An Alternative Application of Magnetic-Activated Cell Sorting: CD45 and CD235a Based Purification of Semen and Testicular Tissue Samples.

Magnetic activated cell sorting (MACS) is a well-known sperm selection technique, which is able to remove apoptotic spermatozoa from semen samples using the classic annexinV based method. Leukocytes and erythrocytes in semen samples or in testicular tissue processed for in vitro fertilization (IVF) could exert detrimental effects on sperm. In the current study, we rethought the aforementioned technique and used magnetic microbeads conjugated with anti-CD45/CD235a antibodies to eliminate contaminating leukocytes and erythrocytes from leukocytospermic semen samples and testicular tissue samples gained via testicular sperm extraction (TESE). With this technique, a 15.7- and a 30.8-fold reduction could be achieved in the ratio of leukocytes in semen and in the number of erythrocytes in TESE samples, respectively. Our results show that MACS is a method worth to reconsider, with more potential alternative applications. Investigations to find molecules labeling high-quality sperm population and the development of positive selection procedures based on these might be a direction of future research.

Oxido-Reduction Potential as a Method to Determine Oxidative Stress in Semen Samples.

There are different estimates for the incidence of infertility. Its occurrence may vary from area to area, but on average, it affects 15% of couples and 10-12% of men worldwide. Many aspects of infertility can be linked to reactive oxygen species (ROS) and the process of oxidative stress (OS). The association between poor semen quality and OS is well known. Unfortunately, there is no accepted protocol for the diagnosis and treatment of OS in andrology. Oxido-reduction potential (ORP) measurement is a new method for determining the ratio between oxidant and antioxidant molecules. Currently, ORP measurement is one of the fastest and most user-friendly methods of andrological OS determination and our goals were to confirm published correlations between ORP values and sperm parameters, examine how sperm concentration influences these results, and investigate whether intracellular ROS formations are also manifested in the ORP values or not after artificial ROS induction. Intracellular ROS formations were induced by menadione (superoxide anion inducer), hydrogen peroxide, and tert-butyl hydroperoxide (lipid peroxidation inducer) treatments; sperm parameters like motility and viability were determined with an SCA Scope system, and ORP changes were recorded by the Mioxsys system. Significant correlations were noticed among the ORP, spermatozoa concentration, motility, progressive motility, and viability. Nevertheless, only the ORP value after normalization with the sperm count correlated with these parameters. Due to normalization, very low and very high sperm concentrations can give misleading results. The means of the non-normalized ORP values were almost the same. All of the applied treatments resulted in decreases in the viability, motility, and progressive motility, and interestingly, altered ORP levels were detected. In addition, it was determined that seminal plasma had a significant protective effect on spermatozoa. The elimination of seminal plasma caused higher sensitivity of spermatozoa against used OS inducers, and higher ORP levels and decreased viabilities and motilities were measured. The ORP level could be a good indicator of male OS; however, in cases of low and high sperm counts, its result can be misleading. Overall, the conclusion can be drawn that ORP determination is a suitable method for detecting intracellular ROS accumulation, but it has limitations that still need to be clarified.

Estrogenic and Non-Estrogenic Disruptor Effect of Zearalenone on Male Reproduction: A Review.

According to some estimates, at least 70% of feedstuffs and finished feeds are contaminated with one or more mycotoxins and, due to its significant prevalence, both animals and humans are highly likely to be exposed to these toxins. In addition to health risks, they also cause economic issues. From a healthcare point of view, zearalenone (ZEA) and its derivatives have been shown to exert many negative effects. Specifically, ZEA has hepatotoxicity, immunotoxicity, genotoxicity, carcinogenicity, intestinal toxicity, reproductive toxicity and endocrine disruption effects. Of these effects, male reproductive deterioration and processes that lead to this have been reviewed in this study. Papers are reviewed that demonstrate estrogenic effects of ZEA due to its analogy to estradiol and how these effects may influence male reproductive cells such as spermatozoa, Sertoli cells and Leydig cells. Data that employ epigenetic effects of ZEA are also discussed. We discuss literature data demonstrating that reactive oxygen species formation in ZEA-exposed cells plays a crucial role in diminished spermatogenesis; reduced sperm motility, viability and mitochondrial membrane potential; altered intracellular antioxidant enzyme activities; and increased rates of apoptosis and DNA fragmentation; thereby resulting in reduced pregnancy.

