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The aim of the research was to compare the impact of nano- and micro-sized-zinc on the kinetics of changes in the level of 3-methyladenine, 7-methylguanine, 7-methylguanosine, O-methylguanosine, 1-methyladenosine, N6-methyl-2’-deoxyguanosine in urine of rats with breast cancer. Female Sprague-Dawley rats divided into 3 groups were used in the study. Animals were fed only a control diet or diets supplemented with the nano and micro-sized zinc particles. To induce the mammary cancer (adenocarcinoma), rats were treated with 7,12-dimethylbenz[a]anthracene (DMBA). Modified nucleosides were determined by a validated high performance liquid chromatography coupled to mass spectrometry method. In the first stage of investigations a synergistic activity of nanosized Zn with DMBA on the growth of the neoplastic process was found. During that time a statistically significant increase in the levels of all six examined markers in the rats’ urine was observed. However, as the experiment continued, the supplementation with nanosized zinc caused inhibition of tumour growth, being followed by regression and remission of tumours, as well as, a statistically significant systematic reduction of the levels of methyl derivatives in the urine. Biopsy images indicated grade 1 tumours with multiple inflammatory infiltrates in the group treated with zinc nanoparticles, whereas, in the other groups, moderately-differentiated grade 2 adenocarcinoma was identified. It was found that the biological activity of zinc depends on the size of applied particles, as the treatment with zinc microparticles has not had much effect on cancer progression.

The purpose of this study was to evaluate the effect of genistein in nano, micro, and macro forms on the intensity of the DMBA-induced tumor process in rats and to understand the mechanisms of this action. The effect of genistein supplementation on the content of selected eicosanoids (HETEs, HODE, and HEPE) in the serum of rats was evaluated. The levels and expression of genes encoding various pro-inflammatory cytokines (IL-1, IL-6) and MMP-9 in the blood of rats were also investigated. The biological material for the study was blood obtained from female rats of the Sprague Dawley strain (n = 32). The animals were randomly divided into four groups: animals without supplementation, and animals supplemented at a dose of 0.2 mg/kg b.w. (0.1 mg/mL) with macro, micro (587 ± 83 nm), or nano (92 ± 41 nm) genistein. To induce mammary neoplasia (adenocarcinoma), rats were given 7,12-dimethyl-1,2-benz[a]anthracene (DMBA). The content of selected eicosanoids was determined by liquid chromatography with UV detection. An immunoenzymatic method was used to determine the content of cytokines and MMP-9. The expression of the IL-6, IL-1beta, and MMP-9 genes was determined with quantitative real-time PCR (qRT-PCR) using TaqMan probes. Based on the study, it was shown that supplementation of animals with genistein in macro, micro, and nano forms increased the intensity of the tumor process in rats. It was shown that the content of 12-HEPE, HODE, and 12-HETE in the serum of genistein-supplemented rats was statistically significantly lower with respect to the content of the aforementioned markers in the serum of rats receiving only a standard diet, devoid of supplementation. It was found that animals supplemented with nano-, micro-, and macrogenistein had higher levels of metalloproteinase-9, MMP-9, compared to animals without supplementation. There was a significant increase in MMP-9 gene expression in the blood of macrogenistein-supplemented animals, relative to the other groups of rats. On the basis of the study, it was shown that supplementation of animals with nano-, micro-, and macrogenistein had an effect on the development of the tumor process. Dietary supplementation with genistein significantly decreased the level of selected eicosanoids, which may have significant impacts on cancer development and progression.

Gelatin-based formulations are utilized in neurosurgical procedures, with Medisponge® serving as an illustration of a secure and biocompatible hemostatic formulation. Noteworthy are combined hemostatic products that integrate pharmacological agents with gelatin. Gelatin matrices, which host biologically active substances, provide a platform for a variety of molecules. Biopolymers function as carriers for chemicals and genes, a facet particularly pertinent in brain cancer therapy, as gene therapy complement conventional approaches. The registration of Zolgensma underscores the efficacy of rAAV vectors in therapeutic gene delivery to the CNS. rAAVs, renowned for their safety, stability, and neuron-targeting capabilities, predominate in CNS gene therapy studies. The effectiveness of rAAV vector therapy varies based on the serotype and administration route. Local gene therapy employing hydrogel (e.g., post-tumor resection) enables the circumvention of the blood-brain barrier and restricts formulation diffusion. This study formulates gelatin rAAV gene formulations and evaluates vector transduction potential. Transduction efficiency was assessed using ex vivo mouse brains and in vitro cancer cell lines. In vitro, the transduction of rAAV vectors in gelatin matrices was quantified through qPCR, measuring the itr and Gfp expression. rAAVDJ and rAAV2 demonstrated superior transduction in ex vivo and in vitro models. Among the cell lines tested (Hs683, B16-F10, NIH:OVCAR-3), gelatin matrix F1 exhibited selective transduction, particularly with Hs683 human glioma cells, surpassing the performance Medisponge®. This research highlights the exploration of local brain cancer therapy, emphasizing the potential of gelatin as an rAAV vector carrier for gene therapy. The functional transduction activity of gelatin rAAV formulations is demonstrated., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Masson SAS.)

