Blockade of CaV3 calcium channels and induction of G0/G1 cell cycle arrest in colon cancer cells by gossypol.

BACKGROUND AND PURPOSE: Gastrointestinal tumours overexpress voltage-gated calcium (CaV3) channels (CaV3.1, 3.2 and 3.3). CaV3 channels regulate cell growth and apoptosis colorectal cancer. Gossypol, a polyphenolic aldehyde found in the cotton plant, has anti-tumour properties and inhibits CaV3 currents. A systematic study was performed on gossypol blocking mechanism on CaV3 channels and its potential anticancer effects in colon cancer cells, which express CaV3 isoforms. EXPERIMENTAL APPROACH: Transcripts for CaV3 proteins were analysed in gastrointestinal cancers using public repositories and in human colorectal cancer cell lines HCT116, SW480 and SW620. The gossypol blocking mechanism on CaV3 channels was investigated by combining heterologous expression systems and patch-clamp experiments. The anti-tumoural properties of gossypol were estimated by cell proliferation, viability and cell cycle assays. Ca2+ dynamics were evaluated with cytosolic and endoplasmic reticulum (ER) Ca2+ indicators. KEY RESULTS: High levels of CaV3 transcripts correlate with poor prognosis in gastrointestinal cancers. Gossypol blockade of CaV3 isoforms is concentration- and use-dependent interacting with the closed, activated and inactivated conformations of CaV3 channels. Gossypol and CaV3 channels down-regulation inhibit colorectal cancer cell proliferation by arresting cell cycles at the G0/G1 and G2/M phases, respectively. CaV3 channels underlie the vectorial Ca2+ uptake by endoplasmic reticulum in colorectal cancer cells. CONCLUSION AND IMPLICATIONS: Gossypol differentially blocked CaV3 channel and its anticancer activity was correlated with high levels of CaV3.1 and CaV3.2 in colorectal cancer cells. The CaV3 regulates cell proliferation and Ca2+ dynamics in colorectal cancer cells. Understanding this blocking mechanism maybe improve cancer therapies.

Aerobic physical training reduces severe asthma phenotype involving kinins pathway.

INTRODUCTION: Aerobic physical training (APT) reduces eosinophilic airway inflammation, but its effects and mechanisms in severe asthma remain unknown. METHODS: An in vitro study employing key cells involved in the pathogenesis of severe asthma, such as freshly isolated human eosinophils, neutrophils, and bronchial epithelial cell lineage (BEAS-2B) and lung fibroblasts (MRC-5 cells), was conducted. Additionally, an in vivo study using male C57Bl/6 mice, including Control (Co; n = 10), Trained (Exe; n = 10), house dust mite (HDM; n = 10), and HDM + Trained (HDM + Exe; n = 10) groups, was carried out, with APT performed at moderate intensity, 5x/week, for 4 weeks. RESULTS: HDM and bradykinin, either alone or in combination, induced hyperactivation in human neutrophils, eosinophils, BEAS-2B, and MRC-5 cells. In contrast, IL-10, the primary anti-inflammatory molecule released during APT, inhibited these inflammatory effects, as evidenced by the suppression of numerous cytokines and reduced mRNA expression of the B1 receptor and ACE-2. The in vivo study demonstrated that APT decreased bronchoalveolar lavage levels of bradykinin, IL-1ß, IL-4, IL-5, IL-17, IL-33, TNF-α, and IL-13, while increasing levels of IL-10, klotho, and IL-1RA. APT reduced the accumulation of polymorphonuclear cells, lymphocytes, and macrophages in the peribronchial space, as well as collagen fiber accumulation, epithelial thickness, and mucus accumulation. Furthermore, APT lowered the expression of the B1 receptor and ACE-2 in lung tissue and reduced bradykinin levels in the lung tissue homogenate compared to the HDM group. It also improved airway resistance, tissue resistance, and tissue damping. On a systemic level, APT reduced total leukocytes, eosinophils, neutrophils, basophils, lymphocytes, and monocytes in the blood, as well as plasma levels of IL-1ß, IL-4, IL-5, IL-17, TNF-α, and IL-33, while elevating the levels of IL-10 and IL-1RA. CONCLUSION: These findings indicate that APT inhibits the severe asthma phenotype by targeting kinin signaling.

