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Cryptococcus gattii is a major cause of life-threatening mycosis in immunocompetent individuals and responsible for the ongoing epidemic outbreak of cryptococcosis in the Pacific Northwest of North America. This deadly fungus is known to evade important host immune responses, including dendritic cell (DC) maturation and concomitant T cell immunity, via immune evasion mechanisms that remain unclear. Here, we demonstrate that primary human DCs phagocytose C. gattii but the maturation of phagosomes to phagolysosomes was blocked as a result of sustained filamentous actin (F-actin) that entrapped and concealed the phagosomes from recognition. Superresolution structured illumination microscopy (SR-SIM) revealed that the persistent phagosomal F-actin formed a cage-like structure that sterically hindered and functionally blocked the fusion of lysosomes. Blocking lysosome fusion was sufficient to inhibit phagosomal acidification and subsequent intracellular fungal killing by DCs. Retention of phagosomal F-actin by C. gattii also caused DC immunoparalysis. Disrupting the retained F-actin cage with cytochalasin D not only restored DC phagosomal maturation but also promoted DC costimulatory maturation and robust T cell activation and proliferation. Collectively, these results reveal a unique mechanism of DC immune evasion that enhances intracellular fungal pathogenicity and may explain suppressed cell-mediated immunity. IMPORTANCE Cryptococcus yeast species typically display characteristics of opportunistic pathogens, with the exception of C. gattii , which can cause life-threatening respiratory and disseminated brain infections in otherwise healthy people. The pathogenesis of C. gattii is not well understood, but an important characteristic is that C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. Here, we report that when virulent C. gattii becomes ingested by a DC, the intracellular compartment containing the fungi is covered by a persistent protein cage structure consisting of F-actin. This F-actin cage acts as a barrier to prevent interaction with other intracellular compartments, and as a result, the DC fails to kill the fungi and activate important cell-mediated immune responses. We propose that this unique immune evasion mechanism permits C. gattii to remain unchallenged within host cells, leading to persistent infection.

Population leakage outside the qubit subspace presents a particularly harmful source of error that cannot be handled by standard error correction methods. Using a trapped ^{171}Yb^{+} ion, we demonstrate an optical pumping scheme to suppress leakage errors in atomic hyperfine qubits. The selection rules and narrow linewidth of a quadrupole transition are used to selectively pump population out of leakage states and back into the qubit subspace. Each pumping cycle reduces the leakage population by a factor of ∼3, allowing for an exponential suppression in the number of cycles. We use interleaved randomized benchmarking on the qubit subspace to show that this pumping procedure has negligible side effects on the qubit subspace, bounding the induced qubit memory error by ≤2.0(8)×10^{-5}  per cycle, and qubit population decay to ≤1.4(3)×10^{-7}  per cycle. These results clear a major obstacle for implementations of quantum error correction and error mitigation protocols.

