Generation of henipavirus nucleocapsid proteins in yeast Saccharomyces cerevisiae

Hendra and Nipah viruses are newly emerged, zoonotic viruses and their genomes have nucleotide and predicted amino acid homologies placing them in the family Paramyxoviridae. Currently these viruses are classified in the new genus Henipavirus, within the subfamily Paramyxovirinae, family Paramyxoviridae. The genes encoding HeV and NiV nucleocapsid proteins were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of expression of these proteins (18-20 mg l(-1) of yeast culture) was obtained. Mass spectrometric analysis confirmed the primary structure of both proteins with 92% sequence coverage obtained using MS/MS analysis. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. The nucleocapsid proteins revealed stability in yeast and can be easily purified by cesium chloride gradient ultracentrifugation. HeV nucleocapsid protein was detected by sera derived from fruit bats, humans, horses infected with HeV, and NiV nucleocapsid protein was immunodetected with sera from, fruit bats, humans and pigs. The development of an efficient and cost-effective system for generation of henipavirus nucleocapsid proteins might help to improve reagents for diagnosis of viruses.     

Thromboprophylaxis with dalteparin in medical patients: which patients benefit?

It is unclear whether thromboprophylaxis produces a consistent risk reduction in different subgroups of medical patients at risk from venous thromboembolism. We performed a retrospective, post hoc analysis of 3706 patients enrolled in the PREVENT study. Patients were at least 40 years old with an acute medical condition requiring hospitalization for at least 4 days and had no more than 3 days of immobilization prior to enrolment. Patients received either subcutaneous dalteparin (5000 IU) or placebo once daily. The primary end point was the composite of symptomatic deep vein thrombosis (DVT), pulmonary embolism, asymptomatic proximal DVT, or sudden death. Primary diagnosis subgroups were acute congestive heart failure, acute respiratory failure, infectious disease, rheumatological disorders, or inflammatory bowel disease. All patients, except those with congestive heart or respiratory failure, had at least one additional risk factor for venous thromboembolism. A risk reduction was shown in patients receiving dalteparin versus placebo. The relative risk (RR) was 0.73 in patients with congestive heart failure, 0.72 for respiratory failure, 0.46 for infectious disease, and 0.97 for rheumatological disorders. The RR was 0.52 in patients aged > or = 75 years, 0.64 in obese patients, 0.34 for patients with varicose veins, and 0.71 in patients with chronic heart failure. No subgroup had a significantly different response from any other. Importantly, multivariate analysis showed that all patient groups benefited from thromboprophylaxis with dalteparin. Our findings, therefore, support the broad application of thromboprophylaxis in acutely ill hospitalized medical patients.     

Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation

INTRODUCTION: Intracellular gene electrotransfer by means of electroporation has been on the increase during the past decade. Significant progress has been achieved both in characterizing mechanisms of gene electrotransfer and in optimizing the protocol in many preclinical trials. Recently this has led to initiation of clinical trials of gene electrotransfer to treat metastatic melanomas. Further progress with the method in various clinical trials requires better understanding of mechanisms of gene electrotransfer. AREAS COVERED: A summary of recent progress in understanding mechanisms of gene electrotransfer, imparting general knowledge of cell electroporation and intracellular molecule electrotransfer. EXPERT OPINION: Gene electrotransfer into cells and tissues is a complex process involving multiple steps that lead to plasmid DNA passage from the extracellular region to the cell nucleus crossing the barriers of the plasma membrane, cytoplasm and nucleus membrane. Electrical parameters of pulses used for gene electrotransfer affect the initial steps of DNA translocation through the plasma membrane and play a crucial role in determining the transfection efficiency. When considering gene electrotransfer into tissues it becomes clear that other nonelectrical conditions are also of primary importance.     

