Nuclear DNA Fragmentation Research Articles

The Arabidopsis DNA glycosylase MBD4L repairs the nuclear genome in vivo.

DNA glycosylases remove mispaired or modified bases from DNA initiating the base excision repair (BER) pathway. The DNA glycosylase MBD4 (methyl-CpG-binding domain protein 4) has been functionally characterized in mammals, but not yet in plants, where it is called MBD4-like (MBD4L). Mammalian MBD4 and Arabidopsis recombinant MBD4L excise U and T mispaired with G, as well as 5-fluorouracil (5-FU) and 5-bromouracil (5-BrU) in vitro. Here, we investigate the ability of Arabidopsis MBD4L to remove some of these substrates from the nuclear genome in vivo in coordination with uracil DNA glycosylase (AtUNG). We found that mbd4l mutants are hypersensitive to 5-FU and 5-BrU, as they displayed smaller size, less root growth, and higher cell death than control plants in both media. Using comet assays, we determined BER-associated DNA fragmentation in isolated nuclei and observed reduced DNA breaks in mbd4l plants under both conditions, but particularly with 5-BrU. The use of ung and ung x mbd4l mutants in these assays indicated that both MBD4L and AtUNG trigger nuclear DNA fragmentation in response to 5-FU. Consistently, we here report the nuclear localization of AtUNG based on the expression of AtUNG-GFP/RFP constructs in transgenic plants. Interestingly, MBD4L and AtUNG are transcriptionally coordinated but display not completely overlapping functions. MBD4L-deficient plants showed reduced expression of BER genes and enhanced expression of DNA damage response (DDR) gene markers. Overall, our findings indicate that Arabidopsis MBD4L is critical for maintaining nuclear genome integrity and preventing cell death under genotoxic stress conditions.

Allergen-stimulated secretion of DNA fragments by the airway epithelium enhances type 2 immune responses

Exposure of human bronchial epithelial cells (hBECs) and mouse airways to allergens from the fungus Alternaria alternata triggers the release of alarmin signaling molecules including ATP, IL-33 and DNA fragments. The goal of this study was to determine the mechanisms that regulate release of genomic DNA fragments and how extracellular DNA (eDNA) contributes to type 2 immunity. Treatment of hBECs with Alternaria extract induces oxidative stress, which leads to ATP release and uptake of Ca 2+ following activation of P2X receptors. DNA release was dependent on a sustained increase in intracellular [Ca 2+ ], which stimulates cleavage of caspase 3 by the proprotein convertase enzyme furin, resulting in activation of caspase 3 and ultimately nuclear DNA fragmentation as shown in comet assays. Some of the fragmented DNA was released into the media under conditions where barrier function of the epithelium was maintained, and nuclear and plasma membranes remained intact. Alternaria-induced DNA release was detected within 30 minutes following exposure, reached its peak at 4 hours and ceased after 12 hours. Pretreatment of hBECs with a calpain inhibitor resulted in continued DNA release and loss of cell viability by 24 hours. This result suggested that calpain activity contributed to termination of Alternaria-evoked DNA release. In mice, intranasal administration of Alternaria extract produced release of DNA fragments into bronchoalveolar lavage (BAL) fluid over a similar time course as observed in hBECs. In caspase 3 deficient mice however, DNA release into BAL fluid was significantly reduced. Furthermore, Alternaria-induced type 2 immune responses were suppressed following pretreatment with a DNA scavenger. Conversely, intranasal administration of mouse genomic DNA with Alternaria amplified IL-5 and IL-13 secretion into BAL fluid while DNA alone had no effect. These findings indicate that DNA release from the airway epithelium is a regulated process involving noncanonical activation of caspase 3 to produce DNA fragmentation and calpain activity, which appears to be essential for the termination of DNA release. Moreover in airways, eDNA appears to function as a secondary alarmin that acts on immune cells to enhance type 2 immunity. This work was supported by grants from the NIH (AI128729 and AI169530) to HK and SMO. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

In Vitro Pro-Apoptotic and Anti-Migratory Effects of Marantodes pumilum (syn. Labisia pumila) Extracts on Human Prostate Cancer Cell Lines: Bioguided Isolation of 5-Henicosene-1-yl-resorcinol.

