A synthetic, closed-looped gene circuit for the autonomous regulation of RUNX2 activity during chondrogenesis.

The transcription factor RUNX2 is a key regulator of chondrocyte phenotype during development, making it an ideal target for prevention of undesirable chondrocyte maturation in cartilage tissue-engineering strategies. Here, we engineered an autoregulatory gene circuit (cisCXp-shRunx2) that negatively controls RUNX2 activity in chondrogenic cells via RNA interference initiated by a tunable synthetic Col10a1-like promoter (cisCXp). The cisCXp-shRunx2 gene circuit is designed based on the observation that induced RUNX2 silencing after early chondrogenesis enhances the accumulation of cartilaginous matrix in ATDC5 cells. We show that the cisCXp-shRunx2 initiates RNAi of RUNX2 in maturing chondrocytes in response to the increasing intracellular RUNX2 activity without interfering with early chondrogenesis. The induced loss of RUNX2 activity in turn negatively regulates the gene circuit itself. Moreover, the efficacy of RUNX2 suppression from cisCXp-shRunx2 can be controlled by modifying the sensitivity of cisCXp promoter. Finally, we show the efficacy of inhibiting RUNX2 in preventing matrix loss in human mesenchymal stem cell-derived (hMSC-derived) cartilage under conditions that induce chondrocyte hypertrophic differentiation, including inflammation. Overall, our results demonstrated that the negative modulation of RUNX2 activity with our autoregulatory gene circuit enhanced matrix synthesis and resisted ECM degradation by reprogrammed MSC-derived chondrocytes in response to the microenvironment of the degenerative joint.

In-vitro antitumor activity of compounds from Glycyrrhiza glabra against C6 glioma cancer cells: identification of natural lead for further evaluation.

A HP20 resin-based unique method was adopted to get an active fraction of the hydroalcoholic extract of G. glabra roots. The fraction showed potent cytotoxicity against cancer cell line and was further subjected to detailed phytochemical investigation to obtain ten biomarkers. The isolated compounds were also tested for the cytotoxicity against the C6 glioma cell line in vitro using MTT assay. Among the isolated compounds, glycyrrhetic acid (1), glabrol (6), and glabridin (9) exhibited significant cytotoxicity. The compounds showed a dose-dependent decrease in cell viability. The active compounds were subjected to molecular docking study against topoisomerase I and topoisomerase II to support the mechanism of antitumor activity.

PSA mimetic 5-nonyloxytryptamine protects cerebellar neurons against glutamate induced excitotoxicity: An in vitro perspective.

PSA-NCAM is a molecule of therapeutic interest for its key role in promoting neuritogenesis and synaptic plasticity. The current study was aimed to investigate the neuroregenerative potential of 5-nonyloxytryptamine (5-NOT) as PSA mimetic compound against glutamate induced excitotoxicity. 2D and 3D cultures of cerebellar neurons challenged with glutamate were used to ascertain the effect of 5-NOT on neurite outgrowth, migration and expression of neuronal plasticity markers. Glutamate excitotoxicity is one of the major underlying pathological factor responsible for neurodegeneration in various neurological disorders such as trauma, stroke, ischemia, epilepsy and neurodegenerative diseases.5-NOT treatment was observed to promote axonal growth and defasiculation in glutamate challenged neurons as well as promoted the migration of cerebellar neurons in both wound scratched area and cerebellar explant cultures. Further, 5-NOT treatment upregulated the expression of synaptic plasticity and cell survival pathway proteins which showed reduced expression after glutamate induced excitotoxicity. Thus, this preliminary data reveals thatPSA-mimetic,5-NOT may prove to be a potential neuroprotective candidate for neurodegenerative diseases.

From ayurvedic folk medicine to preclinical neurotherapeutic role of a miraculous herb, Tinospora cordifolia.

