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Artificial van der Waals heterostructures, obtained by stacking layered two-dimensional materials, represent a novel material platform for investigating physicochemical phenomena and applications. We report here electrochemistry at the one-dimensional edge of a graphene sheet, which is sandwiched between two hexagonal boron nitride (hBN) multilayer flakes. When such an hBN/graphene/hBN heterostructure is immersed in a solution, the basal plane of graphene is protected and isolated by the hBN stack, and the edge of the graphene sheet is exclusively available in the solution. This forms an electrochemical nanoelectrode, which enabled us to investigate electron transfer using several redox probes, e.g., ferrocene(di)methanol, hexaammineruthenium, methylene blue, dopamine and ferrocyanide. Facilitated by the relatively low capacitance of the van der Waals edge electrode, we demonstrate cyclic voltammetry at very high scan rates (up to 1000 V/s). Using fast scan cyclic voltammetry imaging, we show that redox species can be detected voltammetrically down to micromolar concentrations with subsecond time resolution at the sandwiched graphene edge, promoted by the rapid equilibration of analyte species in the diffusion layer. Furthermore, the nanoband nature of the edge electrode allows us to work directly in water in the absence of added electrolyte. Finally, we show that two adjacent edge electrodes can be realized in a redox-cycling format. In all, the van der Waals edge electrode is unique among nanoelectrodes as it enables investigations of all the above-mentioned phenomena in the same device. Due to its versatility, it constitutes a new avenue for nanoscale electrochemistry, which will be useful for studying electron transfer mechanisms as well as for the detection of analyte species in ultralow sample volumes.

Supplementary Materials and Methods. Supplementary Figures: Fig. S1. Sorting strategy and purity. Fig. S2. Comparison of the two batches of NanoString experiments shown in Fig. 1 and Fig. 4. Fig. S3. Melanoma cells respond to CTL attack with gene expression changes. Fig. S4. CTL-induced gene expression changes are driven by IFNgamma and TNFalpha. Fig. S5. Representative histograms of protein expression regulated by cytokines. Fig. S6. Characterization of untreated and CTL-exposed melanocytes. Supplementary Tables: Table S1. Source of the 17 melanoma cell lines used in this study. Table S2. Antibodies (including isotype-matched controls) used to assess protein expression by flow cytometry. Table S3. Differential expression of genes that are part of the enriched gene sets in Supplementary Figs. S3C and S3E. Table S4. Selection of genes for 181-gene panel. Table S5. Differential expression of genes in melanoma cell lines after 24h treatment. Table S6. Protein expression of melanoma cells after 48h culture in presence or absence of IFNgamma and TNFalpha. Table S7. Differential expression of genes in melanocyte cell lines after 24h treatment. Table S8. Genes that have statistically different fold-changes (MelanA-specific CTLs/untreated) between melanoma cell lines and melanocyte lines. Supplementary References.

Although mutations drive cancer, it is less clear to what extent genetic defects control immune mechanisms and confer resistance to T-cell-based immunotherapy. Here, we studied the reactions of malignant and benign melanocyte lines to cytotoxic CD8+ T cells (CTL) using flow cytometry and gene expression analyses. We found rapid and broad upregulation of immune-regulatory genes, essentially triggered by CTL-derived IFNγ and augmented by TNFα. These reactions were predominantly homogenous, independent of oncogenic driver mutations, and similar in benign and malignant cells. The reactions exhibited both pro- and antitumorigenic potential and primarily corresponded to mechanisms that were conserved, rather than acquired, by mutations. Similar results were obtained from direct ex vivo analysis of the tumor microenvironment. Thus, immune regulation in the tumor landscape may often be driven by conserved mechanisms, which may explain why T-cell–based immunotherapy can provide durable benefits with relatively infrequent escape. Cancer Res; 77(7); 1623–36. ©2017 AACR.

Streptococcus pneumoniae is a Gram-positive, encapsulated bacterium that is a significant cause of disease burden in pediatric and elderly populations. The rise in unencapsulated disease-causing strains and antimicrobial resistance in S. pneumoniae has increased the need for developing new antimicrobial strategies.

Practitioners are seeking information and ethical guidance related to the design and utilization of artificial intelligence (AI) across a variety of applications. Developments at Google illustrate the potential good of AI as well as potential concerns. In this article, we use Google to create a context that demonstrates the relevance of virtue for the ethical design and use of AI. We describe a set of ethical challenges encountered by Google and introduce virtue as a framework for ethical decision making that can be applied broadly to numerous organizations. We also examine support for virtue in ethical decision making as well as its power in attracting and retaining the employees who develop AI and the customers who use it. To conclude, we apply the virtue framework to Google’s AI challenges and offer suggestions for its use in other organizations.

