Harnessing the Benefits of Neuroinflammation: Generation of Macrophages/Microglia with Prominent Remyelinating Properties.

Excessive inflammation within the CNS is injurious, but an immune response is also required for regeneration. Macrophages and microglia adopt different properties depending on their microenvironment, and exposure to IL4 and IL13 has been used to elicit repair. Unexpectedly, while LPS-exposed macrophages and microglia killed neural cells in culture, the addition of LPS to IL4/IL13-treated macrophages and microglia profoundly elevated IL10, repair metabolites, heparin binding epidermal growth factor trophic factor, antioxidants, and matrix-remodeling proteases. In C57BL/6 female mice, the generation of M(LPS/IL4/IL13) macrophages required TLR4 and MyD88 signaling, downstream activation of phosphatidylinositol-3 kinase/mTOR and MAP kinases, and convergence on phospho-CREB, STAT6, and NFE2. Following mouse spinal cord demyelination, local LPS/IL4/IL13 deposition markedly increased lesional phagocytic macrophages/microglia, lactate and heparin binding epidermal growth factor, matrix remodeling, oligodendrogenesis, and remyelination. Our data show that a prominent reparative state of macrophages/microglia is generated by the unexpected integration of pro- and anti-inflammatory activation cues. The results have translational potential, as the LPS/IL4/IL13 mixture could be locally applied to a focal CNS injury to enhance neural regeneration and recovery.SIGNIFICANCE STATEMENT The combination of LPS and regulatory IL4 and IL13 signaling in macrophages and microglia produces a previously unknown and particularly reparative phenotype devoid of pro-inflammatory neurotoxic features. The local administration of LPS/IL4/IL13 into spinal cord lesion elicits profound oligodendrogenesis and remyelination. The careful use of LPS and IL4/IL13 mixture could harness the known benefits of neuroinflammation to enable repair in neurologic insults.

Overexpression of WssgtL3.1 gene from Withania somnifera confers salt stress tolerance in Arabidopsis.

KEY MESSAGE: Overexpression of Withania somnifera SGT gene (WssgtL3.1) in transgenic Arabidopsis improves various agronomic and physiological traits and alters conjugated sterol levels to mitigate the effect of salt stress. Sterols are essential constituents of cell membranes that are involved in several biological functions, including response to various biotic and abiotic stresses by altering membrane permeability and signaling pathways. Sterol glycosyltransferases (SGTs) are enzymes that are involved in sterol modification by converting sterols into sterol-conjugates to play essential roles in adaptive responses. However, their roles under abiotic stresses are lesser-known. Among abiotic stresses, salinity imposes serious threat to crop yield worldwide, hence the present study intends to investigate the role of WssgtL3.1-overexpressed Arabidopsis plants under salt stress indicating the crosstalk between SGT gene and salinity to develop improved crop varieties with better stress tolerance ability. The findings revealed that overexpression of WssgtL3.1 gene in A. thaliana improved the resistance against salt stress in the overexpressing lines. Transgenic lines showed significantly higher germination rate, increased plant growth with less chlorophyll damage compared to wild-type (WT) control plants. Moreover, better tolerance also correlated with enhanced osmolytes (proline and soluble sugar), better membrane integrity, decreased H2O2 production and lesser MDA accumulation and Na+/K+ ratio with more negative osmotic potential in overexpressed lines. Additionally, in sterol profiling, significant enhancement in stigmasterol was also observed in transgenic lines than WT plants. Furthermore, in expression profiling, salt responsive genes LEA 4-5, sucrose synthase, and transporter of monosaccharide (ERD) significantly upregulated in overexpressing lines as compared to WT. Thus our data strongly support the defensive role of Withania somnifera SGT gene (WssgtL3.1) against salt stress and contribute to improved salinity tolerance in plants through sterol modulation.

A Review on Osteoarthritis and its Eradication Approaches: An Update.

