α-Synucleinopathies are a group of neurodegenerative disorders characterized by alterations in α-synuclein (α-syn), a protein associated with membrane phospholipids, whose precise function in normal cells is still unknown. These kinds of diseases are caused by multiple factors, but the regulation of the α-syn gene is believed to play a central role in the pathology of these disorders; therefore, the α-syn gene is one of the most studied genes. α-Synucleinopathies are complex disorders that derive from the interaction between genetic and environmental factors. Here, we offer an update on the landscape of the epigenetic regulation of α-syn gene expression that has been linked with α-synucleinopathies. We also delve into the reciprocal influence between epigenetic modifications and other factors related to these disorders, such as posttranslational modifications, microbiota participation, interactions with lipids, neuroinflammation and oxidative stress, to promote α-syn aggregation by acting on the transcription and/or translation of the α-syn gene.

O-GlcNAcylation, a recently discovered post-translational modification of proteins, plays a crucial role in regulating protein structure and function, and is closely associated with multiple diseases. Research has shown that O-GlcNAcylation is abnormally upregulated in most cancers, promoting disease progression. To elucidate the roles of O-GlcNAcylation in cancer, this review summarizes various cancer-associated biological events regulated by O-GlcNAcylation and the corresponding signaling pathways. This work may provide insights for future studies on the function or underlying mechanisms of O-GlcNAcylation in cancer.

The nuclear events of a eukaryotic cell, such as replication, transcription, recombination and repair etc. require the transition of the compactly arranged chromatin into an uncompacted state and vice-versa. This is mediated by post-translational modification of the histones, exchange of histone variants and ATP-dependent chromatin remodeling. The SWI/SNF chromatin remodeling complexes are one of the most well characterized families of chromatin remodelers. In addition to their role in modulating chromatin, they have also been assigned roles in cancer and health-related anomalies such as developmental, neurocognitive, and intellectual disabilities. Owing to their vital cellular and medical connotations, developing an understanding of the structural and functional aspects of the complex becomes imperative. However, due to the intricate nature of higher-order chromatin as well as compositional heterogeneity of the SWI/SNF complex, intra-species isoforms and inter-species homologs, this often becomes challenging. To this end, the present review attempts to present an amalgamated perspective on the discovery, structure, function, and regulation of the SWI/SNF complex.

Several decades of research and clinical trials have conclusively provided proof of concept on the usefulness of monoclonal antibodies in the armamentarium against cancer. There are numerous mAbs approved for both, the treatment of solid tumors as well as hematological malignancies. These have ranked in the top ten best-selling drugs in recent years and one such mAb, pembrolizumab, is slated to be the highest revenue-generating drug by 2024. A large proportion of the mAbs in oncology have been approved by regulatory agencies in just the past decade and many professionals working in the field have been unable to keep abreast with the latest mAbs available and their mechanism of action. In this review, we aim to provide a systematic compilation of the various mAbs in oncology, approved by the US FDA in the past decade. It also elaborates on the mechanism of action of the newly approved mAbs to provide an overall update of the same. For this purpose, we have referred to the Drugs at FDA and relevant articles from PubMed from the year 2010 to date.

Posttranslational modifications (PTMs) play important roles in the regulation of protein function. Acetylation and deacetylation are among the most important PTMs. SIRT7 is a relatively understudied member of the sirtuin family, but recent studies have revealed that it plays a regulatory role in a variety of cellular activities, such as genome stabilization and repair, gene translation, ribosome production and other important processes. Here, we provide a list of the functions and mechanisms of SIRT7 in various organelles and show the important role of SIRT7 in maintaining normal cell function.

A description is given of a sequence of events which would have led to the appearance of the organic compounds and living cells present on Earth, one of which is human cells. The evolutionary events involved are proposed as having taken place in phosphate ion-dominated aqueous pools formed in regions associated with volcanoes. The mechanism involved the unique molecular structure variations and chemical properties of polyphosphoric acid and compounds of this acid producing urea as the first organic compound formed on Earth and derivatives of urea giving rise to DNA and RNA. The occurrence of the process in present times is considered possible.

The reperfusion of coronary artery blood supply is often accompanied by myocardial hypoxia/reperfusion (H/R) injury, and induced cardiomyocytes apoptosis. The activation of p38 can induce apoptosis, thereby aggravating the myocardial H/R injury. Metallothionein-2A (MT2A) has the functions of anti-apoptosis and protective effect through p38. However, it is not clear that MT2A may protect cardiomyocytes from H/R injury through p38 signaling pathway. Here, we constructed an H/R model for H9c2 cardiomyocytes to explore the protective effect of MT2A on cardiomyocytes apoptosis during the process of H/R through p38 signal pathway. The results revealed that both endogenously overexpressed MT2A and exogenously added MT2A can inhibit the active expression of p-p38 and cleaved caspase-3 under H/R. Based on our results, H/R induced cardiomyocytes apoptosis and activation of p38. And, MT2A can inhibit the active expression of caspase-3 and p38. We found that MT2A can protect cardiomyocytes apoptosis from H/R injury through p38 signaling pathway.

Ferroptosis is a newfound mode of regulated cell death that may have potential to associate with prognostic or diagnostic factors in glioma. In this research, 5 genes related to glioma were screened through the FerrDb database, and we analyzed the combination between genes and glioma of survival and prognosis via TCGA, GEPIA, TIMER, and other databases. Survival curve and prognostic analysis showed that the overexpression of NFE2L2 and NOX4, respectively, has a remarkable link with a worse prognosis in glioma. Then, the association between the expression of the two genes and tumor-infiltrating immune cells level was explored based on the GSCA, and the immunity of NFE2L2 and NOX4 based on the TISIDB database was also investigated. In glioma, especially GBM, there is a strong association between gene expression and immune infiltration, even in macrophages, nTreg, and Th2 cells, which play immunosuppressive functions in TME. In conclusion, these results indicate that NFE2L2 and NOX4 could be risk prognosis biomarkers in glioma, and they bound up with immune infiltration and tumor immunity in tumorigenesis.

Extracellular vesicles (EVs) are membranous spheroid organelles secreted by various cells during their development. Previous studies have proved that the elimination of metabolic waste products from the cells is one of the key biological functions of EVs. Besides, recent studies suggest that EVs also promote intercellular information transmission thus further regulating the external environment of cells, especially during the development of cancer. Different EVs are produced by tumor cells and tumor-related cells during the development of tumors. Based on their sources and contents, different EVs may promote the proliferation of tumor cells, interfere with the function of immune cells, or destroy normal tissue barriers. As a landmark component in the occurrence and development of tumors, EVs can be used to solve the biological behaviors that hinder tumor treatment, such as drug resistance and immune escape. Oral cancer is a highly prevalent cancer type in clinic and current therapies often fail to effectively inhibit its deterioration. Based on their essential roles in cancer development, EVs therefore possess great potential to be a target for oral cancer treatment. In this review, we focused on the origin and classification of vesicles in oral cancer tissues around the tumor microenvironment, described their biological functions, and discussed their potential for cancer treatment in combination with existing research methods. In addition, we highlighted the current challenges and recommendations of EVs for the treatment of oral cancer in clinic.

