Recombinase polymerase amplification for fast, selective, DNA-based detection of faecal indicator Escherichia coli.

The bacterium Escherichia coli is commonly associated with the presence of faecal contamination in environmental samples, and is therefore subject to statutory surveillance. This is normally done using a culture-based methodology, which can be slow and laborious. Nucleic acid amplification for the detection of E. coli DNA sequences is a significantly more rapid approach, suited for applications in the field such as a point of sample analysis, and to provide an early warning of contamination. An existing, high integrity qPCR method to detect the E. coli ybbW gene, which requires almost an hour to detect low quantities of the target, was compared with a novel, isothermal RPA method, targeting the same sequence but achieving the result within a few minutes. The RPA technique demonstrated equivalent inclusivity and selectivity, and was able to detect DNA extracted from 100% of 99 E. coli strains, and exclude 100% of 30 non-target bacterial species. The limit of detection of the RPA assay was at least 100 target sequence copies. The high speed and simple, isothermal amplification chemistry may indicate that RPA is a more suitable methodology for on-site E. coli monitoring than an existing qPCR technique.

Medically Defined Infertility Versus Self-Perceived Fertility Problem: Implications of Survey Wording for Assessing Associations with Depressive Symptoms.

Objective: To examine how measures of infertility based on medical criteria and based on self-perception relate to depressive symptoms among women with infertility. Background: Survey-based studies of depressive symptoms have used either measures of self-reported infertility based on meeting medical criteria or measures of self-perceived fertility problems, but seldom both. It is, therefore, not known which type of measure is more closely associated with depressive symptoms. Materials and Methods: Using ordinary least-squares multiple regression, this study compares associations between a measure of meeting medical criteria for infertility and a measure of self-perceived fertility problems with a common measure of depressive symptoms. Data come from the National Survey of Fertility Barriers, a population-based survey of 4,711 U.S. women. Results: Both meeting medical criteria for infertility and self-perception were associated with depressive symptoms after controlling for a number of relevant variables, but the coefficient for the self-perception measure was slightly higher than the coefficient for medical criteria. Conclusion: If possible, both medical criteria and self-perception measures should be used in studies of the consequences of infertility for psychosocial outcomes. If only one measure can be used, self-perception of a fertility problem is an acceptable measure.

Buzz off! An evaluation of ultrasonic acoustic vibration for the disruption of marine micro-organisms on sensor-housing materials.

Biofouling is a process of ecological succession which begins with the attachment and colonization of micro-organisms to a submerged surface. For marine sensors and their housings, biofouling can be one of the principle limitations to long-term deployment and reliability. Conventional antibiofouling strategies using biocides can be hazardous to the environment, and therefore alternative chemical-free methods are preferred. In this study, custom-made testing assemblies were used to evaluate ultrasonic vibration as an antibiofouling process for marine sensor-housing materials over a 28-day time course. Microbial biofouling was measured based on (i) surface coverage, using fluorescence microscopy and (ii) bacterial 16S rDNA gene copies, using Quantitative polymerase chain reaction (PCR). Ultrasonic vibrations (20 KHz, 200 ms pulses at 2-s intervals; total power 16·08 W) significantly reduced the surface coverage on two plastics, poly(methyl methacrylate) and polyvinyl chloride (PVC) for up to 28 days. Bacterial gene copy number was similarly reduced, but the results were only statistically significant for PVC, which displayed the greatest overall resistance to biofouling, regardless of whether ultrasonic vibration was applied. Copper sheet, which has intrinsic biocidal properties was resistant to biofouling during the early stages of the experiment, but inhibited measurements made by PCR and generated inconsistent results later on. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, ultrasonic acoustic vibration is presented as a chemical-free, ecologically friendly alternative to conventional methods for the perturbation of microbial attachment to submerged surfaces. The results indicate the potential of an ultrasonic antibiofouling method for the disruption of microbial biofilms on marine sensor housings, which is typically a principle limiting factor in their long-term operation in the oceans. With increasing deployment of scientific apparatus in aquatic environments, including further offshore and for longer duration, the identification and evaluation of novel antifouling strategies that do not employ hazardous chemicals are widely sought.

A multi-parametric assessment of decontamination protocols for the subglacial Lake Ellsworth probe.

