Hippocampal neurogenesis modulated by Quinic acid: A therapeutic strategy for the neurodegenerative disorders.

Neural progenitor cells (NPCs) reside in the brain and participate in the mechanism of neurogenesis that permits the brain to generate the building blocks for enhancement of cognitive abilities and acquisition of new skills. The existence of NPCs in brain has opened a novel dimension of research to explore their potential for treatment of various neurodegenerative disorders. The present study provides novel insights into the intracellular mechanisms in neuronal cells proliferation, maturation and differentiation regulated by Quinic acid (QA). Furthermore, this study might help in discovery and development of lead molecule that can overcome the challenges in the treatment of neurodegenerative diseases. The growth supporting effect of QA was studied using MTT assay. For that purpose, hippocampal cell cultures of neonatal rats were treated with different concentrations of QA and incubated for 24, 48 and 72 h. Gene and protein expressions of the selected molecular markers nestin, neuron-specific class III beta-tubulin (Tuj-1), neuronal nuclear protein (NeuN), neuronal differentiation 1 (NeuroD1), glial fibrillary acidic protein (GFAP), neuroligin (NLGN) and vimentin were analyzed. QA-induced cell proliferation and differentiation of hippocampal progenitor cells was also accompanied by significantly increased expression of progenitor and immature neuronal marker, mature neuronal marker and differentiating factor, that is, nestin, Tuj-1, NeuN and NeuroD1, respectively. On the other hand, vimentin downregulation and constant GFAP expression were observed following QA treatment. Additionally, the effects of QA on the recovery of stressed cells was studied using in vitro model of oxygen glucose deprivation (OGD). It was observed that hippocampal cells were able to recover from OGD following the treatment with QA. These findings suggest that QA treatment promotes hippocampal neurogenesis by proliferating and differentiating of NPCs and recovers neurons from stress caused by OGD. Thus, the neurogenic potential of QA can be explored for the treatment of neurodegenerative disorders.

Antiseizure Activity of Mitragyna inermis in the Pentylenetetrazol-Induced Seizure Model in Mice: Involvement of Flavonoids and Alkaloids.

Objective: This study aimed to investigate whether Mitragyna inermis (Willd.) Otto Kuntze organic and aqueous extracts are able to control seizures induced by pentylenetetrazol (PTZ) in mice based on flavonoid fingerprints and alkaloidal contents. Materials and Methods: Ethanolic extract and decoction-derived fractions from roots, leaves, and stems were subjected to chromatographic fingerprinting using AlCl3 and screening for their antiseizure effects using PTZ-induced acute seizure model. From the fractions that showed potent bioactivities, plausible antiseizure alkaloids were isolated using thin layer chromatography, and their structures were elucidated using 1H NMR, 2D NMR, 13C NMR, and FAB-HR (+ve or -ve). Results: All fractions, with the exception of the dichloromethane and hexane fractions, revealed remarkable flavonoid fingerprints. An acute PTZ-induced seizure test revealed that ethanolic extract of stem bark [500 mg/kg body weight (bw)], ethyl acetate extract of stem bark (500 mg/kg bw), and aqueous extract of leaves (300 mg/kg bw) significantly delayed the occurrence of hind limb tonic extension (HLTE); however, a non-significant delay was observed in the onset of first myoclonic jerk compared with control animals. Isolation yielded four main alkaloids: that are, pteropodine (1), isopteropodine (2), mitraphylline (3) and corynoxeine (4). Corynoxeine is a new compound derived from M. inermis. Conclusion: This study suggests that flavonoid fingerprints are tracers of M. inermis anticonvulsant ingredients. The stem bark ethanolic and ethyl acetate extracts and leaf aqueous extracts contain anticonvulsant bioactive principles that delay notifying the HLTE occurring in male naval medical research institute mice. Furthermore, alkaloidal contents also remain plausible bioactive anticonvulsant principles. All observations support the traditional use of M. inermis to manage epilepsy. However, further studies are needed to understand the effects of alkaloid fractions, flavonoids, and the isolated compounds as promising antiseizure agents derived from M. inermis in experimental animals.

Urease and human glioblastoma (U87) cells growth inhibitory iridoid glycoside acetates from Nyctanthes arbor-tristis Linn.

