Cell DNA Fragmentation Research Articles

PS-C37-6: IDENTIFICATION OF ALDOSTERONE-DRIVER SOMATIC MUTATIONS IN CELL-FREE DNA FROM ADRENAL VEIN SAMPLES OF PRIMARY ALDOSTERONISM PATIENTS

Objective: Cell-free DNA fragments (cf-DNA) of tumour cells are often found in the blood downstream to the tumour due to the high apoptosis or necrosis rate of the cells. Primary aldosteronism (PA), a curable cause of secondary hypertension, are commonly due to an autonomous aldosterone-producing adenoma that harbours a somatic mutation in an aldosterone-driver gene. We thus aimed to see if genotyping of cf-DNA for mutations in aldosterone-driver genes could be used as a biomarker for APA. Design and Methods: Genotyping of cf-DNA from the adrenal vein samples (AVS) of PA patients was performed using the Agena MassARRAY platform and validated by Sanger sequencing. In this study, six samples of cf-DNA from the left and right adrenals of three PA patients were interrogated. Results: Of the three PA patients, two had left adrenalectomy and one had right adrenalectomy. CTNNB1 S45 mutation was detected by the Agena MassArray platform in the cf-DNA of both right adrenal and left adrenal vein samples of all patients (Patient 1, Patient 2 and Patient 3). In Patient 1, Sanger sequencing confirmed a CTNNB1 S45del mutation in both cf-DNA of right adrenal and left adrenal vein samples. In Patient 3, who was found to have left adrenal hyperplasia, the cf-DNA of the right adrenal and left adrenal vein samples was also detected to have a KCNJ5 G151R mutation by the Agena MassArray platform which was confirmed by Sanger sequencing. Interestingly, only the cf-DNA of the left adrenal was found to have a CACNA1D S672L mutation in Patient 3. Conclusion: These results suggest that genotyping cf-DNA from AVS samples are able to detect aldosterone-driver mutations present in the APA. However, as AVS is an invasive procedure, it would be best if genotyping of cf-DNA from periphery blood can be performed. Therefore, further work is needed to ensure this strategy can be non-invasive as then it can be used as a screening method before AVS.

Morphofunctional characteristics of proliferation and apoptosis of germinal epithelium: pathogenesis of temporary azoospermia after local electron irradiation.

At present, the violation of male reproductive function caused by electron irradiation, leading to a decrease in the proliferative activity of germ cells, as well as the development of methods for its correction are relevant. The effect of leukocyte-poor platelet-rich plasma (LP-PRP) growth factors, which have a high regenerative capacity for the restoration of spermatogenesis, remains poorly understood. Objective of the study: immunohistochemical (IHC) assessment of the proliferation of germinal epithelium after electron irradiation with a dose of 2 Gy. Wistar rats (n=60) were divided into groups (conventional names): (I) control (n=30), which were injected with saline; (II) single local irradiation of the testes with electrons at a dose of 2 Gy (n=30). Animals were gradually withdrawn from the experiment over 11 weeks: 5 animals one week after irradiation, and then 5 animals once every 2 weeks. The testes were studied by histological and IHC methods using antibodies to Ki-67, Bcl-2, and p53. Terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) method was used to study DNA fragmentation in germ cells, which were stained with TdT solution (Thermo Fisher, USA) and incubated for 60 minutes. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) (blue spectrum) (Thermo Fisher), and the luminescence intensity was controlled in a fluorescent microscope with a set of fluorescein isothiocyanate (FITC) filters (green spectrum). IHC analysis of testes after irradiation showed a shift in the proliferative-apoptotic balance towards apoptosis of germ cells: a decrease in the expression levels of Ki-67 (16.3%±1.1%, P<0.05) and Bcl-2 (9.1%±1.1%, P<0.05), an increase in p53-positive cells (74.8%±1.2%, P<0.05) by the end of the experiment. In the experimental model, local irradiation of the testes with electrons at a dose of 2 Gy causes the development of focal hypospermatogenesis, affecting up to 1/8 of the sections of the tubules (in the first week) with a subsequent increase to 1/4 (in the second month), with a tendency to recovery in the third month (temporary azoospermia). The basis of focal hypospermatogenesis is the irradiation-induced proliferative-apoptotic imbalance towards the dominance of apoptosis, which is most pronounced in the pool of spermatogonia.

