Regulation of Keap1-Nrf2 axis in temporal lobe epilepsy-hippocampal sclerosis patients may limit the seizure outcomes.

BACKGROUND: Accumulation of reactive oxygen species (ROS) exacerbates neuronal loss during seizure-induced excitotoxicity. Keap1 (Kelch-like ECH-associated protein1)-nuclear factor erythroid 2-related factor 2 (Nrf2) axis is one of the known active antioxidant response mechanisms. Our study focused on finding the factors influencing Keap1-Nrf2 axis regulation in temporal lobe epilepsy (TLE) associated with hippocampal sclerosis (HS) patients. METHODS: Based on post-surgical follow-up data, patient samples (n = 26) were categorized into class 1 (completely seizure-free) and class 2 (only focal-aware seizures/auras), as suggested by International League Against Epilepsy (ILAE). For molecular analyses, double immunofluorescence assay and Western blot analysis were employed. RESULTS: A significant decrease in expression of Nrf2 (p < 0.005), HO-1; p < 0.02) and NADPH Quinone oxidoreductase1 (NQO1; p < 0.02) was observed in ILAE class 2. Keap1 (p < 0.02) and histone methyltransferases (HMTs) like SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) (p < 0.009) and enhancer of zeste homolog 2 (EZH2; p < 0.02) and methylated histones viz., H3K4me1 (p < 0.001), H3K9me3 (p < 0.001), and H3K27me3 (p < 0.001) was upregulated in ILAE class 2. Nrf2-interacting proteins viz., p21 (p < 0.001) and heat shock protein 90 (HSP90; p < 0.03) increased in class 1 compared to class 2 patients. CONCLUSION: Upregulation of HMTs and methylated histones can limit phase II antioxidant enzyme expression. Also, HSP90 and p21 that interfere with Keap1-Nrf2 interaction could contribute to a marginal increase in HO-1 and NQO1 expression despite histone methylation and Keap1. Based on our findings, we conclude that TLE-HS patients prone to seizure recurrence were found to have dysfunctional antioxidant response, in part, owing to Keap1-Nrf2 axis. The significance of Keap1-Nrf2 signaling mechanism in generation of phase II antioxidant response. Keap1-Nrf2 controls antioxidant response through regulation of phase II antioxidant enzymes like HO-1 (heme oxygenase-1), NQO1 (NADPH-Quinone Oxidoreductase1), and glutathione S-transferase (GST). Release of Nrf2 from negative regulation by Keap1 causes its translocation into nucleus, forming a complex with cAMP response-element binding protein (CBP) and small Maf proteins (sMaf). This complex subsequently binds antioxidant response element (ARE) and elicits and antioxidant response involving expression of phase II antioxidant enzymes. Reactive oxygen species (ROS) modify Cysteine 151 residue, p62 (sequsetosome-1), and interacts with Nrf2- binding site in Keap 1. p21 and HSP90 prevent Nrf2 interaction with Keap1. At transcriptional level, histone methyltransferases like EZH2 (enhancer of zeste homologue2), and SetD7 (SET7/9; SET domain-containing 7 histone lysine methyltransferase) and corresponding histone targets viz., H3K27me3, H3K9me3, and H3K4me1 influence Nrf2 and Keap1 expression respectively.

Surgically Induced Demyelination in Rat Sciatic Nerve.

Demyelination is a common sign of peripheral nerve injuries (PNIs) caused by damage to the myelin sheath surrounding axons in the sciatic nerve. There are not many methods to induce demyelination in the peripheral nervous system (PNS) using animal models. This study describes a surgical approach using a single partial sciatic nerve suture to induce demyelination in young male Sprague Dawley (SD) rats. After the post-sciatic nerve injury (p-SNI) to the sciatic nerve, histology and immunostaining show demyelination or myelin loss in early to severe phases with no self-recovery. The rotarod test confirms the loss of motor function in nerve-damaged rats. Transmission electron microscopic (TEM) imaging of nerve-damaged rats reveals axonal atrophy and inter-axonal gaps. Further, administration of Teriflunomide (TF) to p-SNI rats resulted in the restoration of motor function, repair of axonal atrophies with inter-axonal spaces, and myelin secretion or remyelination. Taken together, our findings demonstrate a surgical procedure that can induce demyelination in the rat sciatic nerve, which is then remyelinated after TF treatment.

