Late Effects of Ionizing Radiation on the Ultrastructure of Hepatocytes and Activity of Lysosomal Enzymes in Mouse Liver Irradiated In Vivo.

The study aimed to investigate late radiation-induced changes in the histology, ultrastructure, and activity of lysosomal enzymes in mouse liver exposed to ionizing radiation. The experiment was conducted on C57BL/6J male mice whose distal part of the liver was exposed occasionally to single doses of radiation (6 MV photons) during targeted heart irradiation; estimated doses delivered to analyzed tissue were 0.025 Gy, 0.25 Gy, 1 Gy, and 2 Gy. Tissues were collected 40 weeks after irradiation. We have observed that late effects of radiation have an adaptive nature and their intensity was dose-dependent. Morphological changes in hepatocytes included an increased number of primary lysosomes and autophagic vacuoles, which were visible in tissues irradiated with 0.25 Gy and higher doses. On the other hand, a significant increase in the activity of lysosomal hydrolases was observed only in tissues exposed to 2 Gy. The etiology of these changes may be multifactorial and result, among others, from unintentional irradiation of the distal part of the liver and/or functional interaction of the liver with an irradiated heart. In conclusion, we confirmed the presence of late dose-dependent ultrastructural and biochemical changes in mouse hepatocytes after liver irradiation in vivo.

The RdRp genotyping of SARS-CoV-2 isolated from patients with different clinical spectrum of COVID-19.

BACKGROUND: The evolution of SARS-CoV-2 has been observed from the very beginning of the fight against COVID-19, some mutations are indicators of potentially dangerous variants of the virus. However, there is no clear association between the genetic variants of SARS-CoV-2 and the severity of COVID-19. We aimed to analyze the genetic variability of RdRp in correlation with different courses of COVID-19. RESULTS: The prospective study included 77 samples of SARS-CoV-2 isolated from outpatients (1st degree of severity) and hospitalized patients (2nd, 3rd and 4th degree of severity). The retrospective analyses included 15,898,266 cases of SARS-CoV-2 genome sequences deposited in the GISAID repository. Single-nucleotide variants were identified based on the four sequenced amplified fragments of SARS-CoV-2. The analysis of the results was performed using appropriate statistical methods, with p < 0.05, considered statistically significant. Additionally, logistic regression analysis was performed to predict the strongest determinants of the observed relationships. The number of mutations was positively correlated with the severity of the COVID-19, and older male patients. We detected four mutations that significantly increased the risk of hospitalization of COVID-19 patients (14676C > T, 14697C > T, 15096 T > C, and 15279C > T), while the 15240C > T mutation was common among strains isolated from outpatients. The selected mutations were searched worldwide in the GISAID database, their presence was correlated with the severity of COVID-19. CONCLUSION: Identified mutations have the potential to be used to assess the increased risk of hospitalization in COVID-19 positive patients. Experimental studies and extensive epidemiological data are needed to investigate the association between individual mutations and the severity of COVID-19.

Molecular mechanism of action of imidazolium carbosilane dendrimers on the outer bacterial membrane - From membrane damage to permeability to antimicrobial endolysin.

The outer bacterial membrane of drug-resistant bacteria is a significant barrier to many antimicrobials. Therefore, the development of new antibacterials primarily focuses on damaging the outer bacterial membrane of Gram-negative bacteria. Among many membrane-disrupting substances, the most promising are cationic dendritic systems. However, the mode of action may vary among different strains due to variations in the lipid compositions of the membrane. Here, we investigated the interaction of two types of cationic imidazolium carbosilane dendrimers: one with a single cationic group (methyl imidazolium) and the other with the same cationic group but attached to a functional group (a pendant pyridyl moiety), capable of establishing interactions with membranes through H-bonding or ion-dipole electrostatic interactions. We used different models of the outer membrane of Gram-negative bacteria - Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Additionally, we assessed the combined effect of the dendrimers and the antibacterial endolysin on P. aeruginosa. Our results show that the mechanism of action depends on the type of dendrimer and the lipid composition of the membrane. We also demonstrate that the alteration of membrane fluidity and permeability to endolysin by the methyl imidazolium and pyridyl imidazolium dendrimers may play a more significant role in antimicrobial activity compared to membrane damage caused by positively charged dendrimers.

