Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize

Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.     

VKORC1 pharmacogenetics and pharmacoproteomics in patients on warfarin anticoagulant therapy: transthyretin precursor as a potential biomarker

BACKGROUND: Recognizing specific protein changes in response to drug administration in humans has the potential for the development of personalized medicine. Such changes can be identified by pharmacoproteomics approach based on proteomic technologies. It can also be helpful in matching a particular target-based therapy to a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism. Warfarin is a commonly prescribed oral anticoagulant in patients with prosthetic valve disease, venous thromboembolism and stroke. METHODS AND FINDING: We used a combined pharmacogenetics and iTRAQ-coupled LC-MS/MS pharmacoproteomics approach to analyze plasma protein profiles of 53 patients, and identified significantly upregulated level of transthyretin precursor in patients receiving low dose of warfarin but not in those on high dose of warfarin. In addition, real-time RT-PCR, western blotting, human IL-6 ELISA assay were done for the results validation. CONCLUSION: This combined pharmacogenomics and pharmacoproteomics approach may be applied for other target-based therapies, in matching a particular marker in a subgroup of patients, in addition to the profile of genetic polymorphism.     

Length-weight relationships of eight elasmobranch species captured along the Coromandel coast of Tamil Nadu,Eastern Indian Ocean

This study reports the length-weight relationships (LWR) for eight elasmobranch species; four shark species (Carcharhiniformes and Orectolobiformes), two species of rays (Myliobatiformes) and two species of guitarfishes (Rhinopristiformes) from the Coromandel Coast of Tamil Nadu, Eastern Indian Ocean. The specimens of these elasmobranch species were collected from the catches of various gears like trawl net, bottom set and drift gill net, hook & line at Chennai—Royapuram Fishing Harbour, Cuddalore Fishing Harbourand Nagapattinam Fishing Harbour situated along this coastal region fortnightly during June 2019–March 2020. The values of the parameter ‘b’ remained within the expected range of 2.5–3.5. Length- weight / Disc-width-weight relationships showed good fit with r² values varying from 0.8923 to 0.9869. This study also reports a new maximum TL length (L_max)for a shark species (Chiloscyllium griseum).     

A novel antithrombotic effect of sulforaphane via activation of platelet adenylate cyclase: ex vivo and in vivo studies

Sulforaphane is a naturally occurring isothiocyanate, which can be found in cruciferous vegetables such as broccoli and cabbage. Sulforaphane was found to have very potent inhibitory effects on tumor growth through regulation of diverse mechanisms. However, no data are available concerning the effects of sulforaphane on platelet activation and its relative issues. Activation of platelets caused by arterial thrombosis is relevant to a variety of cardiovascular diseases. Hence, the aim of this study was to examine the in vivo antithrombotic effects of sulforaphane and its possible mechanisms in platelet activation. Sulforaphane (0.125 and 0.25 mg/kg) was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice. Other in vivo studies also revealed that sulforaphane (0.25 mg/kg) significantly prolonged platelet plug formation in mice. In addition, sulforaphane (15–75 μM) exhibited more-potent activity of inhibiting platelet aggregation stimulated by collagen. Sulforaphane inhibited platelet activation accompanied by inhibiting relative Ca²⁺ mobilization; phosphorylation of phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinases (MAPKs) and Akt; and hydroxyl radical (OH^●) formation. Sulforaphane markedly increased cyclic (c)AMP, but not cyclic (c)GMP levels, and stimulated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. SQ22536, an inhibitor of adenylate cyclase, but not ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxal in-1-one), an inhibitor of guanylate cyclase, obviously reversed the sulforaphane-mediated effects on platelet aggregation; PKC activation, p38 MAPK, Akt and VASP phosphorylation; and OH^● formation. Furthermore, a PI3-kinase inhibitor (LY294002) and a p38 MAPK inhibitor (SB203580) both significantly diminished PKC activation and p38 MAPK and Akt phosphorylation; in contrast, a PKC inhibitor (RO318220) did not diminish p38 MAPK or Akt phosphorylation stimulated by collagen. This study demonstrates for the first time that in addition to it originally being considered as an agent for prevention of tumor growth, sulforaphane possesses potent antiplatelet activity which may initially activate adenylate cyclase/cAMP, followed by inhibiting intracellular signals (such as the PI3-kinase/Akt and PLCγ2-PKC-p47 cascades) and ultimately inhibiting platelet activation. Therefore, this novel role of sulforaphane may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.     

