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1.  Growth Inhibition and Apoptosis Induction by (+)-Cyanidan-3-ol in Hepatocellular Carcinoma 
PLoS ONE  2013;8(7):e68710.
The objective of this study was to evaluate the cytotoxicity of (+)-cyanidan-3-ol (CD-3) in human hepatocellular carcinoma cell line (HepG2) and chemopreventive potential against hepatocellular carcinoma (HCC) in Balb/c mice. The HepG2 cell line was treated with CD-3 at various concentrations and the proliferation of the HepG2 cells was measure by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT), sulforhodamine B (SRB) and lactate dehydrogenase (LDH) assays. Cell apoptosis was detected by Hoechst 33258 (HO), Acridine orange/ethylene dibromide (AO/EB) staining, DNA fragmentation analysis and the apoptosis rate was detected by flow cytometry. The HCC tumor model was established in mice by injecting N-nitrosodiethylamine/carbon tetrachloride (NDEA/CCl4) and the effect of CD-3 on tumor growth in-vivo was studied. The levels of liver injury markers, tumor markers, and oxidative stress were measured. The expression levels of apoptosis-related genes in in-vitro and in vivo models were determined by RT-PCR and ELISA. The CD-3 induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes under fluorescent microscopy and DNA fragmentation analysis. Annexin V/PI assay demonstrated that apoptosis increased with increase in the concentration of CD-3. The expression levels of apoptosis-related genes that belong to bcl-2 and caspase family were increased and AP-1 and NF-κB activities were significantly suppressed by CD-3. Immunohistochemistry data revealed less localization of p53, p65 and c-jun in CD-3 treated tumors as compared to localization in NDEA/CCl4 treated tumors. Taken together, our data demonstrated that CD-3 could significantly inhibit the proliferation of HepG2 cells in-vitro and suppress HCC tumor growth in-vivo by apoptosis induction.
PMCID: PMC3722203  PMID: 23894334
2.  Optimization of Procedures for Isolation of Mycobacteria from Soil and Water Samples Obtained in Northern India 
For isolation of environmental mycobacteria, a decontamination procedure has been standardized by which treatment with 3% sodium dodecyl sulfate plus 4% NaOH (15 and 30 min for rapid and slow growers, respectively) is followed by incubation with 2% cetrimide (5 and 15 min for fast- and slow-growing mycobacteria, respectively); this procedure was found to completely eliminate contamination with other organisms and resulted in the isolation of only mycobacteria.
PMCID: PMC427771  PMID: 15184184
3.  ZKSCAN3 (ZNF306) is a Master Transcriptional Repressor of Autophagy 
Molecular cell  2013;50(1):16-28.
Autophagy constitutes a major cell protective mechanism eliminating damaged components and maintaining energy homoeostasis via recycling nutrients under normal/stressed conditions. Although the core components of autophagy have been well studied, regulation of autophagy at the transcriptional level is poorly understood. Herein, we establish ZKSCAN3, a zinc-finger family DNA-binding protein, as a transcriptional repressor of autophagy. Silencing of ZKSCAN3 induced autophagy and increased lysosome biogenesis. Importantly, we show that ZKSCAN3 represses transcription of a large gene set (>60) integral to, or regulatory for, autophagy and lysosome biogenesis/function and a subset of these genes, including Map1lC3b and Wipi2 represent direct targets. Interestingly, ZKSCAN3 and TFEB are oppositely regulated by starvation and in turn oppositely regulate lysosomal biogenesis and autophagy, suggesting that they act in conjunction. Altogether, our study uncovers an autophagy master-switch regulating the expression of a transcriptional network of genes integral to autophagy and lysosome biogenesis/function.
PMCID: PMC3628091  PMID: 23434374
4.  Experimental Induction of Type 2 Diabetes in Aging-Accelerated Mice Triggered Alzheimer-Like Pathology and Memory Deficits 
Alzheimer’s disease (AD) is an age-dependent neurodegenerative disease constituting ~95% of late-onset non-familial/sporadic AD, and only ~5% accounting for early-onset familial AD. Availability of a pertinent model representing sporadic AD is essential for testing candidate therapies. Emerging evidence indicates a causal link between diabetes and AD. People with diabetes are >1.5-fold more likely to develop AD. Senescence-accelerated mouse model (SAMP8) of accelerated aging displays many features occurring early in AD. Given the role played by diabetes in the pre-disposition of AD, and the utility of SAMP8 non-transgenic mouse model of accelerated aging, we examined if high fat diet-induced experimental type 2 diabetes in SAMP8 mice will trigger pathological aging of the brain. Results showed that compared to non-diabetic SAMP8 mice, diabetic SAMP8 mice exhibited increased cerebral amyloid-β, dysregulated tau-phosphorylating glycogen synthase kinase 3β, reduced synaptophysin immunoreactivity, and displayed memory deficits, indicating Alzheimer-like changes. High fat diet-induced type 2 diabetic SAMP8 mice may represent the metabolic model of AD.
