Commenced in January 2007
Frequency: Monthly
Edition: International
Paper Count: 20

Search results for: glycosylation

20 Common Regulatory Mechanisms Reveals Links between Aberrant Glycosylation and Biological Hallmarks in Cancer

Authors: Jahanshah Ashkani, Kevin J. Naidoo


Glycosylation is the major posttranslational modification (PTM) process in cellular development. In tumour development, it is marked by structural alteration of carbohydrates (glycans) that is the result of aberrant glycosylation. Altered glycan structures affect cell surface ligand-receptor interactions that interfere with the regulation of cell adhesion, migration, and proliferation. The resulting changes in glycan biosynthesis pathways originate from altered expression of glycosyltransferases and glycosidases. While the alteration in glycosylation patterns is a recognized “hallmark of cancer”, the influential overview of the biology of cancer proposes eight hallmarks with no explicit suggestion to connectivity with glycosylation. Recently, we have discovered a connection between the glycosyltransferase gene expression and cancer type and subtype. Here we present an association between aberrant glycosylation and the biological hallmarks of breast cancer by exploring the common regulatory mechanisms at the genomic scale. The result of this study bridges the glycobiological and biological pathways that are accepted hallmarks of cancer by connecting their common regulatory pathways. This is an impetus for further investigation as target therapies of breast cancer are very likely to be uncovered from this.

Keywords: aberrant glycosylation, biological hallmarks, breast cancer, regulatory mechanism

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19 Molecular Portraits: The Role of Posttranslational Modification in Cancer Metastasis

Authors: Navkiran Kaur, Apoorva Mathur, Abhishree Agarwal, Sakshi Gupta, Tuhin Rashmi


Aim: Breast cancer is the most common cancer in women worldwide, and resistance to the current therapeutics, often concurrently, is an increasing clinical challenge. Glycosylation of proteins is one of the most important post-translational modifications. It is widely known that aberrant glycosylation has been implicated in many different diseases due to changes associated with biological function and protein folding. Alterations in cell surface glycosylation, can promote invasive behavior of tumor cells that ultimately lead to the progression of cancer. In breast cancer, there is an increasing evidence pertaining to the role of glycosylation in tumor formation and metastasis. In the present study, an attempt has been made to study the disease associated sialoglycoproteins in breast cancer by using bioinformatics tools. The sequence will be retrieved from UniProt database. A database in the form of a word document was made by a collection of FASTA sequences of breast cancer gene sequence. Glycosylation was studied using yinOyang tool on ExPASy and Differential genes expression and protein analysis was done in context of breast cancer metastasis. The number of residues predicted O-glc NAc threshold containing 50 aberrant glycosylation sites or more was detected and recorded for individual sequence. We found that the there is a significant change in the expression profiling of glycosylation patterns of various proteins associated with breast cancer. Differential aberrant glycosylated proteins in breast cancer cells with respect to non-neoplastic cells are an important factor for the overall progression and development of cancer.

Keywords: breast cancer, bioinformatics, cancer, metastasis, glycosylation

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18 N-Glycosylation in the Green Microalgae Chlamydomonas reinhardtii

Authors: Pierre-Louis Lucas, Corinne Loutelier-Bourhis, Narimane Mati-Baouche, Philippe Chan Tchi-Song, Patrice Lerouge, Elodie Mathieu-Rivet, Muriel Bardor