Dendronized Ag/Au Nanomats: Antimicrobial Scaffold for Wound Healing Bandages.

Electrospun polymer nanofibers, due to high surface area-to-volume ratio, high porosity, good mechanical strength, and ease of functionalization, appear as promising multifunctional materials for biomedical applications. Thanks to their unidirectional structure, imitating the extracellular matrix (ECM), they can be used as scaffolds for cell adhesion and proliferation. In addition, the incorporation of active groups inside nanofiber can give properties for bactericides. The proposed nanomats incorporate nanoparticles templated within the electrospun nanofibers that prevent infections and stimulate tissue regeneration. The generated hybrid electrospun nanofibers are composed of a copolymer of L-lactide-block-ε-caprolactone (PL-b-CL), 70:30, blended with homopolymer polyvinylpyrrolidone (PVP) and gold (Au) nanoparticles. A low cytotoxicity and slightly increased immunoreactivity, stimulated by the nanomat, are observed. Moreover, the decoration of the hybrid nanomat with dendronized silver nanoparticles (Dend-Ag) improves their antibacterial activity against antibiotic-resistant Pseudomonas aeruginosa. The use of Dend-Ag for decorating offers several functional effects; namely, it enhances the antibacterial properties of the produced nanomats and induces a significant increase within macrophages' cytotoxicity. The unidirectional nanostructures of the generated hybrid nanomats demonstrate unique collective physio-chemical and biological properties suitable for a wide range of biomedical applications. Here, the antibacterial properties facilitate an optimal environment, contributing to accelerated wound healing.

Mechanistic insights from high resolution DNA damage analysis to understand mixed radiation exposure.

Cells exposed to densely ionising high and scattered low linear energy transfer (LET) radiation (50 % dose of each) react more strongly than to the same dose of each separately. The relationship between DNA double strand break location inside the nucleus and chromatin structure was evaluated, using high-resolution transmission electron microscopy (TEM) in breast cancer MDA-MB-231 cells at 30 min post 5 Gy. Additionally, response to high and/or low LET radiation was assessed using single (1 ×1.5 Gy) versus fractionated dose delivery (5 ×0.3 Gy). By TEM analysis, the highest total number of γH2AX nanobeads were found in cells irradiated with alpha radiation just prior to gamma radiation (called mixed beam), followed by alpha, then gamma radiation. γH2AX foci induced by mixed beam radiation tended to be surrounded by open chromatin (lighter TEM regions), yet foci containing the highest number of beads, i.e. larger foci representing complex damage, remained in the heterochromatic areas. The γH2AX large focus area was also greater in mixed beam-treated cells when analysed by immunofluorescence. Fractionated mixed beams given daily induced the strongest reduction in cell viability and colony formation in MDA-MB-231 and osteosarcoma U2OS cells compared to the other radiation qualities, as well as versus acute exposure. This may partially be explained by recurring low LET oxidative DNA damage by every fraction together with a delay in recompaction of chromatin after high LET, demonstrated by low levels of heterochromatin marker H3K9me3 at 2 h after the last mixed beam fraction in MDA-MB-231. In conclusion, early differences in response to complex DNA damage may lead to a stronger cell kill induced by fractionated exposure, which suggest a therapeutic potential of combined high and low LET irradiation.

Nano-Sized Selenium Maintains Performance and Improves Health Status and Antioxidant Potential While Not Compromising Ultrastructure of Breast Muscle and Liver in Chickens.

