Publications by authors named "Priyanka Tripathi"

35 Publications

A serum microRNA sequence reveals fragile X protein pathology in amyotrophic lateral sclerosis.

Brain 2021 May;144(4):1214-1229

Department of Neurology, Ulm University, Ulm, Germany.

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS.
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http://dx.doi.org/10.1093/brain/awab018DOI Listing
May 2021

Hsp90-mediated regulation of DYRK3 couples stress granule disassembly and growth via mTORC1 signaling.

EMBO Rep 2021 May 19;22(5):e51740. Epub 2021 Mar 19.

Department of Biomedical, Metabolic and Neural Sciences, Centre for Neuroscience and Nanotechnology, University of Modena and Reggio Emilia, Modena, Italy.

Stress granules (SGs) are dynamic condensates associated with protein misfolding diseases. They sequester stalled mRNAs and signaling factors, such as the mTORC1 subunit raptor, suggesting that SGs coordinate cell growth during and after stress. However, the molecular mechanisms linking SG dynamics and signaling remain undefined. We report that the chaperone Hsp90 is required for SG dissolution. Hsp90 binds and stabilizes the dual-specificity tyrosine-phosphorylation-regulated kinase 3 (DYRK3) in the cytosol. Upon Hsp90 inhibition, DYRK3 dissociates from Hsp90 and becomes inactive. Inactive DYRK3 is subjected to two different fates: it either partitions into SGs, where it is protected from irreversible aggregation, or it is degraded. In the presence of Hsp90, DYRK3 is active and promotes SG disassembly, restoring mTORC1 signaling and translation. Thus, Hsp90 links stress adaptation and cell growth by regulating the activity of a key kinase involved in condensate disassembly and translation restoration.
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http://dx.doi.org/10.15252/embr.202051740DOI Listing
May 2021

Palmitoylation of acetylated tubulin and association with ceramide-rich platforms is critical for ciliogenesis.

J Lipid Res 2021 Jan 7;62:100021. Epub 2021 Jan 7.

Department of Physiology, University of Kentucky College of Medicine, Lexington, KY, USA; Veterans Affairs Medical Center, Lexington, KY, USA. Electronic address:

Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.
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http://dx.doi.org/10.1194/jlr.RA120001190DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7903138PMC
January 2021

Dietary soy lecithin augments antioxidative defense and thermal tolerance but fails to modulate non-specific immune genes in endangered golden mahseer (Tor putitora) fry.

Fish Shellfish Immunol 2021 Feb 4;109:34-40. Epub 2020 Dec 4.

ICAR-Directorate of Coldwater Fisheries Research, Anusandhan Bhawan, Bhimtal, 263136, Uttarakhand, India.

A 70-day experiment was carried out to assess the effect of different levels (0, 1 and 2%) of soy lecithin in the diet on growth, survival, antioxidant defense markers, immune gene expression and thermal tolerance limits of golden mahseer, Tor putitora fry. Percentage weight gain, specific growth rate (SGR %) and survival of mahseer fed lecithin supplemented diets were not significantly different from those of the control group. Also, the mRNA expression levels of different immune related genes such as tnfα, il-1β, il-10, complement-3, interferon-gamma (ifnγ) and tlr4 were unaffected by dietary lecithin supplementation. Nevertheless, superoxide dismutase (SOD) activity was significantly greater in the lecithin-fed groups than the control fish. The glutathione-S-transferase (GST) activity was exceptionally high in the 2% lecithin supplemented group compared to the rest two groups. This increase in antioxidant status with dietary lecithin supplementation, however, was not reflected in the whole body malonaldehyde (MDA) levels, as it did not vary significantly among the dietary groups. Importantly, dietary inclusion of soy lecithin significantly increased upper thermal tolerance limits as evidenced by higher CT and LT values. Likewise, golden mahseer fry fed with lecithin supplemented diets (both 1 and 2%) registered significantly lower critical and lethal thermal minimum (CT and LT) values than the control group, indicating higher cold tolerance capacity. Our results thus demonstrate that the dietary inclusion of soy lecithin could enhance the upper and lower thermal tolerance limits and antioxidant status of golden mahseer fry and failed to enhance immune related gene expression.
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http://dx.doi.org/10.1016/j.fsi.2020.11.031DOI Listing
February 2021

The mono-ADP-ribosyltransferase ARTD10 regulates the voltage-gated K channel Kv1.1 through protein kinase C delta.

BMC Biol 2020 10 15;18(1):143. Epub 2020 Oct 15.

Institute of Physiology, RWTH Aachen University, Pauwelsstrasse 30, 52074, Aachen, Germany.

Background: ADP-ribosylation is a ubiquitous post-translational modification that involves both mono- and poly-ADP-ribosylation. ARTD10, also known as PARP10, mediates mono-ADP-ribosylation (MARylation) of substrate proteins. A previous screen identified protein kinase C delta (PKCδ) as a potential ARTD10 substrate, among several other kinases. The voltage-gated K channel Kv1.1 constitutes one of the dominant Kv channels in neurons of the central nervous system and the inactivation properties of Kv1.1 are modulated by PKC. In this study, we addressed the role of ARTD10-PKCδ as a regulator of Kv1.1.

Results: We found that ARTD10 inhibited PKCδ, which increased Kv1.1 current amplitude and the proportion of the inactivating current component in HeLa cells, indicating that ARTD10 regulates Kv1.1 in living cells. An inhibitor of ARTD10, OUL35, significantly decreased peak amplitude together with the proportion of the inactivating current component of Kv1.1-containing channels in primary hippocampal neurons, demonstrating that the ARTD10-PKCδ signaling cascade regulates native Kv1.1. Moreover, we show that the pharmacological blockade of ARTD10 increases excitability of hippocampal neurons.

