Live Cell Analysis and Mathematical Modeling Identify Determinants of Attenuation of Dengue Virus 2’-O-Methylation Mutant

Dengue virus (DENV) is the most common mosquito-transmitted virus infecting ~390 million people worldwide. In spite of this high medical relevance, neither a vaccine nor antiviral therapy is currently available. DENV elicits a strong interferon (IFN) response in infected cells, but at the same time actively counteracts IFN production and signaling. Although the kinetics of activation of this innate antiviral defense and the timing of viral counteraction critically determine the magnitude of infection and thus disease, quantitative and kinetic analyses are lacking and it remains poorly understood how DENV spreads in IFN-competent cell systems. To dissect the dynamics of replication versus antiviral defense at the single cell level, we generated a fully viable reporter DENV and host cells with authentic reporters for IFN-stimulated antiviral genes. We find that IFN controls DENV infection in a kinetically determined manner that at the single cell level is highly heterogeneous and stochastic. Even at high-dose, IFN does not fully protect all cells in the culture and, therefore, viral spread occurs even in the face of antiviral protection of naïve cells by IFN. By contrast, a vaccine candidate DENV mutant, which lacks 2’-O-methylation of viral RNA is profoundly attenuated in IFN-competent cells. Through mathematical modeling of time-resolved data and validation experiments we show that the primary determinant for attenuation is the accelerated kinetics of IFN production. This rapid induction triggered by mutant DENV precedes establishment of IFN-resistance in infected cells, thus causing a massive reduction of virus production rate. In contrast, accelerated protection of naïve cells by paracrine IFN action has negligible impact. In conclusion, these results show that attenuation of the 2’-O-methylation DENV mutant is primarily determined by kinetics of autocrine IFN action on infected cells.     

Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O₂) than under microaerobic conditions (5% O₂, 10% CO₂), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.Infection with Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world and is often associated with the consumption of undercooked poultry products (19). The United Kingdom Health Protection Agency reported more than 45,000 laboratory-confirmed cases for England and Wales in 2006 alone, although this is thought to be a 5- to 10-fold underestimation of the total number of community incidents (20, 43). The symptoms associated with C. jejuni infection usually last between 2 and 5 days and include diarrhea, vomiting, and stomach pains. Sequelae of C. jejuni infection include more-serious autoimmune diseases, such as Guillain-Barré syndrome, Miller-Fisher syndrome (18), and reactive arthritis (15).Poultry represents a major natural reservoir for C. jejuni, since the organism is usually considered to be a commensal and can reach densities as high as 1 × 10⁸ CFU g of cecal contents⁻¹ (35). As a result, large numbers of bacteria are shed via feces into the environment, and consequently, C. jejuni can spread rapidly through a flock of birds in a broiler house (1). While well adapted to life in the avian host, C. jejuni must survive during transit between hosts and on food products under stressful storage conditions, including high and low temperatures and atmospheric oxygen levels. The organism must therefore have mechanisms to protect itself from unfavorable conditions.Biofilm formation is a well-characterized bacterial mode of growth and survival, where the surface-attached and matrix-encased bacteria are protected from stressful environmental conditions, such as UV radiation, predation, and desiccation (7, 8, 28). Bacteria in biofilms are also known to be >1,000-fold more resistant to disinfectants and antimicrobials than their planktonic counterparts (11). Several reports have now shown that Campylobacter species are capable of forming a monospecies biofilm (21, 22) and can colonize a preexisting biofilm (14). Biofilm formation can be demonstrated under laboratory conditions, and environmental biofilms, from poultry-rearing facilities, have been shown to contain Campylobacter (5, 32, 44). Campylobacter biofilms allow the organism to survive up to twice as long under atmospheric conditions (2, 21) and in water systems (27).Molecular understanding of biofilm formation by Campylobacter is still in its infancy, although there is evidence for the role of flagella and gene regulation in biofilm formation. Indeed, a flaAB mutant shows reduced biofilm formation (34); mutants defective in flagellar modification (cj1337) and assembly (fliS) are defective in adhering to glass surfaces (21); and a proteomic study of biofilm-grown cells shows increased levels of motility-associated proteins, including FlaA, FlaB, FliD, FlgG, and FlgG2 (22). Flagella are also implicated in adhesion and in biofilm formation and development in other bacterial species, including Aeromonas, Vibrio, Yersinia, and Pseudomonas species (3, 23, 24, 31, 42).Previous studies of Campylobacter biofilms have focused mostly on biofilm formation under standard microaerobic laboratory conditions. In this work we have examined the formation of biofilms by motile and nonmotile C. jejuni strains under atmospheric conditions that are relevant to the survival of this organism in a commercial context of environmental and food-based transmission.     

Use of genomic DNA control features and predicted operon structure in microarray data analysis: ArrayLeaRNA – a Bayesian approach

Background  

Microarrays are widely used for the study of gene expression; however deciding on whether observed differences in expression are significant remains a challenge.     

Reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus

Serologic monitoring of sentinel mice exposed to soiled bedding is a common method of detecting viral infections in mice. Because bedding transfer protocols vary, the sensitivity of this method has not been documented sufficiently. We examined the reliability of bedding transfer during various stages of infection with mouse parvovirus (MPV) and mouse hepatitis virus (MHV). Most mice exposed to bedding contaminated with MPV 0, 3, or 7 d previously seroconverted, whereas only mice exposed to bedding contaminated with MHV 4 h previously seroconverted, thus confirming the differing stabilities of these viruses. Index mice were inoculated with 30 times the infectious dose 50 (ID50) of MPV or 300 ID50 of MHV. At 3 d, 1 wk, and 2 wk postinoculation (PI), we transferred 25, 50, or 100 ml of bedding to cages of sentinel mice. Viral infection and shedding by index mice was confirmed by serology and fecal polymerase chain reaction assay. Transfer of soiled bedding between mice in static cages induced seroconversion of sentinel mice most reliably during peak viral shedding (1 wk PI for MPV and 3 d PI for MHV). Soiled bedding transfer between mice in individually ventilated cages induced a higher prevalence of sentinel seroconversion to MPV and MHV than that after transfer between mice in static cages. Our findings indicate that although soiled bedding transfer is an effective method for detecting MHV and MPV under optimal conditions, the method is less than 100% reliable under many conditions in contemporary mouse facilities.     

Field scale evaluation of bovine-specific DNA as an indicator of tissue degradation during cattle mortality composting

Currently, mortality compost is managed by temperature as extent of tissue degradation is difficult to assess. In the present study, field-scale mortality compost was constructed with composted brain tissue (Brain) and compost adjacent to brain tissue (CAB) sampled over 230 d. Following genomic DNA extraction, bovine-specific mitochondrial DNA (Mt-DNA) and bacterial 16S rDNA fragments were quantified using real-time PCR. Genomic DNA yield of Brain and CAB decreased rapidly (89-98%) and stabilized after 7 d. Compared to d 0, Brain Mt-DNA rapidly decreased (84-91% reduction on d 7). In CAB, Mt-DNA dramatically increased until d 28 (up to 34,500 times) thereafter decreasing by 77-93% on d 112. Quantification of bovine Mt-DNA indicates tissue degradation was initially characterized by rapid decomposition and release of cell contents into surrounding compost matrix followed by further degradation of Mt-DNA by flourishing microorganisms. Consequently, bovine Mt-DNA copies in compost matrix were reliable indicators of tissue degradation.     

The SUVR4 histone lysine methyltransferase binds ubiquitin and converts H3K9me1 to H3K9me3 on transposon chromatin in Arabidopsis

Chromatin structure and gene expression are regulated by posttranslational modifications (PTMs) on the N-terminal tails of histones. Mono-, di-, or trimethylation of lysine residues by histone lysine methyltransferases (HKMTases) can have activating or repressive functions depending on the position and context of the modified lysine. In Arabidopsis, trimethylation of lysine 9 on histone H3 (H3K9me3) is mainly associated with euchromatin and transcribed genes, although low levels of this mark are also detected at transposons and repeat sequences. Besides the evolutionarily conserved SET domain which is responsible for enzyme activity, most HKMTases also contain additional domains which enable them to respond to other PTMs or cellular signals. Here we show that the N-terminal WIYLD domain of the Arabidopsis SUVR4 HKMTase binds ubiquitin and that the SUVR4 product specificity shifts from di- to trimethylation in the presence of free ubiquitin, enabling conversion of H3K9me1 to H3K9me3 in vitro. Chromatin immunoprecipitation and immunocytological analysis showed that SUVR4 in vivo specifically converts H3K9me1 to H3K9me3 at transposons and pseudogenes and has a locus-specific repressive effect on the expression of such elements. Bisulfite sequencing indicates that this repression involves both DNA methylation-dependent and -independent mechanisms. Transcribed genes with high endogenous levels of H3K4me3, H3K9me3, and H2Bub1, but low H3K9me1, are generally unaffected by SUVR4 activity. Our results imply that SUVR4 is involved in the epigenetic defense mechanism by trimethylating H3K9 to suppress potentially harmful transposon activity.     

Recovery of time on limits of stability from functional fatigue in Division II collegiate athletes

Health and fitness professionals working with athletes could establish effective and safe practice and training programs if recovery time on dynamic balance from exertion was available. Research investigating the time needed to recover dynamic limits of stability (LOS) from exertion has not been reported. The purpose of this study was to determine the recovery timeline on LOS from functional fatigue in collegiate athletes. Eighteen athletes (11 men, 7 women) from Division II collegiate soccer team who passed prescreening tests to identify their fitness levels were randomly tested on 2 different days by condition (fatigue or nonfatigue). Functional fatigue was determined by using the Borg 15-point rating of perceived exertion (RPE) scale. Subjects were tested on LOS on the Biodex Balance System pre, post, 10, 15, and 20 minutes for each condition. The main effect for condition was not significant (F() = 0.004, p = 0.948), whereas the main effect for time was significant (F(4,64) = 6.167, p < 0.001). The RPE scoring revealed the significant main effect in FATIGUE (F(2.69, 45.73) = 234.8, p < 0.001). In conclusion, 20 minutes of functional activity will likely have a negative influence on dynamic balance, with balance recovery occurring within 10 minutes after the cessation of exercise in Division II collegiate soccer athletes. Moreover, the level of exertion measured by RPE would correspond to athletes' ability to control their center of mass.