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Background: Lymphatic imaging is becoming increasingly important in the management of patients with congenital heart disease. However, the influence of the intravenous contrast agent ferumoxytol on lymphatic imaging is not well understood., Objective: To evaluate the impact of intravenous ferumoxytol on T1-weighted and T2-weighted lymphatic imaging in patients with congenital heart disease., Materials and Methods: We included consecutive patients receiving ferumoxytol-enhanced 3D angiography for congenital heart disease evaluation. The visibility of the thoracic duct was reviewed on the T1-weighted 3D inversion recovery balanced-steady-state free precession (SSFP) with respiratory navigator gating sequence which is routinely used for angiography and the heavily T2-weighted turbo spin echo sequence which is employed for lymphatic imaging. Data on demographics and time interval between contrast administration and imaging were collected. Statistical analyses were performed using t-tests for continuous variables and chi-squared tests for categorical variables., Results: One hundred nineteen consecutive patients with a mean age of 12.46 years±7.7 years were included. Of these, 45 cases underwent both T1-weighted and T2-weighted imaging; the other 74 underwent only T1-weighted imaging. Of the 45 patients, 20 had thoracic duct enhancement on T1-weighted imaging; among the 26 sedated, only 2 showed enhancement, while 18 of 19 non-sedated patients showed enhancement (P<0.001), indicating a strong association between sedation and reduced thoracic duct visibility. If T2-weighted imaging was performed after contrast administration, the thoracic duct was not visible on those images. For all 45 cases of visible thoracic duct in the entire cohort, the time from contrast administration to imaging ranged from 8 min up to 75 min., Conclusion: The enhancement of the thoracic lymphatic duct on T1-weighted imaging, coupled with degradation observed on T2-weighted imaging, suggests that intravenously administered ferumoxytol rapidly enters the lymphatic fluid. To prevent T2 shortening from degrading the imaging results, T2-weighted imaging for lymphatic evaluation should be performed prior to the administration of ferumoxytol. Sedation and, by inference, fasting may influence this property and warrant further investigation in future studies., Competing Interests: Declarations. Ethics approval and consent to participate: This was an IRB-approved retrospective observational cohort study with waiver of consent for use of anonymized data. Consent for publication: Not applicable. Conflicts of interest: J. Greer is an employee of Philips Healthcare. All the other authors were not consultants or employees for Philips Healthcare and had control of inclusion of any data and information that might present a conflict of interest for that author., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Parainfluenza virus type 5 (PIV5) can either have a persistent or a lytic phenotype in cultured cells. We have previously shown that the phenotype is determined by the phosphorylation status of the phosphoprotein (P). Single amino acid substitutions at critical residues, including a serine-to-phenylalanine substitution at position 157 on P, result in a switch between persistent and lytic phenotypes. Here, using PIV5 vectors expressing either mCherry or GFP with persistent or lytic phenotypes, we show that in co-infections the persistent phenotype is dominant. Thus, in contrast to the cell death observed with cells infected solely with the lytic variant, in co-infected cells persistence is immediately established and both lytic and persistent genotypes persist. Furthermore, 10-20 % of virus released from dually infected cells contains both genotypes, indicating that PIV5 particles can package more than one genome. Co-infected cells continue to maintain both genotypes/phenotypes during cell passage, as do individual colonies of cells derived from a culture of persistently infected cells. A refinement of our model on how the dynamics of virus selection may occur in vivo is presented.

Purpose: This study aims to assess the efficacy of current measurement strategies for lung sizing and the feasibility of future use of computed tomography (CT)-derived lung volumes to predict a donor-recipient lung size match during bilateral lung transplants., Methods: We reviewed the data of 62 patients who underwent bilateral lung transplantation for interstitial lung disease and/or idiopathic pulmonary fibrosis from 2018 to 2019. Data for recipients was retrieved from the department's transplant database and medical records, and the donor's data was retrieved from the DonorNet. The data included demographic data, lung heights, measured total lung capacity (TLC) from plethysmography for recipients and estimated TLC for donors, clinical data, and CT-derived lung volumes in both pre- and post-transplant recipients. The post-transplant CT-derived lung volume in recipients was used as a surrogate for donor lung CT volumes due to inadequate or poor donor CT data. Computed tomography-derived lung volumes were calculated using thresholding, region growing, and cutting techniques on Computer-Aided Design and Mimics (Materialise NV, Leuven, Belgium) programs. Preoperative CT-derived lung volumes in recipients were compared with the plethysmography TLC, Frustum Model, and donor-predicted TLC. The ratio of the recipient's pre-and postoperative CT-derived volumes, the ratio of preoperative CT-derived lung volume, and donor-estimated TLC were studied to detect a correlation with 1-year outcomes., Results: The recipient preoperative CT-derived volume correlated with the recipient preoperative plethysmography TLC (Pearson correlation coefficient [PCC] of 0.688) and with the recipient Frustum model volume (PCC of 0.593). The recipient postoperative CT-derived volume correlated with the recipient's postoperative plethysmography TLC (PCC of 0.651). There was no statistically significant correlation between recipients' CT-derived pre- or postoperative volume with donor-estimated TLC. The ratio of preoperative CT-derived volume to donor-estimated TLC correlated inversely with the length of ventilation (P value = .0031). The ratio of postoperative CT-derived volume to preoperative CT-derived volume correlated inversely with delayed sternal closure (P = .0039). No statistically significant correlations were found in evaluating outcomes related to lung oversizing in the recipient (defined as a postoperative to preoperative CT-derived lung volume ratio of >1.2)., Conclusions: Generating CT-derived lung volumes is a valid and convenient method for evaluating lung volumes for transplantation in patients with ILD and/or IPF. Donor-estimated TLC should be interpreted carefully. Further studies should derive donor lung volumes from CT scans for a more accurate evaluation of lung size matching., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Purpose: Janus kinase-1 (JAK1) tyrosine kinase mediates signaling from multiple cytokine receptors, including interferon alpha/beta and gamma (IFN-α/β and IFN-γ), which are important for viral and mycobacterial protection respectively. We previously reported autosomal recessive (AR) hypomorphic JAK1 mutations in a patient with recurrent atypical mycobacterial infections and relatively minor viral infections. This study tests the impact of partial JAK1 deficiency on cellular responses to IFNs and pathogen control., Methods: We investigated the role of partial JAK1 deficiency using patient cells and cell models generated with lentiviral vectors expressing shRNA., Results: Partial JAK1 deficiency impairs IFN-γ-dependent responses in multiple cell types including THP-1 macrophages, Epstein-Barr Virus (EBV)-transformed B cells and primary dermal fibroblasts. In THP-1 myeloid cells, partial JAK1 deficiency reduced phagosome acidification and apoptosis and resulted in defective control of mycobacterial infection with enhanced intracellular survival. Although both EBV-B cells and primary dermal fibroblasts with partial JAK1 deficiency demonstrate reduced IFN-α responses, control of viral infection was impaired only in patient EBV-B cells and surprisingly intact in patient primary dermal fibroblasts., Conclusion: Our data suggests that partial JAK1 deficiency predominantly affects susceptibility to mycobacterial infection through impact on the IFN-γ responsive pathway in myeloid cells. Susceptibility to viral infections as a result of reduced IFN-α responses is variable depending on cell type. Description of additional patients with inherited JAK1 deficiency will further clarify the spectrum of bacterial and viral susceptibility in this condition. Our results have broader relevance for anticipating infectious complications from the increasing use of selective JAK1 inhibitors., Competing Interests: SB has received grant support from the European Union, National Institute of Health Research, UCLH and GOSH/ICH Biomedical Research Centers and CSL Behring and personal fees or travel expenses from Immunodeficiency Canada/IAACI, CSL Behring, Baxalta US Inc and Biotest. VD-C has received travel expenses from CSL Behring and Takeda. IH is employed by Healx. J-LC serves on the board for ADMA and Elixiron Immunotherapeutics and serves as a consultant for Celgene, Kymera TX, Pfizer, Nimbus, Sanofi, Vitae Pharmaceuticals Inc. and Asahi Kasei. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Daza-Cajigal, Albuquerque, Young, Ciancanelli, Moulding, Angulo, Jeanne-Julien, Rosain, Minskaia, Casanova, Boisson-Dupuis, Bustamante, Randall, McHugh, Thrasher and Burns.)