Achieving long term stability of single graphene sheets towards repeated electrochemical hydrogen evolution reaction (HER) cycling has been challenging. Here, we show through appropriate electrode preparation that it is possible to obtain highly durable isolated graphene electrodes, which can survive several hundreds of HER cycles with virtually no damage to the sp

Electrochemical hydrogen evolution reaction (HER) at single graphene sheets has been investigated widely either in its pristine form or after chemical modification. One important challenge is the long-term stability of single graphene sheets on Si/SiO2 substrates under HER. Previous reports have found that due to stress developing under gas evolution, the sheets tend to break apart, with a very low lifetime limited to just a few cycles of HER. Here, we show through appropriate electrode preparation that it is possible to achieve highly durable single graphene electrodes on insulating substrates, which can survive several hundreds of HER cycles with virtually no damage to the sp2-carbon framework. Through systematic investigations including atomic force microscopy, Raman spectroscopy and electroanalysis, we show that even after so many cycles, the sheet is physically intact and the electron transfer capability of the electrodes remain unaffected. This extremely high stability of a single atomic sheet of carbon, when combined with appropriate chemical modification strategies, will pave way for the realization of novel 2D electrocatalysts.

Transition metals are necessary cofactors and structural elements in living systems. Exposure to high concentrations of biologically important transition metals, such as zinc and copper, results in cell toxicity. At the infection site, the immune system deploys metal sorbent proteins (e.g., lactoferrin and calprotectin) to starve pathogens of necessary metals (such as iron), while phagocytes expose engulfed pathogens to high levels of other metals, such as copper and zinc. The opportunistic pathogen Streptococcus pneumoniae (the pneumococcus) encounters macrophages during initial and protracted infections. The pneumococcus employs a copper export pathway, which improves colonization and persistent infection of the nasopharynx and the upper respiratory tract. Because copper is tightly regulated in the host, we instead sought to leverage the localized power of nutritional immunity by identifying small molecules with copper-dependent toxicity (CDT) through a targeted screen of compounds for antibiotic efficacy. We chose to include dithiocarbamates, based on the copper synergy observed in other organisms with 1-(diethylthiocarbamoyldisulfanyl)-N,N-diethyl-methanethioamide (tetraethylthiuram disulfide, disulfiram). We observed CDT of some dithiocarbamates in S. pneumoniae. Only N,N-dimethyldithiocarbamate (DMDC) was consistently toxic across a range of concentrations with copper both in vitro and in vivo against the pneumococcus. We also observed various degrees of CDT in vitro using DMDC in Staphylococcus aureus, Coccidioides posadasii, and Schistosoma mansoni. Collectively, we demonstrate that the compound DMDC is a potent bactericidal compound against S. pneumoniae with antimicrobial efficacy against bacterial and fungal pathogens. IMPORTANCE With the rise of antibiotic resistance, approaches that add new antimicrobials to the current repertoire are vital. Here, we investigate putative and known copper ionophores in an attempt to intoxicate bacteria and use ionophore/copper synergy, and we ultimately find success with N,N-dimethyldithiocarbamate (DMDC). We show that DMDC has in vitro efficacy in a copper-dependent manner and kills pathogens across three different kingdoms, Streptococcus pneumoniae, Coccidioides posadasii, and Schistosoma mansoni, and in vivo efficacy against S. pneumoniae. As such, dithiocarbamates represent a new potential class of antimicrobials and thus warrant further mechanistic investigation.

Nanotechnology is a recent development within construction, however, exposure to nanomaterials entails exposure to emerging hazards and risk, which is exacerbated by the limited knowledge pertaining thereto. A quantitative survey was conducted among medium to large sized general contractors, and architects in the Eastern Cape province, South Africa. The salient findings include: architects and contractors specify and use nanomaterials during the construction process; architects follow ‘designing for safety’ practice to a limited extent; contractors conduct the necessary training, inform their employees of the potential hazards related to the use of nanomaterials, implement safe application techniques when working with nanomaterials, issue employees with the necessary PPE to carry out the work safely, and generally do not analyse the characteristics of materials to a major extent, but do analyse material safety data sheets (MSDSs). Conclusions include: there is a low level of awareness and knowledge with respect to nanomaterials; the responding architects are barely committed to construction H&S; responding contractors are committed to H&S, and there is a need for research to determine the healthiest and safest methods of using nanomaterials. It is recommended that designers and contractors should enhance their awareness of and knowledge pertaining to nanomaterials, including the hazards and risks.

This is the first paper in the symposium Faith in Management Scholarship and Practice. In this role it lays out how a set of naturalistic and materialistic assumptions can be limiting for scholarsh...

Servant leadership is ripe for exploration from both etic and emic perspectives investigating its robustness as a leadership style globally. Through a meta-analysis of data from 139 studies, we examine the relationship of servant leadership with 23 attitudinal, behavioral, and performance outcomes across cultures. Utilizing values and practices approaches to culture, we test the moderating influence of a profile of four GLOBE culture dimensions theoretically linked to servant leadership: assertiveness, institutional collectivism, humane orientation, and power distance. The findings provide evidence of servant leadership's association with workplace outcomes, grant insight into contingencies of cultural practices and values, and offer directions for future research.