Direct measurement and sampling of pristine environments, such as subglacial lakes, without introducing contaminating microorganisms and biomolecules from the surface, represents a significant engineering and microbiological challenge. In this study, we compare methods for decontamination of titanium grade 5 surfaces, the material extensively used to construct a custom-made probe for reaching, measuring and sampling subglacial Lake Ellsworth in West Antarctica. Coupons of titanium were artificially contaminated with Pseudomonas fluorescens bacteria and then exposed to a number of decontamination procedures. The most effective sterilants were (i) hydrogen peroxide vapour, and (ii) Biocleanse™, a commercially available, detergent-based biocidal solution. After each decontamination procedure the bacteria were incapable of proliferation, and showed no evidence of metabolic activity based on the generation of adenosine triphosphate (ATP). The use of ultraviolet irradiation or ethyl alcohol solution was comparatively ineffective for sterilisation. Hydrogen peroxide vapour and ultraviolet irradiation, which directly damage nucleic acids, were the most effective methods for removing detectable DNA, which was measured using 16S rRNA gene copy number and fluorescence-based total DNA quantification. Our results have not only been used to tailor the Ellsworth probe decontamination process, but also hold value for subsequent engineering projects, where high standards of decontamination are required.

Oaths, Promises, and Compulsory Duties: Kant's Response to Mendelssohn's Jerusalem.

This article argues that Kant's essay on enlightenment responds to Moses Mendelssohn's defense of the freedom of conscience in Jerusalem. While Mendelssohn holds that the freedom of conscience as an inalienable right, Kant argues that the use of one's reason may be constrained by oaths. Kant calls such a constrained use of reason the private use of reason. While he also defends the unconditional freedom of the public use of reason, Kant believes that one makes oneself a part of the machinery of the church or state by swearing an oath to and assuming a position within those institutions.

Neck narrowing in resurfacing hip arthroplasty: a vascular insult?

INTRODUCTION: Recent reports show increased failure rates in hip resurfacings that display >10 % neck narrowing. The etiology of neck narrowing remains unknown. METHODS: We assessed 80 hip resurfacings at mean 3.5 years follow-up. RESULTS: The overall rate of significant narrowing was 11.25 %. Neck narrowing occurred in 4 % of patients using an anterolateral approach and 23.3 % using a posterior approach (P = 0.019). Logistic regression showed that both surgical approach and cup inclination angle were the most important risk factors for the development of narrowing. The odds of the presence of narrowing increased for every degree increase in cup abduction angle (P = 0.021). There was no significant association with age, sex, pre-operative diagnosis, pre- and post-operative SF-36 scores, neck shaft angle, femoral or acetabular component sizes. CONCLUSION: We postulate that neck narrowing is a result of damage to the medial circumflex femoral vessel when resurfacing through a posterior approach.

Computer-aided detection for CT colonography: incremental benefit of observer training.

The purpose of this study was to investigate the incremental effect of focused training on observer performance when using computer-assisted detection (CAD) software to interpret CT colonography (CTC). Six radiologists who were relatively inexperienced with CTC interpretation underwent 1 day of focused training before reading 20 patient datasets with the assistance of CAD software (ColonCAR 1.3, Medicsight PLC). Sensitivity, specificity and interpretation times were determined and compared with previous performance when reading the same datasets but without the benefit of focused training, using the binomial exact test and Wilcoxon's signed rank test. Per-polyp sensitivity improved after training by 18% overall (95% confidence interval (CI): 14-24%, p<0.001) and was greatest for polyps of 6-9 mm (26%, 95% CI: 18-34%, p<0.001). Absolute sensitivity was 23% (9-36%), 51% (33-71%) and 74% (44-100%) for polyps of or=10 mm, respectively. Specificity fell significantly after focused training (median of 5.5 false positives per 20 datasets (interquartile range (IQR): 4-6) post-training vs median of 2.5 (IQR: 1-5) pre-training, p = 0.03). Interpretation time also increased significantly after training (from a median of 9.3 min (IQR: 9.3-14.5 min) to a median of 17.1 min (IQR: 15.4-19.4 min), p = 0.03). In conclusion, one day of training increases observer polyp sensitivity when using CAD for CTC at the expense of increased reporting time and reduction in specificity.

Psychological distress by type of fertility barrier.

BACKGROUND: We examined fertility-specific distress (FSD) and general distress by type of fertility barrier (FB). METHODS: In a random sample telephone survey, 580 US women reported their fertility intentions and histories. Six groups of women were identified: (i) no FBs, (ii) infertile with intent, (iii) infertile without intent, (iv) other fertility problems, (v) miscarriages and (vi) situational barriers. Multiple regression analyses were used to compare groups with FBs. RESULTS: Sixty-one percent reported FBs and 28% reported an inability to conceive for at least 12 months. The infertile with intent group had the highest FSD, which was largely explained by (a) self-identification as infertile and (b) seeking medical help for fertility. The no FB group had a mean Center for Epidemiological Studies Depression scale score above the commonly used cut-off of 16, although 23% of the women with FBs did score above 16. CONCLUSIONS: FBs are common. Self-identification as infertile is the largest source of FSD. More women with FBs had elevated general distress than women without FBs; mean general distress was below 16 for all FB groups. It may be that, for some women (even those with children), FBs can have lasting emotional consequences, but many women do heal from the emotional distress that may accompany fertility difficulties.

