Melittin induces autophagy to alleviate chronic renal failure in 5/6-nephrectomized rats and angiotensin II-induced damage in podocytes.

BACKGROUND/OBJECTIVES: Chronic renal failure (CRF) is a complex pathological condition that lacks a cure. Certain Chinese medicines, such as melittin, a major component in bee venom, have shown efficacy in treating CRF patients. On the other hand, the mechanisms underlying the therapeutic effects of melittin are unclear. MATERIALS/METHODS: A 5/6 nephrectomy model (5/6 Nx) of renal failure was established on rats for in vivo assays, and mouse podocyte clone 5 (MPC5) mouse podocyte cells were treated with angiotensin II (AngII) to establish an in vitro podocyte damage model. The 24-h urine protein, serum creatinine, and blood urea nitrogen levels were evaluated after one, 2, and 4 weeks. Hematoxylin and eosin staining, Masson staining, and periodic acid-Schiff staining were used to examine the pathological changes in kidney tissues. A cell counting kit 8 assay was used to assess the cell viability. Reverse transcription polymerase chain reaction and Western blot were used to assess the mRNA and protein levels in the cells, respectively. RESULTS: In the rat 5/6 Nx, melittin reduced the 24-h urinary protein excretion and the serum creatinine and blood urea nitrogen levels. Furthermore, the renal pathology was improved in the melittin-treated 5/6 Nx rats. Melittin promoted podocin, nephrin, Beclin 1, and the LC3II/LC3I ratio and inhibited phosphorylated mammalian target of rapamycin (mTOR)/mTOR in 5/6 Nx-induced rats and AngII-induced MPC5 mouse podocyte cells. Moreover, inhibiting autophagy with 3-MA weakened the effects of melittin on podocin, nephrin, and the LC3II/LC3I ratio in podocytes. CONCLUSION: Melittin may offer protection against kidney injury, probably by regulating podocyte autophagy. These results provide the theoretical basis for applying melittin in CRF therapy.

THADA inhibits autophagy and increases 5-FU sensitivity in gastric cancer cells via the PI3K/AKT/mTOR signaling pathway.

Objectives: 5-Fluorouracil (5-FU) is currently the main drug used in chemotherapy for gastric cancer (GC). The main clinical problems of 5-FU therapy are insensitivity and acquired resistance to 5-FU. The mechanism of GC cell resistance to 5-FU is currently unknown. Materials and Methods: This study employed next-generation sequencing (NGS) to analyze the differentially expressed genes (DEGs) in chemotherapy-sensitive and non-sensitive GC tissues. In addition, a bioinformatics analysis was conducted using the GC dataset of GEO, and further validated and explored through in vitro experiments. Results: Thyroid adenoma-associated gene (THADA) was highly expressed in GC tissues from chemotherapy-sensitive patients and was an independent prognostic factor in GC patients receiving postoperative 5-FU adjuvant chemotherapy. Notably, heightened THADA expression in GC cells was associated with the down-regulation of autophagy-related proteins (LC-3, ATG13, ULK1, and TFEB). Furthermore, the PI3K/AKT/mTOR signaling pathway and mTORC1 signaling pathway were remarkably increased in patients with elevated THADA expression. THADA expression was associated with mTOR, the core protein of the mTOR signaling pathway, and related proteins involved in regulating the mTORC1 signaling pathway (mLST8, RHEB, and TSC2). THADA exhibited inhibitory effects on autophagy and augmented the sensitivity of GC cells to 5-FU through the PI3K/AKT/mTOR signaling pathway. Conclusion: The findings suggest that THADA may be involved in the regulatory mechanism of GC cell sensitivity to 5-FU. Consequently, the detection of THADA in tumor tissues may bring clinical benefits, specifically for 5-FU-related chemotherapy administered to GC patients with elevated THADA expression.

AMPK-mTORC1 pathway mediates hepatic IGFBP-1 phosphorylation in glucose deprivation: a potential molecular mechanism of hypoglycemia-induced impaired fetal growth.