PMCID: PMC3941701  PMID: 24121970
Alzheimer’s disease; amyloid-β; diabetes; glycogen synthase kinase-3β; learning and memory; pathological aging of the brain; senescence-accelerated mice; synaptophysin; tau
5.  Modelling and Optimization Studies on a Novel Lipase Production by Staphylococcus arlettae through Submerged Fermentation 
Enzyme Research  2013;2013:353954.
Microbial enzymes from extremophilic regions such as hot spring serve as an important source of various stable and valuable industrial enzymes. The present paper encompasses the modeling and optimization approach for production of halophilic, solvent, tolerant, and alkaline lipase from Staphylococcus arlettae through response surface methodology integrated nature inspired genetic algorithm. Response surface model based on central composite design has been developed by considering the individual and interaction effects of fermentation conditions on lipase production through submerged fermentation. The validated input space of response surface model (with R2 value of 96.6%) has been utilized for optimization through genetic algorithm. An optimum lipase yield of 6.5 U/mL has been obtained using binary coded genetic algorithm predicted conditions of 9.39% inoculum with the oil concentration of 10.285% in 2.99 hrs using pH of 7.32 at 38.8°C. This outcome could contribute to introducing this extremophilic lipase (halophilic, solvent, and tolerant) to industrial biotechnology sector and will be a probable choice for different food, detergent, chemical, and pharmaceutical industries. The present work also demonstrated the feasibility of statistical design tools integration with computational tools for optimization of fermentation conditions for maximum lipase production.
PMCID: PMC3880713  PMID: 24455210
6.  Evaluation of a New Lipase from Staphylococcus sp. for Detergent Additive Capability 
BioMed Research International  2013;2013:374967.
Lipases are the enzymes of choice for laundry detergent industries owing to their triglyceride removing ability from the soiled fabric which eventually reduces the usage of phosphate-based chemical cleansers in the detergent formulation. In the present study, a partially purified bacterial lipase from Staphylococcus arlettae JPBW-1 isolated from the rock salt mine has been assessed for its triglyceride removing ability by developing a presoak solution so as to use lipase as an additive in laundry detergent formulations. The effects of selected surfactants, commercial detergents, and oxidizing agents on lipase stability were studied in a preliminary evaluation for its further usage in the industrial environment. Partially purified lipase has shown good stability in presence of surfactants, commercial detergents, and oxidizing agents. Washing efficiency has been found to be enhanced while using lipase with 0.5% nonionic detergent than the anioinic detergent. The wash performance using 0.5% wheel with 40 U lipase at 40°C in 45 min results in maximum oil removal (62%) from the soiled cotton fabric. Hence, the present study opens the new era in enzyme-based detergent sector for formulation of chemical-free detergent using alkaline bacterial lipase.
PMCID: PMC3782757  PMID: 24106703
7.  Alterations in mitochondrial DNA copy number and the activities of electron transport chain complexes and pyruvate dehydrogenase in the frontal cortex from subjects with autism 
Translational Psychiatry  2013;3(9):e299-.
Autism is a neurodevelopmental disorder associated with social deficits and behavioral abnormalities. Recent evidence suggests that mitochondrial dysfunction and oxidative stress may contribute to the etiology of autism. This is the first study to compare the activities of mitochondrial electron transport chain (ETC) complexes (I–V) and pyruvate dehydrogenase (PDH), as well as mitochondrial DNA (mtDNA) copy number in the frontal cortex tissues from autistic and age-matched control subjects. The activities of complexes I, V and PDH were most affected in autism (n=14) being significantly reduced by 31%, 36% and 35%, respectively. When 99% confidence interval (CI) of control group was taken as a reference range, impaired activities of complexes I, III and V were observed in 43%, 29% and 43% of autistic subjects, respectively. Reduced activities of all five ETC complexes were observed in 14% of autistic cases, and the activities of multiple complexes were decreased in 29% of autistic subjects. These results suggest that defects in complexes I and III (sites of mitochondrial free radical generation) and complex V (adenosine triphosphate synthase) are more prevalent in autism. PDH activity was also reduced in 57% of autistic subjects. The ratios of mtDNA of three mitochondrial genes ND1, ND4 and Cyt B (that encode for subunits of complexes I and III) to nuclear DNA were significantly increased in autism, suggesting a higher mtDNA copy number in autism. Compared with the 95% CI of the control group, 44% of autistic children showed higher copy numbers of all three mitochondrial genes examined. Furthermore, ND4 and Cyt B deletions were observed in 44% and 33% of autistic children, respectively. This study indicates that autism is associated with mitochondrial dysfunction in the brain.