This study aims to evaluate the in vitro cytotoxic and anti-migratory effects of Marantodes pumilum Blume Kuntze plant extracts on prostate cancer cells, identify the active compound/s, and characterize their mechanism of action. The crude methanolic extract was partitioned into n-hexane (MPh), chloroform (MPc), and aqueous (MPa) extracts. Antiproliferative fractions (IC50 < 30 μg/mL based on SRB staining of LNCaP and PC3 cell lines) were further fractionated. Active compound/s were identified using spectroscopic methods. In vitro mechanistic studies on PC3 cells included: annexin V-FITC staining, mitochondrial membrane potential (MMP) depolarization measurements, the activity of caspases 3 and 7, nuclear DNA fragmentation, cell cycle analysis, modulation of Bax, Bcl-2, Smac/Diablo, Alox-5, VEGF-A, CXCR4, and CXCL12 mRNA gene expression via RT-PCR, 2D migration (scratch assay), and 3D invasion (Boyden chamber). MPc extract was the most active, inducing cell death (p < 0.05) via apoptosis, as evidenced by nuclear DNA fragmentation and an increase in MMP depolarization (p < 0.05) as well as the activation of caspases 3/7 (MPc p < 0.01) in both PC3 and LNCaP cell lines. In addition, MPc upregulated Bax and Smac/DIABLO, downregulated Bcl-2 (p < 0.05), and inhibited ALOX-5 mRNA gene expression (p < 0.001). MPc was not cytotoxic against normal human fibroblast cells (HDFa) at the tested concentrations. Moreover, MPc inhibited migration and invasion of PC3 cells (p < 0.01). These effects were accompanied by the downregulation of both VEGF-A and CXCL-12 gene expressions (p < 0.001). A monounsaturated 5-alkyl resorcinol was isolated as the active compound in the MPc extract and identified as 5-henicosene-1-yl-resorcinol.

TITLE OF THE ARTICLE: EXOINTERACTIVE AMELIORATION AGAINST METHOTREXATE INTOXICATION IN MOUSE SPERMATOZOA

Objective: To assess the synergistic potential using an exointeractive association between Murraya koenigii hydromethanolic leaf extract and the synthetic compound quercetin to offset methotrexate induced toxicity in mouse cauda epididymal spermatozoa. Methods: Healthy adult male albino mice were divided into eight groups (N= 6/group) and were treated with methotrexate intraperitoneally (10mg/kg body weight) at intervals of every 48 hours for 15 days, to combat this toxic effects of the drug, Murraya koenigii hydromethanolic leaf extract (250mg/kg body weight) and quercetin (15mg and 30mg) alone as well as in combination, were given by oral administration. Various sperm parameters such as sperm count, motility and viability followed by Hypo-osmotic Swelling (HOS) test, apoptosis test, Sperm Toroid Integrity (STI) and Sperm Chromatin Dispersion (SCD) assay. Statistical analysis used: Statistical analysis was carried out using Graphpad Prism Version 7 to verify levels of significance at the P &lt; 0.001 value in one-way ANOVA. Results: Methotrexate induced a substantial reduction in sperm count, motility and viability which induces apoptotic changes, also resulted in disruption of sperm toroid protamine-DNA binding, increased fragmentation of nuclear DNA by decreased sperm chromatin dispersion and low percentage of swollen sperm representing membrane integrity loss. whereas Murraya koenigii hydromethanolic leaf extract and quercetin proved effective in reversing the drug induced toxicity, especially with the 15 mg dose of quercetin along with the plant extract brought about significant amelioration than 30mg dose of quercetin due to its pro-oxidant effects. Conclusions: To reduce the toxic manifestations of methotrexate, this exointeractive approach proved efficient.

Unravelling joint cytotoxicity of ibuprofen and oxytetracycline on Chlamydomonas reinhardtii using a programmed cell death-related biomarkers panel

