A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

<p>Abstract</p> <p>Background</p> <p>Hendra virus (HeV) and Nipah virus (NiV) are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new <it>Henipavirus </it>genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4) containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP) gene encoding human immunodeficiency virus type-1 (HIV-1) genome in conjunction with the HeV and NiV fusion (F) and attachment (G) glycoproteins.</p> <p>Results</p> <p>Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2) peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F.</p> <p>Conclusions</p> <p>Together, these results demonstrate that a specific henipavirus entry assay has been developed using NiV or HeV F and G glycoprotein pseudotyped reporter-gene encoding retrovirus particles. This assay can be conducted safely under BSL-2 conditions and will be a useful tool for measuring henipavirus entry and studying F and G glycoprotein function in the context of virus entry, as well as in assaying and characterizing neutralizing antibodies and virus entry inhibitors.</p

Structural analysis and corrosion studies on an ISO 5832-9 biomedical alloy with TiO2 sol–gel layers

The aim of this study was to demonstrate the relationship between the structural and corrosion properties of an ISO 5832-9 biomedical alloy modified with titanium dioxide (TiO2) layers. These layers were obtained via the sol–gel method by acid-catalyzed hydrolysis of titanium isopropoxide in isopropanol solution. To obtain TiO2 layers with different structural properties, the coated samples were annealed at temperatures of 200, 300, 400, 450, 500, 600 and 800 C for 2 h. For all the prepared samples, accelerated corrosion measurements were performed in Tyrode’s physiological solution using electrochemical methods. The most important corrosion parameters were determined: corrosion potential, polarization resistance, corrosion rate, breakdown and repassivation potentials. Corrosion damage was analyzed using scanning electron microscopy. Structural analysis was carried out for selected TiO2 coatings annealed at 200, 400, 600 and 800 C. In addition, the morphology, chemical composition, crystallinity, thickness and density of the deposited TiO2 layers were determined using suitable electron and X-ray measurement methods. It was shown that the structure and character of interactions between substrate and deposited TiO2 layers depended on annealing temperature. All the obtained TiO2 coatings exhibit anticorrosion properties, but these properties are related to the crystalline structure and character of substrate–layer interaction. From the point of view of corrosion, the best TiO2 sol–gel coatings for stainless steel intended for biomedical applications seem to be those obtained at 400 C.This study was supported by Grant No. N N507 501339 of the National Science Centre. The authors wish to express their thanks to J. Borowski (MEDGAL, Poland) for the Rex 734 alloy

Development of an Acute and Highly Pathogenic Nonhuman Primate Model of Nipah Virus Infection

Nipah virus (NiV) is an enigmatic emerging pathogen that causes severe and often fatal neurologic and/or respiratory disease in both animals and humans. Amongst people, case fatality rates range between 40 and 75 percent and there are no vaccines or treatments approved for human use. Guinea pigs, hamsters, cats, ferrets, pigs and most recently squirrel monkeys (New World monkey) have been evaluated as animal models of human NiV infection, and with the exception of the ferret, no model recapitulates all aspects of NiV-mediated disease seen in humans. To identify a more viable nonhuman primate (NHP) model, we examined the pathogenesis of NiV in African green monkeys (AGM). Exposure of eight monkeys to NiV produced a severe systemic infection in all eight animals with seven of the animals succumbing to infection. Viral RNA was detected in the plasma of challenged animals and occurred in two of three subjects as a peak between days 7 and 21, providing the first clear demonstration of plasma-associated viremia in NiV experimentally infected animals and suggested a progressive infection that seeded multiple organs simultaneously from the initial site of virus replication. Unlike the cat, hamster and squirrel monkey models of NiV infection, severe respiratory pathology, neurological disease and generalized vasculitis all manifested in NiV-infected AGMs, providing an accurate reflection of what is observed in NiV-infected humans. Our findings demonstrate the first consistent and highly pathogenic NHP model of NiV infection, providing a new and critical platform in the evaluation and licensure of either passive and active immunization or therapeutic strategies for human use