PMCID: PMC3784762  PMID: 24002085
autism; electron transport chain; mitochondrial DNA; mitochondrial dysfunction; oxidative stress; pyruvate dehydrogenase
8.  Brain Transit and Ameliorative Effects of Intranasally Delivered Anti-Amyloid-β Oligomer Antibody in 5XFAD Mice 
Alzheimer’s disease (AD) is a global health crisis with limited treatment options. Despite major advances in neurotherapeutics, poor brain penetration due to the blood-brain barrier continues to pose a big challenge in overcoming the access of therapeutics to the central nervous system. In that regard, the non-invasive intranasal route of brain targeting is gaining considerable attention. The nasal mucosa offers a large surface area, rapid absorption, and avoidance of first-pass metabolism increasing drug bioavailability with less systemic side effects. Intranasal delivery is known to utilize olfactory, rostral migratory stream, and trigeminal routes to reach the brain. This investigation confirmed that intranasal delivery of oligomeric amyloid-β antibody (NU4) utilized all three routes to enter the brain with a resident time of 96 hours post single bolus intranasal administration, and showed evidence of perikaryal and parenchymal uptake of NU4 in 5XFAD mouse brain, confirming the intranasal route as a non-invasive and efficient way of delivering therapeutics to the brain. In addition, this study demonstrated that intranasal delivery of NU4 antibody lowered cerebral amyloid-β and improved spatial learning in 5XFAD mice.
PMCID: PMC3722071  PMID: 23542865
Alzheimer’s immunotherapy; amyloid-β oligomer antibody; brain transit; cerebral amyloid-β immunocytochemistry; intranasal delivery; olfactory pathway; rostral migratory stream pathway; spatial acquisition learning; trigeminal pathway
9.  Vacuum Assisted Closure Therapy versus Standard Wound Therapy for Open Musculoskeletal Injuries 
Advances in Orthopedics  2013;2013:245940.
Background. This study was performed to evaluate the results of vacuum assisted wound therapy in patients with open musculoskeletal injuries. Study Design and Setting. Prospective, randomized, and interventional at tertiary care hospital, from 2011 to 2012. Materials and Methods. 30 patients of open musculoskeletal injuries underwent randomized trial of vacuum assisted closure therapy versus standard wound therapy around the upper limb and lower limb. Mean patient age was 39 ± 18 years (range, 18 to 76 years). Necrotic tissues were debrided before applying VAC therapy. Dressings were changed every 3 or 4 days. For standard wound therapy, debridement followed by daily dressings was done. Data Management and Statistical Analysis. The results obtained were subjected to statistical analysis. Results. The size of soft tissue defects reduced more than 5 mm to 25 mm after VAC (mean decrease of 26.66%), whereas in standard wound therapy, reduction in wound size was less than 5 mm. A free flap was needed to cover exposed bone and tendon in one case in standard wound therapy group. No major complication occurred that was directly attributable to treatment. Conclusion. Vacuum assisted wound therapy was found to facilitate the rapid formation of healthy granulation tissue on open wounds in the upper limb and lower limb, thus to shorten healing time and minimize secondary soft tissue defect coverage procedures.
PMCID: PMC3710616  PMID: 23878741
10.  Consensus Paper: Pathological Role of the Cerebellum in Autism 
Cerebellum (London, England)  2012;11(3):777-807.
There has been significant advancement in various aspects of scientific knowledge concerning the role of cerebellum in the etiopathogenesis of autism. In the current consensus paper, we will observe the diversity of opinions regarding the involvement of this important site in the pathology of autism. Recent emergent findings in literature related to cerebellar involvement in autism are discussed, including: cerebellar pathology, cerebellar imaging and symptom expression in autism, cerebellar genetics, cerebellar immune function, oxidative stress and mitochondrial dysfunction, GABAergic and glutamatergic systems, cholinergic, dopaminergic, serotonergic, and oxytocin related changes in autism, motor control and cognitive deficits, cerebellar coordination of movements and cognition, gene-environment interactions, therapeutics in autism and relevant animal models of autism. Points of consensus include presence of abnormal cerebellar anatomy, abnormal neurotransmitter systems, oxidative stress, cerebellar motor and cognitive deficits, and neuroinflammation in subjects with autism. Undefined areas or areas requiring further investigation include lack of treatment options for core symptoms of autism, vermal hypoplasia and other vermal abnormalities as a consistent feature of autism, mechanisms underlying cerebellar contributions to cognition, and unknown mechanisms underlying neuroinflammation.
PMCID: PMC3677555  PMID: 22370873
cerebellum; autism
11.  Alternatively-spliced extra domain A of fibronectin promotes acute inflammation and brain injury following cerebral ischemia in mice 
Background and purpose
The fibronectin isoform containing the alternatively-spliced extra domain A (EDA+-FN) is normally absent from the circulation, but plasma levels of EDA+-FN can become markedly elevated in several human pathological conditions associated with inflammation including ischemic stroke. It remains unknown whether EDA+-FN contributes to stroke pathogenesis or is simply an associative marker. Several in vitro studies suggest that EDA+-FN can activate toll-like receptor 4 (TLR4), an innate immune receptor that triggers pro-inflammatory responses. We undertook a genetic approach in mice to investigate the ability of EDA+-FN to mediate inflammatory brain damage in a focal cerebral ischemia/reperfusion injury model.