Burkholderia cepacia complex (Bcc), which includes B. cenocepacia and B. multivorans, pose a life-threatening risk to patients with cystic fibrosis. Eradication of Bcc is difficult due to the high level of intrinsic resistance to antibiotics, and failure of many innate immune cells to control the infection. Because of the pathogenesis of Bcc infections, we wondered if a novel mechanism of microbial host defense involving direct antibacterial activity by natural killer (NK) cells might play a role in the control of Bcc. We demonstrate that NK cells bound Burkholderia, resulting in Src family kinase activation as measured by protein tyrosine phosphorylation, granule release of effector proteins such as perforin and contact-dependent killing of the bacteria. These studies provide a means by which NK cells could play a role in host defense against Bcc infection., (© The Japanese Society for Immunology. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Although often presented as an essential, ahistorical or innate psychological entity, the notion of a 'scientific mind' is ripe for historical analysis. The growing historical interest in the self-fashioning of masculine identities, and more particularly the self-fashioning of the nineteenth-century scientist, has opened up a space in which to probe what was understood by someone being said to possess a 'scientific mind'. This task is made all the more urgent by the recently revived interest of some psychologists in the concept and the highly gendered and culturally conditioned understanding of the scientific mind displayed in some contemporary debates. This article contributes to that task, and fills a rare gap in Darwin studies by making the first detailed exploration of Charles Darwin's understanding of the scientific mind, as revealed in the psychological self-analysis he undertook in his 'Recollections of the development of my mind and character' (1876), and supplemented in his Life of Erasmus Darwin (1879). Drawing upon a broad range of Darwin's published and unpublished works, this article argues that Darwin's understanding of the scientific mind was rooted in his earliest notebooks, and was far more central to his thought than is usually acknowledged. The article further delineates the differences between Darwin's understanding and that of his half-cousin Francis Galton, situates his understanding in relation to his reading of William Whewell and Auguste Comte, and considers what Darwin's view of the scientific mind tells us about his perspective on questions of religion and gender. Throughout, the article seeks to show that the 'scientific mind' is always an agglomeration of historically specific prejudices and presumptions, and concludes that this study of Darwin points to the need for a similarly historical approach to the question of the scientific mind today.

Cryptococcus neoformans is a fungal pathogen that causes fatal meningitis and pneumonia. During host defense to Cryptococcus , NK cells directly recognize and kill C. neoformans using cytolytic degranulation analogous to killing of tumor cells. This fungal killing requires independent activation of Src family kinase (SFK) and Rac1-mediated pathways. Recognition of C. neoformans requires the natural cytotoxicity receptor, NKp30; however, it is not known whether NKp30 activates both signal transduction pathways or whether a second receptor is involved in activation of one of the pathways. We used primary human NK cells and a human NK cell line and found that NKp30 activates SFK → PI3K but not Rac1 cytotoxic signaling, which led to a search for the receptor leading to Rac1 activation. We found that NK cells require integrin-linked kinase (ILK) to activate Rac1 for effective fungal killing. This observation led to our identification of β1 integrin as an essential anticryptococcal receptor. These findings demonstrate that multiple receptors, including β1 integrins and NKp30 and their proximal signaling pathways, are required for recognition of Cryptococcus , which activates a central cytolytic antimicrobial pathway leading to fungal killing., (Copyright © 2018 by The American Association of Immunologists, Inc.)

Cryptococcus is the most important cause of fungal meningitis in immunocompromised individuals. Host defense against Cryptococcus involves direct killing by NK cells. That NK cells from HIV-infected patients fail to polarize perforin to the microbial synapse and kill C. neoformans led us to explore the mechanisms used to reposition and polarize the cytolytic granules to the synapse. Using live-cell imaging, we observed microtubule and granule movements in response to Cryptococcus that revealed a kinesin-dependent event. Eg5-kinesin bound to perforin-containing granules and was required for association with the microtubules. Inhibition of Eg5-kinesin abrogated dynein-dependent granule convergence to the MTOC and granule and MTOC polarization to the synapse and suppressed NK cell killing of Cryptococcus. In contrast, Eg5-kinesin was dispensable for tumor killing. This reveals an alternative mechanism of MTOC repositioning and granule polarization, not used in tumor cytotoxicity, in which Eg5-kinesin is required to initiate granule movement, leading to microbial killing., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cryptococcus gattii is an emerging fungal pathogen on the west coast of Canada and the United States that causes a potentially fatal infection in otherwise healthy individuals. In previous investigations of the mechanisms by which C. gattii might subvert cell-mediated immunity, we found that C. gattii failed to induce dendritic cell (DC) maturation, leading to defective T cell responses. However, the virulence factor and the mechanisms of evasion of DC maturation remain unknown. The cryptococcal polysaccharide capsule is a leading candidate because of its antiphagocytic properties. Consequently, we asked if the capsule of C. gattii was involved in evasion of DC maturation. We constructed an acapsular strain of C. gattii through CAP59 gene deletion by homologous integration. Encapsulated C. gattii failed to induce human monocyte-derived DC maturation and T cell proliferation, whereas the acapsular mutant induced both processes. Surprisingly, encapsulation impaired DC maturation independent of its effect on phagocytosis. Indeed, DC maturation required extracellular receptor signaling that was dependent on TNF-α and p38 MAPK, but not ERK activation, and the cryptococcal capsule blocked this extracellular recognition. Although the capsule impaired phagocytosis that led to pH-dependent serine-, threonine-, and cysteine-sensitive protease-dependent Ag processing, it was insufficient to impair T cell responses. In summary, C. gattii affects two independent processes, leading to DC maturation and Ag processing. The polysaccharide capsule masked extracellular detection and reduced phagocytosis that was required for DC maturation and Ag processing, respectively. However, the T cell response was fully restored by inducing DC maturation., (Copyright © 2016 by The American Association of Immunologists, Inc.)

NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses., (© 2016 S. Karger AG, Basel.)

Undifferentiated spindle cell sarcoma (UDS) is a poorly defined or understood entity, essentially a waste-basket for cases failing to fulfill criteria for better-established diagnoses based on combined histology, immunohistochemistry, and tumor genetic assays. We identified a novel chromosomal translocation t(17;19)(p13;q13) in a pediatric UDS and have characterized this alteration to show rearrangement of the MLL4 and GPS2 genes, resulting in an in-frame fusion gene MLL4-GPS2, the expression of which promotes anchorage-independent growth. MLL4 was previously reported to be similarly rearranged in hepatocellular carcinomas, notably those positive for hepatitis B virus. Isolated reports of individual rearrangements of GPS2 in a prostate carcinoma cell line and in glioblastoma multiforme, each with different partner genes, recently emerged from high-throughput sequencing studies but have not been further evaluated for biological effect., (© 2014 Wiley Periodicals, Inc.)

The Amur leopard is at the point of extinction. At present there are fewer than 35 in the wild. Their natural habitat ranges from China to the North Korean peninsula to Primorsky Krai in Russia. A reintroduction plan has been proposed to increase the population in the wild; however, this proposed plan still has many questions to be answered as to how effective it will be. The main objective is to reintroduce animals from a select group within the Far Eastern leopard programme or the Species Survival programme, which consist of leopards from select populations in the Northern Hemisphere. Zoos are central to the success of this plan, providing suitable breeding pairs to breed animals for reintroduction and also raising much needed funds to finance the project. Zoos are also central in educating the public about the critical status of the Amur leopard and other endangered animals of the world. Veterinary surgeons, by the very nature of their professional skills, are at the forefront of this seemingly endless battle against extinction of thousands of species that are critical to maintaining the balance of our fragile ecosystem. Veterinarians can analyze the health risks and health implications of reintroduction on the animals to be reintroduced and also on the native population. A world without large cats is a world hard to imagine. If we look closer at the implications of extinction, we see the domino effect of their loss and an ecosystem out of control., (© 2013 Published by Elsevier Inc.)

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase-extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.

During adaptive immunity to pathogens, dendritic cells (DCs) capture, kill, process, and present microbial Ags to T cells. Ag presentation is accompanied by DC maturation driven by appropriate costimulatory signals. However, current understanding of the intricate regulation of these processes remains limited. Cryptococcus gattii, an emerging fungal pathogen in the Pacific Northwest of Canada and the United States, fails to stimulate an effective immune response in otherwise healthy hosts leading to morbidity or death. Because immunity to fungal pathogens requires intact cell-mediated immunity initiated by DCs, we asked whether C. gattii causes dysregulation of DC functions. C. gattii was efficiently bound and internalized by human monocyte-derived DCs, trafficked to late phagolysosomes, and killed. Yet, even with this degree of DC activation, the organism evaded pathways leading to DC maturation. Despite the ability to recognize and kill C. gattii, immature DCs failed to mature; there was no increased expression of MHC class II, CD86, CD83, CD80, and CCR7, or decrease of CD11c and CD32, which resulted in suboptimal T cell responses. Remarkably, no increase in TNF-α was observed in the presence of C. gattii. However, addition of recombinant TNF-α or stimulation that led to TNF-α production restored DC maturation and restored T cell responses. Thus, despite early killing, C. gattii evades DC maturation, providing a potential explanation for its ability to infect immunocompetent individuals. We have also established that DCs retain the ability to recognize and kill C. gattii without triggering TNF-α, suggesting independent or divergent activation pathways among essential DC functions.

