Geeta Rai

The importance of cellular localisation of probes: synthesis, photophysical properties, DNA interactions and cellular imaging properties of rhenium dppz complexes with known cellular localisation vectors

The synthesis, photophysical properties, DNA binding, DNA cleavage and cellular imaging behaviour of a range of complexes of the type [Re(CO)3(dppz)(PyR)]+ are reported, where PyR represents a range of substituted pyridines which have previously been studied for cellular localisation of related complexes. Confocal imaging experiments confirm that the complexes retain the variety of cellular localisation behaviour associated with the PyR units in other complexes, and suggest applications as probes for oligonucleotides in specific cellular compartments (e.g. mitochondrial DNA). This study illustrates the importance of considering cellular localisation as a prime consideration in the design of probes for in vivo application.

Distinct Autoantibody Profiles in Systemic Lupus Erythematosus Patients are Selectively Associated with TLR7 and TLR9 Upregulation

PurposeSystemic lupus erythematosus (SLE) patients have a wide array of autoantibodies against nuclear antigens. The two predominant classes of these autoantibodies are directed either against dsDNA or RNA-associated antigens (extractable nuclear antigens; ENA). Nucleic-acid sensing Toll-like receptors (TLRs) that recognize dsDNA and RNA, have been well implicated in some murine models of SLE. We took up this study to identify if unique TLR expression patterns are associated with distinct autoantibody profiles in SLE.MethodsWe segregated the patients into three subsets distinguished on the basis of autoantibody response either against dsDNA or ENA or both. We determined the mRNA expression of TLR3, 7, 8, and 9 by real-time reverse-transcription PCR in peripheral blood leucocytes (PBLs) of the SLE patients of all three subsets. TLR7 and 9 protein expression was determined by western blotting in PBLs and by flow cytometry on B-cells and monocytes. The serum interferon-alpha (IFN-α) and anti-dsDNA/-ENA autoantibodies were detected using enzyme-linked immunosorbant assay.ResultsWe report differential and unique TLR expression patterns associated with different autoantibody profiles. The presence of anti-ENA and anti-dsDNA autoantibodies in SLE patients was associated with elevated levels of TLR7 and TLR9 respectively. The TLR9 mRNA expression was further augmented in SLE patients with Glomerulonephritis. Interestingly, anti-dsDNA+ ENA+ patients displayed higher serum IFN-α and interferon regulatory factor 7 mRNA expression than patients with either anti-dsDNA or anti-ENA autoantibodies alone.ConclusionCharacteristic TLRs expression profile associated with distinct autoantibody repertoire is suggestive of differential immuno-regulatory pathways operative in different subsets of SLE patients.

Differential microRNA Profile and Post-Transcriptional Regulation Exist in Systemic Lupus Erythematosus Patients with Distinct Autoantibody Specificities

PurposeSystemic lupus erythematosus (SLE) patients have anti-nuclear autoantibodies directed against dsDNA and RNA-associated antigens (extractable nuclear antigens; ENA). In this study, we investigated the differences in microRNA (miRNA) expression and its biological implications in SLE patients with distinct autoantibody specificities.MethodsThe SLE patients were grouped into three subsets based on the type of autoantibodies present in their sera (anti-ENA+ group with autoantibodies against ENA alone; anti-dsDNA+ group having autoantibodies against dsDNA only, and anti-ENA+dsDNA+ group having autoantibodies to both dsDNA and ENA). Global miRNA expression profiling was done for each of these three groups using TaqMan® low density miRNA arrays.ResultsWe report that different sets of miRNAs are dysregulated in SLE patients with different autoantibody specificities. Further, Ingenuity pathway analysis (IPA) software revealed specific biological pathways that were targeted by miRNAs dysregulated in different SLE subsets. Molecules involved in cell cycle and cytoskeleton remodeling were the prime targets of miRNAs dysregulated in anti-ENA+ patients whereas miRNAs dysregulated in anti-dsDNA+ patients were found to be implicated in multiple cytokine signaling pathways. IPA analysis of gene targets of miRNAs commonly dysregulated in all three SLE subsets identified several metabolic-, hormone-, and interferon-related pathways to be affected.ConclusionThe differential miRNA expression in patients with distinct autoantibodies is suggestive of different regulatory mechanisms operating among them. Based on these observations, we are hopeful that this ‘sub-grouping’ approach could be used to identify other defective processes associated with varying disease manifestations in SLE and may be considered when designing therapeutic interventions.