The present study was undertaken on the chemical constituents of ethanol extract of aerial parts of Nyctanthes arbor-tristis Linn., and their determination of growth inhibitory activity against glioblastoma multiforme cell line (U87) and urease inhibitory activity. Six constituents were isolated including two new arbortristoside F tetraacetate (1) and arbortristoside G heptaacetate (2) and four known arborside A tetraacetate (3), arborside C pentaacetate (4), 6,7-di-O-acetyl-6ß-hydroxyloganin hexaacetate (5) and nyctanthoside heptaacetate (6) iridoid glycoside acetates. Their structures were elucidated using spectroscopic methods, including 1D and 2D NMR and mass analyses. Compounds 3 and 6 showed significant growth inhibition of U87 cell line (76.41 and 87.62% inhibition) respectively while 2, 4 and 5 showed moderate inhibition. 3 and 6 showed notable urease inhibition (IC50 = 17.2 ± 0.4 and 17.2 ± 0.7 µM) respectively, and IC50 of 2, 4 and 5 were 23.8 - 56.3 µM. Compound 1 was inactive.

Modulation of Apoptotic and Akt/PI3K/mTOR pathways to target Glioblastoma Cells using synthetic compound PGEA-AN.

Objectives: To investigate the anticancer potential of a novel synthetic derivative of a naturally occurring diterpenoid against glioblastoma. METHODS: The in vitro study was conducted at the Ojha Campus of Dow University of Health Sciences, Karachi, from February to December 2021, and comprised U87 cells. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the growth inhibitory effect of 16(R and S) - phenylamino-cleroda3, 13(14) Zdiene- 15, 16 olide and standard drug temozolomide against glioblastoma cells, and half-maximal inhibitory concentration was calculated. Microscopy and immunocytochemistry were used to investigate apoptotic morphology and active caspase-3 and B-cell lymphoma 2 (Bcl-2) expression. Quantitative real time polymerase chain reaction was used to investigate the expression of proliferation markers. Data was analysed using SPSS 21. RESULTS: Both the synthetic derivative and the standard drug significantly inhibited growth of U87 cells (p<0.001) with half-maximal inhibitory concentration of 19µM and 185µM, respectively. Apoptotic morphology and upregulation of active caspase-3 protein expression was observed in cells treated with half-maximal inhibitory concentration doses of both the synthetic derivative (p<0.05) and the standard drug (p<0.001), and Bcl-2 was downregulated in both the synthetic derivative (p<0.01) and the standard drug (p=0.05). However, no significant difference was observed in the expression of proliferation markers (p>0.05). CONCLUSIONS: The synthetic diterpene derivative PGEA-AN showed growth inhibitory actiity against glioblastoma.

Neurogenin-2 induced neuronal differentiation in co-cultured bone marrow stromal and neonatal cortical cells.

Objective: To explore a novel and dynamic role for neurogenin-2 in promoting cortical neurogenesis in cells produced from co-culturing neonatal cortical neural progenitor cells with bone marrow stromal cells. METHODS: The experimental study was conducted from June 2016 to January 2019 at the neuropharmacology laboratory of the Hussein Ebrahim Jamal Research Institute of Chemistry, International Centre for Chemical and Biological Sciences, Karachi. The growth of cells at different stages in harvested cells was determined by 3-(4, 5- dimethyl thiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Immunocytochemistry was used to evaluate the protein expressions of neuronal markers and transcription factors. Data was analysed using SPSS 20. RESULTS: Data showed significant generation of neuronal cells and this was also verified by increased expression of nesting in cortical co-cultures with bone marrow stromal cells. Immunoreactive outcomes showed over expressions in co-cultured chlorotoxin cells. Subsequently, neurogenin-2 was found intermixed with induced expressions of transcriptional factor NeuroD1 and reduced glial fibrillary acidic protein-labelled cells. Conclusion: Better understanding of the mechanisms underlying transcriptional modulation of neurogenic events hold the key for emerging treatment approaches towards neurodegeneration.

Nicotinic acid modulates microglial TREM-2 gene in Phytohaemagglutinin-Induced in vitro model of Alzheimer's disease like pathology.

