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The viral transactivator Tax plays a key role in HTLV-1 reactivation and de novo infection. Previous approaches focused on the histone deacetylase inhibitor (HDACi) Valproate as a latency-reversing agent to boost Tax expression and expose infected cells to the host’s immune response. However, following treatment with Valproate proviral load decreases in patients with HAM/TSP were only transient. Here, we hypothesize that other compounds, including more potent and selective HDACi, might prove superior to Valproate in manipulating Tax expression. Thus, a panel of HDACi (Vorinostat/SAHA/Zolinza, Panobinostat/LBH589/Farydak, Belinostat/PXD101/Beleodaq, Valproate, Entinostat/MS-275, Romidepsin/FK228/Istodax, and MC1568) was selected and tested for toxicity and potency in enhancing Tax expression. The impact of the compounds was evaluated in different model systems, including transiently transfected T-cells, chronically HTLV-1-infected T-cell lines, and freshly isolated PBMCs from HTLV-1 carriers ex vivo. We identified the pan-HDACi Panobinostat and class I HDACi Romidepsin as particularly potent agents at raising Tax expression. qRT-PCR analysis revealed that these inhibitors considerably boost tax and Tax-target gene transcription. However, despite this significant increase in tax transcription and histone acetylation, protein levels of Tax were only moderately enhanced. In conclusion, these data demonstrate the ability of Panobinostat and Romidepsin to manipulate Tax expression and provide a foundation for further research into eliminating latently infected cells. These findings also contribute to a better understanding of conditions limiting transcription and translation of viral gene products.

Breastfeeding is recommended by the World Health Organization for at least 6 months up to 2 years of age, and breast milk protects against several diseases and infections. Intriguingly, few viruses are transmitted via breastfeeding including Human T-cell leukemia virus Type 1 (HTLV-1). HTLV-1 is a highly oncogenic yet neglected retrovirus, which primarily infects CD4+ T-cells in vivo and causes incurable diseases like HTLV-1-associated inflammatory conditions or Adult T-cell leukemia/lymphoma (ATLL) after lifelong viral persistence. Worldwide, at least 5–10 million people are HTLV-1-infected and most of them are unaware of their infection posing the risk of silent transmissions. HTLV-1 is transmitted via cell-containing body fluids such as blood products, semen, and breast milk, which constitutes the major route of mother-to-child transmission (MTCT). Risk of transmission increases with the duration of breastfeeding, however, abstinence from breastfeeding as it is recommended in some endemic countries is not an option in resource-limited settings or underrepresented areas and populations. Despite significant progress in understanding details of HTLV-1 cell-to-cell transmission, it is still not fully understood, which cells in which organs get infected via the oral route, how these cells get infected, how breast milk affects this route of infection and how to inhibit oral transmission despite breastfeeding, which is an urgent need especially in underrepresented areas of the world. Here, we review these questions and provide an outlook how future research could help to uncover prevention strategies that might ultimately allow infants to benefit from breastfeeding while reducing the risk of HTLV-1 transmission.

Human T-cell leukemia virus type 1 (HTLV-1), the cause of adult T-cell leukemia/lymphoma (ATLL), is a retrovirus, which integrates into the host genome and persistently infects CD4+ T-cells. Virus propagation is stimulated by (1) clonal expansion of infected cells and (2) de novo infection. Viral gene expression is induced by the transactivator protein Tax, which recruits host factors like positive transcription elongation factor b (P-TEFb) to the viral promoter. Since HTLV-1 gene expression is repressed in vivo by viral, cellular, and epigenetic mechanisms in late phases of infection, HTLV-1 avoids an efficient CD8+ cytotoxic T-cell (CTL) response directed against the immunodominant viral Tax antigen. Hence, therapeutic strategies using latency reversing agents (LRAs) sought to transiently activate viral gene expression and antigen presentation of Tax to enhance CTL responses towards HTLV-1, and thus, to expose the latent HTLV-1 reservoir to immune destruction. Here, we review strategies that aimed at enhancing Tax expression and Tax-specific CTL responses to interfere with HTLV-1 latency. Further, we provide an overview of LRAs including (1) histone deacetylase inhibitors (HDACi) and (2) activators of P-TEFb, that have mainly been studied in context of human immunodeficiency virus (HIV), but which may also be powerful in the context of HTLV-1.