Conclusions: Our results, for the first time, suggest that MARylation by ARTD10 controls neuronal excitability.
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http://dx.doi.org/10.1186/s12915-020-00878-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7558731PMC
October 2020

Cross-Link/Proximity Ligation Assay for Visualization of Lipid and Protein Complexes in Lipid Rafts.

Methods Mol Biol 2021 ;2187:337-348

Department of Physiology, University of Kentucky Medical School, Lexington, KY, USA.

The detection of protein complexes by coimmunoprecipitation or two-hybrid analysis is often limited to cytosolic and soluble proteins, while interaction between membrane proteins or proteins and lipids is hampered by solubilization artefacts or absence of appropriate antibodies to detect a complex. More recently, the proximity ligation assay (PLA) using antibodies for in situ detection of protein complexes in cells and cross-linkable lipid analogs that can be endowed with molecular tags for pull-down assyas were techniques utilized to identify and monitor interaction between proteins and lipids. We have developed a novel technique termed "cross-link/PLA" combining a cross-linkable ceramide analog with PLA and anti-ceramide antibody to visualize lipid-protein complexes in ceramide-rich platforms (CRPs), a particular type of lipid raft. This chapter will discuss experimental protocols and data analysis to use cross-link/PLA for detection and visualization of lipid-protein complexes in CRPs and other types of lipid rafts.
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http://dx.doi.org/10.1007/978-1-0716-0814-2_20DOI Listing
February 2021

Extracellular Vesicles Containing Ceramide-Rich Platforms: "Mobile Raft" Isolation and Analysis.

Methods Mol Biol 2021 ;2187:87-98

Department of Physiology, University of Kentucky Medical School, Lexington, KY, USA.

Extracellular vesicles (EVs) are secreted by eukaryotic cells and serve as carriers for a variety of cell signaling factors, including RNAs, proteins, and lipids. We described a unique population of EVs, the membrane of which is highly enriched with the sphingolipid ceramide. We suggested that ceramide in the EV membrane is organized in ceramide-rich platforms (CRPs), a type of lipid raft that mediates interaction of ceramide with ceramide-associated proteins (CAPs). Here, we describe methods using anti-ceramide antibody to isolate ceramide-enriched EVs and detect exosomes after uptake into recipient cells. In addition, we discuss methods for EV analysis using nanoparticle tracking and mass spectrometry. The methods can be extended to the isolation of other types of EVs and "mobile rafts" transported by EVs from donor to recipient cells using antibodies against lipids specific for these EVs.
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http://dx.doi.org/10.1007/978-1-0716-0814-2_5DOI Listing
March 2021

In vivo evidence of exosome-mediated Aβ neurotoxicity.

Acta Neuropathol Commun 2020 07 6;8(1):100. Epub 2020 Jul 6.

Department of Physiology, University of Kentucky College of Medicine, 800 Rose Street Room MS519, Lexington, KY, USA.

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http://dx.doi.org/10.1186/s40478-020-00981-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339450PMC
July 2020

Polymer-Coated Hydroxyapatite Nanocarrier for Double-Stranded RNA Delivery.

J Agric Food Chem 2020 Jun 16;68(25):6811-6818. Epub 2020 Jun 16.

Department of Plant and Soil Sciences, University of Kentucky, Lexington, Kentucky 40546, United States.

Conventional synthetic insecticides have limited success due to insect resistance and negative effects on off-target biota and the environment. Although RNA interference (RNAi) is a tool that is becoming more widely utilized in pest control products, naked dsRNA has limited success in most taxa. Nanocarriers have shown promising results in enhancing the efficacy of this tool. In this study, we used a layer-by-layer electrostatic assembly where we synthesized poly(acrylic acid) (PAA)-coated hydroxyapatite (HA) nanoparticles (PAA-HA NPs) as inorganic nanocarriers, which were then coated with a layer of a cationic poly(amino acid), 10 kDa poly-l-arginine (PLR), to allow for binding of a layer of negatively charged dsRNA. Binding of PLR-PAA-HA NPs to dsRNA was found to increase as the mass ratio of NPs to dsRNA increased. In vitro studies with transgenic SF9 cells (from ) expressing the firefly gene showed a significant gene silencing (35% decrease) at a 5:1 NP-to-dsRNA ratio, while naked dsRNA was ineffective at gene silencing. There was a significant concentration-response relationship in knockdown; however, cytotoxicity was observed at higher concentrations. Confocal microscopy studies showed that dsRNA from PLR-PAA-HA NPs was not localized within endosomes, while naked dsRNA appeared to be entrapped within the endosomes. Overall, polymer-functionalized HA nanocarriers enabled dsRNA to elicit gene knockdown in cells, whereas naked dsRNA was not effective in causing gene knockdown.
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http://dx.doi.org/10.1021/acs.jafc.0c02182DOI Listing
June 2020

Association of Aβ with ceramide-enriched astrosomes mediates Aβ neurotoxicity.

Acta Neuropathol Commun 2020 04 28;8(1):60. Epub 2020 Apr 28.

Department of Physiology, University of Kentucky College of Medicine, 800 Rose Street Room MS519, Lexington, KY, 40536, USA.