Magnetic-Activated Cell Sorting as a Method to Improve Necrozoospermia-Related Asthenozoospermic Samples.

According to some statistics, absolute asthenozoospermia affects every 1 in 5000 men. Although this incidence rate does not appear to be too high, it is extremely important to address the phenomenon because it can drastically reduce the chances of pregnancy, even with assisted reproduction. The biggest problem with absolute asthenozoospermia is that it is difficult to distinguish between live and dead sperm cells, and fertilization with non-viable spermatozoa may contribute to the failure of an assisted reproduction cycle. Nowadays, DNA fragmentation (DF) is a crucial parameter of semen analysis, and in this paper, we provide evidence of the correlation between DF and vitality. For this purpose, the main semen parameters were investigated by a CASA system (concentration, motility, progressive motility, vitality and DF). In the necrozoospermic group (vitality < 58%), all the measured parameters showed significant differences compared to normal vitality. Concentration (30.1 M mL−1 vs. 13.6 M mL−1), motility (31.9% vs. 18.3%), and progressive motility (24.3% vs. 12.7%) were significantly decreased, while DF was significantly increased (17.4% vs. 23.7%). Based on the connection between vitality decrement and DF increment, DF lowering methods, such as magnetic-activated cell sorting, have been hypothesized as novel methods for the elimination of dead spermatozoa.

Application of Mass Spectrometry Imaging in Uro-Oncology: Discovering Potential Biomarkers.

A growing need is emerging worldwide for new molecular markers which could enhance the accuracy of diagnostic and therapeutic methods for detecting urogenital cancers. Mass spectrometry imaging (MSI) is a very promising tool in this regard. In this review, we attempt to provide a subjective summary of the latest publications on potential biomarkers of renal, bladder, prostate, and testicular malignancies detected with MSI through the eyes of a clinical urologist.

Elimination of stress factors by continuous embryo culture and its influence on in vitro fertilization outcomes.

Recently, infertility has become one of the most important endemic conditions, affecting approximately 15-20 % of couples worldwide. Among others, the careerist lifestyle, the increasing maternal age and the parallel increment in the aneuploidy rate of embryos play a crucial role in this phenomenon. In this study, embryological parameters and pregnancy outcomes were investigated in IVF cycles using either sequential embryo culture or a single step culture system. By sequential media, oocytes/embryos are needlessly exposed to the potentially negative effects of light exposure, temperature decrement and altered oxygen tension. In comparison with sequential media, single step media induced 1.28, 1.21 and 1.21-fold increments in implantation, biochemical pregnancy and clinical pregnancy rates, respectively. Pregnancy outcomes showed strong maternal age-dependency, so the difference between the two investigated culture systems was equalized by the increasing maternal ages (35-44 years) and the supposed incidence of embryo aneuploidy. Nevertheless, the significant enlargements in the outcomes of the younger ages (25-34) induced by the single step cultures suggest that, beside the resultant maternal aneuploidy, aneuploidy (reduced pregnancy rates) may evolve from exposure to the mentioned environmental stress factors.

Endometriosis Is a Cause of Infertility. Does Reactive Oxygen Damage to Gametes and Embryos Play a Key Role in the Pathogenesis of Infertility Caused by Endometriosis?

Approximately, 10-15% of women of reproductive age are affected by endometriosis, which often leads to infertility. Endometriosis often has an inherited component, and several causative predisposing factors are hypothesized to underlie the pathogenesis of endometriosis. One working hypothesis is the theory of retrograde menstruation. According to the theory of retrograde menstruation, components of refluxed blood, including apoptotic endometrial tissue, desquamated menstrual cells, lysed erythrocytes, and released iron, induce inflammation in the peritoneal cavity. This in turn activates macrophage release of reactive oxygen species (ROS), leading to oxidative stress via the respiratory burst. Refluxed blood promotes the Fenton reaction, terminating in the production of hydroxyl radical, the most potently destructive ROS. In this article, we review the papers that demonstrate decreased quantity and quality of oocytes and embryos retrieved from IVF/ICSI patients with endometriosis. We discuss literature data demonstrating that ROS are generated in endometriotic tissues that have physical proximity to gametes and embryos, and demonstrating adverse impacts on oocyte, sperm and embryo microtubule apparatus, chromosomes, and DNA. Data that addresses the notions that endometriosis causes oocyte and fetal aneuploidy and that these events are mediated by ROS species are also discussed. Literature data are also discussed that employ use of anti-oxidant molecules to evaluate the importance of ROS-mediated oxidative damage in the pathogenesis of endometriosis. Studies are discussed that have employed anti-oxidants compounds as therapeutics to improve oocyte and embryo quality in infertile subjects, and improve fertility in patients with endometriosis.