Properties of mouse connexin 30.2 and human connexin 31.9 hemichannels: implications for atrioventricular conduction in the heart

Four connexins (Cxs), mouse (m)Cx30.2, Cx40, Cx43, and Cx45, determine cell-cell electrical signaling in mouse heart, and Cx43 and Cx45 are known to form unapposed hemichannels. Here we show that mCx30.2, which is most abundantly expressed in sinoatrial and atrioventricular nodal regions of the heart, and its putative human ortholog, human (h)Cx31.9, also form functional hemichannels, which, like mCx30.2 cell-cell channels, are permeable to cationic dyes up to approximately 400 Da in size. DAPI uptake by HeLa cells expressing mCx30.2 was >10-fold faster than that by HeLa parental cells. In Ca(2+)-free medium, uptake of DAPI by HeLaCx30.2-EGFP cells was increased approximately 2-fold, but uptake by parental cells was not affected. Conversely, acidification by application of CO(2) reduced DAPI uptake by HeLaCx30.2-EGFP cells but had little effect on uptake by parental cells. Cells expressing mCx30.2 exhibited higher rates of DAPI uptake than did cells expressing any of the other cardiac Cxs. Cardiomyocytes of 2-day-old rats transfected with hCx31.9-EGFP took up DAPI and ethidium bromide 5-10 times faster than wild-type cardiomyocytes. Mefloquine, a close derivative of quinine and quinidine that exhibits antimalarial and antiarrhythmic properties, reduced conductance of cell-cell junctions and dye uptake through mCx30.2 hemichannels with approximately the same affinity (IC(50) = approximately 10 microM) and increased dependence of junctional conductance on transjunctional voltage. Unitary conductance of mCx30.2 hemichannels was approximately 20 pS, about twice the cell-cell channel conductance. Hemichannels formed of mCx30.2 and hCx31.9 may slow propagation of excitation in the sinoatrial and atrioventricular nodes by shortening the space constant and depolarizing the excitable membrane.     

Novel monoclonal antibodies against Menangle virus nucleocapsid protein

Menangle virus (MenV) is a member of the family Paramyxoviridae isolated in Australia that causes a reproductive disease of pigs. There is a need for specific immunoassays for virus detection to facilitate the diagnosis of MenV infection. Three novel monoclonal antibodies (MAbs) of the IgG1 subtype were generated by immunizing mice with recombinant yeast-expressed MenV nucleocapsid (N) protein self-assembled to nucleocapsid-like structures. One MAb was cross-reactive with recombinant N protein of Tioman virus. The epitopes of MAbs were mapped using a series of truncated MenV N proteins lacking the 29–119 carboxy-terminal amino acid (aa) residues. The epitopes of two MAbs were mapped to aa 430–460 of the MenV N protein, whilst the epitope of one MAb was mapped to residues 460–490. All three MAbs specifically recognized MenV, as indicated by immunohistochemical staining of brain tissue isolated from a field case (a stillborn piglet) of MenV infection. The MAbs against MenV N protein may be a useful tool for immunohistological diagnosis of MenV infection.     

Aggressive primary cutaneous B‐cell lymphomas show increased Angiopoietin‐2‐induced angiogenesis

Primary cutaneous large B‐cell lymphomas, leg type (PCLBCL/LT) are primary cutaneous B‐cell lymphoma (PCBCL) with an intermediate prognosis. Therefore, antracycline‐based polychemotherapy combined with rituximab has been recommended as first‐line treatment. Yet, despite this regimen, the 5‐year survival rate remains 50–66% only. Angiogenesis, the formation of a vascular network, is essential for the pathogenesis of nodal lymphomas. So far, no study has analysed angiogenesis and its key factors in PCLBCL/LT. The present study was aimed at characterizing angiogenesis in PCLBCL/LT to identify the angiogenic molecules as potential therapeutic targets. The intra‐tumoral microvessel density (MVD) was assessed by immunohistochemical studies of CD20 and CD31. The MVD was higher in PCLBCL/LT compared with indolent PCBCL. Analyses of open‐source microarray data showed correlation between the angiogenic molecule angiopoietin‐2 (Ang‐2) and pan‐endothelial cell markers. ELISA studies determined a shift between Ang‐2 and Ang‐1 towards Ang‐2 in the peripheral blood of PCLBCL/LT patients. Immunofluorescence costainings against the Ang receptor Tie2/angiogenic integrins/CD34 revealed that the vasculature in both aggressive and indolent PCBCL tumors harbours an endothelial cell subpopulation with reduced expression of Tie2. In contrast, the alternative Ang‐2 binding partners, angiogenic integrins, are strongly expressed in PCBCL. In line with these findings, downstream targets of Ang‐2‐integrin signalling, that is phosphorylation of focal adhesion kinase at Tyr397, and sprouting angiogenesis are enhanced in PCLBCL/LT. Our data present Ang‐2 as a promising therapeutic target and anti‐angiogenic therapy as a new line in treatment of PCLBCL/LT as a hitherto intractable disease.     