We used genetically modified EDA+/+ mice, which constitutively express EDA+-FN. Extent of injury, neurological outcome and inflammatory mechanisms were assessed following one hour cerebral ischemia/23 hour reperfusion injury and compared to wild-type (WT) mice.
We found that EDA+/+ mice developed significantly larger infarcts and severe neurological deficits that was associated with significant increased neutrophil and macrophage infiltration as quantitated by immunohistochemistry. Additionally, we found upregulation of NF-κB, COX-2, and inflammatory cytokines TNFα, IL-1β and IL-6 in the EDA+/+ mice compared to WT mice. Interestingly, increased brain injury and neurological deficits were largely abrogated in EDA+/+ mice by treatment with a specific TLR4 inhibitor.
These findings provide the first evidence that EDA+-FN promotes inflammatory brain injury following ischemic stroke and suggest that the elevated levels of plasma EDA+-FN observed in chronic inflammatory conditions could worsen injury and outcome in patients following acute stroke.
PMCID: PMC3335936  PMID: 22363055
Fibronectin; Inflammation; Cerebral ischemia; mice
12.  Differences Between the Pattern of Developmental Abnormalities in Autism Associated with Duplications 15q11.2-q13 and Idiopathic Autism 
The purpose of this study was to identify differences in patterns of developmental abnormalities between the brains of individuals with autism of unknown etiology and those of individuals with duplications of chromosome 15q11.2-q13 [dup(15)] and autism, and to identify alterations that may contribute to seizures and sudden death in the latter. Brains of 9 subjects with dup(15), 10 with idiopathic autism, and 7 controls were examined. In the dup(15) cohort, 7 subjects (78%) had autism, 7 (78%) had seizures, and 6 (67%) had experienced sudden unexplained death. Subjects with dup(15) autism were microcephalic, with mean brain weights 300 g less (1,177 g) than those of subjects with idiopathic autism (1,477 g; p < 0.001). Heterotopias in the alveus, CA4, and dentate gyrus and dysplasia in the dentate gyrus were detected in 89% of dup(15) autism cases but in only 10% idiopathic autism cases (p < 0.001). By contrast, cerebral cortex dysplasia was detected in 50% of subjects with idiopathic autism and in no dup(15) autism cases (p < 0.04). The different spectrum and higher prevalence of developmental neuropathological findings in the dup(15) cohort than in cases with idiopathic autism may contribute to the high risk of early onset of seizures and sudden death.
PMCID: PMC3612833  PMID: 22487857
Autism; Chromosome 15q11.2-q13 duplication; Developmental brain alterations; Seizures; Sudden unexpected death
13.  A study of malocclusion and orthodontic treatment needs according to dental aesthetic index among school children of a hilly state of India 
The documentation of magnitude of malocclusion in terms of prevalence and severity has not been done till date in Himachal Pradesh, India.
To assess the prevalence of malocclusion and orthodontic treatment needs (OTNs) among 9-and 12-year-old school children by using the Dental Aesthetic Index (DAI) in the state.
Materials and Methods:
A cross-sectional study was conducted among 1188 children from randomly selected schools. The survey was done according to the Oral Health Assessment Form (modified). DAI was used to assess the severity of malocclusion, along with collection of demographic data.
The overall prevalence of malocclusion was 12.5% and required orthodontic treatment, whereas 87.5% did not require treatment. A severe malocclusion for which treatment was highly desirable was recorded in 3.1%; 8% had a definite malocclusion for which treatment was elective. Only about 1.3% had a handicapping malocclusion that needed mandatory treatment. Almost equal proportions of males and females were affected with malocclusion with the means 20 ± 4.6 and 19.9 ± 4.9, respectively (P < 0.641). The prevalence and severity of malocclusion was more in 12-year age group than in 9-year age group (P = 0.002**). There was an increase in the proportion of malocclusion among older children: In 12-year age group, 15.7% with mean 20.5 ± 5.1 and in 9-year-old children, 8.9% with the mean 19.3 ± 4.1 were in the need of orthodontic treatment.
Severity and treatment needs, both are important factors in public health planning.
PMCID: PMC3894101  PMID: 24478978
Dental Aesthetic Index; malocclusion; orthodontic treatment needs; World Health Organization
14.  Abnormal Intracellular Accumulation and Extracellular Aβ Deposition in Idiopathic and Dup15q11.2-q13 Autism Spectrum Disorders 
PLoS ONE  2012;7(5):e35414.
It has been shown that amyloid ß (Aβ), a product of proteolytic cleavage of the amyloid β precursor protein (APP), accumulates in neuronal cytoplasm in non-affected individuals in a cell type–specific amount.