The aim of the study was to evaluate the effect of selected polyphenolic compounds: epicatechin, apigenin, and naringenin, administered separately or in combination with zinc (Zn), on the growth and development of the neoplastic process induced by 7,12-dimethylbenz[a]anthracene (DMBA) in rats. The impact of supplementation with the above-mentioned compounds on the content of modified derivatives: 1-methyladenosine, N6-methyl-2'-deoxyadenosine, O-methylguanosine, 7-methylguanine, 3-methyladenine, 1-methylguanine, 2-amino-6,8-dihydroxypurine, and 8-hydroxy-2'-deoxyguanosine in the urine of rats with mammary cancer was also assessed. Female Sprague-Dawley rats divided into 7 groups were used in the study: animals without supplementation and animals supplemented with apigenin, epicatechin, and naringenin separately or in combination with zinc. To induce mammary cancer, rats were treated with DMBA. Modified derivatives were determined by a validated high-performance liquid chromatography coupled to mass spectrometry method. Based on the obtained results, it can be said that supplementation of the animals with naringenin inhibits the development and progression of the neoplastic process in rats treated with 7,12-dimethylbenzanthracene. Neoplastic tumors were found in only 2 of 8 rats (incidence: 25%) and were considered to be at most grade 1 malignancy. The first palpable tumors in the group of animals receiving naringenin appeared two-three weeks later when compared to other groups. The combination of zinc with flavonoids (apigenin, epicatechin, and naringenin) seems to stimulate the process of carcinogenesis. The level of N6-methyl-2'-deoxyadenosine and 3-methyladenine in the urine of rats was statistically significantly higher in the groups supplemented with apigenin, epicatechin, and naringenin administered in combination with Zn than in the groups receiving only polyphenolic compounds. In conclusion, supplementation of rats with selected flavonoids administered separately or in combination with Zn has an impact on the development of neoplasms and the level of modified nucleosides in the urine of rats with breast cancer. Our results raise the question of whether simultaneous diet supplementation with more than one anti-cancer agent may reduce/stimulate the risk of carcinogenesis.

(1) Background: Transcriptomic and proteomic studies provide a wealth of new genes potentially involved in red blood cell (RBC) maturation or implicated in the pathogenesis of anemias, necessitating validation of candidate genes in vivo; (2) Methods: We inactivated one such candidate, transmembrane and coiled-coil domain 2 ( Tmcc2 ) in mice, and analyzed the erythropoietic phenotype by light microscopy, transmission electron microscopy (TEM), and flow cytometry of erythrocytes and erythroid precursors; (3) Results: Tmcc2 -/- pups presented pallor and reduced body weight due to the profound neonatal macrocytic anemia with numerous nucleated RBCs (nRBCs) and occasional multinucleated RBCs. Tmcc2 -/- nRBCs had cytoplasmic intrusions into the nucleus and double membranes. Significantly fewer erythroid cells were enucleated. Adult knockouts were normocytic, mildly polycythemic, with active extramedullary erythropoiesis in the spleen. Altered relative content of different stage CD71 + TER119 + erythroid precursors in the bone marrow indicated a severe defect of erythroid maturation at the polychromatic to orthochromatic transition stage; (4) Conclusions: Tmcc2 is required for normal erythropoiesis in mice. While several phenotypic features resemble congenital dyserythropoietic anemias (CDA) types II, III, and IV, the involvement of TMCC2 in the pathogenesis of CDA in humans remains to be determined.

