular-weight (MW 100,000) PAA (FSP2), and then, investigated the effect of the structure of adsorbents and operational parameters on their performance for the lysozyme separation. The type and size of functional molecules have significant effects on the surface concentration of functional carboxyl groups onto the adsorbent particles (increase in the order of FSP2 > FSP1 > FSC), and consequently on the adsorption efficiency (AE) (∼100, 98, and 62%, respectively) and adsorption capacity (AC) (∼1000, 980, and 621 mg·g-1, respectively) of the adsorbents. However, the loss of the antibacterial activity of separated lysozyme molecules due to the molecular conformational change increased in the order of FSP2 > FSP1 = FSC, as compared to the free lysozyme. The application of basic buffer solutions for the elution of adsorbed enzyme molecules resulted in more adverse effects on the enzyme activity. The obtained results recommend that FSP1 can be used as a suitable anionic adsorbent for the isolation of positively charged proteins, owing to its high adsorption capacity, excellent reusability, and structural stability, as well as the high purity, structural stability, and activity recovery of the isolated proteins.

STAT3 inactivation suppresses stemness properties in gastric cancer stem cells and promotes Th17 in Treg/Th17 balance.

Signal transducer and activator of transcription 3 (STAT3) has been recognized with dual effects in provision of cancer; either tumor inductive or immune suppressive. Recent findings considering the role of STAT3 in stem cells and cancer stem cell regulation, but its role in gastric cancer stem cells (GCSCs) and modulating the Th17/Treg balance is unknown. In the present study, we aimed to evaluate the role of activated STAT3 in GCSCs and Th17/ Treg cell paradigm. In completion of our previous results, the findings here indicate that gastro-spheroids, as a model of GCSCs, represent higher level of STAT3 activity, up-regulation of TGF-b and VEGF with downregulation of IL-6. On the other hand, treatment of normal naïve T cells with conditioned medium derived from gastro-spheroids promotes T cell differentiation toward cells with a higher level of FOXP3, TGF-b, and IL-10 expression which is indicative of Treg cells. Suppression of STAT3 activation in cancer cells by using Stattic small molecule treatment, decreases stemness features (i.e. spheroid formation and integrity, stemness gene expression and in vivo tumorigenicity capacity) and downregulates TGF-b in the cancer cells. Furthermore, co-culture of conditioned medium of STAT3 inhibited cancer cells with normal PBMCs leads to reduction in the percentage of Treg accompanied with increase of Th17 cells with a decrease in the secretion of TGF-b and increase in IFN-γ in T cells under differentiation. Therefore, targeting the STAT3 pathway in cancer cells seems to control the tumor formation and also impact on immune cells shifting to antitumor Th17 population.

Evaluation of cellular and humoral immune response in chickens immunized with flagellin-adjuvanted inactivated newcastle disease virus.

The aim of the present study was to evaluate the potential effect of flagellin as adjuvant in Newcastle disease virus (NDV) vaccine on the cellular and humoral immunity in chickens. Fifty-six specific pathogen-free chickens were assigned to seven groups of eight chickens and immunized twice with a two-week interval, intramuscularly. Group 1, received phosphate buffered saline as control (C), groups 2, 3, 4, 5, 6 and 7 were immunized with inactivated NDV [Ag], Ag + full FliC protein [AgF], Ag + truncated Flic protein [AgT], Ag + native Flic protein [AgN], commercial NDV vaccine [Vac] and Vac + N [VacN], respectively. After 45 days, spleen and bursa of Fabricius samples were collected and analyzed by flow cytometry and responses in control/vaccinated chickens were studied by immunophenotyping. Humoral response was also, evaluated by ELISA during the experiment. Results showed that immunized chickens with Ag + flagellin proteins had significantly higher frequency of circulating CD3+, CD4+ and CD8+ T cells in bursa of Fabricius in AgF, AgT and AgN, respectively, compared with other groups. Similar results were observed for spleen; however, the highest frequency of circulating CD3+, CD4+ and CD8+ T cells belonged to AgT and AgF, respectively. ELISA results showed that all flagellin-adjuvanted groups had higher antibody titers than other groups with the highest antibody response in VacN. It can be concluded that flagellin may induce both humoral and cellular immune responses against ND and is suggested for use as an efficient adjuvant.

pH Shock-promoted lysozyme corona for efficient pathogenic infections treatment: Effects of surface chemistry of mesoporous silica nanoparticles and loading method.

The emergence of antibiotic resistant bacteria because of the antibiotics abusement was the motivation to develop the effective alternatives to traditional antibiotics. Hence, various lysozyme corona were prepared through the physical and covalent attachment of lysozyme molecules onto either the bare or carboxyl-functionalized mesoporous silica particles. The prepared samples were characterized by STEM, TGA/DTA, zeta potential, FTIR, UV-vis and CD spectroscopic methods. All the prepared lysozyme-coated particles exhibited an efficient antibacterial activity against Listeria monocytogenes, as a case study, in vitro with no cytotoxicity. The minimal inhibition concentration (MIC) of the lysozyme-physically adsorbed bare and carboxyl-functionalized mesoporous silica nanoparticles (L-MS and L-ads-CMS, respectively) and the lysozyme-covalently attached carboxyl-functionalized MS particles (L-cov-CMS) was 2, 5.3 and 1.7 folds lower than that of the free lysozyme, respectively. Additionally, for the first time, it was reported that the pretreatment of lysozyme corona of L-ads-CMS through inducing a pH-shock can lead to the enhancement of antibacterial properties thereof. This behavior was associated to the controlled release of the immobilized lysozyme molecules and their conformational stability. These natural antibacterial lysozyme-coated silica nanoparticles showing the "pH-shock enhanced activity" could be of utmost interest for design of the highly active enzyme-modified nanoparticles.

Study of Molecular Conformation and Activity-Related Properties of Lipase Immobilized onto Core-Shell Structured Polyacrylic Acid-Coated Magnetic Silica Nanocomposite Particles.

A facile approach for the preparation of core-shell structured poly(acrylic acid) (PAA)-coated Fe3O4 cluster@SiO2 nanocomposite particles as the support materials for the lipase immobilization is reported. Low- or high-molecular-weight (1800 and 100,000, respectively) PAA molecules were covalently attached onto the surface of amine-functionalized magnetic silica nanoacomposite particles. The successful preparation of particles were verified by scanning transmission electron microscopy (STEM), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), thermogravimetric analysis (TGA), zeta potential measurement, and Fourier-transform infrared (FTIR) techniques. Once lipase is covalently immobilized onto the particles with an average diameter of 210 ± 50 nm, resulting from high binding sites concentrations on the low- and high-molecular-weight PAA-coated particles, high lipase immobilization efficiencies (86.2% and 89.9%, respectively), and loading capacities (786 and 816 mg g(-1), respectively) are obtained. Results from circular dichroism (CD) analysis and catalytic activity tests reveal an increase in the ß-sheet content of lipase molecules upon immobilization, along with an enhancement in their activities and stabilities. The lipases immobilized onto the low- and high-molecular-weight PAA-coated particles show maximum activities at 55 and 50 °C, respectively, which are ∼28% and ∼15% higher than that of the free lipase at its own optimum temperature (40 °C), respectively. The immobilized lipases exhibit excellent performance at broader temperature and pH ranges and high thermal and storage stabilities, as well as superior reusability. These prepared magnetic nanocomposite particles can be offered as suitable support materials for efficient immobilization of enzymes and improvement of the immobilized enzymes properties.