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Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder associated with the amyloid beta (Aß) and tau hallmarks. The molecular insights into how neuroinflammation is initially triggered and how it affects neuronal cells are yet at the age of infancy. In this study, SH-SY5Y cells were used as a model for neurons by differentiating and were co-cultured with differentiated THP1 cells (microglia model) as well as treated with Aß(25-35) and with antioxidant FA to study inflammatory, oxidative stress responses and their effects on co-cultured neurons. Neurons co-cultured with microglial cells showed pronounced increase in ROS levels, NOS expression, truncated N-terminal form (34 kDa) of APE1 expression and AIF's translocation in the nucleus. The pre-treatment of FA, on the other hand reversed these effects. It was further evaluated how FA/Aß treatment altered microglial phenotype that in turn affected the neurons. Microglial cells showed M1 phenotype upon Aß(25-35) stress, while FA induced M2 phenotype against Aß stress, suggesting that FA alleviated Aß induced phenotype and its associated effects in the co-cultured neurons by altering the phenotype of microglial cells and induced expression of full length (37 kDa) APE1 enzyme and inhibiting AIF's nuclear translocation, thus inhibiting apoptosis. This is the first study that revealed Aß induced cleavage of APE1 enzyme in differentiated neurons, suggesting that APE1 may be the potential early target of Aß that loses its function and exacerbates AD pathology. FA activated a fully functional form of APE1 against Aß stress. The impaired function of APE1 could be the initial mechanism by which Aß induces oxidative and inflammatory responses and dietary phytochemical FA can be a potential therapeutic strategy in managing the disease by activating APE1 that not only repairs oxidative DNA base damage but also maintains mitochondrial function and alleviates neuroinflammatory responses.

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Alzheimer's disease (AD), a multifactorial disease, is characterized by the accumulation of neurofibrillary tangles (NFTs) and amyloid beta (Aß) plaques. AD is triggered via several factors like alteration in cytoskeletal proteins, a mutation in presenilin 1 (PSEN1), presenilin 2 (PSEN2), amyloid precursor protein (APP), and post-translational modifications (PTMs) in the cytoskeletal elements. Owing to the major structural and functional role of cytoskeletal elements, like the organization of axon initial segmentation, dendritic spines, synaptic regulation, and delivery of cargo at the synapse; modulation of these elements plays an important role in AD pathogenesis; like Tau is a microtubule-associated protein that stabilizes the microtubules, and it also causes inhibition of nucleo-cytoplasmic transportation by disrupting the integrity of nuclear pore complex. One of the major cytoskeletal elements, actin and its dynamics, regulate the dendritic spine structure and functions; impairments have been documented towards learning and memory defects. The second major constituent of these cytoskeletal elements, microtubules, are necessary for the delivery of the cargo, like ion channels and receptors at the synaptic membranes, whereas actin-binding protein, i.e., Cofilin's activation form rod-like structures, is involved in the formation of paired helical filaments (PHFs) observed in AD. Also, the glial cells rely on their cytoskeleton to maintain synaptic functionality. Thus, making cytoskeletal elements and their regulation in synaptic structure and function as an important aspect to be focused for better management and targeting AD pathology. This review advocates exploring phytochemicals and Ayurvedic plant extracts against AD by elucidating their neuroprotective mechanisms involving cytoskeletal modulation and enhancing synaptic plasticity. However, challenges include their limited bioavailability due to the poor solubility and the limited potential to cross the blood-brain barrier (BBB), emphasizing the need for targeted strategies to improve therapeutic efficacy.

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The most widespread neurodegenerative disorder, Alzheimer's disease (AD) is marked by severe behavioral abnormalities, cognitive and functional impairments. It is inextricably linked with the deposition of amyloid ß (Aß) plaques and tau protein in the brain. Loss of white matter, neurons, synapses, and reactive microgliosis are also frequently observed in patients of AD. Although the causative mechanisms behind the neuropathological alterations in AD are not fully understood, they are likely influenced by hereditary and environmental factors. The etiology and pathogenesis of AD are significantly influenced by the cells of the central nervous system, namely, glial cells and neurons, which are directly engaged in the transmission of electrical signals and the processing of information. Emerging evidence suggests that exposure to organophosphate pesticides (OPPs) can trigger inflammatory responses in glial cells, leading to various cascades of events that contribute to neuroinflammation, neuronal damage, and ultimately, AD pathogenesis. Furthermore, there are striking similarities between the biomarkers associated with AD and OPPs, including neuroinflammation, oxidative stress, dysregulation of microRNA, and accumulation of toxic protein aggregates, such as amyloid ß. These shared markers suggest a potential mechanistic link between OPP exposure and AD pathology. In this review, we attempt to address the role of OPPs on altered cell physiology of the brain cells leading to neuroinflammation, mitochondrial dysfunction, and oxidative stress linked with AD pathogenesis.