Conventional and experimental treatment of cerebral malaria.

The most severe complication of Plasmodium falciparum infection is cerebral malaria (CM). Cerebral malaria implies the presence of neurological features, especially impaired consciousness. The treatment of CM is limited to: (i) a few conventional anti-malarial drugs (quinine or artemisinins), (ii) adjunctive treatments (initial stabilisation, blood exchange transfusion, osmotic diuretics and correction of hypoglycaemia, acidosis and hypovolaemia) and (iii) immunomodulation. There are clear procedures concerning treatment of CM, which include the use of the anti-plasmodial drugs. Adjunctive treatments are permissible but there is no single official guideline and immune intervention is a possibility currently being examined in rodent models only. The suggested immunomodulation approach is based on the strong likelihood that CM is the result of an immunopathological process. P. falciparum initiates the multifactorial chain of events leading to lethal CM and, after a certain stage, it is impossible to stop the progression even by using anti-malarial drugs. We present evidence that CM is a result of a dysregulated immune response. Therefore, it might be prevented by early modulation of discrete factors that participate in this process. In experimental systems, some immunomodulators delay or prevent CM without affecting the parasitaemia. Therefore, in the future the ultimate treatment of CM may be a combination of an anti-malarial and an immunomodulator. However, the overall effect of an immunomodulator would need to be carefully examined in view of concomitant infections, especially in malaria endemic areas.

History of affective disorder and the temporal trajectory of fatigue in rheumatoid arthritis.

This study examines whether the general level and rate of change of fatigue over time is different for those rheumatoid arthritis (RA) patients with and those without a history of affective disorder (AD). Four hundred fifteen RA patients from a national panel had yearly telephone interviews to obtain fatigue and distress reports, and a one-time semistructured assessment of the history of depression and generalized anxiety disorder Growth-curve analysis was used to capture variations in initial fatigue levels and changes in fatigue over 7 years for those with and without a history. RA patients with a history of major AD reported levels of fatigue that were 10% higher than those without a history in the 1st year of the study. Their fatigue reports remained elevated over 7 years. Further analysis showed that the effects of a history of AD on fatigue are fully mediated through current distress, although those with a history had a significantly smaller distress-fatigue slope. Thus, a history of AD leaves RA patients at risk for a 7-year trajectory of fatigue that is consistently higher than that of patients without a history. The elevation in fatigue reports is, at least in part, a function of enduring levels of distress.

Depression and the long-term risk of pain, fatigue, and disability in patients with rheumatoid arthritis.

OBJECTIVE: To determine whether a previous episode of major depression leaves a "scar" that places previously depressed patients with rheumatoid arthritis (RA) at risk for experiencing high levels of pain, fatigue, and disability. METHODS: A cohort of 203 patients with RA was randomly selected from a national panel and interviewed by phone about pain, fatigue, depressive symptoms, disability, and history of major depression. RESULTS: Excluding patients who met the criteria for current major depression, patients with both a history of depression and many depressive symptoms at the time of the interview (dysphoria) reported more pain than those without current dysphoria, irrespective of whether they had a history of depression. Dysphoria alone was not reliably related to pain reports. CONCLUSION: An episode of major depression, even if it occurs prior to the onset of RA, leaves patients at risk for higher levels of pain when depressive symptoms persist, even years after the depressive episode.

TNF-alpha and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells.

Vascular cell adhesion molecule-1 (VCAM-1) is expressed on the endothelium and vascular smooth muscle in atherosclerosis, where it is thought to recruit alpha4 integrin-positive leukocytes, which play a role in disease progression. In this study, we show an increase of VCAM-1 expression on vascular smooth muscle cells (VSMC) results in increased adhesion of alpha4 integrin-positive lymphocytes. Additionally, we examine the regulation of VCAM-1 expression by cytokines in cultured VSMC. Previously in endothelial cells, we have demonstrated that TNF-alpha increases transcription of the VCAM-1 gene, whereas IL-4 acts to increase VCAM-1 mRNA stability. The combination of a cytokine that increases transcription with a cytokine that stabilizes mRNA results in a synergistic increase in VCAM-1 expression. In this study, we show that the combination of TNF-alpha with IL-4 also resulted in a synergistic increase in VCAM-1 expression on VSMC; however, the mechanism of cytokine activation differed. In contrast to endothelial cells, IL-4 stimulated VCAM-1 gene transcription in the VSMC, but there was little effect of TNF-alpha alone. Additionally, the synergy between TNF-alpha and IL-4 appears to result, at least in part, from a cooperative transcriptional mechanism.

