Activation of the Listeria monocytogenes Virulence Program by a Reducing Environment.

Upon entry into the host cell cytosol, the facultative intracellular pathogen Listeria monocytogenes coordinates the expression of numerous essential virulence factors by allosteric binding of glutathione (GSH) to the Crp-Fnr family transcriptional regulator PrfA. Here, we report that robust virulence gene expression can be recapitulated by growing bacteria in a synthetic medium containing GSH or other chemical reducing agents. Bacteria grown under these conditions were 45-fold more virulent in an acute murine infection model and conferred greater immunity to a subsequent lethal challenge than bacteria grown in conventional media. During cultivation in vitro, PrfA activation was completely dependent on the intracellular levels of GSH, as a glutathione synthase mutant (ΔgshF) was activated by exogenous GSH but not reducing agents. PrfA activation was repressed in a synthetic medium supplemented with oligopeptides, but the repression was relieved by stimulation of the stringent response. These data suggest that cytosolic L. monocytogenes interprets a combination of metabolic and redox cues as a signal to initiate robust virulence gene expression in vivoIMPORTANCE Intracellular pathogens are responsible for much of the worldwide morbidity and mortality from infectious diseases. These pathogens have evolved various strategies to proliferate within individual cells of the host and avoid the host immune response. Through cellular invasion or the use of specialized secretion machinery, all intracellular pathogens must access the host cell cytosol to establish their replicative niches. Determining how these pathogens sense and respond to the intracellular compartment to establish a successful infection is critical to our basic understanding of the pathogenesis of each organism and for the rational design of therapeutic interventions. Listeria monocytogenes is a model intracellular pathogen with robust in vitro and in vivo infection models. Studies of the host-sensing and downstream signaling mechanisms evolved by L. monocytogenes often describe themes of pathogenesis that are broadly applicable to less tractable pathogens. Here, we describe how bacteria use external redox states as a cue to activate virulence

Partially linear censored quantile regression

Censored regression quantile (CRQ) methods provide a powerful and flexible approach to the analysis of censored survival data when standard linear models are felt to be appropriate. In many cases however, greater flexibility is desired to go beyond the usual multiple regression paradigm. One area of common interest is that of partially linear models: one (or more) of the explanatory covariates are assumed to act on the response through a non-linear function. Here the CRQ approach of Portnoy (J Am Stat Assoc 98:1001–1012, 2003) is extended to this partially linear setting. Basic consistency results are presented. A simulation experiment and unemployment example justify the value of the partially linear approach over methods based on the Cox proportional hazards model and on methods not permitting nonlinearity

Placental syncytiotrophoblast constitutes a major barrier to vertical transmission of Listeria monocytogenes.

Listeria monocytogenes is an important cause of maternal-fetal infections and serves as a model organism to study these important but poorly understood events. L. monocytogenes can infect non-phagocytic cells by two means: direct invasion and cell-to-cell spread. The relative contribution of each method to placental infection is controversial, as is the anatomical site of invasion. Here, we report for the first time the use of first trimester placental organ cultures to quantitatively analyze L. monocytogenes infection of the human placenta. Contrary to previous reports, we found that the syncytiotrophoblast, which constitutes most of the placental surface and is bathed in maternal blood, was highly resistant to L. monocytogenes infection by either internalin-mediated invasion or cell-to-cell spread. Instead, extravillous cytotrophoblasts-which anchor the placenta in the decidua (uterine lining) and abundantly express E-cadherin-served as the primary portal of entry for L. monocytogenes from both extracellular and intracellular compartments. Subsequent bacterial dissemination to the villous stroma, where fetal capillaries are found, was hampered by further cellular and histological barriers. Our study suggests the placenta has evolved multiple mechanisms to resist pathogen infection, especially from maternal blood. These findings provide a novel explanation why almost all placental pathogens have intracellular life cycles: they may need maternal cells to reach the decidua and infect the placenta