Although mutations drive cancer, it is less clear to what extent genetic defects control immune mechanisms and confer resistance to T-cell-based immunotherapy. Here, we studied the reactions of malignant and benign melanocyte lines to cytotoxic CD8+ T cells (CTL) using flow cytometry and gene expression analyses. We found rapid and broad upregulation of immune-regulatory genes, essentially triggered by CTL-derived IFNγ and augmented by TNFα. These reactions were predominantly homogenous, independent of oncogenic driver mutations, and similar in benign and malignant cells. The reactions exhibited both pro- and antitumorigenic potential and primarily corresponded to mechanisms that were conserved, rather than acquired, by mutations. Similar results were obtained from direct ex vivo analysis of the tumor microenvironment. Thus, immune regulation in the tumor landscape may often be driven by conserved mechanisms, which may explain why T-cell–based immunotherapy can provide durable benefits with relatively infrequent escape. Cancer Res; 77(7); 1623–36. ©2017 AACR.

We report the direct exclusive modification of the edge of a single graphene monolayer with nanoparticles or organic functionalities under ambient conditions., We present a versatile and simple method using electrochemistry for the exclusive functionalization of the edge of a graphene monolayer with metal nanoparticles or polymeric amino groups. The attachment of metal nanoparticles allows us to exploit surface-enhanced Raman scattering to characterize the chemistry of both the pristine and the functionalized graphene edge. For the pristine patterned graphene edge, we observe the typical edge-related modes, while for the functionalized graphene edge we identify the chemical structure of the functional layer by vibrational fingerprinting. The ability to obtain single selectively functionalized graphene edges routinely on an insulating substrate opens an avenue for exploring the effect of edge chemistry on graphene properties systematically.

Correction for ‘pH sensitivity of interfacial electron transfer at a supported graphene monolayer’ by Michel Wehrhold et al., Nanoscale, 2019, DOI: 10.1039/c9nr05049c.

Monoclonal antibody-based targeted tumor therapy has greatly improved treatment options for patients. Antibodies against oncogenic receptor tyrosine kinases (RTKs), especially the ErbB receptor family, are prominent examples. However, long-term efficacy of such antibodies is limited by resistance mechanisms. Tumor evasion by a priori or acquired activation of other kinases is often causative for this phenomenon. These findings led to an increasing number of combination approaches either within a protein family, e.g. the ErbB family or by targeting RTKs of different phylogenetic origin like HER1 and cMet or HER1 and IGF1R. Progress in antibody engineering technology enabled generation of clinical grade bispecific antibodies (BsAbs) to design drugs inherently addressing such resistance mechanisms. Limited data are available on multi-specific antibodies targeting three or more RTKs. In the present study, we have evaluated the cloning, eukaryotic expression and purification of tetraspecific, tetravalent Fc-containing antibodies targeting HER3, cMet, HER1 and IGF1R. The antibodies are based on the combination of single-chain Fab and Fv fragments in an IgG1 antibody format enhanced by the knob-into-hole technology. They are non-agonistic and inhibit tumor cell growth comparable to the combination of four parental antibodies. Importantly, TetraMabs show improved apoptosis induction and tumor growth inhibition over individual monospecific or BsAbs in cellular assays. In addition, a mimicry assay to reflect heterogeneous expression of antigens in a tumor mass was established. With this novel in vitro assay, we can demonstrate the superiority of a tetraspecific antibody to bispecific tumor antigen-binding antibodies in early pre-clinical development.

Field-effect transistors (FETs) based on graphene are promising devices for the direct sensing of a range of analytes in solution. We show here that the presence of redox active molecules in the analyte solution leads to the occurrence of heterogeneous electron transfer with graphene generating a Faradaic current (electron transfer) in a FET configuration resulting in shifts of the Dirac point. Such a shift occurs if the Faradaic current is significantly high, e.g. due to a large graphene area. Furthermore, the redox shift based on the Faradaic current, reminiscent of a doping-like effect, is found to be non-Nernstian and dependent on parameters known from electrode kinetics in potentiodynamic methods, such as the electrode area, the standard potential of the redox probes and the scan rate of the gate voltage modulation. This behavior clearly differentiates this effect from other transduction mechanisms based on electrostatic interactions or molecular charge transfer doping effects, which are usually behind a shift of the Dirac point. These observations suggest that large-area unmodified/pristine graphene in field-effect sensors behaves as a non-polarized electrode in liquid. Strategies for ensuring a polarized interface are discussed.