Diverse communities of fungi and bacteria in deadwood mediate wood decay. While rates of decomposition vary greatly among woody species and spatially distinct habitats, the relative importance of these factors in structuring microbial communities and whether these shift over time remains largely unknown. We characterized fungal and bacterial diversity within pieces of deadwood that experienced 6.3-98.8% mass loss while decaying in common garden 'rotplots' in a temperate oak-hickory forest in the Ozark Highlands, MO, USA. Communities were isolated from 21 woody species that had been decomposing for 1-5 years in spatially distinct habitats at the landscape scale (top and bottom of watersheds) and within stems (top and bottom of stems). Microbial community structure varied more strongly with wood traits than with spatial locations, mirroring the relative role of these factors on decay rates on the same pieces of wood even after 5 years. Co-occurring fungal and bacterial communities persistently influenced one another independently from their shared environmental conditions. However, the relative influence of wood construction versus spatial locations differed between fungi and bacteria, suggesting that life history characteristics of these clades structure diversity differently across space and time in decomposing wood., (© 2020 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Chronic periodontitis is one of the most common infectious inflammatory diseases worldwide. Current therapeutic options for the disease are only partially and temporarily successful due to periodontal re-emergence of pathogens such as Porphyromonas gingivalis, a keystone bacterium in the oral microbial communities, which elicits a dysbiosis between the microbiota and the host. Previously, we reported a peptide inhibitor of P. gingivalis (SAPP) that specifically targets P. gingivalis and reduces its virulence potential in vitro. Here, we show that SAPP can modulate the ability of P. gingivalis to suppress the host innate immune system. Using a cytokine array analysis, we found that the levels of several cytokines including IL-6, IL-8, and MCP-1 in the culture media of human oral keratinocytes (HOKs) were significantly diminished in the presence of P. gingivalis. Whereas the levels of these cytokines were restored, at least partially, in the culture media of HOKs by SAPP treatment. Furthermore, we also observed in an ex vivo assay that SAPP efficiently inhibited biofilm primed formation by mixed-species oral bacteria, and significantly dampened the abnormally innate immune responses induced by these bacteria. We also demonstrated, using a mouse model, that SAPP could prevent alveolar bone loss induced by P. gingivalis. Our results suggest that SAPP specifically targets P. gingivalis and its associated bacterial communities and could be envisioned as an emerging therapy for periodontitis., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Vernal pools are ephemeral wetlands that provide critical habitat to many listed species. Pesticide fate in vernal pools is poorly understood because of uncertainties in the amount of pesticide entering these ecosystems and their bioavailability throughout cycles of wet and dry periods. The Pesticide Water Calculator (PWC), a model used for the regulation of pesticides in the US, was used to predict surface water and sediment pore water pesticide concentrations in vernal pool habitats. The PWC model (version 1.59) was implemented with deterministic and probabilistic approaches and parameterized for three agricultural vernal pool watersheds located in the San Joaquin River basin in the Central Valley of California. Exposure concentrations for chlorpyrifos, diazinon and malathion were simulated. The deterministic approach used default values and professional judgment to calculate point values of estimated concentrations. In the probabilistic approach, Monte Carlo (MC) simulations were conducted across the full input parameter space with a sensitivity analysis that quantified the parameter contribution to model prediction uncertainty. Partial correlation coefficients were used as the primary sensitivity metric for analyzing model outputs. Conditioned daily sensitivity analysis indicates curve number (CN) and the universal soil loss equation (USLE) parameters as the most important environmental parameters. Therefore, exposure estimation can be improved efficiently by focusing parameterization efforts on these driving processes, and agricultural pesticide inputs in these critical habitats can be reduced by best management practices focused on runoff and sediment reductions., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Whether global change will drive changing forests from net carbon (C) sinks to sources relates to how quickly deadwood decomposes. Because complete wood mineralization takes years, most experiments focus on how traits, environments and decomposer communities interact as wood decay begins. Few experiments last long enough to test whether drivers change with decay rates through time, with unknown consequences for scaling short-term results up to long-term forest ecosystem projections. Using a 7 year experiment that captured complete mineralization among 21 temperate tree species, we demonstrate that trait effects fade with advancing decay. However, wood density and vessel diameter, which may influence permeability, control how decay rates change through time. Denser wood loses mass more slowly at first but more quickly with advancing decay, which resolves ambiguity about the after-life consequences of this key plant functional trait by demonstrating that its effect on decay depends on experiment duration and sampling frequency. Only long-term data and a time-varying model yielded accurate predictions of both mass loss in a concurrent experiment and naturally recruited deadwood structure in a 32-year-old forest plot. Given the importance of forests in the carbon cycle, and the pivotal role for wood decay, accurate ecosystem projections are critical and they require experiments that go beyond enumerating potential mechanisms by identifying the temporal scale for their effects., (© 2019 John Wiley & Sons Ltd.)

Excessive type I interferon (IFNα/β) activity is implicated in a spectrum of human disease, yet its direct role remains to be conclusively proven. We investigated two siblings with severe early-onset autoinflammatory disease and an elevated IFN signature. Whole-exome sequencing revealed a shared homozygous missense Arg148Trp variant in STAT2 , a transcription factor that functions exclusively downstream of innate IFNs. Cells bearing STAT2 R148W in homozygosity (but not heterozygosity) were hypersensitive to IFNα/β, which manifest as prolonged Janus kinase-signal transducers and activators of transcription (STAT) signaling and transcriptional activation. We show that this gain of IFN activity results from the failure of mutant STAT2 R148W to interact with ubiquitin-specific protease 18, a key STAT2-dependent negative regulator of IFNα/β signaling. These observations reveal an essential in vivo function of STAT2 in the regulation of human IFNα/β signaling, providing concrete evidence of the serious pathological consequences of unrestrained IFNα/β activity and supporting efforts to target this pathway therapeutically in IFN-associated disease., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses., Competing Interests: The authors have declared that no competing interests exist.