Pharmaceutical active compounds (PhACs) are emerging contaminants that pose a growing concern due to their ubiquitous presence and harmful impact on aquatic ecosystems. Among PhACs, the anti-inflammatory ibuprofen (IBU) and the antibiotic oxytetracycline (OTC) are two of the most used compounds whose presence has been reported in different aquatic environments worldwide. However, there is still scarce information about the cellular and molecular alterations provoked by IBU and OTC on aquatic photosynthetic microorganisms as microalgae, even more if we refer to their potential combined toxicity. To test the cyto- and genotoxicity provoked by IBU, OTC and their binary combination on Chlamydomonas reinhardtii, a flow cytometric panel was performed after 24 h of single and co-exposure to both contaminants. Assayed parameters were cell vitality, metabolic activity, intracellular ROS levels, and other programmed cell death (PCD)-related biomarkers as cytoplasmic and mitochondrial membrane potentials and caspase-like and endonuclease activities. In addition, a nuclear DNA fragmentation analysis by comet assay was carried out.For most of the parameters analysed (vitality, metabolic activity, cytoplasmic and mitochondrial membrane potentials, and DNA fragmentation) the most severe damages were observed in the cultures exposed to the binary mixture (IBU+OTC), showing a joint cyto- and genotoxicity effect. Both PhACs and their mixture caused a remarkable decrease in cell proliferation and metabolic activity and markedly increased intracellular ROS levels, parallel to a noticeable depolarization of cytoplasmic and mitochondrial membranes. Moreover, a strong increase in both caspase and endonuclease activities as well as a PCD-related loss of nuclear DNA integrity was observed in all treatments. Results analysis showed that the PhACs caused cell death on this non-target organism, involving mitochondrial membrane depolarization, enhanced ROS production and activation of PCD process. Thus, PCD should be an applicable toxicological target for unraveling the harmful effects of co-exposure to PhACs in aquatic organisms as microalgae.

Rgp1 contributes to craniofacial cartilage development and Rab8a-mediated collagen II secretion.

Rgp1 was previously identified as a component of a guanine nucleotide exchange factor (GEF) complex to activate Rab6a-mediated trafficking events in and around the Golgi. While the role of Rgp1 in protein trafficking has been examined in vitro and in yeast, the role of Rgp1 during vertebrate embryogenesis and protein trafficking in vivo is unknown. Using genetic, CRISPR-induced zebrafish mutants for Rgp1 loss-of-function, we found that Rgp1 is required for craniofacial cartilage development. Within live rgp1-/- craniofacial chondrocytes, we observed altered movements of Rab6a+ vesicular compartments, consistent with a conserved mechanism described in vitro. Using transmission electron microscopy (TEM) and immunofluorescence analyses, we show that Rgp1 plays a role in the secretion of collagen II, the most abundant protein in cartilage. Our overexpression experiments revealed that Rab8a is a part of the post-Golgi collagen II trafficking pathway. Following loss of Rgp1, chondrocytes activate an Arf4b-mediated stress response and subsequently respond with nuclear DNA fragmentation and cell death. We propose that an Rgp1-regulated Rab6a-Rab8a pathway directs secretion of ECM cargoes such as collagen II, a pathway that may also be utilized in other tissues where coordinated trafficking and secretion of collagens and other large cargoes is required for normal development and tissue function.

The potential neuroprotective effects of stingless bee honey.

Tropical Meliponini bees produce stingless bee honey (SBH). Studies have shown beneficial properties, including antibacterial, bacteriostatic, anti-inflammatory, neurotherapeutic, neuroprotective, wound, and sunburn healing capabilities. High phenolic acid and flavonoid concentrations offer SBH its benefits. SBH can include flavonoids, phenolic acids, ascorbic acid, tocopherol, organic acids, amino acids, and protein, depending on its botanical and geographic origins. Ursolic acid, p-coumaric acid, and gallic acid may diminish apoptotic signals in neuronal cells, such as nuclear morphological alterations and DNA fragmentation. Antioxidant activity minimizes reactive oxygen species (ROS) formation and lowers oxidative stress, inhibiting inflammation by decreasing enzymes generated during inflammation. Flavonoids in honey reduce neuroinflammation by decreasing proinflammatory cytokine and free radical production. Phytochemical components in honey, such as luteolin and phenylalanine, may aid neurological problems. A dietary amino acid, phenylalanine, may improve memory by functioning on brain-derived neurotrophic factor (BDNF) pathways. Neurotrophin BDNF binds to its major receptor, TrkB, and stimulates downstream signaling cascades, which are crucial for neurogenesis and synaptic plasticity. Through BDNF, SBH can stimulate synaptic plasticity and synaptogenesis, promoting learning and memory. Moreover, BDNF contributes to the adult brain's lasting structural and functional changes during limbic epileptogenesis by acting through the cognate receptor tyrosine receptor kinase B (TrkB). Given the higher antioxidants activity of SBH than the Apis sp. honey, it may be more therapeutically helpful. There is minimal research on SBH's neuroprotective effects, and the related pathways contribute to it is unclear. More research is needed to elucidate the underlying molecular process of SBH on BDNF/TrkB pathways in producing neuroprotective effects.

