Resistance of the 64K protein of budded Autographa californica nuclear polyhedrosis virus to functional inactivation by proteolysis.

The 64K surface protein of budded Autographa californica nuclear polyhedrosis virus (AcMNPV BV) is known to play a role in the functional entry of AcMNPV BV into Spodoptera frugiperda IPLB-SF-21 cells by adsorptive endocytosis. AcV1, a neutralizing monoclonal antibody, reacts with the 64K protein and in doing so prevents efficient entry. In this communication we report that treatment of AcMNPV BV with either trypsin or proteinase K cleaves the 64K protein into one major fragment of 34.6K and two minor fragments of 36K to 37.2K that are retained with the virus. All of the fragments are glycosylated. Protease treatment does not reduce viral infectivity, but it does result in the destruction of the AcV1-reactive epitope; thus AcV1 is not able to neutralize protease-treated AcMNPV BV. Polyclonal antiserum to BV is able to recognize both cleaved and uncleaved 64K and neutralize both protease-treated and untreated virus. Protease treatment does not diminish the sensitivity of AcMNPV BV to chloroquine, but it does cause the virus to become more susceptible to inactivation by 2-mercaptoethanol (2-ME) even though exposure to 2-ME does not result in dissociation of the fragments from the virus

Budded Autographa californica NPV 64K protein: Further biochemical analysis and effects of postimmunoprecipitation sample preparation conditions.

Previously it was shown that AcV1, a neutralizing monoclonal antibody of the Autographa californica nuclear polyhedrosis virus-budded phenotype reacted with a surface antigen present on infected cells during virus budding, and in the viral envelope (L. E. Volkman, P. A. Goldsmith, R. T. Hess, and P. Faulkner (1984), Virology 133, 354-362). Radioimmune precipitation of solubilized, [35S]methionine-labeled budded virus with AcV1 and analysis on SDS-PAGE revealed four bands consistently: one major band at 64,000 Da, and three minor bands at 127,000, 59,000, and 49,000 Da. The reason for the appearance of four bands instead of one was unclear. Data suggest that two of the bands, 49K and 59K, are aberrant, and are the products of sample preparation conditions. Further, evidence is presented that the 127K band is composed of dimers of the 64K protein, and that under nonreducing conditions, oligomers (trimers and tetramers) of 64K protein can also be detected. BVGP 64 is additionally shown to be phosphorylated and to have an isoelectric point of 3.15. The BVGP 64 epitope reactive with AcV1 is destroyed by interaction with SDS. This could account for the lack of neutralizing activity of antiserum made to the SDS-PAGE purified BVGP 64

Neutralization of budded Autographa californica NPV by a monoclonal antibody: identification of the target antigen.

A neutralizing monoclonal antibody of the budded phenotype of Autographa californica nuclear polyhedrons virus did not react with the occluded form of the virus as determined by neutralization, ELISA, and indirect immunoperoxidase staining. The antibody did react with the surface of infected cells in the prepolyhedra stage of cytopathic effect, the period of active virus budding. Immunoelectron microscopy indicated the antigen(s) reactive with the neutralizing antibody was present all around the viral envelope, but was more highly concentrated at the end with peplomers. Four proteins were immunoprecipitated from solubilized, radiolabeled budded virus preparations with the monoclonal antibody. The major protein had a molecular weight of approximately 64,000, while the other three were approximately 127,000, 59,000, and 49,000. All four proteins could be labeled with N-acetyl-d-[1-3H]glucosamine. This glycosylation reaction could be inhibited by tunicamycin

A Systematic Map of Genetic Variation in Plasmodium falciparum

Discovering novel genes involved in immune evasion and drug resistance in the human malaria parasite, Plasmodium falciparum, is of critical importance to global health. Such knowledge may assist in the development of new effective vaccines and in the appropriate use of antimalarial drugs. By performing a full-genome scan of allelic variability in 14 field and laboratory strains of P. falciparum, we comprehensively identified ≈500 genes evolving at higher than neutral rates. The majority of the most variable genes have paralogs within the P. falciparum genome and may be subject to a different evolutionary clock than those without. The group of 211 variable genes without paralogs contains most known immunogens and a few drug targets, consistent with the idea that the human immune system and drug use is driving parasite evolution. We also reveal gene-amplification events including one surrounding pfmdr1, the P. falciparum multidrug-resistance gene, and a previously uncharacterized amplification centered around the P. falciparum GTP cyclohydrolase gene, the first enzyme in the folate biosynthesis pathway. Although GTP cyclohydrolase is not the known target of any current drugs, downstream members of the pathway are targeted by several widely used antimalarials. We speculate that an amplification of the GTP cyclohydrolase enzyme in the folate biosynthesis pathway may increase flux through this pathway and facilitate parasite resistance to antifolate drugs