Alzheimer's disease (AD) is a multifactorial,neurodegenerative disorder linked withextracellular amyloid beta (Aß) plaques deposition and formation of intracellular neurofibrillary tangles (NFTs). Currently, no effective therapies are available to cure AD. Neuroinflammation isa well-known hallmark in the onset and advancement of AD and triggering receptor expressed on myeloid cells-2 (TREM-2), a microglial gene, is responsible for regulating inflammatory responses and clearance of cellular debris. Loss of TREM-2functionincreases neuroinflammation associated expression of pro-inflammatory markersthus resultingin reduced clearance of Aß that further aid in disease progression.Therefore, targeting neuroinflammation is a good therapeutic approach for AD. This study aimed to determine the neuroprotective effect of nicotinic acid (NA) in vitro model of AD-like pathology induced in F-98 cell line using Phytohemagglutinin (PHA). MTT assay was employed for checking the cell viability as well as the proliferation of the cells following treatment with NA. PHA at the concentration of 10 µg/mL produces maximum plaques. The neuroprotective effect of NA was next evaluated against PHA-induced plaques and it was observed that NA reverses the damages induced by PHA i.e., by inhibiting the clustering of the cells and replacing the damaged cells with the new ones. Further, NA also increased the expression of TREM-2/DAP-12 with parallel decreased in the expression of IL-1ß, TNF-α and iNOS. It also successfully altered disease associated ADAM-10 and BACE-1 compared to PHA control. These findings suggest that NA might be considered as a good therapeutic candidate for the treatment of neurodegenerative disorders like AD.

N-(2-hydroxy phenyl) acetamide ameliorate inflammation and doxorubicin-induced nephrotoxicity in rats.

Doxorubicin (DOX) is an anthracyclin antibiotic used for the treatment of various cancers. Nephrotoxicity is among the serious side effects of DOX, therefore, DOX-induced nephrotoxic model has been widely used to study nephropathies. The objectives of this study is to investigate the possible anti-inflammatory and nephroprotective effects of salicylic acid derivative, N-(2-hydroxy phenyl) acetamide (NA-2), in a rat model of DOX-induced nephrotoxicity. The in vitro anti-inflammatory potential of NA-2 was manifested by whole blood oxidative burst and nitric oxide (NO) assays with no toxicity on normal human fibroblast (BJ) cells, human embryonic kidney (HEK-293) cells, and normal monkey kidney epithelial (Vero) cells. The in vivo study included five groups: Normal control, DOX (6 mg/kg DOX-i.v.via tail vein), NA-2 treated control-i.p., NA-2/DOX treated-i.p., and prednisolone/DOX treated. After 7 days of DOX administration, rats with urinary protein level of >50 mg/kg/day were selected. Treatment group rats received i.p. doses of NA-2 (10 mg/kg/day) for 3 weeks with weekly monitoring of urinary protein excretion and body weights. mRNA expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and kidney injury molecule (KIM)-1 was analyzed by quantitative polymerase chain reaction (qPCR). Protein expressions were analyzed by immunohistochemistry. NA-2 attenuated DOX-induced changes in serum and urine levels, and improved inflammatory profile of the renal tissue. Histopathological findings revealed protective effects of NA-2 showing lesser lesions. We conclude that NA-2 is able to protect against DOX-induced renal damage functionally, biochemically and histopathologically with corresponding improvement in the kidney inflammatory profile.

Combination of metformin and sub-therapeutic dose of valproic acid prevent valproic acid-induced toxicity in animal model of epilepsy.

Valproic acid (VPA) is one of the most prescribed drugs for epilepsy. Extended use of VPA not only induces hepatotoxicity but also impairs the cognitive functions. Metformin has been reported to prevent epileptogenesis and enhance memory. To counter the VPA-induced adverse events, it is hypothesized that combination of sub-therapeutic dose of VPA with metformin may attenuate the toxicity stemming from the therapeutic dose of VPA. Pentylenetetrazole (PTZ)-induced kindling model of epilepsy in mice was used to assess the combined effects of sub-therapeutic dose of VPA (100 mg/kg) and metformin (200 mg/kg). The memory performance was analyzed by passive avoidance test, while alkaline comet assay was used to determine genotoxicity. Histopathological examination and serum biochemical analysis was performed to determine hepatotoxicity. Results showed that combination dose of VPA with metformin reduced seizure scores. VPA (300 mg/kg) administered as a single agent did not enhance memory impairment caused by PTZ, however, combination of sub-therapeutic dose of VPA with metformin enhanced memory function. Furthermore, in alkaline comet assay, combination therapy demonstrated reduced genotoxicity compared to the VPA 300 mg/kg. Histopathological examination of liver and analysis of serum hepatic enzymes revealed that combination therapy (VPA + metformin) reversed the toxicity as seen in case of PTZ or VPA (300 mg/kg) treated animals with no other treatment given. Based on the study data, it is concluded that the combination of sub-therapeutic dose of VPA with metformin might be used for epileptic seizures. This will prevent the hepatotoxicity and enhanced memory functions as compared to the VPA given as a single agent therapy.

