Flow cytometric analysis of apoptosis in cryoconserved chicken primordial germ cells.

Our research aimed to compare the effects of four cryoprotectants and four slow freezing programs on the viability and apoptosis of primordial germ cells (PGCs) in vitro. PGCs were collected from chicken embryonic blood at Hamburger and Hamilton (HH) stages 14-16 and purified by Percoll density gradient centrifugation and then subjected to cryopreservation. We applied microscopy to determine the survival of PGCs after trypan blue staining and flow cytometry to examine apoptosis and viability after annexin V kit staining. We also examined the functionality of cryopreserved PGCs in vivo. Significant differences in viability of PGCs determined via microscopy and flow cytometry were observed. The most unfavorable combination for slow freezing PGCs was program 3 and MIX H (10% DMSO and 5% glycerol in Hank's solution supplemented with 10% FBS) as the cryoprotectant (48.43 and 15.37% live and early apoptotic PGCs, respectively). The highest average percentage of live PGCs (93.1%) and the lowest percentage of early apoptotic PGCs (6.5%) were achieved by slow freezing PGCs in the presence of DMSO F (10% DMSO in FBS) via program 1. Therefore, this method was chosen for the in vivo test. Cryopreserved (group 1) and freshly isolated (group 2) PGCs were transfectedwith a pEGFP-N1 plasmid, cultured under antibiotic selection, and then injected into 3-day-old embryos. After 5 days of incubation, we identified the EGFP marker gene in the gonads of 40 and 45% of recipients in groups 1 and 2, respectively. This is the first study to apply flow cytometry to examine the apoptosis and viability of cryopreserved PGCs. The in vitro and in vivo findings showed that the developed PGC cryoconservation method, depending on slow freezing at the rate of 2°C/min (program 1) in the presence of 10% DMSO F, is an improvement over previous cryoconservation methods and may be a useful tool for the ex situ strategy of poultry biodiversity preservation.

Cryoconservation of Chicken Blastodermal Cells: Effects of Slow Freezing, Vitrification, Cryoprotectant Type and Thawing Method during In Vitro Processing.

Cryoconservation of blastodermal cells (BCs) can preserve genetic material for the future reconstruction of poultry breeds. The aim of our study was to compare the effects of three slow freezing programs and vitrification, different cryoprotectants (5% DMSO, 10% DMSO, or multi-component cryoprotectant (MC) and two thawing methods on the viability of chicken BCs. Significant differences in the survival of slowly frozen BCs using program 3 (2°C/min. to 0.4°C/min.) compared with programs 1 (1°C/min. to 0.3°C/min.) and 2 (4°C/min. to 0.3°C/min.) were observed. The percentage of live BCs was significantly higher after slow freezing in the presence of the MC compared with DMSO. The thawing method did not have a significant effect on the percentage of live BCs. We also observed significant differences in the survival rate of BCs after vitrification (81%) and slow freezing in the presence of 10% DMSO using program 3 (60%). The highest percentage of viable BCs was achieved by slow freezing with the MC using program 2 and thawing with method 1 (94%). The most unfavorable combination for BCs survival was slow freezing in 5% DMSO using program 3 and thawing with method 2 (58.3%). This is the first study to apply MC to the slow freezing of BCs. We also showed successful BCs vitrification.

Changes in Quail Blastodermal Cell Status as a Result of Selection.

Genetic selection over many years has significantly improved the growth rate of broilers and increased the number of eggs laid by egg laying chicken breeds. Selection has improved desired parameters, but has caused some negative effects as well. Adverse effects of selection may negatively affect embryonic development. The number of live and apoptotic blastodermal cells (BCs) at the X stage of embryogenesis may be a good indicator of changes in selected individuals. In this paper, a comparison of the number of live and apoptotic BCs was made for three lines of quail: Pharaoh (F33), meat-type line, selected for body weight; egg laying line (S33), selected for egg number; and laying line (S22), additionally selected (for 17 generations) for high yolk cholesterol content. Apoptotic BCs were separated by the magnetic activated cell sorting (MACS) method. The percentage of live and apoptotic BCs was different (P ≤ 0.01) for F33 (35.8% and 64.2%, respectively) and S33 (60.0% and 36.4%). The number of apoptotic BCs for F33 embryos (45,098) was higher (P ≤ 0.01) compared to the number of apoptotic BCs for S33 embryos (26,667). The selection for high yolk cholesterol content caused an increase (P ≤ 0.01) in the total number of BCs from 78,403 (S33) to 140,139 (S22). The percentage of apoptotic BCs was lower (P ≤ 0.01) in the S22 line (17.1%) compared to the S33 line (36.4%). The results showed that it is possible to evaluate the effects of selection in the early stage of embryonic development.