In Ayurveda, the age-old Indian traditional system of medicine, health is considered to be achieved as equilibrium of physical and mental wellbeing and brain related ailments are recognized as 'Vatavyadi'. Rasayana herbs were mainly used for pharmacological treatment of neurological diseases and Tinospora cordifolia is one of the popular Rasayana herbs of Ayurveda. The traditional claims of therapeutic activity of this herb for treatment of fever, diabetes, anxiety, immunodeficiency, memory deficit and psychological problems have been explored by different research groups using reverse pharmacology and advance technological approaches. The aim of current review is to compile and discuss the neurotherapeutic potential of T. cordifolia in the light of various preclinical and clinical studies from literature. This review summarizes the information about different extracts of this herb and decoctions used for various neuro-related problems such as neurodegenerative diseases, neuroinflammation, sleep disorders, neural cancers, memory and cognition deficits and psychological problems besides other potential activities. The review also provides the knowledge of underlying therapeutic mechanism of T. cordifolia and its active phytoconstituents.

The neuroplasticity marker PSA-NCAM: Insights into new therapeutic avenues for promoting neuroregeneration.

Neuroplastic alterations are the key processes involved in adaptation and rehabilitation after all neurological injuries and pathologies. Being the central contributor to the developmental and adult neuroplasticity, the polysialylated form of Neural Cell Adhesion Molecule (PSA-NCAM) may prove to be a potential target to facilitate repair/regeneration after CNS injury and disease. Over the years, several experimental approaches have been developed to exploit the therapeutic potential of PSA-NCAM. Broadly, the studies focused on cell-transplantation strategies to alter PSA-NCAM properties at the injury site, injection of peptide based as well as synthetic PSA mimetics directly into the injury site or the application of PSA containing hydrogels and scaffolds as biomaterials. A comprehensive understanding of the PSA-based experimental approaches, as well as their pros and cons, is urgently required for successful implementation of this molecule in therapeutics. The current review, therefore, has been designed to give the readers a thorough account of all the diverse roles of PSA in the adult nervous system and the recent progress that has been made in developing PSA-based therapeutic approaches for neuroregeneration.

5-Nonyloxytryptamine oxalate-embedded collagen-laminin scaffolds augment functional recovery after spinal cord injury in mice.

Polysialic acid (PSA) is crucial for the induction and maintenance of nervous system plasticity and repair after injury. In order to exploit the immense therapeutic potential of PSA, previous studies have focused on the identification and development of peptide-based or synthetic PSA mimetics. 5-Nonyloxytryptamine (5-NOT) has been previously reported as a PSA-mimicking compound for promoting functional recovery after spinal cord injury in mice. In order to explore the neuroregeneration potential of 5-NOT, the current study was based on a biomaterial approach using collagen-laminin (C/L) scaffolds. In in vitro studies, 5-NOT was observed to promote neurite outgrowth, migration, and fasciculation in cerebellar neuronal cells, whereas in 3D cell cultures it showed more ramification and complex Sholl profiles. 5-NOT promoted the survival and neurite length of cortical neurons when cocultured with glutamate-challenged astrocytes. In in vivo studies, spinal cord compression injury mice were used with immediate application of C/L hydrogels impregnated with 5-NOT. C/L + 5-NOT-treated mice demonstrated ∼75% of motor recovery 14 days after injury. Furthermore, this effect was shown to be dependent on the ERK-MAPK pathway and augmentation of cell survival. Thus, based on a biomaterial approach, our current study provides new insight for 5-NOT-containing hydrogels as a promising candidate to speed up recovery after central nervous system injuries.

Structural tuning of acridones for developing anticancer agents targeting dihydrofolate reductase.

