[The cell nuclear damage probably induces chronic refractory diseases].

The cell nuclear damage is mainly caused by the radiation and various carcinogenic compounds, the essence of the damage is molecular adhesion fracture and chemical modification. After nuclear damage, the cells whose nuclei may be abnormal in morphology, structure and function, then become a kind of morbid cells or defective cells. The cell nuclear damage can affect gene expression and regulation, leading to dysfunctions or abnormalities of transcription and protein synthesis, which results in aging and induces various chronic refractory diseases, such as hypertension, atherosclerosis, diabetes, Alzheimer's disease, autoimmune diseases, and so on. The cell nuclear damage can also affect the state of cell differentiation and lead to restart of genes related to division and proliferation, thus inducing cancer. The cancer cells are derived from the cells with nuclear abnormalities, and the biological behavior or characteristics of cancer cells (shedding and metastasis, immune tolerance, uncontrolled, loss of contact inhibition, etc.) are derived from cells with nuclear abnormalities. This article reviewed the chronic refractory diseases caused by nuclear damage and their mechanisms, which provided a new idea for occupational health and toxicology research, as well as a new method and strategy for occupational disease prevention and treatment.

Apoptotic M540 bodies present in human semen interfere with flow cytometry-assisted assessment of sperm DNA fragmentation and oxidation.

BACKGROUND: The use of flow cytometry (FC) to evaluate sperm DNA fragmentation via deoxynucleotidyl transferase terminal fluorescein dUTP nick-end labeling (TUNEL) has shown inconsistencies compared with conventional fluorescent microscopic analyses. It has been hypothesized that the observed discrepancies could be attributed to the presence of apoptotic bodies that can be labeled with merocyanine 540, the so-called M540 bodies. In order to verify this hypothesis and determine the accuracy of our in-house FC-assisted evaluation of spermatozoa parameters, we used FC to evaluate both the fragmentation of sperm DNA using the TUNEL assay and the oxidation of sperm DNA using the 8-OHdG assay on semen samples with or without M540 bodies. RESULTS: We show that the presence of M540 bodies lead to underestimation of both the level of sperm DNA fragmentation and sperm DNA oxidation when using FC assisted detection systems. We also observed that this situation is particularly pertinent in semen samples classified as abnormal with respect to the routine WHO semen evaluation as they appear to contain more M540 bodies than normal samples. CONCLUSIONS: We conclude that M540 bodies interfere with both FC-conducted assays designed to evaluate sperm nuclear/DNA integrity. Exclusion of these contaminants in unprepared semen samples should be performed in order to correctly appreciate the true level of sperm DNA/nuclear damage which is known to be a critical male factor for reproductive success.

RéSUMé: CONTEXTE: L'utilisation de la cytométrie en flux (CF) pour évaluer la fragmentation de l'ADN des spermatozoïdes via la technique TUNEL (Terminal transferase dUTP nick-end labelling) a montré des incohérences par rapport aux analyses conventionnelles par microscopie fluorescente. L'hypothèse a été émise que les divergences observées pourraient être attribuées à la présence de corps apoptotiques qui peuvent être marqués à la mérocyanine 540 (corps M540). Afin de vérifier cette hypothèse et de déterminer la précision de notre évaluation interne des paramètres des spermatozoïdes, nous avons mesuré par CF à la fois la fragmentation de l'ADN des spermatozoïdes en utilisant le test TUNEL et l'oxydation de l'ADN des spermatozoïdes en utilisant le test 8-OHdG sur des échantillons de sperme avec ou sans corps M540. RéSULTATS: Nous montrons que la présence des corps M540 entraîne une sous-estimation du niveau de fragmentation et d'oxydation de l'ADN des spermatozoïdes lors de l'utilisation de systèmes de détection assistée par CF. Nous avons également observé que cette situation est exacerbée dans les échantillons de sperme classés comme anormaux (selon les standards de l'OMS), car ces derniers semblent contenir plus de corps M540 que les échantillons normaux. CONCLUSIONS: Nous concluons que les corps M540 interfèrent avec les deux tests conduits par CF et conçus pour évaluer l'intégrité nucléaire des spermatozoïdes. L'exclusion de ces contaminants dans les échantillons de sperme non préparés devrait être considérée afin d'apprécier correctement le véritable niveau de dommages au noyau spermatique qui est connu pour être un facteur critique pour le succès reproductif.