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24–45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26–37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells. Author summary The delta-retrovirus Human T-cell leukemia virus type 1 encodes the accessory protein p8, which is generated by proteolytic cleavage from p12. Earlier work has shown that p8 enhances the formation of cellular conduits between T-cells, is transferred through these conduits to target T-cells and increases HTLV-1 transmission. It was suggested that p8 dampens T-cell responses in target T-cells, thus facilitating HTLV-1 infection. Our work sheds light on the mechanism of p8 transfer to target T-cells. We show that vasodilator-stimulated phosphoprotein (VASP), a novel interaction partner of p8, contributes to transfer of p8 to target T-cells. Mechanistically, VASP is crucial for recruitment of p8 to the cell surface. Since VASP is known to promote elongation of actin filaments by preventing them from capping, interactions of p8 with VASP are an elegant strategy to exploit the host cell machinery for being transported to the cell surface, and as a consequence, to other cells. Given that VASP is also important for cell-to-cell transfer of the HTLV-1 Gag protein, our work proposes that VASP is a new cellular target to counteract HTLV-1 cell-to-cell transmission.

The Human T-cell leukemia virus type 1 (HTLV-1) orf I-encoded accessory protein p8 is cleaved from its precursor p12, and both proteins contribute to viral persistence. p8 induces cellular protrusions, which are thought to facilitate transfer of p8 to target cells and virus transmission. Host factors interacting with p8 and mediating p8 transfer are unknown. Here, we report that vasodilator-stimulated phosphoprotein (VASP), which promotes actin filament elongation, is a novel interaction partner of p8 and important for p8 and HTLV-1 Gag cell-to-cell transfer. VASP contains an Ena/VASP homology 1 (EVH1) domain that targets the protein to focal adhesions. Bioinformatics identified a short stretch in p8 (amino acids (aa) 24–45) which may mediate interactions with the EVH1 domain of VASP. Co-immunoprecipitations confirmed interactions of VASP:p8 in 293T, Jurkat and HTLV-1-infected MT-2 cells. Co-precipitation of VASP:p8 could be significantly blocked by peptides mimicking aa 26–37 of p8. Mutational studies revealed that the EVH1-domain of VASP is necessary, but not sufficient for the interaction with p8. Further, deletion of the VASP G- and F-actin binding domains significantly diminished co-precipitation of p8. Imaging identified areas of partial co-localization of VASP with p8 at the plasma membrane and in protrusive structures, which was confirmed by proximity ligation assays. Co-culture experiments revealed that p8 is transferred between Jurkat T-cells via VASP-containing conduits. Imaging and flow cytometry revealed that repression of both endogenous and overexpressed VASP by RNA interference or by CRISPR/Cas9 reduced p8 transfer to the cell surface and to target Jurkat T-cells. Stable repression of VASP by RNA interference in chronically infected MT-2 cells impaired both p8 and HTLV-1 Gag transfer to target Jurkat T-cells, while virus release was unaffected. Thus, we identified VASP as a novel interaction partner of p8, which is important for transfer of HTLV-1 p8 and Gag to target T-cells., Author summary The delta-retrovirus Human T-cell leukemia virus type 1 encodes the accessory protein p8, which is generated by proteolytic cleavage from p12. Earlier work has shown that p8 enhances the formation of cellular conduits between T-cells, is transferred through these conduits to target T-cells and increases HTLV-1 transmission. It was suggested that p8 dampens T-cell responses in target T-cells, thus facilitating HTLV-1 infection. Our work sheds light on the mechanism of p8 transfer to target T-cells. We show that vasodilator-stimulated phosphoprotein (VASP), a novel interaction partner of p8, contributes to transfer of p8 to target T-cells. Mechanistically, VASP is crucial for recruitment of p8 to the cell surface. Since VASP is known to promote elongation of actin filaments by preventing them from capping, interactions of p8 with VASP are an elegant strategy to exploit the host cell machinery for being transported to the cell surface, and as a consequence, to other cells. Given that VASP is also important for cell-to-cell transfer of the HTLV-1 Gag protein, our work proposes that VASP is a new cellular target to counteract HTLV-1 cell-to-cell transmission.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiologic agent for Adult T-Cell Leukemia/Lymphoma (ATLL) and HTLV-1-Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP). HTLV-1 infects CD4+ T-cells via cell-to-cell transmission requiring reorganization of the cytoskeleton and expression of the viral transactivator and oncoprotein Tax. Viruses spread at the virological synapse (VS), a virus-induced specialized cell-cell contact, by polarized budding into synaptic clefts, and by cell surface transfer of viral biofilms (VBs). Since little is known about Tax’s role in formation of the VB, we asked which component of the VB is regulated by Tax and important for HTLV-1 transmission. Collagens are not only structural proteins of the extracellular matrix and basal membrane but also represent an important component of the VB. Here, we report that among the collagens known to be present in VBs, COL4 is specifically upregulated in the presence of HTLV-1 infection. Further, we found that transient expression of Tax is sufficient to induce COL4A1 and COL4A2 transcripts in Jurkat and CCRF-CEM T-cells, while robust induction of COL4 protein requires continuous Tax expression as shown in Tax-transformed T-cell lines. Repression of Tax led to a significant reduction of COL4A1/A2 transcripts and COL4 protein. Mechanistically, luciferase-based promoter studies indicate that Tax activates the COL4A2 and, to a less extent, the COL4A1 promoter. Imaging showing partial co-localization of COL4 with the viral Gag protein in VBs at the VS and transfer of COL4 and Gag to target cells suggests a role of COL4 in VB formation. Strikingly, in chronically infected C91-PL cells, knockout of COL4A2 impaired Gag transfer between infected T-cells and acceptor T-cells, while release of virus-like particles was unaffected. Taken together, we identified COL4 (COL4A1, COL4A2) as a component of the VB and a novel cellular target of Tax with COL4A2 appearing to impact virus transmission. Thus, this study is the first to provide a link between Tax’s activity and VB formation by hijacking COL4 protein functions.