liver-enriched gene 1a and 1b Encode Novel Secretory Proteins Essential for Normal Liver Development in Zebrafish

liver-enriched gene 1 (leg1) is a liver-enriched gene in zebrafish and encodes a novel protein. Our preliminary data suggested that Leg1 is probably involved in early liver development. However, no detailed characterization of Leg1 has been reported thus far. We undertook both bioinformatic and experimental approaches to study leg1 gene structure and its role in early liver development. We found that Leg1 identifies a new conserved protein superfamily featured by the presence of domain of unknown function 781 (DUF781). There are two copies of leg1 in zebrafish, namely leg1a and leg1b. Both leg1a and leg1b are expressed in the larvae and adult liver with leg1a being the predominant form. Knockdown of Leg1a or Leg1b by their respective morpholinos specifically targeting their 5′-UTR each resulted in a small liver phenotype, demonstrating that both Leg1a and Leg1b are important for early liver development. Meanwhile, we found that injection of leg1-ATGMO, a morpholino which can simultaneously block the translation of Leg1a and Leg1b, caused not only a small liver phenotype but hypoplastic exocrine pancreas and intestinal tube as well. Further examination of leg1-ATGMO morphants with early endoderm markers and early hepatic markers revealed that although depletion of total Leg1 does not alter the hepatic and pancreatic fate of the endoderm cells, it leads to cell cycle arrest that results in growth retardation of liver, exocrine pancreas and intestine. Finally, we proved that Leg1 is a secretory protein. This intrigued us to propose that Leg1 might act as a novel secreted regulator that is essential for liver and other digestive organ development in zebrafish

Arsenic trioxide exerts synergistic effects with cisplatin on non-small cell lung cancer cells via apoptosis induction

<p>Abstract</p> <p>Background</p> <p>Despite multidisciplinary treatment, lung cancer remains a highly lethal disease due to poor response to chemotherapy. The identification of therapeutic agents with synergistic effects with traditional drugs is an alternative for lung cancer therapy. In this study, the synergistic effects of arsenic trioxide (As₂O₃) with cisplatin (DDP) on A549 and H460 non-small cell lung cancer (NSCLC) cells were explored.</p> <p>Methods</p> <p>A549 and H460 human lung cancer cells were treated with As₂O₃and/or DDP. Cell growth curves, cell proliferation, cell cycle, and apoptosis of human cancer cell lines were determined by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) method, clonogenic assay, and flow cytometry (FCM). Apoptosis was further assessed by TUNEL staining. Cell cycle and apoptosis related protein p21, cyclin D1, Bcl-2, bax, clusterin, and caspase-3 were detected by western blot.</p> <p>Results</p> <p>MTT and clonogenic assay showed As₂O₃within 10^-2μM to 10 μM exerted inhibition on the proliferation of NSCLC cells, and 2.5 μM As₂O₃exerted synergistic inhibition on proliferation with 3 μg/ml DDP. The combination indices (CI) for A549 and H460 were 0.5 and 0.6, respectively, as confirmed by the synergism of As₂O₃with DDP. FCM showed As₂O₃did not affect the cell cycle. The G0/G1 fraction ranged from 57% to 62% for controlled A549 cells and cells treated with As₂O₃and/or DDP. The G0/G1 fraction ranged from 37% to 42% for controlled H460 cells and cells treated with As₂O₃and/or DDP. FCM and TUNEL staining illustrated that the combination of As₂O₃and DDP provoked synergistic effects on apoptosis induction based on the analysis of the apoptosis index. Western blotting revealed that the expression of cell cycle related protein p21 and cyclin D1 were not affected by the treatments, whereas apoptosis related protein bax, Bcl-2, and clusterin were significantly regulated by As₂O₃and/or DDP treatments compared with controls. The expression of caspase-3 in cells treated with the combination of As₂O₃and DDP did not differ from that in cells treated with a single agent.</p> <p>Conclusion</p> <p>As₂O₃exerted synergistic effects with DDP on NSCLC cells, and the synergistic effects were partly due to the induction of caspase-independent apoptosis.</p