Apoptosis induction and anti-inflammatory activity of polyherbal drug AS20 on cervical cancer cell lines

Cancer occurs when normal cells convert into tumor cells as a result of the interaction between a patient’s genes and physical, chemical, or biological carcinogens. There are more than 100 types of cancer and each type requires a specific treatment regimen. The traditional treatment regimen for most types of cancer includes surgery, radiotherapy, and chemotherapy. However, treatment methods such as chemotherapy not only kill the cancer cells but also kill healthy cells that divide quickly. Chemotherapy induces widespread senescence, which contributes to local and systemic inflammation. To overcome these problems herbal drug treatments can be used as complementary and alternative medicines (CAMs). We obtained the polyherbal drug AS20 using Amaranthus spinosus leaves and inflorescences, which contain phytochemicals such as saponins, polyphenols, flavonoids, and alkaloids that are known to have anticancer and anti-oxidant activities. We hypothesized that AS20 would show anti-inflammation and induce DNA fragmentation, which is a hallmark of apoptosis in HeLa cells. Furthermore, this study explored the effects of AS20 on the expression of COX2, a marker of inflammation. We found that treatment with AS20 suppressed phorbol 12-myristate 13-acetate (PMA) and 5-flurouracil (5-FU) induction of COX2 expression. We also observed AS20 treated cells showed DNA fragmentation in HeLa cells. This research looks into AS20's possible anti-inflammatory effect on cervical cancer cell lines and its capacity to trigger apoptosis, a programmed cell death mechanism that can restrict cancer cell proliferation. These elements are examined in order to offer light on the therapeutic potential of AS20 and aiding the development of safer and more efficient cervical cancer treatment plans.

About combination of positive and negative outcomes of a subchronic exposure of rats to selenium oxide nanoparticles

Introduction. Occupational contact with selenium and its compounds, including nanoscale forms, occurs in the glass production, rubber industry, metallurgy (metallurgical processes of copper sludge processing, copper pyrite roasting, manganese, selenium and tellurium production). There are scarce data on the toxicity of selenium nanoparticles. Material and methods. Stable suspensions of nanoparticles or deionized water (control group) were administered to male rats 3 times a week for 6 weeks. A single dose of selenium oxide nanoparticles was 0.2 or 1 or 2 mg/kg of body weight). The condition of the animal organism was assessed with a number of indicators of toxic action at the end of the experiment. The statistical significance of intergroup differences was assessed by Student's t-test. Results. Activity of succinate dehydrogenase in blood lymphocytes reflecting the intensity of energy processes in the organism was decreased. The number of eosinophils in smears-imprints of parenchymal organs and mesenteric lymph nodes increased, indicating the ability of selenium nanooxide to trigger signaling cascades in immunocompetent cells. The number of degenerated cells in the proximal and distal tubules in smears of the kidneys was increased. A tendency to a decrease in all hemodynamics parameters was found. A change in the QT duration, together with an increase in the amplitude of the T wave, probably indicates a violation of the processes of myocardial repolarization. The coefficient of fragmentation of genomic DNA in nucleated blood cells decreased. Limitations. The research was limited to the study of indicators of toxic action in only one study using a limited dose range. Conclusion. An ambiguous effect of selenium oxide nanoparticles on rats was found. Along with negative impact of nanoparticles we have demonstrated, for the first time, some beneficial outcomes, in particular, genome -protective action which is in a striking contrast with the genotoxicity of all elemental and element-oxide nanoparticles previously studied in our laboratory.

RNaseH2A downregulation drives inflammatory gene expression via genomic DNA fragmentation in senescent and cancer cells

Cellular senescence caused by oncogenic stimuli is associated with the development of various age-related pathologies through the senescence-associated secretory phenotype (SASP). SASP is mediated by the activation of cytoplasmic nucleic acid sensors. However, the molecular mechanism underlying the accumulation of nucleotide ligands in senescent cells is unclear. In this study, we revealed that the expression of RNaseH2A, which removes ribonucleoside monophosphates (rNMPs) from the genome, is regulated by E2F transcription factors, and it decreases during cellular senescence. Residual rNMPs cause genomic DNA fragmentation and aberrant activation of cytoplasmic nucleic acid sensors, thereby provoking subsequent SASP factor gene expression in senescent cells. In addition, RNaseH2A expression was significantly decreased in aged mouse tissues and cells from individuals with Werner syndrome. Furthermore, RNaseH2A degradation using the auxin-inducible degron system induced the accumulation of nucleotide ligands and induction of certain tumourigenic SASP-like factors, promoting the metastatic properties of colorectal cancer cells. Our results indicate that RNaseH2A downregulation provokes SASP through nucleotide ligand accumulation, which likely contributes to the pathological features of senescent, progeroid, and cancer cells.