Mechanisms underlying limitations in glucose supply that restrict fetal growth are not well established. IGF-1 is an important regulator of fetal growth and IGF-1 bioavailability is markedly inhibited by IGFBP-1 especially when the binding protein is hyperphosphorylated. We hypothesized that the AMPK-mTORC1 pathway increases IGFBP-1 phosphorylation in response to glucose deprivation. Glucose deprivation in HepG2 cells activated AMPK and TSC2, inhibited mTORC1 and increased IGFBP-1 secretion and site-specific phosphorylation. Glucose deprivation also decreased IGF-1 bioavailability and IGF-dependent activation of IGF-1R. AICAR (an AMPK activator) activated TSC2, inhibited mTORC1, and increased IGFBP-1 secretion/phosphorylation. Further, siRNA silencing of either AMPK or TSC2 prevented mTORC1 inhibition and IGFBP-1 secretion and phosphorylation in glucose deprivation. Our data suggest that the increase in IGFBP-1 phosphorylation in response to glucose deprivation is mediated by the activation of AMPK/TSC2 and inhibition of mTORC1, providing a possible mechanistic link between glucose deprivation and restricted fetal growth.

Paraquat and Parkinson's Disease: The Molecular Crosstalk of Upstream Signal Transduction Pathways Leading to Apoptosis.

Parkinson's disease (PD) is a heterogeneous disease involving a complex interaction between genes and the environment that affects various cellular pathways and neural networks. Several studies have suggested that environmental factors such as exposure to herbicides, pesticides, heavy metals, and other organic pollutants are significant risk factors for the development of PD. Among the herbicides, paraquat has been commonly used, although it has been banned in many countries due to its acute toxicity. Although the direct causational relationship between paraquat exposure and PD has not been established, paraquat has been demonstrated to cause the degeneration of dopaminergic neurons in the substantia nigra pars compacta. The underlying mechanisms of the dopaminergic lesion are primarily driven by the generation of reactive oxygen species, decrease in antioxidant enzyme levels, neuroinflammation, mitochondrial dysfunction, and ER stress, leading to a cascade of molecular crosstalks that result in the initiation of apoptosis. This review critically analyses the crucial upstream molecular pathways of the apoptotic cascade involved in paraquat neurotoxicity, including mitogenactivated protein kinase (MAPK), phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT, mammalian target of rapamycin (mTOR), and Wnt/ß-catenin signaling pathways.

Applying a novel kinomics approach to study decidualization and the effects of antigestagens using a canine model.

Maternal decidual cells are crucial for the maintenance of canine pregnancy as they are the only cells expressing the nuclear progesterone (P4) receptor (PGR) in the placenta. Interfering with P4/PGR signaling adversely affects decidual cells and terminates pregnancy. Although immortalized dog uterine stromal (DUS) cells can be decidualized in vitro using cAMP, the involvement of cAMP-dependent kinases in canine decidualization had not been investigated. Therefore, the present project investigated changes in the kinome of DUS cells following in vitro decidualization, using the serine/threonine kinase (STK) PamChip assay (PamGene). Decidualization led to a predicted activation of 85 STKs in DUS cells, including PKA, PKC, ERK1/2 and other MAPKs, CAMKs and Akt1/2. Additionally, blocking PGR with type 2 antigestagens (aglepristone or mifepristone) decreased the activity of virtually all kinases modulated by decidualization. The underlying transcriptional effects were inferred from comparison with available transcriptomic data on antigestagen-mediated effects in DUS cells. In targeted studies, interfering with PKA or MEK1/2 resulted in downregulation of important decidualization markers (e.g. IGF1, PTGES, PRLR, PGR and PTGS2). Conversely, blocking of PKC decreased the mRNA availability of IGF1, PGR and PTGS2, but not of PTGES and PRLR. Moreover, suppressing PKA decreased the phosphorylation of the transcription factors cJUN and CREB, whereas blocking of PKC affected only cJUN. This first kinomics analysis to target decidualization showed an increased activity of a wide range of STKs, which could be hindered by disrupting P4/PGR signaling. Decidualization appears to be regulated in a kinase-dependent manner, with PKA and PKC evoking different effects.

Active compounds of Caodoukou () inhibit the migration, invasion and metastasis of human pancreatic cancer cells by targeting phosphoinosmde-3-kinase/ protein kinase B/mammalian target of rapamycin pathway.

