Visualisation of the Native N-type Calcium Channel

N-type calcium channels (CaV2.2) are important for neurotransmitter release in the central and peripheral nervous system. Immunohistochemical detection of native CaV2.2 has not been possible until now due to the low expression of these channels and lack of suitable antibodies. The present study utilises the recently developed constitutive knock-in (KI) transgenic mouse, expressing CaV2.2 with an epitope tag (2 x haemagglutinin; HA) inserted in the extracellular loop between S3 and S4 of domain II (CaV2.2_HAKIKI). The tag does not affect the function of the channel when expressed in vitro (Cassidy et al., 2014). In the somatosensory nervous system, the data show that CaV2.2_HA is expressed on the cell surface of dorsal root ganglion (DRG) neurons. In the spinal cord, CaV2.2_HA is predominantly in the superficial laminae LIand LII of the dorsal horn, mainly in the primary afferent terminals, since there is a reduction in CaV2.2_HA staining following rhizotomy. Co-cultures between DRG and spinal cord neurons permit the study of CaV2.2_HA at the presynaptic terminal. Super-resolution images of the synapses formed by these co-cultures revealed the regulation of CaV2.2_HA expression at the presynaptic terminal over time in culture. Preliminary studies of TRPV1 activation by capsaicin on cell surface CaV2.2_HA was tested on cultured DRG neurons. Short incubations (20 s) with capsaicin increases CaV2.2_HA expression at the cell membrane of small, medium and large-diameter neurons. However, following longer incubations (2 to 4 min) with capsaicin, there is a substantial decrease of CaV2.2_HA immunoreactivity at the membrane. Nevertheless, further studies are required to determine whether this is mediated by a TRPV1-dependent or -independent mechanism. The CaV2.2_HAKIKI mice will be instrumental in future studies to enhance the understanding of the presynaptic role of endogenous N-type calcium channels in physiological and pathological states

An investigation into the role of intercellular adhesion molecule-2 in neutrophil extravasation using an in vivo murine model

PhDRecruitment of neutrophils into the tissue during inflammation is a crucial component of the immune response. This study aimed to further understand the role of intercellular adhesion molecule-2 (ICAM-2) in this process. Endothelial cell (EC) ICAM-2 has been implicated in neutrophil extravasation however, its precise role in this process is largely unknown. To address this, the current investigation examined the expression and functional role of ICAM-2 in neutrophil-EC interactions in vivo. Analysis of EC ICAM-2 expression was performed in the mouse cremaster muscle using immunofluorescent staining and confocal microscopy. A high EC body expression of ICAM-2 relative to that of EC junctions in post-capillary venules was observed. It was therefore hypothesised that ICAM-2 could potentially be involved in both luminal neutrophil-EC and junctional interactions. This hypothesis was analysed using confocal intravital microscopy (IVM) of cremaster muscles from WT or ICAM-2 KO Lys-eGFP-ki mice (express fluorescent neutrophils) in conjunction with fluorescent labelling of ECs. Neutrophil crawling and transendothelial migration (TEM) dynamics in IL-1β-stimulated post-capillary venules was analysed. A role for ICAM-2 in supporting speed and continuity of crawling and the initiation of TEM was demonstrated. Using functional blocking mAb to MAC-1 in WT and ICAM-2 KOs, the role of ICAM-2 in neutrophil crawling was demonstrated to be governed through a potential interaction with neutrophil MAC-1. It is therefore possible that non-junctional EC ICAM-2 has important roles in regulating neutrophil polarisation during crawling whilst junctional ICAM-2 mediates the opening of EC junctions and/or influencing the site of ‘preferred’ TEM. This study provides the first in vivo evidence for the ability of ICAM-2 to support neutrophil crawling and the initiation of TEM in IL-1β-induced neutrophil extravasation. To extend the above findings in a complex vascular injury model, a cremasteric Shwartzman Reaction, amenable to IVM analysis, was also developed as part of this project.British Heart Foundation (BHF

