Aniline Blue Research Articles

Equilibrium, Kinetic and Thermodynamics Studies of Adsorption of Aniline Blue from Aqueous Media Using Steam-Activated Carbon Prepared from <i>Delonix regia</i> Pod

The adsorption capacity of steam activated carbon prepared from Delonix regia pods SADRC for adsorption of Aniline Blue (AB) from aqueous solution was investigated under various experimental conditions. Batch study was conducted to assess the potential of the activated carbon for the removal of Aniline Blue from aqueous solution. Activated carbon prepared from Delonix regia pods was characterized using Scanning Electron Microscope (SEM) and Fourier Transform Infrared (FTIR) Spectrophotometry before and after adsorption. The FTIR, spectra of SADRC pod before and after Aniline Blue adsorption were compared to study the impact of the Aniline Blue on the activated carbon developed from the Delonix regia pod. The stretching vibration band at 2169.54 cm-1 may be due to strong CN, while the stretching vibration band at around 1580 - 1650 cm-1 may be due to C=C stretching vibration. The bands around 1350 and 426.49 cm-1 are due to C-N and –SO3H group, respectively; this further suggests that some functional groups may be present on the surface of the carbon due to the low temperature of carbonization (300°C) of the adsorbent. Equilibrium isotherm studies were carried out by varying the following four parameters: initial concentration of Aniline Blue dye solution, solution pH and adsorbent dose. The equilibrium data obtained were more fitted to Langmuir than Freundlich isotherm models. The correlation coefficient value (R2) of the pseudo first order kinetics ranged from 0.08 to 0.85 while the R2 of the pseudo second order kinetics ranged from 0.963 to 0.997 at all the temperatures and initial concentrations considered. This suggests that the adsorption kinetics of Aniline Blue onto SADRC can be represented with pseudo second order kinetic model. This study showed that Delonix regia pod could be effectively used as an adsorbent for the removal of Aniline Blue from aqueous solutions over a wide range of concentration and temperature.

Imaging callose at plasmodesmata using aniline blue: quantitative confocal microscopy.

Callose (β-1,3-glucan) is both structural and functional component of plasmodesmata (Pd). The turnover of callose at Pd controls the cell-to-cell diffusion rate of molecules through Pd. An accurate assessment of changes in levels of Pd-associated callose has become a first-choice experimental approach in the research of intercellular communication in plants.Here we describe a detailed and easy-to-perform procedure for imaging and quantification of Pd-associated callose using fixed plant tissue stained with aniline blue. We also introduce an automated image analysis protocol for non-biased quantification of callose levels at Pd from fluorescence images using ImageJ. Two experimental examples of Pd-callose quantification using the automated method are provided as well.

Well-defined bioarchitecture for immobilization of chloroperoxidase on magnetic nanoparticles and its application in dye decolorization

Abstract Molecular orientation of chloroperoxidase (CPO) on surface of Fe 3 O 4 magnetic nanoparticles (MNPs) was achieved through layer-by-layer (LBL) controlled assembly by site specific interactions of avidin and biotin. Enzymatic oxidative decolorization of soluble aniline blue was employed to evaluate the catalytic performance of immobilized CPO (I-CPO). The decolorization efficiency of dye by I-CPO was higher than 90% within 10 min. Moreover, I-CPO displayed improved thermostability and kept higher activity in wider pH range and in the presence of high concentration of oxidant (H 2 O 2 ) compared to that of free enzyme. When incubated at 70 °C for an hour, 42.35% of the activity of immobilized enzyme was reserved compared with that at 25 °C, whereas free CPO remained only 8.46%. The tolerance to H 2 O 2 was improved from 2 mmol L −1 to 4 mmol L −1 , and the suitable pH range become wider. The relative activities of I-CPO in the 20 times use maintained 62% of that in the first run. The enzymatic kinetic constants confirmed that the entrapping CPO into this well-defined architecture could reduce diffusional limitation of reactants and products.