The Human T-cell leukemia virus type 1 (HTLV-1)-encoded accessory protein p8 is cleaved from the precursor protein p12 encoded by the HTLV-1 open reading frame I. Both p12 and p8 are thought to contribute to efficient viral persistence. Mechanistically, p8 induces T-cell conjugates and cellular conduits. The latter are considered to facilitate transfer of p8 to target cells and virus transmission. Transfer of p8 between p8-expressing T-cells and recipient cells has been analyzed by immunofluorescence and live imaging. However, automatic quantitation of p8-transfer between cells has not been studied yet. Here we developed a novel method allowing time saving quantitation of p8 transfer between cells by flow cytometry. After establishing a protocol for the detection of intracellular p8 by flow cytometry and validation of p8 protein expression by western blot and immunofluorescence, we set up a co-culture assay between p8-expressing donor Jurkat T-cells and recipient Jurkat T-cells that had been prestained with a well-retained live cell dye. Upon quantitating the amount of p8 positive recipient cells with regard to the percentage of p8 expressing donor cells, time course experiments confirmed that p8 is rapidly transferred between Jurkat T-cells. We found that p8 enters approximately 5% of recipient T-cells immediately upon co-culture for 5 min. Prolonged co-culture for up to 24 h revealed an increase of relative p8 transfer to approximately 23% of the recipient cells. Immunofluorescence analysis of co-culture experiments and manual quantitation of p8 expression in fluorescence images confirmed the validity of the flow cytometry based assay. Application of the new assay revealed that manipulation of actin polymerization significantly decreased p8 transfer between Jurkat T-cells suggesting an important role of actin dynamics contributing to p8 transfer. Further, transfer of p8 to co-cultured T-cells varies between different donor cell types since p8 transfer could hardly been detected in co-cultures of 293T donor cells with Jurkat acceptor cells. In summary, our novel assay allows automatic and rapid quantitation of p8 transfer to target cells and might thus contribute to a better understanding of cellular processes and dynamics regulating p8 transfer and HTLV-1 transmission.

The retrovirus Human T-lymphotropic virus type 1 (HTLV-1) preferentially infects CD4

Human T-cell leukemia virus type 1 (HTLV-1) infects about 20 million people world-wide. Around 5% of the infected individuals develop adult T-cell leukemia (ATL) or a neurological disease termed tropical spastic paraparesis (TSP) after a clinical latency of years to decades. Through the use of two promoters and alternative splicing HTLV-1 expresses at least 12 different proteins. HTLV-1 establishes a life-long persistent infection by inducing the clonal expansion of infected cells, a property largely ascribed to the viral genes Tax and HBZ. However, the fact that ATL arises in a minority of infected individuals after a long clinical latency suggests the existence of factors counterbalancing the oncogenic potential of HTLV-1 in the context of natural infection.To study the role of the different HTLV-1 gene products in the HTLV-1 life cycle, we optimized a transfection protocol for primary T-cells using an approach based on the electroporation of in vitro-transcribed RNA. Results showed that the RNA transfection technique combines a high transfection efficiency with low toxicity, not only in Jurkat T-cells but also in primary T-cells. These findings suggest that RNA electroporation is preferable for experiments aimed at investigating the role of HTLV-1 gene products in the context of primary T-cells, which represent the main target of HTLV-1 in vivo.