Osteoarthritis (OA) is a disease characterized by degeneration of cartilage or wear and tear. OA is a cause of disability and health issues. It is a disease that affects more than 500 million adults annually worldwide, of which India accounts for about 22 to 39% of OA patients. The most common type of osteoarthritis is knee OA. Pathogenesis of OA requires evolution in basic science and clinical research to enhance our understanding of the pathogenesis and as well as different treatment options. It is mainly classified as primary and secondary OA. The treatment for OA can only reduce the symptoms and cannot cure the disease itself, including pharmacological treatment, like non-steroidal anti-inflammatory drugs (NSAIDs), acting on COX1 (cyclooxygenase 1) and COX2 (cyclooxygenase 2) enzymes. Non-pharmacological treatments for OA include exercise like walking, and aerobic exercise, diet, weight loss, hot and cold therapy, as well as electrotherapy, which improves muscle strength and decreases joint pain. Surgical treatment is the last treatment option for OA patients, which includes arthroscopy and joint replacement therapy. Thus, necessary precautions should be taken for joints to be healthy and disease-free.

Advancements in Nanotechnology for Enhanced Antifungal Drug Delivery: A Comprehensive Review.

Infections caused by fungi can be mildly bothersome or fatal, causing life-threatening conditions or even death. Antifungal drugs have used synthetic chemicals, organic compounds, and phytoconstituents in their formulations to treat fungal infections. Research into novel antifungal drugs has progressed more rapidly than into antibacterial treatments. This can be attributed to the low resistance of fungal infections to antifungal bioactivities and the relatively low incidence of these diseases. Carrier systems based on nanotechnology have generated much interest recently because of the incredible potential of these systems. By using nanoarchitecture as a better carrier and drug delivery system (DDS), we can have greater antifungal effectiveness, bioavailability, targeted action, and less cytotoxicity, a development made possible using nanotechnology. This review discusses various nanocarrier-based technologies in addition to other nanotechnological methods. These include liposomes, transfersomes, ethosomes, niosomes, dendrimers, polymeric nanoparticles, polymer nanocomposites, metallic nanoparticles, carbon nanomaterials, etc. This review focused on general information regarding fungi infections, different antifungal agent types and mechanisms of action, and an overview of formulation strategies such as nanotechnology systems, which are frequently researched for antifungal therapies. We concluded that new drug delivery systems are crucial to delivering antifungal medicines to their target site with the optimum concentration. The researchers also concentrated on these innovative drug delivery systems, which primarily focus on regulating and maintaining the release of antifungal drugs.

Comparative genomic analysis and phylogeny of NAC25 gene from cultivated and wild Coffea species.

Coffee is a high value agricultural commodity grown in about 80 countries. Sustainable coffee cultivation is hampered by multiple biotic and abiotic stress conditions predominantly driven by climate change. The NAC proteins are plants specific transcription factors associated with various physiological functions in plants which include cell division, secondary wall formation, formation of shoot apical meristem, leaf senescence, flowering embryo and seed development. Besides, they are also involved in biotic and abiotic stress regulation. Due to their ubiquitous influence, studies on NAC transcription factors have gained momentum in different crop plant species. In the present study, NAC25 like transcription factor was isolated and characterized from two cultivated coffee species, Coffea arabica and Coffea canephora and five Indian wild coffee species for the first time. The full-length NAC25 gene varied from 2,456 bp in Coffea jenkinsii to 2,493 bp in C. arabica. In all the seven coffee species, sequencing of the NAC25 gene revealed 3 exons and 2 introns. The NAC25 gene is characterized by a highly conserved 377 bp NAM domain (N-terminus) and a highly variable C terminus region. The sequence analysis revealed an average of one SNP per every 40.92 bp in the coding region and 37.7 bp in the intronic region. Further, the non-synonymous SNPs are 8-11 fold higher compared to synonymous SNPs in the non-coding and coding region of the NAC25 gene, respectively. The expression of NAC25 gene was studied in six different tissue types in C. canephora and higher expression levels were observed in leaf and flower tissues. Further, the relative expression of NAC25 in comparison with the GAPDH gene revealed four folds and eight folds increase in expression levels in green fruit and ripen fruit, respectively. The evolutionary relationship revealed the independent evolution of the NAC25 gene in coffee.

A Distinct Hibiscus sabdariffa Extract Prevents Iron Neurotoxicity, a Driver of Multiple Sclerosis Pathology.