This study aimed to evaluate, for the first time, implantable, biodegradable fiducial markers (FMs), which were designed for bimodal, near-infrared fluorescence-based (NIRF) and X-ray-based imaging. The developed FMs had poly(l-lactide- co -caprolactone)-based core-shell structures made of radiopaque (core) and fluorescent (shell) composites with a poly(l-lactide- co -caprolactone) matrix. The approved for human use contrast agents were utilized as fillers. Indocyanine green was applied to the shell material, whereas in the core materials, iohexol and barium sulfate were compared. Moreover, the possibility of tailoring the stability of the properties of the core materials by the addition of hydroxyapatite (HAp) was examined. The performed in situ (porcine tissue) and in vivo experiment (rat model) confirmed that the developed FMs possessed pronounced contrasting properties in NIRF and X-ray imaging. The presence of HAp improved the radiopacity of FMs at the initial state. It was also proved that, in iohexol-containing FMs, the presence of HAp slightly decreased the stability of contrasting properties, while in BaSO 4 -containing ones, changes were less pronounced. A comprehensive material analysis explaining the differences in the stability of the contrasting properties was also presented. The tissue response around the FMs with composite cores was comparable to that of the FMs with a pristine polymeric core. The developed composite FMs did not cause serious adverse effects on the surrounding tissues even when irradiated in vivo . The developed FMs ensured good visibility for NIRF image-supported tumor surgery and the following X-ray image-guided radiotherapy. Moreover, this study replenishes a scanty report regarding similar biodegradable composite materials with a high potential for application.

Experimental allergic encephalomyelitis (EAE) is the animal model of multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (AHSCT) has recently been recognized as the standard treatment for MS. The aim of our experiment was to investigate the effect of AHSCT with the addition of low-dose post-transplantation cyclophosphamide (Cy) on EAE in rats. Low dose post-transplantation Cy is used in haploidentical HSCT to reduce the risk of graft versus host disease. We hypothesized that it could bring additional benefit in autologous HSCT in autoimmune diseases. Rats with evoked EAE were treated with high dose (125 mg/kg) Cy, followed by AHSCT or high dose (125 mg/kg) Cy followed by AHSCT followed by low dose (20 mg/kg) Cy in two-time schedules-with the therapy applied during the pre-symptomatic or symptomatic phase of the disease. Both AHSCT and AHSCT with post-transplantation Cy in accordance with both time schedules reduce the intensity of the inflammatory response in the CNS, in comparison with non-treated EAE rats. The reduction of clinical symptoms was present in all AHSCT treatment protocols, however, it was significantly stronger when post-transplantation Cy was given during the symptomatic phase of the disease. AHSCT with the addition of post HSCT low dose Cy improved the results of AHSCT by not only reducing the intensity of inflammation in the CNS but also by significantly reducing the clinical symptoms in treated animals when compared to AHSCT alone. We provide an experimental rationale that the addition of post-transplantation Cy may improve the outcome of HSCT in MS.

The purpose of this work was to evaluate the effect of the nanosized or microsized zinc (Zn) particles on fatty acid profile, enzyme activity and the level of cholesterol, squalene and oxysterols in rats with breast cancer. Rats (female, n = 24) were divided into the following groups: control, and two test groups, whose diets were enriched with either Zn microparticles (342 nm) or Zn nanoparticles (99 nm). All rats were treated twice with the carcinogenic agent; 7,12-dimethylbenz[a]anthracene. In rats whose diet was enriched with zinc (especially in the form of nanoparticles), the number and sizes of tumors were lower. Diet supplementation also significantly reduced the cholesterol ( p = 0.027) and COPs (cholesterol oxidation products) levels ( p = 0.011) in rats serum. Enriching the diet with Zn microparticles decreased the Δ6-desaturase activity ( p < 0.001). Zn influences fatty acids' profile in rats' serum as well as inhibiting desaturating enzymes. A reduced amount of pro-inflammatory arachidonic acid derivatives may be the expected effect.

The cytotoxic properties of zinc nanoparticles have been evaluated in vitro against several types of cancer. However, there is a lack of significant evidence of their activity in vivo, and a potential therapeutic application remains limited. Herein we report the effective inhibition of tumor growth by zinc nanoparticles in vivo, as the effect of the dietary intervention, after the chemical induction in a rodent model of breast cancer. Biopsy images indicated grade 1 tumors with multiple inflammatory infiltrates in the group treated with zinc nanoparticles, whereas, in the other groups, a moderately differentiated grade 2 adenocarcinoma was identified. Moreover, after the supplementation with zinc nanoparticles, the levels of several metabolites associated with cancer metabolism, important to its survival, were found to have been altered. We also revealed that the biological activity of zinc in vivo depends on the size of applied particles, as the treatment with zinc microparticles has not had much effect on cancer progression.

