Nanoparticle-plant interactions: Physico-chemical characteristics, application strategies, and transmission electron microscopy-based ultrastructural insights, with a focus on stereological research.

Ensuring global food security is pressing among challenges like population growth, climate change, soil degradation, and diminishing resources. Meeting the rising food demand while reducing agriculture's environmental impact requires innovative solutions. Nanotechnology, with its potential to revolutionize agriculture, offers novel approaches to these challenges. However, potential risks and regulatory aspects of nanoparticle (NP) utilization in agriculture must be considered to maximize their benefits for human health and the environment. Understanding NP-plant cell interactions is crucial for assessing risks of NP exposure and developing strategies to control NP uptake by treated plants. Insights into NP uptake mechanisms, distribution patterns, subcellular accumulation, and induced alterations in cellular architecture can be effectively drawn using transmission electron microscopy (TEM). TEM allows direct visualization of NPs within plant tissues/cells and their influence on organelles and subcellular structures at high resolution. Moreover, integrating TEM with stereological principles, which has not been previously utilized in NP-plant cell interaction assessments, provides a novel and quantitative framework to assess these interactions. Design-based stereology enhances TEM capability by enabling precise and unbiased quantification of three-dimensional structures from two-dimensional images. This combined approach offers comprehensive data on NP distribution, accumulation, and effects on cellular morphology, providing deeper insights into NP impact on plant physiology and health. This report highlights the efficient use of TEM, enhanced by stereology, in investigating diverse NP-plant tissue/cell interactions. This methodology facilitates detailed visualization of NPs and offers robust quantitative analysis, advancing our understanding of NP behavior in plant systems and their potential implications for agricultural sustainability.

Human Umbilical Vein Endothelial Cells as a Versatile Cellular Model System in Diverse Experimental Paradigms: An Ultrastructural Perspective.

Human umbilical vein endothelial cells (HUVECs) are primary cells isolated from the vein of an umbilical cord, extensively used in cardiovascular studies and medical research. These cells, retaining the characteristics of endothelial cells in vivo, serve as a valuable cellular model system for understanding vascular biology, endothelial dysfunction, pathophysiology of diseases such as atherosclerosis, and responses to different drugs or treatments. Transmission electron microscopy (TEM) has been a cornerstone in revealing the detailed architecture of multiple cellular model systems including HUVECs, allowing researchers to visualize subcellular organelles, membrane structures, and cytoskeletal elements. Among them, the endoplasmic reticulum, Golgi apparatus, mitochondria, and nucleus can be meticulously examined to recognize alterations indicative of cellular responses to various stimuli. Importantly, Weibel-Palade bodies are characteristic secretory organelles found in HUVECs, which can be easily distinguished in the TEM. These distinctive structures also dynamically react to different factors through regulated exocytosis, resulting in complete or selective release of their contents. This detailed review summarizes the ultrastructural features of HUVECs and highlights the utility of TEM as a pivotal tool for analyzing HUVECs in diverse research frameworks, contributing valuable insights into the comprehension of HUVEC behavior and enriching our knowledge into the complexity of vascular biology.

Cryopreservation of chicken blastodermal cells and their quality assessment by flow cytometry and transmission electron microscopy.

The goal of this study was to evaluate effect of slow freezing and vitrification methods on the viability of chicken blastodermal cells (BCs). Proper aliquot of isolated BCs were diluted in the freezing medium composed of 10% DMSO and frozen in the freezing vessel BICELL to reach desired temperature up to -80°C. Then samples were immersed in liquid nitrogen. Other cell aliquot was vitrified in solution containing 10% DMSO and samples were immediately immersed in the liquid nitrogen. The viability of fresh and frozen/thawed BCs was evaluated using Trypan blue method and flow cytometry. Flow cytometry analysis was provided by DRAQ5 dye in combination with Live-Dead kit. Overall, this technique provides both quantitative and qualitative information about BCs. Results obtained from Trypan blue method showed significant differences (P < 0.05) between control (8.37 ± 1.04%) slow freezing (83.73 ± 2.72%) and vitrification group (84.39 ± 1.77%) in the percentage of Trypan blue positive (necrotic) BCs. Moreover, differences (P < 0.05) between control and slow freezing (5.08 ± 1.94%, 73.31 ± 3.90%) and control and vitrification group (2.97 ± 0.30%, 79.02 ± 1.56%) in results on portion of necrotic cells (DRAQ5+ /LD+ ) analyzed by flow cytometry were also observed. The large percentage of necrotic BCs was found in all freezing methods. However, based on ultrastructural analysis, our study showed, that BCs contain lipid granules which prevent successful freezing even though different methods of cryopreservation were used. Thus, freezing of BCs probably required subsequent culture to eliminate lipid droples and yolk granules in the cells, which could possibly improve the success. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:778-783, 2018.

In vitro effect of various cryoprotectants on the semen quality of endangered Oravka chicken.

We aimed to compare the effect of three different permeating cryoprotectants on the post-thaw spermatozoa quality. Pooled semen from Oravka cock line (n = 6) was diluted in Kobidil+ extender and frozen in cryoprotectant solutions containing 8% dimethylsulfoxide (DMSO), 8% ethylene glycol (EG) or 8% glycerol (GL) in liquid nitrogen vapours before being plunged into the liquid nitrogen. Spermatozoa motility parameters were assessed in vitro after freezing-thawing by a computer-assisted semen analysis (CASA) system and viability status was examined using fluorescent probes. The lower percentage (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing were obtained in all experimental groups (DMSO, EG, GL) compared with the control. Significant (P < 0.05) differences in total motility and progressive movement between GL and DMSO, EG groups were observed. However, the higher number (P < 0.05) of acrosome damaged spermatozoa was found in the DMSO and EG groups and no significant differences were observed in the GL group compared with the control. Differences (P < 0.05) between experimental groups and the control in the results of spermatozoa necrosis were observed. No significant differences in the percentage of apoptotic spermatozoa were found between control and experimental groups. However, significant differences (P < 0.05) in number of live and necrotic spermatozoa between GL and DMSO, EG groups were examined. The findings of the present study indicate that glycerol seems to be suitable for semen cryopreservation in the gene banks. In addition, fertility evaluation in vivo is needed in order to evaluate the possible contribution for the bank of animal genetic resources.