Lipid peroxide-derived short-chain aldehydes promote programmed cell death in wheat roots under aluminum stress.

Lipid peroxidation is a primary event in plant roots exposed to aluminum (Al) toxicity, which leads to the formation of reactive aldehydes. Current evidence demonstrates that the resultant aldehydes are integrated components of cellular damage in plants. Here, we investigated the roles of aldehydes in mediating Al-induced damage, particularly cell death, using two wheat genotypes with different Al resistances. Aluminum treatment significantly induced cell death, which was accompanied by decreased root activity and cell length. Al-induced cell death displayed granular nuclei and internucleosomal fragmentation of nuclear DNA, suggesting these cells underwent programmed cell death (PCD). During this process, caspase-3-like protease activity was extensively enhanced and showed a significant difference between these two wheat genotypes. Further experiments showed that Al-induced cell death was positively correlated with aldehydes levels. Al-induced representative diagnostic markers for PCD, such as TUNEL-positive nuclei and DNA fragmentation, were further enhanced by the aldehyde donor (E)-2-hexenal, but significantly suppressed by the aldehyde scavenger carnosine. As the crucial executioner of Al-induced PCD, the activity of caspase-3-like protease was further enhanced by (E)-2-hexenal but inhibited by carnosine in wheat roots. These results suggest that reactive aldehydes sourced from lipid peroxidation mediate Al-initiated PCD probably through activating caspase-3-like protease in wheat roots.

Качественные характеристики замороженно-оттаянного семени (обычное и разделенное по полу ) у быков-производителей голштинской черно-пестрой породы и возраст полового созревания полученных от них телочек

A study was carried out to assess the quality of frozen-thawed semen using the sex separation method and the traditional method in a comparative aspect, and the authors believe that the work will be of some interest to specialists in zootechnical and biological fields, as well as to veterinary specialists. The work was carried out at the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst and on the basis of LLC “Agrofirma Zarya”, Bogorodsky district, Nizhny Novgorod region in the period from 2017 to 2022. usng the semen of eight sires. To assess the quality of the semen, the index of nuclear DNA fragmentation (nDNA) in chromatin was determined, and the activity of spermatozoa was assessed visually and using the Biola AFS‑500 sperm analyser (ZAO Biola, Moscow). On the basis of the studies conducted, it was found that the morphology of the spermatozoa depends on the individual characteristics of the sires; the content of abnormal spermatozoa varies from 2 to 8% when collected by the traditional method and from 4.10 to 15% when collected by the sexing method. A significant difference between the sires is observed in the iDNA fragmentation index – this indicator varies from 2.66 to 8.62% and from 8.50 to 28.57%, respectively. The division of spermatozoa by sex has a direct effect on the quality indicators, leading to a decrease in their activity. This indicator is 11.2% higher in semen obtained by the traditional method than by the alternative technique. The number of abnormal cells in the semen divided by sex is 2.2% higher than in the semen collected by the traditional method, and the proportion of spermatozoa with fragmented iDNA is 7% higher. The velocity of spermatozoa in the studied doses of semen collected using the technique of sex separation was 96–113 μm/s immediately after thawing, and five hours after incubation at +38°C in a thermostat this indicator decreased to the level of 20–34 μm/s. The age of puberty of their calves obtained from the sexed semen from birth to fruitful insemination is 490 days, which is 16 days more than in group I, where heifers were obtained from the semen frozen in the traditional way (TS). The term of fruiting in heifers born from the use of the sexed semen (SS) was 273 days, while that of heifers born from the use of traditionally frozen semen (TS) was 275 days (2 days more). The age of heifers from birth to first calving in the SS group was 762 days and was 14.4 days more than in the TS group, which was 748 days.

