Assessment of DNA damage and DNA damage response and repair in dormancy-enriched leukemia cells

Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by clonal expansion of abnormal/immature hematopoietic precursor cells in the bone marrow. A side compartment in the BM niche consists of abnormal, quiescent cells, which are called dormant leukemic initiating cells (DLICs). Patients with AML tend to respond well to remission induction chemotherapy, but relapse is common because current therapies cannot completely eradicate leukemic cells. It is widely accepted that CD34+CD38− DLICs are more resistant to chemotherapy and that they contribute to drug resistance and relapse of AML to a greater extent than progenitor CD34+CD38+ cells. DLICs have been extensively characterised, but they remain a critical area of investigation for clinical research because of the low prevalence of DLICs and similarity to normal HSCs. A model of dormancy in vitro that shows most of the features of DLICs had previously been established in the Nottingham Haematology Group. This study used this model and aimed to investigate whether the response to DNA damage was different in dormancy-enriched cells compared to cycling leukemic cells following chemotherapy. The amount of DNA damage was assessed up to 24 hours pre- and post- drug treatment using the neutral Comet assay. Lower levels of damage were observed in dormancy-enriched cells following etoposide (ETO) treatment at 4 hours (p = 0.04), although this switched at the 24 hour time point where accumulated DNA double-stranded breaks (DSBs), in dormancy-enriched KG1a cells were associated with a higher percentage of viable cells. DNA damage response cascade markers in both dormancy-enriched and cycling cells showed phosphorylation by flow cytometry (phospho-H2AX139, pATM-S1981, H2AX142, and pChk-Thr68) in response to conventional AML chemotherapy. Significantly lower levels of cleaved PARP-Asp214 and active caspase 3 were observed in dormancy-enriched cells treated with ara-c (p = 0.0001) or ETO (p = 0.0001) at 24 hours, strongly indicating that survival responses are activated in dormancy-enriched cells. Induction of 53BP1 foci, the hallmark of non-homologous end joining (NHEJ) was observed following treatment with ara-c (p = 0.038) and ETO (p = 0.049) in dormancy-enriched cells, indicating the NHEJ repair pathway is the preferred mechanism for DSB repair. At the molecular level, BTG2 expression was involved in the DNA damage response. Significant induction of BTG2 was detected in cycling treated cells with ETO for 24 hours. In conclusion, this study provides evidence that phosphorylation of H2AX139 and H2AX142 is a key response marker that may explain the mechanism underlying the drug resistance of DLICs and induction of repair. Therefore, results of this study may help in devising novel treatment strategies for AML that target H2AX142 of DLICs to permanently eradicate all leukemic cells and improve overall survival

Utilizing Andrographis paniculata leaves and roots by effective usage of the bioactive andrographolide and its nanodelivery: investigation of antikindling and antioxidant activities through in silico and in vivo studies

To valorise the bioactive constituents abundant in leaves and other parts of medicinal plants with the objective to minimize the plant-based wastes, this study was undertaken. The main bioactive constituent of Andrographis paniculata, an Asian medicinal plant, is andrographolide (AG, a diterpenoid), which has shown promising results in the treatment of neurodegenerative illnesses. Continuous electrical activity in the brain is a hallmark of the abnormal neurological conditions such as epilepsy (EY). This can lead to neurological sequelae. In this study, we used GSE28674 as a microarray expression profiling dataset to identify DEGs associated with andrographolide and those with fold changes >1 and p-value <0.05 GEO2R. We obtained eight DEG datasets (two up and six down). There was marked enrichment under various Kyoto Encyclopaedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms for these DEGs (DUSP10, FN1, AR, PRKCE, CA12, RBP4, GABRG2, and GABRA2). Synaptic vesicles and plasma membranes were the predominant sites of DEG expression. AG acts as an antiepileptic agent by upregulating GABA levels. The low bioavailability of AG is a significant limitation of its application. To control these limitations, andrographolide nanoparticles (AGNPs) were prepared and their neuroprotective effect against pentylenetetrazol (PTZ)-induced kindling epilepsy was investigated using network pharmacology (NP) and docking studies to evaluate the antiepileptic multi-target mechanisms of AG. Andrographolide is associated with eight targets in the treatment of epilepsy. Nicotine addiction, GABAergic synapse, and morphine addiction were mainly related to epilepsy, according to KEGG pathway enrichment analysis (p < 0.05). A docking study showed that andrographolide interacted with the key targets. AG regulates epilepsy and exerts its therapeutic effects by stimulating GABA production. Rats received 80 mg/kg body weight of AG and AGNP, phenytoin and PTZ (30 mg/kg i.p. injection on alternate days), brain MDA, SOD, GSH, GABAand histological changes of hippocampus and cortex were observed. PTZ injected rats showed significantly (***p < 0.001) increased kindling behavior, increased MDA, decreased GSH, SOD, GABA activities, compared with normal rats, while treatment AGNPs significantly reduced kindling score and reversed oxidative damage. Finally, we conclude that the leaves and roots of A. Paniculata can be effectively utilized for its major bioactive constituent, andrographolide as a potent anti-epileptic agent. Furthermore, the findings of novel nanotherapeutic approach claim that nano-andrographolide can be successfully in the management of kindling seizures and neurodegenerative disorders