Percolation effect of a Cu layer on a MWCNT/PP nanocomposite substrate after laser direct structuring and autocatalytic plating

Percolation behavior of a copper (Cu) layer on a multi-walled carbon nanotube/polypropylene (MWCNT/PP) nanocomposite substrate after laser-direct-structuring (LDS) and subsequent autocatalytic Cu deposition (ACD) is presented. The inverse sheet resistance showed percolation type dependence on the area fraction of Cu on MWCNT/PP measured by digital image processing of specimen photos.

Digital image processing of Cu layer on MWCNT/PP nanocomposite substrate after laser direct structuring and autocatalytic plating reveals percolation effect.     

Novel cellulose/hydroxyapatite scaffolds for bone tissue regeneration: In vitro and in vivo study

Cellulose scaffolds containing nano‐ or micro‐hydroxyapatite (nHA or μHA) were prepared by the regeneration of cellulose from its acetylated derivative and the mechanical immobilization of inorganic particles, followed by freeze‐drying. Microtomographic (micro‐computed tomography) evaluation revealed that both scaffolds presented a highly interconnected porous structure, with a mean pore diameter of 490 ± 94 and 540 ± 132 μm for cellulose/nHA and cellulose/μHA, respectively. In vitro and in vivo characterizations of the developed scaffolds were investigated. Commercially available bone allograft was used as a control material. For the in vitro characterization, osteoblastic cell cultures were used and characterized over time to evaluate cell adhesion, metabolic activity, and functional output (alkaline phosphatase activity and osteoblastic gene expression). The results revealed greater spreading cell distribution alongside an increased number of filopodia, higher MTT values, and significantly increased expression of osteoblastic genes (Runx‐2, alkaline phosphatase, and BMP‐2) for cellulose/nHA, compared with cellulose/μHA and the control. The in vivo biocompatibility was evaluated in a rabbit calvarial defect model. The investigated scaffolds were implanted in circular rabbit calvaria defects. Four‐ and 12‐week bone biopsies were investigated using micro‐computed tomography and histological analysis. Although both cellulose/HA scaffolds outperformed the assayed control, a significantly higher amount of newly formed mineralized tissue was found within the defects loaded with cellulose/nHA. Within the limitations of this study, the developed cellulose/HA scaffolds showed promising results for bone regeneration applications. The biological response to the scaffold seems to be greatly dependent on the HA particles' characteristics, with cellulose scaffolds loaded with nHA eliciting an enhanced bone response.     

Loss of endothelial thrombomodulin predicts response to steroid therapy and survival in acute intestinal graft-versus-host disease

Steroid-refractory graft-versus-host disease causes significant morbidity and mortality after allogeneic stem cell transplantation. The pathomechanism of steroid resistance is currently not understood, but it has been suggested that endothelial cell dysfunction plays a role. Endothelial thrombomodulin was quantified along with histological markers of epithelial damage and cytotoxic T cells in colon biopsies from 51 allografted patients, and retrospectively correlated with response to steroids and survival. Loss of endothelial thrombomodulin was the strongest predictor of response to steroids (P=0.02) and nonrelapse mortality (P=0.01) in multivariate analyses adjusting for T-cell infiltrates, histological grading, vessel density, disease status, donor type, and conditioning therapy. Our data provide evidence that at disease onset, loss of endothelial thrombomodulin expression rather than excessive T-cell infiltration associates with steroid-refractory graft-versus-host disease and mortality. Prospective histological investigations are now warranted to improve diagnosis and prognostication of this core complication of stem cell transplantation.Key words: allogeneic stem cell transplantation, acute GvHD, thrombomodulin, endothelial cells, steroid-resistance, cytotoxic T cells, TIA-1