Prevention of Metastasis by Suppression of Stemness Genes Using a Combination of microRNAs.

We propose an original strategy for metastasis prevention using a combination of three microRNAs that blocks the dedifferentiation of cancer cells in a metastatic niche owing to the downregulation of stemness genes. Transcriptome microarray analysis was applied to identify the effects of a mixture of microRNAs on the pattern of differentially expressed genes in human breast cancer cell lines. Treatment of differentiated CD44- cancer cells with the microRNA mixture inhibited their ability to form mammospheres in vitro. The combination of these three microRNAs encapsulated into lipid nanoparticles prevented lung metastasis in a mouse model of spontaneous metastasis. The mixture of three microRNAs (miR-195-5p/miR-520a/miR-630) holds promise for the development of an antimetastatic therapeutic that blocks tumor cell dedifferentiation, which occurs at secondary tumor sites and determines the transition of micrometastases to macrometastases.

The Determination of the Biocompatibility of New Compositional Materials, including Carbamide-Containing Heterocycles of Anti-Adhesion Agents for Abdominal Surgery.

Due to traumatic injuries, including those from surgical procedures, adhesions occur in over 50% of cases, necessitating exclusive surgical intervention for treatment. However, preventive measures can be implemented during abdominal organ surgeries. These measures involve creating a barrier around internal organs to forestall adhesion formation in the postoperative phase. Yet, the effectiveness of the artificial barrier relies on considerations of its biocompatibility and the avoidance of adverse effects on the body. This study explores the biocompatibility aspects, encompassing hemocompatibility, cytotoxicity, and antibacterial and antioxidant activities, as well as the adhesion of blood serum proteins and macrophages to the surface of new composite film materials. The materials, derived from the sodium salt of carboxymethylcellulose modified by glycoluril and allantoin, were investigated. The research reveals that film materials with a heterocyclic fragment exhibit biocompatibility comparable to commercially used samples in surgery. Notably, film samples developed with glycoluril outperform the effects of commercial samples in certain aspects.

Biosynthesis of Functional Silver Nanoparticles Using Callus and Hairy Root Cultures of Aristolochia manshuriensis.

This study delves into the novel utilization of Aristolochia manshuriensis cultured cells for extracellular silver nanoparticles (AgNPs) synthesis without the need for additional substances. The presence of elemental silver has been verified using energy-dispersive X-ray spectroscopy, while distinct surface plasmon resonance peaks were revealed by UV-Vis spectra. Transmission and scanning electron microscopy indicated that the AgNPs, ranging in size from 10 to 40 nm, exhibited a spherical morphology. Fourier-transform infrared analysis validated the abilty of A. manshuriensis extract components to serve as both reducing and capping agents for metal ions. In the context of cytotoxicity on embryonic fibroblast (NIH 3T3) and mouse neuroblastoma (N2A) cells, AgNPs demonstrated varying effects. Specifically, nanoparticles derived from callus cultures exhibited an IC50 of 2.8 µg/mL, effectively inhibiting N2A growth, whereas AgNPs sourced from hairy roots only achieved this only at concentrations of 50 µg/mL and above. Notably, all studied AgNPs' treatment-induced cytotoxicity in fibroblast cells, yielding IC50 values ranging from 7.2 to 36.3 µg/mL. Furthermore, the findings unveiled the efficacy of the synthesized AgNPs against pathogenic microorganisms impacting both plants and animals, including Agrobacterium rhizogenes, A. tumefaciens, Bacillus subtilis, and Escherichia coli. These findings underscore the effectiveness of biotechnological methodologies in offering advanced and enhanced green nanotechnology alternatives for generating nanoparticles with applications in combating cancer and infectious disorders.

Metabolic Homeostasis in Chronic Helminth Infection Is Sustained by Organ-Specific Metabolic Rewiring.