N-glycosylation is a post-translational modification taking place in the Endoplasmic Reticulum and the Golgi apparatus where defined glycan features are added on protein in a very specific sequence Asn-X-Thr/Ser/Cys were X can be any amino acid except proline. Because it is well-established that those N-glycans play a critical role in protein biological activity, protein half-life and that a different N-glycan structure may induce an immune response, they are very important in Biopharmaceuticals which are mainly glycoproteins bearing N-glycans. From now, most of the biopharmaceuticals are produced by mammalian cells like Chinese Hamster Ovary cells (CHO) for their N-glycosylation similar to the human, but due to the high production costs, several other species are investigated as the possible alternative system. In this purpose, the green microalgae Chlamydomonas reinhardtii was investigated as the potential production system for Biopharmaceuticals. This choice was influenced by the facts that C. reinhardtii is a well-study microalgae which is growing fast with a lot of molecular biology tools available. This organism is also producing N-glycan on its endogenous proteins. However, the analysis of the N-glycan structure of this microalgae has revealed some differences as compared to the human. Rather than in Human where the glycans are processed by key enzymes called N-acetylglucosaminyltransferase I and II (GnTI and GnTII) adding GlcNAc residue to form a GlcNAc₂Man₃GlcNAc₂ core N-glycan, C. reinhardtii lacks those two enzymes and possess a GnTI independent glycosylation pathway. Moreover, some enzymes like xylosyltransferases and methyltransferases not present in human are supposed to act on the glycans of C. reinhardtii. Furthermore, the recent structural study by mass spectrometry shows that the N-glycosylation precursor supposed to be conserved in almost all eukaryotic cells results in a linear Man₅GlcNAc₂ rather than a branched one in C. reinhardtii. In this work, we will discuss the new released MS information upon C. reinhardtii N-glycan structure and their impact on our attempt to modify the glycan in a Human manner. Two strategies will be discussed. The first one consisted in the study of Xylosyltransferase insertional mutants from the CLIP library in order to remove xyloses from the N-glycans. The second will go further in the humanization by transforming the microalgae with the exogenous gene from Toxoplasma gondii having an activity similar to GnTI and GnTII with the aim to synthesize GlcNAc₂Man₃GlcNAc₂ in C. reinhardtii.

Keywords: Chlamydomonas reinhardtii, N-glycosylation, glycosyltransferase, mass spectrometry, humanization

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17 Stereoselective Glycosylation and Functionalization of Unbiased Site of Sweet System via Dual-Catalytic Transition Metal Systems/Wittig Reaction

Authors: Mukul R. Gupta, Rajkumar Gandhi, Rajitha Sachan, Naveen K. Khare


The field of glycoscience has burgeoned in the last several decades, leading to the identification of many glycosides which could serve critical roles in a wide range of biological processes. This has prompted a resurgence in synthetic interest, with a particular focus on new approaches to construct the selective glycosidic bond. Despite the numerous elegant strategies and methods developed for the formation of glycosidic bonds, stereoselective construction of glycosides remains challenging. Here, we have recently developed the novel Hexafluoroisopropanol (HFIP) catalyzed stereoselective glycosylation methods by using KDN imidate glycosyl donor and a variety of alcohols in excellent yield. This method is broadly applicable to a wide range of substrates and with excellent selectivity of glycoside. Also, herein we are reporting the functionalization of the unbiased side of newly formed glycosides by dual-catalytic transition metal systems (Ru- or Fe-). We are using the innovative Reverse & Catalyst strategy, i.e., a reversible activation reaction by one catalyst with a functionalization reaction by another catalyst, together with enabling functionalization of substrates at their inherently unreactive sites. As well, we are targeting the diSia derivative synthesis by Wittig reaction. This synthetic method is applicable in mild conditions, functional group tolerance of the dual-catalytic systems and also highlights the potential of the multicatalytic approach to address challenging transformations to avoid multistep procedures in carbohydrate synthesis.

Keywords: KDN, stereoselective glycosylation, dual-catalytic functionalization, Wittig reaction

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16 Correlation between Resistance to Non-Specific Inhibitor and Mammalian Pathogenicity of an Egg Adapted H9N2 Virus

Authors: Chung-Young Lee, Se-Hee Ahn, Jun-Gu Choi, Youn-Jeong Lee, Hyuk-Joon Kwon, Jae-Hong Kim


A/chicken/Korea/01310/2001 (H9N2) (01310) was passaged through embryonated chicken eggs (ECEs) by 20 times (01310-E20), and it has been used for an inactivated oil emulsion vaccine in Korea. After sequential passages, 01310-E20 showed higher pathogenicity in ECEs and acquired multiple mutations including a potential N-glycosylation at position 133 (H3 numbering) in HA and 18aa-deletion in NA stalk. To evaluate the effect of these mutations on the mammalian pathogenicity and resistance to non-specific inhibitors, we generated four PR8-derived recombinant viruses with different combinations of HA and NA from 01310-E2 and 01310-E20 (rH2N2, rH2N20, rH20N2, and rH20N20). According to our results, recombinant viruses containing 01310 E20 HA showed higher growth property in MDCK cells and higher virulence on mice than those containing 01310 E2 HA regardless of NA. The hemagglutination activity of rH20N20 was less inhibited by egg white and mouse lung extract than that of other recombinant viruses. Thus, the increased pathogenicity of 01310-E20 may be related to both higher replication efficiency and resistance to non-specific inhibitors in mice.