OBJECTIVE: To detect the effects of active compounds of Caodoukou () (ACAK) on the proliferation, migration and invasion of pancreatic cancer, and explain the possible molecular mechanism of ACAK interacting with these processes. METHODS: Cell counting kit-8 method, cell scratch repair experiment, Transwell migration and invasion experiment, immunohistochemistry, western blot assay and real-time polymerase chain reaction experiment were used to evaluate the effect of ACAK on the proliferation, migration and invasion of pancreatic cancer cells. The levels of active molecules involved in the phosphoinosmde-3-kinase (PI3K)/Akt/the mammalian target of rapamycin (mTOR) signal transduction were detected by Western blot assay. In addition, the function of ACAK was evaluated by xenotransplantation tumor model in nude mice. RESULTS: The inhibitory effect of ACAK on the proliferation of pancreatic cancer cells showed certain time-dose dependence. The results of scratch repair test, Transwell test, Western blotting and real time polymerase chain reaction assay showed that ACAK could inhibit the migration and invasion of pancreatic cancer cells . In addition, the regulatory effect of ACAK on epithelial-mesenchymal transition (EMT) is partly attributed to PI3K/Akt/mTOR signaling pathway. The experimental results showed that ACAK regulated the development of pancreatic cancer. CONCLUSIONS: ACAK can partly inhibit the activity of EMT and matrix metallopeptidases by down-regulating the downstream proteins of PI3K/Akt/mTOR signal pathway, thus inhibiting the ability of migration and invasion of pancreatic cancer.

Aging inverts the effects of p75NTR -modulated mTOR manipulation on hippocampal neuron synaptic plasticity in male mice.

Age-induced impairments in learning and memory are in part caused by changes to hippocampal synaptic plasticity during aging. The p75 neurotrophin receptor (p75NTR ) and mechanistic target of rapamycin (mTOR) are implicated in synaptic plasticity processes. mTOR is also well known for its involvement in aging. Recently, p75NTR and mTOR were shown to be mechanistically linked, and that p75NTR mediates age-induced impairment of hippocampal synaptic plasticity. Yet the consequences of p75NTR -mTOR interaction on hippocampal synaptic plasticity, and the role of mTOR in age-induced cognitive decline, are unclear. In this study, we utilize field electrophysiology to study the effects of mTOR inhibition and activation on long-term potentiation (LTP) in male young and aged wild-type (WT) mice. We then repeated the experiments on p75NTR knockout mice. The results demonstrate that mTOR inhibition blocks late-LTP in young WT mice but rescues age-related late-LTP impairment in aged WT mice. mTOR activation suppresses late-LTP in aged WT mice while lacking observable effects on young WT mice. These effects were not observed in p75NTR knockout mice. These results demonstrate that the role of mTOR in hippocampal synaptic plasticity is distinct between young and aged mice. Such effects could be explained by differing sensitivity of young and aged hippocampal neurons to changes in protein synthesis or autophagic activity levels. Additionally, elevated mTOR in the aged hippocampus could cause excessive mTOR signaling, which is worsened by activation and alleviated by inhibition. Further research on mTOR and p75NTR may prove useful for advancing understanding and, ultimately, mitigation of age-induced cognitive decline.

Asymmetric Dimethylation of Ribosomal S6 Kinase 2 Regulates Its Cellular Localisation and Pro-Survival Function.

Ribosomal S6 kinases (S6Ks) are critical regulators of cell growth, homeostasis, and survival, with dysregulation of these kinases found to be associated with various malignancies. While S6K1 has been extensively studied, S6K2 has been neglected despite its clear involvement in cancer progression. Protein arginine methylation is a widespread post-translational modification regulating many biological processes in mammalian cells. Here, we report that p54-S6K2 is asymmetrically dimethylated at Arg-475 and Arg-477, two residues conserved amongst mammalian S6K2s and several AT-hook-containing proteins. We demonstrate that this methylation event results from the association of S6K2 with the methyltransferases PRMT1, PRMT3, and PRMT6 in vitro and in vivo and leads to nuclear the localisation of S6K2 that is essential to the pro-survival effects of this kinase to starvation-induced cell death. Taken together, our findings highlight a novel post-translational modification regulating the function of p54-S6K2 that may be particularly relevant to cancer progression where general Arg-methylation is often elevated.

The VEGFR/PDGFR tyrosine kinase inhibitor, ABT-869, blocks necroptosis by targeting RIPK1 kinase.

Necroptosis is a mode of programmed, lytic cell death that is executed by the mixed lineage kinase domain-like (MLKL) pseudokinase following activation by the upstream kinases, receptor-interacting serine/threonine protein kinase (RIPK)-1 and RIPK3. Dysregulated necroptosis has been implicated in the pathophysiology of many human diseases, including inflammatory and degenerative conditions, infectious diseases and cancers, provoking interest in pharmacological targeting of the pathway. To identify small molecules impacting on the necroptotic machinery, we performed a phenotypic screen using a mouse cell line expressing an MLKL mutant that kills cells in the absence of upstream death or pathogen detector receptor activation. This screen identified the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitor, ABT-869 (Linifanib), as a small molecule inhibitor of necroptosis. We applied a suite of cellular, biochemical and biophysical analyses to pinpoint the apical necroptotic kinase, RIPK1, as the target of ABT-869 inhibition. Our study adds to the repertoire of established protein kinase inhibitors that additionally target RIPK1 and raises the prospect that serendipitous targeting of necroptosis signalling may contribute to their clinical efficacy in some settings.

