Differential Activation of Microglia in an in vitro Model of Intracerebral Hemorrhage

An in vitro model of intracerebral hemorrhage was established to examine the protective versus cytotoxic roles of microglia in the context of mild versus severe injury. Co-cultures of microglia, astrocytes, and granule neurons were prepared from the cerebellar cortex of neonatal rats, and grown in standard medium containing fetal bovine serum or, in some cases, a serum-free chemically defined medium. To mimic hemorrhagic stroke, co-cultures grown for 7-8 days in vitro were challenged with two different concentrations of the toxic blood product hemin, corresponding to a mild versus a severe brain bleed. Immunocyto-chemical, real-time RT-PCR, iron deposition, and cell survival assays were performed to assess the effects of hemin, with emphasis on the functional effector states of microglia. In cultures grown in serum-containing medium, the lower concentration of hemin (20 mM) induced significant expression of the protective enzyme, heme oxygenase 1 (HO-1). Consistent with this, iron deposition was localized to the microglia in the cultures. Only a modest induction of the inflammatory molecule TNF-a, and no significant cell death was observed 24 hrs after hemin addition. In contrast, the higher concentration of hemin (100 mM) induced greater expression of HO-1, and iron deposition was detected in all three cell types in the cultures. Moreover, significant and robust induction of the inflammatory molecules TNF-a, COX-2, iNOS, and significant neuronal death were observed. These results suggest that microglia are protective and serve to effectively catabolize hemin when exposed to 20 mM hemin. However, increasing the hemin concentration to 100 mM appears to exceed the capacity of the microglia to effectively catabolize the hemin load, resulting in an inflammatory and cytotoxic environment. Serum proteins, in particular albumin and hemopexin, can bind blood products such as hemin, and slowly release them to be processed by microglia after a brain bleed. To test the possible protective effect of albumin, co-cultures grown in a chemically defined medium lacking serum were exposed to hemin as described above, with or without co-addition of an equivalent amount of albumin. In these cultures, significant neuronal death was observed in response to 20 mM hemin, with essentially total cell death in response to 100 mM hemin. Addition of albumin to the cultures significantly decreased the amount of neuronal death in response to both concentrations of hemin. These data suggest that addition of agents that bind toxic blood products may serve as protective agents after hemorrhagic stroke. In future studies, this in vitro model should prove valuable in characterizing the signaling pathways underlying distinct functional effector states of microglia, and developing strategies to preserve the protective phenotype

Identification and analysis of the synergistic targets of TBX5 and GATA-4

Cardiac development is a complex multi-step process involving a diverse network of genes. Defects in cardiac genes can lead to congenital heart defects (CHDs), and understanding the processes involved in normal cardiogenesis is crucial in elucidating the pathogenesis of disease. Mutations in a number of cardiac transcription factors have been associated with CHDs, including TBX5 and GATA-4. These transcription factors are high in the regulatory hierarchy and form a complex that is thought to direct and synergistically regulate common cardiac pathways. However, little is currently known about their joint targets. This study aimed to identify and analyse genes important in cardiogenesis, with focus on the combined targets of TBX5 and GAT A-4. Microarray expression analysis of TBX5/ GATA-4 double overexpression P19 cell lines led to the identification of a large number of genes, of which seven were selected for study; PA2.26, PETA-3, FUCA 1, FN, TPM1, DES, and RBMS1. Expression of these was confirmed in the embryonic chick heart at Hamburger and Hamilton (HH) stages 12 - 26. The cell cycle regulator and transcription factor RBMS1 was selected for investigation of its role in cardiac development. Morpholino knockdown of RBMS1 expression in the developing chick resulted in defects in cardiac looping and atrial septation. Whilst further work is required to strengthen this data, this is a novel finding and opens up possibilities for future research into cardiac transcriptional networks. Microarray expression analysis using an in ovo model of TBX5 and GATA-4 double knockdown led to the identification of a number of interesting putative targets, including TFAP2B, GPC3, and CRABP1, which have known roles in cardiac development. TFAP2B is of particular interest as mutations in this gene are associated with the heart-hand disorder Char syndrome. LOC420770, a novel gene of unknown function, was also identified and displayed expression indicative of a heart-limb profile. Further studies to attempt elucidation of the function of this gene may provide a novel candidate gene for CHD. Investigation of these genes will form the basis of future research, contributing to our current knowledge of the vast network of genes involved in development of the heart