Regional pressure and temperature differences across the injured human brain : comparisons between intraparenchymal and ventricular measurements

Introduction: Intraparenchymal, multimodality sensors are commonly used in the management of patients with severe traumatic brain injury (TBI). The ‘gold standard’, based on accuracy, reliability and cost for intracranial pressure (ICP) monitoring is within the cerebral ventricle (external strain gauge). There are no standards yet for intracerebral temperature monitoring and little is known of temperature differences between brain tissue and ventricle. The aim of the study therefore was to determine pressure and temperature differences at intraparenchymal and ventricular sites during five days of continuous neurominitoring. Methods: Patients with severe TBI requiring emergency surgery. Inclusion criteria: patients who required ICP monitoring were eligible for recruitment. Two intracerebral probe types were used: a) intraventricular, dual parameter sensor (measuring pressure, temperature) with inbuilt catheter for CSF drainage: b) multiparameter intraparenchymal sensor measuring pressure, temperature and oxygen partial pressure. All sensors were inserted during surgery and under aseptic conditions. Results: Seventeen patients, 12 undergoing neurosurgery (decompressive craniectomy n=8, craniotomy n=4) aged 21–78 years were studied. Agreement of measures for 9540 brain tissue-ventricular temperature ‘pairs’ and 10,291 brain tissue-ventricular pressure ‘pairs’ were determined using mixed model to compare mean temperature and pressure for longitudinal data. There was no significant overall difference for mean temperature (p=0.92) or mean pressure readings (p=0.379) between tissue and ventricular sites. With 95.8% of paired temperature readings within 2SD (−0.4 to 0.4°C) differences in temperature between brain tissue and ventricle were clinically insignificant. For pressure, 93.5% of readings pairs fell within the 2SD range (−9.4756 to 7.8112 mmHg) (Fig. 2). However, for individual patients, agreement for mean tissue-ventricular pressure differences was poor on occasions. Conclusions: There is good overall agreement between paired temperature measurements obtained from deep white matter and brain ventricle in patients with and without early neurosurgery. For paired ICP measurements, 93.5% of readings were within 2SD of mean difference. Whilst the majority of paired readings were comparable (within 10mmHg) clinically relevant tissue-ventricular dissociations were noted. Further work is required to unravel the events responsible for short intervals of pressure dissociation before tissue pressure readings can be definitively accepted as a reliable surrogate for ventricular pressure.</p

Effect of Ca2+ Channel Block on Glycerol Metabolism in Dunaliella salina under Hypoosmotic and Hyperosmotic Stresses

The effect of Ca2+ channel blockers on cytosolic Ca2+ levels and the role of Ca2+ in glycerol metabolism of Dunaliella salina under hypoosmotic or hyperosmotic stress were investigated using the confocal laser scanning microscope (CLSM). Results showed that intracellular Ca2+ concentration increased rapidly when extracellular salinity suddenly decreased or increased, but the increase could be inhibited by pretreatment of Ca2+ channel blockers LaCl3, verapamil or ruthenium red. The changes of glycerol content and G3pdh activity in D. salina to respect to hypoosmotic or hyperosmotic stress were also inhibited in different degrees by pretreatment of Ca2+ channel blockers, indicating that the influx of Ca2+ via Ca2+ channels are required for the transduction of osmotic signal to regulate osmotic responses of D. salina to the changes of salinity. Differences of the three blockers in block effect suggested that they may act on different channels or had different action sites, including influx of Ca2+ from the extracellular space via Ca2+ channels localized in the plasma membrane or from intracellular calcium store via the mitochondrial. Other Ca2+-mediated or non-Ca2+-mediated osmotic signal pathway may exist in Dunaliella in response to hypoosmotic and hyperosmotic stresses

InForm software: A semi-Automated research tool to identify presumptive human hepatic progenitor cells, and other histological features of pathological significance