Production and Characterization of Recombinant Amelogenin Phosphorylation Mimics

Amelogenin is an extracellular matrix protein which has an important role to play in enamel formation during tooth development. The need for amelogenin for research purposes in order to be utilized for its many potential applications have resulted in the rise of the production of recombinant amelogenin. So far, the most optimal expression system used for the production of recombinant amelogenin is Escherichia coli. The only difference between native amelogenin and recombinant amelogenin expressed using E.coli is that the latter lack methionine at the N-terminus and phosphate on Serine-16.The aim of this study was to produce recombinant amelogenin phosphorylation mimics and to investigate the effects of phosphorylation on amelogenin. A total of 16 recombinant amelogenin phosphorylation mimics were successfully constructed by site-directed mutagenesis where serine-16 was mutated to either aspartic acid or glutamic acid. There were difficulties encountered during the purification of the higher positively charged recombinant amelogenin phosphorylation mimics which indicated that factors other than optical density may have a vital effect on their production. No effect of phosphorylation was found on solubility as the phosphorylated amelogenins shared similar solubility profiles to their non-phosphorylated analogues on the pH range of 4 to 8. The solubility of the recombinant amelogenin fusion and phosphorylation mimics were greater than that of the recombinant native amelogenin and its phosphorylation mimic analogue. The analysis of the recombinant amelogenin phosphorylation mimics by dynamic light scattering showed that they too can form nanospheres. There were minor variations in hydrodynamic radii and polydispersity indices of the recombinant phosphorylated and non-phosphorylated amelogenins at both 20oC and 37oC which indicated that phosphorylation had not affected amelogenin self-assembly. It was found that the recombinant amelogenin phosphorylation mimics as well as their non-phosphorylated analogues had similar strong apatite-binding affinity which also showed no effect of phosphorylation on the binding affinity of amelogenin.We use our teeth daily to eat all sorts of food with varying degrees of texture and hardness. The white part of our teeth called the enamel is the hardest tissue of the human body. Have you ever wondered what makes our teeth so hard and strong that we can chew on a chicken bone? Well, the answer is amelogenin which is the main subject of this study. Amelogenin is one of the many proteins that help build the enamel. However, amelogenin is of greatest importance to enamel formation because in its absence scientists have found that it leads to teeth defects. Scientists have also discovered that amelogenin can be utilized in many applications such as teeth defects, wound healing, bone formation and regeneration and many others. So far, there are two amelogenin-based products on the market. These products include (i) Emdogain® for the treatment of periodontitis, an enamel defect due to lack of proper amelogenin and (ii) Xelma® used to treat leg ulcers. As a result of the numerous potential applications of amelogenin, thus, there is increasingly more research carried out on amelogenins. So far, the most optimal production system used for the production of recombinant amelogenin is the bacteria called Escherichia coli. The only difference that existed between native amelogenin and recombinant amelogenin expressed using E.coli is that the latter lack the amino acid methionine at the N-terminus and phosphate group on Serine-16.The aim of this present study was to produce recombinant amelogenin phosphorylation mimics and to investigate the effects of phosphorylation of amelogenin on its properties. The properties of the novel phosphorylated amelogenin mimics with that of non-phosphorylated amelogenins were compared in order to examine the effects of the phosphorylation

Capsaicin-induced endocytosis of endogenous presynaptic Ca_{V}2.2 in DRG-spinal cord co-cultures inhibits presynaptic function

The N-type calcium channel, CaV2.2 is key to neurotransmission from the primary afferent terminals of dorsal root ganglion (DRG) neurons to their post-synaptic targets in the spinal cord. In this study we have utilized CaV2.2_HA knock-in mice, because the exofacial epitope tag in CaV2.2_HA enables accurate detection and localization of endogenous CaV2.2. CaV2.2_HA knock-in mice were used as a source of DRGs to exclusively study the presynaptic expression of N-type calcium channels in co-cultures between DRG neurons and wild-type spinal cord neurons. CaV2.2_HA is strongly expressed on the cell surface, particularly in TRPV1-positive small and medium DRG neurons. Super-resolution images of the presynaptic terminals revealed an increase in CaV2.2_HA expression and increased association with the post-synaptic marker Homer over time in vitro. Brief application of the TRPV1 agonist, capsaicin, resulted in a significant down-regulation of cell surface CaV2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in CaV2.2_HA proximity to and co-localization with the active zone marker RIM 1/2, as well as a lower contribution of N-type channels to single action potential-mediated Ca2+ influx. The mechanism of this down-regulation of CaV2.2_HA involves a Rab 11a-dependent trafficking process, since dominant-negative Rab11a(S25N) occludes the effect of capsaicin on presynaptic CaV2.2_HA expression, and also prevents the effect of capsaicin on action potential induced Ca2+ influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface CaV2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway

PRELIMINARY PHYTOCHEMICAL ANALYSIS AND IN VITRO ANTIOXIDANT ACTIVITY OF ARAUCARIA COLUMNARIS BARK PEEL AND COSMOS SULPHUREUS FLOWERS

Objective: Four different extracts of Araucaria columnaris (bark peel) and Cosmos sulphureus (flowers) were screened for their phytochemical composition, and free radical scavenging activities.Methods: DPPH method was used to test the antioxidant activity for extracts.Results: Among the different extracts tested, the methanol extract of both the plant species showed significant radical scavenging activities. Phytochemical analysis of the extracts revealed that the radical scavenging activities might be due to the presence of flavonoids, tannins and phenolic compounds.Conclusion: The results obtained suggest that Araucaria columnaris (bark peel) and Cosmos sulphureus(flowers) could be exploited in the treatment of various diseases like cancer, cardiovascular diseases and infection diseases. Â

