An easy and cost-effective method for the isolation and culturing of neural stem/progenitor cells from the subventricular (SVZ) and dentate gyrus (DG) of adult mouse brain.

BACKGROUND: Isolation of adult Neural Stem/Progenitor Cells (NSPCs) from their neurogenic niches, is a prerequisite for studies involving culturing of NSPCs as neurospheres or attached monolayers in vitro. The currently available protocols involve the use of multiple animals and expensive reagents to establish the NSPCs culture. NEW METHOD: This unit describes a method to isolate and culture NSPCs from the two neurogenic niches in the mouse brain, the Subventricular Zone (SVZ) and Dentate gyrus (DG)/subgranular zone (SGZ), in an easy and cost-effective manner. RESULTS: NSPCs from SVZ and DG regions of adult mouse brains were isolated and cultured up to passage 15 without losing their stem/progenitor characteristics. These NSPCs could be differentiated into neurons, astrocytes, and oligodendrocytes, revealing its trilineage potential. COMPARISON WITH EXISTING METHODS: This protocol eliminates the need for multiple animals as well as the use of many expensive reagents mentioned in previous protocols, adding to the cost-effectiveness of experiments. In addition, we have effectively reduced the number of steps involved in isolation and propagation, thereby minimizing the chances of contamination. CONCLUSION: Our simplified protocol for the isolation and culturing of adult NSPCs from the SVZ and DG demonstrates a cost-effective and efficient alternative to existing methods, reducing the need for sacrificing many animals and the usage of expensive reagents. This method permits the long-term maintenance of NSPCs' stem/progenitor characteristics and their effective differentiation into the major types of cells in the brain, making it a valuable resource for researchers in the field. BASIC PROTOCOL: Isolation and Culturing of Neural Stem/Progenitor cells from the Sub ventricular Zone and the Dentate Gyrus of the adult mouse brain. SUPPORT PROTOCOL 1: Cryopreservation, and revival of frozen NSPCs. SUPPORT PROTOCOL 2: Preparation of adherent monolayer cultures of neural stem/progenitor cells for the differentiation into multiple lineages SUPPORT PROTOCOL 3: Differentiation of NSPCs to neuronal and glial lineages SUPPORT PROTOCOL 4: Characterization of differentiated cells by immunocytochemistry.

Protection by Nano-Encapsulated Bacoside A and Bacopaside I in Seizure Alleviation and Improvement in Sleep- In Vitro and In Vivo Evidences.

Therapeutic options to contain seizures, a transitional stage of many neuropathologies, are limited due to the blood-brain barrier (BBB). Herbal nanoparticle formulations can be employed to enhance seizure prognosis. Bacoside A (BM3) and bacopaside I (BM4) were isolated from Bacopa monnieri and synthesized as nanoparticles (BM3NP and BM4NP, respectively) for an effective delivery system to alleviate seizures and associated conditions. After physicochemical characterization, cell viability was assessed on mouse neuronal stem cells (mNSC) and neuroblastoma cells (N2a). Thereafter, anti-seizure effects, mitochondrial membrane potential (MMP), apoptosis, immunostaining and epileptic marker mRNA expression were determined in vitro. The seizure-induced changes in the cortical electroencephalogram (EEG), electromyography (EMG), Non-Rapid Eye Movement (NREM) and Rapid Eye Movement (REM) sleep were monitored in vivo in a kainic acid (KA)-induced rat seizure model. The sizes of BM3NPs and BM4NPs were 165.5 nm and 689.6 nm, respectively. They were biocompatible and also aided in neuroplasticity in mNSC. BM3NPs and BM4NPs depicted more than 50% cell viability in N2a cells, with IC50 values of 1609 and 2962 µg/mL, respectively. Similarly, these nanoparticles reduced the cytotoxicity of N2a cells upon KA treatment. Nanoparticles decreased the expression of epileptic markers like fractalkine, HMGB1, FOXO3a and pro-inflammatory cytokines (P < 0.05). They protected neurons from apoptosis and restored MMP. After administration of BM3NPs and BM4NPs, KA-treated rats attained a significant reduction in the epileptic spikes, sleep latency and an increase in NREM sleep duration. Results indicate the potential of BM3NPs and BM4NPs in neutralizing the KA-induced excitotoxic seizures in neurons.