Targeting dihydrofolate reductase, here, we report the tumor growth inhibitory activity of substituted acridones. The screening of the molecules over 60 cell line panel of human cancer cells identified (S)-oxiran-2-ylmethyl 9-oxo-9,10-dihydroacridine-4-carboxylate (19) with average GI50 0.3 µM. The specificity of the compound to CCRF-CEM, MOLT-4 and SR cell lines of leukemia and SW-620, SF268, LOXIMVI, ACHN and MCF7 cancerous cells exhibiting GI50 in the nM range was observed. C6 Glioma cells treated with compound 19 showed differentiated cell morphology and cell cycle arrest in G2/M phase. The interactions of the compound with dihydrofolate reductase were ascertained with the help of enzyme immunoassays, molecular docking and molecular dynamic studies.

Anti-brain cancer activity of chloroform and hexane extracts of Tinospora cordifolia Miers: an in vitro perspective.

BACKGROUND: Plants have been suggested as safest source of therapeutic agents, with multi targeted mode of action and least side effects. Tinospora cordifolia, commonly known as Guduchi in India, is one of the most highly valued herbs in Ayurvedic medicine. It possesses potential anti-cancer, anti-inflammatory, hepatoprotective, anti-diabetic, immune-stimulatory and various other beneficial activities. PURPOSE: The present study was aimed to investigate the differentiation inducing potential of chloroform and hexane extracts of T. cordifolia using U87MG glioblastoma and IMR-32 neuroblastoma cell lines as model system. RESULTS: Chloroform (Chl-TCE) and hexane (Hex-TCE) extracts significantly reduced the rate of proliferation and induced cell differentiation as evidenced by MTT assay and immunostaining for GFAP and MAP-2 in glioblastoma and neuroblastoma, respectively. Further these extracts increased the expression of stress markers HSP70 and Mortalin and induced senescence. Chloroform and hexane extracts also inhibited the migration of U87MG glioblastoma and IMR-32 neuroblastoma as indicated by wound scratch assay and supported by reduced expression of NCAM. Furthermore these extracts are not toxic to normal cells as they showed no inhibitory effects on primary astrocytic and neuronal cultures. CONCLUSIONS: The present study suggests that chloroform and hexane extracts of T. cordifolia retard the rate of proliferation, induce differentiation and inhibit migration of human glioblastomas and neuroblastomas, thus may act as potential phytotherapeutic intervention in treatment of neural cancers.

Low-grade neuroinflammation due to chronic sleep deprivation results in anxiety and learning and memory impairments.

Chronic sleep loss/fragmentation prevalent in the current 24/7 society is associated with irreversible consequences on health and overall wellbeing. Various studies have well documented the ill effects of acute sleep loss on cognitive functions of individuals; however, the underlying mechanism behind the chronic sleep loss is yet to be explored. The present study was aimed to investigate whether chronic sleep deprivation (CSD) triggers anxiety-like behaviour and memory decline in male Wistar rats. Rats were sleep deprived by placing them over slowly rotating drum (2 rpm) for 18 h (between 4 pm and 10 am) followed by 6 h of recovery sleep for 21 consecutive days. Post CSD regimen, rats were subjected to behavioural tests such as elevated plus maze (EPM), Novel Object Recognition (NOR) and Rotarod performance test and then sacrificed to remove brain for further molecular studies. The study demonstrated that CSD rats showed anxiogenic behaviour along with recognition memory decline compared to control rats. CSD rats further showed elevated levels of inflammatory cytokines (TNFα, IL-1ß) along with activation of NFκB and AP1 transcription factors in hippocampus and piriform cortex (PC) regions of brain. These observations were also accompanied by enhanced expression of GFAP and Iba1 in the two brain regions. The data suggest that CSD triggered low-grade neuroinflammation which caused anxiogenic response and recognition memory impairment. The study provides preliminary leads to further explore the role of astrocytes/microglial cells and inflammatory cytokines in mediating these neurobehavioural consequences of chronic sleep loss and to develop effective interventions to combat them.

A naphthalimide-based solid state luminescent probe for ratiometric detection of aluminum ions: in vitro and in vivo applications.