Porphyromonas gingivalis is a keystone bacterium in the oral microbial communities that elicits a dysbiosis between the microbiota and the host. Therefore, inhibition of this organism in dental plaques has been one of the strategies for preventing and treating chronic periodontitis. We previously identified a Streptococcal ArcA derived Anti-P gingivalils Peptide (SAPP) that in vitro, is capable of repressing the expression of several virulence genes in the organism. This leads to a significant reduction in P gingivalis virulence potential, including its ability to colonize on the surface of Streptococcus gordonii, to invade human oral epithelial cells, and to produce gingipains. In this study, we showed that SAPP had minimal cytotoxicity to human oral keratinocytes and gingival fibroblasts. We observed that SAPP directly bound to the cell surface of P gingivalis, and that alterations in the sequence at the N-terminus of SAPP diminished its abilities to interact with P gingivalis cells and repressed the expression of virulence genes. Most strikingly, we demonstrated using an ex-vivo assay that besides its inhibitory activity against P gingivalis colonization, SAPP could also reduce the levels of several other oral Gram-negative bacteria strongly associated with periodontitis in multispecies biofilms. Our results provide a platform for the development of SAPP-targeted therapeutics against chronic periodontitis., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Unlabelled: We have previously shown that IFIT1 is primarily responsible for the antiviral action of interferon (IFN) alpha/beta against parainfluenza virus type 5 (PIV5), selectively inhibiting the translation of PIV5 mRNAs. Here we report that while PIV2, PIV5, and mumps virus (MuV) are sensitive to IFIT1, nonrubulavirus members of the paramyxoviridae such as PIV3, Sendai virus (SeV), and canine distemper virus (CDV) are resistant. The IFIT1 sensitivity of PIV5 was not rescued by coinfection with an IFIT1-resistant virus (PIV3), demonstrating that PIV3 does not specifically inhibit the antiviral activity of IFIT1 and that the inhibition of PIV5 mRNAs is regulated by cis-acting elements. We developed an in vitro translation system using purified human IFIT1 to further investigate the mechanism of action of IFIT1. While the translations of PIV2, PIV5, and MuV mRNAs were directly inhibited by IFIT1, the translations of PIV3, SeV, and CDV mRNAs were not. Using purified human mRNA-capping enzymes, we show biochemically that efficient inhibition by IFIT1 is dependent upon a 5' guanosine nucleoside cap (which need not be N7 methylated) and that this sensitivity is partly abrogated by 2'O methylation of the cap 1 ribose. Intriguingly, PIV5 M mRNA, in contrast to NP mRNA, remained sensitive to inhibition by IFIT1 following in vitro 2'O methylation, suggesting that other structural features of mRNAs may influence their sensitivity to IFIT1. Thus, surprisingly, the viral polymerases (which have 2'-O-methyltransferase activity) of rubulaviruses do not protect these viruses from inhibition by IFIT1. Possible biological consequences of this are discussed., Importance: Paramyxoviruses cause a wide variety of diseases, and yet most of their genes encode structural proteins and proteins involved in their replication cycle. Thus, the amount of genetic information that determines the type of disease that paramyxoviruses cause is relatively small. One factor that will influence disease outcomes is how they interact with innate host cell defenses, including the interferon (IFN) system. Here we show that different paramyxoviruses interact in distinct ways with cells in a preexisting IFN-induced antiviral state. Strikingly, all the rubulaviruses tested were sensitive to the antiviral action of ISG56/IFIT1, while all the other paramyxoviruses tested were resistant. We developed novel in vitro biochemical assays to investigate the mechanism of action of IFIT1, demonstrating that the mRNAs of rubulaviruses can be directly inhibited by IFIT1 and that this is at least partially because their mRNAs are not correctly methylated., (Copyright © 2016 Young et al.)

We have sequenced the parainfluenza virus 5 strain that persistently infects the commonly used AGS human cell line without causing cytopathology. This virus is most closely related to human strains, indicating that it may have originated from biopsy material or from laboratory contamination during generation of the cell line., (Copyright © 2016 Wignall-Fleming et al.)

Type I interferon (IFN-α/β) is a fundamental antiviral defense mechanism. Mouse models have been pivotal to understanding the role of IFN-α/β in immunity, although validation of these findings in humans has been limited. We investigated a previously healthy child with fatal encephalitis after inoculation of the live attenuated measles, mumps, and rubella (MMR) vaccine. By targeted resequencing, we identified a homozygous mutation in the high-affinity IFN-α/β receptor (IFNAR2) in the proband, as well as a newborn sibling, that rendered cells unresponsive to IFN-α/β. Reconstitution of the proband's cells with wild-type IFNAR2 restored IFN-α/β responsiveness and control of IFN-attenuated viruses. Despite the severe outcome of systemic live vaccine challenge, the proband had previously shown no evidence of heightened susceptibility to respiratory viral pathogens. The phenotype of IFNAR2 deficiency, together with similar findings in STAT2-deficient patients, supports an essential but narrow role for IFN-α/β in human antiviral immunity., (Copyright © 2015, American Association for the Advancement of Science.)

Unlabelled: The strain diversity of a rubulavirus, parainfluenza virus 5 (PIV5), was investigated by comparing 11 newly determined and 6 previously published genome sequences. These sequences represent 15 PIV5 strains, of which 6 were isolated from humans, 1 was from monkeys, 2 were from pigs, and 6 were from dogs. Strain diversity is remarkably low, regardless of host, year of isolation, or geographical origin; a total of 7.8% of nucleotides are variable, and the average pairwise difference between strains is 2.1%. Variation is distributed unevenly across the PIV5 genome, but no convincing evidence of selection for antibody-mediated evasion in hemagglutinin-neuraminidase was found. The finding that some canine and porcine, but not primate, strains are mutated in the SH gene, and do not produce SH, raised the possibility that dogs (or pigs) may not be the natural host of PIV5. The genetic stability of PIV5 was also demonstrated during serial passage of one strain (W3) in Vero cells at a high multiplicity of infection, under conditions of competition with large proportions of defective interfering genomes. A similar observation was made for a strain W3 mutant (PIV5VΔC) lacking V gene function, in which the dominant changes were related to pseudoreversion in this gene. The mutations detected in PIV5VΔC during pseudoreversion, and also those characterizing the SH gene in canine and porcine strains, predominantly involved U-to-C transitions. This suggests an important role for biased hypermutation via an adenosine deaminase, RNA-specific (ADAR)-like activity., Importance: Here we report the sequence variation of 16 different isolates of parainfluenza virus 5 (PIV5) that were isolated from a number of species, including humans, monkeys, dogs, and pigs, over 4 decades. Surprisingly, strain diversity was remarkably low, regardless of host, year of isolation, or geographical origin. Variation was distributed unevenly across the PIV5 genome, but no convincing evidence of immune or host selection was found. This overall genome stability of PIV5 was also observed when the virus was grown in the laboratory, and the genome stayed remarkably constant even during the selection of virus mutants. Some of the canine isolates had lost their ability to encode one of the viral proteins, termed SH, suggesting that although PIV5 commonly infects dogs, dogs may not be the natural host for PIV5.

Preparations of parainfluenza virus 5 (PIV5) that are potent activators of the interferon (IFN) induction cascade were generated by high-multiplicity passage in order to accumulate defective interfering virus genomes (DIs). Nucleocapsid RNA from these virus preparations was extracted and subjected to deep sequencing. Sequencing data were analyzed using methods designed to detect internal deletion and "copyback" DIs in order to identify and characterize the different DIs present and to approximately quantify the ratio of defective to nondefective genomes. Trailer copybacks dominated the DI populations in IFN-inducing preparations of both the PIV5 wild type (wt) and PIV5-VΔC (a recombinant virus that does not encode a functional V protein). Although the PIV5 V protein is an efficient inhibitor of the IFN induction cascade, we show that nondefective PIV5 wt is unable to prevent activation of the IFN response by coinfecting copyback DIs due to the interfering effects of copyback DIs on nondefective virus protein expression. As a result, copyback DIs are able to very rapidly activate the IFN induction cascade prior to the expression of detectable levels of V protein by coinfecting nondefective virus.

Severe infectious disease in children may be a manifestation of primary immunodeficiency. These genetic disorders represent important experiments of nature with the capacity to elucidate nonredundant mechanisms of human immunity. We hypothesized that a primary defect of innate antiviral immunity was responsible for unusually severe viral illness in two siblings; the proband developed disseminated vaccine strain measles following routine immunization, whereas an infant brother died after a 2-d febrile illness from an unknown viral infection. Patient fibroblasts were indeed abnormally permissive for viral replication in vitro, associated with profound failure of type I IFN signaling and absence of STAT2 protein. Sequencing of genomic DNA and RNA revealed a homozygous mutation in intron 4 of STAT2 that prevented correct splicing in patient cells. Subsequently, other family members were identified with the same genetic lesion. Despite documented infection by known viral pathogens, some of which have been more severe than normal, surviving STAT2-deficient individuals have remained generally healthy, with no obvious defects in their adaptive immunity or developmental abnormalities. These findings imply that type I IFN signaling [through interferon-stimulated gene factor 3 (ISGF3)] is surprisingly not essential for host defense against the majority of common childhood viral infections.