The poultry industry is looking for the most effective sources of selenium (Se) for commercial use. Over the past five years, nano-Se has attracted a great deal of attention in terms of its production, characterisation and possible application in poultry production. The objective of this study was to evaluate the effects of dietary levels of inorganic and organic Se, selenised yeast and nano forms of selenium on breast meat quality, liver and blood markers of antioxidants, the ultrastructure of tissue and the health status of chickens. A total of 300 one-day-old chicks Ross 308 were divided into 4 experimental groups, in 5 replications, with 15 birds per replication. Birds were fed the following treatments: a standard commercial diet containing inorganic Se in the form of inorganic Se at the level of 0.3 mg/kg diet and an experimental diet with an increased level of Se (0.5 mg/kg diet). The use of other forms of Se (nano-Se) versus sodium selenate significantly influences (p ≤ 0.05) a higher collagen content and does not impair physico-chemical properties in the breast muscle or the growth performance of the chickens. In addition, the use of other forms of selenium at an increased dose versus sodium selenate affected (p ≤ 0.01) the elongation of sarcomeres in the pectoral muscle while reducing (p ≤ 0.01) mitochondrial damage in hepatocytes and improving (p ≤ 0.05) oxidative indices. The use of nano-Se at a dose of 0.5 mg/kg feed has high bioavailability and low toxicity without negatively affecting the growth performance and while improving breast muscle quality parameters and the health status of the chickens.

Mechanistic insight of lysozyme transport through the outer bacteria membrane with dendronized silver nanoparticles for peptidoglycan degradation.

Drug resistance has become a global problem, prompting the entire scientific world to seek alternative methods of dealing with resistant pathogens. Among the many alternatives to antibiotics, two appear to be the most promising: membrane permeabilizers and enzymes that destroy bacterial cell walls. Therefore, in this study, we provide insight into the mechanism of lysozyme transport strategies using two types of carbosilane dendronized silver nanoparticles (DendAgNPs), non-polyethylene glycol (PEG)-modified (DendAgNPs) and PEGylated (PEG-DendAgNPs), for outer membrane permeabilization and peptidoglycan degradation. Remarkably, studies have shown that DendAgNPs can build up on the surface of a bacterial cell, destroying the outer membrane, and thereby allowing lysozymes to penetrate inside the bacteria and destroy the cell wall. PEG-DendAgNPs, on the other hand, have a completely different mechanism of action. PEG chains containing a complex lysozyme resulted in bacterial aggregation and an increase in the local enzyme concentration near the bacterial membrane, thereby inhibiting bacterial growth. This is due to the accumulation of the enzyme in one place on the surface of the bacteria and penetration into it through slight damage of the membrane due to interactions of NPs with the membrane. The results of this study will help propel more effective antimicrobial protein nanocarriers.

The Levels of Anti-SARS-CoV-2 Spike Protein IgG Antibodies Before and After the Third Dose of Vaccination Against COVID-19.

Purpose: The COVID-19 pandemic has been going on for almost three years, and so far, many preventive and therapeutic strategies have been developed. The issue of subsequent booster vaccinations is currently being discussed. We aimed to analyze how the third dose of vaccination against COVID-19 correlates with the dynamics of IgG anti-SARS-CoV-2 spike protein antibody levels in a group of healthy people. Patients and Methods: The prospective study included 93 participants before and after a second booster of COVID-19 vaccination, from whom 4 blood samples were collected at intervals. The levels of IgG anti-SARS-CoV-2 in serum were identified using the chemiluminescent immunoassay specific for the receptor-binding domain (RBD) of the S1 protein. The analysis of the results was performed using appropriate statistical methods, considering p <0.05 as a statistically significant value. Results: The IgG levels were significantly higher and less diverse after the same follow-up time from the second booster vaccination compared to the first booster. The antibody levels were positively correlated with female, healthcare workers, the elderly and participants with a negative COVID-19 history. Furthermore, the increase in IgG antibodies after the second booster vaccination correlated inversely with the baseline level of antibodies before the vaccination. The latest results showed that antibody levels dropped 1.5-fold after approx. 10 months from the second booster vaccination but still remained at a protective level. Conclusion: Booster vaccinations seem to better stimulate immune memory, and in the case of borderline IgG level induces the greatest increase in antibodies. It is worth considering the individual parameters of patients and measuring antibodies before vaccination.