Assessment of DNA damage and DNA damage response and repair in dormancy-enriched leukemia cells

Acute myeloid leukaemia (AML) is a heterogeneous myeloid malignancy characterized by clonal expansion of abnormal/immature hematopoietic precursor cells in the bone marrow. A side compartment in the BM niche consists of abnormal, quiescent cells, which are called dormant leukemic initiating cells (DLICs). Patients with AML tend to respond well to remission induction chemotherapy, but relapse is common because current therapies cannot completely eradicate leukemic cells. It is widely accepted that CD34+CD38− DLICs are more resistant to chemotherapy and that they contribute to drug resistance and relapse of AML to a greater extent than progenitor CD34+CD38+ cells. DLICs have been extensively characterised, but they remain a critical area of investigation for clinical research because of the low prevalence of DLICs and similarity to normal HSCs. A model of dormancy in vitro that shows most of the features of DLICs had previously been established in the Nottingham Haematology Group. This study used this model and aimed to investigate whether the response to DNA damage was different in dormancy-enriched cells compared to cycling leukemic cells following chemotherapy. The amount of DNA damage was assessed up to 24 hours pre- and post- drug treatment using the neutral Comet assay. Lower levels of damage were observed in dormancy-enriched cells following etoposide (ETO) treatment at 4 hours (p = 0.04), although this switched at the 24 hour time point where accumulated DNA double-stranded breaks (DSBs), in dormancy-enriched KG1a cells were associated with a higher percentage of viable cells. DNA damage response cascade markers in both dormancy-enriched and cycling cells showed phosphorylation by flow cytometry (phospho-H2AX139, pATM-S1981, H2AX142, and pChk-Thr68) in response to conventional AML chemotherapy. Significantly lower levels of cleaved PARP-Asp214 and active caspase 3 were observed in dormancy-enriched cells treated with ara-c (p = 0.0001) or ETO (p = 0.0001) at 24 hours, strongly indicating that survival responses are activated in dormancy-enriched cells. Induction of 53BP1 foci, the hallmark of non-homologous end joining (NHEJ) was observed following treatment with ara-c (p = 0.038) and ETO (p = 0.049) in dormancy-enriched cells, indicating the NHEJ repair pathway is the preferred mechanism for DSB repair. At the molecular level, BTG2 expression was involved in the DNA damage response. Significant induction of BTG2 was detected in cycling treated cells with ETO for 24 hours. In conclusion, this study provides evidence that phosphorylation of H2AX139 and H2AX142 is a key response marker that may explain the mechanism underlying the drug resistance of DLICs and induction of repair. Therefore, results of this study may help in devising novel treatment strategies for AML that target H2AX142 of DLICs to permanently eradicate all leukemic cells and improve overall survival

Dual synergistic inhibition of COX and LOX by potential chemicals from Indian daily spices investigated through detailed computational studies