Comparing the Positive Impacts and Stress Induction by Polyethylene Glycol (PEG 6000) Variable Levels on Canola (Brassica napus L.) Growth, Yield, and Oil Contents.

Seed quality (i.e., emergence energy, viability, physical purity, size, weight) is a critical factor that influences the yield of crops. Poor seed quality can lead to reduced germination rates, lower plant populations, and, ultimately, lower crop yields. On the other hand, seed priming is suggested to be an effective technique for improving seeds germination and plant population. In this study, we investigated the effect of seed priming with polyethylene glycol (PEG) on the germination, growth, and yield of two varieties of canola, super canola, and sandal canola. The treatment plan includes five concentrations of PEG (i.e., 5, 10, 15, 20%), distilled water priming, and control (no priming). All of the treatments were applied in 3 replications following a completely randomized design. Our results showed that seed priming with 5%PEG (T2) significantly improved radicle length (50 and 36%), plant height (43 and 34%), chlorophyll a (44 and 43%), chlorophyll b (120 and 208%), and total chlorophyll (83 and 111%) compared to control in super canola and sandal canola, respectively. In particular, seed priming with 5%PEG resulted in the highest increase in protein contents (25 and 1.40%), oleic acid (26 and 40%), and linolenic acid (6 and 6%) compared to control in super canola and sandal canola, respectively. It is concluded that seed priming with 5%PEG is an effective treatment to improve the performance of canola crops in terms of seedling growth, yield, chlorophyll, protein, and oil content. More investigations are recommended as future perspectives using other canola varieties to declare 5% PEG as an effective treatment for canola for improvement in growth, oil, protein, and chlorophyll contents.

Biostimulants alleviate water deficit stress and enhance essential oil productivity: a case study with savory.

Water deficit stress exposure frequently constrains plant and agri-food production globally. Biostimulants (BSs) can be considered a new tool in mitigating water deficit stress. This study aimed to understand how BSs influence water deficit stress perceived by savory plants (Satureja hortensis L.), an important herb used for nutritional and herbal purposes in the Middle East. Three BS treatments, including bio-fertilizers, humic acid and foliar application of amino acid (AA), were implemented. Each treatment was applied to savory plants using three irrigation regimes (low, moderate and severe water deficit stress FC100, FC75 and FC50, respectively). Foliar application of AA increased dry matter yield, essential oil (EO) content and EO yield by 22%, 31% and 57%, respectively. The greatest EO yields resulted from the moderate (FC75) and severe water deficit stress (FC50) treatments treated with AA. Primary EO constituents included carvacrol (39-43%), gamma-terpinene (27-37%), alpha-terpinene (4-7%) and p-cymene (2-5%). Foliar application of AA enhanced carvacrol, gamma-terpinene, alpha-terpinene and p-cymene content by 6%, 19%, 46% and 18%, respectively. Physiological characteristics were increased with increasing water shortage and application of AA. Moreover, the maximum activities of superoxide dismutase (3.17 unit mg-1 min-1), peroxidase (2.60 unit mg-1 min-1) and catalase (3.08 unit mg-1 min-1) were obtained from plants subjected to severe water deficit stress (FC50) and treated with AA. We conclude that foliar application of AA under water deficit stress conditions would improve EO quantity and quality in savory.

PKC-Mediated Orai1 Channel Phosphorylation Modulates Ca2+ Signaling in HeLa Cells.