Mycobacterium tuberculosis and its secreted tyrosine phosphatases.

Tuberculosis is one of the most common infectious diseases and has been a major burden for a long time now. Increasing drug resistance in TB is slowing down the process of disease treatment. Mycobacterium tuberculosis, the causative agent of TB is known to have a cascade of virulence factors to fight with host's immune system. The phosphatases (PTPs) of Mtb plays a critical role as these are secretory in nature and help the survival of bacteria in host. Researchers have been trying to synthesize inhibitors against a lot of virulence factors of Mtb but recently the phosphatases have gained a lot of interest due to their secretory nature. This review gives a concise overview of virulence factors of Mtb with emphasis on mPTPs. Here we discuss the current scenario of drug development against mPTPs.

Everolimus as a therapeutic option in refractory epilepsy in children with tuberous sclerosis: a systematic review / Everolimo como opção terapêutica na epilepsia refratária em crianças com esclerose tuberosa: uma revisão sistemática

Abstract Background Tuberous sclerosis (TS) is a multisystem genetic disease in which epilepsy is a frequent manifestation and is often difficult to control. Everolimus is a drug with proven efficacy in the treatment of other conditions related to TS, and some evidence suggests that its use benefits the treatment of refractory epilepsy in these patients. Objective To evaluate the efficacy of everolimus in controlling refractory epilepsy in children with TS. Methods A literature review was conducted in the Pubmed, BVS, and Medline databases, using the descriptors Tuberous sclerosis, Children, Epilepsy, and Everolimus. Original clinical trials and prospective studies published in Portuguese or English in the last decade that evaluated the use of everolimus as an adjuvant therapy in the control of refractory epilepsy in pediatric patients with TS were included. Results Our search screened 246 articles from electronic databases, 6 of which were chosen for review. Despite the methodological variations between the studies, most patients benefited from the use of everolimus to control refractory epilepsy, with response rates ranging from 28.6 to 100%. Adverse effects were present in all studies leading to dropouts of some patients; however, the majority were of low severity. Conclusion The selected studies suggest a beneficial effect of everolimus in the treatment of refractory epilepsy in children with TS, despite the adverse effects observed. Further studies involving a larger sample in double-blind controlled clinical trials should be performed to provide more information and statistical credibility.

Resumo Antecedentes A esclerose tuberosa (ET) é uma doença genética multissistêmica na qual a epilepsia é a manifestação neurológica mais frequente, sendo muitas vezes de difícil controle. O everolimo é uma droga com eficácia comprovada no tratamento de outras condições relacionadas à ET, e indícios sugerem benefícios de seu uso também no controle da epilepsia refratária nesses pacientes. Objetivo Avaliar a eficácia do everolimo no controle da epilepsia refratária em crianças com ET. Métodos Revisão de literatura nas bases de dados Pubmed, BVS e Medline, utilizando os descritores Tuberous sclerosis, Children, Epilepsy e Everolimus. Incluíram-se ensaios clínicos originais e estudos prospectivos publicados em português ou inglês na última década e que avaliassem o uso do everolimo como terapia adjuvante no controle da epilepsia refratária em pacientes pediátricos com ET. Resultados Nossa busca rastreou 246 artigos nas bases de dados, dos quais 6 foram escolhidos para a revisão. Apesar das variações metodológicas entre os estudos, a maioria dos pacientes tiveram benefício no uso do everolimo para controle da epilepsia refratária, com taxas de resposta variando entre 28.6 e 100%. Os efeitos adversos estiveram presentes em todos os estudos, levando à desistência de alguns pacientes, contudo a maioria foi de baixa gravidade. Conclusão Os estudos selecionados sugerem efeito benéfico do everolimo no tratamento da epilepsia refratária em crianças com ET, apesar dos efeitos adversos observados. Novos estudos envolvendo uma amostra maior em ensaios clínicos controlados duplo-cegos devem ser realizados para fornecer mais informações e credibilidade estatística.