Exceptional preservation of palaeozoic steroids in a diagenetic continuum

The occurrence of intact sterols has been restricted to immature Cretaceous (~125 Ma) sediments with one report from the Late Jurassic (~165 Ma). Here we report the oldest occurrence of intact sterols in a Crustacean fossil preserved for ca. 380 Ma within a Devonian concretion. The exceptional preservation of the biomass is attributed to microbially induced carbonate encapsulation, preventing full decomposition and transformation thus extending sterol occurrences in the geosphere by 250 Ma. A suite of diagenetic transformation products of sterols was also identified in the concretion, demonstrating the remarkable coexistence of biomolecules and geomolecules in the same sample. Most importantly the original biolipids were found to be the most abundant steroids in the sample. We attribute the coexistence of steroids in a diagenetic continuum-ranging from stenols to triaromatic steroids-to microbially mediated eogenetic processes

A High-Throughput Screen for Tuberculosis Progression

One-third of the world population is infected with Mycobacterium tuberculosis and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish Mycobacterium marinum infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study M. tuberculosis, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon M. marinum infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds in vivo

Formative evaluation of the telecare fall prevention project for older veterans

<p>Abstract</p> <p>Background</p> <p>Fall prevention interventions for community-dwelling older adults have been found to reduce falls in some research studies. However, wider implementation of fall prevention activities in routine care has yielded mixed results. We implemented a theory-driven program to improve care for falls at our Veterans Affairs healthcare facility. The first project arising from this program used a nurse advice telephone line to identify patients' risk factors for falls and to triage patients to appropriate services. Here we report the formative evaluation of this project.</p> <p>Methods</p> <p>To evaluate the intervention we: 1) interviewed patient and employee stakeholders, 2) reviewed participating patients' electronic health record data and 3) abstracted information from meeting minutes. We describe the implementation process, including whether the project was implemented according to plan; identify barriers and facilitators to implementation; and assess the incremental benefit to the quality of health care for fall prevention received by patients in the project. We also estimate the cost of developing the pilot project.</p> <p>Results</p> <p>The project underwent multiple changes over its life span, including the addition of an option to mail patients educational materials about falls. During the project's lifespan, 113 patients were considered for inclusion and 35 participated. Patient and employee interviews suggested support for the project, but revealed that transportation to medical care was a major barrier in following up on fall risks identified by nurse telephone triage. Medical record review showed that the project enhanced usual medical care with respect to home safety counseling. We discontinued the program after 18 months due to staffing limitations and competing priorities. We estimated a cost of $9194 for meeting time to develop the project.</p> <p>Conclusions</p> <p>The project appeared feasible at its outset but could not be sustained past the first cycle of evaluation due to insufficient resources and a waning of local leadership support due to competing national priorities. Future projects will need both front-level staff commitment and prolonged high-level leadership involvement to thrive.</p

Mycobacteria counteract a TLR-mediated nitrosative defense mechanism in a zebrafish infection model.

Pulmonary tuberculosis (TB), caused by the intracellular bacterial pathogen Mycobacterium tuberculosis (Mtb), is a major world health problem. The production of reactive nitrogen species (RNS) is a potent cytostatic and cytotoxic defense mechanism against intracellular pathogens. Nevertheless, the protective role of RNS during Mtb infection remains controversial. Here we use an anti-nitrotyrosine antibody as a readout to study nitration output by the zebrafish host during early mycobacterial pathogenesis. We found that recognition of Mycobacterium marinum, a close relative of Mtb, was sufficient to induce a nitrosative defense mechanism in a manner dependent on MyD88, the central adaptor protein in Toll like receptor (TLR) mediated pathogen recognition. However, this host response was attenuated by mycobacteria via a virulence mechanism independent of the well-characterized RD1 virulence locus. Our results indicate a mechanism of pathogenic mycobacteria to circumvent host defense in vivo. Shifting the balance of host-pathogen interactions in favor of the host by targeting this virulence mechanism may help to alleviate the problem of infection with Mtb strains that are resistant to multiple drug treatments

Transcription and Expression of Plasmodium falciparum Histidine-Rich Proteins in Different Stages and Strains: Implications for Rapid Diagnostic Tests

Background: Although rapid diagnostic tests (RDTs) for Plasmodium falciparum infection that target histidine rich protein 2 (PfHRP2) are generally sensitive, their performance has been reported to be variable. One possible explanation for variable test performance is differences in expression level of PfHRP in different parasite isolates. Methods: Total RNA and protein were extracted from synchronised cultures of 7 P. falciparum lines over 5 time points of the life cycle, and from synchronised ring stages of 10 falciparum lines. Using quantitative real-time polymerase chain reaction, Western blot analysis and ELISA we investigated variations in the transcription and protein levels of pfhrp2, pfhrp3 and PfHRP respectively in the different parasite lines, over the parasite intraerythrocytic life cycle. Results: Transcription of pfhrp2 and pfhrp3 in different parasite lines over the parasite life cycle was observed to vary relative to the control parasite K1. In some parasite lines very low transcription of these genes was observed. The peak transcription was observed in ring-stage parasites. Pfhrp2 transcription was observed to be consistently higher than pfhrp3 transcription within parasite lines. The intraerythrocytic lifecycle stage at which the peak level of protein was present varied across strains. Total protein levels were more constant relative to total mRNA transcription, however a maximum 24 fold difference in expression at ring-stage parasites relative to the K1 strain was observed. Conclusions: The levels of transcription of pfhrp2 and pfhrp3, and protein expression of PfHRP varied between different P. falciparum strains. This variation may impact on the detection sensitivity of PfHRP2-detecting RDTs