Airborne Detection and Quantification of Swine Influenza A Virus in Air Samples Collected Inside,Outside and Downwind from Swine Barns

Airborne transmission of influenza A virus (IAV) in swine is speculated to be an important route of virus dissemination, but data are scarce. This study attempted to detect and quantify airborne IAV by virus isolation and RRT-PCR in air samples collected under field conditions. This was accomplished by collecting air samples from four acutely infected pig farms and locating air samplers inside the barns, at the external exhaust fans and downwind from the farms at distances up to 2.1 km. IAV was detected in air samples collected in 3 out of 4 farms included in the study. Isolation of IAV was possible from air samples collected inside the barn at two of the farms and in one farm from the exhausted air. Between 13% and 100% of samples collected inside the barns tested RRT-PCR positive with an average viral load of 3.20E+05 IAV RNA copies/m³ of air. Percentage of exhaust positive air samples also ranged between 13% and 100% with an average viral load of 1.79E+04 RNA copies/m³ of air. Influenza virus RNA was detected in air samples collected between 1.5 and 2.1 Km away from the farms with viral levels significantly lower at 4.65E+03 RNA copies/m³. H1N1, H1N2 and H3N2 subtypes were detected in the air samples and the hemagglutinin gene sequences identified in the swine samples matched those in aerosols providing evidence that the viruses detected in the aerosols originated from the pigs in the farms under study. Overall our results indicate that pigs can be a source of IAV infectious aerosols and that these aerosols can be exhausted from pig barns and be transported downwind. The results from this study provide evidence of the risk of aerosol transmission in pigs under field conditions.     

A Herpes Simplex Virus Scaffold Peptide That Binds the Portal Vertex Inhibits Early Steps in Viral Replication

Previous experiments identified a 12-amino-acid (aa) peptide that was sufficient to interact with the herpes simplex virus 1 (HSV-1) portal protein and was necessary to incorporate the portal into capsids. In the present study, cells were treated at various times postinfection with peptides consisting of a portion of the Drosophila antennapedia protein, previously shown to enter cells efficiently, fused to either wild-type HSV-1 scaffold peptide (YPYYPGEARGAP) or a control peptide that contained changes at positions 4 and 5. These 4-tyrosine and 5-proline residues are highly conserved in herpesvirus scaffold proteins and were previously shown to be critical for the portal interaction. Treatment early in infection with subtoxic levels of wild-type peptide reduced viral infectivity by over 1,000-fold, while the mutant peptide had little effect on viral yields. In cells infected for 3 h in the presence of wild-type peptide, capsids were observed to transit to the nuclear rim normally, as viewed by fluorescence microscopy. However, observation by electron microscopy in thin sections revealed an aberrant and significant increase of DNA-containing capsids compared to infected cells treated with the mutant peptide. Early treatment with peptide also prevented formation of viral DNA replication compartments. These data suggest that the antiviral peptide stabilizes capsids early in infection, causing retention of DNA within them, and that this activity correlates with peptide binding to the portal protein. The data are consistent with the hypothesis that the portal vertex is the conduit through which DNA is ejected to initiate infection.     

Unligated Okazaki Fragments Induce PCNA Ubiquitination and a Requirement for Rad59-Dependent Replication Fork Progression

