Mechanism of Adsorption and Eclipse of Bacteriophage φX174 II. Attachment and Eclipse with Isolated Escherichia coli Cell Wall Lipopolysaccharide

A mixture of aqueous phenol, choloroform, and ether extracts the lipopolysaccharides (LPS) from the phiX174-sensitive strain, Escherichia coli C/1, and resistant strains, C/phiX and K12. Interaction of the C/1 LPS with phiX in a starvation buffer containing 10(-3) M CaCl(2) at 37 C, but not at 15 C, results in a first-order inactivation that is specific for C/1 LPS. After interaction for 60 min at 15 C, followed by centrifugation, 37 and 20% of a (14)C-phiX preparation are bound to the C/1 and C/phiX LPS pellets, respectively. The results for intact cells are 75 and 10%. Supporting the conclusion that this represents specific attachment of phiX to its receptor site in the LPS is the fact that EDTA-borate buffer is required to elute 85% of the (14)C-phiX from the C/1 LPS, whereas starvation buffer elutes the same amount from C/phiX LPS. Moreover, 95% of the PFU are found in the C/1 LPS pellets as compared with 50% in the resistant strain LPS pellets. When the products of interaction between phiX and LPS at 37 C are examined by sucrose density gradients in EDTA-borate, a single 60 to 90S peak is observed in the C/1 sample, and the single peak cosediments with the 120S marker phiX in the C/phiX sample. This change in S(20, w) is very similar to that reported for the eclipse of phiX in vivo. If the inactivation at 37 C is carried out on phiX-LPS complexes first formed at 15 C, the first-order kinetics are biphasic and nearly identical to that observed for the eclipse kinetics of phiX attached to intact cells. Thus, the phiX-LPS system is suitable for in vitro studies on the early events in phiX infection.     

Differential effects of soluble and immobilized fibronectins on aortic endothelial cell proliferation and attachment

Summary We studied the effects of soluble and immobilized forms of plasma fibronectin on bovine aortic endothelial cell (AEC) proliferation and attachment. Soluble fibronectin stimulated AEC growth at 10 μg/ml, but at higher concentrations of soluble fibronectin AEC growth was progressively inhibited. The growth rates of arterial smooth muscle cells (ASMC) and dermal fibroblasts (DF) were not altered by soluble fibronectin concentrations of 10 to 100 μg/ml. Plasma fibronectin, immobilized by attachment to culture dish surfaces, had no significant effects on the proliferation of any of the cell types examined. The attachment rates of AEC were decreased in the presence of 50 μg/ml soluble fibronectin. Immobilized fibronetin increased the rate of AEC attachment, but had no significant effects on ASMC or DF attachment; however, 12 h after plating there was nearly 100% attachment in all groups, whether or not fibronectin was present in the system. That soluble and immobilized fibronectins elicit disparate cellular responses is consistent with published reports of different cell surface receptors for different forms of the protein; in this manner, cells enmeshed in an interstitial matrix containing immobilized fibronectin could still respond to soluble fibronectin in the extracellular milieu. These studies were supported in part by grant EY-0229 from the National Institutes of Health, Bethesda, MD.     

Effect of Chromium(VI) Action on <Emphasis Type="Italic">Arthrobacter oxydans</Emphasis>

Arthrobacter species is of interest because of its high potential for bioremediation. Bacteria can detoxify chromium, by either reduction or accumulation inside the bacteria and/or absorption of chromium(VI) (CrVI) on their surface, and efflux pump. The possible pathway of Cr(VI) reduction by Arthrobacter oxydans isolated from Columbia basalt rocks at a US DOE highly contaminated site (USA) has been considered in the present study. FTIR absorption spectroscopy showed that these bacteria reduce Cr(VI). In the present study the threshold Cr(VI) nontoxic concentration (35 g/mL) for A. oxydans growing in liquid medium was estimated. Complete uptake of this concentration was achieved in about 10 days after chromium addition into the medium. At this concentration an increase in the protein isolated from the cell wall of A. oxydans was observed. This increased protein predominated independently of the growth phase at which Cr(VI) was added. Thermal analysis was used to identify any influence of Cr(VI) on the DNP complex of A. oxydans. According to the data obtained it can be supposed that Cr(VI) reduction predominantly occurs on the bacterial surface and that cell wall represents a permeable barrier for these bacteria at the non-toxic chromium action.     