The role of CaMKK2 in Golgi-associated vesicle trafficking.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a serine/threonine-protein kinase, that is involved in maintaining various physiological and cellular processes within the cell that regulate energy homeostasis and cell growth. CaMKK2 regulates glucose metabolism by the activation of downstream kinases, AMP-activated protein kinase (AMPK) and other calcium/calmodulin-dependent protein kinases. Consequently, its deregulation has a role in multiple human metabolic diseases including obesity and cancer. Despite the importance of CaMKK2, its signalling pathways and pathological mechanisms are not completely understood. Recent work has been aimed at broadening our understanding of the biological functions of CaMKK2. These studies have uncovered new interaction partners that have led to the description of new functions that include lipogenesis and Golgi vesicle trafficking. Here, we review recent insights into the role of CaMKK2 in membrane trafficking mechanisms and discuss the functional implications in a cellular context and for disease.

The Anti-Obesity and Anti-Steatotic Effects of Chrysin in a Rat Model of Obesity Mediated through Modulating the Hepatic AMPK/mTOR/lipogenesis Pathways.

BACKGROUND: Obesity is a complex multifactorial disease characterized by excessive adiposity, and is linked to an increased risk of nonalcoholic fatty liver disease (NAFLD). Flavonoids are natural polyphenolic compounds that exert interesting pharmacological effects as antioxidant, anti-inflammatory, and lipid-lowering agents. In the present study, we investigated the possible therapeutic effects of the flavonoid chrysin on obesity and NAFLD in rats, and the role of AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathways in mediating these effects. METHOD: Thirty-two Wistar male rats were divided into two groups: the control group and the obese group. Obesity was induced by feeding with an obesogenic diet for 3 months. The obese rats were subdivided into four subgroups, comprising an untreated group, and three groups treated orally with different doses of chrysin (25, 50, and 75 mg/kg/day for one month). Results revealed that chrysin treatment markedly ameliorated the histological changes and significantly and dose-dependently reduced the weight gain, hyperglycemia, and insulin resistance in the obese rats. Chrysin, besides its antioxidant boosting effects (increased GSH and decreased malondialdehyde), activated the AMPK pathway and suppressed the mTOR and lipogenic pathways, and stimulated expression of the genes controlling mitochondrial biogenesis in the hepatic tissues in a dose-dependent manner. In conclusion, chrysin could be a promising candidate for the treatment of obesity and associated NAFLD, aiding in attenuating weight gain and ameliorating glucose and lipid homeostasis and adipokines, boosting the hepatic mitochondrial biogenesis, and modulating AMPK/mTOR/SREBP-1c signaling pathways.

PD-L1 Upregulation by the mTOR Pathway in VEGFR-TKI-Resistant Metastatic Clear Cell Renal Cell Carcinoma.

PURPOSE: Tyrosine kinase inhibitors (TKI) targeting vascular endothelial growth factor receptor (VEGFR) signaling pathways have been used for metastatic clear cell renal cell carcinoma (mCCRCC), but resistance to the drug develops in most patients. We aimed to explore the underlying mechanism of the TKI resistance with regard to programmed death-ligand 1 (PD-L1) and to investigate signaling pathway associated with the resistant mechanism. MATERIALS AND METHODS: To determine the mechanism of resistance, 10 mCCRCC patients from whom tumor tissues were harvested at both the pretreatment and the TKI-resistant post-treatment period were included as the discovery cohort, and their global gene expression profiles were compared. A TKI-resistant renal cancer cell line was established by long-term treatment with sunitinib. RESULTS: Among differentially expressed genes in the discovery cohort, increased PD-L1 expression in post-treatment tissues was noted in four patients. Pathway analysis showed that PD-L1 expression was positively correlated with the mammalian target of rapamycin (mTOR) signaling pathway. The TKI-resistant renal cancer cells showed increased expression of PD-L1 and mTOR signaling proteins and demonstrated aggressive tumoral behaviour. Treatment with mTOR inhibitors down-regulated PD-L1 expression and suppressed aggressive tumoral behaviour, which was reversed with stimulation of the mTOR pathway. CONCLUSION: These results showed that PD-L1 expression may be increased in a subset of VEGFR-TKI-resistant mCCRCC patients via the mTOR pathway.

Canonical insulin signaling is not significantly impaired in early stages of depression.