Methodology/Principal Findings
In the present study, we found that the percentage of amyloid-positive neurons increases in subjects diagnosed with idiopathic autism and subjects diagnosed with duplication 15q11.2-q13 (dup15) and autism spectrum disorder (ASD). In spite of interindividual differences within each examined group, levels of intraneuronal Aβ load were significantly greater in the dup(15) autism group than in either the control or the idiopathic autism group in 11 of 12 examined regions (p<0.0001 for all comparisons; Kruskall-Wallis test). In eight regions, intraneuronal Aβ load differed significantly between idiopathic autism and control groups (p<0.0001). The intraneuronal Aβ was mainly N-terminally truncated. Increased intraneuronal accumulation of Aβ17–40/42 in children and adults suggests a life-long enhancement of APP processing with α-secretase in autistic subjects. Aβ accumulation in neuronal endosomes, autophagic vacuoles, Lamp1-positive lysosomes and lipofuscin, as revealed by confocal microscopy, indicates that products of enhanced α-secretase processing accumulate in organelles involved in proteolysis and storage of metabolic remnants. Diffuse plaques containing Aβ1–40/42 detected in three subjects with ASD, 39 to 52 years of age, suggest that there is an age-associated risk of alterations of APP processing with an intraneuronal accumulation of a short form of Aβ and an extracellular deposition of full-length Aβ in nonfibrillar plaques.
The higher prevalence of excessive Aβ accumulation in neurons in individuals with early onset of intractable seizures, and with a high risk of sudden unexpected death in epilepsy in autistic subjects with dup(15) compared to subjects with idiopathic ASD, supports the concept of mechanistic and functional links between autism, epilepsy and alterations of APP processing leading to neuronal and astrocytic Aβ accumulation and diffuse plaque formation.
PMCID: PMC3342283  PMID: 22567102
15.  Curcumin-loaded magnetic nanoparticles for breast cancer therapeutics and imaging applications 
The next generation magnetic nanoparticles (MNPs) with theranostic applications have attracted significant attention and will greatly improve nanomedicine in cancer therapeutics. Such novel MNP formulations must have ultra-low particle size, high inherent magnetic properties, effective imaging, drug targeting, and drug delivery properties. To achieve these characteristic properties, a curcumin-loaded MNP (MNP-CUR) formulation was developed.
MNPs were prepared by chemical precipitation method and loaded with curcumin (CUR) using diffusion method. The physicochemical properties of MNP-CUR were characterized using dynamic light scattering, transmission electron microscopy, and spectroscopy. The internalization of MNP-CUR was achieved after 6 hours incubation with MDA-MB-231 breast cancer cells. The anticancer potential was evaluated by a tetrazolium-based dye and colony formation assays. Further, to prove MNP-CUR results in superior therapeutic effects over CUR, the mitochondrial membrane potential integrity and reactive oxygen species generation were determined. Magnetic resonance imaging capability and magnetic targeting property were also evaluated.
MNP-CUR exhibited individual particle grain size of ~9 nm and hydrodynamic average aggregative particle size of ~123 nm. Internalized MNP-CUR showed a preferential uptake in MDA-MB-231 cells in a concentration-dependent manner and demonstrated accumulation throughout the cell, which indicates that particles are not attached on the cell surface but internalized through endocytosis. MNP-CUR displayed strong anticancer properties compared to free CUR. MNP-CUR also amplified loss of potential integrity and generation of reactive oxygen species upon treatment compared to free CUR. Furthermore, MNP-CUR exhibited superior magnetic resonance imaging characteristics and significantly increased the targeting capability of CUR.
MNP-CUR exhibits potent anticancer activity along with imaging and magnetic targeting capabilities. This approach can be extended to preclinical and clinical use and may have importance in cancer treatment and cancer imaging in the future. Further, if these nanoparticles can functionalize with antibody/ligands, they will serve as novel platforms for multiple biomedical applications.
PMCID: PMC3356199  PMID: 22619526
magnetic nanoparticles; drug delivery systems; magnetic resonance imaging; nanomedicine; cancer therapeutics; biomedical applications
16.  Reduced Activity of Protein Kinase C in the Frontal Cortex of Subjects with Regressive Autism: Relationship with Developmental Abnormalities 
Autism is a neurodevelopmental disorder with unknown etiology. In some cases, typically developing children regress into clinical symptoms of autism, a condition known as regressive autism. Protein kinases are essential for G-protein-coupled receptor-mediated signal transduction, and are involved in neuronal functions, gene expression, memory, and cell differentiation. Recently, we reported decreased activity of protein kinase A (PKA) in the frontal cortex of subjects with regressive autism. In the present study, we analyzed the activity of protein kinase C (PKC) in the cerebellum and different regions of cerebral cortex from subjects with regressive autism, autistic subjects without clinical history of regression, and age-matched control subjects. In the frontal cortex of subjects with regressive autism, PKC activity was significantly decreased by 57.1% as compared to age-matched control subjects (p = 0.0085), and by 65.8% as compared to non-regressed autistic subjects (p = 0.0048). PKC activity was unaffected in the temporal, parietal and occipital cortices, and in the cerebellum in both autism groups, i.e., regressive and non-regressed autism as compared to control subjects. These results suggest brain region-specific alteration of PKC activity in the frontal cortex of subjects with regressive autism. Further studies showed a negative correlation between PKC activity and restrictive, repetitive and stereotyped pattern of behavior (r= -0.084, p = 0.0363) in autistic individuals, suggesting involvement of PKC in behavioral abnormalities in autism. These findings suggest that regression in autism may be attributed, in part, to alterations in G-protein-coupled receptor-mediated signal transduction involving PKA and PKC in the frontal cortex.