Iron deposition in the brain begins early in multiple sclerosis (MS) and continues unabated. Ferrous iron is toxic to neurons, yet the therapies used in MS do not counter iron neurotoxicity. Extracts of Hibiscus sabdariffa (HS) are used in many cultures for medicinal purposes. We collected a distinct HS extract and found that it abolished the killing of neurons by iron in culture; medications used in MS were ineffective when similarly tested. Neuroprotection by HS was not due to iron chelation or anthocyanin content. In free radical scavenging assays, HS was equipotent to alpha lipoic acid, an anti-oxidant being tested in MS. However, alpha lipoic acid was only modestly protective against iron-mediated killing. Moreover, a subfraction of HS without radical scavenging activity negated iron toxicity, whereas a commercial hibiscus preparation with anti-oxidant activity could not. The idea that HS might have altered properties within neurons to confer neuroprotection is supported by its amelioration of toxicity caused by other toxins: beta-amyloid, rotenone and staurosporine. Finally, in a mouse model of MS, HS reduced disability scores and ameliorated the loss of axons in the spinal cord. HS holds therapeutic potential to counter iron neurotoxicity, an unmet need that drives the progression of disability in MS.

Papaya Leaf Curl Virus (PaLCuV) Infection on Papaya (Carica papaya L.) Plants Alters Anatomical and Physiological Properties and Reduces Bioactive Components.

Papaya leaves are used frequently for curing scores of ailments. The medicinal properties of papaya leaves are due to presence of certain bioactive/pharmacological compounds. However, the papaya leaf curl virus (PaLCuV), a geminivirus, is a major threat to papaya cultivation globally. During the present investigation, we observed that PaLCuV infection significantly altered the anatomy, physiology, and bioactive properties of papaya leaves. As compared to healthy leaves, the PaLCuV-infected leaves were found to have reduced stomatal density (76.83%), stomatal conductance (78.34%), photosynthesis rate (74.87%), water use efficiency (82.51%), chlorophyll (72.88%), carotenoid (46.63%), osmolality (48.55%), and soluble sugars (70.37%). We also found lower enzymatic activity (superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT)-56.88%, 85.27%, and 74.49%, respectively). It was found that the size of guard cells (50%), transpiration rate (45.05%), intercellular CO2 concentration (47.81%), anthocyanin (27.47%), proline content (74.17%), malondialdehyde (MDA) (106.65%), and electrolyte leakage (75.38%) was elevated in PaLCuV-infected leaves. The chlorophyll fluorescence analysis showed that the infected plant leaves had a significantly lower value of maximal quantum yield of photosystem II (PSII (Fv/Fm), photochemical quantum yield of photosystem I (PSI (Y(I)), and effective quantum yield of PSII (Y(II)). However, in non-photochemical quenching mechanisms, the proportion of energy dissipated in heat form (Y(NPQ)) was found to be significantly higher. We also tested the bioactivity of infected and healthy papaya leaf extracts on a Caenorhabditis elegans (C. elegans) model system. It was found that the crude extract of papaya leaves significantly enhanced the life span of C. elegans (29.7%) in comparison to virus-infected leaves (18.4%) on application of 100 µg/mL dose of the crude extract. Our research indicates that the PaLCuV-infected leaves not only had anatomical and physiological losses, but that pharmacological potential was also significantly decreased.

Genetic molecular analysis of Coffea arabica (Rubiaceae) hybrids using SRAP markers.

In Coffea arabica (arabica coffee), the phenotypic as well as genetic variability has been found low because of the narrow genetic basis and self fertile nature of the species. Because of high similarity in phenotypic appearance among the majority of arabica collections, selection of parental lines for inter-varietals hybridization and identification of resultant hybrids at an early stage of plant growth is difficult. DNA markers are known to be reliable in identifying closely related cultivars and hybrids. Sequence Related Amplified Polymorphism (SRAP) is a new molecular marker technology developed based on PCR. In this paper, sixty arabica-hybrid progenies belonging to six crosses were analyzed using 31 highly polymorphic SRAP markers. The analysis revealed seven types of SRAP marker profiles which are useful in discriminating the parents and hybrids. The number of bands amplified per primer pair ranges from 6.13 to 8.58 with average number of seven bands. Among six hybrid combinations, percentage of bands shared between hybrids and their parents ranged from 66.29% to 85.71% with polymorphic bands varied from 27.64% to 60.0%. Percentage of hybrid specific fragments obtained in various hybrid combinations ranged from 0.71% to 10.86% and ascribed to the consequence of meiotic recombination. Based on the similarity index calculation, it was observed that F1 hybrids share maximum number of bands with the female parent compared to male parent. The results obtained in the present study revealed the effectiveness of SRAP technique in cultivar identification and hybrid analysis in this coffee species.