The Cytotoxic Effectiveness of Thiourea-Reduced Graphene Oxide on Human Lung Cancer Cells and Fungi.

This study demonstrated the effective reduction of graphene oxide (GO) by employing thiourea as a reducing and stabilizing agent. Two fungi (Aspergillus flavus and Aspergillus fumigatus) were used for anti-fungal assay. Cell viability, cell cycle analysis, DNA fragmentation, and cell morphology were assessed to determine the toxicity of thiourea-reduced graphene oxide (T-rGO) on human lung cancer cells. The results revealed that GO and T-rGO were hazardous to cells in a dose-dependent trend. The viability of both A. fumigatus and A. flavus was affected by GO and T-rGO. The reactive oxygen species produced by T-rGO caused the death of A. flavus and A. fumigatus cells. This study highlighted the effectiveness of T-rGO as an antifungal agent. In addition, T-rGO was found to be more harmful to cancer cells than GO. Thus, T-rGO manifested great potential in biological and biomedical applications.

Naturally Occurring Functional Ingredient from Filamentous Thermophilic Cyanobacterium Leptolyngbya sp. KC45: Phytochemical Characterizations and Their Multiple Bioactivities.

Cyanobacteria are rich in phytochemicals, which have beneficial impacts on the prevention of many diseases. This study aimed to comprehensively characterize phytochemicals and evaluate multifunctional bioactivities in the ethanolic extract of the cyanobacterium Leptolyngbya sp. KC45. Results found that the extract mainly contained chlorophylls, carotenoids, phenolics, and flavonoids. Through LC-ESI-QTOF-MS/MS analysis, 38 phenolic compounds with promising bioactivities were discovered, and a higher diversity of flavonoids was found among the phenolic compounds identified. The extract effectively absorbed the harmful UV rays and showed high antioxidant activity on DPPH, ABTS, and PFRAP. The extract yielded high-efficiency inhibitory effects on enzymes (tyrosinase, collagenase, ACE, and α-glucosidase) related to diseases. Interestingly, the extract showed a strong cytotoxic effect on cancer cells (skin A375, lung A549, and colon Caco-2), but had a much smaller effect on normal cells, indicating a satisfactory level of safety for the extract. More importantly, the combination of the DNA ladder assay and the TUNEL assay proved the appearance of DNA fragmentation in cancer cells after a 48 h treatment with the extract, confirming the apoptosis mechanisms. Our findings suggest that cyanobacterium extract could be potentially used as a functional ingredient for various industrial applications in foods, cosmetics, pharmaceuticals, and nutraceuticals.

Oxineur, a novel peptide from Caspian cobra Naja naja oxiana against HT-29 colon cancer

Colon cancer ranks fourth in mortality. This cancer is still an important clinical challenge worldwide due to its high prevalence and poor prognosis. Proteomic studies revealed that snake venom is a diverse and variable mixture of enzymatic and non-enzymatic proteins and peptides. Despite the toxic effects of these molecules, several proteins and peptides have been isolated that have practical applications and appear to induce apoptosis and prevent cell metastasis. In this study, we worked on cytotoxic effects and anticancer activity of Naja naja oxiana (Iranian Caspian cobra) snake venom components on HT-29 cell line colon cancer. Separated Fraction-5 by FPLC indicated the high cytotoxicity on HT-29 cell line colon cancer by MTT test. Further isolation of F5 by HPLC showed that the purified peak 2, nominated as Oxineur that contains a cytotoxic effect on HT-29 cells and reduces cell viability at 8 μg/ml to 4% in 24 h. Oxineur has the least cytotoxic effect on HEK-293 normal cells. Further studies on Oxineur peptide confirmed the apoptotic effects with high expression of CASP9 gene and DNA fragmentation in cancerous cells. The partial sequence of Oxineur revealed 71% homology with the neurotoxin II from Naja naja oxiana. Since our target molecule is a peptide in the molecular weight range of 7 kDa, it has potentially a therapeutic value.