Keywords: avian influenza virus, egg adaptation, H9N2, N-glycosylation, stalk deletion of neuraminidase

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15 Biotransformation Process for the Enhanced Production of the Pharmaceutical Agents Sakuranetin and Genkwanin: Poised to be Potent Therapeuctic Drugs

Authors: Niranjan Koirala, Sumangala Darsandhari, Hye Jin Jung, Jae Kyung Sohng


Sakuranetin, an antifungal agent and genkwanin, an anti-inflammatory agent, are flavonoids with several potential pharmaceutical applications. To produce such valuable flavonoids in large quantity, an Escherichia coli cell factory has been created. E. coli harboring O-methyltransferase (SaOMT2) derived from Streptomyces avermitilis was employed for regiospecific methylation of naringenin and apigenin. In order to increase the production via biotransformation, metK gene was overexpressed and the conditions were optimized. The maximum yield of sakuranetin and genkwanin under optimized conditions was 197 µM and 170 µM respectively when 200 µM of naringenin and apigenin were supplemented in the separate cultures. Furthermore, sakuranetin was purified in large scale and used as a substrate for in vitro glycosylation by YjiC to produce glucose and galactose derivatives of sakuranetin for improved solubility. We also found that unlike naringenin, sakuranetin effectively inhibits α-melanocyte stimulating hormone (α-MSH)-stimulated melanogenesis in B16F10 melanoma cells. In addition, genkwanin more potently inhibited angiogenesis than apigenin. Based on our findings, we speculate that these compounds warrant further investigation in vivo as potential new therapeutic anti-carcinogenic, anti-melanogenic and anti-angiogenic agents.

Keywords: anti-carcinogenic, anti-melanogenic, glycosylation, methylation

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14 Glyco-Biosensing as a Novel Tool for Prostate Cancer Early-Stage Diagnosis

Authors: Pavel Damborsky, Martina Zamorova, Jaroslav Katrlik


Prostate cancer is annually the most common newly diagnosed cancer among men. An extensive number of evidence suggests that traditional serum Prostate-specific antigen (PSA) assay still suffers from a lack of sufficient specificity and sensitivity resulting in vast over-diagnosis and overtreatment. Thus, the early-stage detection of prostate cancer (PCa) plays undisputedly a critical role for successful treatment and improved quality of life. Over the last decade, particular altered glycans have been described that are associated with a range of chronic diseases, including cancer and inflammation. These glycans differences enable a distinction to be made between physiological and pathological state and suggest a valuable biosensing tool for diagnosis and follow-up purposes. Aberrant glycosylation is one of the major characteristics of disease progression. Consequently, the aim of this study was to develop a more reliable tool for early-stage PCa diagnosis employing lectins as glyco-recognition elements. Biosensor and biochip technology putting to use lectin-based glyco-profiling is one of the most promising strategies aimed at providing fast and efficient analysis of glycoproteins. The proof-of-concept experiments based on sandwich assay employing anti-PSA antibody and an aptamer as a capture molecules followed by lectin glycoprofiling were performed. We present a lectin-based biosensing assay for glycoprofiling of serum biomarker PSA using different biosensor and biochip platforms such as label-free surface plasmon resonance (SPR) and microarray with fluorescent label. The results suggest significant differences in interaction of particular lectins with PSA. The antibody-based assay is frequently associated with the sensitivity, reproducibility, and cross-reactivity issues. Aptamers provide remarkable advantages over antibodies due to the nucleic acid origin, stability and no glycosylation. All these data are further step for construction of highly selective, sensitive and reliable sensors for early-stage diagnosis. The experimental set-up also holds promise for the development of comparable assays with other glycosylated disease biomarkers.

Keywords: biomarker, glycosylation, lectin, prostate cancer

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13 Production of Novel Antibiotics by Importing eryK and eryG Genes in Streptomyces fradiae

