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Breast cancer (BC), the second most common cause of cancer-related deaths, remains a significant threat to the health and wellness of women worldwide. The tumor microenvironment (TME), comprising cellular components, such as cancer-associated fibroblasts (CAFs), immune cells, endothelial cells and adipocytes, and noncellular components such as extracellular matrix (ECM), has been recognized as a critical contributor to the development and progression of BC. The interplay between TME components and cancer cells promotes phenotypic heterogeneity, cell plasticity and cancer cell stemness that impart tumor dormancy, enhanced invasion and metastasis, and the development of therapeutic resistance. While most previous studies have focused on targeting cancer cells with a dismal prognosis, novel therapies targeting stromal components are currently being evaluated in preclinical and clinical studies, and are already showing improved efficacies. As such, they may offer better means to eliminate the disease effectively. In this review, we focus on the evolving concept of the TME as a key player regulating tumor growth, metastasis, stemness, and the development of therapeutic resistance. Despite significant advances over the last decade, several clinical trials focusing on the TME have failed to demonstrate promising effectiveness in cancer patients. To expedite clinical efficacy of TME-directed therapies, a deeper understanding of the TME is of utmost importance. Secondly, the efficacy of TME-directed therapies when used alone or in combination with chemo- or radiotherapy, and the tumor stage needs to be studied. Likewise, identifying molecular signatures and biomarkers indicating the type of TME will help in determining precise TME-directed therapies.

Introduction: Zinc oxide nanoparticles (ZnO NPs) have recently attracted attention as potential anti-cancer agents. To the best of our knowledge, the toxicity of ZnO NPs against human chronic myeloid leukemia cells (K562 cell line) has not been studied using transcriptomics approach. Objective: The goals of this study were to evaluate the capability of ZnO NPs to induce apoptosis in human chronic myeloid leukemia cells (K562 cells) and to investigate the putative mechanisms of action. Methods: We used viability assay and flowcytometry coupled with Annexin V-FITC and propidium iodide to investigate the toxicity of ZnO NPs on K562 cells and normal peripheral blood mononuclear cells. Next we utilized a DNA microarray-based transcriptomics approach to characterize the ZnO NPs-induced changes in the transcriptome of K562 cells. Results: ZnO NPs exerted a selective toxicity (mainly by apoptosis) on the leukemic cells (p≤ 0.005) and altered their transcriptome; 429 differentially expressed genes (DEGs) with fold change (FC)≥ 4 and p≤ 0.008 with corrected p≤ 0.05 were identified in K562 cells post treatment with ZnO NPs. The over-expressed genes were implicated in “response to zinc”, “response to toxic substance” and “negative regulation of growth” (corrected p≤ 0.05). In contrast, the repressed genes positively regulated “cell proliferation”, “cell migration”, “cell adhesion”, “receptor signaling pathway via JAK-STAT” and “phosphatidylinositol 3-kinase signaling” (corrected p≤ 0.05). Lowering the FC to ≥ 1.5 with p≤ 0.05 and corrected p≤ 0.1 showed that ZnO NPs over-expressed the anti-oxidant defense system, drove K562 cells to undergo mitochondrial-dependent apoptosis, and targeted NF-κB pathway. Conclusion: Taken together, our findings support the earlier studies that reported anti-cancer activity of ZnO NPs and revealed possible molecular mechanisms employed by ZnO NPs to induce apoptosis in K562 cells.

Mycotic/fungal keratitis is a suppurative, generally ulcerative infection of the cornea. The filamentous fungi, Aspergillus spp. are the second leading cause of mycotic keratitis, particularly in India. Aspergillus spp. produce a range of extracellular enzymes that are used to break down complex molecules and used for growth and reproduction, also for survival on/in host organism. The current study was designed with an objective to screen in vitro extracellular enzyme activity of Fusarium and Aspergillus isolates from mycotic keratitis patients and to correlate the same as a putative virulence factor. Extracellular enzymes viz., deoxyribonuclease (DNase), protease, lipase, elastase, keratinase, etc., produced by Aspergillus have key role in keratomycosis and hence their (n = 85) in vitro activities were investigated. It was found that, the majority of the Aspergillus isolates produced protease (n = 75; 88% of 85) followed by lipase (n = 59; 69% of 85), DNase (n = 35; 41% of 85), elastase (n = 26; 31% of 85) and keratinase (n = 13; 15% of 85). The enzyme activity indices (EAI) for DNase, elastase, protease and lipase ranged between 1.01 and 1.98, whereas elastase EAI varied between 1.26 and 1.92. DNase, protease and lipase showed a maximum EAI of 1.98 and lowest EAI value of 1.01, respectively. Extracellular enzymes of Aspergillus spp. may have potential role in the onset and progression of keratitis. Keywords: Mycotic keratitis, Aspergillus, Extracellular enzyme activity, Virulence, Corneal ulcer, Eye infections