Aging-Related Changes in the Ultrastructure of Hepatocytes and Cardiomyocytes of Elderly Mice Are Enhanced in ApoE-Deficient Animals.

Biological aging is associated with various morphological and functional changes, yet the mechanisms of these phenomena remain unclear in many tissues and organs. Hyperlipidemia is among the factors putatively involved in the aging of the liver and heart. Here, we analyzed morphological, ultrastructural, and biochemical features in adult (7-month-old) and elderly (17-month-old) mice, and then compared age-related features between wild type (C57Bl/6 strain) and ApoE-deficient (transgenic ApoE-/-) animals. Increased numbers of damaged mitochondria, lysosomes, and lipid depositions were observed in the hepatocytes of elderly animals. Importantly, these aging-related changes were significantly stronger in hepatocytes from ApoE-deficient animals. An increased number of damaged mitochondria was observed in the cardiomyocytes of elderly animals. However, the difference between wild type and ApoE-deficient mice was expressed in the larger size of mitochondria detected in the transgenic animals. Moreover, a few aging-related differences were noted between wild type and ApoE-deficient mice at the level of plasma biochemical markers. Levels of cholesterol and HDL increased in the plasma of elderly ApoE-/- mice and were markedly higher than in the plasma of elderly wild type animals. On the other hand, the activity of alanine transaminase (ALT) decreased in the plasma of elderly ApoE-/- mice and was markedly lower than in the plasma of elderly wild type animals.

Pathogenic Factors Correlate With Antimicrobial Resistance Among Clinical Proteus mirabilis Strains.

Proteus mirabilis is the third most common etiological factor of urinary tract infection. It produces urease, which contributes to the formation of a crystalline biofilm, considered to be one of the most important virulence factors of P. mirabilis strains, along with their ability to swarm on a solid surface. The aim of this study was to analyze the pathogenic properties of two selected groups of clinical P. mirabilis isolates, antimicrobial susceptible and multidrug resistant (MDR), collected from hospitals in different regions in Poland. The strains were examined based on virulence gene profiles, urease and hemolysin production, biofilm formation, and swarming properties. Additionally, the strains were characterized based on the Dienes test and antibiotic susceptibility patterns. It turned out that the MDR strains exhibited kinship more often than the susceptible ones. The strains which were able to form a stronger biofilm had broader antimicrobial resistance profiles. It was also found that the strongest swarming motility correlated with susceptibility to most antibiotics. The correlations described in this work encourage further investigation of the mechanisms of pathogenicity of P. mirabilis.

Breastfeeding and Prevalence of Metabolic Syndrome among Perimenopausal Women.

Little is known about the long-term benefits of breastfeeding for mother's metabolic health. This study aimed to investigate the links between breastfeeding duration and the prevalence of metabolic syndrome (MetS) and its components in perimenopausal women. The analysis included a group of 7621 women aged 55.4 ± 5.4 years. MetS and its components were defined according to the International Diabetes Federation guidelines. Women who breastfed for 13-18 months and beyond 18 months were at lower risk of MetS (odds ratio OR) = 0.76, 95% CI 0.60-0.95; p = 0.017 and OR = 0.79, 95% CI 0.64-0.98; p = 0.030, respectively) than those who never breastfed. Meanwhile, women who breastfed for 7-12 months showed increased glucose concentration (OR = 0.77, 95% CI 0.63-0.94; p = 0.012) compared with those who had never breastfed. The additional analysis involving parity showed that women who had given birth to two babies and breastfed them had lower odds of MetS than those who never breastfed (p < 0.05), although there was no significant difference among women who breastfed for >18 months. Women who had given birth to at least three children and breastfed for 1-6 and 13-18 months had lower odds of MetS and increased triglyceride concentration (p < 0.05). Moreover, participants having breastfed for 1-6 months were found to have a reduced risk of abdominal obesity compared with those who had not breastfed (p < 0.05). Breastfeeding is associated with lower prevalence of MetS in perimenopausal women and can be recommended as a way of reducing the risk of MetS and its components.