PMCID: PMC3432855  PMID: 22949890
Autism; behavior; protein kinase C; protein kinases; regression; signal transduction.
17.  Interaction of curcumin nanoformulations with human plasma proteins and erythrocytes 
Recent studies report curcumin nanoformulation(s) based on polylactic-co-glycolic acid (PLGA), β-cyclodextrin, cellulose, nanogel, and dendrimers to have anticancer potential. However, no comparative data are currently available for the interaction of curcumin nanoformulations with blood proteins and erythrocytes. The objective of this study was to examine the interaction of curcumin nanoformulations with cancer cells, serum proteins, and human red blood cells, and to assess their potential application for in vivo preclinical and clinical studies.
The cellular uptake of curcumin nanoformulations was assessed by measuring curcumin levels in cancer cells using ultraviolet-visible spectrophotometry. Protein interaction studies were conducted using particle size analysis, zeta potential, and Western blot techniques. Curcumin nanoformulations were incubated with human red blood cells to evaluate their acute toxicity and hemocompatibility.
Cellular uptake of curcumin nanoformulations by cancer cells demonstrated preferential uptake versus free curcumin. Particle sizes and zeta potentials of curucumin nanoformulations were varied after human serum albumin adsorption. A remarkable capacity of the dendrimer curcumin nanoformulation to bind to plasma protein was observed, while the other formulations showed minimal binding capacity. Dendrimer curcumin nanoformulations also showed higher toxicity to red blood cells compared with the other curcumin nanoformulations.
PLGA and nanogel curcumin nanoformulations appear to be very compatible with erythrocytes and have low serum protein binding characteristics, which suggests that they may be suitable for application in the treatment of malignancy. These findings advance our understanding of the characteristics of curcumin nanoformulations, a necessary component in harnessing and implementing improved in vivo effects of curcumin.
Video abstract
PMCID: PMC3225220  PMID: 22128249
nanoparticle; curcumin; chemotherapy; cellular uptake; protein binding; hemocompatibility
18.  Brain Region–Specific Decrease in the Activity and Expression of Protein Kinase A in the Frontal Cortex of Regressive Autism 
PLoS ONE  2011;6(8):e23751.
Autism is a severe neurodevelopmental disorder that is characterized by impaired language, communication, and social skills. In regressive autism, affected children first show signs of normal social and language development but eventually lose these skills and develop autistic behavior. Protein kinases are essential in G-protein-coupled, receptor-mediated signal transduction and are involved in neuronal functions, gene expression, memory, and cell differentiation. We studied the activity and expression of protein kinase A (PKA), a cyclic AMP–dependent protein kinase, in postmortem brain tissue samples from the frontal, temporal, parietal, and occipital cortices, and the cerebellum of individuals with regressive autism; autistic subjects without a clinical history of regression; and age-matched developmentally normal control subjects. The activity of PKA and the expression of PKA (C-α), a catalytic subunit of PKA, were significantly decreased in the frontal cortex of individuals with regressive autism compared to control subjects and individuals with non-regressive autism. Such changes were not observed in the cerebellum, or the cortices from the temporal, parietal, and occipital regions of the brain in subjects with regressive autism. In addition, there was no significant difference in PKA activity or expression of PKA (C-α) between non-regressive autism and control groups. These results suggest that regression in autism may be associated, in part, with decreased PKA-mediated phosphorylation of proteins and abnormalities in cellular signaling.
PMCID: PMC3166116  PMID: 21909354
19.  Protective Effects of Walnut Extract Against Amyloid Beta Peptide-Induced Cell Death and Oxidative Stress in PC12 Cells 
Neurochemical Research  2011;36(11):2096-2103.
Amyloid beta-protein (Aβ) is the major component of senile plaques and cerebrovascular amyloid deposits in individuals with Alzheimer’s disease. Aβ is known to increase free radical production in neuronal cells, leading to oxidative stress and cell death. Recently, considerable attention has been focused on dietary antioxidants that are able to scavenge reactive oxygen species (ROS), thereby offering protection against oxidative stress. Walnuts are rich in components that have anti-oxidant and anti-inflammatory properties. The inhibition of in vitro fibrillization of synthetic Aβ, and solubilization of preformed fibrillar Aβ by walnut extract was previously reported. The present study was designed to investigate whether walnut extract can protect against Aβ-induced oxidative damage and cytotoxicity. The effect of walnut extract on Aβ-induced cellular damage, ROS generation and apoptosis in PC12 pheochromocytoma cells was studied. Walnut extract reduced Aβ-mediated cell death assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, and release of lactate dehydrogenase (membrane damage), DNA damage (apoptosis) and generation of ROS in a concentration-dependent manner. These results suggest that walnut extract can counteract Aβ-induced oxidative stress and associated cell death.