Patients with major depression (MD) are at high-risk for insulin resistance (IR), type-2 diabetes, metabolic syndrome, cardiovascular morbidity and mortality. However, our recent study published in this journal [Eur Arch Psychiatry Clin Neurosci. 2019 Jun;269(4):373-377], found no evidence of IR in acutely-ill drug-naive first-episode MD (FEMD) using the homeostatic model assessment of insulin resistance (HOMA-IR). We concluded, that MD may be related to impaired glucose/insulin homeostasis in the long-term but not in early disease stages. Now, we performed a complementary analysis of the canonical insulin signalling pathway containing the set of control and FEMD samples from the study mentioned above. The first node (pS312-IRS-1, pY-IRS-1) and downstream pathway which affects glucose and lipid homeostasis (phosphorylated proteins: pS473-AKT, pS9-GSK3ß, pS2448-mTOR, pT389-p70S6K; total proteins AKT, GSK3ß, mTOR, p70S6K) were analyzed by electrochemiluminescence (ECL) in neuronal extracellular vesicles (nEVs) enriched for L1 neural cell adhesion molecule (L1CAM) expression. No significant diagnosis-related differences were observed for the pS312-IRS-1 / pYIRS-1 ratio (P = 0.093), but the mean ratio was reduced by ~ 70% in FEMD versus controls. Moreover, omnibus analysis of downstream phosphorylated / total signaling protein ratios and respective post-hoc analyses revealed no significant changes in FEMD patients versus controls (P = 0.734). HAMD-21 scores were not correlated with pS312-IRS-1 / pY-IRS-1 or downstream phosphorylated/total signaling protein ratios. In summary, we did not find evidence for altered neuronal insulin signaling in early disease stages of MD. This is in contrast to schizophrenia, where we and other researchers have seen evidence of IR in first-episode patients.

Pulmonary toxicity of mTOR inhibitors. Comparisons of two populations: Solid organ recipients and cancer patients.

INTRODUCTION: Mammalian target of rapamycin (mTOR) inhibitors-associated pneumonitis (mTOR-IP) has long been described in solid organ recipients (T) patients but more recently in cancer (K) patients. Its overall characteristics have never been compared between these 2 populations. The aim of this study was to compare them in terms of presentation, severity and outcome in T and in K patients. MATERIAL AND METHODS: We carried out a retrospective study in a single French tertiary center. Four databases were used to ensure the exhaustive collection of all mTOR-IP cases between 2001 and 2020. All clinical, biological, radiological, pathological and outcome data were reviewed. RESULTS: Thirty-nine patients with mTOR-IP were diagnosed during this period, 24T and 15K patients. The average dosage of everolimus and sirolimus was 2,65mg (±1,78) and 2,75mg (±0,96) in T patients, respectively, versus 8,75mg (±2,26) for everolimus in K patients. The overall prevalence of mTOR-IP was 6.4% with a median time of occurrence of 7 months [IQR 3-35 months]. mTOR-IP were significantly more frequent (P<0.001) and occurred earlier (P<0.001) in cancer patients. No clinical, functional, radiological, pathological nor outcome differences were otherwise observed between the 2 groups. Average everolimus blood levels at the time of mTOR-IP diagnosis were in the range of recommended therapeutic values. CONCLUSION: Our study shows that mTOR-IP is comparable in terms of presentation in T and in K patients but that it occurs significantly earlier after drug introduction in the latter. This raises questions as to the potential role of the higher doses used in K patients as well as that of co-treatments in the pathogeny of the disease.

E. coli Toxin YjjJ (HipH) Is a Ser/Thr Protein Kinase That Impacts Cell Division, Carbon Metabolism, and Ribosome Assembly.

Protein Ser/Thr kinases are posttranslational regulators of key molecular processes in bacteria, such as cell division and antibiotic tolerance. Here, we characterize the E. coli toxin YjjJ (HipH), a putative protein kinase annotated as a member of the family of HipA-like Ser/Thr kinases, which are involved in antibiotic tolerance. Using SILAC-based phosphoproteomics we provide experimental evidence that YjjJ is a Ser/Thr protein kinase and its primary protein substrates are the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. YjjJ activity impacts ribosome assembly, cell division, and central carbon metabolism but it does not increase antibiotic tolerance as does its homologue HipA. Intriguingly, overproduction of YjjJ and its kinase-deficient variant can activate HipA and other kinases, pointing to a cross talk between Ser/Thr kinases in E. coli. IMPORTANCE Adaptation to growth condition is the key for bacterial survival, and protein phosphorylation is one of the strategies adopted to transduce extracellular signal in physiological response. In a previous work, we identified YjjJ, a putative kinase, as target of the persistence-related HipA kinase. Here, we performed the characterization of this putative kinase, complementing phenotypical analysis with SILAC-based phosphoproteomics and proteomics. We provide the first experimental evidence that YjjJ is a Ser/Thr protein kinase, having as primary protein substrates the ribosomal protein RpmE (L31) and the carbon storage regulator CsrA. We show that overproduction of YjjJ has a major influence on bacterial physiology, impacting DNA segregation, cell division, glycogen production, and ribosome assembly.