Fenugreek (Trigonella foenum-graecum L.) Seeds Dietary Supplementation Regulates Liver Antioxidant Defense Systems in Aging Mice.

Fenugreek seeds are widely used in Asia and other places of the world for their nutritive and medicinal properties. In Asia, fenugreek seeds are also recommended for geriatric populations. Here, we evaluated for the first time the effect of fenugreek seed feed supplementation on the liver antioxidant defense systems in aging mice. The study was conducted on 12-months aged mice which were given fenugreek seed dietary supplement. We evaluated the activities of various antioxidant defense enzymes such as superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx), and also estimated the phenolics and free radical scavenging properties in mice liver upon fenugreek supplementation. The estimation of SOD, GPx, and GR activities in aged mice liver revealed a significant (p < 0.01) difference among all the liver enzymes. Overall, this study reveals that fenugreek seed dietary supplementation has a positive effect on the activities of the hepatic antioxidant defense enzymes in the aged mice.

Biochemical and morphological changes in mouse liver induced by mistletoe toxins.

Natural compounds are often characterized by high biological activity and sometimes toxicity. This also applies to compounds contained in the herb mistletoe. The objective of this study was to investigate short-term effects (up to 48 h) of mistletoe toxins on mouse hepatocytes. Standardized mistletoe extract Iscador P was given to female mice as a single injection (0.1 mg/kg b.w., 1 mg/kg b.w., or 2 mg/kg b.w). Activities of lysosomal hydrolases: acid phosphatase, cathepsins D and L, N-acetyl-ß-D-hexosaminidase, ß-D-glucuronidase, ß-D-glucosidase and cytosolic proteases: arginine and leucine aminopeptidases were analyzed in the liver fractions 24 and 48 h after the injection. The morphology of hepatocytes was examined by light and transmission electron microscopy. Iscador P caused a decrease in the activity of all lysosomal hydrolases (except cathepsins) in the lysosomal pellet, and an increase in the activity of both aminopeptidases and ß-D-glucuronidase in the cytosol. However, despite membranotropic properties of the viscotoxins, we did not find a significant labilising effect on the lysosomal membranes. Only ß-D-glucuronidase activity was relocated to the supernatant of lysosomal fraction. Microscopic examinations revealed that hepatocyte mitochondria were enlarged and increased in number, whereas the surface of the rough endoplasmic reticulum was decreased significantly.

Ionizing radiation affects the composition of the proteome of extracellular vesicles released by head-and-neck cancer cells in vitro.

Exosomes and other extracellular vesicles are key players in cell-to-cell communication, and it has been proposed that they are involved in different aspects of the response to ionizing radiation, including transmitting the radiation-induced bystander effect and mediating radioresistance. The functional role of exosomes depends on their molecular cargo, including proteome content. Here we aimed to establish the proteome profile of exosomes released in vitro by irradiated UM-SCC6 cells derived from human head-and-neck cancer and to identify processes associated with radiation-affected proteins. Exosomes and other small extracellular vesicles were purified by size-exclusion chromatography from cell culture media collected 24 h after irradiation of cells with a single 2, 4 or 8 Gy dose, and then proteins were identified using a shotgun LC-MS/MS approach. Exosome-specific proteins encoded by 1217 unique genes were identified. There were 472 proteins whose abundance in exosomes was significantly affected by radiation (at any dose), including 425 upregulated and 47 downregulated species. The largest group of proteins affected by radiation (369 species) included those with increased abundance at all radiation doses (≥2 Gy). Several gene ontology terms were associated with radiation-affected exosome proteins. Among overrepresented processes were those involved in the response to radiation, the metabolism of radical oxygen species, DNA repair, chromatin packaging, and protein folding. Hence, the protein content of exosomes released by irradiated cells indicates their actual role in mediating the response to ionizing radiation.

Changes in activity and structure of lysosomes from liver of mouse irradiated in vivo.

