Anna Wajda

Synthesis, dispersion, and cytocompatibility of graphene oxide and reduced graphene oxide.

The synthesis, characterization, and toxicity of graphene oxide and reduced graphene oxide are reported. Prior to the cytocompatibility tests the stability of the suspensions in a wide range of concentrations (3.125-100 μg/mL) of three different dispersants is studied. Polyethylene glycol (PEG), polyethylene glycol-polypropylene glycol-polyethylene glycol (Pluronic P123), and sodium deoxycholate (DOC) are investigated as the dispersants. The toxicity depends on the type of dispersant and concentration of the nanomaterials in the suspensions. Detailed analysis suggests that graphene oxide functionalized with PEG in the concentration range between 3125 μg/mL and 25 μg/mL exhibits the best biocompatibility with mice fibroblast cells (line L929).

Association of transcription factor 7-like 2 (TCF7L2) gene polymorphism with posttransplant diabetes mellitus in kidney transplant patients medicated with tacrolimus

New onset posttransplant diabetes mellitus (PTDM) has a high incidence after kidney transplantation in patients medicated with tacrolimus. PTDM can adversely affect patient and graft survival. The pathophysiology of PTDM closely mimics type 2 diabetes mellitus (T2DM). One of the possible genetic factors predisposing individuals to PTDM might be a polymorphism in the transcription factor 7-like 2 gene (TCF7L2). This polymorphism has previously been associated with increased risk of T2DM in the general population. Therefore, the present study aimed to evaluate TCF7L2 polymorphisms in PTDM in kidney transplant patients medicated with tacrolimus. Non-diabetic kidney transplant patients medicated with tacrolimus (n = 234) were genotyped for the presence of TCF7L2 gene variants (rs12255372 and rs7903146) using TaqMan probes. Of the 234 patients, 66 patients had developed PTDM and 168 had not. Frequencies of the studied single nucleotide polymorphisms (SNPs) did not differ significantly between the study groups. Moreover, haplotype analyses failed to detect any associations between TCF7L2 haplotypes and PTDM. However, in late-onset PTDM (developed later that 2 weeks from transplantation), frequencies of the rs7903146 TT genotype and T minor allele were significantly increased compared to non-PTDM controls (17.9% vs. 5.9%, p = 0.017, OR: 4.13, 95% CI: 1.19-14.33 for TT genotype, 39.3% vs. 25.9%, p = 0.038 for T allele). If the application of TCF7L2 rs7903146 SNPs as a marker for PTDM is confirmed by further independent studies, replacing tacrolimus with other immunosuppressants could be warranted in patients at high risk of PTDM, as diagnosed by TCF7L2 genotyping.

Mesoporous Silica Nanospheres Functionalized by Tio2 as a Photoactive Antibacterial Agent

In this contribution we present comparative study on synthesis, bio-characterization and antibacterial properties of mesoporous silica nanospheres modified by titanium dioxide. Mesoporous silica nanospheres functionalized by titania were studied as light activated antibacterial agents. The analysis of the antibacterial effects on E. coli ATCC 25922 shows strong enhancement during the visible and ultraviolet light irradiation in respect to the commercial catalyst and sample free from the nanomaterials. In darkness the mesoporous silica/titania nanostructures revealed low antibacterial activity dependent on the stirring intensity of the suspension containing nanomaterials and bacteria. The nanomaterials toxicity was determined on the amount of lactate dehydrogenase released from mouse fibroblast cells L929 with LDH assay. Sample was characterized in details by means of high resolution transmission electron microscopy (HR-TEM), Raman spectroscopy, XRD and BET Isotherm.

Association study of folate-related enzymes (MTHFR, MTR, MTRR) genetic variants with non-obstructive male infertility in a Polish population

Spermatogenesis is a process where an important contribution of genes involved in folate-mediated one-carbon metabolism is observed. The aim of the present study was to investigate the association between male infertility and the MTHFR (677C > T; 1298A > C), MTR (2756A > G) and MTRR (66A > G) polymorphisms in a Polish population. No significant differences in genotype or allele frequencies were detected between the groups of 284 infertile men and of 352 fertile controls. These results demonstrate that common polymorphisms in folate pathway genes are not major risk factors for non-obstructive male infertility in the Polish population.