PMCID: PMC3183245  PMID: 21706234
Alzheimer’s disease; Amyloid beta-protein; Apoptosis; Cytotoxicity; Oxidative stress; Walnut
20.  Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates 
Background & objectives
In drug resistant, especially multi-drug resistant (MDR) tuberculosis, fluoroquinolones (FQs) are used as second line drugs. However, the incidence of FQ-resistant Mycobacterium tuberculosis is rapidly increasing which may be due to extensive use of FQs in the treatment of various other diseases. The most important known mechanism i.e., gyrA mutation in FQ resistance is not observed in a significant proportion of FQ resistant M. tuberculosis isolates suggesting that the resistance may be because of other mechanisms such as an active drug efflux pump. In this study we evaluated the role of the efflux pumps in quinolone resistance by using various inhibitors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil, in clinical isolates of M. tuberculosis.
A total of 55 M. tuberculosis clinical isolates [45 ofloxacin (OFL) resistant and 10 ofloxacin sensitive] were tested by Resazurin microtitre assay (REMA) to observe the changes in ofloxacin minimum inhibitory concentration (MIC) levels in presence of efflux inhibitors as compared to control (without efflux inhibitor).
The MIC levels of OFL showed 2-8 folds reduction in presence of CCCP (16/45; 35.5%), verapamil (24/45; 53.3%) and DNP (21/45; 46.6%) while in case of isolates identified as OFL sensitive these did not show any effect on ofloxacin MICs. In 11 of 45 (24.5%) isolates change in MIC levels was observed with all the three inhibitors. Overall 30 (66.6%) isolates had reduction in OFL MIC after treatment with these inhibitors. A total of eight isolates were sequenced for gyrA gene, of which, seven (87.5%) showed known mutations. Of the eight sequenced isolates, seven (87.5%) showed 2 to 8 fold change in MIC in presence of efflux inhibitors.
Interpretation & conclusions
Our findings suggest the involvement of active efflux pumps of both Major Facilitator Super Family (MFS) family (inhibited by CCCP and DNP) and ATP Binding Cassette (ABC) transporters (inhibited by verapamil) in the development of OFL resistance in M. tuberculosis isolates. Epidemiological significance of these findings needs to be determined in prospective studies with appropriate number of samples / isolates.
PMCID: PMC3121286  PMID: 21623040
Drug resistance; efflux pump inhibitors; MIC; Mycobacterium tuberculosis; ofloxacin; tuberculosis
21.  Increased activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in the frontal cortex and cerebellum of autistic individuals 
Life sciences  2009;85(23-26):788-793.
Na+/K+-ATPase and Ca2+/Mg2+-ATPase are enzymes known to maintain intracellular gradients of ions that are essential for signal transduction. The aim of this study was to compare the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in post-mortem brain samples from the cerebellum and frontal, temporal, parietal, and occipital cortices from autistic and age-matched control subjects.
Main methods
The frozen postmortem tissues from different brain regions of autistic and control subjects were homogenized. The activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase were assessed in the brain homogenates by measuring inorganic phosphorus released by the action of Na+/K+- and Ca2+/Mg2+- dependent hydrolysis of ATP.
Key findings
In the cerebellum, the activities of both Na+/K+-ATPase and Ca2+/Mg2+-ATPase were significantly increased in the autistic samples compared with their age-matched controls. The activity of Na+/K+-ATPase but not Ca2+/Mg2+-ATPase was also significantly increased in the frontal cortex of the autistic samples as compared to the age-matched controls. In contrast, in other regions, i.e., the temporal, parietal and occipital cortices, the activities of these enzymes were similar in autism and control groups.
The results of this study suggest brain-region specific increases in the activities of Na+/K+-ATPase and Ca2+/Mg2+-ATPase in autism. Increased activity of these enzymes in the frontal cortex and cerebellum may be due to compensatory responses to increased intracellular calcium concentration in autism. We suggest that altered activities of these enzymes may contribute to abnormal neuronal circuit functioning in autism.
PMCID: PMC2810556  PMID: 19863947
22.  Gelsolin Levels are increased in the brain as a Function of Age During Normal Development in Children That are further increased in Down Syndrome 
Neuronal dysfunctions in several neurodegenerative diseases such as Down syndrome (DS) have been linked to oxidative stress. In this study, we observed that lipid peroxidation, a marker of oxidative stress, is significantly increased in the frontal cortex of brains of individuals with DS as compared to control subjects. We report here that gelsolin levels are increased in the frontal cortex of individuals with DS as compared to controls during early developmental ages (5–13 years). Interestingly, the levels of gelsolin in the frontal cortex were increased as a function of age in both DS and control subjects. Because cytoplasmic gelsolin has five free thiol groups (cysteine), and its levels are increased in response to oxidative stress, we propose that gelsolin may serve as an anti-oxidant protein.