Идентификация популяций клеток крови рыб на основе сверточной нейронной сети для составления лейкограммы

A study was carried out to assess the quality of frozen-thawed semen using the sex separation method and the traditional method in a comparative aspect, and the authors believe that the work will be of some interest to specialists in zootechnical and biological fields, as well as to veterinary specialists. The work was carried out at the Federal Research Center for Animal Husbandry named after Academy Member L.K. Ernst and on the basis of LLC “Agrofirma Zarya”, Bogorodsky district, Nizhny Novgorod region in the period from 2017 to 2022. usng the semen of eight sires. To assess the quality of the semen, the index of nuclear DNA fragmentation (nDNA) in chromatin was determined, and the activity of spermatozoa was assessed visually and using the Biola AFS‑500 sperm analyser (ZAO Biola, Moscow). On the basis of the studies conducted, it was found that the morphology of the spermatozoa depends on the individual characteristics of the sires; the content of abnormal spermatozoa varies from 2 to 8% when collected by the traditional method and from 4.10 to 15% when collected by the sexing method. A significant difference between the sires is observed in the iDNA fragmentation index – this indicator varies from 2.66 to 8.62% and from 8.50 to 28.57%, respectively. The division of spermatozoa by sex has a direct effect on the quality indicators, leading to a decrease in their activity. This indicator is 11.2% higher in semen obtained by the traditional method than by the alternative technique. The number of abnormal cells in the semen divided by sex is 2.2% higher than in the semen collected by the traditional method, and the proportion of spermatozoa with fragmented iDNA is 7% higher. The velocity of spermatozoa in the studied doses of semen collected using the technique of sex separation was 96–113 μm/s immediately after thawing, and five hours after incubation at +38°C in a thermostat this indicator decreased to the level of 20–34 μm/s. The age of puberty of their calves obtained from the sexed semen from birth to fruitful insemination is 490 days, which is 16 days more than in group I, where heifers were obtained from the semen frozen in the traditional way (TS). The term of fruiting in heifers born from the use of the sexed semen (SS) was 273 days, while that of heifers born from the use of traditionally frozen semen (TS) was 275 days (2 days more). The age of heifers from birth to first calving in the SS group was 762 days and was 14.4 days more than in the TS group, which was 748 days.

A cytotoxic survey on 2-amino-1H-imidazol based synthetic marine sponge alkaloid analogues.

Here, we describe the synthesis and biologic activity evaluation of 20 novel synthetic marine sponge alkaloid analogues with 2-amino-1H-imidazol (2-AI) core. Cytotoxicity was tested on murine 4T1 breast cancer, A549 human lung cancer, and HL-60 human myeloid leukemia cells by the resazurin assay. A total of 18 of 20 compounds showed cytotoxic effect on the cancer cell lines with different potential. Viability of healthy human fibroblasts and peripheral blood mononuclear cells upon treatment was less hampered compared to cancer cell lines supporting tumor cell specific cytotoxicity of our compounds. The most cytotoxic compounds resulted the following IC50 values 28: 2.91 µM on HL-60 cells, and 29: 3.1 µM on 4T1 cells. The A549 cells were less sensitive to the treatments with IC50 15 µM for both 28 and 29. Flow cytometry demonstrated the apoptotic effect of the most active seven compounds inducing phosphatidylserine exposure and sub-G1 fragmentation of nuclear DNA. Cell cycle arrest was also observed. Four compounds caused depolarization of the mitochondrial membrane potential as an early event of apoptosis. Two lead compounds inhibited tumor growth in vivo in the 4T1 triple negative breast cancer and A549 human lung adenocarcinoma xenograft models. Novel marine sponge alkaloid analogues are demonstrated as potential anticancer agents for further development.

Allergen-induced DNA release by the airway epithelium amplifies type 2 immunity

Alternaria alternata and house dust mite exposure evokes IL-33 secretion from the airway epithelium, which functions as an alarmin to stimulate type 2 immunity. Extracellular DNA (eDNA) is also an alarmin that intensifies inflammation in cystic fibrosis, chronic obstructive pulmonary disease, and asthma. We investigated the mechanisms underlying allergen-evoked DNA mobilization and release from the airway epithelium and determined the role of eDNA in type 2 immunity. Human bronchial epithelial (hBE) cells were used to characterize allergen-induced DNA mobilization and extracellular release using comet assays to measure DNA fragmentation, Qubit double-stranded DNA assays to measure DNA release, and DNA sequencing to determine eDNA composition. Mice were used to investigate the role of eDNA in type 2 immunity. Alternaria extract rapidly induces mitochondrial and nuclear DNA release from human bronchial epithelial cells, whereas house dust mite extract induces mitochondrial DNA release. Caspase-3 is responsible for nuclear DNA fragmentation and becomes activated after cleavage by furin. Analysis of secreted nuclear DNA showed disproportionally higher amounts of promotor and exon sequences and lower intron and intergenic regions compared to predictions of random DNA fragmentation. In mice, Alternaria-induced type 2 immune responses were blocked by pretreatment with a DNA scavenger. In caspase-3-deficient mice, Alternaria-induced DNA release was suppressed. Furthermore, intranasal administration of mouse genomic DNA with Alternaria amplified secretion of IL-5 and IL-13 into bronchoalveolar lavage fluid while DNA alone had no effect. These findings highlight a novel, allergen-induced mechanism of rapid DNA release that amplifies type 2 immunity in airways.