2-Hexadecenal (2-HD)-a biologically active long-chain fatty aldehyde formed in organism enzymatically or nonenzymatically in the reaction of free-radical destruction of sphingolipids under the action of hypochlorous acid, producing by myeloperoxidase. This research aimed to study 2-HD effects on polymorphonuclear leukocytes' (PMNLs) functions. It has been shown that at submicromolar concentrations, 2-HD causes an elevation in ROS production by PMNLs. It has been found that such effect is associated with signal transduction pathways modification and expressed in elevation of NADPH oxidase, MPO, and JNK-MAPK contributions to this process. At higher concentrations, 2-HD induces apoptosis, which correlates with a significant increase in free Ca2+ in the cytoplasm, a decrease in ROS production, and a decline in mitochondrial potential. Both of these processes are accompanied by cytoskeleton reorganization.

Increasing evidence suggests that PCSK9 inhibition protects cardiomyocytes against ischemia-reperfusion injury after myocardial infarction. However, it is not clear whether PCSK9 inhibitor (PCSK9i) affects cardiac fibroblasts (CFs) activation after MI. In this study we used SBC-115076, an antagonist of PCSK9, to investigate the role of PCSK9i in the conversion of CFs to cardiac myofibroblasts (CMFs) after MI and provided a basic for its clinical application in cardiac fibrosis after MI. In vivo study, PCSK9i was injected into mice 4 days after MI. Cardiac function and degree of fibrosis were evaluated by echocardiographic and tissue staining after treatment. Western blot showed that PCSK9i treatment decreases expression of α-SMA, collagen and increases expression of Notch1 in border infarct area. Vitro studies showed that PCSK9i decreased the degree of fibrosis, migration, and collagen fiber deposition in CFs. Confocal microscopy imaging also showed that hypoxia contributes to the formation of α-SMA stress filaments, and PCSK9i alleviated this state. Moreover, overexpression of Notch1 further suppress the activation of CFs under hypoxia. These results revealed that SBC-115076 ameliorates cardiac fibrosis and ventricular dysfunction post-myocardial infarction through inhibition of the differentiation of cardiac fibroblasts to myofibroblasts via Notch1/Hes1 signaling.

Programmed cell death (PCD) is a process that occurs naturally in cells in response to different endogenous or exogenous factors and facilitated by specific proteins. The three common pathways are pyroptosis, necroptosis, and apoptosis. Each pathway has its own unique proteins, mechanisms, and byproducts. Dysregulated PCD can lead to abnormal growth of cells causing tumor growth, a hallmark feature of many cancer pathologies. Recently, the PCD pathways have been considered to be activated simultaneously in a combined nature defined as PANoptosis (pyroptosis, apoptosis, and necroptosis). An integral protein, Z-DNA binding protein 1 (ZBP1) aids in the initiation of the NOD-like receptor protein 3 (NLRP3) inflammasome, a known facilitator of pyroptosis. It also is known to bind to a regulator of necroptosis, receptor-interacting protein kinase 3 (RIPK3). A unique binding partner to ZBP1, adenosine deaminase acting on RNA 1 (ADAR1), is involved in RNA editing, stress mechanisms, and disease. In murine bone marrow-derived macrophages (BMDMs) treatment with nuclear export inhibitors (NEIs) has allowed for sequestering of ADAR1 to the nucleus, and increased incidence of cell death. Additionally, the use of interferons (IFNs) to induce ZBP1 has increased the incidence of cell death. Emerging therapies are looking at the efficacy of using a combination of NEI and IFN treatment to rapidly reduce tumor size and growth by inducing PANoptosis. KPT-330 and KPT-8602 are two different NEIs, both of which have shown efficacy in the reduction of tumor size and inhibition of Exportin 1 (XPO1), a transport protein. However, this article posits KPT-8602 as the better of the two. KPT-8602 is more tolerable for the patient and should be pushed to human trials.

Human papillomavirus (HPV) is a risk factor for lung cancer. However, the underlying mechanisms are not known. Long noncoding RNAs (lncRNAs) have been found to play an important role in the occurrence and development of lung cancer due to their particular characteristics. HPV-induced lung carcinogenesis is incompletely defined. We aimed to screen and clarify the functions of lncRNAs that are differentially expressed in HPV-related lung cancer. We found that lncRNA SNHG1 is upregulated in lung cancer cells infected with HPV16 E6 by qRT‒PCR. Further results demonstrated that SNHG1 overexpression facilitates the tube formation of human umbilical vein endothelial cells (HUVECs) in vitro. Our results also indicated that SNHG1 might function in lung cancer by binding with EGFR. Further studies revealed that SNHG1 overexpression could activate the nuclear factor κb (NF-κB) pathway, which increases the expression of interleukin-6 (IL-6). We also found that IL-6 can activate the STAT3 pathway, which promotes VEGF-D expression. These results expanded our understanding of SNHG1 as a new avenue for therapeutic intervention against lung cancer progression. Upregulation of SNHG1 by HPV infection might be an undefined link between lung cancer and HPV.

Colorectal cancer is a malignant tumor with higher morbidity and mortality. The purpose of this study is to investigate whether inhibition of Protein Kinase, Membrane Associated Tyrosine/Threonine 1 (PKMYT1) affects tumor cell proliferation, survival and migration in colon tumors with high Cyclin E1 (CCNE1) expression. PcDNA3.1-CCNE1 vector and si-PKMYT1 were transfected in SW480 cells by Lipofectamine 2000. Q-PCR and western blot assay were processed to detect the expression. Transwell assay and Edu assay were undertaken to verify the migration and proliferation. CCNE1 promotes the proliferation and migration of SW480. Silencing of PKMYT1 inhibited the proliferation of tumor cells. Silencing the expression of PKMYT1 under the premise of overexpression of CCNE1, the level of Cyclin Dependent Kinase 1 (CDK1)-PT14 was reduced, indicating that the cell cycle was blocked. The expression of γH2AX increased significantly, indicating that the DDR pathway of tumor cells was activated and DNA damage accumulated. The results of immunofluorescence microscopy showed significantly increased expression of DNA damage-associated marker (γH2AX: H2AX Variant Histone). In CCNE1 amplificated colorectal tumor cells, knockdown of PKMYT1 reduced cells in S phase, inhibited cell proliferation and promoted cell apoptosis, confirming that PKMYT1 was a potential therapeutic target for colorectal tumor. This study may verify a potential therapeutic target and provide a new idea for the treatment of colorectal cancer in the future.