Analysis of association of TaqI VDR gene polymorphism with the chronic periodontitis in Dravidian ethnicity

AIM:

The aim of this study is to analyze the association of TaqI vitamin D receptor (VDR) gene polymorphism with the chronic periodontitis (CP) in Dravidian ethnicity.

MATERIALS AND METHODS:

A total of 120 subjects were recruited for this study, which included 60 CP and 60 healthy controls. TaqI VDR gene polymorphism was analyzed using specific primers and amplified by polymerase chain reaction (PCR) and visualized under 2% agarose gel.

RESULTS:

Our study results showed that Tt and tt genotype had a higher frequency of occurrence in CP compared with controls. Similarly, t allele was found to be associated with CP.

CONCLUSION:

Our study concludes that TaqI VDR gene polymorphism is associated with CP in Dravidian ethnicity.     

Rationally designed turn promoting mutation in the amyloid-β peptide sequence stabilizes oligomers in solution

Enhanced production of a 42-residue beta amyloid peptide (Aβ(42)) in affected parts of the brain has been suggested to be the main causative factor for the development of Alzheimer's Disease (AD). The severity of the disease depends not only on the amount of the peptide but also its conformational transition leading to the formation of oligomeric amyloid-derived diffusible ligands (ADDLs) in the brain of AD patients. Despite being significant to the understanding of AD mechanism, no atomic-resolution structures are available for these species due to the evanescent nature of ADDLs that hinders most structural biophysical investigations. Based on our molecular modeling and computational studies, we have designed Met35Nle and G37p mutations in the Aβ(42) peptide (Aβ(42)Nle35p37) that appear to organize Aβ(42) into stable oligomers. 2D NMR on the Aβ(42)Nle35p37 peptide revealed the occurrence of two β-turns in the V24-N27 and V36-V39 stretches that could be the possible cause for the oligomer stability. We did not observe corresponding NOEs for the V24-N27 turn in the Aβ(21-43)Nle35p37 fragment suggesting the need for the longer length amyloid peptide to form the stable oligomer promoting conformation. Because of the presence of two turns in the mutant peptide which were absent in solid state NMR structures for the fibrils, we propose, fibril formation might be hindered. The biophysical information obtained in this work could aid in the development of structural models for toxic oligomer formation that could facilitate the development of therapeutic approaches to AD.     

Biocompatible alginate/nano bioactive glass ceramic composite scaffolds for periodontal tissue regeneration

Periodontal regeneration is of utmost importance in the field of dentistry which essentially reconstitutes and replaces the lost tooth supporting structures. For this purpose, nano bioactive glass ceramic particle (nBGC) incorporated alginate composite scaffold was fabricated and characterized using SEM, EDAX, AFM, FTIR, XRD and other methods. The swelling ability, in vitro degradation, biomineralization and cytocompatibility of the scaffold were also evaluated. The results indicated reduced swelling and degradation and enhanced biomineralization and protein adsorption. In addition, the human periodontal ligament fibroblast (hPDLF) and osteosarcoma (MG-63) cells were viable, adhered and proliferated well on the alginate/bioglass composite scaffolds in comparison to the control alginate scaffolds. The presence of nBGC enhanced the alkaline phosphatase (ALP) activity of the hPDLF cells cultured on the composite scaffolds. Thus results suggest that these biocompatible composite scaffolds can be useful for periodontal tissue regeneration.