Self-Assemble Amphiphilic PEO-PPO-PEO Tri-Block Co-Polymeric Methotrexate Nanomicelles to Combat MCF7 Cancer Cells.

BACKGROUND: Methotrexate (MTX) is a water-insoluble, anti-tumor agent that causes adverse effects like bone marrow suppression, chronic interstitial obstructive pulmonary disease, hepatotoxicity, leukopenia, interstitial pneumonitis and nephrotoxicity with slow drug release rate. OBJECTIVE: The present study aimed to successfully incorporate MTX into novel-targeted Pluronic (PEOPPO- PEO tri-block co-polymer) F127 polymeric micelles intended for intravenous administration with improved drug loading and sustained release behavior necessary to achieve better efficacy of MTX. METHODS: MTX-loaded Pluronic F127 micelles were characterized for critical micelle concentration, particle size and zeta potential,1H NMR, drug loading, encapsulation efficiency characterization, cell uptake, in vitro release study along with partition coefficient and solubilization thermodynamics. RESULTS: The micellar formulation resulted in nano size 27.32±1.43nm of PF127/SDS, as compared to Pluronic F127 micelles or PF127/Phosphatidyl choline which were 30.52±1.18nm and 154.35±5.5nm in size, respectively. The uptake of PF127/SDS micellar formulation incorporating Rhodamine 123 in MCF7 cancer cells was found to be higher (84.25%) than PF127/PC, PF127 and MTX i.e. 66.26%, 73.59% and 53% respectively. The in vitro MTX release from PF127, PF127/SDS and PF127/PC polymeric micelles formulations was observed to be 69%, 69.5% and 66% at 12 h whereas 80.89%, 77.67% and 78.54% after 24 h, respectively and revealed a sustained release. MTX-loaded PF127/SDS micelles showed high partition coefficient and negative free energy of solubilization compared to PF127 and PF127/PC which signify self-assembly behavior and thermodynamic stability towards higher dissociation. CONCLUSION: It was finally concluded that MTX-loaded PF127/SDS micelles act as a potential anticancer delivery system in comparison to PF127/PC and PF127 to combat tumor cells by enhancing their cellular uptake targeting with sustained release pattern and reducing the thermodynamic instability. Thus, PF127/SDS micellar formulation can provide a useful alternative dosage form for intravenous administration of MTX.

Guar Gum-Based Floating Microspheres of Repaglinide Using 32 Factorial Design: Fabrication, Optimization, Characterization, and In Vivo Buoyancy Behavior in Albino Rats.

In this present study, floating microspheres of repaglinide were successfully fabricated by the solvent evaporation technique with varying ratios of guar gum, hydroxypropyl methylcellulose, and ethylcellulose with polyvinyl alcohol. Microspheres were characterized by production yield, particle size, in vitro buoyancy, entrapment efficiency, in vitro drug release, and in vivo floating behavior in albino rats. The formulation process was optimized for stirring speed (X1) and concentration of polymer ratio (X2) on dependent variables such as percentage entrapment efficiency, percentage yield, in vitro buoyancy, and percentage of drug release by the 32 factorial Design-Expert® 12, trial version, software. The optimized formulation was characterized by Fourier transform infrared spectroscopy, powder X-ray diffraction, differential scanning calorimetry, and scanning electron microscopy and was successfully formulated with the highest percentage of cumulative drug release (94.26 ± 3.10), entrapment efficiency (74.70% ± 2.16%), and particle size (50.34 ± 3.67 µm) and remains buoyant for 24 h in simulated gastric fluid (0.1N HCL) with high in vitro buoyancy percent (84.90 ± 2.88). When the drug-polymer solution of dichloromethane and ethanol is dropped in polyvinyl alcohol solution, it leads to the formation of a shell and produces cavities, creating the buoyant nature of floating microspheres. X-ray imaging indicates the uniform distribution and buoyant nature of microspheres in the gastric fluid for a 10-h period.