Opisthorchiasis, is a hepatobiliary disease caused by flukes of the trematode family Opisthorchiidae. A chronic form of the disease implies a prolonged coexistence of a host and the parasite. The pathological changes inflicted by the worm to the host's hepatobiliary system are well documented. Yet, the response to the infection also triggers a deep remodeling of the host systemic metabolism reaching a new homeostasis and affecting the organs beyond the worm location. Understanding the metabolic alternation in chronic opisthorchiasis, could help us to pinpoint pathways that underlie infection opening possibilities for the development of more selective treatment strategies. Here, with this report we apply an integrative, multicompartment metabolomics analysis, using multiple biofluids, stool samples and tissue extracts to describe metabolic changes in Opisthorchis felineus infected animals at the chronic stage. We show that the shift in lipid metabolism in the serum, a depletion of the amino acids pool, an alteration of the ketogenic pathways in the jejunum and a suppressed metabolic activity of the spleen are the key features of the metabolic host adaptation at the chronic stage of O. felineus infection. We describe this combination of the metabolic changes as a "metabolically mediated immunosuppressive status of organism" which develops during a chronic infection. This status in combination with other factors (e.g., parasite-derived immunomodulators) might increase risk of infection-related malignancy.

Plasma metabolomics of the time resolved response to Opisthorchis felineus infection in an animal model (golden hamster, Mesocricetus auratus).

BACKGROUND: Opisthorchiasis is a hepatobiliary disease caused by flukes of the trematode family Opisthorchiidae. Opisthorchiasis can lead to severe hepatobiliary morbidity and is classified as a carcinogenic agent. Here we investigate the time-resolved metabolic response to Opisthorchis felineus infection in an animal model. METHODOLOGY: Thirty golden hamsters were divided in three groups: severe infection (50 metacercariae/hamster), mild infection (15 metacercariae/hamster) and uninfected (vehicle-PBS) groups. Each group consisted of equal number of male and female animals. Plasma samples were collected one day before the infection and then every two weeks up to week 22 after infection. The samples were subjected to 1H Nuclear Magnetic Resonance (NMR) spectroscopy and multivariate statistical modelling. PRINCIPAL FINDINGS: The time-resolved study of the metabolic response to Opisthorchis infection in plasma in the main lines agrees with our previous report on urine data. The response reaches its peak around the 4th week of infection and stabilizes after the 10th week. Yet, unlike the urinary data there is no strong effect of the gender in the data and the intensity of infection is presented in the first two principal components of the PCA model. The main trends of the metabolic response to the infection in blood plasma are the transient depletion of essential amino acids and an increase in lipoprotein and cholesterol concentrations. CONCLUSIONS: The time resolved metabolic signature of Opisthorchis infection in the hamster's plasma shows a coherent shift in amino acids and lipid metabolism. Our work provides insight into the metabolic basis of the host response on the helminth infection.

pH-triggered delivery of magnetic nanoparticles depends on tumor volume.

Nowadays there is growing recognition of the fact that biological systems have a greater impact on nanoparticle target delivery in tumors than nanoparticle design. Here we investigate the targeted delivery of Fe3O4 magnetic nanoparticles conjugated with pH-low-insertion peptide (MNP-pHLIP) on orthotopically induced MDA-MB-231 human breast carcinoma xenografts of varying volumes as a model of cancer progression. Using in vivo magnetic resonance imaging and subsequent determination of iron content in tumor samples by inductively coupled plasma atomic emission spectroscopy we found that MNP-pHLIP accumulation depends on tumor volume. Transmission electron microscopy, histological analysis and immunohistochemical staining of tumor samples suggest that blood vessel distribution is the key factor in determining the success of the accumulation of nanoparticles in tumors.

Hemozoin From the Liver Fluke, Opisthorchis felineus, Modulates Dendritic Cell Responses in Bronchial Asthma Patients.

Aims: There is a general, inverse relationship between helminth infection and allergic diseases including bronchial asthma (BA). Proteins and other mediators released from parasitic worms exert cogent downmodulation of atopic and other allergic reactivity. We investigated the immune activities of an immortalized murine dendritic cell (mDC) line (JAWSII) and of primary human dendritic cells (hDCs) collected from study participants with and without BA after Opisthorchis felineus hemozoin (OfHz) treatment. Methods and Results: in vitro, expression of lymphocyte-activating factors-T helper 1 (Th1) induction and anti-inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1ß), IL-10, and IL-12ß-increased significantly in mDCs pulsed with OfHz. In parallel, primary dendritic cells (hDC) from cases clinically diagnosed with BA along with healthy controls were exposed ex vivo to OfHz in combination with lipopolysaccharide (LPS). Whereas no significant change in the cellular maturation markers, CD83, CD86, and CD40, was apparent in BA vs. healthy hDC, pulsing hDC from BA with OfHz with LPS induced significant increases in expression of IL-10 and IL-12ß, although not of TNF-α or tumor growth factor-beta (TGF-ß). Conclusions: Liver fluke hemozoin OfHz stimulated production of Th1 inducer and anti-inflammatory cytokines IL-10 and IL-12ß from BA-hDC pulsed with OfHz, an outcome that enhances our understanding of the mechanisms whereby opisthorchiasis contributes to protection against the atopic disease in liver fluke infection-endemic regions.