A naphthalimide-based fluorescent probe 4 has been designed and synthesized that detects Al3+ ions in solution, solid state and in biological systems, including cells, tissues and nematodes. Further, a portable kit has been developed using probe 4 for the on-site detection of Al3+ ions.

TNF-α and IL-6 inhibitors: Conjugates of N-substituted indole and aminophenylmorpholin-3-one as anti-inflammatory agents.

The conjugates obtained by the combination of indole and aminophenyl morpholinone were screened for TNF-α and IL-6 inhibition in microglial cells. Compound 4 was found to be the most potent anti-inflammatory agent as it reduced LPS induced level of inflammatory cytokines TNF-α and IL-6 by 71% and 53%, respectively. A significant decrease in NO and MMPs release from BV2 cells in culture pretreated with this compound as well as inhibition of nuclear translocation of NF-κB and AP-1 was observed. 75% inhibition of acetic acid induced algesia in swiss albino mice was noticed in the presence of compound 4. Experimental data and molecular docking studies indicate that the compounds are targeting TNF-α, iNOS and IL-6.

A Highly Selective Fluorescent Probe for Detection of Hydrogen Sulfide in Living Systems: In Vitro and in Vivo Applications.

A fluorescein-based fluorescent probe has been designed and synthesised that selectively detects H2 S in aqueous medium, among various analytes tested. This fluorescein-based fluorescent probe has also been successfully utilised for real-time imaging of exo- and endogenously produced H2 S in cancer cells and normal cells. Moreover, the probe can also detect H2 S in the rat brain hippocampus at variable depths and in living nematodes.

The polysialic acid mimetics idarubicin and irinotecan stimulate neuronal survival and neurite outgrowth and signal via protein kinase C.

Polysialic acid (PSA) is a large, negatively charged, linear homopolymer of alpha2-8-linked sialic acid residues. It is generated by two polysialyltransferases and attached to N- and/or O-linked glycans, and its main carrier is the neural cell adhesion molecule (NCAM). PSA controls the development and regeneration of the nervous system by enhancing cell migration, axon pathfinding, synaptic targeting, synaptic plasticity, by regulating the differentiation of progenitor cells and by modulating cell-cell and cell-matrix adhesions. In the adult, PSA plays a role in the immune system, and PSA mimetics promote functional recovery after nervous system injury. In search for novel small molecule mimetics of PSA that are applicable for therapy, we identified idarubicin, an antineoplastic anthracycline, and irinotecan, an antineoplastic agent of the topoisomerase I inhibitor class, as PSA mimetics using a competition enzyme-linked immunosorbent assay. Idarubicin and irinotecan compete with the PSA-mimicking peptide and colominic acid, the bacterial analog of PSA, for binding to the PSA-specific monoclonal antibody 735. Idarubicin and irinotecan stimulate neurite outgrowth and survival of cultured cerebellar neurons after oxidative stress via protein kinase C and Erk1/2 in a similar manner as colominic acid, whereas Fyn, casein kinase II and the phosphatase and tensin homolog are only involved in idarubicin and irinotecan-stimulated neurite outgrowth. These novel results show that the structure and function of PSA can be mimicked by the small organic compounds irinotecan and idarubicin which trigger the same signaling cascades as PSA, thus introducing the possibility of retargeting these drugs to treat nervous system injuries.

A lysosome targetable fluorescent probe for endogenous imaging of hydrogen peroxide in living cells.

A lysosome targetable naphthalimide based fluorescent probe (LyNC) has been designed and synthesized which detects hydrogen peroxide (H2O2) with high selectivity and sensitivity in brain tissues and in living nematodes among various ROS/RNS tested. Further, the probe LyNC was successfully employed in exogenous and endogenous imaging of H2O2 in living cell lines.

Nicotine-based surface active ionic liquids: Synthesis, self-assembly and cytotoxicity studies.