Concurrent impairment of nucleus and mitochondria for synergistic inhibition of cancer metastasis.

Cancer metastasis, which increases the mortality in a short period of time, has been considered as the main challenge in tumor treatment. However, tumor growth suppression also should not be ignored in cancer metastasis treatment. Recently, accumulating evidences have suggested that mitochondria play an important role in mitigating caner metastasis. Nucleus, as the repository of genetic information, plays a key role in cell proliferation. However, it remains elusive that the concurrent impairment of nucleus and mitochondria may achieve better anti-tumor and anti-metastatic effects. Here, we designed a mitochondria-penetrating peptide modified doxorubicin (MPP-Dox) loaded N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer conjugates (PM), as well as a nuclear accumulating HPMA copolymer Dox conjugates (PN) by the nuclear tendency of Dox. After co-delivering the two copolymers (abbreviation for PMN), PM promoted cell apoptosis and inhibited tumor metastasis by damaging mitochondria, whereas PN suppressed cell proliferation and promoted apoptosis by destroying nucleus. Importantly, PM and PN complemented each other as expected. The mitochondrial dysfunction and tumor metastasis inhibition of PM was improved by PN, while cell proliferation suppression and apoptosis by nucleus destroying of PN was enhanced by PM. As a result, tumor growth of breast cancer 4T1 cells in vivo was significantly restrained and lung metastasis was potently decreased and almost eradicated, fully reflecting the advantages of organelle targeting combination therapy. As a consequence, our work showed that concurrent impairment of nucleus and mitochondria was feasible and beneficial to metastatic cancer treatment.

Heat shock protein 70 and the extent of nuclear damage in periodontal ligament compression side cells with orthodontic force application - An ex-vivo evaluation.

OBJECTIVE: Increased levels of heat shock proteins after several types of stress play a central role in cellular homeostasis allowing for continued cell survival. This study was aimed at quantitatively analysing the expression pattern of major damage associated molecular patterns (DAMPs) - HSP70, and the amount of nuclear damage incident in periodontal ligament compression side cells. MATERIAL AND METHODS: Sixteen subjects with bimaxillary dentoalveolar protrusion requiring extraction of all first premolars as part of orthodontic treatment were selected. Extractions were carried out pretreatment from control group. In the experimental group, a buccally directed spring, with force range of 70-120gms promoting bodily movement of maxillary first premolars was placed. Periodontal ligament was scraped from the middle third of the root from the compression side, the cells were isolated and cultured followed by HSP70 protein estimation with western blot analysis and the extend of nuclear damage was assessed with COMET assay. RESULTS: Western Blot analysis of HSP70 revealed a statistically significant increased expression of HSP70 (P<0.05; CI=95%) in the force applied group compared with the control group. COMET assay could demonstrate significant amount of nuclear fragmentation in the compression side periodontal ligament cells in comparison to control group (P<0.05; CI=95%). CONCLUSIONS: All these findings demonstrated, for the first time, that orthodontic force application augments release of HSP70 from periodontal ligament cells as a measure to restore tissue homeostasis. Further the study demonstrated that orthodontic forces induce DNA fragmentation, which is quantified more than double the amount observed in the control group.

Oxidation of Sperm DNA and Male Infertility.