Abstract Cyclooxygenase (COX) and Lipoxygenase (LOX) are essential enzymes for arachidonic acid (AA) to eicosanoids conversion. These AA-derived eicosanoids are essential for initiating immunological responses, causing inflammation, and resolving inflammation. Dual COX/5-LOX inhibitors are believed to be promising novel anti-inflammatory agents. They inhibit the synthesis of prostaglandins (PGs) and leukotrienes (LTs), but have no effect on lipoxin formation. This mechanism of combined inhibition circumvents certain limitations for selective COX-2 inhibitors and spares the gastrointestinal mucosa. Natural products, i.e. spice chemicals and herbs, offer an excellent opportunity for drug discovery. They have proven anti-inflammatory properties. However, the potential of a molecule to be a lead/ drug candidate can be much more enhanced if it has the property of inhibition in a dual mechanism. Synergistic activity is always a better option than the molecule's normal biological activity. Herein, we have explored the dual COX/5-LOX inhibition property of the three major potent phytoconsituents (curcumin, capsaicin, and gingerol) from Indian spices using in silico tools and biophysical techniques in a quest to identify their probable inhibitory role as anti-inflammatory agents. Results revealed the dual COX/5-LOX inhibitory potential of curcumin. Gingerol and capsaicin also revealed favorable results as dual COX/5-LOX inhibitors. Our results are substantiated by target similarity studies, molecular docking, molecular dynamics, energy calculations, DFT, and QSAR studies. In experimental inhibitory (in vitro) studies, curcumin exhibited the best dual inhibitory activities against COX-1/2 and 5-LOX enzymes. Capsaicin and gingerol also showed inhibitory potential against both COX and LOX enzymes. In view of the anti-inflammatory potential these spice chemicals, this research could pave the way for more scientific exploration in this area for drug discovery

Quantitative Evaluation of Soil Quality Using Principal Component Analysis: The Case Study of El-Fayoum Depression Egypt

Soil quality assessment is the first step towards precision farming and agricultural management. In the present study, a multivariate analysis and geographical information system (GIS) were used to assess and map a soil quality index (SQI) in El-Fayoum depression in the Western Desert of Egypt. For this purpose, a total of 36 geo-referenced representative soil samples (0–0.6 m) were collected and analyzed according to standardized protocols. Principal component analysis (PCA) was used to reduce the dataset into new variables, to avoid multi-collinearity, and to determine relative weights (Wi) and soil indicators (Si), which were used to obtain the soil quality index (SQI). The zones of soil quality were determined using principal component scores and cluster analysis of soil properties. A soil quality index map was generated using a geostatistical approach based on ordinary kriging (OK) interpolation. The results show that the soil data can be classified into three clusters: Cluster I represents about 13.89% of soil samples, Cluster II represents about 16.6% of samples, and Cluster III represents the rest of the soil data (69.44% of samples). In addition, the simulation results of cluster analysis using the Monte Carlo method show satisfactory results for all clusters. The SQI results reveal that the study area is classified into three zones: very good, good, and fair soil quality. The areas categorized as very good and good quality occupy about 14.48% and 50.77% of the total surface investigated, and fair soil quality (mainly due to salinity and low soil nutrients) constitutes about 34.75%. As a whole, the results indicate that the joint use of PCA and GIS allows for an accurate and effective assessment of the SQI

Neuroprotective effect of endophytic fungal antioxidant polyphenols on cerebral ischemic stroke-induced Albino rats; memory impairments, brain damage, and upregulation of metabolic proteins