Deficiency in DNA ligase I, encoded by CDC9 in budding yeast, leads to the accumulation of unligated Okazaki fragments and triggers PCNA ubiquitination at a non-canonical lysine residue. This signal is crucial to activate the S phase checkpoint, which promotes cell cycle delay. We report here that a pol30-K107 mutation alleviated cell cycle delay in cdc9 mutants, consistent with the idea that the modification of PCNA at K107 affects the rate of DNA synthesis at replication forks. To determine whether PCNA ubiquitination occurred in response to nicks or was triggered by the lack of PCNA-DNA ligase interaction, we complemented cdc9 cells with either wild-type DNA ligase I or a mutant form, which fails to interact with PCNA. Both enzymes reversed PCNA ubiquitination, arguing that the modification is likely an integral part of a novel nick-sensory mechanism and not due to non-specific secondary mutations that could have occurred spontaneously in cdc9 mutants. To further understand how cells cope with the accumulation of nicks during DNA replication, we utilized cdc9-1 in a genome-wide synthetic lethality screen, which identified RAD59 as a strong negative interactor. In comparison to cdc9 single mutants, cdc9 rad59Δ double mutants did not alter PCNA ubiquitination but enhanced phosphorylation of the mediator of the replication checkpoint, Mrc1. Since Mrc1 resides at the replication fork and is phosphorylated in response to fork stalling, these results indicate that Rad59 alleviates nick-induced replication fork slowdown. Thus, we propose that Rad59 promotes fork progression when Okazaki fragment processing is compromised and counteracts PCNA-K107 mediated cell cycle arrest.     

Causes,temporal trends,and the effects of urbanization on admissions of wild raptors to rehabilitation centers in England and Wales

Data from wildlife rehabilitation centers (WRCs) can provide on‐the‐ground records of causes of raptor morbidity and mortality, allowing threat patterns to be explored throughout time and space. We provide an overview of native raptor admissions to four WRCs in England and Wales, quantifying the main causes of morbidity and mortality, trends over time, and associations between threats and urbanization between 2001 and 2019. Throughout the study period, 14 raptor species were admitted totalling 3305 admission records. The Common Buzzard (Buteo buteo; 31%) and Tawny Owl (Strix aluco; 29%) were most numerous. Relative to the proportion of breeding individuals in Britain and Ireland, Peregrine Falcons (Falco peregrinus), Little Owls (Athene noctua), and Western Barn Owls (Tyto alba) were over‐represented in the admissions data by 103%, 73%, and 69%, respectively. Contrastingly Northern Long‐eared Owls (Asio otus), Western Marsh Harriers (Circus aeruginosus), and Merlin (Falco columbarius) were under‐represented by 187%, 163%, and 126%, respectively. Across all species, vehicle collisions were the most frequent anthropogenic admission cause (22%), and orphaned young birds (10%) were most frequent natural cause. Mortality rate was highest for infection/parasite admissions (90%), whereas orphaned birds experienced lowest mortality rates (16%). For one WRC, there was a decline in admissions over the study period. Red Kite (Milvus milvus) admissions increased over time, whereas Common Buzzard and Common Kestrel admissions declined. There were significant declines in the relative proportion of persecution and metabolic admissions and an increase in orphaned birds. Urban areas were positively associated with persecution, building collisions, and unknown trauma admissions, whereas vehicle collisions were associated with more rural areas. Many threats persist for raptors in England and Wales, however, have not changed substantially over the past two decades. Threats associated with urban areas, such as building collisions, may increase over time in line with human population growth and subsequent urban expansion.     

Synthesis and maturation of cross-reactive glycoprotein in fibroblasts deficient in arylsulfatase a activity

The biosynthesis of arylsulfatase A was studied in cultured fibroblasts by pulse-chase labeling with [2-³H]mannose; the enzyme was isolated by immunoprecipitation and denaturing polyacrylamide gel electrophoresis. In normal fibroblasts, and in fibroblasts from a patient with multiple sulfatase deficiency, the enzyme was synthesized as a glycoprotein of apparent molecular weight of 59,000; half of it was processed over a period of 4 days to M_r= 57,000. The precursor chain of M_r= 59,000 was secreted in the presence of 10 mM NH₄Cl. An immunoprecipitable glycoprotein of normal size was synthesized by fibroblasts from two unrelated patients with metachromatic leukodystrophy, but this material disappeared within twenty hours. In fibroblasts from an individual with pseudodeficiency of arylsulfatase A, the immunoprecipitable precursor glycoprotein was smaller (M_r= 56,000). The synthesis of cross-reactive proteins with altered properties supports the concept of allelic mutations as the genetic basis of metachromatic leukodystrophy and of arylsulfatase A pseudodeficiency.     