Effects of IL-18 and IL-10 pre-treatment on the alteration of endogenous cytokines in liver and spleen of mice with experimental endotoxemia.

Mechanisms of interleukin-18 (IL-18) and interleukin-10 (IL-10) in lipopolysaccharide (LPS) induced endotoxemia are not clear; their protective role is being investigated so that they may effectively modulate the host cytokine levels during endotoxemia. The aim of the study was to evaluate protective effects of IL-18 and IL-10 in experimentally induced endotoxemia in mice correlating the changes in tissue anti-oxidant enzymes and circulating cytokines. Liver injury was determined by estimation of serum glutamate oxalate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT), serum nitric oxide (NOx), hepatic anti-oxidant enzyme and cytokine content in LPS (250 microg/kg) induced endotoxemic mice receiving either IL-18 (500 ng/mouse) or IL-10 (600 ng/mouse) treatment. Mice (87% of IL-10 treated and 74% of IL-18 treated) survived when administered prior to LPS challenge. Pre-treatment of mice with either IL-10 or IL-18 followed by LPS, lead to reduction in SGPT and SGOT level, serum NOx, and altered hepatic anti-oxidant enzymes activity and myeloperoxidase activity than the only LPS treated group. Marked reduction in the amounts of LPS-induced hepatic and splenic TNF-u content has been observed after IL-10 pre-treatment. Results suggested that attenuating the induction of TNF-alpha and IFN-gamma and subsequent induction of nitric oxide formation in response to LPS may in part account for efficient protection by IL-18 and IL-10 in the reduction of LPS-induced liver injury.

Tobacco carcinogen induces microglial activation and subsequent neuronal damage.

4-Methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) is a tobacco-specific procarcinogen. We have investigated whether NNK causes inflammatory upheaval in the brain by activation of resident microglia and astrocyte and result in bystander neuronal damage. We have carried out the work in both in vitro and in vivo models. We have found that treatment with NNK causes significant activation of mouse microglial (BV2) cell line as evident by increase in reactive oxygen species and nitric oxide level. Western blot analysis has showed increase in proinflammatory signaling proteins, proinflammatory effector proteins, and other stress-related proteins. Interestingly, increased levels of proinflammatory cytokines like interleukin (IL)-6, tumor necrosis factor-alpha, monocyte chemoattractant protein 1 (MCP1), and IL-12p70 are also detected. Work from our in vivo studies has demonstrated similar increase in proinflammatory signaling and effector molecules along with the proinflammatory cytokine levels, following NNK treatment. Immunohistochemical staining of the brain sections of NNK-treated mice reveals massive microglial and astrocyte activation along with distinct foci of neuronal damage. Both in vitro and in vivo results provide strong indication that NNK causes significant upheaval of the inflammatory condition of brain and inflicts subsequent neuronal damage.

Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells.

BACKGROUND: Chemokines are involved in the homing of various cancer cells, including those of ovarian cancer (OvCa), to distant organs. They may also promote or inhibit cancer progression and metastasis. Hypoxia, a common phenomenon in malignant tumors, promotes cell proliferation regulated by HIF-1α. Hypoxia-induced genes are involved in metastasis-associated functions and in the epithelial-to-mesenchymal transition (EMT). RESULTS: Tissue microarrays of human OvCa showed elevated expression of CX3CR1 and HIF-1α compared to normal cells, and their levels were higher in adenocarcinoma stages II and III. To substantiate these observations, we performed studies using OvCa cells. Following exposure to hypoxia, OVCAR-3, SW 626, and TOV-112D cells showed high expression of CX3CR1, a transmembrane protein involved in the adhesion and migration of leukocytes, causing an increased chemotactic response to CX3CL1, the ligand for CX3CR1. As determined by flow cytometry, immunofluorescence, RT-PCR, and western blots, there were higher expressions of CX3CR1 and HIF-1α in OvCa cell lines exposed to hypoxia. Further, OvCa cells expressing CX3CR1 were sensitive to the CX3CL1 ligand. Chemotaxis based on chemokine receptors was influential in elevating the expression of EMT markers and matrix metalloproteinases, which are involved in the progression and metastasis of cancer cells. CONCLUSIONS: In OvCa cells, CX3CR1 was upregulated in a process involving hypoxia-mediated regulation of HIF-1α. The elevated levels of CX3CR1, which were sensitive to CX3CL1, increased EMT markers that led to the progression and metastasis of OvCa. Thus, CX3CR1 and HIF-1α are suitable targets for treatment of OvCa.