Forms of capital play a significant role in the innovation and performance of start–up firms. Current entrepreneurial research has focused on the role of financial, human, and social forms of capital. We build on a large body of theory and research in sociology and economics, proposing spiritual capital as an additional influence where institutional voids are greater in the development contexts studied. Results from microcredit entrepreneurs in Kenya and Indonesia indicate significant relationships between entrepreneurs’ spiritual capital and business innovation and performance, even after accounting for other forms of capital.

The immune system defends us and combats many microbes, like the viruses that cause the common cold or bacteria that enter wounds. Our defenses can also learn to protect us from more difficult situations—and they can do so with the lessons learned from vaccines. For example, the poliovirus vaccine teaches the immune system to recognize and eliminate the poliovirus, in case it ever enters the body. In recent years, scientists have found that the immune system can also be taught to attack another type of disease: cancer. Cancer is a big mistake in our body. It happens when cells lose control and start to grow without limits. The good news is that we can educate the immune system in cancer patients—this is called immunotherapy. In this article, we will explain how our defenses can learn to attack and eliminate cancer.

Disputes over the connection between beliefs and behaviors, coupled with the relative paucity of survey measures of specific theological beliefs related to work and its association with financial success, motivated the development of two beliefs scales: beliefs about work as a means to honor God and beliefs about God promising prosperity for believers. Analyses demonstrated these two scales to be distinct from one another and reliable. Using a national sample of over 1,000 working adults, we assessed the relationship of the specific belief scales to Lynn et al.’s (J Bus Ethics 85(2):227–241, 2009) faith at work scale, Mirels and Garrett’s (J Consul Clin Psychol 36(1):40–44, 1971) Protestant Work Ethic scale, two personality scales (Conscientiousness and Neuroticism), entrepreneurial behavior, helping behavior, and religious tradition. Results indicate convergent and divergent validity and demonstrate the potential utility of these beliefs scales for predicting work-related attitudes and practices.

O1 IL-15 primes an mTOR-regulated gene-expression program to prolong anti-tumor capacity of human natural killer cells #### Andreas Lundqvist1, Vincent van Hoef1, Xiaonan Zhang1, Erik Wennerberg2, Julie Lorent1, Kristina Witt1, Laia Masvidal Sanz1, Shuo Liang1, Shannon Murray3, Ola Larsson1

Background Children with HIV will be on antiretroviral therapy (ART) longer than adults, and therefore the durability of first-line ART and timing of switch to second-line are key questions. We assess the long-term outcome of protease inhibitor and non-nucleoside reverse transcriptase inhibitor (NNRTI) first-line ART and viral load switch criteria in children. Methods In a randomised open-label factorial trial, we compared effectiveness of two nucleoside reverse transcriptase inhibitors (NRTIs) plus a protease inhibitor versus two NRTIs plus an NNRTI and of switch to second-line ART at a viral load of 1000 copies per mL versus 30,000 copies per mL in previously untreated children infected with HIV from Europe and North and South America. Random assignment was by computer-generated sequentially numbered lists stratified by age, region, and by exposure to perinatal ART. Primary outcome was change in viral load between baseline and 4 years. Analysis was by intention to treat, which we defined as all patients that started treatment. This study is registered with ISRCTN, number ISRCTN73318385. Findings Between Sept 25, 2002, and Sept 7, 2005, 266 children (median age 6.5 years; IQR 2.8-12.9) were randomly assigned treatment regimens: 66 to receive protease inhibitor and switch to second-line at 1000 copies per mL (PI-low), 65 protease inhibitor and switch at 30,000 copies per mL (PI-higher), 68 NNRTI and switch at 1000 copies per mL (NNRTI-low), and 67 NNRTI and switch at 30,000 copies per mL (NNRTI-higher). Median follow-up was 5.0 years (IQR 4.2-6.0) and 188 (71%) children were on first-line ART at trial end. At 4 years, mean reductions in viral load were -3.16 log(10) copies per mL for protease inhibitors versus -3.31 log(10) copies per mL for NNRTIs (difference -0.15 log(10) copies per mL, 95% CI -0.41 to 0.11; p=0.26), and -3.26 log(10) copies per mL for switching at the low versus -3.20 log(10) copies per mL for switching at the higher threshold (difference 0.06 log(10) copies per mL, 95% CI -0.20 to 0.32; p=0.56). Protease inhibitor resistance was uncommon and there was no increase in NRTI resistance in the PI-higher compared with the PI-low group. NNRTI resistance was selected early, and about 10% more children accumulated NRTI mutations in the NNRTI-higher than the NNRTI-low group. Nine children had new CDC stage-C events and 60 had grade 3/4 adverse events; both were balanced across randomised groups. Interpretation Good long-term outcomes were achieved with all treatments strategies. Delayed switching of protease-inhibitor-based ART might be reasonable where future drug options are limited, because the risk of selecting for NRTI and protease-inhibitor resistance is low. Funding Paediatric European Network for Treatment of AIDS (PENTA) and Pediatric AIDS Clinical Trials Group (PACTG/IMPAACT).