Effect of alpha-lactalbumin and lactoferrin oleic acid complexes on chromatin structural organization.

This work focuses on the study of multimeric alpha-lactalbumin oleic acid and lactoferrin oleic acid complexes. The purpose of the research is to study possible mechanisms involved in their pro-apoptotic activities, as seen in some tumor cell cultures. Complexes featuring oleic acid (OA) with human alpha-lactalbumin (hAl) or with bovine alpha-lactalbumin (bAl), and human lactoferrin (hLf) were investigated using small-angle neutron scattering (SANS). It was shown that while alpha-lactalbumin protein complexes were formed on the surface of polydisperse OA micelles, the lactoferrin complexes comprised a monodisperse system of nanoscale particles. Both hAl and hLf complexes appeared to interact with the chromatin of isolated nuclei affecting chromatin structural organization. The possible roles of these processes in the specific anti-tumor activity of these complexes are discussed.

Imbalance in the glutathione system in Opisthorchis felineus infected liver promotes hepatic fibrosis.

Although data on oxidative stress during liver fluke infection have been previously presented, a comprehensive study of the glutathione system that plays a crucial role in scavenging of reactive oxygen species (ROS) and detoxification of primary and secondary oxidation products has not been addressed yet. In the present study, the hepatic glutathione system was investigated in a hamster model of experimental opisthorchiasis infection. It was shown that chronic oxidative stress in an Opisthorchis felineus infected liver, evidenced by abundant hydroperoxide accumulation, leads to strong imbalance in the hepatic glutathione system, namely the depletion of reduced form of glutathione (GSH), lowering of the GSH/GSSG ratio, and a decrease in the glutathione peroxidase and glyoxalase 1 activity. O. felineus infection provokes hepatocellular damage that results in the progression of liver fibrosis, accompanied by an increase in collagen deposition in the hepatic tissue. Modulation of hepatic GSH levels in the O. felineus infected liver through N-acetylcysteine (NAC) or l-buthionine-S, R-sulfoxinine (BSO) treatments lead to changes in expression and activity of glutathione S-transferase and glyoxalase I as well as markedly decreases or increases collagen content in the O. felineus infected liver and the severity of liver fibrosis, respectively. Thus, the glutathione system can be considered as a target for liver protection from O. felineus-induced injury.

PMIDA-Modified Fe3O4 Magnetic Nanoparticles: Synthesis and Application for Liver MRI.

The surface modification of Fe3O4-based magnetic nanoparticles (MNPs) with N-(phosphonomethyl)iminodiacetic acid (PMIDA) was studied, and the possibility of their use as magnetic resonance imaging contrast agents was shown. The effect of the added PMIDA amount, the reaction temperature and time on the degree of immobilization of this reagent on MNPs, and the hydrodynamic characteristics of their aqueous colloidal solutions have been systematically investigated for the first time. It has been shown that the optimum condition for the modification of MNPs is the reaction at 40 °C with an equimolar amount of PMIDA for 3.5 h. The modified MNPs were characterized by X-ray diffraction, transmission electron microscopy, Fourier transform infrared spectroscopy, thermogravimetric, and CHN elemental analyses. The dependence of the hydrodynamic characteristics of the MNP colloidal solutions on the concentration and pH of the medium was studied by the dynamic light scattering method. On the basis of the obtained data, we can assume that the PMIDA molecules are fixed on the surface of the MNPs as a monomolecular layer. The modified MNPs had good colloidal stability and high magnetic properties. The calculated relaxivities r2 and r1 were 341 and 102 mmol-1 s-1, respectively. The possibility of using colloidal solutions of PMIDA-modified MNPs as a T2 contrast agent for liver studies in vivo (at a dose of 0.6 mg kg-1) was demonstrated for the first time.