Objectives In preterm infants, hyperbilirubinemia is common and can impair the central nervous system. The tests available for measuring bilirubin is to collect blood from heel pricking and occasionally taking blood samples from inserted cannulas, which is painful. Therefore, there is a need to develop a non-invasive device to detect bilirubin levels in newborns and interpret the severity of jaundice. Methods We conducted a cross-sectional study of 100 neonates. Patient data was collected between June 2015 and December 2016 from King Khalid Hospital at Al-Majma'ah, Saudi Arabia, and Alpine Hospital, Gurgaon, India. The mean gestational age of neonates was 39.0 weeks. Total bilirubin was measured using a transcutaneous bilirubinometer on the forehead and obtaining optical imaging through scanning of conjunctiva of eyes, also referred to as BiliChek and BiliCapture, respectively. Later the blood samples were obtained from these patients and tested in the laboratory to determine total serum bilirubin (TSB) levels. Results The concentration of bilirubin as measured from serum, BiliChek, and BiliCapture were 10.7±2.0, 11.6±2.7, and 13.1±2.3 mg/dL, respectively. Correlation was high between TSB and BiliChek (r2 = 0.88) and between TSB and BiliCapture (r2 = 0.73). The Bland-Altman plots showed good agreement when comparing bilirubin values for both BiliChek and BiliCapture devices. Bilirubin measurement was further checked for the sensitivity and specificity and was 88.0% and 76.0% using BiliChek and 92.0% and 75.6% using BiliCapture, respectively. Conclusions The optical imaging of conjunctiva for bilirubin assay is a safe alternative to a laboratory bilirubin assay and transcutaneous bilirubinometer BiliChek.

Much recent attention has been given to traditional medicines and natural products with potential and promising anti-inflammatory properties. Leaf extract of Ginkgo biloba GbE-761, possesses several clinical beneficial effects and is now used in a broad spectrum of pharmacological actions. However, mechanisms associated with its anti-inflammatory effect are not very much clear. Previously we established that GbE-761 exerts a neuroprotective effect against ischemic brain injury through an anti-apoptotic mechanism. In this study we investigated the anti-inflammatory effect of GbE-761 in LPS-activated murine macrophage. Our results demonstrate that GbE-761 potently inhibited LPS-induced NO and PGE2 production and these observations were consistent with the inhibition in the protein and mRNA expression levels of inducible iNOS and COX-2 enzymes in a dose dependent manner. Not only this, GbE-761 attenuated the production of pro-inflammatory cytokines like IL-1 , IL-6 and TNFin LPS-activated murine macrophages in concentration dependent manner. In addition we reported inhibition in the protein and mRNA expression levels of IL-1 , IL-6 and TNF. Furthermore, GbE-761 inhibited the NFB activation induced by LPS. Together these results suggest that anti-inflammatory properties of GbE-761 extract might be the results of inhibition of iNOS and COX-2 by upregulating anti-oxidative enzymes and inhibition of pro-inflammatory cytokines (IL-1 , IL-6 and TNF) expression through the down-regulation of NF-kB activation in murine macrophages.