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Small non-coding RNAs (miRNAs) regulate gene expression by binding to mRNA and mediating its degradation or inhibiting translation. Since miRNAs can regulate the expression of several genes, they have multiple roles to play in biological processes and human diseases. The majority of miRNAs are known to be expressed in the brain and are involved in synaptic functions, thus marking their presence and role in major neurodegenerative disorders, including Alzheimer's disease (AD). In AD, amyloid beta (Aß) plaques and neurofibrillary tangles (NFTs) are known to be the major hallmarks. The clearance of Aß and tau is known to be associated with miRNA dysregulation. In addition, the ß-site APP cleaving enzyme (BACE 1), which cleaves APP to form Aß, is also found to be regulated by miRNAs, thus directly affecting Aß accumulation. Growing evidences suggest that neuroinflammation can be an initial event in AD pathology, and miRNAs have been linked with the regulation of neuroinflammation. Inflammatory disorders have also been associated with AD pathology, and exosomes associated with miRNAs are known to regulate brain inflammation, suggesting for the role of systemic miRNAs in AD pathology. Several miRNAs have been related in AD, years before the clinical symptoms appear, most of which are associated with regulating the cell cycle, immune system, stress responses, cellular senescence, nerve growth factor (NGF) signaling, and synaptic regulation. Phytochemicals, especially polyphenols, alter the expression of various miRNAs by binding to miRNAs or binding to the transcriptional activators of miRNAs, thus control/alter various metabolic pathways. Awing to the sundry biological processes being regulated by miRNAs in the brain and regulation of expression of miRNAs via phytochemicals, miRNAs and the regulatory bioactive phytochemicals can serve as therapeutic agents in the treatment and management of AD.

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Synaptic mitochondria are crucial for maintaining synaptic activity due to their high energy requirements, substantial calcium (Ca2+) fluctuation, and neurotransmitter release at the synapse. To provide a continuous energy supply, neurons use special mechanisms to transport and distribute healthy mitochondria to the synapse while eliminating the damaged mitochondria from the synapse. Along the neuron, mitochondrial membrane potential (ψ) gradient exists and is highest in the somal region. Lower ψ in the synaptic region renders mitochondria more vulnerable to oxidative stress-mediated damage. Secondly, mitochondria become susceptible to the release of cytochrome c, and mitochondrial DNA (mtDNA) is not shielded from the reactive oxygen species (ROS) by the histone proteins (unlike nuclear DNA), leading to activation of caspases and pronounced oxidative DNA base damage, which ultimately causes synaptic loss. Both synaptic mitochondrial dysfunction and synaptic failure are crucial factors responsible for Alzheimer's disease (AD). Furthermore, amyloid beta (Aß) and hyper-phosphorylated Tau, the two leading players of AD, exaggerate the disease-like pathological conditions by reducing the mitochondrial trafficking, blocking the bi-directional transport at the synapse, enhancing the mitochondrial fission via activating the mitochondrial fission proteins, enhancing the swelling of mitochondria by increasing the influx of water through mitochondrial permeability transition pore (mPTP) opening, as well as reduced ATP production by blocking the activity of complex I and complex IV. Mild cognitive impairment (MCI) is also associated with decline in cognitive ability caused by synaptic degradation. This review summarizes the challenges associated with the synaptic mitochondrial dysfunction linked to AD and MCI and the role of phytochemicals in restoring the synaptic activity and rendering neuroprotection in AD.