Authors: Neda Gegar Goshe, Hossein Rassi


The antibacterial properties of macrolide antibiotics (such as erythromycin and tylosin) depend ultimately on the glycosylation of otherwise inactive polyketide lactones. Among the sugars commonly found in such macrolides are various 6-deoxyhexoses including the 3-dimethylamino sugars mycaminose and desosamine (4-deoxymycaminose). Some macrolides (such as tylosin) possess multiple sugar moieties, whereas others (such as erythromycin) have two sugar substituents. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors-malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA-used by modular PKS, and is a host that is amenable to genetic manipulation. As patterns of glycosylation markedly influence a macrolide's drug activity, there is considerable interest in the possibility of using combinatorial biosynthesis to generate new pairings of polyketide lactones with sugars, especially 6-deoxyhexoses. Here, we report a successful attempt to alter the aminodeoxyhexose-biosynthetic capacity of Streptomyces fradiae (a producer of tylosin) by importing genes from the erythromycin producer Saccharopolyspora erythraea. The biotransformation of erythromycin-D into the desired major component erythromycin-A involves two final enzymatic reactions, EryK-catalyzed hydroxylation at the C-12 position of the aglycone and EryG-catalyzed O methylation at the C-3 position of macrose .This engineered S. fradiae produced substantial amounts of two potentially useful macrolides that had not previously been obtained by fermentation.

Keywords: Streptomyces fradiae, eryK and eryG genes, tylosin, antibiotics

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12 Production of Novel Antibiotics of Tylosin by Importing eryK and eryG Genes in Streptomyces fradiae

Authors: Neda Gegar Goshe, M. Moradi, Hossein Rassi


The antibacterial properties of macrolide antibiotics (such as erythromycin and tylosin) depend ultimately on the glycosylation of otherwise inactive polyketide lactones. Among the sugars commonly found in such macrolides are various 6-deoxyhexoses including the 3-dimethylamino sugars mycaminose and desosamine (4-deoxymycaminose). Some macrolides (such as tylosin) possess multiple sugar moieties, whereas others (such as erythromycin) have two sugar substituents. Streptomyces fradiae is an ideal host for development of generic polyketide-overproducing strains because it contains three of the most common precursors-malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA-used by modular PKS, and is a host that is amenable to genetic manipulation. As patterns of glycosylation markedly influence a macrolide's drug activity, there is considerable interest in the possibility of using combinatorial biosynthesis to generate new pairings of polyketide lactones with sugars, especially 6-deoxyhexoses. Here, we report a successful attempt to alter the aminodeoxyhexose-biosynthetic capacity of Streptomyces fradiae (a producer of tylosin) by importing genes from the erythromycin producer Saccharopolyspora erythraea. The bio transformation of erythromycin-D into the desired major component erythromycin-A involves two final enzymatic reactions, EryK-catalyzed hydroxylation at the C-12 position of the aglycone and EryG-catalyzed O methylation at the C-3 position of macrose. This engineered S. fradiae produced substantial amounts of two potentially useful macrolides that had not previously been obtained by fermentation.

Keywords: tylosin, eryK and eryG genes, streptomyces fradiae

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11 Free Radical Scavenging, Antioxidant Activity, Phenolic, Alkaloids Contents and Inhibited Properties against α-Amylase and Invertase Enzymes of Stem Bark Extracts Coula edulis B

Authors: Eric Beyegue, Boris Azantza, Judith Laure Ngondi, Julius E. Oben


Background: It is clearly that phytochemical constituents of plants in relation exhibit free radical scavenging, antioxidant and glycosylation properties. This study investigated the in vitro antioxidant and free radical scavenging, inhibited activities against α-amylase and invertase enzymes of stem bark extracts C. edulis (Olacaceae). Methods: Four extracts (hexane, dichloromethane, ethanol and aqueous) from the barks of C. edulis were used in this study. Colorimetric in vitro methods were using for evaluate free radical scavenging activity DPPH, ABTS, NO, OH, antioxidant capacity, glycosylation activity, inhibition of α-amylase and invertase activities, phenolic, flavonoid and alkaloid contents. Results: C. edulis extracts (CEE) had a higher scavenging potential on the 2, 2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), nitrite oxide (NO), 2, 2-azinobis (3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) radicals and glucose scavenging with the IC50 varied between 41.95 and 36694.43 µg/ml depending on the solvent of extraction. The ethanol extract of C. edulis stem bark (CE EtOH) showed the highest polyphenolic (289.10 + 30.32), flavonoid (1.12 + 0.09) and alkaloids (18.47 + 0.16) content. All the tested extracts demonstrated a relative high inhibition potential against α-amylase and invertase digestive enzymes activities. Conclusion: This study suggests that CEE exhibited higher antioxidant potential and significant inhibition potential against digestive enzymes.