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.

Clear cell sarcoma of kidney (CCSK) is classified as a tumour of unfavourable histology by the National Wilms' Tumor Study Group. It has worse clinical outcomes than Wilms' tumour. Virtually nothing is known about CCSK biology, as there have been very few genetic aberrations identified to act as pointers in this cancer. Three cases of CCSK bearing a chromosomal translocation, t(10;17)(q22;p13), have been individually reported but not further investigated to date. The aim of this research was to characterize t(10;17)(q22;p13) in CCSK to identify the genes involved in the translocation breakpoints. Using fluorescently labelled bacterial artificial chromosomes (BACs) and a chromosome-walking strategy on an index case of CCSK with t(10;17)(q22;p13) by karyotype, we identified the chromosomal breakpoints on 17p13.3 and 10q22.3. The translocation results in rearrangement of YWHAE on chromosome 17 and FAM22 on chromosome 10, producing an in-frame fusion transcript of ∼3 kb, incorporating exons 1-5 of YWHAE and exons 2-7 of FAM22, as determined by RT-PCR using YWHAE- and FAM22-specific primers. The YWHAE-FAM22 transcript was detected in six of 50 further CCSKs tested, therefore showing an overall incidence of 12% in our cohort. No transcript-positive cases presented with stage I disease, despite this being the stage for 31% of our cohort. Tumour cellularity was significantly higher in the cases that were transcript-positive. Based on the chromosome 10 breakpoint identified by FISH and the sequences of the full-length transcripts obtained, the FAM22 members involved in the translocation in these CCSK cases include FAM22B and FAM22E. Elucidation of the role of YWHAE-FAM22 in CCSK will assist development of more efficient and targeted therapies for this childhood cancer, which currently has poor outcomes., (Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Ectomesenchymoma is a rare mesenchymal malignancy occurring mainly in the pediatric population. The hallmark diagnostic features are a combination of sarcoma, usually rhabdomyosarcoma (RMS) with admixed ganglion cells. The lesion arises either in soft tissues or the cranial cavity, and outcomes vary considerably. Current knowledge about the genetics and biology of ectomesenchymoma is extremely limited with only 4 published karyotypes, showing overlaps only in trisomies 2, 8, and 11. Here, we describe a case with genetic findings that, in conjunction with preexisting observations, offer some additional insights into the genetic aberrations of ectomesenchymoma., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Nonribosomal peptide synthesis (NRPS) is a documented virulence factor for the opportunistic pathogen Aspergillus fumigatus and other fungi. Secreted or intracellularly located NRP products include the toxic molecule gliotoxin and the iron-chelating siderophores triacetylfusarinine C and ferricrocin. No structural or immunologically relevant NRP products have been identified in the organism. We investigated the function of the largest gene in A. fumigatus, which encodes the NRP synthetase Pes3 (AFUA&#95;5G12730), by targeted gene deletion and extensive phenotypic analysis. It was observed that in contrast to other NRP synthetases, deletion of pes3 significantly increases the virulence of A. fumigatus, whereby the pes3 deletion strain (A. fumigatus Δpes3) exhibited heightened virulence (increased killing) in invertebrate (P < 0.001) and increased fungal burden (P = 0.008) in a corticosteroid model of murine pulmonary aspergillosis. Complementation restored the wild-type phenotype in the invertebrate model. Deletion of pes3 also resulted in increased susceptibility to the antifungal, voriconazole (P < 0.01), shorter germlings, and significantly reduced surface β-glucan (P = 0.0325). Extensive metabolite profiling revealed that Pes3 does not produce a secreted or intracellularly stored NRP in A. fumigatus. Macrophage infections and histological analysis of infected murine tissue indicate that Δpes3 heightened virulence appears to be mediated by aberrant innate immune recognition of the fungus. Proteome alterations in A. fumigatus Δpes3 strongly suggest impaired germination capacity. Uniquely, our data strongly indicate a structural role for the Pes3-encoded NRP, a finding that appears to be novel for an NRP synthetase.