Potential antianxiety activity of Fumaria indica: A preclinical study

Background: In the view of diverse CNS modulating properties of Fumaria indica, present study was planned to evaluate its putative anxiolytic activity in behavioural models of rats, followed by elucidation of mechanism of observed activity through biochemical estimations. Materials and Methods: Effects of seven daily 100, 200 and 400 mg/kg oral doses of a Fumaria indica extract (FI) was compared with those of an acute oral dose (5 mg/kg) of lorazepam in a battery of rat models consisting of open-field, elevated plus and zero maze, social interaction, and novelty induced feeding tests. Results: Dose dependant antianxiety effects of FI observed in all tests were qualitatively similar to those of the reference anxiolytic drug. Although FI treatments did not alter the concentrations of noradrenaline and serotonin in hippocampus and hypothalamus, concentrations of both these monoamines were dose dependently elevated in prefrontal cortex of FI treated animals. Flunitrazepam binding in brain frontal cortex was also elevated by the extract. Moreover, higher levels of brain expressions of the cytokines TNF-α, IL-1β, and IL-10 observed in animals with prior experience on elevated plus maze were almost completely reversed by the lowest dose of FI tested in the behavioral models. Conclusion: Taken together, these observations strongly suggest that FI is a functionally novel type of antianxiety agent, and that inhibition of cytokine expressions in the brain could be involved in its mode of action.

Decreased Pattern Recognition Receptor Signaling, Interferon-Signature, and Bactericidal/Permeability-Increasing Protein Gene Expression in Cord Blood of Term Low Birth Weight Human Newborns

Background Morbidity and mortality rates of low birth weight (LBW) newborns at term are higher than rates in normal birth weight (NBW) newborns. LBW newborns are at greater risk to acquire recurrent bacterial and viral infections during their first few weeks of life possibly as an outcome of compromised innate immune functions. As adaptive immunity is in a naive state, increased risk of infection of LBW as compared to NBW newborns may reflect impairments in innate immunity. Methodology To characterize the increased susceptibility to infections in LBW newborns we used microarray technology to identify differences in gene expression in LBW newborns (n = 8) compared to NBW newborns (n = 4) using cord blood. The results obtained from the microarray study were validated on a larger number of samples using real time RT-PCR (LBW = 22, NBW = 18) and western blotting (LBW = 12, NBW = 12). The Interferome database was used to identify interferon (IFN) signature genes and ingenuity pathway analysis identified canonical pathways and biological functions associated with the differentially expressed genes in LBW newborns. ELISAs for IFNs and bactericidal/permeability-increasing protein were performed in both LBW and NBW newborns and in adults (LBW = 18, NBW = 18, Adults  = 8). Principal Findings Upon microarray analysis, we identified 1,391 differentially expressed genes, of which, 1,065 genes were down-regulated and 326 genes were up-regulated in the LBW compared to NBW newborns. Of note, 70 IFN-signature genes were found to be significantly down-regulated in LBW compared to NBW newborns. Ingenuity pathway analysis revealed pattern recognition receptors signaling including Toll-Like Receptors (TLRs) -1, -5, and -8 genes and IFN signaling as the most significantly impacted pathways. Respiratory infectious diseases were the most significantly affected bio-functions in LBW newborns. Conclusion and Significance Diminished PRRs, IFN-signature, and BPI gene expression raises the possibility that impairments in these pathways contribute to the susceptibility of LBW term infants to infection.

Beneficial effects of an Andrographis paniculata extract and andrographolide on cognitive functions in streptozotocin-induced diabetic rats.