The retrovirus Human T-lymphotropic virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission, while cell-free infection of T-cells is inefficient. Substantial insights into the different routes of transmission have largely been obtained by imaging techniques or by flow cytometry. Recently, strategies to quantify infection events with HTLV-1 improved. In this chapter, we present two different methods to quantitate virus transmission. Both methods are based on measuring gene activity of luciferase with a cost-saving in-house luciferase assay. First, we established a reporter Jurkat T-cell line carrying a luciferase gene under the control of the HTLV-1 core promoter U3R. Upon co-culture with chronically HTLV-1-infected T-cell lines, reporter cells are infected, and upon expression of the viral transactivator Tax, the viral promoter is activated resulting in enhanced luciferase activity. However, this assay as presented here does not exclude cell fusion as the mechanism allowing intracellular Tax-dependent activation of luciferase gene expression. Therefore, we describe a second method, the single-cycle replication-dependent reporter system developed by Mazurov et al. (PLoS Pathog 6:e1000788, 2010) that allows quantitation of HTLV-1 infection in co-cultured cells. Taken together, both methods facilitate quantitation of HTLV-1 transmission and will help to unravel pathways required for cell-to-cell transmission on a quantitative basis.

The tumorvirus human T-cell lymphotropic virus type 1 (HTLV-1), a member of the delta-retrovirus family, is transmitted via cell-containing body fluids such as blood products, semen, and breast milk. In vivo, HTLV-1 preferentially infects CD4+ T-cells, and to a lesser extent, CD8+ T-cells, dendritic cells, and monocytes. Efficient infection of CD4+ T-cells requires cell-cell contacts while cell-free virus transmission is inefficient. Two types of cell-cell contacts have been described to be critical for HTLV-1 transmission, tight junctions and cellular conduits. Further, two non-exclusive mechanisms of virus transmission at cell-cell contacts have been proposed: (1) polarized budding of HTLV-1 into synaptic clefts; and (2) cell surface transfer of viral biofilms at virological synapses. In contrast to CD4+ T-cells, dendritic cells can be infected cell-free and, to a greater extent, via viral biofilms in vitro. Cell-to-cell transmission of HTLV-1 requires a coordinated action of steps in the virus infectious cycle with events in the cell-cell adhesion process; therefore, virus propagation from cell-to-cell depends on specific interactions between cellular and viral proteins. Here, we review the molecular mechanisms of HTLV-1 transmission with a focus on the HTLV-1-encoded proteins Tax and p8, their impact on host cell factors mediating cell-cell contacts, cytoskeletal remodeling, and thus, virus propagation.

Fluorescence microscopy is an essential tool to examine hostpathogen interactions such as the influence of Fascin on cell-cell contacts between infected and uninfected cells. Manual analysis of fluorescence microscopy images is prone to errors leading to inter- and intra-observer variability. To increase reproducibility and objectivity, automated and semi-automated image processing methods are required. For a reliable segmentation of touching and overlapping cells, we propose an active contours algorithm extended by an energy term based on an active shape model. The algorithm is evaluated on confocal cell image data labeled by a human expert.

Die Erforschung des Zusammenhangs zwischen obstruktivem Schlafapnoe-Syndrom und Beeintrachtigungen im Bereich der exekutiven Funktionen lieferte bisher inhomogene Ergebnisse. Gestorter Nachtschlaf bedingt u. a. Beeintrachtigungen auf der kognitiven Ebene. Bei 32 Patienten wurde die Arbeitsgedachtnisleistung mittels N-back-Aufgaben vor und nach erfolgter CPAP-Therapie gemessen. Die untersuchte Klientel wies eine signifikante Steigerung der Arbeitsgedachtnisleistung nach erfolgter CPAP-Therapie auf. Anfanglich waren 50% durch unterdurchschnittliche Werte der Arbeitsgedachtnisleistung charakterisiert, nach erfolgreicher CPAP-Beatmung nur noch 19%. Klinisch leiden die Patienten v. a. unter erhohter Tagesschlafrigkeit und Konzentrationsproblemen. Die Studie zeigt einen Zusammenhang zwischen dem Schlafapnoesyndrom und der Aufmerksamkeitsleistung und zeigt, dass eine nachtliche Atemhilfstherapie gestorte Arbeitsgedachtnisleistungen verbessern kann. Als Konsequenz fur den offentlichen Diskurs wird das erhohte Risiko von Arbeits- und Verkehrsunfallen aufgrund verminderter Aufmerksamkeitsleistung diskutiert.