Enhancement and integration for CINDI system

The CINDI system is an assembly of inter-related subsystems, working together as a digital library for academic documents in the field of computer science. These subsystems include the CINDI Robot, which downloads scientific documents including theses, technical reports, FAQ's, academic papers and discussion groups, the CINDI Conference system and the CINDI Registration and Upload subsystem, where authors upload academic documents. In addition, there is the Gleaning subsystem that converts the non-PDF documents to PDF format and filters out the documents that are more appropriate, the Automatic Semantic Header Generator which locates information about the author, title, keywords, subject and abstract from the documents, and the CINDI Search subsystem which enables users to search for resources in the CINDI repository, This thesis is based on the techniques that were used for the integration of subsystems, which includes porting of the Document Converter from the Windows platform to Linux. Enhancements were made to the Registration and Upload subsystem to allow multiple file uploads and improvements were made to the Graphical User Interface. The CINDI Search subsystem was redesigned to improve functionality and its interface was made more user-friendly. We have also developed an Annotation subsystem allowing users to make comments on documents in the CINDI repository

Involvement of CaV2.2 channels and α2δ-1 in homeostatic synaptic plasticity in cultured hippocampal neurons

In the mammalian brain, presynaptic CaV2 channels play a pivotal role for synaptic transmission by mediating fast neurotransmitter exocytosis via influx of Ca2+ into the active zone of presynaptic terminals. However, the distribution and modulation of CaV2.2 channels at plastic hippocampal synapses remains to be elucidated. Here, we assess CaV2.2 channels during homeostatic synaptic plasticity, a compensatory form of homeostatic control preventing excessive or insufficient neuronal activity during which extensive active zone remodelling has been described. We show that chronic silencing of neuronal activity in mature hippocampal cultures resulted in elevated presynaptic Ca2+ transients, mediated by increased levels of CaV2.2 channels at the presynaptic site. This work focussed further on the role of α2δ-1 subunits, important regulators of synaptic transmission and CaV2.2 channel abundance at the presynaptic membrane. We find that α2δ-1-overexpression reduces the contribution of CaV2.2 channels to total Ca2+ flux without altering the amplitude of the Ca2+ transients. Levels of endogenous α2δ-1 decreased during homeostatic synaptic plasticity, whereas the overexpression of α2δ-1 prevented homeostatic synaptic plasticity in hippocampal neurons. Together, this study reveals a key role for CaV2.2 channels and novel roles for α2δ-1 during synaptic plastic adaptation

Secure Image Transmission using Visual Steganography

In today’s information age, information sharing and transfer has increased exponentially. The threat of an intruder accessing secret information has been an ever existing concern for all. Cryptography and Steganography are the most widely used techniques to overcome these threats. Steganography is a branch of security technique which involves the art of hiding the existence of the message between sender and the intended recipient. Here, steganography has been used to hide digital images. Cryptography involves converting the message text into an unreadable cipher. To overcome this predictability, we propose the concept of visual cryptographic steganography where slices of original secret image upon encryption are embedded within a cover image. The resultant stego image is then decrypted to recover the original image

Study of patients with liver dysfunction during pregnancy and their maternal and perinatal outcomes

Background: Liver dysfunction in pregnancy can be associated with maternal and perinatal morbidity and mortality, therefore early recognition and timely management is of paramount importance to improve the outcome. The studies related to liver dysfunction in pregnancy and its outcome are sparse from this part of India and are retrospective in nature, so present study was planned.Methods: A total of 80 pregnant patients with liver dysfunction were enrolled as per the inclusion criteria after taking informed consent. Patients were investigated depending on the symptoms and pregnancy related complications with an aim to know the probable cause of liver dysfunction. Maternal and perinatal outcomes were noted in these patients.Results: Intrahepatic cholestasis of pregnancy was the most common cause of deranged liver function tests (71.3%) followed by HELLP syndrome (21.3%), viral hepatitis (6.3%) and AFLP (1.3%) respectively. The most common maternal complication seen was preterm labour (33.8%) followed by thrombocytopenia (11.3%), postpartum hemorrhage (7.5%), vaginal wall hematoma (7.5%) and coagulopathy (3.8%). 2 patients (2.5%) required ICU admission and both patients expired due to fulminant hepatic failure. The most common fetal complication was prematurity (33.8%). Intrauterine fetal demise occurred in 10% of the patients and there were 12.5% perinatal deaths observed in our study.Conclusions: The commonest cause of liver dysfunction in our study was IHCP (71.3%) followed by HELLP syndrome (21.3%). In spite of multidisciplinary approach, liver dysfunction during pregnancy was associated with high maternal and perinatal morbidity and mortality