Biocompatible, Europium-Doped Fluorapatite Nanoparticles as a Wide-Range pH Sensor.

The development of a simple, biocompatible, pH sensor with a wide range of detection, using a single fluorescent probe is highly important in the medical field for the early detection of diseases related to the pH change of tissues and body fluids. For this purpose, europium-doped fluorapatite (FAP: Eu) nanoparticles were synthesized using the coprecipitation method. Doping with the rare earth element europium (Eu) makes the non-luminescent phosphate mineral fluorapatite, luminescent. The luminous response of the sample upon dissolution in hydrochloric acid (HCl), in highly acidic to weakly basic media, makes it a potential pH sensor. A linear variation was observed with an increase in pH, in both the total intensity of emission and the R-value or the asymmetry ratio. The ratiometric pH sensing enabled by the variation in R-value makes the sensor independent of external factors. The structural, optical, and photoluminescent (PL) lifetime analysis suggests a particle size-dependent pH sensing mechanism with the changes in the coordinated water molecules around the Eu3+ ion in the nanoparticle. Given its exceptional biocompatibility and pH-dependent fluorescence intensity for a wide range of pH from 0.83 to 8.97, the probe can be used as a potential candidate for pH sensing of biological fluid.

Biological and Sensing Applications of a Few 1,3,4-Oxadiazole Based Donor-Acceptor Systems.

1,3,4-Oxadiazole pharmacophore is still considered a viable biologically active scaffold for the synthesis of more effectual and broad-spectrum antimicrobial agents. Therefore, the present study is based on five 1,3,4-oxadiazole target structures, viz., CAROT, CAROP, CARON (D-A-D-A systems) and NOPON and BOPOB (D-A-D-A-D systems) bearing various bioactive heterocyclic moieties relevant to potential biological activities. Three of the compounds, CARON, NOPON and BOPOB were assessed in-vitro for their efficacy as antimicrobial agents against gram positive (Staphylococcus aureus and Bacillus cereus) and gram negative (Escherichia coli and Klebsiella pneumonia) bacteria; and two fungi, Aspergillus niger and Candida albicans; also, as an anti-tuberculosis agent against Mycobacterium tuberculosis. Most of the tested compounds displayed promising antimicrobial activity, especially CARON which was then analyzed for the minimum inhibitory concentration (MIC) studies. Similarly, NOPON portrayed the highest anti-TB activity among the studied compounds. Consequently, to justify the detected anti-TB activity of these compounds and to recognize the binding mode and important interactions between the compounds and the ligand binding site of the potential target, these compounds were docked into the active binding site of cytochrome P450 CYP121 enzyme of Mycobacterium tuberculosis, 3G5H. The docking results were in good agreement with the result of in-vitro studies. In addition, all the five compounds were tested for their cell viability and have been investigated for cell labeling applications. To conclude, one of the target compounds, CAROT was used for the selective recognition of cyanide ion by 'turn-off' fluorescent sensing technique. The entire sensing activity was examined by spectrofluorometric method and MALDI spectral studies. The limit of detection obtained was 0.14 µM.

Structural and optical properties of dysprosium-doped hydroxyapatite nanoparticles and the use as a bioimaging probe in human cells.

In this work, the hydroxyapatite nanoparticles doped with trivalent dysprosium ions were synthesized by a co-precipitation method. The characterization techniques such as X-ray diffraction (XRD), transmission electron microscopy (TEM), energy dispersive X-ray spectroscopy (EDX) were carried out to determine the crystalline and structural properties. The Rietveld structural refinement of the XRD patterns confirmed the purity of the phase formation of the synthesized nanoparticles. The photoluminescence emission spectra exhibited intense emissions in the blue region at 450 nm and 476 nm along with less intense yellow emission at 573 nm which can be attributed to the magnetic dipole and electric dipole transitions of dysprosium respectively. In order to evaluate the colour tunability of the emitted light CIE chromaticity coordinate values were calculated. The intense blue emissions from the synthesized sample were found to be favourable for bioimaging. The images obtained from the fluorescence microscopy revealed that the dysprosium-doped hydroxyapatite nanoparticles are potential bioimaging probes in human cells.

Eggshell Derived Europium Doped Hydroxyapatite Nanoparticles for Cell Imaging Application.