Using Sperm chromatin Staining Techniques as a Predictive Diagnostic Tool for Male Infertility

Background: It is a scientific fact today that routine seminal analysis is only a poor predictor of male reproductive potentiality, so that there is an increased need for finding out new functional tests in the assessment of male infertility. Objective: The aim of the present study was to investigate the predictive value of sperm chromatin integrity assays as a complementary tool to assist in diagnosis of infertility. Methodology: This study was carried out between January 2013 and November 2013 including one hundred (100)selected infertile men who attended to fertility Center in Al-Sadr Medical City. The study also included twenty (20) healthy fertile volunteers as a control. Aniline blue (AB) staining was used to differentiate between normal condensed chromatin (unstained) and abnormal decondensed chromatin (blue), while toluidine blue (TB) was use to differentiate between normal intact sperm DNA (blue) and abnormal damaged sperm DNA (purple), this is the first study in Iraq that employed (TB) to assess sperm DNA fragmentation. Results: The results showed that there was a high significant difference (p<0.01) in sperm chromatin decondensation and sperm DNA damage between infertile and fertile men. The study also revealed a significantdifference (p<0.05) in sperm DNA damage between normozoospermic and fertile men, but no significant difference (p>0.05) was found in the sperm chromatin condensation. Both sperm chromatin condensation and sperm DNA damage showed a significant positive correlation with sperm morphology, and a significant negative correlation with sperm concentration, motility and progressive motility. Conclusions: The study concluded that sperm DNA fragmentation (by TB staining) has good predictive value in discriminating infertile and fertile men over seminal fluid analysis. Recommendations: TB and AB staining of sperm chromatin can be used in the course of infertility diagnosis. However, more investigations may be needed to confirm the discriminating power of sperm chromatin condensation assays (by AB staining) to be ready to use in the routine workup in the fertility clinics.

Salinity-induced changes in the root development of a northern woody species, Cornus sericea

To study the salt-induced changes in root development, the roots of Cornus sericea L. exposed to NaCl (0, 50, and 100 mmol·L−1) for 4 weeks were sectioned at 1, 3, 5, 10, 15, and 25 cm from the tip, after having determined their increase in length. Observations were made on longitudinal and transverse sections from embedded tissues (stained with toluidine blue) and fresh tissues (examined under UV light or stained with Sudan red 7B or phloroglucinol-HCl). A modified outermost cortical layer was observed in C. sericea, suggesting the presence of a hypodermis or exodermis. While suberin was identified in the wall of the hypodermal cells, the presence of Casparian bands in these walls could not be clearly detected in sections stained with or without berberine-hemisulphate and aniline blue. Although the overall sequence of development of root tissues in C. sericea did not appear to be modified in salt-treated plants, the development of the hypodermis and endodermis occurred closer to the root tip in salt-treated plants. These altered root characteristics could have contributed to improve the salinity tolerance of the plants by limiting the amount of Na+ and (or) Cl− reaching the shoots. Our results will provide useful information for the selection of boreal forest plants adequate for land reclamation, a major challenge for the oil sand industry faced with large areas of salt-affected land.

Vitamin C attenuates detrimental effects of diabetes mellitus on sperm parameters, chromatin quality and rate of apoptosis in mice

ObjectivesThe main goal was to examine the protective effects of vitamin C on sperm parameters, sperm chromatin condensation and apoptosis in experimentally-induced diabetic mice. Study design28 adult Syrian mice were divided into 4 groups. In Group1, the mice were diabetic that received a single dose of Streptozocin (STZ) (200mg/kg) intra-peritoneally (ip). Group 2 was included diabetic mice that received vitamin C (10mg/kg/daily, ip). Mice in group 3 were received vitamin C and group 4 was considered as control. After 35 days, sperm analysis was done accordingly. To asses sperm chromatin and DNA quality, we used aniline blue (AB), toluidine blue (TB), chromomycin A3 (CMA3), acridine orange (AO) and terminal transferase mediated deoxyuridine triphosphate biotin end labeling (TUNEL) tests. ResultsAll of the sperm parameters (count, motility, morphology and viability) had significant reduction in diabetic mice but, the data showed a significant increase in all of the sperm parameters in diabetic+vitamin C when compared with diabetic and control animals (P<0.05). There were significant differences (P<0.001) between groups regarding to TB staining (48.8±5.92 vs 34.3±4.13), AO test (35.9±6.11 vs 20.8±2.89) and TUNEL test (39.42±7.18 vs 22.00±3.65) in diabetic and diabetic+vitamin C groups, respectively. Nevertheless, in CMA3 and AB staining assays, there were not any significant differences between different groups. ConclusionVitamin C, as a potent antioxidant, can attenuate detrimental effects of diabetes mellitus on the sperm parameters, chromatin quality and apoptosis in an experimental model.