PIMT regulates hepatic gluconeogenesis in mice.

The physiological and metabolic functions of PIMT/TGS1, a third-generation transcriptional apparatus protein, in glucose homeostasis sustenance are unclear. Here, we observed that the expression of PIMT was upregulated in the livers of short-term fasted and obese mice. Lentiviruses expressing Tgs1-specific shRNA or cDNA were injected into wild-type mice. Gene expression, hepatic glucose output, glucose tolerance, and insulin sensitivity were evaluated in mice and primary hepatocytes. Genetic modulation of PIMT exerted a direct positive impact on the gluconeogenic gene expression program and hepatic glucose output. Molecular studies utilizing cultured cells, in vivo models, genetic manipulation, and PKA pharmacological inhibition establish that PKA regulates PIMT at post-transcriptional/translational and post-translational levels. PKA enhanced 3'UTR-mediated translation of TGS1 mRNA and phosphorylated PIMT at Ser656, increasing Ep300-mediated gluconeogenic transcriptional activity. The PKA-PIMT-Ep300 signaling module and associated PIMT regulation may serve as a key driver of gluconeogenesis, positioning PIMT as a critical hepatic glucose sensor.

New class of fused [3,2-b][1,2,4]triazolothiazoles for targeting glioma in vitro.

Glioma is aggressive malignant tumor with limited therapeutic interventions. Herein we report the synthesis of fused bicyclic 1,2,4-triazolothiazoles by a one-pot multi-component approach and their activity against C6 rat and LN18 human glioma cell lines. The target compounds 2-(6-phenylthiazolo[3,2-b][1,2,4]triazol-2-yl) isoindoline-1,3-diones and (E)-1-phenyl-N-(6-phenylthiazolo[3,2-b][1,2,4]triazol-2-yl) methanimines were obtained by the reaction of 5-amino-4H-1,2,4-triazole-3-thiol with substituted phenacyl bromide, phthalic anhydride, and different aromatic aldehydes in EtOH/HCl under reflux conditions. In C6 rat glioma cell lines, compounds 4g and 6i showed good cytotoxic activity with IC50 values of 8.09 and 8.74 µM, respectively, resulting in G1 and G2-M phase arrest of the cell cycle and activation of apoptosis by modulating phosphorylation of ERK and AKT pathway.

Docosahexaenoic acid is potent against the growth of mature stages of Plasmodium falciparum; inhibition of hematin polymerization a possible target.

The present study investigates the potential effect of externally added unsaturated fatty acids on P. falciparum growth. Our results indicate that polyunsaturated fatty acids (PUFAs) inhibit the growth of Plasmodium in proportional to their degree of unsaturation. At higher concentration the PUFA Docosahexaenoic acid (DHA) induces pyknotic nuclei in infected erythrocytes. When Plasmodium stages were treated transiently with DHA, the ring stage culture recovered from the drug effect and parasitemia was increased post DHA removal with delayed growth of 12 h, compared to untreated control. Schizont stage treated culture displayed a 36 h delay in growth to infect fresh erythrocytes signifying its recovery is less than the ring stage. However the trophozoite stage failed to recover and showed a decrease in parasitemia, similar to that of continuous treated culture. PUFAs inhibited ß- hematin polymerization by binding to free heme derived from hemoglobin degradation. Digestive vacuole neutral lipid bodies, which are pivotal for ß- hematin polymerization, decreased and subsequently abrogated with increasing concentration of DHA in trophozoite stage treated culture. Our study concludes that DHA interacts with heme monomers and inhibits the ß- hematin polymerization and growth of mature stages i.e., trophozoite and schizont stages of plasmodium.

NADPH Oxidase: a Possible Therapeutic Target for Cognitive Impairment in Experimental Cerebral Malaria.