Background: We previously identified increased tissue localization of monomeric C-reactive protein (mCRP) in the infarcted cortical brain tissue of patients following ischaemic stroke. Here, we investigated the relationship of mCRP expression in haemorrhagic stroke, and additionally examined the capacity of mCRP to travel to or appear at other locations within the brain that might account for later chronic neuroinflammatory or neurodegenerative effects. Methods: Immunohistochemistry was performed on Formalin-fixed, paraffin-embedded archived brain tissue blocks obtained at autopsy from stroke patients and age-matched controls. We modelled mCRP migration into the brain after haemorrhagic stroke by infusing mCRP (3.5 µg) into the hippocampus of mice and localized mCRP with histological and immunohistochemistry methods. Results: On human tissue in the early stages of haemorrhage, there was no staining of mCRP. However, with increasing post-stroke survival time, mCRP immunostaining was associated with some parenchymal brain cells, some stroke-affected neurons in the surrounding areas and the lumen of large blood vessels as well as brain capillaries. Further from the peri-haematoma region, however, mCRP was detected in the lumen of micro-vessels expressing aquaporin 4 (AQP4). In the hypothalamus, we detected clusters of neurons loaded with mCRP along with scattered lipofuscin-like deposits. In the peri-haematoma region of patients, mCRP was abundantly seen adjacent to AQP4 immunoreactivity. When we stereotactically injected mCRP into the hippocampus of mice, we also observed strong expression in distant neurones of the hypothalamus as well as cortical capillaries. Conclusions: mCRP is abundantly expressed in the brain after haemorrhagic stroke, directly impacting the pathophysiological development of the haematoma. In addition, it may have indirect effects, where the microcirculatory system appears to be able to carry it throughout the cortex as far as the hypothalamus, allowing for long-distance effects and damage through its capacity to induce inflammation and degenerate neuronal perivascular compartments., This work was supported by a grant of the Romanian National Authority for Science Research and Innovation, CNCS—UEFISCDI, project number TE-41/2020 and from a grant from the Competitiveness Operational Programme 2014-2020: C-reactive protein therapy for stroke-associated dementia: ID_P_37_674, My SMIS code: 103432 contract 51/05.09.2016, and also and a grant from the Spanish Ministry of Science and Innovation SAF2016-77703 to CS. This research was also supported by the Stroke Chair-(project number R-1441-164).

Both in malignant and non-malignant disorders stem cell based therapies are increasingly being utilized with promising results. hematopoietic reconstitution comprises mainly of 3 types of cells bone marrow (BM), peripheral blood (PB), and umbilical cord blood (UCB). The readily available and abundant resource of stem cell is umbilical cord blood. However, the typical single UCB unit could fetch relatively low numbers of hematopoietic stem- and progenitor cells (HSPCs) and the associated delay in procuring them restrict its routine applicability. In the past decade, the clinical applications of UCBbased cell therapies have broadened with a growing number of diseases treated with hematopoietic stem cell (HSC) transplantation. The assessment of UCB unit hematopoietic stem cells (HSCs), such as CD34+ cells or CFUs (Colony forming units), may provide a more direct estimate of the hematopoietic potential of the unit than the TNC count. As interlaboratory standardization of these assays has been achieved, the selection of UCB units for banking based on quantitation of hematopoietic progenitors is currently not feasible. However, CFU assays provide the only reliable assessment of the viability of the CB unit, although great efforts have been made to develop flow cytometry methods for viability staining. Despite of extreme efforts to find strategies that would enable the ex vivo amplification of stem cells for transplantation globally, it has been proven difficult to culture the HSCs in the labs. Therefore, it is imperative to have clear understanding regarding integration of HSC self-renewal, proliferation, and differentiation, molecules participating in their regulation and clinical benefit after their modification. Attempts have been made to improve the quality of UCB units through e.g. standardization of bank procedures, of stem cell enumeration, and of the assessment of HPCs viability. Over 4000000 UCB units are currently available in international registries. However, a significant proportion of patients is still left without a suitable donor, necessitating further development of UCB banking process.

Introduction: Stroke is a multifactorial and heterogeneous disorder, correlates with heritability and considered as one of the major diseases. The prior reports performed the variable models such as genome-wide association studies (GWAS), replication, case-control, cross-sectional and meta-analysis studies and still, we lack diagnostic marker in the global world. There are limited studies were carried out in Saudi population, and we aim to investigate the molecular association of single nucleotide polymorphisms (SNPs) identified through GWAS and meta-analysis studies in stroke patients in the Saudi population. Methods: In this case-control study, we have opted gender equality of 207 cases and 207 controls from the capital city of Saudi Arabia in King Saud University Hospital. The peripheral blood (5 ml) sample will be collected in two different vacutainers, and three mL of the coagulated blood will be used for lipid analysis (biochemical tests) and two mL will be used for DNA analysis (molecular tests). Genomic DNA will be extracted with the collected blood samples, and specific primers will be designed for the opted SNPs (SORT1-rs646218 and OLR1-rs11053646 polymorphisms) and PCR-RFLP will be performed and randomly DNA sequencing will be carried out to cross check the results. Results: The rs646218 and rs11053646 polymorphisms were significantly associated with allele, genotype and dominant models with and without crude odds ratios (OR’s) and Multiple logistic regression analysis (p