Sperm concentration, hyaluronic acid-binding capacity, aneuploidy and persistent histones in testicular cancer.

Are the quantitative and qualitative characteristics of semen samples of patients with testicular cancer (TC), prior to anticancer therapy, different from infertile oligozoospermic (IO) and normozoospermic (NZ) age-matched men? Sperm concentration in TC patients was significantly decreased with no difference in estimated numerical chromosome aberrations and nuclear decondensation compared with NZ men, while the infertile, oligozoospermic men had significantly poorer sperm qualitative characteristics versus the TC group overall and oligozoospermic patients with TC. Spermatogenesis is altered in TC patients at the time of diagnosis. However, the mechanism responsible for the decreased semen quantity in patients with TC is not well understood. Anticancer treatment may have gonadotoxic side effects and post-treatment fertility cannot be predicted. Before commencing anticancer treatment, cryopreservation may be suggested to preserve fertility but there are no data regarding the risk of genetic aberrations in these sperms. This is a cross-sectional study examining semen from 28 patients with TC, 20 IO and 20 NZ age-matched men attending the Andrology Center and the Sperm Cryopreservation Laboratory of the Medical and Health Science Center, University of Debrecen. Semen samples from patients with TC were collected after orchidectomy, but prior to anticancer treatment. Semen samples from TC patients recruited over a period of 4 years were studied. Based on their sperm concentration, TC patients were subgrouped into an oligozoospermic TC (TCO) and a normozoospermic TC group. For statistical analysis, the normal group (NZ + IO) comprised non-tumorous NZ and IO men. The ejaculates were assessed as per World Health Organization guidelines. Hyaluronic acid (HA)-binding capacity was the functional test. To determine the numerical chromosome aberrations, we used multi-color fluorescence in situ hybridization. Aniline blue (AB) staining was performed as a nuclear decondensation marker test. The results did not reveal any significant difference in disomy of sex chromosomes and chromosome 17, diploidy and estimated numerical chromosome aberrations and AB staining results upon comparing the NZ and TC groups, although the sperm concentration (P < 0.001) and HA-binding capacity (P < 0.001) were lower in the TC group. Estimated numerical chromosome aberrations (P < 0.001), AB staining (P < 0.001) and HA-binding capacity (P = 0.019) were lower in the infertile, oligozoospermic group when compared with the patients with TC. The TCO group had significantly better results in every examined parameter than the infertile, oligozoospermic group. In the non-tumorous control group (NZ + IO), a significant (P < 0.001) correlation (Spearman's rho = r) was found between sperm concentration and aneuploidy rate (r = -0.642), AB staining (r = -0.876) and HA binding (r = 0.842); the HA-binding capacity was related to the aneuploidy rate (r = -0.678) and the AB staining (r = -0.811); and there was significant correlation between aneuploidy and AB staining (r = 0.559). In the TC group, apart from the negative correlation between sperm concentration and estimated chromosomal aberrations (r = -0.642), no other correlations were observed. Data on confounders influencing sperm characteristics, such as smoking, occupational or environmental hazards, alcoholism, co-morbidities and other andrological conditions, were not collected. This is the first study to demonstrate that sperm qualitative characteristics in anticancer therapy naïve oligozoospermic TC patients differ significantly from those in IO men and do not differ from those in NZ men. Our results need to be validated in similar groups of men and in other patient groups with cancer where cryopreservation is advisable. This research was supported by the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of TÁMOP-4.2.4.A/2-11/1-2012-0001 'National Excellence Program'. The authors have no conflict of interest to declare.

A double-blinded comparison of in situ TUNEL and aniline blue versus flow cytometry acridine orange for the determination of sperm DNA fragmentation and nucleus decondensation state index

The impact of sperm DNA fragmentation on assisted reproductive technology (ART) successes, in terms of outcome, is now established. High levels of DNA strand breaks severely affect the probability of pregnancy. The importance of sperm nucleus condensation in early embryogenesis and, subsequently, on the quality of the conceptus is now emerging. In this article we have compared in situ analyses with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) (for DNA fragmentation) with aniline blue (AB) (for nucleus decondensation), versus flow cytometry (FC) after acridine orange staining, in a double-blinded analysis. In our hands, TUNEL and acridine orange give perfectly comparable results. For decondensation the results are also comparable, but the double-stranded green fluorescence obtained with acridine orange seems to slightly underestimate the decondensation status obtained with AB.

Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet give rise to the world's largest flowers

Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis. Serial sectioning and staining with non-specific dyes, periodic-Schiff's reagent and aniline blue were employed in order to characterize the structure of the endophyte across a phylogenetically diverse sampling. A previously identified difference in the nuclear size between Rafflesiaceae endophytes and their hosts was used to investigate the morphology and development of the endophytic body. The endophytes generally comprise uniseriate filaments oriented radially within the host root. The emergence of the parasite from the host during floral development is arrested in some cases by an apparent host response, but otherwise vegetative growth does not appear to elicit suppression by the host. Rafflesiaceae produce greatly reduced and modified vegetative bodies even when compared with the other holoparasitic angiosperms once grouped with Rafflesiaceae, which possess some vegetative differentiation. Based on previous studies of seeds together with these findings, it is concluded that the endophyte probably develops directly from a proembryo, and not from an embryo proper. Similarly, the flowering shoot arises directly from the undifferentiated endophyte. These filaments produce a protocorm in which a shoot apex originates endogenously by formation of a secondary morphological surface. This degree of modification to the vegetative body is exceptional within angiosperms and warrants additional investigation. Furthermore, the study highlights a mechanical isolation mechanism by which the host may defend itself from the parasite.

Comparison of different chromatin staining techniques for bull sperm

Abstract. Morphological analysis of semen is a very important step in fertility assessment, but many semen defects are not detectable at the morphological level. These include pathological changes in sperm chromatin structure. During mammalian spermiogenesis, histone proteins associated with DNA structure are replaced by specific protamines, with which chromatin does not form nucleosomal complexes. In the fully developed, mature sperm, the histones are replaced with protamines. Disruptions of nucleoprotein structure can be treated as possible indicators of the biological value of spermatozoa. The experimental material consisted of sperm from one-and-a-half-year-old bulls, isolated post mortem from the tail of the epididymis. The smears were stained with silver nitrate (AgNO3), acridine orange (AO), aniline blue (AB) and chromomycin A3 (CMA3). Sperm dimensions largely depend on individual variability among the bulls. In most cases, differences in sperm dimensions were identified between individuals, which was confirmed in the statistics. Sperm with elevated, abnormal histone levels were proportionally quite scarce (1.4 %). Studies of nuclear proteins in the context of infertility demonstrate the important influence of normal chromatin structure on sperm functions.

Investigation of the association between the outcomes of sperm chromatin condensation and decondensation tests, and assisted reproduction techniques.

The main purpose of this prospective study is to examine possible influences of abnormalities of sperm nuclear condensation and chromatin decondensation with sodium dodecyl sulphate (SDS)-EDTA on outcomes of intrauterine insemination (IUI) or intracytoplasmic sperm injection (ICSI) cycles. Semen samples from 122 IUI and 236 ICSI cycles were evaluated. Before semen preparation for IUI or ICSI, basic semen analysis was performed and a small portion from each sample was spared for fixation. The condensation of sperm nuclear chromatin was evaluated with acidic aniline blue, followed by sperm chromatin decondensation by SDS-EDTA and evaluation under light microscope. Ongoing pregnancy rate was 24% and 26.2% in the IUI and ICSI groups respectively. The chromatin condensation rate was significantly higher in the ongoing pregnancy-positive group compared to the negative group, both in IUI (P=0.042) and ICSI groups (P=0.027), and it was positively correlated with ongoing pregnancy rate in both IUI and ICSI groups (P=0.015, r=0.214 and P=0.014, r=0.312 respectively). Chromatin decondensation rates were not significantly different in neither of the groups. These results indicate that IUI and ICSI outcome is influenced by the rate of spermatozoa with abnormal chromatin condensation. Sperm chromatin condensation with aniline blue is useful for selecting assisted reproduction techniques (ART) patients.