Lead and mercury are the ubiquitous heavy metals triggering toxicity and initiating apoptosis in cells. Though the toxic effects of heavy metals on various organs are known, there is a paucity of information on the mechanisms that instigate the current study. A plausible role of phospholipid scramblase 3 (PLSCR3) in Pb2+ and Hg2+ induced apoptosis was investigated with human embryonic kidney (HEK 293) cells. After 12 h of exposure, ~30-40% of the cells were in the early stage of apoptosis with increased reactive oxygen species (ROS), decreased mitochondrial membrane potential, and increased intracellular calcium levels. Also, ~20% of the cardiolipin localized within the inner mitochondrial membrane was translocated to the outer mitochondrial membrane along with the mobilization of truncated Bid (t-Bid) to the mitochondria and cytochrome c from the mitochondria. The endogenous expression levels of PLSCR3, caspase 8, and caspase 3 were upregulated in Pb2+ and Hg2+ induced apoptosis. The activation and upregulation of PLSCR3 mediate CL translocation playing a potential role in initiating the heavy metal-induced apoptosis. Therefore, PLSCR3 could be the linker between mitochondria and heavy metal apoptosis.

Aerobic glycolysis, also known as the Warburg effect, has emerged as a hallmark of cancer and is associated with tumor progression and unfavorable clinical outcomes in cancer patients. PP2A is a highly conserved eukaryotic serine/threonine protein phosphatase that functions as a tumor suppressor in a variety of human cancers. However, the relationship between PP2A and the Warburg effect in gastric cancer has yet to be fully understood. In this study, the expression profile of two endogenous inhibitors of PP2A, SET and CIP2A, in gastric cancer, were analyzed by real-time quantitative polymerase chain reaction. Loss-of-function and gain-of-function studies were performed to investigate the roles of PP2A in gastric cancer cell proliferation and glycolysis. Cell biological, molecular, and biochemical approaches were employed to uncover the underlying mechanisms. The results showed that SET and CIP2A were overexpressed in gastric cancer and associated with a decreased PP2A activity. Pharmacological activation of PP2A with FTY-720 and DT-061 in two gastric cancer cell lines significantly reduced gastric cancer cell proliferation and glycolytic ability. Importantly, inhibition of PP2A activity by genetic silencing of PPP2R5A resulted in a growth advantage, which can be largely compromised by the addition of the glycolysis inhibitor 2-Deoxy-D-glucose, suggesting a glycolysis-dependent effect of PP2A in gastric cancer. Mechanistically, the well-known transcription factor and glycolysis regulator c-Myc was discovered as the functional mediator of PP2A in regulating cell glycolysis. Ectopic expression of a phosphorylation-mutant c-Myc resistant to PP2A (MycT58A) restored the inhibitory effect of FTY-720 and DT-061 on lactate production and glucose uptake. Furthermore, there was a close association between SET and CIP2A expression and c-Myc gene signatures in gastric cancer samples. Collectively, this study provides strong evidence of the involvement of PP2A in the Warburg effect and indicates that it could be a novel antitumor strategy to target tumor metabolism in gastric cancer.

Microglia, the macrophage-like glial cells, behave as sentinels against exogenous pathogens invading the neural tissue. Their commitment is not only confined to the defensive function, but they also perform balancing trophic activities such as neuronal postnatal development, remodeling and pruning of synapses. Likewise, microglia-derived extracellular vesicles (EVs) can play strategic roles in maintaining a healthy brain by modulating neuronal activity and by controlling neurite outgrowth as well as innate immune response. Nevertheless, strong evidence also points to their role in the development of neurodegenerative pathologies such as Alzheimer's disease (AD). Here, we explored EV protein content released by BV2 microglial cells in a resting state and after stimulation with beta-amyloid peptides (Aβ), mimicking conditions occurring in AD. In the resting BV2 cells, we extended the list of proteins present in mouse microglia EV cargo with respect to those reported in the Vesiclepedia exosome database while, in amyloid-triggered microglia, we highlighted a pronounced drop in EV protein content. Focusing on Rab11A, a key factor in the recycling routes of amyloid species, we observed a dramatic decrease of this protein in Aβ-treated microglia EV cargo with respect to the EVs from the untreated sample. This decrease might affect the delivery of Rab11A to neurons thus increasing the harmful amyloid burden in neuronal cells that eventually may lead to their death. We tentatively proposed that alterations observed in EVs derived from Aβ-treated microglia may represent molecular features that, among others, shape the disease-associated microglial phenotype, a recently proposed subset of microglial population, present in neurodegenerative pathologies.

The ubiquitin-specific protease 14 (USP14) is a deubiquitinating enzyme, its inhibitor was reported could alleviate the ischemia/reperfusion (I/R)-stimulated cerebral neuronal damage. However, its specific role in I/R-induced acute kidney injury (AKI) remains unclear. We established hypoxia/reoxygenation (H/R)-induced HK-2 cell injury model in vitro and I/R-induced kidney injury mice model in vivo. The expression or activity of USP14 was inhibited by siRNA or IU1, a small molecule inhibitor of USP14. ROS were scavenged by N-acetyl-cysteine (NAC). Biochemical index analysis and hematoxylin & eosin (H&E) staining were performed to evaluate renal injury. The results indicated that USP14 was upregulated in H/R-induced HK-2 cells and kidney tissues of I/R mice. Inhibition of USP14 suppressed the cell death, inflammatory, oxidative stress and reactive oxygen species (ROS)-dependent ferroptosis of H/R-induced HK-2 cells. What's more, IU1 and NAC effectively alleviated renal injury of I/R mice. In summary, this study suggested that inhibition of USP14 protected renal from I/R injury.

The most important environmental factor that can play a key role in the development of asthma in the obese is overproduction of reactive oxygen species (ROS). The aim of the study was to examine changes in the concentration of oxidative stress parameters in the lungs, bronchoalveolar lavage (BAL) fluid and blood of mice in models of asthma or/and obesity caused by high-fat diet (HFD). The concentrations of 4-HNE and isoprostanes in the lungs of the animals were measured. BAL fluid levels of hydrogen peroxide were marked. Additionally, thiobarbituric acid reactive substances (TBARS) and ferric reducing ability of plasma (FRAP) were used as biomarkers of oxidative stress in the blood. Administration of lipoic acid (LA), a probiotic with standard-fat diet (SFD, 10% fat) and low-fat diet (LFD, 5% fat) significantly decreased the concentration of 4-HNE as compared to the OVA (ovalbumin) + HFD group (p < 0.05). Treatment with low-fat diet or LFD in combination with apocynin insignificantly decreased H2O2 values as compared to the OVA + HFD group. Supplementation of probiotic with SFD and LFD significantly decreased the concentration of TBARS as compared to the OVA + SFD and saline + HDF groups (p < 0.05). Significantly lower concentrations of TBARS were also observed in the LA plus LFD group (p < 0.05) as compared to the OVA + HFD group. Low-fat diet with probiotic significantly increased the concentration of FRAP as compared to the obese mice (p = 0.017). Treatment with LFD in combination with LA significantly increased FRAP values as compared to the obese and obese asthmatic mice (p < 0.001).