Gender, paid work, and symptoms of emotional distress in rheumatoid arthritis patients.

OBJECTIVE: To evaluate the relative contribution of gender-related work conditions, gender-related socialization practices, and disease characteristics to the explanation of emotional distress in men and women with rheumatoid arthritis (RA). METHODS: Three hundred sixty-nine RA patients who were employed outside the home were recruited from a national randomized sample of rheumatology practices. Data on paid work and disease characteristics were obtained by telephone interview. Emotional distress was measured by the Center for Epidemiological Studies Depression (CES-D) scale. Hierarchical ordinary least-squares regression was used to assess the relationship of sex, class, work characteristics, and disease characteristics to both the CES-D summary scale and the CES-D factor structure. RESULTS: Differences in emotional distress were explained best by functional ability and pain and secondarily by the characteristics of paid work, with no independent effect for sex. Distress increased with decreasing functional ability, increasing pain, and exposure to such work characteristics as low autonomy, low income, and high demands. No sex differences in any of the CES-D subscales remained after controlling for disease and work variables. CONCLUSION: Among employed RA patients with high levels of functional disability and exposure to stressful work characteristics, men and women are at equal risk of experiencing emotional distress.

Predictors of work disability in rheumatoid arthritis patients. A five-year followup.

OBJECTIVE: To evaluate regression models that include social, attitudinal, work structure, health status, and family characteristics, with regard to their prediction of work disability in a national sample of patients with rheumatoid arthritis (RA). METHODS: Four hundred ninety-eight employed RA patients were recruited from a national sample of private rheumatology practices. Three hundred ninety-two remained in the study after 5 years. Data were collected from patients by telephone interview, and patients' physicians provided written clinical assessments. Only variables on which information was obtained in year 1 were used to predict work status in year 5, using hierarchical multiple logistic regression analysis. RESULTS: The significant predictors of work disability were age (odds ratio [OR] 1.04), number of deformed joints (OR 1.26), number of joints with flare (OR 1.23), the complexity of working with things at work (OR 0.88), and the desire to remain employed (OR 2.3). The risk of work disability increased with increasing age, more severe disease, greater complexity of involvement with things at work, reduced work hours, and desire to not be working outside the home. CONCLUSION: The risk of becoming work disabled in 5 years was predicted more by clinical status at entry into the study than by work structure. These results, which contradict previous research on work disability in arthritis, prompt a rethinking of future studies of work disability in RA.

Control of vascular cell adhesion molecule-1 gene promoter activity during neural differentiation.

Here we demonstrate that vascular cell adhesion molecule-1 (VCAM-1) is expressed in the developing central nervous system on neuroepithelial cells, which are the precursors of neurons and glia. As these cells differentiate, VCAM-1 is restricted to a subset of the glial population. An understanding of mechanisms responsible for this restricted pattern could provide insights into how lineage-specific gene expression is maintained during neural differentiation. As a model of neural differentiation, we turned to the P19 embryonic carcinoma cell line, which in response to retinoic acid will differentiate along a neural pathway. We show that VCAM-1 expression on the differentiating P19 cells resembles that in the central nervous system. Transfection of VCAM-1 gene promoter constructs into P19 cells revealed that the VCAM-1 gene is controlled sequentially by negative and positive elements during differentiation. We present evidence that early during differentiation, POU proteins block VCAM-1 gene activity; however, later in differentiation coincident with the appearance of VCAM-1 the pattern of POU proteins changes and the VCAM-1 gene promoter is activated. This activation is mediated through the NF kappa B/rel complex p50/p65, which forms during P19 cell differentiation.

Vascular cell adhesion molecule 1: contrasting transcriptional control mechanisms in muscle and endothelium.