The overexpression of the Orai1 channel inhibits SOCE when using the Ca2+ readdition protocol. However, we found that HeLa cells overexpressing the Orai1 channel displayed enhanced Ca2+ entry and a limited ER depletion in response to the combination of ATP and thapsigargin (TG) in the presence of external Ca2+. As these effects require the combination of an agonist and TG, we decided to study whether the phosphorylation of Orai1 S27/S30 residues had any role using two different mutants: Orai1-S27/30A (O1-AA, phosphorylation-resistant) and Orai1-S27/30D (O1-DD, phosphomimetic). Both O1-wt and O1-AA supported enhanced Ca2+ entry, but this was not the case with O1-E106A (dead-pore mutant), O1-DD, and O1-AA-E106A, while O1-wt, O1-E106A, and O1-DD inhibited the ATP and TG-induced reduction of ER [Ca2+], suggesting that the phosphorylation of O1 S27/30 interferes with the IP3R activity. O1-wt and O1-DD displayed an increased interaction with IP3R in response to ATP and TG; however, the O1-AA channel decreased this interaction. The expression of mCherry-O1-AA increased the frequency of ATP-induced sinusoidal [Ca2+]i oscillations, while mCherry-O1-wt and mCherry-O1-DD decreased this frequency. These data suggest that the combination of ATP and TG stimulates Ca2+ entry, and the phosphorylation of Orai1 S27/30 residues by PKC reduces IP3R-mediated Ca2+ release.

Application of humic acid and biofertilizers changes oil and phenolic compounds of fennel and fenugreek in intercropping systems.

The study investigated the effect of organic/biofertilizers in intercropping patterns on seed yield and yield components and essential oil, fatty acid, and phenolic compounds of fennel (Foeniculum vulgare L.) and fenugreek (Trigonella foenum-graecum L.). Experimental treatments included the application of humic acid (HA), biofertilizers (BFS), and the unfertilized control in five planting patterns [1 row fennel + 2 rows fenugreek intercropping (1F:2FG), 2 rows fennel + 2 rows fenugreek intercropping (2F:2FG), 2 rows fennel + 4 rows fenugreek intercropping (2F:4FG), and sole cropping of each species]. Sole cropping with BFS produced the highest seed yields for fennel (2233 kg ha-1) and fenugreek (1240 kg ha-1). In contrast, the 2F:2FG intercropping ratio with BFS yielded the maximum fixed oil content for fennel (17.4%) and fenugreek (8.3%). Application of HA and BFS enhanced oil yields by 66% and 75% in fennel and 40% and 57% in fenugreek, respectively. The 2F:2FG intercropping ratio with BFS produced the maximum essential oil constituents [(E)-anethole, estragole, and fenchone] in fennel. In addition, 2F:4FG with BFS and 1F:1FG with HA produced the highest unsaturated fatty acid (oleic and linoleic acids) concentration in both species. The 2F:2FG intercropping ratio with BFS and HA produced the highest chlorogenic acid and quercetin contents, respectively, in fennel. In contrast, the 2F:4FG intercropping ratio with HA produced the highest chlorogenic acid and caffeic acid contents in fenugreek. Intercropping fennel/fenugreek with BFS or HA improved the essential oil content (fennel only), fixed oil quality and quantity, and phenolic compounds and created a more sustainable cultivation system than sole cropping systems for both species under low-input conditions.

Altered non-coding RNA expression profile in F1 progeny 1 year after parental irradiation is linked to adverse effects in zebrafish.

Gamma radiation produces DNA instability and impaired phenotype. Previously, we observed negative effects on phenotype, DNA methylation, and gene expression profiles, in offspring of zebrafish exposed to gamma radiation during gametogenesis. We hypothesize that previously observed effects are accompanied with changes in the expression profile of non-coding RNAs, inherited by next generations. Non-coding RNA expression profile was analysed in F1 offspring (5.5 h post-fertilization) by high-throughput sequencing 1 year after parental irradiation (8.7 mGy/h, 5.2 Gy total dose). Using our previous F1-γ genome-wide gene expression data (GSE98539), hundreds of mRNAs were predicted as targets of differentially expressed (DE) miRNAs, involved in pathways such as insulin receptor, NFkB and PTEN signalling, linking to apoptosis and cancer. snRNAs belonging to the five major spliceosomal snRNAs were down-regulated in the F1-γ group, Indicating transcriptional and post-transcriptional alterations. In addition, DEpiRNA clusters were associated to 9 transposable elements (TEs) (LTR, LINE, and TIR) (p = 0.0024), probable as a response to the activation of these TEs. Moreover, the expression of the lincRNAs malat-1, and several others was altered in the offspring F1, in concordance with previously observed phenotypical alterations. In conclusion, our results demonstrate diverse gamma radiation-induced alterations in the ncRNA profiles of F1 offspring observable 1 year after parental irradiation.