Green synthesis of silver nanoparticles using transgenic Nicotiana tabacum callus culture expressing silicatein gene from marine sponge Latrunculia oparinae.

In the present investigation, transgenic tobacco callus cultures and plants overexpressing the silicatein gene LoSilA1 from marine sponge Latrunculia oparinae were obtained and their bioreduction behaviour for the synthesis of silver nanoparticles (AgNPs) was studied. Synthesized nanoparticles were characterized using UV-visible spectroscopy, Fourier transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), atomic flame electron microscopy (AFM) and nanoparticle tracking analysis (NTA). Our measurements showed that the reduction of silver nitrate produced spherical AgNPs with diameters in the range of 12-80 nm. The results of XRD analysis proved the crystal nature of the obtained AgNPs. FTIR analysis indicated that particles are reduced and stabilized in solution by the capping agent, which is likely to be proteins present in the callus extract. Interestingly, the reduction potential of LoSiLA1-transgenic callus line was increased three-fold compared with the empty vector-transformed calli. The synthesized AgNPs were found to exhibit strong antibacterial activity against Escherichia coli and Agrobacterium rhizogenes. The present study reports the first evidence for using genetic engineering for activation of the reduction potential of plant cells for synthesis of biocidal AgNPs.

Exploratory metabolomics study of the experimental opisthorchiasis in a laboratory animal model (golden hamster, Mesocricetus auratus).

BACKGROUND: Opisthorchiasis is a parasitic infection caused by the liver flukes of the Opisthorchiidae family. Both experimental and epidemiological data strongly support a role of these parasites in the etiology of the hepatobiliary pathologies and an increased risk of intrahepatic cholangiocarcinoma. Understanding a functional link between the infection and hepatobiliary pathologies requires a detailed description a host-parasite interaction on different levels of biological regulation including the metabolic response on the infection. The last one, however, remains practically undocumented. Here we are describing a host response on Opisthorchiidae infection using a metabolomics approach and present the first exploratory metabolomics study of an experimental model of O. felineus infection. METHODOLOGY AND PRINCIPAL FINDINGS: We conducted a Nuclear Magnetic Resonance (NMR) based longitudinal metabolomics study involving a cohort of 30 animals with two degrees of infection and a control group. An exploratory analysis shows that the most noticeable trend (30% of total variance) in the data was related to the gender differences. Therefore further analysis was done of each gender group separately applying a multivariate extension of the ANOVA-ASCA (ANOVA simultaneous component analysis). We show that in the males the infection specific time trends are present in the main component (43.5% variance), while in the females it is presented only in the second component and covers 24% of the variance. We have selected and annotated 24 metabolites associated with the observed effects and provided a physiological interpretation of the findings. CONCLUSIONS: The first exploratory metabolomics study an experimental model of O. felineus infection is presented. Our data show that at early stage of infection a response of an organism unfolds in a gender specific manner. Also main physiological mechanisms affected appear rather nonspecific (a status of the metabolic stress) the data provides a set of the hypothesis for a search of the more specific metabolic markers of the Opisthorchiidae infection.

Magnetic resonance imaging and spectroscopy for differential assessment of liver abnormalities induced by Opisthorchis felineus in an animal model.

BACKGROUND: European liver fluke Opisthorchis felineus, causing opisthorchiasis disease, is widespread in Russia, Ukraine, Kazakhstan and sporadically detected in the EU countries. O. felineus infection leads to hepatobiliary pathological changes, cholangitis, fibrosis and, in severe cases, malignant transformation of bile ducts. Due to absence of specific symptoms, the infection is frequently neglected for a long period. The association of opisthorchiasis with almost incurable bile duct cancer and rising international migration of people that increases the risk of the parasitic etiology of liver fibrosis in non-endemic regions determine high demand for development of approaches to opisthorchiasis detection. METHODOLOGY/PRINCIPAL FINDINGS: In vivo magnetic resonance imaging and spectroscopy (MRI and MRS) were applied for differential assessment of hepatic abnormalities induced by O. felineus in an experimental animal model. Correlations of the MR-findings with the histological data as well as the data of the biochemical analysis of liver tissue were found. MRI provides valuable information about the severity of liver impairments induced by opisthorchiasis. An MR image of O. felineus infected liver has a characteristic pattern that differs from that of closely related liver fluke infections. 1H and 31P MRS in combination with biochemical analysis data showed that O. felineus infection disturbed hepatic metabolism of the host, which was accompanied by cholesterol accumulation in the liver. CONCLUSIONS: A non-invasive approach based on the magnetic resonance technique is very advantageous and may be successfully used not only for diagnosing and evaluating liver damage induced by O. felineus, but also for investigating metabolic changes arising in the infected organ. Since damages induced by the liver fluke take place in different liver lobes, MRI has the potential to overcome liver biopsy sampling variability that limits predictive validity of biopsy analysis for staging liver fluke-induced fibrosis.

