Detection of Emerging and Re-Emerging Pathogens in Surface Waters Close to an Urban Area

Current knowledge about the spread of pathogens in aquatic environments is scarce probably because bacteria, viruses, algae and their toxins tend to occur at low concentrations in water, making them very difficult to measure directly. The purpose of this study was the development and validation of tools to detect pathogens in freshwater systems close to an urban area. In order to evaluate anthropogenic impacts on water microbiological quality, a phylogenetic microarray was developed in the context of the EU project µAQUA to detect simultaneously numerous pathogens and applied to samples from two different locations close to an urban area located upstream and downstream of Rome in the Tiber River. Furthermore, human enteric viruses were also detected. Fifty liters of water were collected and concentrated using a hollow-fiber ultrafiltration approach. The resultant concentrate was further size-fractionated through a series of decreasing pore size filters. RNA was extracted from pooled filters and hybridized to the newly designed microarray to detect pathogenic bacteria, protozoa and toxic cyanobacteria. Diatoms as indicators of the water quality status, were also included in the microarray to evaluate water quality. The microarray results gave positive signals for bacteria, diatoms, cyanobacteria and protozoa. Cross validation of the microarray was performed using standard microbiological methods for the bacteria. The presence of oral-fecal transmitted human enteric-viruses were detected using q-PCR. Significant concentrations of Salmonella, Clostridium, Campylobacter and Staphylococcus as well as Hepatitis E Virus (HEV), noroviruses GI (NoGGI) and GII (NoGII) and human adenovirus 41 (ADV 41) were found in the Mezzocammino site, whereas lower concentrations of other bacteria and only the ADV41 virus was recovered at the Castel Giubileo site. This study revealed that the pollution level in the Tiber River was considerably higher downstream rather than upstream of Rome and the downstream location was contaminated by emerging and re-emerging pathogens.

Cardiac rehabilitation uptake following myocardial infarction: cross-sectional study in primary care

Background Policies suggest that primary care should be more involved in delivering cardiac rehabilitation. However, there is a lack of information about what is known in primary care regarding patients' invitation or attendance. Aim To determine, within primary care, how many patients are invited to and attend rehabilitation after myocardial infarction (MI), examine sociodemographic factors related to invitation, and compare quality of life between those who do and do not attend. Design of study Review of primary care paper and computer records; cross-sectional questionnaire. Setting Northern Ireland general practices (38); stratified sample, based on practice size and health board area. Method Patients, identified from primary care records, 12-16?weeks after a confirmed diagnosis of MI, were posted questionnaires, including a validated MacNew post-MI quality-of-life questionnaire. Practices returned anonymised data for non-responders. Results Information about rehabilitation was available for 332 of the 432 patients identified (76.9%): 162 (37.5%) returned questionnaires. Of the total sample, 54.4% (235/432) were invited and 37.0% (160/432) attended; of those invited, 68.1% (160/235) attended. Invited patients were younger than those not invited (mean age 63?years [standard deviation SD 16] versus 68.5?years [SD 16]); mean difference 5.5?years (95% confidence interval [CI] = 1.7 to 9.3). Among questionnaire responders, those who attended were younger and reported better emotional, physical, and social functioning than non-attenders (P = 0.01; mean differences 0.44 (95% CI = 0.11 to 0.77), 0.48 (95% CI = 0.10 to 0.85) and 0.54 (95% CI = 0.15 to 0.94) respectively). Conclusion Innovative strategies are needed to improve cardiac rehabilitation uptake, integration of hospital and primary care services, and healthcare professionals' awareness of patients' potential for health gain after MI.

Erythropoietin-Induced Activation of the JAK2/STAT5, PI3K/Akt, and Ras/ERK Pathways Promotes Malignant Cell Behavior in a Modified Breast Cancer Cell Line

Abstract Erythropoietin (Epo), the major regulator of erythropoiesis, and its cognate receptor (EpoR) are also expressed in nonerythroid tissues, including tumors. Clinical studies have highlighted the potential adverse effects of erythropoiesis-stimulating agents when used to treat cancer-related anemia. We assessed the ability of EpoR to enhance tumor growth and invasiveness following Epo stimulation. A benign noninvasive rat mammary cell line, Rama 37, was used as a model system. Cell signaling and malignant cell behavior were compared between parental Rama 37 cells, which express few or no endogenous EpoRs, and a modified cell line stably transfected with human EpoR (Rama 37-28). The incubation of Rama 37-28 cells with pharmacologic levels of Epo led to the rapid and sustained increases in phosphorylation of signal transducers and activators of transcription 5, Akt, and extracellular signal-regulated kinase. The activation of these signaling pathways significantly increased invasion, migration, adhesion, and colony formation. The Epo-induced invasion capacity of Rama 37-28 cells was reduced by the small interfering RNA-mediated knockdown of EpoR mRNA levels and by inhibitors of the phosphoinositide 3-kinase/Akt and Ras/extracellular signal-regulated kinase signaling pathways with adhesion also reduced by Janus-activated kinase 2/signal transducers and activators of transcription 5 inhibition. These data show that Epo induces phenotypic changes in the behavior of breast cancer cell lines and establishes links between individual cell signaling pathways and the potential for cancer spread.