Sperm-cell DNA fragmentation prediction using label-free quantitative phase imaging and deep learning.

In intracytoplasmic sperm injection (ICSI), a single sperm cell is selected and injected into an egg. The quality of the chosen sperm and specifically its DNA fragmentation have a significant effect on the fertilization success rate. However, there is no method today to measure the DNA fragmentation of live and unstained cells during ICSI. We present a new method to predict the DNA fragmentation of sperm cells using multi-layer stain-free imaging data, including quantitative phase imaging, and lightweight deep learning architectures. The DNA fragmentation ground truth is achieved by staining the cells with acridine orange and imaging them via fluorescence microscopy. Our prediction model is based on the MobileNet convolutional neural network architecture combined with confidence measurement determined by distances between vectors in the latent space. Our results show that the mean absolute error for cells with high prediction confidence is 0.05 and the 90th percentile mean absolute error is 0.1, where the range of DNA fragmentation score is [0,1]. In the future, this model may be applied to improve cell selection by embryologists during ICSI.

Size distribution analysis of residual host cell DNA fragments in lentivirus by CGE-LIF.

During the production of cell and gene therapy products, residual host cell DNA (HCD) could cause safety risks of the biological products, and the longer the residual HCD fragment, the greater the risk to the human body. For this reason, it was necessary to develop an effective method for the size distribution analysis of residual HCD fragments with high accuracy and sensitivity. In this study, capillary gel electrophoresis with laser-induced fluorescence detector (CGE-LIF) was used to analyze the size distribution of residual HCD fragments in lentivirus products. The results confirmed that lentiviral RNA genome could interfere with the size distribution analysis of residual HCD fragments. By optimizing the amount of RNase I and digestion time in sample pretreatment process, the interfere of RNA genome could be avoided. The specificity, precision, accuracy, linear range, the detection of limit (LOD), and the quantification of limit (LOQ) of CGE-LIF method were also validated. The results showed that the CGE-LIF method had a good performance both in terms of specificity and reproducibility. The intra- and inter-day relative standard deviations of migration time and corrected peak area were all less than 1% and 2%, respectively. The 200bp DNA marker had a good linearity between 50 and 1000pg/ml. The LOD and LOQ of 200bp DNA marker were 2.59 and 8.64pg/ml, respectively. In addition, this method was successfully used to analyze the size distribution analysis of residual HCD fragments in lentivirus products with different production processes.

Genotoxicity, DNA damage and sperm defects induced by vinblastine

BackgroundThe treatment with chemotherapy may develop secondary tumors as a result of chemo genotoxicity. Sperm defects is another complication associated with chemo treatment. In this study the genotoxicity of vinblastine (VB) was estimated in both somatic and germ cells.Materials85 mice were taken. Four single doses of VB at 3, 4.5, 6 and 10 mg/kg and three successive doses at 3, 4.5 and 6 mg/kg were taken for estimation of chromosomal aberrations (CAs). Four single doses of VB were involved in estimating the DNA fragmentation, and comet assay. For sperm abnormalities mice were injected with three successive doses of VB at 3, 4.5, and 6 mg/kg.ResultsThe results demonstrated a significant frequency of DNA fragmentation in spleen cells and in the percentage of CAs in bone marrow. Numerical and structural aberrations were recorded with a pronounced number of polyploidy metaphases which reached (11.60%) after treatment with 6 mg/kg for three successive days vs zero for control. VB also induced a significant percentage of CAs in spermatocytes in the form of univalent. Sperm defects in the form of coiled tail, absence of acrosome and shapeless head and a significant DNA damage in the testes were recorded. The frequency of sperm abnormalities reached 11.06 ± 0.14 after treatment with highest tested dose (6 mg/kg) vs 3.04 ± 0.19 for control.ConclusionVB is genotoxic in somatic and germ cells. Sperm defects induced by VB are of serious concern to future generations and may affect the fertility of cancer survivors.