Bromhexine (BH), expectorant used in the treatment of respiratory disorders associated with viscid or excessive mucus, is not permitted for use in the competing horse by many authorities in horseracing and Olympic disciplines. Metabolic studies are of the great importance in anti-doping field because they allow for updating the selection of the most appropriate markers for prohibited substances, such as metabolites present at higher concentration levels and/or lasted for a longer period of time in biological samples than a parent drug. This study describes LC-MS/MS-based method for simultaneous determination of BH and its metabolites, including 4-(2-amino-3,5-dibromobenzylamino)cyclohexanol (4-HDMB), 3-(2-amino-3,5-dibromobenzylamino)cyclohexanol (3-HDMB), in equine serum samples. The 2-(2-amino-3,5-dibromobenzylamino)cyclohexanol (2-HDMB) was monitored as well. The assay was validated in terms of linearity (R 2  greater than 0.9951), intra- and inter-assay accuracy (91.6 - 109.1%) and precision (CV < 9.6%) as well as recovery (94.8 - 105.65%). The LODs were 0.0052, 0.0053, 0.0056 and 0.0043 ng/mL for BH, 2-HDMB, 3-HDMB and 4-HDMB, respectively. The developed method was applied to determine the time curses of BH and its metabolites concentrations in equine serum collected for 95.25 h following a single oral administration of BH to two healthy mares (in dose of 0.8 mg/kg). The parent drug was found at higher concentration levels than 3-HDMB (major metabolite) and 4-HDMB (minor metabolite), however, both BH metabolites lasted for a longer period of time in equine serum than the parent drug. Thus, both metabolites of BH can be considered as BH abuse markers., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