Recognition of TLR agonists involves a complex interplay among a variety of serum and cell membrane molecules, including mCD14 and sCD14 that is not fully understood. TLR activation results in downstream signaling that induces inflammatory cytokine production in response to pathogenic molecules, such as ExoS, which is a TLR2 and TLR4 agonist produced by the opportunistic pathogen Pseudomonas aeruginosa. We reasoned that responses to ExoS, a protein, might differ from canonical TLR agonists such as LPS. Stimulating the expression of mCD14 with vitamin D3 enhanced the response to ExoS and LPS. Also, blocking anti-CD14 antibody or removing mCD14 using PLC reduced responses to ExoS and LPS. Furthermore, CD14-deficient cells were unable to bind and respond to ExoS, which was restored by stable transfection of mCD14, indicating that mCD14 was required for the response to ExoS. However, addition of sCD14 to culture enhanced responsiveness to LPS but not ExoS. Moreover, the addition of serum did not alter the response to ExoS but enhanced the response to LPS. Despite differences of adaptor molecule use between ExoS and LPS, lipid antagonists that compete for LPS binding to CD14 also inhibited the response to ExoS. These results highlight a fundamental difference between TLR agonists in their requirements for CD14 and serum components. These results suggest that understanding the dissimilarities and targeting overlapping sites of interaction on CD14 may yield a synergistic, clinical benefit during infections where a variety of TLR agonists are present.

Osteosarcoma is a genetically complex malignancy, predominantly afflicting the adolescent population and associated still with relatively poor long-term outcomes. Although there has been some improvement in the understanding of osteosarcoma biology, this has not yet translated particularly well into therapeutic advances. By using a whole-genome tiling path array for comparative genomic hybridization analysis, we sought to evaluate DNA copy number changes in 22 osteosarcoma tumor samples. Regions of most frequent gains or losses generated by Genomic Identification of Significant Targets in Cancer analysis were evaluated for genes of interest. Correlation of the copy number data with preexisting expression data for these genes yielded not only targets known to be important in osteosarcoma but also novel targets, notably cyclin E1. Fluorescence in situ hybridization and immunohistochemical analysis confirmed the findings. Overexpression of cyclin E1 has potential prognostic and therapeutic implications that are discussed herein., (Copyright © 2011 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

No data exist on the ability of thiolation domains from fungal non-ribosomal peptide synthetases to undergo 4'-phosphopantetheinylation, using either biotinylated or fluorescently labeled coenzyme A analogues, mediated by 4'-phosphopantetheinyl transferases (PPTase). Yet, this is a key requirement to confirm the amino acid recognition function, and coding potential, of either non-ribosomal peptide synthetases or recombinantly expressed regions of these enzymes (e.g., didomains or modules). Moreover, determination of 4'-phosphopantetheinylation activity remains cumbersome. Here, we demonstrate that a recombinant fungal PPTase catalyzes the solution-phase transfer of either biotin- or fluorescein-labeled 4'-phosphopantetheine region of coenzyme A to a fungal thiolation domain, which is either part of a non-ribosomal peptide synthetase didomain (72 kDa), derived from Aspergillus fumigatus, or fused to a non-native protein (glutathione s-transferase). Significantly, we demonstrate that this reaction can unexpectedly occur when the target protein (4.4 pmol) is immobilized on a solid surface. These findings (i) confirm that thiolation domains of fungal origin, in native or non-native configuration, can accept modified 4'-phosphopantetheine residues via PPTase-mediated labeling and (ii) illustrate a novel, high-throughput method to determine PPTase activity.