One important reason for male infertility is oxidative stress and its destructive effects on sperm structures and functions. The particular composition of the sperm membrane, rich in polyunsaturated fatty acids, and the easy access of sperm DNA to oxidative damage due to sperm cell specific cytologic and metabolic features (no cytoplasm left and cells unable to mount stress responses) make it the cell type in metazoans most susceptible to oxidative damage. In particular, oxidative damage to the spermatozoa genome is an important issue and a cause of male infertility, usually associated with single- or double-strand paternal DNA breaks. Various methods of detecting sperm DNA fragmentation have become important diagnostic tools in the prognosis of male infertility and such assays are available in research laboratories and andrology clinics. However, to date, there is not a clear consensus in the community as to their respective prognostic value. Nevertheless, it is important to understand that the effects of oxidative stress on the sperm genome go well beyond DNA fragmentation alone. Oxidation of paternal DNA bases, particularly guanine and adenosine residues, the most sensitive residues to oxidative alteration, is the starting point for DNA damage in spermatozoa but is also a danger for the integrity of the embryo genetic material independently of sperm DNA fragmentation. Due to the lack of a spermatozoa DNA repair system and, if the egg is unable to correct the sperm oxidized bases, the risk of de novo mutation transmission to the embryo exists. These will be carried on to every cell of the future individual and its progeny. Thus, in addition to affecting the viability of the pregnancy itself, oxidation of the DNA bases in sperm could be associated with the development of conditions in young and future adults. Despite these important issues, sperm DNA base oxidation has not attracted much interest among clinicians due to the lack of simple, reliable, rapid and consensual methods of assessing this type of damage to the paternal genome. In addition to these technical issues, another reason explaining why the measurement of sperm DNA oxidation is not included in male fertility is likely to be due to the lack of strong evidence for its role in pregnancy outcome. It is, however, becoming clear that the assessment of DNA base oxidation could improve the efficiency of assisted reproductive technologies and provide important information on embryonic developmental failures and pathologies encountered in the offspring. The objective of this work is to review relevant research that has been carried out in the field of sperm DNA base oxidation and its associated genetic and epigenetic consequences.

Combined Effect of Vaccinium nilgiriensis Bark Extract and 680nm Laser Irradiation in Inducing Breast Cancer Cell Death.

BACKGROUND: Cancer refers to a collection of diseases where cells begin to multiply uncontrollably. Breast cancer is the most predominant malignancy in women. Herbal medicine is one of the important health care systems in most developing countries. Many studies have shown that naturally occurring compounds may support the prevention and treatment of various diseases, including cancer. Some of the plant extracts and isolated compounds show photosensitizing activities and reduce cell proliferation whereas some have revealed photoprotective effects. OBJECTIVES: The biological properties and medicinal uses of extracts and bioactive compounds from V. nilgiriensis have not been investigated. This study aims to evaluate the cytotoxic effects of V. nilgiriensis in combination with 680nm laser irradiation on MCF-7 breast cancer cells. METHODS: The inverted microscopy, ATP and LDH assay were used to analyze the cellular morphology, proliferation, cytotoxicity respectively after the treatment with V. nilgiriensis bark extract. The diode laser of wavelength 680nm and 15 J/cm2 fluency has been used for laser irradiation. The activity of apoptotic proteins was studied using ELISA and nuclear damage by Hoechst staining. RESULTS: The exposure of V. nilgiriensis extracts with laser irradiation at 680nm increases the cytotoxicity and decreases the proliferation of MCF-7 cells. The results of the Hoechst stain indicated nuclear damage. Our study proved that V. nilgiriensis holds a strong cytotoxic effect on breast cancer cells alone and in combination with laser irradiation by upregulating the expression of apoptotic proteins such as caspase 3, p53 and Bax. CONCLUSION: The results from this study showed that the bark ethyl acetate of V. nilgiriensis and in combination with laser is effective in preventing breast cancer cell proliferation in vitro. Further work is warranted to isolate the bioactive compounds from V. nilgiriensis bark extract and study the effect of compounds in the cell death induction. Due to the cytotoxic properties, V. nilgiriensis can be considered as a potent therapeutic agent for the treatment of cancer.

Antifungal Effects of Fusion Puroindoline B on the Surface and Intracellular Environment of Aspergillus flavus.

Aspergillus flavus infection is a major issue for safe food storage. In this study, we constructed an efficient prokaryotic expression system for puroindoline B (PINB) protein to detect its antifungal activity. The Puroindoline b gene was cloned into pET-28a (+) vector and expressed in Escherichia coli. Treatment with fusion PINB revealed that it inhibits mycelial growth of A. flavus, a common grain mold. Moreover, fusion PINB-treated A. flavus mycelium withered and exhibited a sunken spore head. As fusion PINB concentration increased, electrical conductivity in mycelium also increased, indicative of cell membrane damage. Furthermore, intracellular malate dehydrogenase and succinate dehydrogenase activity decreased, revealing a disruption in the tricarboxylic acid cycle. Moreover, the dampened activity of the ion pump Na+K+-ATPase negatively affected the intracellular regulation of both ions. Catalase and superoxide dismutase activity decreased, thus reducing antioxidant capacity, a result confirmed with an increase in malondialdehyde content. Changes to the GSH/GSSG ratio indicated a shift to an intracellular oxidative state. At the same time, laser scanning confocal microscopy assay showed the accumulation of reactive oxygen species and nuclear damage. Therefore, the PINB fusion protein may have the potential to control A. flavus in grain storage and food preservation.