Minocycline neuroprotects, reduces microglial activation, inhibits caspase 3 induction, and viral replication following Japanese encephalitis.

Minocycline is broadly protective in neurological disease models featuring inflammation and cell death and is being evaluated in clinical trials. Japanese encephalitis virus (JEV) is one of the most important causes of viral encephalitis worldwide. There is no specific treatment for Japanese encephalitis (JE) and no effective antiviral drugs have been discovered. Studies indicate that JE involves profound neuronal loss as well as secondary inflammation caused because of cell death. Minocycline is a semisynthetic second-generation tetracycline that exerts anti-inflammatory and antiapoptotic effects that are completely separate from its antimicrobial action. Because tetracycline treatment is clinically well tolerated, we investigated whether minocycline protects against experimental model of JE. Intravenous inoculation of GP78 strain of JEV in adult mice results in lethal encephalitis and caused primarily because of neuronal death and secondary inflammation caused because of cell death. Minocycline confers complete protection in mice following JEV infection (p < 0.0001). Neuronal apoptosis, microglial activation, active caspase activity, proinflammatory mediators, and viral titer were markedly decreased in minocycline-treated JEV infected mice on ninth day post-infection. Treatment with minocycline may act directly on brain cells, because neuronal cell line Neuro2a were also salvaged from JEV-induced death. Our data suggest that minocycline may be a candidate to consider in human clinical trials for JE patients.

Novel strategy for treatment of Japanese encephalitis using arctigenin, a plant lignan.

UNLABELLED: OBJECTIVES; To evaluate therapeutic efficacy of arctigenin in an experimental model of Japanese encephalitis (JE). METHODS: Four- to 5-week-old BALB/c mice of either sex were infected intravenously with lethal dose of 3 x 10(5) pfu of Japanese encephalitis virus (JEV). By the 9th day post-infection, all untreated animals succumbed to the infection. Arctigenin was dissolved in DMSO at a concentration of 0.5 mg/mL and stored at 4 degrees C. After one day following virus inoculation, animals were given arctigenin intraperitoneally, twice daily (10 mg/kg of body weight) for next 7 days. RESULTS: Treatment with arctigenin provided complete protection against experimental JE. Arctigenin's neuroprotective effect was associated with marked decreases in: (i) viral load; (ii) active caspase-3 activity; (iii) reactive oxygen species and reactive nitrogen species; (iv) microgliosis and proinflammatory cytokines; (v) levels of stress-associated signalling molecules; and (vi) neuronal death. Furthermore, treatment with arctigenin also improves the behavioural outcome following JE. CONCLUSIONS: In conclusion, our findings provide a novel mechanistic insight into the actions of arctigenin in JE. Results from our in vivo and in vitro experiments clearly indicate that arctigenin reduced (i) viral load and viral replication within the brain, (ii) neuronal death and (iii) secondary inflammation and oxidative stress resulting from microglial activation, thereby suggesting its potential for treating JE. The antiviral, neuroprotective, anti-inflammatory and antioxidative effects of arctigenin successfully reduced the severity of disease induced by JEV.

Japanese Encephalitis Virus infection induces IL-18 and IL-1beta in microglia and astrocytes: correlation with in vitro cytokine responsiveness of glial cells and subsequent neuronal death.

IL-1beta and IL-18 are members of the IL-1 family of ligands, and their receptors are members of the IL-1 receptor family. Although several biological properties overlap for these cytokines, differences exist. In order to assess functional importance of these two cytokines in viral encephalitis, we have exploited an experimental model of Japanese Encephalitis (JE) and subsequent in vitro cell culture system. We report for the first time that in Japanese Encephalitis, microglia and astrocytes both produce IL-18 and IL-1beta. In vitro, these two cytokines differentially activate microglia and astrocyte, and also alter the by stander neuronal survival following treatment with these two cytokines.