Counties selecting public health priorities--a "bottom-up" approach (Croatian experience)

The subject of this paper is how to incorporate a multi-disciplinary and inter-sectored approach into development of public health policy and plans at the local (county) level in Croatia by educational program. Method used was the public health capacity building program "Health--Plan for it", which was developed with the aim to assist the counties to overcome recognized weaknesses and introduce more effective and efficient local public health practices. Two main instruments were used: Local Public Health Practice Performance Measures Instrument, and Basic Priority Rating System. This program has helped counties to asses population health needs in a participatory manner, to plan for health and, ultimately, assure provision of the right kind and quality of services (better tailored to population health needs). This program's benefits are going beyond and above the county level. It provides support for the Healthy Cities project locally, and facilitates changes in national policymaking body's mindset that a "one-size-fits-all" approach is sufficient.     

Flow cytometry analysis of single-strand DNA damage in neuroblastoma cell lines using the F7-26 monoclonal antibody.

The F7-26 monoclonal antibody (Mab) has been reported to be specific for single-strand DNA damage (ssDNA) and to also identify cells in apoptosis. We carriedout studies to determine if F7-26 binding measured by flow cytometry was able to specifically identify exogenous ssDNA as opposed to DNA damage from apoptosis. Neuroblastoma cells were treated with melphalan (L-PAM), fenretinide, 4-hydroperoxycyclophosphamide (4-HC)+/-pan-caspase inhibitor BOC-d-fmk, topotecan or with 10Gy gamma radiation+/-hydrogen peroxide (H2O2) and fixed immediately postradiation. Cytotoxicity was measured by DIMSCAN digital imaging fluorescence assay. The degree of ssDNA damage was analyzed by flow cytometry using Mab F7-26, with DNA visualized by propidium iodide counterstaining. Flow cytometry was used to measure apoptosis detected by terminal deoxynucleotidyltransferase (TUNEL) assay and reactive oxygen species (ROS) by carboxy-dichlorofluorescein diacetate. Irradiated and immediately fixed neuroblastoma cells showed increased ssDNA, but not apoptosis by TUNEL (TUNEL-negative). 4-HC or L-PAM+/-BOC-d-fmk increased ssDNA (F7-26-positive), but BOC-d-fmk prevented TUNEL staining. Fenretinide increased apoptosis by TUNEL but not ssDNA damage detected with F7-26. Enhanced ssDNA in neuroblastoma cells treated with radiation+H2O2 was associated with increased ROS. Topotecan increased both ssDNA and cytotoxicity in 4-HC-treated cells. These data demonstrate that Mab F7-26 recognized ssDNA due to exogenous DNA damage, rather than apoptosis. This assay should be useful to characterize the mechanism of action of antineoplastic drugs.     

Multivariate analysis of vegetational changes in permanent woodland plots with different sewage sludge treatments

Vegetation data from an experiment on the impact of sewage sludge on woodland vegetation dynamics are analysed by ordination to examine the reaction of a forest community to sludge disturbance. Two different kinds of vegetational response are discussed in relation to horizontal patchiness of vegetation. It is suggested that the species-poor component of the vegetation mosaic observed reveals quicker recovery from sludge disturbance than the species-rich component, which is characterized by a more complicated network of interspecific relations. A high correlation between plot-scores on the first ordination axis and sludge dose is found, indicating that in the altered community the main vegetational gradient reflects the intensity of disturbance. An attempt is made to interpret the main gradients of vegetational variation in terms of ecological indicator values of species. It is concluded that the modified competitive ability of species in a changed environment plays the most important role in building up a new community structure.     

Identification of a novel senolytic agent,navitoclax, targeting the Bcl‐2 family of anti‐apoptotic factors

Clearing senescent cells extends healthspan in mice. Using a hypothesis‐driven bioinformatics‐based approach, we recently identified pro‐survival pathways in human senescent cells that contribute to their resistance to apoptosis. This led to identification of dasatinib (D) and quercetin (Q) as senolytics, agents that target some of these pathways and induce apoptosis preferentially in senescent cells. Among other pro‐survival regulators identified was Bcl‐xl. Here, we tested whether the Bcl‐2 family inhibitors, navitoclax (N) and TW‐37 (T), are senolytic. Like D and Q, N is senolytic in some, but not all types of senescent cells: N reduced viability of senescent human umbilical vein epithelial cells (HUVECs), IMR90 human lung fibroblasts, and murine embryonic fibroblasts (MEFs), but not human primary preadipocytes, consistent with our previous finding that Bcl‐xl siRNA is senolytic in HUVECs, but not preadipocytes. In contrast, T had little senolytic activity. N targets Bcl‐2, Bcl‐xl, and Bcl‐w, while T targets Bcl‐2, Bcl‐xl, and Mcl‐1. The combination of Bcl‐2, Bcl‐xl, and Bcl‐w siRNAs was senolytic in HUVECs and IMR90 cells, while combination of Bcl‐2, Bcl‐xl, and Mcl‐1 siRNAs was not. Susceptibility to N correlated with patterns of Bcl‐2 family member proteins in different types of human senescent cells, as has been found in predicting response of cancers to N. Thus, N is senolytic and acts in a potentially predictable cell type‐restricted manner. The hypothesis‐driven, bioinformatics‐based approach we used to discover that dasatinib (D) and quercetin (Q) are senolytic can be extended to increase the repertoire of senolytic drugs, including additional cell type‐specific senolytic agents.     