Conflicting reports exist regarding the requirement for virus replication in interferon (IFN) induction by paramyxoviruses. Our previous work has demonstrated that pathogen-associated molecular patterns capable of activating the IFN-induction cascade are not normally generated during virus replication, but are associated instead with the presence of defective interfering (DI) viruses. We demonstrate here that DIs of paramyxoviruses, including parainfluenza virus 5, mumps virus and Sendai virus, can activate the IFN-induction cascade and the IFN-β promoter in the absence of virus protein synthesis. As virus protein synthesis is an absolute requirement for paramyxovirus genome replication, our results indicate that these DI viruses do not require replication to activate the IFN-induction cascade.

It is generally thought that pathogen-associated molecular patterns (PAMPs) responsible for triggering interferon (IFN) induction are produced during virus replication and, to limit the activation of the IFN response by these PAMPs, viruses encode antagonists of IFN induction. Here we have studied the induction of IFN by parainfluenza virus type 5 (PIV5) at the single-cell level, using a cell line expressing GFP under the control of the IFN-β promoter. We demonstrate that a recombinant PIV5 (termed PIV5-VΔC) that lacks a functional V protein (the viral IFN antagonist) does not activate the IFN-β promoter in the majority of infected cells. We conclude that viral PAMPs capable of activating the IFN induction cascade are not produced or exposed during the normal replication cycle of PIV5, and suggest instead that defective viruses are primarily responsible for inducing IFN during PIV5 infection in this system., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The infection of cells by RNA viruses is associated with the recognition of virus PAMPs (pathogen-associated molecular patterns) and the production of type I interferon (IFN). To counter this, most, if not all, RNA viruses encode antagonists of the IFN system. Here we present data on the dynamics of IFN production and response during developing infections by paramyxoviruses, influenza A virus and bunyamwera virus. We show that only a limited number of infected cells are responsible for the production of IFN, and that this heterocellular production is a feature of the infecting virus as opposed to an intrinsic property of the cells., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Although the Enders strain of mumps virus (MuV) encodes a functional V protein that acts as an interferon (IFN) antagonist, in multi-cycle growth assays MuV Enders grew poorly in naïve ('IFN-competent' Hep2) cells but grew to high titres in 'IFN-compromised' Hep2 cells. Even so, the growth rate of MuV Enders was significantly slower in 'IFN-compromised' Hep2 cells when compared with its replication rate in Vero cells and with the replication rate of parainfluenza virus type 5 (a closely related paramyxovirus) in both naïve and 'IFN-compromised' Hep2 cells. This suggests that a consequence of slower growth is that the IFN system of naïve Hep2 cells can respond quickly enough to control the growth of MuV Enders. This is supported by the finding that rapidly growing variants of MuV Enders that were selected on 'IFN-compromised' Hep2 cells (i.e. in the absence of any selection pressure exerted by the IFN response) also grew to high titres on naïve Hep2 cells. Sequencing of the complete genome of one of these variants identified a single point mutation that resulted in a substitution of a conserved asparagine by histidine at position 498 of the haemagglutinin-neuraminidase protein, although this mutation was not present in all rapidly growing variants. These results support the concept that there is a race between the ability of a cell to detect and respond to virus infection and the ability of a virus to block the IFN response. Importantly, this emphasizes that factors other than viral IFN antagonists influence the sensitivity of viruses to IFN.

Although the replication cycle of parainfluenza virus type 5 (PIV5) is initially severely impaired in cells in an interferon (IFN)-induced antiviral state, the virus still targets STAT1 for degradation. As a consequence, the cells can no longer respond to IFN and after 24-48 h, they go out of the antiviral state and normal virus replication is established. Following infection of cells in an IFN-induced antiviral state, viral nucleocapsid proteins are initially localized within small cytoplasmic bodies, and appearance of these cytoplasmic bodies correlates with the loss of STAT1 from infected cells. In situ hybridization, using probes specific for the NP and L genes, demonstrated the presence of virus genomes within these cytoplasmic bodies. These viral cytoplasmic bodies do not co-localize with cellular markers for stress granules, cytoplasmic P-bodies or autophagosomes. Furthermore, they are not large insoluble aggregates of viral proteins and/or nucleocapsids, as they can simply and easily be dispersed by 'cold-shocking' live cells, a process that disrupts the cytoskeleton. Given that during in vivo infections, PIV5 will inevitably infect cells in an IFN-induced antiviral state, we suggest that these cytoplasmic bodies are areas in which PIV5 genomes reside whilst the virus dismantles the antiviral state of the cells. Consequently, viral cytoplasmic bodies may play an important part in the strategy that PIV5 uses to circumvent the IFN system.

Efficient herpes simplex virus type 1 (HSV-1) infection of human fibroblasts (HFs) is highly dependent on the viral immediate-early regulatory protein ICP0 unless the infection is conducted at a high multiplicity. ICP0-null mutant HSV-1 exhibits a plaque-forming defect of up to 3 orders of magnitude in HFs, whereas in many other cell types, this defect varies between 10- and 30-fold. The reasons for the high ICP0 requirement for HSV-1 infection in HFs have not been established definitively. Previous studies using other cell types suggested that ICP0-null mutant HSV-1 is hypersensitive to interferon and that this sensitivity is dependent on the cellular promyelocytic leukemia (PML) protein. To investigate the roles of two important aspects of interferon signaling in the phenotype of ICP0-null mutant HSV-1, we isolated HFs depleted of STAT-1 or interferon regulatory factor 3 (IRF-3). Surprisingly, plaque formation by the mutant virus was not improved in either cell type. We found that the sensitivity to interferon pretreatment of both ICP0-null mutant and wild-type (wt) HSV-1 was highly dependent on the multiplicity of infection. At a low multiplicity in virus yield experiments, both viruses were extremely susceptible to interferon pretreatment of HFs, but the sensitivity of the wild type but not the mutant could be overcome at higher multiplicities. We found that both wt and ICP0-null mutant HSV-1 remained sensitive to interferon in PML-depleted HFs albeit to an apparently lesser extent than in control cells. The data imply that the substantial reduction in ICP0-null HSV-1 infectivity at a low multiplicity in HFs does not occur through the activities of STAT-1- and IRF-3-dependent pathways and cannot be explained solely by enhanced sensitivity to interferon. We suggest that antiviral activities induced by interferon may be separable from and additive to those resulting from PML-related intrinsic resistance mechanisms.

Whilst screening various cell lines for their ability to respond to interferon (IFN), we noted that in comparison to other tissue culture cells AGS tumour cells, which are widely used in biomedical research, had very low levels of STAT1. Subsequent analysis showed that the reason for this is that AGS cells are persistently infected with parainfluenza virus type 5 (PIV5; formally known as SV5), a virus that blocks the interferon (IFN) response by targeting STAT1 for proteasome-mediated degradation. Virus protein expression in AGS is altered in comparison to the normal pattern of virus protein synthesis observed in acutely infected cells, suggesting that the AGS virus is defective. We discuss the relevance of these results in terms of the need to screen cell lines for persistent virus infections that can alter cellular functions.