Characterization and sorting of flow cytometric populations in human semen

Human semen is a complex biological matrix. It contains mature spermatozoa, immature germ cells, residual apoptotic bodies and, in some cases, epithelial cells and leucocytes. Hence, one of the challenges in applying flow cytometry in spermatology is the correct recognition of spermatozoa and their separation from signals of other semen cells/elements. In this study, we show that semen spermatozoa are included in a well-defined, flame-shaped FSC/SSC region (FR), by demonstrating that the count of the spermatozoa contained in such region overlaps that obtained by microscopy in the same samples. In FR, nuclear staining of semen samples reveals three different populations: unstained, brighter and dimmer. Unstained elements were previously characterized as apoptotic bodies of testis origin and the brighter elements represent the majority of semen spermatozoa, whereas the composition and the origin of the population with a lower nuclear staining is less clear, albeit we have previously shown that all the elements constituting it are positive for TUNEL. In this study, we sorted all the elements contained in FR region and demonstrated that the dimmer elements are spermatozoa. To further characterize dimmer spermatozoa, we evaluated apoptotic caspases and chromatin immaturity, the latter detected by aniline blue (AB) and chromomycin A (CMA3) staining. We found that caspases were much more expressed in the dimmer spermatozoa (71.4 ± 18.8%) than in the brighter (46.7 ± 15.1%), whereas similar amounts of spermatozoa with chromatin immaturity were found in both populations (brighter, AB: 48.2 ± 19.5%; CMA3: 48.5 ± 20.4% and dimmer, AB: 43.4 ± 19.8%; CMA3: 36.1 ± 18.0%). Hence, the role of apoptosis in generating dimmer spermatozoa and their DNA fragmentation appears clear, whereas the involvement of defects during the chromatin packaging remains elusive.

Spectroscopy of Charge Transfer Complexes of Aniline Blue

Charge transfer (CT) complexes of aniline blue (AB) were prepared with standard organic acceptors such as TCNQ, TCNE, DDQ, and chloranil and studied with UV-VIS-NIR spectra showing σ→π* and π→π* transitions along with free-carrier absorption due to scattering of light particles which has been found involving phonon–photon, electron–photon, and electron–electron scattering. Infrared spectra contain half power beta density due to hopping conduction and an asymmetric band corresponding to A(k) = A0k exp(−bk) as absorption associated with diffraction of IR light from the crystalline particles.

The impact of semen quality, occupational exposure to environmental factors and lifestyle on recurrent pregnancy loss

This study aimed to investigate the effect of male semen quality, occupational exposure, and lifestyle on recurrent pregnancy loss (RPL). Information on couples' occupational exposure and lifestyle was collected using a detailed questionnaire from 68 RPL couples and 63 randomly selected healthy controls. Semen parameters were estimated by computer-assisted sperm analysis, and sperm nuclear status was detected with aniline blue (AB) staining. Patients in the RPL group had significantly lower viability, normal morphology, and total progressive motility of sperm, and a higher mean percentage of AB staining positive sperm compared with those of controls (P < 0.05). There were no differences in sperm concentration, and motility between the groups (P > 0.05). Significant odds ratio (OR) was found when occupational exposure and unhealthy habits were superimposed (OR: 11.965, P = 0.005). In addition to standard female factors for evaluating the risk for RPL, the use of male factors should also be taken into consideration. We found that sperm quality, occupational exposure, and lifestyle are factors that affect RPL. Consequently, occupational exposure and lifestyle factors should constitute an important section of questionnaires given to patients, and these factors should be evaluated by a clinician or trained staff.

Association of sperm-nucleoprotein transition with sperm parameters and embryo development