Studies on DNA fragmentation and cell integrity of rapidly hydroxyl radical inactivated Microcystis aeruginosa

Microcystis aeruginosa (M. aeruginosa) blooms occur frequently in freshwater lakes and reservoirs, posing a great threaten to drinking water safety. When using conventional oxidants remedy toxic algae, there is an unavoidable shortage leading to cell rupture and release of intracellular organic matter (IOM), thus causing more serious hazard to water safety. In this study, with the synergistic effect of strong ionisation discharge and hydrodynamic cavitation, hydroxyl radical (•OH) were generated and injected into cells to inactivate M. aeruginosa within 3 s. •OH-inactivated M. aeruginosa was intact without rupture and exhibited a 39 % increase in dissolved organic carbon (DOC) due to the degradation of the components of the cell wall and extracellular matrix. For comparison, ClO2 inactivated M. aeruginosa after 10 min of exposure, which induced cell rupture and a 113 % increase in DOC due to the release of IOM. Meanwhile, in •OH-inactivated M. aeruginosa, intracellular •OH was increased to 2 times according to the detection by fluorescence probe and the DNA oxidative damage biomarker 8-hydroxy-2′-deoxyguanosine increased to 1.4 times according to the enzyme-linked immunosorbent assay, confirming the direct action of •OH on the DNA. And •OH induced DNA fragmentation in cells of M. aeruginosa was identified by comet assay and TUNEL assay, finding a 4636 % increase in the DNA tail length, and 96.71 % of the cells showed DNA phosphatediester bond cleavage. In summary, the main reason for the rapid •OH-inactivation of M. aeruginosa is DNA fragmentation, which may represent a new method for the safely and efficient control of harmful algal blooms.

The effects of transpositions of functional I retrotransposons depend on the conditions and dose of parental exposure

Purpose Transposable elements (TEs) cause destabilization of animal genomes. I retrotransposons of Drosophila melanogaster, as well as human LINE1 retrotransposons, are sources of intra- and interindividual diversity and responses to the action of internal and external factors. The aim of this study was to investigate the response to irradiation for the offspring of Drosophila melanogaster with the increased activity of inherited functional I elements. Materials and methods The material used was dysgenic Drosophila females with active I retrotransposons obtained as a result of crossing irradiated/non-irradiated parents of a certain genotype. Non-dysgenic females (without functional I elements) were used as controls. The effects of different conditions (irradiation of both parents simultaneously or separately) and doses (1–100 Gy) of parental irradiation have been assessed by analyzing SF-sterility, DNA damage and lifespan. The presence of full-size I retrotransposons was determined by PCR analysis. Results The maternal exposure and exposure of both parents are efficient in contrast with paternal exposure. Irradiation of mothers reduces the reproductive potential and viability of their female offspring which undergo high activity of functional I retrotransposons. Though I retrotranspositions negatively affect the female gonads, irradiation of the paternal line can increase the lifespan of SF-sterile females. Radiation stress in the range of 1–100 Gy increases DNA fragmentation in both somatic and germ cells of the ovaries with high I-retrotransposition. Conclusions These results allow for the specificity of the radiation-induced behavior of I retrotransposons and their role in survival under conditions of strong radiation stress.

Detection of apoptotic cells based on in situ hybridization chain reaction using specific hairpins.

There are an increasing number of experiments to study programmed cell death/apoptosis, one of the characteristics of which is DNA fragmentation. The only current method for in situ detection of DNA fragmentation is Terminal deoxynucleotidyl transferase mediated-dUTP Nick End Labeling, TUNEL. In this study, a new method for in situ detection of apoptotic DNA fragments, namely In Situ Hybridization Chain Reaction, isHCR, was established. The principle of the assay is that the sticky end sequence of the apoptotic cell DNA fragment non-specifically initiates a hybridization chain reaction that specifically detects the apoptotic cell. The results of the combined TUNEL and isHCR method demonstrated that the majority of isHCR-positive cells were also labeled by TUNEL. In situ HCR often detect DNA fragments in the cytoplasm that the classical TUNEL method couldnot, and these cells may be in the early stages of apoptosis. It also indicates that DNA fragments are transferred to the cytoplasm during apoptosis. Because the staining process does not require terminal deoxynucleotidyl transferase as TUNEL staining does, isHCR staining cost low and can be performed on a large number of tissue specimens. It is believed that isHCR has the potential to detect DNA fragmentation of apoptotic cells in situ.