Alpha terpineol directs bone marrow mesenchymal stem cells toward neuronal lineage through regulation of wnt signaling pathway.

Central nervous system anomalies give rise to neuropathological consequences with immense damage to the neuronal tissues. Cell based therapeutics have the potential to manage several neuropathologies whereby the differentiated cells are explored for neuronal regeneration. The current study analyzes the effect of a bioactive compound, alpha terpineol (AT) on the differentiation of rat bone marrow derived mesenchymal stem cells (BM-MSCs) toward neuronal lineage, and explores regulation of differentiation process through the study of Wnt pathway mediators. BM-MSCs were cultured and characterized based on their surface markers and tri-lineage differentiation. Safe dose of AT as optimized by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium bromide assay, was used for the treatment of MSCs. Treated cells were analyzed for the neuronal, astroglial and germ layer transition markers at the gene and protein levels, by quantitative polymerase chain reaction and immunocytochemistry, respectively. Temporal expression of Wnt pathway genes was assessed during the course of neuronal differentiation. AT treated group showed significant upregulation of neuron specific (NSE, MAP2, Tau, Nestin, and NefL) and astroglial (GFAP) genes with positive expression of late neuronal markers. Germ layer transition analysis showed the overexpression of ectodermal markers (NCAM, Nestin, and Pax6), whereas endodermal (AFP, MixL1, and Sox17), and mesodermal (Mesp1 and T Brachyury) markers were also found to be upregulated. Wnt signaling pathway was activated during the initial phase (30 min) of differentiation, which later was downregulated at 1, 3, and 5 h. AT efficiently induces neuronal differentiation of BM-MSCs by regulating Wnt signaling. Overexpression of both early and late neuronal markers indicate their neuro-progenitor state and thus can be utilized as a promising approach in cellular therapeutics to treat various neurodegenerative ailments. In addition, exploration of the molecular pathways may be helpful to understand the mechanism of cell-based neuronal regeneration.

In-vitro inhibition of NLRP3 inflammasome by 3,6-dihydroxyflavone (3,6-DHF): a therapeutic strategy for the treatment of chronic inflammatory and autoimmune diseases.

Nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 3 (NLRP3) inflammasome complex has an important role in immune system and its abnormal activation is associated with the pathogenesis of various inflammatory and auto-immune diseases. The study reveals the anti-inflammatory effects of 3,6-dihydroxyflavone (3,6-DHF). Here, we aimed to determine the inhibitory effects of 3,6-DHF on NLRP3 inflammasome and its associated components, thereby determining the signaling pathways involved in the inhibition. Reactive oxygen species (ROS) and nitric oxide (NO) were quantified by chemiluminescence and Griess methods, respectively. Inflammatory cell model was induced in human leukemic monocytes (THP-1). mRNA levels were estimated through real-time RT-PCR, protein expressions were evaluated by protein slot blot and immunocytochemistry, MTT and alamar blue assays were employed for toxicity studies. The compound 3,6-DHF was found to be the potent inhibitor of NLRP3 inflammasome by targeting the molecules involve in its activation pathway. Anti-inflammatory effects were revealed by inhibition of ROS and NO, reduction in the transcription of caspase-1, ASC, IL-1ß and TLR-4 was observed along with the marked inhibition of NLRP3, IL-18, NF-κB and pNF-κB at translational level. 3,6-DHF was non-toxic on normal human fibroblast (BJ) and THP-1 cells and, could be a potential therapeutic agent in NLRP3 inflammasome driven diseases.

Levetiracetam ameliorates epileptogenesis by modulating the adenosinergic pathway in a kindling model of epilepsy in mice.