Objective To investigate the correlation between sperm-nucleporotein transition and sperm parameters and embryo development,also to evaluate the influence of pregnancy out comes of assisted reproductive technology (ART).Methods Sperm-nucleoprotein transition assay of a total of 676 patients underwent ART treatment were detected by aniline blue staining,and the correlation analysis between spermnucleoprotein transition and sperm parameters,DNA damage,acrosin activity,fertilization rate,cleavage rate,quality of early embryo development as well as blastocyst formation rate was performed.Results The sperm concentration,(a+b) ％ sperm,sperm count and acrosin activity was (66.5±4.6) × 109/L,(149.2±9.9)×109/L,(51.2±1.3)％ and (72.2±3.3) mU/106 sperm in abnormal group,and (91.9±2.7) ×109/L,(240.0±8.0) ×109/L,(57.3±0.8)％ and (85.7±1.9) mU/106 sperm in normal group,which reached significant difference (P＜0.01).DNA fragmentation index (DFI) was (17.3± 1.0)％ in abnormal group,which was significantly higher than (14.6±0.5)％ in normal group.The cleavage rate of 95.0％,D3/D5 high quality embryo rates of 34.2％ and 1.28％,D5 blastocyst formation rate and the total rate of blastocyst formation rate of 22.4％ and 38.6％ in abnormal group,which were significantly lower than that in normal group (96.9％,38.2％,2.70％,27.9％ and 46.4％) (P＜0.01).The rate of spontaneous abortion was 12.3％ in abnormal group,which was significantly higher than that in normal group (4.7％) (P＜0.01).However,there was no significant difference in biochemical pregnancy rate and ectopic pregnancy rate between the 2 groups (P＞0.05).Conclusions Sperm-nucleoprotein transition was positively related with sperm parameters,DNA damage,acrosin activity,and also has an adverse effect on embryo development and the outcomes of ART.It is suggested that the sperm-nucleoprotein transition should be detected before ART. Key words: Sperm-nucleoprotein transition; Sperm parameters ; Embryo development; Pregnancy outcome

γ-Aminobutyric acid (GABA) homeostasis regulates pollen germination and polarized growth in Picea wilsonii

γ-Aminobutyric acid (GABA) is a four-carbon non-protein amino acid found in a wide range of organisms. Recently, GABA accumulation has been shown to play a role in the stress response and cell growth in angiosperms. However, the effect of GABA deficiency on pollen tube development remains unclear. Here, we demonstrated that specific concentrations of exogenous GABA stimulated pollen tube growth in Picea wilsonii, while an overdose suppressed pollen tube elongation. The germination percentage of pollen grains and morphological variations in pollen tubes responded in a dose-dependent manner to treatment with 3-mercaptopropionic acid (3-MP), a glutamate decarboxylase inhibitor, while the inhibitory effects could be recovered in calcium-containing medium supplemented with GABA. Using immunofluorescence labeling, we found that the actin cables were disorganized in 3-MP treated cells, followed by the transition of endo/exocytosis activating sites from the apex to the whole tube shank. In addition, variations in the deposition of cell wall components were detected upon labeling with JIM5, JIM7, and aniline blue. Our results demonstrated that calcium-dependent GABA signaling regulates pollen germination and polarized tube growth in P. wilsonii by affecting actin filament patterns, vesicle trafficking, and the configuration and distribution of cell wall components.

Molecular morphology and function of bull spermatozoa linked to histones and associated with fertility

Sub-par fertility in bulls is influenced by alterations in sperm chromatin, and it might not be solved with increased sperm concentration in artificial insemination. Appropriate histone retention during sperm chromatin condensation plays critical roles in male fertility. The objective of this study was to determine failures of sperm chromatin condensation associated with abnormal persistence or accessibility of histones by aniline blue (ANBL) test, expression levels, and cellular localizations of one variant and two core histones (H3.3, H2B, and H4 respectively) in the spermatozoa of low-fertility (LF) vs high-fertility (HF) bulls. The expression levels and cellular localizations of histones in spermatozoa were studied using immunoblotting, immunocytochemistry, and staining methods. The bioinformatics focused on the sequence identity and evolutionary distance of these proteins among three mammalian species: bovine, mouse, and human. We demonstrated that ANBL staining was different within the LF (1.73 (0.55, 0.19)) and HF (0.67 (0.17, 0.06)) groups (P<0.0001), which was also negatively correlated with in vivo bull fertility (r=-0.90, P<0.0001). Although these histones were consistently detectable and specifically localized in bull sperm cells, they were not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and were evolutionarily conserved in bulls, mice and humans. The H2B variants were more conserved between bulls and humans, than in mice. In conclusion, we showed that H2B, H3.3, and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related to bull fertility.