As a result of numerous, highly publicized, ethical breaches, firms and their agents are under ongoing scrutiny. In an attempt to improve both their image and their ethical performance, some firms have adopted ethical codes of conduct. Past research investigating the effects of ethical codes of conduct on behavior and ethical attitudes has yielded mixed results. In this study, we again take up the question of the effect of ethical codes on ethical attitudes and find strong evidence to suggest that business professionals employed at firms with ethical codes of conduct are significantly less accepting of ethically questionable behavior toward most stakeholders. One notable exception relates to ethical actions toward customers.

Binding of hemagglutinin-neuraminidase proteins (HN) to sialylated receptors initiates the infection process of several paramyxoviruses, whereas later in the viral life cycle, the neuramindase (NA) activity of newly synthesized HN destroys all receptors. Prior to NA action, expressed HN has to bind the receptor. To evaluate this HN-receptor complex with respect to receptor inactivation, three temperature-sensitive Sendai virus HN mutants carrying amino acid exchanges at positions 262, 264 and/or 461 were created that uncoupled NA activity from receptor binding at 39 degrees Celsius. Interestingly, at elevated temperature, when there is no detectable neuramindase activity, all infected cells are protected against homologous superinfection. Mutated HN protein on the cell surface is mainly bound to sialylated cell-surface components but can be released by treatment with NA. Thus, continuous binding to HN already inactivates the receptors quantitatively. Furthermore, mutant HN bound to receptors is prevented from being incorporated into virus particles in the absence of NA. It is shown here for the first time that during paramyxoviral infection, quantitative receptor inactivation already occurs due to binding of receptors to expressed HN protein without involvement of NA and is independent of NA activity of viral progeny. NA subsequently functions in the release of HN from the complex, coupled with desialysation of receptors. These findings could have implications for further antiviral drug development.

Whereas most free-riding research has focused on limiting free-riding and on understanding free-riding motives, we were concerned with capturing the psychological processes used by observers to interpret the poor performance behavior of a team member. Using a sample of 268 observers of a simulated team interaction, we found that peer reactions to a poor performance could be explained through attribution theory (AT). Poor performers perceived as possessing high general cognitive ability (g) and low conscientiousness traits were more likely to be rated high on free-riding when compared to those perceived as possessing low g/low conscientiousness traits. In addition, AT helped explain observer anger and intent to punish free-riders.

For a long time, Sendai virus (SeV) was used as a model paramyxovirus to study the virus life cycle. Upon infection of a host cell, different stages are completed during replication of the negatively oriented single-stranded RNA genome with its six genes before progeny virus particles are assembled. In the beginning, only one nucleocapsid (NC) is present in the cell. The NC is composed of the RNA genome encapsulated by nucleocapsid (N) proteins (ribonucleoproteins [RNP]) and 30 to 50 associated viral RNA-dependent RNA polymerase molecules (vRdRp) consisting of a P protein tetramer and one L protein. This minimal replication unit is capable of starting viral transcription. During these first transcriptional events, termed primary transcription, the genome is transcribed by the vRdRp and de novo protein synthesis begins. As soon as sufficient amounts of N proteins have been translated, the vRdRp is thought to switch to a replicative mode to synthesize antigenomes. From that template, new genomes are replicated that are concomitantly encapsidated with N proteins (22). Thus, new templates that also can be transcribed before they are assembled into progeny viral particles are present in the cell. This phase of transcription is called secondary transcription and is much more effective than primary transcription. During in vitro studies, viral transcription can be reproduced by incubating purified RNPs with cell extracts containing SeV P and L proteins (14). For genome replication, additional expression of viral N protein is necessary, which, when bound to a P protein (termed N0-P), serves as a substrate for encapsidation of nascent RNA genomes and antigenomes (20). However, different puzzling observations have led to an ongoing discussion about other possible functions and tasks for the P protein. During infections, the synthesis of viral P protein is disproportionately high compared to that of the other proteins (23, 26). Furthermore, the P protein is observed in 4 to 10 clusters on the NC in infected cells. In contrast, NCs derived from virions show a uniform distribution of the P protein (27, 28). This difference is proposed to be related to different functions of the P protein. Inside virions, the P protein may stabilize the NC, the half-life of which is estimated to be only 1 to 2 days in a cellular environment (24). On the other hand, the P protein may enhance the processivity of the vRdRp during infections. When RNPs are incubated in the presence of a 5- to 10-fold elevated amount of P protein compared to the level of P as part of vRdRps, only a twofold increase in mRNA synthesis is detected (12). Interestingly, in vitro RNA synthesis studies have shown that mutations in P inhibit viral transcription, even though the interactions between P and L, P and P, and P-L and the template RNA were not impaired (4). These results also may indicate an additional function of the P protein independent of binding to L or to itself. There have been several observations indicating that the P protein is involved in other undescribed processes during viral replication; no concrete evidence exists for an additional function. However, these observations all were made under in vitro conditions with about 1011 NCs employed per assay. Under these conditions, it is impossible to estimate the transcriptive power of single NCs inside cells. However, the intent of our studies here was to investigate, for the first time, the influence of SeV P protein on viral gene expression under conditions of a normal infection, i.e., with about one virus particle per cell using specifically designed mutants. We therefore constructed SeV mutants that lacked the P open reading frame (ORF) (SeV ΔP) or the N ORF (SeV ΔN). Without de novo-synthesized P protein, we expected to elucidate additional functions of this protein, e.g., a possible influence on biphasic mRNA synthesis rates as described by Plumet and colleagues (25); without N protein synthesis, we tried to suppress genome replication. During infection experiments, we could show that de novo synthesis of viral P protein is necessary to bring preliminary primary viral transcription to normal levels. Very surprising and never reported before, we also found that viral N protein not only was necessary for viral replication but also was essential for normal viral primary transcription.