Panicle Apical Abortion 7 Regulates Panicle Development in Rice (Oryza sativa L.).

The number of grains per panicle significantly contributes to rice yield, but the regulatory mechanism remains largely unknown. Here, we reported a loss-of-function mutant, panicle apical abortion 7 (paa7), which exhibited panicle abortion and degeneration of spikelets on the apical panicles during the late stage of young panicle development in rice. High accumulations of H2O2 in paa7 caused programmed cell death (PCD) accompanied by nuclear DNA fragmentation in the apical spikelets. Map-based cloning revealed that the 3 bp “AGC” insertion and 4 bp “TCTC” deletion mutation of paa7 were located in the 3′-UTR regions of LOC_Os07g47330, which was confirmed through complementary assays and overexpressed lines. Interestingly, LOC_Os07g47330 is known as FRIZZY PANICLE (FZP). Thus, PAA7 could be a novel allele of FZP. Moreover, the severe damage for panicle phenotype in paa7/lax2 double mutant indicated that PAA7 could crosstalk with Lax Panicle 2 (LAX2). These findings suggest that PAA7 regulates the development of apical spikelets and interacts with LAX2 to regulate panicle development in rice.

Possibilities for implementing of anti-fibrotic and anti-inflammatory effects of metabolic therapy in acute alcoholic disorders under experimental conditions

The aim of the work was to study antifibrotic and anti-inflammatory effects of metabolic therapy and mechanisms of regeneration in acute alcoholic liver damage (AALD) in rats under experimental conditions. Materials and methods. The experiment involved 66 white non-linear male rats with a mass of 120–130 g, which were divided into 5 groups: 1 – intact animals (n = 10); 2 – animals with AALD (n = 20), 3 – animals (n = 12) with AALD and intraperitoneally injected with Corvitin, 4 – animals (n = 12) with AALD and injected with Glutargin, 5 – аnimals with AALD (n = 12) and injected with Corvitin and Glutargin. The pro-inflammatory cytokines and cell cycle phases were analyzed. Results. The level of IGF-1 was significantly 24.1 % higher in group 2 compared to the control. In animals of group 3, the level of IGF-1 was 20.2 % decreased compared with group 2. The level of IGF-1 was significantly 9.7 % decreased in group 5 animals compared with group 2. There was a 31.6% increase in the level of TGF-β in animals of group 2 in comparison with the control ones. The level of TGF-β was 22.8 % decreased in group 3 compared with group 2. In group 5 animals, the value was 12.0 % lower than in group 2. The percentage of cell nuclei in the presynthetic phase in group 2 rats was 8.3 % higher than in controls. In animals of group 2, the number of cell nuclei in the phase of DNA synthesis were 33.3 % larger than in group 1. The rate of DNA fragmentation in AALD exceeded the corresponding value in control group by 27.5 %. Conclusions. AALD was accompanied by an increase in the serum IGF-1 and TGF-β in animals. The administration of corvitin decreased the level of IGF-1 in rats with AALD, and the use of glutargine mainly decreased the level of TGF-β. Combined use of the drugs did not show significant effectiveness. Compensatory regeneration mechanisms were activated in AALD processes and apoptotic cell death was evidenced by the increased indicators of nuclear DNA fragmentation.