Amyloid-β (Aβ) associates with extracellular vesicles termed exosomes. It is not clear whether and how exosomes modulate Aβ neurotoxicity in Alzheimer's disease (AD). We show here that brain tissue and serum from the transgenic mouse model of familial AD (5xFAD) and serum from AD patients contains ceramide-enriched and astrocyte-derived exosomes (termed astrosomes) that are associated with Aβ. In Neuro-2a cells, primary cultured neurons, and human induced pluripotent stem cell-derived neurons, Aβ-associated astrosomes from 5xFAD mice and AD patient serum were specifically transported to mitochondria, induced mitochondrial clustering, and upregulated the fission protein Drp-1 at a concentration corresponding to 5 femtomoles Aβ/L of medium. Aβ-associated astrosomes, but not wild type or control human serum exosomes, mediated binding of Aβ to voltage-dependent anion channel 1 (VDAC1) and subsequently, activated caspases. Aβ-associated astrosomes induced neurite fragmentation and neuronal cell death, suggesting that association with astrosomes substantially enhances Aβ neurotoxicity in AD and may comprise a novel target for therapy.
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http://dx.doi.org/10.1186/s40478-020-00931-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7189561PMC
April 2020

Aggregates of RNA Binding Proteins and ER Chaperones Linked to Exosomes in Granulovacuolar Degeneration of the Alzheimer's Disease Brain.

J Alzheimers Dis 2020 ;75(1):139-156

Institute of Neuropathology, RWTH Aachen University Medical School, Aachen, Germany.

Granulovacuolar degeneration (GVD) occurs in Alzheimer's disease (AD) brain due to compromised autophagy. Endoplasmic reticulum (ER) function and RNA binding protein (RBP) homeostasis regulate autophagy. We observed that the ER chaperones Glucose - regulated protein, 78 KDa (GRP78/BiP), Sigma receptor 1 (SigR1), and Vesicle-associated membrane protein associated protein B (VAPB) were elevated in many AD patients' subicular neurons. However, those neurons which were affected by GVD showed lower chaperone levels, and there was only minor co-localization of chaperones with GVD bodies (GVBs), suggesting that neurons lacking sufficient chaperone-mediated proteostasis enter the GVD pathway. Consistent with this notion, granular, incipient pTau aggregates in human AD and pR5 tau transgenic mouse neurons were regularly co-localized with increased chaperone immunoreactivity, whereas neurons with mature neurofibrillary tangles lacked both the chaperone buildup and significant GVD. On the other hand, APP/PS1 (APPswe/PSEN1dE9) transgenic mouse hippocampal neurons that are devoid of pTau accumulation displayed only few GVBs-like vesicles, which were still accompanied by prominent chaperone buildup. Identifying a potential trigger for GVD, we found cytoplasmic accumulations of RBPs including Matrin 3 and FUS as well as stress granules in GVBs of AD patient and pR5 mouse neurons. Interestingly, we observed that GVBs containing aggregated pTau and pTDP-43 were consistently co-localized with the exosomal marker Flotillin 1 in both AD and pR5 mice. In contrast, intraneuronal 82E1-immunoreactive amyloid-β in human AD and APP/PS1 mice only rarely co-localized with Flotillin 1-positive exosomal vesicles. We conclude that altered chaperone-mediated ER protein homeostasis and impaired autophagy manifesting in GVD are linked to both pTau and RBP accumulation and that some GVBs might be targeted to exocytosis.
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http://dx.doi.org/10.3233/JAD-190722DOI Listing
January 2020

VAPB ER-Aggregates, A Possible New Biomarker in ALS Pathology.

Cells 2020 01 9;9(1). Epub 2020 Jan 9.

Department of Medical Surgical and Experimental Sciences, University of Sassari, Viale San Pietro 8, 07100 Sassari, Italy.

A point mutation (P56S) in the gene-encoding vesicle-associated membrane-protein-associated protein B (VAPB) leads to an autosomal-dominant form of amyotrophic lateral sclerosis (ALS), classified as ALS-8. The mutant VAPB is characterized by ER-associated aggregates that lead to a complete reorganization of ER structures. Growing evidences suggest VAPB involvement in ALS pathomechanisms. In fact, numerous studies demonstrated VAPB alteration also in sporadic ALS (sALS) and showed the presence of its aggregates when others ALS-related gene are mutant. Recently, the identification of new biomarkers in peripheral blood mononuclear cells (PBMCs) has been proposed as a good noninvasive option for studying ALS. Here, we evaluated VAPB as a possible ALS pathologic marker analyzing PBMCs of sALS patients. Immunofluorescence analysis (IFA) showed a peculiar pattern of VAPB aggregates in sALS, not evident in healthy control (HC) subjects and in Parkinson's disease (PD) PBMCs. This specific pattern led us to suppose that VAPB could be misfolded in sALS. The data indirectly confirmed by flow cytometry assay (FCA) showed a reduction of VAPB fluorescent signals in sALS. However, our observations were not associated with the presence of a genetic mutation or altered gene expression of VAPB. Our study brings further evidences of the VAPB role in ALS as a diagnostic biomarker.
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http://dx.doi.org/10.3390/cells9010164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017080PMC
January 2020

Visualization of Ceramide-Associated Proteins in Ceramide-Rich Platforms Using a Cross-Linkable Ceramide Analog and Proximity Ligation Assays With Anti-ceramide Antibody.

Front Cell Dev Biol 2019 16;7:166. Epub 2019 Aug 16.

Department of Physiology, University of Kentucky, Lexington, KY, United States.