SUMMARYAn unknown Trypanosoma species was isolated from an axenic culture of intact skin from a domestic dog captured in Rio de Janeiro, Brazil, which was co-infected with Leishmania (Viannia) braziliensis. Giemsa-stained smears of cultures grown in different media revealed the presence of epimastigotes, trypomastigotes, spheromastigotes, transitional stages, and dividing forms (epimastigotes or spheromastigotes). The highest frequency of trypomastigotes was observed in RPMI (15·2%) and DMEM (9·2%) media containing 5% FCS, with a mean length of these forms of 43·0 and 36·0 μm, respectively. Molecular analysis by sequential application of PCR assays indicated that this trypanosome differs from Trypanosoma cruzi and T. rangeli when specific primers were applied. On the other hand, a PCR strategy targeted to the D7 domain of 24sα rDNA, using primers D75/D76, amplified products of about 250 bp in that isolate (stock A-27), different from the amplification products obtained with T. cruzi and T. rangeli. This organism differs from T. cruzi mainly by the size of its trypomastigote forms and kinetoplasts and the absence of infectivity for macrophages and triatomine bugs. It is also morphologically distinct from salivarian trypanosomes reported in Brazil. Isoenzyme analysis at 8 loci demonstrated a very peculiar banding pattern clearly distinct from those of T. rangeli and T. cruzi. We conclude that this isolate is a new Trypanosoma species. The name T. caninum is suggested.

An optimization tool is proposed to determine the optimal operation schedule for distributed combined heat and power plants which participate in electricity spot markets. It was tested and analysed using realistic data for heat and electricity demands and their corresponding prices. The results show substantial cost savings compared with the benchmarked alternatives.

Adult T-cell leukemia/lymphoma is a highly infiltrative neoplasia of CD4+ T-lymphocytes that occurs in about 5% of carriers infected with the deltaretrovirus human T-cell leukemia virus type 1 (HTLV-1). The viral oncoprotein Tax perturbs cellular signaling pathways leading to upregulation of host cell factors, amongst them the actin-bundling protein Fascin, an invasion marker of several types of cancer. However, transcriptional regulation of Fascin by Tax is poorly understood. In this study, we identified a triple mode of transcriptional induction of Fascin by Tax, which requires (1) NF-κB-dependent promoter activation, (2) a Tax-responsive region in the Fascin promoter, and (3) a promoter-independent mechanism sensitive to the Src family kinase inhibitor PP2. Thus, Tax regulates Fascin by a multitude of signals. Beyond, using Tax-expressing and virus-transformed lymphocytes as a model system, our study is the first to identify the invasion marker Fascin as a novel target of PP2, an inhibitor of metastasis.

Experiments on sputter deposition of TiN-coatings were carried out under two dynamic conditions which are commonly used in industrial applications: linear periodic motion of the substrates beneath one target or rotating substrates between two targets. The anisotropic stresses in the TiN-films were determined by means of phase sensitive interferometry. A strong stress anisotropy was found when the substrates were subjected to linear motion with a controlled velocity. The ratio of the stress components σ 1 in the direction of motion and σ 2 perpendicular to it was up to σ 1 / σ 2 = 2 . 1. The stresses can be related to a periodic undulated wavy like fibre structure in the films which is induced by the periodic motion of the substrate beneath the target. The film structure is characterized by an undulation period λ = t / n , with t the total thickness of the film and n the number of cycles. The anisotropy effects were confirmed by X-ray diffraction. A distinct structure with a preferential direction parallel to the direction of motion combined with a high stress anisotropy was found for large undulation periods, whereas an almost isotropic structure with a poor crystalline order and almost isotropic stresses was observed for small undulation periods.

We report on high-speed impact nonequilibrium molecular dynamics simulations (NEMD) of brittle cohesive materials. A two-dimensional mesoscopic network of particles with a failure threshold is used for capturing the main mesostructural features. We find a good agreement between the initiation and propagation of cracks in our model and the experimental findings in impact experiments with different brittle materials. The failure dynamics in the material as a function of impact speed is discussed for standard load experiments and compared with experimental results.