1Department of Microbiology, MR Government Arts College, Mannargudi 614 001, India 2Department of Microbiology, Dr. GR Damodaran College of Science, Coimbatore – 641 014, India. 3Faculty of Science and Informatics, Department of Microbiology, University of Szeged, Szeged, Hungary. 4Department of Medical Laboratory, Applied Medical Sciences College, Majmaah University, Kingdom of Saudi Arabia. 5Department of Microbiology, Aravind Eye Hospital and Postgraduate Institute of Ophthalmology, Coimbatore 641 014, Tamilnadu, India

Total cholesterol monitoring in human blood serum is one of the most important routine analysis performed in clinical laboratory. Epidemiological studies have shown a strong correlation between coronary heart disease and blood cholesterol level. There is need for a method that is sufficiently flexible to yield good results in clinical laboratory. Numbers of cholesterol biosensors have been developed over the past 30 years. Fibre-optic fluorescence, Fibre-optic luminescence, Potentiometric, Spectrophotometric and Fluorometric biosensors, which determine cholesterol enzymatically. Some of these methods suffer from interference from other substances found in the blood such as ascorbic acid and uric acid. Therefore amperometric biosensor was designed based on titanium oxide nano particle with Advanced RISC (Reduced Instruction Set Computing) Machine processor to determine the cholesterol level in human blood.

Tuberculosis (TB) also known as white plague is a major infectious disease and a global emergency. It is one of the oldest recorded human afflictions and still continues to cause widespread morbidity and mortality in children and adults worldwide, despite the extensive use of a live attenuated vaccine and several antibiotics. One third of the world’s population is already infected and about 3 million people die and 8 million people develop the active disease each year. In the last decade there is a sharp rise in the cases of TB mainly due to emergence of multi-drug resistant (MTD), extensively drug-resistant (XDR) and totally drugresistant (TDR) strains of Mycobacterium Tuberculosis, which are virtually untreatable.

© 2016 Wen-Hui Fang et al.The potential use of stem cells as therapeutics in disease has gained momentum over the last few years and recently phase-I clinical trials have shown favourable results in treatment of a small cohort of acute stroke patients. Similarly, they have been used in preclinical models drug-loaded for the effective treatment of solid tumours. Here we have characterized uptake and release of a novel p5-cyclin-dependent kinase 5 (CDK5) inhibitory peptide by mesenchymal stem cells and showed release levels capable of blocking aberrant cyclin-dependent kinase 5 (CDK5) signaling pathways, through phosphorylation of cyclin-dependent kinase 5 (CDK5) and p53. These pathways represent the major acute mechanism stimulating apoptosis after stroke and hence its modulation could benefit patient recovery. This work indicates a potential use for drug-loaded stem cells as delivery vehicles for stroke therapeutics and in addition as anticancer receptacles particularly, if a targeting and/or holding mechanism can be defined.

Context: Didactic (lecture) method is a popular way of delivering the information in class among teachers amidst the other multiple novel methods, but there are very few researches published related to exploring the perception of teachers and students about effective didactic teaching. Aims: The aim of this survey is to find the best way to deliver a lecture as per the opinion of students and teachers in the College of Applied Health Sciences. Settings and Design: A cross-sectional survey through a questionnaire was obtained from a previous study on medical students with original questionnaire consisting of 17 items. In addition to the 17 items in the questionnaire, 4 new items were included through a pilot study on students and teachers in the College of Applied Medical Sciences. Subjects and Methods: Students (n = 110) and teachers (n = 41) participated in the cross-sectional study. Good Teaching Questionnaire was used to obtain opinion and views about ways to make didactic teaching better and effective. Statistical Analysis Used: Nonparametric tests were used to analyze data obtained from the questionnaire. Results: Almost 100% of teachers and students agreed to include mental rehearsal and smart board method of teaching in didactic method. Nearly 100% of students and 29.2% (12) of teachers agreed to include multiple-choice questions in the lecture. Conclusions: Smart board presentation is preferred over the other modes of delivery of information. Mental rehearsal at the end of class is a useful tool to enhance learning and content retention. Teachers must implement students' suggestions such as ideal duration of class, time of class, lot of relevant examples, and little bit of fun would make learning enjoyable and effective.