Long-term cognitive impairment associated with seizure-induced hippocampal damage is the key feature of cerebral malaria (CM) pathogenesis. One-fourth of child survivors of CM suffer from long-lasting neurological deficits and behavioral anomalies. However, mechanisms on hippocampal dysfunction are unclear. In this study, we elucidated whether gp91phox isoform of nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) (a potent marker of oxidative stress) mediates hippocampal neuronal abnormalities and cognitive dysfunction in experimental CM (ECM). Mice symptomatic to CM were rescue treated with artemether monotherapy (ARM) and in combination with apocynin (ARM + APO) adjunctive based on scores of Rapid Murine Come behavior Scale (RMCBS). After a 30-day survivability period, we performed Barnes maze, T-maze, and novel object recognition cognitive tests to evaluate working and reference memory in all the experimental groups except CM. Sensorimotor tests were conducted in all the cohorts to assess motor coordination. We performed Golgi-Cox staining to illustrate cornu ammonis-1 (CA1) pyramidal neuronal morphology and study overall hippocampal neuronal density changes. Further, expression of NOX2, NeuN (neuronal marker) in hippocampal CA1 and dentate gyrus was determined using double immunofluorescence experiments in all the experimental groups. Mice administered with ARM monotherapy and APO adjunctive treatment exhibited similar survivability. The latter showed better locomotor and cognitive functions, reduced ROS levels, and hippocampal NOX2 immunoreactivity in ECM. Our results show a substantial increase in hippocampal NeuN immunoreactivity and dendritic arborization in ARM + APO cohorts compared to ARM-treated brain samples. Overall, our study suggests that overexpression of NOX2 could result in loss of hippocampal neuronal density and dendritic spines of CA1 neurons affecting the spatial working and reference memory during ECM. Notably, ARM + APO adjunctive therapy reversed the altered neuronal morphology and oxidative damage in hippocampal neurons restoring long-term cognitive functions after CM.

The Tumor Suppressor MTUS1/ATIP1 Modulates Tumor Promotion in Glioma: Association with Epigenetics and DNA Repair.

Glioblastoma (GBM) is a highly aggressive brain tumor. Resistance mechanisms in GBM present an array of challenges to understand its biology and to develop novel therapeutic strategies. We investigated the role of a TSG, MTUS1/ATIP1 in glioma. Glioma specimen, cells and low passage GBM sphere cultures (GSC) were analyzed for MTUS1/ATIP1 expression at the RNA and protein level. Methylation analyses were done by bisulfite sequencing (BSS). The consequence of chemotherapy and irradiation on ATIP1 expression and the influence of different cellular ATIP1 levels on survival was examined in vitro and in vivo. MTUS1/ATIP1 was downregulated in high-grade glioma (HGG), GSC and GBM cells and hypermethylation at the ATIP1 promoter region seems to be at least partially responsible for this downregulation. ATIP1 overexpression significantly reduced glioma progression by mitigating cell motility, proliferation and facilitate cell death. In glioma-bearing mice, elevated MTUS1/ATIP1 expression prolonged their survival. Chemotherapy, as well as irradiation, recovered ATIP1 expression both in vitro and in vivo. Surprisingly, ATIP1 overexpression increased irradiation-induced DNA-damage repair, resulting in radio-resistance. Our findings indicate that MTUS1/ATIP1 serves as TSG-regulating gliomagenesis, progression and therapy resistance. In HGG, higher MTUS1/ATIP1 expression might interfere with tumor irradiation therapy.

Aberrant Dopamine Receptor Signaling Plays Critical Role in the Impairment of Striatal Neurons in Experimental Cerebral Malaria.