To survive in today's ultra-competitive business environment, organizations must better understand the factors that cause managers to fail to achieve desired results. To that end, focus group data was collected from 1040 managers from over 100 different U.S. manufacturing and service organizations experiencing large scale organizational change in order to help identify the primary causes of managerial failure. This article discusses the 15 primary causes of managerial failure identified in the study, along with their perceived consequences to managerial and organizational performance. Ultimately, it is hoped that this will provide a guide for improving the effectiveness of both individual managers and the organizations they serve.

Coaching is a frequently cited, but less often effectively implemented, developmental tool for enhancing managerial performance at all levels. The purpose of this paper is to identify and explore the practices junior managers consider to be most critical for senior managers to employ when implementing coaching as a performance improvement practice. In this research, we engaged 45 focus groups consisting of 225 middle managers from over twenty different US organizations to explore the issue of managerial coaching and its implications for organizations and individual managers.

Delivery of Ags to dendritic cells (DCs) plays a pivotal role in the induction of efficient immune responses ranging from immunity to tolerance. The observation that certain viral pathogens are able to infect DCs has led to a concept in which applications of recombinant viruses are used for Ag delivery with the potential benefit of inducing potent Ag-specific T cell responses directed against multiple epitopes. As a prerequisite for such an application, the infection of DCs by recombinant viruses should not interfere with their stimulatory capacity. In this context, we could show that an emerging negative-strand RNA viral vector system based on the Sendai virus (SeV) is able to efficiently infect monocyte-derived human DCs (moDCs). However, after infection with SeV wild type, both the response of DCs to bacterial LPS as a powerful mediator of DC maturation and the allostimulatory activity were severely impaired. Interestingly, using various recombinant SeV vectors that were devoid of single viral genes, we were able to identify the SeV matrix (M) protein as a key component in moDC functional impairment after viral infection. Consequently, use of M-deficient SeV vectors preserved the allostimulatory activity in infected moDCs despite an efficient expression of all other virally encoded genes, thereby identifying M-deficient vectors as a highly potent tool for the genetic manipulation of DCs.

Induction of apoptosis during Sendai virus (SeV) infection has previously been documented to be triggered by initiator caspases (for strain F) or by a contribution of the cellular protein TIAR (T-cell-activated intracellular antigen-related) (for strain Z). Here, evidence was provided that both TIAR and caspases are simultaneously involved in apoptosis induction as a result of infection with SeV strain F. SeV F infection induced death in all tested cell lines, which could only be partially prevented through the pan-caspase inhibitor z-VAD-fmk. However, infection of seven different cell lines with the SeV mutant Fctr48z overexpressing a TIAR-sequestering RNA from the modified leader resulted in a cell type-dependent reduced cytopathic effect (CPE); in an earlier study a similar mutant derived from SeV Z was shown to prevent the induction of any CPE. Finally, blocking of caspases through z-VAD-fmk combined with Fctr48z infection led to complete abrogation of CPE, clearly demonstrating the existence of two separate mechanisms inducing cell death during SeV F infections. Interestingly, a cell type-specific interference between these two mechanisms could be detected during infection with the mutant virus Fctr48z: RNA transcribed from the mutated leader was able to trans-dominantly inhibit caspase-mediated apoptosis. Thus, virus-expressed factors enabling a well-balanced ratio of suppression and triggering of apoptosis seem to be essential for optimal virus replication.