Oxidative stress is defined as an injury resulting from a disturbance in the dynamic equilibrium of the redox environment due to the overproduction of active/radical oxygen exceeding the antioxidative ability of the body. This is a key step in the development of various diseases. Oxidative stress is modulated by different factors and events, including the modification of histones, which are the cores of nucleosomes. Histone modification includes acetylation and deacetylation of certain amino acid residues; this process is catalyzed by different enzymes. Histone deacetylase 6 (HDAC6) is a unique deacetylating protease that also catalyzes the deacetylation of different nonhistone substrates to regulate various physiologic processes. The intimate relationship between HDAC6 and oxidative stress has been demonstrated by different studies. The present paper aims to summarize the data obtained from a mechanistic study of HDAC6 and oxidative stress to guide further investigations on mechanistic characterization and drug development.

We investigated the mechanisms underlying the effects of Ziziphora clinopodioides Lam. (ZCL) on atherosclerosis (AS) using network pharmacology and in vitro validation.We collected the active components of ZCL and predicted their targets in AS. We constructed the protein-protein interaction, compound-target, and target-compound-pathway networks, and performed GO and KEGG analyses. Molecular docking of the active components and key targets was constructed with Autodock and Pymol software. Validation was performed with qRT-PCR, ELISA, and Western blot.We obtained 80 components of ZCL. The network analysis identified that 14 components and 37 genes were involved in AS. Then, 10 key nodes in the PPI network were identified as the key targets of ZCL because of their importance in network topology. The binding energy of 8 components (Cynaroside, α-Spinasterol, Linarin, Kaempferide, Acacetin, Genkwanin, Chrysin, and Apiin) to 4 targets (MMP9, TP53, AKT1, SRC) was strong and <-1 kJ/mol. In addition, 13 of the 14 components were flavonoids and thus total flavonoids of Ziziphora clinopodioides Lam. (ZCF) were used for in vitro validation. We found that ZCF reduced eNOS, P22phox, gp91phox, and PCSK9 at mRNA and protein levels, as well as the levels of IL-1β, TNF-α, and IL-6 proteins in vitro (P < 0.05).We successfully predicted the active components, targets, and mechanisms of ZCL in treating AS using network pharmacology. We confirmed that ZCF may play a role in AS by modulating oxidative stress, lipid metabolism, and inflammatory response via Cynaroside, Linarin, Kaempferide, Acacetin, Genkwanin, Chrysin, and Apiin.

Genetically engineered stem cells, not only acting as vector delivering growth factors or cytokines but also exhibiting improved cell properties, are promising cells for periodontal tissue regeneration. Sema3A is a power secretory osteoprotective factor. In this study, we aimed to construct Sema3A modified periodontal ligament stem cells (PDLSCs) and evaluated their osteogenic capability and crosstalk with pre-osteoblasts MC3T3-E1. First, Sema3A modified PDLSCs was constructed using lentivirus infection system carrying Sema3A gene and the transduction efficiency was analyzed. The osteogenic differentiation and proliferation of Sema3A-PDLSCs was evaluated. Then, MC3T3-E1 was directly co-cultured with Sema3A-PDLSCs or cultured in condition medium of Sema3A-PDLSCs and the osteogenic ability of MC3T3-E1 was assessed. The results showed that Sema3A-PDLSCs expressed and secreted upregulated Sema3A protein, which confirmed successful construction of Sema3A modified PDLSCs. After osteogenic induction, Sema3A-PDLSCs expressed upregulated ALP, OCN, RUNX2, and SP7 mRNA, expressed higher ALP activity, and produced more mineralization nodes, compared with Vector-PDLSCs. Whereas, there was no obvious differences in proliferation between Sema3A-PDLSCs and Vector-PDLSCs. MC3T3-E1 expressed upregulated mRNA of ALP, OCN, RUNX2, and SP7 when directly co-cultured with Sema3A-PDLSCs than Vector-PDLSCs. MC3T3-E1 also expressed upregulated osteogenic markers, showed higher ALP activity, and produced more mineralization nodes when cultured using condition medium of Sema3A-PDLSCs instead of Vector-PDLSCs. In conclusion, our results indicated that Sema3A modified PDLSCs showed enhanced osteogenic capability, and also facilitated differentiation of pre-osteoblasts.

Muscarinic acetylcholine receptor subtype 3 (M3 receptor) is a G Protein-Coupled Receptor (GPCR) that mediates many important physiological functions. Currently, most M3 receptor drugs also have high affinity for other subtypes of muscarinic acetylcholine receptors (mAChRs) and produce the risk of side effects. Therefore, in order to find M3 receptor drugs with high specificity, high activity and low side effects, we established a cell model and method for efficient and sensitive screening of M3 receptor based on calcium-activated chloride channels (CaCCs), and this method is also suitable for the screening of other GPCR drugs. This screening model consists of Fischer rat thyroid follicular epithelial (FRT) cells that endogenously express M3 receptors, CaCCs, and the indicator YFP-H148Q/I152L. We verified that the model can sensitively detect changes in intracellular Ca2+ concentration using fluorescence quenching kinetics experiments, confirmed the screening function of the model by applying available M3 receptor drugs, and also evaluated the good performance of the model in high-throughput screening.

Lysosomes are known to influence cholesterol trafficking into endoplasmic reticulum (ER) membranes. Though intracellular cholesterol levels are known to influence the lipid biosynthetic responses in ER, the specific effects of lysosomal modulation on these outcomes is not known. To demonstrate this, C2C12 cells were treated with chloroquine, a lysosomotropic agent, and its effects on cellular biosynthetic capacity, structural and functional status of ER was determined. In addition to its known effects on autophagy reduction, chloroquine treatment induced accumulation of total cellular lipid and ER-specific cholesterol content. It was also observed that chloroquine caused an increase in smooth-ER content with defects in overall protein turnover. Further, since ER and mitochondria function in close association through ER membrane contact sites, it is likely that lysosomal modulation also brings about associated changes in mitochondria. In this regard, we found that chloroquine reduces mitochondrial membrane potential and mitochondrial dynamics. Collectively, the differential biosynthetic response of rise in lipid content, but not protein content, cannot be accounted by merely considering that chloroquine induced suppression of autophagy causes defects in organelle function. In this defective autophagy scenario, both biosynthetic responses such as lipid and protein synthesis are expected to be reduced rather than only the latter, as observed with chloroquine. These findings suggest that cholesterol trafficking/distribution within cellular organelles could act as an intracellular mediator of differential biosynthetic remodelling in interconnected organelles.

Mucopolysaccharidosis type II (MPS II) is a disorder caused by a deficient activity of iduronate-2-sulfatase, a lysosomal enzyme responsible for degrading glycosaminoglycans (GAGs). The abnormal storage of GAGs within lysosomes disrupts cellular homeostasis and leads to a severe symptomatology. Patients present neuropsychiatric impairment characterized by mental retardation and impaired cognition. The aim of this study was to quantify four neurodegeneration biomarkers in plasma: brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF-AA), neural cell adhesion molecule (NCAM) and cathepsin-D, as well as to identify possible correlations with urinary GAGs in seven patients undergoing treatment with ERT (Elaprase® 0.5 mg/kg of body weight). Patients with both severe and attenuated forms of MPS II showed signs of neurodegeneration in neuroimaging exams. Patients have a decrease in BDNF and PDGF-AA concentrations, and an increase in NCAM level compared to controls. No alterations in cathepsin-D concentration were seen. GAGs levels were higher in patients than in controls, but no significant correlations between GAGs and biomarkers were observed. These results evidence that patients have neurodegeneration and that monitoring these biomarkers might be useful for assessing this process. To this date, this is the first work to analyze these plasmatic markers of neurodegeneration in patients.