Induction of SM-20 in PC12 cells leads to increased cytochrome c levels,accumulation of cytochrome c in the cytosol,and caspase-dependent cell death

Sympathetic neurons deprived of nerve growth factor (NGF) release cytochrome c into the cytosol and undergo caspase-dependent cell death through a process that requires de novo gene expression. Expression of the SM-20 gene increases after NGF withdrawal, and ectopic SM-20 expression induces cell death in NGF-maintained neurons. To further evaluate the mechanism by which SM-20 promotes cell death, we developed a PC12-derived cell line in which SM-20 expression can be induced by addition of doxycycline to the culture medium. Induction of SM-20 in either undifferentiated or NGF-differentiated cells resulted in cell death. Cell death was accompanied by an increase in caspase activity and was inhibited by the caspase inhibitor zVAD-FMK. Analysis of cytochrome c in cytosolic and mitochondria-enriched subcellular fractions revealed that induction of SM-20 led to the accumulation of cytochrome c in the cytosol. Surprisingly, SM-20 expression also resulted in a selective increase in the total amount of cytochrome c protein. Thus, induction of SM-20 expression appears to affect both the amount and subcellular localization of cytochrome c in PC12 cells. These results suggest that SM-20 promotes caspase-dependent cell death through a mechanism involving cytochrome c.     

Bracketed generic inactivation of rodent retroviruses by low pH treatment for monoclonal antibodies and recombinant proteins

Viral safety is a predominant concern for monoclonal antibodies (mAbs) and other recombinant proteins (RPs) with pharmaceutical applications. Certain commercial purification modules, such as nanofiltration and low-pH inactivation, have been observed to reliably clear greater than 4 log(10) of large enveloped viruses, including endogenous retrovirus. The concept of "bracketed generic clearance" has been proposed for these steps if it could be prospectively demonstrated that viral log(10) reduction value (LRV) is not impacted by operating parameters that can vary, within a reasonable range, between commercial processes. In the case of low-pH inactivation, a common step in mAb purification processes employed after protein A affinity chromatography, these parameters would include pH, time and temperature of incubation, the content of salts, protein concentration, aggregates, impurities, model protein pI, and buffer composition. In this report, we define bracketed generic clearance conditions, using a prospectively defined bracket/matrix approach, where low-pH inactivation consistently achieves >or=4.6 log(10) clearance of xenotropic murine leukemia virus (X-MLV), a model for rodent endogenous retrovirus. The mechanism of retrovirus inactivation by low-pH treatment was also investigated.     

Global nutritional profiling for mutant and chemical mode-of-action analysis in filamentous fungi

We describe a method for gene function discovery and chemical mode-of-action analysis via nutrient utilization using a high throughput Nutritional Profiling platform suitable for filamentous microorganisms. We have optimized the growth conditions for each fungal species to produce reproducible optical density growth measurements in microtiter plates. We validated the Nutritional Profiling platform using a nitrogen source utilization assay to analyze 21 Aspergillus nidulans strains with mutations in the master nitrogen regulatory gene, areA. Analysis of these data accurately reproduced expected results and provided new data to demonstrate that this platform is suitable for fine level phenotyping of filamentous fungi. Next, we analyzed the differential responses of two fungal species to a glutamine synthetase inhibitor, illustrating chemical mode-of-action analysis. Finally, a comparative phenotypic study was performed to characterize carbon catabolite repression in four fungal species using a carbon source utilization assay. The results demonstrate differentiation between two Aspergillus species and two diverse plant pathogens and provide a wealth of new data on fungal nutrient utilization. Thus, these assays can be used for gene function and chemical mode-of-action analysis at the whole organism level as well as interspecies comparisons in a variety of filamentous fungi. Additionally, because uniform distribution of growth within wells is maintained, comparisons between yeast and filamentous forms of a single organism can be performed.Electronic Supplementary Material Supplementary material is available in the online version of this article at The revised version of the PDF file was published online in January 2004. The figures are now in color.An erratum to this article can be found at     