One-fourth survivors of cerebral malaria (CM) retain long-term cognitive and behavioral deficits. Structural abnormalities in striatum are reported in 80% of children with CM. Dopamine receptors (D1 and D2) are widely expressed in striatal medium spiny neurons (MSNs) that regulate critical physiological functions related to behavior and cognition. Dysregulation of dopamine receptors alters the expression of downstream proteins such as dopamine- and cAMP-regulated phosphoprotein (DARPP), Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα), and p25/cyclin-dependent kinase 5 (cdk5). However, the role of dopamine receptor signaling dysfunction on the outcome of striatal neuron degeneration is unknown underlying the pathophysiology of CM. Using experimental CM (ECM), the present study attempted to understand the role of aberrant dopamine receptor signaling and its possible relation in causing MSNs morphological impairment. The effect of antimalarial drug artemether (ARM) rescue therapy was also assessed after ECM on the outcome of dopamine receptors downstream signaling. ECM was induced in C57BL/6 mice (male and female) infecting with Plasmodium berghei ANKA (PbA) parasite that reiterates the clinical setting of CM. We demonstrated that ECM caused a significant increase in the expression of D1, D2 receptors, phosphorylated DARPP, p25, cdk5, CaMKIIα, and D1-D2 heteromers. A substantial increase in neuronal damage observed in the dorsolateral striatum region of ECM brains (particularly in MSNs) as revealed by increased Fluoro-Jade C staining, reduced dendritic spine density, and impaired dendritic arborization with varicosities. While the ARM rescue therapy significantly altered the effects of ECM induced dopamine receptor signaling dysfunction and neurodegeneration. Overall, our data suggest that dysregulation of dopamine receptor signaling plays an important role in the degeneration of MSNs, and the ARM rescue therapy might provide better insights to develop effective therapeutic strategies for CM.

Molecular targets in cerebral malaria for developing novel therapeutic strategies.

Cerebral malaria (CM) is the severe neurological complication associated with Plasmodium falciparum infection. In clinical settings CM is predominantly characterized by fever, epileptic seizures, and asexual forms of parasite on blood smears, coma and even death. Cognitive impairment in the children and adults even after survival is one of the striking consequences of CM. Poor diagnosis often leads to inappropriate malaria therapy which in turn progress into a severe form of disease. Activation of multiple cell death pathways such as Inflammation, oxidative stress, apoptosis and disruption of blood brain barrier (BBB) plays critical role in the pathogenesis of CM and secondary brain damage. Thus, understanding such mechanisms of neuronal cell death might help to identify potential molecular targets for CM. Mitigation strategies for mortality rate and long-term cognitive deficits caused by existing anti-malarial drugs still remains a valid research question to ask. In this review, we discuss in detail about critical neuronal cell death mechanisms and the overall significance of adjunctive therapy with recent trends, which provides better insight towards establishing newer therapeutic strategies for CM.

Synthesis of thiazolyl hydrazonothiazolamines and 1,3,4-thiadiazinyl hydrazonothiazolamines as a class of antimalarial agents.

Novel thiazolyl hydrazonothiazolamines and 1,3,4-thiadiazinyl hydrazonothiazolamines were synthesized by a facile one-pot multicomponent approach by the reaction of 2-amino-4-methyl-5-acetylthiazole, thiosemicarbazide or thiocarbohydrazide and phenacyl bromides or 3-(2-bromoacetyl)-2H-chromen-2-ones in acetic acid with good to excellent yields. These new compounds were screened in vitro for their antimalarial activity; among them, four compounds, 4h, 4i, 4k, 4l, showed moderate activity with half-maximal inhibitory concentration (IC50 ) values of 3.2, 2.7, 2.7, and 2.8 and 3.2, 3.2, 3.1, and 3.5 µM against chloroquine-sensitive and -resistant strains of Plasmodium falciparum, respectively. Compound 4l inhibited the ring stage growth of P. falciparum 3D7 at an IC90 concentration of 12.5 µM in a stage-specific assay method, where the culture is incubated with specific stages of P. falciparum for 12 hr, and no activity was found against the trophozoite and schizont stages, confirming that 4l may have potent action against the ring stage of P. falciparum.

Statins exacerbate glucose intolerance and hyperglycemia in a high sucrose fed rodent model.

Statins are first-line therapy drugs for cholesterol lowering. While they are highly effective at lowering cholesterol, they have propensity to induce hyperglycemia in patients. Only limited studies have been reported which studied the impact of statins on (a) whether they can worsen glucose tolerance in a high sucrose fed animal model and (b) if so, what could be the molecular mechanism. We designed studies using high sucrose fed animals to explore the above questions. The high sucrose fed animals were treated with atorvastatin and simvastatin, the two most prescribed statins. We examined the effects of statins on hyperglycemia, glucose tolerance, fatty acid accumulation and insulin signaling. We found that chronic treatment with atorvastatin made the animals hyperglycemic and glucose intolerant in comparison with diet alone. Treatment with both statins lead to fatty acid accumulation and inhibition of insulin signaling in the muscle tissue at multiple points in the pathway.