Cancer is a complex genetic disorder due to uncontrolled growth of abnormal cells in the body, causes damage to the immune system, and may lead to life-threatening situations. Common approaches to cancer treatment includes chemotherapy, hormone therapy, immunotherapy, radiation therapy etc. Development of novel and natural chemotherapeutic agents is highly demanded because of the side effects of synthetic drugs. Essential oils from aromatic plants exhibited antioxidant, antimutagenic, antiproliferative and immunomodulating activities. Mechanism of multidrug resistance and synergistic action of these volatile constituents are responsible for their chemopreventive properties. These oils primarily comprising of terpenoid constituents and are characterized by volatility, aroma, low molecular weight etc. The chemical composition of these oils varies depending on the environmental condition, species, plant part and geographical region. Literature analysis revealed that plant essential oils play an important role in cancer prevention and treatment. Cancer patients exposed to essential oils via inhaler devices were found to have less anxiety, stress, and nausea and insomnia. Nowadays, there is an increasing demand for investigating the biological properties of aromatic plants due to their availability, chemical diversity, and low toxicity. In aromatherapy, Citrus essential oils repress cancer related pain and enhance immune system. Current review summarizes existing variability of the chemical composition of Citrus essential oils and its molecular level anticancer mechanism against various human cancer cell lines. Citrus essential oils enhance cytotoxicity, antiproliferative and apoptotic behavior of cancer cell lines. Since essential oils exhibiting significant anticancer potential is worthy of further investigation for cancer chemoprevention. The findings of various research activities can be exploited by cancer researchers world wide for the development of anticancer drugs which can relieve cancer symptoms.

This study was conducted to compare the efficacy of the mouse hepatic and renal antioxidant systems against inflammation-induced oxidative stress. Increased Il-1 and Il-6 expressions, markers of inflammation, were represented by inflammation models in mouse liver and kidney tissues injected intraperitoneally with LPS. After establishing the model, the GSH level and the GSH/GSSG ratio, which are oxidative stress markers, were investigated in both tissues treated with LPS and the control group. The expression of Trx1, TrxR, and Txnip genes increased in the liver tissues of LPS-treated mice. In the kidney tissue, while Trx1 expression decreased, no change was observed in TrxR1 expression, and Txnip expression increased. In the kidneys, TRXR1 and GR activities decreased, whereas GPx activity increased. In both tissues, the TRXR1 protein expression decreased significantly, while TXNIP expression increased. In conclusion, different behaviors of antioxidant system members were observed during acute inflammation in both tissues. Additionally, it can be said that the kidney tissue is more sensitive and takes earlier measures than the liver tissue against cellular damage caused by inflammation and inflammation-induced oxidative stress.

Recent studies revealed that NOP2/Sun RNA methyltransferase 5 (NSUN5) - ferritin heavy chain (FTH1) pathway is associated with ferroptosis in stem cells, whereas its roles in gastric cancer are still unclear. Our study aims to investigate the roles of the NSUN5-FTH1 axis in gastric cancer (GC) and its molecular mechanisms. Stable cell lines were constructed on SGC7901 cells by using shRNAs and pcDNA3.1 expression vectors, respectively. CCK-8 kits were used to determine cell viability. Biochemicals assays were used to detect lipid reactive oxygen species (ROS) and intracellular Fe2+ levels. RNA immunoprecipitation assay, qPCR, and Western blotting were used to determine the changes in biomarkers. GC xenograft mouse model was established to confirm the observation in vivo. An elevation of NSUN5 was observed in GC tumor tissues. NSUN5 inhibited ferroptosis including decreasing cell viability and increasing levels of lipid ROS and Fe2+ in GC cells. Besides, a positive correlation was also observed between NSUN5 and FTH1. Interestingly, NSUN5 regulated the levels of FTH1, instead of FTH1 regulating NSUN5 in GC cells. NSUN5-FTH1 axis regulated erastin-induced ferroptosis in SGC7901 cells. Consistently, silencing NSUN5 or FTH1 inhibited the growth of the SGC7901 tumor in vivo. NSUN5-FTH1 axis promoted the growth of GC cells in part by the regulation of ferroptosis.

This study aimed to investigate the role of PPARγ and underlying mechanisms in myocardial ischemia/reperfusion injury (IRI). IRI was surgically induced in mice and neonatal rat cardiomyocytes (NRCM) were exposed to oxygen-glucose deprivation and reoxygenation (OGD/R). Quantitative genetic analysis and western blotting were performed to assess mRNA and protein levels, respectively, of PPARγ, as well as of different inflammatory, fibrosis, and apoptosis markers in cells and tissues. PPARγ was overexpressed in the heart of mice and NRCMs by viral transfection. Apoptosis and fibrosis were detected by TUNEL and Masson's trichrome staining, respectively. Enzyme-linked immunosorbent assay was performed to detect M1 and M2 macrophage-related inflammatory factors present in mouse sera. PPARγ overexpression significantly inhibited OGD/R- and IRI-induced cardiomyocyte apoptosis and fibrosis in vitro and in vivo. Moreover, PPARγ overexpression inhibited IRI-induced secretion of M1-related proinflammatory factors, whereas it supported the secretion of M2-related anti-inflammatory factors. Notably, these events were found to be mediated by the JAK/STAT pathway. In conclusion, PPARγ regulates macrophage polarization upon IRI via the JAK/STAT pathway, which will in turn prevent myocardial apoptosis and fibrosis. Hence, PPARγ may represent a valuable target for myocardial IRI treatment.

Colorectal cancer is associated with significant morbidity and mortality worldwide. Egypt, as a developing country, has a high-rise incidence of cancer. The current study objective was to investigate the antitumor influences of ellagitannin-loaded CS-PEG-decorated PLGA nano-prototypes against human colorectal cancer cell lines (HCT 116 as well as Caco-2) in vitro. Doxorubicin (DOX), punicalin (PN), and punicalagin (PNG)-encapsulated chitosan-polyethylene glycol-decorated PLGA (PLGA-CS-PEG) nanoparticles (NPs) were described. The cytotoxicity of each preparation was evaluated using MTT assays in HCT 116 as well as Caco-2 cells during G0, G1, S, and G2 cell cycle phases. Cell cycle-related gene expression and protein levels were measured after treatment. Reactive oxygen species (ROS) levels were also measured. Both PN and PNG PLGA-CS-PEG NPs induce colon cancer cell death with cell cycle arrest in the G1 phase in vitro. Caco-2 cells were more sensitive to the nano-therapy than HCT 116 cells. Upon treatment, the ratio of Bax to Bcl-2 expression was increased following nano-therapy, with increased levels of Cas-3 and decreased expression of Bcl-2, PI3k, and NF-ĸB compared to control. The nitric oxide level (NO), a marker of ROS, was increased following nano-therapy compared to control. In conclusion, ROS-mediated cell cycle arrest can be induced by PN as well as PNG nano-therapy in cell lines of colorectal cancer.