Nuclear factor E2-related factor-2 (Nrf2) expression and regulation in male reproductive tract

BACKGROUND Nuclear factor E2-related factor-2 (Nrf2, Nfe2l2) plays an important, protective role in many tissues. However, information on molecular mechanisms of detoxification and drug metabolism regulated by Nrf2/NRF2 in testis and epididymis is scarce, but it may help to better characterize the function of blood-testis and epididymis barriers. METHODS Constitutive gene expression was analyzed by real time PCR with TaqMan Assay using ΔCT-method. Additionally, gene expression after treatment with oltipraz- specific Nrf2 inducer was evaluated using ΔΔCT-method. Cellular localization of the Nrf2 was visualized by immunohistochemical reaction. RESULTS The study showed that Nrf2 mRNA level in rat epididymis was higher than in testis. In human tissues, both testis and epididymis demonstrated similar expression levels of NRF2. Immunohistochemical analysis revealed NRF2/Nrf2 protein expression in testis and epididymis, which in the case of testis was dependant on spermatogenesis stage. Both in human and rat tissues constitutive expression of NQO1/Nqo1 was slightly higher in epididymis than in testis. Other Nrf2 regulated genes: GCLC/Gclc and UGT1A6/Ugt1a6 showed different ratios of testis/epididymis/liver expression levels. Treatment with oltipraz (Nrf2 inducer) resulted in significant induction of Nrf2 expression solely in corpus of epididymis. CONCLUSIONS Components of the Nrf2/NRF2 system along with coordinated genes are expressed in testis and epididymis. Moreover, some interspecies differences between rat and human were observed, which may impact extrapolation of experimental data into clinical findings. Studies on animal model showed that corpus of epididymis is the most responsive part of the male reproductive tract to oltipraz exposure at the gene expression level.

Antibacterial performance of nanocrystallined titania confined in mesoporous silica nanotubes

In this paper, we study synthesis and characteristics of mesoporous silica nanotubes modified by titanium dioxide, as well as their antimicrobial properties and influence on mitochondrial activity of mouse fibroblast L929. Nanocrystalized titania is confined in mesopores of silica nanotubes and its light activated antibacterial response is revealed. The analysis of the antibacterial effect on Escherichia coli. (ATCC 25922) shows strong enhancement during irradiation with the artificial visible and ultraviolet light in respect to the commercial catalyst and control sample free from the nanomaterials. In darkness, the mesoporous silica/titania nanostructures exhibited antibacterial activity dependent on the stirring speed of the suspension containing nanomaterials. Obtained micrograph proved internalization of the sample into the microorganism trough the cell membrane. The analysis of the mitochondrial activity and amount of lactate dehydrogenase released from mouse fibroblast cells L929 in the presence of the sample were determined with LDH and WST1 assays, respectively. The synthesized silica/titania antibacterial agent also exhibits pronounced photoinduced inactivation of the bacterial growth under the artificial visible and UV light irritation in respect to the commercial catalyst. Additionally, mesoporous silica/titania nanotubes were characterized in details by means of high resolution transmission electron microscopy (HR-TEM), XRD and BET Isotherm.

Cell and region specificity of Aryl hydrocarbon Receptor (AhR) system in the testis and the epididymis

Aryl hydrocarbon receptor (AhR) plays multiple important functions in adaptive responses. Exposure to AhR ligands may produce an altered metabolic activity controlled by the AhR pathways, and consequently affect drug/toxin responses, hormonal status and cellular homeostasis. This research revealed species-, cell- and region-specific pattern of the AhR system expression in the rat and human testis and epididymis, complementing the existing knowledge, especially within the epididymal segments. The study showed that AhR level in the rat and human epididymis is higher than in the testis. The downregulation of AhR expression after TCDD treatment was revealed in the spermatogenic cells at different stages and the epididymal epithelial cells, but not in the Sertoli and Leydig cells. Hence, this basic research provides information about the AhR function in the testis and epididymis, which may provide an insight into deleterious effects of drugs, hormones and environmental pollutants on male fertility.