Treatment by gene replacement is critical in the field of gene therapy. Suitable vectors for the delivery of therapeutic genes have to be generated and tested in preclinical settings. Recently, extraordinary features for a local gene delivery by Sendai virus vectors (SeVV) have been reported for different tissues. Here we show that direct intravenous application of SeVV in mice is not only feasible and safe, but it results in the secretion of therapeutic proteins to the circulation, for example, human clotting Factor IX (hFIX). In vitro characterization of first-generation SeVV demonstrated that secreted amounts of hFIX were at least comparable to published results for retroviral or adeno-associated viral vectors. Furthermore, as a consideration for application in humans, SeVV transduction led to efficient hFIX synthesis in primary human hepatocytes, and SeVV-encoded hFIX proteins could be shown to be functionally active in the human clotting cascade. In conclusion, our investigations demonstrate for the first time that intravenous administration of negative-strand RNA viral vectors may become a useful tool for the wide area of gene replacement requirements.

The Sendai virus RNA polymerase is a complex of two virus-encoded proteins, the phosphoprotein (P) and the large (L) protein, where L is believed to possess all the enzymatic activities necessary for viral transcription and replication. The alignment of amino acid sequences of L proteins from negative-sense RNA viruses shows six regions, designated domains I–VI, of good conservation which have been proposed to be important for the various enzymatic activities of the polymerase. To directly address the role(s) of domains II and III, site-directed mutations were constructed by the substitution of multiple amino acids at 13 highly or mostly conserved residues. Analysis of in vitro viral transcription and replication showed that the majority of the mutations completely inactivated the L protein for all aspects of RNA synthesis, thus conservation correlated with the essential nature of the amino acid. At some positions different phenotypes, from inactivation to partial activities, were observed which depended on the nature of the amino acid that was substituted. Two mutants, K543R and K666V, could synthesize some leader RNA, but were defective in mRNA synthesis and replication. K666R and G737E had significantly reduced replication compared to transcription in vitro, but replicated genome RNA much more efficiently in vivo. K666A gave transcription, but no replication. Representative inactive L mutants, however, were still able to bind P protein and the polymerase complex was capable of binding nucleocapsids, so the defect appeared to be in the initiation of RNA synthesis.

Apoptotic cell death is of central importance in the pathogenesis of viral infections. Activation of a cascade of cysteine proteases, i.e. caspases, plays a key role in the effector phase of virus-induced apoptosis. However, little is known about pathways leading to the activation of initiator caspases in virus-infected host cells. Recently, we have shown that Sendai virus (SeV) infection triggers apoptotic cell death by activation of the effector caspase-3 and initiator caspase-8. We now investigated mechanisms leading to the activation of another initiator caspase, caspase-9. Unexpectedly we found that caspase-9 cleavage is not dependent on the presence of active caspases-3 or -8. Furthermore, the presence of caspase-9 in mouse embryonic fibroblast (MEF) cells was a prerequisite for Sendai virus-induced apoptotic cell death. Caspase-9 activation occurred without the release of cytochrome c from mitochondria and was not dependent on the presence of Apaf-1 or reactive oxygen intermediates. Our results therefore suggest an alternative mechanism for caspase-9 activation in virally infected cells beside the well characterized pathways via death receptors or mitochondrial cytochrome c release.

The influence of religion on work has not been fully explored, and, in particular, the relationship between religion and entrepreneurship as a specific type of work. This study explores the link between entrepreneurial behavior and religion. The study finds that religion, for entrepreneurs, is highly individualized, leading to the initial impression that religion and work have no relationship. Upon closer inspection, however, the study finds that religion does shape entrepreneurial activity. Entrepreneurial activity is impacted by a need for the entrepreneurs to reinterpret their work in religious terms, ending the tension for them between faith and work.

The etiopathogenesis of otosclerosis is still largely unexplained and remains controversial. Morphologic examinations have shown the presence of a chronic inflammation in otosclerotic tissue. Among the proposed explanations for this inflammation are an immunologic reaction against collagen, mutations of collagen gene 1A1, and a viral infection. In this paper, we focus on the role of measles virus in otosclerosis, and we review the current literature, devoting particular attention to a suspected paramyxoviral etiopathogenesis in Paget's disease. Our examination of footplate fragments by reverse transcription polymerase chain reaction testing in 95 patients with otosclerosis revealed the presence of measles virus RNA in 83% of cases. Quantification of measles virus immunoglobulin G (IgG) in otosclerosis patients indicated that the ratio of antimeasles virus IgG in total IgG was higher in perilymph than in serum. Furthermore, an almost identical incidence of otosclerosis and measles virus-caused mortality in women suggests that women are more susceptible to measles virus infection. Finally, since the introduction of the measles virus vaccination program in Europe, there has been a decline in the incidence of otosclerosis. Moreover, the average age of patients at diagnosis and surgery at our hospital has increased to 54 years. Our findings, when they are considered along with findings regarding the presence of paramyxoviral RNA in Paget's disease, support the hypothesis that measles virus is involved in the etiopathogenesis of otosclerosis.