Fitting censored quantile regression by variable neighborhood search

Quantile regression is an increasingly important topic in statistical analysis. However, fitting censored quantile regression is hard to solve numerically because the objective function to be minimized is not convex nor concave in regressors. Performance of standard methods is not satisfactory, particularly if a high degree of censoring is present. The usual approach is to simplify (linearize) estimator function, and to show theoretically that such approximation converges to optimal values. In this paper, we suggest a new approach, to solve optimization problem (nonlinear, nonconvex, and nondifferentiable) directly. Our method is based on variable neighborhood search approach, a recent successful technique for solving global optimization problems. The presented results indicate that our method can improve quality of censored quantizing regressors estimator considerably

Apoptosis induced in vivo by photodynamic therapy in normal brain and intracranial tumour tissue

The apoptotic response of normal brain and intracranial VX2 tumour following photodynamic therapy (PDT) mediated by 5 different photosensitizers (Photofrin, 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX), chloroaluminium phthalocyanine (AlCIPc), Tin Ethyl Etiopurpurin (SnET 2), and meta-tetra(hydroxyphenyl)chlorin (m THPC)) was evaluated following a previous analysis which investigated the necrotic tissue response to PDT at 24 h post treatment. Free DNA ends, produced by internucleosomal DNA cleavage in apoptotic cells, were stained using a TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labelling) assay. Confocal laser scanning microscopy (CLSM) was used to quantify the local incidence of apoptosis and determine its spatial distribution throughout the brain. The incidence of apoptosis was confirmed by histopathology, which demonstrated cell shrinkage, pyknosis and karyorrhexis. At 24 h post PDT, AlClPc did not cause any detectable apoptosis, while the other photosensitizers produced varying numbers of apoptotic cells near the region of coagulative necrosis. The apoptotic response did not appear to be related to photosensitizer dose. These results suggest that at this time point, a minimal and fairly localized apoptotic effect is produced in brain tissues, the extent of which depends largely on the particular photosensitizer. © 2000 Cancer Research Campaig

Transcriptomic Events Involved in Melon Mature-Fruit Abscission Comprise the Sequential Induction of Cell-Wall Degrading Genes Coupled to a Stimulation of Endo and Exocytosis

Background: Mature-fruit abscission (MFA) in fleshy-fruit is a genetically controlled process with mechanisms that, contrary to immature-fruit abscission, has not been fully characterized. Here, we use pyrosequencing to characterize the transcriptomes of melon abscission zone (AZ) at three stages during AZ-cell separation in order to understand MFA control at an early stage of AZ-activation. Principal Findings: The results show that by early induction of MFA, the melon AZ exhibits major gene induction, while by late induction of MFA, melon AZ shows major gene repression. Although some genes displayed similar regulation in both early and late induction of abscission, such as EXT1-EXT4, EGase1, IAA2, ERF1, AP2D15, FLC, MADS2, ERAF17, SAP5 and SCL13 genes, the majority had different expression patterns. This implies that time-specific events occur during MFA, and emphasizes the value of characterizing multiple time-specific abscission transcriptomes. Analysis of gene-expression from these AZs reveal that a sequential induction of cell-wall-degrading genes is associated with the upregulation of genes involved in endo and exocytosis, and a shift in plant-hormone metabolism and signaling genes during MFA. This is accompanied by transcriptional activity of small-GTPases and synthaxins together with tubulins, dynamins, V-type ATPases and kinesin-like proteins potentially involved in MFA signaling. Early events are potentially controlled by down-regulation of MADS-box, AP2/ERF and Aux/IAA transcription-factors, and up-regulation of homeobox, zinc finger, bZIP, and WRKY transcription-factors, while late events may be controlled by up-regulation of MYB transcription-factors. Significance: Overall, the data provide a comprehensive view on MFA in fleshy-fruit, identifying candidate genes and pathways associated with early induction of MFA. Our comprehensive gene-expression profile will be very useful for elucidating gene regulatory networks of the MFA in fleshy-fruit

Određivanje alergena Asp f 1 (Aspergillus fumigatus) u peradarniku enzimimunokemijskom metodom