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The present study examined the wheat protein gliadin-induced oxidative and nitrosative stress and its downstream responses in human intestinal HCT-116 and HT-29 cells. The beneficial role of dietary phytochemical curcumin and role of multifunctional enzyme Apurinic/aprymidinic endonuclease 1 (APE1) a major player involved in the base excision repair (BER)-pathway in gliadin intolerant intestinal HCT-116 and HT-29 cell lines were evaluated as an in vitro model study. The cultured cells were exposed to gliadin protein, H2 O2 , and curcumin followed by the assessment of oxidative stress and the consequences were measured using spectrophotometric, PCR, flow cytometer, Western blotting, confocal microscopy, and other methods. Results demonstrate that a 3 h pretreatment of curcumin, followed by the treatment of gliadin protein for 24 h time period protected both the HCT-116 and HT-29 cells via: (i) decreasing the ROS/RNS, restoring the mitochondrial transmembrane potential; (ii) re-establishing the cellular antioxidant defense system (superoxide dismutase, catalase, and GSH); (iii) enhancing the functions of APE1 viz. endonuclease activity and redox activation of transcription factor Nrf-2, the later binds with the antioxidant response elements (ARE) and activates downstream targets involved in cell survival. The cross-talk between APE1 and Nrf-2 was also established using immunofluorescence imaging and co-immunoprecipitation assays. In conclusion, gliadin protein induces oxidative/nitrosative stress, mitochondrial dysfunction and it damages cellular biomolecules in the intestinal cells. Hence it can be attributed to the tissue damage and disease pathogenesis in wheat intolerance-associated intestinal diseases. The gliadin-induced stress and its consequences are significantly reduced by the pretreatment of curcumin via BER-pathway and ARE-pathway; which is evident through the interaction between these two essential proteins. Hence suggesting for the intervention of curcumin and other natural dietary phytochemicals-based disease management and treatment of gliadin intolerance associated intestinal diseases like celiac disease.

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Oxidative stress has been implicated in various human diseases, including cancer, mainly through the generation of reactive nitrogen species (RNS), such as nitric oxide (NO), nitrite, nitroxyl, s-nitrosothiols, and reactive oxygen species (ROS) such as peroxides, superoxide, and hydroxyl radicals. NO being the main player among RNS induced altered cellular molecules and metabolisms, thus making it important to understand and detect the generation of NO in biological samples. There are many methods for direct and indirect detection of NO; out of these most commonly used are spectrophotometric-based Griess assay and fluorescence probe-based assays. In this chapter, we summarize these routinely used methods to detect NO and various challenges associated with these methods.

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Reactive oxygen species (ROS) overproduction results in oxidative stress leading to genomic instability via the generation of small base lesions in the genome, and this unrepaired DNA base damage leads to various cellular consequences. The oxidative stress-mediated DNA base damage is involved in various human disorders like cancer, cardiovascular, ocular, and neurodegenerative diseases. Base excision repair (BER) pathway, one of the DNA repair pathways, is majorly involved in the repair of oxidative DNA base lesions, which utilizes a different set of enzymes, including endonuclease viz Apurinic/apyrimidinic endonuclease 1 (APE1). APE1 is a well-known multifunctional enzyme with DNA repair, REDOX regulatory, and protein-protein interaction/cross-talk functions associated with the cell survival mechanisms. APE1 acts as an important player in both normal and cancerous cell survival; thus, evaluating its endonuclease activity in the biological samples provide useful readout of the DNA repair capacity/ability, which can be used to tune for the development of therapeutic candidates via either stimulating or blocking its DNA repair function in normal vs. cancer cells, respectively. This chapter enlists two methods used for the determination of APE1's endonuclease activity by oligonucleotide-based radioactive P32-labeled and nonradioactive fluorescence dyes using the cell extracts and recombinant APE1 protein.

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Doxorubicin (DOX) is a boon for cancer-suffering patients. However, the undesirable effect on health on vital organs, especially the heart, is a limiting factor, resulting in an increased number of patients with cardiac dysfunction. The present review focuses on the contractile machinery and associated factors, which get affected due to DOX toxicity in chemo-patients for which they are kept under life-long investigation for cardiac function. DOX-induced oxidative stress disrupts the integrity of cardiac contractile muscle proteins that alter the rhythmic mechanism and oxygen consumption rate of the heart. DOX is an oxidant and it is further discussed that oxidative stress prompts the damage of contractile components and associated factors, which include Ca2+ load through Ca2+ ATPase, SERCA, ryanodine receptor-2, phospholamban, and calsequestrin, which ultimately results in left ventricular ejection and dilation. Based on data and evidence, the associated proteins can be considered as clinical markers to develop medications for patients. Even with the advancement of various diagnosing tools and modified drugs to mitigate DOX-induced cardiotoxicity, the risk could not be surmounted with survivors of cancer.