Linalool-induced oxidative stress processes in the human pathogen Candida albicans.

The present study investigated the linalool (Lol)-induced effects in acute toxicity tests in the human pathogen Candida albicans (C. albicans). Lol treatments induced reduced germ tube formation of the pathogen, which plays a crucial role in the virulence. In comparison with the untreated control, the exposure of 107 cells ml-1 to 0.7 mM or 1.4 mM Lol for one hour induced 20% and 30% decrements, respectively, in the colony-forming ability. At the same time, these treatments caused dose-dependent decrease in the levels of superoxide anion radical and total reactive oxygen species, while there was 1.5 and 1.8-fold increases in the concentrations of peroxides and lipid peroxides, respectively, indicating oxidative stress induction in the presence of Lol. Lol treatments resulted in different adaptive modifications of the antioxidant system. In 0.7 mM-treated cells, decreased specific activities of superoxide dismutase and catalase were detected, while exposure to 1.4 mM Lol resulted in the up-regulation of catalase, glutathione reductase and glutathione peroxidases.

Effects of clary sage oil and its main components, linalool and linalyl acetate, on the plasma membrane of Candida albicans: an in vivo EPR study.

The effects of clary sage (Salvia sclarea L.) oil (CS-oil), and its two main components, linalool (Lol) and linalyl acetate (LA), on cells of the eukaryotic human pathogen yeast Candida albicans were studied. Dynamic and thermodynamic properties of the plasma membrane were investigated by electron paramagnetic resonance (EPR) spectroscopy, with 5-doxylstearic acid (5-SASL) and 16-SASL as spin labels. The monitoring of the head group regions with 5-SASL revealed break-point frequency decrease in a temperature dependent manner of the plasma membrane between 9.55 and 13.15 °C in untreated, in CS-oil-, Lol- and LA-treated membranes. The results suggest a significant increase in fluidity of the treated plasma membranes close to the head groups. Comparison of the results observed with the two spin labels demonstrated that CS-oil and LA induced an increased level of fluidization at both depths of the plasma membrane. Whereas Lol treatment induced a less (1 %) ordered bilayer organization in the superficial regions and an increased (10 %) order of the membrane leaflet in deeper layers. Acute toxicity tests and EPR results indicated that both the apoptotic and the effects exerted on the plasma membrane fluidity depended on the composition and chemical structure of the examined materials. In comparison with the control, treatment with CS-oil, Lol or LA induced 13.0, 12.3 and 26.4 % loss respectively, of the metabolites absorbing at 260 nm, as a biological consequence of the plasma membrane fluidizing effects. Our results confirmed that clary sage oil causes plasma membrane perturbations which leads to cell apoptosis process.

Regulation of the antioxidant system in cells of the fission yeast Schizosaccharomyces pombe after combined treatment with patulin and citrinin.

The effects of combined treatment with patulin (PAT) and citrinin (CTN) on Schizosaccharomyces pombe cells were investigated in acute toxicity tests. In comparison with the controls the exposure of fission yeast cells (10(7) cells ml(-1)) to PAT + CTN (250 µM each) for 1 h at a survival rate of 66.6% significantly elevated the concentration of total reactive oxygen species (ROS) via increased levels of peroxides without affecting the concentrations of superoxides or the hydroxyl radical. This treatment induced a 3.08-fold increase in the specific concentration of glutathione and elevated specific activities of catalase and glutathione S-transferase, while at the same time the activity of glutathione reductase decreased. The pattern of the ROS was the same as that induced by CTN (Máté et al., 2014), while the presence of PAT in the PAT + CTN combination treatment modified the activities of the antioxidant system (Papp et al., 2012) in comparison with the individual PAT or CTN treatment, suggesting toxin-specific regulation of glutathione and the enzymes of the antioxidant system and the possibility that the transcription factor (pap1 and atf1) -regulated processes might be influenced directly by ROS.

The influence of the combination of carboxylate and phosphinate pendant arms in 1,4,7-triazacyclononane-based chelators on their 68Ga labelling properties.