PURPOSE: Lysosomes may have an important role in response to ionizing radiation. Moreover, radiation could affect autophagy, which process involves the activity of lysosomal enzymes. In the present study, the effect of ionizing radiation on the lysosomal compartment of mouse liver was investigated after in vivo exposure. MATERIALS AND METHODS: Morphology and ultrastructure of hepatocytes were assessed by light and electron microscopy, and activities of selected lysosomal enzymes were assessed in 12, 36 and 120 h after exposure to the mean dose of 1 Gy. The levels of autophagy-related proteins LC3-II and p62 were compared by Western blotting between untreated and irradiated animals (120 h after exposure). RESULTS: Increased number of autophagic vacuoles in hepatocytes from exposed animals was documented in the ultrastructural study; destroyed mitochondria were the dominant component of such vacuoles. Moreover, an increased activity of lysosomal hydrolases was observed after exposure. However, levels of autophagy substrates LC3-II and p62 were barely affected in exposed animals 120 h after irradiation when the accumulation of autophagic vacuoles was observed. CONCLUSION: Effects of irradiation included an increased number of autophagic vacuoles, especially of autophagosomes, and increased activity of lysosomal enzymes. However, putative markers of autophagic flux were not observed, which suggested suppression of the completion of the radiation-mediated autophagy pathway.

Long-term effects of low-dose mouse liver irradiation involve ultrastructural and biochemical changes in hepatocytes that depend on lipid metabolism.

The aim of the study was to investigate long-term effects of radiation on the (ultra)structure and function of the liver in mice. The experiments were conducted on wild-type C57BL/6J and apolipoprotein E knock-out (ApoE-/-) male mice which received a single dose (2 or 8 Gy) of X-rays to the heart with simultaneous exposure of liver to low doses (no more than 30 and 120 mGy, respectively). Livers were collected for analysis 60 weeks after irradiation and used for morphological, ultrastructural, and biochemical studies. The results show increased damage to mitochondrial ultrastructure and lipid deposition in hepatocytes of irradiated animals as compared to non-irradiated controls. Stronger radiation-related effects were noted in ApoE-/- mice than wild-type animals. In contrast, radiation-related changes in the activity of lysosomal hydrolases, including acid phosphatase, ß-glucuronidase, N-acetyl-ß-D-hexosaminidase, ß-galactosidase, and α-glucosidase, were observed in wild type but not in ApoE-deficient mice, which together with ultrastructural picture suggests a higher activity of autophagy in ApoE-proficient animals. Irradiation caused a reduction of plasma markers of liver damage in wild-type mice, while an increased level of hepatic lipase was observed in plasma of ApoE-deficient mice, which collectively indicates a higher resistance of hepatocytes from ApoE-proficient animals to radiation-mediated damage. In conclusion, liver dysfunctions were observed as late effects of irradiation with an apparent association with malfunction of lipid metabolism.

Ionizing radiation affects protein composition of exosomes secreted in vitro from head and neck squamous cell carcinoma.

Exosomes are membrane vesicles of endocytic origin that participate in inter-cellular communication. Environmental and physiological conditions affect composition of secreted exosomes, their abundance and potential influence on recipient cells. Here, we analyzed protein component of exosomes released in vitro from cells exposed to ionizing radiation (2Gy dose) and compared their content with composition of exosomes released from control not irradiated cells. Exosomes secreted from FaDu cells originating from human squamous head and neck cell carcinoma were analyzed using LC-MS/MS approach. We have found that exposure to ionizing radiation resulted in gross changes in exosomal cargo. There were 217 proteins identified in exosomes from control cells and 384 proteins identified in exosomes from irradiated cells, including 148 "common" proteins, 236 proteins detected specifically after irradiation and 69 proteins not detected after irradiation. Among proteins specifically overrepresented in exosomes from irradiated cells were those involved in transcription, translation, protein turnover, cell division and cell signaling. This indicated that exosomal cargo reflected radiation-induced changes in cellular processes like transient suppression of transcription and translation or stress-induced signaling.