Background: Levetiracetam (LEV) has been found to have an antihyperalgesic effect via acting on the adenosine system. However, the effects of LEV on the modulation of the adenosine system in the brain have not been elucidated in the prevention of seizures and epilepsy. The present study aimed to explore the possible LEV mechanisms of action in the adenosine signaling systems in an animal model of epilepsy. Methodology: A docking study was initially performed to determine the possible interaction of LEV with adenosine A1 receptors (A1Rs) and equilibrative nucleoside transporters-1 (ENT1). The experimental study was divided into an acute seizure test (32 mice distributed into 4 groups) and a chronic kindling model study (40 mice distributed into 5 groups), followed by gene expression analysis and immunohistochemistry. The kindling model lasted 26 days and took 13 subconvulsive doses of pentylenetetrazole (PTZ) to completely kindle the mice in the PTZ control group. Gene expression changes in the A1Rs, potassium inwardly-rectifying channel 3.2 (Kir3.2), and ENT1 in the brain tissue samples of the mice following treatment with LEV were analyzed using reverse transcription-quantitative polymerase chain reaction, and immunohistochemistry was performed for the A1R protein expression. Results: Docking studies predicted a significant interaction of LEV with A1Rs and ENT1 proteins. Results from the acute testing revealed that caffeine (100 mg/kg) and 8-cyclopentyl-1,3-dipropylxanthine (25 mg/kg) significantly reversed the antiseizure effects of LEV by reversing the percent protection and shortening the onset of the first myoclonic jerk (FMJ) and generalized clonic seizures (GCSs). In the PTZ-induced kindling, LEV demonstrated an increased gene expression of A1Rs and Kir3.2 in the brain. LEV also significantly reduced the gene expression of ENT1. Furthermore, the immunohistochemical analysis showed that LEV increased the protein expression of A1Rs in the brain. Conclusion: Based on these results, it can be concluded that LEV modulates epileptogenesis by acting on the adenosine pathway in the central nervous system.

Design, Synthesis and Characterization of [G10a]-Temporin SHa Dendrimers as Dual Inhibitors of Cancer and Pathogenic Microbes.

As the technologies for peptide synthesis and development continue to mature, antimicrobial peptides (AMPs) are being widely studied as significant contributors in medicinal chemistry research. Furthermore, the advancement in the synthesis of dendrimers' design makes dendrimers wonderful nanostructures with distinguishing properties. This study foregrounds a temporin SHa analog, [G10a]-SHa, and its dendrimers as globular macromolecules possessing anticancer and antibacterial activities. These architectures of temporin SHa, named as [G10a]-SHa, its dendrimeric analogs [G10a]2-SHa and [G10a]3-SHa, and [G10a]2-SHa conjugated with a polymer molecule, i.e., Jeff-[G10a]2-SHa, were synthesized, purified on RP-HPLC and UPLC and fully characterized by mass, NMR spectroscopic techniques, circular dichroism, ultraviolet, infrared, dynamic light scattering, and atomic force microscopic studies. In pH- and temperature-dependent studies, all of the peptide dendrimers were found to be stable in the temperature range up to 40-60 °C and pH values in the range of 6-12. Biological-activity studies showed these peptide dendrimers possessed improved antibacterial activity against different strains of both Gram-positive and Gram-negative strains. Together, these dendrimers also possessed potent selective antiproliferative activity against human cancer cells originating from different organs (breast, lung, prostate, pancreas, and liver). The high hemolytic activity of [G10a]2-SHa and [G10a]3-SHa dendrimers, however, limits their use for topical treatment, such as in the case of skin infection. On the contrary, the antibacterial and anticancer activities of Jeff-[G10a]2-SHa, associated with its low hemolytic action, make it potentially suitable for systemic treatment.

Serum Stable and Low Hemolytic Temporin-SHa Peptide Analogs Disrupt Cell Membrane of Methicillin-Resistant Staphylococcus aureus (MRSA).