Our study aimed to investigate key molecular targets in the pathogenesis of AMI, and provide new strategy for the treatment. In this work, the myocardial ischemia and hypoxia model was constructed by using HL-1 mouse cardiomyocytes. The over-expressing POSTN wild-type, mutant and negative control lentiviruses (GV492-POSTNWT,GV492-POSTN-MUT, GV492-NC) was conducted and transfected. Cardiomyocytes were examined for cell proliferation and apoptosis to explore the effects of POSTN and its alternative splicing. The endoplasmic reticulum stess-related apoptosis proteins were selected and detected. We found that POSTN could promote the proliferation of normal and hypoxic cardiomyocytes and inhibit their apoptosis. The mechanism by which POSTN inhibited cardiomyocyte apoptosis may be through inhibiting the GRP78-eIF2α-ATF4-CHOP pathway of endoplasmic reticulum stress. Alternative splicing of POSTN could inhibit the apoptosis of ischemic and hypoxic cardiomyocytes, and its mechanism needs to be confirmed by further studies. We drawed the conclusion that POSTN might be a potential therapeutic target for AMI.

Activation of mixed lineage kinase 3 (MLK3) by phosphorylation at Thr277/Ser281 stimulates downstream apoptotic pathways and ultimately leads to cell injury. MLK3 is reported to localize to both the cytoplasm and nucleus in human ovarian cancer cells and immortalized ovarian epithelial cells (T80 and T90 cells), and phosphorylation at Thr477 is required for the cytoplasmic retention of MLK3 in T80 cells. However, the subcellular distribution of MLK3 in other cell types has rarely been reported, and whether phosphorylation of MLK3 at Thr277/Ser281 affects its subcellular distribution is unknown. Here, our bioinformatics analysis predicted that MLK3 was mainly distributed in the cytoplasm and nucleus. In the human HEK293T embryonic kidney cell line and murine HT22 hippocampal neuronal cell line, endogenous MLK3 was more abundant in the cytoplasm and less abundant in the nucleus. In addition, overexpressed Myc-tagged MLK3 and EGFP-tagged MLK3 were also observed to localize mainly to the cytoplasm. MLK3 that was activated by phosphorylation at Thr277/Ser281 was mainly distributed in the cytoplasm, and phosphorylation deficient (T277A/S281A) and mimic (T277E/S281E) mutants both showed distributions similar to that of wild type (wt) MLK3, further proving that phosphorylation at Thr277/Ser281 was not involved in regulating MLK3 subcellular localization. In HEK293T cells, H2O2 stimulation accelerated MLK3 phosphorylation (activation), and this phosphorylation was reduced by the antioxidant N-acetylcysteine in a dose-dependent manner. Overexpressing wt MLK3 promoted the production of intracellular reactive oxygen species and increased cell apoptosis, both of which were enhanced by the phosphorylation-mimic (T277E/S281E) MLK3 variant but not by the phosphorylation-deficient (T277A/S281A) MLK3 variant. These findings provided additional evidence for the cytoplasmic and nuclear distribution of MLK3 in HEK293T cells or HT22 cells and revealed the pivotal role of MLK3 in the positive feedback loop of oxidative stress injury.

The tubulin-microtubule system is a major target for a variety of small molecules which can interfere in cell cycle progression. Therefore, it serves as a prospective to control the incessant division of cancer cells. To identify novel inhibitors of the tubulin-microtubule system, a group of estrogen derivatives has been tested with tubulin as a target since literature surveys portray coveted behaviour from the same. Out of them, β-Estradiol-6-one 6- (O-carboxy methyl Oxime) abbreviated as Oxime, disrupts the cytoskeleton network and induces apoptosis with nuclei fragmentation. It has been revealed from the work that Oxime targets the colchicine binding site and binds tubulin in an entropy-driven manner. This suggests that structural variation might play a key role in modulating the anti-mitotic role of estrogen derivatives. Our work reveals that Oxime might serve as a lead molecule to nurture anti-cancer research, having the potential for recovery of the vast cancer population.

In proso millet, in certain circumstances, drought stress greatly influences growth and metabolisms. Thus, the present study was aimed to examine morphological, biochemical and ROS mechanisms between plant and drought stress in Panicum miliaceum L. To create the drought condition, water irrigation was done at different time intervals including 4, 7, 10, 13 days and control. All the experiments were carried out at different maturity stages such as 30, 50, and 70 days (after sowing). The results demonstrated that the root length, proline, glycine betaine, amino acid and superoxide dismutase, catalase and peroxidase activities were boosted in all treatments as compared with control. As the proso millet matured, the length of shoots and the amount of chlorophyll pigment in the leaves reduced in all treatments as compared to control. Induced reduction of shoot growth, chlorophyll estimation and increases of root growth, osmolyte accumulations, antioxidant enzymes, were found to be drought-tolerant adaptative mechanisms in this study.

Autophagy plays various roles at different stages of ischemia reperfusion (I/R) injury in cardiomyocytes. It has been reported that tissue factor pathway inhibitor (TFPI) has a protective effect on I/R injury. This study aimed to determine the roles of TFPI in autophagy during the I/R injury process in cardiomyocytes and the possible mechanisms. An isolated hypoxia/reoxygenation (H/R) pattern of cardiomyocytes was established by the MIC101 system. The cell viability and oxidative stress of cardiomyocytes were detected by an MTT assay and ROS assay, respectively. The autophagy level was measured by Ad-mCherry-GFP-LC3B and MDC. We detected the expression levels of autophagy-related proteins by western blotting. After 2 h of hypoxia and 12 h of reoxygenation, the cardiomyocyte viability in the H/R group was significantly lower than that in the control group (p < 0.05) than in the H/R group. According to intracellular ROS production, the fluorescence intensity in the H/R group was enhanced compared with that in the negative control group, and it was weaker in the H/R + rTFPI group compared with the H/R group. The level of autophagy and the expression levels of autophagy-related proteins (LC3-II/LC3-I, Beclin-1 and PI3K) were markedly increased in the H/R group compared to the control group (p < 0.05) whereas the levels were markedly decreased in the H/R + rTFPI group compared to the H/R group (p < 0.05). TFPI could relieve cardiomyocyte injury by inhibiting the Class III PI3K/Beclin-1 pathway and oxidative stress; thus, TFPI decreased autophagy and protected cardiomyocytes induced by H/R injury. In conclusion, TFPI may be a new direction for the prevention of myocardial I/R injury.