Effects of simvastatin on nuclear receptors, drug metabolizing enzymes and transporters expression in Human Umbilical Vein Endothelial Cells

BACKGROUND Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS HUVEC were exposed to TCDD (10nM), oltipraz (100μM) and simvastatin (1μM) for 24h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.BACKGROUND Vascular endothelial cells (EC) are constantly exposed to endo- and exogenous compounds, which may disturb EC function. One of the protecting mechanisms against chemicals consists of drug metabolizing enzymes and transporter proteins regulated by nuclear receptors and transcription factors. Therefore, the aim of the current study was to assess the regulation of nuclear receptors and their coordinated genes in Human Umbilical Vein Endothelial Cells (HUVEC). METHODS HUVEC were exposed to TCDD (10 nM), oltipraz (100 μM) and simvastatin (1 μM) for 24 h. Gene expressions were evaluated using quantitative real-time PCR. The protein expression levels were determined by Western blotting. Enzymatic activity of CYP1A1/CYP1B1 was assessed by luciferin-labelled CYPs substrate. RESULTS Our study confirmed that nuclear receptor AhR and nuclear factor Nrf2 are highly expressed in HUVECs. Treatment of HUVECs with TCDD (AhR inducer) resulted in a significant induction of AHR target genes CYP1A1, CYP1B1 and NQO1. Oltipraz (Nrf2 inducer) also markedly increased expression of NQO1 but did not affect Nrf2 mRNA nor protein levels. Under simvastatin stimulation PXR and NRF2 target transcripts were not altered, however AHR-regulated genes: CYP1A1, CYP1B1 and MDR1 were significantly induced. Western blot analysis confirmed CYP1B1 induction in TCDD-treated HUVECs, but not in the simvastatin group. Moreover, HUVEC exposure to TCDD resulted in induction of CYP1A1/CYP1B1 enzymatic activity. CONCLUSIONS This study revealed functional expression of AhR and Nrf2 in HUVECs. Moreover, it was defined that simvastatin induced AhR and its related genes.

Single Nucletide Polymorphisms in Gene of IL-1Beta in Bronchial Asthma

ABSTRACT Bronchial asthma is a common chronic lung disease that is driven by abnormal inflammatory reactions in the airways in response to the complex interaction between genetics and environmental factors. The underlying inflammation in asthma is manifested by prominent eosinophil infiltration and Th2 inflammatory mediators; however the pro-inflammatory cytokines, particularly IL-1β, have also been found in increased amount in the sputum and BAL in individuals with bronchial asthma, especially in more severe state. Therefore, IL-1β is considered to be of importance in pathogenesis of this condition as the protein level of the cytokine is finely regulated by variety of factors, including genetics. The IL1B gene displays many SNPs both in promoter and coding regions, which have been associated with IL-1β production. In the current case-control study we investigated -511C>T (rs16944) promoter polymorphism and +3953C>T (rs1143634) silent polymorphism in exon 5 of IL1B and their haplotypes as candidate risk factors of Bronchial asthma in Bulgarian population. We genotyped 47 patients with bronchial asthma and 174 control individuals using Taqman genotyping assay for IL1B -511C>T SNP and PCR-RFLP-based method for +3953C>TSNP. We did not observe statistically significant differences in genotype frequencies of IL1B -511C>T and IL1B +3953C>T between controls and patients with asthma (p=0.065 and p=0.987). However, the minor T allele of IL1B -511C>T was less frequently found in the controls (0.305) compared to the patients with asthma (0.415, p=0.0002). Carriers of IL1B -511T allele (TT or TC genotypes) appeared to have 2.25-fold higher risk for Bronchial asthma (95% CI: 1.127–4.498, p=0.019). The performed estimations of IL1B haplotypes did not reveal any difference in the haplotype frequencies between controls and patients with asthma (p=0.270). However, the T_C haplotype, constructed by alleles found to determine enhanced expression of IL-1β, appeared to be associated with higher risk of asthma (OR 1.78, 1.04–3.03, p=0.035) compared to the most common C_C haplotype. Based on the results of the current study we suggest that the -511C>T promoter polymorphism and +3953C>T silent polymorphism in exon 5 of IL1B may influence the genetic predisposition of Bronchial asthma in Bulgarian population, as the carriers of alleles and haplotypes supposed to define higher IL-1β protein levels are more susceptible for this lung diseases.