Morphologische und biochemische Untersuchungen von verschiedenen Autoren haben gezeigt, dass die Otosklerose eine Masernvirus-assoziierte, entzündliche Erkrankung des menschlichen Felsenbeins ist. Das Ziel unserer Untersuchungen war es, Informationen über die Auswirkungen der Masernvirusinfektion im Felsenbein auf das Immunsystem des Innenohres zu erhalten und zu überprüfen, ob sich in Präosteoblasten, die aus dem Otoskleroseherd kultiviert wurden, Masernvirusgenom befindet. Während Stapedektomien wurde neben Fussplatten und Kontrollgewebe für die Präosteoblastenkultur Perilymphe entnommen und mit Westernblot auf Antikörperexpressionsmuster hin überprüft. Aus den Präosteoblasten konnte keine Masernvirus-RNA amplifiziert werden. In der Perilymphe konnten IgG gegen Masernviren nachgewiesen werden und das Expressionsmuster wies in 2 von 10 Fällen einen Unterschied im Vergleich zum Expressionsmuster im Serum des gleichen Patienten auf. Diese Ergebnisse sind im Einklang mit der Hypothese, dass in der Otosklerose eine Masernviruspersistenz vorliegt.

Mixed infection of cells with both Moloney murine leukemia virus (MoMLV) and related or heterologous viruses produces progeny pseudotype virions bearing the MoMLV genome encapsulated by the envelope of the other virus. In this study, pseudotype formation between MoMLV and the prototype parainfluenza virus Sendai virus (SV) was investigated. We report for the first time that SV infection of MoMLV producer cells results in the formation of MoMLV(SV) pseudotypes, which display a largely extended host range compared to that of MoMLV particles. This could be associated with SV hemagglutinin-neuraminidase (SV-HN) glycoprotein incorporation into MoMLV envelopes. In contrast, solitary incorporation of the other SV glycoprotein, SV fusion protein (SV-F), resulted in a distinct and narrow extension of the MoMLV host range to asialoglycoprotein receptor (ASGP-R)-positive cells (e.g., cultured human hepatoma cells). Since stably ASGP-R cDNA-transfected MDCK cells, but not parental ASGP-R-negative MDCK cells, were found to be transduced by MoMLV(SV-F) pseudotypes and transduction of ASGP-R-expressing cells was found to be inhibited by ASGP-R antiserum, a direct proof for the ASGP-R-restricted tropism of MoMLV(SV-F) pseudotypes was provided. Cultivation of ASGP-R-positive HepG2 hepatoma cells on Transwell-COL membranes led to a significant enhancement of MoMLV(SV-F) titers in subsequent flowthrough transduction experiments, thereby suggesting the importance of ASGP-R accessibility at the basolateral domain for MoMLV(SV-F) pseudotype transduction. The availability of such ASGP-R-restricted MoMLV(SV-F)-pseudotyped vectors opens up new perspectives for future liver-restricted therapeutic gene transfer applications.

Biochemical evidence suggests that the asialoglycoprotein receptor (ASGP-R) can be used as an alternative receptor for a temperature-sensitive Sendai virus (SV) mutant. We now have investigated this possible alternative route of infection for SV wild-type (SV-wt) strain Fushimi by using a pair of cell lines which differ only with regard to ASGP-R expression. Infection studies after enzymatic destruction of conventional sialic acid-containing SV receptors (SA-R) revealed that only ASGP-R-expressing cells could be infected by SV-wt. This alternative route of cell entry could be completely blocked by incubation of cells with ASGP-R-specific antibodies prior to infection. Furthermore, cleavage of SV-F0 precursor protein into the subunits F1 and F2 was necessary to establish infection via ASGP-R, suggesting a fusion-mediated cell entry after binding of SV-wt to the ASGP-R on host cells. Interestingly, infection via ASGP-R was found to be nearly as efficient as infection via conventional sialic acid-containing SV receptors. A possible physiological role of the ASGP-R-mediated route of SV infection is discussed.

Due to late diagnosis and a pronounced chemoresistance, most patients with hepatocellular carcinoma (HCC) have an overall poor prognosis. Measles vaccine viruses (MeV) have been shown to possess anti-tumor properties and their efficacy has been enhanced by arming with suicide genes. To test armed MeV for the treatment of HCC, we equipped it with the suicide gene Super-cytosine deaminase (SCD) and tested the efficacy in cell culture and in a mouse xenograft model of human HCC. Prodrug conversion was investigated in cell culture and quantified by high-performance liquid chromatography. We observed a strong oncolytic activity of MeV-SCD against human HCC in vitro and in vivo. The prodrug was efficiently converted in infected cells leading to a significant enhancement of the cytotoxic effect. Treatment of HCC xenografts with MeV caused long-term virus replication in tumor tissue. We show that the suicide gene therapy induces an apoptosis-like cell death but is not dependent on intact apoptosis pathways. These results demonstrate that MeV-based suicide gene therapy is a promising novel therapy regimen for HCC overcoming resistance towards conventional therapy. The independence from apoptosis raises hopes for the treatment of patients whose tumor cells exert defects in this cell death mechanism.