@article {8740, title = {Neem leaf glycoprotein binding to Dectin-1 receptors on dendritic cell induces type-1 immunity through CARD9 mediated intracellular signal to NFκB. [Mass Spectrometry - Proteomics Facility]}, journal = {Cell Commun Signal}, volume = {22}, year = {2024}, month = {2024 Apr 23}, pages = {237}, abstract = {

BACKGROUND: A water-soluble ingredient of mature leaves of the tropical mahogany {\textquoteright}Neem{\textquoteright} (Azadirachta indica), was identified as glycoprotein, thus being named as {\textquoteright}Neem Leaf Glycoprotein{\textquoteright} (NLGP). This non-toxic leaf-component regressed cancerous murine tumors (melanoma, carcinoma, sarcoma) recurrently in different experimental circumstances by boosting prime antitumor immune attributes. Such antitumor immunomodulation, aid cytotoxic T cell (T)-based annihilation of tumor cells. This study focused on identifying and characterizing the signaling gateway that initiate this systemic immunomodulation. In search of this gateway, antigen-presenting cells (APCs) were explored, which activate and induce the cytotoxic thrust in T cells.

METHODS: Six glycoprotein-binding C-type lectins found on APCs, namely, MBR, Dectin-1, Dectin-2, DC-SIGN, DEC205 and DNGR-1 were screened on bone marrow-derived dendritic cells from C57BL/6\ J mice. Fluorescence microscopy, RT-PCR, flow cytometry and ELISA revealed Dectin-1 as the NLGP-binding receptor, followed by verifications through RNAi. Following detection of β-Glucans in NLGP, their interactions with Dectin-1 were explored in silico. Roles of second messengers and transcription factors in the downstream signal were studied by co-immunoprecipitation, western blotting, and chromatin-immunoprecipitation. Intracellularization of FITC-coupled NLGP was observed by processing confocal micrographs of DCs.

RESULTS: Considering extents of hindrance in NLGP-driven transcription rates of the cytokines IL-10 and IL-12p35 by receptor-neutralization, Dectin-1 receptors on dendritic cells were found to bind NLGP through the ligand{\textquoteright}s peripheral β-Glucan chains. The resulting signal phosphorylates PKCδ, forming a trimolecular complex of CARD9, Bcl10 and MALT1, which in turn activates the canonical NFκB-pathway of transcription-regulation. Consequently, the NFκB-heterodimer p65:p50 enhances Il12a transcription and the p50:p50 homodimer represses Il10 transcription, bringing about a cytokine-based systemic-bias towards type-1 immune environment. Further, NLGP gets engulfed within dendritic cells, possibly through endocytic activities of Dectin-1.

CONCLUSION: NLGP{\textquoteright}s binding to Dectin-1 receptors on murine dendritic cells, followed by the intracellular signal, lead to NFκB-mediated contrasting regulation of cytokine-transcriptions, initiating a pro-inflammatory immunopolarization, which amplifies further by the responding immune cells including T cells, alongside their enhanced cytotoxicity. These insights into the initiation of mammalian systemic immunomodulation by NLGP at cellular and molecular levels, may help uncovering its mode of action as a novel immunomodulator against human cancers, following clinical trials.

}, keywords = {Animals, Azadirachta, CARD Signaling Adaptor Proteins, Dendritic Cells, Lectins, C-Type, Mice, Mice, Inbred C57BL, NF-kappa B, Plant Leaves, Protein Binding, Signal Transduction}, issn = {1478-811X}, doi = {10.1186/s12964-024-01576-z}, author = {Ganguly, Nilanjan and Das, Tapasi and Bhuniya, Avishek and Guha, Ipsita and Chakravarti, Mohona and Dhar, Sukanya and Sarkar, Anirban and Bera, Saurav and Dhar, Jesmita and Dasgupta, Shayani and Saha, Akata and Ghosh, Tithi and Das, Juhina and Sk, Ugir Hossain and Banerjee, Saptak and Laskar, Subrata and Bose, Anamika and Baral, Rathindranath} } @article {8898, title = {Shotgun proteomics provides an insight into pathogenesis-related proteins using anamorphic stage of the biotroph, Erysiphe pisi pathogen of garden pea [Mass Spectrometry - Proteomics Facility]}, journal = {Microbiol Res}, volume = {222}, year = {2019}, month = {2019 May}, pages = {25-34}, abstract = {

E. pisi is an ascomycete member causing powdery mildew disease of garden pea. It is a biotrophic pathogen, requiring a living host for its survival. Our understanding of molecular mechanisms underlying its pathogenesis is limited. The identification of proteins expressed in the pathogen is required to gain an insight into the functional mechanisms of an obligate biotrophic fungal pathogen. In this study, the proteome of the anamorphic stage of E. pisi pathogen has been elucidated through the nano LC-MS/MS approach. A total of 328 distinct proteins were detected from Erysiphe isolates infecting the susceptible pea cultivar, Arkel. The proteome is available via ProteomeXchange with identifier PXD010238. The functional classification of protein accessions based on Gene Ontology revealed proteins related to signal transduction, secondary metabolite formation and stress which might be involved in virulence and pathogenesis. The functional validation carried through differential expression of genes encoding G-protein beta subunit, a Cyclophilin (Peptidyl prolyl cis-transisomerase) and ABC transporter in a time course study confirmed their putative role in pathogenesis between resistant and susceptible genotypes, JI2480 and Arkel. The garden pea-powdery mildew pathosystem is largely unexplored, therefore, the identified proteome provides a first-hand information and will form a basis to analyze mechanisms involving pathogen survival, pathogenesis and virulence.