The elastic and plastic behaviour of an open-cell aluminium foam is investigated numerically. Finite element discretisations are used that have been derived from real foam specimens by computer tomography data. By different combinations of boundary velocities in the three directions in space, various multiaxial stress states are realised in the foam. This is done for nine foam discretisations with varying relative density. Thus, the elastic constants, the yield surface and the plastic potential of the foam are determined depending on the relative density. The results of the numerical simulations are: (1) the variation of the Young's modulus in terms of the relative density can be described by a power law relationship. (2) The elastic Poisson's ratio does not depend on the relative density. (3) The yield surface is not rotationally symmetric with regard to the hydrostatic axis. (4) The dimensions of the yield surface vary with a power law relationship in terms of the relative density. (5) The plastic potential is approximately associated to the yield surface for all stress states.

Different methods of photostabilization are presented for the very light sensitive molsidomine tablets. The incorporation of photostabilizers such as light absorber or pigments into the tablets considerably improved the photostability. Nevertheless, photodegradation was still detected after 12 h of intense light stress. Pigments are superior to colorants or ultraviolet absorbers. The use of titanium dioxide needs to be considered carefully. Preblending the pigment with the drug substance is very helpful for taking full advantage of its photostabilizing properties. Surface-treated titanium dioxide with reduced photocatalytic activity was less suitable than untreated. That was due to a change of particle agglomeration and adhesion behavior, which was demonstrated by scanning electron microscopy pictures. However, only the protection of the tablets by a cover, either by blistering or film coating, gave a photostable drug product.

A modified Shell four-ball apparatus was used to determine failure mechanisms and estimate time to failure of hybrid ball bearings using silicon nitride and zirconia balls. The machine was set up to hold a hybrid bearing containing three ceramic balls, instead of the usual 14 in order to increase contact pressure. Five kinds of silicon nitride ceramics, which differed in terms of surface roughness, porosity as well as the amount and chemical composition of additives were investigated. The goal of this study was to establish a quality criterion for silicon nitride for industrial use in hybrid ball bearings. Lifetime and failure mechanisms varied between the five bearings with silicon nitride balls and a dependance was found on the porosity and chemical composition of the materials, whereas surface roughness did not seem to influence their performance.

A series of experiments using steel or hybrid balls in an SNFA VEX25 type bearing was conducted, at a rotational speed of 50,000 rpm. The race material was 100Cr6 steel, whereas for the balls various steels, coatings, and ceramics were used. The different materials used for the balls as well as the method of lubrication (air/oil or grease) strongly influenced the surface degradation of the ball bearings. Hybrid bearings with ceramic balls showed very little wear of either the balls or the steel races, and so offer potential for high-speed applications. The degradation of the bearings was examined using scanning electron microscopy, atomic force microscopy, and laser scanning profilometry. The disadvantages and advantages of these methods are given, along with the results of surface roughness measurements.

In many sputtering processes the substrate is rotated or periodically moved with respect to the target in order to obtain a homogeneous film deposition. In that case anisotropic conditions of film growth exist which lead to anisotropic mechanical stresses. The stress anisotropy depends on the carrier velocity and can be attributed to the film structure. The stress measurements, therefore, will be related to measurements of composition by SNMS, to determination of stoichiometry by ellipsometry and to investigations of structure by X-ray diffraction.

OBJECTIVE To measure the 12-year incidence (1982–1994) of nontraumatic lower extremity amputations (LEAs) in Nauruans, a population at high risk for NIDDM, and to determine the risk factors for amputation in Nauruans with diabetes. RESEARCH DESIGN AND METHODS Amputation data were abstracted from operating theater records in Nauru, hospital databases in Australia, and Nauru government records. Baseline characteristics of a cohort of 1,564 Nauruans aged ≥ 20 years examined during a population-based survey in 1982 were used to determine risk factors for first LEAs. RESULTS Over this 12-year period, 46 first LEAs were performed on people with NIDDM, of whom 30 were members of the 1982 study cohort. The incidence of first LEAs in Nauruans aged ≥ 25 years with NIDDM was 8.1 per 1,000 person-years in the study cohort and an estimated 7.6 per 1,000 person-years nationally. Amputations were associated significantly with lower BMI, lower blood pressure, higher fasting plasma glucose (FPG) level, and longer mean duration of diabetes at baseline, but levels of other risk factors, including cigarette smoking, plasma triglycerides, and plasma cholesterol, were also elevated in amputees. There were no amputations among individuals with baseline FPG levels < 7.8 mmol/l, irrespective of diabetes duration. FPG, baseline diabetes duration, and male sex were independent risk factors for first amputation using the Cox proportional hazards model. There was a decrease in the incidence of amputations after the commencement of a national foot care health education and prevention campaign in June 1992. CONCLUSIONS The incidence of LEAs in diabetic Nauruans was higher than in other populations after adjusting for age and duration. Given the apparent success of the Nauruan footcare program in reducing amputation rates, other populations with high rates of NIDDM and LEAs should consider population-wide prevention strategies.