Keywords: Coula edulis, antioxidant, scavenging activity, amylase, invertase

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10 Analytical Similarity Assessment of Bevacizumab Biosimilar Candidate MB02 Using Multiple State-of-the-Art Assays

Authors: Marie-Elise Beydon, Daniel Sacristan, Isabel Ruppen


MB02 (Alymsys®) is a candidate biosimilar to bevacizumab, which was developed against the reference product (RP) Avastin® sourced from both the European Union (EU) and United States (US). MB02 has been extensively characterized comparatively to Avastin® at a physicochemical and biological level using sensitive orthogonal state-of-the-art analytical methods. MB02 has been demonstrated similar to the RP with regard to its primary and higher-order structure, post- and co-translational profiles such as glycosylation, charge, and size variants. Specific focus has been put on the characterization of Fab-related activities, such as binding to VEGF A 165, which directly reflect the bevacizumab mechanism of action. Fc-related functionality was also investigated, including binding to FcRn, which is indicative of antibodies' half-life. The data generated during the analytical similarity assessment demonstrate the high analytical similarity of MB02 to its RP.

Keywords: analytical similarity, bevacizumab, biosimilar, MB02

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9 Facile Synthetic Process for Lamivudine and Emtricitabine

Authors: Devender Mandala, Paul Watts


Cis-Nucleosides mainly lamivudine (3TC) and emtricitabine (FTC) are an important tool in the treatment of Human immune deficiency virus (HIV), Hepatitis B virus (HBV) and Human T-Lymotropoic virus (HTLV). Lamivudine and emtricitabine are potent nucleoside analog reverse transcriptase inhibitors (nRTI). These two drugs are synthesized by a four-stage process from the starting materials: menthyl glyoxylate hydrate and 1,4-dithane-2,5-diol to produce the 5-hydroxy oxathiolane which upon acetylation with acetic anhydride to yield 5-acetoxy oxathiolane. Then glycosylation of this acetyl product with silyl protected nucleoside to produce the intermediate. The reduction of this intermediates can provide the final targets. Although there are several different methods reported for the synthesis of lamivudine and emtricitabine as a single enantiomer, we required an efficient route, which was suitable for large-scale synthesis to support the development of these compounds. In this process, we successfully prepared the intermediates of lamivudine and emtricitabine without using any solvents and catalyst, thus promoting the green synthesis. All the synthesized compound were confirmed by TLC, GC, Mass, NMR and 13C NMR spectroscopy.

Keywords: emtricitabine, green synthesis, lamivudine, nucleoside

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8 Introducing α-Oxoester (COBz) as a Protecting Group for Carbohydrates

Authors: Atul Kumar, Veeranjaneyulu Gannedi, Qazi Naveed Ahmed


Oligosaccharides, which are essential to all cellular organisms, play vital roles in cell recognition, signaling, and are involved in a broad range of biological processes. The chemical synthesis of carbohydrates represents a powerful tool to provide homogeneous glycans. In carbohydrate synthesis, the major concern is the orthogonal protection of hydroxyl groups that can be unmasked independently. Classical protecting groups include benzyl ethers (Bn), which are normally cleaved through hydrogenolysis or by means of metal reduction, and acetate (Ac), benzoate (Bz) or pivaloate esters, which are removed using base promoted hydrolysis. In present work a series of α-Oxoester (COBz) protected saccharides, with divergent base sensitivity profiles against benzoyl (Bz) and acetyl (Ac), were designed and KHSO₅/CH₃COCl in methanol was identified as an easy, mild, selective and efficient deprotecting reagent for their removal in the perspective of carbohydrate synthesis. Timely monitoring of later reagent was advantageous in establishing both sequential as well as simultaneous deprotecting of COBz, Bz, and Ac. The salient feature of our work is its ease to generate different acceptors using designed monosaccharides. In summary, we demonstrated α-Oxoester (COBz) as a new protecting group for carbohydrates and the application of this group for the synthesis of Glycosylphosphatidylinositol (GPI) anchor are in progress.