PMCID: PMC2788096  PMID: 19561443
gelsolin; development; Down syndrome; oxidative stress
23.  Experimental posterolateral spinal fusion with beta tricalcium phosphate ceramic and bone marrow aspirate composite graft 
Indian Journal of Orthopaedics  2010;44(4):402-408.
Beta tricalcium phosphate is commonly used in metaphyseal defects but its use in posterolateral spinal fusion remains controversial. There are very few published animal studies in which use of beta tricalcium phosphate has been evaluated in the posterolateral lumbar arthrodesis model. Hence we conducted a study to evaluate the potential of composite graft of beta tricalcium phosphate and bone marrow aspirate in comparison to autologous bone graft, when used for posterolateral spinal fusion.
Materials and Methods:
Single level posterolateral lumbar fusion was performed in 40 adult male Indian rabbits, which were assigned randomly into one of the four groups based on graft materials implanted; a) 3 gm beta tricalcium phosphate plus 3 ml bone marrow aspirate (Group I); b) 3 ml bone marrow aspirate alone (Group II); c) 3 gm beta tricalcium phosphate (Group III) and d) 3 gm autologous bone graft (Group IV). Each group had 10 rabbits. Half of the rabbits were sacrificed by injecting Phenobarbitone intraperitoneally after eight weeks and the remaining after 24 weeks, and were evaluated for fusion by X-rays, computed tomography (CT) scans, manual palpation test and histology.
Beta tricalcium phosphate used with bone marrow aspirate produced best results when compared to other groups (P =.0001). When beta tricalcium phosphate was used alone, fusion rates were better as compared to fusion achieved with autologous iliac crest bone graft though statistically not significant (P =0.07). Autologous bone graft showed signs of new bone formation. However, the rate of new bone formation was comparatively slow.
Composite graft of beta tricalcium phosphate and bone marrow aspirate can be used as an alternative to autologous iliac crest bone graft.
PMCID: PMC2947727  PMID: 20924481
Beta tricalcium phosphate; bone marrow aspirate; posterolateral spinal fusion
24.  In Vitro Evaluation of Antibacterial Potential of Dry FruitExtracts of Elettaria cardamomum Maton (Chhoti Elaichi) 
Emergence of resistance among pathogenic bacteria against available antibiotics is posing a great challenge to the current world. Thus, there is a great need to discover novel antibiotics. Traditional plants have been proved to be novel source in the search of antimicrobial compounds. The current study pertained to the susceptibilities of some clinically significant bacterial species to various crude extracts of Elettaria cardamomum Maton (Chhoti elaichi) dry fruits by agar well diffusion assay. Minimum inhibitory concentrations (MIC) of extracts were further evaluated against these bacteria. The study indicated that antibacterial activity of this plant is dependent on the type of extract and the organism evaluated. Ethanol extract was found to have comparatively higher activity than other organic and aqueous extracts. Gram-positive bacteria showed competent but variable susceptibilities to all the tested extracts. MIC data showed hopeful results as some of the extracts exhibited significant inhibitions of bacteria even at concentrations as low as 512 μg/mL. Overall, E. cardamomum seems to have significant antibacterial activity and to be very useful in the discovery of novel antibiotic.
PMCID: PMC3863444  PMID: 24363739
Antibacterial activity; Elettaria cardamomum; Chhoti elaichi; Minimum inhibitory concentration
25.  In vitro antibacterial evaluation of Anabaena sp. against several clinically significant microflora and HPTLC analysis of its active crude extracts 
Indian Journal of Pharmacology  2010;42(2):105-107.
The present study was conducted to evaluate the possible antibacterial activity of Anabaena extracts. Anabaena was isolated from a natural source and cultured in vitro. after suitable growth, cyanobacterial culture was harvested using different solvents. Extracts, thus prepared, were evaluated for their antibacterial potential by agar-well diffusion assay against bacterial species of clinical significance. MIC values were determined further to check the concentration ranges for significant inhibition. HPTLC analysis was done to separate the components of active crude extract in an attempt to identify the bio-active chemical entity. Methanol extract exhibited more potent activity than that of hexane and ethyl acetate extracts. No inhibitory effect was found against Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumoniae. Staphylococcus aureus required about 256 µg/ml of the crude methanol extract for effective inhibition. HPTLC evaluation at λ 254 nm was performed for the separation of a complex mixture of the methanol extract. The results provide evidence that Anabaena sp. extracts might indeed be potential sources of new antibacterial agents.
PMCID: PMC2907006  PMID: 20711376
Blue-green algae; drug-resistance; biologically active substances; bacterial species

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