Gamma radiation induces locus specific changes to histone modification enrichment in zebrafish and Atlantic salmon.

Ionizing radiation is a recognized genotoxic agent, however, little is known about the role of the functional form of DNA in these processes. Post translational modifications on histone proteins control the organization of chromatin and hence control transcriptional responses that ultimately affect the phenotype. The purpose of this study was to investigate effects on chromatin caused by ionizing radiation in fish. Direct exposure of zebrafish (Danio rerio) embryos to gamma radiation (10.9 mGy/h for 3h) induced hyper-enrichment of H3K4me3 at the genes hnf4a, gmnn and vegfab. A similar relative hyper-enrichment was seen at the hnf4a loci of irradiated Atlantic salmon (Salmo salar) embryos (30 mGy/h for 10 days). At the selected genes in ovaries of adult zebrafish irradiated during gametogenesis (8.7 and 53 mGy/h for 27 days), a reduced enrichment of H3K4me3 was observed, which was correlated with reduced levels of histone H3 was observed. F1 embryos of the exposed parents showed hyper-methylation of H3K4me3, H3K9me3 and H3K27me3 on the same three loci, while these differences were almost negligible in F2 embryos. Our results from three selected loci suggest that ionizing radiation can affect chromatin structure and organization, and that these changes can be detected in F1 offspring, but not in subsequent generations.

The Trans Golgi Region is a Labile Intracellular Ca2+ Store Sensitive to Emetine.

The Golgi apparatus (GA) is a bona fide Ca2+ store; however, there is a lack of GA-specific Ca2+ mobilizing agents. Here, we report that emetine specifically releases Ca2+ from GA in HeLa and HL-1 atrial myocytes. Additionally, it has become evident that the trans-Golgi is a labile Ca2+ store that requires a continuous source of Ca2+ from either the external milieu or from the ER, to enable it to produce a detectable transient increase in cytosolic Ca2+. Our data indicates that the emetine-sensitive Ca2+ mobilizing mechanism is different from the two classical Ca2+ release mechanisms, i.e. IP3 and ryanodine receptors. This newly discovered ability of emetine to release Ca2+ from the GA may explain why chronic consumption of ipecac syrup has muscle side effects.

Ionizing radiation induces transgenerational effects of DNA methylation in zebrafish.

Ionizing radiation is known to cause DNA damage, yet the mechanisms underlying potential transgenerational effects of exposure have been scarcely studied. Previously, we observed effects in offspring of zebrafish exposed to gamma radiation during gametogenesis. Here, we hypothesize that these effects are accompanied by changes of DNA methylation possibly inherited by subsequent generations. We assessed DNA methylation in F1 embryos (5.5 hours post fertilization) with whole genome bisulfite sequencing following parental exposure to 8.7 mGy/h for 27 days and found 5658 differentially methylated regions (DMRs). DMRs were predominantly located at known regulatory regions, such as gene promoters and enhancers. Pathway analysis indicated the involvement of DMRs related to similar pathways found with gene expression analysis, such as development, apoptosis and cancers, which could be linked to previous observed developmental defects and genomic instability in the offspring. Follow up of 19 F1 DMRs in F2 and F3 embryos revealed persistent effects up to the F3 generation at 5 regions. These results indicate that ionizing radiation related effects in offspring can be linked to DNA methylation changes that partly can persist over generations. Monitoring DNA methylation could serve as a biomarker to provide an indication of ancestral exposures to ionizing radiation.