Anti-microbial peptides (AMPs) have attracted major attention due to their potential bio-activities against some multidrug resistant pathogens. The present study evaluated the mechanism of actions of highly potent AMP temporin-SHa analogs, i.e., [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa, against methicillin-resistant Staphylococcus aureus (MRSA) NCTC (13277) with minimum inhibitory concentrations (MICs) of 14.35, 7.16, and 3.58 µM, respectively. These analogs exhibited significant anti-MRSA activity at physiological salt concentration, 30% fetal bovine serum, and 30% human serum. [G4a]-SHa and [G7a]-SHa were non-hemolytic and non-cytotoxic to normal mouse fibroblast 3T3 cell and human Caco-2 cell line. Atomic force microscopy revealed that these analogs have profound effect on the morphological changes in MRSA surface with significant leakage of cell cytoplasmic content. Propidium iodide uptake kinetic assay and (bis-(1,3-dibutylbarbituric acid) trimethine oxonol) DiBAC4(3) membrane depolarization assay demonstrated that these analogs display a membrane disrupting property, characterized by elevation of plasma membrane permeability and rapid transmembrane potential depolarization. [G10a]-SHa showed a significant anti-biofilm activity against biofilm forming S. aureus (ATCC 6538). Acute in vivo toxicity studies revealed that [G10a]-SHa possesses some toxic effect at 100-mg/kg dose. While [G4a]-SHa at 100 mg/kg, i.p. has no toxic effect even after 48 h, [G7a]-SHa also did not show any toxic effect at the dose of 100 mg/kg, i.p. during 24-h observation of animals. In conclusion, [G4a]-SHa, [G7a]-SHa, and [G10a]-SHa show improved activity against MRSA and stability compared to SHa peptide. Although highly potent, [G10a]-SHa, due to its hemolytic activity, might be more suitable for topical application, whereas [G4a]-SHa and [G7a]-SHa have potential to be used for systemic application.

Cepharanthine action in preventing obesity and hyperlipidemia in rats on a high-fat high sucrose diet.

Background: It was demonstrated that cepharanthine (CEP), derived from Stephania cepharantha hayata, is a potent inhibitor of the ABCC10 transmembrane protein. It is approved to be a natural product or remedy. The present study focuses on investigating whether cepharanthine effectively reduces hyperlipidemia and obesity in an experimental hyperlipidemic rat model. Method: Four groups of Wistar rats were assigned randomly to the following groups: a high-fat high sucrose diet (HFHS), normal-fat diet (NFD), HFHS plus cepraranthine (10 mg/kg) (HFHS-C), and a HFHS diet with atorvastatin (HFHS-A). The responses of rats were observed on the basis of serum and hepatic biochemical parameters, food intake, and body weight after CEP treatment, and assessing the histopathological modifications by the optical microscope in the liver and its cells. Results: Significant improvement in the serum total cholesterol (TC), serum triglycerides (TG), and serum low-density lipoprotein (LDL) levels were observed following CEP treatment. We have also observed significant improvement in the structure of liver tissue and reduced-fat droplets in the cytoplasm. Moreover, CEP had a significant effect in preventing the gain in body weight of animals, and food intake was not significantly affected. Conclusion: Our research results revealed that CEP significantly improved dyslipidemia and prevented the accumulation of fatty deposits in the rats' liver tissue fed an HFHS diet. In addition, CEP exerted an anti-obesity effect.

Opuntiol Inhibits Growth and Induces Apoptosis in Human Glioblastoma Cells by Upregulating Active Caspase 3 Expression.

BACKGROUND: Glioblastoma Multiforme (GBM) is a deadly tumor with poor prognosis. Resistance to apoptosis considered as an important factor in treatment failure. Therefore, identification of new compounds that facilitates apoptosis is crucial. Natural Anti-inflammatory compounds have emerged as potential anti-cancer agents and should be explored for their apoptotic activity against GBM. Therefore, the present study aims to evaluate growth inhibitory and apoptotic activity of a natural anti-inflammatory compound "Opuntiol" against GBM cell line U87. METHODS: MTT assay was performed to determine the effect of Temozolomide and Opuntiol on growth inhibition of U87 cell. While, TUNEL assay was used to assess their apoptotic activity. To further assess apoptosis, nuclear condensation and nuclear area factor (NAF) was evaluated through DAPI staining. Whereas, active caspase-3 protein expression determined using immunocytochemistry. RESULTS: Significant growth inhibition was observed in U87 cells treated with Temozolomide (IC50 380 µM) and Opuntiol (IC50 357 µM). Temozolomide (p<0.001) and Opuntiol (p<0.001) significantly improved rate of apoptosis when compared to control group. A significant decrease in NAF was also observed in Temozolomide (p < 0.05) and Opuntiol (p < 0.05) treated cells. There was a significant increase in active caspase-3 expression when observed in Temozolomide (p<0.001) and Opuntiol (p<0.05) treated groups as compared to control. CONCLUSION: In conclusion our findings suggests, Opuntiol repress cell viability and possess strong apoptotic activity against GBM cell line U-87. However, further mechanistic studies will be required to confirm whether it can be develop as a potential drug against GBM.