Paramyxoviruses enter cells by fusing their envelopes with the plasma membrane, a process that occurs at neutral pH. Recently, it has been found that there is an exception to this dogma in that a porcine isolate of the paramyxovirus parainfluenza virus 5 (PIV5), known as SER, requires a low-pH step for fusion (S. Seth, A. Vincent, and R. W. Compans, J. Virol. 77: 6520-6527, 2003). As a low-pH activation mechanism for fusion would greatly facilitate biophysical studies of paramyxovirus-mediated membrane fusion, we have reexamined the triggering of the PIV5 SER fusion protein. Using multiple assays, we could not find a requirement for low-pH triggering of PIV5 SER fusion. The challenge of discovering how the paramyxovirus receptor binding protein (HN, H, or G) activates the metastable fusion protein to cause membrane fusion at neutral pH remains.

Previous work has documented that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation, whilst the V protein of human parainfluenza virus type 2 (hPIV2) targets STAT2. Here, it was shown that the processes of ubiquitination and degradation could be reconstructed in vitro by using programmed rabbit reticulocyte lysates. Using this system, the addition of bacterially expressed and purified SV5 V protein to programmed lysates was demonstrated to result in the polyubiquitination and degradation of in vitro-translated STAT1, but only if human STAT2 was also present. Surprisingly, in the same assay, purified hPIV2 V protein induced the polyubiquitination of both STAT1 and STAT2. In the light of these in vitro results, the specificity of degradation of STAT1 and STAT2 by SV5 and hPIV2 in tissue-culture cells was re-examined. As previously reported, STAT1 could not be detected in human cells that expressed SV5 V protein constitutively, whilst STAT2 could not be detected in human cells that expressed hPIV2 V protein, although the levels of STAT1 may also have been reduced in some human cells infected with hPIV2. In contrast, STAT1 could not be detected, whereas STAT2 remained present, in a variety of animal cells, including canine (MDCK) cells, that expressed the V protein of either SV5 or hPIV2. Thus, the V protein of SV5 appears to be highly specific for STAT1 degradation, but the V protein of hPIV2 is more promiscuous.

Most paramyxoviruses circumvent the IFN response by blocking IFN signaling and limiting the production of IFN by virus-infected cells. Here we report that the highly conserved cysteine-rich C-terminal domain of the V proteins of a wide variety of paramyxoviruses binds melanoma differentiation-associated gene 5 (mda-5) product. mda-5 is an IFN-inducible host cell DExD/H box helicase that contains a caspase recruitment domain at its N terminus. Overexpression of mda-5 stimulated the basal activity of the IFN-beta promoter in reporter gene assays and significantly enhanced the activation of the IFN-beta promoter by intracellular dsRNA. Both these activities were repressed by coexpression of the V proteins of simian virus 5, human parainfluenza virus 2, mumps virus, Sendai virus, and Hendra virus. Similar results to the reporter assays were obtained by measuring IFN production. Inhibition of mda-5 by RNA interference or by dominant interfering forms of mda-5 significantly inhibited the activation of the IFN-beta promoter by dsRNA. It thus appears that mda-5 plays a central role in an intracellular signal transduction pathway that can lead to the activation of the IFN-beta promoter, and that the V proteins of paramyxoviruses interact with mda-5 to block its activity.

Obtaining representative physiological samples for glucose analysis remains a challenge especially when developing less invasive glucose monitoring systems for diabetic patients. In the present study the glucose content of the stratum corneum was compared with the amount of glucose obtained by short aqueous extractions from a site on the dorsal wrist, using high pressure liquid chromatography with pulsed amperometric detection. Ten successive aqueous 1-minute extractions of the site yielded a total of 60 ng cm(-2). The total glucose content of the stratum corneum of the site, determined from 30 successive tape-strippings of the site, was 360 ng cm(-2). After tape-stripping, the transcutaneous aqueous extraction rate was 86 +/- 13 ng cm(-2) min(-1), compared with rates of 80-600 ng cm(-2) min(-1) obtained with suction effusion or microdialysis after tape-stripping. Glucose on the surface of the skin and within the stratum corneum should be considered as sources of extraneous glucose contamination during testing of less invasive glucose monitoring devices.

The V protein of the paramyxovirus simian virus 5 blocks interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. Here we report on the isolation of human cell lines that express the V protein and can no longer respond to IFN. A variety of viruses, particularly slow-growing wild-type viruses and vaccine candidate viruses (which are attenuated due to mutations that affect virus replication, virus spread, or ability to circumvent the IFN response), form bigger plaques and grow to titers that are increased as much as 10- to 4,000-fold in these IFN-nonresponsive cells. We discuss the practical applications of using such cells in vaccine development and manufacture, virus diagnostics and isolation of newly emerging viruses, and studies on host cell tropism and pathogenesis.

The V protein of simian virus 5 (SV5) blocks interferon signaling by targeting STAT1 for proteasome-mediated degradation. Here we present three main pieces of evidence which demonstrate that the p127 subunit (DDB1) of the UV damage-specific DNA binding protein (DDB) plays a central role in this degradation process. First, the V protein of an SV5 mutant which fails to target STAT1 for degradation does not bind DDB1. Second, mutations in the N and C termini of V which abolish the binding of V to DDB1 also prevent V from blocking interferon (IFN) signaling. Third, treatment of HeLa/SV5-V cells, which constitutively express the V protein of SV5 and thus lack STAT1, with short interfering RNAs specific for DDB1 resulted in a reduction in DDB1 levels with a concomitant increase in STAT1 levels and a restoration of IFN signaling. Furthermore, STAT1 is degraded in GM02415 (2RO) cells, which have a mutation in DDB2 (the p48 subunit of DDB) which abolishes its ability to interact with DDB1, thereby demonstrating that the role of DDB1 in STAT1 degradation is independent of its association with DDB2. Evidence is also presented which demonstrates that STAT2 is required for the degradation of STAT1 by SV5. These results suggest that DDB1, STAT1, STAT2, and V may form part of a large multiprotein complex which leads to the targeted degradation of STAT1 by the proteasome.

Key factors and practical limits of blood extraction from lancet wounds on body sites other than the finger were determined by testing a large number of conditions. During these tests, the pain associated with lancing alternate body sites was rated as less painful than a fingerstick 98% of the time. Vacuum combined with skin stretching was effective in extracting an adequate volume of blood from the forearm for glucose testing, up to an average of 16 microl in 30 s. The amount of blood extracted increases with the application of heat or vacuum before lancing, the level of vacuum, the depth of lancing, the time of collection, and the amount of skin stretching. Vacuum and skin stretching led to significant increases, up to fivefold in the perfusion of blood in the skin as measured by laser Doppler. Our observations suggest that vacuum combined with skin stretching increases blood extraction at alternate sites by increasing the lancet wound opening, increasing the blood available for extraction by vasodilatation, and reducing the venous return of blood through capillaries.

Human cell lines were isolated that express the V protein of either simian virus 5 (SV5) or human parainfluenza virus type 2 (hPIV2); the cell lines were termed 2f/SV5-V and 2f/PIV2-V, respectively. STAT1 was not detectable in 2f/SV5-V cells, and the cells failed to signal in response to either alpha/beta interferons (IFN-alpha and IFN-beta, or IFN-alpha/beta) or gamma interferon (IFN-gamma). In contrast, STAT2 was absent from 2f/PIV2-V cells, and IFN-alpha/beta but not IFN-gamma signaling was blocked in these cells. Treatment of both 2f/SV5-V and 2f/PIV2-V cells with a proteasome inhibitor allowed the respective STAT levels to accumulate at rates similar to those seen in 2fTGH cells, indicating that the V proteins target the STATs for proteasomal degradation. Infection with SV5 can lead to a complete loss of both phosphorylated and nonphosphorylated forms of STAT1 by 6 h postinfection. Since the turnover of STAT1 in uninfected cells is longer than 24 h, we conclude that degradation of STAT1 is the main mechanism by which SV5 blocks interferon (IFN) signaling. Pretreatment of 2fTGH cells with IFN-alpha severely inhibited both SV5 and hPIV2 protein synthesis. However, and in marked contrast, pretreatment of 2fTGH cells with IFN-gamma had little obvious effect on SV5 protein synthesis but did significantly reduce the replication of hPIV2. Pretreament with IFN-alpha or IFN-gamma did not induce an antiviral state in 2f/SV5-V cells, indicating either that the induction of an antiviral state is completely dependent on STAT signaling or that the V protein interferes with other, STAT-independent cell signaling pathways that may be induced by IFNs. Even though SV5 blocked IFN signaling, the addition of exogenous IFN-alpha to the culture medium of 2fTGH cells 12 h after a low-multiplicity infection with SV5 significantly reduced the subsequent cell-to-cell spread of virus. The significance of the results in terms of the strategy that these viruses have evolved to circumvent the IFN response is discussed.