Parental exposure to gamma radiation causes progressively altered transcriptomes linked to adverse effects in zebrafish offspring.

Ionizing radiation causes a variety of effects, including DNA damage associated to cancers. However, the effects in progeny from irradiated parents is not well documented. Using zebrafish as a model, we previously found that parental exposure to ionizing radiation is associated with effects in offspring, such as increased hatching rates, deformities, increased DNA damage and reactive oxygen species. Here, we assessed short (one month) and long term effects (one year) on gene expression in embryonic offspring (5.5 h post fertilization) from zebrafish exposed during gametogenesis to gamma radiation (8.7 or 53 mGy/h for 27 days, total dose 5.2 or 31 Gy) using mRNA sequencing. One month after exposure, a global change in gene expression was observed in offspring from the 53 mGy/h group, followed by embryonic death at late gastrula, whereas offspring from the 8.7 mGy/h group was unaffected. Interestingly, one year after exposure newly derived embryos from the 8.7 mGy/h group exhibited 2390 (67.7% downregulated) differentially expressed genes. Overlaps in differentially expressed genes and enriched biological pathways were evident between the 53 mGy/h group one month and 8.7 mGy/h one year after exposure, but were oppositely regulated. Pathways could be linked to effects in adults and offspring, such as DNA damage (via Atm signaling) and reproduction (via Gnrh signaling). Comparison with gene expression analysis in directly exposed embryos indicate transferrin a and cytochrome P450 2x6 as possible biomarkers for radiation response in zebrafish. Our results indicate latent effects following ionizing radiation exposure from the lower dose in parents that can be transmitted to offspring and warrants monitoring effects over subsequent generations.

Dose-dependent effects of gamma radiation on the early zebrafish development and gene expression.

Ionizing radiation from natural sources or anthropogenic activity has the potential to cause oxidative stress or genetic damage in living organisms, through the ionization and excitation of molecules and the subsequent production of free radicals and reactive oxygen species (ROS). The present work focuses on radiation-induced biological effects using the zebrafish (Danio rerio) vertebrate model. Changes in developmental traits and gene expression in zebrafish were assessed after continuous external gamma irradiation (0.4, 3.9, 15 and 38 mGy/h) with corresponding controls, starting at 2.5 hours post fertilization (hpf) and lasting through embryogenesis and the early larval stage. The lowest dose rate corresponded to recommended benchmarks at which adverse effects are not expected to occur in aquatic ecosystems (2-10 mGy/day). The survival observed at 96 hours post fertilization (hpf) in the 38 mGy/h group was significantly lower, while other groups showed no significant difference compared to controls. The total hatching was significantly lower from controls in the 15 mGy/h group and a delay in hatching onset in the 0.4 mGy/h group was observed. The deformity frequency was significantly increased by prolonged exposure duration at dose rates ≥ 0.4 mGy/h. Molecular responses analyzed by RNA-seq at gastrulation (5.5 hpf transcriptome) indicate that the radiation induced adverse effects occurred during the earliest stages of development. A dose-response relationship was found in the numbers of differentially regulated genes in exposure groups compared to controls at a total dose as low as 1.62 mGy. Ingenuity Pathway Analysis identified retinoic acid receptor activation, apoptosis, and glutathione mediated detoxification signaling as the most affected pathways in the lower dose rate (0.54 mGy/h), while eif2 and mTOR, i.e., involved in the modulation of angiogenesis, were most affected in higher dose rates (5.4 and 10.9 mGy/h). By comparing gene expression data, myc was found to be the most significant upstream regulator, followed by tp53, TNF, hnf4a, TGFb1 and cebpa, while crabp2b and vegfab were identified as most frequent downstream target genes. These genes are associated with various developmental processes. The present findings show that continuous gamma irradiation (≥ 0.54 mGy/h) during early gastrula causes gene expression changes that are linked to developmental defects in zebrafish embryos.