In this study, we aimed to analyze the proteomics of the liver in rabbits on a high cholesterol diet (HCD). We randomly divided New Zealand white rabbits into the normal diet group and the HCD group. We established the atherosclerosis model and measured plasma cholesterol and triglycerides. The model was successfully established using ultrasound examination and histopathological staining of the intima of aorta and liver of the two groups of rabbits. The differential proteins in the rabbit liver were analyzed using Tandem Mass Tags proteomic analysis technology. Finally, we used western blot to verify the reliability of proteomics. The results showed that compared with the control group, the serum lipid levels of rats in the HCD group was significantly increased, and the pathological sections showed the formation of atherosclerotic plaques in the aorta, inflammation, and adipose lesions in the liver. Proteomic analysis of the liver revealed 149 differences in HCD-expressed protein, which is mainly involved in inflammation and regulation of lipid and sugar metabolism. In addition, we verified differentially expressed liver proteins in the HCD group using western blot. We found that HCD caused lipid accumulation, abnormal glucose metabolism, and inflammatory response in the liver.

The interaction of protein and peptide amyloid oligomers with membranes is thought to be one of the mechanisms contributing to cellular toxicity. However, techniques to study these interactions in the complex membrane environment of live cells are lacking. Spectral phasor analysis is a recently developed biophysical technique that can enable visualisation and analysis of membrane-associated fluorescent dyes. When the spectral profile of these dyes changes as a result of changes to the membrane microenvironment, spectral phasor analysis can localise those changes to discrete membrane regions. In this study, we investigated whether spectral phasor analysis could detect changes in the membrane microenvironment of live cells in the presence of fibrillar aggregates of the disease-related Aβ42 peptide or the functional amyloid neurokinin B. Our results show that the fibrils cause distinct changes to the microenvironment of nile red associated with both the plasma and the nuclear membrane. We attribute these shifts in nile red spectral properties to changes in membrane fluidity. Results from this work suggest that cells have mechanisms to avoid or control membrane interactions arising from functional amyloids which have implications for how these peptides are stored in dense core vesicles. Furthermore, the work highlights the utility of spectral phasor analysis to monitor microenvironment changes to fluorescent probes in live cells.

Previous studies have confirmed that Platycodon grandiflorus polysaccharide (PGPSt) has the effects of regulating immunity and anti-apoptosis, but its effect on mitochondrial damage and apoptosis caused by PRV infection is still unclear. In this research, the effects of PGPSt on the cell viability, mitochondria morphology, mitochondrial membrane potential and apoptosis caused by PRV based on PK-15 cells were respectively examined by CCK-F assay, Mito-Tracker Red CMXRos, JC-1 staining method and Western blot etc. CCK-F test results showed that PGPSt had a protective effect on the decrease of cell viability caused by PRV. The results of morphological observation found that PGPSt can improve mitochondrial morphology damage, mitochondrial swelling and thickening, and cristae fracture. Fluorescence staining test results showed that PGPSt alleviated the decrease of mitochondrial membrane potential and apoptosis in infected cells. The expression of apoptosis-related proteins showed that PGPSt down-regulated the expression of the pro-apoptotic protein Bax and up-regulated the expression of the anti-apoptotic protein Bcl-2 in infected cells. These results indicated that PGPSt protected against PRV-induced PK-15 cell apoptosis by inhibiting mitochondrial damage.

The study investigates the movement of sodium ions inside the ligand-binding pocket of the class-A GPCR serotonin receptor (5HT2BR), a primary target for modern drugs. The available PDBs are mutant chimeras, so a 3D structure is modeled and validated by structural similarity (84.05%), Ramachandran favorable residues (93.01%), and clash score. Using MD simulations (500 ns), the ion active site is tracked in the presence and absence of ions and ligands. The ions enter the active site along helices III, VI, and VII, and the primary residue (ASP3.32) interacts with ions via H-bond (stronger- ~2.4 Å). The radial distribution function around ASP3.32 rises promptly at intermediate distances (2 Å < r < 4 Å), suggesting a higher probability of finding sodium ions at these distances. The ions stabilize the receptor at a better RMSD and promote stronger interactions (3-H-bonds, 1-π-bond~3.35 Å) with the agonist, and not the antagonist (no H-bond). Simulating unrestrained ligands further confirms this pattern, suggesting that ions might promote agonist binding but not be a prerequisite for antagonist action. The study highlights the mechanistic evaluation of sodium ions mobility in 5HT2BR modulation and ligand binding, showing potential in drug development.

CONTEXT: Gastric cancer (GC) is a significant contributor to global mortality and is recognized for its elevated prevalence and fatality rates. Nitric Oxide (NO) plays a role in multiple aspects of cancer metastasis and progression. CS-NO is a polysaccharide-based biomaterial with NO-releasing properties that shows promising therapeutic potential. Nonetheless, the action mechanism of CS-NO in GC is still largely unclear. METHODS: The present study employed various experimental techniques, including CCK-8 assay, colony formation assay, EdU staining, and transwell assays, to evaluate the proliferation, migration, and invasion of GC cells. Additionally, ELISA was utilized to measure glucose uptake, lactate production, and cellular ATP levels in GC cells. In vivo investigations on nude mice were conducted to validate the in vitro results. OBJECTIVE: The present study aimed to examine the potential anti-tumor properties of CS-NO on GC through in vitro and in vivo investigations, while also exploring the underlying mechanisms involved. RESULTS: Our data suggested that CS-NO might prevent GC cell invasion and migration. Decreased expressions of GLUT1, HK2, and LDHA further demonstrated that CS-NO significantly suppressed aerobic glycolysis in GC cells. The administration of CS-NO resulted in a significant reduction of YAP and TAZ levels in GC cells. Our data further show that CS-NO treatment could inhibit GC cancer growth in mice, consistent with the significant decrease in Ki67, GLUT1 and YAP expression levels. DISCUSSION AND CONCLUSION: These findings could reveal the good effects of CS-NO therapy on inhibiting GC.

Overstimulation of pancreatic β-cells can lead to dysfunction and death, prior to the clinical manifestations of type 2 diabetes (T2D). The excessive consumption of carbohydrates induces metabolic alterations that can affect the functions of the β-cells and cause their death. We analyzed the role of p53 in pancreatic β cell death in carbohydrate-supplemented Sprague Dawley rats. For four months, the animals received drinking water containing either 40% sucrose or 40% fructose. The glucose tolerance test was performed at week 15. Apoptosis was assessed with the TUNEL assay (TdT-mediated dUTP-nick end-labeling). Bax, p53, and insulin were assessed by Western blotting, immunofluorescence, and real-time quantitative PCR. Insulin, triacylglycerol, and serum glucose and fatty acids in pancreatic tissue were measured. Carbohydrate consumption promotes apoptosis and mobilization of p53 from the cytosol to rat pancreatic β-cell mitochondria before blood glucose rises. An increase in p53, miR-34a, and Bax mRNA was also detected (P < 0.001) in the sucrose group. As well as hypertriglyceridemia, hyperinsulinemia, glucose intolerance, insulin resistance, visceral fat accumulation, and increased pancreatic fatty acids in the sucrose group. Carbohydrate consumption increases p53 and its mobilization into β-cell mitochondria and coincides with the increased rate of apoptosis, which occurs before serum glucose levels rise.