This overview deals with very high impact velocities, where complete vaporization of an impacting cosmic dust particle is to be expected upon expansion from the high pressure high temperature state behind the stopping shock (v > 15 km/s). The topics discussed are the mechanics and thermodynamics of compression, adiabatic release, equation of state and nonequilibrium states upon expansion. The case of very high particle porosity (ϱ ≪ 1 g/cm3) and the case of very small dust masses (m < 10−17 g) are discussed from what one presently knows. The possibility of three body collisions in the expanding gas phase is discussed briefly. The effect of oblique impact is discussed with respect to its relevance to the ionization process. The numbers communicated are up to the highest “experimental” impact velocities (80 km/s. Halley mission). As one goes to lower impact velocities (20 < v < 30 km/s) there is still complete vaporization of the dust particle but ionization out of the bulk of the particle becomes low. Other than thermal processes may become important. Ideas are outlined to understand their physical nature.

The delta-retrovirus Human T-cell leukemia virus type 1 (HTLV-1) preferentially infects CD4+ T-cells via cell-to-cell transmission. Viruses are transmitted by polarized budding and by transfer of viral biofilms at the virological synapse (VS). Formation of the VS requires the viral Tax protein and polarization of the host cytoskeleton, however, molecular mechanisms of HTLV-1 cell-to-cell transmission remain incompletely understood. Recently, we could show Tax-dependent upregulation of the actin-bundling protein Fascin (FSCN-1) in HTLV-1-infected T-cells. Here, we report that Fascin contributes to HTLV-1 transmission. Using single-cycle replication-dependent HTLV-1 reporter vectors, we found that repression of endogenous Fascin by short hairpin RNAs and by Fascin-specific nanobodies impaired gag p19 release and cell-to-cell transmission in 293T cells. In Jurkat T-cells, Tax-induced Fascin expression enhanced virus release and Fascin-dependently augmented cell-to-cell transmission to Raji/CD4+ B-cells. Repression of Fascin in HTLV-1-infected T-cells diminished virus release and gag p19 transfer to co-cultured T-cells. Spotting the mechanism, flow cytometry and automatic image analysis showed that Tax-induced T-cell conjugate formation occurred Fascin-independently. However, adhesion of HTLV-1-infected MT-2 cells in co-culture with Jurkat T-cells was reduced upon knockdown of Fascin, suggesting that Fascin contributes to dissemination of infected T-cells. Imaging of chronically infected MS-9 T-cells in co-culture with Jurkat T-cells revealed that Fascin’s localization at tight cell-cell contacts is accompanied by gag polarization suggesting that Fascin directly affects the distribution of gag to budding sites, and therefore, indirectly viral transmission. In detail, we found gag clusters that are interspersed with Fascin clusters, suggesting that Fascin makes room for gag in viral biofilms. Moreover, we observed short, Fascin-containing membrane extensions surrounding gag clusters and clutching uninfected T-cells. Finally, we detected Fascin and gag in long-distance cellular protrusions. Taken together, we show for the first time that HTLV-1 usurps the host cell factor Fascin to foster virus release and cell-to-cell transmission., Author Summary Human T-cell leukemia virus type 1 (HTLV-1) is the only human retrovirus causing cancer and is transmitted via breast feeding, sexual intercourse, and cell-containing blood products. Efficient infection of CD4+ T-cells occurs via polarized budding of virions or via cell surface transfer of viral biofilms at a tight, specialized cell-cell contact, the virological synapse (VS). The viral protein Tax and polarization of the host cell cytoskeleton are crucial for formation of the VS, however, only little is known about the link between Tax and remodeling of the cytoskeleton to foster viral spread. The actin-bundling protein Fascin has evolved as a therapeutic target in several types of cancer. Here, we show that Fascin is also crucial for release and transmission of the tumorvirus HTLV-1. Since Fascin is a transcriptional target gene of Tax in T-cells, our work provides a link between Tax’s activity and virus transmission. Visualization of cell-cell contacts between infected and uninfected T-cells suggests a role of Fascin in viral transmission potentially by facilitating the transport of viral proteins to budding sites. Thus, Fascin is not only crucial for metastasis of tumors, but also for transmission of HTLV-1 and is a new cellular target to counteract HTLV-1.