Hepatic progenitor cells (HPCs) play an important regenerative role in acute and chronic liver pathologies. Liver disease research often necessitates the grading of disease severity, and pathologists' reports are the current gold-standard for assessment. However, it is often impractical to recruit pathologists in large cohort studies. In this study we utilise PerkinElmer's "InForm" software package to semi-Automate the scoring of patient liver biopsies, and compare outputs to a pathologist's assessment. We examined a cohort of eleven acute hepatitis samples and three non-Alcoholic fatty liver disease (NAFLD) samples, stained with HPC markers (GCTM-5 and Pan Cytokeratin), an inflammatory marker (CD45), Sirius Red to detect collagen and haematoxylin/eosin for general histology. InForm was configured to identify presumptive HPCs, CD45 +ve inflammatory cells, areas of necrosis, fat and collagen deposition (p &lt; 0.0001). Hepatitis samples were then evaluated both by a pathologist using the Ishak-Knodell scoring system, and by InForm through customised algorithms. Necroinflammation as evaluated by a pathologist, correlated with InForm outputs (r 2 = 0.8192, p &lt; 0.05). This study demonstrates that the InForm software package provides a useful tool for liver disease research, allowing rapid, and objective quantification of the presumptive HPCs and identifies histological features that assist with assessing liver disease severity, and potentially can facilitate diagnosis

Tocotrienols are good adjuvants for developing cancer vaccines

<p>Abstract</p> <p>Background</p> <p>Dendritic cells (DCs) have the potential for cancer immunotherapy due to their ability to process and present antigens to T-cells and also in stimulating immune responses. However, DC-based vaccines have only exhibited minimal effectiveness against established tumours in mice and humans. The use of appropriate adjuvant enhances the efficacy of DC based cancer vaccines in treating tumours.</p> <p>Methods</p> <p>In this study we have used tocotrienol-rich fraction (TRF), a non-toxic natural compound, as an adjuvant to enhance the effectiveness of DC vaccines in treating mouse mammary cancers. In the mouse model, six-week-old female BALB/c mice were injected subcutaneously with DC and supplemented with oral TRF daily (DC+TRF) and DC pulsed with tumour lysate from 4T1 cells (DC+TL). Experimental mice were also injected with DC pulsed with tumour lysate and supplemented daily with oral TRF (DC+TL+TRF) while two groups of animal which were supplemented daily with carrier oil (control) and with TRF (TRF). After three times vaccination, mice were inoculated with 4T1 cells in the mammary breast pad to induce tumour.</p> <p>Results</p> <p>Our study showed that TRF in combination with DC pulsed with tumour lysate (DC+TL+TRF) injected subcutaneously significantly inhibited the growth of 4T1 mammary tumour cells as compared to control group. Analysis of cytokines production from murine splenocytes showed significant increased productions of IFN-γ and IL-12 in experimental mice (DC+TL+TRF) compared to control, mice injected with DC without TRF, mice injected with DC pulsed with tumour lysate and mice supplemented with TRF alone. Higher numbers of cytotoxic T cells (CD8) and natural killer cells (NK) were observed in the peripheral blood of TRF adjuvanted DC pulsed tumour lysate mice.</p> <p>Conclusion</p> <p>Our study show that TRF has the potential to be an adjuvant to augment DC based immunotherapy.</p

Current CONtrolled Transmit And Receive Coil Elements (C2ONTAR) for Parallel Acquisition and Parallel Excitation Techniques at High-Field MRI

A novel intrinsically decoupled transmit and receive radio-frequency coil element is presented for applications in parallel imaging and parallel excitation techniques in high-field magnetic resonance imaging. Decoupling is achieved by a twofold strategy: during transmission elements are driven by current sources, while during signal reception resonant elements are switched to a high input impedance preamplifier. To avoid B0 distortions by magnetic impurities or DC currents a resonant transmission line is used to relocate electronic components from the vicinity of the imaged object. The performance of a four-element array for 3 T magnetic resonance tomograph is analyzed by means of simulation, measurements of electromagnetic fields and bench experiments. The feasibility of parallel acquisition and parallel excitation is demonstrated and compared to that of a conventional power source-driven array of equivalent geometry. Due to their intrinsic decoupling the current-controlled elements are ideal basic building blocks for multi-element transmit and receive arrays of flexible geometry