}, keywords = {Ascomycota, Chromatography, Liquid, Disease Resistance, Fungal Proteins, Gardens, Gene Ontology, Genes, Plant, Genotype, Host-Pathogen Interactions, Pisum sativum, Plant Diseases, Plant Leaves, Proteome, Proteomics, Tandem Mass Spectrometry}, issn = {1618-0623}, doi = {10.1016/j.micres.2019.02.006}, author = {Bheri, Malathi and M Bhosle, Sheetal and Makandar, Ragiba} } @article {498, title = {Genome sequencing of herb Tulsi (Ocimum tenuiflorum) unravels key genes behind its strong medicinal properties.[Mass spectrometry - Metabolomics]}, journal = {BMC Plant Biol}, volume = {15}, year = {2015}, month = {2015 Aug 28}, pages = {212}, abstract = {

BACKGROUND: Krishna Tulsi, a member of Lamiaceae family, is a herb well known for its spiritual, religious and medicinal importance in India. The common name of this plant is {\textquoteright}Tulsi{\textquoteright} (or {\textquoteright}Tulasi{\textquoteright} or {\textquoteright}Thulasi{\textquoteright}) and is considered sacred by Hindus. We present the draft genome of Ocimum tenuiflurum L (subtype Krishna Tulsi) in this report. The paired-end and mate-pair sequence libraries were generated for the whole genome sequenced with the Illumina Hiseq 1000, resulting in an assembled genome of 374\ Mb, with a genome coverage of 61\ \% (612\ Mb estimated genome size). We have also studied transcriptomes (RNA-Seq) of two subtypes of O. tenuiflorum, Krishna and Rama Tulsi and report the relative expression of genes in both the varieties.

RESULTS: The pathways leading to the production of medicinally-important specialized metabolites have been studied in detail, in relation to similar pathways in Arabidopsis thaliana and other plants. Expression levels of anthocyanin biosynthesis-related genes in leaf samples of Krishna Tulsi were observed to be relatively high, explaining the purple colouration of Krishna Tulsi leaves. The expression of six important genes identified from genome data were validated by performing q-RT-PCR in different tissues of five different species, which shows the high extent of urosolic acid-producing genes in young leaves of the Rama subtype. In addition, the presence of eugenol and ursolic acid, implied as potential drugs in the cure of many diseases including cancer was confirmed using mass spectrometry.

CONCLUSIONS: The availability of the whole genome of O.tenuiflorum and our sequence analysis suggests that small amino acid changes at the functional sites of genes involved in metabolite synthesis pathways confer special medicinal properties to this herb.

}, keywords = {Gene Expression Regulation, Plant, Genome, Plant, India, Ocimum, Plant Leaves, Plants, Medicinal}, issn = {1471-2229}, doi = {10.1186/s12870-015-0562-x}, author = {Upadhyay, Atul K and Chacko, Anita R and Gandhimathi, A and Ghosh, Pritha and Harini, K and Joseph, Agnel P and Joshi, Adwait G and Karpe, Snehal D and Kaushik, Swati and Kuravadi, Nagesh and Lingu, Chandana S and Mahita, J and Malarini, Ramya and Malhotra, Sony and Malini, Manoharan and Mathew, Oommen K and Mutt, Eshita and Naika, Mahantesha and Nitish, Sathyanarayanan and Pasha, Shaik Naseer and Raghavender, Upadhyayula S and Rajamani, Anantharamanan and Shilpa, S and Shingate, Prashant N and Singh, Heikham Russiachand and Sukhwal, Anshul and Sunitha, Margaret S and Sumathi, Manojkumar and Ramaswamy, S and Gowda, Malali and Sowdhamini, Ramanathan} } @article {506, title = {Simple Detection Methods for Antinutritive Factor β-ODAP Present in Lathyrus sativus L. by High Pressure Liquid Chromatography and Thin Layer Chromatography. [Protein Technology Core]}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0140649}, abstract = {

Lathyrus sativus L. (Grass pea) is the source for cheap and nutritious food choice in drought and famine susceptible zones in greater part of North India and Africa. The non-protein amino acid β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP) has been known for decades for its potent neurotoxic effect, causing irreversible neurodegenerative disease "neurolathyrism", present in both seed and leaf of Lathyrus sativus L. and other species in varying proportions. It is crucial to establish a rapid as well as reliable detection methodology for β-ODAP content in various Lathyrus plants. Currently available HPLC based methods involve multi-step derivatization of the sample. To overcome this, we have developed β-ODAP analysis method by HPLC without any prior derivatization. This method is statistically significant in the range of 2 to 100μg/ml and exhibited linear response with r2 \> 0.99. Limit of detection and quantitation of the later method was determined to be 5.56 μg/ml and 16.86 μg/ml, respectively. In addition to this, a TLC based method has also been developed. The limit of detection of β-ODAP is 0.6μg and for its substrate, L-1,2-diaminopropionic acid is 5μg. Both HPLC and TLC methods were validated by conducting in-vitro bioconversion test to detect the presence of biocatalyst in plant extract. This method is economical, rapid and simple.

}, keywords = {Africa, Amino Acids, Diamino, Chromatography, High Pressure Liquid, Chromatography, Thin Layer, India, Lathyrus, Neurotoxins, Plant Extracts, Plant Leaves, Seeds}, issn = {1932-6203}, doi = {10.1371/journal.pone.0140649}, author = {Ghosh, Bidisha and Mitra, Joy and Chakraborty, Saikat and Bhattacharyya, Jagannath and Chakraborty, Anirban and Sen, Soumitra Kumar and Neerathilingam, Muniasamy} }