Chromatin structure analysis of spermatozoa from reciprocal chromosome translocation (RCT) carriers with known meiotic segregation patterns

The presence of reciprocal chromosome translocations (RCTs), as well as sperm chromatin disturbances, is known to exert negative influence on male fertility. The aim of this study was to identify an association between chromosome structural rearrangements in male RCT carriers and sperm seminological parameters (concentration, motility, morphology), chromatin status (fragmentation and maturity), meiotic segregation pattern and observed chromosomal hyperhaploidy. Sperm samples originated from ten male RCT carriers with reproductive failure/success. TUNEL assay (DNA fragmentation) and chromomycin A3 (CMA3)/aniline blue (AB) staining (chromatin maturity) were used to analyze sperm chromatin status while fluorescent in situ hybridization (FISH) was applied to observe meiotic segregation patterns and hyperhaploidy in spermatozoa. We found that the mean level of sperm DNA fragmentation in the RCT carrier group (18.0±11.9%) was significantly higher (p=0.0006) than the mean of the control group (7.5±4.3%). There was no correlation observed between sperm DNA fragmentation levels (5.6–38.0%) and the frequency of genetically normal/balanced gametes (34.3–62.4%), sperm seminological quality or revealed reproductive failure. In contrast, a correlation between the frequencies of genetically normal/balanced spermatozoa and of gametes with mature chromatin was observed (CMA3: R=0.4524, p=0.2604; AB: R=0.5238, p=0.1827). A statistically significant increase in the hyperhaploidy level of selected chromosomes in all analyzed RCT carriers was documented but was not correlated to sperm seminology or fertility status. Further evaluation and additional assays toward sperm chromatin quality assessment in RCT carriers is suggested to explain the complexity of genomic structural rearrangements and its possible relevance to reproductive success or failure.

Optimization, equilibrium, kinetic modeling, and thermodynamic studies of biosorption of aniline blue by the dead biomass of Aspergillus fumigatus

The effect of biosorbent concentration, initial pH, temperature, agitation rate, adsorption time, and initial dye concentration was studied for the biosorption of aniline blue dye using dead fungal biomass of Aspergillus fumigatus. The maximum biosorption of aniline blue was observed at the sorbent concentration of 1 g/L, initial pH 10, temperature 30°C, agitation rate 160 rpm, and initial dye concentration of 50 mg/L. The experimental data were analyzed using Freundlich, Temkin, and Scatchard equilibrium isotherm models out of which the Freundlich isotherm (R2 = 0.98) was found to best fit the experimental data. Thermodynamic parameters such as enthalpy change (−3.08 kJ/mol) and entropy change (8.32 J/mol K) were also calculated. The biosorption kinetics of Aniline blue was found to obey pseudo-second-order kinetic model (R2 = 0.967). The results showed that biosorption was favorable and spontaneous, thus, indicating the positive affinity of the dye towards the adsorbent. The characteristics of the fungal biosorbent were studied using Fourier transform-infrared spectroscopy (FTIR) and scanning electron microscopic (SEM).

Bees’ subtle colour preferences: how bees respond to small changes in pigment concentration

Variability in flower colour of animal-pollinated plants is common and caused, inter alia, by inter-individual differences in pigment concentrations. If and how pollinators, especially bees, respond to these small differences in pigment concentration is not known, but it is likely that flower colour variability impacts the choice behaviour of all flower visitors that exhibit innate and learned colour preferences. In behavioural experiments, we simulated varying pigment concentrations and studied its impact on the colour choices of bumblebees and honeybees. Individual bees were trained to artificial flowers having a specific concentration of a pigment, i.e. Acridine Orange or Aniline Blue, and then given the simultaneous choice between three test colours including the training colour, one colour of lower and one colour of higher pigment concentration. For each pigment, two set-ups were provided, covering the range of low to middle and the range of middle to high pigment concentrations. Despite the small bee-subjective perceptual contrasts between the tested stimuli and regardless of training towards medium concentrations, bees preferred neither the training stimuli nor the stimuli offering the highest pigment concentration but more often chose those stimuli offering the highest spectral purity and the highest chromatic contrast against the background. Overall, this study suggests that bees choose an intermediate pigment concentration due to its optimal conspicuousness. It is concluded that the spontaneous preferences of bees for flower colours of high spectral purity might exert selective pressure on the evolution of floral colours and of flower pigmentation.