Prognostic Significance of Anatomic Origin and Evaluation of Survival Statistics of Astrocytoma Patients-a Tertiary Experience.

Astrocytoma constitutes the most noted malignancies of the central nervous system with worse clinical outcomes in grade IV astrocytoma or glioblastoma multiforme. Owing to poor clinical outcomes with existing therapeutic regime, there is a need to revisit the initial course of treatment. Statistical information of clinicopathological parameters could be used to understand the spread of disease and, in turn, to formulate updated treatment management. In the present study, we have seen anatomic distribution of astrocytoma subtypes in a group of 479 patients and correlated it with survival outcomes. Anatomic location was confirmed by MRI (magnetic resonance imaging) images. A registry of patients was maintained with clinicopathological details as tumor type, location, age/sex, and survival after surgery. We have observed overall survival particulars in patients diagnosed with astrocytoma. Our findings highlight that in total cases, tumor location was anatomically dominated by frontal and temporal lobes. Survival analysis in high-grade (grade III, p = 0.03; grade IV, p = 0.01) astrocytic tumors confirms poor outcomes with temporal, parietal, and occipital location as compared to frontal lobe. Overall survival study demonstrates glioblastoma multiforme (GBM) was associated with worse prognosis as compared to astrocytoma subtypes (p < 0.0001). In high-grade astrocytomas, anaplastic astrocytoma was found with 34 months of median survival age while 14 months in the case of patients with glioblastoma multiforme. In conclusion, we report dismal prognosis in parietal, temporal, and occipital lobes in grade II, grade III, and grade IV astrocytoma patients. Among astrocytoma subtypes, patients with glioblastoma multiforme were associated with worse survival outcomes. We uniquely feature the survival of astrocytoma patients for the first time and observe GBM patients have slightly longer survival.

Roscovitine effectively enhances antitumor activity of temozolomide in vitro and in vivo mediated by increased autophagy and Caspase-3 dependent apoptosis.

Gliomas are incurable solid tumors with extremely high relapse rate and definite mortality. As gliomas readily acquire resistance to only approved drug, temozolomide (TMZ), there is increasing need to overcome drug resistance by novel therapeutics or by repurposing the existing therapy. In the current study, we investigated antitumor efficacy of roscovitine, a Cdk inhibitor, in combination with TMZ in vitro (U87, U373, LN 18 and C6 cell lines) and in vivo (orthotopic glioma model in Wistar rats) glioma models. We observed that TMZ treatment following a pre-treatment with RSV significantly enhanced chemo-sensitivity and suppressed the growth of glioma cells by reducing Cdk-5 activity and simultaneous induction of autophagy and Caspase-3 mediated apoptosis. Additionally, reduced expression of Ki67, GFAP and markers of angiogenesis (CD31, VEGF) was observed in case of TMZ + RSV treatments. Also, presence of reactive astrocytes in peri-tumoral areas and in areas around blood vessels was completely diminished in TMZ + RSV treated brain sections. Taken together, results in the current study provide evidence that RSV in conjunction with TMZ restricts glioma growth, reduces angiogenesis and also eliminates reactive astrocytes thereby preventing the spread of glioma to adjacent healthy brain tissues and thus might be more potent therapeutic option for glioma.

Expression and clinicopathological significance of Nck1 in human astrocytoma progression.