Antibody Targeting of Camptothecin-Loaded PLGA Nanoparticles to Tumor Cells

Antibody targeting of drug substances can improve the efficacy of the active molecule, improving distribution and concentration of the drug at the site of injury/disease. Encapsulation of drug substances into polymeric nanoparticles can also improve the therapeutic effects of such compounds by protecting the molecule until its action is required. In this current study, we have brought together these two rationales to develop a novel immunonanoparticle with improved therapeutic effect against colorectal tumor cells. This nanoparticle comprised a layer of peripheral antibodies (Ab) directed toward the Fas receptor (CD95/Apo-1) covalently attached to poly(lactide-co-glycolide) nanoparticles (NP) loaded with camptothecin. Variations in surface carboxyl density permitted up to 48.5 mu g coupled Ab per mg of NP and analysis of nanoparticulate cores showed efficient camptothecin loading. Fluorescence visualization studies confirmed internalization of nanoconstructs into endocytic compartments of HCT 116 cells, an effect not evident in NP without superficial Ab. Cytotoxicity studies were then carried out against HCT116 cells. After 72 h, camptothecin solution resulted in an IC50 of 21.8 ng mL(-1). Ab-directed delivery of NP-encapsulated camptothecin was shown to be considerably more effective with an IC50 of 0.37 ng mL(-1). Calculation of synergistic ratios for these nanoconstructs demonstrated synergy of pharmacological relevance. Indeed, the results in this paper suggest that the attachment of anti-Fas antibodies to camptothecin-loaded nanoparticles may result in a therapeutic strategy that could have potential in the treatment of tumors expressing death receptors.

The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.

Missing Links, Cultural Modernity and the Dead: Anatomically Modern Humans in the Great Cave of Niah (Sarawak, Borneo)

The Great Cave of Niah in Sarawak (northern Borneo) came into the gaze of Western Science through the work of Alfred Russell Wallace, who came to Sarawak in the 1850s to search for ‘missing links’ in his pioneering studies of evolution and the natural history of Island Southeast Asia and Australasia. The work of Tom and Barbara Harrisson in the 1950s and 1960s placed the Great Cave, and particularly their key find, the ‘Deep Skull’, at the nexus of the evolving archaeological framework for the region: for decades the skull, dated in 1958 by adjacent charcoal to c.40,000 BP, was the oldest fossil of an anatomically modern human anywhere in the world and thus critical to ideas about human evolution and dispersal. Although several authorities later questioned the provenance and antiquity of the Deep Skull, renewed investigations of the Harrisson excavations since 2000 have shown that it can be attributed securely to a specific location in the Pleistocene stratigraphy, with direct U-series dating on a piece of the skull indicating an age for it of c.37,500 BP and the first evidence for associated human activity at the site going back to c.50,000 BP. The new work also indicates that the skull is part of a cultural deposit, perhaps a precursor to the long tradition in Borneo of processing of the dead and secondary burial. These indicators of cultural complexity chime with the complexity of the subsistence behaviour of the early users of the caves discussed by Philip Piper and Ryan Rabett in chapter ten of this volume.

Interobserver Reliability of Radiologists' Interpretations of Mobile Chest Radiographs for Nursing Home-Acquired Pneumonia

Objectives: To determine the interobserver reliability of radiologists' interpretations of mobile chest radiographs for nursing home-acquired pneumonia. Design: A cross-sectional reliability study. Setting: Nursing homes and an acute care hospital. Participants: Four radiologists reviewed 40 mobile chest radiographs obtained from residents of nursing homes who met a clinical definition of lower respiratory tract infections. Measurements: Radiologists were asked to interpret radiographs with respect to the film quality; presence, pattern, and extent of an infiltrate; and the presence of a pleural effusion or adenopathy. Interrater reliability was evaluated using the intraclass correlation coefficient derived from a 2-way random effects model. Results: On average the radiologists reported that 6 of the 40 films were of very good or excellent quality and 16 of the 40 were of fair or poor quality. When the finding of an infiltrate was dichotomized (0 = no; 1 = possible, probable, or definite) all 4 radiologists agreed on 21 of the 37 chest radiographs. The intraclass correlation coefficient for the presence or absence of infiltrates was 0.54 (95% confidence intervals [CI] 0.38 to 0.69). For the 14 radiographs where infiltrates were observed by all radiologists, intraclass correlation coefficients for the presence of pleural effusions was 0.08 (95% CI -0.10 to 0.41), hilar adenopathy 0.54 (95% CI 0.29 to 0.79), and mediastinal adenopathy 0.49 (95% CI 0.21 to 0.76). Conclusion: In conclusion, the interrater agreement among radiologists for mobile chest radiographs in establishing the presence or absence of an infiltrate can be judged to be "fair." Treatment decisions need to include clinical findings and should not be made based on radiographic findings alone. © 2006 American Medical Directors Association.