In order to compare the coordination properties of 1,4,7-triazacyclononane (tacn) derivatives bearing varying numbers of phosphinic/carboxylic acid pendant groups towards 68Ga, 1,4,7-triazacyclononane-7-acetic-1,4-bis(methylenephosphinic) acid (NOPA) and 1,4,7- triazacyclononane-4,7-diacetic-1-[methylene(2-carboxyethyl)phosphinic] acid (NO2AP) were synthesized using Mannich reactions with trivalent or pentavalent forms of H-phosphinic acids as phosphorus components. Stepwise protonation constants logK1-3 12.06, 3.90 and 1.95, and stability constants with GaIII and CuII, logKGaL 24.01 and logKCuL 16.66, were potentiometrically determined for NOPA. Both ligands were labelled with 68Ga and compared with NOTA (tacn-N,N',N″-triacetic acid) and NOPO, a TRAP-type [tacn-N,N',N″- tris(methylenephosphinic acid)] chelator. At pH 3, NOPO and NOPA showed higher labelling efficiency (binding with lower ligand excess) at both room temperature and 95 °C, compared to NO2AP and NOTA. Labelling efficiency at pH = 0-3 correlated with a number of phosphinic acid pendants: NOPO >> NOPA > NO2AP >> NOTA; however, it was more apparent at 95 °C than at room temperature. By contrast, NOTA was found to be labelled more efficiently at pH > 4 compared to the ligands with phosphinic acids. Overall, replacement of a single phosphinate donor with a carboxylate does not challenge 68Ga labelling of TRAP-type chelators. However, the presence of carboxylates facilitates labelling at neutral or weakly acidic pH.

2'[(18)F]-fluoroethylrhodamine B is a promising radiotracer to measure P-glycoprotein function.

In vivo detection of the emergence of P-glycoprotein (Pgp) mediated multidrug resistance in tumors could be beneficial for patients treated with anticancer drugs. PET technique in combination with appropriate radiotracers could be the most convenient method for detection of Pgp function. Rhodamine derivatives are validated fluorescent probes for measurement of mitochondrial membrane potential and also Pgp function. The aim of this study was to investigate whether 2'[(18)F]-fluoroethylrhodamine B ((18)FRB) a halogenated rhodamine derivative previously synthesized for PET assessment of myocardial perfusion preserved its Pgp substrate character. ATPase assay as well as accumulation experiments carried out using Pgp(+) and Pgp(-) human gynecologic (A2780/A2780(AD) and KB-3-1/KB-V1) and a mouse fibroblast cell pairs (NIH 3T3 and NIH 3T3 MDR1) were applied to study the interaction of (18)FRB with Pgp. ATPase assay proved that (18)FRB is a high affinity substrate of Pgp. Pgp(-) cells accumulated the (18)FRB rapidly in accordance with its lipophilic character. Dissipation of the mitochondrial proton gradient by a proton ionophore CCCP decreased the accumulation of rhodamine 123 (R123) and (18)FRB into Pgp(-) cells. Pgp(+) cells exhibited very low R123 and (18)FRB accumulation (around 1-8% of the Pgp(-) cell lines) which was not sensitive to the mitochondrial proton gradient; rather it was increased by the Pgp inhibitor cyclosporine A (CsA). Based on the above data we conclude that (18)FRB is a high affinity Pgp substrate and consequently a potential PET tracer to detect multidrug resistant tumors as well as the function of physiological barriers expressing Pgp.

In vivo imaging of Aminopeptidase N (CD13) receptors in experimental renal tumors using the novel radiotracer (68)Ga-NOTA-c(NGR).