3-Aminopropylsilane-modified iron oxide nanoparticles for contrast-enhanced magnetic resonance imaging of liver lesions induced by Opisthorchis felineus.

PURPOSE: Liver fluke causes severe liver damage in an infected human. However, the infection often remains neglected due to the lack of pathognomonic signs. Nanoparticle-enhanced magnetic resonance imaging (MRI) offers a promising technique for detecting liver lesions induced by parasites. MATERIALS AND METHODS: Surface modification of iron oxide nanoparticles produced by coprecipitation from a solution of Fe3+ and Fe2+ salts using 3-aminopropylsilane (APS) was carried out. The APS-modified nanoparticles were characterized by transmission electron microscopy, fourier transform infrared spectroscopy, and thermogravimetric analysis. Magnetic resonance properties of MNPs were investigated in vitro and in vivo. RESULTS: The amount of APS grafted on the surface of nanoparticles (0.60±0.06 mmol g-1) was calculated based on elemental analysis and infrared spectroscopy data. According to transmission electron microscopy data, there were no essential changes in the structure of nanoparticles during the modification. The APS-modified nanoparticles exhibit high magnetic properties; the calculated relaxivity r2 was 271 mmol-1 s-1. To obtain suspension with optimal hydrodynamic characteristics, amino groups on the surface of nanoparticles were converted into an ionic form with HCl. Cellular uptake of modified nanoparticles by rat hepatoma cells and human monocytes in vitro was 74.1±4.5 and 10.0±3.7 pg [Fe] per cell, respectively. Low cytotoxicity of the nanoparticles was confirmed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and Annexin V/7-aminoactinomycin D flow cytometry assays. For the first time, magnetic nanoparticles were applied for contrast-enhanced MRI of liver lesions induced by Opisthorchis felineus. CONCLUSION: The synthesized APS-modified iron oxide nanoparticles showed high efficiency as an MRI contrast agent for the evaluation of opisthorchiasis-related liver damage.

Ascorbic acid glucoside reduces neurotoxicity and glutathione depletion in mouse brain induced by nitrotriazole radiosensitazer.

AIM: To investigate the potential of the anti-oxidant ascorbic acid glucoside (AA-2G) to modulate neurotoxicity induced by high doses of nitrotriazole radiosensitizer. MATERIALS AND METHODS: Male and female C56Bl/6xCBA hybrid mice aged 8-14 weeks (weight 18-24 g) were used. Nitrotriazole drug radiosensitizer sanazole at a high dose of 2, 1 g/kg was per os administered to induce neurotoxicity at mice. Ascorbic acid glucoside was given 30 min before the sanazole administration. Serum ascorbic acid, brain glutathione level, as well as behavioral performance using open field apparatus were measured. RESULTS: Administration of high (non-therapeutic) doses of the nitrotriazole drug sanazole results in neurotoxicity in mice as evidenced from behavioral performance, emotional activity and depletion of the cellular antioxidant, glutathione, in the brain. The serum levels of ascorbic acid was also found reduced in high dose sanazole treated animals. Per os administration of ascorbic acid glucoside significantly reduced the neurotoxicity. This effect was associated with the prevention of glutathione depletion in mouse brain and restoring the ascorbic acid level in serum. CONCLUSION: Administration of ascorbic acid glucoside, but not ascorbic acid, before sanazole administration protected from sanazole-induced neurotoxicity by preventing the decrease in the brain reduced glutathione level and providing high level of ascorbic acid in plasma.