Background: Stroke is one of the leading causes of mortality and disability throughout the world. Recently, antioxidant therapies were attempted to reduce apoptotic cell death in cerebral ischemia animal model. Purpose: To study the neuroprotective properties of polyphenol derived from fungal endophyte analyzed on experimental Albino rat. Methods: Polyphenols producing endophytic fungi was initially isolated from the seeds of Moringa oleifera Lam. The endophytes were cultured in potato dextrose broth and the potent strain Simplicillium sp. ED7 produced maximum phenolic content (86.42 ± 5.3 mg GAE/g) than other fungi. Polyphenols were extracted with solvent and used for the determination of neuroprotective properties. Results: Isoflurane was used to induce stroke in Albino rat and treated polyphenols showed reduced neurological deficits and improved neuroprotective properties. The ischemic Albino rats treated with polyphenols restored memory loss. The increased dosage of polyphenol improved the biosynthesis of more antioxidant enzymes than lower dosages. Central artery occlusion evoked about 2.28-fold increase in reactive oxygen species in brain tissue and the generation of reactive oxygen species was decreased in polyphenol treated animal. Conclusion: Albino rats treated with different doses of polyphenol had decrease ROS amount than sham group. The elevated level of cytochrome revealed mitochondrial damage in stroke induced control Albino rat. After 24 h of reperfusion on Albino rat, upregulation of total p65 and phospho-p65 were determined. The present finding revealed that polyphenl has a neuroprotective property in ischemia and regulate metabolic enzymes and restore brain injury

Table9_A protein–miRNA biomic analysis approach to explore neuroprotective potential of nobiletin in human neural progenitor cells (hNPCs).DOCX

Chemical-induced neurotoxicity is increasingly recognized to accelerate the development of neurodegenerative disorders (NDs), which pose an increasing health burden to society. Attempts are being made to develop drugs that can cross the blood–brain barrier and have minimal or no side effects. Nobiletin (NOB), a polymethoxylated flavonoid with anti-oxidative and anti-inflammatory effects, has been demonstrated to be a promising compound to treat a variety of NDs. Here, we investigated the potential role of NOB in sodium arsenate (NA)-induced deregulated miRNAs and target proteins in human neural progenitor cells (hNPCs). The proteomics and microRNA (miRNA) profiling was done for different groups, namely, unexposed control, NA-exposed, NA + NOB, and NOB groups. Following the correlation analysis between deregulated miRNAs and target proteins, RT-PCR analysis was used to validate the selected genes. The proteomic analysis showed that significantly deregulated proteins were associated with neurodegeneration pathways, response to oxidative stress, RNA processing, DNA repair, and apoptotic process following exposure to NA. The OpenArray analysis confirmed that NA exposure significantly altered miRNAs that regulate P53 signaling, Wnt signaling, cell death, and cell cycle pathways. The RT-PCR validation studies concur with proteomic data as marker genes associated with autophagy and apoptosis (HO-1, SQSTM1, LC-3, Cas3, Apaf1, HSP70, and SNCA1) were altered following NA exposure. It was observed that the treatment of NOB significantly restored the deregulated miRNAs and proteins to their basal levels. Hence, it may be considered one of its neuroprotective mechanisms. Together, the findings are promising to demonstrate the potential applicability of NOB as a neuroprotectant against chemical-induced neurotoxicity.</p

Table7_A protein–miRNA biomic analysis approach to explore neuroprotective potential of nobiletin in human neural progenitor cells (hNPCs).DOCX

Chemical-induced neurotoxicity is increasingly recognized to accelerate the development of neurodegenerative disorders (NDs), which pose an increasing health burden to society. Attempts are being made to develop drugs that can cross the blood–brain barrier and have minimal or no side effects. Nobiletin (NOB), a polymethoxylated flavonoid with anti-oxidative and anti-inflammatory effects, has been demonstrated to be a promising compound to treat a variety of NDs. Here, we investigated the potential role of NOB in sodium arsenate (NA)-induced deregulated miRNAs and target proteins in human neural progenitor cells (hNPCs). The proteomics and microRNA (miRNA) profiling was done for different groups, namely, unexposed control, NA-exposed, NA + NOB, and NOB groups. Following the correlation analysis between deregulated miRNAs and target proteins, RT-PCR analysis was used to validate the selected genes. The proteomic analysis showed that significantly deregulated proteins were associated with neurodegeneration pathways, response to oxidative stress, RNA processing, DNA repair, and apoptotic process following exposure to NA. The OpenArray analysis confirmed that NA exposure significantly altered miRNAs that regulate P53 signaling, Wnt signaling, cell death, and cell cycle pathways. The RT-PCR validation studies concur with proteomic data as marker genes associated with autophagy and apoptosis (HO-1, SQSTM1, LC-3, Cas3, Apaf1, HSP70, and SNCA1) were altered following NA exposure. It was observed that the treatment of NOB significantly restored the deregulated miRNAs and proteins to their basal levels. Hence, it may be considered one of its neuroprotective mechanisms. Together, the findings are promising to demonstrate the potential applicability of NOB as a neuroprotectant against chemical-induced neurotoxicity.</p