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ETHNOPHARMACOLOGICAL RELEVANCE: Doxorubicin (DOX) is an effective anti-neoplastic drug, however; it has downside effects on cardiac health and other vital organs. The herbal remedies used in day to day life may have a beneficial effect without disturbing the health of the vital organs. Glycyrrhiza glabra L. is a ligneous perennial shrub belonging to Leguminosae/Fabaceae/Papilionaceae family growing in Mediterranean region and Asia and widespread in Turkey, Italy, Spain, Russia, Syria, Iran, China, India and Israel. Commonly known as mulaithi in north India, G. glabra has glycyrrhizin, glycyrrhetic acid, isoliquiritin, isoflavones, etc., which have been reported for several pharmacological activities such as anti-demulcent, anti-ulcer, anti-cancer, anti-inflammatory and anti-diabetic. AIM OF THE STUDY: The objective of the present study is to investigate the interaction between the molecular factors like PPAR-α/γ and SIRT-1 during cardiac failure arbitrated by DOX under in vitro conditions and role of Glycyrrhiza glabra (Gg) root extract in alleviating these affects. MATERIALS AND METHODS: In the present study, we have examined the DOX induced responses in H9c2 cardiomyocytes and investigated the role of phytochemical Glycyrrhiza glabra in modulating these affects. MTT assay was done to evaluate the cell viability, Reactive Oxygen Species (ROS)/Reactive Nitrogen Species (RNS) levels, mitochondrial ROS, mitochondrial membrane potential was estimated using fluorescent probes. The oxidative stress in terms of protein carbonylation, lipid peroxidation and DNA damage was detected via spectrophotometric methods and immune-fluorescence imaging. The cardiac markers and interaction between SIRT-1 and PPAR-α/γ was measured using Real-Time PCR, Western blotting and Co-immunoprecipitation based studies. RESULTS: The Glycyrrhiza glabra (Gg) extracts maintained the membrane integrity and improved the lipid homeostasis and stabilized cytoskeletal element actin. Gg phytoextracts attenuated aggravated ROS level, repaired the antioxidant status and consequently, assisted in repairing the DNA damage and mitochondrial function. Further, the expression of hypertrophic markers in the DOX treated cardiomyocytes reconciled the expression factors both at the transcriptional and translational levels after Gg treatment. SIRT-1 mediated pathway and its downstream activator PPARs are significant in maintaining the cellular functions. It was observed that the Gg extract allows regaining the nuclear SIRT-1 and PPAR-γ level which was otherwise reduced with DOX treatment in H9c2 cardiomyocytes. The co-immunoprecipitation (Co-IP) documented that SIRT-1 interacts with PPAR-α in the untreated control H9c2 cardiomyocytes whereas DOX treatment interferes and diminishes this interaction however the Gg treatment maintains this interaction. Knocking down SIRT-1 also downregulated expression of PPAR-α and PPAR-γ in DOX treated cells and Gg treatment was able to enhance the expression of PPAR-α and PPAR-γ in SIRT-1 knocked down cardiomyocytes. CONCLUSIONS: The antioxidant property of Gg defend the cardiac cells against the DOX induced toxicity via; 1) reducing the oxidative stress, 2) maintaining the mitochondrial functions, 3) regulating lipid homeostasis and cardiac metabolism through SIRT-1 pathway, and 4) conserving the cardiac hypertrophy and hence preserving the cardiomyocytes health. Therefore, Gg can be recommended as a healthy supplement with DOX towards cancer therapeutics associated cardiotoxicity.

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The low concentrations of cancer biomarkers in the blood have limited the utility of quantitative bioassays developed for the purpose. The advent of nicking endonucleases (NEases) as signal amplification tools have greatly enhanced the detection efficiency and provided a multi-optional platform to design target specific detection methods. The present review focuses on the prominent features of NEases, modified DNA probes (such as hairpin (HP) probes, molecular beacons, and G- quadruplex) that mediate cyclic cascade and role of helper enzymes. Application of NEase assisted signal amplification (NESA) has been discussed for diagnosis of two prominent cancer biomarkers viz. DNA methyl transferase (Dam MTase) and microRNA (miRNA). NESA mediated techniques such as rolling circle amplification (RCA), strand displacement amplification (SDA) and isothermal exponential amplification (EXPAR), have been compared in light of their future applications in clinical diagnosis. Significance of nanomaterials to achieve further amplification and NESA assays for simultaneous detection of miRNAs has also been conversed. It is anticipated that the information gained from the analyses of the prospects and limitations of NESA-based assays will be useful towards understanding the applications, and improvement of efficient isothermal exponential amplification strategies for highly sensitive and selective detection of cancer biomarkers.

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