Antimicrobial and biofilm inhibiting potential of an amide derivative [N-(2', 4'-dinitrophenyl)-3ß-hydroxyurs-12-en-28-carbonamide] of ursolic acid by modulating membrane potential and quorum sensing against colistin resistant Acinetobacter baumannii.

Acinetobacter baumannii is Gram-negative, an opportunistic pathogen responsible for life-threatening ventilator-associated pneumonia. World Health Organization (WHO) enlisted it as a priority pathogen for which therapeutic options need speculations. Biofilm further benefits this pathogen and aids 100-1000 folds more resistant against antimicrobials and the host immune system. In this study, ursolic acid (1) and its amide derivatives (2-4) explored for their antimicrobial and antibiofilm potential against colistin-resistant A. baumannii (CRAB) reference and clinical strains. Viability, crystal violet, microscopic, and gene expression assays further detailed the active compounds' antimicrobial and biofilm inhibition potential. Compound 4 [N-(2',4'-dinitrophenyl)-3ß-hydroxyurs-12-en-28-carbonamide)], a synthetic amide derivate of ursolic acid significantly inhibits bacterial growth with MIC in the range of 78-156 µg/mL against CRAB isolates. This compound failed to completely kill the CRAB isolates even at 500 µg/mL concentration, suggesting the compound's anti-virulence and bacteriostatic nature. Short and prolonged exposure of 4 inhibited or delayed the bacterial growth at sub MIC, MIC, and 2× MIC, as evident in time-kill and post-antibacterial assay. It significantly inhibited and eradicated >70% of biofilm formation at MIC and sub MIC levels compared to colistin required in high concentrations. Microscopic analysis showed disintegrated biofilm after treatment with the 4 further strengthened its antibiofilm potential. Atomic force microscopy (AFM) hinted the membrane disrupting effect of 4 at MIC's. Further it was confirmed by DiBAC4 using fluorescence-activating cells sorting (FACS), suggesting a depolarized membrane at MIC. Gene expression analysis also supported our data as it showed reduced expression of biofilm-forming (bap) and quorum sensing (abaR) genes after treatment with sub MIC of 4. The results suggest that 4 significantly inhibit bacterial growth and biofilm mode of colistin-resistant A. baumannii. Thus, further studies are required to decipher the complete mechanism of action to develop 4 as a new pharmacophore against A. baumannii.

A Novel Variant in Dopamine Receptor Type 2 Gene is Associated with Schizophrenia.

BACKGROUND: Being the primary target of antipsychotic therapy, dopamine receptor type 2 (DRD2) remains a point of interest in schizophrenia pathology. Polymorphisms in DRD2 have been shown to alter patients' response to antipsychotics. DRD2 SNP rs6275 (C>T) have found to be associated with schizophrenia in different populations; however, data remains inconsistent. AIM OF THE STUDY: Keeping in view the genetic diversity the present study was aimed to explore association of rs6275 with schizophrenia in population from Pakistan. METHOD: Using Diagnostic and statistical Manual 5 (DSM 5) criteria, 100 schizophrenia cases and 100 controls (individuals without any psychiatric illness) were enrolled in the study. Severity of illness was determined using PANSS score. Genotyping was done via Sanger sequencing. MEGA-X was used to align the sequences, Expasy translate tool was used to translate nucleotide sequences. Difference in genotype and allele frequencies between cases and controls was determined using χ2 test. RESULT: No significant difference in genotype or allele frequencies of rs6275 (p >0.0.5) was found between cases and controls. Interestingly, a novel SNP (C>A, Pro297Thr) was spotted during electropherogram analysis at position chr11:113412805. Significant difference was found in genotype and allele frequency of this novel SNP among schizophrenia cases and controls (p = 0.003). CONCLUSION: No association of rs6275 was observed with schizophrenia in Pakistani population. However, the study found significant association of a novel missense SNP of DRD2 at chr11:113412805 (C>T) with schizophrenia in Pakistani population. A large-scale multicenter study will be required to confirm the association of this novel SNP with schizophrenia.