Poultry farms contain high levels of allergenic fungi, and Aspergillus spp. is the most common genus of moulds. Aspergillus fumigatus antigens are responsible for the development of several respiratory diseases including asthma. The aim of this study was to measure the mass fraction of Asp f 1, a major allergen of Asperillus fumigatus in 37 indoor dust samples collected from four poultry farms in a rural area of the Zagreb County (Croatia) using the enzyme-linked immunosorbent assay. More than 62 % of dust samples had detectable Asp f 1 levels (limit of detection 3.6 ng g-1). The overall mean Asp f 1 level was 17.9 ng g-1 [range (3.8 to 72.4) ng g-1]. Satisfactory results were obtained for analytical within-run imprecision (6.7 %), between-run imprecision (10.5 %), and accuracy (91 % to 115 %). Microclimate parameters (air temperature, relative humidity, and velocity) were within the recommended ranges in all poultry farms. This study has shown that Asp f 1 settles on dust at poultry farms and that occupational exposure to this allergen deserves monitoring in livestock buildings.Peradarnici sadržavaju veliku koncentraciju alergenih plijesni, a rod Aspergillus najčešće je zastupljen. Antigeni soja Aspergillus fumigatus odgovorni su za nastanak nekoliko respiratornih bolesti uključujući astmu. Cilj ovoga rada bio je odrediti masenu frakciju Asp f 1, glavnog alergena soja Asperillus fumigatus u 37 uzoraka prašine uzorkovanih u četiri peradarnika sa šireg područja Zagrebačke županije rabeći enzimimunokemijsku metodu. Više od 62 % uzoraka prašine u tri peradarnika imalo je mjerljivu koncentraciju Asp f 1 (granica detekcije = 3.6 ng g-1). Ukupni srednji maseni udio Asp f 1 iznosio je 17.9 ng g-1 (raspon od 3.8 ng g-1 do 72.4 ng g-1). Dobiveni su zadovoljavajući rezultati za analitičku nepreciznost u seriji (6.7 %), nepreciznost iz dana u dan (10.5 %) i točnost (91 % do 115 %). Mikroklimatski parametri (temperatura zraka, relativna vlaga i protok zraka) u svim peradarnicima bili su u okviru preporučenih vrijednosti. Rezultati ovoga rada pokazuju da Asp f 1 sedimentira na prašinu u peradarnicima te da profesionalnu izloženost tom alergenu treba pratiti u jedinicama za uzgoj stoke

These aren’t the loci you’re looking for: Principles of effective SNP filtering for molecular ecologists

Sequencing reduced‐representation libraries of restriction site‐associated DNA (RADseq) to identify single nucleotide polymorphisms (SNPs) is quickly becoming a standard methodology for molecular ecologists. Because of the scale of RADseq data sets, putative loci cannot be assessed individually, making the process of filtering noise and correctly identifying biologically meaningful signal more difficult. Artefacts introduced during library preparation and/or bioinformatic processing of SNP data can create patterns that are incorrectly interpreted as indicative of population structure or natural selection. Therefore, it is crucial to carefully consider types of errors that may be introduced during laboratory work and data processing, and how to minimize, detect and remove these errors. Here, we discuss issues inherent to RADseq methodologies that can result in artefacts during library preparation and locus reconstruction resulting in erroneous SNP calls and, ultimately, genotyping error. Further, we describe steps that can be implemented to create a rigorously filtered data set consisting of markers accurately representing independent loci and compare the effect of different combinations of filters on four RAD data sets. At last, we stress the importance of publishing raw sequence data along with final filtered data sets in addition to detailed documentation of filtering steps and quality control measures

Ageing vision and falls: a review

Background: Falls are the leading cause of accidental injury and death among older adults. One of three adults over the age of 65 years falls annually. As the size of elderly population increases, falls become a major concern for public health and there is a pressing need to understand the causes of falls thoroughly. Main body of the abstract: While it is well documented that visual functions such as visual acuity, contrast sensitivity, and stereo acuity are correlated with fall risks, little attention has been paid to the relationship between falls and the ability of the visual system to perceive motion in the environment. The omission of visual motion perception in the literature is a critical gap because it is an essential function in maintaining balance. In the present article, we first review existing studies regarding visual risk factors for falls and the effect of ageing vision on falls. We then present a group of phenomena such as vection and sensory reweighting that provide information on how visual motion signals are used to maintain balance. Conclusion: We suggest that the current list of visual risk factors for falls should be elaborated by taking into account the relationship between visual motion perception and balance control