Japanese encephalitis virus differentially modulates the induction of multiple pro-inflammatory mediators in human astrocytoma and astroglioma cell-lines.

Astrocytes become activated in response to many CNS pathologies. The process of astrocyte activation remains rather enigmatic and results in so-called reactive gliosis, a reaction with specific structural and functional characteristics. Astrocytes play a vital role in regulating aspects of inflammation and in the homeostatic maintenance of the CNS. However, the responses of different human astroglial cell-lines in viral encephalitis mediated inflammation are not well documented. We have shown that Japanese encephalitis virus (JEV) infection causes morphological and functional changes in astrocytic cell-lines. We have demonstrated that besides reactive oxygen species (ROS) JEV infection differentially regulated the induction pattern of IL-6, IL-1 beta and IL-8. IP-10, MCP-1, MIG and RANTES secretions in different astroglial cell-lines. The expression of different proteins such as astrocyte-specific glial fibrillary acidic protein (GFAP), the glutamate aspartate transporter/essential amino acid transporter-1 (GLAST/EAAT-1), glutamate transporter-1/essential amino acid transporter-2 (GLT-1/EAAT-2), Ceruloplasmin and Thioredoxin (TRX) expression level also differ in different human astrocyte cell-lines following infection.

Kaempferol induces apoptosis in glioblastoma cells through oxidative stress.

Despite recent advances in understanding molecular mechanisms involved in glioblastoma progression, the prognosis of the most malignant brain tumor continues to be dismal. Because the flavonoid kaempferol is known to suppress growth of a number of human malignancies, we investigated the effect of kaempferol on human glioblastoma cells. Kaempferol induced apoptosis in glioma cells by elevating intracellular oxidative stress. Heightened oxidative stress was characterized by an increased generation of reactive oxygen species (ROS) accompanied by a decrease in oxidant-scavenging agents such as superoxide dismutase (SOD-1) and thioredoxin (TRX-1). Knockdown of SOD-1 and TRX-1 expression by small interfering RNA (siRNA) increased ROS generation and sensitivity of glioma cells to kaempferol-induced apoptosis. Signs of apoptosis included decreased expression of Bcl-2 and altered mitochondrial membrane potential with elevated active caspase-3 and cleaved poly(ADP-ribose) polymerase expression. Plasma membrane potential and membrane fluidity were altered in kaempferol-treated cells. Kaempferol suppressed the expression of proinflammatory cytokine interleukin-6 and chemokines interleukin-8, monocyte chemoattractant protein-1, and regulated on activation, normal T-cell expressed and secreted. Kaempferol inhibited glioma cell migration in a ROS-dependent manner. Importantly, kaempferol potentiated the toxic effect of chemotherapeutic agent doxorubicin by amplifying ROS toxicity and decreasing the efflux of doxorubicin. Because the toxic effect of both kaempferol and doxorubicin was amplified when used in combination, this study raises the possibility of combinatorial therapy whose basis constitutes enhancing redox perturbation as a strategy to kill glioma cells.

A rapid and efficient SDS-based RNA isolation protocol from different tissues of coffee.

Isolation of high-quality RNA from coffee is challenging because of high level of polysaccharides, polyphenols and other secondary metabolites. In the present study, a rapid and efficient RNA extraction protocol from different tissues of coffee was optimized. Sufficiently high quality and quantity (225.6-454.8 µg/g) of RNA was obtained by using the optimized protocol. The presence of two distinct bands of 28S rRNA and 18S rRNA in agarose gel proved the intactness of the RNA samples. The average spectrophotometric values of the isolated RNA ranged from 1.96 to 2.02 (A260/280) and 1.95 to 2.14 (A260/230), indicating the high quality of RNA devoid of polyphenols, polysaccharides and protein contamination. In the optimized protocol, addition of PVPP to the extraction buffer and a brief incubation of samples at 65 °C and subsequent purification with potassium acetate resulted in good-quality RNA isolation. The suitability of RNA for downstream processing was confirmed by PCR amplification with cytochrome c oxidase gene-specific primers. The amplification of a single 392 bp fragment using cDNA and 1.5 kb fragment using genomic DNA samples confirmed the absence of DNA contamination. The present protocol is rapid and yielded good quality and quantity of RNA suitable for functional genomics studies.