Hen's eggshell, a biological waste product, was turned into a cell imaging probe: europium doped hydroxyapatite (HAp: Eu) nanoparticle using hydrothermal method. Luminescence of the synthesized nanoparticle was studied for various doping concentrations of the lanthanide ion europium (Eu3+). Eu doped HAp showed a hexagonal crystal structure and rod-shaped morphology. Well-defined emission peaks of europium, corresponding to the substitution of Eu3+ at the Ca2+(I) site of HAp, were confirmed from the samples' photoluminescence (PL) spectra. Good biocompatibility up to 500 µg/mL of the samples indicates their potential applications in bioimaging. Synthesized nanoparticles were internalized and used for in vitro imaging of the PC12 cells without any surface modification. The materials' use as a potential in vivo imaging agent is proposed from the haemolysis study.

HSP72 protects cells from ER stress-induced apoptosis via enhancement of IRE1alpha-XBP1 signaling through a physical interaction.

Endoplasmic reticulum (ER) stress is a feature of secretory cells and of many diseases including cancer, neurodegeneration, and diabetes. Adaptation to ER stress depends on the activation of a signal transduction pathway known as the unfolded protein response (UPR). Enhanced expression of Hsp72 has been shown to reduce tissue injury in response to stress stimuli and improve cell survival in experimental models of stroke, sepsis, renal failure, and myocardial ischemia. Hsp72 inhibits several features of the intrinsic apoptotic pathway. However, the molecular mechanisms by which Hsp72 expression inhibits ER stress-induced apoptosis are not clearly understood. Here we show that Hsp72 enhances cell survival under ER stress conditions. The UPR signals through the sensor IRE1alpha, which controls the splicing of the mRNA encoding the transcription factor XBP1. We show that Hsp72 enhances XBP1 mRNA splicing and expression of its target genes, associated with attenuated apoptosis under ER stress conditions. Inhibition of XBP1 mRNA splicing either by dominant negative IRE1alpha or by knocking down XBP1 specifically abrogated the inhibition of ER stress-induced apoptosis by Hsp72. Regulation of the UPR was associated with the formation of a stable protein complex between Hsp72 and the cytosolic domain of IRE1alpha. Finally, Hsp72 enhanced the RNase activity of recombinant IRE1alpha in vitro, suggesting a direct regulation. Our data show that binding of Hsp72 to IRE1alpha enhances IRE1alpha/XBP1 signaling at the ER and inhibits ER stress-induced apoptosis. These results provide a physical connection between cytosolic chaperones and the ER stress response.

Sensitization of taxol-induced apoptosis by curcumin involves down-regulation of nuclear factor-kappaB and the serine/threonine kinase Akt and is independent of tubulin polymerization.

Taxol is the best anticancer agent that has ever been isolated from plants, but its major disadvantage is its dose-limiting toxicity. In this study, we report with mechanism-based evidence that curcumin, a nontoxic food additive commonly used by the Indian population, sensitizes tumor cells more efficiently to the therapeutic effect of Taxol. A combination of 5 nm Taxol with 5 microm curcumin augments anticancer effects more efficiently than Taxol alone as evidenced by increased cytotoxicity and reduced DNA synthesis in HeLa cells. Furthermore, our results reveal that this combination at the cellular level augments activation of caspases and cytochrome c release. This synergistic effect was not observed in normal cervical cells, 293 cells (in which Taxol down-regulates nuclear factor-kappaB (NF-kappaB)), or HeLa cells transfected with inhibitor kappaBalpha double mutant (IkappaBalpha DM), although the transfection itself sensitized the cells to Taxol-induced cytotoxicity. Evaluation of signaling pathways common to Taxol and curcumin reveals that this synergism was in part related to down-regulation of NF-kappaB and serine/threonine kinase Akt pathways by curcumin. An electrophoretic mobility shift assay revealed that activation of NF-kappaB induced by Taxol is down-regulated by curcumin. We also noted that curcumin-down-regulated Taxol induced phosphorylation of the serine/threonine kinase Akt, a survival signal which in many instances is regulated by NF-kappaB. Interestingly, tubulin polymerization and cyclin-dependent kinase Cdc2 activation induced by Taxol was not affected by curcumin. Altogether, our observations indicate that Taxol in combination with curcumin may provide a superior therapeutic index and advantage in the clinic for the treatment of refractory tumors.