The development and investigation of reactive sandwich targets and their interaction with shaped charge jets is one of the many research areas, where important contributions were made by Manfred Held. Such reactive sandwiches are known to be extremely effective against jets, but have a number of disadvantages. In this work, the interaction of the copper jet from a shaped charge calibre 136 mm with a double sandwich system, each sandwich consisting of a non-reacting layer between outer steel plates, is investigated. In 10 experiments with identical geometrical setup, only the material of the non-reacting layer is changed. Using flash X-ray pictures, the interaction of the jet with the sandwich system is investigated. Evaluation of these pictures as well as the comparison of the measured residual penetrations behind the sandwich target clearly demonstrate the influence of the material of the inner sandwich layer. The deformation and movement of the steel plates, caused by the penetrating jet, can be seen in the X-ray pictures. It is shown that it is possible to distort a shaped charge jet so that its penetration capability behind the target is reduced to a minimum. A fully three-dimensional hydrocode simulation of the experiments enabled the jet/sandwich interaction and steel plate deformation and rupture to be studied in detail.

Bei der Herstellung von Hartstoffschichten durch reaktives Zerstauben ist es sinnvoll, die Zusammensetzung der Schicht bereits in-situ wahrend des Aufwachsens zu kontrollieren. Dieses Ziel wurde durch einen experimentellen Aufbau erreicht, bei dem die Analyse mit Hilfe der charakteristischen Rontgenstrahlung erfolgt, die wahlweise durch Elektronen- oder Rontgenanregung der Schicht ausgelost werden kann. Das System wurde am Beispiel von Ti1−xNx-Schichten erprobt, bei deren Herstellung die Zerstaubungsparameter systematisch verandert wurden. Die Struktur von Hartstoffschichten wird durch Rontgenbeugung nach der Herstellung untersucht; die Besonderheit des hier angewandten Verfahrens ist, das durch streifenden Einfall die Oberflache empfindlich vermessen werden kann. Es konnen Aussagen uber die Mikrokristallitgrose und die Mikrodehnung der Schicht, aber auch uber den Eigenspannungszustand gemacht werden. An diversen Hartstoffschichten wird der Einflus der Herstellungsparameter Stickstoffdurchflus und Vorspannung auf die Schichtstruktur gezeigt. Investigation of hard coatings prepared by RF sputtering An analysis system has been developed to control the composition of thin films in situ during growth. The analysis is based on the characteristic X-ray emission excited either by an electron beam or by X-ray radiation. As an example, the system has been tested on Ti1−xNx films prepared by reactive sputtering. The structure of hard layers is examined by X-ray diffraction after the preparation. Grazing incidence is employed which means enhanced sensitivity for radiation from the surface. The size of microcrystallites and the microstrain as well as intrinsic macrostrains are examined. For several layer systems the influence of preparation parameters such as nitrogen flow and biasvoltage is demonstrated.

Recent changes in legislation governing water quality management of receiving water bodies have led to a reappraisal of wastewater land disposal techniques. However, more stringent regulations have also necessitated the development of a multi-disciplinary planning approach, to ensure that land based wastewater disposal is functionally and environmentally sustainable in the long-term. Of principal concern are the long term impact of nutrients, salt and other potential contaminants on the soils of the receiving site and on downstream water quality. Assessment of hydrological, soil physical and geological characteristics, together with civil construction and service considerations, assist in the determination of receiving-site selection, application area and balance storage volume, irrigation method, environmental monitoring system specification etc. Analysis and interpretation of wastewater and soil chemical characteristics determines the pre-application water treatment required, and aliows long-term monitoring of the effect of wastewater disposal on the receiving-site soils. Two case-studies are presented. One describes the planning and design of a recently commissioned land-disposal system using industrial wastewater from a chemical process plant to irrigate a Eucalypt plantation in western metropolitan Melbourne. The other reports on the on-going assessment and planning of a large-scale land-disposal system proposed to accommodate the treated sewage effluent from a large north-west Victorian regional city.