Previous work has demonstrated that the V protein of simian virus 5 (SV5) targets STAT1 for proteasome-mediated degradation (thereby blocking interferon [IFN] signaling) in human but not in murine cells. In murine BF cells, SV5 establishes a low-grade persistent infection in which the virus fluxes between active and repressed states in response to local production of IFN. Upon passage of persistently infected BF cells, virus mutants were selected that were better able to replicate in murine cells than the parental W3 strain of SV5 (wild type [wt]). Viruses with mutations in the Pk region of the N-terminal domain of the V protein came to predominate the population of viruses carried in the persistently infected cell cultures. One of these mutant viruses, termed SV5 mci-2, was isolated. Sequence analysis of the V/P gene of SV5 mci-2 revealed two nucleotide differences compared to wt SV5, only one of which resulted in an amino acid substitution (asparagine [N], residue 100, to aspartic acid [D]) in V. Unlike the protein of wt SV5, the V protein of SV5 mci-2 blocked IFN signaling in murine cells. Since the SV5 mci-2 virus had additional mutations in genes other than the V/P gene, a recombinant virus (termed rSV5-V/P N(100)D) was constructed that contained this substitution alone within the wt SV5 backbone to evaluate what effect the asparagine-to-aspartic-acid substitution in V had on the virus phenotype. In contrast to wt SV5, rSV5-V/P N(100)D blocked IFN signaling in murine cells. Furthermore, rSV5-V/P N(100)D virus protein synthesis in BF cells continued for significantly longer periods than that for wt SV5. However, even in cells infected with rSV5-V/P N(100)D, there was a late, but significant, inhibition in virus protein synthesis. Nevertheless, there was an increase in virus yield from BF cells infected with rSV5-V/P N(100)D compared to wt SV5, demonstrating a clear selective advantage to SV5 in being able to block IFN signaling in these cells.

A vacuum-lancet device was applied to the forearm for the purpose of obtaining capillary blood samples for glucose monitoring with minimal pain. In four clinical trials, a total of 215 individuals aged 12-77 years were tested four times using standard conditions and four times with either a different depth of lancing, different brand of lancet or a larger-sized device. The volume of blood collected using one-half atmosphere of vacuum in 40 sec was measured. The sensation and visual appearance of each lancet puncture on the forearm was recorded. Glucose was measured in forearm and in conventional fingerstick blood samples. The distribution of volumes was skewed to higher values with median values for each trial in the range of 3-10 microL. Ninety-five percent of the lancet sticks were judged as less painful than a fingerstick. Redness and bruising around the lanced sites were noted in some patients but disappeared within a few days. Overall correlation of the forearm versus fingerstick glucose values was 0.96. The vacuum-lancet device was very successful in obtaining capillary blood samples for glucose testing in a relatively painless manner. Incorporation of a glucose measuring system into the device might improve testing compliance among those who fear pain or the sight of blood.

STAT1 and STAT2 are cellular transcription factors involved in interferon (IFN) signaling and are thus critical for the IFN-induced antiviral state. We have previously shown that the paramyxovirus Simian Virus 5 (SV5) blocks both type I and type II interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. To determine whether this is a feature common to all Paramyxoviridae, we examined the abilities of SV5, Sendai virus (SeV), human respiratory syncytial virus (RSV), and human parainfluenza viruses types 2 and 3 (hPIV2 and hPIV3, respectively) to block interferon signaling. The results showed that in reporter assays SV5, SeV, and hPIV3 blocked both type I and type II IFN-signaling; hPIV2 blocked type I but not type II IFN-signaling; and RSV failed to block either type I or type II IFN-signaling. In agreement with these results, SV5 and SeV inhibited the formation of the ISGF3 and GAF transcription complexes (essential for type I and type II signaling, respectively). Surprisingly, although hPIV3 inhibited IFN-induction of the ISGF3 complex, GAF complexes were detected in hPIV3-infected cells. hPIV2 also blocked the formation of the ISGF3 complex but not the GAF complex, whereas RSV failed to block the induction of either complex. SV5 was the only virus that caused the degradation of STAT1. Indeed, in SeV- and hPIV3-infected cells STAT1 was phosphorylated on tyrosine 701 (Y701), a characteristic of IFN receptor activation. However, consistent with these viruses blocking IFN signaling downstream of receptor activation, there was a specific reduction in the levels of serine 727 (S727)-phosphorylated forms of STAT1alpha in SeV- and hPIV3-infected cells. In contrast both (Y701)- and (S727)-phosphorylated forms of STAT1 were detected in hPIV2-infected cells but there was a specific loss of STAT2. Both STAT1 (including Y701- and S727-phosphorylated forms) and STAT2 could readily be detected in RSV-infected cells. Despite not being able to block type I or type II IFN signaling, RSV was able to replicate in human cells that produce and respond to IFN, suggesting that RSV must have an alternative method(s) for circumventing the IFN response. These results demonstrate that, although interference with IFN signaling is a common strategy among Paramyxovirinae, distinct virus-specific mechanisms are used to achieve this end., (Copyright 2000 Academic Press.)

To replicate in vivo, viruses must circumvent cellular antiviral defense mechanisms, including those induced by the interferons (IFNs). Here we demonstrate that simian virus 5 (SV5) blocks IFN signalling in human cells by inhibiting the formation of the IFN-stimulated gene factor 3 and gamma-activated factor transcription complexes that are involved in activating IFN-alpha/beta- and IFN-gamma-responsive genes, respectively. SV5 inhibits the formation of these complexes by specifically targeting STAT1, a component common to both transcription complexes, for proteasome-mediated degradation. Expression of the SV5 structural protein V, in the absence of other virus proteins, also inhibited IFN signalling and induced the degradation of STAT1. Following infection with SV5, STAT1 was degraded in the absence of virus protein synthesis and remained undetectable for up to 4 days postinfection. Furthermore, STAT1 was also degraded in IFN-pretreated cells, even though the cells were in an antiviral state. Since pretreatment of cells with IFN delayed but did not prevent virus replication and protein synthesis, these observations suggest that following infection of IFN-pretreated cells, SV5 remains viable within the cells until they eventually go out of the antiviral state.