Optimizing multiplex SNP-based data analysis for genotyping of Mycobacterium tuberculosis isolates

Background

Multiplex ligation-dependent probe amplification (MLPA) is a powerful tool to identify genomic polymorphisms. We have previously developed a single nucleotide polymorphism (SNP) and large sequence polymorphisms (LSP)-based MLPA assay using a read out on a liquid bead array to screen for 47 genetic markers in the Mycobacterium tuberculosis genome. In our assay we obtain information regarding the Mycobacterium tuberculosis lineage and drug resistance simultaneously. Previously we called the presence or absence of a genotypic marker based on a threshold signal level. Here we present a more elaborate data analysis method to standardize and streamline the interpretation of data generated by MLPA. The new data analysis method also identifies intermediate signals in addition to classification of signals as positive and negative. Intermediate calls can be informative with respect to identifying the simultaneous presence of sensitive and resistant alleles or infection with multiple different Mycobacterium tuberculosis strains.

Results

To validate our analysis method 100 DNA isolates of Mycobacterium tuberculosis extracted from cultured patient material collected at the National TB Reference Laboratory of the National Center for Tuberculosis and Lung Diseases in Tbilisi, Republic of Georgia were tested by MLPA. The data generated were interpreted blindly and then compared to results obtained by reference methods. MLPA profiles containing intermediate calls are flagged for expert review whereas the majority of profiles, not containing intermediate calls, were called automatically. No intermediate signals were identified in 74/100 isolates and in the remaining 26 isolates at least one genetic marker produced an intermediate signal.

Conclusion

Based on excellent agreement with the reference methods we conclude that the new data analysis method performed well. The streamlined data processing and standardized data interpretation allows the comparison of the Mycobacterium tuberculosis MLPA results between different experiments. All together this will facilitate the implementation of the MLPA assay in different settings.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-572) contains supplementary material, which is available to authorized users.     

Comparison of HIV-1 Genotypic Resistance Test Interpretation Systems in Predicting Virological Outcomes Over Time

Background

Several decision support systems have been developed to interpret HIV-1 drug resistance genotyping results. This study compares the ability of the most commonly used systems (ANRS, Rega, and Stanford''s HIVdb) to predict virological outcome at 12, 24, and 48 weeks.

Methodology/Principal Findings

Included were 3763 treatment-change episodes (TCEs) for which a HIV-1 genotype was available at the time of changing treatment with at least one follow-up viral load measurement. Genotypic susceptibility scores for the active regimens were calculated using scores defined by each interpretation system. Using logistic regression, we determined the association between the genotypic susceptibility score and proportion of TCEs having an undetectable viral load (<50 copies/ml) at 12 (8–16) weeks (2152 TCEs), 24 (16–32) weeks (2570 TCEs), and 48 (44–52) weeks (1083 TCEs). The Area under the ROC curve was calculated using a 10-fold cross-validation to compare the different interpretation systems regarding the sensitivity and specificity for predicting undetectable viral load. The mean genotypic susceptibility score of the systems was slightly smaller for HIVdb, with 1.92±1.17, compared to Rega and ANRS, with 2.22±1.09 and 2.23±1.05, respectively. However, similar odds ratio''s were found for the association between each-unit increase in genotypic susceptibility score and undetectable viral load at week 12; 1.6 [95% confidence interval 1.5–1.7] for HIVdb, 1.7 [1.5–1.8] for ANRS, and 1.7 [1.9–1.6] for Rega. Odds ratio''s increased over time, but remained comparable (odds ratio''s ranging between 1.9–2.1 at 24 weeks and 1.9–2.2 at 48 weeks). The Area under the curve of the ROC did not differ between the systems at all time points; p = 0.60 at week 12, p = 0.71 at week 24, and p = 0.97 at week 48.

Conclusions/Significance

Three commonly used HIV drug resistance interpretation systems ANRS, Rega and HIVdb predict virological response at 12, 24, and 48 weeks, after change of treatment to the same extent.