Effect of metformin preconditioning on AMPK/PINK1 signaling pathway during myocardial ischemia-reperfusion injury in diabetic rats / 中华麻醉学杂志

Objective:To evaluate the effect of metformin preconditioning on adenosine monophosphate-activated protein kinase(AMPK)/PTEN-induced putative protein kinase(PINK1) signaling pathway during ischemia-reperfusion (I/R) injury in diabetic rats.Methods:Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 6 weeks, weighing 120-160 g, were divided into 3 groups ( n=12 each) by the random number table method: diabetic sham operation group (DS group), diabetic myocardial I/R group (DI/R group) and diabetic myocardial I/R+ metformin preconditioning group(DI/R+ Met group). After 4 weeks of feeding a high-fat and high-glucose diet, the model of type 2 diabetes mellitus was induced by a single intraperitoneal injection of 1% streptozotocin 40 mg/kg. The myocardial I/R injury was induced by blocking the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion in anesthetized animals. In DI/R+ Met group, metformin 200 mg/kg was given by intragastric gavage once a day within 1 week before myocardial ischemia. Blood samples from the femoral vein were collected at 120 min of reperfusion for determination of the serum creatine kinase isoenzymes (CK-MB) and cardiac troponin I (cTnI) concentrations by enzyme-linked immunosorbent assay. Then the rats were sacrificed and myocardial tissues were obtained for examination of the pathological changes(by HE staining) and for determination of the percentage of myocardial infarct size (by the double staining of Ewan blue and TTC) and expression of myocardial autophagy-related protein Beclin-1, PTEN-induced putative kinase 1 (PINK1), phosphorylated 5′-adenosine monophosphate-activating protein kinase (p-AMPK), and ratio of microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ (LC3Ⅱ/Ⅰ) (by Western blot). Results:Compared with DS group, the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly increased, the expression of Beclin-1, p-AMPK and PINK1 in myocardial tissues was up-regulated, the ratio of LC3II/I was increased( P<0.05), and the pathological changes were aggravated in DI/R group and DI/R+ Met group. Compared with DI/R group, the percentage of myocardial infarct size and serum CK-MB and cTnI concentrations were significantly decreased, the expression of Beclin-1, p-AMPK and PINK1 in myocardial tissues was up-regulated, the ratio of LC3Ⅱ/Ⅰ was increased ( P<0.05), and the pathological changes were significantly reduced in DI/R+ Met group. Conclusions:The mechanism by which metformin preconditioning reduces myocardial I/R injury is related to activation of AMPK/PINK1 signaling pathway and up-regulation of mitochondrial autophagy in diabetic rats.

Role of PI3K/Akt/mTOR signaling pathway in edaravone-induced reduction of postoperative cognitive dysfunction in aged rats / 中华麻醉学杂志