Intermediate structural states involved in MRP1-mediated drug transport. Role of glutathione

Human multidrug resistance protein 1 (MRP1) is a member of the ATP-binding cassette transporter family and transports chemotherapeutic drugs as well as diverse organic anions such as leukotriene LTC(4). The transport of chemotherapeutic drugs requires the presence of reduced GSH. By using hydrogen/deuterium exchange kinetics and limited trypsin digestion, the structural changes associated with each step of the drug transport process are analyzed. Purified MRP1 is reconstituted into lipid vesicles with an inside-out orientation, exposing its cytoplasmic region to the external medium. The resulting proteoliposomes have been shown previously to exhibit both ATP-dependent drug transport and drug-stimulated ATPase activity. Our results show that during GSH-dependent drug transport, MRP1 does not undergo secondary structure changes but only modifications in its accessibility toward the external environment. Drug binding induces a restructuring of MRP1 membrane-embedded domains that does not affect the cytosolic domains, including the nucleotide binding domains, responsible for ATP hydrolysis. This demonstrates that drug binding to MRP1 is not sufficient to propagate an allosteric signal between the membrane and the cytosolic domains. On the other hand, GSH binding induces a conformational change that affects the structural organization of the cytosolic domains and enhances ATP binding and/or hydrolysis suggesting that GSH-mediated conformational changes are required for the coupling between drug transport and ATP hydrolysis. Following ATP binding, the protein adopts a conformation characterized by a decreased stability and/or an increased accessibility toward the aqueous medium. No additional change in the accessibility toward the solvent and/or the stability of this specific conformational state and no change of the transmembrane helices orientation are observed upon ATP hydrolysis. Binding of a non-transported drug affects the dynamic changes occurring during ATP binding and hydrolysis and restricts the movement of the drug and its release.     

Biosynthesis,glycosylation, and enzymatic processing in vivo of human tripeptidyl-peptidase I

Human tripeptidyl-peptidase I (TPP I, CLN2 protein) is a lysosomal serine protease that removes tripeptides from the free N termini of small polypeptides and also shows a minor endoprotease activity. Due to various naturally occurring mutations, an inherited deficiency of TPP I activity causes a fatal lysosomal storage disorder, classic late infantile neuronal ceroid lipofuscinosis (CLN2). In the present study, we analyzed biosynthesis, glycosylation, transport, and proteolytic processing of this enzyme in stably transfected Chinese hamster ovary cells as well as maturation of the endocytosed proenzyme in CLN2 lymphoblasts, fibroblasts, and N2a cells. Human TPP I was initially identified as a single precursor polypeptide of approximately 68 kDa, which, within a few hours, was converted to the mature enzyme of approximately 48 kDa. Compounds affecting the pH of intracellular acidic compartments, those interfering with the intracellular vesicular transport as well as inhibition of the fusion between late endosomes and lysosomes by temperature block or 3-methyladenine, hampered the conversion of TPP I proenzyme into the mature form, suggesting that this process takes place in lysosomal compartments. Digestion of immunoprecipitated TPP I proenzyme with both N-glycosidase F and endoglycosidase H as well as treatment of the cells with tunicamycin reduced the molecular mass of TPP I proenzyme by approximately 10 kDa, which indicates that all five potential N-glycosylation sites in TPP I are utilized. Mature TPP I was found to be partially resistant to endo H treatment; thus, some of its N-linked oligosaccharides are of the complex/hybrid type. Analysis of the effect of various classes of protease inhibitors and mutation of the active site Ser(475) on human TPP I maturation in cultured cells demonstrated that although TPP I zymogen is capable of autoactivation in vitro, a serine protease that is sensitive to AEBSF participates in processing of the proenzyme to the mature, active form in vivo.