Purification and Transfection Methods of Chicken Primordial Germ Cells.

Primordial germ cells (PGCs) play a special role in the vertebrate life cycle since they are the precursors of germ cells through which the genome is passed to the next generations. PGCs are found in different locations and variable numbers in the chick embryo, as in other species, depending on the developmental stages. Here, we describe in detail a method based on the Percoll gradient, routinely used in our laboratory, allowing us to obtain from blood and gonad anlages significant numbers of viable PGCs which can be successfully cultured or efficiently genetically modified.

Assessing cytotoxicity and endoplasmic reticulum stress in human blood-brain barrier cells due to silver and copper oxide nanoparticles.

In recent years, it has been generally accepted that metal-based nanoparticles (NPs) may induce stress in the endoplasmic reticulum (ER), a key organelle where protein folding occurs. We examined ER stress in immortalized human cerebral microvascular cells (hCMEC/D3) after exposure to silver-NPs (Ag-NPs)- and copper oxide-NPs (CuO-NPs) induced toxicity at < 10 nm and < 40 nm or < 50 nm diameters, respectively. In cytotoxicity assessments, cells were exposed to different CuO-NPs (5-400 µg/mL) or Ag-NPs (1-10 µg/mL) concentration ranges for 24 h and 72 h, and tetrazole salt reduction assays (EZ4U) were performed. Also, Ag-NP or CuO-NP effects on cell proliferation, apoptosis (caspase 3/7 assays), and ER stress and cell morphology were evaluated. In ER stress assessments, RNA-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1a), and others stress factor mRNA levels were determined after 24 h treatment using Real-Time PCR. Increased stress sensors (IRE1a, PERK, and ATF6) mRNA levels were observed after exposure to Ag-NPs (< 10 and < 40 nm) or CuO-NPs (< 50 nm). We investigated the expression of tight junction (TJ) proteins (barrier junctions) and showed that both types of NP reduced of OCLN gene expression. Morphological changes were observed after Ag-NP or CuO-NP exposure using holotomographic microscopy. Our data suggest that Ag- and CuO-NPs should undergo future in vitro and in vivo toxicology studies, especially for downstream biomedical application and occupational risk assessments.

[The exposure routes of micro- and nanoplastics and their potential toxic effects on human health]. / Drogi ekspozycji mikro- i nanoplastiku oraz potencjalne toksyczne efekty ich dzialania na zdrowie czlowieka.

This article discusses the classification of micro- and nanoplastics (MNP), the routes of their exposure and the effects of MNP on the reproductive, respiratory, digestive and immune systems based on in vitro and in vivo studies, as well as available epidemiological data. The MNP can enter our body through inhalation, food or skin. The presence of microplastics (MP) in tap, bottled and deep sea water, as well as in sea salt, fruit and vegetables has been demonstrated. Due to their small size, MNP can be absorbed and easily distributed through the blood and lymphatic vessel system to tissues and organs. Recent studies have provided evidence of the accumulation of MNP in human lungs and even in the placenta. The accumulation of MNP in the body may have long-term effects and lead to health problems in humans, such as bronchitis, development of asthma, pulmonary fibrosis, inflammation and cancer. The information included in the article gives partial insight into how MNP may affect the human body. However, to fully assess the toxicity of MNP, comprehensive research is necessary, including standardization of MNP detection techniques and determination of the MNP content in food and water. It is also advisable to assess toxicokinetic parameters, as well as to determine the daily dose of exposure and interaction of MNP with various cells. Insufficient data on direct exposure to MNP in the work environment, as well as in other public places, constitutes a factor hindering the establishment of appropriate legal standards. In 2024, work on establishing the first act of EU law enabling the monitoring of MP in drinking water should be completed, which raises great hopes that in the future limit values for MNP in water and food and in workplaces will also be established. Med Pr Work Health Saf. 2024;75(1):81-96.

[Risk assessment and toxicity effects of materials used during additive manufacturing with FDM technology]. / Ocena zagrozen i dzialanie toksyczne materialów stosowanych podczas drukowania przestrzennego w technologii FDM.