Table8_A protein–miRNA biomic analysis approach to explore neuroprotective potential of nobiletin in human neural progenitor cells (hNPCs).DOCX

Chemical-induced neurotoxicity is increasingly recognized to accelerate the development of neurodegenerative disorders (NDs), which pose an increasing health burden to society. Attempts are being made to develop drugs that can cross the blood–brain barrier and have minimal or no side effects. Nobiletin (NOB), a polymethoxylated flavonoid with anti-oxidative and anti-inflammatory effects, has been demonstrated to be a promising compound to treat a variety of NDs. Here, we investigated the potential role of NOB in sodium arsenate (NA)-induced deregulated miRNAs and target proteins in human neural progenitor cells (hNPCs). The proteomics and microRNA (miRNA) profiling was done for different groups, namely, unexposed control, NA-exposed, NA + NOB, and NOB groups. Following the correlation analysis between deregulated miRNAs and target proteins, RT-PCR analysis was used to validate the selected genes. The proteomic analysis showed that significantly deregulated proteins were associated with neurodegeneration pathways, response to oxidative stress, RNA processing, DNA repair, and apoptotic process following exposure to NA. The OpenArray analysis confirmed that NA exposure significantly altered miRNAs that regulate P53 signaling, Wnt signaling, cell death, and cell cycle pathways. The RT-PCR validation studies concur with proteomic data as marker genes associated with autophagy and apoptosis (HO-1, SQSTM1, LC-3, Cas3, Apaf1, HSP70, and SNCA1) were altered following NA exposure. It was observed that the treatment of NOB significantly restored the deregulated miRNAs and proteins to their basal levels. Hence, it may be considered one of its neuroprotective mechanisms. Together, the findings are promising to demonstrate the potential applicability of NOB as a neuroprotectant against chemical-induced neurotoxicity.</p

Table3_A protein–miRNA biomic analysis approach to explore neuroprotective potential of nobiletin in human neural progenitor cells (hNPCs).DOCX

Chemical-induced neurotoxicity is increasingly recognized to accelerate the development of neurodegenerative disorders (NDs), which pose an increasing health burden to society. Attempts are being made to develop drugs that can cross the blood–brain barrier and have minimal or no side effects. Nobiletin (NOB), a polymethoxylated flavonoid with anti-oxidative and anti-inflammatory effects, has been demonstrated to be a promising compound to treat a variety of NDs. Here, we investigated the potential role of NOB in sodium arsenate (NA)-induced deregulated miRNAs and target proteins in human neural progenitor cells (hNPCs). The proteomics and microRNA (miRNA) profiling was done for different groups, namely, unexposed control, NA-exposed, NA + NOB, and NOB groups. Following the correlation analysis between deregulated miRNAs and target proteins, RT-PCR analysis was used to validate the selected genes. The proteomic analysis showed that significantly deregulated proteins were associated with neurodegeneration pathways, response to oxidative stress, RNA processing, DNA repair, and apoptotic process following exposure to NA. The OpenArray analysis confirmed that NA exposure significantly altered miRNAs that regulate P53 signaling, Wnt signaling, cell death, and cell cycle pathways. The RT-PCR validation studies concur with proteomic data as marker genes associated with autophagy and apoptosis (HO-1, SQSTM1, LC-3, Cas3, Apaf1, HSP70, and SNCA1) were altered following NA exposure. It was observed that the treatment of NOB significantly restored the deregulated miRNAs and proteins to their basal levels. Hence, it may be considered one of its neuroprotective mechanisms. Together, the findings are promising to demonstrate the potential applicability of NOB as a neuroprotectant against chemical-induced neurotoxicity.</p