Abstract Context Andrographolide containing Andrographis paniculata (Burm. F.) Wall. Ex Nees (Acanthaceae) extracts is often used for treatments of diabetes and other inflammatory disorders commonly accompanying cognitive and other psychiatric disorders. Objective To compare the efficacies of a standardised A. paniculata extract (AP) and pure andrographolide on cognitive functions, oxidative stress and cholinergic function in diabetic rats. Materials and methods Streptozotocin-induced diabetic Charles Foster albino rats treated orally with a hydro-methanolic A. paniculata leaf extract (50, 100 and 200 mg/kg/day), or with pure andrographolide (15, 30 and 60 mg/kg/day) for 10 consecutive days, were subjected to Morris water maze test. After the test, acetylcholinesterase, superoxide dismutase (SOD), and catalase (CAT) activities and lipid peroxidation (LPO) in brain tissues were assessed. Results Acetylcholinesterase activity in pre-frontal cortex and hippocampus of diabetic rats was 2.1 and 2.6 times higher compared to nondiabetic rats. LPO was 1.6 times higher and decreased SOD (56.3%) and CAT (44.9%) activities in pre-frontal cortex of diabetic rats compared to nondiabetic rats. AP or andrographolide treatments dose dependently attenuated cognitive deficits, reduced acetylcholinesterase activity, oxidative stress, improved diabetic hyperglycemia and insulin deficiency. All observed effects of AP were quantitatively almost equal to those expected from its analytically quantified andrographolide content. Discussion and conclusion Reported observations are the very first ones suggesting beneficial effects of andrographolide against diabetes associated cognitive deficits, increased acetylcholinesterase activity and deteriorated antioxidative status. Efforts to exploit A. paniculata extracts enriched in andrographolide as preventive measures against such disorders can be warranted.

RNA-seq Analysis Reveals Unique Transcriptome Signatures in Systemic Lupus Erythematosus Patients with Distinct Autoantibody Specificities

Systemic lupus erythematosus (SLE) patients exhibit immense heterogeneity which is challenging from the diagnostic perspective. Emerging high throughput sequencing technologies have been proved to be a useful platform to understand the complex and dynamic disease processes. SLE patients categorised based on autoantibody specificities are reported to have differential immuno-regulatory mechanisms. Therefore, we performed RNA-seq analysis to identify transcriptomics of SLE patients with distinguished autoantibody specificities. The SLE patients were segregated into three subsets based on the type of autoantibodies present in their sera (anti-dsDNA+ group with anti-dsDNA autoantibody alone; anti-ENA+ group having autoantibodies against extractable nuclear antigens (ENA) only, and anti-dsDNA+ENA+ group having autoantibodies to both dsDNA and ENA). Global transcriptome profiling for each SLE patients subsets was performed using Illumina® Hiseq-2000 platform. The biological relevance of dysregulated transcripts in each SLE subsets was assessed by ingenuity pathway analysis (IPA) software. We observed that dysregulation in the transcriptome expression pattern was clearly distinct in each SLE patients subsets. IPA analysis of transcripts uniquely expressed in different SLE groups revealed specific biological pathways to be affected in each SLE subsets. Multiple cytokine signaling pathways were specifically dysregulated in anti-dsDNA+ patients whereas Interferon signaling was predominantly dysregulated in anti-ENA+ patients. In anti-dsDNA+ENA+ patients regulation of actin based motility by Rho pathway was significantly affected. The granulocyte gene signature was a common feature to all SLE subsets; however, anti-dsDNA+ group showed relatively predominant expression of these genes. Dysregulation of Plasma cell related transcripts were higher in anti-dsDNA+ and anti-ENA+ patients as compared to anti-dsDNA+ ENA+. Association of specific canonical pathways with the uniquely expressed transcripts in each SLE subgroup indicates that specific immunological disease mechanisms are operative in distinct SLE patients’ subsets. This ‘sub-grouping’ approach could further be useful for clinical evaluation of SLE patients and devising targeted therapeutics.

Microarray to deep sequencing: transcriptome and miRNA profiling to elucidate molecular pathways in systemic lupus erythematosus.