Autophagy alleviates indium-induced programmed cell death in wheat roots

Indium released in agroecosystems is becoming an emerging plant stressor, causing cellular damage and consequently crop yield losses. Previous studies have focused on indium-induced toxicity in plants, while plant adaptive responses to such emerging metal xenobiotics are poorly understood. Here, we explored the relationship of autophagy and programmed cell death (PCD) in wheat roots under indium stress. Indium treatment significantly decreased root activity and cell viability, and suppressed the length of root epidermal cells in the elongation zones. These symptoms may be associated with indium-induced PCD, as indium-stressed wheat roots displayed condensed and granular nuclei, increased number of TUNEL-positive nuclei, enhanced nuclear DNA fragmentation and caspase-3-like protease activity compared to untreated roots. Accordingly, indium enhanced the expression levels of TaMCA1 and TaMCA4, two major metacaspase genes mediated PCD in wheat plants. The enhanced expression of autophagy genes and formation of autophagosomes indicate that autophagy could regulate metabolic adaptation and repair stress-induced damage in wheat roots. Furthermore, reinforcing autophagy by activator rapamycin significantly decreased the number of TUNEL-positive nuclei and the activity of caspase-3-like protease, whereas inhibition of autophagy by 3-methyladenine aggravated diagnostic markers for PCD. These results together suggest that autophagy suppresses indium-induced PCD in wheat roots.

Nuclear DNA Fragmentation in Boar Spermatozoa: Measurement Methods and Reproductive Performance Implications

The aim of this research was to compare the different techniques to measure sperm nuclear DNA fragmentation (sDF) and to check its relations to boar reproductive value, classical spermiogram parameters, and reproductive results of the doses in sows. Sperm chromatin stability assay (SCSA), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and sperm chromatin dispersion test (SCD, Halomax®) results were compared, finding a statistically significant correlation only between SCSA and TUNEL results. The fertility direct boar effect (DBE) index, calculated from the whole productive life of the boar, was not correlated (p > 0.05) with sDF (measured by any technique). Total or progressive sperm motility was not correlated with sDF, while it found a positive correlation between TUNEL measure and abnormal acrosomes (%) and between SCD measure and total sperm morphological abnormalities (%). No significant correlations were obtained between fertility or prolificacy results and sDF results with the different techniques. However, in the case of total born and SCSA measure, the correlation was close to significance (r partial = −0.095; p = 0.066), appointing to a tendency; as SCSA increases, the number of total piglets born decreases. In conclusion, although the different techniques for the sDF seem not to target exactly the same DNA events and the relationship between their values and the reproductive results and the classical spermiogram results is still to be elucidated, the studied sDF techniques may offer extra information that could be useful for the management of AI studs.

Growth-Suppressive and Apoptosis-Inducing Effects of Tetrandrine in SW872 Human Malignant Liposarcoma Cells via Activation of Caspase-9, Down-Regulation of XIAP and STAT-3, and ER Stress.

Liposarcoma is a rare and heterogeneous soft tissue malignant tumor and has a significant impact on mortality with a poor prognosis. To date, there is no effective treatment for liposarcoma, whereas surgical resection is only the gold treatment with numerous adverse effects. Here we investigated whether tetrandrine inhibits the growth of SW872 human malignant liposarcoma cells. Of note, tetrandrine at 10 μM vastly inhibited growth and induced apoptosis, as evidenced by increased nuclear DNA fragmentation and sub-G1 population of SW872 cells. Mechanistically, treatment with tetrandrine led to activation of caspase-9/3 in SW872 cells, and z-VAD-fmk, a pan-caspase inhibitor, attenuated the tetrandrine-induced apoptosis and growth suppression in SW872 cells. In addition, tetrandrine treatment resulted in down-regulation of XIAP andSTAT-3 in SW872 cells, and importantly knockdown of STAT-3 caused a significant reduction of the cell survival. Tetrandrine also had abilities to up-regulate not only the expression of GRP78 and ATF-4 but also the phosphorylation of eIF-2α in SW872 cells. In summary, these results demonstrated that tetrandrine has strong growth-suppressive and apoptosis-inducing effects on SW872 cells, which are mediated through control of the intrinsic caspase pathway, down-regulation of XIAP and STAT-3, and triggering ER stress.

Anti-Growth, Anti-Angiogenic, and Pro-Apoptotic Effects by CX-4945, an Inhibitor of Casein Kinase 2, on HuCCT-1 Human Cholangiocarcinoma Cells via Control of Caspase-9/3, DR-4, STAT-3/STAT-5, Mcl-1, eIF-2α, and HIF-1α.