OBJECTIVES: Astrocytoma represents most noted malignancy of the brain. The overall survival rate of patients with progressive form remains dismal despite of the present clinical advancements. Search for biomarkers can open new avenues of therapeutic measures to curb the progressive astrocytic tumors. Nck1 is reported to be involved in actin cytoskeleton rearrangement and neuronal migration. Here, we have determined prognostic importance of Nck1 protein in astrocytoma progression. Temporal lobe epilepsy tissues were used as control. METHODS: Real time PCR was used to analyze Nck1 transcript expression while western blotting and immunohistochemistry techniques were used to study expression on translational levels. Protein expression in western blots was categorized as Nck1 positive and Nck1 negative. We further seen the prognostic significance of Nck1 in 246 glioblastoma tissue samples as visible from the TCGA database. RESULTS: We find Nck1 RNA and protein was upregulated significantly in high grade tissues as compared to low grade and control tissue samples (p < 0.05). Logrank test and Kaplan-Meier analysis signified the use of Nck1 as independent prognostic marker for astrocytoma progression and its expression levels were correlated with poor survival in surgically resected human tissue samples (Chi square = 10.7, p = 0.001). Further, glioblastoma was noticed to be predominant at frontal and temporal lobe. CONCLUSION: On account of it's over expression, Nck1 appears as possible biomarker for astrocytoma progression and may serve as an important therapeutic target. Prominent origin of glioblastoma at frontal and temporal lobe suggests possible involvement of tissue specific developmental or transcriptional factors in origin of tumors.

Profiling of microRNAs modulating cytomegalovirus infection in astrocytoma patients.

Astrocytoma is recognized as the most common neoplasm of the brain with aggressive progression. The therapeutic regime for glioblastoma, the most aggressive astrocytoma, often consists of aggressive chemo and radiotherapy. The present holistic approaches, however, have failed to influence the quality life of patients. Therefore, it is necessary to understand the underlying mechanisms of its progression for updated therapeutic evaluation. Human cytomegalovirus (HCMV) is reported to be associated with glioblastoma progression. The hypothesis still remains controversial due to the lack of concrete evidences. Here, we report the profile of miRNAs encoded by human host and the cytomegalovirus (CMV) involved in modulation of CMV infection in surgically resected human astrocytoma tissue samples of various malignancy grades (n = 24). Total RNA from the control brain and tumor tissues was extracted by TriZol reagent. The expression levels of the mature form of miRNA were detected by real-time PCR. Primarily, we found the upregulation of miR-210-3p, miR-155-5p, miR-UL-112-3p, miR-183-5p, and miR-223-5p in high-grade astrocytic tumors as compared with low-grade tumor tissues. miR-214-3p is significantly expressed in control brain tissues and its expression decreased with astrocytoma grade progression. This miRNA was reported to be associated with antiviral proprieties. Among CMV-encoded miRNA, miR-UL-112-3p was significantly upregulated in glioblastoma tissue samples and may be involved in providing immune escape to the virus as well as involved in modulating the immune microenvironment of glioblastoma. Taken together, we conclude the possible involvement of miRNAs in modulating the CMV dependent astrocytoma progression.

Synthesis of pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazoles and their evaluation as potential anticancer agents.

A series of hybrid aza heterocycles containing pyrazolo[3,4-d]pyrimidin-4(5H)-ones tethered to 1,2,3-triazole scaffold were synthesized from 1,3-dipolar cycloaddition reaction of pyrazolopyrimidinone based alkyne with azides using Cu(II) catalyst in presence of sodium ascorbate and evaluated for their anticancer efficacy in vitro against C6 rat and U87 human glioma cell lines. These compounds induced a concentration dependent inhibition of C6 rat and U87 human glioma cell proliferation. Compound 5f arrested the cells at S-phase of the cell cycle and induced apoptosis in U87 GBM cell lines. Further, apoptosis was evidenced by the cleavage of Caspase-3, PARP and up regulation of p53. In silico docking studies reveal that the compounds 5a, 5f and 5l were more effective in binding with TGFBR2 than other compounds.

2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid (Activator-3) is a potent activator of AMPK.