Patients with cystic fibrosis suffer recurrent pulmonary infections that are characterized by an overactive yet ineffective and destructive inflammatory response that is associated with respiratory infections by Pseudomonas aeruginosa, a pathogen that produces a number of phlogistic molecules. To better understand this process, we used exoenzyme S (ExoS), one of the key P. aeruginosa-secreted exoproducts, which is known to stimulate cells via the Toll-like receptor (TLR) pathway. We found that ExoS induced proinflammatory cytokine production via the NF-kappaB, Erk1/2, and Src kinase pathways. Because Src kinases are concentrated within cholesterol-containing, detergent-resistant membrane microdomains (DRM) (also called lipid rafts) and DRM act as signaling platforms and amplifiers on the surface of cells, we addressed the role of DRM in ExoS signaling. ExoS bound directly to a subset of DRM and induced the phosphorylation of multiple proteins within DRM, including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-kappaB and Erk 1/2 activation and TNF-alpha production in response to ExoS. Activation of monocytic cells by other TLR and Nod-like receptor agonists, such as lipoteichoic acid, lipopolysaccharide, and peptidoglycan, were also dependent on DRM, and disruption prevented TNF-alpha production. Disruption of DRM did not prevent ExoS binding but did release the Src kinase, Lyn, from the DRM fraction into the detergent-soluble fraction, a site in which Src kinases are not active. These studies show that ExoS, a TLR agonist, requires direct binding to DRM for optimal signaling, which suggests that DRM are possible therapeutic targets in cystic fibrosis.

Background: Despite extensive research with families raising children with or at risk for developmental delay (DD), it is not clear whether primary caregivers of these children are at increased risk for depressive symptoms. Discrepant findings in the literature may be owing to heterogeneity of child problems. More research is needed on child, parent and family variables that may increase risk for, or resilience to, caregiver depressive symptoms. Some studies have found that parental resources (e.g. social support and coping strategies) may buffer the effects of parental distress, while other studies have highlighted the role of parental self-efficacy., Methods: We examined Beck Depression Inventory (BDI) scores in 178 primary caregivers (mainly biological mothers) who had 2-year-old children with or at risk for DD owing to: (a) low birthweight, prematurity or multiple birth (n = 58), (b) other known reasons (e.g. Down syndrome, spina bifida) (n = 67), or (c) unknown reasons (n = 69)., Results: We found that 20% (n = 35) of the caregivers scored above the BDI clinical cut-off for depression. Analysis of variance revealed that caregivers with elevated BDI scores had higher child behaviour problem and escape-avoidance coping scores, and lower social support and parent self-efficacy, compared with caregivers without depressive symptoms. Caregivers with children who had DD for unknown reasons had higher BDI scores than caregivers of the other two groups of children. Regression analyses showed that child behaviour problems, escape-avoidance coping strategies and social support predicted caregiver BDI scores, but caregiver self-efficacy only did so when entered independently of social support. Only social support mediated and (marginally) moderated the relationship between child behaviour problems and caregiver depressive symptoms., Conclusions: These findings suggest that early intervention programmes should carefully consider the interaction of child characteristics (e.g. Diagnosis and behaviour problems), caregiver resources (e.g. coping strategies and social support), and parental mental health and mood when planning and tailoring services for families of children with or at risk for DD.