Combination Therapy of Curcumin and Disulfiram Synergistically Inhibits the Growth of B16-F10 Melanoma Cells by Inducing Oxidative Stress

Oxidative stress plays a central role in the pathophysiology of melanoma. Curcumin (CUR) is a polyphenolic phytochemical that stimulates reactive oxygen species (ROS) production, while disulfiram (DSS) is a US FDA-approved drug for the treatment of alcoholism that can act by inhibiting the intracellular antioxidant system. Therefore, we hypothesized that they act synergistically against melanoma cells. Herein, we aimed to study the antitumor potential of the combination of CUR with DSS in B16-F10 melanoma cells using in vitro and in vivo models. The cytotoxic effects of different combination ratios of CUR and DSS were evaluated using the Alamar Blue method, allowing the production of isobolograms. Apoptosis detection, DNA fragmentation, cell cycle distribution, and mitochondrial superoxide levels were quantified by flow cytometry. Tumor development in vivo was evaluated using C57BL/6 mice bearing B16-F10 cells. The combinations ratios of 1:2, 1:3, and 2:3 showed synergic effects. B16-F10 cells treated with these combinations showed improved apoptotic cell death and DNA fragmentation. Enhanced mitochondrial superoxide levels were observed at combination ratios of 1:2 and 1:3, indicating increased oxidative stress. In vivo tumor growth inhibition for CUR (20 mg/kg), DSS (60 mg/kg), and their combination were 17.0%, 19.8%, and 28.8%, respectively. This study provided data on the potential cytotoxic activity of the combination of CUR with DSS and may provide a useful tool for the development of a therapeutic combination against melanoma.

75P Biosynthesis and characterization of selenium nanoparticles using marine algae Sargassum wightii: Understanding its anticancer activity through ROS-mediated p53 and Akt signaling pathways in A549 cells

75P Biosynthesis and characterization of selenium nanoparticles using marine algae Sargassum wightii: Understanding its anticancer activity through ROS-mediated p53 and Akt signaling pathways in A549 cells

Cryopreservation of Roughscale Sole (Clidoderma asperrimum) Sperm: Effects of Cryoprotectant, Diluent, Dilution Ratio, and Thawing Temperature.

Simple SummaryThe roughscale sole, Clidoderma asperrimum, is found in the East and West Seas of Korea, waters north of Hokkaido in Japan, as well as the East China Sea, the East Pacific, and the Canadian Maritimes. In 2021, this fish was categorized as an endangered species. Thus, there is a need to maintain gametes by freezing sperm. This study investigated the impacts of the cryoprotective agent, diluent, dilution ratio, and thawing temperature on the cryopreservation of fish sperm. In this investigation, sperm dilution 1:1 with a mixture of 10% dimethyl sulfoxide + Stein’s solution and thawing at 10 °C provided the most effective DNA damage prevention. These results support the development of a roughscale sole sperm cryopreservation procedure.The roughscale sole, Clidoderma asperrimum is categorized as an endangered species. Sperm freezing is essential for preserving gametes. This study examined the CPA concentration, diluent, dilution ratio, and thawing temperature to design a sperm cryopreservation protocol for roughscale sole. The variables examined included sperm motility and kinematics, cell survival, fertilization, and DNA fragmentation. Sperm motility parameters were assessed via computer-assisted sperm analysis using a CEROS II instrument. Cell survival rate and DNA damage were assessed using the Cell Counting Kit-8 and single-cell gel electrophoresis assay, respectively. Sperm preservation was tested using several CPAs, including ethylene glycol, dimethyl sulfoxide (DMSO), glycerol, propylene glycol, and methanol. The diluents tested were 300 mM sucrose, 300 mM glucose, Stein’s solution, Ringer’s solution, and Hank’s solution. The optimal conditions for sperm cryopreservation were 10% DMSO + Stein’s solution. After thawing, sperm motility was highest with a 1:1 dilution ratio (sperm to CPA + diluent), at 69.20 ± 0.32%; thawing at 10 °C was optimal for post-thaw motility (72.03 ± 0.95%). The highest fertilization rate (40.00 ± 1.22%) was obtained using DMSO. The fresh sperm had the lowest tail DNA, followed by 10% DMSO + Stein’s solution. The developed cryopreservation methods can be used in roughscale sole hatcheries.

Anti-Apoptotic and Anti-Inflammatory Effects of an Ethanolic Extract of Lycium chinense Root against Particulate Matter 10-Induced Cell Death and Inflammation in RBL-2H3 Basophil Cells and BALB/c Mice.