AMPK is considered as a potential high value target for metabolic disorders. Here, we present the molecular modeling, in vitro and in vivo characterization of Activator-3, 2-[2-(4-(trifluoromethyl)phenylamino)thiazol-4-yl]acetic acid, an AMP mimetic and a potent pan-AMPK activator. Activator-3 and AMP likely share common activation mode for AMPK activation. Activator-3 enhanced AMPK phosphorylation by upstream kinase LKB1 and protected AMPK complex against dephosphorylation by PP2C. Molecular modeling analyses followed by in vitro mutant AMPK enzyme assays demonstrate that Activator-3 interacts with R70 and R152 of the CBS1 domain on AMPK γ subunit near AMP binding site. Activator-3 and C2, a recently described AMPK mimetic, bind differently in the γ subunit of AMPK. Activator-3 unlike C2 does not show cooperativity of AMPK activity in the presence of physiological concentration of ATP (2 mM). Activator-3 displays good pharmacokinetic profile in rat blood plasma with minimal brain penetration property. Oral treatment of High Sucrose Diet (HSD) fed diabetic rats with 10 mg/kg dose of Activator-3 once in a day for 30 days significantly enhanced glucose utilization, improved lipid profiles and reduced body weight, demonstrating that Activator-3 is a potent AMPK activator that can alleviate the negative metabolic impact of high sucrose diet in rat model.

ERK1/2 activated PHLPP1 induces skeletal muscle ER stress through the inhibition of a novel substrate AMPK.

Nutritional abundance associated with chronic inflammation and dyslipidemia impairs the functioning of endoplasmic reticulum (ER) thereby hampering cellular responses to insulin. PHLPP1 was identified as a phosphatase which inactivates Akt, the master regulator of insulin mediated glucose homeostasis. Given the suggestive role of PHLPP1 phosphatase in terminating insulin signalling pathways, deeper insights into its functional role in inducing insulin resistance are warranted. Here, we show that PHLPP1 expression is enhanced in skeletal muscle of insulin resistant rodents which also displayed ER stress, an important mediator of insulin resistance. Using cultured cells and PHLPP1 knockdown mice, we demonstrate that PHLPP1 facilitates the development of ER stress. Importantly, shRNA mediated ablation of PHLPP1 significantly improved glucose clearance from systemic circulation with enhanced expression of glucose transporter 4 (GLUT-4) in skeletal muscle. Mechanistically, we show that endogenous PHLPP1 but not PP2Cα interacts with and directly dephosphorylates AMPK Thr172 in myoblasts without influencing its upstream kinase, LKB1. While the association between endogenous PHLPP1 and AMPK was enhanced in ER stressed cultured cells and soleus muscle of high fat diet fed mice, the basal interaction between PP2Ac and AMPK was minimally altered. Further, we show that PHLPP1α is phosphorylated by ERK1/2 at Ser932 under ER stress which is required for its ability to interact with and dephosphorylate AMPK and thereby induce ER stress. Taken together, our data position PHLPP1 as a key regulator of ER stress.

Ameliorating the in vivo antimalarial efficacy of artemether using nanostructured lipid carriers.

Cerebral malaria (CM) is a fatal neurological complication of Plasmodium falciparum infection that affects children (below five years old) in sub-Saharan Africa and adults in South-East Asia each year having the fatality rate of 10-25%. The survivors of CM also have high risk of long term neurological or cognitive deficits. The objective of the present investigation was to develop optimised nanostructured lipid carriers (NLCs) of artemether (ARM) for enhanced anti-malarial efficacy of ARM. NLCs of ARM were prepared by a combination of high speed homogenisation (HSH) and probe sonication techniques. Preliminary solubility studies for ARM showed highest solubility in trimyristin (solid lipid), capmul MCM NF (liquid lipid) and polysorbate 80 (surfactant). Trimyristin and capmul showed superior miscibility at a ratio of 70:30.The optimised NLC formulation has the particle size (PS) of: 48.59 ± 3.67 nm, zeta potential (ZP) of: -32 ± 1.63 mV and entrapment efficiency (EE) of: 91 ± 3.62%. In vitro cell line (human embryonic kidney fibroblast cell line (HEK 293 T)) cytotoxicity studies showed that prepared formulation was non-toxic. The results of in vivo studies in CM induced mice prevented the recrudescence of parasite after administration of NLCs of ARM. Additionally, NLCs of ARM showed better parasite clearance, higher survival (60%) in comparison to ARM solution (40%). Also it was observed that lesser entrapment of Evans blue stain (prepared in PBS as solution) in the NLCs of ARM treated brains of C57BL/6 mice than ARM solution treated mice. Hence NLCs of ARM may be a better alternative for improving therapeutic efficacy than ARM solution.