The objective of the present study was to determine the influence of dietary supplementation with pomegranate seed oil (PSO) and/or an aqueous extract of dried bitter melon fruits (BME) on breast cancer risk and fatty acid profile in serum of female rats with chemical carcinogen-inflicted mammary tumours. Sprague-Dawley rats (n = 96) were fed control diet or experimental diets supplemented with 0.15 ml PSO/day, BME or jointly PSO and BME. After 21 weeks mammary tumours were subjected to histopathological examination and in serum fatty acids, 8-isoprostaglandin F 2α content and indices of desaturases activity were analysed. Supplementation of the diet with PSO and BME did not inhibit the breast cancer formation. Conjugated linolenic acids (CLnA), present in PSO, were converted into cis-9, trans-11 conjugated linoleic acid (CLA), however, its content was lower in groups treated with a carcinogen. A similar tendency was observed for the content of SFA, MUFA, PUFA, 8-iso PGF 2α and the activity of Δ 6 -desaturase. Enhanced pro-carcinogenic effect of 7,12-dimethylbenz[a]anthracene (DMBA), caused by applied supplements, may be a result of their influence on DMBA metabolism., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Ibuprofen is widely used in human and veterinary medicine for the treatment of chronic pain as well as rheumatic and musculoskeletal disorders. However, the analgesic and anti-inflammatory properties of Ibuprofen have contributed to frequent drug abuse in equestrian sports. A sensitive and rapid gas chromatography with tandem mass spectrometry based method with a simple liquid-liquid extraction and derivatization requiring 200 μL volume of sample and 2 mL of extraction solvent for the simultaneous determination of ibuprofen and its metabolites was developed. The proposed procedure was optimized and validated according to the principles for bioanalytical methods. The assay achieved satisfactory validation parameters, namely, recovery (92.2-105%), interday accuracy (92.5-106%), and precision (0.3-4.4%) for all investigated compounds as well as limits of quantification of 50 ng/mL for ibuprofen, 2-hydroxyibuprofen, and carboxyibuprofen, 25 ng/mL for 1-hydroxyibuprofen and 100 ng/mL for 3-hydroxyibuprofen. The applicability of the method was evaluated by the analysis of five real urine samples collected from different horses after drug administration. In view of the low limits of quantification, high selectivity, repeatability, and recovery, the procedure can be utilized for laboratory applications, including the control of ibuprofen abuse in equestrian sports for anti-doping purposes and drug/pharmaceutical mentality investigations., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A novel assay for the simultaneous determination of ibuprofen (IBU) and its four probable metabolites, 1-hydroxyibuprofen (1-OH IBU), 2-hydroxyibuprofen (2-OH IBU), 3-hydroxyibuprofen (3-OH IBU) and carboxyibuprofen (CBX IBU) in equine urine samples with the application of Gas Chromatography-Electron Ionization-Mass Spectrometry (GC-EI-MS) has been developed and elaborated. The new approach for sample preparation including minimizing matrix effects by the application of weak cation exchange solid-phase extraction together with strong cation exchange solid-phase extraction has been applied. The GC-EI-MS method was validated to demonstrate specificity, matrix effect, linearity, limit of detection (LOD) and quantification (LOQ), precision, trueness, carry-over and stability by using the matrix-matched quality control samples. Additionally, extraction yield was evaluated. The assay achieved the LOQ of 1.75 μg mL -1 , 0.62 μg mL -1 , 4.15 μg mL -1 , 0.58 μg mL -1 and 4.04 μg mL -1 for IBU, 1-OH IBU, 2-OH IBU, 3-OH IBU and CBX IBU, respectively. The developed method has been successfully applied to the excretion study in horses, in which a single oral IBU dose was administered to twelve horses (mares and geldings) and equine urine samples were collected for 5 or 6 days after the drug administration. Data on the detection and determination of three IBU metabolites, 2-OH IBU, 3-OH IBU and CBX IBU in equine urine samples has been presented for the first time. The obtained results indicated the rapid excretion of IBU and its metabolites that were detectable only in the first day after the drug administration. IBU was mainly the most abundant compound detected in equine urine samples (with two exceptions in the case of samples collected from two horses, for which the highest instrumental responses were obtained for CBX IBU). The received results have indicated that two major IBU metabolites, CBX IBU and 2-OH IBU can be important markers for the IBU abuse in view of doping control in equestrian sports., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Multiple sclerosis (MS) is an autoimmunological disease leading to neurodegeneration. The etiology of the disease remains unknown, which strongly impedes the development of effective therapy. Most MS treatments focus on modulating the activity of the immune system. Dimethyl fumarate (DMF) exerts a broad spectrum of action, such as modulating immune cell differentiation towards anti-inflammatory subtypes, influencing cytokine production, regulating immune cell migration into the central nervous system, and activating intracellular antioxidant mechanisms. It is well established that activation of the nuclear factor E2 (Nrf2)-dependent pathway, leading to expression of the second-phase antioxidant enzymes, is influenced by DMF. In our experiments we used female Lewis rats in an animal model of MS - experimental allergic encephalomyelitis (EAE). The rats were fed with dimethyl fumarate to test the expression of heme oxygenase-1 (HO-1), one of the second-phase antioxidant enzymes, at specific time points of the symptomatic phases of the disease: on the first day of the occurrence of clinical symptoms (10th day post immunization, DPI); at the peak of clinical symptoms (14th DPI); and at the end of the relapse (21st DPI). The results showed that HO-1 expression, at both the mRNA and protein level, is influenced by DMF administration only at the very beginning of the symptomatic phase of EAE, and not at the peak of clinical symptoms, nor at the end of the relapse. This indicates that the regulation of the Nrf2-dependent antioxidant pathway by DMF occurs at a certain time interval (early EAE/MS) and strongly underlines the importance of the earliest introduction of the therapy to the patient. .

Neospora caninum is a protozoan parasite which causes abortion in cattle as well as reproduction problems and neurological disorders in dogs. To assess the prevalence of the parasite in urban dogs in the Mazovian Voivodeship, Central Poland, serum samples from 257 dogs were analyzed for the presence of specific IgG antibodies. The examined dogs visited three private veterinary clinics located in Warsaw due to control tests, vaccinations, or other reasons not directly connected with neosporosis. Using ELISA and Western blot, antibodies against the parasite were detected in 56 out of 257 dogs, giving a prevalence of 21.7%. A greater prevalence was observed in female dogs than in males, 28% and 17.3%, respectively, and the differences were statistically significant (p<0.05). There were no significant differences in seroprevalence of Neospora infection within the age groups (p>0.05). This study indicates the presence of N. caninum in the Mazovian Voivodeship, in dogs which live in urban areas and exposure of these dogs to the parasite. The fact that seropositive dogs had no contact with cattle confirms the important role of dogs in the parasite's epidemiology.