Cannabis consumption might exert deleterious effects on sperm nuclear quality in infertile men.

RESEARCH QUESTION: Can cannabis consumption alter sperm nuclear integrity in infertile men? DESIGN: A retrospective cross-sectional study conducted between July 2003 and December 2013, which included 54 men who consulted for male-factor infertility. Twenty-seven infertile men who were regular cannabis users were matched to 27 infertile men who were cannabis non-users. To complement the conventional semen parameter and plasma hormone level assessments, sperm nuclear alterations were explored using fluorescence in-situ hybridization to assess numerical chromosomal abnormalities, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling to investigate DNA fragmentation, aniline blue staining to examine chromatin condensation and a motile sperm organelle morphology examination to detect vacuoles in sperm heads. RESULTS: The rates of sperm aneuploidy (P = 0.0044), diploidy (P = 0.037), total chromosome abnormalities (P = 0.0027) and DNA fragmentation (P = 0.027) were significantly higher in cannabis users than in non-cannabis users. CONCLUSIONS: Cannabis consumption might have deleterious effects on sperm nuclear quality in infertile men by increasing numerical chromosome abnormalities and DNA fragmentation. Cannabis consumption induces these detrimental effects on the progression of spermatogenesis from meiotic stages to spermiogenesis and potentially on post-testicular sperm maturation in infertile men. Any potential findings, however, need to be validated with larger sample size, and our data are only exploratory findings.

Russell's viper venom affects regulation of small GTPases and causes nuclear damage.

Russell's viper with its five sub-species is found throughout the Indian subcontinent. Its venom is primarily hemotoxic. However, its envenomation causes damage to several physiological systems. The present work was aimed to study the dose and time dependent cytotoxic effects of Russell's viper venom (RVV) on human A549 cells grown in vitro. Time dependent changes have been observed in cellular morphology following exposure to RVV. Presence of stress granules, rounding-off of the cells, and formation of punctate structure and loss of cell-cell contact characterized the cellular effects. Fluorescence microscopic studies revealed that apoptotic cell population increased on exposure to RVV. Further to understand the mechanism of these effects, status of small GTPase (smGTPases) expression were studied by Western blot and RT-PCR; as smGTPases play pivotal roles in deciding the cellular morphology, polarity, cell movement and overall signaling cascade. It was shown for the first time that expression patterns of Rac, Rho and CDC42 genes are altered on exposure to RVV. Similarly, significant difference in the expression pattern of HSP70 and p53 at the mRNA levels were noted. Our results confirmed that RVV induces apoptosis in A549 cells; this was further confirmed by AO/EtBr staining as well as caspase-3 assay. All experiments were compared using RVV unexposed cells. We propose for the first time that RVV induces morphological changes in human A549 cells through modulation of smGTPase expression and affects the cellular-nuclear architecture which in turn interferes in proliferation and migration of these cells along with apoptosis.

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on a HAB causing Alexandrium tamarense.

Toxicity of algicidal extracts from Mangrovimonas yunxiaonensis strain LY01 on Alexandrium tamarense were measured through studying the algicidal procedure, nuclear damage and transcription of related genes. Medium components were optimized to improve algicidal activity, and characteristics of algicidal extracts were determined. Transmission electron microscope analysis revealed that the cell structure was broken. Cell membrane integrity destruction and nuclear structure degradation were monitored using confocal laser scanning microscope, and the rbcS, hsp and proliferating cell nuclear antigen (PCNA) gene expressions were studied. Results showed that 1.0% tryptone, 0.4% glucose and 0.8% MgCl2 were the optimal nutrient sources. The algicidal extracts were heat and pH stable, non-protein and less than 1kD. Cell membrane and nuclear structure integrity were lost, and the transcription of the rbcS and PCNA genes were significantly inhibited and there was up-regulation of hsp gene expression during the exposure procedure. The algicidal extracts destroyed the cell membrane and nuclear structure integrity, inhibited related gene expression and, eventually, lead to the inhibition of algal growth. All the results may elaborate firstly the cell death process and nuclear damage in A. tamarense which was induced by algicidal extracts, and the algicidal extracts could be potentially used as bacterial control of HABs in future.