In fungi, nonribosomal peptide synthetases (NRP synthetases) are large multi-functional enzymes containing adenylation, thiolation (or peptidyl carrier protein, PCP) and condensation domains. These enzymes are often encoded within gene clusters. Multiple NRP synthetase ORFs have also been identified in fungi (14 in Aspergillus fumigatus). LeaA, a methyltransferase, is involved in secondary metabolite gene cluster regulation in Aspergillus spp. The NRP synthetases GliP and FtmA respectively direct the biosynthesis of the toxic metabolites gliotoxin and brevianamide F, a precursor of bioactive prenylated alkaloids. The NRP synthetase Pes1 has been shown to mediate resistance to oxidative stress, and in plant-pathogenic ascomycetes (e.g. Cochliobolus heterostrophus) an NRP synthetase, encoded by the NPS6 gene, significantly contributes to virulence and resistance to oxidative stress. Adenylation (A) domains within NRP synthetases govern the specificity of amino acid incorporation into nonribosomally synthesized peptides. To date there have only been limited demonstrations of A domain specificity (e.g. A. fumigatus GliP and in Beauveria bassiana) in fungi. Indeed, only in silico prediction data are available on A domain specificity of NRP synthetases from most fungi. NRP synthetases are activated by 4'-phosphopantetheinylation of serine residues within PCP domains by 4'-phosphopantetheinyl transferases (4'-PPTases). Coenzyme A acts as the 4'-phosphopantetheine donor, and labelled coenzyme A can be used to affinity-label apo-NRP synthetases. Emerging fungal gene disruption and gene cluster expression strategies, allied to proteomic strategies, are poised to facilitate a greater understanding of the coding potential of NRP synthetases in fungi.

Some bacterial products possess multiple immunomodulatory effects and thereby complex mechanisms of action. Exogenous administration of an important Pseudomonas aeruginosa virulence factor, exoenzyme S (ExoS) induces potent monocyte activation leading to the production of numerous proinflammatory cytokines and chemokines. However, ExoS is also injected directly into target cells, inducing cell death through its multiple effects on signaling pathways. This study addresses the mechanisms used by ExoS to induce monocyte activation. Exogenous administration resulted in specific internalization of ExoS via an actin-dependent mechanism. However, ExoS-mediated cellular activation was not inhibited if internalization was blocked, suggesting an alternate mechanism of activation. ExoS bound a saturable and specific receptor on the surface of monocytic cells. ExoS, LPS, and peptidoglycan were all able to induce tolerance and cross-tolerance to each other suggesting the involvement of a TLR in ExoS-recognition. ExoS activated monocytic cells via a myeloid differentiation Ag-88 pathway, using both TLR2 and the TLR4/MD-2/CD14 complex for cellular activation. Interestingly, the TLR2 activity was localized to the C-terminal domain of ExoS while the TLR4 activity was localized to the N-terminal domain. This study provides the first example of how different domains of the same molecule activate two TLRs, and also highlights the possible overlapping pathophysiological processes possessed by microbial toxins.

Unlabelled: Intrathecal (IT) morphine provides excellent postoperative analgesia but may result in many side effects, including postoperative nausea and vomiting, pruritus, and respiratory depression, particularly at larger doses. Older patients may be at particular risk. The optimal dose of spinal morphine in older patients undergoing hip arthroplasty is not known. We designed this prospective, randomized, controlled, double-blinded study to evaluate the analgesic efficacy and side effect profile of 50-200 microg of IT morphine in older patients undergoing elective hip arthroplasty. Sixty patients older than 65 years undergoing elective hip arthroplasty were enrolled. Patients were randomized to receive spinal anesthesia with 15 mg of bupivacaine and IT morphine in four groups: 1). 0 microg, 2). 50 microg, 3). 100 microg, and 4). 200 microg. IT morphine 100 and 200 microg produced effective pain relief and decreased the postoperative requirement for morphine compared with control. IT morphine 50 microg did not provide effective pain relief. Both 100 and 200 microg of IT morphine provided comparable levels of postoperative analgesia. There were no between-group differences in postoperative nausea and vomiting, sedation, respiratory depression, or urinary retention. Pruritus was significantly more frequent with 200 microg of IT morphine. In conclusion, 100 microg of IT morphine provided the best balance between analgesic efficacy and side effect profile in older patients undergoing hip arthroplasty., Implications: The dosage of intrathecal morphine that provides the best balance between analgesic efficacy and side effect profile in the older patient undergoing hip arthroplasty is not known. This prospective, randomized, controlled, double-blinded clinical trial demonstrates that a dose of 100 microg of intrathecal morphine provides the best balance between efficacy and side effects, compared with doses of 0, 50, and 200 microg of morphine, in this patient population.