Objective:To evaluate the role of phosphatidylinositol 3-kinase (PI3K)/serine threonine protein kinase (Akt)/mammalian rapamycin target protein (mTOR) signaling pathway in edaravone-induced reduction of postoperative cognitive dysfunction in aged rats.Methods:Sixty healthy male Sprague-Dawley rats, aged 20 months, weighing 600-700 g, were divided into 4 groups ( n=15 each) using a random number table method: control group (group C), operation group (group O), edaravone group (group E) and PI3K inhibitor LY294002 group (group LY). The rats received laparotomy under 3% sevoflurane anesthesia in O, E and LY groups. Edaravone 3 mg/kg was intraperitoneally injected at 30 min before operation in E and LY groups, and LY294002 0.3 mg/kg was simultaneously injected via the tail vein in group LY. Open field test was performed at 3 days after surgery to evaluate the spontaneous activity of rats, then Morris water maze test was performed to evaluate the cognitive function of rats. The rats were sacrificed after the end of behavioral experiment to isolate hippocampal tissues for determination of the expression of phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-Akt), phosphorylated mTOR (p-mTOR), synaptophysin (SYP) and postsynaptic density protein 95 (PSD 95) (by Western blot ) and dendrite length in hippocampal CA1 area (using Golgi staining). The density of dendrites was calculated. Results:There were no statistically significant differences in exercise speed, distance, and time of staying at the center between the four groups ( P>0.05). Compared with group C, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-PI3K, p-Akt, p-mTOR, SYP and PSD-95 was down-regulated, the dendritic length of neurons in hippocampal CA1 region was shortened, and the density of neurons in hippocampal CA1 region was decreased in group O ( P<0.05). Compared with group O, the escape latency was significantly shortened, the number of crossing the original platform was increased, the expression of p-PI3K, p-Akt, p-mTOR, SYP and PSD-95 was up-regulated, the dendritic length of neurons in hippocampal CA1 region was prolonged, and the density of neurons in hippocampal CA1 region was increased in group E ( P<0.05). Compared with group E, the escape latency was significantly prolonged, the number of crossing the original platform was reduced, the expression of p-PI3K, p-Akt, p-mTOR, SYP and PSD-95 was down-regulated, and the dendritic length of neurons in hippocampal CA1 region was shortened, and the density of neurons in hippocampal CA1 region was decreased in group LY ( P<0.05). Conclusions:The mechanism by which edaravone reduces postoperative cognitive dysfunction is related to activating PI3K/Akt/mTOR signaling pathway and improving synaptic plasticity in aged rats.

Effect of esketamine on long-term cognitive dysfunction induced by propofol anesthesia in developing rats and the role of PI3K/Akt signaling pathway / 中华麻醉学杂志

Objective:To evaluate the effect of esketamine on long-term cognitive dysfunction induced by propofol anesthesia in the developing rats and the role of phosphatidylinositol-3-kinase (PI3K)/serine-threonine protein kinase (Akt) signaling pathway.Methods:Forty-eight clean-grade healthy Sprague-Dawley rats of either sex, aged 7 days, weighing 10-15 g, were divided into 4 groups ( n=12 each) using a random number table method: fat emulsion group (C group), propofol group (P group), esketamine + propofol group (EP group), and PI3K inhibitor LY294002 + esketamine + propofol group (LYEP group). Medium/long-chain fat emulsion injection 100 mg/kg was intraperitoneally injected in C group. Propofol was intraperitoneally injected at a dose of 50 mg/kg, followed by an additional dose of 50 mg/kg after the righting reflex was restored (40-60 min later) in P group. In group EP, esketamine 10 mg/kg was intraperitoneally injected, followed by propofol administration using the same method as previously described in P group. In LYEP group, LY294002 25 μg was injected via the lateral ventricle, 30 min later ketamine 10 mg/kg was intraperitoneally injected, and then propofol was given using the same method as previously described in P group. Six rats in each group were randomly sacrificed at 2 h after emergence for microscopic examination of pathological changes of hippocampal neurons and for determination of Akt, phosphorylated Akt (p-Akt), Bax, and cleaved caspase-3 in the hippocampal tissues (using Western blot). The remaining 6 rats in each group were subjected to Y-maze test to evaluate their learning and memory abilities at 30 days after birth. The p-Akt/Akt ratio was calculated. Results:Compared with C group, the p-Akt/Akt ratio in the hippocampal tissues was significantly decreased, the expression of Bax and cleaved caspase-3 was up-regulated, the number of training sessions required for learning was increased, the correct response rate was decreased ( P<0.05), and the pathological damage to neurons in hippocampal CA1 region was found in P, EP and LYEP groups. Compared with P group, the p-Akt/Akt ratio in the hippocampal tissues was significantly increased, the expression of Bax and cleaved caspase-3 was down-regulated, the number of training sessions required for learning was decreased, the correct response rate was increased ( P<0.05), and the pathological damage to neurons in hippocampal CA1 region was significantly attenuated in EP and LYEP groups. Compared with EP group, the p-Akt/Akt ratio in the hippocampal tissue was significantly decreased, and the expression of Bax and cleaved caspase-3 was up-regulated, the number of training sessions required for learning was increased, the correct response rate was decreased ( P<0.05), and the pathological damage to neurons in hippocampal CA1 region was aggravated in LYEP group. Conclusions:Esketamine can alleviate long-term cognitive impairment caused by propofol anesthesia in the developing rats, and the mechanism may be related to activation of the PI3K/Akt signaling pathway and inhibition of apoptosis in neurons.