PURPOSE: Aminopeptidase N (APN/CD13) plays an important role in tumor neoangiogenic process and the development of metastases. Furthermore, it may serve as a potential target for cancer diagnosis and therapy. Previous studies have already shown that asparagine-glycine-arginine (NGR) peptides specifically bind to APN/CD13. The aim of the study was to synthesize and investigate the APN/CD13 specificity of a novel (68)Ga-labeled NOTA-c(NGR) molecule in vivo using miniPET. METHODS: c[KNGRE]-NH2 peptide was conjugated with p-SCN-Bn-NOTA and was labeled with Ga-68 ((68)Ga-NOTA-c(NGR)). Orthotopic and heterotopic transplanted mesoblastic nephroma (NeDe) bearing Fischer-344 rats were prepared, on which biodistribution studies and miniPET scans were performed for both (68)Ga-NOTA-c(NGR) and ανß3 integrin selective (68)Ga-NODAGA-[c(RGD)]2 tracers. APN/CD13 receptor expression of NeDe tumors and metastases was analyzed by western blot. RESULTS: (68)Ga-NOTA-c(NGR) was produced with high specific activity (5.13-5.92GBq/µmol) and with excellent radiochemical purity (95%<), at all cases. Biodistribution studies in normal rats showed that uptake of the (68)Ga-NOTA-c(NGR) was significantly (p⩽0.05) lower in abdominal organs in comparison with (68)Ga-NODAGA-[c(RGD)]2. Both radiotracers were mainly excreted from the kidney. In NeDe tumor bearing rats higher (68)Ga-NOTA-c(NGR) accumulation was found in the tumors than that of the (68)Ga-NODAGA-[c(RGD)]2. Using orthotopic transplantation, metastases were developed which showed specific (68)Ga-NOTA-c(NGR) uptake. Western blot analysis confirmed the presence of APN/CD13 expression in NeDe tumors and metastases. CONCLUSION: Our novel radiotracer (68)Ga-NOTA-c(NGR) showed specific binding to the APN/CD13 expressed ortho- and heterotopic transplanted NeDe tumors. Therefore, (68)Ga-NOTA-c(NGR) is a suitable tracer for the detection of APN/CD13 positive tumors and metastases in vivo.

Regulation of oxidative stress-induced cytotoxic processes of citrinin in the fission yeast Schizosaccharomyces pombe.

In this study, the citrinin (CTN)-induced accumulation of reactive oxygen species (ROS) and the regulation of the activities of antioxidant enzymes were investigated in acute toxicity tests in Schizosaccharomyces pombe. 30% of the CTN was accumulated by the cells in 1000 µM CTN solution. In comparison with the control, exposure of 10(7) cells ml(-1) to 1000 µM CTN for 60 min at pH = 4.5 induced significantly (p < 1%) elevated levels of peroxides and total ROS, but not of superoxide or hydroxyl radicals, while there was a 3-fold increase in the concentration of glutathione. ROS-induced adaptation processes at cell and molecular levels via activation of the redox-sensitive transcription factors Pap1 and (in part) Atf1 resulted in significantly increased specific activities of glutathione peroxidases, glucose-6-phosphate dehydrogenase and glutathione S-transferase and in decreased levels of catalase and glutathione reductase, but no changes were detected in the activities of superoxide dismutases. This treatment caused a G2/M cell cycle arrest and elevated the number of fragmented nuclei, which is one of the markers of apoptosis. Comparison of these results with those for the positive control, 200 µM H2O2, suggested that CTN induced a medium level of oxidative stress.

tert-Butyl hydroperoxide-induced differing plasma membrane and oxidative stress processes in yeast strains BY4741 and erg5Δ.

The molecular mechanism of tert-butyl hydroperoxide (t-BuOOH) elicited cytotoxicity and the background of t-BuOOH sensitivity were studied in the Saccharomyces cerevisiae ergosterol-less gene deletion mutant erg5Δ and its parental strain BY4741. In comparison to BY4741, untreated erg5Δ cells exhibited alterations in sterol and fatty acid compositions of the plasma membrane, as reflected by the inherent amphotericin B resistance, an elevated level (31%) of plasma membrane rigidity and a decreased uptake of glycerol. Surprisingly, the untreated erg5Δ cells exhibited an unbalanced intracellular redox state, accompanied by the continuous upregulation of the antioxidant enzymes Mn superoxide dismutase, catalase, and glutathione S-transferase, which resulted in decreased specific concentrations of superoxide and peroxides and elevated levels of the hydroxyl radical and thiols. The 2.5-fold sensitivity of erg5Δ to t-BuOOH suggested that the oxidative stress adaptation processes of the mutant could not restore the redox homeostasis of the cells and there is an overlap between sterol and redox homeostases. t-BuOOH treatment of both strains induced adaptive modification of the sterol and fatty acid compositions, increased the plasma membrane fluidity and elevated the specific activities of most antioxidant enzymes through specific regulation processes in a strain-dependent manner.

Synthesis of fluorescent ristocetin aglycon derivatives with remarkable antibacterial and antiviral activities.

Isoindole and benzoisoindole derivatives of ristocetin aglycon have been prepared by reaction with o-phthalaldehyde or naphthalene-2,3-dialdehyde and various thiols. The new compounds exhibited potent antibacterial and anti-influenza virus activity. The cluster forming and fluorescent properties of the aglycon derivatives were also studied.