M. Slevin et al., Alzheimer's disease (AD) increases dramatically in patients with ischaemic stroke. Monomeric C-reactive protein (mCRP) appears in the ECM of ischaemic tissue after stroke, associating with microvasculature, neurons and AD-plaques, Aβ, also, being able to dissociate native-CRP into inflammatory, mCRP in vivo. Here, mCRP injected into the hippocampal region of mice was retained within the retrosplenial tract of the dorsal 3rd ventrical and surrounding major vessels. Mice developed behavioural/cognitive deficits within 1 month, concomitant with mCRP staining within abnormal looking neurons expressing p-tau and in beta-amyloid 1-42-plaque positive regions. mCRP co-localised with CD105 in microvessels suggesting angiogenesis. Phospho-arrays/Western blotting identified signalling activation in endothelial cells and neurons through p-IRS-1, p-Tau and p-ERK1/2-which was blocked following pre-incubation with mCRP-antibody. mCRP increased vascular monolayer permeability and gap junctions, increased NCAM expression and produced haemorrhagic angiogenesis in mouse matrigel implants. mCRP induced tau244-372 aggregation and assembly in vitro. IHC study of human AD/stroke patients revealed co-localization of mCRP with Aβ plaques, tau-like fibrils and IRS-1/P-Tau positive neurons and high mCRP-levels spreading from infarcted core regions matched reduced expression of Aβ/Tau. mCRP may be responsible for promoting dementia after ischaemia and mCRP clearance could inform therapeutic avenues to reduce the risk of future dementia., We would like to thank the Fundacion BBVA for their generous support of Professor Mark Slevin through the award of BBVA Chair in Clinical Biomedicine at the ICCC, St Pau Hospital, Barcelona. This work was also supported by the Spanish Ministry of Science (SAF2009-01237 to J.K.), Spanish Ministry Project-CSD2010-00045 to C.S. and RyC (RyC2007-01466 to M.B.-P.) and the Almajmaah University Stroke Chair

Alzheimer's disease (AD) and vascular dementia (VaD) are the most common cause of dementia. Cerebral ischemia is a major risk factor for development of dementia. (123)I-FP-CIT SPECT (DaTScan) is a complementary tool in the differential diagnoses of patients with incomplete or uncertain Parkinsonism. Additional application of DaTScan enables the categorization of Parkinsonian disease with dementia (PDD), and its differentiation from pure AD, and may further contribute to change the therapeutic decision. The aim of this study was to analyze the vascular contribution towards dementia and mild cognitive impairment (MCI). We evaluated the utility of DaTScan for the early diagnosis of dementia in patients with and without a clinical vascular component, and the association between neuropsychological function, vascular component and dopaminergic function on DaTScan. One-hundred and five patients with MCI or the initial phases of dementia were studied prospectively. We developed an initial assessment using neurologic examination, blood tests, cognitive function tests, structural neuroimaging and DaTScan. The vascular component was later quantified in two ways: clinically, according to the Framingham Risk Score (FRS) and by structural neuroimaging using Wahlund Scale Total Score (WSTS). Early diagnosis of dementia was associated with an abnormal DaTScan. A significant association was found between a high WSTS and an abnormal DaTScan (p < 0.01). Mixed AD was the group with the highest vascular component, followed by the VaD group, while MCI and pure AD showed similar WSTS. No significant associations were found between neuropsychological impairment and DaTScan independently of associated vascular component. DaTScan seems to be a good tool to discriminate, in a first clinical assessment, patients with MCI from those with established dementia. There was bigger general vascular affectation observable in MRI or CT in patients with abnormal dopaminergic uptake seen on DaTScan.

Jaundice is a yellowish pigmentation of the skin, the conjunctiva, and other mucous membranes caused by increased levels of bilirubin in blood beyond normal levels (hyperbilirubinemia). Jaundice occurs in neonates in 2 forms: Pathological and Physiological Jaundice. In Pathological Jaundice, high bilirubin levels can result in severe neurotoxicity, brain damage, hearing loss, muscular disorders and physical abnormalities. So measuring the exact level of bilirubin in blood in neonates is very crucial. In the past, serum bilirubin has been the preferred method of detecting hyperbilirubinemia in newborns. The ordering of serum bilirubin in neonates is based on visual evaluation by either physicians or nursing staff. Skin puncture collection of blood exposes the neonate to trauma and risk of infection. A noninvasive device for predicting serum bilirubin levels in newborns diminishes the need to do skin punctures. There are numerous studies demonstrating the transcutaneous bilirubin measurements reasonably close to serum bilirubin performed by clinical laboratories. These devices use spectrophotometry (light absorption measurements at standardized wavelengths of light) to calculate measurements. All existing methods involve transcutaneous detection of bilirubin, which is not error free, as there are various factors affecting its accuracy, like presence of hemoglobin and melanin, and maturity of skin tissue and fibres. So this gave an urge for us to develop a new technique of quantitating bilirubin level noninvasively. This study was done by analyzing conjunctival images that would imitate the blood level of bilirubin by calculating the intensity of yellow color. Machine learning techniques were applied in order to develop a system capable of predicting the bilirubin level.

Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition. © 2013 Bosutti et al.

Analysis of blood substances such as hemoglobin, glucose, protein, cholesterol, etc. are important parameters for health condition monitoring. Traditionally, blood sample is taken using a needle. Some chemical process such as enzyme reaction is required before the final value for concentration is read. Reagents involved in such processes are expensive for both the hospital and patient. Non-invasive measurement is much more attractive by its evident advantages such as real time monitoring and infection free operation. The Hemoglobin (Hb) in human blood is measured and analyzed using Advanced RISC (Reduced Instruction Set Computing) machine processor non-invasively. This investigation helps in ruling out the diseases like anemia (a low hemoglobin level) and polycythemia vera (a high hemoglobin level). At present, the procedures used to measure the Hb concentration in blood are invasive. For this purpose blood is taken and analyzed. The hindrance of this method is the delay between the blood collection and its analysis. The portable smart non-invasive measurement system, discussed in this paper, gives a detailed idea about the hemoglobin concentration in the blood.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License.-- et al., [Background}: Citicoline is one of the neuroprotective agents that have been used as a therapy in stroke patients. There is limited published data describing the mechanisms through which it acts. [Methods]: We used in vitro angiogenesis assays: migration, proliferation, differentiation into tube-like structures in Matrigel™ and spheroid development assays in human brain microvessel endothelial cells (hCMEC/D3). Western blotting was performed on protein extraction from hCMEC/D3 stimulated with citicoline. An analysis of citicoline signalling pathways was previously studied using a Kinexus phospho-protein screening array. A staurosporin/calcium ionophore-induced apoptosis assay was performed by seeding hCMEC/D3 on to glass coverslips in serum poor medium. In a pilot in vivo study, transient MCAO in rats was carried out with and without citicoline treatment (1000 mg/Kg) applied at the time of occlusion and subsequently every 3 days until euthanasia (21 days). Vascularity of the stroke-affected regions was examined by immunohistochemistry. [Results]: Citicoline presented no mitogenic and chemotactic effects on hCMEC/D3; however, it significantly increased wound recovery, the formation of tube-like structures in Matrigel™ and enhanced spheroid development and sprouting. Citicoline induced the expression of phospho-extracellular-signal regulated kinase (ERK)-1/2. Kinexus assays showed an over-expression of insulin receptor substrate-1 (IRS-1). Knock-down of IRS-1 with targeted siRNA in our hCMEC/D3 inhibited the pro-angiogenic effects of citicoline. The percentage of surviving cells was higher in the presence of citicoline. Citicoline treatment significantly increased the numbers of new, active CD105-positive microvessels following MCAO. [Conclusions]: The findings demonstrate both a pro-angiogenic and protective effect of citicoline on hCMEC/D3 in vitro and following middle cerebral artery occlusion (MCAO) in vivo., This study was supported from research grant from Ferrer International.

Several prokaryotic and eukaryotic expression systems have been used for in vitro production of viruses' proteins. However eukaryotic expression system was always the first choice for production of proteins that undergo post-translational modification such as glycosylation. Recombinant baculoviruses have been widely used as safe vectors to express heterologous genes in the culture of insect cells, but the manipulation involved in creating, titrating, and amplifying viral stocks make it time consuming and laborious. Therefore, to facilitate rapid expression in insect cell, a plasmid based expression system was used to express herpes simplex type 1 glycoprotein D (HSV-1 gD) and varicella zoster glycoprotein E (VZV gE). Recombinant plasmids were generated, transfected into insect cells (SF9), and both glycoproteins were expressed 48 h post-infection. A protein with approximately molecular weight of 64-kDa and 98-kDa for HSV-1 gD and VZV gE respectively was expressed and confirmed by SDS. Proteins were detected in insect cells cytoplasm and outer membrane by immunofluorescence. The antigenicity and immunoreactivity of each protein were confirmed by immunoblot and ELISA. Results suggest that this system can be an alternative to the traditional baculovirus expression for small scale expression system in insect cells.