New ester-functionalized surface active ionic liquids (SAILs) based on nicotine, [CnENic][Br] (n=8, 10 and 12), with bromide counterions have been synthesized, characterized and investigated for their self-assembly behavior in aqueous medium. Conductivity measurements in aqueous solutions of the investigated SAILs have provided information about their critical micelle concentration (cmc), and degree of counterion binding (ß), where cmc was found to be 2-3-fold lower than homologous SAILs or conventional cationic surfactants. The inherent fluorescence of SAILs in the absence of any external fluorescent probe have shed light on cmc as well as interactions prevailing between the monomers in micelle at molecular level. The thermodynamic parameters related to micellization have been deduced from isothermal titration calorimetry (ITC) and conductivity measurements. 1H NMR, spin-lattice (T1) relaxation time and 2D 1H-IH ROESY measurements have been exploited to get detailed account of internal structure of micelle. The size and shape of the micelles have been explored using dynamic light scattering (DLS) and transmission electron microscopy (TEM) measurements. The synthesized SAILs have been found to be non-cytotoxic towards C6-Glioma cell line, which adds to the possible utility of these SAILs for diverse biological applications.

Aqueous Leaf Extract of Withania somnifera as a Potential Neuroprotective Agent in Sleep-deprived Rats: a Mechanistic Study.

Modern lifestyle and sustained stress of professional commitments in the current societal set up often disrupts the normal sleep cycle and duration which is known to lead to cognitive impairments. In the present study, we report whether leaf extract of Withania somnifera (Ashwagandha) has potential neuroprotective role in acute stress of sleep deprivation. Experiments were performed on three groups of adult Wistar rats: group 1 (vehicle treated-undisturbed sleep [VUD]), group 2 (vehicle treated-sleep deprived [VSD]), and group 3 (ASH-WEX treated-sleep deprived [WSD]). Groups 1 and 2 received single oral feeding of vehicle and group 3 received ASH-WEX orally (140 mg/kg or 1 ml/250 g of body weight) for 15 consecutive days. Immediately after this regimen, animals from group 1 were allowed undisturbed sleep (between 6 a.m. and 6 p.m.), whereas rats of groups 2 and 3 were deprived of sleep during this period. We observed that WSD rats showed significant improvement in their performance in behavioral tests as compared to VSD group. At the molecular level, VSD rats showed acute change in the expression of proteins involved in synaptic plasticity, cell survival, and apoptosis in the hippocampus region of brain, which was suppressed by ASH-WEX treatment thus indicating decreased cellular stress and apoptosis in WSD group. This data suggest that Ashwagandha may be a potential agent to suppress the acute effects of sleep loss on learning and memory impairments and may emerge as a novel supplement to control SD-induced cognitive impairments.

Efficacy of Anti-Epileptic Drugs in the Treatment of Tumor and Its Associated Epilepsy: An in vitro Perspective.

The change in the therapeutic targets from neuron to glia has proved beneficial in the treatment of many psychiatric disorders. The anti-epileptic drugs (AEDs) have been widely prescribed for the treatment of partial and complete seizures, bipolar disorder among others. The current study was carried out to explore the efficacy of some conventional and novel AEDs for the treatment of tumor-associated epilepsy which develops in 29-49% of the patients diagnosed with brain tumors. We used C6 glioma cell line as model system to study the effect of selected AEDs, viz., gabapentin (GBP), valproic acid (VPA) and topiramate (TPM). Morphometry, cell cycle analysis, apoptosis, expression of different protein markers, viz., GFAP, HSP70 and nuclear factor-κB (NFκB) were studied in AED-treated cultures. The study was further extended to rat hypothalamic primary explant cultures, and cell migration and expression of plasticity markers - neural cell adhesion molecule (NCAM) and polysialylation of NCAM (PSA-NCAM) - were studied in the explants. TPM was observed to show more pronounced increase in apoptosis of glioblastoma cells accompanied by significant downregulation in the expression of HSP70 and NFκB. TPM-treated explants also showed highest process ramification and cellular migration accompanied by intense expression of the plasticity markers as compared to those treated with GBP and VPA. Among the 3 AEDs tested, TPM was observed to show more promising effects on cytoprotection and plasticity of C6 glioma cells.