Sendai virus (SeV) is highly pathogenic for mice. In contrast, mice (including SCID mice) infected with simian virus 5 (SV5) showed no overt signs of disease. Evidence is presented that a major factor which prevented SV5 from productively infecting mice was its inability to circumvent the interferon (IFN) response in mice. Thus, in murine cells that produce and respond to IFN, SV5 protein synthesis was rapidly switched off. In marked contrast, once SeV protein synthesis began, it continued, even if the culture medium was supplemented with alpha/beta IFN (IFN-alpha/beta). However, in human cells, IFN-alpha/beta did not inhibit the replication of either SV5 or SeV once virus protein synthesis was established. To begin to address the molecular basis for these observations, the effects of SeV and SV5 infections on the activation of an IFN-alpha/beta-responsive promoter and on that of the IFN-beta promoter were examined in transient transfection experiments. The results demonstrated that (i) SeV, but not SV5, inhibited an IFN-alpha/beta-responsive promoter in murine cells; (ii) both SV5 and SeV inhibited the activation of an IFN-alpha/beta-responsive promoter in human cells; and (iii) in both human and murine cells, SeV was a strong inducer of the IFN-beta promoter, whereas SV5 was a poor inducer. The ability of SeV and SV5 to inhibit the activation of IFN-responsive genes in human cells was confirmed by RNase protection experiments. The importance of these results in terms of paramyxovirus pathogenesis is discussed.

A method of conjugating poly(glutamic acid) poly(phosphorothioate)-cross-linked alkaline phosphatase to maleimide-derivatized immunoglobulin is described. Intramolecular autocatalyzed cross-linking of alkaline phosphatase at 2:1 to 4:1 polymer:enzyme ratios introduced 32-68 thiolates on the surface of the enzyme. Depending on the stoichiometry of polymer to enzyme, the cross-linked alkaline phosphatase retained 75-90% of its native catalytic activity. The cross-linked thiolate-functionalized alkaline phosphatase was conjugated to maleimide-derivatized immunoglobulin. Compared to a control prepared using non-cross-linked alkaline phosphatase, these conjugates were smaller in size and more stable to heat. The enzymatic activity of the cross-linked conjugates after incubation at 45 degrees C and pH 7.5 for 25 days was 35% higher than those of the highest-activity control conjugates. The conjugation process could be controlled by varying the stoichiometries of poly(glutamic acid) poly(phosphorothioate), alkaline phosphatase, and immunoglobulin.

We report the synthesis of polymeric cross-linking agents, poly(glutamic acid) poly(phosphorothioates), and their use in the cross-linking and stabilization of proteins upon treatment with alkaline phosphatase. We have shown that poly(phosphorothioates) are excellent substrates of alkaline phosphatase, yielding thiolated polymers which react covalently with electrophilic groups introduced into the proteins. Three proteins of different structure and function were cross-linked using this method: calf intestinal alkaline phosphatase, glucose oxidase (Aspergillus niger), and (R)-phycoerythrin. The cross-linking of alkaline phosphatase is self-catalyzed since this enzyme catalyzes the hydrolysis of phosphates, unmasking thiolates which react with the maleimide prederivatized alkaline phosphatase. Incubation of buffered solutions of native alkaline phosphatase at 45 degreesC for 7-14 days resulted in a 35% higher loss of enzymatic activity compared to that of cross-linked enzyme. The effect of cross-linking glucose oxidase is even more notable, ranging from 800% stabilization at 37 degrees C and pH 9.0 to 3000% at 37 degrees C and pH 7.4. (R)-Phycoerythrin cross-linked with 1-3 equiv of poly(phosphorothioates) and incubated at 45 degrees C for 45 days was 20% more fluorescent than the native (R)-phycoerythrin subjected to the same conditions. The stabilizing effect of cross-linking was confirmed by comparing the rate of loss of quaternary structure of the cross-linked (R)-phycoerythrin with that of the native protein.

Seven right-handed participants performed bimanual circling movements in either a symmetrical or an asymmetrical coordination mode. Movements were paced with an auditory metronome at predetermined frequencies corresponding to transition frequency, where asymmetrical patterns became unstable, or at two-thirds transition frequency where both symmetrical and asymmetrical patterns were stable. The pacing tones were presented in either a high (1000 Hz) or low (500 Hz) pitch, and the percentage of high-pitched tones during a 20 s trial varied between 0% and 70%. Participants were instructed to count the number of high-pitched pacing tones that occurred during a trial of bimanual circling. Overall, the symmetrical pattern was more stable than the asymmetrical pattern at both frequencies. Errors on the tone-counting task were significantly higher during asymmetrical circling than symmetrical circling but only at the transition movement frequency. The results suggest that cognitive processes play a role in maintaining coordination patterns within regions of instability.

A series of experiments were undertaken to examine how interferon and neutralizing antibodies influence the ability of simian virus 5 (SV5) (strain W3) to establish and maintain persistent infections in murine cells. In contrast to the rapid decline in SV5 protein synthesis observed in murine BALB/c fibroblasts (BF cells), which produce and respond to interferon, between 24 and 48 h postinfection there was no inhibition of virus protein synthesis in MSFI- cells, skin fibroblasts derived from alpha/beta-interferon receptor knockout BALB/c mice. Furthermore, the addition of anti-interferon antibodies to the culture medium of infected BF cells significantly reduced the observed decline in virus protein synthesis. Following infection of untreated BF cells, the majority replicated virus but survived the infection and eventually cleared the virus after 8 to 15 days. However, not all the cells were cured, and the cultures became persistently infected. Upon passage of persistently infected cultures, the virus fluxed between active and repressed states as a consequence of interferon production. This resulted in a balance being reached in which only 5 to 20% of the cells were infected at any one time. After 30 passages of the persistently infected cells, highly fusogenic virus variants arose (one of which was isolated and termed W3-f). W3-f remained as sensitive to interferon as the parental W3 isolate but, in the absence of interferon, spread much more rapidly than the parental W3 strain through BF cell monolayers. Sequence analysis revealed no deduced amino acid differences between the F proteins of W3 and W3-f. BF cell cultures persistently infected with W3-f were rapidly cleared of virus by the addition of virus-neutralizing antibodies to the culture medium. In contrast, neutralizing antibodies had little effect on the numbers of cells persistently infected with W3 over several passages. These results suggest that the ability of paramyxoviruses to cause cell-cell fusion may be selected for in vivo as a consequence of their adaptation to the interferon response rather than their need to escape from neutralizing antibodies. The significance of these observations with regard to persistent parainfluenza virus infections in vivo is further discussed.

The P, V, and NP genes of the paramyxovirus simian virus 5 (SV5) were cloned such that their expression was regulated by the tetracycline-controlled transactivator (M. Gossen and H. Bujard, Proc. Natl. Acad. Sci. USA 89:5547-5551, 1992), and mammalian cell lines that inducibly expressed individually the P, V, or NP protein or coexpressed the P plus NP or V plus NP proteins were isolated. A plasmid that expresses the tetracycline-controlled transactivator linked, via the foot-and-mouth disease virus 2A cleavage peptide sequence, to the neomycin aminoglycoside phosphotransferase gene was constructed. Cells were cotransfected with this plasmid, and the appropriate responder plasmids and clonies were selected on the basis of their resistance to Geneticin (via the neomycin aminoglycoside phosphotransferase gene). The properties of these cell lines, in terms of the induction of the P, V, and NP genes, are described in detail. Both the P and V proteins were phosphorylated when expressed alone. In immunoprecipitation studies using a monoclonal antibody that recognizes both the P and V proteins, a nonphosphorylated host cell protein with an estimated molecular weight of 150,000 was coprecipitated with V but not P. Immunofluorescence data demonstrated that when expressed separately, the P protein had a diffuse cytoplasmic distribution, but the related V protein had both a nuclear and cytoplasmic distribution. The NP protein had a granular cytoplasmic distribution, giving rise to punctate and granular fluorescence. Coexpression of the NP and P proteins resulted in the accumulation of large cytoplasmic inclusion aggregates, similar to those visualized at late times in SV5-infected cells. Coexpression of V with NP led to a partial redistribution of the NP protein in that the NP protein had both a diffuse cytoplasmic and nuclear distribution in the presence of V, but no NP-V aggregates or inclusion bodies were visualized. Direct binding studies also revealed that NP bound to both P and V. For SV5, these studies suggest that V may have a role in keeping NP soluble prior to encapsidation.

