The mechanistic target of rapamycin (mTOR) pathway coordinates the metabolic activity of eukaryotic cells through environmental signals, including nutrients, energy, growth factors, and oxygen. In the nervous system, the mTOR pathway regulates fundamental biological processes associated with neural development and neurodegeneration. Intriguingly, genes that constitute the mTOR pathway have been found to be germline and somatic mutation from patients with various epileptic disorders. Hyperactivation of the mTOR pathway due to said mutations has garnered increasing attention as culprits of these conditions : somatic mutations, in particular, in epileptic foci have recently been identified as a major genetic cause of intractable focal epilepsy, such as focal cortical dysplasia. Meanwhile, epilepsy models with aberrant activation of the mTOR pathway have helped elucidate the role of the mTOR pathway in epileptogenesis, and evidence from epilepsy models of human mutations recapitulating the features of epileptic patients has indicated that mTOR inhibitors may be of use in treating epilepsy associated with mutations in mTOR pathway genes. Here, we review recent advances in the molecular and genetic understanding of mTOR signaling in epileptic disorders. In particular, we focus on the development of and limitations to therapies targeting the mTOR pathway to treat epileptic seizures. We also discuss future perspectives on mTOR inhibition therapies and special diagnostic methods for intractable epilepsies caused by brain somatic mutations.
Biological Processes ; Brain ; Drug Resistant Epilepsy ; Epilepsies, Partial ; Epilepsy ; Eukaryotic Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; Malformations of Cortical Development ; Nervous System ; Oxygen ; Sirolimus

OBJECTIVES: The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed. METHODS: Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually. RESULTS: Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order. CONCLUSION: Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.
Agar ; Bacteria ; Bacteriophages ; Caudovirales ; Classification ; DNA ; Electrophoresis ; Eukaryotic Cells ; Head ; Humans ; Livestock ; Membranes ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Methods ; Microscopy, Electron, Scanning Transmission ; Microscopy, Electron, Transmission ; Sewage ; Siphoviridae ; Staphylococcal Infections ; Tail

The Endoplasmic reticulum (ER), an indispensable sub-cellular component of the eukaryotic cell carries out essential functions, is critical to the survival of the organism. The chaperone proteins and the folding enzymes which are multi-domain ER effectors carry out 3-dimensional conformation of nascent polypeptides and check misfolded protein aggregation, easing the exit of functional proteins from the ER. Diverse conditions, for instance redox imbalance, alterations in ionic calcium levels, and inflammatory signaling can perturb the functioning of the ER, leading to a build-up of unfolded or misfolded proteins in the lumen. This results in ER stress, and aiming to reinstate protein homeostasis, a well conserved reaction called the unfolded protein response (UPR) is elicited. Equally, in protracted cellular stress or inadequate compensatory reaction, UPR pathway leads to cell loss. Dysfunctional ER mechanisms are responsible for neuronal degeneration in numerous human diseases, for instance Alzheimer's, Parkinson's and Huntington's diseases. In addition, mounting proof indicates that ER stress is incriminated in psychiatric diseases like major depressive disorder, bipolar disorder, and schizophrenia. Accumulating evidence suggests that pharmacological agents regulating the working of ER may have a role in diminishing advancing neuronal dysfunction in neuropsychiatric disorders. Here, new findings are examined which link the foremost mechanisms connecting ER stress and cell homeostasis. Furthermore, a supposed new pathogenic model of major neuropsychiatry disorders is provided, with ER stress proposed as the pivotal step in disease development.
Apoptosis ; Biological Psychiatry ; Bipolar Disorder ; Calcium ; Depressive Disorder, Major ; Endoplasmic Reticulum Stress* ; Endoplasmic Reticulum* ; Eukaryotic Cells ; Homeostasis ; Humans ; Neurons ; Neuropsychiatry ; Oxidation-Reduction ; Peptides ; Proteostasis Deficiencies ; Schizophrenia ; Unfolded Protein Response