Particulate matters (PMs) from polluted air cause diverse pulmonary and cardiovascular diseases, including lung inflammation. While the fruits (Goji) of Lycium trees are commonly consumed as traditional medicine and functional food ingredients, the majority of their roots are discarded as by-products. To enhance the industrial applicability of Lycium roots, we prepared an ethanol extract (named GR30) of L. chinense Miller roots and evaluated its potential protective effects against particulate matter 10 (PM10)-induced inflammation and immune cell death. The GR30 treatment (0–500 μg/mL) significantly attenuated the PM10-induced cell cycle arrest, DNA fragmentation and mitochondria-dependent apoptosis in RBL-2H3 basophil cells. GR30 also significantly antagonized the PM10-induced expression of proinflammatory cytokines (IL-4, IL-13, and TNF-α) and COX2 expression through downregulation of MAPKs (ERK and JNK) signalling pathway. Oral administration of GR30 (200–400 mg/kg) to PM10 (20 mg/mL)-challenged mice significantly reduced the serum levels of IgE and the expression of TNF-α and Bax in lung tissues, which were elevated by PM10 exposure. These results revealed that the ethanolic extract (GR30) of L. chinense Miller roots exhibited anti-inflammatory and cyto-protective activity against PM10-induced inflammation and basophil cell death, and thus, it would be useful in functional food industries to ameliorate PM-mediated damage to respiratory and immune systems.

Proteomic profiling reveals neuronal ion channel dysregulation and cellular responses to DNA damage-induced cell cycle arrest and senescence in human neuroblastoma SH-SY5Y cells exposed to cypermethrin

Cypermethrin (CYP), a synthetic pyrethroid of class II, is widely used as a pesticide worldwide. The primary target of cypermethrin is a voltage-gated sodium channel. The neurotoxicity of CYP has been extensively studied in terms of affecting neuronal development, increasing cellular oxidative stress, and apoptosis. However, little is known about how it affects the expression of channel proteins involved in synaptic transmission, as well as the effects of cypermethrin on DNA damage and cell cycle processes. We found that the ligand and voltage-gated calcium channels and proteins involved in synaptic transmission including NMDA 1 receptor subunit, alpha 1A-voltage-dependent calcium channel, synaptotagmin-17, and synaptojanin-2 were downregulated in CYP-treated cells. After 48 h of CYP exposure, cell viability was reduced with flattened and enlarged morphology. The levels of 23 proteins regulating cell cycle processes were altered in CYP-treated cells, according to a proteomic study. The cell cycle analysis showed elevated G0/G1 cell cycle arrest and DNA fragmentation at the sub-G0 stage after CYP exposure. CYP treatment also increased senescence-associated β-galactosidase positive cells, DNA damage, and apoptotic markers. Taken together, the current study showed that cypermethrin exposure caused DNA damage and hastened cellular senescence and apoptosis via disrupting cell cycle regulation. In addition, despite its primary target sodium channel, CYP might cause synaptic dysfunction via the downregulation of synaptic proteins and dysregulation of synapse-associated ion channels.

Cytotoxicity, apoptosis inducing activity and Western blot analysis of tanshinone derivatives from Stachys parviflora on prostate and breast cancer cells.

Cytotoxic activities of methanolic crude extract of Stachys parviflora (Lamiaceae family) and its sub-fractions were primarily evaluated against human breast adenocarcinoma (MCF-7 and MDA-MB-231) and prostate (PC3) cell lines. The methanolic extract exhibited the highest activity, and was chosen for the isolation procedure. Four diterpenoid quinones, namely miltirone [1], tanshinone IIA [2], 1-hydroxy-tanshinone IIA [3], and cryptotanshinone [4] were isolated. Notably, this is the first report on the isolation and/or characterization of the mentioned diterpenoids from the Stachys genus. In this study, 1-hydroxy-tanshinone IIA [3] displayed the highest cytotoxicity among the isolated compounds. The mechanism of the cytotoxicity of methanolic extract and isolated compounds was further investigated by the utilization of propidium iodide staining (PI) assay. The results showed that the methanolic extract and 1-hydroxy-tanshinone IIA [3] enhanced DNA fragmentation in PC3 and MCF-7 cells. Moreover, the western blotting analysis demonstrated increasing and decreasing protein levels of Bax and Bcl2, respectively, and cleaved poly ADP-ribose polymerase (PARP). Further bioassay-guided phytochemical assessments of S. parviflora can be suggested as a promising approach for discovering potent bioactive secondary metabolites.