Ceramide-rich platforms (CRPs) mediate association of proteins with the sphingolipid ceramide and may regulate protein interaction in membrane contact sites to the cytoskeleton, organelles, and infectious pathogens. However, visualization of ceramide association to proteins is one of the greatest challenges in understanding the cell biology of ceramide. Here we introduce a novel labeling technique for ceramide-associated proteins (CAPs) by combining photoactivated cross-linking of a bioorthogonal and bifunctional ceramide analog, pacFACer with proximity ligation assays (PLAs). pacFACer cross-linked to CAPs is covalently attached to a fluorophore using click chemistry. PLAs use antibodies to: (1) the candidate CAP and the fluorophore (PLA1); and (2) the CAP and ceramide (PLA2). PLA1 shows the subcellular localization of a particular CAP that is cross-linked to pacFACer, while PLA2 tests if the cross-linked CAP forms a complex with endogenous ceramide. Two proteins, tubulin and voltage-dependent anion channel 1 (VDAC1), were cross-linked to pacFACer and showed PLA signals for a complex with ceramide and pacFACer, which were predominantly colocalized with microtubules and mitochondria, respectively. Binding of tubulin and VDAC1 to ceramide was confirmed by coimmunoprecipitation assays using anti ceramide antibody. Cross-linking to pacFACer was confirmed using click chemistry-mediated attachment of biotin and streptavidin pull-down assays. Inhibition of ceramide synthases with fumonisin B1 (FB1) reduced the degree of pacFACer cross-linking and complex formation with ceramide, while it was enhanced by amyloid beta peptide (Aβ). Our results show that endogenous ceramide is critical for mediating cross-linking of CAPs to pacFACer and that a combination of cross-linking with PLAs (cross-link/PLA) is a novel tool to visualize CAPs and to understand the regulation of protein interaction with ceramide in CRPs.
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http://dx.doi.org/10.3389/fcell.2019.00166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6706757PMC
August 2019

Ceramide regulates interaction of Hsd17b4 with Pex5 and function of peroxisomes.

Biochim Biophys Acta Mol Cell Biol Lipids 2019 10 5;1864(10):1514-1524. Epub 2019 Jun 5.

Department of Physiology, University of Kentucky, Lexington, KY, United States of America. Electronic address:

The sphingolipid ceramide regulates beta-oxidation of medium and long chain fatty acids in mitochondria. It is not known whether it also regulates oxidation of very long chain fatty acids (VLCFAs) in peroxisomes. Using affinity chromatography, co-immunoprecipitation, and proximity ligation assays we discovered that ceramide interacts with Hsd17b4, an enzyme critical for peroxisomal VLCFA oxidation and docosahexaenoic acid (DHA) generation. Immunocytochemistry showed that Hsd17b4 is distributed to ceramide-enriched mitochondria-associated membranes (CEMAMs). Molecular docking and in vitro mutagenesis experiments showed that ceramide binds to the sterol carrier protein 2-like domain in Hsd17b4 adjacent to peroxisome targeting signal 1 (PTS1), the C-terminal signal for interaction with peroxisomal biogenesis factor 5 (Pex5), a peroxin mediating transport of Hsd17b4 into peroxisomes. Inhibition of ceramide biosynthesis induced translocation of Hsd17b4 from CEMAMs to peroxisomes, interaction of Hsd17b4 with Pex5, and upregulation of DHA. This data indicates a novel role of ceramide as a molecular switch regulating interaction of Hsd17b4 with Pex5 and peroxisomal function.
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http://dx.doi.org/10.1016/j.bbalip.2019.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679753PMC
October 2019

FUS pathology in ALS is linked to alterations in multiple ALS-associated proteins and rescued by drugs stimulating autophagy.

Acta Neuropathol 2019 07 1;138(1):67-84. Epub 2019 Apr 1.

Technische Universität Dresden, Center for Regenerative Therapies Dresden, Fetscherstr. 105, 01307, Dresden, Germany.

Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by motor neuron degeneration and associated with aggregation of nuclear RNA-binding proteins (RBPs), including FUS. How FUS aggregation and neurodegeneration are prevented in healthy motor neurons remain critically unanswered questions. Here, we use a combination of ALS patient autopsy tissue and induced pluripotent stem cell-derived neurons to study the effects of FUS mutations on RBP homeostasis. We show that FUS' tendency to aggregate is normally buffered by interacting RBPs, but this buffering is lost when FUS mislocalizes to the cytoplasm due to ALS mutations. The presence of aggregation-prone FUS in the cytoplasm causes imbalances in RBP homeostasis that exacerbate neurodegeneration. However, enhancing autophagy using small molecules reduces cytoplasmic FUS, restores RBP homeostasis and rescues motor function in vivo. We conclude that disruption of RBP homeostasis plays a critical role in FUS-ALS and can be treated by stimulating autophagy.
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http://dx.doi.org/10.1007/s00401-019-01998-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570784PMC
July 2019

Carbomer gel bearing methotrexate loaded lipid nanocontainers shows improved topical delivery intended for effective management of psoriasis.

Int J Biol Macromol 2018 Dec 29;120(Pt A):1322-1334. Epub 2018 Aug 29.