Overexpression of casein kinase 2 (CK2) has an oncogenic and pro-survival role in many cancers. CX-4945 (Silmitasertib) is a CK2 inhibitor with anti-cancerous and anti-angiogenic effects. Up to date, the anti-cancer effect and mechanism of CX-4945 on human cholangiocarcinoma (CCA) remain unclear. This study investigated whether CX-4945 inhibits growth and induces apoptosis of HuCCT-1 cells, a human CCA cell line. Of note, treatment with CX-4945 at 20 μM markedly reduced survival and induced apoptosis of HuCCT-1 cells, as evidenced by nuclear DNA fragmentation, PARP cleavage, activation of caspase-9/3, and up-regulation of DR-4. Although CX-4945 did not affect the phosphorylation and expression of CK2, it vastly inhibited the phosphorylation of CK2 substrates, supporting the drug’s efficacy in inhibiting CK2 and its downstream pathway. Importantly, knockdown of CK2 that partially suppressed the phosphorylation of CK2 substrates resulted in a significant reduction of HuCCT-1 cell survival. In addition, CX-4945 reduced the phosphorylation and expression of STAT-3 and STAT-5 in HuCCT-1 cells, and pharmacological inhibition or respective knockdown of these proteins resulted in significant growth suppression of HuCCT-1 cells. CX-4945 also had abilities to decrease Mcl-1 expression while increasing eIF-2α phosphorylation in HuCCT-1 cells. Furthermore, there was a time-differential negative regulation of HIF-1α expression by CX-4945 in HuCCT-1 cells, and knockdown of HIF-1α caused a significant reduction of the cell survival. In summary, these results demonstrated that CX-4945 has anti-growth, anti-angiogenic, and pro-apoptotic effects on HuCCT-1 cells, which are mediated through control of CK2, caspase-9/3, DR-4, STAT-3/5, Mcl-1, eIF-2α, and HIF-1α.

Evaluation of selected semen parameters and biomarkers of male infertility - preliminary study.

Background: Because the etiopathogenesis of male infertility is multifactorial our study was designed to clarify the relationship between standard semen parameters, testicular volume, levels of reproductive hormones and the fragmentation of sperm nuclear DNA (SDF). Methods: Patients (n = 130) were clustered as subjects: 1) with an abnormal volume (utrasonography) of at least one testis (<12 mL) or with a normal volume of testes and 2) with abnormal levels of at least one of the reproductive hormones (FSH, LH, PRL, TSH, total T - electrochemiluminescence method) or with normal hormonal profiles and 3) with high level of SDF (>30%), moderate (>15-30%) or low (≤15%) (sperm chromatin dispersion test). Results: In subjects with a decreased testicular volume and in subjects with abnormal levels of reproductive hormones, decreased basic semen parameters were found. Participants with abnormal testicular volume had a higher percentage of SDF and a higher level of FSH (Mann-Whitney U test). In turn, men with a high level of SDF had lower testicular volume and conventional sperm parameters than men with a low level of SDF (Kruskal-Wallis test). Conclusions: We showed that spermatogenesis disorders coexisted with decreased testicular volume and increased FSH levels. The disorders of spermatogenesis were manifested by reduced basic sperm characteristics and a high level of sperm nuclear DNA damage.

Oxime derivative TFOBO promotes cell death by modulating reactive oxygen species and regulating NADPH oxidase activity in myeloid leukemia

Several derivatives derived from the oxime structure have been reported as potential anticancer agents in various cancers. Here, we first tested a novel oxime-containing derivative of 2-((2,4,5-trifluorobenzyl)oxy)benzaldehyde oxime (TFOBO) to evaluate its anticancer effect in myeloid leukemic cells. Compared to (2-((2,4,5-trifluorobenzyl)oxy)phenyl)methanol (TFOPM), the oxime derivative TFOBO suppresses leukemic cell growth by significantly increasing reactive oxygen species (ROS) levels and cell death. Leukemic cells treated with TFOBO displayed apoptotic cell death, as indicated by nuclear condensation, DNA fragmentation, and annexin V staining. TFOBO increases Bax/Bcl2 levels, caspase9, and caspase3/7 activity and decreases mitochondrial membrane potential. ROS production was reduced by N-acetyl-l-cysteine, a ROS scavenger, diphenyleneiodonium chloride, a nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, after exogenous TFOBO treatment. ROS inhibitors protect leukemic cells from TFOBO-induced cell death. Thus, our study findings suggest that TFOBO promotes apoptosis by modulating ROS and regulating NADPH oxidase activity. Collectively, the oxime-containing derivative TFOBO is a novel therapeutic drug for myeloid leukemia.