Keywords: α-Oxoester, oligosaccharides, new protecting group, acceptor synthesis, glycosylation

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7 Development of Lectin-Based Biosensor for Glycoprofiling of Clinical Samples: Focus on Prostate Cancer

Authors: Dominika Pihikova, Stefan Belicky, Tomas Bertok, Roman Sokol, Petra Kubanikova, Jan Tkac


Since aberrant glycosylation is frequently accompanied by both physiological and pathological processes in a human body (cancer, AIDS, inflammatory diseases, etc.), the analysis of tumor-associated glycan patterns have a great potential for the development of novel diagnostic approaches. Moreover, altered glycoforms may assist as a suitable tool for the specificity and sensitivity enhancement in early-stage prostate cancer diagnosis. In this paper we discuss the construction and optimization of ultrasensitive sandwich biosensor platform employing lectin as glycan-binding protein. We focus on the immunoassay development, reduction of non-specific interactions and final glycoprofiling of human serum samples including both prostate cancer (PCa) patients and healthy controls. The fabricated biosensor was measured by label-free electrochemical impedance spectroscopy (EIS) with further lectin microarray verification. Furthermore, we analyzed different biosensor interfaces with atomic force microscopy (AFM) in nanomechanical mapping mode showing a significant differences in the altitude. These preliminary results revealing an elevated content of α-2,3 linked sialic acid in PCa patients comparing with healthy controls. All these experiments are important step towards development of point-of-care devices and discovery of novel glyco-biomarkers applicable in cancer diagnosis.

Keywords: biosensor, glycan, lectin, prostate cancer

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6 Rapid Weight Loss in Athletes: A Look at Suppressive Effects on Immune System

Authors: Nazari Maryam, Gorji Saman


For most competitions, athletes usually engage in a process called rapid weight loss (RWL) and subsequent rapid weight gain (RWG) in the days preceding the event. Besides the perfection of performance, weight regulation mediates a self-image of being “a real athlete” which is mentally important as a part of the pre-competition preparation. This feeling enhances the focus and commitment of the athlete. There is a large body of evidence that weight loss, particularly in combat sports, results in several health benefits. However, intentional weight loss beyond normal levels might have unknown negative special effects on the immune system. As the results show, a high prevalence (50%) of RWL is happening among combat athletes. It seems that energy deprivation and intense exercise to reach RWL results in altered blood cell distribution through modification of body composition that, in turn, changes B and T-Lymphocyte and/or CD4 T-Helper response. Moreover, it may diminish IgG antibody levels and modulate IgG glycosylation after this course. On the other hand, some studies show suppression of signaling and regulation of IgE antibody and chemokine production are responsible for immunodeficiency following a period of low-energy availability. Some researchers hypothesize that severe glutamine depletion, which occurs during exercise and calorie restriction, is responsible for this immune system weakness. However, supplementation by this amino acid is not prescribed yet. Therefore, weight loss is achieved not only through chronic strategies (body fat losses) but also through acute manipulations prior to competition should be supervised by a sports nutritionist to minimize side effects on the immune system and other body systems.

Keywords: athletes, immune system, rapid weight loss, weight loss strategies

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5 Evaluation of Certain Medicinal Plants for in vitro Anti-Oxidant and Anti-Glycation Activities

Authors: K. Shailaja


The advanced glycation end products (AGEs) formed between the reducing sugar and protein as a result of Oxidative stress and non-enzymatic glycosylation play an important role in pathogenesis of diabetes and aging complication. Glycation results in the production of free radicals. The oxidation process is believed to play an important role in AGEs formation. Thus agents with antioxidative property and antiglycation activity may retard the process of AGEs formation. Selected medicinal plants for the present study include Catharanthus roseus, Bougainvillea spectabilis (pink flowers), Cinnamomum tamala, Cinnamomum zeylanica, Abutilon indicum, Asparagus racemosus, and Sapindus emarginatus. The crude ethanolic extracts of the selected medicinal plants at varying concentrations ranging from 1-100 mg/ml were evaluated for in vitro antioxidant and protein glycation activities by FRAP and glucose-BSA assay respectively. Among all the plants tested, Bougainvillea spectabilis, Catharanthus roseus and Abutilon indicum showed strong antioxidant activity The antioxidant activity was expressed as mg of Gallic acid/ gm sample which was found to be 4.3 mg, 1.3mg, and 1.3mg respectively for Bougainvillea spectabilis, Catharanthus roseus and Abutilon indicum. The results of inhibition of the initial glycation product i.e., fructosamine was found to be 35% for Asparagus racemosus, Cinnamomum tamala and Abutilon indicum followed by the other plant extracts. The results indicate that these plants are potential sources of natural antioxidants which have free radical scavenging activity and might be used not only for reducing oxidative stress in diabetes but also open a new research avenues in the field of Natural Products.

Keywords: in vitro antioxidant activity, anti-glycation activity, ethanol extracts, polyphenols, Catharanthus roseus, Cinnamomum tamala

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4 Cotton Fiber Quality Improvement by Introducing Sucrose Synthase (SuS) Gene into Gossypium hirsutum L.