Shri RNS College of Pharmacy, Gormi, Bhind, MP 477660, India. Electronic address:

The present investigation reports the evaluation of potential use of Carbomer gel bearing methotrexate loaded nanostructured lipid carriers for topical application of methotrexate for possible therapy of psoriasis in comparison to solid lipid nanoparticles. These were evaluated for various parameters such as particle size, surface charge, entrapment efficiency, shape and surface morphology, thermal analysis, in-vitro drug release through skin (Franz diffusion cell) and drug deposition study, fluorescence microscopy, particle-skin interaction study, skin-irritation testing and storage stability. The formulation (NLC) showed the best entrapment efficiency (62.72 ± 0.94%) while SLN showed only 26.84 ± 0.64% with particle size of 221 ± 14nm and 212 ± 11nm, respectively. Skin permeation study of MTX loaded SLN and NLC hydrogels showed prolonged drug release up to 24 h. The skin drug deposition study showed the greatest deposition of drug enriched NLC hydrogel (28.8%) when compared to plain drug enriched hydrogel (11.4%) and drug enriched SLN hydrogel (18.6%). Fluorescence microscopy suggested the localization effect of these lipid based systems to deeper skin region. The primary skin irritation studies indicated that MTX loaded SLN or NLC hydrogels resulted no erythema. It can be concluded that NLC represents a promising particulate carrier having prolonged drug release, improved skin permeation.
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http://dx.doi.org/10.1016/j.ijbiomac.2018.08.136DOI Listing
December 2018

Hexadecylphosphocholine (Miltefosine) stabilized chitosan modified Ampholipospheres as prototype co-delivery vehicle for enhanced killing of L. donovani.

Int J Biol Macromol 2017 Dec 15;105(Pt 1):625-637. Epub 2017 Jul 15.

Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031, India; Academy of Scientific and Innovative Research (AcSIR), New Delhi 110001, India. Electronic address:

Lipid nanoparticles are stable, biodegradable and biocompatible carriers offering excellent therapeutic efficacy. Here, a novel effort has been made to develop Miltefosine (HePC- hexadecylphosphocholine) stabilized chitosan anchored nanostructured lipid carriers (NLC) of Amphotericin B (AmB) as co-delivery vehicle to enhance killing of L. donovani. The entrapment efficiency of AmB was achieved upto 85.3% for HePC-AmB-CNLCs with mean particle size of 150.8±8.4nm, and zeta potential value of +28.2±1.1mV, respectively. The cumulative amount of AmB released at even after the 24h was less than 65% from HePC-AmB-CNLCs and Tween-80-AmB-CNLCs. Intravenous administration of HePC-AmB-CNLCs revealed the significantly increased localization of AmB in both liver and spleen when estimated. FACS study represented enhanced uptake of FITC-HePC-CNLCs over FITC-HePC-NLCs in J774A.1 cell lines. Highly significant in vitro and in vivo anti-leishmanial activity (p<0.05 compared with Tween 80-AmB-CNLCs) was observed with HePC-AmB-CNLCs when tested against VL in Leishmania donovani-infected hamsters. The haemolysis and cytotoxicity studies showed the safety of HePC-AmB-CNLCs and Tween 80-AmB-CNLCs. The findings suggested that it would be preferable to deliver AmB through HePC stabilized chitosan anchored nanostructured lipid carriers for rapid and effective treatment with decreased adverse effects.
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http://dx.doi.org/10.1016/j.ijbiomac.2017.07.076DOI Listing
December 2017

Pharmacokinetics and bioavailability assessment of Miltefosine in rats using high performance liquid chromatography tandem mass spectrometry.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Sep 26;1031:123-130. Epub 2016 Jul 26.

Pharmacokinetics and Metabolism Division, CSIR-Central Drug Research Institute, Lucknow, India; Academy of Scientific and Innovative Research (AcSIR), New Delhi, India. Electronic address:

Miltefosine (MFS) is the first effective oral drug for treatment of visceral, mucosal and cutaneous leishmaniasis. In this study, liquid chromatography coupled mass spectrometry (LC-MS/MS) method of MFS was validated in rat plasma and its practical utilization to pharmacokinetic studies in rats for the first time. A rapid, selective and sensitive LC-MS/MS method for MFS in rat plasma was linear over the calibration range of 1-500ng/mL. MFS and Phenacetin (internal standard) were separated on Phenomenex Luna 3μ HILIC 200A (150×4.6mm) column under isocratic condition using methanol: 0.1% formic acid in triple distilled water, 90:10 (v/v) mobile phase at a flow rate of 0.8mL/min. The total chromatographic run time was 4.0min. The intra- and inter-day assay accuracy was observed between 99.45-102.88% and 99.92-101.58%, respectively. The intra- and inter-day assay precision was observed between 2.68-5.54% and 2.35-5.94%, respectively. The validated assay was practically applied to determine the plasma concentrations after oral and intravenous administration of MFS to rats. After oral administration, MFS showed Cmax (3200.00±95.39ng/mL) was observed at 12.00h (tmax) and t1/2 was 102.36±16.65h. The absolute bioavailability of MFS was 60.33±2.32%.
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http://dx.doi.org/10.1016/j.jchromb.2016.07.042DOI Listing
September 2016

Computing symmetrical strength of N-grams: a two pass filtering approach in automatic classification of text documents.

Springerplus 2016 30;5(1):942. Epub 2016 Jun 30.

Department of Computer Engineering and Applications, National Institute of Technical Teachers Training & Research Bhopal, Bhopal, MP India.