OBJECTIVE: To investigate the anti-leukemia effect of total saponins of Rubus parvifolius L. (TSRP) on K562 cell xenografts in nude mice and the mechanisms of action. METHODS: The K562 cell xenografts in nude mice were established, and then randomly divided into 5 groups, the control group, the cytosine arabinoside group(Ara-c) and 3 TSRP groups (20, 40 and 100 mg/kg). The tumor volume and mass of each group of nude mice were measured and the anti-tumor rates of TSRP were calculated subsequently. The apoptosis status of tumor cells was detected by hematoxylin-eosin (HE) and terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining analysis. Finally, the activities of apoptosis related signaling of signal transducer and activator of transcription 3 (STAT3), eukaryotic initiation factor 4E (eIF4E) and B-cell lymphoma-2 (bcl-2) were determined with immunohistochemistry tests. RESULTS: Subcutaneous injection of K562 cells induced tumor formation in nude mice, and the TSRP treated group showed a signifificant inhibitory effect on tumor formation. The nude mice treated with TSRP showed a signifificant decrease in tumor growth rate and tumor weight in comparison to the control group (all P<0.05). The HE staining and TUNEL assay showed that TSRP induced cell death by apoptosis. The immunohistochemical assay showed down-regulation of the bcl-2 gene in the TSRP treated cells. The phosphorylation levels of eIF4E and STAT3 were decreased obviously after the treatment of TSRP. CONCLUSION TSRP had an excellent tumor-suppressing effect on K562 cells in the nude mice xenograft model, suggesting that TSPR can be developed as a promising anti-chronic myeloide leukemia drug.
Animals ; Apoptosis ; drug effects ; Eukaryotic Initiation Factor-4E ; physiology ; Humans ; K562 Cells ; Leukemia ; drug therapy ; pathology ; Male ; Mice ; Rubus ; chemistry ; STAT3 Transcription Factor ; physiology ; Saponins ; pharmacology ; Signal Transduction ; drug effects ; Xenograft Model Antitumor Assays

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.
Acanthamoeba castellanii* ; Acanthamoeba* ; Amino Acids ; Clone Cells ; Cytoplasm ; DNA, Complementary ; Epigenomics ; Eukaryotic Cells ; Protein-Arginine N-Methyltransferases* ; RNA, Small Interfering

Mitochondria play essential role in eukaryotic cells including in the oxidative phosphorylation and generation of adenosine triphosphate via the electron-transport chain. Therefore, defects in mitochondrial DNA (mtDNA) can result in mitochondrial dysfunction which leads to various mitochondrial disorders that may present with various neurologic and non-neurologic manifestations. Mutations in the nuclear gene polymerase gamma (POLG) are associated with mtDNA depletions, and Alpers-Huttenlocher syndrome is one of the most severe manifestations of POLG mutation characterized by the clinical triad of intractable seizures, psychomotor regression, and liver failure. The hepatic manifestation usually occurs late in the disease's course, but in some references, hepatitis was reportedly the first manifestation. Liver transplantation was considered contraindicated in Alpers-Huttenlocher syndrome due to its poor prognosis. We acknowledged a patient with the first manifestation of the disease being hepatic failure who eventually underwent liver transplantation, and whose neurological outcome improved after cocktail therapy.
Adenosine Triphosphate ; Diffuse Cerebral Sclerosis of Schilder* ; DNA, Mitochondrial ; Eukaryotic Cells ; Hepatitis ; Humans ; Liver Failure* ; Liver Transplantation* ; Liver* ; Mitochondria ; Mitochondrial Diseases ; Oxidative Phosphorylation ; Prognosis ; Seizures

Mitochondria play essential role in eukaryotic cells including in the oxidative phosphorylation and generation of adenosine triphosphate via the electron-transport chain. Therefore, defects in mitochondrial DNA (mtDNA) can result in mitochondrial dysfunction which leads to various mitochondrial disorders that may present with various neurologic and non-neurologic manifestations. Mutations in the nuclear gene polymerase gamma (POLG) are associated with mtDNA depletions, and Alpers-Huttenlocher syndrome is one of the most severe manifestations of POLG mutation characterized by the clinical triad of intractable seizures, psychomotor regression, and liver failure. The hepatic manifestation usually occurs late in the disease's course, but in some references, hepatitis was reportedly the first manifestation. Liver transplantation was considered contraindicated in Alpers-Huttenlocher syndrome due to its poor prognosis. We acknowledged a patient with the first manifestation of the disease being hepatic failure who eventually underwent liver transplantation, and whose neurological outcome improved after cocktail therapy.
Adenosine Triphosphate ; Diffuse Cerebral Sclerosis of Schilder* ; DNA, Mitochondrial ; Eukaryotic Cells ; Hepatitis ; Humans ; Liver Failure* ; Liver Transplantation* ; Liver* ; Mitochondria ; Mitochondrial Diseases ; Oxidative Phosphorylation ; Prognosis ; Seizures