Authors: Ahmad Ali Shahid, Mukhtar Ahmed


The demand for long staple fiber having better strength and length is increasing with the introduction of modern spinning and weaving industry in Pakistan. Work on gene discovery from developing cotton fibers has helped to identify dozens of genes that take part in cotton fiber development and several genes have been characterized for their role in fiber development. Sucrose synthase (SuS) is a key enzyme in the metabolism of sucrose in a plant cell, in cotton fiber it catalyzes a reversible reaction, but preferentially converts sucrose and UDP into fructose and UDP-glucose. UDP-glucose (UDPG) is a nucleotide sugar act as a donor for glucose residue in many glycosylation reactions and is essential for the cytosolic formation of sucrose and involved in the synthesis of cell wall cellulose. The study was focused on successful Agrobacterium-mediated stable transformation of SuS gene in pCAMBIA 1301 into cotton under a CaMV35S promoter. Integration and expression of the gene were confirmed by PCR, GUS assay, and real-time PCR. Young leaves of SuS overexpressing lines showed increased total soluble sugars and plant biomass as compared to non-transgenic control plants. Cellulose contents from fiber were significantly increased. SEM analysis revealed that fibers from transgenic cotton were highly spiral and fiber twist number increased per unit length when compared with control. Morphological data from field plants showed that transgenic plants performed better in field conditions. Incorporation of genes related to cotton fiber length and quality can provide new avenues for fiber improvement. The utilization of this technology would provide an efficient import substitution and sustained production of long-staple fiber in Pakistan to fulfill the industrial requirements.

Keywords: agrobacterium-mediated transformation, cotton fiber, sucrose synthase gene, staple length

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3 Identification and Characterization of Polysaccharide Biosynthesis Protein (CAPD) of Enterococcus faecium

Authors: Liaqat Ali, Hubert E. Blum, Türkân Sakinc


Enterococcus faecium is an emerging multidrug-resistant nosocomial pathogen increased dramatically worldwide and causing bacteremia, endocarditis, urinary tract and surgical site infections in immunocomprised patients. The capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system are also involved in hindering leukocyte killing of enterococci. The gene cluster (enterococcal polysaccharide antigen) of E. faecalis encoding homologues of many genes involved in polysaccharide biosynthesis. We identified two putative loci with 22 kb and 19 kb which contained 11 genes encoding for glycosyltransferases (GTFs); this was confirmed by using genome comparison of already sequenced strains that has no homology to known capsule genes and the epa-locus. The polysaccharide-conjugate vaccines have rapidly emerged as a suitable strategy to combat different pathogenic bacteria, therefore, we investigated a polysaccharide biosynthesis CapD protein in E. faecium contains 336 amino acids and had putative function for N-linked glycosylation. The deletion/knock-out capD mutant was constructed and complemented by homologues recombination method and confirmed by using PCR and sequencing. For further characterization and functional analysis, in-vitro cell culture and in-vivo a mouse infection models were used. Our ΔcapD mutant shows a strong hydrophobicity and all strains exhibited biofilm production. Subsequently, the opsonic activity was tested in an opsonophagocytic assay which shows increased in mutant compared complemented and wild type strains but more than two fold decreased in colonization and adherence was seen on surface of uroepithelial cells. However, a significant higher bacterial colonialization was observed in capD mutant during animal bacteremia infection. Unlike other polysaccharides biosynthesis proteins, CapD does not seems to be a major virulence factor in enterococci but further experiments and attention is needed to clarify its function, exact mechanism and involvement in pathogenesis of enteroccocal nosocomial infections eventually to develop a vaccine/ or targeted therapy.

Keywords: E. faecium, pathogenesis, polysaccharides, biofilm formation

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2 Comprehensive Longitudinal Multi-omic Profiling in Weight Gain and Insulin Resistance

Authors: Christine Y. Yeh, Brian D. Piening, Sarah M. Totten, Kimberly Kukurba, Wenyu Zhou, Kevin P. F. Contrepois, Gucci J. Gu, Sharon Pitteri, Michael Snyder