Study Objectives: To compare the prophylactic antiemetic efficacy of the combination of ondansetron and droperidol with that of droperidol alone in patients undergoing elective laparoscopic cholecystectomy., Design: Randomized, double-blind controlled trial. University affiliated teaching hospital after induction of standardized general anesthesia., Patients: 64 ASA physical status I or II patients aged 18 to 80 years, undergoing elective laparoscopic cholecystectomy., Intervention: Following induction of general anesthesia, patients received either droperidol 1.25 mg intravenously (IV; n = 30; Group D) or the combination of droperidol 1.25 mg IV and ondansetron 4 mg IV (n = 34; Group D+O)., Measurements: Number and severity of nausea episodes, number of emetic episodes, total analgesic consumption, and rescue antiemetic administration were assessed at 1, 3, and 24 hours after admission to the recovery room. Data were analyzed using Fisher's Exact test and unpaired Student's t-test; a p-value <0.05 was considered significant., Results: The proportions of patients who experienced nausea (70% and 53% for D and D+O groups, respectively) and vomiting (30% and 19% for D and D+O groups, respectively) were similar in the two groups. The frequency of moderate and severe nausea (requiring administration of antiemetic) was less in group D + O (7%) compared with group D (19%; p < 0.05)., Conclusions: Patients who received the combination of droperidol and ondansetron experienced less severe nausea compared with patients who received droperidol alone., (Copyright 2002 by Elsevier Science Inc.)

Molecular dosimetry of depurinating DNA adducts of benzo[alpha]pyrene (BP) is a promising new approach to measurement of cancer risk associated with exposure to polycyclic aromatic hydrocarbons (PAH). Depurinating adducts of BP are spontaneously released from DNA and can be detected in urine. As a first step toward developing a monoclonal antibody (MAb)-based molecular dosimetry for depurinating DNA adducts of BP, a MAb (MAb CB53) has been produced with high specific affinity for 7-(benzo[alpha]pyren-6-yl)guanine (BP-6-N7Gua), a major depurinating adduct of BP. Production of this MAb was dependent on the successful synthesis of an effective immunogen consisting of the hydrophobic BP-6-N7Gua coupled to carrier protein via a rigid spacer arm. A competitive enzyme-linked immunosorbent assay (ELISA) for BP-6-N7Gua has been developed with MAb CB53 and has been applied to evaluation of MAb binding and to quantitation of BP-6-N7Gua in a biological sample. The MAb binds with high affinity to BP-6-N7Gua (Ka = 1.4 x 10(8) M-1) and to BP-6-N7Ade (Ka = 0.7 x 10(8) M-1), another major depurinating DNA adduct of BP, but discriminates well between BP and BP-6-N7Gua. BP-6-N7Gua produces 50% inhibition at 750 fmol in the competitive ELISA, whereas BP produces 50% inhibition at 960 000 fmol. Binding affinities to selected PAH, BP-DNA adducts, and BP metabolites indicate significant contributions of the hydrophobic region C-3, C-4, and C-5 of BP and the polar oxygen of guanine to MAb/adduct binding. In a preliminary test of the utility of the competitive ELISA for quantitation of BP-6-N7Gua in urine samples, the assay (sensitivity: 200 fmol per well) produced an accurate determination of the adduct added to normal human urine.