The contiguous sequences of the terms (N-grams) in the documents are symmetrically distributed among different classes. The symmetrical distribution of the N-Grams raises uncertainty in the belongings of the N-Grams towards the class. In this paper, we focused on the selection of most discriminating N-Grams by reducing the effects of symmetrical distribution. In this context, a new text feature selection method named as the symmetrical strength of the N-Grams (SSNG) is proposed using a two pass filtering based feature selection (TPF) approach. Initially, in the first pass of the TPF, the SSNG method chooses various informative N-Grams from the entire extracted N-Grams of the corpus. Subsequently, in the second pass the well-known Chi Square (χ(2)) method is being used to select few most informative N-Grams. Further, to classify the documents the two standard classifiers Multinomial Naive Bayes and Linear Support Vector Machine have been applied on the ten standard text data sets. In most of the datasets, the experimental results state the performance and success rate of SSNG method using TPF approach is superior to the state-of-the-art methods viz. Mutual Information, Information Gain, Odds Ratio, Discriminating Feature Selection and χ(2).
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http://dx.doi.org/10.1186/s40064-016-2573-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4929121PMC
July 2016

Development of 4-sulfated N-acetyl galactosamine anchored chitosan nanoparticles: A dual strategy for effective management of Leishmaniasis.

Colloids Surf B Biointerfaces 2015 Dec 24;136:150-9. Epub 2015 Aug 24.

Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow 226031, India. Electronic address:

The present investigation reports the modification of chitosan nanoparticles with a ligand 4-sulfated N-acetyl galactosamine (4-SO4GalNAc) for efficient chemotherapy in leishmaniasis (SCNPs) by using dual strategy of targeting. These (SCNPs) were loaded with amphotericin B (AmB) for specific delivery to infected macrophages. Developed AmB loaded SCNPs (AmB-SCNPs) had mean particle size of 333 ± 7 nm, and showed negative zeta potential (-13.9 ± 0.016 mV). Flow cytometric analysis revealed enhanced uptake of AmB-SCNPs in J774A.1, when compared to AmB loaded unmodified chitosan NPs (AmB-CNPs). AmB-SCNPs provide significantly higher localization of AmB in liver and spleen as compared to AmB-CNPs after i.v. administration. The study stipulates that 4-SO4GalNAc assures of targeting, resident macrophages. Highly significant anti-leishmanial activity (P<0.05 compared with AmB-CNPs) was observed with AmB-SCNPs, causing 75.30 ± 3.76% inhibition of splenic parasitic burdens. AmB-CNPs and plain AmB caused only 63.89 ± 3.44% and 47.56 ± 2.37% parasite inhibition, respectively, in Leishmania-infected hamsters (P<0.01 for AmB-SCNPs versus plain AmB and AmB-CNPs versus plain AmB).
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http://dx.doi.org/10.1016/j.colsurfb.2015.08.037DOI Listing
December 2015

A taxonomic revision of some previously described species of monogenoideans found on Indian Siluriform fishes.

J Parasit Dis 2015 Sep 8;39(3):355-60. Epub 2013 Oct 8.

Department of Zoology, University of Lucknow, Lucknow, 226007 Uttar Pradesh India.

A Taxonomic revision of. Bychowskyella bagariusi Sharma, 1983 and Bychowskyella singhi Rajeshwari & Kulkarni, 1983 Jain, 1952 found on Indian Siluriform fishes is being done in the present work. These species were considered as species inquirenda or nomen nudum, (Lim et al. Syst Parasitol 50:159-197, 2001) and thus, their taxonomical clarification was required Among them, B. bagariusi Sharma, 1983 from Bagarius bagarius (Hamilton) is considered as valid and B. singhi Rajeshwari & Kulkarni, 1983 from Wallago attu (Bloch & Schneider), belongs to the genus Thaparocleidus Jain, 1952.
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http://dx.doi.org/10.1007/s12639-013-0359-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4554568PMC
September 2015

Development of docetaxel nanocapsules for improving in vitro cytotoxicity and cellular uptake in MCF-7 cells.

Drug Dev Ind Pharm 2015 10;41(11):1759-68. Epub 2015 Sep 10.

a Pharmaceutics Division , CSIR-Central Drug Research Institute , Lucknow , Uttar Pradesh , India .

The aim of this study was to fabricate docetaxel loaded nanocapsules (DTX-NCs) with a high payload using Layer-by-Layer (LbL) technique by successive coating with alternate layers of oppositely charged polyelectrolytes. Developed nanocapsules (NCs) were characterized in terms of morphology, particle size distribution, zeta potential (ζ-potential), entrapment efficiency and in vitro release. The morphological characteristics of the NCs were assessed using transmission electron microscopy (TEM) that revealed coating of polyelectrolytes around the surface of particles. The developed NCs successfully attained a submicron particle size while the ζ-potential of optimized NCs alternated between (+) 34.64 ± 1.5 mV to (-) 33.25 ± 2.1 mV with each coating step. The non-hemolytic potential of the NCs indicated the suitability of the developed formulation for intravenous administration. A comparative study indicated that the cytotoxicity of positively charged NCs (F4) was significant higher (p < 0.05) rather than negative charged NCs (F3), plain drug (DTX) and marketed preparation (Taxotere®) when evaluated in vitro on MCF-7 cells. Furthermore, cell uptake studies evidenced a higher uptake of positive NCs (≥1.2 fold) in comparison to negative NCs. In conclusion, formulated NCs are an ideal vehicle for passive targeting of drugs to tumor cells that may result in improved efficacy and reduced toxicity of encapsulated drug moiety.
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http://dx.doi.org/10.3109/03639045.2014.1003220DOI Listing
July 2016

Arteether nanoemulsion for enhanced efficacy against Plasmodium yoelii nigeriensis malaria: an approach by enhanced bioavailability.

Colloids Surf B Biointerfaces 2015 Feb 7;126:467-75. Epub 2015 Jan 7.