The polysialic acid mimetics 5-nonyloxytryptamine and vinorelbine facilitate nervous system repair.

Polysialic acid (PSA) is a large negatively charged glycan mainly attached to the neural cell adhesion molecule (NCAM). Several studies have shown that it is important for correct formation of brain circuitries during development and for synaptic plasticity, learning and memory in the adult. PSA also plays a major role in nervous system regeneration following injury. As a next step for clinical translation of PSA based therapeutics, we have previously identified the small organic compounds 5-nonyloxytryptamine and vinorelbine as PSA mimetics. Activity of 5-nonyloxytryptamine and vinorelbine had been confirmed in assays with neural cells from the central and peripheral nervous system in vitro and shown to be independent of their function as serotonin receptor 5-HT1B/1D agonist or cytostatic drug, respectively. As we show here in an in vivo paradigm for spinal cord injury in mice, 5-nonyloxytryptamine and vinorelbine enhance regain of motor functions, axonal regrowth, motor neuron survival and remyelination. These data indicate that 5-nonyloxytryptamine and vinorelbine may be re-tasked from their current usage as a 5-HT1B/1D agonist or cytostatic drug to act as mimetics for PSA to stimulate regeneration after injury in the mammalian nervous system.

Tinospora cordifolia ameliorates anxiety-like behavior and improves cognitive functions in acute sleep deprived rats.

Sleep deprivation (SD) leads to the spectrum of mood disorders like anxiety, cognitive dysfunctions and motor coordination impairment in many individuals. However, there is no effective pharmacological remedy to negate the effects of SD. The current study examined whether 50% ethanolic extract of Tinospora cordifolia (TCE) can attenuate these negative effects of SD. Three groups of adult Wistar female rats - (1) vehicle treated-sleep undisturbed (VUD), (2) vehicle treated-sleep deprived (VSD) and (3) TCE treated-sleep deprived (TSD) animals were tested behaviorally for cognitive functions, anxiety and motor coordination. TSD animals showed improved behavioral response in EPM and NOR tests for anxiety and cognitive functions, respectively as compared to VSD animals. TCE pretreatment modulated the stress induced-expression of plasticity markers PSA-NCAM, NCAM and GAP-43 along with proteins involved in the maintenance of LTP i.e., CamKII-α and calcineurin (CaN) in hippocampus and PC regions of the brain. Interestingly, contrary to VSD animals, TSD animals showed downregulated expression of inflammatory markers such as CD11b/c, MHC-1 and cytokines along with inhibition of apoptotic markers. This data suggests that TCE alone or in combination with other memory enhancing agents may help in managing sleep deprivation associated stress and improving cognitive functions.

A bodipy based fluorescent probe for evaluating and identifying cancer, normal and apoptotic C6 cells on the basis of changes in intracellular viscosity.

The applications of a bodipy based probe 1 for the identification of diseased cell population out of normal cells on the basis of changes in intracellular viscosity have been explored. Probe 1 works on the principle of restriction of rotation in viscous medium and the molecular rotor nature of probe 1 is supported by low temperature 1H NMR and variable dihedral angle DFT and TD-DFT studies. More importantly, probe 1 is the first probe which shows its practical application in monitoring micro-viscosity changes in a cell based model system of undifferentiated, differentiated and apoptotic C6 glial cells. Further, probe 1 can effectively monitor the apoptosis pathway by showing an increase in fluorescence intensity from cancerous cells to apoptotic cells via real time live-cell video imaging. Moreover, the viscosity changes in living cells were proved by fluorescence lifetime imaging (FLIM) studies, flow cytometry using Annexin-V and Bcl-xl expression by immunocytofluorescence (ICC) and western blot analysis.