BackgroundHerpes simplex virus type-1 (HSV-1) infections can cause a number of diseases ranging from simple cold sores to dangerous keratitis and lethal encephalitis. The interaction between virus and host cells, critical for viral replication, is being extensively investigated by many laboratories. In this study, we tested the hypothesis that HSV-1 lytic infection triggers the expression of important multi-functional transcription factor Egr1. The mechanisms of induction are mediated, at least in part, by signaling pathways such as NFκB and CREB.MethodsSIRC, VERO, and 293HEK cell lines were infected with HSV-1, and the Egr-1 transcript and protein were detected by RT-PCR and Western blot, respectively. The localization and expression profile of Egr-1 were investigated further by immunofluorescence microscopy analyses. The recruitment of transcription factors to the Egr-1 promoter during infection was studied by chromatin immunoprecipitation (ChIP). Various inhibitors and dominant-negative mutant were used to assess the mechanisms of Egr-1 induction and their effects were addressed by immunofluorescence microscopy.ResultsWestern blot analyses showed that Egr-1 was absent in uninfected cells; however, the protein was detected 24-72 hours post treatment, and the response was directly proportional to the titer of the virus used for infection. Using recombinant HSV-1 expressing EGFP, Egr-1 was detected only in the infected cells. ChIP assays demonstrated that NFкB and cAMP response element binding protein (CREB) were recruited to the Egr-1 promoter upon infection. Additional studies showed that inhibitors of NFкB and dominant-negative CREB repressed the Egr-1 induction by HSV-1 infection.ConclusionCollectively, these results demonstrate that Egr-1 is expressed rapidly upon HSV-1 infection and that this novel induction could be due to the NFкB/CREB-mediated transactivation. Egr-1 induction might play a key role in the viral gene expression, replication, inflammation, and the disease progression.
Animal manure provides benefits to agriculture but may contain pathogens that contaminate ready-to-eat produce. U.S. Department of Agriculture National Organic Program standards include 90- or 120-day intervals between application of manure and harvest of crop to minimize risks of pathogen contamination of fresh produce. Data on factors affecting survival of Escherichia coli in soils under greenhouse conditions are needed. Three separate studies were conducted to evaluate survival of nonpathogenic E. coli (gEc) and attenuated E. coli O157:H7 (attO157) inoculated at either low (4 log CFU/ml) or high (6 log CFU/ml) populations over 56 days. Studies involved two pot sizes (small, 398 cm(3); large, 89 liters), three soil types (sandy loam, SL; clay loam, CL; silt loam, SIL), and four amendments (poultry litter, PL; dairy manure liquids, DML; horse manure, HM; unamended). Amendments were applied to the surface of the soil in either small or large containers. Study 1, conducted in regularly irrigated small containers, showed that populations of gEc and attO157 (2.84 to 2.88 log CFU/g) in PL-amended soils were significantly (P < 0.05) greater than those in DML-amended (0.29 to 0.32 log CFU/g [dry weight] [gdw]) or unamended (0.25 to 0.28 log CFU/gdw) soils; soil type did not affect E. coli survival. Results from study 2, in large pots with CL and SIL, showed that PL-amended soils supported significantly higher attO157 and gEc populations compared with HM-amended or unamended soils. Study 3 compared results from small and large containers that received high inoculum simultaneously. Overall, in both small and large containers, PLamended soils supported higher gEc and attO157 populations compared with HM-amended and unamended soils. Populations of attO157 were significantly greater in small containers (1.83 log CFU/gdw) than in large containers (0.65 log CFU/gdw) at week 8, perhaps because small containers received more regular irrigation than large pots. Regular irrigation of small pots may have affected E. coli persistence in manure-amended soils. Overall, PL-amended soils in both small and large containers supported E. coli survival at higher populations compared with DML-, HM-, or unamended soils.
Aims To understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication. Study Design Rabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection. Methodology Egr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays. Results Our results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus. Conclusion Together these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.
Cardiopulmonary resuscitation (CPR) skills are fundamental to the function of health professionals, but studies have shown them to be inadequate and outdated. This week, Nursing Standard launches a three-part weekly series on resuscitation. Parts two and three will address the associated ethical issues and measures of outcomes and accountability. The series begins with a survey in a district general hospital which aimed to establish nurses' levels of awareness on the current recommendations for CPR laid down by the Resuscitation Council of the UK. The results show a poor knowledge level and recommendations are offered.
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