Identification and analysis of the synergistic targets of TBX5 and GATA-4

Cardiac development is a complex multi-step process involving a diverse network of genes. Defects in cardiac genes can lead to congenital heart defects (CHDs), and understanding the processes involved in normal cardiogenesis is crucial in elucidating the pathogenesis of disease. Mutations in a number of cardiac transcription factors have been associated with CHDs, including TBX5 and GATA-4. These transcription factors are high in the regulatory hierarchy and form a complex that is thought to direct and synergistically regulate common cardiac pathways. However, little is currently known about their joint targets. This study aimed to identify and analyse genes important in cardiogenesis, with focus on the combined targets of TBX5 and GAT A-4. Microarray expression analysis of TBX5/ GATA-4 double overexpression P19 cell lines led to the identification of a large number of genes, of which seven were selected for study; PA2.26, PETA-3, FUCA 1, FN, TPM1, DES, and RBMS1. Expression of these was confirmed in the embryonic chick heart at Hamburger and Hamilton (HH) stages 12 - 26. The cell cycle regulator and transcription factor RBMS1 was selected for investigation of its role in cardiac development. Morpholino knockdown of RBMS1 expression in the developing chick resulted in defects in cardiac looping and atrial septation. Whilst further work is required to strengthen this data, this is a novel finding and opens up possibilities for future research into cardiac transcriptional networks. Microarray expression analysis using an in ovo model of TBX5 and GATA-4 double knockdown led to the identification of a number of interesting putative targets, including TFAP2B, GPC3, and CRABP1, which have known roles in cardiac development. TFAP2B is of particular interest as mutations in this gene are associated with the heart-hand disorder Char syndrome. LOC420770, a novel gene of unknown function, was also identified and displayed expression indicative of a heart-limb profile. Further studies to attempt elucidation of the function of this gene may provide a novel candidate gene for CHD. Investigation of these genes will form the basis of future research, contributing to our current knowledge of the vast network of genes involved in development of the heart

Mouse Gesture Recognition for Human Computer Interaction

In the field of Computer Science and Information Technology, it goes without saying that the focus has been shifted from the System Oriented software to the User Oriented software. Naturally, for such software applications, the importance of User Experience has gained a paradigm shift. The paper highlights the significance of a seamless interaction between the user and the computer by proposing a reliable algorithm for performing basic operations by drawing gestures with a mouse. It aims to embrace simplicity and quick access using gestures and providing effortless interaction for the uniquely abled users. The core of the algorithm comes from the Hidden Markov Model, which is emblematic of a probabilistic approach for gesture recognition. DOI: 10.17762/ijritcc2321-8169.15050

PHYTOCHEMICAL SCREENING OF SAPTAPARNA (ALSTONIA SCHOLARIS R. Br.) BARK

The Ayurvedic Pharmaceutical Industries use barks of many herbal drugs like Neema, Vata, Udumbara, Parisha, Saptaparna, Plaksha, Baboola etc. for the manufacturing of different herbal medicines. The stem bark of Alstonia scholaris R. Br. commonly known as Saptaparna in Ayurved, is in demand, due to its antipyretic, galactogogue, cardiotonic, anticancer, anti-helminthic etc. activities. But, the identification and separation of these barks drugs from each other is very difficult. Hence, a preliminary study has been done to ensure the basic phytochemical profile of A. scholaris R. Br. stem bark for identification of herbal drug. Physicochemical parameters, preliminary phytochemical screening, quantitative estimation of alkaloid and Thin Layer Chromatography (TLC) were carried out in the present study. Physicochemical data revealed, there is more amount of water soluble extractive value (27.10% w/w) than alcohol soluble extractive value (7.40% w/w). Facts of phytochemical screening showed presents of alkaloid, carbohydrate, tannin, saponin, flavanoids and cardiotonic glycosides in the sample. Result of TLC identification showed 7, 8, and 6 spots under short UV, long UV and after spray reagent respectively. The present study on phytochemical investigation of A. scholaris R. Br. bark will be helpful in developing standards for quality, purity and sample identification of this plant

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy

Utilization of Amul Dairy effluent for agriculture practices

ABSTRACT Pot experiments were conducted to evaluate the impact of Amul dairy effluent on certain physico-chemical properties of soil and on growth, and quality of Lady's finger (Abelomoschus esculentus) and Guar (Cymopsis tetragonoloba.). The effluent used in different concentration 20%, 40%, 60%, 80% and 100%. The pH of the waste water was near about neutral but it contained an enough amount of nitrogen, phosphate, chloride, calcium, carbonates, bicarbonates and suspended and dissolved solids when compared with fresh water. Soil receiving the waste water showed no significant changes in water soluble salts, electrical conductivity, cation exchange capacity, pH, total organic carbon etc. Moreover, waste water irrigation resulted in increased growth and nutrients of both the crops

Phytonanofabrication of iron oxide particles from the Acacia jacquemontii plant and their potential application for the removal of brilliant green and Congo red dye from wastewater