Neuronal transcription program induced in hippocampal cells cocultured with bone marrow derived mesenchymal cells.

Several approaches have been applied to harvest bone marrow stromal cells (BMSCs) and to differentiate into neurons or neuronal-like cells through chemical stimulation or exposing to growth factors. To date, the data regarding induction or regulation of neuronal transcription program in neuronal-like cells derived from BMSCs is yet unknown. The objective of this study is to co-culture BMSCs with neonatal hippocampal cells and generate neuronal-like cells by direct cell-to-cell contact system without using neuronal growth factors or neurobasal medium. Here, we proposed a role for NeuroD1 and Neurogenin -2 bHLH family of transcription factors implicated in onset of neurogenesis and differentiation of cells into neurons in promoting the interaction of hippocampal cells with BMSCs and their differentiation in to neurons. The proliferation of the cells was assessed with MTT assay and the role of neuronal induction and differentiation transcription regulators NeuroD1 and Neurogenin-2 in cocultured cells was determined through immunocytochemical analysis. We observed activation and expression of the neurogenic transcription factors NeuroD1 and NGN-2 associated with neuronal activation program to initiate the onset of neurogenesis in cocultured cells. Further, our results have shown a significant expression of neuronal progenitor and immature neuronal marker i.e., nestin and tubulin respectively in cocultured cells endorsing the initiation of neuronal activation.

Attenuation of cisplatin-induced acute kidney injury by N-(2-Hydroxyphenyl) acetamide and its gold conjugated nano-formulations in mice.

The attenuation of cisplatin-induced acute kidney injury (AKI) in mice by N-(2-hydroxyphenyl) acetamide (NA-2) and NA-2-conjugated gold nanoparticles (NA2-AuNPs) was investigated. Male BALB/c mice (n = 54) were divided into nine groups having six animals in each group. Animals in groups 3-9 were pre-treated for 5 days with test compounds, whereas, animals in group 1 and 2 received normal saline. On day 4, animals in groups 2, 3, 4, 5, 6 and 9 were given single intra-peritoneal injection of CP at the dose of 5 mg/kg. After 72 hours of CP injection, all animals were sacrificed. Blood was collected for serum urea and creatinine estimation, and kidneys were harvested for histo-pathological examinations and qPCR studies for nuclear factor-κB p50, (NFκB) ; inducible nitric oxide synthase (iNOS); hemeoxygenase-1 (HO-1); and interleukin-6 (IL-6).NA-2 and NA2-AuNPs was observed to decrease the serum urea and creatinine levels. Both the test compounds reduced kidney injury damage score and improved histological architecture in the treated animals in dose dependent manner. Furthermore, the mRNA expressions of NFkB p50, iNOS and IL-6 genes were down-regulated, and HO-1 gene was up-regulated in the animals treated with the test compounds. It is concluded that NA-2 and NA2-AuNPs attenuates CP-induced AKI in mice models through anti-inflammatory and anti-oxidant mechanisms.

Synthesis of lactobionic acid based bola-amphiphiles and its application as nano-carrier for curcumin delivery to cancer cell cultures in-vitro.

Curcumin is highly effective against various types of cancers; however, its low aqueous solubility, high metabolism and non-specificity hinder its efficacy. This study reports the synthesis of three lactobionic acid containing bola-amphiphiles and their investigation for curcumin nano-vesicular delivery into cancer cells. Synthesized bola-amphiphiles were capable of forming nano-vesicles and curcumin loading in a lipophilicity dependent manner. Bola-amphiphile with higher lipophilicity (C12) caused 89.55 ± 5.52% drug encapsulation in its spherical shape nano-vesicles (195.90 ± 0.83 nm). Bola-amphiphile resulting increased curcumin encapsulation with minimum vesicles size was further investigated for cellular uptake and in-vitro anticancer activity. Anticancer activity of curcumin significantly increased against the tested cancer cells upon loading in bola-amphiphile nano-vesicles. Furthermore, nano-vesicular drug delivery of curcumin enhanced its cellular uptake even at the lowest concentration of 1.25 µg/mL.It is concluded that the synthesized bola-amphiphile based nano-vesicles can efficiently deliver curcumin to the tested cancer cells and needs to be tested for established anticancer drugs against different cancer cell lines for effective treatment of cancer.