Detection of macrophages in rabbit semen and their relationship with semen quality.

We aimed at the evaluating the occurrence of macrophages in rabbit semen and finding possible relationship between macrophage concentration and spermatozoa quality. The concentration of macrophages in semen samples from broiler rabbit males of lines M91 and P91 (n = 30) without overt evidence of genital tract infections was determined using monocyte/macrophage lineage antigen CD14 and flow cytometry. Then the rabbits were assigned into three groups according to the macrophage concentration in semen (MΦ1 group with less than 1 × 106 macrophages/mL, MΦ2 group with 1.5-3.5 × 106 macrophages/mL and MΦ3 group with more than 8 × 106 macrophages/mL). Spermatozoa viability parameters such as occurrence of apoptotic (Yo-Pro-1) and dead/necrotic (propidium iodide) spermatozoa and plasma membrane integrity (PNA-Fluos) were evaluated using flow cytometry. Sperm motility parameters were determined by CASA (Computer Assisted Semen Analysis). Ultrastructural detection of macrophages was performed using transmission electron microscopy. Spermatozoa fertility potential was examined after intravaginal artificial insemination of rabbit doses. Significantly higher proportions of the apoptotic and necrotic spermatozoa and spermatozoa with lower plasma membrane integrity were revealed in the MΦ3 group compared to MΦ1 and MΦ2 groups. The percentage value of total motility and progressive movement was significantly highest in the MΦ1 group, whilst lowest in the MΦ3 group. The conception rate and the kindling rate were significantly decreased in the group with the highest macrophage concentration (MΦ3). Based on our results we can conclude that the abundance of seminal macrophages in the rabbit semen may be closely associated with poor spermatozoa quality.

Factors Influencing Non-Persistence with Antiplatelet Medications in Elderly Patients After Ischaemic Stroke.

OBJECTIVES: This study investigated the extent of, and patient-related characteristics for, non-persistence with antiplatelet therapy during follow-up in elderly patients after their first ischaemic non-cardioembolic stroke. METHODS: A database of the largest health insurance provider in the Slovak Republic was used to assemble the study cohort of 4319 patients (56.8% were women) aged ≥65 years in whom antiplatelet therapy was initiated following a hospital-based diagnosis of stroke during the period 1 January 2010 to 31 December 2010. Patients were followed for 3 years from the date on which the first prescription of antiplatelet medication was recorded. Patients with a 6-month treatment gap without antiplatelet medication prescription were designated as non-persistent, and the Cox proportional hazards model was used to identify predictors of non-persistence. RESULTS: At the end of the 3-year follow-up period, 1184 (27.4%) patients were considered non-persistent with antiplatelet medication. In 1244 (28.8%) patients, a switch in the use of a particular antiplatelet drug was registered during this follow-up period. Female sex (hazard ratio [HR] 1.25) was associated with increased risk of non-persistence. In contrast, factors associated with lower probability of non-persistence were age ≥75 years (HR 0.72), switch in antiplatelet medication use (HR 0.76), diabetes mellitus (HR 0.81), dementia (HR 0.69) and epilepsy (HR 0.69). CONCLUSIONS: Our results suggest that women, patients aged <75 years, and patients without certain comorbid conditions may need improved assistance in secondary prevention management after an ischaemic stroke.

The cryoprotective effect of Ficoll on the rabbit spermatozoa quality.

The aim of this study was to evaluate the effect of the addition of Ficoll 70 into the cryopreservation medium containing sucrose and dimethyl sulfoxide (DMSO) on rabbit spermatozoa characteristics following freezing/thawing. This large molecular weight polymer elevates the viscosity of medium and, therefore, could better protect spermatozoa during the freezing process. Only ejaculates of good initial motility (>80%) were used in the experiments. Heterospermic pools were diluted in a freezing medium composed of commercial diluent, 16% dimethyl sulphoxide (DMSO) and 2% sucrose (control) or in the same medium enriched with 4% Ficoll 70 (Ficoll) and frozen in liquid nitrogen vapours for 10 min before being plunged in liquid nitrogen. The quality of fresh and frozen/thawed spermatozoa samples was evaluated in vitro using the Computer Assisted Semen Analysis (CASA) system, fluorescent probes (peanut agglutinin (PNA)-Alexa Fluor®; annexin V-FLOUS) and by electron microscopy. Better cryoprotective effect was observed when Ficoll 70 was added, compared with the semen cryopreserved with sucrose and DMSO only. The higher values (P < 0.05) of motile and progressively moving spermatozoa immediately after thawing and at 30 min following incubation at 37°C were obtained in the Ficoll group. Moreover, the higher number (P < 0.05) of acrosome intact sperm was found in the Ficoll compared with the control group. Furthermore, no significant differences in kindling rates and number of pups born between frozen/thawed and fresh semen group were found. In conclusion, our study showed that the addition of Ficoll 70 might improve several characteristics of rabbit spermatozoa measured in vitro following freezing/thawing.