Pharmaceutics Division, Central Drug Research Institute, Lucknow 226031, India. Electronic address:

The present work is focused on the preparation of nanoemulsions (NEs) loaded with arteether (ART) for its enhanced efficacy against malaria parasites. ART-NEs have been prepared using high pressure homogenization (HPH) technique with the aim of improving its solubility and thus its bioavailability. ART-NEs were optimized in terms of pressure and number of cycles. Globule size and size distributions were chosen as quality parameters. The maximum drug loading was achieved up to 93 ± 7.4% with globule size 156 ± 10.2 nm and zeta potential of -23.3 ± 3.4 mV. The developed ART-NEs were found to be stable in terms of globule size and size distribution at different pH. The in vitro release profile of the ART-NEs showed 62% drug release within 12h. The percentage cell viability of blank NEs were within acceptable limits. A sensitive assay method for the determination of ART in rat plasma by liquid chromatography-mass spectrometry (LC-MS) was employed after oral administration of ART-NEs. The pharmacokinetic study showed significantly enhanced bioavailability of ART in ART-NE-V. The area under curve (AUC) of ART-NE-V was AUC0-t 1988.411 ± 119.66 h ng/ml which was significantly higher (p<0.05) than ART in ground nut oil (GNO) AUC0-t 671.852 ± 187.05 h ng/ml. The Cmax of ART-NE-V (1506 ± 161.22 ng/ml) was also significantly higher (p<0.05) than ART in GNO (175.2 ± 16.54 ng/ml) and ART given intramuscularly (IM) (278.05 ± 38.59 ng/ml). The ART-NE-V was having significantly high antimalarial efficacy and survival rate of mice giving 80% cure rate at 12.5 mg/kg for 5 days in comparison to 30% cure rate of ART in GNO at the same daily dose and it was also comparable to the 100% cure rate at 12.5 mg/kg for 5 days for ART given intramuscularly. In conclusion ART-NE can be a promising oral delivery system for ART.
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http://dx.doi.org/10.1016/j.colsurfb.2014.12.052DOI Listing
February 2015

Dual substrate strategy to enhance butanol production using high cell inoculum and its efficient recovery by pervaporation.

Bioresour Technol 2014 19;152:377-83. Epub 2013 Nov 19.

Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India. Electronic address:

The present study deals with the development of an efficient ABE fermentation process using mixed substrate strategy for butanol production wherein no acetone was produced. For this, glucose was supplemented in the medium containing glycerol as main substrate which leads to a higher butanol production of 17.75 g/L in 72 h by Clostridium acetobutylicum KF158795. Moreover, the high cell inoculum also resulted in an increased ABE productivity of 0.46 g/L/h. Further, industrial scalability of the process was also successfully validated in a 300 L fermenter. Furthermore, potential of the Polymeric (PolyRMem) and Zeolite (ZeoMem) membranes for separation of butanol from fermentation broth was also studied by testing the pervaporation performance through which the butanol was successfully recovered.
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http://dx.doi.org/10.1016/j.biortech.2013.11.022DOI Listing
August 2014

An interactive study of influential parameters for shikimic acid production using statistical approach, scale up and its inhibitory action on different lipases.

Bioresour Technol 2013 Sep 4;144:675-9. Epub 2013 Jul 4.

Department of Microbiology, University of Delhi South Campus, New Delhi, India.

Shikimic acid is the promising candidate as a building block for the industrial synthesis of drug Tamiflu used for the treatment of Swine flu. The fermentative production process using microbes present an excellent and even more sustainable alternative to the traditional plants based extraction methods. In the present study, the fermentative production of shikimic acid by Citrobacter freundii GR-21 (KC466031) was optimized by process engineering using a statistical modeling approach and a maximum amount of 16.78 g L(-1) was achieved. The process was also scaled up to 14L bioreactor to validate the production of shikimic acid. Further, the potential of anti-enzymatic nature of purified shikimic acid was evaluated for different lipases wherein, shikimic acid inhibited the hydrolysis of triglycerides by 55-60%. Shikimic acid also profoundly inhibited pancreatic lipase activity by 66%, thus providing another valuable therapeutic aspect for treating diet induced obesity in humans.
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http://dx.doi.org/10.1016/j.biortech.2013.06.113DOI Listing
September 2013

Expanding horizons of shikimic acid. Recent progresses in production and its endless frontiers in application and market trends.

Appl Microbiol Biotechnol 2013 May 5;97(10):4277-87. Epub 2013 Apr 5.

Department of Microbiology, University of Delhi South Campus, New Delhi 110021, India.

Shikimic acid is an industrially important chiral compound used as a key ingredient in formulation of drug Oseltamivir phosphate (Tamiflu) for the treatment of swine/avian flu. The high cost and limited availability of shikimic acid isolated from plants has detained the use of this valuable building block of the drug. It is a versatile compound having many characteristic properties for many synthetic reactions particularly in pharmaceuticals and cosmetic industries. By virtue of being a natural product, the relevant biochemical pathway in microorganisms can be harnessed into fermentation processes to produce shikimic acid. This is an excellent alternative for the sustainable and efficient production of shikimic acid over the tedious and cumbersome process of plant based extraction methods. Various strategies of shikimic acid production are reviewed and an account of comparison of their challenges, promises and restraint is presented. Furthermore, present review attempts to focus on the market trend of shikimic acid due to its high demand with particular emphasis laid on the pandemics of swine flu. This review not only covers the recent advances in shikimic acid production but also highlights the versatile applications and its market scenario. The concluding remarks and its potential as a commercial bulk chemical are discussed in the light of current research.
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http://dx.doi.org/10.1007/s00253-013-4840-yDOI Listing
May 2013

Fermentative production of shikimic acid: a paradigm shift of production concept from plant route to microbial route.