This paper discusses the potential of additive printing, the risks it poses to users' health (including 3D printer operators) and the effects of chemical substances released during the printing based on the available in vitro and in vivo studies. It was shown that substances emitted during printing with the commonly used acrylonitrile butadiene styrene filament in additive manufacturing might have carcinogenic, hepatotoxic and teratogenic effects, as well as toxic effect on the respiratory system. The latest research on the mechanism of formation of particles and volatile organic compounds during 3D printing, the parameters affecting their potential emission, and trends in reducing these hazards are indicated. The need for the design of more environmentally friendly and less emissive printing materials, as well as strategies for prevention and individual and collective protection measures are emphasized. Users of 3D printers should be familiar with all possible aspects of the threats associated with the printing process. Insufficient data on direct exposure to chemicals and particles released during the use of filaments makes it difficult to build awareness of safe working practices. Of particular concern is the health impact of emitted chemicals and particles from thermally treated materials in one of the most popular technologies for 3D printing, i.e., fused deposition modelling. Exposure of the users to, e.g., plasticizers added to filaments occurs through a variety of routes, by absorption through the skin, by inhalation or ingestion. Available epidemiological data, as well as current experimental works, indicate that such exposure is a high risk of cardiovascular diseases, atherosclerosis in adults, and cardiac problems and metabolic disorders in children. This review, by identifying potential risk factors, may contribute to reducing the health loss of printer users and improving working conditions and safety, especially in enterprises where additive manufacturing technology is used. Med Pr Work Health Saf. 2024;75(2):159-171.

The effects of co-exposure to methyl paraben and dibutyl phthalate on cell line derived from human skin.

Data on the cumulative effects of chemical substances are necessary for the proper risk assessment, but their availability is still insufficient. The aim of the study was to evaluate the cytotoxic effect of methyl paraben (MePB) and dibutyl phthalate (DBP) on the cells of the skin line (A431) and to compare the cytotoxic effects of the tested substances after single application to A431 cells with the effects of an equimolar/equitoxic (1:1) binary mixture of these compounds as well as their mixtures in ratio 1:3: and 3:1. On the basis of the obtained results, it was found that there were interactions between the tested compounds in terms of cytotoxic effect on A431, assessed on the basis of metabolic activity of cells (MTT test) and integrity of their cell membranes (NRU test). The obtained values of synergy coefficients (SI) and isobolographic analysis indicate that between the tested chemicals in a two-component equimolar mixture (1:1) there is a synergism of action, which, at a high DBP content in the mixture (> 50%) turned into antagonism. Observations using a holotomographic microscope show morphological changes in A431 cells after exposure to both DBP and MePB separately and binary mixtures of these compounds, compared to untreated cells. The observed changes in cell morphology seem to be more pronounced when the cells are exposed to the binary mixtures of DBP and MePB than when exposed to these substances individually, which may confirm the synergy of cytotoxic activity between them (this phenomenon was observed for the higher of the tested concentrations in all tested proportions). It is important to consider such effects when considering the effects of cumulative exposure in the risk assessment in order not to underestimate the risk of adverse effects associated with exposure to chemical mixtures.

Cryoconservation of embryonic cells and gametes as a poultry biodiversity preservation method.

A report of the Food and Agriculture Organization of the United Nations (FAO) from 2000 claims that 9% of the global farm animal population is in a critical condition and 39% is threatened with extinction. Production efficiency, exploitation and conservation of animal genetic resources are crucial not only for the global economy, but also for the environment. As many as 30% of poultry breeds are threatened with extinction and 9% have already gone extinct. To preserve the genetic resources in situ methods are used, however, they need to be supported by an ex situ strategy. This includes the storage of genetic material in liquid nitrogen under a deep freeze. This process can be performed by using electronically-controlled programs or vitrification. Data shows that usage of electronically-controlled programs leads increases cell viability. A good technique of cell culture and freezing methods will give a broad perspective for unlimited storage of genetic recourses, which in the future can be useful for the restoration of extinct species/breeds.

The in vitro toxicity evaluation of halloysite nanotubes (HNTs) in human lung cells.