A small 14 amino acid oligopeptide tag (termed SV5-Pk) was fused onto the carboxy-terminus of simian immunodeficiency virus gp160 expressed from a recombinant baculovirus. The presence of the Pk tag had no obvious effect on the expression and glycosylation of gp160 and did not interfere either with CD4 binding or with cleavage at its maturation site by the protease furin. The presence of the Pk tag did, however, facilitate the simplified purification of full-length gp160 and its incorporation into immunogenic solid matrix-antibody-antigen (SMAA) complexes.

There has been a long history of unexplained anomalous absorption of solar radiation by clouds. Collocated satellite and surface measurements of solar radiation at five geographically diverse locations showed significant solar absorption by clouds, resulting in about 25 watts per square meter more global-mean absorption by the cloudy atmosphere than predicted by theoretical models. It has often been suggested that tropospheric aerosols could increase cloud absorption. But these aerosols are temporally and spatially heterogeneous, whereas the observed cloud absorption is remarkably invariant with respect to season and location. Although its physical cause is unknown, enhanced cloud absorption substantially alters our understanding of the atmosphere's energy budget.

Following infection of BALB/c fibroblastic (BF) cells with simian virus 5 (SV5) only low levels of infectious virus were produced and the majority of cells survived the infection. However at 1 day post-infection (p.i.), near normal levels of all the virus proteins were synthesized and the virus genome was replicated. RNA analysis of the infected cells revealed that the levels of viral genomic RNA remained high over 5 days of infection, but that viral mRNA levels were significantly reduced by 3 days p.i. There was no evidence for the accumulation of defective genomes over this period. The reduction in mRNA levels was reflected by a concomitant decrease in the rate of ongoing viral protein synthesis. Despite the apparent decrease in viral transcription, comparative measurements of the relative levels of the different virus proteins at various times p.i. revealed that the levels of the P and NP proteins were similar at 1 and 5 days p.i. but the levels of V, M and F declined. Immunofluorescence analysis supported this data showing that at later times p.i., although there were some cells which were positive for all the viral proteins, a high proportion of cells were strongly positive for NP and P but negative for M, F and HN proteins. In these cells, NP and P were often located in discrete cytoplasmic foci. A series of cell lines were established from BF cells that had been infected at high multiplicity. Immunofluorescence studies showed that only a minority of cells in these cell lines were infected. This suggests that upon cell division, in a proportion of cells, virus replication was not taking place; otherwise it would be expected that all the daughter cells would remain infected. However, upon co-cultivation of these cells with Vero cells (cells that are fully permissive for SV5 replication), non-defective virus could be recovered. Virus cytoplasmic inclusion bodies could still be detected in a small proportion of BF cells that had been infected at high m.o.i. and passaged 10 times over a 12 week period, and again low levels of infectious virus could be recovered from these cells. It is proposed that in these persistently infected cells, the majority of virus genomes reside in an inactive form in cytoplasmic inclusion bodies but from which virus may occasionally be reactivated.(ABSTRACT TRUNCATED AT 400 WORDS)

We report the syntheses of two new fluorescein derivatives, 3',6'-dihydroxy-3-oxo-2-[(phosphonothio)-acetyl]spiro[isobenzof uran- 1(3H),9'-9H-xanthene]-6-carboxylic acid hydrazide, disodium salt, a phosphorothioate fluorescein, and 3',6'-dihydroxy-3-oxo-2-(mercaptoacetyl)spiro[isobenzofuran-1(3H), 9'-9H- xanthene]-6-carboxylic acid hydrazide, a mercaptoacetyl fluorescein. The latter is derived from the first compound by hydrolysis of the phosphate. Direct nonenzymatic labeling of the maleimide-derivatized IgG molecule by the novel mercaptoacetyl fluorescein is discussed. We also present a new method of bioconjugating phosphorothioate-functionalized fluorophores to a maleimide-derivatized protein, based on the alkaline phosphatase-catalyzed hydrolysis of the S-P bond of the phosphorothioate and the concomitant liberation of the fluorophore thiolate. This last species reacts in situ with the maleimide on the protein. A high degree of conjugation control is achieved in that modulation of the stoichiometry of the label and enzyme results in incorporation from seven to eight fluorophores per protein, depending on the ratio of the phosphorothioate fluorescein to alkaline phosphatase. The quantum yield of the mercaptoacetyl fluorescein relative to 6-carboxyfluorescein is 0.22 and lambda exc = 494 nm and lambda em = 517 nm.

A direct systolic cardiac augmentation device, called a heart cuff, has been developed. The device is designed to be placed around a "weakened" heart to improve cardiac function. The heart cuff forcibly squeezes the heart during ventricular systole by rhythmically inflating two balloons; one is placed over the left ventricle and the other over the right ventricle. An outer, adjustable, non-distensible cylindrical shell with a liquid-filled liner (bladder) holds the balloons in place. The conformable outer shell and liner inflation allow the device to fit on several different sized and shaped hearts. A purse-string nylon "dome", which is wrapped around the base of the heart and secured in the atrio-ventricular groove, is used to keep the heart cuff in position during assistance. An intra-aortic balloon pump system is used to apply the alternating positive and negative pressures to the balloons. The timing circuitry contained in the system allows for proper inflation/deflation of the balloons corresponding to the contraction/relaxation of the heart using the ECG. Studies show that the heart cuff is capable of being quickly and easily slipped into position, remain in position, and supplement heart function during assistance on normal beating canine hearts.

We describe a chromogenic detection system for beta-lactamase which yields water-insoluble colored products. The assay is based on kinetic measurement of the appearance of color due to the beta-lactamase-initiated redox reaction. The substrates are C3' thiolate-substituted cephalosporins, which, after enzyme-catalyzed hydrolysis of the beta-lactam ring, undergo elimination of the thiolate ion. This thiolate, in a postenzymatic step, reduces the tetrazolium salts, which are water-soluble colorless compounds, to a colored water-insoluble precipitate of formazan. Our model in this study was a beta-lactamase Enterobacter cloacae P-99-catalyzed reaction of thiolacetate cephalosporin with several tetrazolium salts. We found that the reaction rate is dependent on the concentration of the electron carrier 5-methyl phenazinium methyl sulfate, the pKa of the C3' thiolate substituent of the cephalosporin substrate, and the reduction potential of the tetrazolium salts. A kinetic study of this system yielded a rate law for the reaction. We present a mechanism of the reaction and determination of the kinetic parameters for the process. The sensitivity of this kinetic assay is very high; we detect 3 x 10(-10) M beta-lactamase P-99, which is approximately 30 mIU. The assay times are very short, lasting from 2 to 5 min. The new assay system is particularly suitable for a rapid detection of beta-lactamases in bacterial colonies and in enzyme immunoassays where beta-lactamase may be used as the label.

A very sensitive and rapid heterogeneous sandwich enzyme immunoassay for human chorionic gonadotropin (hCG) is described. The assay is based on the application of the novel chromogenic redox substrate system for beta-lactamase which is used as label. The chromogen system consists of a thioacetylcephalosporin beta-lactamase substrate, which upon turnover by the enzyme label releases the thiolate with the concomitant reduction of the tetrazolium salt to a colored formazan. The concentration of the formazan is directly related to the amount of the hormone in the sample and is read spectrophotometrically. The enzyme-antibody conjugates, produced through use of heterobifunctional maleimide crosslinker, maintain 90% of the enzyme activity after 30 days at 25 degrees C. Concentrations of the hormone as low as 5 mIU/ml, equivalent to 25 fmol/ml, are detectable in 3 h.