Phytonanofabrication is one of the most promising areas that has drawn the attention of scientists worldwide due to its eco-friendly nature and biocompatibility. In the current investigation, we reported the phyto-assisted formation of iron oxide nanoparticles (IONPs) from a rare species of Acacia (Acacia jacquemontii). First, ethanolic extracts of the stem powder were analyzed by high-performance thin-layer chromatography (HPTLC) for the identification of phytochemicals in the stem sections of Acacia. Furthermore, IONPs were synthesized by a chemical co-precipitation method by using the stem extract. The phytonanofabricated iron oxide particles were investigated by UV–Vis spectroscopy, Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), field emission scanning electron microscopy (FESEM), and Energy-dispersive X-ray spectroscopy (EDS) for elemental analysis. HPTLC confirmed the presence of several phenols and terpenoids in the ethanolic extracts of the stem. UV–Vis spectroscopy exhibited an absorbance peak at 380 nm, indicating the formation of IONPs, while FTIR spectroscopy showed the typical bands for Fe-O in the range of 599–1,000 cm−1 in addition to several functional groups of organic molecules at 1,596 cm−1, 2,313 cm−1, and 3,573 cm−1. XRD exhibits the amorphous nature of IONPs with peaks at 30.7, 35.5, and 62.7 nm. The IONPs were spherical-shaped, whose size varies from 10 to 70 nm, as confirmed by FESEM. EDS exhibited the presence of Fe, O, C, and NaCl. Finally, the phytonanofabricated iron oxide particles were utilized for the removal of brilliant green (BG) and Congo red (CR) dye from the aqueous solution. The removal efficiency of BG dye was up to 54.28%, while that of Congo red dye was up to 36.72% in 120 min and 60 min, respectively. Furthermore, the effect of pH and contact time was also assessed on both the dyes, where CR exhibited maximum removal at acidic pH, i.e., 47.5%, while BG showed maximum removal at pH 10, i.e., 76.59%

Intestinal toxicity to CTLA-4 blockade driven by IL-6 and myeloid infiltration

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Identification of Immunoglobulin IgZ and IgM and the Role of Innate Immune Signaling on their Synthesis in Catla Catla

This thesis illustrates the existence of newly identified immunoglobulin (Ig) heavy chain isotype, IgZ, universally known isotype, IgM and their functional basis against pathogenic manifestation in the most primitive vertebrate model of fish. The study elucidates the molecular crosstalk between TLR mediated immune signalling in immunoglobulin activation involving crucial role of MAP kinase cascade. Further, it provides insights into the therapeutic activities of an immunomodulator from lactic acid bacterial group in facilitation of immunoprophylaxis for fish health management. Partial IgZ and IgM heavy chain constant region sequences of three different heterologous species-Ctenopharygodon idella, Danio rerio and Cyprinus carpio were obtained from NCBI database to identify Ig isotypes in the Indian major carp, Catla catla. Bioinformatic and phylogenetic analysis of 417bp nucleotide sequence of Catla catla (CcIgZ) and 615bp of Catla catla (CcIgM) with a putative 139 and 205 amino acid sequence respectively showed homology with corresponding constant region domains of other teleosts. 5´ and 3´ RACE cloning was performed using nested primers to extend newly identified partial CcIgZ nucleotide sequence upto 933bp. Transcript expression of IgZ and IgM was evaluated in immunologically relevant tissues in response to Aeromonas hydrophila, Streptococcus uberis, Argulus sp. and rhabdoviral antigen stimulation in a time dependent manner. Basal level of both CcIgZ and CcIgM mRNA expression was found to be relatively higher in kidney. Bacterial induction triggered significant increase in CcIgZ mRNA expression in intestine (P<0.001) followed by spleen (P<0.01), whereas Argulus heavily infected groups showed higher expression in gill (P<0.01) and blood (P<0.01). However, rhabdoviral antigenic stimulation triggered relatively higher IgZ expression in intestine followed by gill. CcIgM was relatively up-regulated in blood (P<0.001) on bacterial and parasitic stimulations. Thus, the thesis provides the first evidence of existence of IgZ ortholog in C. catla and its comparative expression analysis with IgM against pathogenic assault. Further, to delineate the role of TLR dependent pathway in Ig activation, C. catla fingerlings were induced with various pathogen associated molecular patterns (PAMPs). A significant upregulation (P<0.001, One-way ANOVA) of different TLRs (TLR2, TLR3, TLR4 and TLR5) followed by activation of MyD88 dependent and independent pathway was observed on PAMPs induction. Subsequent stimulation of ERK, NF-κB mediated cytokine production enhanced expression of IgZ and IgM evident by qRT-PCR analysis, flow cytometry, immunoblotting and ELISA. Pretreatment with ERK inhibitor (UO126) antagonized PAMPs mediated TLR stimulation leading to sequential downregulation of NF-κB/cytokines via interrupting ERK/NF-κB signaling axis. Together these results demonstrated that TLR stimulation triggered IgZ and IgM production via activation of ERK and NF-κB in C. catla, indicating that NF-κB mediated cytokine production and ERK1/2 signaling is not only functional in fish, but may be crucial for generation of Ig repertoire in lower vertebrates against pathogenic infiltration. The findings provide avenues for designing better prophylactic interventions in fish health management. Increased use of chemotherapeutants and antibiotics has led to resurgence of antimicrobial resistance in aquaculture practices. Thus, screening of immunomodulators such as probiotics and their mechanism of attenuation of potent fish pathogen, A. hydrophila-induced mortality in C. catla was investigated. The thesis reports that A. hydrophila markedly induced cell injuries, increased ROS/RNS and cytokine production, DNA damage leading to apoptosis as evidenced from increased apoptotic fractions in flow cytometry using Annexin V/PI assay in Catla Thymus Macrophage (CTM) cells. Probiotic, Lactobacillus acidophilus in CTM cells and in in-vivo treatment attenuated A. hydrophila-induced apoptosis by abrogating ROS/RNS production and regulated TLR-mediated Ig expression