Halloysite nanotubes (HNTs) have been increasingly used in many industrial and biomedical fields. Therefore, the assessment of risk and consequences of exposure to HNTs is very important to better protect human safety. This study aims to investigate the short- (24 or 72 h) and long-term (7 days) cytotoxic effects of HNTs at doses 10-200 µg/mL on human alveolar carcinoma epithelial cells (A549) and human bronchial epithelial cells (BEAS-2B). The effect of HNTs on cell viability, apoptosis, cell proliferation, oxidative/antioxidative status and cell morphology was evaluated. Our results showed that cytotoxicity of HNTs is dependent on dose, cell model and time of exposure. During the time of exposition toxic effects were intensified. To the best of our knowledge, this is the first study to use holo-tomographic microscopy (HTM) to visualise changes in cell morphology due to exposure from HNTs. We observed cells contraction, changes in the size and shape, cell surface folding and cytoplasmic vacuolization, peripheral arrangement of cell nuclei and even increase number of nucleus, which undoubtedly confirmed cytotoxic effect of HNTs at low doses (5 µg/mL and 25 µg/mL). Our results demonstrated that HTM technique provides a new insight into the assessment of HNTs toxicity. Further studies with different cell models are recommended to assess the toxic effect of HNTs on whole human body.

CRISPR/Cas9 gene editing in a chicken model: current approaches and applications.

Improvements in genome editing technology in birds using primordial germ cells (PGCs) have made the development of innovative era genome-edited avian models possible, including specific chicken bioreactors, production of knock-in/out chickens, low-allergenicity eggs, and disease-resistance models. New strategies, including CRISPR/Cas9, have made gene editing easy and highly efficient in comparison to the well-known process of homologous recombination. The clustered regularly interspaced short palindromic repeats (CRISPR) technique enables us to understand the function of genes and/or to modify the animal phenotype to fit a specific scientific or production target. To facilitate chicken genome engineering applications, we present a concise description of the method and current application of the CRISPR/Cas9 system in chickens. Different strategies for delivering sgRNAs and the Cas9 protein, we also present extensively. Furthermore, we describe a new gesicle technology as a way to deliver Cas9/sgRNA complexes into target cells, and we discuss the advantages and describe basal applications of the CRISPR/Cas9 system in a chicken model.

A Comparison of Empirical Correlations of Viscosity and Thermal Conductivity of Water-Ethylene Glycol-Al2O3 Nanofluids.

Because of their superb thermal conductivity, nanofluids are seen as new generation of cooling mediums in many engineering applications. It is well established that even a small amount of nanoparticles mixed with a base fluid may result in distinct thermal conductivity enhancement. On the other hand, addition of nanoparticles to the base fluid results in its substantial viscosity increase. Therefore, it is very difficult to evaluate the relative importance of viscosity and thermal conductivity of the nanofluid on convective heat transfer performance. In order to estimate such resultant impact properly, it is necessary to develop reliable correlation equations for predictions of these two thermophysical properties of nanofluids. In this paper, the thermal conductivity and dynamic viscosity of five fluids, i.e., pure water, ethylene glycol (EG) and three mixtures of water and EG with volume ratio of 40:60, 50:50 and 60:40 have been experimentally determined. The aforementioned fluids served as base fluids in nanofluids with Al2O3 nanoparticles at the concentration of 0.01%, 0.1% and 1% by weight. A set of 20 correlations for prediction of thermal conductivity and dynamic viscosity of base fluids and corresponding nanofluids has been developed. Moreover, present results have been confronted with literature data and predictions made by use of carefully selected recognized literature correlations.

Domain mapping of the Rad51 paralog protein complexes.

The five human Rad51 paralogs are suggested to play an important role in the maintenance of genome stability through their function in DNA double-strand break repair. These proteins have been found to form two distinct complexes in vivo, Rad51B-Rad51C-Rad51D-Xrcc2 (BCDX2) and Rad51C-Xrcc3 (CX3). Based on the recent Pyrococcus furiosus Rad51 structure, we have used homology modeling to design deletion mutants of the Rad51 paralogs. The models of the human Rad51B, Rad51C, Xrcc3 and murine Rad51D (mRad51D) proteins reveal distinct N-terminal and C-terminal domains connected by a linker region. Using yeast two-hybrid and co-immunoprecipitation techniques, we have demonstrated that a fragment of Rad51B containing amino acid residues 1-75 interacts with the C-terminus and linker of Rad51C, residues 79-376, and this region of Rad51C also interacts with mRad51D and Xrcc3. We have also determined that the N-terminal domain of mRad51D, residues 4-77, binds to Xrcc2 while the C-terminal domain of mRad51D, residues 77-328, binds Rad51C. By this, we have identified the binding domains of the BCDX2 and CX3 complexes to further characterize the interaction of these proteins and propose a scheme for the three-dimensional architecture of the BCDX2 and CX3 paralog complexes.

Obtaining chicken primordial germ cells used for gene transfer: in vitro and in vivo results.