Bioprocess Biosyst Eng 2013 Nov 31;36(11):1665-73. Epub 2013 Mar 31.

Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.

Different physiological and nutritional parameters affect the fermentative production of shikimic acid. In our study, Citrobacter freundii initially produced 0.62 g/L of shikimic acid in 72 h. However, when process optimization was employed, 5.11 g/L of shikimic acid was produced in the production medium consisting of glucose (5.0 %), asparagine (4.5 %), CaCO3 (2.0 %), at pH 6.0, when inoculated with 6 % inoculum and incubated at 30 ± 1 °C, 200 rpm for 60 h. Preliminary fed-batch studies have resulted in the production of 9.11 g/L of shikimic acid on feeding the production medium by 20 g/L of glucose at 24 h of the fermentation run. Production of similar amount of shikimic acid was observed when the optimized conditions were employed in a 10-L bioreactor as obtained in shake flask conditions. A total of 9.11 g/L of shikimic acid was produced in 60 h. This is approximately 14.69-fold increase in shikimic acid production when compared to the initial un-optimized production conditions. This has also resulted in the reduction of the production time. The present study provides useful information to the industrialists seeking environmentally benign technology for the production of bulk biomolecules through manipulation of various chemical parameters.
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http://dx.doi.org/10.1007/s00449-013-0940-4DOI Listing
November 2013

A natural isolate producing shikimic acid: isolation, identification, and culture condition optimization.

Appl Biochem Biotechnol 2013 Apr 27;169(8):2290-302. Epub 2013 Feb 27.

Department of Microbiology, University of Delhi South Campus, New Delhi, 110021, India.

Shikimic acid has wide use in pharmaceuticals due to its application in the synthesis of drug Tamiflu used in the treatment of Swine flu. The high cost and limited availability of shikimic acid isolated from plants has impeded the use of this valuable building block of the drug. In this context, fermentation route to produce shikimic acid from renewable resources has become increasingly attractive. The present study was embarked upon isolation of wild-type microorganisms able to produce shikimic acid. Out of the 42 isolates obtained from the soil, isolate GR-21 was selected as the best with initial production of 0.54 g/L shikimic acid and later identified as Citrobacter sp. The process optimization resulted in 14-fold increase in the shikimic acid production, thereby claiming this process to be a sustainable alternative for the production of this important biomolecule. The process was further scaled up to 14 L bioreactor to validate the production of shikimic acid. Further, the product formed is shikimic acid was confirmed by FTIR analysis. The current studies suggest that the selected isolate could be used as a promising agent to fulfill the worldwide demand of shikimic acid.
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http://dx.doi.org/10.1007/s12010-013-0150-1DOI Listing
April 2013

Exploiting 4-sulphate N-acetyl galactosamine decorated gelatin nanoparticles for effective targeting to professional phagocytes in vitro and in vivo.

J Drug Target 2012 Dec 1;20(10):883-96. Epub 2012 Oct 1.

Pharmaceutics Division, CSIR-Central Drug Research Institute, Lucknow, India.

The present study was focused on the development of surface modified gelatin nanoparticles (SGNPs) using novel ligand 4-sulfated N-acetyl galactosamine (4-SO(4)GalNAc) for specific targeting to macrophages. The gelatin has been modified with the potential targeting moiety 4-SO(4)GalNAc, which was further used for the preparation of modified nanoparticles. The nanoparticles have been prepared by two step desolvation method. The SGNPs and unmodified gelatin nanoparticles (GNPs) were loaded with doxorubicin (DxR) and its targeting potential was compared. Developed DxR-loaded SGNPs (DxR-SGNPs) were found to have negative zeta potential (-19.8 ± 0.22 mV) whereas DxR-loaded GNPs (DxR-GNPs) have the positive zeta potential of around +12.2 ± 0.36 mV. The mean particle size of DxR-SGNPs and DxR-GNPs was found to be 283 ± 7 and 134 ± 5 nm, respectively. Flow cytometric data confirmed the enhanced uptake of DxR-SGNPs in J774A.1 and PBMC when compared with DxR-GNPs. Intracellular localization studies indicate that the fluorescence intensity of DxR-SGNPs was significantly higher when compared to DxR-GNPs. DxR-SGNPs rendered significantly higher localization of DxR in liver and spleen as compared to DxR-GNPs after i.v. administration. The study stipulates that 4-SO(4)GalNAc assures for targeting resident macrophages.
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http://dx.doi.org/10.3109/1061186X.2012.725169DOI Listing
December 2012

Anti-inflammatory activity of Abutilon indicum extract.

Nat Prod Res 2012 14;26(17):1659-61. Epub 2011 Oct 14.

Shri Ramnathsingh Mahavidyalaya-Pharmacy, Gormi, Bhind 477660, Madhya Pradesh, India.

Abutilon indicum Linn. had been broadly used for its reported biological activities in indigenous system of medicine. The ethanolic extract of the whole plant of A. indicum Linn. was evaluated for its anti-inflammatory activity at doses 250, 500 and 750 mg kg⁻¹ using the carrageenan-induced paw oedema in healthy Wistar albino rats. Results of in vivo activity led to the conclusion that the ethanolic extract of A. indicum showed predominantly significant activity in a dose-dependent manner, which is comparable to the reference standard ibuprofen. The results prove the traditional use of plant in the treatment of inflammation.
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http://dx.doi.org/10.1080/14786419.2011.616508DOI Listing
December 2012