Interaction between vascular cell adhesion molecule 1 (VCAM-1), which appears on the surface of endothelial cells in response to inflammation, and its integrin counter receptor, alpha 4 beta 1, on immune cells is responsible for targeting these immune cells to cytokine-stimulated endothelium. In addition to its role in the immune system, VCAM-1 is also expressed in a developmentally specific pattern on differentiating skeletal muscle, where it mediates cell-cell interactions important for myogenesis through interaction with alpha 4 beta 1. In contrast to endothelium, there is high basal expression of VCAM-1 in skeletal muscle cells and the expression is not cytokine-responsive. Here, we examine the molecular basis for these contrasting patterns of expression in muscle and endothelium, using VCAM-1 promoter constructs in a series of transfection assays. In endothelial cells, octamer binding sites act as silencers that prevent VCAM-1 expression in unstimulated cells. Tumor necrosis factor alpha overcomes the negative effects of these octamers and activates the promoter through two adjacent NF-kappa B binding sites. In muscle cells, a position-specific enhancer located between bp -21 and -5 overrides the effect of other promoter elements, resulting in constitutive VCAM-1 expression. A nuclear protein binds the position-specific enhancer in muscle but not endothelial cells; thus the pattern of expression of this protein could control enhancer activity.

Characterization of the promoter for vascular cell adhesion molecule-1 (VCAM-1).

Vascular cell adhesion molecule-1 (VCAM-1) was first identified as a protein that appears on the surface of endothelial cells after exposure to inflammatory cytokines. Through interaction with its integrin counter receptor VLA-4, VCAM-1 mediates cell-cell interactions important for immune function. We have cloned and begun characterization of the promoter for the VCAM-1 gene. In a series of transfection assays into human umbilical vein endothelial cells (HUVECs), we find that silencers between positions -1.641 kilobases and -288 base pairs restrict promoter activity, and that treatment with tumor necrosis factor-alpha overcomes this inhibition and activates the promoter through two NF kappa B sites located at positions -77 and -63 base pairs of the VCAM-1 gene. This responsiveness appears cell-specific since constructs containing the VCAM-1 NF kappa B sites are not responsive to tumor necrosis factor alpha in the T-cell line Jurkat. The two VCAM-1 NF kappa B sites, which differ slightly in their sequence, form distinct complexes in gel retardation assays, suggesting that they interact with different NF kappa B-site binding proteins. The distribution of these proteins could then control activity of the NF kappa B sites. We conclude that the pattern of VCAM-1 expression in HUVECs is controlled by a combination of these silencers and NF kappa B sites.

Identification of a protein that interacts with the nuclear factor-1 (NF-1) binding site in cells that do not express NF-1: comparison to NF-1, cellular distribution, and effect on transcription.

We examined expression of nuclear factor-1 (NF-1) in different cell lines. Expression was low or undetectable in T and B lymphocyte cell lines, whereas fibroblasts and other adherent cell lines generally had a relatively high level of NF-1 mRNA. In cell lines that did not express NF-1, gel retardation assays, nevertheless, indicated complexes between a protein or proteins and the NF-1 site. These complexes were less abundant than those formed with NF-1, they migrated more slowly, and they appeared as single species instead of the multiple species observed with NF-1. NF-1 site-binding proteins were compared in the fibrosarcoma cell line HT-1080 (expressed the highest level of NF-1 in our study) and the B cell line Raji (does not express NF-1). UV-crosslinking studies indicated that the NF-1 site-binding proteins in both cell lines were similar in size. Proteolytic clipping band shift assays suggested that the Raji protein and NF-1 share structural similarity in their DNA binding domains, but are distinct proteins. The NF-1 site mediated transcriptional stimulation in cell lines where NF-1 is expressed; however, this element did not affect transcription in cell lines that do not express NF-1, suggesting that the NF-1 site-binding protein in these cells is functionally distinct from NF-1.

The alpha 5 beta 1 fibronectin receptor. Characterization of the alpha 5 gene promoter.

A genomic clone containing the gene for the alpha 5 subunit of the human alpha 5 beta 1 integrin complex was isolated by screening a human genomic library with the previously described alpha 5 cDNA (Argraves, W. S., Suzuki, S., Arai, H., Thompson, K., Pierschbacher, M. D., and Ruoslahti, E. (1987) J. Cell Biol 105, 1183-1190). The alpha 5 gene 5'-flanking region lacks both TATA and CCAAT boxes, and it is located in a CpG island. This region was an active promoter in transfection assays using the HT-1080 cell line (fibrosarcoma), which expresses alpha 5, but was inactive in the Raji cell line (B cell), which does not express alpha 5. These results indicate that the alpha 5 gene 5'-flanking region acts as a promoter that exhibits the expected cell-type specificity. Deletion of alpha 5 promoter sequences from position -657 to -178 caused transcription stimulation, suggesting that a silencer is located between these sites. Successive 5' deletions from position -178 decreased promoter activity until activity was essentially eliminated on deletion to position -27. Isolation of a functional promoter for the alpha 5 gene is a first step in understanding how expression of this gene is controlled at the molecular level.