Recently, several attempts have been made to create a generation of transgenic chickens via chimeric intermediates produced by primordial germ cells (PGCs) transfer. This study aimed to compare the influences of different chicken PGCs isolated from circulating blood (bPGCs) or gonads (gPGCs), purification (ACK, Percoll or trypsin) and transfection methods (electroporation or lipofection) on the expression of transgenes in vitro and the migration of modified donor cells to the recipient gonads. The highest average frequency of pEGFP-N1 plasmid-transfected bPGCs (75.8%) was achieved with Percoll density gradient centrifugation and electroporation. After ammonium chloride-potassium (ACK) treatment and lipofection, in vitro transgene expression was only detected in 35.2% of bPGCs. Chimeric chickens were produced from these purified, transfected and cultured cells, and the transgene was detected in the gonads of 44 and 42% of the recipient embryos that had been injected with bPGCs and gPGCs, respectively. These data confirmed that the combination of PGC purification via Percoll centrifugation and electroporation was an effective method for producing transgenic chickens. Subsequently, we used this method with expression vectors for gene hIFNα 2a/hepatitis B virus surface antigen (HBsAg) under the control of the ovalbumin promoter to generate G0 transgenic chickens. Consequently, we observed that 4.9% of the hens and 3.5% of the roosters carried the hIFNα 2a gene, whereas 16.7% of the hens and 2.4% of the roosters carried the HBsAg gene, thus undisputedly confirming the exceptional effectiveness of the applied methods.

Two processivity clamp interactions differentially alter the dual activities of UmuC.

DNA polymerases of the Y family promote survival by their ability to synthesize past lesions in the DNA template. One Escherichia coli member of this family, DNA pol V (UmuC), which is primarily responsible for UV-induced and chemically induced mutagenesis, possesses a canonical beta processivity clamp-binding motif. A detailed analysis of this motif in DNA pol V (UmuC) showed that mutation of only two residues in UmuC is sufficient to result in a loss of UV-induced mutagenesis. Increased levels of wild-type beta can partially rescue this loss of mutagenesis. Alterations in this motif of UmuC also cause loss of the cold-sensitive and beta-dependent synthetic lethal phenotypes associated with increased levels of UmuD and UmuC that are thought to represent an exaggeration of a DNA damage checkpoint. By designing compensatory mutations in the cleft between domains II and III in beta, we restored UV-induced mutagenesis by a UmuC beta-binding motif variant. A recent co-crystal structure of the 'little finger' domain of E. coli pol IV (DinB) with beta suggests that, in addition to the canonical beta-binding motif, a second site of pol IV ((303)VWP(305)) interacts with beta at the outer rim of the dimer interface. Mutational analysis of the corresponding motif in UmuC showed that it is dispensable for induced mutagenesis, but that alterations in this motif result in loss of the cold-sensitive phenotype. These two beta interaction sites of UmuC affect the dual functions of UmuC differentially and indicate subtle and sophisticated polymerase management by the beta clamp.

The DNA-binding domain of the ultraspiracle drives deformation of the response element whereas the DNA-binding domain of the ecdysone receptor is responsible for a slight additional change of the preformed structure.

Ecdysteroids control molting and metamorphosis in insects via a heterodimeric complex of two nuclear receptors, the ecdysone receptor (EcR) and ultraspiracle protein (Usp). We used fluorescence resonance energy transfer (FRET) to study the topology of the natural pseudopalindromic element from the hsp27 gene (hsp27pal) in complex with the DNA-binding domains of Usp and EcR (UspDBD and EcRDBD, respectively). Steady-state data revealed shortening of the end-to-end distance of the hsp27pal-derived probe. For the 70.8 +/- 0.6 A distance obtained for the UspDBD-complexed DNA a bend of about 23.1 +/- 2.9 degrees was measured. Nearly the same value (23.0 +/- 3.4 degrees) was obtained for the DNA complexed with the UspDBD/EcRDBD heterodimer. The respective bend angles estimated using fluorescence decay measurements were 19.0 +/- 2.1 degrees and 20.9 +/- 3.6 degrees . Thus, the FRET data suggest for the first time that the UspDBD defines the architecture of the UspDBD/EcRDBD heterocomplex due to the significant deformation of the hsp27pal. This suggestion has been further reinforced using gel retardation experiments, which, in conjunction with high-resolution DNase I footprinting, indicate that the main contribution to the observed bend is given by the UspDBD itself, while binding of the EcRDBD molecule brings on a slight additional change of the preformed structure.