Autophagy is a principle catabolic process mediated by lysosomes in eukaryotic cells. This is an intracellular homeostatic mechanism crucial for degradation in acidic lysosomal compartments of waste components from the cytoplasm. Autophagy research was initially focused on its degradation mechanism, but focus is now shifting to its effects on immunity. It contributes to detection and removal of pathogens as well as regulation of inflammasomes and neutrophil extracellular traps. Moreover, it is pivotal in antigen presentation and immune cell maturation, survival and homeostasis. The importance of autophagic pathways in normal and dysregulated immunity has become increasingly recognized in the past several years. Dysregulation of the autophagic pathway is implicated in the pathogenesis of several rheumatic diseases. In this review, we summarize the immunological function of autophagy in innate and adaptive immunity, and the functions of autophagy in the pathogenesis of rheumatic diseases.
Adaptive Immunity ; Antigen Presentation ; Arthritis, Rheumatoid ; Autophagy* ; Cytoplasm ; Eukaryotic Cells ; Extracellular Traps ; Homeostasis ; Inflammasomes ; Lupus Erythematosus, Systemic ; Lysosomes ; Rheumatic Diseases*

OBJECTIVEPERK/eIF2α/CHOP is a major signaling pathway mediating endoplasmic reticulum (ER) stress related with atherosclerosis. Oxidized LDL (ox-LDL) also induces endothelial apoptosis and plays a vital role in the initiation and progression of atherosclerosis. The present study was conducted to explore the regulatory effect of ox-LDL on PERK/eIF2α/CHOP signaling pathway in vascular endothelial cells.

METHODSThe effects of ox-LDL on PERK and p-eIF2α protein expression of primary human umbilical vein endothelial cells (HUVECs) were investigated by Western blot analysis. PERK gene silencing and selective eIF2α phosphatase inhibitor, salubrinal were used to inhibit the process of ox-LDL induced endothelial cell apoptosis, caspase-3 activity, and CHOP mRNA level.

RESULTSOx-LDL treatment significantly increased the expression of PERK, PERK-mediated inactivation of eIF2α phosphorylation, and the expression of CHOP, as well as the caspase-3 activity and apoptosis. The effects of ox-LDL were markedly decreased by knocking down PERK with stable transduction of lentiviral shRNA or by selective eIF2α phosphatase inhibitor, salubrinal.

CONCLUSIONThis study provides the first evidence that ox-LDL induces apoptosis in vascular endothelial cells mediated largely via the PERK/eIF2α/CHOP ER-stress pathway. It adds new insights into the molecular mechanisms underlying the pathogenesis and progression of atherosclerosis.

Apoptosis ; Endoplasmic Reticulum Stress ; Eukaryotic Initiation Factor-2 ; genetics ; metabolism ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipoproteins, LDL ; genetics ; metabolism ; Signal Transduction ; Transcription Factor CHOP ; genetics ; metabolism ; eIF-2 Kinase ; genetics ; metabolism

OBJECTIVEThis study aimed to construct a eukaryotic expression vector pEGFP-N1-EMP1 of epithelial mem-brane protein 1 (EMP1) and investigate its influence on migration and invasion of human oral tongue squamous carcinoma cells.

METHODSThe human EMP1 gene was amplified by reverse transcription polymerase chain reaction and then ligated into the pEGFP-N1 vector by double restriction endonuclease digestion to construct pEGFP-N1-EMP1 recombinant plasmid. After sequencing identification, pEGFP-N1-EMP1 recombinant plasmid and pEGFP-N1 plasmid were transfected into human oral tongue squamous carcinoma Tb3.1 cell line. The expression of green fluorescent protein in cells was observed after transfection using an inverted fluorescence microscope. The overexpression of EMP1 mRNA was identified at 24, 48, and 72 h after transfection by real-time fluorescence quantitative polymerase chain reaction. The effect of EMP1 overexpression on migration and invasion of Tb3.1 cells was detected by Transwell assay.

RESULTSThe full-length EMP1 gene sequence was successfully obtained. Sequence analysis showed that the EMP1 gene was inserted into the pEGFP-N1 vector correctly. Green fluorescence was observed in the transfected cells under fluorescence microscopy. The results of real-time fluorescence quantitative polymerase chain reaction indicated that the expression of EMP1 at 24 h after pEGFP-N1-EMP1 transfection was significantly higher than the other groups. Transwell assays indicated that overexpression of the EMP1 gene could significantly inhibit the migration and invasion ability of Tb3.1 cells.

CONCLUSIONSThe eukaryotic expression vector of EMP1 was successfully constructed, and EMP1 overexpression was confirmed to inhibit the migration and inva-sion of oral tongue squamous carcinoma cells in vitro. This study laid a foundation for further investigation on the influence of the EMP1 gene on the metastasis of oral tongue squamous carcinoma and its molecular mechanism.
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Carcinoma, Squamous Cell ; Cell Line, Tumor ; Cell Movement ; Eukaryotic Cells ; Genetic Vectors ; Green Fluorescent Proteins ; Humans ; Neoplasm Proteins ; Plasmids ; Receptors, Cell Surface ; Tongue Neoplasms ; Transfection