Heavy metals from slag waste (HMSWs) have attracted much attention because of their serious toxicity to the environment and human organs, especially hepatotoxicity. The aim of this study was to explore the effects of different HMSWs exposure on mitochondrial lipid peroxidation, microsomal drug metabolizing enzyme activities as well as their relationship in the rat liver injury. Based on toxicogenomic analysis, heavy metals including iron, copper, cobalt, nickel and manganese, might interfere with pathophysiological processes such as oxidative stress, cell death, and energy metabolism regulation in vivo, and participate in the regulation of HIF-1 signaling pathway, peroxisomes, drug metabolism-cytochrome P450, ferroptosis, and other signaling pathways. HMSWs exposure caused weight loss, and significantly increased lactate dehydrogenase (LDH), malondialdehyde (MDA), alanine transaminase (ALT), and aspartate transaminase (AST) in different groups of rat liver, suggesting the presence of mitochondrial lipid peroxidation damage. In addition, the ratios of AST/ALT and ALT/LDH were down-regulated, especially the ALT/LDH ratios were less than 1, indicating that hepatic ischemic injury occurred in the process of liver injury. The superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) activities in rats also showed significant decreases, indicating the occurrence of hepatic oxidative/antioxidant dysfunction imbalance. Further decision tree analysis of live biochemical abnormalities suggested that AST > 58.78 U/gprot and MDA > 173.2 nmol/mgprot could be used for hepatotoxicity warning. Liver microsomal cytochrome P4501A2 (CYP1A2) and 3A1 (CYP3A1) enzymes were also involved in the hepatotoxic process of heavy metals. These results suggest that lipid peroxidation damage and metabolic damage in liver mitochondria and peroxisomes, may be one of the key events in heavy metal-induced liver injury.

Atomoxetine (ATX) is a presynaptic norepinephrine transporter (NET) inhibitor widely prescribed for attention-deficit/hyperactivity disorder (ADHD) due to its low abuse potential and absence of psychostimulant effects. While NET inhibition is implicated in the clinical response, several additional pharmacoactivities may contribute to clinical efficacy or unwanted side effects. We recently reported that ATX can dose-dependently alter mitochondrial function and cellular redox status. Here, we assessed potential alterations in mitochondrial biogenesis, mitochondrial dynamics and cellular antioxidant capacity following high- and low-dose ATX treatment of differentiated human neuroblastoma cells. Human SH-SY5Y neuroblastoma cells were treated with ATX (1, 5, 10, 20 and 50 μM) for 7 days under differentiation culture conditions. Changes in the expression levels of protein markers for mitochondrial biogenesis, fusion and fission as well as of antioxidant proteins were analysed by Western blot. High-dose ATX (50 μM) reduced while low-dose ATX (10 μM) increased mitochondrial biogenesis as evidenced by parallel changes in SDHA, COX-I, PGC1α and TFAM expression. High-dose ATX also reduced mitochondrial fusion as evidenced by OPA1 and MFN2 downregulation, and mitochondrial fission as indicated by DRP1 and Fis1 downregulation. In contrast, ATX did not alter expression of the antioxidant enzymes SOD1 and catalase, the phase II transcription factor Nfr2, or the Nfr2-regulated antioxidant enzyme NQO1. Clinical responses and side effects of ATX may be mediated by dose-dependent modulation of mitochondrial biogenesis and dynamics as well as NET inhibition.

Pex30 is a peroxisomal protein whose role in peroxisome biogenesis via the endoplasmic reticulum has been established. It is a 58 KDa multi-domain protein that facilitates contact site formation between various organelles. The present study aimed to investigate the role of various domains of the protein in its sub-cellular localization and regulation of peroxisome number. For this, we created six truncations of the protein (1-87, 1-250, 1-352, 88-523, 251-523 and 353-523) and tagged GFP at the C-terminus. Biochemical methods and fluorescence microscopy were used to characterize the effect of truncation on expression and localization of the protein. Quantitative analysis was performed to determine the effect of truncation on peroxisome number in these cells. Expression of the truncated variants in cells lacking PEX30 did not cause any effect on cell growth. Interestingly, variable expression and localization of the truncated variants in both peroxisome-inducing and non-inducing medium was observed. Truncated variants depicted different distribution patterns such as punctate, reticulate and cytosolic fluorescence. Interestingly, lack of the complete dysferlin domain or C-Dysf resulted in increased peroxisome number similar to as reported for cells lacking Pex30. No contribution of this domain in the reticulate distribution of the proteins was also observed. Our results show an interesting role for the various domains of Pex30 in localization and regulation of peroxisome number.

Nordihydroguaiaretic acid (NDGA), a dicatechol and phytochemical polyphenolic antioxidant and an established inhibitor of human arachidonic acid (AA) 5-lipoxygenase (LOX) and 15-LOX, is widely used to ascertain the role of LOXs in vascular endothelial cell (EC) function. As the modulatory effect of NDGA on phospholipase D (PLD), an important lipid signaling enzyme in ECs, thus far has not been reported, here we have investigated the modulation of PLD activity and its regulation by NDGA in the bovine pulmonary artery ECs (BPAECs). NDGA induced the activation of PLD (phosphatidic acid formation) in cells in a dose- and time-dependent fashion that was significantly attenuated by iron chelator and antioxidants. NDGA induced the formation of reactive oxygen species (ROS) in cells in a dose- and time-dependent manner as evidenced from fluorescence microscopy and fluorimetry of ROS and electron paramagnetic resonance spectroscopy of oxygen radicals. Also, NDGA caused a dose-dependent loss of intracellular glutathione (GSH) in BPAECs. Protein tyrosine kinase (PTyK)-specific inhibitors significantly attenuated NDGA-induced PLD activation in BPAECs. NDGA also induced a dose- and time-dependent phosphorylation of tyrosine in proteins in cells. NDGA caused in situ translocation and relocalization of both PLD1 and PLD2 isoforms, in a time-dependent fashion. Cyclooxygenase (COX) inhibitors were ineffective in attenuating NDGA-induced PLD activation in BPAECs, thus ruling out the activation of COXs by NDGA. NDGA inhibited the AA-LOX activity and leukotriene C4 (LTC4) formation in cells. On the other hand, the 5-LOX-specific inhibitors, 5, 8, 11, 14-eicosatetraynoic acid and kaempferol, were ineffective in activating PLD in BPAECs. Antioxidants and PTyK-specific inhibitors effectively attenuated NDGA cytotoxicity in BPAECs. The PLD-specific inhibitor, 5-fluoro-2-indolyl deschlorohalopemide (FIPI), significantly attenuated and protected against the NDGA-induced PLD activation and cytotoxicity in BPAECs. For the first time, these results demonstrated that NDGA, the classic phytochemical polyphenolic antioxidant and LOX inhibitor, activated PLD causing cytotoxicity in ECs through upstream oxidant signaling and protein tyrosine phosphorylation.