Three million deaths worldwide are attributed to obesity. However, the biomolecular mechanisms that describe the link between adiposity and subsequent disease states are poorly understood. Insulin resistance characterizes approximately half of obese individuals and is a major cause of obesity-mediated diseases such as Type II diabetes, hypertension and other cardiovascular diseases. This study makes use of longitudinal quantitative and high-throughput multi-omics (genomics, epigenomics, transcriptomics, glycoproteomics etc.) methodologies on blood samples to develop multigenic and multi-analyte signatures associated with weight gain and insulin resistance. Participants of this study underwent a 30-day period of weight gain via excessive caloric intake followed by a 60-day period of restricted dieting and return to baseline weight. Blood samples were taken at three different time points per patient: baseline, peak-weight and post weight loss. Patients were characterized as either insulin resistant (IR) or insulin sensitive (IS) before having their samples processed via longitudinal multi-omic technologies. This comparative study revealed a wealth of biomolecular changes associated with weight gain after using methods in machine learning, clustering, network analysis etc. Pathways of interest included those involved in lipid remodeling, acute inflammatory response and glucose metabolism. Some of these biomolecules returned to baseline levels as the patient returned to normal weight whilst some remained elevated. IR patients exhibited key differences in inflammatory response regulation in comparison to IS patients at all time points. These signatures suggest differential metabolism and inflammatory pathways between IR and IS patients. Biomolecular differences associated with weight gain and insulin resistance were identified on various levels: in gene expression, epigenetic change, transcriptional regulation and glycosylation. This study was not only able to contribute to new biology that could be of use in preventing or predicting obesity-mediated diseases, but also matured novel biomedical informatics technologies to produce and process data on many comprehensive omics levels.

Keywords: insulin resistance, multi-omics, next generation sequencing, proteogenomics, type ii diabetes

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1 Evolutionary Analysis of Influenza A (H1N1) Pdm 09 in Post Pandemic Period in Pakistan

Authors: Nazish Badar


In early 2009, Pandemic type A (H1N1) Influenza virus emerged globally. Since then, it has continued circulation causing considerable morbidity and mortality. The purpose of this study was to evaluate the evolutionary changes in Influenza A (H1N1) pdm09 viruses from 2009-15 and their relevance with the current vaccine viruses. Methods: Respiratory specimens were collected with influenza-like illness and Severe Acute Respiratory Illness. Samples were processed according to CDC protocol. Sequencing and phylogenetic analysis of Haemagglutinin (HA) and neuraminidase (NA) genes was carried out comparing representative isolates from Pakistan viruses. Results: Between Jan2009 - Feb 2016, 1870 (13.2%) samples were positive for influenza A out of 14086. During the pandemic period (2009–10), Influenza A/ H1N1pdm 09 was the dominant strain with 366 (45%) of total influenza positives. In the post-pandemic period (2011–2016), a total of 1066 (59.6%) cases were positive Influenza A/ H1N1pdm 09 with co-circulation of different Influenza A subtypes. Overall, the Pakistan A(H1N1) pdm09 viruses grouped in two genetic clades. Influenza A(H1N1)pdm09 viruses only ascribed to Clade 7 during the pandemic period whereas viruses belong to clade 7 (2011) and clade 6B (2015) during the post-pandemic years. Amino acid analysis of the HA gene revealed mutations at positions S220T, I338V and P100S specially associated with outbreaks in all the analyzed strains. Sequence analyses of post-pandemic A(H1N1)pdm09 viruses showed additional substitutions at antigenic sites; S179N,K180Q (SA), D185N, D239G (CA), S202A (SB) and at receptor binding sites; A13T, S200P when compared with pandemic period. Substitution at Genetic markers; A273T (69%), S200P/T (15%) and D239G (7.6%) associated with severity and E391K (69%) associated with virulence was identified in viruses isolated during 2015. Analysis of NA gene revealed outbreak markers; V106I (23%) among pandemic and N248D (100%) during post-pandemic Pakistan viruses. Additional N-Glycosylation site; HA S179N (23%), NA I23T(7.6%) and N44S (77%) in place of N386K(77%) were only found in post-pandemic viruses. All isolates showed histidine (H) at position 275 in NA indicating sensitivity to neuraminidase inhibitors. Conclusion: This study shows that the Influenza A(H1N1)pdm09 viruses from Pakistan clustered into two genetic clades, with co-circulation of some variants. Certain key substitutions in the receptor binding site and few changes indicative of virulence were also detected in post-pandemic strains. Therefore, it is imperative to continue monitoring of the viruses for early identification of potential variants of high virulence or emergence of drug-resistant variants.

Keywords: Influenza A (H1N1) pdm09, evolutionary analysis, post pandemic period, Pakistan

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