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease with diverse clinical manifestations and autoantibody repertoires. The etiology of SLE is multifactorial involving genetic, epigenetic and environmental factors. This complexity leads to poor prognosis, which poses major challenges in the treatment of SLE. Understanding the complex genetic pathways and regulatory mechanisms operative in SLE was feasible by utilizing several highly efficient molecular biological tools during the past few years. In this perspective, DNA microarray technology offered a high-throughput platform in unraveling SLE-associated genes. Additionally, extensive microarray analysis had demonstrated aberrant DNA methylation pattern and differential microRNAs, thus contributing to the knowledge of epigenetic modulators and posttranscriptional regulatory machinery in SLE. It was through the aid of these technologies that interferon signature was identified as an important contributor in SLE pathogenesis along with dysregulation of cytokine-, chemokine- and apoptosis-related genes. The emergence of next-generation sequencing technologies such as RNA sequencing has added new dimensions in understanding the dynamics of the disease processes. Compared with microarrays, deep sequencing has provided higher resolution in gene expression measurement along with identification of different splicing events, noncoding RNAs and novel loci in SLE. The focus, therefore, has now been shifted toward the identification of novel gene loci and their isoforms, and their implication in disease pathogenesis. This advancement in the technology from microarray to deep sequencing has helped in deciphering the molecular pathways involved in pathogenesis of SLE and opens new avenues to develop novel treatment strategies for SLE.

Effects of ethanolic extract of Fumaria indica L. on rat cognitive dysfunctions.

Fumaria indica L. in Ayurveda is known as Parpat and traditionally used to calm the brain. Due to lack of scientific validation, 50% ethanolic extract of F. indica L. (FI) was evaluated for putative cognitive function modulating effects. Suspension of FI in 0.3% carboxymethyl cellulose (CMC) was orally administered to rats during the entire experimental period of 16 days at dose levels of 100, 200, and 400 mg/kg/day. Piracetam was used as standard nootropic. Behavioral models of learning and memory used were modified elevated plus-maze (M-EPM) and passive avoidance (PA) tests. Scopolamine (1 mg/kg, s.c.), sodium nitrite (25 mg/kg, i.p.), and electroconvulsive shock (150 mA, 0.2 sec) were used to induce amnesia . Acetylcholinesterase (AChE) activity, muscarinic receptor density, oxidative status, and cytokine expressions [tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-10] were also assessed. Piracetam (500 mg/kg/day)-like memory-enhancing and anti-amnesic activity of the extract was observed. FI showed dose-dependent decrease in brain AChE activity and increase in muscarinic receptor density, and such was also the case for its observed beneficial effects on the brain antioxidative status. FI also inhibited the scopolamine-induced overexpression of the three tested cytokines observed in rat′s brain. FI possesses nootropic-like beneficial effects on cognitive functions.

Differential clearance mechanisms, neutrophil extracellular trap degradation and phagocytosis, are operative in systemic lupus erythematosus patients with distinct autoantibody specificities.

Systemic lupus erythematosus (SLE) patients are generally presented with autoantibodies against either dsDNA or RNA-associated antigens (also known as extractable nuclear antigens, ENA) or both. However, the mechanisms and processes that lead to this distinctive autoantibody profile are not well understood. Defects in clearance mechanism i.e. phagocytosis may lead to enhanced microbial and cellular debris of immunogenic potential. In addition to defective phagocytosis, impaired neutrophil extracellular trap (NET) degradation has been recently reported in SLE patients. However, the extent to which both these clearance processes (NET-degradation and phagocytosis) are operative in serologically distinguished subsets of SLE patients is not established. Therefore, in this report, we evaluated NET-degradation and phagocytosis efficiency among SLE patients with different autoantibody specificities. SLE patients were classified into three subsets based on their autoantibody profile (anti-dsDNA, anti-ENA or both) as determined by ELISA. NET-degradation by SLE and control sera was assessed by sytox orange-based fluorescence assay. Neutrophil-mediated phagocytosis in the presence of SLE and control sera was determined by flowcytometry. The segregation of SLE patients revealed significant differences in NET-degradation and phagocytosis in SLE patients with autoantibodies against dsDNA and ENA. We report that NET-degradation efficiency was significantly impaired in SLE patients with anti-dsDNA autoantibodies and not in those with anti-ENA autoantibodies. In contrast to NET-degradation, neutrophil-mediated phagocytosis was impaired in all three subsets independent of autoantibody specificity. These observations suggest that varying clearance mechanisms are operative in SLE subsets with anti-dsDNA or anti-ENA autoantibodies. The results outlined in this manuscript also suggest that sub-grouping of SLE patients could be useful in delineating the molecular and pathological processes that are often missed when SLE patients are studied as a single group. Further, it will be imperative to propose that therapies targeted at improving NET clearance can be effective in anti-dsDNA(+) SLE patients.